Cell Plasticity Research Articles

Cell-fate conversion of intestinal cells in adult Drosophila midgut by depleting a single transcription factor

The manipulation of cell identity by reprograming holds immense potential in regenerative medicine, but is often limited by the inefficient acquisition of fully functional cells. This problem can potentially be resolved by better understanding the reprogramming process using in vivo genetic models, which are currently scarce. Here we report that both enterocytes (ECs) and enteroendocrine cells (EEs) in adult Drosophila midgut show a surprising degree of cell plasticity. Depleting the transcription factor Tramtrack in the differentiated ECs can initiate Prospero-mediated cell transdifferentiation, leading to EE-like cells. On the other hand, depletion of Prospero in the differentiated EEs can lead to the loss of EE-specific transcription programs and the gain of intestinal progenitor cell identity, allowing cell cycle re-entry or differentiation into ECs. We find that intestinal progenitor cells, ECs, and EEs have a similar chromatin accessibility profile, supporting the concept that cell plasticity is enabled by pre-existing chromatin accessibility with switchable transcription programs. Further genetic analysis with this system reveals that the NuRD chromatin remodeling complex, cell lineage confliction, and age act as barriers to EC-to-EE transdifferentiation. The establishment of this genetically tractable in vivo model should facilitate mechanistic investigation of cell plasticity at the molecular and genetic level.

Small-Cell Lung Cancer: Is Liquid Biopsy a New Tool Able to Predict the Efficacy of Immunotherapy?

Small-cell lung cancer (SCLC) cases represent approximately 15% of all lung cancer cases, remaining a recalcitrant malignancy with poor survival and few treatment options. In the last few years, the addition of immunotherapy to chemotherapy improved clinical outcomes compared to chemotherapy alone, resulting in the current standard of care for SCLC. However, the advantage of immunotherapy only applies to a few SCLC patients, and predictive biomarkers selection are lacking for SCLC. In particular, due to some features of SCLC, such as high heterogeneity, elevated cell plasticity, and low-quality tissue samples, SCLC biopsies cannot be used as biomarkers. Therefore, the characterization of the tumor and, subsequently, the selection of an appropriate therapeutic combination may benefit greatly from liquid biopsy. Soluble factors, circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and extracellular vesicles (EVs) are now useful tools in the characterization of SCLC. This review summarizes the most recent data on biomarkers detectable with liquid biopsy, emphasizing their role in supporting tumor detection and their potential role in SCLC treatment choice.

Abstract 6935: Non-genetic routes of lung cancer evolution

Abstract Cancer cell plasticity underlies the progression of several solid tumors. In lung adenocarcinoma, morphological changes characterizing the transition from indolent to aggressive disease exhibit profound epigenetic and transcriptional reprogramming1. However, the extent, features, and drivers of cancer cell plasticity in the progression of human lung adenocarcinoma is still largely unexplored. Here, we assembled and analyzed a meta-cohort of 119 human lung tumors analyzed by single-cell RNA-sequencing, for a total of ~730,000 cells. Cancer cell heterogeneity was the result of differential activation of 17 transcriptional programs associated with common cancer phenotypes, such as proliferation, cell migration, and response to hypoxia, as well as alveolar lineage identity (AT2). In particular, AT2-specific expression of MHC-II was down-regulated with the progression of the disease and markedly reduced in KEAP1 and/or STK11 mutated samples, providing a rationale for the association between these mutations and immunotherapy resistance. Co-activation of transcriptional programs clustered tumor cells in 4 major cell states, which emerged with the progression of the disease. To explore spatial distribution of different cancer cell states, we generated and analyzed multiple spatial transcriptomics and proteomics lung adenocarcinoma cohorts. To this purpose we developed a new algorithm called CellCharter2. CellCharter identified distinct cancer-enriched niches within individual samples, corresponding to the cancer cell states that we discovered in the scRNA-seq cohort and exhibited specific interactions with the tumor microenvironment (TME). In particular, we identified a cancer cell state characterized by response-to-hypoxia and migratory phenotypes, which spatially segregated from highly proliferative cancer cell niches and formed specific interactions with tumor associated neutrophils. This cell state was enriched in advanced primary samples, it uniquely characterized metastatic samples, and was an independent marker of poor prognosis across multiple independent datasets. Overall, our work uncovered cell plasticity trajectories associated with the progression of lung adenocarcinoma and shed new light into the tumor-TME interactions at their origin. 1 Tavernari et al., Cancer Discovery, 20212 Varrone et al, Nat. Genetics, (in press) Citation Format: Giovanni Ciriello, Daniele Tavernari, Marco Varrone. Non-genetic routes of lung cancer evolution [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 6935.

Abstract 1146: Pan-cancer characterization of cancer cell state and plasticity using spatially resolved transcriptomics

Abstract Cancer cell state heterogeneity constitutes a foundational characteristic of cancer, posing a formidable barrier to the discovery of biomarkers and the efficacy of therapeutic interventions. However, current understanding of cancer cell heterogeneity and plasticity remains limited, especially in the spatial context. Previous studies underscored that cancer cell states are not strictly governed by genetics, but rather display a remarkable degree of plasticity. Recent pan-cancer single-cell studies have unveiled dozens of recurrent cancer cell states. Nevertheless, the relationship between these cancer cell states and tumor microenvironment (TME) remains poorly understood, especially when considering the diversity across different tumor types. Recent advancements in spatial transcriptomics (ST) have paved the way for a novel approach to spatially profile cell location, organization, and interaction within the tumor microenvironment. In this study, we curated ST data (Visium, 10x Genomics) from public repositories and in-house datasets, encompassing a total of >200 tissue sections across 11 cancer types. By incorporating histological annotations and inferred copy number variations, we systematically investigated the spatial heterogeneity of cancer cell states and inferred clonal architectures of cancer cell-enriched spots. Non-negative matrix factorization was applied to identify highly recurrent cancer states and transcriptional hallmarks associated with intra-tumor heterogeneity. By employing this approach, we uncover a diverse curated gene set of meta-programs. Notably, we observe a high level of reproducibility of these meta-programs when compared to the findings from single-cell level. Furthermore, we observe significant variations of cell states across different cancer types, even among two cancer subtypes within a specific tissue. We noted a unique distribution of 'hypoxia' and 'epithelial-mesenchymal transition' in the basal lineage tumor. Following this, we perform single-cell and ST co-embedding using available tools, including CellTrek and CytoSPACE. We noticed the cell type composition of tumor’s neighbor spots play a significant role in influencing tumor states, especially for myofibroblastic CAFs and the tumor state in epithelial-mesenchymal transition. These findings contribute valuable insights into the relationship between cancer cell state plasticity and interactions with the TME, offering potential targets for further exploration and therapeutic intervention. Citation Format: Guangsheng Pei, Kyung S. Cho, Yunhe Liu, Serrano Alejandra, Rossana Lazcano, Enyu Dai, Guangchun Han, Fuduan Peng, Daiwei Zhang, Yanshuo Chu, Ansam F. Sinjab, Jiahui Jiang, Mingyao Li, Cassian Yee, Andrew Futreal, Alex Lazar, Humam Kadara, Jianjun Gao, Luisa M. Soto, Anirban Maitra, Jaffer Ajani, Linghua Wang. Pan-cancer characterization of cancer cell state and plasticity using spatially resolved transcriptomics [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 1146.

