Mast cells are heterogeneous tissue-resident immune cells, with connective tissue mast cells (CTMCs) and mucosal mast cells in rodents exhibiting distinct phenotypes and activation profiles. Cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily adhesion molecule, has been implicated in mast cell-nerve and mast cell-stromal interactions as well as in the pathogenesis of atopic dermatitis. Here, we investigated CADM1 function in CTMCs using a monoclonal antibody against the extracellular domain of CADM1, termed 3E1. CTMCs were differentiated from bone marrow-derived mast cells (BMMCs) by fibroblast coculture, where CADM1 expression was markedly upregulated compared with BMMCs. Treatment with 3E1 downregulated CADM1 expression and suppressed β-hexosaminidase release from IgE-sensitized, antigen-stimulated CTMCs by 18%, whereas BMMCs were unaffected. In addition, 3E1 reduced FM4-64-positive granule formation in activated CTMCs by 34.2% and 27.3% at 5 and 60 min, respectively. Confocal analysis further showed that 3E1 pretreatment decreased F-actin rearrangement in activated CTMCs by 66.2% at 5 min. In a passive cutaneous anaphylaxis model, intravenous 3E1 reduced dermal mast cell degranulation by 10.4%. These findings identify CADM1 as a regulator of IgE-mediated degranulation in CTMCs and demonstrate 3E1 as a valuable tool for dissecting mast cell heterogeneity in relation to tissue localization.

Intra-abdominal epithelioid neoplasm with EWSR1/FUS::CREB fusions is an emerging entity characterized by a broad age distribution, epithelioid morphology, variable epithelial marker expression, prominent lymphoplasmacytic infiltration, and systemic inflammation. A few ovarian cases have been reported. We describe a 63-yr-old woman who presented with anemia and elevated C-reactive protein. She underwent surgery for a 14-cm right ovarian mass. Grossly, the tumor was solid with cystic change and hemorrhage, and had a light tan cut surface. Histologically, it consisted of uniform sheets of epithelioid cells with ample pale eosinophilic cytoplasm, divided by fibrotic septa with dense lymphoplasmacytic infiltration. Immunohistochemically, the tumor was positive for EMA, WT1, and vimentin; focally positive for CAM5.2; and negative for AE1/AE3, estrogen and progesterone receptors, PAX8, sex cord markers, desmin, HMB45, and Melan A. The Ki-67 labeling index was 20%. The differential diagnoses, including poorly differentiated carcinoma, sex cord-stromal tumors, perivascular epithelioid cell tumor, and inflammatory myofibroblastic tumor, were considered. Whole-genome sequencing revealed a FUS::CREM gene fusion. Based on clinicopathologic and genomic features, the tumor was classified as an ovarian example of EWSR1/FUS::CREB fusion-associated epithelioid neoplasm. Inflammation-related laboratory abnormalities resolved postoperatively. No adjuvant therapy was administered, and the patient remained disease-free at 12 mo. This represents the third reported ovarian tumor with FUS::CREM fusion and the seventh adnexal tumor with EWSR1/FUS::CREB family fusion. Prognostic information on these adnexal tumors is limited, but given the aggressive nature of analogous extra-adnexal and testicular tumors, cautious management and further studies are warranted.

BACKGROUND: Stroke-prone spontaneously hypertensive rats (SHRSP) exhibit slow-twitch muscle-specific hypotrophy compared with normotensive Wistar-Kyoto rats (WKY). Because slow-twitch muscles are prone to disuse atrophy, SHRSP may experience both disuse atrophy and impaired recovery from it. This study investigated the response of SHRSP to disuse atrophy and subsequent recovery, using WKY as a control. RESULTS: WKY and SHRSP were subjected to a 7-day tail suspension followed by reloading for 1, 3, and 7 days. The soleus of WKY and SHRSP showed similar atrophic rates following tail suspension; however, the recovery after reloading was delayed in SHRSP. Moreover, WKY, but not SHRSP, exhibited sarcomere structure disruption after tail suspension, followed by necrosis, inflammatory cell infiltration, and edema upon reloading. Phosphorylation of ribosomal protein S6 (RPS6), an indicator of protein translation, was significantly higher in tail-suspended WKY-but not SHRSP-than those in non-tail-suspended groups after reloading. p70-S6 kinase 1 (S6K1), an upstream protein of RPS6, was phosphorylated at Thr389 in a mechanistic target of rapamycin complex 1-dependent manner to the same extent in both WKY and SHRSP; however, the expression of p60-S6K1-a shorter isoform of p70-S6K1 that activates RPS6 without p60-S6K1 phosphorylation-significantly increased only in tail-suspended WKY compared with those in non-tail-suspended WKY and tail-suspended SHRSP. Previously, p60-S6K1 protein expression was thought to result from an alternative translation of the full-length S6K1 transcript that also produces other S6K1 isoforms. However, recent studies have identified a p60-S6K1-specific transcript, and our PCR results showed that this p60-S6K1-specific transcript, but not the full-length S6K1 transcript, was significantly increased only in tail-suspended WKY corresponding with the increase of p60-S6K1 protein expression. CONCLUSIONS: SHRSP exhibited different phenotypes in disuse atrophy and recovery from it compared with WKY, which could be related to suppressed RPS6 phosphorylation associated with the lack of upregulation in p60-S6K1 expression. These findings suggest that p60-S6K1, in cooperation with p70-S6K1, activates RPS6 and promotes rapid recovery from disuse atrophy by enhancing the transcription of the p60-S6K1-specific transcript. The study also suggests a potential involvement of hypertension in disuse atrophy and its recovery.

BACKGROUND: B7-H3 and delta-like ligand 3 (DLL3) are novel therapeutic targets in extensive-stage small cell lung cancer (ES-SCLC). We aimed to assess the impact of B7-H3 and DLL3 expression on the tumor immune microenvironment (TME) and on the therapeutic efficacy of programmed cell death-ligand 1 (PD-L1) blockade for ES-SCLC. PATIENTS AND METHODS: A total of 146 ES-SCLC patients who received platinum-based chemotherapy either with (Chemo + ICI cohort) or without (Chemo cohort) an immune checkpoint inhibitor was analyzed. Progression-free survival (PFS) and overall survival (OS) were evaluated in each cohort according to B7-H3 or DLL3 expression status as detected by immunohistochemistry. The relation of B7-H3 or DLL3 expression to characteristics of the TME was assessed by immune-related gene expression profiling (irGEP). RESULTS: In the Chemo + ICI cohort, patients with high B7-H3 expression showed a shorter PFS (median of 4.3 vs. 5.4 months; HR of 2.11 with a 95 % Cl of 1.08-4.10; P = 0.03) and OS (median of 8.4 vs. 14.2 months; HR of 1.83 with a 95 % CI of 0.91-3.69; P = 0.09) than those with low B7-H3 expression. In the Chemo cohort, there was no apparent difference in survival outcomes between the high and low B7-H3 expression groups. The irGEP analysis revealed that the effector function of CD8+ T cells was impaired in tumors with high B7-H3 expression. No clear association was apparent between therapeutic efficacy and DLL3 expression status. CONCLUSIONS: High B7-H3 expression may serve as a resistance mechanism for PD-L1 antibody therapy by promoting T cell dysfunction in ES-SCLC.

UNLABELLED:

Objective: Cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member, is abundantly expressed on nerve fibers. Recently, the anti-CADM1 ectodomain antibody 3E1 has been shown to be potentially useful as a local anesthetic due to its high affinity for subcutaneous nerve fibers. When injected intrathecally into mice, where 3E1 accumulates, and whether it serves as an analgesic was examined. METHODS: The 3E1 injection was detected using indocyanine green-based imaging and immunohistochemistry. As a neuropathic pain model, mechanical allodynia-affected mice were created by spinal nerve ligation (SNL). These mice were administered intrathecally with 3E1, control antibody, or pregabalin, and then the pain threshold of the hind paw was measured using von Frey monofilaments. After SNL, followed by intrathecal injection of 3E1, real-time PCR was conducted to examine the expression of pain-related genes, interleukin (IL)-1β, IL-2, IL-6, IL-10, tumor necrosis factor-α, chemokine (C-C motif) ligand (CCL) 2, CCL7, and CCL11. RESULTS: Intrathecally, injected 3E1 was localized mainly on the dorsal root ganglion cell bodies and spinal dorsal horn nerve fibers, with no motor side effects. In the von Frey test, 3E1-injected mice exhibited higher pain thresholds for up to 2 weeks compared to control mice. Pregabalin also raised the threshold, but the effect dissipated within 1 day. While SNL increased mRNA expression of all pain-related genes examined in spinal dorsal horn neurons, 3E1 injection suppressed the increased expression of IL-6. CONCLUSION: 3E1 was suggested to be a potential ganglion-blocking analgesic and spinal anesthetic that acts ten or more times longer than pregabalin.

.

DNA is frequently damaged by genotoxic stresses such as ionizing radiation, reactive oxygen species, and nitrogen species. DNA damage is a key contributor to cancer initiation and progression, and thus the precise and timely repair of these harmful lesions is required. Recent studies revealed transcription as a source of genome instability, and transcription-coupled DNA damage has been a focus in cancer research. Impaired mRNA export is closely related to DNA damage through R-loop formation. The molecular machineries of transcription-coupled DNA damage have been extensively analyzed in Saccharomyces cerevisiae. However, the molecular basis of these phenomena in higher eukaryotes remains elusive. In this review, we focus on the relationship between deregulated mRNA export through the transcription-export-2 (TREX-2) complex and cancer development. Particularly, the expression of germinal center-associated nuclear protein (GANP), a molecular scaffold in the TREX-2 complex, is highly associated with tumorigenesis in mice and humans. Although the deregulated expression of other components in the TREX-2 complex might affect cancer development, we have directly demonstrated the significance of GANP in tumorigenesis using genetically modified mice. Additionally, we describe recent evidence for medical applications demonstrating that the downregulation of the other components may be a good candidate for a chemotherapeutic target in terms of reducing the side effects.

AIMS: Cell adhesion molecule 1 (CADM1) is a member of the immunoglobulin superfamily and is abundantly expressed on nerve fibers. Recently, the anti-CADM1 ectodomain antibody 3E1 has proven useful as a drug delivery vector for CADM1-expressing cells in vitro. When injected subcutaneously into mice, whether 3E1 accumulates on nerve fibers and serves as an analgesic was examined. MAIN METHODS: Injected 3E1 was detected by immunohistochemistry and double immunofluorescence. Analgesic effects were verified by a formalin-induced chemical-inflammatory pain test and video-recorded behavior analysis that were performed 6, 12, and 24 h after antibody injection. Primary cultures of mouse dorsal root ganglion (DRG) cells were incubated with 3E1 and expressions of CADM1 and its key downstream molecules were examined by Western blot analyses and live cell imaging. DRG cells were loaded with a Ca2+ fluorescent indicator Fluo-8 and a femtosecond laser pulse was irradiated near the cell body to mechanically stimulate the nerves. KEY FINDINGS: Subcutaneously injected 3E1 was widely localized almost exclusively on peripheral nerve fibers in the dermis. In formalin tests, 3E1-injected mice exhibited less pain-related behavior than control mice. When 3E1 was added to DRG cell cultures, it localized to neurites and resulted in decreased expression of CADM1, increased phosphorylation of Src and Akt, and CADM1-3E1 complex formation. Femtosecond laser-induced stimulation transmission along neurites was clearly visualized by Fluo-8 fluorescence in control cells, whereas it was markedly suppressed in 3E1-treated cells. SIGNIFICANCE: 3E1 was suggested to be a potential long-acting analgesic based on its high affinity for CADM1.

Most elements of filamentous fungi seen in human tissue by pathologists are hyphae, and encountering other elements may interfere with diagnosis. Sporangia and chlamydospores are such elements that have been described in only a few case reports. We present an autopsy case with the extremely rare coexistence of Mucorales sporangia and chlamydospores in the lung. These fungal elements must be recognized and identified accurately because they can easily be mistaken for other fungi, microorganisms, or degenerated tissue structures.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the alimentary tract. The prognosis depends on the primary site, and small intestinal GISTs have a worse prognosis than gastric GISTs. Molecularly targeted drugs to inhibit tyrosine kinase activity of KIT were used for unresectable or recurrent GISTs. However, secondary resistance to the drugs is often acquired, and treatments based on other mechanisms are needed. Previously, we reported that cell adhesion molecule 1 (CADM1) was highly expressed in most of small intestinal GISTs but not in most of gastric GISTs. In the present study, we examined whether the antibody-drug conjugate (ADC) with anti-CADM1 antibody and monomethyl auristatin E (anti-CAD-ADC) shows anti-tumor effect on CADM1-expressing human GIST cells. The ADC adhibited in this study was previously used for CADM1-expressing human mesothelioma cells and showed anti-tumor effect for them in vitro. GIST-T1 cell line of gastric origin which scarcely expresses CADM1 and GIST-T1 cells transfected with CADM1 cDNA (GIST-T1-CAD cells) which highly expresses CADM1 and represents small intestinal GIST were used. In vitro, anti-CAD-ADC showed remarkable cytotoxic activity on GIST-T1-CAD cells, but control ADC did not. Both anti-CAD-ADC and control ADC did not show anti-tumor effect on original GIST-T1 cells. When GIST-T1-CAD cells were subcutaneously injected to the nude mice, intravenous administration of anti-CAD-ADC showed inhibitory effect for tumor enlargement. Tumor of GIST-T1 cells grew even after anti-CAD-ADC injection. When GIST-T1-CAD cells were injected into peritoneal cavity of the SCID mice, intraperitoneal administration of anti-CAD-ADC showed reduction of the peritoneal tumor. On the other hand, peritoneal tumor grew after control ADC administration. Tissue and organ damage due to administration of anti-CAD-ADC was not apparent by macroscopic and histological examinations in mice. These results indicate that anti-CAD-ADC could have apparent anti-tumor effect on CADM1-expressing human GIST cells both in in vitro and in vivo mouse models.

Cell adhesion molecule 1 (CADM1), a single-pass transmembrane protein, is involved in oncogenesis. We previously demonstrated the therapeutic efficacy of anti-CADM1 ectodomain monoclonal antibodies against mesothelioma; however, the underlying mechanism is unclear. In the present study, we explored the molecular behavior of anti-CADM1 antibodies in CADM1-expressing tumor cells. Sequencing analyses revealed that the anti-CADM1 chicken monoclonal antibodies 3E1 and 9D2 are IgY and IgM isotype antibodies, respectively. Co-administration of 3E1 and 9D2 altered the subcellular distribution of CADM1 from the detergent-soluble fraction to the detergent-resistant fraction in tumor cells. Using recombinant chicken-mouse chimeric antibodies that had been isotype-switched from IgG to IgM, we demonstrated that the combination of the variable region of 3E1 and the constant region of IgM was required for CADM1 relocation. Cytochemical studies showed that 3E1 colocalized with late endosomes/lysosomes after co-administration with 9D2, suggesting that the CADM1-antibody complex is internalized from the cell surface to intracellular compartments by lipid-raft mediated endocytosis. Finally, 3E1 was conjugated with the antimitotic agent monomethyl auristatin E (MMAE) via a cathepsin-cleavable linker. Co-administration of 3E1-monomethyl auristatin E and 9D2 suppressed the growth of multiple types of tumor cells, and this anti-tumor activity was confirmed in a syngeneic mouse model of melanoma. 3E1 and 9D2 are promising drug delivery vehicles for CADM1-expressing tumor cells.

INTRODUCTION: Immune checkpoint inhibitors have recently been approved for the treatment of early-stage NSCLC in the perioperative setting on the basis of phase 3 trials. However, the characteristics of such patients who are susceptible to recurrence after adjuvant chemotherapy or who are likely to benefit from postoperative immunotherapy have remained unclear. METHODS: This biomarker study (WJOG12219LTR) was designed to evaluate cancer stem cell markers (CD44 and CD133), programmed death-ligand 1 (PD-L1) expression on tumor cells, CD8 expression on tumor-infiltrating lymphocytes, and tumor mutation burden in completely resected stage II to IIIA NSCLC with the use of archived DNA and tissue samples from the prospective WJOG4107 trial. Tumors were classified as inflamed or noninflamed on the basis of the PD-L1 tumor proportion score and CD8+ tumor-infiltrating lymphocyte density. The association between each potential biomarker and relapse-free survival (RFS) during adjuvant chemotherapy was assessed by Kaplan-Meier analysis. RESULTS: A total of 117 patients were included in this study. The median RFS was not reached (95% confidence intervals [CI]: 22.4 mo-not reached; n = 39) and 23.7 months (95% CI: 14.5-43.6; n = 41) in patients with inflamed or noninflamed adenocarcinoma, respectively (log-rank p = 0.02, hazard ratio of 0.52 [95% CI: 0.29-0.93]). Analysis of the combination of tumor inflammation category and TP53 mutation status revealed that inflamed tumors without TP53 mutations were associated with the longest RFS. CONCLUSIONS: PD-L1 expression on tumor cells, CD8+ T cell infiltration, and TP53 mutation status may help identify patients with early-stage NSCLC susceptible to recurrence after adjuvant chemotherapy.

