Expression Of Cell Adhesion Molecules Research Articles

#1449 Maturation of tertiary lymphoid structures in progressive kidney damage due to chronic phosphate load

Abstract Background and Aims We have recently shown that a high phosphate diet (HPD) in mice leads to an increase in FGF23 levels and causes progressive tubular damage and interstitial fibrosis with increased accumulation of macrophages and concomitant perivascular immune cell aggregates in the corticomedullary zone. Such tertiary lymphoid structures (TLS) can develop as a result of chronic inflammation and have already been described in lupus nephritis and IgA nephropathy, among others. In our model, the question arose to what extent damage to the proximal tubule is temporally associated with the development and maturation of TLS and which factors favor TLS development. Method Wild-type mice were fed HPD for 1, 2, 3, 4 and 6 months and the kidneys of each were compared with those of mice on a normal phosphate diet (NPD). To evaluate the effect of high FGF23 ± phosphate, Fgf23 was overexpressed in mice on NPD and HPD using an adeno-associated virus (AAV-Fgf23), and hypophosphatemic Hyp mice were used. Results An increased tubular damage score was seen after only two months of HPD. In parallel, perivascular immune cell clusters developed in the corticomedullary zone and in the renal cortex, which were characterized by aggregates of CD45R+ B cells and CD3+ T cells, as well as LYVE1+ lymphatic vessels after three months of HPD and were accompanied by increased expression of venous markers (Plvap, Aplnr, EphB4) and cell adhesion molecules (Sell, Vcam1, Madcam1). Further analyses showed a time-dependent induction of the immunofibroblast-derived chemokines Cxcl13 and Ccl19, as well as the lymphotoxins Lta and Ltb, which are important for the differentiation of immunofibroblasts into follicular dendritic cells (FDC) and follicular reticular cells (FRC). Already after four months of HPD, proliferating B-cell clusters with FDCs, FRCs located in T-cell clusters, podoplanin+ networks, high endothelial venules, plasma cells and increased IgD synthesis indicated fully mature TLS, while tubular damage and fibrosis continued to increase until six months of HPD. Compared to AAV-Fgf23 mice on NPD, only AAV-Fgf23 animals on HPD developed tubular damage with fibrosis and fully mature TLS. Hypophosphatemic Hyp mice with high FGF23 showed neither signs of tubular damage nor the development of TLS. Conclusion The development of renal TLS due to chronic high phosphate load coincides with the development of tubular damage, with earlier maturation of TLS and progression of tubular damage.

Epithelial Cell Adhesion Molecule (EpCAM) Expression in Human Tumors: A Comparison with Pan-Cytokeratin and TROP2 in 14,832 Tumors.

EpCAM is expressed in many epithelial tumors and is used for the distinction of malignant mesotheliomas from adenocarcinomas and as a surrogate pan-epithelial marker. A tissue microarray containing 14,832 samples from 120 different tumor categories was analyzed by immunohistochemistry. EpCAM staining was compared with TROP2 and CKpan. EpCAM staining was detectable in 99 tumor categories. Among 78 epithelial tumor types, the EpCAM positivity rate was ≥90% in 60 categories-including adenocarcinomas, neuroendocrine neoplasms, and germ cell tumors. EpCAM staining was the lowest in hepatocellular carcinomas, adrenocortical tumors, renal cell neoplasms, and in poorly differentiated carcinomas. A comparison of EpCAM and CKpan staining identified a high concordance but EpCAM was higher in testicular seminomas and neuroendocrine neoplasms and CKpan in hepatocellular carcinomas, mesotheliomas, and poorly differentiated non-neuroendocrine tumors. A comparison of EpCAM and TROP2 revealed a higher rate of TROP2 positivity in squamous cell carcinomas and lower rates in many gastrointestinal adenocarcinomas, testicular germ cell tumors, neuroendocrine neoplasms, and renal cell tumors. These data confirm EpCAM as a surrogate epithelial marker for adenocarcinomas and its diagnostic utility for the distinction of malignant mesotheliomas. In comparison to CKpan and TROP2 antibodies, EpCAM staining is particularly common in seminomas and in neuroendocrine neoplasms.

Angiogenic potential in periodontal stem cells from upper and lower jaw: A pilot study.

