Progranulin and Inflammation-Induced Cancer: An Important Player in the Tumor Microenvironment?

e14553 Background: The tumor microenvironment (TME) plays a crucial role in tumor growth and progression and has a significant influence on response to therapy. The TME consists of myeloid-derived cells, stroma (e.g. fibroblasts and extracellular matrix (ECM)), and the vasculature that together support tumor growth and progression. Studies in animal models and in humans show that myeloid- derived cells such as myeloid derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs) engage in activities that support tumor growth and progression. These cells also promote immune suppression in the TME that blunts the efficacy of the anti-cancer drugs and immune-targeted therapies such as immune checkpoint inhibitors. In addition to MDSCs, cancer-associated fibroblasts (CAFs) in the TME support tumor progression via production of pro-tumor and pro-angiogenic growth-factors, remodeling of the ECM via production of proteases, and suppression of anti-tumor immune function. CAF abundance in the TME is a negative prognostic factor for several solid tumors and is associated with negative outcomes and poor response to immune-targeted therapies like immune checkpoint inhibitors. ONP-302 nanoparticles fabricated from biodegradable poly (lactic-co-glycolic acid)(PLGA) polymer have been previously described in the literature for the treatment of acute inflammatory conditions via immuno-modulatory effects on myeloid derived cells. Here, we evaluated the efficacy of ONP-302 nanoparticles at inhibiting tumor growth via targeted inhibition of myeloid-derived cells and reshaping of the TME. Methods: ONP-302 anti-tumor efficacy was evaluated in syngeneic mouse tumor models using both immunocompetent and immunodeficient mice. We examined the effect of ONP-302 treatment tumor growth kinetics and effects on the major cellular constituents of the TME such as myeloid-derived cells and CAFs. Results: Therapeutic treatment with ONP-302 in vivo resulted in a marked delay in tumor growth in three different syngeneic tumor models in immunocompetent mice. ONP- 302 efficacy persisted with depletion of CD8+ T cells in immunocompetent mice and also was effective in immune deficient mice. We found ONP-302 treatment caused a gene expression shift in TAMs toward the pro-inflammatory M1 type and substantially inhibited the expression of genes associated with the pro-tumorigenic function of CAFs. ONP-302 also induced apoptosis in CAFs in the TME. Conclusions: Our data indicate that the slowing of tumor growth after ONP-302 treatment is due to disruptions in known signaling pathways involving TAMs and CAFs, pathways typically supporting tumor growth. These data taken in concert indicate the activity of ONP-302 is pleotropic and affects multiple pathways.

PGRN mediates the crosstalk between ovarian cancer cells and CAFs and reshapes tumor immune microenvironment.

Myeloid-derived suppressor cells (MDSC) accumulate in patients and animals with cancer where they mediate systemic immune suppression and obstruct immune-based cancer therapies. We have previously demonstrated that inflammation, which frequently accompanies tumor onset and progression, increases the rate of accumulation and the suppressive potency of MDSC. To determine how inflammation enhances MDSC levels and activity we used mass spectrometry to identify proteins produced by MDSC induced in highly inflammatory settings. Proteomic pathway analysis identified the Fas pathway and caspase network proteins, leading us to hypothesize that inflammation enhances MDSC accumulation by increasing MDSC resistance to Fas-mediated apoptosis. The MS findings were validated and extended by biological studies. Using activated caspase 3 and caspase 8 as indicators of apoptosis, flow cytometry, confocal microscopy, and Western blot analyses demonstrated that inflammation-induced MDSC treated with a Fas agonist contain lower levels of activated caspases, suggesting that inflammation enhances resistance to Fas-mediated apoptosis. Resistance to Fas-mediated apoptosis was confirmed by viability studies of MDSC treated with a Fas agonist. These results suggest that an inflammatory environment, which is frequently present in tumor-bearing individuals, protects MDSC against extrinsic-induced apoptosis resulting in MDSC with a longer in vivo half-life, and may explain why MDSC accumulate more rapidly and to higher levels in inflammatory settings.

