Pancreatic Ductal Adenocarcinoma Tumor Microenvironment Research Articles

Single-cell RNA sequencing and influences of necroptosis-associated myeloid lineages in immune microenvironment of PDAC.

e16261 Background: Tumor-infiltrating myeloid cells (TIMs) are abundant in tumor stroma and emerging evidence indicate that the presence of these cells influences clinical outcomes in many cancer types. TIMs play important roles in tumor progression and specific subsets may exhibit divergent anti-tumor or tumor-promoting functions. Currently, the molecular characteristics of TIMs in pancreatic ductal adenocarcinoma (PDAC) largely remain unknown. Like other programmed cell death procedures such as ferroptosis and pyroptosis, necroptosis has recently emerged as an important cellular event that modulates tumorigenesis and tumor progression. Combination of necroptosis and TIMs is of great interest to study the molecular mechanism of clinical outcomes for PDAC patients. Methods: This study enrolled 16 PDAC patients. The scRNA-seq data were obtained from a published dataset and each patient included primary tumor, metastatic tumor, primary tumor-adjacent and PBMC samples. All myeloid cells comprising granulocytes, monocytes, macrophages, and dendritic cells from enrolled patients were extracted for further analysis. We performed comprehensive analysis about the characteristics of myeloid lineages, differentially expressed genes, pathway enrichment analysis and novel cell subtype identification. The necroptosis-associated novel TIMs cell sub-clusters were elucidated in detail. Results: To comprehensively analyze the heterogeneous TIMs microenvironment in PDAC patients using scRNA-seq dataset, we firstly obtained consistently upregulated genes among granulocytes, monocytes, macrophages, and dendritic cell types between tumor samples and non-tumor samples. Those uniquely expressed genes represent different functions in necroptosis-associated TIMs cells. In total, we identified 10 necroptosis-associated upregulated genes in PDAC tumors and 5 upregulated genes in tumor-adjacent and peripheral blood samples, respectively. Based on the cells expressed those necroptosis-related genes, we identified three novel cell populations, including GLUL-SQSTM1- RTM, HSP90AA1+HSP90AB1+ mast cells, and JAK3+TLR4+ CD16 monocytes, which were regulators of immunity either tumor-promoters or tumor-suppressors. Clinical outcomes further supported their function roles in the PDAC tumor microenvironment. Conclusions: This study comprehensively studied the heterogeneity of myeloid microenvironment in PDAC by scRNA-Seq. Several necroptosis-associated genes and myeloid cell sub-clusters were identified and they have prognostic clinical values in PDAC. These preliminary results were helpful for further understanding the molecular mechanism of PDAC treatment strategies.

Phase II trial of BXCL701 and pembrolizumab in patients with metastatic pancreatic ductal adenocarcinoma (EXPEL-PANC).

TPS4194 Background: Pancreatic ductal adenocarcinoma (PDAC) has limited therapeutic options and is thought to be a “cold” tumor that does not respond to immunotherapy, due to a tumor microenvironment (TME) consisting of a desmoplastic stroma and poor T cell infiltrate. BXCL701 is an oral synthetic dipeptide that competitively inhibits dipeptidyl peptidases DPP4, DPP8, DPP9 and fibroblast activation protein (FAP). BXCL701 exerts antitumor activity via inhibition of DPP8/9, which is associated with induction of proinflammatory cytokines, as well as inhibition of FAP, which disrupts tumor-stromal interactions. Preclinical xenograft models demonstrate synergy between BXCL701 and an anti-PD-1 monoclonal antibody, reducing tumor growth and promoting an increase in intratumoral CD4+ and CD8+ T cells and NK cells. The combination of BXCL701 and pembrolizumab is already being studied in a phase II trial in patients with prostate cancer (NCT03910660) and a multi-cohort basket study (NCT04171219). Methods: This is a phase II trial of BXCL701 administered at 0.2 mg PO BID days 1-7 and 0.3 mg BID days 8-14 during cycle 1 (21 days) followed by 0.3 mg BID days 1-14 every 21 days in all subsequent cycles, given with pembrolizumab 200 mg IV every 21 days (all cycles). The primary objective is to determine the 18-week progression-free survival rate (PFS18weeks) in patients with metastatic PDAC treated in the second-line setting. We estimate that historical 2nd-line PFS18weeks is 30% or less; using a Simon’s two-stage (minimax) design, a type I error rate of 0.05 and power of 80% when the true response rate of 50%, we will need 19 patients in stage 1 and 20 in stage 2 (a total of 39). We plan to enroll 43 patients to account for a predicted 10% drop out of unevaluable patients. Eligible patients must have metastatic PDAC and disease progression or intolerance to only 1 line of therapy for metastatic disease, without exposure to prior immunotherapy. Patients must have measurable disease amenable to serial biopsies. Correlative pharmacodynamic studies include imaging mass cytometry to examine 34 markers of the PDAC TME in tissue biopsies, as well as blood-based analyses of KRAS circulating tumor DNA, circulating markers of fibrosis, and IL-6. Enrollment began in Q1 2023. Clinical trial information: NCT05558982 .

