Impaired Adhesion Research Articles

The Hexosamine Biosynthetic Pathway alters the cytoskeleton to modulate cell proliferation and migration in metastatic prostate cancer.

Castration-resistant prostate cancer (CRPC) progresses despite androgen deprivation therapy, as cancer cells adapt to grow without testosterone, becoming more aggressive and prone to metastasis. CRPC biology complicates the development of effective therapies, posing challenges for patient care. Recent gene-expression and metabolomics studies highlight the Hexosamine Biosynthetic Pathway (HBP) as a critical player, with key components like GNPNAT1 and UAP1 being downregulated in metastatic CRPC. GNPNAT1 knockdown has been shown to increase cell proliferation and metastasis in CRPC cell lines, though the mechanisms remain unclear. To investigate the cellular basis of these CRPC phenotypes, we generated a CRISPR-Cas9 knockout model of GNPNAT1 in 22Rv1 CRPC cells, analyzing its impact on metabolomic, glycoproteomic, and transcriptomic profiles of cells. We hypothesize that HBP inhibition disrupts the cytoskeleton, altering mitotic progression and promoting uncontrolled growth. GNPNAT1 KO cells showed reduced levels of cytoskeletal filaments, such as actin and microtubules, leading to cell structure disorganization and chromosomal mis-segregation. GNPNAT1 inhibition also activated PI3K/AKT signaling, promoting proliferation, and impaired cell adhesion by mislocalizing EphB6, enhancing migration via the RhoA pathway and promoting epithelial-to-mesenchymal transition. These findings suggest that HBP plays a critical role in regulating CRPC cell behavior, and targeting this pathway could provide a novel therapeutic approach.

The Actin Motor Protein Myosin 6 Contributes to Cell Migration and Expression of GIPC1 and Septins in Breast Cancer Cells.

Breast cancer is highly metastatic. One protein that may participate in breast cancer cell migration is the actin motor protein myosin 6 (MYO6), which is likely regulated by the GIPC1 protein. Additionally, septins (SEPTs) appear to participate in breast cancer motility. Here, we investigated the effects of loss of MYO6 on cell morphology, migration, and expression of GIPC1, SEPT2, and SEPT7 in two breast cancer cell lines. The research material consisted of two breast cancer cell lines, MCF-7 and MDA-MB-231, in which the level of MYO6 was reduced and the effect of knockdown on the migration potential and the expression of GIPC1, SEPT2 and SEPT7 was determined. The levels of these proteins were also analyzed in silico. siRNA-mediated knock down of MYO6 altered the morphology of MCF-7 cells and reduced the expression of GIPC1 and SEPT7 in both MCF-7 and MDA-MB-231 cells. In in silico data, GIPC1, SEPT2, and SEPT7 were all overexpressed in breast cancer tissue samples from patients. Finally, MYO6 knock down impaired migration and adhesion in both MCF-7 and MDA-MB-231 cells. Our study substantiates that downregulation of MYO6 diminishes the migratory abilities of breast cancer cell lines with varying invasiveness. Furthermore, we have demonstrated that decreased MYO6 protein leads to reduced expression of GIPC1, SEPT2, and SEPT7 in breast cancer cells. These findings contribute to a more comprehensive understanding of the pathways influencing breast cancer cell migration, a critical aspect of metastasis.

Tensin-2 interactomics reveals interaction with GAPDH and a phosphorylation-mediated regulatory role in glycolysis

Integrin adaptor proteins, like tensin-2, are crucial for cell adhesion and signaling. However, the function of tensin-2 beyond localizing to focal adhesions remain poorly understood. We utilized proximity-dependent biotinylation and Strep-tag affinity proteomics to identify interaction partners of tensin-2 in Flp-In 293 T-REx cells. Interactomics linked tensin-2 to known focal adhesion proteins and the dystrophin glycoprotein complex, while also uncovering novel interaction with the glycolytic enzyme GAPDH. We demonstrated that Y483-phosphorylation of tensin-2 regulates the glycolytic rate in Flp-In 293 T-REx and MEF cells and found that pY483 tensin-2 is enriched in adhesions in MEF cells. Our study unveils novel interaction partners for tensin-2 and further solidifies its speculated role in cell energy metabolism. These findings shed fresh insight on the functions of tensin-2, highlighting its potential as a therapeutic target for diseases associated with impaired cell adhesion and metabolism.