Integrated molecular analysis demonstrated that colorectal cancer can be classified into four molecular subtypes (proliferative, immunomodulatory, immunosuppressed, and immune-excluded subtypes), providing valuable insight into the intricate relationship between tumor microenvironment heterogeneity and various clinical phenotypes.

It is well established that multifactorial drug resistance hinders successful cancer treatment. Tumor cell interactions with the tumor microenvironment (TME) are crucial in epithelial-mesenchymal transition (EMT) and multidrug resistance (MDR). TME-induced factors secreted by cancer cells and cancer-associated fibroblasts (CAFs) create an inflammatory microenvironment by recruiting immune cells. CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSCs) and inflammatory tumor associated macrophages (TAMs) are main immune cell types which further enhance chronic inflammation. Chronic inflammation nurtures tumor-initiating/cancer stem-like cells (CSCs), induces both EMT and MDR leading to tumor relapses. Pro-thrombotic microenvironment created by inflammatory cytokines and chemokines from TAMs, MDSCs and CAFs is also involved in EMT and MDR. MDSCs are the most common mediators of immunosuppression and are also involved in resistance to targeted therapies, e.g. BRAF inhibitors and oncolytic viruses-based therapies. Expansion of both cancer and stroma cells causes hypoxia by hypoxia-inducible transcription factors (e.g. HIF-1α) resulting in drug resistance. TME factors induce the expression of transcriptional EMT factors, MDR and metabolic adaptation of cancer cells. Promoters of several ATP-binding cassette (ABC) transporter genes contain binding sites for canonical EMT transcription factors, e.g. ZEB, TWIST and SNAIL. Changes in glycolysis, oxidative phosphorylation and autophagy during EMT also promote MDR. Conclusively, EMT signaling simultaneously increases MDR. Owing to the multifactorial nature of MDR, targeting one mechanism seems to be non-sufficient to overcome resistance. Targeting inflammatory processes by immune modulatory compounds such as mTOR inhibitors, demethylating agents, low-dosed histone deacetylase inhibitors may decrease MDR. Targeting EMT and metabolic adaptation by small molecular inhibitors might also reverse MDR. In this review, we summarize evidence for TME components as causative factors of EMT and anticancer drug resistance.

CD73, an ecto-5′-nucleotidase, is the major extracellular adenosine-generating enzyme that converts AMP to immunosuppressive adenosine (ADO). Previous studies demonstrated that CD73-mediated ADO production in the tumor microenvironment (TME) is an immune evasion mechanism explored by tumors, myeloid-derived suppressor cell (MDSCs), and regulatory T cells. Targeted suppression of CD73, either via CD73 blocking antibody or inhibitors, suppresses tumor progression and metastasis. Given the complexity of the TME and crucial involvement of cancer-associated fibroblasts (CAFs) in immunosuppression and tumor progression, we explored the contribution of CD73 in CAFs to tumor progression. Using a CD73-negatvie EL4 tumor in C57BL/6 WT and CD73KO mice, we observed a high level of CD73 expression in CAFs, associated with their higher capacity of converting extracellular AMP to ADO than that of lymphocytes and myeloid cells in culture. Interestingly, as tumor progresses, CAFs in the TME of WT mice expended extensively to form an interconnected network and the percentage of CD73+ CAFs increases, while that of tumor infiltrating lymphocytes, especially CD8 T cells, decreases. However, in CD73KO mice, EL4 tumor progression was greatly delayed, associated with their significantly higher level of tumor-infiltrating CD8+ cytotoxic T cells. Functional analysis of fibroblastic stromal cells confirmed that CD73 inactivation greatly diminished their ability to generate extracellular ADO and subsequent suppression of T cell activation. Our results suggest that CD73 also represents one of the important mechanisms of CAF-mediated immunosuppression. We will discuss the potential environmental factors that enforce CD73 expression in CAFs.

