Human Pancreatic Ductal Adenocarcinoma Tumors Research Articles

Abstract C007: Targeting vasoactive intestinal peptide receptor signaling enhances T-cell mediated anti-tumor response to immune checkpoint therapy in pancreatic ductal adenocarcinoma

Abstract Current immune checkpoint therapies including anti-CTLA-4 and anti-PD1 have limited efficacy in the treatment of pancreatic ductal adenocarcinoma (PDAC), largely due to a paucity of effector T cells within the tumor microenvironment (TME). In our study, we show that vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, is over-expressed in human PDAC tumors. Thus, we hypothesized that paracrine production of VIP by tumor cells within the PDAC TME constitutes an immune checkpoint pathway that limits the antitumor activity of VIP-receptor (VIP-R) expressing T cells. We first evaluated whether inhibiting VIP-R signaling by peptide-based VIP-R antagonist affects the growth of PDAC cells in vitro. Treatment of PDAC cells with increasing concentrations of VIP-R antagonist did not affect the viability of PDAC cell lines in vitro, except for a transient effect in MT5 albeit not significant. Furthermore, depletion of VIP receptor 2 (VPAC2) in a PDAC cell line, Panc02, by CRISPR-Cas9 KO had similar growth rates in vitro with a slight growth delay in vivo compared to the parental cell line. These data suggest that the expression of VPAC2 on PDAC cells may not confer a significant autocrine effect on tumor growth impacted by VIP signaling. On the contrary, human T cells cultured with VIP-R antagonists had enhanced activation, decreased proportions of Tregs, and exhausted T cells co-expressing PD-1, Tim-3, and Lag-3. Next, we performed pharmacological inhibition with VIP-R antagonists in tumor-bearing mice to model T cell homing, activation, and antitumor responses in preclinical murine models of PDAC. Daily subcutaneous injections of VIP-R antagonists had synergistic antitumor activity when combined with anti-PD-1 therapy with decreased tumor burden and improved survival. In contrast to the limited single-agent activity of anti-PD1 or VIP-R antagonists, combination therapy eliminated tumors in up to 40% of animals. Anti-tumor responses were T cell-dependent, as the combination therapy failed to improve survival in CD4 or CD8 deficient mice using knock-out strains and antibody depletion. In addition, tumor-free mice were resistant to tumor re-challenge, indicating the generation of anti-cancer immunological memory. In correspondent to these data, combination therapy led to enhanced T cell activation in vivo with increased antigen-specific T cells, decreased frequencies of Tregs as well as enhanced T cell infiltration to the tumor site as observed by infiltration of adoptively transferred GFP+ T cells into PDAC tumors. Taken together, our findings show that VIP-VIPR signaling is a targetable mechanism of immune escape in PDAC. Inhibiting VIP-R signaling improves T cell effector properties and enhances the responsiveness of PDAC to immune checkpoint therapy. Further pharmacokinetic and toxicology studies are underway. Citation Format: Tenzin Passang, Sruthi Ravindranathan, Jian-Ming Li, Shuhua Wang, Michael Brandon Ware, Hanwen Zhang, Gregory B. Lesinski, Edmund K. Waller. Targeting vasoactive intestinal peptide receptor signaling enhances T-cell mediated anti-tumor response to immune checkpoint therapy in pancreatic ductal adenocarcinoma [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 C007.

Abstract C058: Remodeling the tumor microenvironment by targeting integrin alpha V beta 3 (αvβ3) expressing cells in pancreatic cancer

