Tumor Cell Heterogeneity Research Articles

Unveiling drug resistance pathways in high-grade serous ovarian cancer(HGSOC): recent advances and future perspectives

High-Grade Serous Ovarian Carcinoma (HGSOC) represents the most prevalent and lethal subtype of ovarian cancer, with approximately 225,000 new cases reported globally each year and a five-year survival rate of merely 49.1%. The clinical management of HGSOC encounters substantial challenges, primarily attributable to its intricate drug resistance mechanisms, which involve multiple biological processes, including tumor cell heterogeneity, microenvironment remodeling, gene mutations, and drug efflux. This study systematically reviews the most recent advancements in HGSOC drug resistance research, concentrating on the molecular biological foundations of resistance mechanisms, innovative detection strategies, and potential therapeutic approaches. The research indicates that HGSOC drug resistance constitutes a complex process characterized by multifactorial interactions, involving aberrant cell signaling pathways, dynamic alterations in the tumor microenvironment, and specific expressions of molecular markers. In this review, we systematically analyzed and investigated the intricate biological behaviors associated with HGSOC drug resistance, which not only enhances the understanding of disease progression but also provides essential theoretical foundations for the development of more precise and effective targeted therapies. This review firstly illustrated the detailed drug resistance cellular and molecular mechanisms underlying HGSOC chemotherapy, which can pave the way for future studies in HGSOC drug resistance practices.

Abstract 5388: Metastasis suppressor RKIP modulates mitochondrial metabolism in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) remains the most aggressive subtype of breast cancer, with a high propensity for metastasis. Tumor cell heterogeneity has been closely linked to metastatic progression, therapy resistance, and disease recurrence in solid tumors, including TNBC. Within tumors, metabolic heterogeneity emerges as cancer cells adapt to diverse environmental pressures, further driving tumor aggressiveness. However, the role of metastasis suppressors, such as Raf Kinase Inhibitory Protein (RKIP), in regulating metabolic heterogeneity is not yet fully understood. Analysis of RNA sequencing data from breast cancer patients revealed a strong correlation between RKIP expression and oxidative phosphorylation (OXPHOS) genes. Together with this, by performing gene set enrichment analysis of bulk RNA sequencing of mouse xenografts we observed an enrichment of hallmark of OXPHOS in RKIP-overexpressing tumors compared to controls. Consistent with these findings, RKIP was shown to modulate oxygen consumption rates in vitro. Using stable isotope tracing with [U-13C] glucose in vivo, we observed higher glucose contribution to citrate labeling in RKIP-overexpressing tumors compared to controls. Through single-cell RNA sequencing, we demonstrate that RKIP reduces overall transcriptional heterogeneity, particularly by decreasing the variability of genes involved in mitochondrial respiration. Importantly, treatment with the OXPHOS inhibitor metformin selectively inhibited the growth of RKIP-overexpressing tumors, while control tumors were unaffected. This study suggests that RKIP or drugs that mimic its action have the potential to promote OXPHOS in tumor cells, presenting an opportunity to suppress tumor growth by subsequent therapeutic targeting of mitochondrial respiration in TNBC. Citation Format: Joana Pinheiro, Dongbo Yang, Madeline Henn, Long Chi Nguyen, Olga Martinho, Brandon Faubert, Marsha Rich Rosner. Metastasis suppressor RKIP modulates mitochondrial metabolism in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5388.

Abstract 5302: Mapping the tumor microenvironment at single-cell resolution with Stereo-seq v1.3

