Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion.

Pancreatic Ductal Adenocarcinoma (PDAC) is an aggressive disease characterized by a prominent desmoplastic stromal reaction and deregulated metabolism. The role of stroma in PDAC biology is complex and has been shown to play critical roles that may differ depending on the biological context. The intense stromal reaction also impacts the vasculature, leading to a highly hypoxic and nutrient poor environment. As such, these tumors must adapt how nutrients are captured and utilized to support their metabolic needs. In this talk, I will describe how stromal-associated pancreatic stellate cells (PSCs) are critical for PDAC metabolism through the secretion of non-essential amino acids (NEAA). Specifically, we uncovered an undescribed role for alanine, which outcompetes glucose and glutamine-derived carbon in PDAC to fuel the tricarboxylic acid (TCA) cycle, and thus NEAA and lipid biosynthesis. This shift in fuel sources decreases the dependence on glucose and serum-derived nutrients, which are limiting in the pancreatic tumor microenvironment. Moreover, we demonstrated that PSC alanine secretion is dependent on PSC autophagy, a process stimulated by the cancer cells. Thus, our results demonstrate a novel metabolic crosstalk between PSCs and cancer cells, with PSC-derived alanine as an alternative carbon source, and highlight a previously unappreciated metabolic network within pancreatic tumors where diverse fuel sources are utilized to promote growth in an austere tumor microenvironment. Citation Format: Costas A. Lyssiotis. Stromal support of pancreatic tumor metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 437. doi:10.1158/1538-7445.AM2017-437

Desmoplasia of Pancreatic Ductal Adenocarcinoma

Cell-cell interactions within the tumor microenvironment play critical roles in tumor progression and its response to various therapies. Specifically in the case of pancreatic cancer, pancreatic cancer cells (PCCs) act on pancreatic stellate cells (PSCs), a major stromal cell type within the pancreatic tumor microenvironment, to provoke their transformation into and sustain an activated state, while activated PSCs interact with PCCs to promote tumor progression and confer a favorable environment for PCC resistance to immunosuppression and therapeutic treatment, setting up a vicious cycle. Therefore, effectively attacking the stromal PSCs alone or together with tumor cells may offer a new therapeutic route for pancreatic ductal adenocarcinoma (PDA) treatment. To identify key signaling molecules or pathways activated as a result of PSC-PCC interactions that might be targeted in pancreatic cancer as novel therapies, we developed unbiased quantitative mass spectrometry (MS)-based proteomics strategies to perform systemic analysis. Our comprehensive molecular and cellular analysis of in vitro PSC and PSC co-culture unraveled many interesting secreted proteins, either known or unknown previously, involved in the paracrine PCC-PSC crosstalk. We mainly focused on an intriguing signaling loop, in which PCCs secrete PDGFs that act on PSCs to promote the production of inflammatory cytokines by PSCs, including IL-6 family members, and especially LIF. These cytokines then act on PCCs to stimulate STAT3 activation, a known critical event for PDA initiation and progression. Significantly increased production of the cytokines and expression of corresponding receptors in both mouse and human PDA tumor contexts at both mRNA and protein levels were confirmed using various techniques. To further evaluate the functional significance of LIF in paracrine regulation of PDA progression in vivo, we exploited therapeutic blockage of the signaling using neutralizing mAbs and specific small molecule inhibitors in the classical KPC mouse PDA model. Our preclinical studies revealed significant efficacy of an anti-LIF mAb in increasing sensitivity to the standard-of-care chemotherapeutic gemcitabine, reducing tumor progression and prolonging survival of KPC mice. Currently, the molecular mechanisms underlying this sensitization are being investigated. Genetic studies using LIF receptor conditional knockout mice were also performed and showed significant beneficial effects. Furthermore, potential correlations between tissue or serum LIF and IL-6 cytokine levels and overall survival of human patients are also being evaluated. Citation Format: Yu Shi, Ruijun Tian, Tony Hunter.{Authors}. Interdicting the cytokine-mediated paracrine communication between pancreatic cancer and stellate cells as a new treatment approach for pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B27.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC, also known as pancreatic cancer) is one of the deadliest tumor types, characterized by poor prognosis, profound chemoresistance and overall low survival. Gemcitabine remains the standard of care for all stages of PDAC, however, with poor clinical benefits which is considered to be due to reduced drug availability in tumor cells. Gemcitabine-induced cytotoxicity depends upon sufficient drug uptake followed by intracellular activation. Pancreatic stellate cells (PSCs), a major stromal component of PDAC, were recently reported to scavenge active metabolites of gemcitabine, thereby making it unavailable for cancer cells. Gemcitabine uptake and processing in both tumor cells and PSCs, as well as expression analysis of its molecular metabolic regulators, was investigated in this study. We observed heterogeneous gemcitabine-induced cytotoxicity in different pancreatic cancer cells whereas it was absent in PSCs. The gemcitabine-induced cytotoxicity in pancreatic cancer cells was driven by differential expression of its molecular regulators.Gemcitabine resistance in pancreatic ductal adenocarcinoma (PDAC) is attributed to cancer cell-intrinsic drug processing and the impact of the tumor microenvironment, especially pancreatic stellate cells (PSCs). This study uses human PDAC-derived paired primary cancer cells (PCCs) and PSCs from four different tumors, and the PDAC cell lines BxPC-3, Mia PaCa-2, and Panc-1, to assess the fate of gemcitabine by measuring its cellular uptake, cytotoxicity, and LC-MS/MS-based metabolite analysis. Expression analysis and siRNA-mediated knockdown of key regulators of gemcitabine (hENT1, CDA, DCK, NT5C1A) was performed. Compared to PSCs, both the paired primary PCCs and cancer cell lines showed gemcitabine-induced dose-dependent cytotoxicity, high uptake, as well as high and variable intracellular levels of gemcitabine metabolites. PSCs were gemcitabine-resistant and demonstrated significantly lower drug uptake, which was not influenced by co-culturing with their paired PCCs. Expression of key gemcitabine regulators was variable, but overall strong in the cancer cells and significantly lower or undetectable in PSCs. In cancer cells, hENT1 inhibition significantly downregulated gemcitabine uptake and cytotoxicity, whereas DCK knockdown reduced cytotoxicity. In conclusion, heterogeneity in gemcitabine processing among different pancreatic cancer cells and stellate cells results from the differential expression of molecular regulators which determines the effect of gemcitabine.

