Patient-derived Pancreatic Ductal Adenocarcinoma Cell Research Articles

Cytokine-armed vaccinia virus promotes cytotoxicity toward pancreatic carcinoma cells via activation of human intermediary CD56dimCD16dim natural killer cells.

Pancreatic ductal adenocarcinoma (PDAC) remains a particularly aggressive disease with few effective treatments. The PDAC tumor immune microenvironment (TIME) is known to be immune suppressive. Oncolytic viruses can increase tumor immunogenicity via immunogenic cell death (ICD). We focused on tumor-selective (vvDD) and cytokine-armed Western-reserve vaccinia viruses (vvDD-IL2 and vvDD-IL15) and infected carcinoma cell lines as well as patient-derived primary PDAC cells. In co-culture experiments, we investigated the cytotoxic response and the activation of human natural killer (NK). Infection and virus replication were assessed by measuring virus encoded YFP. We then analyzed intracellular signaling processes and oncolysis via in-depth proteomic analysis, immunoblotting and TUNEL assay. Following the co-culture of mock or virus infected carcinoma cell lines with allogenic PBMCs or NK cell lines, CD56+ NK cells were analyzed with respect to their activation, cytotoxicity and effector function. Both, dose- and time-dependent release of danger signals following infection weremeasured. Viruses effectively entered PDAC cells, emitted YFP signals and resulted in concomitant oncolysis. The proteome showed reprogramming of normally active core signaling pathways in PDAC (e.g., MAPK-ERK signaling). Danger-associated molecular patterns were released upon infection and stimulated co-cultured NK cells for enhanced effector cytotoxicity. NK cell subtyping revealed enhanced numbers and activation of a rare CD56dimCD16dim population. Tumor cell killing was primarily triggered via Fas ligands rather than granule release, resulting in marked apoptosis. Overall, the cytokine-armed vaccinia viruses induced NK cell activation and enhanced cytotoxicity toward human PDAC cells in vitro. We could show that cytokine-armed virus targets the carcinoma cells and thus hasgreat potential to modulate the TIME in PDAC.

Effect of oncolytic Newcastle disease virus harboring PTEN gene on pancreatic adenocarcinoma growth through inhibition of PI3K/mTOR signaling and promotion of apoptosis.

49 Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and intractable cancer, warranting the need for more effective therapies. Cell proliferation, progression, and severity in PDAC are highly activated owing to KRAS mutations and low PTEN expression. Methods: A recombinant Newcastle disease virus (rNDV) harboring PTEN (rNDV-PTEN) was constructed to infect PDAC cells to artificially induce PTEN expression. The cancer killing effect of the rNDV-PTEN virus was observed in an in vitro assay, and 107.0 pfu/dose(0.1 ml) of the rNDV-PTEN virus was inoculated through intravenous injection into a PANC-1 cell transplanted mouse to prove the tumor growth inhibition effect. Apoptotic signaling-related proteins were analyzed with Western blotting and qPCR assays based on the mRNA. During the treatment, biomarkers were also analyzed. Results: Factitious PTEN expression in KRAS-mutated PDAC cells following rNDV-PTEN infection lowered PI3K/AKT/mTOR signaling, induced PDAC cell death, and suppressed tumor growth. PTEN overexpression promoted apoptotic and autophagic signaling pathways in PANC-1 cells and orthotopic xenograft mice. rNDV-PTEN has substantial cancer cell-killing effects and inhibits tumor growth. rNDV-PTEN injection into animals did not elicit an immune response against rNDV and improved blood parameters, such as glucose, triglyceride, and total cholesterol levels. Conclusions: rNDV-PTEN showed a strong tumor-suppressive effect on PDAC, which was caused by an abnormally activated PI3K/AKT/mTOR signaling pathway due to KRAS mutations and PTEN loss. We also showed that rNDV-PTEN is relatively safe for use in cancer therapy. Further studies using patient-derived PDAC cells with the same genetic background, as well as more extensive animal safety tests, are required to demonstrate rNDV-PTEN as a PDAC therapeutic. However, PTEN gene-containing rNDV may be a very promising therapeutic candidate for pancreatic cancer treatment.

Abstract 382: Pancreatic cancer-derived organoids alter muscle fiber type and increased energy consumption leading to cachexia

