Pancreatic Ductal Adenocarcinoma Tumor Microenvironment Research Articles

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.

Expression Profiles of Cuproptosis-Related Genes Determine Distinct Subtypes of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent subtype of pancreatic cancer and one of the most malignant tumors worldwide. Due to the heterogeneity of its genomics and proteomics, the prognosis of PDAC remains disappointing despite advances in surgery and medicines. Recently, a novel form of programmed cell death, cuproptosis, was proposed, although its role in PDAC has not been investigated. This study aimed to quantify the expression of cuproptosis-related genes and characterize the novel subtypes of PDAC. To evaluate the pattern of cuproptosis in PDAC, the gene expression data and clinical information of 372 samples were collected from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. A consensus cluster analysis was performed using the transcriptional levels, genetic alterations, and individual prognostic values of seven pre-selected cuproptosis-related genes (DLAT, LIPT1, FDX1, DLD, PDHB, PDHA1, and LIAS) to identify the novel subtypes associated with cuproptosis in PDAC. A univariate Cox regression analysis was used to determine the significant prognostic indicators and cuproptosis scores among the differentially expressed genes (DEGs) between the dividing subclusters, followed by a principal component analysis. The prognostic values, immune profiles, treatment sensitivities, and cuproptosis scores were evaluated between the different subgroups. Seven cuproptosis-related genes showed aberrant expression levels and genetic alterations in the PDAC tumor microenvironment. Among them, LIPT1, LIAS, DLAT, PDHA1, and DLD were significantly correlated with overall survival. Based on the expression profiles of the seven cuproptosis-related genes, three cuproptosis clusters (Clusters A, B, and C) were identified, which were represented by different clinicopathologic features, gene expression levels, and biological processes. A total of 686 DEGs were identified among the three cuproptosis clusters, of which 35 prognosis-related DEGs were selected to further classify the PDAC samples into two subgroups with different survival rates, clinicopathologic features, immune infiltration levels, and drug sensitivities. Higher cuproptosis scores were associated with a significantly poorer prognosis. The cuproptosis subtypes, scores, and relevant genes represent valuable information for assessing the heterogeneity, treatment, and prognosis of PDAC.

Abstract IA013: Age-related changes in pancreatic fibroblasts drive pancreatic cancer growth and progression

Abstract Aging is an independent risk factor for the development of pancreatic ductal adenocarcinoma (PDAC). However, the ways in which aging alters cell populations that will ultimately comprise the PDAC tumor microenvironment (TME) are incompletely understood. PDAC tumors are highly desmoplastic, and fibroblasts alter the behavior of pancreatic cancer cells and impact treatment responses. Here, we show that normal pancreatic fibroblasts acquire tumor-promoting properties during aging using both in vitro and in vivo models. To determine if PDAC tumors may be influenced by an aged microenvironment, we performed orthotopic KPC cell injections into the pancreata of aged (>52-week-old) and young (6-8-week-old) syngeneic C57Bl/6J mice. Aged mice have significantly larger tumors, increased incidence of liver metastases, and decreased survival compared to young mice. We next queried whether aged pancreatic fibroblasts 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 aged human pancreatic fibroblasts significantly increases the proliferation of PDAC cells, suggesting that secreted factors produced by aged fibroblasts enhance cancer cell growth. Next, we evaluated changes in the invasiveness of PDAC cells in a 3D co-culture system with either aged or young pancreatic fibroblasts. PDAC spheroids containing cancer cells and aged fibroblasts exhibit a significant increase in invasiveness compared to those containing young fibroblasts. Aged (>52-week-old) compared to young (6-8-week-old) mouse pancreatic fibroblasts also increase the proliferation and invasiveness of KPC cells in vitro, providing evidence that aged fibroblasts may contribute to the phenotypes observed in vivo experiments using aged mouse models. 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. We hypothesize that factors secreted by pancreatic fibroblast change during aging and that this aged secretome may serve to establish a tumor-promoting niche for pancreatic cancer cells. Citation Format: Daniel Zabransky, Elizabeth Jaffee, Ashani Weeraratna. Age-related changes in pancreatic fibroblasts drive pancreatic cancer growth and progression [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr IA013.