Translocation of Oral Microbiota into the Pancreatic Ductal Adenocarcinoma Tumor Microenvironment.

Oral dysbiosis has long been associated with pancreatic ductal adenocarcinoma (PDAC). In this work, we explore the relationship between the oral and tumor microbiomes of patients diagnosed with PDAC. Salivary and tumor microbiomes were analyzed using a variety of sequencing methods, resulting in a high prevalence and relative abundance of oral bacteria, particularly Veillonella and Streptococcus, within tumor tissue. The most prevalent and abundant taxon found within both saliva and tumor tissue samples, Veillonella atypica, was cultured from patient saliva, sequenced and annotated, identifying genes that potentially contribute to tumorigenesis. High sequence similarity was observed between sequences recovered from patient matched saliva and tumor tissue, indicating that the taxa found in PDAC tumors may derive from the mouth. These findings may have clinical implications in the care and treatment of patients diagnosed with PDAC.

Activation of the integrated stress response (ISR) pathways in response to Ref-1 inhibition in human pancreatic cancer and its tumor microenvironment.

Pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC) is characterized by a profound inflammatory tumor microenvironment (TME) with high heterogeneity, metastatic propensity, and extreme hypoxia. The integrated stress response (ISR) pathway features a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) and regulate translation in response to diverse stress conditions, including hypoxia. We previously demonstrated that eIF2 signaling pathways were profoundly affected in response to Redox factor-1 (Ref-1) knockdown in human PDAC cells. Ref-1 is a dual function enzyme with activities of DNA repair and redox signaling, responds to cellular stress, and regulates survival pathways. The redox function of Ref-1 directly regulates multiple transcription factors including HIF-1α, STAT3, and NF-κB, which are highly active in the PDAC TME. However, the mechanistic details of the crosstalk between Ref-1 redox signaling and activation of ISR pathways are unclear. Following Ref-1 knockdown, induction of ISR was observed under normoxic conditions, while hypoxic conditions were sufficient to activate ISR irrespective of Ref-1 levels. Inhibition of Ref-1 redox activity increased expression of p-eIF2 and ATF4 transcriptional activity in a concentration-dependent manner in multiple human PDAC cell lines, and the effect on eIF2 phosphorylation was PERK-dependent. Treatment with PERK inhibitor, AMG-44 at high concentrations resulted in activation of the alternative ISR kinase, GCN2 and induced levels of p-eIF2 and ATF4 in both tumor cells and cancer-associated fibroblasts (CAFs). Combination treatment with inhibitors of Ref-1 and PERK enhanced cell killing effects in both human pancreatic cancer lines and CAFs in 3D co-culture, but only at high doses of PERK inhibitors. This effect was completely abrogated when Ref-1 inhibitors were used in combination with GCN2 inhibitor, GCN2iB. We demonstrate that targeting of Ref-1 redox signaling activates the ISR in multiple PDAC lines and that this activation of ISR is critical for inhibition of the growth of co-culture spheroids. Combination effects were only observed in physiologically relevant 3D co-cultures, suggesting that the model system utilized can greatly affect the outcome of these targeted agents. Inhibition of Ref-1 signaling induces cell death through ISR signaling pathways, and combination of Ref-1 redox signaling blockade with ISR activation could be a novel therapeutic strategy for PDAC treatment.

FAP-targeted CAR-T suppresses MDSCs recruitment to improve the antitumor efficacy of claudin18.2-targeted CAR-T against pancreatic cancer

PurposeThe claudin 18.2 (CLDN18.2) antigen is frequently expressed in malignant tumors, including pancreatic ductal adenocarcinoma (PDAC). Although CLDN18.2-targeted CAR-T cells demonstrated some therapeutic efficacy in PDAC patients, further improvement is needed. One of the major obstacles might be the abundant cancer-associated fibroblasts (CAFs) in the PDAC tumor microenvironment (TME). Targeting fibroblast activation protein (FAP), a vital characteristic of CAFs provides a potential way to overcome this obstacle. In this study, we explored the combined antitumor activity of FAP-targeted and CLDN18.2-targeted CAR-T cells against PDAC.MethodsNovel FAP-targeted CAR-T cells were developed. Sequential treatment of FAP-targeted and CLDN18.2-targeted CAR-T cells as well as the corresponding mechanism were explored in immunocompetent mouse models of PDAC.ResultsThe results indicated that the priorly FAP-targeted CAR-T cells infusion could significantly eliminate CAFs and enhance the anti-PDAC efficacy of subsequently CLDN18.2-targeted CAR-T cells in vivo. Interestingly, we observed that FAP-targeted CAR-T cells could suppress the recruitment of myeloid-derived suppressor cells (MDSCs) and promote the survival of CD8+ T cells and CAR-T cells in tumor tissue.ConclusionIn summary, our finding demonstrated that FAP-targeted CAR-T cells could increase the antitumor activities of sequential CAR-T therapy via remodeling TME, at least partially through inhibiting MDSCs recruitment. Sequential infusion of FAP-targeted and CLDN18.2-targeted CAR-T cells might be a feasible approach to enhance the clinical outcome of PDAC.

