EpCAM Expression Research Articles

MYL9 binding with MYO19 suppresses epithelial-mesenchymal transition in non-small cell lung cancer.

The elusive function of myosin light chain 9 (MYL9) in cancer is an area ripe for further investigation. Bioinformatics was utilized to compare the expression levels of MYL9 in non-small cell lung cancer (NSCLC) and normal tissues. Gene set enrichment analysis (GSEA) was employed to investigate the pathways associated with MYL9. BioGRID database was utilized to screen for potential targets of MYL9. The expression of MYL9 and myosin 19 (MYO19) mRNA was quantified using quantitative reverse transcriptase PCR. Cell migration was assessed using a scratch wound healing assay. Protein levels of MYL9, MYO19, and epithelial-mesenchymal transition (EMT) biomarkers were examined using western blot (WB). EpCAM expression in different cell groups was profiled using flow cytometry analysis. Co-immunoprecipitation assays were performed to determine the binding affinity between MYL9 and MYO19. Additionally, direct protein interaction between MYL9 and MYO19 was explored using a GST-pull-down assay. In NSCLC patients, MYL9 was significantly downregulated in vivo and in cell cultures, showing high enrichment in the EMT pathway. Scratch assays indicated its inhibitory effect on cell migration. Western blotting revealed that MYL9 suppresses EMT marker protein expression in NSCLC cells. Flow cytometry showed that MYL9 reduced EpCAM levels on the cell surface. MYO19 was identified as a potential target of MYL9 through CoIP and GST-pull-down assays. Rescue experiments demonstrated that MYO19 enhances cell migration, EMT marker expression, and EpCAM levels, but these effects were countered by MYL9 overexpression. MYL9 impedes the migration and EMT in NSCLC cells by binding to MYO19.

The regenerative capacity of hepatocyte organoids following long-term cryopreservation in Republic of Korea

Background: Hepatocyte organoids (HOs) are more functionally versatile than bile duct epithelial cell organoids, but have a shorter lifespan. This limitation has been improved by optimizing culture methods, but could remain a barrier to their wider application in research and therapy, especially for use at the appropriate time.Methods: This study aimed to prolong the lifespan of pig HOs and assess their viability and functionality after a year of cryopreservation. Genes involved in apoptosis (TP53, P21, CASP8) and liver function (ALB, CYP3A29, EPCAM) were analyzed to evaluate the impact of adipose-derived mesenchymal stem cell (A-MSC) co-culture before and after freezing on cryoresistance. HOs were cut into fragments, cryopreserved for a year, and cultured alone or co-cultured with A-MSCs in Matrigel post-thawing.Results: After thawing, co-cultured HOs exhibited a higher development rate than those cultured alone. P21 expression increased irrespective of pre-freezing culture conditions. The ALB and CYP3A29 expression patterns resembled those of non-frozen HOs, with similar effects from co-culture. EPCAM expression surged post-freezing.Conclusion: This study demonstrates that HOs maintain liver function post-preservation, showing increased EPCAM expression and enhanced regenerative capacity against cryoinjury. These findings suggest that HOs may be a valuable cell source for drug development in animals and research on medications for human diseases.

Markers for egg quality in European eel derived from offspring of females subjected to different gonadotropic treatments

Broodstock management techniques in general, and gonadotropic treatments for vitellogenesis induction in particular, can influence the maternal provisioning of different compounds to the eggs, thus affecting embryo and larval competence. Among these, the abundance of certain transcripts as well as lipid content and fatty acid composition have been used as egg quality markers alongside other egg morphological parameters, such as egg size and the occurrence of abnormal cleavages during the first stages of embryonic development. Here, we evaluated the use of different markers of egg quality from farmed-raised European eel, Anguilla anguilla, broodstock reared on the same formulated diet but subjected to different pituitary extract administration schemes: weekly injections of salmon pituitary extract (SPE) in a constant dose or weekly injections of carp pituitary extract (CPE) at a stepwise increasing dose. Egg batches were categorized in two quality groups (high and low) according to their survival to the first feeding larval stage. In agreement with previous studies, unfertilized eggs and 4-h post fertilization embryos (16-cell stage) from SPE treated females had higher epcam expression levels than CPE ones, which is a molecule involved in cell-cell adhesion. When comparing treatments, batches from the SPE treatment had a lower incidence of cleavage abnormalities which could originate from compromised cell adhesion. On the other hand, this difference was not observed when comparing high-quality batches (i.e. those surviving up to the first-feeding stage) and low-quality ones. From the five genes analysed: epcam, dcbld1, plec, cenpf, and cenpk, only the latter had a differential expression between high- and low-quality groups both in unfertilized eggs and 4 h-post-fertilization embryos. This gene, cenpk, is involved in kinetochore assemblage during cell division and appears to be promising as egg quality marker in European eel in combination with epcam and dcbld1. Common biomarkers, such as egg size, total protein and lipid content, were similar between groups. However, although fatty acid composition was similar between quality groups, DHA and EPA content in unfertilized eggs was affected by the gonadotropic treatment applied to the female broodstock, and higher for the CPE treated females than the SPE ones. This entails that the hormonal treatments applied for vitellogenesis induction can influence the amount of specific fatty acids being transferred to the eggs, even for female broodstock that were fed the same high-quality diet.