Abstract Tumor cell heterogeneity and the tumor microenvironment are fundamental questions that heavily influence the process of cancer development, disease progression, and the response to treatments. Knowledge of cancer biology facilitates understanding of pharmacological effects and mechanism of action to improve clinical outcomes. Spatial transcriptomics technology, particularly Stereo-seq, makes the groundbreaking advancement in cancer research with its outstanding performance with high capture efficiency and single cell resolution. It allows scientists to visualize and analyze the complexity of tumors at an unprecedented level of detail. In this study, we mapped tumor heterogeneity and microenvironment of fresh frozen section from colorectal cancer tissue (CRC) at a single-cell resolution with Stereo-seq v1.3 kit. We identified10 cell populations using cell2location, including B cells, fibroblasts, CD4+ T cells, CD8+ T cells, dendritic cells, endothelial cells, epithelial cells, macrophages, mast cells and NK cells. In situ mapping and portion analysis of cell identification revealed a B-cell dominated area which was confirmed by H&E staining of adjacent section. To comprehensively understand the immune activities, we quantified the interactions among different cell types (by StereoSite framework), revealing biologically meaningful intercellular communications mediated by GZMA-PARD3 and CCR10- CCL28 between B cells and epithelial cells. Further pathway enrichment analysis indicated cooperated molecular activities and underlying functions in CRC, which showed interaction activities significantly associated with EGF or BMP signaling pathways. Patteren recognition method identified how functional units assocatied with pathways. Take EGF pathway as an example, results revealed CD4+ T cells and macrophages may coordinated to impact EGF pathway by communication activities. Analysis in this study showed that spatial transcriptomic data at single-cell resolution is vital for exploring intercellular communications, molecular activities, and functional units by providing spatial context. Stereo-seq possess the ability to efficiently gathering evidence of mode of action and resistance mechanims by deeply mapping the spatial profiling in TME. It paves the ways for drug development by removing uncertainties and reducing time and cost. Implementation of Stereo-seq workflow could not only offer impetus to solve unmet needs in area of oncological research, but also bring new findings from lab to patients faster. Citation Format: Jiajun Zhang, Xing Liu, Zexian Zeng, Rui Zhang, Tibby Tianbi Duan, Jing Guo, Liang Wu, Sha Liao, Ao Chen. Mapping the tumor microenvironment at single-cell resolution with Stereo-seq v1.3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5302.

Abstract 2081: Tissue-niche-based and cell-type-selective proteomics

Abstract Background: The spatial organization of cell populations within tissues is crucial for maintaining physiological function and understanding the mechanisms underlying various diseases. Proteins are the functional components that drive essential processes such as tissue homeostasis, disease progression, and therapeutic response. Spatial proteomics integrates molecular profiling with cellular location data, offering a powerful approach to decode these mechanisms. Here, we present an innovative spatial proteomics (SP) tool that combines widely adopted methodologies in biological research, enabling cell type-specific and comprehensive proteomic insights within tissue niches. Method: Our approach utilizes thick tissue macrosections (≥300 μm) to precisely select regions of interest (ROIs) based on 3D multiplexed microscopy images. We perform microscopic photobleaching on the immunofluorescence-stained cells in ROIs to create a distinguishable fluorescent label for capturing their spatial context. These labeled cells are subsequently dissociated and collected via flow sorting. Lastly, we conducted LC-MS/MS analysis for a comprehensive spatial proteomic analysis of the collected cells. Result: We confirmed that our SP technology does not significantly alter the protein expression in the samples. No significant changes in protein expression were observed in the heatmap and the volcano plot compared to the control samples. Using this technology, we investigated dendritic cell (DC) subsets in mouse spleen during inflammation. The result reveals significant proteome differences among three splenic DC subsets, categorized by their locations (“inside” or “outside” spleen T-cell zones) and control DCs. Our results align with published proteome data on inflammatory splenic DCs, demonstrating the feasibility and reproducibility of our technology. Importantly, we identified 50 proteins, including cytoskeletal proteins FSCN1 and SH3BP1, with differential expression between the “outside” and “inside” DC subsets in the inflamed spleen. These findings suggest varying migration capabilities between these subsets, providing potential therapeutic targets for inflammatory diseases. Additionally, we adapted this technology to study T cell-macrophage interaction in mouse peripheral T cell lymphoma and explore tumor cell heterogeneity in human tumors. Conclusion: Our SP technology provides extensive protein profiling tailored to cell-type-specific spatial proteomics. It also demonstrates a broad applicability across diverse tissue specimens in various diseases. These findings hold promise for identifying novel cell types and potential drug targets to enhance treatment strategies for inflammatory diseases and cancers. Citation Format: Yi-Chien Wu, Elie Abi Khalil, Aditi Upadhye, Jalees Rehman, Nermin Kady, Ryan Wilcox, Steve Lee. Tissue-niche-based and cell-type-selective proteomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2081.