EnvZ/OmpR Controls Protein Expression and Modifications in Cronobacter sakazakii for Virulence and Environmental Resilience.

Cronobacter sakazakii is a notorious foodborne opportunistic pathogen, particularly affecting vulnerable populations such as premature infants, and poses significant public health challenges. This study aimed to elucidate the role of the envZ/ompR genes in environmental tolerance, pathogenicity, and protein regulation of C. sakazakii. An envZ/ompR knockout mutant was constructed and assessed for its impact on bacterial growth, virulence, environmental tolerance, and protein regulation. Results demonstrate that deletion of envZ/ompR genes leads to reduced growth rate and attenuated virulence in animal models. Additionally, the knockout strain exhibited compromised environmental tolerance, particularly in desiccation and oxidative stress conditions, along with impaired adhesion and invasion abilities in epithelial cells. Proteomic analysis revealed significant alterations in protein expression and phosphorylation patterns, highlighting potential compensatory mechanisms triggered by gene deletion. Furthermore, investigation into protein deamidation and glucose metabolism uncovered a link between envZ/ompR deletion and energy metabolism dysregulation. Interestingly, the downregulation of MalK and GrxC proteins was identified as contributing factors to altered desiccation tolerance and disrupted redox homeostasis, respectively, providing mechanistic insights into the phenotypic changes observed. Overall, this study enhances understanding of the multifaceted roles of envZ/ompR in C. sakazakii physiology and pathogenesis, shedding light on potential targets for therapeutic intervention and food safety strategies.

Adhesion of pancreatic tumor cell clusters by desmosomal molecules enhances early liver metastases formation

Desmosomes are intercellular adhesion complexes providing mechanical coupling and tissue integrity. Previously, a correlation of desmosomal molecule expression with invasion and metastasis formation in several tumor entities was described together with a relevance for circulating tumor cell cluster formation. Here, we investigated the contribution of the desmosomal core adhesion molecule desmoglein-2 (DSG2) to the initial steps of liver metastasis formation by pancreatic cancer cells using a novel ex vivo liver perfusion mouse model. We applied the pancreatic ductal adenocarcinoma cell line AsPC-1 with and without a knockout (KO) of DSG2 and generated mouse lines with a hepatocyte-specific KO of the known interacting partners of DSG2 (DSG2 and desmocollin-2). Liver perfusion with DSG2 KO AsPC-1 cells led to smaller circulating cell clusters and a reduced number of cells adhering to murine livers compared to control cells. While this was independent of the expression levels of desmosomal adhesion molecules in hepatocytes, we show that increased cluster size of cancer cells, which correlates with stronger cell–cell adhesion and expression of desmosomal molecules, is a major factor contributing to the early phase of metastatic spreading. In conclusion, impaired desmosomal adhesion results in reduced circulating cell cluster size, which is relevant for seeding and attachment of metastatic cells to the liver.

Abstract Tu032: LRRC8 complex regulates platelet activation and thrombosis

During activation, platelets undergo both shape and volumetric changes which are mechanical events essential for thrombosis. Volume-Regulated Anion Channels (VRACs) are ubiquitously expressed mechanoresponsive, heterohexameric anion channels comprised of leucine-rich repeat containing protein 8 (LRRC8) A (essential for channel activity) in combination with B, C, D and/or E subunits. In this study, we identify single-nucleotide polymorphisms (SNPs) in three LRRC8 subunits (LRRC8A, C, and D) which are expressed in human platelets and are independently associated with altered mean platelet volume in human genetic studies – implicating the volume-sensing LRRC8 channel complex as a regulator of platelet function in humans. We show that LRRC8A is required for functional LRRC8/VRAC in megakaryocytes (MKs). MK-specific LRRC8A conditional knockout (cKO) mice give rise to LRRC8A-null platelets with larger mean volumes. LRRC8A-null platelets and CRISPR-edited human LRRC8A KO MKs (hiPSC MKs) have reduced agonist-stimulated P-selectin exposure and aIIbb3 integrin activation. LRRC8A-null platelets exhibit impaired adhesion to collagen-coated surfaces and reduced agonist-stimulated aggregation and ATP secretion. Additionally, VRAC in MKs mediate ATP currents which are abolished in LRRC8A-null MKs. In vivo, MK-specific LRRC8A cKO mice have reduced platelet thrombus formation in laser-induced cremaster arteriolar thrombosis, and prolonged occlusion times in FeCl 3 -induced carotid arterial thrombosis, compared to control mice, without affecting tail bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced ATP release to amplify agonist-stimulated calcium influx and platelet aggregation. Small-molecule LRRC8 channel inhibitors largely recapitulate the defects observed in LRRC8A-null platelets. These studies identify the mechanoresponsive LRRC8 channel complex as an ATP release channel in platelets which regulates platelet function and arterial thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