BACKGROUND: Immune-related adverse events (irAEs) have been found to predict PD-L1 inhibitor efficacy in metastatic NSCLC. However, the relation of irAEs to clinical outcome for nonmetastatic NSCLC has remained unknown. METHODS: In this multicenter prospective study of Stage III NSCLC treated with PACIFIC regimen, the relation of irAEs to PFS was evaluated by 8-week landmark analysis to minimise lead-time bias as well as by multivariable analysis adjusted for baseline factors. irAEs were categorised as mild or nonmild according to whether they were treated with systemic steroid. RESULTS: Median PFS was 16.0 months, not reached, and 9.7 months for patients without (85 cases) or with mild (21 cases) or nonmild (21 cases) irAEs, respectively. Multivariable analysis indicated that nonmild irAEs were associated with poor PFS, with HRs of 3.86 (95% CI, 1.31-11.38) compared with no irAEs and 11.58 (95% CI, 2.11-63.63) compared with mild irAEs. This pattern was consistent after irAE grade, the number of durvalumab doses and immune profiles (PD-L1 score, CD8+ tumour-infiltrating lymphocyte density, and tumour mutation burden) were taken into consideration. CONCLUSIONS: The development of mild irAEs might predict a better survival outcome, whereas immunosuppressive steroid-treated irAEs were associated with a worse outcome, regardless of baseline clinical and immune profiles.

Cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member, is expressed in endometrial glandular cells highly during the proliferative phase but lowly during the secretory phase. Previously, a CADM1-targeting antibody-drug conjugate (ADC) was generated, in which a humanized anti-CADM1 ectodomain antibody h3E1 was linked with monomethyl auristatin E (h3E1-MMAE ADC). The present study aimed at probing whether this ADC could be useful for the treatment of endometrial neoplasm. Firstly, immunohistochemistry for CADM1 was conducted on proliferative-phase endometrium (n = 13), endometrial hyperplasia (n = 35), and endometrioid carcinoma at various stages (n = 166). CADM1 immunostaining intensity was highest in atypical endometrial hyperplasia and endometrioid carcinoma confined within the endometrium and was decreased stepwise as the carcinoma stage progressed. Next, h3E1-MMAE ADC was examined for its cytotoxicity in vitro using human endometrial adenocarcinoma cell lines expressing CADM1; HEC-1B, HEC-50B, JHUM-3, and OMC-2. The ADC killed these cells in a dose-dependent manner with half maximal inhibitory concentration (IC50) of 12.02 nM for HEC-1B and 2.04 nM for HEC-50B. Collectively, h3E1-MMAE ADC may serve as a noninvasive alternative to simple hysterectomy in the treatment of endometrioid carcinoma confined within the endometrium.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first infects the host nasal mucosa, where the viral spike protein binds to angiotensin-converting enzyme 2 (ACE2) on the mucosal cells. This study aimed at searching host cell surface molecules that could contribute to the infection in two views; abundance on host cells and affinity to the spike protein. Since the nasal mucosa is lined by respiratory and olfactory epithelia, and both express an immunoglobulin superfamily member cell adhesion molecule 1 (CADM1), whether CADM1 would participate in the spike protein binding was examined. Immunohistochemistry on the mouse nasal cavity detected CADM1 strongly in the olfactory epithelium at cell–cell contacts and on the apical surface but just faintly in the respiratory epithelium. In contrast, ACE2 was detected in the respiratory, not olfactory, epithelium. When mice were administered intranasally with SARS-CoV-2 S1 spike protein and an anti-CADM1 ectodomain antibody separately, both were detected exclusively on the olfactory, not respiratory, epithelium. Then, the antibody and S1 spike protein were administered intranasally to mice in this order with an interval of 1 hour. After 3 hours, S1 spike protein was detected as a protein aggregate floating in the nasal cavity. Next, S1 spike protein labeled with fluorescein was added to the monolayer cultures of epithelial cells exogenously expressing ACE2 or CADM1. Quantitative detection of fluorescein bound to the cells revealed that S1 spike protein bound to CADM1 with affinity half as high as to ACE2. Consistently, docking simulation analyses revealed that S1 spike protein could bind to CADM1 three quarters as strongly as to ACE2 and that the interface of ACE2 was similar in both binding modes. Collectively, intranasal S1 spike protein appeared to prefer to accumulate on the olfactory epithelium, and CADM1 was suggested to contribute to this preference of S1 spike protein based on the molecular abundance and affinity.

INTRODUCTION: The PACIFIC regimen of consolidation therapy with the programmed cell death-ligand 1 inhibitor durvalumab after definitive concurrent chemoradiation therapy has become a standard of care for individuals with unresectable stage III NSCLC. Nevertheless, approximately half of the treated patients experience disease progression within 1 year, with the mechanisms of treatment resistance being poorly understood. We here performed a nationwide prospective biomarker study to explore the resistance mechanisms (WJOG11518L:SUBMARINE). METHODS: A total of 135 patients with unresectable stage III NSCLC who received the PACIFIC regimen were included for comprehensive profiling of the tumor microenvironment by immunohistochemistry, transcriptome analysis, and genomic sequencing of pretreatment tumor tissue and flow cytometric analysis of circulating immune cells. Progression-free survival was compared on the basis of these biomarkers. RESULTS: The importance of preexisting effective adaptive immunity in tumors was revealed for treatment benefit regardless of genomic features. We also identified CD73 expression by cancer cells as a mechanism of resistance to the PACIFIC regimen. Multivariable analysis of immunohistochemistry data with key clinical factors as covariables indicated that low CD8+ tumor-infiltrating lymphocyte density and the high CD73+ cancer cells were independently associated with poor durvalumab outcome (hazard ratios = 4.05 [95% confidence interval: 1.17-14.04] for CD8+ tumor-infiltrating lymphocytes; 4.79 [95% confidence interval: 1.12-20.58] for CD73). In addition, whole-exome sequencing of paired tumor samples suggested that cancer cells eventually escaped immune pressure as a result of neoantigen plasticity. CONCLUSIONS: Our study emphasizes the importance of functional adaptive immunity in stage III NSCLC and implicates CD73 as a promising treatment target, thus providing insight forming a basis for development of a new treatment approach in NSCLC.

Abstract Aims programmed death-1 (PD-1) is a negative regulator of immune responses. Upon deletion of PD-1 in mice, symptoms of autoimmunity developed only after they got old. In a model experiment in cancer immunotherapy, PD-1 was shown to prevent cytotoxic T lymphocytes from attacking cancer cells that expressed neoantigens derived from genome mutations. Furthermore, the larger number of genome mutations in cancer cells led to the more robust anti-tumor immune responses after the PD-1 blockade. In order to understand the common molecular mechanisms underlying these findings, we hypothesize that we might have acquired PD-1 during evolution in order to avoid/suppress autoimmune reactions against neoantigens derived from mutations in the genome of aged individuals. Main methods: to test the hypothesis, we introduced random mutations into the genome of young PD-1^-/-and PD-1^+/+mice. We employed two different procedures of random mutagenesis: administration of a potent chemical mutagen N-ethyl-N-nitrosourea (ENU) into the peritoneal cavity of mice and deletion ofMSH2, which is essential for the mismatch-repair activity in the nucleus and, therefore, for the suppression of accumulation of random mutations in the genome. Key findings we observed granulomatous inflammatory changes in the liver of the ENU-treated PD-1 knockout (KO) mice, but not in the wild-type (WT) counterparts. Such lesions also developed in the PD-1/MSH2 double KO mice, but not in the MSH2 single KO mice. Significance: the results we obtained support our hypothesis: PD-1 probably functions to avoid/suppress inflammatory responses against neoantigens derived from genome mutations in aged individuals.

Abstract Aldosterone-producing adenomas (APAs) are the commonest curable cause of hypertension. Most have gain-of-function somatic mutations of ion channels or transporters. Herein we report the discovery, replication and phenotype of mutations in the neuronal cell adhesion gene CADM1. Independent whole exome sequencing of 40 and 81 APAs found intramembranous p.Val380Asp or p.Gly379Asp variants in two patients whose hypertension and periodic primary aldosteronism were cured by adrenalectomy. Replication identified two more APAs with each variant (total, n = 6). The most upregulated gene (10- to 25-fold) in human adrenocortical H295R cells transduced with the mutations (compared to wildtype) was CYP11B2 (aldosterone synthase), and biological rhythms were the most differentially expressed process. CADM1 knockdown or mutation inhibited gap junction (GJ)-permeable dye transfer. GJ blockade by Gap27 increased CYP11B2 similarly to CADM1 mutation. Human adrenal zona glomerulosa (ZG) expression of GJA1 (the main GJ protein) was patchy, and annular GJs (sequelae of GJ communication) were less prominent in CYP11B2-positive micronodules than adjacent ZG. Somatic mutations of CADM1 cause reversible hypertension and reveal a role for GJ communication in suppressing physiological aldosterone production.

OBJECTIVE: Immune checkpoint inhibitors (ICIs) have become a key therapeutic modality for advanced non-small cell lung cancer (NSCLC), but most patients experience primary or acquired resistance to these drugs. We here explored the mechanisms underlying both types of ICI resistance by analysis of the tumor immune microenvironment (TME). MATERIALS AND METHODS: Four patients who experienced a long-term response to ICI treatment (progression-free survival [PFS] of ≥12 months) followed by disease progression, after which a rebiopsy was immediately performed (cohort-A), as well as four patients who experienced early tumor progression during ICI treatment (PFS of <9 weeks, cohort-B) were enrolled in this retrospective study. The pretreatment TME was evaluated by 16- or 17-color multiplex immunohistochemistry (mIHC)-based spatial profiling at the single-cell level for both cohorts. In cohort-A, changes in the TME after disease progression during ICI treatment were also investigated by mIHC analysis and transcriptomic analysis. RESULTS: Pretreatment tumor tissue from cohort-B manifested poor infiltration of tumor-reactive CD8+ T cells characterized by CD39 and CD103 expression or by programmed cell death-1 expression, implicating insufficient recognition of tumor cells by CD8+ T cells as a mechanism of primary ICI resistance. Analysis of the paired tumor specimens from cohort-A revealed various changes in the TME associated with acquired ICI resistance, including substantial infiltration of myeloid-derived suppressor cells and M2-type tumor-associated macrophages without a marked decline in the number of tumor-reactive CD8+ T cells; a decrease in the number of tumor-reactive CD8+ T cells; and an apparent decrease in neoantigen presentation by tumor cells. CONCLUSION: The presence of intratumoral tumor-reactive CD8+ T cells may be a prerequisite for a long-term response to ICI treatment in advanced NSCLC, but it is not sufficient for cancer cell eradication. Various TME profiles are associated with acquired ICI resistance, suggesting that patient-specific strategies to overcome such resistance may be necessary.

Introduction: Despite a considerable benefit of adding immune checkpoint inhibitors (ICIs) to platinum-based chemotherapy for patients with extensive-stage SCLC (ES-SCLC), a durable response to ICIs occurs in only a small minority of such patients. Methods: A total of 135 patients with ES-SCLC treated with chemotherapy either alone (chemo-cohort, n = 71) or together with an ICI (ICI combo-cohort, n = 64) was included in this retrospective study. Tumors were classified pathologically as inflamed or noninflamed on the basis of programmed death-ligand 1 expression and CD8+ tumor-infiltrating lymphocyte density. Immune-related gene expression profiling was performed, and predicted neoantigen load was determined by whole-exome sequencing. Results: Among patients in the ICI combo-cohort, median progression-free survival was 10.8 and 5.1 months for those with inflamed (n = 7) or noninflamed (n = 56) tumors, respectively (log-rank test p = 0.002; hazard ratio of 0.26). Among the 89 patients with immune-related gene expression profiling data available, inflamed tumors had a higher T cell-inflamed GEP score than did noninflamed tumors (-0.18 versus -0.58, p < 0.001). The 12-month progression-free survival rate was 16.1% and 0% for patients in the ICI combo-cohort harboring tumors with a high (n = 26) or low (n = 18) frameshift neoantigen load, respectively. A high-frameshift neoantigen load was associated with up-regulation of gene signatures related to antigen presentation and costimulatory signaling. A durable clinical benefit of ICI therapy was observed only in patients with inflamed tumors and a high-frameshift neoantigen load. Conclusions: Expression of programmed death-ligand 1, CD8+ T cell infiltration, and a high-frameshift neoantigen load are associated with clinical benefit of ICI therapy in ES-SCLC. Clinical trial registration: UMIN000041056.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its S1 spike protein to bind to angiotensin-converting enzyme 2 (ACE2) on human cells in the first step of cell entry. Tryptanthrin, extracted from leaves of the indigo plant, Polygonum tinctorium, using d-limonene (17.3 µg/ml), is considered to inhibit ACE2-mediated cell entry of another type of coronavirus, HCoV-NL63. The current study examined whether this extract could inhibit the binding of the SARS-CoV-2 spike protein to ACE2. Binding was quantified as cell-bound fluorescence intensity in live cell cultures in which canine kidney MDCK cells overexpressing ACE2 were incubated with fluorescein-labeled S1 spike protein. When indigo extract, together with S1 protein, was added at 8,650x and 17,300x dilutions, fluorescence intensity decreased in a dose- and S1 extract-dependent manner, without affecting cell viability. When 4.0-nM tryptanthrin was added instead of the indigo extract, fluorescence intensity also decreased, but to a lesser degree than with indigo extract. Docking simulation analyses revealed that tryptanthrin readily bound to the receptor-binding domain of the S1 protein, and identified 2- and 7-amino acid sequences as the preferred binding sites. The indigo extract appeared to inhibit S1-ACE2 binding at high dilutions, and evidently contained other inhibitory elements as well as tryptanthrin. This extract may be useful for the prevention or treatment of SARS-CoV-2 infection.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the human gastrointestinal tract. Small intestinal GISTs appear to be associated with poorer prognosis and higher metastasis rate than gastric GISTs of the same size and mitotic index. Recently, we reported that cell adhesion molecule 1 (CADM1) is expressed specifically in most small intestinal GISTs, but not in most gastric GISTs, suggesting that this difference in CADM1 expression between gastric GISTs and small intestinal GISTs might influence the difference in clinical behavior between them. The aim of the present study was to examine whether high CADM1 expression affected proliferation, migration, invasion, adhesion to endothelial cells and transendothelial migration of cultured GIST cells by comparing original GIST-T1 cells with very low CADM1 expression with GIST-T1 cells with high CADM1 expression induced by CADM1 cDNA transfection (GIST-T1-CAD cells). GIST-T1-CAD cells had reduced ability to proliferate, migrate and invade compared with the original GIST-T1 cells, but showed significantly higher ability to adhere to human umbilical vein endothelial cells and migrate through endothelial cell monolayers. Thus, CADM1 may contribute to higher metastasis rates in small intestinal GISTs facilitating tumor cell adhesion to vascular endothelial cell and transendothelial migration of tumor cells. CADM1 might serve as a potential target for inhibition of metastasis in small intestinal GISTs.

BACKGROUND: Cancer of unknown primary (CUP) has a poor prognosis. Given the recent approval of immune checkpoint inhibitors for several cancer types, we carried out a multicenter phase II study to assess the efficacy of nivolumab for patients with CUP. PATIENTS AND METHODS: Patients with CUP who were previously treated with at least one line of systemic chemotherapy constituted the principal study population. Previously untreated patients with CUP were also enrolled for exploratory analysis. Nivolumab (240 mg/body) was administered every 2 weeks for up to 52 cycles. The primary endpoint was objective response rate in previously treated patients as determined by blinded independent central review according to RECIST version 1.1. RESULTS: Fifty-six patients with CUP were enrolled in the trial. For the 45 previously treated patients, objective response rate was 22.2% [95% confidence interval (CI), 11.2% to 37.1%], with a median progression-free survival and overall survival of 4.0 months (95% CI, 1.9-5.8 months) and 15.9 months (95% CI, 8.4-21.5 months), respectively. Similar clinical benefits were also observed in the 11 previously untreated patients. Better clinical efficacy of nivolumab was apparent for tumors with a higher programmed death-ligand 1 expression level, for those with a higher tumor mutation burden, and for microsatellite instability-high tumors. In contrast, no differences in efficacy were apparent between tumor subgroups based on estimated tissue of origin. Adverse events were consistent with the known safety profile of nivolumab. No treatment-related death was observed. CONCLUSIONS: Our results demonstrate a clinical benefit of nivolumab for patients with CUP, suggesting that nivolumab is a potential additional therapeutic option for CUP.

Only four cases of colorectal adenocarcinoma with a yolk sac tumor (YST) component have been reported in the English literature. No genetic investigation has been performed in these cases. We report a case of colorectal adenocarcinoma in which the recurrent tumor had a YST component. A 49-year-old woman presented with a pelvic tumor three years after endoscopic mucosal resection of sigmoid colon adenocarcinoma. The pelvic tumor consisted of an undifferentiated carcinoma component and a YST component. The serum alpha-fetoprotein level was elevated to 42 ng/mL. Treatment as conventional colorectal carcinoma produced some anticancer effects, but the patient died 14 months after the recurrence and 49 months after the EMR. With the help of the next-generation sequencing results of the recurrent tumor, APC c.835 - 8A > G and TP53 c.524G > A (p.R175H) mutations were identified by direct sequencing in both the primary and the recurrent tumors, confirming the relationship between the two metachronous tumors.