Abstract 5276: Comprehensive spatial mapping uncovers immune ecotypes linked to tumor heterogeneity in HCC lung metastasis

Abstract Tumor immune microenvironment (TME) heterogeneity, shaped by diverse immune and stromal cells, significantly influences tumor metastasis, yet the specific immune ecotypes involved and their relationship to intra-tumor heterogeneity remain unclear. In this study, we present a comprehensive spatial and cellular multi-omics analysis of 44 primary and 54 lung metastatic lesions from 34 HCC patients. By integrating public single-cell RNA sequencing data from 187, 290 tumor cells across 146 HCC cases, we define HCC cellular states and uncover their organization in high-dimensional spatial expression profiles from paired HCC primary and metastatic lung lesions. Our integrated analysis, incorporating data with three independent cohorts, uncovers three pro-metastatic modules—Cell cycle(G2/M), Cellular Stress and Hypoxia —that contribute to early HCC lung metastasis. Notably, the first two modules are specific to lung metastasis, while the latter one is common across all extrahepatic organs. We show how tumor cells with distinct modules interact with various immune cell types, facilitating metastasis through different mechanisms. Additionally, these tumor cells form multi-layered structures of varying sizes, with specific state pairs preferentially clustering across multiple scales, all within a 55 µm limit. In vivo studies further reveal that interactions between G2/M-high tumor cells and Angio-macrophages and T3-neutrophils accelerate lung dissemination. Treatment with the G2/M inhibitor paclitaxel effectively disrupts this process, reducing metastasis. These findings provide critical insights into how tumor cell heterogeneity reshapes the TME to drive metastasis, underscoring the importance of spatially informed features for personalized cancer therapies. Our work emphasizes the potential of tailored strategies to prevent and manage metastasis based on tumor-immune interactions. Citation Format: Jinghan Zhu, Guangzhen Cai, Bin Guo, Xiaoping Chen, Huilan Zhang, Hong Zhu, Wanguang Zhang. Comprehensive spatial mapping uncovers immune ecotypes linked to tumor heterogeneity in HCC lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5276.

Abstract 1269: Development of humanized PDX mouse model for high grade serous ovarian cancer

Abstract Introduction: High-grade serous ovarian cancer (HGSOC) is one the most lethal gynecological cancer, accounting for 70% deaths and with a 5-year survival rate below 50%. Traditionally, HGSOC are considered 'immune-cold' tumors, characterized by low immune infiltration and poor response rates to single agent immune checkpoint blockers (ICB). To improve the therapeutic potential of immunotherapies in HGSOC, combination treatments are being applied such as the Phase 2 MEDIOLA trial, which aims to identify mechanisms of response and resistance. A major challenge in this field remains the lack of suitable preclinical models for ICB-based treatments. Although patient-derived tumor xenografts (PDX) are valuable for developing new therapies, they are limited by the absence of an immunocompetent host. Here we present humanized mouse models incorporating human immune cells. Methods: Female NRG-W41-3GS mice (ENW) aged 7-8 weeks were used to generate the humanized PDX model. HGSOC tumors originally sourced from human patients and maintained in our laboratory’s previous PDX models using NRG mice, were injected subcutaneously into ENW mice. After 3 weeks, 5x104 human hematopoietic CD34+ cells collected from umbilical cord blood of healthy donors were intravenously injected via the tail vein. To monitor human cell engraftment, bone marrow (BM) aspiration was performed at week 6 post-CD34+ cell injection, and peripheral blood (PB) samples were collected from saphenous vein starting from week 7 until the experimental endpoint. BM and PB samples were analyzed by flow cytometry using antibodies against human CD45 and mouse CD45 to determine humanization level. Tumors were harvested at the endpoint, embedded in paraffin, and remaining tumor tissues were cryopreserved for future. To confirm human immune cell infiltration into the tumors, fresh frozen paraffin embedded tumor sections were immunohistochemically stained with rabbit anti-human CD45 antibodies. Results: We successfully generated sixteen huENW PDX mice. Robust engraftment of human CD34+ cells was confirmed in each mouse, with human CD45+ cells ranging from 35% to 90% (mean ± SD: 76.2 ± 18.5%) in BM samples and from 7% to 35% in PB samples. The huENW PDX model have revealed strong evidence of human immune infiltration with CD45+ cells on immunohistochemistry of the PDX tumors and spleen. Conclusion: Humanized mouse models offer a valuable resource for advancing HGSOC research. Our humanized PDX HGSOC mouse model demonstrated significant engraftment of human immune infiltration into HGSOC tumors. Future works will focus on characterizing tumor cell heterogeneity, analyzing spatial gene expression patterns and human immune cells distribution using Xenium in situ coupled with scRNA seq. We believe our model has the potential to enhance our understanding of HGSOC clonal diversity, microenvironment interactions, and contributing to the development of new therapies. Citation Format: Tsz Yin (Jacky) Lam, Farhia Kabeer, Shary Chen, Makoto Kishida, Yuchen Ding, Lorena Zoltan, Yvette Drew, David Huntsman. Development of humanized PDX mouse model for high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1269.