Mechanistic Insights into Self-Reinforcing Processes Driving Abnormal Histogenesis During the Development of Pancreatic Cancer

Pancreatic cancer has a very poor prognosis, largely due to its propensity for early local and distant spread. Histopathologically, most pancreatic cancers are characterized by a prominent stromal/fibrous reaction in and around tumor tissue. The aims of this study were to determine whether (1) the cells responsible for the formation of the stromal reaction in human pancreatic cancers are activated pancreatic stellate cells (PSCs) and (2) an interaction exists between pancreatic cancer cells and PSCs that may facilitate local and distant invasion of tumor. Serial sections of human pancreatic cancer tissue were stained for desmin and glial fibrillary acidic protein (stellate cell selective markers) and alpha-smooth muscle actin (alphaSMA), a marker of activated PSC activation, by immunohistochemistry, and for collagen using Sirius Red. Correlation between the extent of positive staining for collagen and alphaSMA was assessed by morphometry. The cellular source of collagen in stromal areas was identified using dual staining methodology, ie, immunostaining for alphaSMA and in situ hybridization for procollagen alpha1I mRNA. The possible interaction between pancreatic cancer cells and PSCs was assessed in vitro by exposing cultured rat PSCs to control medium or conditioned medium from 2 pancreatic cancer cell lines (PANC-1 and MiaPaCa-2) for 24 hours. PSC activation was assessed by cell proliferation and alphaSMA expression. Stromal areas of human pancreatic cancer stained strongly positive for the stellate cell selective markers desmin and GFAP (indicating the presence of PSCs), for alphaSMA (suggesting that the PSCs were in their activated state) and for collagen. Morphometric analysis demonstrated a close correlation (r = 0.77; P < 0.04; 8 paired sections) between the extent of PSC activation and collagen deposition. Procollagen mRNA expression was localized to alphaSMA-positive cells in stromal areas indicating that activated PSCs were the predominant source of collagen in stromal areas. Exposure of PSCs to pancreatic cancer cell secretions in vitro resulted in PSC activation as indicated by significantly increased cell proliferation and alphaSMA expression. Activated PSCs are present in the stromal reaction in pancreatic cancers and are responsible for the production of stromal collagen. PSC function is influenced by pancreatic cancer cells. Interactions between tumor cells and stromal cells (PSCs) may play an important role in the pathobiology of pancreatic cancer.

