Malignant Epithelial Cells Research Articles

Tumor Cell-Derived Complement Component C1r Acts as a Prognostic Biomarker and Promotes Esophageal Squamous Cell Carcinoma Progression.

Mounting evidence indicates that complement components play a crucial role in cancer progression. Recent findings indicate that certain complement components display a significant rise in expression within esophageal squamous cell carcinoma (ESCC). However, the specific tumorigenic functions of these components remain unclear. This study focuses on investigating the expression pattern of C1r, elucidating a role for C1r in ESCC, as well as exploring underlying mechanisms controlled by C1r. The expression of C1r in ESCC tissues, malignant epithelial cells, and its relationship with survival were analyzed using the Gene Expression Omnibus (GEO) database and tissue microarrays. Single-cell RNA sequencing (scRNA-seq) was used to study the expression of C1r in malignant epithelial cells. C1r knockdown or C1r overexpression in cultured ESCC cells were used to assess the effects of C1r on proliferation, migration, invasion, cell-matrix adhesion, apoptosis, and growth of xenografted tumors in immunocompromised (nude) mice. Western blotting was used to detect the expression of MMP-1 and MMP-10 in C1r knockdown or C1r overexpressing ESCC cells. C1r was highly expressed in ESCC tissues, malignant epithelial cells, and cultured ESCC cell lines. High C1r expression indicated a poor prognosis. Knockdown of C1r significantly suppressed the proliferation, migration, invasion, cell-matrix adhesion, and promoted apoptosis in cultured ESCC cells. Additionally, knockdown of C1r markedly inhibited tumor growth in nude mice. Overexpression of C1r had the opposite effects. C1r induced the expression of MMP-1 and MMP-10. C1r is highly expressed in ESCC and promotes the progression of this tumor type. Our findings suggest that C1r may serve as a novel prognostic biomarker and therapeutic target in ESCC.

Integrated analysis of scRNA-seq and bulk RNA-seq identifies FBXO2 as a candidate biomarker associated with chemoresistance in HGSOC

BackgroundHigh-grade serous ovarian carcinoma (HGSOC) is the most prevalent and aggressive histological subtype of epithelial ovarian cancer. Around 80% of individuals will experience a recurrence within five years because of resistance to chemotherapy, despite initially responding well to platinum-based treatment. Biomarkers associated with chemoresistance are desperately needed in clinical practice. MethodsWe jointly analyzed the transcriptomic profiles of single-cell and bulk datasets of HGSOC to identify cell types associated with chemoresistance. Copy number variation (CNV) inference was performed to identify malignant cells. We subsequently analyzed the expression of candidate biomarkers and their relationship with patients' prognosis. The enrichment analysis and potential biological function of candidate biomarkers were explored. Then, we validated the candidate biomarker using in vitro experiments. ResultsWe identified 8871 malignant epithelial cells in a single-cell RNA sequencing dataset, of which 861 cells were associated with chemoresistance. Among these malignant epithelial cells, FBXO2 (F-box protein 2) is highly expressed in cells related to chemoresistance. Moreover, FBXO2 expression was found to be higher in epithelial cells from chemoresistance samples compared to those from chemosensitivity samples in a separate single-cell RNA sequencing dataset. Patients exhibiting elevated levels of FBXO2 experienced poorer outcomes in terms of both overall survival (OS) and progression-free survival (PFS). FBXO2 could impact chemoresistance by influencing the PI3K-Akt signaling pathway, focal adhesion, and ECM-receptor interactions and regulating tumorigenesis. The 50% maximum inhibitory concentration (IC50) of cisplatin decreased in A2780 and SKOV3 ovarian carcinoma cell lines with silenced FBXO2 during an in vitro experiment. ConclusionsWe determined that FBXO2 is a potential biomarker linked to chemoresistance in HGSOC by combining single-cell RNA-seq and bulk RNA-seq dataset. Our results suggest that FBXO2 could serve as a valuable prognostic marker and potential target for drug development in HGSOC.

Ferroptosis biomarkers predict tumor mutation burden's impact on prognosis in HER2-positive breast cancer.

Ferroptosis has recently been associated with multiple degenerative diseases. Ferroptosis induction in cancer cells is a feasible method for treating neoplastic diseases. However, the association of iron proliferation-related genes with prognosis in HER2+ breast cancer (BC) patients is unclear. To identify and evaluate fresh ferroptosis-related biomarkers for HER2+ BC. First, we obtained the mRNA expression profiles and clinical information of HER2+ BC patients from the TCGA and METABRIC public databases. A four-gene prediction model comprising PROM2, SLC7A11, FANCD2, and FH was subsequently developed in the TCGA cohort and confirmed in the METABRIC cohort. Patients were stratified into high-risk and low-risk groups based on their median risk score, an independent predictor of overall survival (OS). Based on these findings, immune infiltration, mutations, and medication sensitivity were analyzed in various risk groupings. Additionally, we assessed patient prognosis by combining the tumor mutation burden (TMB) with risk score. Finally, we evaluated the expression of critical genes by analyzing single-cell RNA sequencing (scRNA-seq) data from malignant vs normal epithelial cells. We found that the higher the risk score was, the worse the prognosis was (P < 0.05). We also found that the immune cell infiltration, mutation, and drug sensitivity were different between the different risk groups. The high-risk subgroup was associated with lower immune scores and high TMB. Moreover, we found that the combination of the TMB and risk score could stratify patients into three groups with distinct prognoses. HRisk-HTMB patients had the worst prognosis, whereas LRisk-LTMB patients had the best prognosis (P < 0.0001). Analysis of the scRNA-seq data showed that PROM2, SLC7A11, and FANCD2 were significantly differentially expressed, whereas FH was not, suggesting that these genes are expressed mainly in cancer epithelial cells (P < 0.01). Our model helps guide the prognosis of HER2+ breast cancer patients, and its combination with the TMB can aid in more accurate assessment of patient prognosis and provide new ideas for further diagnosis and treatment.