Abstract ProAgio is a therapeutic cytotoxin that targets and kills integrin αvβ3-expressing cells, resulting in reduced tumor collagen, decreased formation of new blood vessels in the tumor microenvironment (TME), and prolonged survival in mouse models of pancreatic ductal adenocarcinoma (PDAC). ProAgio is currently being tested in a Phase 1 dose-finding study in patients with solid tumors including pancreas cancers. The cell subsets within the PDAC TME that express integrin αvβ3 and could be subject to ProAgio-mediated killing have not been previously studied. Using the KPC genetically engineered mouse model of PDAC, we found that ProAgio (20 mg/kg daily, x7 days) restrained tumor growth by 20% compared to vehicle treatment without changing the cancer cell proliferation rate. We subsequently re-analyzed publicly available scRNA-seq PDAC datasets to identify cells present in human and mouse PDAC tumors that co-express integrins αv and β3 and could be targets of ProAgio. We found that specific subsets of cancer-associated fibroblasts (CAFs), monocytes and macrophages expressed both integrins. Flow cytometry was performed on cells dissociated from orthotopically implanted KPC-derived tumors treated for 15 days with ProAgio or vehicle to identify TME subsets affected by ProAgio treatment. There was no significant difference in the myofibroblast (myCAF) subset, but an increase in the percentage of inflammatory CAFs (iCAFs) was seen. ProAgio treatment also significantly increased the percentage of conventional dendritic cells which play a pivotal role in antigen recognition and T cell priming. T cell abundance was unchanged, but a decrease in B cells was observed. These data demonstrate that ProAgio treatment modifies specific immune and fibroblast subsets within the PDAC TME. Ongoing studies seek to further delineate the cell subtypes affected by ProAgio and the cytokine and chemokine mediators responsible for these changes. Citation Format: Mayrel Palestino Dominguez, Philip Homan, Xianyu Zhang, Sandra Navas Reyes, Theresa Guerin, Laura Bassel, Zhi-ren Liu, Serguei Kozlov, Christine Alewine. Remodeling the tumor microenvironment by targeting integrin alpha V beta 3 (αvβ3) expressing cells in 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 C058.

Targeting vasoactive intestinal peptide-mediated signaling enhances response to immune checkpoint therapy in pancreatic ductal adenocarcinoma

A paucity of effector T cells within tumors renders pancreatic ductal adenocarcinoma (PDAC) resistant to immune checkpoint therapies. While several under-development approaches target immune-suppressive cells in the tumor microenvironment, there is less focus on improving T cell function. Here we show that inhibiting vasoactive intestinal peptide receptor (VIP-R) signaling enhances anti-tumor immunity in murine PDAC models. In silico data mining and immunohistochemistry analysis of primary tumors indicate overexpression of the neuropeptide vasoactive intestinal peptide (VIP) in human PDAC tumors. Elevated VIP levels are also present in PDAC patient plasma and supernatants of cultured PDAC cells. Furthermore, T cells up-regulate VIP receptors after activation, identifying the VIP signaling pathway as a potential target to enhance T cell function. In mouse PDAC models, VIP-R antagonist peptides synergize with anti-PD-1 antibody treatment in improving T cell recruitment into the tumors, activation of tumor-antigen-specific T cells, and inhibition of T cell exhaustion. In contrast to the limited single-agent activity of anti-PD1 antibodies or VIP-R antagonist peptides, combining both therapies eliminate tumors in up to 40% of animals. Furthermore, tumor-free mice resist tumor re-challenge, indicating anti-cancer immunological memory generation. VIP-R signaling thus represents a tumor-protective immune-modulatory pathway that is targetable in PDAC.

Abstract 2177: Myeloid-derived arginase depletes microenvironmental arginine in PDAC tumors and leads to activation of arginine de novo biosynthesis in cancer cells