Abstract 5241: Mapping spatial 3D genome landscape of breast cancer cells in different tumor immune microenvironments using onco-immune co-culture as a model

One of the leading causes of tumor cell heterogeneity is the complex nature of the tumor immune microenvironment involving crosstalk between different signaling pathways of cancer cells and immune cells. For cancer cells to grow into a primary tumor and then metastasize into secondary tumors, they must avoid or overcome the attack of different types of immune cells, including macrophages, natural killer (NK) cells, and T lymphocytes. For example, cancer cells can polarize macrophages from “tumor killing” to “tumor promoting” type using specific signaling molecules. Such tumor immune crosstalk is dependent on the clonal and sub-clonal heterogeneity in genomic, transcriptomic and protein signaling networks across different cancer cell sub-populations, immune cells and its surrounding microenvironment. Since structural aberrations affect gene dysregulation, the study of the 3D- genome organization in single cells can help in better understanding of disease progression and prognosis. The genome is organized and packaged in a highly structured manner inside the nucleus of mammalian cells and exhibits dynamic reorganization throughout disease progression. Understanding this 3D organization at the single-cell level with high spatial resolution is crucial for immune-oncology disease research such as investigating tumor-immune cell interactions. In this abstract, we present a novel jebFISHTM protocol on the PaintScapeTM platform that can be used to investigate immune cell modulated differences in global 3D genome organization among different localized and metastatic breast cancer cell lines at single cell, sub-population and population level. Using a genome wide panel of chromosomal targets on an onco-immune co-culture system involving different breast cancer cell lines and CD4+ T-cells or macrophages, we will show disruption in chromosome territory, radius of gyration, chromosomal instability between localized and metastatic breast cancer cells at single cell and sub-population level. We observed signatures of specific 3D genome structural alterations and interchromosomal interactions in cancer cells and/or immune cells, related to the effect of different types of immune response. We will show how the 3D architecture of key genomic loci and its associated regulatory elements belonging to major cancer signaling pathways exhibit specific signatures including differential folding, chromosomal instability, differential interchromosomal interaction and structural variation patterns at single cell, sub-chromosomal and sub-population level. We envision this study will improve our understanding on breast cancer tumor immune microenvironment and provide deeper insight into the underlying genomic heterogeneity of single cancer cells and sub-populations which might help to design better treatment options in the future. Citation Format: Jude Dunne, Huy Nguyen, Shyamtanu Chattoraj. Mapping spatial 3D genome landscape of breast cancer cells in different tumor immune microenvironments using onco-immune co-culture as a model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5241.

Abstract 2484: A novel algorithm to expand mechanistic computational models of biochemical reaction networks to heterogeneous tumor cell communities

Abstract 2484: A novel algorithm to expand mechanistic computational models of biochemical reaction networks to heterogeneous tumor cell communities

Abstract 7479: Single-cell OncoTreat: Novel therapeutic candidates by high resolution inference and validation of drug sensitivities in heterogeneous cholangiocarcinoma tumors