The Cell Biology of Metastatic Invasion in Pancreatic Cancer: Updates and Mechanistic Insights

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related mortality worldwide. This is largely due to the lack of routine screening protocols, an absence of symptoms in early-stage disease leading to late detection, and a paucity of effective treatment options. Critically, the majority of patients either present with metastatic disease or rapidly develop metastatic disease. Thus, there is an urgent need to deepen our understanding of metastasis in PDAC. During metastasis, tumor cells escape from the primary tumor, enter the circulation, and travel to a distant site to form a secondary tumor. In order to accomplish this relatively rare event, tumor cells develop an enhanced ability to detach from the primary tumor, migrate into the surrounding matrix, and invade across the basement membrane. In addition, cancer cells interact with the various cell types and matrix proteins that comprise the tumor microenvironment, with some of these factors working to promote metastasis and others working to suppress it. In PDAC, many of these processes are not well understood. The purpose of this review is to highlight recent advances in the cell biology of the early steps of the metastatic cascade in pancreatic cancer. Specifically, we will examine the regulation of epithelial-to-mesenchymal transition (EMT) in PDAC and its requirement for metastasis, summarize our understanding of how PDAC cells invade and degrade the surrounding matrix, and discuss how migration and adhesion dynamics are regulated in PDAC to optimize cancer cell motility. In addition, the role of the tumor microenvironment in PDAC will also be discussed for each of these invasive processes.

Abstract 5851: Cancer associated fibroblast crosstalk through VEGF increases tumor cell proliferation in human pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a classically heterogeneous tumor for which tumor cells comprise less than 40% of the total tumor mass. To better understand mechanisms of intercellular interactions driving tumor response or resistance to chemotherapy, it is crucial to account for the complex tumor microenvironment (TME). Cancer associated fibroblasts (CAFs) have been implicated as key drivers supporting mechanisms that both promote and restrain tumor growth. Using our model system that investigates interactions between CAFs and cancer cells, we show a novel intercellular interaction through VEGF signaling that drives proliferation. Methods: We interrogate intracellular crosstalk in the PDAC TME using a novel 3-dimensional (3D), patient-matched coculture system of patient-derived organoids (PDO) and CAFs, obtained from patients undergoing surgical resection. Molecular characterization is performed by profiling with a 1200 analyte multiplex ELISA screen, multiparameter flow cytometry, bulk RNA sequencing of CAF and PDO cocultures, imaging mass cytometry (IMC) on patient samples, and qPCR. Results: Secretome profiling of 7 patient-derived CAF lines demonstrated patient-specific heterogeneity in proteins such as: FGF-12, FGF3, and CD79a. Common CAF-derived signaling proteins with the potential to regulate intercellular crosstalk that were seen across all samples include CRP, IL-6R, R-Spondin and others. To better understand how factors change with intercellular interactions, we set up direct CAF-PDO cocultures over 4 days to identify global transcriptional changes using bulk RNAseq. Phenotypically, coculture drives gene expression changes in both PDO subtype (basal vs classical) and CAF subtype (prevalence of iCAFs vs myCAFs). We used these transcriptome data to identify pathways modulated by CAF-PDO crosstalk. Profiling coculture supernatant, we identified upregulation in VEGF secretion. In tandem, when we investigate cellular surface marker changes, we see upregulation of VEGFR2 on the surface of CAFs by flow cytometry. This interaction is accompanied by an increase in PDO proliferation seen in the coculture conditions. Ongoing experiments aim to investigate the impact of this relationship spatially using matched patient tissue. Conclusions: Interactions between cancer cells and CAFs compound the complexity of the biology of the TME and contribute to poor patient outcomes; therefore, we have put forth a model that better represents these interactions through patient matched PDO - CAF cocultures. Using this model, we demonstrate a novel interaction through VEGF that enhances tumor cell proliferation. We introduce a targeted approach to investigating the complex biology of the TME to inform the mechanisms driving cancer biology in individual patients that can also be used to develop novel therapeutic targets. Citation Format: Samantha Guinn, Joseph Tandurella, Jignasha Patel, Richard Burkhart, Jacquelyn Zimmerman, Elizabeth Jaffee. Cancer associated fibroblast crosstalk through VEGF increases tumor cell proliferation in human pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5851.