Abstract Arginine is an amino acid critical for various cellular processes, not only protein synthesis but also metabolism of other essential metabolites, like polyamines, as well as a signaling factor for pathways such as the growth regulator mTOR. Previously, our group measured arginine levels in the interstitial fluid of tumors (TIF) of pancreatic ductal adenocarcinoma (PDAC) murine models and found extremely low arginine levels (2-5 uM) in the tumor microenvironment (TME). Despite near complete absence of this critical nutrient in the TME, pancreatic tumors exhibit aggressive growth. We have sought to understand both how the PDAC TME becomes arginine limited and how PDAC cells adapt to proliferate in the absence of arginine. Using genetically engineered mice, we find that arginase activity in the myeloid compartment of PDAC tumors is responsible for arginine depletion in the TME. Staining of Arg1+ myeloid populations in human PDAC samples suggest a similar mechanism reduces arginine availability in human PDAC tumors as well. We then leveraged our newfound knowledge of PDAC TIF composition to develop a novel ex vivo cell culture media formulation with physiologically relevant nutrient levels and monitored arginine acquisition pathways using isotope tracing and metabolomics assays to determine how PDAC cells cope with arginine deprivation. Under TME nutrient conditions, PDAC cells consume available citrulline and use it to produce arginine by de novo synthesis. Starving cells of citrulline or genetically perturbing arginosuccinate synthase (ASS1), key enzyme in arginine biosynthesis, significantly reduces PDAC cellular arginine and proliferative capacity. Immunohistochemical analysis of both human and mouse PDAC tumors indicates that the de novo arginine synthesis pathway is highly expressed in PDAC but not in untransformed pancreas, suggesting a key role for this pathway in PDAC progression. Altogether, we find that myeloid-derived arginase challenges PDAC cells by limiting arginine availability and suggest that de novo arginine synthesis may be a critical metabolic pathway that enables PDAC tumors to cope with this metabolic challenge. Citation Format: Juan Apiz-Saab, Alex Muir. Myeloid-derived arginase depletes microenvironmental arginine in PDAC tumors and leads to activation of arginine de novo biosynthesis in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2177.

Abstract A029: Identification of tumor microenvironment components in patient-derived pancreatic ductal adenocarcinoma organoids

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is highly drug resistant with no change in therapeutic approach for the last decade. The PDAC tumor microenvironment (TME) is highly desmoplastic and associated with altered treatment response. Our group previously identified 9 distinct cell types in human PDAC tumors using single-cell RNA-sequencing, demonstrating a diverse TME. However, the TME of in vitro organoid PDAC models remains underexplored. We initiated characterization of the TME from PDAC patient derived organoids (PDOs) to establish a working model for accurate drug sensitivity testing. Methods: We performed immunofluorescence (IF) staining on whole-mount early passage (<P4) PDO lines and corresponding paraffin-embedded primary PDAC specimens to evaluate for activated cancer-associated fibroblasts (CAFs) using antibodies against α-smooth muscle actin (α-SMA) and fibroblast activated protein (FAP); and to evaluate for T-regulatory (Treg) cells with antibodies against interleukin-2 receptor α (IL-2Rα) and forkhead box protein P3 (FOXP3). Results: CAFs promote an immunosuppressive TME in PDAC and have recently been subclassified into inflammatory fibroblasts and myofibroblasts, which can both restrain and promote tumor progression. In this study, we identified three distinct subtypes of CAFs in PDOs, which are consistent with the subtypes found in matched clinical tumor specimens: α-SMA+, which has been reported to be indicative of pancreatic stellate cell activation and associated with increased desmoplasia; FAP+, which influences cancer cell motility, invasion, and cycle progression, extracellular matrix deposition, and angiogenesis within the TME; and α-SMA+/FAP+, which is a myofibroblast subtype. We additionally identified Treg populations in PDOs with peripheral IL-2Rα and central FOXP3 expression in the organoid spheres. Tregs have previously been shown to be overexpressed in the PDAC TME, making this finding clinically relevant. Conclusions: In this preliminary study, we show that PDAC PDOs demonstrate TME components that mirror primary tumor specimens, including CAFs and Tregs. The demonstration of a tumor-matched, immunocompetent TME in PDAC PDOs provides a more clinically relevant in vitro tumor model for patient-matched drug sensitivity testing. Citation Format: Mei Gao, Charles J. Bailey, Megan M. Harper, Michael J. Cavnar, Prakash Pandalai, Shadi A. Qasem, Joseph Kim. Identification of tumor microenvironment components in patient-derived pancreatic ductal adenocarcinoma organoids [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A029.