Abstract Cachexia, marked by continuous skeletal muscle mass loss and severe metabolic disturbances, presents a substantial challenge for pancreatic ductal adenocarcinoma (PDAC) patients. Despite its prevalence and the adverse impact on quality of life and survival rates, effective clinical treatments for cachexia remain elusive. This study aims to bridge this gap by employing innovative in vitro models to investigate the underlying mechanisms of cachexia and identify potential therapeutic targets. To enhance the relevance of cell culture models, modifications were introduced to replicate in vivo conditions, where the muscle is exposed to the ongoing kinetics of constant tumor secretion of active factors. Patient-derived PDAC cells were cultured in a dense 3D stiffness extracellular matrix (ECM) to mimic the hypoxic desmoplastic tumor microenvironment. The conditioned medium from prolonged cultured patient-derived organoids (PDOs) was then used to supplement C2C12-derived myotubes. The investigation focuses on dynamic adaptations in muscle fiber type and metabolism when exposed to tumor paracrine factors. Metabolomic investigations of PDO-conditioned medium (PDO CM) on myotubes revealed distinct metabolic differences compared to non-treatment controls. Discriminatory metabolites included those associated with glycolysis, lipid, amino acid, and fatty acid metabolism. PDO CM induced a significant shift in muscle fiber type from slow-twitch to fast-twitch phenotypes, accompanied by de novel expression of embryonic and neonatal Myosin Heavy Chain (MyHC) isoforms. Fast fiber (F59) staining also confirmed the protein expression. Concurrently, PGC-1a, a master regulator of mitochondrial biogenesis and oxidative metabolism, significantly decreased, along with reduced ATP and Mito tracker staining, indicating a metabolic switch under the influence of tumor paracrine factors. Metabolome results on C2C12 myotubes treated with PDO CM also revealed significantly changed ABC transporter-related pathways, an adaptive process required to optimize substrate use under the influence of tumor secretome. However, the genes involved in the proteasome degradation process, such as MAFbx and MuRF1, did not show altered expression. The establishment of an in vitro cachexia model, focusing on changes in energy metabolism and utilizing muscle fiber type alterations as markers, will deepen our understanding of the mechanisms of cancer-associated cachexia. The insights gained may pave the way for identifying potential therapeutic targets to mitigate muscle wasting in PDAC patients and improve overall outcomes. Citation Format: Bo Han, Shuqing Zhao, zhi Yang, Jose Trevino, Steven Grossman, Heinz-Josef Lenz, Ba Xuan Hoang. Pancreatic cancer-derived organoids alter muscle fiber type and increased energy consumption leading to cachexia [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 382.

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 B059: A novel pancreatic cancer mouse model with human immunity

Abstract The iRGD tumor-penetrating peptide delivers chemically linked drugs and even co-injected free drugs selectively to extravascular tumor tissue by targeting the αvβ5 integrin and neuropilin-1 (NRP1). A phase 1b clinical trial showed that iRGD appears to safely double the response rate of standard chemotherapy in patients with stage 4 pancreatic ductal adenocarcinoma (PDAC). We recently found that long-term iRGD treatment, even as a monotherapy, reduced regulatory T cells (Tregs) selectively in the PDAC tissue, which led to the expansion of CD8+ T cells and improved efficacy of immunotherapy in syngeneic PDAC mice. This finding led to the discovery of αvβ5 integrin being a novel targetable marker for tumor-resident Tregs in PDAC mice. Our recent data show that αvβ5 integrin+ Tregs are also present in human PDAC tissue. To study the significance of αvβ5 integrin+ human Tregs as a therapeutic target for iRGD, we developed a humanized PDAC (huPDAC) mouse model by transplanting human PDAC cells into human immune system (HIS) mice, which harbor fully matured human CD8+ and CD4+ T cells, natural killer T cells, B cells, and dendritic cells. The CD8+ T cells are human leukocyte antigen (HLA)-restricted and show a tumor antigen-specific response. We orthotopically implanted HLA-matched human PDAC cells into HLA-A2-positive HIS mice. Both an established human PDAC cell line (PANC-1) and patient-derived PDAC cells (0779E) successfully formed orthotopic tumors and liver metastases. The tumors were infiltrated by various human immune cells at a ratio similar to that found in patient-derived PDAC tissues. Of note, αvβ5 integrin+ human Tregs were also noted in the tumors but not in the spleen. iRGD monotherapy increased the ratio of CD8+ T cells per Tregs in the tumors, suggesting that iRGD will likely function as an immunomodulator in humans. The first patient was recently dosed in a new phase 1b/2a clinical trial (iLSTA Trial: ACTRN12623000223639), which assesses the safety and preliminary efficacy of iRGD in combination with standard-of-care chemotherapy and immunotherapy in locally advanced PDAC patients. Citation Format: Norio Miyamura, Kodai Suzuki, Richard A. Friedman, Aristidis Floratos, Yuki Kunisada, Kazuya Masuda, Andrew M. Lowy, Moriya Tsuji, Kazuki N. Sugahara. A novel pancreatic cancer mouse model with human immunity [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 B059.

USP10 promotes pancreatic ductal adenocarcinoma progression by attenuating FOXC1 protein degradation to activate the WNT signaling pathway.