Abstract 1328: Building a 3D co-culture model for metastatic pancreatic ductal cancer using Curiochips microphysiological system

Abstract Despite the use of multi-agent conventional chemotherapy regimens, metastatic pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal solid tumors with a 5-year survival of 9%. PDAC is characterized by a complex tumor microenvironment with multilayered interplay between neoplastic and stromal cells and the natural extracellular matrix (ECM). To date, models that recapitulate the complexity of these features are only partially reproduced ex vivo and lack the capacity to recreate PDAC tumor microenvironment physiology, fluid flow and the ECM architecture. To address these issues, we developed Curiochips MPS platform for metastatic PDAC on-a-chip. To replicate an accurate PDAC complex tumor microenvironment, we investigated human pancreatic cancer cell lines with/without SMAD4 mutation as a test model. SMAD4 is among the most frequently altered genes in PDAC and its mutation accelerates pancreatic cancer cell migration. To tailor cell-ECM interactions and recreate natural biochemical environment of tumors, we examined various ECM conditions. ECM substitutes such as type I collagen and fibrin gel offer mechanical properties to closely mimic the tumor ECM. We accessed the effect of ECM condition on the pancreatic stellate cells (PSC) activation and pancreatic cancer cell migration using PANC-1 (SMAD4-WT) and BxP3 (SMAD4-null) cell lines. Notably, type I collagen induced more PSC activation than fibrin gel. Interestingly BxP3 spheroids migrated toward to the endothelial compartment compared to PANC-1. Angiogenesis of endothelial cells was more prominent in co-culture with BxP3 than PANC-1. Furthermore, we are building the metastatic model using PDAC patient derived organoids and co-culture with primary PSC in different types of ECM. Preliminary analysis showed functional responses such as 3D organoid migration and aggregation, PSC activation and migration controlled by specific properties of EMC. Our data highlight the importance of the ECM composition on PDAC migration, PSC activation and angiogenesis in the context of genetic heterogeneity and presence/absence oncogenic mutation. The 3D co-culture platform, Curiochips offers suitable and tailored solution to investigate tumor-stroma interaction and potentially can be used as a platform for drug discovery and precision oncology to target PDAC desmoplastic tumor microenvironment. Citation Format: Sumin Kim, Yeongmin Choi, Jihye Baek, Bharti Garg, Jan Pencik, Andrew M. Lowy, Herve Tiriac, Sanghee Yoo. Building a 3D co-culture model for metastatic pancreatic ductal cancer using Curiochips microphysiological system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1328.

Abstract 1337: Aged pancreatic fibroblasts enhance pancreatic cancer growth and progression

Abstract Advancing age is a major independent risk factor for the development of pancreatic ductal adenocarcinoma (PDAC). However, the ways in which aging alters cells that eventually comprise the PDAC tumor microenvironment (TME) are incompletely understood. Fibroblasts comprise a large part of PDAC tumors, can alter the behavior of cancer cells, and impact treatment responses. Our studies investigate how normal pancreatic fibroblasts acquire tumor-promoting properties during aging and alter the tumorigenic properties of PDAC both in vitro and in vivo. To determine if PDAC tumor growth is altered by an aged microenvironment, we performed orthotopic injection of KPC and Panc02 PDAC cells into the pancreata of aged (>64-week-old) and young (6-8-week-old) C57Bl/6J mice. Tumors in aged mice are significantly larger compared to those in young mice, and there is increased incidence of liver metastases in the aged mouse cohort. We next queried whether aged pancreatic fibroblasts may contribute to this increase in tumor growth. We generated a panel of aged (donors >55 years old) and young (donors <35 years old) normal human pancreatic fibroblasts to determine the effects of healthy aging fibroblasts on PDAC cell behavior. Conditioned media from or co-culture with aged human pancreatic fibroblasts increases the proliferation, migration, and invasion of PDAC cells compared to young fibroblasts. Aged fibroblast conditioned media activated AKT signaling and enhances features of EMT and stemness. Proteomic analysis of conditioned media reveals a significantly altered secretome in aged fibroblasts, with a number of known tumor-promoting proteins released at higher levels from aged compared to young fibroblasts. We examined the contribution of GDF-15, secreted by aged fibroblasts at relatively high levels, as a paracrine modulator of PDAC growth and progression. Addition of recombinant GDF-15 to young fibroblast conditioned media increases proliferation and invasion of PDAC cells to levels similar to aged fibroblast conditioned media. Treatment of young mice with recombinant GDF-15 increases tumor growth and AKT phosphorylation in vivo. Additionally, inhibition of AKT reduces tumor size in aged but not young mice, suggesting that the aged microenvironment at least in part exerts its pro-tumor effects through AKT. In conclusion, we show that aged, non-cancer-associated pancreatic fibroblasts have the potential to promote growth, migration, and invasiveness in multiple models of pancreatic ductal adenocarcinoma. Further studies examining fibroblast-secreted factors and other changes in the aged TME are ongoing. Citation Format: Daniel J. Zabransky, Yash Chhabra, Mitchell Fane, Emma Kartalia, James Leatherman, Jacquelyn Zimmerman, Elizabeth Jaffee, Ashani Weeraratna. Aged pancreatic fibroblasts enhance pancreatic cancer growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1337.