The serine-rich repeat glycoprotein Srr2 mediates Streptococcus agalactiae interaction with host fibronectin

BackgroundGroup B Streptococcus (GBS) is a commensal of healthy adults and an important pathogen in newborns, the elderly and immunocompromised individuals. GBS displays several virulence factors that promote colonisation and host infection, including the ST-17 strain-specific adhesin Srr2, previously characterised for its binding to fibrinogen. Another common target for bacterial adhesins and for host colonization is fibronectin, a multi-domain glycoprotein found ubiquitously in body fluids, in the extracellular matrix and on the surface of cells.ResultsIn this study, fibronectin was identified as a novel ligand for the Srr2 adhesin of GBS. A derivative of the ST-17 strain BM110 overexpressing the srr2 gene showed an increased ability to bind fibrinogen and fibronectin, compared to the isogenic wild-type strain. Conversely, the deletion of srr2 impaired bacterial adhesion to both ligands. ELISA assays and surface plasmon resonance studies using the recombinant binding region (BR) form of Srr2 confirmed a direct interaction with fibronectin with an estimated Kd of 92 nM. Srr2-BR variants defective in fibrinogen binding also exhibited no interaction with fibronectin, suggesting that Srr2 binds this ligand through the dock-lock-latch mechanism, previously described for fibrinogen binding. The fibronectin site responsible for recombinant Srr2-BR binding was identified and localised in the central cell-binding domain of the protein. Finally, in the presence of fibronectin, the ability of a Δsrr2 mutant to adhere to human cervico-vaginal epithelial cells was significantly lower than that of the wild-type strain.ConclusionBy combining genetic and biochemical approaches, we demonstrate a new role for Srr2, namely interacting with fibronectin. We characterised the molecular mechanism of this interaction and demonstrated that it plays a role in promoting the adhesion of GBS to human cervico-vaginal epithelial cells, further substantiating the role of Srr2 as a factor responsible for the hypervirulence of GBS ST-17 strains. The discovery of the previously undescribed interaction between Srr2 and fibronectin establishes this adhesin as a key factor for GBS colonisation of host tissues.

HGG-52. MULTI-INTEGRIN TARGETING EFFECTIVELY SUPPRESSES DIFFUSE MIDLINE GLIOMA MIGRATION

Abstract Diffuse midline gliomas (DMGs) are aggressive tumors characterized by infiltration into normal midline brain tissue, hindering surgical resection and contributing to overall morbidity and mortality. We established a novel two-step pooled whole genome CRISPR-Migration screen by using serum-free conditions and laminin-dependent cell attachment, mimicking cell migration on brain vasculature scaffolds, a common feature in glioblastoma (GBM). Comparing DMG and GBM primary cell lines (n=5), genes involved in focal adhesion (ITGB1, CRKL, PARVA, PTK2, FERMT2) were identified as significantly restricting migration in all cell lines. Multi-institutional transcriptomic data revealed an association of expression of these focal adhesion genes with survival primarily in K27M-DMG and pediatric high-grade glioma compared to lower-grade brain tumors, and ITGB1 expression increased with glioma grade. Single-cell RNA-seq and spatial transcriptomic data demonstrated higher expression of ITGB1 in OPC-like/Mesenchymal cells associated with leading edge and microvasculature in both K27M-DMG and GBM tumors. Unexpectedly, knockout of ITGB1 (ITGB1-KO), primarily responsible for cell adhesion to laminin, led to the decreased expression of multiple mitochondrial NADH dehydrogenase genes, and reduced levels of glycolysis and oxidative phosphorylation (OXPHOS) activity. Preliminary mechanistic data linked this phenotype to impaired mitochondrial fusion. Orthotopic implantation of control and ITGB1-KO models of H3K27M-DMG and adult GBM in mouse brains revealed two distinct phenotypes: (1) ITGB1-KO reducing proliferation and significantly increasing survival; and (2) ITGB1-KO leading to upregulation of ITGB3 and ITGB5 in vivo without impacting survival. Cilengitide (αvβ3 and αvβ5 inhibitor) ablated migration of all ITGB1-KO models on vitronectin (secreted by pericytes surrounding blood vessels) in vitro. Ongoing combinatorial therapy with an experimental anti-Integrin Beta 1 antibody (AIIB2, CNS delivered) and Cilengitide is underway with promising early results. In summary, multi-integrin inhibition provides a feasible avenue to curb glioma spread in the brain through impaired focal adhesion as well as blunting mitochondrial glucose metabolism.