Teeth and supporting oral tissues are attractive and accessible sources of stem cells. Periodontal ligament stem cells (PDLSC) are readily isolated from extracted third molars, and exhibit the ability to self-renew and differentiate into multiple mesodermal cell fates. Clinical experience suggests that the exact location of periodontal defects affects the oral bone remodeling and wound healing. Compared to the mandible, the maxilla heals quicker and more efficiently. Angiogenesis is key in tissue regeneration including dental tissues, yet few studies focus on the angiogenic potential of PDLSC, none of which considered the differences between upper and lower jaw PDLSC (u-PDLSC and l-PDLSC, respectively). Here we studied the angiogenic potential of u-PDLSC and l-PDLSC and compared the results to well-established mesenchymal stem cells (MSC). Cells were characterized in terms of surface markers, proliferation, and vascular endothelial growth factor (VEGF) secretion, and angiogenic assays were performed. Newly formed capillaries were stained with CD31, and their expression of platelet endothelial cell adhesion molecule (PECAM-1), angiopoietin 2 (ANGPT2), and vascular endothelial growth factor receptor 1 and 2 (VEGFR-1, VEGFR-2) were measured. Periodontal stem cells from the upper jaw showed a higher proliferation capacity, secreted more VEGF, and formed capillary networks faster and denser than l-PDLSC. Gene expression of angiogenesis-related genes was significantly higher in u-PDLSC than in l-PDLSC or MSC, given that culture conditions were suitable. The oral cavity is a valuable source of stem cells, particularly PDLSC, which are promising for oral tissue engineering due to their robust growth, lifelong accessibility, low immunogenicity, and strong differentiation potential. Notably, u-PDLSC exhibit higher VEGF secretion and accelerate capillary formation compared to l-PDLSC or MSC. This study suggests a potential molecular mechanism in capillary formation, emphasizing the significance of precise location isolation of PDLSC.

Content and tissue expression of Collagen I, Collagen IV, and Laminin in the Extracellular Matrix in Prostate Adenocarcinoma.

Prostate cancer is one of the most common neoplasm in the male population. It is not known why some tumors become more aggressive than others. Although most studies show changes in the expression of cell adhesion molecules and the extracellular matrix correlated with the Gleason score, no study has objectively measured the tissue content of these molecules. This study aims to measure the content and tissue expression of collagen type I and IV and laminin in the extracellular matrix of patients with prostate adenocarcinoma and correlate these findings with the Gleason score and clinical characteristics. Forty-one patients who underwent radical prostate surgery at the Urology Department of a reference Hospital in Brazil between January 2015 and December 2020 were studied. The tissue protein content was estimated under light microscopy at a final magnification of 200 × . The mean collagen I score in prostate adenocarcinoma tissue samples was 7.16 ± 1.03 pixels/field. The mean type IV collagen score was 3.44 ± 0.61 pixels/field. The mean laminin score was 5.19 ± 0.79 pixels/field. The total Gleason score was correlated with both collagen and laminin. All the correlations were negative, which shows that the higher the collagen/laminin expression was, the lower the total Gleason score (p-value < 0,05). According to the Pearson correlation analysis, age has no statistical relationship with collagen and laminin content. PSA, in turn, showed a correlation only with laminin, but r = -0.378 (p = 0.015). Among the associated diseases and lifestyle habits, there is only statistical significance in the comparison of alcoholism for collagen I. For collagen IV and laminin, no statistical significance was obtained with the clinical variables analyzed.

Transgenic human C-reactive protein affects oxidative stress but not inflammation biomarkers in the aorta of spontaneously hypertensive rats

BackgroundC-reactive protein (CRP) is an acute inflammatory protein detected in obese patients with metabolic syndrome. Moreover, increased CRP levels have been linked with atherosclerotic disease, congestive heart failure, and ischemic heart disease, suggesting that it is not only a biomarker but also plays an active role in the pathophysiology of cardiovascular diseases. Since endothelial dysfunction plays an essential role in various cardiovascular pathologies and is characterized by increased expression of cell adhesion molecules and inflammatory markers, we aimed to detect specific markers of endothelial dysfunction, inflammation, and oxidative stress in spontaneously hypertensive rats (SHR) expressing human CRP. This model is genetically predisposed to the development of the metabolic syndrome.MethodsTransgenic SHR male rats (SHR-CRP) and non-transgenic SHR (SHR) at the age of 8 months were used. Metabolic profile (including serum and tissue triglyceride (TAG), serum insulin concentrations, insulin-stimulated incorporation of glucose, and serum non-esterified fatty acids (NEFA) levels) was measured. In addition, human serum CRP, MCP-1 (monocyte chemoattractant protein-1), and adiponectin were evaluated by means of ELISA, histological analysis was used to study morphological changes in the aorta, and western blot analysis of aortic tissue was performed to detect expression of endothelial, inflammatory, and oxidative stress markers.ResultsThe presence of human CRP was associated with significantly decreased insulin-stimulated glycogenesis in skeletal muscle, increased muscle and hepatic accumulation of TAG and decreased plasmatic cGMP concentrations, reduced adiponectin levels, and increased monocyte chemoattractant protein-1 (MCP-1) levels in the blood, suggesting pro-inflammatory and presence of multiple features of metabolic syndrome in SHR-CRP animals. Histological analysis of aortic sections did not reveal any visible morphological changes in animals from both SHR and SHR-CRP rats. Western blot analysis of the expression of proteins related to the proper function of endothelium demonstrated significant differences in the expression of p-eNOS/eNOS in the aorta, although endoglin (ENG) protein expression remained unaffected. In addition, the presence of human CRP in SHR in this study did not affect the expression of inflammatory markers, namely p-NFkB, P-selectin, and COX2 in the aorta. On the other hand, biomarkers related to oxidative stress, such as HO-1 and SOD3, were significantly changed, indicating the induction of oxidative stress.ConclusionsOur findings demonstrate that CRP alone cannot fully induce the expression of endothelial dysfunction biomarkers, suggesting other risk factors of cardiovascular disorders are necessary to be involved to induce endothelial dysfunction with CRP.