Gα13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin.

SUMMARYGα13 transduces signals from G-protein-coupled receptors. While Gα13 functions as a tumor suppressor in lymphomas, it is not known whether Gα13 is pro-tumorigenic or tumor suppressive in genetically engineered mouse (GEM) models of epithelial cancers. Here, we show that loss of Gα13 in the Kras/Tp53 (KPC) GEM model promotes well-differentiated tumors and reduces survival. Mechanistically, tumors developing in KPC mice with Gα13 loss exhibit increased E-cadherin expression and mTOR signaling. Importantly, human pancreatic ductal adenocarcinoma (PDAC) tumors with low Gα13 expression also exhibit increased E-cadherin expression and mTOR signaling. Treatment with the mTOR inhibitor rapamycin decreases the growth of syngeneic KPC tumors with Gα13 loss by promoting cell death. This work establishes a tumor-suppressive role of Gα13 in pancreatic tumorigenesis in the KPC GEM model and suggests targeting mTOR in human PDAC tumors with Gα13 loss.

Modulation of the Human Pancreatic Ductal Adenocarcinoma Immune Microenvironment by Stereotactic Body Radiotherapy.

Stereotactic body radiotherapy (SBRT) is an emerging treatment modality for pancreatic ductal adenocarcinoma (PDAC), which can effectively prime cytotoxic T cells by inducing immunogenic tumor cell death in preclinical models. SBRT effects on human PDAC have yet to be thoroughly investigated; therefore, this study aimed to characterize immunomodulation in the human PDAC tumor microenvironment following therapy. Tumor samples were obtained from patients with resectable PDAC. Radiotherapy was delivered a median of 7 days prior to surgical resection, and sections were analyzed by multiplex IHC (mIHC), RNA sequencing, and T-cell receptor sequencing (TCR-seq). Analysis of SBRT-treated tumor tissue indicated reduced tumor cell density and increased immunogenic cell death relative to untreated controls. Radiotherapy promoted collagen deposition; however, vasculature was unaffected and spatial analyses lacked evidence of T-cell sequestration. Conversely, SBRT resulted in fewer tertiary lymphoid structures and failed to lessen or reprogram abundant immune suppressor populations. Higher percentages of PD-1+ T cells were observed following SBRT, and a subset of tumors displayed more clonal T-cell repertoires. These findings suggest that SBRT augmentation of antitumor immunogenicity may be dampened by an overabundance of refractory immunosuppressive populations, and support the continued development of SBRT/immunotherapy combination for human PDAC.

Metabolic reprogramming by driver mutation-tumor microenvironment interplay in pancreatic cancer: new therapeutic targets.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers globally with a mortality rate exceeding 95% and very limited therapeutic options. A hallmark of PDAC is its acidic tumor microenvironment, further characterized by excessive fibrosis and depletion of oxygen and nutrients due to poor vascularity. The combination of PDAC driver mutations and adaptation to this hostile environment drives extensive metabolic reprogramming of the cancer cells toward non-canonical metabolic pathways and increases reliance on scavenging mechanisms such as autophagy and macropinocytosis. In addition, the cancer cells benefit from metabolic crosstalk with nonmalignant cells within the tumor microenvironment, including pancreaticstellate cells, fibroblasts, and endothelial and immune cells. Increasing evidence shows that this metabolic rewiring is closely related to chemo- and radioresistance and immunosuppression, causing extensive treatment failure. Indeed, stratification of human PDAC tumors into subtypes based on their metabolic profiles was shown to predict disease outcome. Accordingly, an increasing number of clinical trials target pro-tumorigenic metabolic pathways, either as stand-alone treatment or in conjunction with chemotherapy. In this review, we highlight key findings and potential future directions of pancreatic cancer metabolism research, specifically focusing on novel therapeutic opportunities.