Abstract 1578: RAS-GTP inhibition modulates Hedgehog signaling, suppressing myCAFs and promoting iCAFs in pancreatic ductal adenocarcinoma

Abstract More than 90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS. RMC-7977 is a potent inhibitor of GTP-bound RAS proteins (RAS(ON), including both wild type and mutant variants of KRAS, NRAS, and HRAS. The related investigational agent, RMC-6236, is a first-in-class, potent, orally bioavailable, RASMULTI(ON) inhibitor currently in Phase 1/1b clinical trials (NCT05379985). We performed preclinical studies in a range of models of PDAC, including the highly chemoresistant K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) genetically engineered mouse. RMC-7977 exhibited broad anti-tumor activity at tolerable doses, provoking radiographic responses in KPC pancreatic tumors and extending overall survival by 3-fold, the largest response observed yet in this model. RMC-7977 dosing produced a metronomic effect on RAS signaling in tumor and normal tissues. This yielded tumor-selective effects on apoptosis and proliferation, consistent with RAS oncogene addition in PDAC. PDAC tumors are characterized by an expansive stroma that harbors multiple subtypes of cancer associated fibroblasts (CAFs) that manufacture a fibrotic, inflammatory extracellular matrix. CAF proliferation is driven by signals emanating from the malignant epithelial cells, such as Sonic Hedgehog (SHH), a secreted ligand that drives downstream Hedgehog pathway signaling in myofibroblastic CAFs (myCAFs). To understand the impact of RAS-GTP inhibition on paracrine signaling to CAFs in PDAC, To understand the dynamic responses of malignant epithelial cells following GTP-RAS inhibition, we performed single cell RNA sequencing (scRNAseq) on KPC pancreatic tumors treated with RMC-7977 or vehicle at multiple timepoints. Using the ARACNe and VIPER algorithms, we performed single cell regulatory network analysis on the expression profiles of &amp;gt;210,000 cells to calculate the activities of thousands of transcriptional regulatory protein in each cell. We found that the RAS-GTP inhibition blocked SHH expression in malignant epithelial cells, resulting in loss of downstream Hh pathway activity in CAFs within 24 hours of treatment. By one week of treatment, myCAFs were significantly depleted whereas inflammatory CAFs (iCAFs) were significantly accumulated. Strikingly, CAF heterogeneity was restored in KPC pancreatic tumors that acquired resistance to RMC-7977, despite continued suppression of Hedgehog pathway signaling. Consistent with prior studies of Smoothened inhibitors, the depletion of myCAFs from RAS-GTP inhibition was associated with the rapid opening and hypersprouting of intra-tumoral blood vessels. Ongoing studies using the mutant-selective KRASG12D inhibitor RM-044 should discriminate between direct effects of wild type RAS inhibition in CAFs versus the paracrine consequences of mutant RAS inhibition in malignant epithelial cells. Citation Format: Marie Hasselluhn, Lorenzo Tomassoni, Urszula Wasko, Alvaro Curiel-Garcia, Tanner Dalton, Steve A. Sastra, Carmine Palermo, Andrea Califano, Kenneth P. Olive. RAS-GTP inhibition modulates Hedgehog signaling, suppressing myCAFs and promoting iCAFs in pancreatic ductal adenocarcinoma [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 1578.

Abstract 1697: Phase separation of BHLHE40 up-regulates SREBF1 and inhibits ferroptosis in pancreatic cancer cells

Abstract Pancreatic cancer (PCa) is one of the most lethal malignancies in human cancers. The enhanced infiltration of stromal tissue of PCa tumor microenvironment limits the identification of key tumor-specific transcription factors and epigenomic abnormalities in malignant epithelial cells. Integrated transcriptome and epigenetic multi-omics analysis of paired PCa organoids indicated that the basic helix-loop-helix transcription factor 40 (BHLHE40) was significantly upregulated in tumor samples. Increased chromatin accessibility at promoter region and enhanced mTOR pathway activity contribute to the elevated expression of BHLHE40. Integrated analysis of chromatin immunoprecipitation (ChIP)-seq, RNA-seq and high-through chromosome conformation capture (Hi-C) data together with chromosome conformation capture (3C) assay indicate that BHLHE40 not only regulates the sterol regulatory element-binding factor 1 (SREBF1) transcription as a classic transcription factor, but also links the enhancer and promoter regions of SREBF1 via forming liquid-liquid phase-separated droplets. Then, we find the BHLHE40-SREBF1-stearoyl-CoA desaturase (SCD1) axis protects PCa cells from ferroptosis, leading to the reduced accumulation of lipid peroxidation in cells. Moreover, SREBF1 inhibitor, Fatostatin, significantly suppresses the cell growth of PCa tumors with higher expression of BHLHE40. This study highlights the important roles of BHLHE40 mediated lipid peroxidation in inducing ferroptosis of PCa cells and provides a novel mechanism underlying the SREBF1 overexpression in PCa. Citation Format: Yizhi Cao, Xuelong Wang, Yang Liu, Pengyi Liu, Jiejie Qin, Youwei Zhu, Shuyu Zhai, Minmin Shi, Weishen Wang, Xiaxing Deng, Chenghong Peng, Hao Chen, Ruibao Ren, Lingxi Jiang, Baiyong Shen. Phase separation of BHLHE40 up-regulates SREBF1 and inhibits ferroptosis in pancreatic 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 1697.