Abstract 257: Plasticity of preneoplastic cells drives esophageal squamous cell cancer initiation

Abstract Esophageal squamous cell carcinoma (ESCC) accounts for over 80% of esophageal cancer, with a poor prognosis mainly due to a lack of early-stage symptoms. Early detection of ESCC is challenging due to its origin in the basal cell layer and invasion into the lamina propria upon becoming neoplastic. Thus, understanding the transition mechanism from preneoplasia to neoplasia is crucial for early-stage detection of ESCC before dissemination. To dissect the mechanisms of ESCC preneoplasia and neoplasia, we utilized both spatial and temporal datasets of ESCC in conjunction with a genetically engineered organoid model. For spatial datasets, we conducted comparative analyses of single-cell transcriptomes from healthy esophageal tissues, normal tissue adjacent to cancer (NAC), and paired ESCC tumors to identify distinguishable preneoplastic cells. Notably, unlike normal or ESCC, NAC harbors a distinct preneoplastic cell cluster characterized by the exclusive expression of the CELF2 (CUGBP Elav-Like Family Member 2) gene, high gene copy number variations, cell hyperproliferation, and apoptosis, reminiscent of a 'crisis state'. Consistently, we detected a significant presence of CELF2 positive (+) cells at the stage of inflammation, a very early lesion preceding neoplasia in a temporal model of ESCC development. Copy number variations were also already abundant from the inflammation stage, as we observed in the NAC dataset. Interestingly, both spatial and temporal preneoplastic cells showed decreased scores of TP53 and CDKN2A signaling pathways, of which mutations are observed in 68% of ESCC patients. To recapitulate this phenotype, we employed genetically engineered esophageal organoids and found that the loss of Trp53 and Cdkn2a (PC) generated preneoplastic CELF2+ cells. Intriguingly, these preneoplastic CELF2+ cells exhibited remarkable cellular plasticity in two ways. Firstly, CELF2+ cells of PC display increased stemness mediated by SOX2-driven reprogramming with disrupted normal esophageal cell lineage. Second, CELF2+ cells of PC undergo epithelial-mesenchymal plasticity to generate fibroblast-like cells, releasing cytokines involved in immune remodeling. Consequently, preneoplastic CELF2+ cells developed significant tumors compared to CELF2-negative cells in transplantation assays. Our study identified preneoplastic CELF2+ cells driven by TP53 and CDKN2A loss as pivotal drivers in ESCC initiation via cell-autonomous (cellular reprogramming) as well as non-cell autonomous (immune niche remodeling) processes, proposing CELF2+ cells and their molecular signatures as potential biomarkers and therapeutic targets for early-stage ESCC. Citation Format: Kyung Pil Ko, Jie Zhang, Sohee Jun, Jae-Il Park. Plasticity of preneoplastic cells drives esophageal squamous cell cancer initiation [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 257.

Abstract 5412: In vitro plasticity profiling of metastatic cancer cells with an imaging-based pipeline

Abstract Different types of cancer cells exhibit varying levels of invasiveness, migration behavior and ability to resume growth at distant sites. In order to classify cell lines for these fundamentally important traits of metastasis, we sought to generate an image-based in vitro assay that quantifies morphological and functional features of cancer cell plasticity. We developed an Imaging-Based Pipeline as an R Package for in vitro cell monitoring using the breast cancer cell lines MDA-MB-231 and MCF-7 that represent high-metastatic and low-metastatic cells in vivo, respectively, to identify relevant feature variables. Live cell imaging via the zenCELL owl system captured images for labeling and segmentation. Pre-trained neural network models from Cellpose, combined with user-calibrations, accurately segmented cells for tracking. The tracking pipeline calculated similarity coefficients, Jaccard index, between consecutive frames for cell trajectory analysis. Geometric and morphological features, speed, and distance measurements were derived for assessing structural motility and proliferation-associated changes. The analysis of two cell line models revealed significant variations in morphological changes, with MDA-MB-231 showing more rapid changes between round and elongated shape compared to MCF-7 cells. Moreover, the analysis of cell motility, consistent with the morphological changes, revealed that TGF-beta1 induced migration in MDA-MB-231 cells much stronger than in MCF-7 cells. Likewise, differences in proliferation induction upon addition of growth factor was monitored, showing that MCF-7 cells are more responsive to the growth stimulation than MDA-MB-231 cells. Consequently, the morphological plasticity of cancer cells, closely linked to the epithelial-to-mesenchymal transition (EMT) state, emerges as a defining factor for the in vitro plasticity of these cells. Quantified proliferation rate and migration speed of the cells was then used to establish an in vitro "plasticity index" that captures a cell line’s ability to switch between migration and proliferation. This index will undergo validation using cell lines with well-established metastatic propensities. In conclusion, we developed an Imaging-Based Pipeline that can effectively explore cancer cell plasticity in vitro and monitor the impact of genetic or pharmacologic perturbations on this critical characteristic of aggressive cancer cells. Citation Format: Saba Sameri, Durdam Das, Stefanie Michaelis, Joachim Wegener, Hedayatollah Hosseini, Martin Hoffmann. In vitro plasticity profiling of metastatic cancer cells with an imaging-based pipeline [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 5412.