Mechanical behaviors and meso analysis of asphalt mixtures under moisture damage induced by hydrodynamic impacts

Moisture damage significantly affects the performance and integrity of asphalt pavement. The water immersion method commonly used in laboratory is considered as a static water intrusion technique to simulate the moisture damage on asphalt mixtures. However, field conditions mainly involve dynamic water impacting due to moving tires and pressurized pore water. In this study, the in-situ moisture damage induced by repeated traffics was recurred with the Moisture Induced Sensitivity Tester (MIST), and their mechanisms and impacts on the void structure, viscoelastic properties, and fracture performance of the asphalt mixture were assessed. The findings revealed that hydrodynamic impacts with various loading cycle (0, 2000, 4000, 6000 cycles) could lead to an increase in the porosity of the mixture, with isolated voids eventually merged under water intrusion and erosion. The growth rate of the connected voids was more drastic in the initial stage and gradually decreased with the increased loading cycle. Besides, the dynamic water damage could enlarge the overall permeability of the asphalt mixtures and aggrandize the complexity of the water flow paths in a non-linear manner. The dynamic water damage factor (Dm) reflecting the variation of porosity was introduced to characterize the damage severity of the mixture. The results indicated that a severer damage was normally observed for the mixture with a higher initial porosity. Furthermore, significant effects of hydrodynamic damage on the dynamic modulus of the asphalt mixtures, especially in the high temperature (low frequency) domain, were also observed on the MIST-conditioned specimens. The CT-scanning analysis implied that the impaired adhesion between asphalt and aggregates and mechanical damage from pore water pressures were the primary causes of the damage. A degradation in crack resistance is observed from the Semi-Circular Bending (SCB) tests considering the hydrodynamic damage at various MIST-conditioning cycle.

Darier–White’s follicular dyskeratosis. Experience of successful therapy

Darier–White’s follicular dyskeratosis is an autosomal dominant hereditary dermatosis, which is based on pathological keratinization due to impaired adhesion between keratinocytes, manifested mainly in the form of hyperkeratotic follicular papules on seborrheic areas of the skin. Therapy of rare genodermatosis still presents difficulties in the tactics of choosing a treatment method for each individual patient. Currently, there are no regulated methods of treating this disease. Isotretinoin is a well-studied drug, the profile points of application of which are moderate and severe forms of acne, rosacea, but recently indications for the use of isotretinoin have been increasing. In this article presents a clinical case of Darier–White’s follicular dyskeratosis and demonstrates the successful experience of therapy of rare genodermatosis with isotretinoin.

Phage-Bacterial Interaction Alters Phenotypes Associated with Virulence in Acinetobacter baumannii.

Bacteriophages exert strong selection on their bacterial hosts to evolve resistance. At the same time, the fitness costs on bacteria following phage resistance may change their virulence, which may affect the therapeutic outcomes of phage therapy. In this study, we set out to assess the costs of phage resistance on the in vitro virulence of priority 1 nosocomial pathogenic bacterium, Acinetobacter baumannii. By subjecting phage-resistant variant Ev5-WHG of A. baumannii WHG40004 to several in vitro virulence profiles, we found that its resistance to phage is associated with reduced fitness in host microenvironments. Also, the mutant exhibited impaired adhesion and invasion to mammalian cells, as well as increased susceptibility to macrophage phagocytosis. Furthermore, the whole-genome sequencing of the mutant revealed that there exist multiple mutations which may play a role in phage resistance and altered virulence. Altogether, this study demonstrates that resistance to phage can significantly alter phenotypes associated with virulence in Acinetobacter baumannii.

Functional study of a rare L1CAM gene c.1759G>C variant prove its pathogenicity.