Abstract 3718: Deciphering the role of TREM2+ tumor-associated macrophages as mediators of immunoresistance in micro-satellite stable colorectal metastases

Abstract Introduction: Most Colorectal Cancer (CRC)-related deaths are caused by metastatic disease, and more than 95% of CRC metastases (mCRC) are microsatellite stable (MSS) which respond poorly to Immune checkpoint inhibitors (ICI). Tumor-associated macrophages (TAMs) have been recognized as major drivers of immunoresistance in primary CRC; however, their role in mCRC remains poorly understood. We analyzed primary CRC and major metastatic sites (Liver, Peritoneum, Lungs) using single-cell RNA-sequencing (scRNAseq) to elucidate the TAM driven mechanisms of immunoresistance in the mCRC tumor microenvironment (TME) and used a novel TAM directed targeting strategy to improve responses to ICI in a preclinical orthotopic model of MSS-mCRC. Methods: Using scRNA-seq, we analyzed 22 samples from 14 patients with mCRC. Target antigens identified were validated using flow cytometry and immunofluorescence (IF). The antitumor activity of anti-TREM2 and anti-PD1 mABs was evaluated in-vivo using CT26 CRC cell line in BALB-C mice, assessing tumor immune response via flow cytometry. Results: We characterized the TME in MSS-mCRC and identified 17 clusters of malignant epithelial, immune, and stromal cells. Our sub-clustering analysis revealed 6 unique sub-populations of TAMs which were differentially distributed across liver, lung, and peritoneal metastases (PM).Two subsets of TAMs, 5 and 6, were enriched in PM and malignant ascites. TAMs 5/6 expressed canonical lipid-associated genes; TREM2, APOE, and PLTP. TREM2+ TAMs were also enriched in immunosuppressive and pro-angiogenic pathways, and expressed genes known to drive EMT and ECM remodeling in the TME. Thus, TREM2 was identified as a potential therapeutic target. We validated our transcriptomic findings using flow cytometry and IF analysis of MSS-mCRC showing an abundance of TREM2+ TAMs in PM. TREM2+ TAMs co-expressed CD163, known marker for M2-TAMs and stained positive for BODIPY. In an aggressive PM mouse model, treatment with anti-PD1, anti-TREM2, or combination anti-PD1/anti-TREM2 was insufficient to reduce tumor burden. However, a trend towards a reduction in total intratumoral F4/80+CD11b+ TAMs was observed in the combination treatment compared to other groups. All three treatments trended towards increased CD8+ intratumoral T-cell infiltration compared to control mice. Conclusion: Our study provides new insights into the TME of MSS-CRC metastases at a single-cell level. Our findings indicate that TREM2+ TAMs contribute to the immunosuppressive milieu of MSS-CRC metastases. Targeting TREM2 in combination with anti-PD1 resulted in a modulation of TME via reduction of TAMs paralleled by an increased infiltration of CD8+ T cells. This reprogramming of TME suggests that inhibiting TREM2 might augment response to ICI in MSS-CRC metastases, traditionally resistant to ICI. Citation Format: Muhammad Bilal Mirza, Matthew Alexander Cottam, Mustafa Raoof, Kamran Idrees. Deciphering the role of TREM2+ tumor-associated macrophages as mediators of immunoresistance in micro-satellite stable colorectal metastases [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 3718.

Abstract 4219: Establishing CAF-like and TAM-like transformation induced by TNBC culture supernatant in 3D in-vitro culture as a model for targeted drug testing