Increasing evidence has suggested that ubiquitin-specific protease 10 (USP10), a deubiquitinating enzyme, plays an essential role in targeted protein degradation and participates in cancer progression. However, the relationship between USP10 and pancreatic ductal adenocarcinoma (PDAC) is poorly understood. Here, we developed a USP-targeting siRNA library, combining a loss-of-function experimental screen in patient-derived PDAC cells. This approach identified USP10 as a master regulator of PDAC cell migration. High USP10 expression levels were observed in PDAC patient tissues, which were associated with poor prognosis. Furthermore, knockdown of USP10 expression inhibited PDAC cell proliferation and migration in vivo and in vitro. Mechanistically, USP10 increased FOXC1 protein stability via deubiquitination. The phosphorylation of FOXC1 at S272A was dependent on USP10-mediated deubiquitination of FOXC1. Additionally, USP10 promoted FOXC1 protein localization in the nucleus. Interestingly, FOXC1 could increase USP10 mRNA expression levels by transcriptional activation. Our data suggest that a positive feedback loop exists between USP10 and FOXC1 that can activate WNT signaling, thus facilitating PDAC malignant progression. Therefore, USP10 represents an exciting therapeutic target that could support new strategies for treating PDAC.

FAK suppresses antigen processing and presentation to promote immune evasion in pancreatic cancer

ObjectiveImmunotherapy for the treatment of pancreatic ductal adenocarcinoma (PDAC) has shown limited efficacy. Poor CD8 T-cell infiltration, low neoantigen load and a highly immunosuppressive tumour microenvironment contribute to this lack...

Incorporation of human iPSC-derived stromal cells creates a pancreatic cancer organoid with heterogeneous cancer-associated fibroblasts

The aggressiveness of pancreatic ductal adenocarcinoma (PDAC) is affected by the tumor microenvironment (TME). In this study, to recapitulate the PDAC TME exvivo, we cocultured patient-derived PDAC cells with mesenchymal and vascular endothelial cells derived from human induced pluripotent stem cells (hiPSCs) to create a fused pancreatic cancer organoid (FPCO) in an air-liquid interface. FPCOs were further induced to resemble two distinct aspects of PDAC tissue. Quiescent FPCOs were drug resistant, likely because the TME consisted of abundant extracellular matrix proteins that were secreted from the various types of cancer-associated fibroblasts (CAFs) derived from hiPSCs. Proliferative FPCOs could re-proliferate after anticancer drug treatment, suggesting that this type of FPCO would be useful for studying PDAC recurrence. Thus, we generated PDAC organoids that recapitulate the heterogeneity of PDAC tissue and are a potential platform for screening anticancer drugs.

Establishment and Molecular Characterization of Two Patient-Derived Pancreatic Ductal Adenocarcinoma Cell Lines as Preclinical Models for Treatment Response.

The prognosis of pancreatic ductal adenocarcinoma (PDAC) is exceedingly poor. Although surgical resection is the only curative treatment option, multimodal treatment is of the utmost importance, as only about 20% of tumors are primarily resectable at the time of diagnosis. The choice of chemotherapeutic treatment regimens involving gemcitabine and FOLFIRINOX is currently solely based on the patient's performance status, but, ideally, it should be based on the tumors' individual biology. We established two novel patient-derived primary cell lines from surgical PDAC specimens. LuPanc-1 and LuPanc-2 were derived from a pT3, pN1, G2 and a pT3, pN2, G3 tumor, respectively, and the clinical follow-up was fully annotated. STR-genotyping revealed a unique profile for both cell lines. The population doubling time of LuPanc-2 was substantially longer than that of LuPanc-1 (84 vs. 44 h). Both cell lines exhibited a typical epithelial morphology and expressed moderate levels of CK7 and E-cadherin. LuPanc-1, but not LuPanc-2, co-expressed E-cadherin and vimentin at the single-cell level, suggesting a mixed epithelial-mesenchymal differentiation. LuPanc-1 had a missense mutation (p.R282W) and LuPanc-2 had a frameshift deletion (p.P89X) in TP53. BRCA2 was nonsense-mutated (p.Q780*) and CREBBP was missense-mutated (p.P279R) in LuPanc-1. CDKN2A was missense-mutated (p.H83Y) in LuPanc-2. Notably, only LuPanc-2 harbored a partial or complete deletion of DPC4. LuPanc-1 cells exhibited high basal and transforming growth factor (TGF)-β1-induced migratory activity in real-time cell migration assays, while LuPanc-2 was refractory. Both LuPanc-1 and LuPanc-2 cells responded to treatment with TGF-β1 with the activation of SMAD2; however, only LuPanc-1 cells were able to induce TGF-β1 target genes, which is consistent with the absence of DPC4 in LuPanc-2 cells. Both cell lines were able to form spheres in a semi-solid medium and in cell viability assays, LuPanc-1 cells were more sensitive than LuPanc-2 cells to treatment with gemcitabine and FOLFIRINOX. In summary, both patient-derived cell lines show distinct molecular phenotypes reflecting their individual tumor biology, with a unique clinical annotation of the respective patients. These preclinical ex vivo models can be further explored for potential new treatment strategies and might help in developing personalized (targeted) therapy regimens.