Phase I Clinical Evaluation of Designed Ankyrin Repeat Protein [99mTc]Tc(CO)3-(HE)3-Ec1 for Visualization of EpCAM-Expressing Lung Cancer.

A high level of EpCAM overexpression in lung cancer makes this protein a promising target for targeted therapy. Radionuclide visualization of EpCAM expression would facilitate the selection of patients potentially benefiting from such treatment. Single-photon computed tomography (SPECT) using 99mTc-labeled engineered scaffold protein DARPin Ec1 has shown its effectiveness in imaging tumors with overexpression of EpCAM in preclinical studies, providing high contrast just a few hours after injection. This first-in-human study aimed to evaluate the safety and distribution of [99mTc]Tc(CO)3-(HE)3-Ec1 in patients with primary lung cancer. Twelve lung cancer patients were injected with 300.7 ± 103.2 MBq of [99mTc]Tc(CO)3-(HE)3-Ec1. Whole-body planar imaging (at 2, 4, 6 and 24 h after injection) and SPECT/CT of the lung (at 2, 4, and 6 h) were performed. The patients' vital signs and possible side effects were monitored up to 7 days after injection. The patients tolerated the injection of [99mTc]Tc(CO)3-(HE)3-Ec1 well, and their somatic condition remained normal during the entire follow-up period. There were no abnormalities in blood and urine tests after injection of [99mTc]Tc(CO)3-(HE)3-Ec1. The highest absorbed doses were in the kidneys, liver, pancreas, thyroid, gallbladder wall, and adrenals. There was also a relatively high accumulation of [99mTc]Tc(CO)3-(HE)3-Ec1 in the small and large intestines, pancreas and thyroid. According to the SPECT/CT, accumulation of [99mTc]Tc(CO)3-(HE)3-Ec1 in the lung tumor was found in all patients included in the study. Intensive accumulation of [99mTc]Tc(CO)3-(HE)3-Ec1 was also noted in regional metastases. [99mTc]Tc(CO)3-(HE)3-Ec1 can potentially be considered a diagnostic tracer for imaging EpCAM expression in lung cancer patients and other tumors with overexpression of EpCAM.

Prognostic and Predictive Significance of EpCAM and SOX2 expression in Serous Ovarian Carcinoma

Prognostic and Predictive Significance of EpCAM and SOX2 expression in Serous Ovarian Carcinoma

Role of HNF4alpha-cMyc interaction in liver regeneration after partial hepatectomy.

Hepatocyte nuclear factor 4 alpha (HNF4α) is the master regulator of hepatic differentiation. Recent studies have also revealed the role of HNF4α in hepatocyte proliferation via negatively regulating the expression of proto-mitogenic genes, including cMyc. Here, we aimed to study the interaction between HNF4α-cMyc during liver regeneration after partial hepatectomy (PHX). Wild-type (WT), hepatocyte-specific knockout of HNF4α (HNF4α-KO), cMyc (cMyc-KO), and HNF4α-cMyc double knockout (DKO) mice were subjected to PHX to induce liver regeneration. Blood and liver tissue samples were collected at 0h, 24h, 48h, 7D, and 14D after PHX for further analysis. WT, HNF4α-KO, cMyc-KO and DKO mice regained liver weight by 14 days after PHX. The deletion of cMyc did not affect liver regeneration, which was similar to the WT mice. WT and cMyc-KO mice started regaining liver weight as early as 24 hours after PHX, with a peak proliferation response at 48 hours after PHX. HNF4α- KO and DKO showed a delayed response with liver weight increase by day 7 after PHX. The overall hepatocyte proliferation response by DKO mice following PHX was lower than that of other genotypes. Interestingly, the surviving HNF4α-KO and DKO mice showed re-expression of HNF4α at mRNA and protein levels on day 14 after PHX. This was accompanied by a significant increase in the expression of Krt19 and Epcam, hepatic progenitor cell markers, in the DKO mice on day 14 after PHX. These data indicate that, in the absence of HNF4α, cMyc contributes to hepatocyte-driven proliferation to compensate for the lost tissue mass. Furthermore, in the absence of both HNF4α and cMyc, HPC-driven proliferation occurs to support liver regeneration.

3D Porous Polymer Scaffold-Conjugated KGF-Mimetic Peptide Promotes Functional Skin Regeneration in Chronic Diabetic Wounds.