Abstract Several previous studies have highlighted the significance of cell-to-cell communication, or crosstalk, between tumor cells and non-tumorigenic cells in the context of cancer progression and metastasis. Within the tumor microenvironment (TME), these interactions have the potential to alter the phenotypes and behaviors of normal cells. The use of 2D in-vitro cultures has been limited due to their inability to accurately replicate the intricate in-vivo TME. Conditioned medium (CM) derived from cultured cancer cells contains secreted factors that may influence the phenotype and functionality of normal cells. In our investigation, the exposure of normal murine fibroblast NIH3T3 and macrophage RAW 264.7 cells to conditioned medium (CM) obtained from malignant mammary epithelial 4T1 cells (4T1CM) resulted in a modified phenotype with enhanced cell viability. Treatment with 4T1CM led to the upregulation of genes, including α-smooth muscle actin (αSMA), IL-10, CD206, and vascular endothelial growth factor (VEGF), in NIH3T3 and RAW 264.7 cells compared to their respective control cells. Additionally, NIH3T3 cells treated with 4T1CM exhibited an epithelial-mesenchymal transition (EMT) phenotype, as indicated by the regulation of EMT markers such as E-cadherin, β-catenin, N-cadherin, and Vimentin. Notably, RAW 264.7 cells treated with 4T1CM showed an upregulation of cyclooxygenase-2 (COX-2) and programmed death-ligand 1 (PDL-1), suggesting a propensity for an inhibitory immune response. Moreover, NIH3T3 cells conditioned with 4T1CM demonstrated an upregulation of stemness markers, including sex determining region Y-box 2 and Aldehyde dehydrogenase. In summary, our study highlights the potential role of 4T1CM in transforming normal NIH3T3 and RAW 264.7 cells into cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Ongoing proteomics analysis aims to map differentially expressed proteins potentially regulating the 4T1CM-induced transformation. To further investigate, a series of experiments using mono- and co-cultures of these cells are underway, specifically targeting the transformed macrophages and fibroblasts with the aim of either re-educating or eliminating them. This treatment involves drug combinations that specifically target the identified signaling pathway intermediates, offering novel therapeutic intervention strategies. Citation Format: Biplov Sapkota, Naveen Chintalaramulu, Abhishek Pandit, Shilpa Thota, Rizwana Begum, Amirsalar Mansouri, Jiri Adamec, Joseph Francis. Establishing CAF-like and TAM-like transformation induced by TNBC culture supernatant in 3D in-vitro culture as a model for targeted drug testing [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 4219.

Abstract 1592: Single-cell transcriptomics reveals unique stromal and metabolic heterogeneity in liver metastatic pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is the 3rd leading cause of cancer related death in the US. This mortality is strongly linked to the complex PDA tumor microenvironment that drives resistance to chemotherapy and immune suppression. Most patients succumb to metastatic disease, with liver being the most frequently colonized organ. Despite this, most pre-clinical studies have focused on primary PDA models to study the disease. Accordingly, the extent that the liver metastatic niche promotes immune evasion and resistance to treatment remains unclear.To directly compare primary vs. metastatic tumors in PDA tumors, we have optimized a syngeneic experimental model to contrast primary vs. metastatic lesions. To define the similarities and differences between cell types present in these tumors, we employed a single-cell transcriptomics approach. Our analysis has identified shared populations of malignant epithelial, stromal, and immune cells between lesions, allowing for a direct comparison of programming. Here, we have observed that the dominant populations of PDA cells in pancreas vs. liver tumors engage in distinct metabolic programs that suggest different actionable vulnerabilities.Beyond these, we also observed fibroblasts and myeloid sub-populations that are exclusive to liver tumors. These data further support our hypothesis that alternative mechanisms of metabolic, immune, and stromal interactions take place in the metastatic tumor microenvironment. To define these crosstalk networks, we employed the interactome tool CellChat, which has uncovered unique signaling interactions between the CAF and cancer cell populations in pancreatic or liver tumors. Overall, these data have the potential to lead to new avenues to directly target cancer cells or engage an effective immune response that can be designed to treat metastatic pancreatic cancer- an urgent clinical need. Citation Format: Rima Singh, Christopher Halbrook. Single-cell transcriptomics reveals unique stromal and metabolic heterogeneity in liver metastatic pancreatic 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 1592.

Single cell deciphering of progression trajectories of the tumor ecosystem in head and neck cancer

Head and neck squamous cell carcinoma is the sixth most common cancer worldwide and has high heterogeneity and unsatisfactory outcomes. To better characterize the tumor progression trajectory, we perform single-cell RNA sequencing of normal tissue, precancerous tissue, early-stage, advanced-stage cancer tissue, lymph node, and recurrent tumors tissue samples. We identify the transcriptional development trajectory of malignant epithelial cells and a tumorigenic epithelial subcluster regulated by TFDP1. Furthermore, we find that the infiltration of POSTN+ fibroblasts and SPP1+ macrophages gradually increases with tumor progression; their interaction or interaction with malignant cells also gradually increase to shape the desmoplastic microenvironment and reprogram malignant cells to promote tumor progression. Additionally, we demonstrate that during lymph node metastasis, exhausted CD8+ T cells with high CXCL13 expression strongly interact with tumor cells to acquire more aggressive phenotypes of extranodal expansion. Finally, we delineate the distinct features of malignant epithelial cells in primary and recurrent tumors, providing a theoretical foundation for the precise selection of targeted therapy for tumors at different stages. In summary, the current study offers a comprehensive landscape and deep insight into epithelial and microenvironmental reprogramming throughout initiation, progression, lymph node metastasis and recurrence of head and neck squamous cell carcinoma.