Abstract B011: Identifying druggable lipid synthesis targets as novel therapeutics for pancreatic cancer

Abstract Lipid metabolic reprogramming is a hallmark of cancer that contributes to tumorigenesis by supporting energy metabolism and the synthesis of signaling molecules, cellular membranes, and protein modifications. Cells modulate lipid metabolic genes through the regulation of sterol regulatory element-binding proteins (SREBPs), which serve as transcriptional activators of genes responsible for fatty acid and cholesterol synthesis and uptake. Despite efforts demonstrating that the SREBP pathway is an attractive chemotherapeutic target, a potent bioavailable inhibitor of the pathway has not yet been discovered. Here, we identify druggable SREBP target genes as potential targets in pancreatic ductal adenocarcinoma (PDAC). Results: To discover candidate genes, we constructed a CRISPR knockout library containing 148 known SREBP target genes. We used a patient-derived PDAC cell line to conduct the knockout screens in vivo (n = 7 tumors) by injecting library-infected cells into the pancreas of nude mice. After 4 weeks of growth, mouse PDAC tumors showed a statistically significant depletion of guide RNAs targeting 8 genes within the mevalonate pathway, which is responsible for synthesizing cholesterol and isoprenoids such as farnesyl-diphosphate and geranyl-diphosphate. One of the most significant hits was HMG-CoA reductase (HMGCR), which catalyzes the formation of mevalonate. We chose to investigate statins, which are a well-characterized class of FDA-approved drugs that inhibit HMGCR. Using flow cytometry, we stained for annexin-V as a marker for apoptosis and found minimal cell death induced by Fluvastatin alone. Given that inhibiting HMGCR would disrupt lipid homeostasis and in turn activate the SREBP pathway, we hypothesized that a combination therapy with an SREBP inhibitor, TMDP, would potentiate statin-induced cell death. We observed minimal cell death with TMDP alone, but striking synergy between Fluvastatin and TMDP, showing a five-fold increase in annexin-V staining after 48 hours. Mevalonate and geranylgeranyl-diphosphate both fully rescued this cell death to basal levels. Conclusions: In vivo CRISPR screening is a powerful tool for the identification of novel therapeutic targets for cancer. We found that the mevalonate pathway plays a significant role in pancreatic tumor growth in vivo and that existing FDA-approved drugs can be used in combination with an SREBP inhibitor to dramatically enhance cell killing in vitro. Citation Format: Casie S. Kubota, Chiaki T. Ishida, Stephanie Myers, Peter J. Espenshade. Identifying druggable lipid synthesis targets as novel therapeutics for pancreatic cancer [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 B011.

Abstract C065: Dissecting the role of CXCL16 downstream of FAK inhibition in the anti-tumor immune response against pancreatic cancer

Abstract Background: Unfortunately, the impact of immunotherapy in pancreatic cancer has, to date, been limited. Nonetheless, despite lack of success with checkpoint blockade alone, there is hope that combinations of agents may unlock the potential for immunotherapy success in pancreatic cancer. FAK (Focal Adhesion Kinase) is a non-receptor tyrosine kinase that is elevated in human Pancreatic Ductal Adenocarcinoma (PDAC) tissue and correlates with high levels of fibrosis and poor CD8 cytotoxic T cell infiltration. We, and others, have already demonstrated a role for FAK in promoting tumor evasion by inducing an immunosuppressive microenvironment, specifically by regulation of cytokines such as CCL5. This has led to trials of the FAK inhibitor (defactinib) in conjunction with immunotherapy. Here we set out to improve our understanding of the role of FAK in regulating chemokine/cytokine mediated signaling in PDAC with a view to identify clinically targetable pathways with potential therapeutic benefit to pancreatic cancer patients. Methods: We combined CRISPR, NanoString, Forward Phase Protein Arrays (FPPA) and ELISA with in vivo experiments using a transplantable KrasG12Dp53R172H mouse model of Pancreatic Cancer and validated findings using human patient-derived PDAC cell lines. Results: We used CRISPR-Cas9 gene editing to deplete FAK gene expression in Panc47 cells, a syngeneic cell line isolated from PDAC arising in fully back-crossed C57BL/6 KPC mice, and reconstituted wild-type FAK (FAK-WT) expression into a clonal Panc47 FAK null (FAK-/-) cell line. Subsequent NanoString and FPPA analyses of secreted cytokines in Panc47 FAK-WT and Panc47 FAK-/- cell lines identified CXCL16 as one of the most significantly increased chemokines upon FAK depletion. Similar results were observed when CRISPR mediated FAK depletion was performed in patient-derived PDAC cell lines. Previous work in our lab has shown that depletion of Signal Transducer and Activator of Transcription-3 (STAT3), a key mediator of immune evasion, in combination with FAK depletion in orthotopically implanted PDAC tumors significantly increases CD8 T-cell infiltration into murine tumors when compared with FAK-/- tumors alone. Here we identify that dual FAK/STAT3 depletion further elevates CXCL16 expression and show that CXCR6, the receptor for CXCL16, is associated with both murine and human CD8 T-cells. Conclusions: These findings identify a FAK-dependent CXCL16:CXCR6 paracrine signaling axis associated with elevated recruitment of CD8 T-cells into PDAC tumors. This requires the dual targeting of FAK and STAT3, identifying a novel therapeutic strategy that warrants further investigation. Citation Format: Jane L. Parry, Marta Canel, Alan Serrels. Dissecting the role of CXCL16 downstream of FAK inhibition in the anti-tumor immune response against pancreatic cancer [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 C065.