Runx3 Regulates CD8+ T Cell Local Expansion and CD43 Glycosylation in Mice by H1N1 Influenza A Virus Infection.

We have recently reported that transcription factor Runx3 is required for pulmonary generation of CD8+ cytotoxic T lymphocytes (CTLs) that play a crucial role in the clearance of influenza A virus (IAV). To understand the underlying mechanisms, we determined the effects of Runx3 knockout (KO) on CD8+ T cell local expansion and phenotypes using an inducible general Runx3 KO mouse model. We found that in contrast to the lungs, Runx3 general KO promoted enlargement of lung-draining mediastinal lymph node (mLN) and enhanced CD8+ and CD4+ T cell expansion during H1N1 IAV infection. We further found that Runx3 deficiency greatly inhibited core 2 O-glycosylation of selectin ligand CD43 on activated CD8+ T cells but minimally affected the cell surface expression of CD43, activation markers (CD44 and CD69) and cell adhesion molecules (CD11a and CD54). Runx3 KO had a minor effect on lung effector CD8+ T cell death by IAV infection. Our findings indicate that Runx3 differently regulates CD8+ T cell expansion in mLNs and lungs by H1N1 IAV infection. Runx3 is required for CD43 core 2 O-glycosylation on activated CD8+ T cells, and the involved Runx3 signal pathway may mediate CD8+ T cell phenotype for pulmonary generation of CTLs.

LncRNA MALAT1 suppresses monocyte-endothelial cell interactions by targeting miR-30b-5p and enhancing ATG5-mediated autophagy

BackgroundMonocyte-endothelial cell (EC) interactions are one of the earliest events in the development of atherosclerosis and play a crucial role in atherosclerotic plaque formation. Although attempts have been made to modulate this interaction, the underlying molecular signalling mechanisms remain unclear. This study aimed to investigate the role of long non-coding RNA MALAT1 in monocyte-EC interactions. MethodsThe expression of MALAT1, ICAM-1, VCAM-1, P-selectin, CCL2 and CXCL1 was evaluated in ApoE-/- mouse aortic tissues and inflamed human umbilical vein endothelial cells (HUVECs). The regulatory impact of MALAT1 on cell adhesion molecules, monocyte-EC adhesion, and autophagy was assessed. The interactions between MALAT1 and microRNAs (miRNAs) were evaluated using dual-luciferase reporter and RNA pull-down assays. ResultsMALAT1 expression decreased in ApoE-/- mouse aortic tissues and inflammatory HUVECs. MALAT1 overexpression suppressed the expression of ICAM-1, VCAM-1 and CXCL1, and reduced the migration and adhesion of monocytes to ECs. Inhibition of MALAT1 promoted cell adhesion molecule expression and monocyte-EC interactions. Mechanistically, MALAT1 binds directly to miR-30b-5p and decreases its effective expression by functioning as an endogenous sponge, thereby increasing the expression of autophagy-related gene 5 (ATG5) and stimulates endothelial autophagy. ConclusionsOur findings suggest that MALAT1 suppresses monocyte-EC interactions by targeting miR-30b-5p and enhancing ATG5-mediated endothelial autophagy. These data imply that MALAT1 may play a protective role at the early stages of the atherosclerotic process.

Inflammation-induced TRPV4 channels exacerbate blood–brain barrier dysfunction in multiple sclerosis

BackgroundBlood–brain barrier (BBB) dysfunction and immune cell migration into the central nervous system (CNS) are pathogenic drivers of multiple sclerosis (MS). Ways to reinstate BBB function and subsequently limit neuroinflammation present promising strategies to restrict disease progression. However, to date, the molecular players directing BBB impairment in MS remain poorly understood. One suggested candidate to impact BBB function is the transient receptor potential vanilloid-type 4 ion channel (TRPV4), but its specific role in MS pathogenesis remains unclear. Here, we investigated the role of TRPV4 in BBB dysfunction in MS.Main textIn human post-mortem MS brain tissue, we observed a region-specific increase in endothelial TRPV4 expression around mixed active/inactive lesions, which coincided with perivascular microglia enrichment in the same area. Using in vitro models, we identified that microglia-derived tumor necrosis factor-α (TNFα) induced brain endothelial TRPV4 expression. Also, we found that TRPV4 levels influenced brain endothelial barrier formation via expression of the brain endothelial tight junction molecule claudin-5. In contrast, during an inflammatory insult, TRPV4 promoted a pathological endothelial molecular signature, as evidenced by enhanced expression of inflammatory mediators and cell adhesion molecules. Moreover, TRPV4 activity mediated T cell extravasation across the brain endothelium.ConclusionCollectively, our findings suggest a novel role for endothelial TRPV4 in MS, in which enhanced expression contributes to MS pathogenesis by driving BBB dysfunction and immune cell migration.