Abstract 4335: High-resolution characterization of age-specific cell type composition changes and signaling events in HPV-negative HNSCC tumors

Abstract This study is aimed at testing the hypothesis that increased patient age fosters conditions that are favorable for head and neck squamous cell carcinomas (HNSCC) of the oral cavity development. There is a well-documented increase in cancer incidence with age observed across diverse cancer types, where elderly patients exhibit diminished survival outcomes compared to their younger counterparts. While the convergence of aging and cancer hallmarks offers valuable insights, further work is needed to elucidate the age-specific mechanisms influencing HNSCC, beyond the shared characteristics of these biological processes. To this end, we have assembled a high-quality human single-cell RNA-sequencing HNSCC atlas profiling more than 230,000 cells across more than 50 patients with ages ranging between 18 and 89, which provides a resource to investigate age-associated changes in the disease heterogeneity. For this library, we integrated six publicly available single-cell RNAseq datasets from 54 HPV-negative patients to create the atlas. Cells were clustered, classified, characterized by gene set enrichment analysis, both in the epithelial cell compartment and in the tumor microenvironment (TME). Differential cell type proportion analysis was performed to identify cell types enriched in young (&amp;lt;49 years) or old (&amp;gt;70 years) patients. Cell-cell communication analysis was performed to identify signaling events occurring between different populations, and how these and specific ligand-receptor pairs differed in patients of different ages. CNV analyses were performed to identify cancer subclones and assess level of variation across tumors. Interestingly, we identified distinct cell populations and signaling events that associate with age, and we were able to compare, validate and recapitulate these observations in the 4MOSC1 OSCC isograft tumor model in old and young mice. Additional notable findings include a differential enrichment for cytotoxic CD8 T cells over dysfunctional CD8 T cells in young patients when compared to old, suggesting younger patients have a more effective immune TME. We also found an enrichment for cancer-associated fibroblast populations in old patients (myCAFs) that send collagen signals to malignant epithelial cells, a result we have recapitulated using the 4MOSC1 model, and hypothesize may play a role in ECM stiffening, EMT, and metastasis. Further analyses are ongoing, and we plan to validate the hypotheses generated, specifically the presence of differential abundance of specific cell populations, and age-specific ligand-receptor signaling events that lead to tumor growth. Citation Format: Lina Kroehling, Anthony Spinella, Maria Kukuruzinska, Xaralabos Varelas, Stefano Monti. High-resolution characterization of age-specific cell type composition changes and signaling events in HPV-negative HNSCC tumors [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 4335.

The immunomodulatory impact of naturally derived neem leaf glycoprotein on the initiation progression model of 4NQO induced murine oral carcinogenesis: a preclinical study.

Murine tumor growth restriction by neem leaf glycoprotein (NLGP) was established in various transplanted models of murine sarcoma, melanoma and carcinoma. However, the role of NLGP in the sequential carcinogenic steps has not been explored. Thus, tongue carcinogenesis in Swiss mice was induced by 4-nitroquinoline-1-oxide (4NQO), which has close resemblance to human carcinogenesis process. Interventional role of NLGP in initiation-promotion protocol established during 4NQO mediated tongue carcinogenesis in relation to systemic immune alteration and epithelial-mesenchymal transition (EMT) is investigated. 4NQO was painted on tongue of Swiss mice every third day at a dose of 25µl of 5mg/ml stock solution. After five consecutive treatment with 4NQO (starting Day7), one group of mice was treated with NLGP (s.c., 25µg/mice/week), keeping a group as PBS control. Mice were sacrificed in different time-intervals to harvest tongues and studied using histology, immunohistochemistry, flow-cytometry and RT-PCR on different immune cells and EMT markers (e-cadherin, vimentin) to elucidate their phenotypic and secretory status. Local administration of 4NQO for consecutive 300 days promotes significant alteration in tongue mucosa including erosion in papillae and migration of malignant epithelial cells to the underlying connective tissue stroma with the formation of cell nests (exophytic-hyperkeratosis with mild dysplasia). Therapeutic NLGP treatment delayed pre-neoplastic changes promoting normalization of mucosa by maintaining normal structure. Flow-cytometric evidences suggest that NLGP treatment upregulated CD8+, IFNγ+, granzyme B+, CD11c+ cells in comparison to 4NQO treated mice with a decrease in Ki67+ and CD4+FoxP3+ cells in NLGP treated cohort. RT-PCR demonstrated a marked reduction of MMP9, IL-6, IL-2, CD31 and an upregulation in CCR5 in tongues from 4NQO+NLGP treated mice in comparison to 4NQO treated group. Moreover, 4NQO mediated changes were associated with reduction of e-cadherin and simultaneous up-regulation of vimentin expression in epithelium that was partially reversed by NLGP. Efficacy of NLGP was tested first time in sequential carcinogenesis model and proved effective in delaying the initial progression. NLGP normalizes type 1 immunity including activation of the CD8+T effector functions, reduction of regulatory T cell functions, along with changes in EMT to make the host systemically alert to combat the carcinogenic threat.