Matrix metalloproteinase 14 (MMP14) expression is implicated in progression of colorectal cancer, but its role in the tumor microenvironment (TME) has been unclear. The relevance of MMP14 to colorectal cancer progression was explored by analysis of transcriptomic data for colorectal adenocarcinoma patients (n = 592) in The Cancer Genome Atlas. The role of MMP14 in the TME was investigated in a retrospective analysis of tumor samples from 86 individuals with stage III colorectal cancer by single cell-based spatial profiling of MMP14 expression as performed by 12-color multiplex immunohistochemistry (mIHC). Analysis of gene expression data revealed that high MMP14 expression was associated with tumor progression and implicated both cancer-associated fibroblasts (CAFs) and tumor-associated macrophages in such progression. Spatial profiling by mIHC revealed that a higher percentage of MMP14+ cells among intratumoral CAFs (MMP14+ CAF/CAF ratio) was associated with poorer relapse-free survival. Multivariable analysis including key clinical factors identified the MMP14+ CAF/CAF ratio as an independent poor prognostic factor. Moreover, the patient subset with both a high MMP14+ CAF/CAF ratio and a low tumor-infiltrating lymphocyte density showed the worst prognosis. Our results suggest that MMP14+ CAFs play an important role in progression of stage III colorectal cancer and may therefore be a promising therapeutic target.

Plasmablastic lymphoma is a mature B-cell neoplasm with plasmablastic differentiation, often associated with human immunodeficiency virus (HIV) infection and other forms of immunosuppression. Although it is usually an aggressive disease, spontaneous regression has been seen in a few cases. Plasmablastic lymphoma of the uterus is rare. We report a case of atypical lymphoplasmacytic proliferation resembling plasmablastic lymphoma associated with pyometra that disappeared completely as the pyometra resolved. A 76-year-old HIV-negative woman presented with abnormal vaginal bleeding. Ultrasound and MRI findings were consistent with pyometra diagnosis. Endometrial biopsy revealed large plasmablastoid cells with abundant cytoplasm and prominent nucleoli proliferating in the endometrium. Immunohistochemistry showed that large cells stained positive for CD138, CD79a, and MUM1, and negative for CD20, PAX5, CD3, and CD5. Ki67 labelled at least 80% of the large cells. Epstein-Barr virus was detected in a small number of cells. The histologic picture was highly indicative of lymphoma, especially plasmablastic lymphoma, though the clinical context was unusual. As the pyometra was treated and resolved, the intrauterine abnormality disappeared completely. The patient has been well after 16 months with no sign of recurrent disease. This case underscores the sometimes blurry distinction between benign inflammation and lymphoma.

Malignant pleural mesothelioma (MPM) is a highly aggressive malignant tumor, and the effective therapeutic drugs are limited. Thus, the establishment of novel therapeutic method is desired. Considerable proportion of MPMs are shown to express cell adhesion molecule 1 (CADM1), and to use CADM1 to bind to and proliferate on the pleural mesothelial surface, suggesting that CADM1 is a possible therapeutic target. Here, anti-CADM1 ectodomain chicken monoclonal antibodies, 3E1 and 9D2, were examined for their possible therapeutic utility. The full-length form of CADM1 was expressed in eight out of twelve human MPM cell lines. MPM cell lines were cultured on a confluent monolayer of mesothelial MeT-5A cells in the presence of 9D2, the neutralizing antibody. 9D2 suppressed the cell growth of CADM1-positive MPM cells with the loss and aggregation of CADM1 molecules on the MPM cell membrane, but not of CADM1-negative MPM cells. Co-addition of 3E1, lacking the neutralizing action, enhanced the growth-suppressive effect of 9D2. The two antibodies were tested as drug delivery vectors. 3E1 was converted into a humanized antibody (h3E1) and conjugated with monomethyl auristatin E (MMAE), a tubulin polymerization inhibitor. When the resulting h3E1-MMAE antibody-drug conjugate (ADC) was added to the standard cultures of CADM1-positive MPM cells, it suppressed the cell growth in a dose-dependent manner. Co-addition of 9D2 enhanced the growth-suppressive effect of h3E1-MMAE ADC. Anti-CADM1 ectodomain antibodies were suggested to serve as both antibody drugs and drug vectors in the treatment of MPM.

BACKGROUND: Tumour burden (TB) is implicated in resistance to programmed cell death-1/PD-L1 inhibitor (immune checkpoint inhibitor [ICI]) therapy. However, whether TB contributes to such resistance in non-small-cell lung cancer (NSCLC) has remained unknown. METHODS: A total of 260 treatment-naïve patients with advanced NSCLC who started ICI monotherapy (ICI cohort), platinum-doublet therapy (Chemo cohort) or ICI and platinum-doublet therapy (ICI+Chemo cohort) as first-line treatment were consecutively included. TB was estimated on the basis of the sum of the diameters of measurable target lesions as per Response Evaluation Criteria in Solid Tumours. Progression-free survival (PFS) in the ICI cohort was evaluated as per TB as a preplanned primary objective, with the analysis based on propensity score-weighted survival curves and estimation of restricted mean survival time (RMST). The Chemo cohort served as a control to determine whether TB is predictive of ICI treatment outcomes. The ICI+Chemo cohort was exploratory. The relation of TB to tumour immune status was assessed by immune-related gene expression profiling (irGEP) of pretreatment tumour tissue. RESULTS: In the ICI cohort, patients with a low TB showed a significantly longer PFS than did those with a high TB (median, 17.9 vs 4.3 months; weighted hazard ratio, 0.32 [95% confidence interval, 0.19-0.53]). No such difference was apparent in the other cohorts. A significant difference in overall survival was also observed only in the ICI cohort. RMST-based analysis confirmed these results. The irGEP analysis implicated M2-type macrophages, angiogenesis and transforming growth factor-β as well as protumourigenic signalling pathways in ICI resistance conferred by a high TB. CONCLUSION: A high TB was associated with a poor outcome of ICI therapy for advanced NSCLC as a result of immunosuppressive phenotypes. Development of combination or novel treatment strategies for such disease is thus warranted.

AIMS: Cell adhesion molecule 1 (CADM1) mediates interepithelial adhesion and is upregulated in crowded epithelial monolayers. This study aimed to examine CADM1 expression in the human endometrium of proliferative and secretory phases, and its transcriptional regulation in terms of estrogen stimuli and higher cellularity. MAIN METHODS: CADM1 immunohistochemistry was conducted on endometrial tissues from women in their 40s and adult mice subcutaneously injected with estradiol following ovariectomy. Dual-luciferase reporter assays were conducted using human endometrial HEC-50B and HEC-1B cells and reporter plasmids harboring the human CADM1 3.4-kb promoter and its deleted and mutated forms. Cells were transfected with estrogen receptor α cDNA and reporter plasmids, and treated with estradiol before luciferase activity measurement. KEY FINDINGS: Immunohistochemistry revealed that CADM1 was clearly expressed on the lateral membranes of the simple columnar glandular cells in the proliferative phase, but not in the secretory phase, from both women and the mouse model. The glandular cell density increased two-fold in the proliferative phase. Reporter assays identified three Sp1-binding sites as estradiol-responsive elements in the proximal region (from -223 to -84) of the transcription start site (+1) in HEC-50B cells. When the cell culture was started at eight-fold higher cell density, the CADM1 3.4-kb promoter was transactivated at a two-fold higher level in HEC-50B cells. This cell density effect was not detected for the CADM1 2.3-kb or 1.6-kb promoter. SIGNIFICANCE: Two (proximal and distal) promoter regions are suggested to function additively to transactivate CADM1 in endometrial glandular cells that crowd in the proliferative phase.

BACKGROUND: Implementation of personalized medicine requires the accessibility of tumor molecular profiling in order to allow prioritization of appropriate targeted therapies for individual patients. Our aim was to study the role of comprehensive genomic profiling assays that may inform treatment recommendations for patients with solid tumors. MATERIALS AND METHODS: We performed a prospective study to evaluate the feasibility of application of the FoundationOne CDx panel-which detects substitutions, insertions and deletions, and copy number alterations in 324 genes, select gene rearrangements, and genomic signatures including microsatellite instability and tumor mutation burden (TMB)-to patients with advanced or recurrent solid tumors before its approval in Japan. RESULTS: A total of 181 samples were processed for genomic testing between September 2018 and June 2019, with data being successfully obtained for 175 of these samples, yielding a success rate of 96.7%. The median turnaround time was 41 days (range, 21-126 days). The most common known or likely pathogenic variants were TP53 mutations (n = 113), PIK3CA mutations (n = 33), APC mutations (n = 32), and KRAS mutations (n = 29). Among the 153 patients assessed for TMB, the median TMB was 4 mutations/Mb, and tumors with a high TMB (≥10 mutations/Mb) were more prevalent for lung cancer (11/32) than for other solid tumor types (9/121, Fisher's exact test p < .01). No clear trend toward increased efficacy for immune checkpoint inhibitor (ICI) monotherapy or ICI combination chemotherapy in patients with a high programmed cell death-ligand 1 tumor proportion score or a high TMB was apparent. Among the 174 patients found to harbor known or likely pathogenic actionable alterations, 24 individuals (14%) received matched targeted therapy. CONCLUSION: The FoundationOne CDx assay was performed with formalin-fixed, paraffin-embedded tumor specimens with a success rate of >95%. Such testing may inform the matching of patients with cancer with investigational or approved targeted drugs. IMPLICATIONS FOR PRACTICE: This prospective cohort study was initiated to investigate the feasibility and utility of clinical application of FoundationOne CDx. A total of 181 samples were processed for genomic testing between September 2018 and June 2019, with data being successfully obtained for 175 of these samples, yielding a success rate of 96.7%, and 24 individuals (14%) received matched targeted therapy.

Adult T-cell leukemia/leukemia (ATLL) is an aggressive peripheral T-cell malignancy, caused by infection with the human T-cell leukemia virus type 1 (HTLV-1). We have recently shown that cell adhesion molecule 1 (CADM1), a member of the immunoglobulin superfamily, is specifically and consistently overexpressed in ATLL cells, and functions as a novel cell surface marker. In this study, we first show that a soluble form of CADM1 (sCADM1) is secreted from ATLL cells by mainly alternative splicing. After developing the Alpha linked immunosorbent assay (AlphaLISA) for sCADM1, we showed that plasma sCADM1 concentrations gradually increased during disease progression from indolent to aggressive ATLL. Although other known biomarkers of tumor burden such as soluble interleukin-2 receptor α (sIL-2Rα) also increased with sCADM1 during ATLL progression, multivariate statistical analysis of biomarkers revealed that only plasma sCADM1 was selected as a specific biomarker for aggressive ATLL, suggesting that plasma sCADM1 may be a potential risk factor for aggressive ATLL. In addition, plasma sCADM1 is a useful marker for monitoring response to chemotherapy as well as for predicting relapse of ATLL. Furthermore, the change in sCADM1 concentration between indolent and aggressive type ATLL was more prominent than the change in the percentage of CD4+CADM1+ ATLL cells. As plasma sCADM1 values fell within normal ranges in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients with higher levels of serum sIL-2Rα, a measurement of sCADM1 may become a useful tool to discriminate between ATLL and other inflammatory diseases, including HAM/TSP.

The clinicopathological, immunohistochemical, and molecular characteristics of α-fetoprotein (AFP)-producing endometrial carcinoma (AFP+ EC) are poorly understood. From 284 cases of endometrial carcinoma in our pathology archive, we identified five cases (1.8%) of AFP+ EC with fetal gut-like (4/5) and/or hepatoid (2/5) morphology. All cases exhibited lymphovascular infiltration. In addition, 24 cases of endometrial carcinoma with elevated serum AFP levels were retrieved from the literature. The patient age ranged from 44 to 86 years (median: 63). Of 26 cases whose FIGO (International Federation of Gynecology and Obstetrics) stage and follow-up information was available (mean follow-up 24 months), 15 were stage I or II and 11 were stage III or IV. Even in stage I or II disease, death or relapse occurred in more than half of the patients (8/15). Detailed analysis of our five cases revealed that, on immunohistochemistry, AFP+ EC was positive for SALL4 (4/5), AFP (3/5), and HNF1β (4/5) in >50% of neoplastic cells and negative for estrogen and progesterone receptors (5/5), PAX8 (4/5), and napsin A (5/5). Four cases exhibited aberrant p53 immunohistochemistry and were confirmed to harbor TP53 mutations by direct sequencing. No mutation was found in POLE, CTNNB1, or KRAS. In conclusion, AFP+ EC merits recognition as a distinct subtype of endometrial carcinoma, which occurs in 1.8% of endometrial carcinoma cases, are associated with TP53 abnormalities, exhibit lymphovascular infiltration, and can show distant metastasis even when treated in early stage.

Elevation of intraocular pressure is a major risk factor for glaucoma development, which causes the loss of retinal ganglion cells (RGCs). Lipocalin 2 (Lcn2) is upregulated in glaucomatous retinae; however, whether Lcn2 is directly involved in glaucoma is debated. In this study, retinal explant cultures were subjected to increased water pressure using a two-chamber culture device, and Lcn2 protein levels were examined by immunoblotting. In situ TdT-mediated dUTP nick and labeling (TUNEL) and glial fibrillary acidic protein (GFAP) immunohistochemical assays were performed to assess apoptosis and gliosis, respectively. The neurotoxicity of Lcn2 in the retinal explant culture was determined with exogenous administration of recombinant Lcn2. The Lcn2 protein levels, percentage of TUNEL-positive cells, and GFAP-positive area were significantly higher in retinae cultured under 50 cm H2O pressure loads compared to those cultured under 20 cm H2O. We found that Lcn2 exhibited neurotoxicity in retinae at dose of 1 μg/ml. The negative effects of increased hydrostatic pressure were attenuated by the iron chelator deferoxamine. This is the first report demonstrating the direct upregulation of Lcn2 by elevating hydrostatic pressure. Modulating Lcn2 and iron levels may be a promising therapeutic approach for retinal degeneration.

Gastrointestinal stromal tumor (GIST), the most common mesenchymal tumor of the human gastrointestinal tract, differentiating toward the interstitial cell of Cajal (ICC), arises predominantly in the stomach and small intestine. Small intestinal GISTs appear to have worse prognosis than gastric GISTs. In a pilot study of a cDNA expression chip using several GISTs, we found that Cell Adhesion Molecule 1 (CADM1), which could contribute to tumor growth and infiltration, is expressed more strongly in small intestinal GISTs than gastric GISTs. In the present study, we examined CADM1 expression in GISTs of different sites and with different gene abnormalities using a large number of gastric and small intestinal GISTs. First, immunoblotting confirmed significantly higher CADM1 expression in small intestinal GISTs with exon 11 c-kit mutation than gastric GISTs with exon 11 c-kit mutation. Real-time PCR also revealed that small intestinal GISTs with exon 11 c-kit mutation showed significantly higher CADM1 mRNA than gastric GISTs with exon 11 c-kit mutation. Although most small intestinal GISTs showed high CADM1 mRNA expression regardless of gene abnormality types, different CADM1 expression was detected between gastric GISTs with c-kit mutation and those with PDGFRA mutation. Immunohistochemistry showed that many small intestinal GISTs were CADM1-positive but most gastric GISTs CADM1-negative or -indefinite. In the normal gastric and small intestinal walls, immunoreactivity of CADM1 was detected only in nerves, but neither in gastric ICCs nor small intestinal ICCs, indicating that the high CADM1expression in small intestinal GISTs might be acquired during tumorigenesis. Different CADM1 expression between gastric and small intestinal GISTs might be related to different prognoses between them. Further functional experiments are needed to elucidate the role of CADM1 on GIST biology, and there is a possibility that targeting therapy against CADM1 has a preventive effect for tumor spreading in small intestinal GISTs.

Cell adhesion molecules act as tumor suppressors primarily by cell attachment activity, but additional mechanisms modifying signal transduction are suggested in some cases. Cell adhesion molecule 1 (CADM1), a membrane-spanning immunoglobulin superfamily, mediates intercellular adhesion by trans-homophilic interaction and acts as a tumor suppressor. Here, we investigated CADM1-associated proteins comprehensively using proteomic analysis of immune-precipitates of CADM1 by mass spectrometry and identified a transmembrane adaptor protein, Csk-binding protein (Cbp), known to suppress Src-mediated transformation, as a binding partner of CADM1. CADM1 localizes to detergent-resistant membrane fractions and co-immunoprecipitated with Cbp and c-Src. Suppression of CADM1 expression using siRNA reduces the amount of co-immunoprecipitated c-Src with Cbp and activates c-Src in colon cancer cells expressing both CADM1 and Cbp. On the other hand, co-replacement of CADM1 and Cbp in colon cancer cells lacking CADM1 and Cbp expression suppresses c-Src activation, wound healing and tumorigenicity in nude mice. Furthermore, expression of Cbp and CADM1 was lost in 55% and 83% of human colon cancer, respectively, preferentially in tumors with larger size and/or lymph node metastasis. CADM1 would act as a colon tumor suppressor by intervening oncogenic c-Src signaling through binding with Cbp besides its authentic cell adhesion activity.