The dendritic cell (DC) -based vaccine immunotherapy has been a promising cancer immunotherapy, but has been insufficient to eradicate the tumor in patients with advanced cancer. This can result from the complicated tumor microenvironment (TME) that is implicated in suppression of anti-tumor immune responses. Several immune cell types in TME, such as Tregs, myeloid derived suppressor cells (MDSC) and tumor-associated macrophages (TAMs), have been reported to regulate anti-tumor immune responses negatively. Cancer-associated fibroblasts (CAFs) are also primary stromal cells in TME, and contribute to tumor growth and metastases through the secretion of TGF-β and stromal cell-derived factor-1 (SDF-1). We considered that TME-targeted strategies should be innovated for the development of the potent cancer immunotherapy. On the basis of these viewpoints, we focused on the role of CAFs in TME, and hypothesized that inhibition of CAFs would lead to improvement of systemic anti-tumor immune responses and enhancement of the potency of the DCs-based vaccine immunotherapy. In this study, we applied tranilast in order to inhibit CAFs, the anti-fibrotic and -allergic agent that is used clinically and has been shown to inhibit fibroblast in the scar tissue. In in vitro studies, we examined effects of tranilast on CAFs that were isolated from established EG7 (mouse lymphoma cells) tumors. As results, tranilast was able to suppress the proliferation of CAFs, and decrease the production of SDF-1 as well as TGF-β from CAFs. Regarding the effect on Tregs, tranilast was able to decrease the induction of them from spleen cells of normal mice. Based on these results, we confirmed that tranilast could inhibit the function of CAFs. Next, we examined the association between inhibition of CAFs and anti-tumor immune responses in tumor-bearing mouse model. C57BL/6 mice bearing EG7 were administered tranilast into the established tumor in combination with tumor antigen-loaded DCs vaccination, and were evaluated anti-tumor immune responses. As results, the population of CAFs was decreased by targeting them, leading to lower expressions of TGF-β as well as SDF-1 in TME. Inhibition of CAFs in TME resulted in the decreased distributions of Tregs in TME and tumor-draining lymph nodes (TDLs). On the induction of effector cells, antigen-specific CD8+ cells producing IFN-γ were significantly increased in TDLs and spleen through inhibition of CAFs in TME. In these mice, systemic antigen-specific cytotoxic responses were augmented, leading to suppression of tumor growth as compared with mice in control groups. These results demonstrate that CAFs are associated with immune suppression, and inhibition of CAFs functions in TME can augment systemic anti-tumor immune responses. Our mouse models provide a new rationale with TME-targeted strategies for enhancing the potency of the DCs-based vaccine immunotherapy. Citation Format: Yasuhiko Ohshio, Ryosuke Kaku, Keiko Ishida, Masayuki Hashimoto, Shoji Kitamura, Koji Teramoto, Jun Hanaoka, Noriaki Tezuka. The cancer-associated fibroblasts-targeted strategycan augment the potency of the dendritic cell-based vaccine immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4945. doi:10.1158/1538-7445.AM2013-4945