Abstract 440: BAP1 deubiquitinates ACLY to promote lipogenesis yet predisposing cholangiocarcinoma to iron overload and ferroptosis

Abstract Background: Cholangiocarcinoma (CCA) is a highly lethal and aggressive epithelial cell malignancy of the liver and biliary tract. CCA survival remains poor and response to chemotherapy or immunotherapy is limited. We AIM to identify metabolic vulnerabilities that render CCA susceptible to ferroptosis, an iron-dependent and caspase-independent cell death driven by lipid peroxidation. Materials and Methods: We treated human and mouse CCA cell lines with ferroptosis inducers (RSL3, Erastin), inhibitors (Fer-1, Lip-1), necroptosis inhibitor and a pan-caspase inhibitor. Cell viability, intracellular and mitochondrial iron, lipid peroxidation, mitochondrial structure, and function by cell-based and biochemical assays. Gain- and loss-of-function strategies, TURBO proximity-dependent biotin identification, and metabolic techniques were used for mechanistic studies. In vivo, orthotopically implanted C57B/6 mice were treated with RSL3 or vehicle intraperitoneally, followed by assessment of tumor burden and immunophenotyping analysis. Epcam-aptamer coated nanoparticles (tMNPs) packaged with siRNA against GPX4 were used for selective ferroptosis induction. Results: We identified ferroptosis-sensitive and -resistant human and mouse CCA cells. Ferroptosis-sensitive cells, rescued by ferroptosis inhibitors but not by necroptosis or pan-caspase inhibitors, exhibited elevated intracellular free iron and mitochondrial iron overload. This led to significant mitochondrial dysfunction and structural changes associated with ferroptosis. Mitochondrial free iron triggered reactive oxygen species generation and lipid peroxidation. We identified BRCA-associated protein 1 (BAP1) as a potential driver of ferroptosis sensitivity. BAP1, mutated in 22-25% of human CCAs, is a tumor suppressor and deubiquitinase (DUB). BAP1 knockdown by siRNA protected cells from ferroptosis, while re-expression of BAP1 sensitized cells to ferroptosis. Subcellular fractionation identified BAP1 in extranuclear compartments, including mitochondria and cytosol. We found that BAP1 interacts and deubiquitinates ATP citrate lyase (ACLY), which converts iron chelator citrate to oxaloacetate and acetyl-CoA. Inhibition of mitochondrial citrate export, protected cells from iron overload and ferroptosis. In vivo, RSL3-induced ferroptosis significantly reduced tumor burden, as compared to vehicle-treated mice. RSL3-induced ferroptosis promoted an immunogenic tumor immune microenvironment. Lastly, tMNPs with siGPX4 induced marked ferroptosis of mouse CCA cells and showed maximal enrichment in liver orthotopic tumors in vivo. Conclusions: Our findings indicate that BAP1 deubiquitination of ACLY predisposes CCA cells to iron overload by exhausting citrate to support lipogenesis. CCA vulnerability to ferroptosis opens up novel therapeutic opportunities for CCA. Citation Format: Peyton Classon, Sophia Jaramillo, Danielle Carlson, Irene Yan, Tushar Patel, Sumera I. Ilyas, Rory L. Smoot, Gregory J. Gores, Davide Povero. BAP1 deubiquitinates ACLY to promote lipogenesis yet predisposing cholangiocarcinoma to iron overload and ferroptosis [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 440.

Sebaceous carcinoma in a 54-year-old Black African man after cancer chemotherapy: a case report

BackgroundSebaceous carcinoma is a very rare malignant skin adnexal tumor that is occasionally aggressive. We have not seen a case of sebaceous carcinoma in our center in the last 10 years. It is extremely rare in Black Africans.Case presentationWe described the case of a 55-year-old man African man who presented to our ophthalmologist with complaints of growth on the right upper eyelid for 8 months. He had surgery and chemotherapy for rectal carcinoma 6 years prior to presentation and received his last dose of chemotherapy 5 years before seeing our ophthalmologist. There was a history of spontaneous unprovoked bleeding from the lesion. He subsequently underwent surgical excision under general anesthesia. Histology of the mass showed an effaced architecture due to proliferating malignant epithelial cells disposed as trabecules, solid nests, and tongues. The microscopic features of widespread multivacuolated cytoplasm of the neoplastic cells led us to conclude that the tumor was a sebaceous carcinoma. The patient is alive and well.ConclusionSebaceous carcinoma is a rare malignant skin adnexal tumor in Black Africans. It can present as an eyelid mass with spontaneous bleeding. It can follow cancer chemotherapy either because of its association with other tumors in Muir–Torre syndrome or because of mutagenic effects of chemotherapeutic agents.

Tumor suppressive role of the epigenetic master regulator BRD3 in colorectal cancer.

Bromodomain and extraterminal domain (BET) family proteins are epigenetic master regulators of gene expression via recognition of acetylated histones and recruitment of transcription factors and co-activators to chromatin. Hence, BET family proteins have emerged as promising therapeutic targets in cancer. In this study, we examined the functional role of bromodomain containing 3 (BRD3), a BET family protein, in colorectal cancer (CRC). Invitro and vivo analyses using BRD3-knockdown or BRD3-overexpressing CRC cells showed that BRD3 suppressed tumor growth and cell cycle G1/S transition and induced p21 expression. Clinical analysis of CRC datasets from our hospital or The Cancer Genome Atlas revealed that BET family genes, including BRD3, were overexpressed in tumor tissues. In immunohistochemical analyses, BRD3 was observed mainly in the nucleus of CRC cells. According to single-cell RNA sequencing in untreated CRC tissues, BRD3 was highly expressed in malignant epithelial cells, and cell cycle checkpoint-related pathways were enriched in the epithelial cells with high BRD3 expression. Spatial transcriptomic and single-cell RNA sequencing analyses of CRC tissues showed that BRD3 expression was positively associated with high p21 expression. Furthermore, overexpression of BRD3 combined with knockdown of, a driver gene in the BRD family, showed strong inhibition of CRC cells invitro. In conclusion, we demonstrated a novel tumor suppressive role of BRD3 that inhibits tumor growth by cell cycle inhibition in part via induction of p21 expression. BRD3 activation might be a novel therapeutic approach for CRC.