Abstract Cholangiocarcinoma is an aggressive biliary adenocarcinoma with limited treatment options. Stromal infiltration and intratumoral heterogeneity in this tumor type prevents identification of actionable dependencies from bulk-tissue profiles. While cholangiocarcinoma illustrates these challenges, they are common across many other aggressive tumors.To address these challenges, we introduce a single-cell RNA-Sequencing (scRNA-Seq) framework for prioritization of drugs to treat heterogeneous tumor cell subpopulations. Single-cell analyses can bypass limitations of sample size in a precision medicine approach by providing large tumor cell “cohorts” even from individual patients, with the advantage that cell subpopulations can be effectively distinguished. Unfortunately, >80% of genes in each cell profiled by scRNA-Seq are typically undetectable, limiting discoverability of therapeutic targets and drug sensitivities. The VIPER algorithm addresses this by inferring the transcriptional activity of a protein from expression of its downstream target genes. By then identifying aberrantly activated proteins, each cell can be matched to an empiric database of cell line drug perturbation RNA-Seq, ranking drugs which empirically inhibit the proteins aberrantly activated in each cell. We refer to this approach as single-cell OncoTreat.Analysis of tumor cells across six patients by this approach has revealed three distinct sub-phenotypes of cholangiocarcinoma cells, with conserved proteomic signatures, and distinct predicted drug sensitivities. A seventh patient recapitulated these phenotypes, with drug predictions validated in a low-passage, patient-derived xenograft (PDX) model. The top-ranked drug by integrated analysis of all tumor cells was plicamycin, which at sub-cluster level only two of three clusters were predicted to respond to. Conversely, dacinostat was inferred to be active against all three tumor-cell sub-phenotypes. Tumor growth studies in PDX demonstrated growth rate control with both plicamycin and dacinostat, but much improved survival time with dacinostat, outperforming gemcitabine + cisplatin standard of care. Post-treatment scRNA-Seq confirmed predicted subpopulation-specific effects, such that dacinostat depleted all three tumor sub-phenotypes, while plicamycin depleted only the two predicted, allowing the third to reconstitute a treatment-resistant tumor. To our knowledge, this is the first report of dacinostat as an HDAC inhibitor effective in cholangiocarcinoma treatment, in vivo. Ultimately, this approach suggests promising candidates for follow-up trials in cholangiocarcinoma, and is highly generalizable to elucidate complementary dependencies of other aggressive, heterogeneous tumors. Citation Format: Aleksandar Obradovic, Lorenzo Tomassoni, Daoqi You, Charles Drake, Filemon Dela Cruz, Andrew Kung, Andrea Califano. Single-cell OncoTreat: Novel therapeutic candidates by high resolution inference and validation of drug sensitivities in heterogeneous cholangiocarcinoma tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7479.

Precancerous Cells Initiate Glioblastoma Evolution and Contribute to Intratumoral Heterogeneity.

Neural stem cells (NSCs) in the subventricular zone (SVZ) are identified as cells-of-origin harboring driver mutations in glioblastoma (GBM), which is the most devastating brain tumor with highly heterogeneous nature. However, the sequential transformation of a limited number of mutation-harboring NSCs into a distant tumor with high intratumoral heterogeneity remains poorly understood. In this study, we have identified transcriptionally distinct types of mutation-harboring precancerous cells in our spontaneous, somatic mouse model recapitulating human GBM evolution as well as in tumor-free SVZ tissues from patients. These precancerous cells emerge via oligodendrocyte lineage specification, exhibiting unique transcriptional programs involving dysregulated translations and extracellular matrix remodeling. Subsequently, they give rise to heterogeneous tumor cell populations by activating multiple programs crucial for gliomagenesis. Our findings highlight the pivotal role of precancerous cells in tumor evolution and intratumoral heterogeneity, suggesting their potential as a novel therapeutic target for GBM.

Joint analysis of chromatin accessibility and gene expression in the same single cells reveals cancer-specific regulatory programs.

Biological analyses conducted at the single-cell scale have revealed profound impacts of heterogeneity and plasticity of chromatin states and gene expression on physiology and cancer. Here, we developed Parallel-seq, a technology for simultaneously measuring chromatin accessibility and gene expression in the same single cells. By combining combinatorial cell indexing and droplet overloading, Parallel-seq generates high-quality data in an ultra-high-throughput fashion and at a cost two orders of magnitude lower than alternative technologies (10× Multiome and ISSAAC-seq). We applied Parallel-seq to 40 lung tumor and tumor-adjacent clinical samples and obtained over 200,000 high-quality joint scATAC-and-scRNA profiles. Leveraging this large dataset, we characterized copy-number variations (CNVs) and extrachromosomal circular DNA (eccDNA) heterogeneity in tumor cells, predicted hundreds of thousands of cell-type-specific regulatory events, and identified enhancer mutations affecting tumor progression. Our analyses highlight Parallel-seq's power in investigating epigenetic and genetic factors driving cancer development at the cell-type-specific level and its utility for revealing vulnerable therapeutic targets.