Background: The pancreatic ductal adenocarcinoma tumor microenvironment plays an important role in promoting the epithelial to mesenchymal transition (EMT), an early event in pancreatic cancer, involved in cancer invasiveness and in tumor progression. Among the stromal components the cancer-associated fibroblasts (CAFs) are responsible for the peculiar pancreatic tumor microenvironment and are known to be linked to the induction of EMT. The EMT process requires a dynamic remodeling of the actin cytoskeleton and we have suggested that the splicing program of hMENA, an actin regulator, play a role in EMT. Two alternatively expressed isoforms, hMENA11a and hMENAΔv6, with opposite functions in invasiveness have been described in breast cancer (Di Modugno et al PNAS 2012). hMENA expression has not been detected in normal pancreatic ducts, whereas expressed in the human pancreatic ductal adenocarcinoma (PDAC) samples, but no data are available on hMENA alternative isoform expression in this neoplasia. The aim of this study is to investigate whether TGFβ1-mediated EMT in pancreatic cancer cells is affected by hMENA overexpression and splicing and how CAFs affect this process in cancer cell lines and in human tissues. Methods: hMENA isoform expression was evaluated in PDAC tissues by immunohistochemistry using isoform specific antibodies. hMENA isoforms and EMT markers expression were characterized in human PDAC cell lines, TGFβ1-treated or untreated, by qRT-PCR and WB analysis. The effects of either hMENA isoform specific knockdown or overexpression in the TGFβ1-induced EMT were also evaluated. Pancreatic CAFs were isolated from human tissues of resected PDAC patients. The effect of the conditioned medium of cultured CAFs was evaluated on hMENA expression. In parallel, the role of CAF-cancer cell interaction on the expression of the different hMENA isoforms was analysed using a co-culture system. Results: Freshly explanted CAFs expressed the “mesenchymal” hMENAΔv6, and not hMENA11a and secreted paracrine factors involved in the induction of hMENA isoforms in tumor cells. In a panel of pancreatic cancer cell lines, hMENA11a expression correlated with an epithelial phenotype, while hMENAΔv6 expression was correlated with a mesenchymal phenotype. Interestingly, the expression of the invasive hMENAΔv6 isoform is specifically up-regulated by TGFβ1 treatment. hMENA isoform expression levels influenced molecular changes induced by TGFβ1. Thus, the hMENA11a specific silencing led to E-cadherin down-regulation that is more evident in TGFβ1 treated cells. On the contrary, hMENA11a overexpression led to a reduction of vimentin expression and to E-cadherin up-regulation. Knockdown of the endogenous hMENA/hMENAΔv6 isoform expression prevented the activation of TGFβ1 signaling and up-regulation of mesenchymal markers. In addition, hMENA/hMENAΔv6 isoform depletion impaired the TGFβ1-induced invasiveness, migration and production of MMPs. IHC analysis of PDAC tissues revealed that the epithelial hMENA11a is rarely expressed in primary pancreatic tumour, while high levels of hMENA and hMENAΔv6 isoforms were found in 75% of primary tumours analysed. Conclusions: This data suggests that the lack of the epithelial hMENA11a isoform is an early event in pancreatic cancer, provides new insights into the role of hMENA splicing in TGFβ1-mediated EMT and highlights hMENA splicing program as an attractive pathway for the development of new therapies in PDAC. Citation Format: Roberta Melchionna, Pierluigi Iapicca, Francesca Di Modugno, Paola Trono, Novella Gualtieri, Maria Grazia Diodoro, Marcella Mottolese, Gian Luca Grazi, Matteo Fassan, Aldo Scarpa, Mina J. Bissell, Paola Nisticò. The hMENA Splicing Program: An important regulator of TGFβ1-driven EMT and invasiveness in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A60.

PGRN promotes migration and invasion of epithelial ovarian cancer cells through an epithelial mesenchymal transition program and the activation of cancer associated fibroblasts

Immune checkpoint inhibitors show insufficient efficacy against pancreatic ductal adenocarcinoma (PDAC). The tumor microenvironment (TME) has a remarkable influence on responsiveness to cancer immunotherapy. The aim of this study was to investigate immunosuppressive characteristics of TME in PDAC tissues. The flow cytometry (FCM) of PDAC surgical specimens revealed that the profile of tumor-infiltrating leukocytes was classified into myeloid cell- and T-cell-dominant subtypes; the myeloid subtype was associated with poorer patient outcomes. Myeloid-derived suppressor cells (MDSCs) showed the highest hazard ratio among various myeloid cell types. Single-cell RNA sequencing and FCM revealed that most MDSCs, but not lymphocytes, in PDAC tissues characteristically express CD74. Macrophage migration inhibitory factor (MIF), a CD74 ligand, was highly expressed in cancer-associated fibroblasts (CAFs) and cancer cells. Spatial transcriptomics demonstrated that the MIF-CD74+ myeloid cell interaction was recognized in CAF-dominant areas in PDAC tissue. CAFs expressing immune suppressor molecules such as MFAP5 and LRRC15 were consistent with MIF+ CAFs. Furthermore, MIF+ CAFs enhanced the migratory activity of MDSCs and promoted MDSC induction and activation. In the murine model, MDSCs were significantly increased in MIF-expressing PDAC tumors, as were CD74+ M-MDSCs per M-MDSC, confirming in vivo interaction between CD74 and MIF. MDSCs play a crucial role in creating an immunosuppressive TME in PDAC; the MIF-CD74 axis drives interactions between MDSCs and CAFs.