Comparative analysis of single-cell transcriptome reveals heterogeneity and commonality in the immune microenvironment of colorectal cancer and inflammatory bowel disease.

During aging, chronic inflammation can promote tumor development and metastasis. Patients with chronic inflammatory bowel diseases (IBD) are at an increased risk of developing colorectal cancer (CRC). However, the molecular mechanism underlying is still unclear. We conducted a large-scale single-cell sequencing analysis comprising 432,314 single cells from 92 CRC and 24 IBD patients. The analysis focused on the heterogeneity and commonality of CRC and IBD with respect to immune cell landscape, cellular communication, aging and inflammatory response, and Meta programs. The CRC and IBD had significantly different propensities in terms of cell proportions, differential genes and their functions, and cellular communication. The progression of CRC was mainly associated with epithelial cells, fibroblasts, and monocyte-macrophages, which displayed pronounced metabolic functions. In particular, monocyte-macrophages were enriched for the aging and inflammation-associated NF-κB pathway. And IBD was enriched in immune-related functions with B cells and T cells. Cellular communication analysis in CRC samples displayed an increase in MIF signaling from epithelial cells to T cells, and an increase in the efferent signal of senescence-associated SPP1 signaling from monocyte-macrophages. Notably, we also found some commonalities between CRC and IBD. The efferent and afferent signals showed that the pro-inflammatory cytokine played an important role. And the activity of aging and inflammatory response with AUCell analysis also showed a high degree of commonality. Furthermore, using the Meta programs (MPs) with the NMF algorithm, we found that the CRC non-malignant cells shared a substantial proportion of the MP genes with CRC malignant cells (68% overlap) and IBD epithelial cells (52% overlap), respectively. And it was extensively involved in functions of cell cycle and immune response, revealing its dual properties of inflammation and cancer. In addition, CRC malignant and non-malignant cells were enriched for the senescence-related cell cycle G2M phase transition and the p53 signaling pathway. Our study highlights the characteristics of aging, inflammation and tumor in CRC and IBD at the single-cell level, and the dual property of inflammation-cancer in CRC non-malignant cells may provide a more up-to-date understanding of disease transformation.

Association of preoperative aspartate aminotransferase to platelet ratio index with outcomes and tumour microenvironment among colorectal cancer with liver metastases

This study aims to investigate applicable robust biomarkers that can improve prognostic predictions for colorectal liver metastasis (CRLM) patients receiving simultaneous resection. A total of 1323 CRLM patients from multiple centres were included. The preoperative aspartate aminotransferase to platelet ratio index (APRI) level from blood of patients were obtained. Patients were stratified into a high APRI group and a low APRI group, and comparisons were conducted by analyzing progression-free survival (PFS), overall survival (OS) and postoperative early recurrence. Tumour samples of CRLM were collected to perform single-cell RNA sequencing and multiplex immunohistochemistry/immunofluorescence (mIHC/IF) to investigate the association of APRI levels and the tumour microenvironment of CRLM. Compared with APRI <0.33, PFS disadvantage (IPTW-adjusted HR = 1.240, P = 0.015) and OS disadvantage (IPTW- adjusted HR = 1.507, P = 0.002) of APRI ≥0.33 were preserved in the IPTW-adjusted Cox hazards regression analyses. An APRI ≥0.25 was associated with a significantly increased risk of postoperative early recurrence after adjustment (IPTW-adjusted OR = 1.486, P = 0.001). The external validation showed consistent results with the training cohort. In the high APRI group, the single-cell RNA sequencing results revealed a heightened malignancy of epithelial cells, the enrichment of inflammatory-like cancer-associated fibroblasts and SPP1+ macrophages associated with activation of malignant cells and fibrotic microenvironment, and a more suppressed-function T cells; mIHC/IF showed that PD1+ CD4+ T cells, FOXP3+ CD4+ T cells, PD1+ CD8+ T cells, FOXP3+ CD8+ T cells, SPP1+ macrophages and iCAFs were significantly increased in the intratumoral region and peritumoral region. This study contributed valuable evidence regarding preoperative APRI for predicting prognoses among CRLM patients receiving simultaneous resection and provided underlying clues supporting the association between APRI and clinical outcomes by single-cell sequencing bioinformatics analysis and mIHC/IF.