Fungal mycobiome-mediated immune response: a non-negligible promoter in pancreatic oncogenesis and chemoresistance.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers in humans due to late diagnosis and poor response to treatments. The tumor microenvironment (TME) of PDAC is characterized by a distinctive, suppressive immune profile, which inhibits the protective functions of anti-tumor immunity and thereby contributes to PDAC progression. Recently, the study of Alam et al. discovered for the first time that the intratumoral fungal mycobiome could contribute to the recruitment and activation of type 2 immune cells in the TME of PDAC via enhancing the secretion of a chemoattractant, interleukin (IL-) 33. In this article, we reviewed the important findings of this study. Together with our findings, we synthetically discussed the role of the fungal mycobiome in orchestrating the immune response and thereby modulating tumor progression.

Prognostic value of tumor-associated N1/N2 neutrophil plasticity in patients following radical resection of pancreas ductal adenocarcinoma

BackgroundAs an integral part of the tumor microenvironment (TME), tumor-associated neutrophils play a crucial role in tumor development. The objective of this study was to investigate the plasticity of tumor-associated...

Pancreatic ductal adenocarcinoma holds unique features to form an immunosuppressive microenvironment: a narrative review

Pancreatic ductal adenocarcinoma (PDAC) is the most common type and composes about 90% of pancreatic cancer featured with high intra-tumoral heterogeneity and poor prognosis, which has been considered the least immunogenic cancer for decades. However, this characterization might be over-simplistic, and more sophisticated approaches are needed to develop effective treatment strategies. In this review, we aim to summarize studies involving PDAC immunity in different aspects to provide a multidimensional recognition and comprehensively understanding of the mechanisms underlying the tumor microenvironment (TME) of PDAC. A database search of peer-reviewed articles published in English between 2003 and 2022 in PubMed and the Web of Science was performed. Original articles and review articles relevant to the topic were selected. We emphasized the importance of investigating tumor-infiltrating lymphocytes (TILs) in pancreatic cancer, especially focusing on CD8+ T cells, along with indicating potential therapeutic strategies to turn the immune-cold PDACs into the immune-hot ones.

Abstract PR008: Overcoming PDAC T cell therapy barriers with CD47-targeted costimulatory fusion proteins

Abstract T cell therapy is a promising immunotherapy treatment option that uses genetically modified immune cells (T cells) to eliminate tumors. This approach uses the patients’ own immune cells to generate a “living drug” and can avoid the toxic side effects of other common therapies, such as radiation or chemotherapies. However, the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) can establish several obstacles to protect the tumor from T cells, including delivery of inhibitory and death signals that shut down T cells, usurping metabolic nutrients, and recruiting and/or converting immune cells to inhibitory phenotypes that block the T cell response. CD47 is upregulated in PDAC tumors, and patients with CD47hi tumors exhibit worse survival. CD47 serves as a “don’t eat me” signal by binding to the SIRPα receptor on macrophages to block phagocytosis. CD47 expression also inhibits T cell-mediated tumor rejection by blocking cross-presentation of tumor antigens by SIRPα+ dendritic cells. Reagents that interfere with SIRPα-CD47 signaling, such as monoclonal antibodies, are currently in clinical trials and have been shown to synergize with chemotherapy in PDA models, but native CD47 expression can produce erythrocyte and platelet depletion. Costimulatory signals delivered by surface-bound receptors can initiate gene expression programs that address multiple issues in the PDAC TME by mechanisms such as lowering the threshold of activation, altering metabolic programming, and reducing exhaustion. We develop engineering strategies to deliver costimulatory signals to engineered T cells. Immunomodulatory fusion proteins (IFPs) combine the ectodomain of an inhibitory T cell receptor with an intracellular costimulatory signaling domain and we have shown in proof-of-concept studies that this effectively “replaces a brake with an accelerator” in CD8 T cells. We have found that generating IFPs is not as simple as indiscriminately fusing any two proteins together and we have developed critical design concepts that inform the selection of the fusion site, ectodomain and costimulatory signaling domain pairing, and immunological synapse localization to enhance signaling. To develop a CD47-targeted IFP, we combined the SIRPa ectodomain with the CD28 signaling endodomain and tested this technology with our mesothelin-targeted TCR-T cell platform. In in vitrostudies, SIRPa-CD28 T cells exhibited enhanced proliferation, accumulation, and tumor cytotoxicity. SIRPa IFP-T cells exhibited increased intratumoral accumulation and therapeutic efficacy in the KrasLSL-G12D/+; Trp53LSL-R172H/+;p48Cre/+ (KPC) model, without toxicity or erythrocyte depletion. Here we describe how a thoughtfully engineered fusion protein can “armor” T cells against multiple obstacles and significantly improve therapeutic efficacy against PDAC. Citation Format: Shannon Oda, Leah Schmidt, Ashley Thelen, Cody Jenkins, Edison Chiu, Aitong Ruan, Philip Greenberg. Overcoming PDAC T cell therapy barriers with CD47-targeted costimulatory fusion proteins [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 PR008.