When epithelial cells in vivo are stimulated to proliferate, they crowd and often grow in height. These processes are likely to implicate dynamic interactions among lateral membranous proteins, such as cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member. Pulmonary epithelial cell lines that express CADM1, named NCI-H441 and RLE-6TN, were grown to become overconfluent in the polarized 2D culture system, and were examined for the expression of CADM1. Western analyses showed that the CADM1 expression levels increased gradually up to 3 times in a cell density-dependent manner. Confocal microscopic observations revealed dense immunostaining for CADM1 on the lateral membrane. In the overconfluent monolayers, CADM1 knockdown was achieved by two methods using CADM1-targeting siRNA and an anti-CADM1 neutralizing antibody. Antibody treatment experiments were also done on 6 other epithelial cell lines expressing CADM1. The CADM1 expression levels were reduced roughly by half, in association with cell height decrease by half in 3 lines. TUNEL assays revealed that the CADM1 knockdown increased the proportion of TUNEL-positive apoptotic cells approximately 10 folds. Increased expression of CADM1 appeared to contribute to cell survival in crowded epithelial monolayers.

PURPOSE: The impact of EGFR tyrosine kinase inhibitors (TKI) on the tumor immune microenvironment (TME) in non-small cell lung cancer (NSCLC) is unclear. EXPERIMENTAL DESIGN: We retrospectively identified 138 patients with EGFR-mutated NSCLC who underwent rebiopsy after progression during EGFR-TKI treatment. PD-L1 and CD73 expression in tumor cells and tumor-infiltrating lymphocyte (TIL) density at baseline and after progression were determined by IHC. Tumor mutation burden (TMB) was determined by next-generation sequencing. RESULTS: The proportion of patients with a PD-L1 expression level of ≥50% (high) increased from 14% before to 28% after EGFR-TKI (P = 0.0010). Whereas CD8+ and FOXP3+ TIL densities were significantly lower after EGFR-TKI treatment than before, CD8+ TIL density was maintained in tumors with a high PD-L1 expression level. Expression of CD73 in tumor cells after EGFR-TKI treatment was higher than that before in patients with a high PD-L1 expression level. TMB tended to be higher after EGFR-TKI treatment than before (3.3→4.1 mutations/Mbp, P = 0.0508). Median progression-free survival for subsequent treatment with antibodies to PD-1 was longer for patients with a high than for those with a low PD-L1 expression after EGFR-TKI (7.1 vs. 1.7 months, P = 0.0033), and two of five patients whose PD-L1 expression level changed from low to high after EGFR-TKI treatment achieved a PFS of >6 months. CONCLUSIONS: EGFR-TKI treatment was associated with changes in the TME of EGFR-mutated NSCLC, and such changes may provide clues for optimization of subsequent PD-1 inhibitor treatment.

AIMS: Stroke-prone spontaneously hypertensive rats (SHRSP) show significantly lower body weight than normotensive Wistar-Kyoto rats (WKY). Our hypotheses are as follows: weight loss of the skeletal muscle is related to hypertension-related diseases, and muscle hypotrophy is useful as a therapeutic target for hypertension and hypertension-related diseases. In this study, we aimed to investigate the pathophysiological characteristics of muscle hypotrophy in SHRSP to determine the therapeutic target molecule(s). MAIN METHODS: The difference in skeletal muscles in the lower leg between WKY and SHRSP was evaluated mainly through weight/tibial length, histological, gene expression, and protein expression analyses. KEY FINDINGS: SHRSP had a significantly lower weight/tibial length in soleus and gastrocnemius, but not in plantaris and tibialis anterior, indicating that muscles consisting of a relatively high amount of slow muscle fiber were affected. This result was confirmed by the histological analysis of soleus, showing that type I fiber mainly decreased the fiber size. Microarray and protein expression analyses showed that the muscle-specific ubiquitin ligase, muscle RING finger 1 (MuRF1), but not atrogin-1, was highly expressed in soleus, but not in plantaris, in SHRSP. TNF-like weak inducer of apoptosis receptor (TWEAKR) was predicted as a MuRF1 up-regulator by Ingenuity Pathway Analysis and immunostained only in type II fiber in WKY but in both type I and II fibers in SHRSP. SIGNIFICANCE: TWEAKR is a type II-specific receptor in the skeletal muscle. Ectopic TWEAKR expression in type I fiber of SHRSP is most likely involved in slow muscle-specific hypotrophy through MuRF1 overexpression.

Previously, we identified molecules involved in human invasive lung adenocarcinoma, and guanylate-binding protein 1 (GBP-1) was selected for further analysis. RT-PCR of normal lung and invasive lung adenocarcinoma tissue samples showed that the relative GBP-1 expression levels normalized to GAPDH for invasive lung adenocarcinoma were three-fold higher than those for normal lung samples (P < 0.05). GBP-1 gene and protein expression levels were also higher in mesenchymal-like than in epithelial-like lung adenocarcinoma cell lines. To determine whether GBP-1 participates in lung adenocarcinoma invasion, we performed migration and wound healing assays using RERF-LC-OK cells transfected with various siRNAs. The relative migration of transfected GBP1-siRNA1 and GBP1-siRNA2 cells was significantly lower than that of transfected control-siRNA cells. The relative wound healing capacities 6 and 12 h after cells transfected with GBP1-siRNA1 and GBP1-siRNA2 were scratched were significantly lower than those of the control-siRNA cells. Immunohistochemistry of 80 patients with Stage I lung adenocarcinoma revealed that non-invasive cells were GBP-1 negative in all cases. Invasive cells were GBP-1 positive in 10 cases (12.5%) and GBP-1 negative in 70 cases (87.5%). Lymphatic-vascular invasion was positive in 20 patients (25%) and positively correlated with GBP-1 expression (P < 0.05). In conclusion, GBP-1 may enhance lung adenocarcinoma invasiveness by promoting cell motility, and control of GBP-1 expression has the potential to contribute to the development of new therapeutic strategies for lung adenocarcinoma.

The correct name of the corresponding author should be ''Takahiro Mimae'', and not ''Emiko Hiraoka'' as given in the original publication of the article.

PURPOSE: Pseudopodia are actin-rich ventral protrusions associated with cell motility and cancer cell invasion. We previously applied our established method of using excimer laser cell etching to isolate pseudopodial proteins from MDA-MB-231 breast cancer cells. We later identified 14-3-3γ as an oncogenic molecule among 46 candidate proteins that are specific to pseudopodia. The present study aimed to determine the function of 14-3-3γ in the motility of breast cancer cells. METHODS: MDA-MB-231 cells were cultured on 3-µm porous membranes and double stained to localize 14-3-3γ and phalloidin in pseudopodia using confocal imaging. We assessed pseudopodia numbers and length, as well as migration and wound healing in MDA-MB-231 cells with knockdown and forced expression of 14-3-3γ to determine 14-3-3γ involvement in cell motility. We also immunohistochemically analyzed 14-3-3γ in human breast cancer tissues with high-grade lymphatic invasion. RESULTS: We specifically located 14-3-3γ in pseudopodia of MDA-MB-231 cells. Knockdown and forced expression of 14-3-3γ, respectively, decreased and increased pseudopodial formation and elongation. Migration and wound healing assays also showed that 14-3-3γ knockdown and forced expression, respectively, decreased and increased the number of underside cells and acellular areas in MDA-MB-231 breast cancer cells. More 14-3-3γ was expressed in sites of lymphatic invasion, than in the center and periphery of human breast cancer tissues. CONCLUSION: The role of 14-3-3γ in breast cancer invasiveness might be to promote cell motility. Inhibition of 14-3-3γ could, therefore, become a novel target of therapy to prevent invasion and metastasis in patients with breast cancers expressing 14-3-3γ.

Medical oncologists are challenged to personalize medicine with scientific evidence, drug approvals, and treatment guidelines based on sequencing of clinical samples using next generation sequencer (NGS). Knowledge-based curation systems have the potential to help address this challenge. We report here the results of examining the level of evidence regarding treatment approval and clinical trials between recommendations made by Watson for Genomics (WfG), QIAGEN Clinical Insight Interpret (QCII), and Oncomine knowledge-based reporter (OKR). The tumor samples obtained from the solid cancer patients between May to June 2018 at Kindai University Hospital. The formalin-fixed paraffin-embedded tumor samples (n = 31) were sequenced using Oncomine Comprehensive Assay v3. Variants including copy number alteration and gene fusions identified by the Ion reporter software were used commonly on three curation systems. Curation process of data were provided for 25 solid cancers using three curation systems independently. Concordance and distribution of curated evidence levels of variants were analyzed. As a result of sequencing analysis, nonsynonymous mutation (n = 58), gene fusion (n = 2) or copy number variants (n = 12) were detected in 25 cases, and subsequently subjected to knowledge-based curation systems (WfG, OKR, and QCII). The number of curated information in any systems was 51/72 variants. Concordance of evidence levels was 65.3% between WfG and OKR, 56.9% between WfG and QCII, and 66.7% between OKR and QCII. WfG provided great number of clinical trials for the variants. The annotation of resistance information was also observed. Larger differences were observed in clinical trial matching which could be due to differences in the filtering process among three curation systems. This study demonstrates knowledge-based curation systems (WfG, OKR, and QCII) could be helpful tool for solid cancer treatment decision making. Difference in non-concordant evidence levels was observed between three curation systems, especially in the information of clinical trials. This point will be improved by standardized filtering procedure and enriched database of clinical trials in Japan.

Gene fusions play an important role in the carcinogenesis of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for precision medicine. We used formalin-fixed, paraffin-embedded tissue samples of non-small cell lung cancer from 150 EGFR mutation-negative cases and 10 fusion status-known cases and compared the performance of the Oncomine Dx Fusion Transcript Test (ODxFT) with FISH break-apart for the detection of ALK, RET, and ROS1 fusion genes. RNA was extracted from the paraffin-embedded tissue samples with or without macrodissection under hematoxylin and eosin staining, and the ALK fusion gene was independently determined using these assays. Fusion detection analyses were successfully carried out using ODxFT in 150 cases, with only one invalid case. ALK fusion genes were detected at a frequency of 7.3% (11/150) in the lung cancer specimens. Concordance rate between the ODxFT and ALK-FISH analyses was 99.3% (148/149). Sensitivity and specificity were 91.7% and 99.3%, respectively. All the samples with a known fusion status were accurately matched between the two assays. Our results show a high concordance rate between the ODxFT and ALK-FISH analyses. ODxFT was thus validated as an effective method for detecting clinically significant ALK fusion genes in paraffin-embedded tissue samples.

非小細胞肺癌の細胞株や患者由来検体を実験材料とし、上皮成長因子受容体(EGFR)チロシンキナーゼ阻害薬(TKI)がMHCクラスIの発現に及ぼす影響について調査した。EGFR遺伝子にT790M二次的突然変異などの変異がある細胞株を適切なEGFR-TKIで処理するとMHCクラスIのmRNA/蛋白質発現は亢進した。細胞外シグナル調節キナーゼ(ERK)キナーゼであるMEKの阻害薬で処理した場合もMHCクラスI発現が亢進したことから、EGFR活性化に応答してみられるMHCクラスI発現の下方制御はMEK-ERK経路が媒介していることが示唆された。EGFR-TKI治療を施行したEGFR変異非小細胞肺癌患者から得た検体の免疫組織化学解析からも、疾患進行後においては、リン酸化EGFRやリン酸化ERKの下方制御が、MHC-Iの上方制御、CD8+浸潤T細胞の増加、およびPD-1リガンド1発現亢進と関連していることが明らかになった。これらの結果から、非小細胞癌においてEGFRが変異活性化するとMEK-ERK経路を通じてMHCクラスI発現が阻害され、免疫療法に不応となるのに寄与することが示唆された。

OBJECTIVES: Immune-checkpoint inhibitors (ICIs) are now an established therapeutic option for advanced non-small cell lung cancer (NSCLC). It has remained unclear, however, whether cytotoxic chemotherapy affects the immune microenvironment in NSCLC wild type for EGFR and ALK. MATERIALS AND METHODS: We retrospectively evaluated changes in programmed cell death 1-ligand 1 (PD-L1) expression, tumor mutation burden (TMB), and CD8+ tumor-infiltrating lymphocyte (TIL) density in NSCLC patients who underwent rebiopsy at the site of recurrence after postoperative platinum-based adjuvant chemotherapy, or in those who underwent rebiopsy after one or more chemotherapeutic regimens at the advanced stage. The PD-L1 tumor proportion score (TPS) and CD8+ TIL density were determined by immunohistochemistry. TMB was estimated by next-generation sequencing with a cancer gene panel (409 genes). RESULTS: Seventeen patients with NSCLC wild type for EGFR and ALK were enrolled. Although PD-L1 TPS tended to be increased in rebiopsy samples compared with initial biopsy tissue, this difference was not significant (P =  0.113). Seven patients showed an increase in PD-L1 TPS, with this change being pronounced in four. Two cases in which PD-L1 TPS increased from 0 to 90% or from 0 to 95% after cytotoxic chemotherapy also showed a durable response to subsequent treatment with an ICI. No substantial correlation between PD-L1 TPS and TMB was apparent either before (R = 0.112) or after (R = 0.101) chemotherapy. A moderate correlation was detected between PD-L1 TPS and CD8+ TIL density before chemotherapy (R = 0.517) and a negligible correlation after (R = 0.0219). CONCLUSION: Cytotoxic chemotherapy may change the biological characteristics of tumors including PD-L1 expression level and TMB.

The efficacy of programmed cell death-1 (PD-1) blockade in patients with non-small cell lung cancer (NSCLC) positive for epidermal growth factor receptor (EGFR) gene mutations has been found to be limited, but the underlying mechanisms for this poor response have remained obscure. Given that the recognition by T cells of tumor antigens presented by major histocompatibility complex class I (MHC-I) molecules is essential for an antitumor immune response, we examined the effects of EGFR tyrosine kinase inhibitors (TKIs) on MHC-I expression in NSCLC cell lines. Appropriate EGFR-TKIs increased MHC-I expression at the mRNA and cell surface protein levels in NSCLC cells positive for EGFR mutations including those with the T790M secondary mutation. Trametinib, an inhibitor of the extracellular signal-regulated kinase (ERK) kinase MEK, also increased MHC-I expression, whereas the phosphatidylinositol 3-kinase (PI3K) inhibitor buparlisib did not, suggesting that the MEK-ERK pathway mediates the down-regulation of MHC-I expression in response to EGFR activation. Immunohistochemical analysis of EGFR-mutated NSCLC specimens obtained before and after EGFR-TKI treatment also revealed down-regulation of phosphorylated forms of EGFR and ERK in association with up-regulation of MHC-I, an increased number of infiltrating CD8+ T cells, and increased PD-1 ligand 1 expression after such treatment. Our results thus suggest that mutational activation of EGFR inhibits MHC-I expression through the MEK-ERK pathway in NSCLC and thereby contributes to the poor response of such tumors to immunotherapy. Further studies are warranted to evaluate the relation between EGFR-MEK-ERK signaling in and the immune response to EGFR-mutated NSCLC. .

AIMS: To determine cellular distribution of cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member, in the human oxyntic gastric mucosa, and to explore possible involvement in the development and peritoneal dissemination of signet ring cell (SRC) gastric carcinoma, which often develops in the oxyntic mucosa. MAIN METHODS: Immunohistochemistry and double immunofluorescence were conducted on surgical specimens of normal and SRC-bearing stomachs and peritoneal metastatic foci of SRCs. KATO-III (lacking CADM1) and HSC-43 (expressing CADM1) SRC cell lines were cocultured on a Met-5A mesothelial or TIG-1 fibroblastic cell monolayer. KEY FINDINGS: In the oxyntic gland, some neck and nearly all base glandular cells were CADM1-positive, and mucin 5AC-positive cells were CADM1-negative, while some mucin 6-positive neck cells were CADM1-positive. Foveolar-epithelial, parietal, and endocrine cells were CADM1-negative. CADM1 was negative in all SRC carcinomas that were confined within the submucosa (n = 11) and all but one of those invading deeper (n = 15). In contrast, peritoneal metastatic foci of SRCs were CADM1-positive in five out of eleven cases (P < 0.01). In the cocultures, exogenous CADM1 made KATO-III cells adhere more and grow faster on a Met-5A monolayer, not on TIG-1 monolayers. HSC-43 cells adhered more and grew faster on Met-5A than on TIG-1 monolayers, which were partly counteracted by a function-neutralizing anti-CADM1 antibody. SIGNIFICANCE: Nearly all chief cells and a part of mucous neck cells express CADM1. SRC gastric carcinoma appears to emerge as a CADM1-negative tumor, but CADM1 may help SRCs develop peritoneal dissemination through promoting their adhesion and growth in the serosal tissue.