Low expression of lysosome-related genes KCNE1, NPC2, and SFTPD promote cancer cell proliferation and tumor associated M2 macrophage polarization in lung adenocarcinoma

BackgroundRecent research has shown that lysosomes play a critical role in the onset and progression of malignancy by regulating tumor cell death through several mechanisms. Nevertheless, the involvement of lysosome-associated genes (LSAGs) in lung adenocarcinoma (LUAD) is still not well understood. MethodsLSAGs were identified in malignant lung epithelial cells, as well as biologically and functionally annotated by the comprehensive integration of single-cell and bulk RNA-sequencing data. Prognostic characterization of LSAGs was established, of which the accuracy and reliability were assessed by one-way Cox and LASSO regression. Correlations between LSAG properties and immune cell infiltration, chemotherapy, and immunotherapy were analyzed by integrated omics data. Finally, we characterized the expression of three LSAGs (KCNE1, NPC2, and SFTPD) in malignant lung epithelium and assessed their impact on tumor malignancy related phenotypes. ResultsWe identified 18 LSAGs associated with prognosis, of which 3 LSAGs were used to construct prognostic models. High-risk patients had worse survival and the model predicted it better than other clinical indicators. Based on the functional enrichment analyses, LSAGs were associated with binding and molecular activity functions, inhibition of DNA damage repair and tumor growth, IL7 signaling pathway, and glycolysis. M0 macrophages and M1 macrophages were substantially enriched in high-risk patients. Conversely, there was a considerable enrichment of resting dendritic cells and M2 macrophages in patients at low risk. We also found that risk scores predicted the outcome of immunotherapy. In vitro, we found that KCNE1, NPC2, and SFTPD were lowly expressed in malignant epithelial cells and patients with low expression of KCNE1, NPC2, and SFTPD had a higher percentage of M2 macrophage infiltration. Overexpression of KCNE1, NPC2, and SFTPD suppressed the proliferation and invasion of malignant cells, and M0 macrophages remarkably reduced M2 macrophage polarization and cellular secretion of pro-tumor cytokines. ConclusionsWe used three LASGs—KCNE1, NPC2, and SFTPD—to develop and validate a predictive signature for LUAD patients. Furthermore, we found that low expression of KCNE1, NPC2, and SFTPD promotes lung cancer cell proliferation and invasion and M2 macrophage polarization. Our study may provide fresh perspectives for customized immunotherapy.

Epstein-Barr virus-associated lymphoepithelioma-like intrahepatic cholangiocarcinoma: A report of 3 cases and literature review.

Hepatic lymphoepithelioma-like carcinoma (LELC) is an extremely rare malignant tumor characterized by undifferentiated malignant epithelial cells and significant lymphatic infiltration. Hepatic LELC mainly includes lymphoepithelioma-like hepatocellular carcinoma (LEL-HCC) and lymphoepithelioma-like intrahepatic cholangiocarcinoma (LEL-CC). Epstein-Barr virus (EBV) infection is considered as an important factor in LELC carcinogenesis. Since 2005, Xiangya Hospital of Central South University has treated a total of 3 patients with EBV-associated LEL-CC, which all showed liver masses by CT scans. After surgical resection, the EBV encoded RNA (EBER) and CK19 expression in all 3 patients were positive, and pathological examination confirmed EBV-associated LEL-CC. Two patients had a good postoperative prognosis, while 1 patient received relevant immunotherapy and chemotherapy after surgery. Based on the analysis of existing literature, the author believes that hepatic LELC can be included in the classification of liver tumors, which will provide new ideas for the accurate diagnosis and treatment of hepatic LELC.

Stroma-specific gene expression signature identifies prostate cancer subtype with high recurrence risk

Current prognostic tools cannot clearly distinguish indolent and aggressive prostate cancer (PC). We hypothesized that analyzing individual contributions of epithelial and stromal components in localized PC (LPC) could improve risk stratification, as stromal subtypes may have been overlooked due to the emphasis on malignant epithelial cells. Hence, we derived molecular subtypes of PC using gene expression analysis of LPC samples from prostatectomy patients (cohort 1, n = 127) and validated these subtypes in two independent prostatectomy cohorts (cohort 2, n = 406, cohort 3, n = 126). Stroma and epithelium-specific signatures were established from laser-capture microdissection data and non-negative matrix factorization was used to identify subtypes based on these signatures. Subtypes were functionally characterized by gene set and cell type enrichment analyses, and survival analysis was conducted. Three epithelial (E1-E3) and three stromal (S1-S3) PC subtypes were identified. While subtyping based on epithelial signatures showed inconsistent associations to biochemical recurrence (BCR), subtyping by stromal signatures was significantly associated with BCR in all three cohorts, with subtype S3 indicating high BCR risk. Subtype S3 exhibited distinct features, including significantly decreased cell-polarity and myogenesis, significantly increased infiltration of M2-polarized macrophages and CD8 + T-cells compared to subtype S1. For patients clinically classified as CAPRA-S intermediate risk, S3 improved prediction of BCR. This study demonstrates the potential of stromal signatures in identification of clinically relevant PC subtypes, and further indicated that stromal characterization may enhance risk stratification in LPC and may be particularly promising in cases with high prognostic ambiguity based on clinical parameters.

Adamantinoma: About 4 Cases

Adamantinoma is a rare primary malignant bone tumor of the tibia in young adults. Histologically, it comprises malignant epithelial cells within a fibrous or osteofibrous stroma. Adamantinoma poses a problem of differential diagnosis with osteofibrous dysplasia and osteofibrodysplasia. the evolution of this low-grade sarcoma is slow. Treatment is surgical, involving resection of the tumour and reconstruction of the bone. Metastases may occur in the lungs, lymph nodes or bones. We report 4 cases of adamantinoma.