Pancreatic stellate cells (PSCs) are a key component of tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) and contribute to drug resistance. c-MET receptor tyrosine kinase activation plays an important role in tumorigenesis in different cancers including PDAC. In this study, effects of PSC conditioned medium (PCM) on c-MET phosphorylation (by immunocytochemistry enzyme-linked immunosorbent assay (ELISA)) and drug response (by sulforhodamine B assay) were investigated in five primary PDAC cells. In novel 3D-spheroid co-cultures of cyan fluorescence protein (CFP)-firefly luciferase (Fluc)-expressing primary human PDAC cells and green fluorescence protein (GFP)-expressing immortalized PSCs, PDAC cell growth and chemosensitivity were examined by luciferase assay, while spheroids’ architecture was evaluated by confocal microscopy. The highest phospho-c-MET expression was detected in PDAC5 and its subclone sorted for “stage specific embryonic antigen-4” (PDAC5 (SSEA4)). PCM of cells pre-incubated with PDAC conditioned medium, containing increased hepatocyte growth factor (HGF) levels, made PDAC cells significantly more resistant to gemcitabine, but not to c-MET inhibitors. Hetero-spheroids containing both PSCs and PDAC5 (SSEA4) cells were more resistant to gemcitabine compared to PDAC5 (SSEA4) homo-spheroids. However, c-MET inhibitors (tivantinib, PHA-665752 and crizotinib) were equally effective in both spheroid models. Experiments with primary human PSCs confirmed the main findings. In conclusion, we developed spheroid models to evaluate PSC–PDAC reciprocal interaction, unraveling c-MET inhibition as an important therapeutic option against drug resistant PDAC.

The ATP‐gated receptor P2X7 (P2X7R) is involved in regulation of cell survival and has been of interest in cancer field. Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer and new markers and therapeutic targets are needed. PDAC is characterized by a complex tumour microenvironment, which includes cancer and pancreatic stellate cells (PSCs), and potentially high nucleotide/side turnover. Our aim was to determine P2X7R expression and function in human pancreatic cancer cells in vitro as well as to perform in vivo efficacy study applying P2X7R inhibitor in an orthotopic xenograft mouse model of PDAC. In the in vitro studies we show that human PDAC cells with luciferase gene (PancTu‐1 Luc cells) express high levels of P2X7R protein. Allosteric P2X7R antagonist AZ10606120 inhibited cell proliferation in basal conditions, indicating that P2X7R was tonically active. Extracellular ATP and BzATP, to which the P2X7R is more sensitive, further affected cell survival and confirmed complex functionality of P2X7R. PancTu‐1 Luc migration and invasion was reduced by AZ10606120, and it was stimulated by PSCs, but not by PSCs from P2X7‐/‐ animals. PancTu‐1 Luc cells were orthotopically transplanted into nude mice and tumour growth was followed noninvasively by bioluminescence imaging. AZ10606120‐treated mice showed reduced bioluminescence compared to saline‐treated mice. Immunohistochemical analysis confirmed P2X7R expression in cancer and PSC cells, and in metaplastic/neoplastic acinar and duct structures. PSCs number/activity and collagen deposition was reduced in AZ10606120‐treated tumours.

Aim of the study: The aim of this study was to validate a novel 3D spheroid co-culture model of pancreatic ductal adenocarcinoma (PDAC) cells and pancreatic stellate cells (PSCs), and to use said model as to investigate the effects of PDAC cell and PSC cross-talk on cell proliferation, cell death, migration and chemo-resistance. Methods: An eGFP-positive PSC line and PDAC cell lines were cultured in 3D spheroid mono- and co-cultures. Activation of the PSCs within the co-culture model was investigated by real time PCR for whole spheroids and spheroid populations sorted by fluorescence activated cell sorting (FACS). To investigate cell proliferation the cell numbers within the cultures were followed over time and the proportions of the different cell types within the co-cultures were monitored by FACS. Results: The mRNA expression of the PSC activation marker ASMA as well as the ECM proteins collagen I, laminin and lumican, the PDGFbeta receptor (PDGFRb) and the transforming growth factor beta1 (TGFb-1) downstream effector connective tissue growth factor (CTGF), were increased in PSCs within co-cultures compared to mono-cultures. PSCs were initially expanding but then diminished within 3D spheroid co-cultures together with two separate PDAC cell lines (Panc-1 and HPAF-II). Panc-1 cell numbers were seemingly unaffected by the presence of PSCs, but with higher proportions of PSCs the Panc1 cell numbers within the co-cultures initially were lagging behind but then caught up to the numbers within the mono-cultures. This growth switch was correlated with the opposite switch seen for the PSCs. The proliferation of HPAF-II cells, was on the other hand induced within the co-cultures compared to mono-cultures. Conclusions: PSCs are activated within the 3D spheroid co-cultures. Both tumor cell and PSC proliferation is influenced by co-culturing the cells in the 3D spheroid model. Different PDAC cell – PSC combinations are affected differently. We currently are investigating the effects on cell death and apoptosis, migration and chemo-resistance. Citation Format: Jessica K. Norberg, Salvatore Nania, Rainer L. Heuchel, Matthias J. Löhr. Pancreatic cancer and stroma cell cross-talk affects cell proliferation in a 3D spheroid co-culture model. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C40.