Abstract PR-001: Ex vivo co-culture system with patient-derived organoids to assess CXCR4 inhibitor as an immune modulating agent for human pancreas adenocarcinoma

Abstract Introduction: Immune checkpoint blockade either alone or in combination with chemotherapy has been ineffective in pancreatic ductal adenocarcinoma (PDAC) likely due to underlying immunosuppressive pathways. The C-X-C chemokine receptor type 4 (CXCR4)/chemokine (C-X-C motif) ligand 12 (CXCL12) axis is a key immunosuppressive pathway where CXCL12 released by cancer-associated fibroblasts (CAF) binds to its receptor CXCR4 expressed by cytotoxic CD8+ T cells (CTL) which results in their decreased abundance within tumors. Encouraging preclinical and clinical data from mouse PDAC models and human PDAC tumor sections have shown T cell tumor accumulation after co-inhibition of CXCR4 and PD1/PDL1, however number of tumor regressions were limited. Results: To further explore the mechanism by which inhibition of the CXCR4/CXCL12 pathway modulates the immune TME in triggering an anti-tumor immune response in human PDAC, we generated an autologous co-culture system with patient-derived tumor organoids (PDTO) and peripheral blood mononuclear cells (PBMC). PDAC tumor tissue from surgical resection was used to generate lines of PDTO that recapitulate the histological and genetic characteristics of the original tumor. We demonstrate that tumor-specific reactive T cells can be obtained in PDAC by co-culture with autologous PDTO, with up to 5% of CD8+ T cells producing IFNγ after tumor organoid stimulation. No T cell reactivity was observed against normal pancreatic organoids, and blocking tumor MHC I/MHC II decreased the proportion of activated T cells. Ex vivo migration assays were established to better understand the role of CXCL12 in PDAC tumor T cell exclusion. Tumor organoids attracted autologous PBMC and addition of recombinant CXCL12 decreased PBMC migration, while treatment of PBMC with the CXCR4 inhibitor AMD3100, rescued the number of PBMCs that were able to migrate towards the tumor organoids. PBMC infiltrated PDTO-containing but not empty Matrigel domes. Addition of AMD3100 increased PBMC infiltration within the Matrigel dome containing PDTOs. Finally, we found that pre-treatment of PBMC with AMD3100 increased T cell reactivity against PDTO, and that T cell activation was further increased when stimulated with PDTO pre-treated with chemotherapy. Conclusions: We found that CXCL12 modulates the migration of T cells towards tumor organoids generated from pancreatic cancer tissue, and that inhibition of CXCR4 not only increases the migration potential, but also increases tumor-specific T cell activation. The use of ex vivo autologous co-culture and migration assays provide a unique strategy to identify modulators that may enhance T cell mediated neoplastic cell death to guide therapeutic intervention in PDAC. Citation Format: Emily Alouani, Ilenia Pellicciotta, Winston Wong, Alexander S. Thomas, Michael D. Kluger, Anna M. Chiarella, Anil K. Rustgi, Gulam A. Manji. Ex vivo co-culture system with patient-derived organoids to assess CXCR4 inhibitor as an immune modulating agent for human pancreas adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-001.

USP21 deubiquitinase elevates macropinocytosis to enable oncogenic KRAS bypass in pancreatic cancer.

Activating mutations in KRAS (KRAS*) are present in nearly all pancreatic ductal adenocarcinoma (PDAC) cases and critical for tumor maintenance. By using an inducible KRAS* PDAC mouse model, we identified a deubiquitinase USP21-driven resistance mechanism to anti-KRAS* therapy. USP21 promotes KRAS*-independent tumor growth via its regulation of MARK3-induced macropinocytosis, which serves to maintain intracellular amino acid levels for anabolic growth. The USP21-mediated KRAS* bypass, coupled with the frequent amplification of USP21 in human PDAC tumors, encourages the assessment of USP21 as a novel drug target as well as a potential parameter that may affect responsiveness to emergent anti-KRAS* therapy.

Targeting Tumor-Stromal IL6/STAT3 Signaling through IL1 Receptor Inhibition in Pancreatic Cancer.