L1 syndrome, a neurological disorder with an X-linked inheritance pattern, mainly results from mutations occurring in the L1 cell adhesion molecule (L1CAM) gene. The L1CAM molecule, belonging to the immunoglobulin (Ig) superfamily of neurocyte adhesion molecules, plays a pivotal role in facilitating intercellular signal transmission across membranes and is indispensable for proper neuronal development and function. This study identified a rare missense variant (c.1759G>C; p.G587R) in the L1CAM gene within a male fetus presenting with hydrocephalus. Due to a lack of functional analysis, the significance of the L1CAM mutation c.1759G>C (p.G587R) remains unknown. We aimed to perform further verification for its pathogenicity. Blood samples were obtained from the proband and his parents for trio clinical exome sequencing and mutation analysis. Expression level analysis was conducted using western blot techniques. Immunofluorescence was employed to investigate L1CAM subcellular localization, while cell aggregation and cell scratch assays were utilized to assess protein function. The study showed that the mutation (c.1759G>C; p.G587R) affected posttranslational glycosylation modification and induced alterations in the subcellular localization of L1-G587R in the cells. It resulted in the diminished expression of L1CAM on the cell surface and accumulation in the endoplasmic reticulum. The p.G587R altered the function of L1CAM protein and reduced homophilic adhesion capacity of proteins, leading to impaired adhesion and migration of proteins between cells. Our findings provide first biological evidence for the association between the missense mutation (c.1759G>c; p.G587R) in the L1CAM gene and L1 syndrome, confirming the pathogenicity of this missense mutation.

Abstract 1047: Cerebral Cavernous Malformation 2 Knockout Hinders Murine Atherosclerosis And Lowers Vascular Inflammation

Introduction: Genome-wide association studies (GWAS) have identified >300 loci associated with coronary artery disease (CAD). The chr7p13 locus harbors several variants associated with decreased CAD risk, which are within the exon or the potential regulatory element of cerebral cavernous malformation 2 (CCM2, odds ratio = 0.95, p=1.53e-08). While CCM2 has been studied in venous endothelial cells (ECs) in the context of cerebral cavernous malformation, the role of CCM2 in aortic ECs and coronary atherosclerosis remains unknown. Methods and Results: To test whether CCM2 regulates atherosclerosis development, CCM2 heterozygous knockout (CCM2 +/-) mice and their control littermates (CCM2 +/+) were injected with AAV-PCKS9-D377Y and subjected to a high-fat, high cholesterol diet for 12 weeks. Atherosclerotic lesions were quantified at three different aortic sites (aortic root, minor aortic arch, and the brachiocephalic artery). Notably, heterozygous CCM2 knockout mice exhibited reduced atherosclerosis lesion in the aortic root in male mice (p=0.03), but not in female mice. Next, we sought to understand the mechanism by which CCM2 regulates the development of atherosclerosis. Single cell RNA-seq was conducted using aorta from CCM2 +/- mice and CCM2 +/+ mice. Gene set enrichment analysis on the EC population suggested that CCM2 heterozygous knockout suppressed inflammation pathways especially the TNF-alpha signaling pathway. Concordantly, after 12-weeks of high-fat, high cholesterol diet, the number of monocytes in the aorta were decreased in CCM2 +/- mice when compared to CCM2 +/+ mice. Furthermore, in vitro, ECs lacking CCM2 showed impaired THP-1 monocyte adhesion (p=0.001) and decreased expression of MCP-1 (p=3e-4). Conclusions: Our findings suggest that CCM2 heterozygous knockout hinders the development of atherosclerosis. This is likely achieved by lowering MCP-1 expression and decreasing monocyte recruitment. Further investigations are being conducted to functionally identify the causal variant in the CCM2 locus, specifically under disease-relevant stimuli, to further our understanding of CCM2’s potential as a diagnostic and therapeutic target in CAD.

Abstract 119: LRRC8 Complex Is An ATP Release Channel Regulating Platelet Activation And Thrombosis