The re-epithelialization process gets severely dysregulated in chronic nonhealing diabetic foot ulcers/wounds. Keratinocyte growth factor (KGF or FGF-7) is the major modulator of the re-epithelialization process, which regulates the physiological phenotypes of cutaneous keratinocytes. The existing therapeutic strategies of growth factor administration have several limitations. To overcome these, we have designed a KGF-mimetic peptide (KGFp, 13mer) based on the receptor interaction sites in murine KGF. KGFp enhanced migration and transdifferentiation of mouse bone marrow-derived MSCs toward keratinocyte-like cells (KLCs). A significant increase in the expression of skin-specific markers Bnc1 (28.5-fold), Ck5 (14.6-fold), Ck14 (26.1-fold), Ck10 (187.7-fold), and epithelial markers EpCam (23.3-fold) and Cdh1 (64.2-fold) was associated with the activation of ERK1/2 and STAT3 molecular signaling in the KLCs. Further, to enhance the stability of KGFp in the wound microenvironment, it was conjugated to biocompatible 3D porous polymer scaffolds without compromising its active binding sites followed by chemical characterization using Fourier transform infrared spectroscopy, field-emission scanning electron microscopy, dynamic mechanical analysis, and thermogravimetry. In vitro evaluation of the KGFp-conjugated 3D polymer scaffolds revealed its potential for transdifferentiation of MSCs into KLCs. Transplantation of allogeneic MSCGFP using KGFp-conjugated 3D polymer scaffolds in chronic nonhealing type 2 diabetic wounds (db/db transgenic, 50-52 weeks old male mice) significantly enhanced re-epithelialization-mediated wound closure rate (79.3%) as compared to the control groups (Untransplanted -22.4%, MSCGFP-3D polymer scaffold -38.5%). Thus, KGFp-conjugated 3D porous polymer scaffolds drive the fate of the MSCs toward keratinocytes that may serve as potential stem cell delivery platform technology for tissue engineering and transplantation.

Longitudinal analysis of circulating tumor cell numbers improves tracking metastatic breast cancer progression

Hormone-responsive breast cancer represents the most common type and has the best prognosis, but still approximately 40% of patients with this type can develop distant metastases, dramatically worsening the patient’s survival. Monitoring metastatic breast cancer (mBC) for signs of progression is an important part of disease management. Circulating tumor cell (CTC) detection and molecular characteristics gain importance as a diagnostic tool, but do not represent a clinical standard and its value as a predictor of progression is not yet established. The main objective of this study was to estimate the prognostic value of not only the CTC numbers, but also the dynamics of the CTC numbers in the same patient during the continuous evaluation of CTCs in patients with advanced breast cancer. The other objective was to assess the molecular changes in CTCs compared to primary tumor samples by genetic analysis of the seven genes associated with estrogen signaling pathway, mutations in which are often responsible for the resistance to endocrine therapy, and subsequent progression. This approach was taken to evaluate if genetic analysis of CTCs can be used in tracking the resistance, signaling that hormonal therapy should be replaced. Consequently, this report presents the results of a longitudinal CTC study based on three subsequent blood collections from 135 patients with metastatic breast cancer, followed by molecular analysis of the isolated single CTCs. CTCs were detected and isolated using an image-based, EpCAM-independent system CytoTrack; this approach allowed evaluation of EpCAM expression in detected CTCs. Isolated CTCs were subjected to NGS analysis to assess mutational changes. The results confirm the importance of the status of the CTC for progression-free survival and overall survival and provide new data on the dynamics of the CTC during a long monitoring period and in relation to clinical progression, highlighting the advantage of constant monitoring over the single count of CTC. Furthermore, high genetic and phenotypic inter- and intrapatient heterogeneity observed in CTCs suggest that metastatic lesions are divergent. High genetic heterogeneity in the matching CTC/primary tumor samples may indicate early dissemination. The tendency towards the accumulation of activating/oncogenic mutation in CTCs, leading to anti-estrogen resistant disease, was not confirmed in this study.

IGFBP6 regulates extracellular vesicles formation via cholesterol abundance in MDA-MB-231 cells

Breast cancer recurrence is associated with the growth of disseminated cancer cells that separate from the primary tumor before surgical treatment and hormonal therapy and form a metastatic niche in distant organs. We previously demonstrated that IGFBP6 expression is associated with the risk of early relapse of luminal breast cancer. Knockdown of IGFBP6 in MDA-MB-231 breast cancer cells increased their invasiveness, proliferation, and metastatic potential. In addition, the knockdown of IGFBP6 leads to impaired lipid metabolism. In this study, we demonstrated that the knockdown of the IGFBP6 gene, a highly selective inhibitor of IGF-II, led to a significant decline in the number of secreted extracellular vesicles (EVs) and altered cholesterol metabolism in MDA-MB-231 cells. Knockdown of IGFBP6 led to a decrease in the essential proteins responsible for the biogenesis of cholesterol LDLR and LSS, which reduced the amount by more than 13 times. In addition, the knockdown of IGFBP6 led to a possible change in the profile of adhesion molecules on the surface of EVs. The expression of L1CAM, IGSF3, EpCAM, CD24, and CD44 decreased, and the expression of EGFR increased. We can conclude that the negative prognostic value of low expression of this gene could be associated with increased activity of IGF2 in tumor-associated fibroblasts due to low secretion of IGFBP6 by tumor cells. In addition, changing the profile of adhesion molecules on the surface of tumor EVs may contribute to the more efficient formation of metastatic niches.