Abstract 1398: The role of ST8SIA2 &amp; ST8SIA4 in melanoma progression and metastasis

Abstract Melanoma is the deadliest form of skin cancer; while early detection affords patients a 5-year survival rate greater than 90 percent, survival for metastatic patients is poor. Metastatic dissemination is reliant on melanoma’s ability to escape immune surveillance and migrate to distant organs. These processes are heavily influenced by cell surface proteins, which themselves can be regulated by post-translational modifications (PTM) like protein glycosylation. An understudied glycosylation that may affect immune evasion and cancer progression is polysialylation (polySia). Here, a long-chained polymer of sialic acid moieties is formed by the polysialyltransferases ST8SIA2 and ST8SIA4. In silico analysis suggests that compared to normal skin, primary cutaneous melanoma has elevated expression of both ST8SIA2 and ST8SIA4. Interestingly, although ST8SIA4 is upregulated in metastatic samples, its expression is associated with increased survival. Further analysis revealed a positive correlation between CD8+ T cell infiltrates and ST8SIA4 expression. Furthermore, analysis of ST8SIA2 expression shows an inverse correlation with survival and T Cell CD8+ cells infiltration, suggesting ST8SIA2 and -4 differentially regulate the tumor immune microenvironment. One potential polySia modified cell surface protein, mediating these effects is Cell Adhesion Molecule 1 (CADM1). CADM1 is a target of ST8SIA2 and may also be modified by ST8SIA4. Loss of CADM1 increases the invasive properties of melanoma cells and our recent point mutational analysis reveals that the adhesive function of CADM1 might be regulated by polySia at Asp 113. In addition to its role in cancer migration and invasiveness, CADM1 expression may also affect the tumor immune environment. Further investigations are needed to untangle the complex relationship between PolySia modification and melanoma immune evasion and metastasis. Citation Format: Amirali Amirfallah, Kayla Gallant, Maria Cavallo, Zongguan Huang, Edward Hartsough. The role of ST8SIA2 &amp; ST8SIA4 in melanoma progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1398.

Abstract 5512: Cerebral endothelial FAK paradoxically inhibits brain metastasis in vivo

Abstract Introduction: In cancer metastasis in the trunk, the secreted proteins from tumors, such as VEGFs and TNF-α, activate focal adhesion kinase (FAK) in vascular endothelial cells (VECs). The activated FAK promotes VE-cadherin phosphorylation and the expression of cell adhesion molecules, and it facilitates the extravasation of cancer cells. In the cerebral blood vessels, the VECs maintain very strong cell adhesion supported by the surrounding pericytes and astrocytes. The strong barrier of cerebral VECs is called the blood-brain barrier (BBB) and is regulated differently from trunk VECs. Although the role of FAK in trunk metastasis is shown in many studies, its importance in brain metastasis is not verified. In this study, we focused on the role of the cerebral endothelial FAK and compared the role of FAK in brain metastasis with that in trunk metastasis. Methods: We generated tamoxifen-inducible VEC-specific FAK knockout (FAK-cKO) mice (VEcad-CreERT2; Ptk2-flox/flox) and brain metastatic (BrM) cell lines of Ex3LL (murine lung cancer cell line). We intracardially injected BrM cells of Ex3LL into the FAK-cKO mice and analyzed the tumor development using in vivo bioluminescence imaging and a CUBIC tissue clearing system. We also analyzed the intracranially injected mice of the parental and BrM cell lines to evaluate the tumor angiogenesis, which is reported to be promoted by the endothelial FAK. Results: Although FAK-cKO in VECs has been reported to inhibit lung metastasis, brain metastasis was accelerated in FAK-cKO mice compared with control mice. The number of brain metastatic sites increased in FAK-cKO mice, but there was no difference in the individual tumor size. It suggests that the brain metastasis was promoted by FAK-cKO, but that the proliferation of brain metastasis was not promoted by FAK-cKO. Blood vessel density was decreased with irregular shape in the brain tumors of FAK-cKO mice, suggesting that the angiogenesis was inhibited by FAK-cKO. It indicates that the angiogenic function of endothelial FAK in the brain vasculature was not different from that in the trunk. Conclusion: FAK-cKO in VECs exacerbated the brain metastasis of the BrM cells although it has been reported to inhibit the metastasis in the trunk. FAK-cKO in VECs decreased tumor angiogenesis both in the brain and the trunk. It suggests that the regulatory mechanism of cell adhesion and the FAK function in the brain vasculature is partly different from that in the other organs in the body trunk. We need further investigation to elucidate the function of endothelial FAK in brain metastasis. Citation Format: Shoko Noda-Narita, Atsu Aiba. Cerebral endothelial FAK paradoxically inhibits brain metastasis in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5512.