The identification of the anthracycline aclarubicin as an effective cytotoxic agent for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer, mainly due to its delayed diagnosis and lack of effective therapeutic options. Therefore, it is imperative to find novel treatment options for PDAC. Here, we tested a series of conventional chemotherapeutics together with anthracycline compounds as single agents or in combination, determining their effectivity against established commercial and patient-derived, low-passage PDAC cell lines. Proliferation and colony formation assays were performed to determine the anticancer activity of anthracyclines; aclarubicin and doxorubicin, on commercial and patient-derived, low-passage PDAC cell lines. In addition, the effect of standard-of-care drugs gemcitabine and individual components of FOLFIRINOX were also investigated. To evaluate which mechanisms of cell death were involved in drug response, cleavage of poly(ADP-ribose)polymerase was evaluated by western blot. Aclarubicin showed superior antitumor activity compared to other anthracyclines and standard of care drugs (gemcitabine and individual components of FOLFIRINOX) in a patient-derived, low-passage PDAC cell line and in commercial cell lines. Importantly, the combination of gemcitabine and aclarubicin showed a synergistic effect at a dose range where the single agents by themselves were ineffective. In parallel, evaluation of the antitumor activity of aclarubicin demonstrated an apoptotic effect in all PDAC cell lines. Aclarubicin is cytotoxic for commercial and patient-derived low-passage PDAC cell lines, at doses lower than peak serum concentrations for patient treatment. Our findings support a (re)consideration of aclarubicin as a backbone of new combination regimens for pancreatic cancer patients.

CEACAM7 Is an Effective Target for CAR T-cell Therapy of Pancreatic Ductal Adenocarcinoma.

To investigate whether CEACAM7 represents a novel therapeutic target for treating pancreatic ductal adenocarcinoma (PDAC) and to generate CEACAM7-targeting CAR T cells to test this hypothesis. We identified CEACAM7 (CGM2), a member of the CEA family of proteins with expression restricted to the colon and pancreas, as a potential CAR T-cell target for PDAC. We probed a panel of PDAC tumor sections as well as patient-derived PDAC cell cultures for CEACAM7 expression. We generated CAR-targeting CEACAM7, and assessed antitumor efficacy of CEACAM7 CAR T cells using in vitro and in vivo models. We show here that CEACAM7 is expressed in a large subset of PDAC tumors, with low to undetectable expression in all normal tissues tested. CEACAM7 is also expressed in primary PDAC cultures isolated from patient-derived tumors, with high expression within the cancer stem cell-enriched subset. CAR T cells targeting CEACAM7 are capable of targeting antigen-expressing tumor cells, and mediate remission in patient-derived xenograft tumors. We identify CEACAM7 as a potential therapeutic target in PDAC and describe the development of CEACAM7-targeted CAR T cells with efficacy against PDAC.

Abstract PO-009: Genetically engineered exosomes enable active pancreatic cancer targeting and evading mononuclear phagocytic system

Abstract Introduction: Exosomes are emerging as promising nanocarriers for genes and drugs; however, major challenges exist including the absence of active tumor targeting and a high proportion of clearance by mononuclear phagocytic system (MPS) in liver and spleen. To overcome these obstacles currently preventing the effective application of nanodelivery technology in the field of cancer biology, we generate a novel exosome that recognizes both pancreatic ductal adenocarcinoma (PDAC) specific cell surface marker avb3 and the signal regulatory protein (SIRPa) on macrophages. Using this novel exosome, we have achieved active PDAC targeting and MPS evasion. Methods: We engineered the CD9, an exosome marker, to display a HA tagged RGD peptide at the E174 site of CD9 (CD9-HA-RGD). We overexpressed CD9-HA-RGD and a Flag tagged CD47 (CD47-Flag) in HEK 293 cells. CD47, a “don’t eat me” protein signal, mediates inhibition of phagocytosis through interacting with SIRPa on macrophages. Exosomes overexpressing CD9-HA-RGD and CD47-Flag were harvested from HEK 293 cell culture media by ultracentrifugation followed by purification with size exclusion chromatography (SEC). Exosomes displaying RGD and CD47 (Exo-RGD/CD47) were characterized in vitro using ELISA, western blot (WB), flow cytometry (FCM), PDAC cell uptake, and macrophage phagocytosis assays. Exo-RGD/CD47 was also evaluated in vivo in patient-derived PDAC cell line (PDCL5) orthotopic xenograft mouse model. Results: RGD and CD47 were demonstrated to be successfully displayed on exosome surface by analysis of HA and Flag expression using ELISA and FCM. Engineered Exo-RGD/CD47 exosomes achieved enhanced cell binding and increased uptake capacities in an avb3 expression dependent manner in PDAC cells. This was confirmed using excess RDG peptide in a competition assay. Control exosomes demonstrated low uptake efficiency by PDAC cells. Exo-RGD/CD47 resulted in 71.5% reduction of macrophage phagocytosis when compared to control exosomes as evaluated by THP1 cells phagocytosis assay. Using DiR stained exosomes, we demonstrated that Exo-RGD/CD47 systemic delivery significantly reduced liver accumulation up to 80.6% and 65.8% after 1h and 6h after injection, respectively, as compared to control group (p<0.05). Similarly, Exo-RGD/CD47 delivery resulted in 70.1% reduction of splenic accumulation as compared to control at 6h after application (p<0.05). Exo-RGD/CD47 systemic delivery also showed significant tumor accumulation in the PDCL5 xenograft tumor model as compared to control (p<0.05). Conclusions: These data demonstrate that CD9 is an editable exosome biomarker by which peptides can be effectively displayed on the surface of an exosome. Displaying RGD and CD47 on the exosome surface results in a novel exosome that enables active PDAC targeting and evasion of phagocytosis by MPS. These findings suggest the newly designed exosome may have a better translational potential in PDAC diagnosis and therapy than traditional exosomes. Citation Format: Shi-He Liu, Justin Creeden, F. Charles Brunicardi, JianTing Zhang. Genetically engineered exosomes enable active pancreatic cancer targeting and evading mononuclear phagocytic system [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-009.