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the presence of a dense, desmoplastic stroma and the consequent altered interactions between cancer cells and their surrounding tumor microenvironment (TME) that promote disease progression, metastasis, and chemoresistance. We have previously shown that IL6 secreted from pancreatic stellate cells (PSC) stimulates the activation of STAT3 signaling in tumor cells, an established mechanism of therapeutic resistance in PDAC. We have now identified the tumor cell-derived cytokine IL1α as an upstream mediator of IL6 release from PSCs that is involved in STAT3 activation within the TME. Herein, we show that IL1α is overexpressed in both murine and human PDAC tumors and engages with its cognate receptor IL1R1, which is strongly expressed on stromal cells. Further, we show that IL1R1 inhibition using anakinra (recombinant IL1 receptor antagonist) significantly reduces stromal-derived IL6, thereby suppressing IL6-dependent STAT3 activation in human PDAC cell lines. Anakinra treatment results in significant reduction in IL6 and activated STAT3 levels in pancreatic tumors from Ptf1aCre/+;LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice. Additionally, the combination of anakinra with cytotoxic chemotherapy significantly extends overall survival compared with vehicle treatment or anakinra monotherapy in this aggressive genetic mouse model of PDAC. These data highlight the importance of IL1 in mediating tumor-stromal IL6/STAT3 cross-talk in the TME and provide a preclinical rationale for targeting IL1 signaling as a therapeutic strategy in PDAC.

TGF-β Alters the Proportion of Infiltrating Immune Cells in a Pancreatic Ductal Adenocarcinoma

PurposeImmunotherapy, such as checkpoint inhibitors against anti-programmed death-ligand 1 (PD-L1), has not been successful in treating patients with pancreatic ductal adenocarcinoma (PDAC). Tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs), and the TGF-β cytokine are critical in anti-cancer immunity. We hypothesized that TGF-β enhances the immunosuppressive effects of TAM, MDSC, and DC presence in tumors. MethodsUsing a murine PDAC cell line derived from a genetically engineered mouse model, we orthotopically implanted treated cells plus drug embedded in Matrigel into immunocompetent mice. Treatments included saline control, TGF-β1, or a TGF-β receptor 1 small molecule inhibitor, galunisertib. We investigated TAM, MDSC, DC, and TAM PD-L1 expression with flow cytometry in tumors. Separately, we used the TIMER2.0 database to analyze TAM and PD-L1 gene expression in human PDAC tumors in TCGA database. ResultsTGF-β did not alter MDSC or DC frequencies in the primary tumors. However, in PDAC metastases to the liver, TGF-β decreased the proportion of MDSCs (P=0.022) and DCs (P=0.005). TGF-β significantly increased the percent of high PD-L1 expressing TAMs (32 ± 6 % vs. 12 ± 5%, P=0.013) but not the proportion of TAMs in primary and metastatic tumors. TAM PD-L1 gene expression in TCGA PDAC database was significantly correlated with tgb1 and tgfbr1 gene expression (P<0.01). ConclusionsTGF-β is important in PDAC anti-tumor immunity, demonstrating context-dependent impact on immune cells. TGF-β has an overall immunosuppressive effect mediated by TAM PD-L1 expression and decreased presence of DCs. Future investigations will focus on enhancing anti-cancer immune effects of TGF-β receptor inhibition.

Fungi, host immune response, and tumorigenesis.