Pulmonary emphysema usually arises in cigarette smokers, and often progresses after smoking cessation and even in ex-smokers. Lung-epithelial cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member, is extracellularly shed to produce a proapoptotic C-terminal fragment (CTF) within the cell and contribute to the development of emphysema. Here, we made an ex-smoker model using C57BL/6 mice; mice (6-week-old; 5 mice per group) were exposed to passive smoke of eight cigarettes twice a day 5 days a week until 18 weeks of age, and were then left untreated until 30 weeks of age. We calculated the mean linear intercept (Lm) and the alveolar septal thickness in the lung histologic sections to estimate the alveolar space dilatation. At 18 weeks of age, Lm was marginally enlarged (P = 0.023) with a marked increase in the septal thickness (P < 0.001) in comparison with age-matched control mice (5 mice per group), while at 30 weeks, the increase in Lm was much more prominent (P = 0.006) and the septal thickness was normalized, suggesting that emphysema progressed with septal remodeling during smoking cessation. Western blot analyses of the lungs were performed for CADM1, a possible CADM1 sheddase ADAM10, an epithelial marker pan-cytokeratin, and a myofibroblastic marker α-smooth muscle actin to estimate the expression levels of CTF and ADAM10 per epithelial cell and the levels of pan-cytokeratin and αSMA per tissue. CADM1 shedding was increased in the treated mice than in control mice at both ages, in association with an increase in the CTF level at 30 weeks (P = 0.021). In total of the treated and control mice of 30 weeks of age, Lm was positively correlated with the CTF and ADAM10 levels, and pan-cytokeratin was negatively correlated with CTF, suggesting an involvement of CADM1 shedding in emphysema progression. Positive correlations were also found between CTF and ADAM10, and between ADAM10 and αSMA, suggesting that increased septal myofibroblasts might be involved in increased CADM1 shedding. Taken together, persisting increase in ectodomain shedding of CADM1 appeared to contribute to the progression of emphysema in ex-smokers, and might be accounted for by alveolar septal remodeling.

A disintegrin and metalloproteinases (ADAMs) are a Zn2+-dependent transmembrane and secreted metalloprotease superfamily, so-called "molecular scissors," and they consist of an N-terminal signal sequence, a prodomain, zinc-binding metalloprotease domain, disintegrin domain, cysteine-rich domain, transmembrane domain and cytoplasmic tail. ADAMs perform proteolytic processing of the ectodomains of diverse transmembrane molecules into bioactive mediators. This review summarizes on their most well-known members, ADAM10 and 17, focusing on the kidneys. ADAM10 is expressed in renal tubular cells and affects the expression of specific brush border genes, and its activation is involved in some renal diseases. ADAM17 is weakly expressed in normal kidneys, but its expression is markedly induced in the tubules, capillaries, glomeruli, and mesangium, and it is involved in interstitial fibrosis and tubular atrophy. So far, the various substrates have been identified in the kidneys. Shedding fragments become released ligands, such as Notch and EGFR ligands, and act as the chemoattractant factors including CXCL16. Their ectodomain shedding is closely correlated with pathological factors, which include inflammation, interstitial fibrosis, and renal injury. Also, the substrates of both ADAMs contain the molecules that play important roles at the plasma membrane, such as meaprin, E-cadherin, Klotho, and CADM1. By being released into urine, the shedding products could be useful for biomarkers of renal diseases, but ADAM10 and 17 per se are also notable as biomarkers. Furthermore, ADAM10 and/or 17 inhibitions based on various strategies such as small molecules, antibodies, and their recombinant prodomains are valuable, because they potentially protect renal tissues and promote renal regeneration. Although temporal and spatial regulations of inhibitors are problems to be solved, their inhibitors could be useful for renal diseases.

NEW FINDINGS: What is the central question of this study? An inverse correlation between circulating adiponectin and many diseases has been reported, but some studies have found no correlation. To evaluate this controversy, we investigated the relationship between heart-bound adiponectin and hypertension or cardiac hypertrophy, compared with serum adiponectin. What is the main finding and its importance? Using hypertensive and normotensive rats, we found that heart-bound adiponectin was inversely correlated with cardiac hypertrophy, suggesting that heart-bound adiponectin has a more important function in preventing cardiac hypertrophy than circulating adiponectin. Our study provides new insights regarding the role of adiponectin in diseases. The inverse correlation between circulating adiponectin concentration and hypertension or cardiac hypertrophy is still controversial. In addition to circulating adiponectin, adiponectin is also bound to tissues such as the heart and skeletal muscle. In this study, we investigated the relationship of serum adiponectin and heart-bound adiponectin with hypertension and cardiac hypertrophy. Four types of hypertensive rats presenting different blood pressure levels were used at different ages, as follows: normotensive Wistar-Kyoto rats (WKYs); two sub-strains (strains C and B2, having low and high blood pressure, respectively) of spontaneously hypertensive rats (SHRs); and stroke-prone SHRs (SHRSPs). Blood pressure, heart-to-body weight ratio, serum adiponectin and heart-bound adiponectin were determined. Histopathological analysis of the heart was carried out to evaluate the relationship with heart-bound adiponectin. Serum adiponectin concentration was not inversely correlated with blood pressure or heart-to-body weight ratio. In contrast, heart-bound adiponectin levels were significantly lower in SHRSPs than in other strains at respective ages. This resulted from a decrease in T-cadherin expression, which induced adiponectin binding to tissues. No significant difference in heart-bound adiponectin among WKYs and SHRs (C and B2) was detected, indicating that heart-bound adiponectin is not related to hypertension. In addition, differences in heart-bound adiponectin did not affect AMP-activated protein kinase in the traditional adiponectin activation cascade. Histopathological analysis revealed that heart-bound adiponectin was inversely correlated with cardiomyocyte hypertrophy and left ventricular wall thickness and, in part, with cardiac fibrosis. These results suggest that the decreased level of heart-bound adiponectin in SHRSPs is more related to their cardiac hypertrophy than circulating adiponectin.

Cell adhesion mediated by adhesion molecules is of central importance in the maintenance of tissue homeostasis. Therefore, altered expression of adhesion molecules leads to the development of various tissue disorders involving cell activation, degeneration, and apoptosis. Nevertheless, it still remains unclear what initiates the altered expression of adhesion molecules and how the subsequent pathological cascades proceed. In this regard, cell adhesion molecule 1 (CADM1) is one of the candidates that is involved in the development of pathological lesions; it is an intercellular adhesion molecule that is expressed in various types of cells such as pulmonary cells, neurons, and mast cells. Recent studies have revealed that alterations in the transcriptional or post-transcriptional expressions of CADM1 correlate with the pathogenesis of pulmonary diseases and allergic diseases. In this review, we specifically focus on how CADM1 is involved in the development of pathological lesions in pulmonary emphysema and atopic dermatitis.

The heparan sulfate sulfotransferase gene family catalyzes the transfer of sulfate groups to heparan sulfate and regulates various growth factor-receptor signaling pathways. However, the involvement of this gene family in cancer biology has not been elucidated. It was demonstrated that the heparan sulfate D-glucosaminyl 6-O-sulfotransferase-2 (HS6ST2) gene is overexpressed in colorectal cancer (CRC) and its clinical significance in patients with CRC was investigated. The mRNA levels of HS6ST2 in clinical CRC samples and various cancer cell lines were assessed using a microarray analysis and quantitative RT-PCR, respectively. An immunohistochemical (IHC) analysis of the HS6ST2 protein was performed using 102 surgical specimens of CRC. The correlations between the HS6ST2 expression status and clinicopathological characteristics were then evaluated. HS6ST2 mRNA was significantly overexpressed by 37-fold in CRC samples compared to paired colonic mucosa. High levels of HS6ST2 mRNA expression were also observed in colorectal, esophageal and lung cancer cell lines. The IHC analysis demonstrated that HS6ST2 was expressed in the cytoplasmic region of CRC cells, but not in normal colonic mucosal cells. Positive staining for HS6ST2 was detected in 40 patients (39.2%). There was no significant association between the clinicopathological characteristics and HS6ST2 expression. However, positive staining for HS6ST2 was associated with a poor survival (P=0.074, log-rank test). In conclusion, HS6ST2 was found to be overexpressed in CRC and its expression tended to be a poor prognostic factor, although the correlation was not significant. These findings indicate that HS6ST2 may be a novel cancer-related marker that may provide insight into the glycobiology of CRC.

背景：病理組織実習における学生の積極性を引き出す方法が模索されていた。携帯電話やスマートフォン内蔵デジタルカメラを用いた顕微鏡画像撮影を勧奨することは学生の積極性を引き出すのに役立つかどうか観察した。
方法：学生に自由に内臓デジタルカメラで顕微鏡画像を接眼部から撮影させた。病理組織実習に対する学生の態度の変化を観察記録した。
結果：学生は顕微鏡画像撮影に積極的であった。撮影された画像を画面上で見ながら質問・説明が可能であった。学生は自ら撮影した画像を整理して復習に利用した。
結論：学生一人一人が自ら顕微鏡画像を撮影することで病理組織実習への積極的な参加を誘導できた。

AIMS: An immunohistochemical screen for mouse embryos showed that cell adhesion molecule 1 (CADM1), which is an immunoglobulin superfamily member, was expressed in developing bones. Here, we determined the cell types expressing CADM1 and examined its usefulness in the differential diagnosis of osteosarcoma. MAIN METHODS: Serial sections of murine developing mandibles were stained with anti-CADM1 antibody, by a coloring substrate reactive to alkaline phosphatase (ALP), a broad osteoblastic marker for preosteoblasts to osteoblasts, and by in situ hybridization for osteopontin (OPN), a marker for mature osteoblasts. CADM1 immunohistochemistry was also performed on human remodeling bones, osteosarcomas and other soft tissue tumors. KEY FINDINGS: CADM1 immunohistochemistry for the mandible revealed that morphologically identifiable osteoblasts expressed CADM1 on their plasma membranes, but neither osteocytes nor bone lining cells did. At the mandibular margin, not only OPN-positive cells but also OPN-negative, ALP-positive cells were CADM1-positive, whereas inside the mandible, OPN-positive cells were often CADM1-negative. Clear membranous staining was detected in the majority of osteosarcomas (46/57), whereas only 13% (6/46) of the other soft tissue tumors were CADM1-positive (P<0.001). SIGNIFICANCE: These results indicated that CADM1 was a novel osteoblastic adhesion molecule that is expressed transiently during osteoblastic maturation, and a useful diagnostic marker for osteosarcoma cells.

Takayasu’s arteritis (TA), a form of vasculitis (angiitis), is a chronic inflammatory disease involving the large blood vessels. This study reports the identification of genes showing increased mRNA levels in peripheral blood mononuclear cells (PBMCs) from TA patients regardless of symptoms or disease activity/inactivity. Of these, mRNA
for Ficolin 1 (FCN1) showed a four-fold increase in all eight TA patients examined. Increased FCN1 mRNA levels observed in cDNA microarrays were confirmed by quantitative reverse transcription polymerase chain reaction. Increased FCN1 protein expression was also observed in inflamed regions of the surgical aorta specimens. Moreover, most FCN1-positive cells were also positive for CD68, indicating the presence of monocytic cells, such as macrophages or dendritic cells, which attack infectious agents within the inflamed regions. Taken together, the results suggest that FCN1 mRNA levels in peripheral blood samples may be a diagnostic marker for TA.

There are a plethora of important biological events that are regulated by cellular interactions among heterotypic cell types. Recent biological achievements have identified many molecules that control heterotypic cell-cell interactions. Although our understandings on these events have lately made remarkable advances at molecular levels, physical aspects of cellular adhesion have not been fully examined yet. Cell Adhesion Molecule-1, CADM1, is a member of the immunoglobulin superfamily, and has multiple functions involved in tumor suppression, synaptogenesis, and spermatogenesis. CADM1 plays a key role as not only simple glue among cells, but also a conductor or promoter of heterotypic intercellular communications. Interestingly, it is now being revealed that the efficiency of CADM1-mediated intercellular communication is closely correlated with the kinetic strength of CADM1-mediated intercellular adhesion, by applying the latest laser technique.

PURPOSE: The applicability of absorbable materials as ligatures of pulmonary vessels has not been described. The present study compares tissue reactions around sites of pulmonary arteries ligated with absorbable material (Vicryl) and with nonabsorbable material (silk). METHODS: Beagle dogs underwent thoracotomy and the pulmonary artery branches were ligated with silk or Vicryl under general anesthesia. The ligated arterial tissues were obtained at 4 and 8 weeks after thoracotomy and processed for pathological analysis. RESULTS: The arteries ligated using Vicryl or silk were clinically completely sealed at 4 weeks after ligation. More inflammation and granuloma were evident at tissues surrounding ligations made with silk than with Vicryl at 8 weeks. Hyperplasia of the arterial intima continued at 8 weeks after ligation with both Vicryl and silk sutures, although some hyperplasia similar to that in nonligated arterial intima appeared at 4 weeks after ligation. CONCLUSION: Less inflammation and granuloma are caused at arterial tissues around ligations accomplished with absorbable Vicryl than those done with nonabsorbable silk sutures, although both are equally effective. Absorbable sutures might be suitable for ligating pulmonary arteries.

Vasculitis (angiitis) is a systemic autoimmune disease that often causes fatal symptoms. We aimed to isolate cDNA markers that would be useful for diagnosing not only vasculitis but also other autoimmune diseases. For this purpose, we used stepwise subtractive hybridization and cDNA microarray analyses to comprehensively isolate the genes whose expressions are augmented in peripheral blood mononuclear cells (PBMCs) pooled from vasculitis patients. Subsequently, we used quantitative real-time polymerase chain reaction (qRT-PCR) to examine the mRNA levels of each candidate gene in individual patients. These analyses indicated that seven genes exhibit remarkably augmented expression in many vasculitis patients. Of these genes, we analyzed G0/G1 switch gene 2 (G0S2) further because G0S2 expression is also enhanced in the PBMCs of patients with systemic lupus erythematodes (SLE). We generated G0S2 transgenic mice that ubiquitously overexpress human G0S2. Although we did not observe any obvious vasculitis-related histopathologic findings in these mice, these mice are unhealthy as they produce only few offspring and showed elevated serum levels of two autoimmunity-related antibodies, anti-nuclear antibody, and anti-double strand DNA antibody. Thus, our large-scale gene profiling study may help finding sensitive and specific DNA markers for diagnosing autoimmune diseases including vasculitis and SLE.

Mast cells are a native composer of connective tissue of the skin dermis and intestinal and respiratory mucosa. Independent lines of accumulated evidence indicate the existence of an intensive bidirectional crosstalk between mast cells and sensory nerves and suggest that mast cells and sensory nerves may be viewed as a functional unit, which could be of crucial importance in neuroimmunological pathways. Mast cells appear to have a property of influencing smooth muscle function via not only such nerve-mast cell effects, but also direct pathways. In bronchial asthma, mast cells infiltrate the airway smooth muscle layer, and interact directly with smooth muscle cells, suggesting pathogenic roles for mast cells in airway obstruction. Current studies on mast cell biology identified a novel adhesion molecule of mast cells, namely cell adhesion molecule-1, CADM1. This molecule is unique, because it serves as not only simple glue but also appears to promote functional communication between nerve and mast cells and between smooth muscle and mast cells.

BACKGROUND: Malignant pleural mesothelioma is a challenging disease with regard to diagnosis and treatment; early and accurate diagnosis would lead to appropriate therapeutic strategies, including extrapleural pneumonectomy. Immunohistochemistry has proven valuable for the diagnosis of the most common epithelioid mesothelioma, although it is often difficult to differentiate it from pulmonary or metastatic adenocarcinoma with absolute certainty if a single antibody is employed. The current study was designed to identify an immunodiagnostic panel for pleural mesothelioma. METHODS: Large surgical specimens from 66 cases with pleural mesothelioma and 66 with lung adenocarcinoma were immunohistochemically reevaluated under uniform conditions. The antibodies examined were directed against the novel mesothelial marker D2-40, as well as calretinin, CEA, and TTF-1. RESULTS: For mesothelioma the sensitivities of D2-40 and calretinin were 84.8% and 87.9%, respectively, and their specificities were both 95.5%. For adenocarcinoma, the sensitivities of CEA and TTF-1 were 95.5% and 92.4%, respectively, and their specificities were both 100%. Immunoreactivity to D2-40 and calretinin was observed in most areas of epithelioid differentiation in mesothelioma. Western blots also showed higher levels of D2-40 antigen in pleura invaded by epithelioid mesothelioma as compared with unaffected pleura. CONCLUSIONS: These data strongly suggest the significant usefulness of D2-40 and calretinin as positive markers, and of CEA and TTF-1 as negative markers, for pleural mesothelioma. The 4-antibody immunohistochemical panel showed high sensitivity and specificity with regard to differentiation of epithelioid mesothelioma from lung adenocarcinoma.