BackgroundActivated pancreatic stellate cells (PaSCs) are the key cellular source of cancer-associated fibroblasts in the pancreatic stroma of patients with pancreatic ductal adenocarcinoma (PDAC), however, the activation mechanism of PaSCs is not yet known. The Notch signaling pathway, components of which are expressed in stromal cells, is involved in the fibrosis of several organs, including the lung and liver. In the current study, we investigated whether Notch signal transduction is involved in PaSC activation in PDAC.MethodsThe expression of Notch signaling pathway components in human PDAC was examined via immunohistochemical staining and assessed in mouse PaSCs using RT-qPCR and western blotting. Notch3 expression in both PDAC stromal cells and activated mouse PaSCs was evaluated using immunofluorescence, RT-qPCR and western blotting. The impact of siRNA-mediated Notch3 knockdown on PaSC activation was detected with RT-qPCR and western blotting, and the impact on PaSC proliferation and migration was detected using CCK-8 assays and scratch experiments. The effect of conditioned medium from PaSCs activated with Notch3 siRNA on pancreatic cancer (LTPA) cells was also detected with CCK-8 assays and scratch experiments. The data were analyzed for statistical significance using Student’s t-test.ResultsNotch3 was overexpressed in both human PDAC stromal cells and activated mouse PaSCs, and Notch3 knockdown with Notch3 siRNA decreased the proliferation and migration of mouse PaSCs. The levels of markers related to PaSC activation, such as α-smooth muscle actin (α-SMA), collagen I and fibronectin, decreased in response to Notch3 knockdown, indicating that Notch3 plays an important role in PaSC activation. Furthermore, we confirmed that inhibition of PaSC activation via Notch3 siRNA reduced the proliferation and migration of PaSC-induced mouse pancreatic cancer (LTPA) cells.ConclusionsNotch3 inhibition in PaSCs can inhibit the activation, proliferation and migration of PaSCs and reduce the PaSC-induced pro-tumorigenic effect. Therefore, Notch3 silencing in PaSCs is a potential novel therapeutic option for patients with PDAC.