Abstract 6944: Transcriptional programs underpinning reoccurring cancer cell states, plasticity and tumour phylogenies in clear cell kidney cancer

Abstract Intratumor heterogeneity (ITH) is a substrate for tumor evolution and confers amenability to stressors and therapies. Sources of ITH include subclonal mutations, microenvironmental factors, and plasticity whereby a cell has the ability to adopt different cell states. Clear cell kidney cancer (ccRCC) has a well-characterized genetic initiating event - VHL inactivation - resulting in activation of HIF-1 and HIF-2 transcription factors. Although VHL inactivation is consistently clonal in nature, ccRCC nonetheless exhibits extensive genetic and morphological ITH. This makes ccRCC an excellent model in which to study ITH. We performed single cell RNA-seq on multiregional samples from kidney tumors and macroscopically normal kidney tissue. We obtained 55 samples from 16 tumors and used the 10x Genomics platform to obtain gene expression data from ~150,000 single cells, including ~80,000 cancer and normal epithelial cells. We identified three cancer cell states, co-existing in all clear cell kidney cancer tumors analyzed and marked by different gene expression programs: 1) differentiated-like cells marked by proximal tubule cell genes characteristic of the cancer cell of origin; 2) epithelial-mesenchymal transition (EMT)-like cells and 3) injured-like cells. Differentiated markers and EMT markers were associated with good and poor prognosis, respectively, indicating that these cell subpopulations are clinically important. Notably, these cell states are also present in the normal proximal tubule although in different proportions to the tumor samples, which contain more cells in the EMT-like and injured-like states. This suggests that the core features of ITH are inherent to the cell of origin of the cancer, but that the cell state trajectories are dysregulated in the tumor. Furthermore, in tumors these states exist as a continuum, whereby transitional cells in between states appear to exist, suggesting that cancer cells are continually and dynamically transitioning between states. Lastly, we used chromosomal copy number variation (CNV) patterns to study genetic ITH. We examined the effect of 14q loss, which is associated with ccRCC metastasis, on gene expression. We found that 14q loss is associated with downregulation of HIF-1 transcriptional activity together with a reciprocal increase in HIF-2 activity, MYC activity and oxidative phosphorylation, highlighting potential mechanisms by which 14q loss may driver ccRCC metastasis. Citation Format: Olivia Lombardi, Ran Li, Lisa Browning, Faiz Jabbar, Hannah Evans, Peter J. Ratcliffe, David R. Mole. Transcriptional programs underpinning reoccurring cancer cell states, plasticity and tumour phylogenies in clear cell kidney 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 6944.

Abstract 2580: Targeting chromatin in ovarian cancer stem cells

Abstract The dynamic organization of chromatin, governed by epigenetic modifiers, critically regulates cell plasticity and determines cell fate in both normal and malignant stem cells. We hypothesized that chromatin architecture plays a role in regulating transcriptional heterogeneity in ovarian cancer stem cells (OCSCs) to maintain stemness and chemoresistance. PWS microscopy showed that ALDH+ OCSCs derived displayed higher nuclear fractal dimension D signals (P<0.05) than ALDH- non-OCSCs, indicating that chromatin in OCSCs possesses higher packing compared to non-OCSCs. Cut & TAG Sequencing showed that of the 3606-histone repressive H3K27m3 enriched regions (P<0.05, fold change >2), 3208 were detected in OCSCs while 398 peaks were present in non-OCSCs, consistent with findings supporting high D features in OCSCs. The active mark H3K4me3 was enriched at the promoter of stemness-associated genes, including SOX2, ALDH1A3, and FZD7 in OCSCs; and the repressive histone mark H3K27me3 was enriched at the promoter of the epithelial cell marker CDH1 in OCSCs. These results support that the chromatin of OCSCs contains open genomic regions, corresponding to stemness-associated genes and repressed regions associated with genes related to differentiation. RNA sequencing indicated that the coefficient of variance (COV) reflecting transcriptional heterogeneity in OCSCs was higher than in non-OCSCs at baseline. In response to platinum, genes with low baseline expression in OCSCs were most upregulated, supporting increased transcriptional heterogeneity in response to chemotherapy. Inhibitors of epigenetic modifiers (DNMTi, EZH2i, and Dot1Li) induced chromatin re-organization in OCSCs and promoted OCSCs transition toward differentiation, supporting that stemness-associated chromatin structure is targetable. We demonstrated that a Dot1L inhibitor (Dot1Li) targeted OCSCs by decreasing chromatin packing D, resulting in decreased transcriptional heterogeneity. In conclusion, OCSCs harbor highly packed chromatin, contributing to chemoresistance and cell plasticity. Epigenetic modifiers (such as Dot1Li) inhibit stemness by regulating chromatin organization and transcriptional malleability. Citation Format: Yinu Wang, Jane Frederick, Yaqi Zhang, Elizabeth Thomas Bartom, Greta Bauer, Edward Tanner, Vadim Backman, Daniela Matei. Targeting chromatin in ovarian cancer stem 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 2580.

Abstract 1577: Cancer associated fibroblast - tumor cell crosstalk enhances epithelial to mesenchymal transition and promotes a classical to basal switch in human pancreatic ductal adenocarcinoma