Abstract 6995: Functional characterization of the novel cell adhesion molecule MPZL3 in ovarian cancer

Abstract Ovarian cancer (OVCA) is the most lethal gynecological malignancy in the United States often detected at advanced stages. OVCA progression involves transcoelomic metastasis, where cells disseminate into the peritoneal fluid, adhere to form multicellular aggregates that promote anchorage-independent survival and facilitate metastatic colonization of the peritoneum. To meet the requirements of each stage of this detachment, aggregation, and re-attachment cycle, OVCA cells have been shown to dynamically regulate the expression of multiple cell adhesion molecules (CAMs) throughout their progression. Moreover, targeting cell-cell adhesion molecules has shown to be an effective method to slow/inhibit the progression of OVCA. In this study, we characterize the novel role of the understudied CAM MPZL3 in OVCA. Myelin protein zero-like 3 (MPZL3) is a transmembrane protein with homology to other immunoglobulin-like (Ig) family of CAMs. While it has been reported that altered Ig-CAM expression plays a role in ovarian cancer, the function of MPZL3 has not been investigated. Interestingly, TCGA data demonstrates that chromosomal loss of the MPZL3 locus (11q23.3) is frequently detected in several cancer types, including 22% of clear cell and 30% of high grade serous ovarian cancers (HGSOC), implying that loss of genes located in this area has tumorigenic consequences. On the other hand, few studies showed that MPZL3 contains an oncogenic potential in breast and MET-amplified cancer cell lines, suggesting that the role of MPZL3 in cancer is context dependent. To study the function of MPZL3 in OVCA, we used shRNA mediated MPZL3 knockdown in OVCAR4 human HGSOC cells and examined transcriptome-wide effects by RNA-sequencing. The subsequent Gene Set Enrichment Analysis indicated that pathways involved in regulating cell growth, death, adhesion, and epithelial-mesenchymal transition were significantly altered by loss of MPZL3. To validate the candidate pathways, we knocked down or over-expressed MPZL3 in OVCA cells to test its importance in regulating OVCA cell growth, apoptosis, adhesion, and migration. We found that loss of MPZL3 resulted in decreased cell growth with a concomitant resistance to both Cisplatin and Olaparib treatments, both of which are commonly used for treating OVCA. Moreover, we show that knockdown of MPZL3 alters the homotypic adhesive-capacity of OVCA cells. Ongoing studies are underway to further characterize the role played by MPLZ3 in the mentioned contexts. Additionally, to elucidate the underlying molecular mechanism and identify novel interactors of MPZL3, we will conduct co-immunoprecipitation experiments in OVCA cells expressing V5-tagged MPZL3. Understanding the novel role of MPZL3 will provide further insight into OVCA progression, thus generating opportunities of developing new treatments for patients with low MPZL3 expression as an approach for precision cancer medicine. Citation Format: Ya-Yun Cheng, Beth Worley, Zaineb Javed, Amal T. Elhaw, Priscilla W. Tang, Nadine Hempel. Functional characterization of the novel cell adhesion molecule MPZL3 in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6995.

Abstract 2364: Anti-CADM1 chimeric antigen receptor (CAR) is a novel therapeutic strategy against small cell lung cancer

Abstract Small cell lung cancer (SCLC) is a highly aggressive form accounting for 15% of all lung cancers. SCLC is characterized by high proliferative rates, early metastasis, and poor prognosis. The median survival is less than 2 years even in patients with early-stage disease and less than a year in patients with metastatic disease which highlights the need for new therapeutic strategies. Cell Adhesion Molecule (CADM) 1 encodes a glycoprotein which belongs to an immunoglobulin superfamily of cell adhesion molecules responsible for epithelial cell adhesion through its homophilic trans-interaction between the adjacent cells. CADM1 is highly expressed on the cell surface of 80% of the SCLC cells, associated with its neuroendocrine features, along with the expression of Neural Cell Adhesion Molecule (NCAM). CADM1 is known to act as tumor promoter through its interactions with actin binding proteins. Consistently, we observed CADM1 expression in H69, H82, and H510 SCLC cell lines as assessed by western immuno blot. More importantly they were all susceptible to NK mediated cytotoxicity when assessed against NK cell line, NK92. We previously demonstrated that CADM1 mediates NK-mediated immune surveillance in non-small cell lung cancer, suggesting that CADM1 could be a potential immunotherapeutic target for SCLC. Here we report the development of an anti-CADM1 chimeric antigen receptor (CAR) specific for T and NK cells engineered with T and NK cell specific adaptor domains. Assessed the specificity of CARs by expressing them in NK92 cells and testing them against CADM1 expressing and CADM1-null A549 cells as targets. Finally, we evaluated the efficacy of anti-CADM1 CAR-T and CAR-NK cells against SCLC targets and observed a robust killing, largely proportionate to the levels of CADM1 expression in them. We are further assessing the efficacy of anti-CADM1 CAR-T and CAR-NK products in inhibiting tumor growth in NSG mice against SCLC xenografts. Together, these observations demonstrate that targeting CADM1 with CAR-T and CAR-NK cells is a potential new therapeutic strategy for SCLC. Citation Format: Shiva Krishna Katkam, Sergei Zolov, Delaney Shield, Sergei Chuikov, Venkateshwar G Keshamouni. Anti-CADM1 chimeric antigen receptor (CAR) is a novel therapeutic strategy against small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2364.