Abstract IA-02: Pancreatic cancer heterogeneity and plasticity: The mix of seed and soil

Abstract A series of transcriptomic analyses of human pancreatic ductal adenocarcinoma (PDAC) have consistently demonstrated multiple cancer subtypes with different functional behavior in preclinical models and patient outcomes in clinical studies. The two major molecular subtypes described in PDAC are the classical epithelial (E) and the quasi-mesenchymal (QM) subtypes, otherwise known as the basal-like or squamous subtype. Although a model of E and QM subtypes to dichotomize PDAC tumor behavior affords the simplicity of use in biomarker and functional studies, there has been increasing evidence of epithelial mesenchymal transition (EMT) transcriptional plasticity of PDAC cells in model systems as well as evidence from single cell RNA-seq studies. This suggests that the E and QM states are on a continuum that can result in interconverting cell types. We have utilized single cell technologies, functional model systems, and human tumor analysis to characterize the landscape of PDAC heterogeneity and the factors involved with cellular plasticity between E and QM states. Initial single cell RNA-seq studies in PDAC mouse models revealed that partial EMT is enriched in circulating tumor cells (CTCs) compared to primary tumor cells (Ting DT et al. Cell Reports 2014). These CTCs were also highly enriched for extracellular matrix (ECM) gene expression that were shared with stromal fibroblasts in the primary tumor indicating that the “seeds” of metastasis produce their own “soil”. Functional studies in patient derived PDAC cell lines and cancer associated fibroblasts (CAFs) revealed EMT heterogeneity was partially driven by the relative amounts of stromal CAFs (Ligorio M*, Sil S* et al. Cell 2019). Using RNA in situ hybridization (RNA-ISH) in human primary PDAC tumors and high content digital image analysis, we classified individual tumor cells for EMT and proliferative (PRO) phenotypes that were linked with relative stromal content and characterize the significant inter- and intra-tumoral heterogeneity of these cell types within distinct tumor glands. In addition, analysis of PDAC tumorspheres revealed a significant induction of EMT changes in response to FOLFIRINOX, which was also found in patient tumors that were resected after neoadjuvant FOLFIRINOX (Porter RL et al. PNAS 2019). Finally, we have recently characterized human PDAC CTCs that has identified 4 different stem cell genes (WNT5A/KLF4, LGALS3, LIN28B) that are enriched in these metastatic precursors and cluster separately between patients (Franses JW*, Philipp J* et al. Nature Communications 2020). Each of these CTC phenotypes are all associated with EMT features suggestive of different pathways that can achieve similar metastatic behavior. Altogether, single cell heterogeneity of PDAC is functionally linked with tumor stromal interactions, response to chemotherapy, and the ability to seed distant metastases. Citation Format: David T. Ting. Pancreatic cancer heterogeneity and plasticity: The mix of seed and soil [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr IA-02.

FOLFIRINOX Versus Gemcitabine-based Therapy for Pancreatic Ductal Adenocarcinoma: Lessons from Patient-derived Cell Lines.

FOLFIRINOX [fluorouracil (5-FU), irinotecan, oxaliplatin] and gemcitabine plus nab-paclitaxel are standard treatments for patients with pancreatic ductal adenocarcinoma (PDAC). Despite efficacy rates of less than 32%, evidence is lacking to guide the use of one drug over the other. Herein, we compared the sensitivity of patient-derived PDAC cell lines to each of these regimens. Changes in the growth of 19 low-passage patient-derived PDAC cell lines were evaluated in response to treatment with FOLFIRINOX and gemcitabine plus paclitaxel (Gem-Pac). Six cell lines exhibited optimal sensitivity (high EMax and low GI50) to FOLFIRINOX and three cell lines exhibited optimal sensitivity to Gem-Pac. Several cell lines that were optimally sensitive to one drug regimen exhibited very poor response to the other. Further characterization of cancer cells exhibiting preferential sensitivity to each of these regimens may allow the identification of biomarkers to guide the selection of appropriate chemotherapy for a given patient.