Abstract 5173: BET inhibition reprograms pancreatic cancer associated fibroblasts to promote anti-tumor immunity

Abstract BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the most malignant neoplasm of the pancreas, and is refractory to most therapeutic strategies, including immunotherapy. We have previously demonstrated that loss of CDKN2A in PDAC tumor cells promotes an immunologically-cold tumor microenvironment, which can be counteracted through the use of bromodomain and extraterminal domain inhibitors (BETi). The immunosuppressive PDAC tumor microenvironment is also characterized by a dense desmoplastic stroma, rich in cancer associated fibroblasts (CAFs). These CAFs are a highly plastic population of cells, comprised of myofibroblastic, inflammatory, and antigen-presenting CAFs (myCAFs, iCAFs, and apCAFs, respectively). Previously research has suggested BETi can alter the function of CAFs, suggesting this therapeutic strategy may be effective at targeting both the tumor and stromal compartments of PDAC. Here we hypothesize that BETi enhances anti-tumor immunity in CDKN2A-deficient PDAC by modulating the phenotype and function of PDAC CAFs. METHODS: Isogenic wild-type and CDKN2A-deficient KPCluc pancreatic cancer cell lines were used in conjunction with KPCluc-derived murine CAF cells. CAF spheroids were used to evaluate the effects of BETi on CAF phenotype (as measured by flow cytometry) and function (as measured by NanoString analysis, checkpoint ligand expression by flow cytometry, and T cell migration assays). Additionally, the impact of BETi-induced modulation of CAF phenotype and function on wild-type and CDKN2A-deficient tumor cell spheroids was measured by flow cytometry. RESULTS: Treatment of pancreatic CAFs with BETi induced expression of genes associated with DNA damage responses and type I IFN expression. Additionally, while CAF supernatants suppressed T cell migration in vitro, treatment with BETi restored T cell migration across a transwell. This was due in part to DNA damage/STING/NF-κB signaling induced in PDAC CAF following BETi. Supernatants from BETi-treated PDAC CAFs were found to modulate PD-L1 expression on PDAC tumor cells. Direct treatment of CAFs with BETi induced a dramatic dose- and time-dependent polarization of CAF spheroids towards a myCAF phenotype. However, while BETi induced DNA damage/STING/NF-κB signaling in CAF cells, this signaling did not contribute towards this myCAF polarization, as treatment of CAF spheroids with BETi and STING or NF-κB inhibitors did not impact myCAF polarization. Additional mechanisms of polarization are currently being evaluated and will be updated at presentation. CONCLUSIONS: BET inhibition modulates CAF cell phenotype and function to promote anti-tumor immune responses. Taken together with the impact of BET inhibition on CDKN2A-deficient PDAC tumor cells, these data suggest BETi is a rational treatment approach to promote anti-tumor immunity in this genetically defined immunotherapy resistant patient population. Citation Format: Trisha Minocha, Alison Thomas, Brian M. Olson. BET inhibition reprograms pancreatic cancer associated fibroblasts to promote anti-tumor immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5173.

Pancreatic Ductal Adenocarcinoma and Immune Checkpoint Inhibitors: The Gray Curtain of Immunotherapy and Spikes of Lights

Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with a poor 5-year overall survival rate (~10%). The revolution of immunotherapy in clinical oncology has not substantially changed clinical outcome for patients with PDAC. Despite outstanding efforts, neither immune checkpoint inhibitors (ICIs) alone, nor in combination with chemotherapy or targeted therapies have shown encouraging results. This failure mirrors the lack of knowledge about the real key players of immune system senescence and the complexity of the tumor microenvironment in PDAC. However, some hope can be derived from PARP-inhibitor combinations, vaccines, and CAR-T-cells therapy. In this review, we comprehensively summarize the latest updates about the use of ICIs in PDAC, focusing on clinical evidence and ongoing studies highlighting explanations for the failure of immunotherapy and possible solutions.