Advances in -omics analyses have tremendously enhanced our understanding of the role of the microbiome in human health and disease. Most research is focused on the bacteriome, but scientists have now realized the significance of the virome and microbial dysbiosis as well, particularly in noninfectious diseases such as cancer. In this review, we summarize the role of mycobiome in tumorigenesis, with a dismal prognosis, and attention to pancreatic ductal adenocarcinoma (PDAC). We also discuss bacterial and mycobial interactions to the host's immune response that is prevalently responsible for resistance to cancer therapy, including immunotherapy. We reported that the Malassezia species associated with scalp and skin infections, colonize in human PDAC tumors and accelerate tumorigenesis via activating the C3 complement-mannose-binding lectin (MBL) pathway. PDAC tumors thrive in an immunosuppressive microenvironment with desmoplastic stroma and a dysbiotic microbiome. Host-microbiome interactions in the tumor milieu pose a significant threat in driving the indolent immune behavior of the tumor. Microbial intervention in multimodal cancer therapy is a promising novel approach to modify an immunotolerant ("cold") tumor microenvironment to an immunocompetent ("hot") milieu that is effective in eliminating tumorigenesis.

Cancer-Associated Fibroblasts in Pancreatic Ductal Adenocarcinoma Determine Response to SLC7A11 Inhibition.

Cancer-associated fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through protumor signaling and the generation of fibrosis, the latter of which creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumor stroma and its prognostic significance. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poorer overall survival. Orthogonal approaches showed that PDAC-derived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis and that SLC7A11 inhibition significantly decreases CAF proliferation, reduces their resistance to oxidative stress, and inhibits their ability to remodel collagen and support PDAC cell growth. Importantly, specific ablation of SLC7A11 from the tumor compartment of transgenic mouse PDAC tumors did not affect tumor growth, suggesting the stroma can substantially influence PDAC tumor response to SLC7A11 inhibition. In a mouse orthotopic PDAC model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle gene-silencing drug against SLC7A11, developed by our laboratory, reduced PDAC tumor growth, incidence of metastases, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth. SIGNIFICANCE: This study demonstrates that SLC7A11 in PDAC stromal cells is important for the tumor-promoting activity of CAFs and validates a clinically translatable nanomedicine for therapeutic SLC7A11 inhibition in PDAC.

Recent, Bioelectricity-Related Articles Selected by Ann M. Rajnicek, Media Editor of Bioelectricity.

Recent, Bioelectricity-Related Articles Selected by Ann M. Rajnicek, Media Editor of Bioelectricity.

Pancreatic cancer is suppressed by fibroblast-derived collagen I.

Clinical implementation of anti-stromal therapies in pancreatic cancer has been delayed by unanticipated tumor-restraining properties of the desmoplastic stroma. In confronting these challenges, Chen etal. demonstrate in this issue of Cancer Cell that fibroblast-specific deletion of collagen I, in the background of oncogenic Kras-induced spontaneous murine pancreatic ductal adenocarcinoma, enhances immune suppression and accelerates progression of disease.

Preclinical Models of Pancreatic Ductal Adenocarcinoma and Their Utility in Immunotherapy Studies

Simple SummaryImmune checkpoint blockade has provided durable clinical responses in a number of human malignancies, but not in patients with pancreatic cancer. Efforts to understand mechanisms of resistance and increase efficacy of immune checkpoint blockade in pancreatic cancer require the use of appropriate preclinical models in the laboratory. Here, we discuss the benefits, caveats, and potentials for improvement of the most commonly used models, including murine-based and patient-derived models.The advent of immunotherapy has transformed the treatment landscape for several human malignancies. Antibodies against immune checkpoints, such as anti-PD-1/PD-L1 and anti-CTLA-4, demonstrate durable clinical benefits in several cancer types. However, checkpoint blockade has failed to elicit effective anti-tumor responses in pancreatic ductal adenocarcinoma (PDAC), which remains one of the most lethal malignancies with a dismal prognosis. As a result, there are significant efforts to identify novel immune-based combination regimens for PDAC, which are typically first tested in preclinical models. Here, we discuss the utility and limitations of syngeneic and genetically-engineered mouse models that are currently available for testing immunotherapy regimens. We also discuss patient-derived xenograft mouse models, human PDAC organoids, and ex vivo slice cultures of human PDAC tumors that can complement murine models for a more comprehensive approach to predict response and resistance to immunotherapy regimens.