Abstract A48: Quantifying stroma-tumor cell interactions in three-dimensional cell culture systems

Abstract Background: In cancer research, studying cell motility is fundamental to investigate cancer invasion and drug resistance. In solid tumors constituted by a huge stroma component, such as pancreatic ductal adenocarcinoma (PDAC), the ability to quantify cell movement and interactions is mandatory to better understand the complex crosstalk between cancer and stroma cells. In PDAC, the pancreatic stellate cells (PSCs) are the principal source of dense fibrotic stroma: these closely interact with the PDAC cells to create a facilitating tumor microenvironment that supports local and distant tumor progression through the secretion and/or the response to a number of cytokines that globally increase cancer invasiveness; moreover, the dense tumor microenvironment contributes to resistance to chemotherapy and radiation therapy. Recent studies have established that targeting the stromal compartment in PDAC may lead to promising outcomes. Unfortunately, significant improvements in the overall survival of patients have not been realized in more than four decades, in part because of the lack of relevant preclinical models. Methods: To infer interactions existing between stromal and cancer cells in complex 3D environments, we developed a novel platform that combines time-lapse fluorescence microscopy, automated image detection, and extensive statistical analysis. To better mimic in situ PDAC, we created a hydrogel-based (Matrigel, collagen) model that allows cell tracking in 4 dimensions (x, y, z, t), not possible in 2D, using a mixed population of L3.6PL pancreatic cancer cells and PSCs at a ratio of 25% and 75%, respectively. To precisely identify the two populations and facilitate automatic cell detection, cells were transfected with GFP and mCherry vectors. Once in the 3D conformation, the cocultures were monitored through time-lapse confocal microscopy (CLSM) in controlled conditions, and the dataset containing the temporal evolution of the cells was processed by statistical tools. Results: Key to our approach was the generation of new machine learning-driven automated inferential protocols that resulted in the high-resolution imaging of the strength of the interactions between cells, as well as the potential presence of local chemokine gradients. In our ongoing studies, both commercial PDAC cell lines and pancreatic stellate cells were used to establish the model system. We are currently adopting the model to take full advantage of patient-derived PDAC cells established using the conditionally reprogrammed cells technique, mixed with patient-matched stellate cells. Since cell migration is a hallmark of cancer, we believe that this platform could be used as a reliable and reproducible approach for studying single-cell migration and invasion also in patient-derived models. The research leading to these results received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 759959, ERC-StG “INTERCELLMED”). Citation Format: Marta Maria Cavo, Francesco Alemanno, Donatella Delle Cave, Eliana D'Amone, Adriano Barra, Enza Lonardo, Erika Parasido, Chris Albanese, Loretta Laureana del Mercato. Quantifying stroma-tumor cell interactions in three-dimensional cell culture systems [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A48.

Gemcitabine Exposure Induces Epigenomic Remodeling in Pancreatic Cancer Cells during Resistance Development

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer‐related deaths in the United States with a devastating overall five‐year survival rate of less than five percent. Chemotherapy is a mainstay for PDAC treatment, with Gemcitabine (Gem) as a part of a first‐line regimen. Although Gem has shown clinical benefit, it possesses limited control over cancer progression, due to the major obstacle of chemoresistance. Consequently, there is an urgent need to further improve our understanding of molecular mechanisms underlying resistance development to identify innovative therapeutic targets. Our laboratory is focused on the use of epigenomic drugs as novel therapeutic approaches. Our experimental design involves developing chemoresistance in patient derived PDAC cells to identify epigenomic alterations and gene expression changes that can participate in the initiation and establishment of resistance to chemotherapeutic agents. Identifying such modifications can provide valid druggable targets to enhance the effect or reverse resistance to anti‐cancer drugs. In the present study, we induced resistance to Gem on a naïve patient‐derived PDAC cell line and evaluated epigenomic landscape remodeling through changes of histone post‐translational modifications. In addition, we characterized phenotypic changes in response to Gem exposure. Using the Incucyte® imaging system, we determined that exposure to increasing concentrations of Gem induced resistance on naïve patient‐derived PDAC cells. Gem resistant cells showed a reduced basal proliferation rate and apoptotic response compared to parental cells, as measured by activation of caspases 3 and 7. Additionally, cells developed morphological changes towards an epithelial state, coincident with the increase of Keratin 17/19 and E‐Cadherin. Moreover, treated cells demonstrated reduced sensitivity to the well‐known DNA damaging agents cisplatin and 5‐Fluorouracil. Evaluation of histone post‐translational modifications on these cells revealed reduction in the global amount of repressive marks (H3K9Me1, 2 and 3; H3K27Me2 and 3), as well as an increase of the activating histone mark H3K27Ac, associated with super enhancers. Furthermore, immunofluorescence‐based microscopy showed re‐distribution of the H3K9Me1 and H3K27Me3 repressive marks from the nuclear periphery to the whole nucleus, suggesting epigenomic remodeling of pancreatic cancer cells in response to the development of Gem resistance. In summary, these data demonstrate that Gem treatment induces chromatin remodeling and that targeting key epigenomic pathways may hold promise for novel strategies toward re‐sensitizing or preventing resistance of pancreatic cancer to chemotherapeutic agents.