A drug that induces the microRNA miR-124 enables differentiation of retinoic acid-resistant neuroblastoma cells.

Tumor cell heterogeneity in neuroblastoma, a pediatric cancer arising from neural crest-derived progenitor cells, presents clinical challenges. Unlike adrenergic (ADRN) neuroblastoma cells, neuroblastoma cells with a mesenchymal (MES) identity are resistant to chemotherapy and retinoid therapy, which contributes to relapses and treatment failures. We explored whether up-regulation of the neurogenic, tumor suppressor microRNA miR-124 could promote the differentiation of retinoic acid-resistant MES neuroblastoma cells. Leveraging our screen for miRNA-modulatory small molecules, we identified and validated the tyrosine and phosphoinositide kinase inhibitor PP121 as a robust inducer of miR-124. Combining PP121 and BDNF-activating bufalin synergistically arrested proliferation and promoted the sustained differentiation of MES/heterogeneous SK-N-AS cells over several weeks. This protocol also resulted in the differentiation of multiple MES neuroblastoma and glioblastoma cell lines. RNA-seq analysis of differentiated MES/heterogeneous SK-N-AS cells revealed the replacement of the ADRN core regulatory circuitry with circuitries associated with chromaffin cells and Schwann cell precursors. Furthermore, differentiation was associated with inhibition of the CDK4/CDK6 pathway and activation of a transcriptional program that correlated with improved outcomes for patients with neuroblastoma. Our findings suggest an approach with translational potential to induce the differentiation of therapy-resistant cancers of the nervous system. Moreover, these long-lived, differentiated cells could be used to study mechanisms underlying cancer biology and therapies.

Advances in strategies to improve the immunotherapeutic efficacy of chimeric antigen receptor-T cell therapy for lymphoma.

Chimeric antigen receptor-T (CAR-T) cell therapy is a precise immunotherapy for lymphoma. However, its long-term efficacy faces many challenges related to tumor cell heterogeneity, interference from immunosuppressive microenvironments, CAR-T cell exhaustion, and unmanageable adverse events. Diverse modifications have been introduced into conventional CAR-T cells to overcome these obstacles; examples include addition of recognition sites to prevent immune escape, coupling of cytokine domains to enhance killing ability, blocking of immune checkpoint signals to resist tumor microenvironments, and inclusion of suicide systems or safety switches to improve safety and flexibility. With increasing understanding of the importance of metabolism and epigenetics in cancer and cytotherapy, glycolysis, methylation, and acetylation have become crucial CAR-T cell therapeutic targets. Universal and in situ CAR-T cells are also expected to be used in clinical applications, thus providing hope to patients with relapsed/refractory lymphomas.

Single-cell and spatial RNA sequencing identify divergent microenvironments and progression signatures in early- versus late-onset prostate cancer.

The clinical and pathological outcomes differ between early-onset (diagnosed in men ≤55 years of age) and late-onset prostate cancer, potentially attributed to the changes in hormone levels and immune activities associated with aging. Exploring the heterogeneity therein holds potential for developing age-specific precision interventions. Here, through single-cell and spatial transcriptomic analyses of prostate cancer tissues, we identified that an androgen response-related transcriptional meta-program (AR-MP) might underlie the age-related heterogeneity of tumor cells and microenvironment. APOE+ tumor-associated macrophages infiltrated AR-MP-activated tumor cells in early-onset prostate cancer, potentially facilitating tumor progression and immunosuppression. By contrast, inflammatory cancer-associated fibroblasts in late-onset prostate cancer correlated with downregulation of AR-MP of tumor cells and increased epithelial-to-mesenchymal transition and pre-existing castration resistance, which may also be linked to smoking. This study provides potential insights for tailoring precision treatments by age groups, emphasizing interventions that include targeting AR and tumor-associated macrophages in young patients but anchoring epithelial-to-mesenchymal transition and inflammatory cancer-associated fibroblasts in old counterparts.