Spermatogenic immunoglobulin superfamily (SgIGSF) expressed on nerve and mast cells, binds homophilically between both in culture. In the steady-state mesentery of mice, the proportion of morphologically degranulating mast cells was approximately 20%, and it increased nearly two-fold when the mesenteric nerve root was stimulated electrically. In contrast, there was no significant increase detectable in the mesentery of MITF-mutants, from which bone marrow-derived cultured mast cells (BMMCs) lack SgIGSF. BMMCs from SgIGSF-knockout mice transplanted to the mesentery of mast cell-deficient W/W(v) mice did not degranulate in response to the mesenteric nerve stimulation, whereas transfection with SgIGSF cDNA restored those responses. SgIGSF appeared to promote communication between nerves and mast cells in the murine mesentery.

Reduced expression of connexins (Cxs), gap junction proteins, is frequently reported in malignant cell lines and tumors, whereas recent studies suggested that C x 26, a subtype of Cxs, might help tumor cells acquire malignant phenotypes. To examine this suggestion in the clinical setting, 50 lung squamous cell carcinomas (SCCs) were stained with the anti-C x 26 antibody. No C x 26-specific signals were detectable in 34 tumors (group I; 68%), whereas the remaining 16 were judged positive for C x 26 (group II; 32%). In 14 tumors of group II, C x 26-specific signals were detected not in all SCC cells but in SCC cells facing the tumor stroma or capsule, in which the signals were localized on the plasma membrane. Involved lymph nodes of group-II patients often contained metastatic foci consisting of all C x 26-positive cells. The proportion of C x 26-positive to C x 26-negative SCC cells in the metastatic nodes was larger than that in the corresponding primary tumors. C x 26-positive SCC cells seemed to be more invasive and metastatic than negative ones. Consistently, the 5-year cancer-specific survival rate of group-II patients was significantly lower than that of group-I patients (12.5 vs 38.9%; P=0.0391). Multivariate analysis demonstrated that C x 26 expression (P=0.0448) as well as pathological stage (P=0.0338) and vascular invasion (P=0.0191) were independent, significant prognostic predictors. These results suggest that C x 26 may represent an essential effector for controlling the biological aggressiveness of lung SCC tumor.

SgIGSF (spermatogenic immunoglobulin superfamily) is a recently identified intercellular adhesion molecule of the immunoglobulin superfamily. In a mast-cell cDNA library, we found a clone that resulted from the retention of intron 7 within the mature SgIGSF message. This clone was predicted to encode a soluble isoform of SgIGSF (sSgIGSF) with 336 amino-acid residues because its open reading frame ended just before the transmembrane domain. We constructed a plasmid expressing sSgIGSF fused to the human IgG Fc fragment at its C-terminus (sSgIGSF-Fc), and transfected it into COS-7 cells. The fusion protein was readily detectable in the culture supernatant. Solid-phase binding assay showed that sSgIGSF interacted directly the extracellular domain of membrane-bound SgIGSF (mSgIGSF). We next examined whether this interaction inhibited homophilic binding of mSgIGSF by aggregation assays using L cells that did not express mSgIGSF. A stable L-cell clone that overexpressed mSgIGSF aggregated with each other but not with mock-transfected L cells, indicating that a homophilic interaction of mSgIGSF mediated the aggregation. Addition of sSgIGSF-Fc inhibited the aggregation of L cells overexpressing mSgIGSF in a dose-dependent manner. Moreover, FACScan analyses revealed the specific binding of sSgIGSF-Fc to mSgIGSF expressed in L cells. Binding of sSgIGSF-Fc to mSgIGSF appeared to inhibit homophilic interactions of mSgIGSF.

Spermatogenic immunoglobulin superfamily (SgIGSF) is a recently identified adhesion molecule, and the microphthalmia transcription factor (MITF) was essential for its expression in mast cells. Since the tg mutant allele is practically a null mutation of the MITF gene, cultured mast cells (CMCs) derived from (WB×C57BL/6)F1 (F1)-tg/tg mice did not express SgIGSF whereas CMCs from F1-wild-type (+/+) mice expressed it abundantly. When cocultured with NIH/3T3 fibroblasts, F1-tg/tg CMCs showed poor adhesion to NIH/3T3 fibroblasts. When injected intraperitoneally, F1-tg/tg CMCs showed poor survival in the peritoneal cavity of mast cell-deficient F1-W/Wv mice. SgIGSF was expressed in tg/tg CMCs ectopically through retroviral transfection and through expression of a transgene. The resulting tg/tg CMCs showed not only a better adhesion to NIH/3T3 fibroblasts but also a better survival in the peritoneal cavity than control F1-tg/tg CMCs. SgIGSF-mediated adhesion seemed to play a role in the survival of CMCs in the peritoneal cavity. © 2004 Published by Elsevier Inc.

BACKGROUND: Recently, the TSLC1 (tumor suppressor in lung cancer 1) gene has been identified as a novel tumor suppressor in human nonsmall cell lung carcinoma. To the authors' knowledge, the clinical relevance of TSLC1 gene expression has not been studied using patient data and surgical samples. The current study was designed to evaluate whether the TSLC1 gene can serve as a target for the prognostic determination of patients with pulmonary adenocarcinoma. METHODS: A total of 38 patients who were surgically treated for proven primary lung adenocarcinoma were enrolled in the current study. Surgical specimens were examined for TSLC1 protein expression immunohistochemically and by Western blot analysis. The correlation between levels of TSLC1 expression and pathologic characteristics, as well as prognosis, was investigated. RESULTS: All patients underwent a potentially curative resection of their tumor. TSLC1 antigen expression as evaluated by immunohistochemistry was confirmed by immunoblotting. The expression of TSLC1 protein was found to be inversely correlated with advanced disease stage, lymph node involvement, lymphatic permeation, and vascular invasion. The 4-year overall survival rates of patients with a tumor demonstrating high (> 70% positive cells [n = 14 patients]), intermediate (20-70% positive cells [n = 10 patients]), and low (< 20% positive cells [n = 14 patients]) expression of the TSLC1 antigen were 84%, 28%, and 7%, respectively. In addition, the disease-free survival of patients with a tumor that demonstrated a high percentage of TSLC1 protein-positive cells was reported to be significantly better than that of patients with a tumor that showed a low percentage of TSLC1 protein-positive cells. CONCLUSIONS: The loss or reduction of TSLC1 expression in resected lung adenocarcinoma cases was associated with a poor prognosis, indicating that TSLC1 represents a central effector gene for controlling the biologic aggressiveness of the tumor and that it is an essential biomarker for predicting patient prognosis. These data may help to detect those patients at high risk for recurrence who might benefit from additional therapeutic strategies such as adjuvant therapy.

Toriello-Carey syndrome comprises agenesis of the corpus callosum, telecanthus, small palpebral fissures, Pierre Robin sequence, abnormal ears, and cardiac defects. We report a boy who has some additional findings, including a severe respiratory failure and intestinal dysmotility. The boy died of these two disorders at age 13 months. Histological examination revealed pulmonary immaturity and a defect of smooth muscle cells in the longitudinal muscle coat of the intestinal musculature, both of which might explain some aspects of the pathophysiology of the patient.

Cyclin G1 is one of the target genes of the transcription factor p53, and is induced in a p53-dependent manner in response to DNA damage. Although cyclin G1 has been implicated in a range of biological phenomena, its precise function remains unclear. Here we present an analysis of the physiological role of cyclin G1 using mice homozygous for a targeted disruption of the cyclin G1 gene. In order to clarify the role of cyclin G1 in the p53 pathway, downstream events such as apoptosis, cell growth and cell cycle checkpoint control were analysed in thymocytes and embryonic fibroblasts derived from cyclin G1-disrupted mice. No difference was detected in induction of apoptosis between mouse embryo fibroblasts (MEFs) derived from cyclin G1+/+ and cyclin G1-/- mice. Following irradiation, cyclin G1-/- MEFs proliferated more slowly and reached lower cell densities in culture dishes than cyclin G1+/+ MEFs. Analysis of cell survival showed that cyclin G1-/- MEFs were about twice as sensitive as cyclin G1+/+ MEFs to gamma radiation or UV radiation. Cyclin G1-/- mice were more sensitive to gamma radiation than wild-type mice. Flow cytometeric analysis revealed that the number of cyclin G1-/- ME

アルツハイマー病の老人斑を形成するβアミロイド蛋白の前駆体であるamyloid precursor protein(APP)は脳外傷により長期にわたり発現する。しかしながら脳外傷後におけるAPPの機能は不明な点が多い。そこで今回、我々はAPPの影響を検討するためにラット実験的脳損傷後の損傷部位に抗APP抗体を直接投与し、脳機能評価と形態学的変化を検討した。抗APP抗体投与により脳機能評価で有意な改善が認められた。抗APP抗体投与により損傷部位の面積に有意な減少を示した。抗APP抗体投与により多数の大型のアストロサイトとGFAP陽性面積の有意な増加が認められた。抗APP抗体投与によりMAP-2陽性細胞数が有意に増加した。これらのことから脳損傷後、APPが損傷周囲のアストロサイトや神経細胞に対して障害性に働くと考えられた。(英文）

レーザーマイクロダイセクションは，病理組織切片にレーザーを照射することにより，組織内に存在する特定の細胞(癌細胞や炎症細胞)を選択的に採取する方法で，採取した細胞や組織から核酸（ＤＮＡやＲＮＡ）あるいはタンパク質を抽出することにより，病変内の特定の細胞における遺伝子やタンパク質発現の異常を検出することができる．

肺真菌症は日和見感染症の１つとして増加している。その診断には真菌形態を病理組織標本中に検出することが重要である。病理標本中に認められる糸状菌の菌要素は菌糸がほとんどである。今回厚膜胞子が気管支内に認められ，診断時に他の真菌との異同が問題になると考えられた症例を経験したので報告した。

目的；脳卒中自然発症stroke-prone spontaneously hypertensive rats (SHRSP)を使って、病変周囲組織と脳室周囲において神経幹細胞と新生神経細胞が出現することを報告した。さらにstromal cell-derived factor-1α (SDF-1α) / CXCR4が脳外傷後の神経幹細胞の出現に関与していることを報告した。しかしながら脳梗塞後の神経幹細胞の出現とSDF-1α/CXCR4についての報告はない。 方法； SHRSPを使って脳卒中発症後の病変周囲組織において神経幹細胞の出現とSDF-1α/CXCR4系について免疫組織学的及び生化学的に調べた。結果；発症周囲組織においてSDF-1α mRNAと蛋白合成の発現増加は認められなかった。発症後、発症周囲組織からSDF-1αが漏出し、周囲組織への拡散が見られ、脳脊髄液中でSDF-1α量の有意な増加が認められた。発症周囲組織ではCXCR4 mRNAと蛋白合成の発現の有意な増加が認められた。発症周囲組織ではnestin陽性細胞像とCXCR4陽性細胞像が一致した。 結論；このことから脳卒中発症後に障害を受けた細胞より逸

目的；脳卒中自然発症stroke-prone spontaneously hypertensive rats (SHRSP)を使って、病変周囲組織と脳室周囲において神経幹細胞と新生神経細胞が出現することを報告した。さらにstromal cell-derived factor-1α (SDF-1α) / CXCR4が脳外傷後の神経幹細胞の出現に関与していることを報告した。しかしながら脳梗塞後の神経幹細胞の出現とSDF-1α/CXCR4についての報告はない。方法； SHRSPを使って脳卒中発症後の病変周囲組織において神経幹細胞の出現とSDF-1α/CXCR4系について免疫組織学的及び生化学的に調べた。結果；発症周囲組織においてSDF-1α mRNAと蛋白合成の発現増加は認められなかった。発症後、発症周囲組織からSDF-1αが漏出し、周囲組織への拡散が見られ、脳脊髄液中でSDF-1α量の有意な増加が認められた。発症周囲組織ではCXCR4 mRNAと蛋白合成の発現の有意な増加が認められた。発症周囲組織ではnestin陽性細胞像とCXCR4陽性細胞像が一致した。結論；このことから脳卒中発症後に障害を受けた細胞より逸脱した

To date, the cell aggregation assay has been widely used to evaluate intercellular attachment in suspension culture. We successfully dissociated the cell aggregates by focusing femtosecond laser pulses (fsLP) at the cell-cell interface, and estimated the binding force of cell-cell adhesion from the laser energy minimally required for the dissociation. Interestingly, this estimation was quite well correlated with that provided by the cell aggregation assay. The fsLP was applied to leukocyte-endothelial cell coculture: HL-60 leukocytes were seeded onto a monolayer of HUVEC endothelial cells, and fsLP was repeatedly focused at the ventral side of a HUVEC-adherent HL-60 cell in its vicinity. When the fsLP was adjusted to an appropriate energy, the HL-60 cell moved 1-3 um after every fsLP shot. The fsLP appeared to be a powerful tool to reveal adhesive properties of a variety of circulating cells, such as leukocytes and cancer cells, which can interact with endothelial cells.

接着分子に関する理解は分子レベルにおいて近年急速に進歩したが、細胞間接着の力学的側面についてはあまり調べられて来なかった。最近我々はフェムト秒レーザー（fs-L）を用いてこの問題に取り組んだ。fs-Lを培養液中に集光すると？mのスケールで衝撃力が伝搬するので、この衝撃力によって細胞間の接着を乖離させ、その際の集光点と標的細胞までの距離を測定することにより、細胞間接着力を見積もることに成功した。また、レーザー技術を細胞微細構造採取に応用した。高転移性癌細胞が作る浸潤突起を選択的に回収することに成功した。回収された蛋白量は微量であったが、それをマス解析に供することで、浸潤突起のプロテオミクスへと展開した。

接着分子に関する理解は分子レベルにおいて近年急速に進歩したが、細胞間接着の力学的側面についてはあまり調べられて来なかった。最近我々はフェムト秒レーザー（fs-L）を用いて細胞間接着力を見積もることに成功したので報告する。fs-Lを培養液中に集光すると？mのスケールで衝撃力が伝搬するので、この衝撃力によって細胞間の接着を乖離させ、その際の集光点と標的細胞までの距離を測定した。一方、集光したfs-Lの集光点における衝撃力の大きさは原子間力顕微鏡の応用により力積単位（Ns = Newton x second）で測定した（奈良先端大学細川らによる）。この２つの測定値から細胞間接着力を見積った。実例として、（１）HL-60白血球とHUVEC内皮細胞間の接着力、及び（２）MDCK上皮細胞間の接着力を見積もった。（１）ではHUVEC単層培養の上からHL-60細胞を播種し30分後にHL-60細胞の側方にfs-Lを集光した所、HL-60細胞はHUVEC上を数？mスリップした。（２）ではMDCK細胞を穴あきフィルター

本研究の目的は、術前化学療法を受ける進行食道癌患者対象に治療開始前FMISO PET/CTで治療効果予測が可能か病理学的評価を踏まえて検討することである。さらにFMISO集積で腫瘍内低酸素やPD-L1やCD8T細胞が発現する腫瘍免疫環境を予測可能か前向き研究で検討する。 科研費交付決定後に近畿大学医学部倫理委員会に研究計画書を提出し、一括申請を行った。倫理委員会で研究実施実施計画が承認された後に食道癌の病理組織標本に免疫染色行うための抗体やFMISOの薬剤合成にかかる消耗品の購入、FMISO PET/CTの検査が円滑に進められるように研究体制を整えた。倫理委員会承認後本研究を開始し、共同研究を行う上部消化管外科と密接に連携して、研究に参加可能な対象者を集め始めた。初年度は研究体制整備が主だった実績となった。 今年度は10症例程度を目標に予定していたが、医学部倫理委員会に申請する研究計画書の作成やその審査にかなり時間を要し、計画に遅れが生じた。次年度からは研究に該当する食道癌患者の収集を第一に努め、収集症例数をあげることである。また、食道癌の術前化学療法後、手術を行った患者の病理染色標本の染色および患者の経過観察に関して円滑に進めていけるように関係各所（近畿大学上部消化管外科、近畿大学病理学講座、久留米大学病院病理部病理診断科、大阪大学大学院医学系研究科情報統合医学講座医学統計学）との連携を再確認して、随時研究の打ち合わせの実施を行う。

抗PD-1/PD-L1抗体と細胞障害性抗がん薬もしくはその他の免疫チェックポイント阻害薬併用療法を使用した肺がんを中心とした固形がんの臨床検体を使用した免疫関連の遺伝子発現測定を行い、遺伝子発現情報からの分類を行う（免疫プロファイリング）。免疫プロファイリングによるサブグループと抗PD-1/PD-L1 抗体薬＋細胞障害性抗がん薬の併用療法の有効性についての関連を検討する。肺がん等の治療として、免疫チェックポイント阻害薬と細胞障害性抗がん薬併用療法治療が有用（もしくは無効）と考えられる集団を同定する。 1） 抗PD-1/PD-L1抗体およびその細胞障害性抗がん薬の併用療法を受けた進行非小細胞肺がんや小細胞肺がんなどの臨床検体を使用した遺伝子発現、遺伝子異常の解析を行うNanoString Panel(GX PanCancer Immune Profiling Panel等)を用いて腫瘍免疫に関連する770遺伝子の発現解析を行う。Whole exome sequencingやTargeted sequencingにより遺伝子異常を包括的に評価する。 2）肺癌臨床検体を使用して、腫瘍微小環境における免疫関連因子の発現を免疫染色により病理学的に評価する・PD-L1の腫瘍における発現およびCD4、CD8、FOXP3等の免疫関連因子を病理学的に免疫染色等の手法を用いて評価する。 3）これらの遺伝子解析、病理学的解析および臨床データを使用したクラスター解析を行い、非小細胞肺がんにおける免疫プロファイル毎のサブグループ分類を確立する。 4)上記サブグループ分類を元に、抗PD-1/PD-L1抗体と細胞障害性抗がん薬もしくは抗PD-1/PD-L1抗体単剤の有効性、安全性の差異との関連に関して検討を行う。