Development of a novel co-culture system using human pancreatic cancer cells and human iPSC-derived stellate cells to mimic the characteristics of pancreatic ductal adenocarcinoma in vitro

Pancreatic ductal adenocarcinoma (PDAC) is a serious disease with poor prognosis and prone to chemotherapy resistance. It is speculated that the tumor microenvironment (TME) of PDAC contributes to these characteristics. However, the detailed mechanisms of interactions between pancreatic cancer cells and stroma in the TME are unclear. Therefore, the aim of this study was to establish a co-culture system that mimics the TME, using cancer cells derived from PDAC patient specimens and stellate cells from human induced pluripotent stem cells as stromal cells. We succeeded in observing the interaction between cancer cells and stellate cells and reproduced some features of PDAC in vitro using our co-culture systems. In addition, we demonstrated the applicability of our co-culture system for drug treatment in vitro. To conclude, we propose our co-culture system as a novel method to analyze cell-cell interactions, especially in the TME of PDAC.

Pancreatic cancer: Emerging field of regulatory B-cell-targeted immunotherapies.

Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer, is characterized by a high mortality rate and poor prognosis. Current treatments for PDAC, are ineffective due to a prominent immunosuppressive PDAC tumor microenvironment (TME). Although B lymphocytes are highly infiltrated into PDAC, the importance of B lymphocytes in tumorigenesis is largely neglected. B cells play a dual role in the PDAC tumor microenvironment, acting as either anti-tumorigenic or pro-tumorigenic depending on where they are localized. Tumor-infiltrating B cells, which reside in ectopic lymph nodes, namely tertiary lymphoid structures (TLS), produce anti-tumor antibodies and present tumor antigens to T cells to contribute to cancer immunosurveillance. Alternatively, regulatory B cells (Bregs), dispersed inside the TME, contribute to the dampening of anti-tumor immune responses by secreting anti-inflammatory cytokines (IL-10 and IL-35), which promote tumor growth and metastasis. Determining the role of Bregs in the PDAC microenvironment is thus becoming increasingly attractive for developing novel immunotherapeutic approaches. In this minireview, we shed light on the emerging role of B cells in PDAC development and progression, with an emphasis on regulatory B cells (Bregs). Furthermore, we discussed the potential link of Bregs to immunotherapies in PDAC. These current findings will help us in understanding the full potential of B cells in immunotherapy.

The Role of Exosomes in Pancreatic Ductal Adenocarcinoma Progression and Their Potential as Biomarkers

Pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic malignancy, is an aggressive and lethal cancer with a dismal five-year survival rate. Despite remarkable improvements in cancer therapeutics, the clinical outcome of PDAC patients remains poor due to late diagnosis of the disease. This highlights the importance of early detection, wherein biomarker evaluation including exosomes would be helpful. Exosomes, small extracellular vesicles (sEVs), are cell-secreted entities with diameters ranging from 50 to 150 nm that deliver cellular contents (e.g., proteins, lipids, and nucleic acids) from parent cells to regulate the cellular processes of targeted cells. Recently, an increasing number of studies have reported that exosomes serve as messengers to facilitate stromal-immune crosstalk within the PDAC tumor microenvironment (TME), and their contents are indicative of disease progression. Moreover, evidence suggests that exosomes with specific surface markers are capable of distinguishing patients with PDAC from healthy individuals. Detectable exosomes in bodily fluids (e.g., blood, urine, saliva, and pancreatic juice) are omnipresent and may serve as promising biomarkers for improving early detection and evaluating patient prognosis. In this review, we shed light on the involvement of exosomes and their cargos in processes related to disease progression, including chemoresistance, angiogenesis, invasion, metastasis, and immunomodulation, and their potential as prognostic markers. Furthermore, we highlight feasible clinical applications and the limitations of exosomes in liquid biopsies as tools for early diagnosis as well as disease monitoring. Taking advantage of exosomes to improve diagnostic capacity may provide hope for PDAC patients, although further investigation is urgently needed.

FES null mice demonstrate a reduction in neutrophil dependent pancreatic cancer metastatic burden.

Patients with pancreatic ductal adenocarcinoma (PDAC) have a dismal 5-year survival rate of less than 10%, predominantly due to delayed diagnosis and a lack of effective treatment options. In the PDAC tumor microenvironment (TME), neutrophils are among the immune cell types that are most prevalent and are linked to a poor clinical prognosis. However, treatments that target tumor-associated neutrophils are limited despite recent developments in our understanding of neutrophil function in cancer. The feline sarcoma oncogene (FES) is a nonreceptor tyrosine kinase previously associated with leukemia and hematopoietic homeostasis. Here we describe a newly derived FES null mouse with no distinct phenotype and no defects in hematopoietic homeostasis including neutrophil viability. The immune cell composition and neutrophil population were analyzed with flow cytometry, colony-forming unit (CFU) assay, and a neutrophil viability assay, while the response to PDAC was examined with an in vivo cancer model. In an experimental metastasis model, the FES null model displayed a reduced PDAC hepatic metastatic burden and a reduction in neutrophils granulocytes. Accordingly, our results indicate FES as a potential target for PDAC TME modulation.