PD-L1 siRNA incorporation into a cationic liposomal tumor mRNA vaccine enhances cytotoxic T cell activation and prevents immune evasion.

PD-L1 siRNA incorporation into a cationic liposomal tumor mRNA vaccine enhances cytotoxic T cell activation and prevents immune evasion.

DNA methylation memory of pancreatic acinar-ductal metaplasia transition state altering Kras-downstream PI3K and Rho GTPase signaling in the absence of Kras mutation

BackgroundA critical area of recent cancer research is the emergence of transition states between normal and cancer that exhibit increased cell plasticity which underlies tumor cell heterogeneity. Pancreatic ductal adenocarcinoma (PDAC) can arise from the combination of a transition state termed acinar-to-ductal metaplasia (ADM) and a gain-of-function mutation in the proto-oncogene KRAS. During ADM, digestive enzyme-producing acinar cells acquire a transient ductal epithelium-like phenotype while maintaining their geographical acinar organization. One route of ADM initiation is the overexpression of the Krüppel-like factor 4 gene (KLF4) in the absence of oncogenic driver mutations. Here, we asked to what extent cells acquire and retain an epigenetic memory of the ADM transition state in the absence of oncogene mutation.Methods We profiled the DNA methylome and transcriptome of KLF4-induced ADM in transgenic mice at various timepoints during and after recovery from ADM. We validated the identified DNA methylation and transcriptomic signatures in the widely used caerulein model of inducible pancreatitis.ResultsWe identified differential DNA methylation at Kras-downstream PI3K and Rho/Rac/Cdc42 GTPase pathway genes during ADM, as well as a corresponding gene expression increase in these pathways. Importantly, differential methylation persisted after gene expression returned to normal. Caerulein exposure, which induces widespread digestive system changes in addition to ADM, showed similar changes in DNA methylation in ADM cells. Regions of differential methylation were enriched for motifs of KLF and AP-1 family transcription factors, as were those of human pancreatic intraepithelial neoplasia (PanIN) samples, demonstrating the relevance of this epigenetic transition state memory in human carcinogenesis. Finally, single-cell spatial transcriptomics revealed that these ADM transition cells were enriched for PI3K pathway and AP1 family members.ConclusionsOur comprehensive study of DNA methylation in the acinar-ductal metaplasia transition state links epigenetic memory to cancer-related cell plasticity even in the absence of oncogenic mutation.

Characterizing resistant cellular states in nasopharyngeal carcinoma during EBV lytic induction.

The pervasive occurrence of nasopharyngeal carcinoma (NPC) is intricately linked to Epstein-Barr virus (EBV) infection, making EBV and its associated pathways promising therapeutic targets for NPC and other EBV-related cancers. Lytic induction therapy, an emerging virus-targeted therapeutic strategy, capitalizes on the presence of EBV in tumor cells to specifically induce cytotoxicity against EBV-associated malignancies. Despite the expanding repertoire of compounds developed to induce EBV lytic reactivation, achieving universal induction across all infected cells remains elusive. The inherent heterogeneity of tumor cells likely contributes to this variability. In this study, we used the NPC43 cell line, an EBV-positive NPC in vitro model, and single-cell transcriptomics to characterize the diverse cellular responses to EBV lytic induction. Our longitudinal monitoring revealed a distinctive lytic induction non-responsive cellular state characterized by elevated expression of SOX2 and NTRK2. Cells in this state exhibit phenotypic similarities to cancer stem cells (CSCs), and we verified the roles of SOX2 and NTRK2 in manifesting these phenotypes. Our findings reveal a significant challenge for lytic induction therapy, as not all tumor cells are equally susceptible. These insights highlight the importance of combining lytic induction with therapies targeting CSC-like properties to enhance treatment efficacy for NPC and other EBV-associated cancers.

Annexin A1 Is Involved in the Antitumor Effects of 5-Azacytidine in Human Oral Squamous Carcinoma Cells.