Abstract Introduction: Cancer associated fibroblasts (CAF) drive a complex tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) through incompletely understood interactions. Two major CAF subtypes, myofibroblastic (myCAF) and inflammatory (iCAF), dominate the tumor landscape and have been defined by gene expression profiles. There is significant plasticity between subtypes and alterations in CAF behavior likely drive changes in tumorigenicity and immune evasion. Additionally, PDAC tumor cells usually present with a predominant classical or basal phenotype that is associated with clinical outcomes. We hypothesized that CAF phenotype impacts tumor cell gene expression and alters the clinically-relevant tumor subtyping. Methods: We interrogated cellular crosstalk in the PDAC TME using a novel three-dimensional, patient-matched coculture system of patient-derived organoids (PDO) and CAFs, established from 12 patients undergoing surgical resection. Molecular characterization included bulk RNA sequencing, proteome profiling (107 factor), flow cytometry, multiplex RNA Scope, imaging mass cytometry (IMC), and RT-qPCR. Results: PDO gene expression analysis from bulk RNAseq demonstrated CAF coculture induced phenotypic shifts in the Moffitt subtype from a classical to a basal phenotype. Further, CAF coculture enhanced epithelial to mesenchymal transition (EMT) across all patients, even those with a baseline basal phenotype. We hypothesized this could be due to both cell-cell crosstalk and secreted factors. We therefore treated PDO with CAF-conditioned media (CM) and demonstrated that CM is sufficient to drive the observed classical-to-basal shift by RT-qPCR. Secretome analysis of CM derived from 3 CAF lines identified HGF, FGF19, and IL-8 as factors contributing to this transcriptional change. Next, we used spatial - omics (transcriptomics and proteomics) to examine the validity of these findings in source patient tissue. Using RNA Scope, we discovered distinct tumor neighborhoods expressing basal mRNAs KRT17 and KRT6a surrounded by CAFs compared to those expressing classical genes TFF1 and GATA6. We then used IMC to further explore CAF subtypes in patient tumors and found FAP+ myCAF regions corresponded to basal tumor regions, while CXCL12+ iCAFs were found in classical regions. Conclusions: PDAC tumor biology is likely driven by interactions between cancer cells and CAFs. Potential mediators of these interactions include both cell-cell contact and secreted factors. We introduce a novel approach combining in vitro coculture with tissue-based assays, demonstrating that modern culture techniques preserve cell type heterogeneity and plasticity. Together, these data demonstrate that a CAF-rich TME drives tumor cell plasticity not only towards EMT but also a more basal disease phenotype. Citation Format: Samantha Guinn, Brayan Perez, Jignasha Patel, Jae Lee, Daniel Zabransky, Won Ho, Elana J. Fertig, Emma Kartalia, Joseph Tandurella, Richard Burkhart, Jacquelyn W. Zimmerman, Elizabeth Jaffee. Cancer associated fibroblast - tumor cell crosstalk enhances epithelial to mesenchymal transition and promotes a classical to basal switch in human 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 1577.

Abstract 251: MCAM controls mammary cell state in luminal progenitor-derived breast cancer and alveologenesis

Abstract To establish and maintain functional and properly formed tissues, cells must integrate cues from their environment into fate decisions. External cues—from other cells and the extracellular matrix for instance—must be met with appropriate responses to ensure cell fate decisions that control lineage plasticity and favor proper development, maintenance, or regeneration of tissues while suppressing neoplastic transformation. MCAM (CD146; MUC18; S-endo-1; and Gicerin) is an integral membrane glycoprotein whose misexpression is associated with a variety of epithelial cancers, including breast cancer. However, the mechanisms by which MCAM conveys extracellular cues into malignant cellular outputs is not well understood. Our observation that MCAM is normally expressed in transplantable fetal and adult mammary stem cells (MaSCs) with multilineage potential, suggested it could control mammary cell state plasticity in both neoplastic and normal mammary tissue. Using a plastic, mouse mammary tumor cell line derived from the MMTV-PyMT mouse (Py230) that has been reported to exhibit mammary stem cell and luminal progenitor features, we tested the role of Mcam in controlling the tumorigenicity and lineage/cell-state relationships of mammary tumor cells. We found that Mcam knockdown in Py230 cells increases STAT3 expression, activation, and downstream target transcription, leading to a shift in the predominant cell state away from a luminal progenitor state toward more differentiated alveolar and basal phenotypes. Mechanistically, these changes involve altered activity of casein kinase II (CK2) against multiple substrates including STAT3 and PTEN. This effect may be mediated by direct coupling and sequestration of CK2 to various substrates via the cytoplasmic tail of MCAM. We have also found that inhibition of Ck2 or Stat3 reversed the transcriptional state change elicited by Mcam knockdown. In grafted tumors in vivo, this state switch correlates with reduced overall tumorigenicity and attenuated expression of Sox10, a gatekeeper of neural crest-like invasive features. In the normal gland, we hypothesize that this activity regulates multilineage stem cell potential upon transplantation and multilineage regenerative potential across lactation/involution cycles as we found reciprocal expression patterns for MCAM and STAT3 in alveolar mammary epithelial cells. These data point to an integral role for MCAM in lineage plasticity of normal mammary epithelial cells that is co-opted during transformation to potentiate tumor cell plasticity, progression, and evasion of targeted cancer therapies. Citation Format: Brooke L. Gates, Ozlen Balcioglu, David W. Freeman, Berhane M. Hagos, Benjamin T. Spike. MCAM controls mammary cell state in luminal progenitor-derived breast cancer and alveologenesis [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 251.

Abstract 4436: Phenotypic heterogeneity in pancreatic ductal adenocarcinoma revealed by single-cell lipidomics

Abstract Introduction: The high chemoresistance of pancreatic ductal adenocarcinoma (PDAC) is partly attributed to the phenotypic plasticity and high adaptability of PDAC cells to the challenges of the harsh microenviroment. Phenotypic presentation of the PDAC cell is also determined by the lipid composition of the plasma membrane and lipid species that organize the rigid or more flexible lipid bilayer and determine the cellular mobility and plasticity. Lipids are also important energy and signaling molecules that contribute to the aggressiveness of the cell. Here we focus on differences in lipid profile of PDAC cells in epithelial and mesenchymal cell state using single-cell lipidomics approach (SpaceM). Methods: Lipid profile of 2D cultures of eight conventional and ten primary patient derived cell lines of different source (patient derived xenograft, ascites, primary tumor, organoid derived) was investigated. Epithelial (E) or mesenchymal (M) cell state was evaluated by gene and protein expression of EMT markers. Matrix-Assisted Laser Desorption/Ionization (MALDI)-imaging MS was used for data acquisition on positive ion mode at 25 µm step size and m/z range of 600-1000. For annotating lipids in MALDI-imaging data, we used the METASPACE cloud software. For constructing single-cell lipidomic profiles we used the SpaceM method. In total, around 150.000 cells were analyzed and 1400 lipid ions detected. Results: Gene expression analysis suggested prominent metabolism of structural lipids in the conventional epithelial cells. In UMAP analysis of single cell lipidomics data, each cell line, conventional and primary, presented a unique lipid fingerprint with however prominent clustering into the two phenotypes, epithelial and mesenchymal. We detected enrichment of alkyl-acyl phosphatidylcholines, alkyl-acyl phospatidylethanolamines, ceramides and sphingolipids in the epithelial phenotype. Mesenchymal phenotype was enriched for lipids involved in lateral diffusion and low bilayer thickness supporting the increased fluidity of the mesenchymal cell membrane. Furthermore, mesenchymal phenotype was enriched for lipid droplets and energy storing triacylglycerols, also confirmed by histological lipid droplet staining. Conclusions: Using the single cell lipid information, a successful grouping to epithelial or mesenchymal subtype could be performed regardless of sample source, suggesting a conserved lipid profile typical for the two states. Further detailed investigation of single cell lipidomics data aims to unveil the prominent epithelial-mesenchymal state lipid markers and open a niche for functional investigations of cell-state specific markers and potential cell-state targeting. Citation Format: Marija Trajkovic-Arsic, Jeany Delafiori, Mohammed Shahraz, Corinna Münch, Smiths S. Lueong, Sven T. Liffers, Doris Hellerschmied, Theodore Alexandrov, Jens T. Siveke. Phenotypic heterogeneity in pancreatic ductal adenocarcinoma revealed by single-cell lipidomics [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 4436.