Curcumin-loaded liposomes modulating the synergistic role of EpCAM and estrogen receptor alpha in lung cancer management

Lung cancer (LC) remains a leading cause of cancer-related mortality worldwide, necessitating the exploration of innovative therapeutic strategies. This study delves into the in vitro potential of liposomal therapeutics utilizing Curcumin-loaded PlexoZome® (CUR-PLXZ) in targeting EpCAM/TROP1 and Estrogen Receptor Alpha (ERα) signalling pathways for LC management. The prevalence of LC, particularly non-small cell lung cancer (NSCLC), underscores the urgent need for effective treatments. Biomarkers like EpCAM/TROP1 and ERα/NR3A1 play crucial roles in guiding targeted therapies and influencing prognosis. EpCAM plays a key role in cell-cell adhesion and signalling along with ERα which is a nuclear receptor that binds estrogen and regulates gene expression in response to hormonal signals. In LC, both often get overexpressed and are associated with tumour progression, metastasis, and poor prognosis. Curcumin, a phytochemical with diverse therapeutic properties, holds promise in targeting these pathways. However, its limited solubility and bioavailability necessitate advanced formulations like CUR-PLXZ. Our study investigates the biological significance of these biomarkers in the A549 cell line and explores the therapeutic potential of CUR-PLXZ, which modulates the expression of these two markers. An in vitro analysis of the A549 human lung adenocarcinoma cell line identified that CUR-PLXZ at a dose of 5 μM effectively inhibited the expression of EpCAM and ERα. This finding paves the way for targeted intervention strategies in LC management.

Characterization of 3D Organotypic Culture of Mouse Adipose-Derived Stem Cells.

Although stem cells are a promising avenue for harnessing the potential of adipose tissue, conventional two-dimensional (2D) culture methods have limitations. This study explored the use of three-dimensional (3D) cultures to preserve the regenerative potential of adipose-derived stem cells (ADSCs) and investigated their cellular properties. Flow cytometric analysis revealed significant variations in surface marker expressions between the two culture conditions. While 2D cultures showed robust surface marker expressions, 3D cultures exhibited reduced levels of CD44, CD90.2, and CD105. Adipogenic differentiation in 3D organotypic ADSCs faced challenges, with decreased organoid size and limited activation of adipogenesis-related genes. Key adipocyte markers, such as lipoprotein lipase (LPL) and adipoQ, were undetectable in 3D-cultured ADSCs, unlike positive controls in 2D-cultured mesenchymal stem cells (MSCs). Surprisingly, 3D-cultured ADSCs underwent mesenchymal-epithelial transition (MET), evidenced by increased E-cadherin and EpCAM expression and decreased mesenchymal markers. This study highlights successful ADSC organoid formation, notable MSC phenotype changes in 3D culture, adipogenic differentiation challenges, and a distinctive shift toward an epithelial-like state. These findings offer insights into the potential applications of 3D-cultured ADSCs in regenerative medicine, emphasizing the need for further exploration of underlying molecular mechanisms.

A comprehensive review of the botany, phytochemistry, pharmacology, and toxicology of Murrayae Folium et Cacumen.

Ethnopharmacological relevance: Murrayae Folium et Cacumen (MFC) is a plant considered to be a traditional Chinese medicine with culinary value as well. The dry leaves and twigs of Murraya paniculata and M. exotica are used to treat stomach aches, rheumatism, toothaches, swelling, and insect and snake bites. They are also used to prepare spicy chicken dishes. Aim of the review: This review comprehensively summarizes the available information on the botanical characterization, phytochemistry, pharmacological activities, pharmacodynamics, pharmacokinetics, and toxicity of MFC. Methods: Relevant scientific literature up to August 2023 was included in the study. Chinese and English studies on MFC were collected from databases, including PubMed, Elsevier, Web of Science, Springer, Science Direct, Wiley, ACS, and CNKI (Chinese). Doctoral and Master's dissertations were also included. Results: In total, 720 compounds have been identified and reported in the literature, including flavonoids, coumarins, alkaloids, sterols, phenylpropenols, organic acids, spirocyclopentenones, and volatile oils. Flavonoids and coumarins are the two most important bioactive compounds responsible for these pharmacological activities. MFC has anti-inflammatory, anti-bacterial, anti-microbial, anti-diabetic, anti-tumor, anti-oxidant, anti-depressant, potential anti-Alzheimer's disease, chondroprotective, and analgesic properties. The pharmacological effects include interrupting the STAT3/NF-κB/COX-2 and EGFR signaling pathways, downregulating EpCAM expression, inhibiting NF-κB and ERK signals, inhibiting the EP/cAMP/PKA signaling pathway and miR-29a/Wnt/β-catenin signaling activity, and upregulating Foxo3a expression. Conclusion: This review demonstrates that the chemical constituents, pharmacological activities, pharmacodynamics, pharmacokinetics, and toxicity of MFC support its use in traditional Chinese botanical medicines. MFC contains a wide range of chemical compounds. Flavonoids and coumarins promote strong pharmacological activity and, are low-toxicity natural phytomedicines that are widely used in medicine, food, ornamentation, and cosmetics, making MFC a promising compound for development and use in the treatment of several medical conditions.