Despite constant efforts to improve treatment, the prognosis remains poor, with an overall survival rate of 6% (ranges from 2% to 9%) due to lack of efficient chemotherapy, radiotherapy, and targeted therapy. Immune checkpoint inhibitors (ICIs) has been reported to be effective in various solid cancers, such as melanoma, lung cancer, and renal cell carcinoma. However, pancreatic ductal adenocarcinoma (PDAC) is resistant against ICIs. The responsiveness of immune therapy is mainly determined by immune tumor microenvironment (TME) composed of tumor-infiltrating lymphocytes (TIL) and stromal cells. PDAC possesses unique TME, which is abundant of fibrosis, termed of desmoplasia, and the role of fibrosis in constructing immune suppressive TME remains unclear. First, in this study, to examine the immunological characteristics of TME, we constructed an integrated data base of TIL profiling, RNA-seq and whole exome seq using 31 fresh resected tissues of PDAC. An unsupervised clustering of based on numbers and percentages of 12 TIL types analyzed by flow cytometry showed that PDAC was divided into 2 types of cluster (myeloid cell-, T cell-dominant type), and the prognosis of myeloid type was significantly poorer than T cell-type. Myeloid type contained the high frequency of myeloid cell lineage including monocytes, macrophages and myeloid-derived suppressor cells (MDSC). Only MDSCs were significantly associated with poor prognosis among various immune cell types. Furthermore, gene enrichment analysis identified activated and suppressed pathways in each immune type, and the immune-related pathways were significantly down-regulated in myeloid cell-dominant type. Then, to understand the characteristics of MDSCs infiltrated into pancreatic cancer tissues, single cell RNA-seq with surgical specimens was performed, and the analysis showed that a specific subtype of MDSCs infiltrated into cancer tissues. In addition, activation status of cancer-associated fibroblasts (CAFs) was related with MDSC recruitment and activation, indicating that the interaction between MDSCs and CAFs is crucial to create MDSC-rich TME. Inhibition of interaction between MDSCs and CAFs is expected to overcome immunosuppressive TME can be a novel immunotherapy in PDAC. Citation Format: Kazunori Aoki, Hironori Fukuda, Eri Hashimoto, Kosuke Arai. Molecular basis of immune suppressive microenvironment specified by cancer-associated fibroblasts in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A020.