Abstract 4294: The emerging role of deubiquitinase enzymes in lung cancer plasticity

Abstract Lung cancer is characterized by a high degree of genetic and molecular heterogeneity. Lineage plasticity has emerged as a source of intratumoral heterogeneity and drug resistance, resulting in poor prognosis and treatment failure. The molecular mechanisms driving lung cancer plasticity remain unclear. Ubiquitin-Specific Peptidase 13 (USP13), a deubiquitinating enzyme, is one of the most amplified genes in lung squamous cell carcinoma (LUSC). We developed a USP13 knock-in overexpressing mouse model in KrasLSL-G12D/+; Trp53fl/fl (KP) background (KPU mice). USP13 overexpression resulted in aggressive tumorigenesis and a shortened survival in Kras/Trp53-driven lung cancer. Notably, while KP mice developed lung adenocarcinoma (LUAD), KPU mice developed LUSC and LUAD. LUSC in KPU mice faithfully recapitulated the key pathohistological, molecular features, and cellular pathways of human LUSC. Bulk RNA-sequencing analysis showed that KPU tumors were heterogeneous and enriched in lineage reprogramming pathways including basal cell signaling, stem cell pluripotency, and epithelial-mesenchymal transition (EMT). Using cell-type-restricted adenoviral Cre to target cells expressing surfactant protein C (SPC) or club cell antigen 10 (CC10), we identified bronchiolar secretory club cells as the predominant cell origin of LUSC. USP13 altered the levels of lineage transcription factors such as TTF-1 and SOX2 in club cells during early tumorigenesis. Altered expression of these factors reinforced the fate of CC10+ club cells to squamous carcinoma development rather than adenocarcinoma. In addition, USP13 directly acted on the K48-linked ubiquitination of c-Myc and increased its protein stability, contributing to the elevation of squamous gene expression (SOX2, CK5, P40) in the primary mouse and advanced human lung cancer cells. These results suggest that USP13 promotes lineage plasticity in club cells during the early stage of cancer development and drives reprogramming into LUSC. We also found a potential functional association between USP13 and EMT pathway. Notably, irrespective of cell origin, USP13 overexpression enhanced EMT marker expression including N-cadherin, SNAI1, and ZEB1 in lung cancer cells, promoting cell migration and invasion. USP13 increased tumorigenic and metastatic abilities of lung cancer cells in 3D organoid culture and syngeneic mouse models. Collectively, our research highlights the pivotal significance of USP13 in unleashing lineage plasticity during lung cancer progression, suggesting the potential role of deubiquitinase enzymes in regulating lineage plasticity, which may lead to novel therapeutics for treating lung cancer. Citation Format: Juntae Kwon, Jinmin Zhang, Boram Mok, Samuel Allsup, Cecil Han. The emerging role of deubiquitinase enzymes in lung cancer plasticity [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 4294.

Abstract 6940: The transcriptional and epigenetic regulation of epithelial-mesenchymal plasticity in patient-derived pancreatic cancer cell lines

Abstract Epithelial-mesenchymal transition (EMT) is a hallmark of pancreatic ductal adenocarcinoma (PDAC) invasion. In general, cellular plasticity is a major contributor to both tumor progression and therapy resistance. The major goals of our study are to 1) directly and quantitatively measure plasticity in PDAC3 cell lines, and 2) identify candidate genes that could alter the plasticity of cancer cells. By understanding the regulation of tumor cell plasticity, we may find ways to perturb cells and prevent the transition towards treatment resistant cell states. A strict definition of plasticity in cancer has yet to be established, however, and is imperative to our understanding of cell state regulation and for identifying vulnerabilities in plastic cells. We argue that in order to prove plasticity occurs within a population of cells, one must either 1) demonstrate that a new cell state has emerged that was previously not present, or 2) trace the lineage of a cell to show it can adopt multiple cell states. For our study, we profile 12 patient-derived PDAC cell lines to find convergent EMT programs and perform lineage tracing on these cell lines using a modified CROP-seq vector, called ClonMapper, to quantitatively measure plasticity. Towards this goal, we perform single-cell RNA-seq (scRNA) and single-cell multiomic measurements of 1000 uniquely barcoded cells expanded over a time course of 2, 3 and 4 weeks. We refer to cells that have the same lineage barcode - originating from the same initial cell - as families. We found that ~50% of families are composed of cells that are epithelial and mesenchymal, thus proving plasticity must exist, as it shows that a given parental cell of unknown state can give rise to progeny that are in multiple states. We then develop mathematical models to show that families have different levels of plasticity, defined by the rates of transition between distinct cell states. We next leverage our barcode-resolution time course data to identify factors at early time points that may influence plasticity at later time points. We show there is differential accessibility of several of these factors across families with different plasticity properties, thus proposing a role for epigenetic regulation of EMT. Importantly, we find that specific factors drive gene regulatory networks important for epithelial and mesenchymal programs, and therefore propose candidates for the modulation of EMT for therapeutic benefit. Citation Format: Deepika Yeramosu, Lynn Bi, Abigail Collins, Mike Bogaev, Martin Jankowiak, Aziz Al’Khafaji, Milan Parikh, Mehrtash Babadi, Alex Bloemendal, Surya Nagaraja, Ray Jones, Jay Shendure, David T. Ting, Andrew Aguirre, Nir Hacohen, Dana Pe’er, Eric S. Lander, Arnav Mehta. The transcriptional and epigenetic regulation of epithelial-mesenchymal plasticity in patient-derived pancreatic cancer cell lines [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 6940.