緑内障や水頭症において病的内圧上昇は神経変性の一因と考えられているが、分子機序は未だ解明されていない。これまでに申請者らは、特殊な加圧装置を必要とせず且つ通常の培養環境において静的圧負荷が可能な細胞培養装置を開発し、圧力上昇に伴って神経接着分子であるCell adhesion molecule 1 (CADM1)の細胞外領域の酵素的切断(shedding)が亢進し軸索変性を惹起することを明らかにした。その過程でCADM1のshedding産物にはアスパラギン酸に富む配列があり、shedding産物は圧負荷によって神経軸索上で凝集することを見出した。申請者が独自に樹立した水柱下2-チャンバー培養装置を用いて、マウスの上頸神経節細胞を初代培養し、30～50センチ水柱の圧負荷を加えた。抗D体化アスパラギン酸抗体にてウエスタンブロットしたところ、分子量約70 kDa付近に特異的なシングルバンドが検出された。本バンドは圧負荷検体にてシグナル強度が明瞭に上昇し、非圧負荷検体にても弱い反応性を示した。当該蛋白質は神経軸索内にて非圧負荷の状態にてアスパラギン酸残基にある程度のD体化が生じており、圧負荷にてD体化が亢進するとの可能性が考えられた。当該バンドを質量分析に供した。いくつかの候補分子が選出された中、ウエスタンブロット法等にて上述の特異的バンドはアネキシンA6と同定された。 一方、接着分子CADM1については、その細胞外切断後に細胞側に残るC末断片（CTF）については、上述の抗D体化アスパラギン酸抗体によるウエスタンブロットでは特異的抗体反応が検出されなかった。現時点で、CADM1切断産物のアスパラギン酸残基にD体化が生じているとの明確な証拠は得られていないが、CTFの一部（15アミノ酸長）を合成し、LC-MSにてアスパラギン酸残基のD体化の検出を試みる予定である。

この研究の目的は11C-methionine (MET) PETと神経膠腫で手術を受けた患者を対象に、アミノ酸代謝や細胞増殖シグナル伝達因子（LAT1, mTOR, HIF1-α，VEGF, EGFR)とMET集積との相関性を明らかにし、MET集積パラメータ(T/N ratio,SUVmax, SUVmean, TLA, MTV)と、細胞増殖シグナル伝達因子を用いて予後予測因子を前向き研究で明らかし、脳腫瘍の腫瘍増殖とアミノ酸代謝の関係について病理学的解明も試みる。令和元年度も当施設の脳神経外科と密接に連携し、研究参加可能な脳腫瘍患者の登録を継続している。年度末までにMET PET/CTを受けた32例の脳腫瘍患者を登録できた。これらの患者は当施設で手術や放射線治療などの集学的治療やその後の経過観察も行っている。現在、そのうち17例の病理標本染色を共同研究機関である久留米大学の病院病理部で開始し、染色を行っている途中である。また、収集したMET PET/CTデータのパラメータに関する解析も同時に遂行中で、その結果の一部を第79回日本医学放射線学会総会で発表する予定である。来年度も対象患者のデータ収集を第一に努めていきたいと考える。PET/CTデータ収集と解析、手術を行った患者の病理標本の染色に関して円滑に進めていけるよう、関係各所との連携を再確認し、随時打ち合わせを実施する。次年度は最終年度に該当するため、データのとりまとめを行っていく予定である。

腔内圧（眼内圧や尿路内圧等）の上昇は神経変性や粘膜変性を惹起する。その閾値は30 cm水柱程度と比較的低値であり一定である。細胞にこの程度の圧を負荷できるtwo-chamber培養装置を新規に考案した。初代培養神経細胞は30 cm水柱以上の圧負荷で軸索変性を来すことがわかった。この際には軸索の細胞膜上に存在するIgCAM型接着分子CADM1の細胞外切断が亢進し、その細胞側産物の蓄積が変性誘因の１つであった。一方、円柱状の上皮細胞では30 cm水柱以上の圧負荷で形状は扁平化するとともに、増殖が抑制されることがわかった。本研究は軽微な圧負荷が病変を形成するに至る病態生理の解明に寄与するものである。

政府の重要政策のひとつである個人消費の拡大への寄与を目的に、商品・サービスのより良い品揃えのための情報になる、世帯の家族構成が分かる世帯分類を開発した。さらに、この新しい世帯分類によって世帯分布の時系列分析を行い、1980年から2010年に至る30年の間に、世帯の家族構成別分布に大きな変化が起こっていること、及びその要因を明らかにした。

ハプテン誘導型アトピー性皮膚炎マウスモデルにおける神経-マスト細胞間相互作用の実態とIgCAM型接着分子CADM1の役割を解析した。病変内のマスト細胞数は健常対照の約５倍で、病変内の各マスト細胞におけるCADM1 mRNA発現は健常対照の約３倍であった。マスト細胞と後根神経節細胞との共生培養系において、マスト細胞と神経突起との接着力、及び神経脱分極に応答するマスト細胞の割合は、マスト細胞におけるCADM1の発現レベルと相関した。アトピー性皮膚炎ではマスト細胞におけるCADM1の発現上昇の結果神経-マスト細胞相互作用が増強しているため、精神的なストレスによって症状が増悪する可能性が示唆された。

細胞接着分子群の解析に基づくがんの個性診断法の開発を目指した研究を行い、細胞接着分子CADM1のスプライシング・バリアントが小細胞肺がんに特異的に発現し、診断マーカー候補となること、また、CADM1がMETシグナル経路を抑制し、肺腺がん細胞のゲフィティニブ耐性獲得に関わり、耐性予測マーカーとなる可能性を見出した。

アジア域内の9か国の各国統計局から所得分布関連世帯統計のミクロデータとメタデータの提供を受け、それらミクロデータのファイル変換とメタデータの整備を行った上で、各国がこれまで実施してこなかった集計分析を行った。それらを例示として、データ提供国の統計局職員を対象に国際ワークショップを開催し、研究成果のデータ提供国への還元及びデータ提供国職員の分析能力の向上を図った。

高強度の近赤外フェムト秒レーザー光を顕微鏡下で細胞培養液に集光すると、効率的な多光子吸収により集光点で爆発現象が引き起こされる。この爆発現象により発生する衝撃力は、ミクロンオーダーの領域に局在するため、1細胞の局所領域に機械刺激を加えられる。本研究では、筋芽細胞と神経細胞に、このフェムト秒レーザー誘起衝撃力を作用させ、カルシウムインディケーターにより1細胞レベルの細胞活性変化を解析する新手法を開発した。レーザー強度により機械刺激の大きさや作用領域を自在に変えられる特徴を駆使して、1細胞内で刺激が活性化されるメカニズムを検討した。

がん浸潤部において高発現が確認されたNotch2とSix1について機能解析を行い、Notch2-Six1転写カスケードとして2分子が協調的に一連の遺伝子群の発現を活性化し、肺上皮細胞においてepithelial-mesenchymal transition, EMTや核の腫大を促進させることにより肺腺癌の悪性化が進展する可能性を明らかにした。新たにGGO/ Solid混在の肺腺癌症例において2分子の免疫組織染色による発現パターンと臨床データとの関連性の検討を行い、2分子が浸潤部で高発現を認める肺腺癌症例は浸潤部で高発現を認めない症例よりも悪性度が高い可能性が示唆された

神経とマスト細胞の両者に発現するIgCAM型接着分子CADM1は神経-マスト細胞相互作用を促進する。ハプテン誘導型アトピー性皮膚炎マウスモデルの病変皮膚及び正常皮膚から、レーザーマイクロダイセクション法によりマスト細胞だけを選択的に採取した。RT-PCRによって病変内マスト細胞におけるCADM1の発現上昇が検出された。従って、アトピー性皮膚炎では神経-マスト細胞相互作用が増強し、マスト細胞の脱顆粒が誘発される可能性が示唆された。

マウスの神経節から初代培養神経細胞を神経成長因子の存在下で培養し、神経細胞と免疫細胞(マスト細胞)との共存培養システムを確立した。次いで、分子イメージンング法を用いて両細胞の相互作用を追究した。神経細胞からのサブスタンスPがマスト細胞を活性化し、また、マスト細胞からのATPが神経細胞を活性化していることを解明した。さらに、細胞間相互作用には、接着分子CADM1が重要な役割を果たしていることを明らかにした。

悪性黒色腫は早期に転移をきたす予後不良の腫瘍である。これまでにわれわれは悪性黒色腫の転移においてコネキシン26(Cx26)が重要な役割を担っていることを見いだした。本研究では悪性黒色腫の転移を抑制する分子標的治療薬の開発を目指して、Cx26分子の細胞外ドメインに対する抗体の作製を試み、マウス悪性黒色腫転移モデルを用いて、この抗体の転移阻止効果を調べた。その結果、作製した抗体は不完全ではあるが悪性黒色腫の転移抑制作用を示した。

がん細胞と宿主の遺伝子解析を行い、がんの分子診断に有用な技術、標的分子を明らかにした。まず、我々が開発したアレル当りの遺伝子発現の高度定量的解析法であるRNA Difference Plot法を用いて、家族性腫瘍の原因遺伝子のアレル当りの発現が、その変異保因者間の表現型の予測に有用であることを見出した。次に、非小細胞肺がん、腎淡明細胞がんでは、CADM1、並びにその結合タンパク質である4.1Bががん抑制遺伝子として働くことを見出した。一方、成人T細胞性白血病ではCADM1が特異的に高発現し、下流ではTiam1が結合し、RACタンパク質を活性化して細胞浸潤能を亢進すること、従ってこの経路が浸潤抑制の標的分子経路となることを見出した。

SgIGSF(spermatogenic immunoglobulin superfamily)/TSLC1(tumor suppressor in lung cancer-1)は、免疫グロブリン・スーパーファミリーに属する細胞膜1回貫通型の接着分子で、様々な細胞に発現しており、精子形成やシナプス形成など種々の機能を有するが、本分子の機能制御の仕組みについてはよくわかっていない。本研究課題では、SgIGSF/TSLC1の発現制御機構として転写調節(splicingによる分泌型分子の産生)と転写後調節(sheddingによる細胞外ドメイン断片の産生)が存在することを明らかにした。加えて、分泌型分子は細胞外に放出された後、神経突起上に発現する膜貫通型分子とtrans-ホモフィリックに結合することにより、神経突起の伸長方向の制御に関わることがわかった。

[緒言]Cell adhesion molecule 1 (CADM1;旧名称SgIGSF)は免疫グロブリンスーパーファミリーに属するマスト細胞接着分子で、マスト細胞と腸間膜中皮細胞との接着を媒介する。胸膜中皮腫は胸膜面に瀰漫性病変を形成し、しばしば肺や心臓を完全に包囲するが、肺実質内への浸潤傾向は著しく乏しい。従って中皮腫細胞は胸膜、特にその表面を覆う中皮細胞と相互作用する特別な道具を持っていると推測される。本研究ではCADM1がその道具の1つである可能性を検討した。[結果1]免疫組織化学及びウエスタン法によりヒト悪性胸膜中皮腫57症例におけるCADM1の発現を調べた所、少なくとも1/4の症例においてCADM1が腫瘍細胞の細胞膜上に高発現していることを見出した。一方、非腫瘍性中皮細胞は反応性異型を伴うものでもCADM1の発現は検出されなかった。中皮腫細胞はCADM1発現を獲得するものと考えられるが、その機序として細胞外ドメインshedding機構の異常を想定された。臨床病理学的な緒因子についてCADM1陽性症例と陰性症例との間に有意差はなかった。[結果2]5種の胸膜中皮腫細胞株ではCADM1の発現があるものが3つ、ないものが2つであった。両者を初代中皮細胞又は肺線維芽細胞単層培養上で共生培養して比較した。中皮細胞単層培養上では、CADM1陽性細胞は中皮細胞との良好な接着性を示し、陰性細胞より早く増殖した。CADM1陰性細胞は接着性に乏しくpile upする傾向が強かったが、CADM1全長cDNAを遺伝子導入すると陽性細胞と同様の接着性・増殖性を獲得した。一方、肺線維芽細胞単層培養上では陽性・陰性細胞間に大きな表現型の差はなく、いずれの細胞も中皮細胞単層培養上より増殖速度が著しく遅かった。[結語]胸膜中皮腫細胞に発現するCADM1は中皮腫細胞と中皮細胞との接着を媒介するだけでなく中皮細胞上での増殖も促進することで、胸膜中皮腫に特徴的な胸膜面彌漫性病変形成に関与している可能性が示唆された。

神経とマスト細胞との間の解剖学的及び機能的な連関は古くから知られているが、この連関を支持する分子は不明であった。最近研究代表者は接着分子SgIGSF(Spermatogenic Immunoglobulin Superfamily)を新規マスト細胞接着分子として単離した。SgIGSFは免疫グロブリンスーパーファミリーの一員で、細胞外に3つの免疫グロブリン様ループを、細胞内には細胞骨格系と連結するモチーフ配列を持つ。構造的にはNCAM(Neural Cell Adhesion Molecule)-1やNCAM-2に似ている。この分子には他にTSLC1(Tumor Suppressor in Lung Cancer-1)とSynCAM(Synaptic Cell Adhesion Molecule)の名がある。即ち、この分子は、1)精原細胞に発現し、セルトリー細胞への接着を媒介する(SgIGSF)、2)肺癌で発現の消失が高頻度に見られ、肺癌細胞に強制発現させると造腫瘍性が失われる(TSLC1)、3)神経細胞に発現し、神経細胞間にシナプスの形成を誘導する(SynCAM)、という3つの機能を持つことが他のグループにより報告された。興味深いことに、この接着分子はホモフィリックに結合できる。従って、マスト細胞と神経との相互作用がこの分子のホモフィリックな結合によって媒介される可能性がある。この接着分子の神経-マスト細胞間相互作用への寄与を検討するため、上頸神経節細胞とマスト細胞を共生培養し、(1)神経突起に接着するマスト細胞の数、及び(2)サソリ毒による神経特異的刺激に対して応答(細胞内Caイオン濃度の一過性上昇)するマスト細胞の割合を調べた。SgIGSFを発現しないマスト細胞(MITF転写因子変異マウス由来培養マスト細胞やIC-2マスト細胞株)はSgIGSFを発現するマスト細胞(野生型マウス由来培養マスト細胞)に比して、(1)接着細胞数は3分の1、(2)応答率は2分の1であったが、外来性にSgIGSFを発現させるといずれのパラメーターも正常化した。免疫染色では野生型マウス由来培養マスト細胞と神経突起との接点にSgIGSFの集積した局在が確認された。以上の結果より、SgIGSFはそのホモフィリックな結合により神経-マスト細胞間の接着及び機能的な相互作用を媒介する分子であることが示唆された。

代表者は最近SgIGSF(Spermatogenic Immunoglobulin Superfamily)をマスト細胞の新規接着分子として単離し、その発現にはmicrophthalmia転写因子(MITF)が必須であることを示した。即ち、野生型マウス由来培養マスト細胞(+/+-CMC)はSgIGSFを豊富に発現するが、MITF遺伝子の実質的なnull変異のホモtg/tgマウス由来の培養マスト細胞(tg/tg-CMC)はSgIGSFを発現しない。CMCをNIH/3T3線維芽細胞と共生培養すると、+/+-CMCは相当数が線維芽細胞に接着するが、tg/tg-CMCの接着細胞数は4分の1程度である。またCMCをマスト細胞欠損W/W^Vマウスに腹腔内投与すると、+/+-CMCは生存するが、tg/tg-CMCは全て死滅する。この2つの欠陥がSgIGSFの発現低下によるものかを調べるため、外来性にSgIGSFを発現するtg/tg-CMCを、1)レトロウイルスベクターを用いた遺伝子導入、2)SgIGSFトランスジェニックマウスの作製とtg/tgマウスとの交配により樹立した。これら2種類の、外来性にSgIGSFを発現するtg/tg-CMCはいずれも線維芽細胞への接着能が正常化し、W/W^Vマウスの腹腔内での生存能についても有意な改善を示した。従って、SgIGSFが媒介するCMCの接着が腹腔内での生存に寄与すると考えられた。