Despite constant efforts to improve treatment, the prognosis remains poor, with an overall survival rate of 6% (ranges from 2% to 9%) due to lack of efficient chemotherapy, radiotherapy, and targeted therapy. Immune checkpoint inhibitors (ICIs) has been reported to be effective in various solid cancers, such as melanoma, lung cancer, and renal cell carcinoma. However, pancreatic ductal adenocarcinoma (PDAC) is resistant against ICIs. The responsiveness of immune therapy is mainly determined by immune tumor microenvironment (TME) composed of tumor-infiltrating lymphocytes (TIL) and stromal cells. PDAC possesses unique TME, which is abundant of fibrosis, termed of desmoplasia, and the role of fibrosis in constructing immune suppressive TME remains unclear. First, in this study, to examine the immunological characteristics of TME, we constructed an integrated data base of TIL profiling, RNA-seq and whole exome seq using 31 fresh resected tissues of PDAC. An unsupervised clustering of based on numbers and percentages of 12 TIL types analyzed by flow cytometry showed that PDAC was divided into 2 types of cluster (myeloid cell-, T cell-dominant type), and the prognosis of myeloid type was significantly poorer than T cell-type. Myeloid type contained the high frequency of myeloid cell lineage including monocytes, macrophages and myeloid-derived suppressor cells (MDSC). Only MDSCs were significantly associated with poor prognosis among various immune cell types. Furthermore, gene enrichment analysis identified activated and suppressed pathways in each immune type, and in myeloid cell-dominant type, the immune-related pathways were significantly down-regulated. Then, to understand the characteristics of MDSCs infiltrated into pancreatic cancer tissues, single cell RNA-seq with surgical specimens was performed, and the analysis showed that a specific subtype of MDSCs infiltrated into cancer tissues. In addition, activation status of cancer-associated fibroblasts (CAFs) was related with MDSC recruitment and activation, indicating that the interaction between MDSCs and CAFs is crucial to create MDSC-rich TME. Inhibition of interaction between MDSCs and CAFs is expected to overcome immunosuppressive TME can be a therapy in PDAC. Citation Format: Kazunori Aoki, Hironori Fukuda, Yukihiro Mizoguchi, Kosuke Arai. Intercellular network in immune suppressive microenvironment of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1593.

This systematic review assesses the role of the tumor microenvironment (TME) in cancer progression and therapy resistance by defining drug-microenvironment interactions and determining the molecular determinants in the TME that could help improve the efficacy of administered treatments and alleviate existing adverse effects. This systematic review follows the PRISMA protocol and the PICOS selection framework to retrieve studies from PubMed/MEDLINE, Web of Science, Scopus, and the Cochrane Library. Only original human-related research published in English between 2008 and 2023 was used to explore the reciprocal relation between tumor cells and TME components. The ROBINS-I tool assessed the risk of bias. Out of 258 articles initially identified, 15 met the inclusion criteria for this review. The results showed that TMEs significantly influence treatment outcomes in cancer progression, metastasis, and drug resistance. Focusing on TMEs like CAFs, immune cells, and ECM enhances drug efficacy. The study highlighted potential strategies to improve drug delivery, suppress metastatic processes, and restore immune function, ultimately leading to better outcomes for cancer patients. Original evidence suggests that Cancer-Associated Fibroblasts (CAFs), immune cells, and Extracellular Matrix (ECM) contribute to therapeutic resistance and metastasis within the TME. They also promote metastasis by inducing Epithelial- Mesenchymal Transition (EMT) and affecting Cancer Stem Cell (CSC) populations. Moreover, the immunosuppressive TME consists of regulatory T cells and myeloidderived suppressor cells that allow tumors to evade the immune system, a concern for immunotherapy. The TME plays a vital role in cancer development, metastasis formation, and therapy failure. The perspectives for innovative ECM-modulating treatments and interventions targeting the direct interactions between TME and cancer cells can be revolutionary and suggest better outcomes for treatment-naïve and refractory cancers. Future research should use these results as inputs to apply clinical and therapy studies to enhance cancer management outcomes.