Abstract 6395: Metabolic dysregulation as a determinant of prognosis and response to sorafenib in liver cancer

Abstract Background & Aims: Elevated metabolic activity is a defining characteristic of liver cancer. Nevertheless, our understanding of the molecular underpinnings of high metabolic activity in liver cancer and its impact on clinical outcomes and treatment responses remains limited. We aimed to elucidate the metabolic features of liver cancer associated with clinical outcomes, particularly overall survival and responsiveness to sorafenib treatment. Methods: Utilizing cross-species comparisons of genomic data, we integrated gene expression profiles from liver tissues in metabolically challenged mice and from patients with cirrhosis and liver cancer. To categorize patients, we developed a genomic predictor and applied it to gene expression data from a cirrhosis cohort (n=216) and large liver cancer cohorts (n=1390). Comprehensive statistical and informatics analyses were conducted to evaluate clinical significance, including overall survival and responses to standard treatments. Moreover, we integrated genomic data derived from patient-derived xenograft (PDX) models into our analytical framework. Results: Our integrated data analysis unveiled three distinct metabolic subtypes of tissues: high, moderate, and low metabolic subtypes. In the cirrhosis cohort, the high metabolic subtype was significantly associated with early liver cancer development. Among liver cancer cohorts, the high metabolic subtype exhibited characteristics such as poor survival, heightened hepatic stem cell features, increased genomic instability, and elevated expression of AFP, KRT19, EPCAM, and SALL4. Notably, when liver cancer patients undergoing systemic sorafenib treatment were stratified using our genomic predictor, those with low and moderate metabolic activity derived significant benefits from sorafenib therapy. Conversely, the high metabolic subtype displayed substantial resistance to sorafenib, suggesting that metabolic activity may dictate the response of liver cancer cells to sorafenib treatment. Gene network analysis of the metabolic expression signature identified multiple signaling pathways that potentially contribute to sorafenib resistance in liver cancer cells. Our analysis predicted the activation of pathways such as the TGF-β pathway (TGFB1), consistent with previous findings of TGF-β pathway activation in highly metabolic cancer cells. Additionally, our analysis identified YAP1, HIF1A, and MAPK pathways as activated pathways in sorafenib-resistant liver cancer. Conclusions: This study identifies clinically and metabolically distinct subtypes of liver cancer, biomarkers associated with these subtypes, and mechanisms underlying metabolic-mediated resistance of liver cancer cells to sorafenib. Furthermore, the metabolic subtypes observed in PDX models provide valuable tools for selecting appropriate preclinical models for future research endeavors. Citation Format: Hyewon Park, Sowon Park, Sung-Hwan Lee, Yun Seong Jeong, Bohwa Sohn, Sun Young Yim, Ju-Seog Lee. Metabolic dysregulation as a determinant of prognosis and response to sorafenib in liver 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 6395.

Abstract 5479: Spatially resolved molecular features in the tumor-immune microenvironment of germline BRCA1/2 mutated versus BRCA wild type high grade serous ovarian cancers

Abstract High-grade serous ovarian cancer (HGSC) is the most common and aggressive subtype of epithelial ovarian cancer. Importantly, germline mutations in BRCA1/2 give rise to HGSC. Because most HGSC are diagnosed at stage III/IV, outcomes are poor and more effective therapies are needed. Although immune checkpoint inhibitors show promise for a number of cancer types, efficacy has been variable in HGSC. To help provide new clues to the molecular features of HGSC, we set out to elucidate the tumor microenvironment of HGSC using spatial transcriptomics approaches. Specifically, we applied 10x Genomics Visium Spatial Transcriptomics (ST) technology to 48 samples representing primary HGSC tumors from 24 patients, including 15 BRCA1/2 mutated cases and 9 BRCA non-mutated cases. In addition to individual sample analysis, we normalized data from 64,784 total features across all sections and performed an integrated cluster analysis to get an overall picture of the cellular architecture of HGSC, but to also directly investigate differences in the tumor microenvironment based on BRCA mutation status. The resulting 17 integrated clusters were subjected to molecular annotation using GSEA, ESTIMATE, and CIBERSORTx, which revealed feature clusters representing important cell types defined by marker genes. Analysis of the entire dataset identified a primary tumor cluster defined by the expression of known ovarian cancer genes including WFDC2 and “don’t eat me” marker genes CD24 and CD47. However, the predominant tumor cell population in BRCA mutated cases was marked by the expression of MUC1, EPCAM, CLDN3, and CD24, while BRCA non-mutated tumor cell cluster was marked by EFNA1, ID1, LSR, and CD9. Importantly, both groups revealed the presence of a specialized monocyte derived macrophage cluster defined by the expression of LYZ and genes involved in cholesterol metabolism including APOE and APOC1. These lipid-associated macrophages are M2 polarized and shown to be associated with poor outcome. Additional analyses are underway to determine spatial dependencies of various tumor and immunological cell compartments. These data provide novel insights into the HGSC tumor microenvironment that may influence HGSC’s vulnerability to immunological therapies. Citation Format: Jing Qian, Lynda D. Roman, Rania Bassiouni, Seeta Rajpara, Monica Neuman, Varun Khetan, David W. Craig, Joseph W. Carlson, John D. Carpten. Spatially resolved molecular features in the tumor-immune microenvironment of germline BRCA1/2 mutated versus BRCA wild type high grade serous ovarian cancers [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 5479.