Platelet shape and volume changes are early mechanical events in platelet activation proposed to contribute to arterial thrombosis. Here, we identify single-nucleotide polymorphisms (SNPs) in four leucine-rich repeat containing protein subunits (LRRC8A, B, C, and D) expressed in human platelets that are independently associated with increased mean platelet volume in human genetic studies - implicating the volume-sensing LRRC8 channel complex as regulating platelet function in humans. We show LRRC8A is required for functional LRRC8/VRAC in megakaryocytes (MKs). MK-specific LRRC8A conditional knockout mice give rise to LRRC8A-null platelets with larger mean volumes. LRRC8A-null platelets and CRISPR-edited human LRRC8A KO MKs (hiPSC MKs) have reduced agonist -stimulated P-selectin exposure, and αIIbβ3 integrin activation. LRRC8A-null platelets exhibit impaired adhesion to collagen-coated surfaces, agonist-induced platelet aggregation and ATP secretion. MK-specific LRRC8A conditional knockout mice have reduced platelet thrombus formation in laser-induced cremaster arteriolar thrombosis and prolonged occlusion times in FeCl 3 -induced carotid arterial thrombosis, compared to control mice, without affecting tail bleeding times. Mechanistically, LRRC8A mediates swelling-induced ATP release to amplify agonist-stimulated calcium influx and aggregation in platelets. Treatment of platelets with small-molecule LRRC8 channel inhibitors recapitulate the defects in LRRC8A-null platelets. These studies identify the mechanoresponsive LRRC8 channel complex as a regulator of platelet function and thrombosis - providing a proof-of-concept for a novel anti-thrombotic drug target.

Autocrine VEGF drives neural stem cell proximity to the adult hippocampus vascular niche.

The vasculature is a key component of adult brain neural stem cell (NSC) niches. In the adult mammalian hippocampus, NSCs reside in close contact with a dense capillary network. How this niche is maintained is unclear. We recently found that adult hippocampal NSCs express VEGF, a soluble factor with chemoattractive properties for vascular endothelia. Here, we show that global and NSC-specific VEGF loss led to dissociation of NSCs and their intermediate progenitor daughter cells from local vasculature. Surprisingly, though, we found no changes in local vascular density. Instead, we found that NSC-derived VEGF supports maintenance of gene expression programs in NSCs and their progeny related to cell migration and adhesion. In vitro assays revealed that blockade of VEGF receptor 2 impaired NSC motility and adhesion. Our findings suggest that NSCs maintain their own proximity to vasculature via self-stimulated VEGF signaling that supports their motility towards and/or adhesion to local blood vessels.

Abstract 1261: In vitro medulloblastoma leptomeningeal metastasis models reveal adhesion signaling as a therapeutic vulnerability

Abstract Introduction: How tumor cells colonize an inherently distinct environment and survive the pressures of therapeutic treatment during metastasis is incompletely understood. In patients with medulloblastoma, the most common malignant pediatric brain tumor, mortality is almost exclusively dictated by metastasis to the leptomeninges, the inner covering of the brain and spinal cord. We aim to mechanistically understand the molecular drivers of leptomeningeal colonization, including how leptomeningeal adhesion mediates therapy resistance. Experimental approach: Progress in leptomeningeal metastasis research has been hindered by the scarcity of matched primary and metastatic tissue from patients given the lack of clinical benefit to obtaining metastatic tissue. Furthermore, end points from pre-clinical human xenograft mouse models are often driven by primary cerebellar tumors, not leptomeningeal dissemination. These barriers underscore the necessity for novel in vitro leptomeningeal metastasis models that allow for rigorous experimental analysis. These models must simultaneously recapitulate tumor cell-host and tumor cell-environment interactions driving colonization to produce translatable findings. Accordingly, our lab has developed two novel in vitro leptomeningeal metastasis models that allow for cell-based experimental analysis: a primary meningeal cell-medulloblastoma organotypic co-culture and an acellular leptomeningeal matrix mimetic monoculture. Results: Proteomic analysis identified that leptomeningeal cells promote medulloblastoma chemotactic honing to the leptomeninges through CXCL12-CXCR4 signaling. Additionally, loss of CXCL12/CXCR4 signaling impaired medulloblastoma adhesion to the leptomeninges. CXCL12/CXCR4 are known to mediate adhesion of stem cells in stromal niches via activation of integrin receptor ITGA4/ITGB1 (VLA4) binding to stromal VCAM1. Indeed, the VLA4-VCAM1 axis is required for medulloblastoma leptomeningeal adhesion and requires activation by CXCL12/CXCR4 signaling. From a translational perspective, we found that leptomeningeal adhesion promotes therapy resistance and that inhibition of adhesion signaling sensitized adherent medulloblastoma cells to therapy. Conclusions: These findings provide proof-of-concept that appropriate in vitro models of metastasis can be developed and utilized to define the biology driving adhesion and chemotherapy resistance in medulloblastoma metastasis. Furthermore, we show that medulloblastoma leptomeningeal adhesion is mediated by a CXCL12/CXCR4-VLA4/VCAM1 axis. Finally, we demonstrate the therapeutic potential of targeting this axis in sensitizing medulloblastoma metastases to current therapies. Citation Format: Leyre Jimenez Garcia, Amy C. Gross, Ryan D. Roberts, James B. Reinecke. In vitro medulloblastoma leptomeningeal metastasis models reveal adhesion signaling as a therapeutic vulnerability [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 1261.