Abstract 5458: Plasticity of CAF-like states in Ewing sarcoma is mediated by TGF-beta

Abstract Transcriptional plasticity of cancer cells promotes tumor progression and treatment resistance via generation of phenotypically distinct cell states. We previously showed that Ewing sarcoma (EwS) cells exhibit inter- and intra-tumoral transcriptional heterogeneity and that they undergo state changes in response to cues from the tumor microenvironment (TME). More recently, we identified phenotypically distinct EwS tumor cell subpopulations in vivo that deposit pro-tumorigenic extracellular matrix (ECM) proteins, adopting similar functions to cancer-associated fibroblasts (CAFs). Here, we sought to identify the upstream regulators of these CAF-like EwS cells with the goal of defining molecular mechanisms that control EwS plasticity and creation of ECM-remodeling states. Integration of single cell proteogenomic sequencing (CITE-seq) with immunofluorescence and digital spatial profiling of cell line xenografts and patient tumors validated the cell-surface protein CD73 (NT5E) as a biomarker of ECM-secreting EwS cell subpopulations in vitro and in vivo. The transcriptomes of CD73+ CAF-like cells were compared to isogenic CD73- cells (n=9 cell lines) and highly significant enrichment of gene ontology terms related to TGF-beta signaling (p.adj = <4.4x10-14) was noted. EwS cells were previously reported to be non-responsive to TGF-beta due to profound transcriptional repression of the TGF-beta type 2 receptor (TGFBR2) by EWS::FLI1. In support of this, TGFBR2 expression was low in bulk measurements of EwS cells. However, TGFBR2 mRNA and surface protein expression were significantly increased in CD73+ subpopulations. Exposure of EwS cells to TGF-beta ligands (TGFB1 or TGFB2) strongly induced the CAF-like gene signature, including ECM genes BGN, COL1A1, SPARC, and TNC (p. adj < 8.5x10-5), and this was augmented in CD73+ cell populations. Transduction of EwS cells with dominant negative-TGFBR2 inhibited both basal and TGF-beta-induced expression of CAF-like signature genes and blocked SMAD2 phosphorylation. To test the functional significance of the TGF-beta response, unsorted EwS cells were plated in 2D or as tumor spheroids in collagen I-rich 3D cultures and exposed to TGFB1 or SB-505124, a TGF-beta inhibitor. TGFB1 treatment had no effect on cell viability or proliferation in 2D but increased invasion into collagen in 3D. Inhibition of TGF-beta reduced CAF-like gene expression in 2D cultures and diminished both viability and invasion in 3D. Finally, our studies revealed that NT5E (CD73) is a TGF-beta induced target in EwS cells and that CD73+ cells upregulate expression of TGFB2. Thus, an autocrine signaling circuit is created in which TGF-beta activity in CAF-like tumor cell subpopulations is sustained by CD73+ tumor cell-derived ligand. Together these studies demonstrate that TGF-beta is a key determinant of cell plasticity and ECM remodeling in EwS. Citation Format: Emma Wrenn, April A. Apfelbaum, Katherine Braun, Erin R. Rudzinski, Xuemei Deng, Aya Miyaki, Trisha Lipson, Nicolas M. Garcia, Virginia J. Hoglund, Elizabeth R. Lawlor. Plasticity of CAF-like states in Ewing sarcoma is mediated by TGF-beta [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 5458.

Abstract 1721: Epigenetic heterogeneity of osteosarcoma reveals distinct cellular states driven by enhancer regulation

Abstract Osteosarcoma (OS) is characterized by structural rearrangements and copy number changes which drive considerable heterogeneity. What is less clear is how these genetic rearrangements alter are in turn regulated at the epigenetic level to drive gene expression. Defining the epigenetic circuitry of osteosarcoma could help to understand the role of chromatin accessibility in determining cell plasticity. Chromatin accessibility can prime gene expression and define a cellular state. As chromatin accessibility is a major determinant of epigenetic transcriptional regulation, understanding the variability of chromatin accessibility could identify clinically-relevant subtypes and biomarkers of therapy response in Osteosarcoma, a disease that has seen few advances in therapy for over 40 years. Using ATAC-seq, we identified two epigenetic subtypes (EC1 and EC2) of OS with distinct chromatin accessibility. These studies were done in a panel of 11 PDX-derived cell lines and 9 established OS cell lines. We also performed ATACseq on the matching PDX for the 11 PDX-derived lines as well as 8 primary tumor samples. Analysis of transcription factor (TF) motifs and footprints of ATAC-seq peaks identified transcription factors distinctly activated in these two subtypes. For example, EC1 is characterized by high level of activity of developmental transcription factors including RUNX2, MEF2C and homeobox TFs. In contrast, EC2 is characterized by high activity of AP1 transcription factors such as FOSL1 and FOSL2. In order to understand the underlying transcriptional circuitry of these cell states, we performed CUT&RUN assay for the complete panel of PDX-derived cell lines. We found that the subtypes are driven by enhancer regulation (H3K27ac). We defined the specific Core Regulatory Circuitry (CRC) per subtype by defining the superenhancers by ROSE. We overlapped ATAC-seq data in the acetylated regions to demarcate TF binding regions dramatically and reduce the search space for TF cis-regulatory sites. In parallel, we used our PDX-derived cell line panel to screen cluster-specific targeted therapies and found differential responses dependent on cellular states defined by chromatin accessibility. For example, cell lines in the EC2 cluster are marked by upregulation of AP1 transcription factors, have evidence of ERK activation and are highly responsive to ERK inhibitors. In contrast cell lines in the EC1 cluster are sensitive to AURKA and AURKB inhibitors. Single-cell RNAseq analysis indicates that these subtypes may coexist within a single tumor. In summary, we discovered two epigenetically distinct cell states that are controlled by a state-specific collection of transcription factors, creating a gene signature that may aid in distinguishing osteosarcoma subclasses and predicting treatment response. Citation Format: Eunice Lopez Fuentes, Andrew Clugston, Alex Lee, Leanne Sayles, Maria Pons, Alejandro Sweet-Cordero. Epigenetic heterogeneity of osteosarcoma reveals distinct cellular states driven by enhancer regulation [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 1721.