Abstract 2769: Long non coding RNA H19/cell adhesion molecules circuitry as novel target and diagnostic tool for metastatic prostate cancer

Abstract In the complex tumor biology of prostate cancer (PCa), we previously contributed by demonstrating that estrogen receptor and hypoxia signaling promoted transcription of progression-associated genes. Notably, combined treatment, estrogens plus hypoxia, caused a significant down regulation of the long noncoding RNA H19, one of the most sensitive to these pro-tumoral stimuli. Mechanistically, H19 acts as transcriptional repressor: H19 over-expression decreases E-cadherin (CDH1) level causing epithelial-to-mesenchymal transition whereas H19 reduction promotes the expression of E-cadherin and β4 integrin (ITGB4) switching the tumor dissemination program toward an alternative mechanism, specifically the cohesive metastatic phenotype. We focused on this H19/cell adhesion molecules circuitry by investigating its role as: 1.Novel prognostic biomarker at the time of biopsies. eNOS, HIF2a and β4 integrin expression was evaluated by immunohistochemistry, H19 and CDH1 transcripts by Droplet Digital PCR on FFPE-biopsies in a retrospective cohort of PCa patients [n = 30; Gleason score GS≤ 7(3+4) n= 17, GS ≥7(4+3) n= 13; biochemical, local, or metastatic recurrence defining disease progression (n=8/30; time of recurrence: range 2-6 years)]. Importantly, multivariate analysis of combined biomarkers, named “Bioscore”, defined by the presence of at least 3 among HIF-2α, eNOS, CDH1, β4 integrin ≥cut off or H19≤cut off revealed its prognostic value for progression and Progression-Free Survival determined using the ROC analysis (p=0.003) and Kaplan Meier curve (p= 0.005), respectively. 2.Master regulator of metastatic program in vivo and ex vivo PCa experimental models. Upon H19 silencing, mimicking combined estrogens plus hypoxia treatment, luciferase-positive PC-3 and 22Rv1 cells led to increased E-cadherin and β4 integrin expression, enhanced tumor growth and metastasis formation at bone, lung, and liver in PCa mouse models. Further, treatment with H3K27 demethylase inhibitor, GSK-J4, rescued cell adhesion molecules expression by modulating H3K27me3 level at gene promoters, reduced metastasis number and size, and induced cell death in PCa-derived Organotypic Slice Cultures. Additionally, treatment with JQ1 and dBET6 BET protein family inhibitors) markedly reduced both E-cadherin and β4 integrin levels, cell proliferation and in vivo tumor growth. Intriguingly, H19 silencing increased BRD3 and BRD4 levels, implicating H19 in BRD transcription. Overall, this research enhances our understanding of the molecular mechanisms driving aggressive prostate cancer phenotypes, providing a foundation for new diagnostic and therapeutic strategies in prostate cancer management. Citation Format: Valeria Pecci, Aurora Aiello, Sara De Martino, Cristian Ripoli, Dante Rotili, Francesco Pierconti, Francesco Pinto, Claudio Grassi, Carlo Gaetano, Alfredo Pontecorvi, Lidia Strigari, Antonella Farsetti, Simona Nanni. Long non coding RNA H19/cell adhesion molecules circuitry as novel target and diagnostic tool for metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2769.

CD56/NCAM mediates cell migration of human NK cells by promoting integrin-mediated adhesion turnover.

Natural killer (NK) cells patrol tissue to mediate lysis of virally infected and tumorigenic cells. Human NK cells are typically identified by their expression of neural cell adhesion molecule (NCAM, CD56), yet despite its ubiquitous expression on NK cells, CD56 remains a poorly understood protein on immune cells. CD56 has been previously demonstrated to play roles in NK cell cytotoxic function and cell migration. Specifically, CD56-deficient NK cells have impaired cell migration on stromal cells and CD56 is localized to the uropod of NK cells migrating on stroma. Here, we show that CD56 is required for NK cell migration on ICAM-1 and is required for the establishment of persistent cell polarity and unidirectional actin flow. The intracellular domain of CD56 (NCAM-140) is required for its function and the loss of CD56 leads to enlarged actin foci and sequestration of phosphorylated Pyk2 accompanied by increased size and frequency of activated LFA-1 clusters. Together, these data identify a role for CD56 in regulating human NK cell migration through modulation of actin dynamics and integrin turnover.