A growing number of studies have shown that in addition to adaptive immune cells such as CD8 + T cells and CD4 + T cells, various other cellular components within prostate cancer (PCa) tumor microenvironment (TME), mainly tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs) and myeloid-derived suppressor cells (MDSCs), have been increasingly recognized as important modulators of tumor progression and promising therapeutic targets. In this review, we aim to delineate the mechanisms by which TAMs, CAFs and MDSCs interact with PCa cells in the TME, summarize the therapeutic advancements targeting these cells and discuss potential new therapeutic avenues. We searched PubMed for relevant studies published through December 10 2023 on TAMs, CAFs and MDSCs in PCa. TAMs, CAFs and MDSCs play a critical role in the tumorigenesis, progression, and metastasis of PCa. Moreover, they substantially mediate therapeutic resistance against conventional treatments including anti-androgen therapy, chemotherapy, and immunotherapy. Therapeutic interventions targeting these cellular components have demonstrated promising effects in preclinical models and several clinical trials for PCa, when administrated alone, or combined with other anti-cancer therapies. However, the lack of reliable biomarkers for patient selection and incomplete understanding of the mechanisms underlying the interactions between these cellular components and PCa cells hinder their clinical translation and utility. New therapeutic strategies targeting TAMs, CAFs, and MDSCs in PCa hold promising prospects. Future research endeavors should focus on a more comprehensive exploration of the specific mechanisms by which these cells contribute to PCa, aiming to identify additional drug targets and conduct more clinical trials to validate the safety and efficacy of these treatment strategies.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a heterogeneous tumor comprised of epithelial tumor, endothelial, immune, and importantly, cancer associated fibroblasts (CAFs) cells. CAFs drive a complex tumor microenvironment (TME) through mechanisms of intratumoral interactions that are incompletely understood. It’s crucial to account for the complex role of CAFs in modern ex-vivo tumor systems as regulators of both promoting and restraining tumor growth, providing growth factor support, reprogramming immune cells, and altering the TME. Using our patient-derived organoid (PDO) and CAF coculture, we identify novel intercellular interactions between CAF and PDO that promote tumor cell proliferation, impair response to therapy, and alter transcriptional phenotype. Methods: We interrogate mechanisms of cellular crosstalk in the PDAC TME using a novel three-dimensional, patient-matched coculture of PDO and CAFs, established from patients undergoing surgical resection. Molecular characterization included bulk RNA sequencing-based transcriptomics, comprehensive proteome profiling for 107 secreted proteins with curated validation by ELISA, cellular phenotyping by multiparameter flow cytometry, and qPCR for validation of curated gene lists. Results: Bulk RNAseq of FACS sorted CAF – PDO cocultures from 12 patients demonstrate Moffitt classification transcriptional changes in 42% of samples compared to PDO monoculture, suggesting CAFs drive basal tumor phenotype. To investigate protein level changes, we compared the secreted proteome of PDOs that transitioned to a basal phenotype after coculture to PDOs that did not undergo transition. Proteome analysis from coculture supernatant revealed that HGF, GDF15, TFF3, and VEGF-A are significantly increased in samples that undergo classical to basal transition. Pathway analysis comparing transcriptional data from classical and basal PDOs identifies significant upregulation of pathways associated with epithelial-to-mesenchymal transition (EMT), inflammation, and NF-kB in basal PDOs. Further, coculture leads to decreased expression of E-cadherin and increased expression of N-cadherin on the surface of PDOs while CAF markers remain unchanged. This interaction is accompanied by an increase in PDO proliferation in coculture, suggesting that CAF presence enhances tumorgenicity and changes epithelial tumor cell fitness. Ongoing experiments will investigate tumor cell-CAF interactions spatially using matched patient tissue to complement the in vitro coculture system. Conclusions: Interactions between cancer cells and CAFs drive overall tumor biology via a complex TME and contribute to poor PDAC patient outcomes. Using our patient matched PDO - CAF coculture, we demonstrate that a CAF competent TME drives PDO plasticity towards a more basal transcriptional phenotype and enhanced EMT. We introduce an elegant approach combining in vitro coculture experiments and high dimensional assays to investigate the complex biology of the TME and inform the mechanisms driving cancer biology in individual patients with PDAC. Citation Format: Samantha Guinn, Joseph Tandurella, Jae W. Lee, Daniel J. Zabransky, Mili Ramani, Jignasha Patel, Elana J. Fertig, Elizabeth M. Jaffee, Richard A. Burkhart, Jacquelyn W. Zimmerman. Cancer associated fibroblasts drive transcriptional changes in tumor cells from classical to basal phenotype and promote epithelial-to-mesenchymal transition in human pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A042.