Abstract 1100: Fascin-1 inhibitor decreases hepatoblastoma cells tumorigenicity via YAP 1

Abstract Introduction: Hepatoblastoma (HB) is a liver tumor that arises in children. It's a sporadic malignancy that is often overly aggressive. The current treatment consists of chemotherapy. However, chemotherapy in young patients has disastrous and long-term side effects such as ototoxicity, cardiomyopathy, and infertility. Thus, alternative strategies are needed. One hint is to target the most common mutations in HB. It has been demonstrated that 90% of HB tumors are mutated for the Wnt pathway effector ß-catenin. This mutation leads to an aberrant constitutive activation of Wnt/ß-catenin signaling. Here, we investigate one of ß-catenin transcriptional targets, Fascin-1 that is found up-regulated in many tumors. Fascin1 affects actin organization into bundles, and this leads to cell migration and invasion. Whereas Fascin-1 is absent from normal hepatocytes, we found its expression associated to the poor prognosis C2 subtype of HB. In both human and murine HB samples, Fascin-1 is associated to undifferentiated tumor cells. We further demonstrated that Fascin-1 expression modulates tumor hepatocyte differentiation status through gene expression. In this study, we investigate how Fascin-1 can regulate tumor cell plasticity and whether Fascin-1 is a druggable target in HB tumors. Methods: We use two classical HB model cells Huh6 and HepG2 and 3 Patient-Derived-Xenograft cell lines. We explore the effect of Fascin-1 actin-bundling activity impairment by using inhibitors, on invasion and migration using Trans-well and wound-healing assays. We follow proliferation and cell death by Flow cytometry and investigate gene expression by PCR and reporter assay. We investigate Fascin modulation of the kinome with PamGene technology. Results: We show that the inhibition of Fascin actin-binding activity decreases cell invasion and migration as well as proliferation. We show an increase of cell death in Huh6/HepG2 cells but not in the PDX models. A cell cycle arrest is induced, and the cells become senescent. Differentiation genes are overexpressed and EMT genes are repressed. Yap expression is downregulated; Yap promoter activity is downregulated, and Yap is found translocated into the cytoplasm upon Fascin-1 inhibition. These data suggest that Fascin inhibition decreases tumors cell invasion, induces cell senescence, and favors a switch towards a differentiated and these effects on the cells are mediated via the Hippo pathway. The kinome is also modulated. Conclusion: Fascin-1 is an interesting target in hepatoblastoma, commercialized phase-2 drugs are available, and this study will confirm the potential use of those drugs in HB treatment and elucidate by which mechanism Fascin-1 inhibition impacts tumors. Citation Format: Lydia DIF, Amandine Martin, Sara Basbous, Gregoire Manaud, Violaine Moreau. Fascin-1 inhibitor decreases hepatoblastoma cells tumorigenicity via YAP 1 [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 1100.

Abstract 1786: Metabolic rewiring induced by protein or calorie restriction provides unique therapeutic vulnerabilities in translocation and select clear cell renal cell carcinomas

Abstract While the approval of several combinations with immune checkpoint inhibitors have significantly improved the outcome of kidney cancer patients, advanced clear cell and non-clear cell renal cell carcinoma (RCC) remain a clinical challenge. There has been a longstanding interest in using dysregulated metabolism as a therapeutic target due to its association with the pathogenesis and progression of several cancer types. As one of these highly metabolic tumors, RCC has the potential to greatly benefit from strategies targeting tumor metabolism. Dietary interventions are an innovative adjunct therapy that has the potential to induce a wide range of anti-cancer effects by disrupting metabolism due to the role diet plays in providing tumors with macromolecules for energy metabolism. Unfortunately, tumor cell plasticity, intratumor heterogeneity, and a lack of understanding regarding metabolic vulnerabilities in tumors limit the efficacy of implementing these strategies and a fundamental gap in the field remains as to how basic metabolic requirements of the tumor can be targeted with specific dietary interventions. In this study, the inherent metabolic phenotypes of translocation RCC (tRCC) and clear cell RCC (ccRCC) were characterized by integrating in vivo metabolomics and transcriptomics data, assessing mitochondrial and glycolytic activity in vitro via Seahorse, and performing immunohistochemical analyses of patient samples. Interestingly, the respective mitochondrial and glycolytic dependencies in tRCC and ccRCC patient-derived xenograft (PDX) models appear to influence the response to nutrient deprivation as seen by in vitro assays conducted after amino acid or glucose restriction. Importantly, these results indicate that dietary interventions may be able to selectively target metabolic vulnerabilities in tRCC and ccRCC. Ongoing combination studies with either protein or calorie restriction and targeted therapies, such as standard of care receptor tyrosine-kinase inhibitors, suggest the benefit of using dietary interventions as an adjunct therapy in RCC. Overall, these studies reveal that selective dietary interventions alter the metabolic landscape of RCC and expose vulnerabilities that can be exploited with targeted therapy. As dietary interventions are a safe, affordable, and potentially well tolerated adjunct therapy, we anticipate that the results of these studies will provide the biologic and mechanistic support for translating this strategy to advanced RCC patients. Citation Format: Sabrina A. Orsi, Christopher Rupert, Sean Colligan, Musea Chang, Abbas Jawadwala, Ilaria Delle Fontane, Filomena de Nigris, Roberto Pili. Metabolic rewiring induced by protein or calorie restriction provides unique therapeutic vulnerabilities in translocation and select clear cell renal cell carcinomas [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 1786.

Abstract 6938: Identification of distinct tumor-TME ecomodules in glioma from neurofibromatosis type 1