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.

Abstract PR010: Single-cell sequencing elucidates the effects of chemotherapy on cancer cell heterogeneity and the tumor microenvironment of human pancreatic adenocarcinoma

Abstract The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) is a complex ecosystem that drives tumor progression. Improving our understanding of the PDAC TME and its role in response to therapy via in-depth single cell characterization will have broad clinical implications for biomarker development and therapeutic design. In this study, we performed single-cell RNA sequencing on freshly collected human PDAC samples of primary (n=16) or metastatic (n=11) origin, either before (n=20) or after (n=7) chemotherapy. We found a heterogeneous mixture of basal and classical Moffitt cancer cell subtypes in all samples, along with distinct cancer-associated fibroblast (CAF) and tumor-associated macrophage (TAM) subpopulations. We identified the major CAF subpopulations as inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs); within these subpopulations were a very few cells expressing immunogenic features which have previously been associated with antigen presenting CAFs (apCAFs). Tumor-associated macrophages (TAMs) could be categorized into two major subpopulations, C1QC+ TAMs or SPP1+ TAMs, each with distinct functional characteristics. For example, phagocytosis-associated gene sets were enriched in C1QC+ TAMs, while angiogenesis-associated gene sets were enriched in SPP1+ TAMs. Comparison of naïve and chemotherapy treated primary PDAC samples revealed that classical and basal-like cancer cells exhibited similar transcriptional responses to chemotherapy; this contrasts with some previous reports which posited a shift towards a basal-like transcriptional program among treated samples. We further noted that treated samples evinced fewer ligand-receptor interactions, particularly between TIGIT on CD8+ T cells and its ligand on cancer cells. We also identified TIGIT, not PD1, as the major inhibitory checkpoint molecule of CD8+ T cells in the PDAC TME. Altogether, our results suggest that chemotherapy impacts the PDAC TME and may further reduce response to immunotherapy. Citation Format: Daniel Weissinger, Emily A. Kawaler, Gregor Werba, Ende Zhao, Despoina Kalfakakou, Surajit Dhara, Grace Oh, Xiaohong Jing, Nina Beri, Lauren Khanna, Tamas Gonda, Paul E. Oberstein, Cristina Hajdu, Cynthia Loomis, Adriana Heguy, Mara H. Sherman, Amanda W. Lund, Theodore H. Welling, Igor Dolgalev, Aristotelis Tsirigos, Diane M. Simeone. Single-cell sequencing elucidates the effects of chemotherapy on cancer cell heterogeneity and the tumor microenvironment of human pancreatic 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 PR010.