Stromal heterogeneity in pancreatic cancer and chronic pancreatitis

Introduction: Stromal cells associated with cancer cells are considered an important component of tumor microenvironment of pancreatic cancer. The intratumoral desmoplasia characteristic of pancreatic cancer is a result of growing carcinoma paracrine action on surrounding normal tissue cells. Selective targeting of tumor stroma cells can markedly increase the effectiveness of currently used chemotherapeutic agents against pancreatic cancer. Therefore, a search for new targets in stromal cells is an important part of developing new combined therapies of this disease. Our work was aimed at comparative studying of gene expression in stromal cells of normal pancreas, pancreatitis and pancreatic ductal adenocarcinoma tissues. Methods: Cultured stromal cells were obtained from samples of normal pancreatic (n=3), pancreatitis (n=3) and ductal adenocarcinoma tissues (n=4). The cell cultures obtained were preliminary characterized by immunofluorescence, RT-PCR and western blotting. A full genome gene expression analysis was performed using a modified SAGE technique. Expression of selected dysregulated genes was analyzed by quantitative PCR. Results: Morphological and immunofluorescent analyses of the obtained primary stromal cultures indicated that they could be assigned to pancreatic stellate cells (PSC). The SAGE analysis allowed to quantitatively estimate expression of 9860 genes in normal PSC, 8744 PSC genes in pancreatitis, and 9311 genes in tumor PSC. A comparison of normal and tumor PSC revealed statistically significant (P&amp;lt;0.001) differences in expression of 270 genes. Of them, 146 genes were upregulated and 124 genes downregulated in tumor PSC as compared to normal PSC. An expression analysis of selected dysregulated genes by RT-PCR detected changes in the activity of some genes involved in tumor progression of pancreatic cancer. In particular, the genes of two chemokines (CXCL12 and CX3CL1) and the JAG1 Notch ligand were upregulated in tumor PSC. A similar comparison of tumor PSC and PSC from pancreatitis tissue detected 68 differentially expressed genes (P&amp;lt;0.001), 40 of which were upregulated and 28 downregulated in tumor PSC as compared to pancreatitis PSC. Expression of 23 genes was upregulated in tumor PSC as compared with PSC both from pancreas and pancreatitis. On the whole, the expression profile of tumor PSC was markedly more similar to that of PSC from pancreatitis tissues than from normal PSC. Conclusions: The results obtained in our work revealed statistically significant changes in the expression of some PSC genes in stroma of ductal pancreatic adenocarcinoma as compared to normal PSC. The similarity of tumor and pancreatitis PSC expression profiles may suggest mutual mechanisms of forming the fibrous tissue of pancreatitis and tumor stroma of pancreatic carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4277. doi:1538-7445.AM2012-4277

Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a transmembrane glycoprotein that is involved in tumor progression and metastasis. In the present study, the expression and functional role of ALCAM in pancreatic cancer cells and pancreatic stellate cells (PSCs) was investigated. Tissue specimens were obtained from patients with pancreatic ductal adenocarcinoma (n=56) or chronic pancreatitis (CP; n=10), who underwent pancreatic resection, and from normal pancreatic tissue samples (n=10). Immunohistochemistry was used to analyze the localization and expression of ALCAM in pancreatic tissues. Subsequently, reverse transcription-quantitative polymerase chain reaction and immunoblotting were applied to assess the expression of ALCAM in pancreatic cancer Panc-1 and T3M4 cells, as well as in PSCs. An enzyme-linked immunosorbent assay was used to measure ALCAM levels in cell culture medium stimulated by hypoxia, tumor necrosis factor (TNF)-α and transforming growth factor-β. Silencing of ALCAM was performed using ALCAM small interfering (si)RNA and immunocytochemistry was used to analyze the inhibition efficiency. An invasion assay and a cell interaction assay were performed to assess the invasive ability and co-cultured adhesive potential of Panc-1 and T3M4 cells, as well as PSCs. Histologically, ALCAM expression was generally weak or absent in pancreatic cancer cells, but was markedly upregulated in PSCs in pancreatic cancer tissues. ALCAM was highly expressed in PSCs from CP tissues and PSCs surrounding pancreatic intraepithelial neoplasias, as well as in pancreatic cancer cells. ALCAM mRNA was highly expressed in PSCs, with a low to moderate expression in T3M4 and Panc-1 cells. Similar to the mRNA expression, immunoblotting demonstrated that ALCAM protein levels were high in PSCs and T3M4 cells, but low in Panc-1 cells. The expression of TNF-α increased, while hypoxia decreased the secretion of ALCAM in pancreatic cancer Panc-1 and T3M4 cells, and also in PSCs. Silencing of ALCAM by siRNA revealed no significant alteration in the invasion of pancreatic cancer cells, however, it inhibited the invasive ability of PSCs, and decreased the interaction between Panc-1 cells and PSCs. In conclusion, ALCAM is upregulated in PSCs of pancreatic cancer tissues, suggesting a potential role of ALCAM in regulating pancreatic cancer cell-PSC interactions.