Abstract Neurofibromatosis type 1 (NF-1) is the most common cancer predisposition syndrome in which 15-20% of affected individuals develop glioma. Large scale DNA and RNA bulk profiling showed the molecular complexity of NF-1 glioma with the tumor cellular ecosystem constituted by multiple malignant phenotypes and heterogenous immune microenvironment. However, the composition and function of infiltrating cells was hidden in the bulk tumor, and the extended granularity of NF-1 glioma tumor microenvironment (TME) remained still unexplored. Here, we collected glioma samples from 46 NF-1 patients including 22 high-grade (HGG) and 24 low-grade (LGG) tumors, and we analyzed their gene expression by single nuclei RNA sequencing. A total of 239,044 single cells were classified into tumor and non-tumor components by integrating multiple computational approaches (including genomic copy number inference, gene signature enrichment, and clustering). We defined the pattern of intra-tumor heterogeneity of NF-1 glioma cells using non-negative matrix factorization and derived 7 malignant meta-programs (MPs) that we respectively defined as Neuronal-like, EMT, Astrocyte-like, Dividing Radial Glia-like, Ependymal-like, Immune, and Glycolytic/Hypoxic-like. These MPs recapitulated normal brain cell subtypes, thereby reflecting broad cell plasticity. The non-tumor cell compartment (121,364 cells, 51%) was dissected for the characterization of the cell types that populate the TME of NF-1 glioma. We identified different subpopulations exhibiting specific immune functions within myeloid and lymphoid components. Different glioma ecomodules were highlighted by comparing the relative composition of the TME across the tumors. Recruitment and activation of cytotoxic CD8+ T cells and natural killers by an active crosstalk with dendritic and pro-inflammatory myeloid cells defined an immune-supportive phenotype that could mediate a potential anti-tumor response in low-grade NF-1 glioma (LGG immune high). Conversely, regulatory T cell infiltration and effector T cell exhaustion induced immune suppression in a low-grade glioma immune dysfunctional ecomodule. The absence of lymphocytes characterized a large set of cold tumors, mostly including high-grade glioma. Together, the complex interplay of tumor cell states with different TME compartments elucidated the existence of separate ecomodules in NF-1 glioma, with the LGG immune high TME associated with Neuronal-like and the LGG immune dysfunctional with Ependymal-like tumor cells. The Ependymal-like state also exhibits maximal association with brain-specific normal cells, including oligodendrocytes, neurons and astrocytes, whereas the HGG are enriched with Dividing Radial Glia- and Glycolytic/Hypoxic-like tumor cell states. The elucidation of different ecomodules provides novel insights for the application of targeted therapies in NF-1 glioma patients. Citation Format: Luciano Garofano, Fulvio D'Angelo, Michael Oh, Michele Ceccarelli, Franck Bielle, Marc Sanson, Anna Lasorella, Antonio Iavarone. Identification of distinct tumor-TME ecomodules in glioma from neurofibromatosis type 1 [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 6938.

Abstract 6958: CRIPTO-regulated extracellular vesicles mediate heterotypic cellular crosstalk in triple negative breast cancers

Abstract Tumor progression involves dynamic interaction between various cell types within the tumor microenvironment. Fibroblasts, which normally participate in wound healing, reciprocally communicate with breast cancer cells to promote tumor cell plasticity and heterogeneity. Gaining insights into the molecular mechanisms underlying this cell-to-cell communication, could unlock novel therapeutic avenues for inhibiting cellular plasticity, breast cancer progression and metastasis. The small GPI-anchored adaptor protein CRIPTO is a key player in this context though important molecular details remain to be elucidated. CRIPTO orchestrates TGF-β pathway signaling as well as growth factor-like signaling pathways that activate c-Src/MEK/AKT, in an autocrine and paracrine manner. CRIPTO is expressed during mammary development, wound healing, and breast cancer but is largely undetectable in homeostatic tissue, making it a promising therapeutic target. Our previous studies have demonstrated that the activity of CRIPTO is contingent upon cellular stress, with pronounced influence within pro-fibrotic, hypoxic domains of tumors. Importantly, we have shown that inhibition of CRIPTO using the soluble recombinant protein antagonist A4Fc effectively impedes the progression of triple negative breast cancer (TNBC) in murine xenograft models. Building upon these findings, we have been dissecting CRIPTO’s role in coordinating fibroblast activation and cell-state reprogramming in TNBC. We find that inhibition of CRIPTO with A4Fc suppresses cancer associated fibroblast (CAF) activation and collagen remodeling in vivo. Further, by modeling tumor cell:fibroblast crosstalk in vitro, we have demonstrated mechanistically that CRIPTO expression in tumor cells mediates subsequent extracellular vesicle (EV) uptake by fibroblasts, as well as fibroblast SMAD pathway activation and priming for reciprocal signaling back to tumor cells to promote transcriptional reprogramming and cellular invasion.The significance of these findings lies in their elucidation of key EV control mechanisms that could be exploited to intervene in essential tumor cell:fibroblast crosstalk or to enhance targeting of therapeutic moieties based on EV architecture and function in cancer and/or normative regenerative processes. Citation Format: David W. Freeman, Brooke L. Gates, Mauri D. Spendlove, Benjamin T. Spike. CRIPTO-regulated extracellular vesicles mediate heterotypic cellular crosstalk in triple negative breast 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 6958.

Abstract 6626: Functional and molecular interrogation of a high-plasticity cell state in lung cancer

Abstract Despite recent advancements in lung adenocarcinoma (LUAD) treatment, mortality remains high. Thus, a need exists for novel therapeutic concepts to enhance treatment efficacy and improve patient survival. Cancer cell plasticity, the capacity for cancer cells to differentiate and adapt to cell-extrinsic pressures, drives LUAD progression and enables treatment resistance. We discovered a molecularly distinct "high-plasticity cell state" (HPCS), which emerges during the initial stages of tumor development and is maintained in established tumors, comprising 10-15% of the cancer cell pool. We hypothesized that the HPCS is critical for tumor progression and drug resistance. To functionally interrogate this cell state, we engineered a novel genetic system that enables both in situ ablation (via a suicide gene) and longitudinal lineage tracing (via secreted luciferases) of the HPCS in an autochthonous genetically engineered mouse LUAD model. Lineage-tracing revealed the HPCS harbors high differentiation potential (plasticity) and growth potential in situ. Strikingly, ablation of the HPCS led to robust tumor regression and growth suppression. Motivated by these findings, we sought to identify molecular drivers of the HPCS using an in vivo multiplexed gene perturbation approach coupled to single-cell RNA sequencing. This effort led to the identification of central transcriptional plasticity drivers. This study motivates the therapeutic targeting of the HPCS in lung cancer and uncovers candidate targets. Citation Format: Jason Earl Chan, Chun-Hao Pan, Carleigh Sussman, Hannah Styers, Emma Brown, Griffin Hartmann, Brigita Meškauskaitė, Melissa Blum, Selena Ding, Stefan Torborg, Zhuxuan Li, Simon Joost, Eliud Rivas-Hernandez, Thomas Norman, Ronan Chaligne, Yan Yan, Tuomas Tammela. Functional and molecular interrogation of a high-plasticity cell state in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6626.