S100A14 promotes progression and gemcitabine resistance in pancreatic cancer

S100 calcium binding protein A14 (S100A14) plays an important role in the progression of several types of cancer. However, its roles in pancreatic ductal adenocarcinoma (PDAC) are largely unexplored. Here, we characterized the functional roles of S100A14 in the progression and chemoresistance of PDAC. Gene expression microarray identified that S100A14 was significantly highly expressed in four pairs of human PDAC tumor compared with corresponding non-tumor tissues genes. Quantitative reverse transcription PCR (qRT-PCR), western blotting and immunohistochemical staining (IHC) showed that S100A14 was frequently overexpressed in PDAC cell lines and tissues. Moreover, expression level of S100A14 was positively correlated to advanced cancer stages. Further, Kaplan-Meier survival analysis suggested that PDAC patients with low S100A14 expression had longer overall survival in TCGA PDAC datasets. Transient overexpressing of S100A14 promoted cell proliferation, anchorage-independent colony formation, cell migration and invasion in cell lines with low endogenous S100A14 levels, while transient silencing of S100A14 inhibited cell proliferation, anchorage-independent colony formation, cell migration and invasion in cell lines with high endogenous S100A14 levels. Persistent knockdown of S100A14 by transducing shRNAs carrying lentivirus inhibited subcutaneous tumor formation in nude mice, and sensitized the PDAC cells to gemcitabine treatment. Taken together, S100A14 exhibited oncogenic properties by promoting cell proliferation, transformation, migration and invasion, and enhanced in vivo tumor growth. More importantly, inhibition of S100A14 could effectively abrogate the cancerous properties of the PDAC cells. Our study indicated that S100A14 was a valuable target for the development of therapeutic strategy, as well as a diagnostic and prognosis biomarker for PDAC patients.

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.

Metabolic programming of distinct cancer stem cells promotes metastasis of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) metastasizes to distant organs, which is the primary cause of mortality; however, specific features mediating organ-specific metastasis remain unexplored. Emerging evidence demonstrates that cancer stem cells (CSCs) and cellular metabolism play a pivotal role in metastasis. Here we investigated the role of distinct subtypes of pancreatic CSCs and their metabolomic signatures in organ-specific metastatic colonization. We found that PDAC consists of ALDH+/CD133+ and drug-resistant (MDR1+) subtypes of CSCs with specific metabolic and stemness signatures. Human PDAC tissues with gemcitabine treatment, autochthonous mouse tumors from KrasG12D; Pdx1-Cre (KC) and KrasG12D; Trp53R172H; Pdx-1 Cre (KPC) mice, and KPC- Liver/Lung metastatic cells were used to evaluate the CSC, EMT (epithelial-to-mesenchymal transition), and metabolic profiles. A strong association was observed between distinct CSC subtypes and organ-specific colonization. The liver metastasis showed drug-resistant CSC- and EMT-like phenotype with aerobic glycolysis and fatty acid β-oxidation-mediated oxidative (glyco-oxidative) metabolism. On the contrary, lung metastasis displayed ALDH+/CD133+ and MET-like phenotype with oxidative metabolism. These results were obtained by evaluating FACS-based side population (SP), autofluorescence (AF+) and Alde-red assays for CSCs, and Seahorse-based oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and fatty acid β-oxidation (FAO)-mediated OCR assays for metabolic features along with specific gene signatures. Further, we developed in vitro human liver and lung PDAC metastasis models by using a combination of liver or lung decellularized scaffolds, a co-culture, and a sphere culture methods. PDAC cells grown in the liver-mimicking model showed the enrichment of MDR1+ and CPT1A+ populations, whereas the PDAC cells grown in the lung-mimicking environment showed the enrichment of ALDH+/CD133+ populations. In addition, we observed significantly elevated expression of ALDH1 in lung metastasis and MDR1/LDH-A expression in liver metastasis compared to human primary PDAC tumors. Our studies elucidate that distinct CSCs adapt unique metabolic signatures for organotropic metastasis, which will pave the way for the development of targeted therapy for PDAC metastasis.