Abstract B50: Keratin 17 drives tumor aggression and could be targeted for treatment of pancreatic ductal adenocarcinoma

Abstract Our aims are to uncover the molecular mechanisms through which keratin 17 (K17), a prognostic biomarker, drives tumor aggression and to target these mechanisms to provide more effective treatment for pancreatic ductal adenocarcinoma (PDAC). In murine orthotopic xenografts, we found that K17-positve PDACs survive for a shorter interval than controls. Prompted by previous reports that post-translational modifications (PTMs) regulate intermediate filament dynamics, we established in vitro that phosphorylated K17 detaches from the cytoskeleton and enters the nucleus, where it promotes tumor growth by targeting tumor suppressor proteins, including p27, for nuclear export and degradation. To further understand the events that control K17 solubilization, we sequenced K17 from primary PDACs by liquid chromatography-mass spectrometry and identified serine sites within the N-terminus that are phosphorylated only in soluble K17. Furthermore, phosphorylation is required to maintain K17 solubility and soluble K17 accumulates in the nucleus of PDAC cells. By an unbiased screen of 80 small-molecule kinase inhibitors in PDAC, we determined that SYK kinase inhibitors, already in clinical trials for other malignancies, abrogated K17 solubilization. Prompted by our finding that K17 serves as a nuclear shuttle of p27, we identified two amino acid sequences in K17 that have similar polarity to sequences that are used by cyclins to dock to p27. Point mutations in two of these domain key residues blocked K17-mediated degradation of nuclear p27, and we identified similar effects in the background of wild-type and oncogenic KrasG12D PDAC cells. Current studies are under way to find additional protein and RNA targets for potential therapeutic intervention. Using patient-derived organoids, human and murine PDAC cells, we determined that K17-expressing PDAC cells are more than twice as resistant as isogenic K17-negative cells to gemcitabine (Gem) and 5-fluorouracil (5-FU), two key components of current chemotherapeutic regimens. By unbiased liquid chromatography-coupled tandem mass spectrometry metabolomics, RNA-sequencing analyses (TCGA), and in vivo magnetic resonance spectroscopy, we found that K17 induces metabolic reprogramming by increasing glycolysis and pyrimidine biosynthesis, pathways that have been linked to chemoresistance. We are extending this work to determine if disruption of K17-mediated metabolic rewiring by small-molecule inhibitors will resensitize tumor cells to pyrimidine analogues. In conclusion, K17 undergoes key post-translational modifications that enable solubilization and nuclear translocation, the targeting of tumor suppressor proteins, and enhanced pyrimidine biosynthesis to drive chemoresistance. Uncovering these mechanisms could ultimately lead to the identification of novel approaches to target the oncogenic functions of K17, and thereby, to enable the development of more effective treatment options for PDAC. Citation Format: Kenneth R. Shroyer, Luisa Escobar-Hoyos, Cindy Leiton, Chun-Hao Pan, Ryan Kawalerski, Lucia Roa-Peña, Sruthi Babu. Keratin 17 drives tumor aggression and could be targeted for treatment of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B50.

Xenografts Derived From Patients' Ascites Recapitulate the Gemcitabine Resistance Observed in Pancreatic Cancer Patients.

Most patient-derived pancreatic ductal adenocarcinoma (PDAC) xenografts have been established from surgical specimens of patients who have not received chemotherapy. However, xenografts have rarely been established from chemotherapy-resistant, advanced PDACs, because such cases are usually inoperable. The purpose of this study is to establish patient-derived xenografts using PDAC cells refractory to chemotherapy. Clinical PDAC cells obtained from ascites of patients who had received continuous chemotherapy were implanted into the flanks of immunocompromised mice. Growth and histological features of the xenografts with and without gemcitabine treatment were then analyzed. Ascites-derived PDAC cells were successfully expanded through serial xenograft passage without changes in histological appearance. While treatment with gemcitabine substantially inhibited the growth of all PDAC xenografts tested, the tumor volume gradually increased, and the tumors showed marked regrowth even under continued gemcitabine treatment. These findings are consistent with the actual clinical course of the corresponding patients for each xenograft. Ascites-derived xenograft models represent a valuable experimental system for testing the efficacy of currently available therapeutic compounds on chemotherapy-resistant PDAC cells and for elucidation of the mechanisms underlying chemotherapy resistance.