L1 Cell Adhesion Molecule (L1CAM) Expression and Molecular Alterations Distinguish Low-Grade Oncocytic Tumor From Eosinophilic Chromophobe Renal Cell Carcinoma

Renal low-grade oncocytic tumor (LOT) is a recently recognized renal cell neoplasm designated within the “other oncocytic tumors” category in the 2022 World Health Organization classification system. Although the clinicopathologic, immunohistochemical, and molecular features reported for LOT have been largely consistent, the data are relatively limited. The morphologic overlap between LOT and other low-grade oncocytic neoplasms, particularly eosinophilic chromophobe renal cell carcinoma (E-chRCC), remains a controversial area in renal tumor classification. To address this uncertainty, we characterized and compared large cohorts of LOT (n = 67) and E-chRCC (n = 69) and revealed notable differences between the 2 entities. Clinically, LOT predominantly affected women, whereas E-chRCC showed a male predilection. Histologically, although almost all LOTs were dominated by a small-nested pattern, E-chRCC mainly showed solid and tubular architectures. Molecular analysis revealed that 87% of LOT cases harbored mutations in the tuberous sclerosis complex (TSC)-mTOR complex 1 (mTORC1) pathway, most frequently in MTOR and RHEB genes; a subset of LOT cases had chromosomal 7 and 19q gains. In contrast, E-chRCC lacked mTORC1 mutations, and 60% of cases displayed chromosomal losses characteristic of chRCC. We also explored the cell of origin for LOT and identified L1 cell adhesion molecule (L1CAM), a collecting duct and connecting tubule principal cell marker, as a highly sensitive and specific ancillary test for differentiating LOT from E-chRCC. This distinctive L1CAM immunohistochemical labeling suggests the principal cells as the cell of origin for LOT, unlike the intercalated cell origin of E-chRCC and oncocytoma. The ultrastructural analysis of LOT showed normal-appearing mitochondria and intracytoplasmic lumina with microvilli, different from what has been described for chRCC. Our study further supports LOT as a unique entity with a benign clinical course. Based on the likely cell of origin and its clinicopathologic characteristics, we propose that changing the nomenclature of LOT to “Oncocytic Principal Cell Adenoma of the Kidney” may be a better way to define and describe this entity.

The CHD Protein Kismet Restricts the Synaptic Localization of Cell Adhesion Molecules at the Drosophila Neuromuscular Junction.

The appropriate expression and localization of cell surface cell adhesion molecules must be tightly regulated for optimal synaptic growth and function. How neuronal plasma membrane proteins, including cell adhesion molecules, cycle between early endosomes and the plasma membrane is poorly understood. Here we show that the Drosophila homolog of the chromatin remodeling enzymes CHD7 and CHD8, Kismet, represses the synaptic levels of several cell adhesion molecules. Neuroligins 1 and 3 and the integrins αPS2 and βPS are increased at kismet mutant synapses but Kismet only directly regulates transcription of neuroligin 2. Kismet may therefore regulate synaptic CAMs indirectly by activating transcription of gene products that promote intracellular vesicle trafficking including endophilin B (endoB) and/or rab11. Knock down of EndoB in all tissues or neurons increases synaptic FasII while knock down of EndoB in kis mutants does not produce an additive increase in FasII. In contrast, neuronal expression of Rab11, which is deficient in kis mutants, leads to a further increase in synaptic FasII in kis mutants. These data support the hypothesis that Kis influences the synaptic localization of FasII by promoting intracellular vesicle trafficking through the early endosome.

Acid-sensing receptor GPR4 plays a crucial role in lymphatic cancer metastasis.

Cancer tissues exhibit an acidic microenvironment owing to the accumulation of protons and lactic acid produced by cancer and inflammatory cells. To examine the role of an acidic microenvironment in lymphatic cancer metastasis, gene expression profiling was conducted using human dermal lymphatic endothelial cells (HDLECs) treated with a low pH medium. Microarray and gene set enrichment analysis revealed that acid treatment induced the expression of inflammation-related genes in HDLECs, including genes encoding chemokines and adhesion molecules. Acid treatment-induced chemokines C-X3-C motif chemokine ligand 1 (CX3CL1) and C-X-C motif chemokine ligand 6 (CXCL6) autocrinally promoted the growth and tube formation of HDLECs. The expression of vascular cell adhesion molecule 1 (VCAM-1) increased in HDLECs after acid treatment in a time-dependent manner, which, in turn, enhanced their adhesion to melanoma cells. Among various acid-sensing receptors, HDLECs basally expressed G protein-coupled receptor 4 (GPR4), which was augmented under the acidic microenvironment. The induction of chemokines or VCAM-1 under acidic conditions was attenuated by GPR4 knockdown in HDLECs. In addition, lymph node metastases in a mouse melanoma model were suppressed by administering an anti-VCAM-1 antibody or a GPR4 antagonist. These results suggest that an acidic microenvironment modifies the function of lymphatic endothelial cells via GPR4, thereby promoting lymphatic cancer metastasis. Acid-sensing receptors and their downstream molecules might serve as preventive or therapeutic targets in cancer.