Dysregulated miRNAs modulate tumor microenvironment associated signaling networks in pancreatic ductal adenocarcinoma.

The desmoplastic and complex tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) has presented tremendous challenges for developing effective therapeutic strategies. Strategies targeting tumor stroma, albeit with great potential, have met with limited success due to the lack of knowledge on the molecular dynamics within the tumor microenvironment (TME). In pursuit of a better understanding of the influence of miRNAs on TME reprogramming and to explore circulating miRNAs as diagnostic and prognostic biomarkers for PDAC, using RNA-seq, miRNA-seq, and single-cell RNA-seq (scRNA-seq), we investigated the dysregulated signaling pathways in PDAC TME modulated by miRNAs from plasma and tumor tissue. Our bulk RNA-seq in PDAC tumor tissue identified 1445 significantly differentially expressed genes with extracellular matrix and structure organization as the top enriched pathways. Our miRNA-seq identified 322 and 49 abnormally expressed miRNAs in PDAC patient plasma and tumor tissue, respectively. We found many of the TME signaling pathways were targeted by those dysregulated miRNAs in PDAC plasma. Combined with scRNA-seq from patient PDAC tumor, our results revealed that these dysregulated miRNAs were closely associated with extracellular matrix (ECM) remodeling, cell-ECM communication, epithelial-mesenchymal transition, as well as immunosuppression orchestrated by different cellular components of TME. The findings of this study could assist the development of miRNA-based stromal targeting biomarkers or therapy for PDAC patients.

Targeted therapy for pancreatic ductal adenocarcinoma: Mechanisms and clinical study.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal malignancy with a high rate of recurrence and a dismal 5-year survival rate. Contributing to the poor prognosis of PDAC is the lack of early detection, a complex network of signaling pathways and molecular mechanisms, a dense and desmoplastic stroma, and an immunosuppressive tumor microenvironment. A recent shift toward a neoadjuvant approach to treating PDAC has been sparked by the numerous benefits neoadjuvant therapy (NAT) has to offer compared with upfront surgery. However, certain aspects of NAT against PDAC, including the optimal regimen, the use of radiotherapy, and the selection of patients that would benefit from NAT, have yet to be fully elucidated. This review describes the major signaling pathways and molecular mechanisms involved in PDAC initiation and progression in addition to the immunosuppressive tumor microenvironment of PDAC. We then review current guidelines, ongoing research, and future research directions on the use of NAT based on randomized clinical trials and other studies. Finally, the current use of and research regarding targeted therapy for PDAC are examined. This review bridges the molecular understanding of PDAC with its clinical significance, development of novel therapies, and shifting directions in treatment paradigm.

Single-cell RNA sequencing reveals the effects of chemotherapy on human pancreatic adenocarcinoma and its tumor microenvironment

The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) is a complex ecosystem that drives tumor progression; however, in-depth single cell characterization of the PDAC TME and its role in response to therapy is lacking. Here, we perform single-cell RNA sequencing on freshly collected human PDAC samples either before or after chemotherapy. Overall, we find a heterogeneous mixture of basal and classical cancer cell subtypes, along with distinct cancer-associated fibroblast and macrophage subpopulations. Strikingly, classical and basal-like cancer cells exhibit similar transcriptional responses to chemotherapy and do not demonstrate a shift towards a basal-like transcriptional program among treated samples. We observe decreased ligand-receptor interactions in treated samples, particularly between TIGIT on CD8 + T cells and its receptor on cancer cells, and identify TIGIT as the major inhibitory checkpoint molecule of CD8 + T cells. Our results suggest that chemotherapy profoundly impacts the PDAC TME and may promote resistance to immunotherapy.

The utility of PD-L1 as a prognostic marker in pancreatic ductal adenocarcinoma (PDAC).