Abstract C063: Smad4 deletion alters the composition of the tumor microenvironment in pancreatic cancer

Abstract Rationale: Pancreatic ductal adenocarcinoma (PDAC) tumors are notorious for their extensive non-malignant stroma in which the most abundant cells are cancer-associated fibroblasts (CAFs). CAFs are comprised of distinct subtypes with seemingly diverse roles within the PDAC tumor microenvironment (TME) and have been shown to influence disease progression and therapy response. However, most insights into PDAC CAF heterogeneity have been based on KPC (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx1-Cre) mouse models, which recapitulate the pathophysiology of human PDAC but not its genetic complexity, representing only ~5% of patient tumor mutation profiles. We hypothesized that further understanding of CAF heterogeneity in PDAC tumors with commonly occurring mutations is essential for improving patient stratification and treatment strategies, and consequently, survival of this disease. To start exploring this, we developed and analyzed new PDAC models with deletion of Smad4, which is inactivated in ~30% of PDAC cases, in the context of Kras and Trp53 mutations. Methods: Smad4 deletion was performed using CRISPR-Cas9 technology in pancreatic cancer organoids derived from KPC tumors to generate matched KPC Smad4 knockout organoids. To assess whether this mutation profile modulates the PDAC TME, we cultured pancreatic stellate cells (PSCs), a precursor of CAFs, in media conditioned from KPC and KPC Smad4 knockout organoids and assessed the activation of inflammatory CAF (iCAF) and myofibroblastic CAF (myCAF) markers by RT-qPCR. We also established co-cultures of PSCs and organoids and performed bulk RNA-sequencing on flow-sorted CAFs and cancer cells. Finally, orthotopically-grafted organoid (OGO) transplantation mouse models were generated and pancreatic tumors were evaluated by ultrasound-based imaging, single-cell RNA-sequencing, immunohistochemistry, and flow cytometry. Results: We found that Smad4 deletion in PDAC cells, in the context of Kras and Trp53 mutations, promotes PDAC progression, as expected, and alters the PDAC TME. In particular, Smad4 deletion leads to a decrease in matrix-producing myCAFs, CD105-positive CAFs and antigen-presenting apCAFs, while increasing potentially immunosuppressive iCAFs. Additionally, pancreatic tumors with Smad4 deletion are depleted for macrophages, while enriched for neutrophils. Conclusions and Significance: This study demonstrates how distinct cancer genetics differentially shape the PDAC TME and establishes a workflow for ongoing investigation of additional relevant mutation profiles. This approach could lead to better PDAC patient stratification based on the profile of both cancer and stromal cells for more effective and targeted therapies. Citation Format: Eloise G. Lloyd, Gianluca Mucciolo, Muntadher Jihad, Judhell Sandoval Manansala, Sara Pinto Teles, Giulia Biffi. Smad4 deletion alters the composition of the tumor microenvironment 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 C063.

Abstract C085: Spatial arrangements of immune cells of the pancreatic ductal adenocarcinoma tumor microenvironment correlated with outcomes in the phase 3 APACT trial