Abstract 7500: Distribution and prognostic significance of circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tdEVs) in patients (pts) with lobular metastatic breast cancer (MBC)

Abstract Background: Invasive lobular carcinoma (ILC) has distinctive clinical and molecular features compared with invasive ductal carcinoma (IDC). CTCs and tdEVs are independent prognostic factors in MBC, however, liquid biopsy studies focusing on ILC are to date scarce. Higher CTC levels have been observed in ILC than IDC. One study including 28 ILC pts suggested the use of higher CTC cutoff for prognosis stratification. The clinical significance of tdEVs in ILC is unexplored. This study aimed to assess differences in the distribution and prognostic value of CTCs and tdEVs between ILC and IDC. Methods: Blood samples were collected from 304 pts with MBC before starting a new line of therapy at Northwestern University (Chicago, IL) between 2016 and 2021 (NU16B06 trial). Blood was processed with the CellSearch® system. The ACCEPT software was applied to CellSearch® images to automatically enumerate CTCs and tdEVs. Association of CTCs and tdEVs with overall survival (OS) was tested in ILC and IDC pts. A count of CTCs ≥ 5 was considered high. The cutoff levels for tdEVs were < 20 (low), 20-79 (intermediate), and ≥ 80 (high), as previously reported. Additional exploratory cutoffs for CTCs (≥ 20, ≥ 50, ≥ 80, and ≥ 100) and tdEVs (based on quartiles of tdEV distribution in the overall population and ILC) were also evaluated. Results: Of the 304 pts, 47 had ILC while 257 had IDC. 56% of pts received first-line therapy. Median CTC count was 8 [interquartile range (IQR) 2-35] in ILC and 1 (IQR 0-8) in IDC pts (P <.001). A significantly higher median tdEVs count was observed in ILC (36; IQR 18-115) than in IDC (11; IQR 2-86) pts (P =.002). High levels of CTCs (≥5) were not associated with OS in ILC (P =.88), even when higher cutoffs (≥ 20, ≥ 50, and ≥ 80) were used. Only the detection of ≥ 100 CTCs was significantly associated with OS in ILC pts (median OS 36 vs 10 months; HR 3.4; P =.028). In contrast, the prognostic effect of CTCs ≥ 5 was strong in IDC and maintained for all cutoffs. Similarly, while the prognostic value for tdEVs was confirmed for IDC using both 20-80 and exploratory cutoffs (3-105 and 18-155), no association between tdEV levels and OS was observed in ILC regardless of the cutoffs used. Conclusion: ILC is associated with higher CTCs and tdEVs compared to IDC, probably due to the impaired cell-cell adhesion that characterizes ILC. Unlike IDC, the prognostic effect for the established cutoff of ≥ 5 CTC was not observed in ILC. A higher CTC cutoff could be considered for this subtype of BC. For tdEV no association with OS was observed, regardless of the cutoff used. These findings emphasize that ILC is a distinct entity also in the liquid biopsy features. Because of the low prevalence of ILC, research efforts should be directed at combining data from ILC pts to better characterize this subtype of BC and understand the reason behind the different associations with OS as compared to IDC. Citation Format: Eleonora Nicolo, Lorenzo Gerratana, Lorenzo Foffano, Laura Munoz-Arcos, Mara S. Serafini, Maroua Manai, Letizia Pontolillo, Nadia Bayou, Elisabetta Molteni, Amanda K. Strickland, Caterina Gianni, Youbin Zhang, Paolo D'Amico, Andrew A. Davis, Jeannine Donahue, Huiping Liu, William J. Gradishar, Ami N. Shah, Giuseppe Curigliano, Carolina Reduzzi, Massimo Cristofanilli. Distribution and prognostic significance of circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tdEVs) in patients (pts) with lobular metastatic breast cancer (MBC) [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 7500.

Epidermolysis Bullosa: Practical Clinical Tips From the Field.