Background: the treatment of squamous cell carcinomas of the oral cavity (OSCCs) is limited by the lack of reliable diagnostic/prognostic, and predictive markers, as well as by intrinsic tumor cell heterogeneity. 5-azacytidine (5-AZA) offers opportunities for cancer cell reprogramming to develop new target-specific treatments. The protein annexin A1 (ANXA1) is downregulated in head and neck squamous cell carcinoma (HNSCC), correlated with pathological differentiation grade. Objectives: this work aimed to further investigate the role of ANXA1 in OSCC progression based on 5-AZA activity. Methods: we used CAL27 and CAL33 cell lines, which differ in drug sensitivity and differentiation status. Results: CAL27 showed a higher expression of the stemness markers compared to CAL33 cells, but this positivity was lost after treatment with 5-AZA. This drug also decreased CAL27 cell motility, promoting a less aggressive phenotype. Moreover, 5-AZA increased ANXA1 expression only in CAL27. After siRNA-mediated downmodulation, we witnessed a significant rise in cell motility and the inversion of E-/N-cadherin expression, which was reverted again by 5-AZA. To investigate the role of exogenous ANXA1 derived from the tumor microenvironment, we treated CAL27 with Ac2-26, an ANXA1 mimetic peptide. Interestingly, we found that this peptide alone showed impacts similar to 5-AZA in reversing the aggressive phenotype. All these effects were not evidenced in CAL33 cells. Finally, to prove the loop of the exogenous protein, we detected increased expression of its receptors, formyl peptide receptors (FPRs), and their activation, leading to oncosuppressor effects. Conclusions: we propose that ANXA1 mediates the effects of 5-AZA only in poorly differentiated stemlike CAL27 cell lines. This suggests the relevance of ANXA1 as a diagnostic/prognostic biomarker in OSCCs, paving the way for personalized therapies to overcome treatment difficulties.

Abstract B035: Evaluating drug sensitivity heterogeneity in cancer organoids derived from circulating tumor cells

Abstract Background: Drug resistance is a major challenge in cancer treatment, often driven by tumor cell heterogeneity. We cultivated circulating tumor cells (CTCs) from the blood of patients with solid tumors, including those preserved as frozen PBMCs, into cancer organoids. Two organoid lines, derived from renal and colon cancers, have been maintained and used extensively to assess drug sensitivity. During these experiments, viability assays revealed that a subset of organoids displayed resistance to certain drugs, resuming growth after initial inhibition. This prompted us to investigate this heterogeneity using specialized assays. Methods: To study variability in drug responses among CTC-derived organoids, we employed three complementary approaches: 1. Microcavity Plates: Corning's Elplasia plates with microcavities were used to segregate organoids, facilitating high-resolution, kinetic imaging for monitoring individual organoid growth and survival during drug treatment. 2. Image Analysis: Morphological changes in organoids during drug exposure were tracked using image analysis, allowing for the quantitation of resistant organoids and providing insights into variations in growth patterns and morphological changes. 3. Transcriptome Profiling: Using individual organoids, each with sufficient RNA for analysis, allowed detailed transcriptomic profiling and provided a comprehensive assessment of genetic responses in drug-resistant organoids. Results: Preliminary results from a limited set of one renal and one colon cancer organoids indicate that the three methods we employed show promise in detecting heterogeneity in drug sensitivity. The use of Elplasia plates allowed for consistent kinetic monitoring of individual organoid survival, and image analysis effectively captured morphological changes associated with varying drug responses. Initial transcriptomic profiling hinted at distinct genetic signatures in drug-resistant organoids, suggesting a potential reflection of physiological heterogeneity seen in patient tumors, potentially including metastatic ones. While these findings are encouraging, further studies with more samples are needed to confirm these observations and refine our approach. Conclusion and Future Directions: Our integrated approach appears to be promising in identifying and characterizing drug sensitivity heterogeneity in CTC-derived cancer organoids. We plan to expand this study to include more patient samples and cancer types, aiming to confirm our findings and identify potential biomarkers of resistance. Citation Format: Yi-chun Han, David Hsieh, Shian-Jiun Shih. Evaluating drug sensitivity heterogeneity in cancer organoids derived from circulating tumor cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr B035.

Immunosuppressive microenvironment of liver restrains chemotherapeutic efficacy in triple-negative breast cancer

BackgroundPatients with liver metastases of triple-negative breast cancer (TNBC) show poor prognosis compared with other metastases. Chemotherapy is the primary treatment for advanced TNBC. Tumor cell diversity and the tumor...