Pancreatic ductal adenocarninoma (PDA) is one of the most aggressive human cancers and is characterized by a predominant stromal compartment mainly composed of fibroblastic and immune cells. Pancreatic stellate cells (PSCs) are the main fibroblastic cells in the pancreatic tumor microenvironment. During tumor progression, they evolve into activated and proliferative myofibroblast-like cells that synthesize copious amounts of extracellular matrix (ECM) proteins and have diminished lipid storage capacity. They produce a dense fibrous stroma that obstructs intratumoral vasculature to create physical and biological therapeutic barriers. These observations suggest that strategies that normalize the stroma may enhance anti-tumor treatment strategies. Activated PSCs in pancreatic cancers encode wild type p53, raising the possibilities that p53 activation could prevent their proliferation, and induce other paracrine anti-tumor effects. Nutlin-3a is a small-molecule p53 agonist that blocks the binding of its major negative regulator, Mdm2. As Nutlin derivatives are currently in clinical trials, we tested the consequences of activating p53 by Nutlin-3a in PSCs. The data to be presented show that Nutlin-3a induces transcriptional regulation of genes that induce multiple changes consistent with quiescence. Using PSCs from patient samples, we show that p53 activation induces lipid droplet formation, decreases expression of αSMA (ACTA2), and produces growth arrest. We also observed that p53 activation inhibits TGFβ-induced transcription of genes involved in PSC activation. To assess the genome-wide effects of p53, we performed transcriptome analysis of PSCs grown in the presence or absence of Nutlin-3a. This analysis revealed that p53 inhibits the expression of genes associated with the activation of PSCs such as ECM components and induces the expression of genes implicated in lipid metabolism characteristic of the quiescent state. P53 activation also decreased expression of genes with tumor-supporting potential including cytokines, growth factors and signaling molecules. Ongoing investigations are designed to determine the consequences of stromal p53 activation on cancer cells using in vitro co-culture systems and mouse models of pancreatic cancer. Citation Format: Maya Ridinger, Annabelle Kraus, Miranda Cox, Mark Wade, Christopher Liddle, Michael Downes, Ron Evans, Geoffrey Wahl. Function and therapeutic implications of p53 in the stroma of 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 B10.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a 5-year survival rate of only 10%. One hallmark of PDAC’s tumor microenvironment is dense desmoplasia, due to abnormal accumulation of extracellular matrix and proliferative fibroblasts. During tumorigenesis, pancreatic stellate cells (PSCs) obtain myofibroblast-like signatures and contribute to the fibrotic environment of PDAC. Desmoplasia-induced hypovascularity severely limits oxygen and nutrient delivery. Our laboratory revealed that hypoxia is prominent even at the pancreatic intraepithelial neoplasia (PanIN) stage, a dominant precursor lesion of PDAC. Hypoxia inhibits oxygen-dependent metabolic processes. For example, unsaturated lipid biosynthesis relies on oxygen to maintain desaturase enzymatic activities. Hypoxia also stimulates lipid uptake. Previous studies demonstrated that RAS-transformed cells preferentially import unsaturated lysophosphatidylcholines (LPCs) from culture media. However, in a nutrient-limited fibrotic environment, the potential source of lipids is unclear. Interestingly, a lipidomic study of a PSC secretome displayed a significant amount of LPCs, suggesting that PSCs are the potential lipid source in the PDAC microenvironment. Our data indicate that exogenous unsaturated lipids sustain PDAC cell viability under hypoxia and nutrient deficiency. Moreover, we find PDAC cell survival under tumor-like stress is enhanced in PSC conditioned medium, but not in delipidated conditioned medium. Strikingly, we show that cancer-associated fibroblasts do not experience hypoxic stress as much as adjacent malignant epithelium cells in vivo, suggesting cancer-associated fibroblasts provide unsaturated (and other) lipids to PDAC cells. Based on our lipidomic mass spectra of PSC conditioned medium, we determined that LPCs are actively utilized by PDAC cells for phosphatidylcholine and triglyceride regeneration. To discover novel therapeutic strategies, we performed small molecule screens to identify potential drugs that disrupt LPC uptake and metabolism by PDAC cells. Our overall hypothesis is that activated PSCs (and potentially other fibroblasts) support PDAC cell survival by providing lipids, particularly LPCs, and inhibition of LPC utilization by cancer cells or unsaturated lipid synthesis by PSCs induces PDAC cell death under metabolically challenging conditions. In summary, this project aims to demonstrate metabolic crosstalk between fibroblasts and PDAC cells under stress conditions. The key advance of my research is that activated PSCs suppress PDAC cell death by supplying tumor cells with unsaturated LPCs for the maintenance of lipid homeostasis, in a hypoxic and nutrient-poor environment. If true, our findings will reveal novel metabolic targets for developing combinatorial therapy of PDAC. Citation Format: Xu Han, Michelle Burrows, Yanqing Jiang, Clementina Mesaros, David Schultz, Brian Keith, Celeste Simon. Investigating lipid homeostasis in pancreatic ductal adenocarcinoma under tumor-like stress [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR024.