転写因子MITFがマスト細胞の分化にどのように関係しているのか総合的に調べる計画を立てて4年間研究を行い以下の成果を得た。1)MITFをコードするmi遺伝子座には多くの突然変異が知られている。突然変異の構造と転写因子としての機能の間にはどのような関係があるのか調べた。MITFのDNA結合部分であるbasic領域の点突然変異の結果、DNA結合能を欠くMITFは転写能を欠くばかりでなく他の転写因子の機能を妨害する(inhibitory mutation)。一方MITFを全く産生しないか、構造上きわめて大きな異常を持つMITFを産生する突然変異の場合、MITFの転写能が存在しないだけであり、ホモ・マウスの症状はinhibitory mutationの場合より軽い(null mutation)。2)MITFを発現していないtg/tgマウスを、マスト細胞の最も重要な増殖因子stem cell factor(SCF)の受容体であるc-kit receptor tyrosine kinase(KIT)の機能喪失性突然変異マウスW/Wvと同じ遺伝的背景で作成した。このtg/tgマウスはほぼ正常に成長するのでin vivoの実験を容易に行うことができた。X線照射したW/Wvマウスの静脈内に+/+マウスの骨髄細胞を注射すると赤血球、好中球、マクロファージ、B細胞、T細胞は+/+マウス由来の細胞に置き換わり、W/Wvマウスで欠損していたマスト細胞も全身の組織で出現する。一方tg/tgマウスの骨髄細胞をW/Wvマウスに注射した場合には、赤血球、好中球、マクロファージ、B細胞、T細胞はtg/tg由来の細胞に置き換わるものの、マスト細胞はどの組織でも出現しない。MITFは成熟したマウスの組織内でマスト細胞がその前駆細胞から分化するために必須の転写因子であると考えられた。

マスト細胞は骨髄で最終分化を終えぬまま末梢血中移動し、末梢組織において血管外へ浸潤し最終分化を行う。マスト細胞は全身の皮膚や消化管、腹腔内などに生理的に存在するが、血管内に存在するマスト細胞前駆細胞が末梢組織内へ浸潤する機構についてはよくわかっていない。MITF遺伝子のプロモーター領域に挿入されたトランスジーンをホモに持つtg/tgマウスでは皮膚以外の全組織でマスト細胞が欠損する。ところが、このマウスの骨髄細胞や脾臓細胞をインターロイキン3存在下で培養すると、野生型マウスの場合と同じように培養マスト細胞(CMC)を得ることが出来る。従って、tg/tgマウスではマスト細胞の前駆細胞は存在するが、その細胞が組織へ浸潤することが出来ない可能性、更にはMITFが転写活性化を行う遺伝子の中にマスト細胞の組織浸潤に関与する分子が含まれている可能性が考えられる。tg/tgマウスの腹腔内におけるマスト細胞欠損について調べるため、CMCをtg/tgマウスの腹腔内に注射した。野生型マウス由来のCMC(+/+-CMC)は腹腔内で生存し続けたが、tg/tgマウス由来CMC(tg/tg-CMC)は死滅した。この現象はin vitroでも再現され、+/+-CMCは線維芽細胞との共生培養において線維芽細胞に接着し増殖したが、tg/tg-CMCは接着せず死滅し、tg/tg-CMCの接着能の欠陥が明らかになった。次にtg/tg-CMCにおいて発現が著しく低下し、かつ接着に関与する可能性がある遺伝子の単離をサブトラクション法により試みた。出来上がったライブラリーから600クローンを単離し、その中にSgIGSF (Spermatogenic Immunoglobulin Superfamily)をコードするクローンを見つけた。SgIGSFは細胞外に3つの免疫グロブリン様構造を持つ接着分子である。tg/tg-CMCにSgIGSFを遺伝子導入すると、線維芽細胞への接着能が正常化した。以上の結果より、SgIGSFはマスト細胞が線維芽細胞に接着する時に使われる接着分子で、その転写障害がMITF変異マウスにおける腹腔マスト細胞欠損の一因である可能性が考えられた。

マウスを用いた転移実験モデルにおいて足底部皮下接種あるいは尾静脈内接種の後の肺転移は、それぞれ自然転移あるいは実験転移と呼ばれる。B16マウス・メラノーマ細胞の亜株F10とBL6は同程度の実験転移能を有するが、自然転移能はBL6にあってF10にはほとんどない。従って、BL6には皮下組織における浸潤・血管内侵入能があるのにF10にはないと推測される。cDNAライブラリーを用いたサブトラクション法の改良を行った結果、F10とBL6との間で発現に差のあるクローンを20数種単離することに成功した。 そのうち最大の発現差異を示したクローンの1つは、コネキシン26遺伝子で、BL6細胞でこの遺伝子に変異はなかった。F10、BL6細胞への野生型及びドミナント・ネガティブ型コネキシン26遺伝子導入実験により、コネキシン26はメラノーマ細胞と内皮細胞とのギャップ結合を媒介するとともに、メラノーマ細胞の自然転移能を規定する分子であることを示した。最近報告されたギャップ結合阻害物質オレアミドは生理的に脳脊髄液内に存在する脂肪酸でふる。オレアミドをマウスの腹腔内に連日投与したが、明らかな副作用は認められなかった。媒体としてはオリーブ油が適していた。マウスにメラノーマ細胞を接種後オレアミドを連日投与して、メラシーマ細胞の転移形質に対する抑制効果を判定すれば、オレアミドが副作用の少ない初めての抗転移化学療法薬となりうるものと考えられた。

突然変異マウスmi/miマウスの責任遺伝子座にはbHLH-Zip型の転写因子MITFがコードされている。mi変異遺伝子座から作られる変異型転写因子mi-MITFは塩基領域の4連続アルギニンのうちの1つが欠失しており、このためDNA結合能や核移行能が障害され転写因子として機能できない。申請者はこれまでにmi/miマウス由来培養マスト細胞において転写が傷害されている遺伝子群の単離をcDNAライブラリーのサブトラクション法により行ってきた。その結果MITFによって転写制御を受ける遺伝子の1つとしてグランザイムBを同定した。グランザイムBは主にnatural killer(NK)細胞、細胞傷害性T細胞の細胞質顆粒内に存在し、これらの細胞が示す細胞傷害活性に必須なセリン・プロテアーゼである。興味あることに、mi/miマウスでは細胞傷害活性の低下が報告されている。その原因としてNK細胞数の減少が考えられていたが、申請者が得た知見はその原因がNK細胞におけるグランザイムB遺伝子の転写障害である可能性を示唆している。 本研究において、mi/miマウスのNK細胞におけるグランザイムB遺伝子の転写障害の有無、細胞傷害活性の低下との関連、及びmi-MITFが転写に及ぼす影響を調べた。mi/miマウスのNK細胞においてグランザイムB遺伝子の転写障害はなかった。その代わりパーフォリン遺伝子の発現レベルが低下していた。パーフォリンはグランザイムBと並んでNK細胞の細胞傷害活性に必須の分子であり、パーフォリンの発現低下がmi/miマウスのNK活性低下の原因と考えられた。発現低下の分子機序は、mi-MITFがパーフォリン遺伝子転写活性化因子の核移行を障害するためであることを示した。

mi遺伝子座がコードするbasic-helix-loop-helix leucine zipper型転写因子(MITF)によるマスト細胞特異的遺伝子発現機構を、突然変異マウスを用いて解析した。mi遺伝子座には多くの突然変異マウスが報告され、分子レベルで異常点が明らかとなっているものも多い。一つの転写因子でこれほど多くの突然変異マウスの存在する例は他になく、転写因子の機能をin vivoで解析できるよい系である。mi遺伝子座の突然変異マウスのうち、mi/miマウスは異常なMITF(mi-MITF)を正常量発現するのに対し、tg/tgマウスはプロモーター領域への挿入突然変異によりMITFを発現しない。mi/miマウスとtg/tgマウスのマスト細胞の表現型を比較すると、mi/miマウスのマスト細胞でより異常の程度が強かった。このことは、mi-MITFが転写に対して抑制的に働くことを示唆する。今回、zipper domainを欠損するmi^<・ce>/mi^<・ce>マウスのマスト細胞をtg/tgマウスと比較し、MITFの機能におけるzipper domainの重要性を検討した。その結果、mi^<・ce>/mi^<・ce>マウスのマスト細胞の異常の程度は、tg/tgマウスのマスト細胞の異常の程度と同程度で、zipper domainがMITFの機能に重要であることがわかった。また、以前mi/miマウスの皮膚マスト細胞でヘパリン含量の減少を報告したが、その機構として、ヘパリンの糖鎖硫酸化に関与するN-deacetylase-N-sulfotransferase2(NDST-2)をコードする遺伝子の発現に転写因子GABPが関与すること、更にmi-MITFがGABPの活性を抑制してNDST-2遺伝子の転写レベルを低下させることを明らかにし、MITFを中心とした転写因子ネットワークがマスト細胞の分化に重要であることも明らかにした。

マウスのmi遺伝子座にコードされる転写因子MITFはマスト細胞の分化に強く関係している。最初に発見されたミュータントであるmi/miマウスではbasic領域のアルギニンが1個欠失するためDNA結合能と転写活性化能を欠くmi-MITFが正常量産生されるが、mi伝子のプロモーター領域にトランス・ジンが挿入されたtg/tgマウスではMITFはまったく産生されない。我々はmi/miミュータント・マウスとtg/tgミュータント・マウスから得た培養マスト細胞で発現している遺伝子を正常(+/+)マウスの培養マスト細胞で発現している遺伝子と比較する事によりmi-MITFがどのような機能を持っているか調べることにした。各々の遺伝子型を持った培養マスト細胞からcDNAライブラリーを作製し、続いて(+/+-mi/mi)と(+/+-tg/tg)のサブトラクション・ライブラリーを作製した。予想に反して(+/+-mi/mi)サブトラクション・ライブラリーの方が(+/+-tg/tg)サブトラクション・ライブラリーよりも有意に多くのクローンを含んでいた。つまりmi-MITFが存在する場合の方がMITFが全く存在しない場合より培養マスト細胞で発現する遺伝子を減らすのである。実際+/+マスト細胞で発現していることが分かっている種々の遺伝子についてmi/mi培養マスト細胞とtg/tg培養マスト細胞で比べてみると、大部分の遺伝子ではmi/mi培養マスト細胞における発現量はtg/tg培養マスト細胞における発現量よりも多かった。以上の結果はmi-MITFの転写抑制効果を示している。さらにmi-MITFの転写抑制の機序についても研究を進め、MITF以外の蛋白との相互作用を介している事を示唆する結果を得た。相互作用の相手となる蛋白は転写しようとする遺伝子により異なる。

mi転写因子(MITF)遺伝子変異マウスのうち、NK活性を欠くmi/miマウスとNK活性の正常なtg/tgマウスを比較し、NK細胞が持つ2つの主要な細胞障害活性機能分子、グランザイムBとパーフォリンの転写調節におけるMITFの機能を解析し、以下の点を明らかにした。 1)NK細胞におけるグランザイムB遺伝子の発現に対してはMITFは関与しない。 2)NK細胞におけるパーフォリン遺伝子の発現に対して正常型MITFは関与しないが、mi変異型MITFは転写抑制効果を示す。その機序はパーフォリン遺伝子転写活性化に重要な他の転写因子に対する核移行障害作用と考えられた。 3)mi/miマウスは細胞障害活性を有する2系統の細胞種、マスト細胞とNK細胞において異なる遺伝子発現障害を示す変異動物である。即ち、マスト細胞においてはグランザイムBの、NK細胞においてはパーフォリンの遺伝子発現が障害されている。 (考察)tg/tgマウスのNK1.1陽性大リンパ球では、NF-P結合因子がNF-Pモチーフを活性化し、パーフォリン遺伝子の転写が行われる。その翻訳産物、パーフォリン分子はアズール顆粒に貯蔵され、細胞障害活性が正常に備わる。mi/miマウスでもNKL1陽性大リンパ球の数は正常であった。しかし、核移行能が不全なmi型MITFが存在するために、その相互作用パートナーであるNF-Pモチーフ結合因子の一部の核移行が阻害され、パーフォリン遺伝子の転写活性化が低下する。さらに細胞質内アズール顆粒形成も障害されていた。その結果としてmi/miマウスではNK活性が低下していると考えられた。

マウス・メラノーマ細胞株B16の亜株のうち、実験的転移能(静注後肺転移)と自然転移能(皮下注後肺転移)を有するBL6細胞と、実験的転移能のみを有するF10細胞との間でcDNAライブラリーのサブトラクションを行った。BL6細胞において高発現を示す遺伝子として、コネキシン26と、プロテイン・フォスファターゼ2A(PP2A)制御サブユニットB56γ変異型アイソフォームΔγ1を単離した。コネキシン26は表皮細胞、肝細胞のがん化過程においてはその発現が低下する。一方メラノサイト及びその良性・悪性腫瘍細胞においてコネキシン26の発現はまだ調べられていない。Δγ1はPP2AB56遺伝子におけるレトロトランスポゾン挿入がもたらす変異型アイソフォームで、B56γ1アイソフォームのN末の100アミノ酸が欠失している。PP2Aの阻害剤オカダ酸が発がん作用を持つことから、PP2Aは発がん抑制物質として期待される。実験結果:1)メラノーマ細胞はコネキシン26を介して血管内皮細胞とヘテロなギャップ結合を形成できる。2)F10及びBL6細胞においては、ギャップ結合形成能と自然転移能とはよく相関した。3)パキシリンとPP2Aとの接着斑における共局在を見つけた。4)PP2Aの細胞骨格再構成に対する抑制的な機能はパキシリンのリン酸化制御を介していた。5)Δγ1はPP2Aによるセリン脱リン酸化を抑制し、パキシリンの接着斑リクルートを亢進した。考察:1)自然転移能獲得、特に血管内侵入・血管外浸潤において、腫瘍細胞と血管内皮細胞との間のヘテロなギャップ結合形成が重要な役割を果たしている可能性が示された。2)Δγ1はPP2Aによるパキシリン機能制御を破壊し、細胞骨格再構成が迅速化した。発がん抑制におけるPP2Aの作用点の1つがパキシリンである可能性が示された。

マウス・メラノーマ細胞株B16の亜株のうち、実験的転移能と自然転移能のいずれをも有するBL6細胞と、実験的転移能は有するが自然転移能は有しないF10細胞との間でcDNAライフラリーのサブトラクションを行って、自然転移能獲得に正の働きをしている遺伝子群の単離を試みた。BL6細胞において高発現する遺伝子を現在までに20数種単離した。そのうち次の遺伝子について解析を進めている。コネキシン26、プロテイン・フォスファターゼ2Aの制御サブユニット、Phakinin(中間径フィラメント)、アセチルCoAトランスポーター、b-Myc、I-κbに結合する酵母遺伝子のマウス・ホモローグ、Zn・フィンガー・モチーフを持つ新規遺伝子。 現在までの成果を以下に列挙する。 1) コネキシンは自然転移能獲得に正の働きをしうる。この機能はコネキシンのサブタイプによる特異性があり、正常細胞(血管内皮細胞等)とのheterotypicなギャップ・ジャンクション形成能に依存するものと思われた。 2) ヒト肺癌症例のスクリーニングで、転移ステージとコネキシン遺伝子発現との間に正の相関が見い出された。 3) プロテイン・フォスファターゼ2A制御サブユニットの発現亢進は、BL6細胞のゲノム上第一イントロンにレトロトランスポゾンの挿入があるからで、そのアリルに由来する翻訳産物はN末に約70アミノ酸の欠失を生じていた。 4) このフォスファターゼは焦点接着構成蛋白(インテグリンやパキシリン)と結合した。 5) このフォスファターゼは細胞外基質に対するF10及びBL6細胞のspreading、migration、invasion能の評価において抑制性に機能した。即ち、細胞外基質-インテグリ系を介して細胞内に入るシグナルに対してフォスファターゼが負の制御を行っているものと考えられた。

マウスmi遺伝子座にはbasic-helix-loop-helix-leucine zipper型転写囚子MITFがコードされている。mi遺伝子座におけるmi変異のホモ個体mi/miマウスでは塩基領域の1アルギニンが欠失した変異型MITFが発現している。この蛋白は転写因子として機能できないため、mi/miマウスではMITFによる遺伝子の転写活性化が起こらない。これら転写障害を受ける遺伝子を単離するために、野生型(+/+)及びmi/miマウス由来培養マスト細胞からcDNAライブラリーを作製し、(+/+-mi/mi)のサブトラクションを行った。cDNAプローブによるサザン法で約400クローンをスクリーニングし、mi/miマウス由来培養マスト細胞で転写障害を示す遺伝子20種を単離した。塩基配列を決定した所、12種は未知の遺伝子で、8種はグランザイムB、トリプトファン水酸化酵素、gp49、ST2L、ビメンチン、E25、マウスマスト細胞プロテアーゼ5及び6であった。グランザイムBとトリプトファン水酸化酵素についてはMITFの直接的な転写標的遺伝子であることをゲル・シフト法及びルシフェラーゼ・アッセイにより示した。これら2遺伝子のプロモーター領域内に存在するそれぞれ3つ及び1つのCANNTGモチーフがMITFによる転写活性化に重要であった。またこれら2遺伝子はそれぞれ細胞障害活性及びセロトニン合成に重要な蛋白をコードしているが、mi/miマウス由来培養マスト細胞ではYAC-1細胞に対する細胞障害活性及び細胞当たりのセロトニンの含量が有意に低下していた。