The Role of Innate and Adaptive Immune System in the Pathogenesis of Schizophrenia.

Schizophrenia is one of the most severely debilitating mental disorders that affects 1.1% of the world's population. The exact cause of the disease is not known, but genetics, environmental factors (such as infectious agents, season and region of birth, exposure to viruses, low birth weight, advanced paternal age, and tobacco), and immune system dysfunction can all contribute to the development of schizophrenia. Recently, the role of the immune system in schizophrenia has received much attention. Both acquired and innate immune systems are involved in the pathogenesis of schizophrenia and facilitate the disease's progression. Almost all cells of the immune system including microglia, B cells, and T cells play an important role in the blood-brain barrier damage, inflammation, and in the progression of this disease. In schizophrenia, the integrity of the blood-brain barrier is reduced and then the immune cells are recruited into the endothelium following an increase in the expression of cell adhesion molecules. The entry of immune cells and cytokines leads to inflammation and antibody production in the brain. Accordingly, the results of this study strengthen the hypothesis that the innate and acquired immune systems are involved in the pathogenesis of schizophrenia.

Quantitative Evaluation by Digital Pathology of Immunohistochemical Expression of CK7, CK19, and EpCAM in Advanced Stages of NASH.

(1) Background: Nonalcoholic Steatohepatitis/Nonalcoholic Fatty Liver Disease (NASH/NAFLD) is the most recurrent chronic liver disease. NASH could present with a cholestatic (C) or hepatic (H) pattern of damage. Recently, we observed that increased Epithelial Cell Adhesion Molecule (EpCAM) expression was the main immunohistochemical feature to distinguish C from H pattern in NASH. (2) Methods: In the present study, we used digital pathology to compare the quantitative results of digital image analysis by QuPath software (Q-results), with the semi-quantitative results of observer assessment (S-results) for cytokeratin 7 and 19, (CK7, CK19) as well as EpCAM expression. Patients were classified into H or C group on the basis of the ratio between alanine transaminase (ALT) and alkaline phosphatase (ALP) values, using the "R-ratio formula". (3) Results: Q- and S-results showed a significant correlation for all markers (p < 0.05). Q-EpCAM expression was significantly higher in the C group than in the H group (p < 0.05). Importantly ALP, an indicator of hepatobiliary disorder, was the only biochemical parameter significantly correlated with Q-EpCAM. Instead, Q-CK7, but not Q-CK19, correlated only with γGlutamyl-Transferase (γGT). Of note, Stage 4 fibrosis correlated with Q-EpCAM, Q-CK19, and ALP but not with γGT or ALT. Conclusions: Image analysis confirms the relation between cholestatic-like pattern, associated with a worse prognosis, with increased ALP values, EpCAM positive biliary metaplasia, and advanced fibrosis. These preliminary data could be useful for the implementation of AI algorithms for the assessment of cholestatic NASH.

Association of α-SMA and EpCAM Expressions with Recurrence Risk Based on Histopathological Subtypes of Basal Cell Carcinoma

Background Basal cell carcinoma (BCC) is the most common skin malignancy in the world with a proportion 70%. Recurrent and aggressive variants are still challenging in diagnosis and treatment. Histopathologically, there are two group of BCC risk of recurrence, i.e low risk and high risk. Assessment the expression of alpha-smooth muscle actin (α-SMA) as a biomarker of cancer associated fibroblast cell in stroma and the expression of epithelial cell adhesion molecule (EpCAM) in tumor cells probably have role in the pathomechanism of BCC progression, suggest its can distinguish the risk of BCC recurrence. This research aims to determine the assosiation between the expression of α-SMA and EpCAM with the recurrence risk group of BCC histopathological subtypes. Methods This was a cross sectional study using 48 samples, with 24 low and high risk groups each. Histopathological subtypes were determined from Hematoxylin and eosin slide. The expression of α-SMA and EpCAM was examined by the immunohistochemical method which was assessed semiquantitatively. Statistical analysis was performed using the Chi-square test with p&lt;0.05 was considered significant. Results Expression of α-SMA with the score 3 was found more in high risk BCC (85.7%), while a score of 1 was more in low risk BCC (100%). Loss of EpCAM expression was mainly found in high risk BCC (82.8%). Statistical analysis showed that there was a significant assosiation between the expression of α-SMA and EpCAM and the recurrence risk group of BCC histopathological subtypes with p value =0.000 for each. Conclusions This study concluded that increased of α-SMA expression and loss of EpCAM expression were associated with a high recurrence risk group of BCC histopathological subtypes.