Abstract 6939: Residual disease after HER2 inhibition is driven by a primary tumor subpopulation expressing an ERBB2-low associated transcriptional program

Abstract Background: In HER2+ breast cancer (BC), residual disease (RD) after neoadjuvant HER2 inhibition is associated with increased risk of recurrence. However, the genetic and non-genetic resistance mechanisms underlying RD remain incompletely characterized. Here we used single nuclei (sn) RNA and DNA sequencing to profile genetic and transcriptional changes in growing, residual, and recurrent tumors from an inducible mouse tumor model of HER2+ breast cancer. Methods: We used mammary tumors/tumor bed (n=2-3/time point) from female MMTV-rtTA; TetO-ERBB2 mice at 9 weeks DoxOn (HER2on), 7 days DoxOff (HER2off) and 12-21 weeks Doxoff (recurrent). Samples were snap frozen and single nuclei isolation was performed. snRNAseq was performed using 10 × 5’ v2 kits for library preparation and sequencing. snDNAseq was performed using a custom amplicon panel targeting the mouse genome and human ERBB2 locus via the MissionBio Tapestri platform. Results: For snRNAseq analysis, tumor cell clusters were identified by Epcam and ERBB2 gene expression. Tumor cells were further subclustered into 18 unique clusters that separated by ERBB2 expression. Seven subclusters of HER2on tumor cells were characterized by low ERRB2 expression: 1.38 vs 2.94 (median normalized log counts) in HER2on-ERBB2high cells. Top 5 differentially expressed genes (DEG) per condition (log2 fold change >1.5, adjusted p value < 0.05) were: HER2on: Erbb2, Csn3, Lcn2, Csn2, Wfdc18; HER2on-ERBB2high: Csn3, Igfbp5, Csn2, Lcn2, Trf; HER2on-ERBB2low: Lars2, Gm47283, Cmss1, Gm42418, Neat1; HER2off: Lars2, Hexb, Cmss1, Gphn, Gm42418; Recurrent: Capza2, Vim, Lgals1, Crabp1, S100a6. HER2on-ERBB2low cells were more closely related in transcriptional space to HER2off residual tumor cells than HER2on-ERBB2high tumor cells. Trajectory analysis suggested that HER2off RD evolved from HER2on-ERRB2low cells and formed the basis of recurrent disease. To determine clonal evolution over time, we used common top 10 DEGs between HER2on vs HER2off vs HER2low snRNAseq data and intersected that set with significantly enriched single nucleotide variants (SNVs) from our snDNAseq data. We identified 16 clones containing at least 1 SNV at >1% frequency across all samples. We observed clonal persistence, relative enrichment and extinguishment between HER2on, HER2off and recurrent samples but without clonal dominance. Conclusion: Time-series analysis of genetic and transcriptional evolution in a mouse model of HER2+ BC RD suggests that a tumor cell subpopulation characterized by an ERBB2low-associated transcriptional program drives RD after HER2 inhibition. Citation Format: Sheheryar K. Kabraji, Weihua Wang, Yali Zhang, Johann Bergholz, Tao Jiang, Vittal Kurisetty, Maia Homsi, Jianmin Wang, Jean Zhao. Residual disease after HER2 inhibition is driven by a primary tumor subpopulation expressing an ERBB2-low associated transcriptional program [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 6939.

Abstract 2743: Molecular characterization and prognostic evaluation of disseminated cancer cell subpopulations detected in bone marrow of non-small cell lung cancer patients

Abstract Introduction: Disseminated cancer cells (DCC) can be detected years before metastatic manifestation in bone marrow of lung cancer patients. Although a consensus protocol has been established, the impact of markers and methods for DCC detection are still not settled. We recently found that EpCAM+ DCC detected by immunofluorescence (IF) outperform the classical immunocytologic (IC) detection by cytokeratin (CK) antibodies regarding prognostic impact. However, it remained unclear whether this difference resulted from biological traits associated with EpCAM+ vs CK+ DCC or from methodological reasons. We therefore performed a side-by-side comparison of IC vs IF for CK and EpCAM. Methods: We prospectively collected bone marrow (BM) aspirates from 56 non-metastasized NSCLC patients that underwent tumor resection with curative intention. After density gradient centrifugation, we analyzed the mononucleated cells by (i) IC using the anti-CK antibody A45-B/B3 and (ii) by IF using the anti-CK antibody A45-B/B3 and the anti-EpCAM antibody HEA-125. IF signals were captured and objectively quantified using ImageJ, whereas IC was evaluated by two independent observers. Marker-positive cells were isolated and subjected to Whole Genome Amplification (WGA) and whole genome sequencing for assessment of copy number alterations (CNA). Survival analysis was performed for progression-free survival (PFS), tumor-specific survival (TSS) and overall-survival (OS) after a median follow-up time of 72.5, 78.7, 78.7 months, respectively. Results: IC and IF revealed the presence of CK+ DCCs in 37.5% and 41.1% of cases, respectively (P=0.34). IF identified EpCAM+ DCCs in 55.4% of patients, resulting in a 63.8% DCC-positivity rate found by IF, when combined with CK. The average number of DCCs per million MNC, the DCC density (DCCD), was 0.92, 0.93 and 1.44 for CKIC and CKIF and for EpCAMIF, respectively. No association was found between CKIC + DCCs and PFS, TSS or OS. In contrast, detection of EpCAMIF + and/or CKIF + DCCs showed a trend towards reduced PFS (P=0.055), TSS (P=0.225) and OS (P=0.155). Subgroup analysis revealed that both EpCAMIF + and CKIF + cells were correlated with worse PFS (P=0.016 and P=0.040, respectively) and TSS (P=0.033 and P=0.008, respectively). CKIF-positivity was also associated with worse OS (P=0.014), whereas EpCAM was not. CKIF + DCCs remained an independent prognostic variable for TSS and OS upon multivariate testing (hazard ratio: 6.45 and 3.31, respectively), while EpCAMIF + was not. The IF-marker-defined cell phenotypes exhibited comparable frequencies and distributions of CNAs across the entire genome (~50% of cells). Conclusions: Detection of CK+ DCC by quantified IF analysis apparently outperforms CK detection by IC in survival analysis. The impact of CK and EpCAM expression levels on outcome are under investigation. Citation Format: Daniel Spitzl, Tobias Mederer, Julius Zank, Severin Gütter, Michael Ried, Hans-Stefan Hofmann, Christoph A. Klein. Molecular characterization and prognostic evaluation of disseminated cancer cell subpopulations detected in bone marrow of non-small cell lung cancer patients [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 2743.