753 Background: The PDAC tumor microenvironment is notoriously immune suppressive, and PD-L1 expression is one mechanism by which tumor cells evade immune surveillance. In PDAC, clinical trials evaluating immunotherapy have not been successful in microsatellite stable tumors, and PD-L1 has not been shown to be predictive of immunotherapy response. Studies suggest that high PD-L1 expression may be associated with worse clinical outcomes and more advanced disease. We aimed to evaluate PD-L1 as a prognostic marker in routine clinical practice using a real-world population of patients with PDAC. Methods: We conducted a retrospective study of patients with PDAC at Mount Sinai Health System from 5/2017 to 12/2021 for whom PD-L1 status by immunohistochemistry (22c3) and combined positive score (CPS) were reported. We assessed the association between PD-L1 expression and overall survival (OS) using Kaplan-Meier estimates and multivariable Cox proportional hazards regression models. Chi-squared tests were used to evaluate the association between PD-L1 expression and mutations detected by routine next generation sequencing (NGS) using a standard panel. Results: 107 patients were evaluable. At diagnosis, median age was 68 years, 43% of patients were male, 83% had ECOG performance status 0-1, 38% had resectable disease, 14% had borderline resectable disease, and 48% had unresectable disease. Ultimately, 45 patients (42%) underwent surgery. 44% patients had gemcitabine-based initial therapy, perioperatively or as first-line, and 56% had 5FU-based initial therapy. 35% of patients had radiation therapy (RT) at any time. In the entire cohort, 44 (41%) patients had PD-L1 CPS < 1, and 63 patients had CPS ≥ 1. 93 patients had NGS, but no associations were found between PD-L1 status and commonly mutated genes, such as KRAS and SMAD4. For patients who underwent surgery, the median OS (mOS) of patients with PD-L1 CPS ≥ 1 was 46.1 months, and mOS of PD-L1 CPS < 1 was 29.8 months ( P = 0.13). For patients who did not receive surgery, mOS was 12.3 months for CPS ≥ 1 and 15.8 months for CPS < 1 ( P = 0.07). When adjusted for age, gender, ECOG score, stage, type of chemotherapy, RT, and baseline CA 19-9 level, patients who received surgery and had a CPS ≥ 1 had better OS than those who had CPS < 1 (HR 0.22, 95% CI 0.06-0.77, P = 0.02). For patients without resection, CPS ≥ 1 was not associated with OS in the adjusted model (HR 1.52, 95% CI 0.73-3.15, P = 0.26). However, patients with PD-L1 CPS ≥ 10 without surgery had worse OS compared to patients with CPS < 1 in the adjusted model (HR 11.02, 95% CI 2.89-41.96, P < 0.001). Chemotherapy type and receipt of RT did not modify OS based on PD-L1 expression. Conclusions: In resected PDAC, PD-L1 CPS ≥ 1 was independently associated with improved OS, whereas in unresectable PDAC, PD-L1 CPS ≥ 10 was independently prognostic of worse OS. PD-L1 expression is a potential prognostic factor in PDAC and may be a useful and context-dependent biomarker.

Effects of losartan and vitamin D on outcomes in metastatic pancreatic adenocarcinoma.

723 Background: Active modification of surrounding stroma is a critical mechanism of tumor cell growth in pancreatic ductal adenocarcinoma (PDAC). There is some early translational evidence that losartan (L) and vitamin D (D) may affect the tumor microenvironment and supportive stroma in PDAC to help restore a physically and biologically tumor-suppressive stroma. These effects of potentially stroma-modifying drugs have not been established in the clinical management of metastatic PDAC (mPDAC), where there have been limited advances in treatment and prognosis remains poor. Methods: This is a single center, retrospective analysis of patients with mPDAC treated at Fox Chase Cancer Center from 2010 to 2019. All data including L and D use were extracted from the medical record. Patient characteristics were compared across subgroups using Chi-square tests, Fisher’s exact tests, and Wilcoxon rank sum tests. The primary outcome was overall survival (OS) from the time of metastatic diagnosis. Kaplan-Meier curves and log-rank tests were used to compare unadjusted OS across subgroups. Cox proportional hazards models were used to characterize OS adjusted for age at metastatic diagnosis, sex, initial stage at diagnosis, performance status, Charlson Comorbidity Index (CCI), hypertension, and renal disease. Results: 518 patients were included; a majority were male (54.1%), White (82.4%), and diagnosed with de novo mPDAC (54.6%). About half of patients had hypertension (53.5%) and few had renal dysfunction (5.6%). 153 (29.5%) patients were taking D and 41 (8%) patients were taking L. Women were more likely to take D than men (p < 0.001) and White patients were more likely to take L than other races/ethnicities (p = 0.006). Median OS since metastatic diagnosis for patients taking L was 11.4 months compared to 9.6 months for patients not taking L (p = 0.07). Median OS for patients taking D was 11.3 months compared to 8.7 months for those not taking D (p = 0.10). A Cox proportional hazards model adjusting for confounders, however, showed that both L (HR 0.66, p = 0.02) and D (HR 0.81, p = 0.04) were significantly associated with improved OS from time of metastatic diagnosis. Conclusions: This large retrospective study demonstrates potential clinical and survival benefit of L and D in conjunction with mPDAC treatment. L and D are safe, well tolerated, and widely used in general medical practice. These results support further investigation of the stroma-modifying potential of L and D, as well as conducting prospective trials with these agents to further validate these findings.