Epidermolysis bullosa (EB) is a rare genetic condition characterized by fragile skin caused by impaired adhesion between the dermis and epidermis. EB is present at or near birth. There is no cure and treatments are supportive. Children with EB are at elevated risk of squamous cell cancer. Under ideal circumstances, EB patients benefit from interdisciplinary care teams who can offer state-of-the-art treatments. In reality and particularly in less-developed nations, care can be limited.In all cases, families dealing with a member with EB face great challenges in caregiving, much of which is managed at home, and incur great financial expenses for dressings, equipment, transportation, and out-of-pocket expenses. While research groups are working to find a cure for EB, clinicians working with EB patients around the worldhave found practical and relatively inexpensive tips to make life easier for people with EB. NoBabyBlisters.org, a nonprofit organization actively supplying monthly medical supplies for EB children on five continents and working on EB research, has innovated, developed, collected, and now offers here seven such practical and actionable items learned from its experience in the real world assisting children in less-developed nations, typically with hot climates. These are based on real-world clinical experience dealing with a complex disorder under challenging circumstances. The goal of this short paper is to provide advice to EB caregivers and their loved ones that may make things easier and enhance quality of life, including blister and pain reduction.

Levamisole Modulation of Podocytes' Actin Cytoskeleton in Nephrotic Syndrome.

Podocytes play a central role in glomerular diseases such as (idiopathic) nephrotic syndrome (iNS). Glucocorticoids are the gold standard therapy for iNS. Nevertheless, frequent relapses are common. In children with iNS, steroid-sparing agents are used to avoid prolonged steroid use and reduce steroid toxicity. Levamisole is one of these steroid-sparing drugs and although clinical effectiveness has been demonstrated, the molecular mechanisms of how levamisole exerts its beneficial effects remains poorly studied. Apart from immunomodulatory capacities, nonimmunological effects of levamisole on podocytes have also been suggested. We aimed to elaborate on the effects of levamisole on human podocytes in iNS. RNA sequencing data from a human podocyte cell line treated with levamisole showed that levamisole modulates the expression of various genes involved in actin cytoskeleton stabilization and remodeling. Functional experiments showed that podocytes exposed to puromycin aminonucleoside (PAN), lipopolysaccharides (LPS), and NS patient plasma resulted in significant actin cytoskeleton derangement, reduced cell motility, and impaired cellular adhesion when compared to controls, effects that could be restored by levamisole. Mechanistic studies revealed that levamisole exerts its beneficial effects on podocytes by signaling through the glucocorticoid receptor and by regulating the activity of Rho GTPases. In summary, our data show that levamisole exerts beneficial effects on podocytes by stabilizing the actin cytoskeleton in a glucocorticoid receptor-dependent manner.

Abstract 17747: Gynecological Manifestations in a Patient With Arrhythmogenic Right Ventricular Cardiomyopathy: A Desmosomal Link?

Introduction: Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is inherited cardiomyopathy which is caused by desmosomal protein gene mutations, primarily in the desmoplakin (DSP), plakophilin-2 (PKP2), and desmoglein-2 (DSG2) genes. It is characterized pathologically by myocardial atrophy, fibrofatty replacement, and fibrosis, precipitating ventricular arrhythmias (VA). Desmosomal mutations have been linked to impaired follicular development and leiomyoma formation. However, there is little information about the association of their expression in myocardial with ovarian or uterine tissues. As incidences of ARVC/D in women increase, it is crucial to determine if women with the gynecological manifestations of these mutations are at a higher risk of developing ARCV/D. Case: We report a case of a 32-year-old female who presented with exercise-induced palpitations and syncopal episodes. She has a history of severe dysmenorrhea but has no medical conditions otherwise. On monitoring, she was found to have a high burden of ventricular premature complexes (VPCs). VAs were thought to have precipitated her syncopal episodes. She was started on antiarrhythmics. Echocardiogram demonstrated right ventricular dilatation and wall thinning. The patient later underwent genetic testing, which confirmed ARVC-associated mutations. Her dysmenorrhea was managed with combined oral contraceptives and nonsteroidal anti-inflammatory drugs. Pelvic ultrasound revealed leiomyomas, which were surgically removed. Discussion: This case supports the possible complex interplay between cardiovascular and gynecological conditions in patients with desmosomal mutations. Desmosomes are specialized protein complexes crucial in maintaining tissue's structural and mechanical stability. Mutations in desmosome genes disrupt the normal functioning of these protein complexes, leading to impaired cell-cell adhesion and increased vulnerability to tissue damage. The next step is to conduct studies to better understand the risk of developing cardiovascular disease in patients presenting with these gynecological conditions and vice versa to provide early monitoring and treatment.