Abstract 4309: Establishment of intratumor heterogeneity using patient derived lung cancer cells

Abstract Overcoming cancer treatment resistance is a problem that must be solved to improve the prognosis of cancer patients. In recent years, genome analysis has revealed the existence of multiple subgroups with different properties within a single tumor, and intratumor heterogeneity is a cause of treatment resistance. It has also been reported that. However, the mechanism of intratumor heterogeneity remains unclear. Previous research on intratumor heterogeneity has mainly focused on analyzing patient cancer cells, classifying subgroups, and estimating characteristic factors. This method has two drawbacks. One is limitations in including all subgroups as research subjects, and other is lack of verification of whether characteristic factors are actually involved in intratumor heterogeneity in patients. Therefore, it is necessary to use a model of "early intratumor heterogeneity" in which monoclonal cells are divided into subgroups with different properties, and to verify the discovered factors using patient-derived cancer cell lines. In alectinib-resistant cell line (H2228-AR1S) created from monoclonal ALK-positive lung cancer cell line (H2228) by exposing to alectinib in vitro, existence of two fractions with different appearances. Using flow cytometry, it was revealed that H2228-AR1S bimodally expresses EpCAM, and the EpCAM-low fraction has high migration ability. In EpCAM-low fraction, epithelial markers were decreased, and mesenchymal markers were increased using immunoblotting. It suggests that EpCAM low subpopulation has undergone epithelial-mesenchymal transition (EMT). Additionally, we established a BRAF-positive lung cancer cell line (KTOR83) collected with consent from patients attending Kyoto University Hospital. Cells after in vitro dabrafenib exposure to KTOR83 showed bimodal EpCAM expression, and the EpCAM-low fraction has high migration ability. We established an intratumor heterogeneous model using commercially available cells and a patient-derived lung cancer cell line. Furthermore, we will explore factors common to the two models and examine factors involved in intratumor heterogeneity. Citation Format: Tomoko Funazo, Hiroaki Ozasa, Takahiro Tsuji, Yusuke Shima, Keiichiro Suminaga, Kentaro Hashimoto, Hiroshi Yoshida, Tatsuya Ogimoto, Kazutaka Hosoya, Hitomi Ajimizu, Takashi Nomizo, Hironori Yoshida, Toyohiro Hirai. Establishment of intratumor heterogeneity using patient derived lung cancer cells [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 4309.

Abstract 3689: Optimizing CTC isolation techniques for molecular characterization of circulating tumor cells in clear cell renal cell carcinoma: A comparative study of EpCAM-based and density-based methods

Abstract Introduction: Renal cell carcinoma (RCC) belongs to the most invasive cancers with the high molecular intrapatient heterogeneity. Circulating tumor cells (CTCs) are cells that detach from the tumor, reflecting the activity of the metastatic procedure. The most popular CTCs isolation methods are based on EpCAM signal detection on epithelial tumor cells. The clear cell RCC (ccRCC) originate from epithelial entity, since that is the EpCAM-based method suitable to isolate CTCs in this cancer. However, the prone of CTCs to EMT is known, which may in consequence downregulate the EpCAM expression and change the cell profile (hybrid phenotype). This is one of the points challenging the CTCs isolation by RCC disease. In our study, we compare the effectivity of antibody-based and density-based isolations technics in ccRCC patients to detect potential markers in CTCs. Purpose: Our aim was to find a precise and costs effective method to isolate and characterize CTCs from ccRCC patients. Materials and Methods: Prospective blood samples (3mL) from pulmonary and/or osseous metastatic ccRCC patients were collected during the immunotherapy and from the healthy donors. CTCs were isolated using EpCAM beads (RCC n=13; healthy n=15) or density-based (RCC n=13; healthy n=15) method. Expression of potential RCC biomarkers (mucin-1 (Muc1), androgen receptor (AR)) was identified using qPCR (Real-Time PCR) followed by dPCR (digital PCR), the results status received from both methods was evaluated. Summary: Based on our prior results, we first isolated CTCs based on EpCAM beads. Using qPCR we analyzed expression of genes, which we previously associated to the RCC (Muc1, PD-L1, AR). The results revealed differences in Muc1 (40% of tested patients positive but none of the healthy donors), when PD-L1 did not differed and AR was not detected. Applying more precise dPCR did neither show any expression. We continued our investigations with isolation of CTCs using density-based method. We confirmed more frequent expression of Muc1 by RCC Patients than by donors, as in our EpCAM cohort. PD-L1 was similar in both groups. Results of AR obtained using qPCR were confusing, showing deviations within same patients. The use of dPCR displayed significant differences in expression between RCC patients (77%; median [range] 0.28 [0.00 - 0.62] and donors (60%; 0.04 [0.00 - 0.51] confirming dPCR as optimal tool for molecular CTC characterization. Conclusions: Based on our results we demonstrate higher expression of Muc1 and AR in CTCs of metastatic ccRCC patients. The isolation technic should be adapted to the disease status and treatment choice. Further investigations confirming Muc1 and AR as ccRCC biomarkers are required. Citation Format: Joanna Bialek, Anne Muthe, Stefan Yankulov, Felix Kawan, Georgios Gakis, Gerit Theil. Optimizing CTC isolation techniques for molecular characterization of circulating tumor cells in clear cell renal cell carcinoma: A comparative study of EpCAM-based and density-based methods [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 3689.

Biallelic EPCAM deletions induce tissue-specific DNA repair deficiency and cancer predisposition

We report a case of Mismatch Repair Deficiency (MMRD) caused by germline homozygous EPCAM deletion leading to tissue-specific loss of MSH2. Through the use of patient-derived cells and organoid technologies, we performed stepwise in vitro differentiation of colonic and brain organoids from reprogrammed EPCAMdel iPSC derived from patient fibroblasts. Differentiation of iPSC to epithelial-colonic organoids exhibited continuous increased EPCAM expression and hypermethylation of the MSH2 promoter. This was associated with loss of MSH2 expression, increased mutational burden, MMRD signatures and MS-indel accumulation, the hallmarks of MMRD. In contrast, maturation into brain organoids and examination of blood and fibroblasts failed to show similar processes, preserving MMR proficiency. The combined use of iPSC, organoid technologies and functional genomics analyses highlights the potential of cutting-edge cellular and molecular analysis techniques to define processes controlling tumorigenesis and uncovers a new paradigm of tissue-specific MMRD, which affects the clinical management of these patients.

Role of sodium/iodide symporter overexpression in inhibiting thyroid cancer cell invasion and stem cell maintenance by inhibiting the β-catenin/LEF-1 pathway

BackgroundIn thyroid cancers, a reduction in the expression of the sodium/iodide symporter (NIS) is observed concomitant with a diminution in cancer cell differentiation. The β-catenin/LEF-1 pathway emerges as a crucial regulatory pathway influencing the functional expression of NIS in human thyroid cancer cells. Further research is required to comprehensively elucidate the role of NIS overexpression in impeding the progression of thyroid cancer cells. MethodsHuman papillary thyroid carcinoma (PTC) cell lines, specifically PTC-1 and KTC-1, were subjected to Scratch and Transwell assays, colony formation, and tumor sphere formation tests to investigate invasion and migration, focusing on the impact of NIS overexpression. The assessment involved the use of western blot to analyze the expression levels of β-catenin, NIS, CD133, SRY-related HMG box2 (Sox2), lymphoid enhancer-binding factor 1 (LEF-1), NANOG, octamer-binding transcription factor 4 (Oct4), aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), and epithelial cellular adhesion molecule (EpCAM). Statistical analysis was conducted using SPSS version 20.0, and the graphs were developed using GraphPad Prism 7 (GraphPad Software, Inc.). ResultsOur observations revealed that Nthy-ori-3-1 cell lines exhibited notably higher average expression levels of NIS, yet significantly lower levels of LEF-1 and β-catenin compared to PTC-1 and KTC-1 cell lines. Furthermore, the overexpression of β-catenin resulted in reduced binding of LEF-1 to NIF promotion but concurrently increased the expression of NIS. The downregulation of NIS markedly enhanced the expression of ALDH1A1, CD133, OCT4, Nanog, SOX2, and EpCam—all of which are targets within the Wnt/β-catenin signaling pathway. Conversely, the upregulation of NIS suppressed the expression of these proteins. Moreover, cells treated with β-catenin activators demonstrated an increased capability to form more spheroids and displayed heightened aggressiveness. Conversely, the NIS overexpression (OE) group exhibited suppressed abilities in invasion and colony formation. ConclusionThyroid cancer cells exhibit diminished expression of NIS, and the invasion and maintenance of stem cells in thyroid cancer cells were hindered by NIS OE through the inhibition of the β-catenin/LEF-1 pathway. Further research is warranted to comprehensively assess this outcome, which holds promise as a potential targeted treatment for thyroid cancer.