Abstract Introduction The newly diagnosed pancreatic ductal adenocarcinoma (PDAC) population has a high unmet need for effective treatments, with median survival of < 1 year. The phase 3 APACT (Adjuvant Pancreatic Adenocarcinoma Clinical Trial) evaluated the use of adjuvant nab-paclitaxel plus gemcitabine vs. gemcitabine in 866 patients with surgically resected PDAC. We explored the tumor microenvironment (TME) of >500 baseline resected APACT tumors to identify TME features that are associated with adverse outcomes. Methods Biopsy analyses included RNA-seq, DNA-seq, and multiplexed immunohistochemistry (mIHC). We quantified, for 533 patient biopsies, via mIHC the spatial arrangement of 7 immune cell types and 2 checkpoint markers relative to tumor and nontumor regions, and pairwise colocalization relative to each other. We identified combinations of biomarkers that correlate significantly with molecular signatures, predefined patient subsets, and overall survival (OS). Hazard ratios (HR) and p-values were computed via a Cox proportional hazards regression model which included resection and lymph node status as covariates. Significantly differentially-expressed transcripts were associated with biomarkers derived from the mIHC via unpaired t-test. Results Higher densities of CD8+ T cells within tumor regions correlated with longer OS (hazard ratio HR=0.76; p=0.03), and higher overall densities of CD163+ macrophages correlated with shorter OS (HR=1.44; p=0.006). We furthermore identified a patient subset (n=72) with a combination of higher CD8+ T cell and lower CD163+ macrophage densities that had a strikingly significant decreased HR of 0.46 (p=0.009). Moreover, patients with a high degree of spatial colocalization between CD8+ T cells and dual-positive CMAF+CD163+ M2-like macrophages observed a significant increased HR of 1.51 (p=0.0006), a biomarker only surpassed by nodal status in significance of correlation with OS (HR=1.9; p=0.00015). While this cellular colocalization was computed to be independent of cell densities, we found that the association of this colocalized pair of cell types with shorter OS was most significant for patients with lower overall CD8+ T cell densities (HR=1.84; p=0.0004). We further identified differentially regulated transcripts associated with this interaction and found specific putative ligand-receptor pairs that were also associated with lower OS. Conclusion A combination of greater infiltration of CD8+ cytotoxic T cells and lower infiltration of macrophages is associated with longer OS in the APACT trial. Moreover, the spatial colocalization between CD8+ T cells and CMAF+ M2 macrophages is associated with shorter OS. These findings were observed across both treatment arms of the study. Future investigation of this apparent interaction, and associated differentially expressed transcripts, may inform management of patients with pancreatic adenocarcinoma and increase effectiveness of PDAC therapies. Citation Format: David J. Reiss, Andrew Browne, Brian Fox, Alexander V. Ratushnyy, Maria Wang, Andrew V. Biankin, Thomas Lila. Spatial arrangements of immune cells of the pancreatic ductal adenocarcinoma tumor microenvironment correlated with outcomes in the phase 3 APACT trial [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 C085.

Abstract C036: Cancer associated fibroblasts are regulators of the tumor microenvironment 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). Specifically, cancer associated fibroblasts (CAFs) have been implicated as both a tumor promoting and tumor restraining entity through disease progression and have therefore been a focus of our evolving model system. Methods: Our group created a 3-dimensional (3D), matched coculture system of patient-derived organoids (PDO), CAFs, and immune cells obtained from patients undergoing surgical resection. PDOs grown in 3D preserves the nascent tumor architecture and better replicates the disease in vitro than traditional 2D cell lines. The fully patient-matched coculture model of human PDAC allows for mechanistic interrogation into the PDAC TME by utilizing multiparameter flow cytometry, cellular sorting, and qPCR. Additionally, pharmacotyping experiments using a 5-drug regimen were used to assess chemotherapeutic resistance allowing for targeted questions exploring patient response to standard of care chemotherapy. Results: Using a combination of direct CAF-PDO coculture and PDO with CAF-conditioned media, we examined the direct (coculture) and indirect (conditioned media) impact of CAFs on PDOs. Using flow cytometry, we showed that CAF-conditioned human complete organoid media drives increased proliferation of PDOs when compared to PDOs grown in standard media. This effect was likely due to factors that are secreted from CAFs having a synergistic effect with compounds in the human complete organoid media, as this phenotype was not replicated in traditional serum free conditioned media. In direct coculture experiments of PDOs and CAFs, PDO numbers increased over time from day 2 to day 7 when compared to monoculture of PDOs, likely due to CAF presence. Lastly, to explore how CAFs may alter chemotherapeutic efficacy, PDOs were cocultured with CAFs and assessed for chemotherapeutic resistance. Pharmacotyping results showed that PDOs sorted from the PDO-CAF coculture exhibited a change in IC50 value in comparison to those PDOs that were not cocultured with CAFs. Conclusions: Here we put forth a model of PDO and CAF coculture and demonstrated its application for examining the impact of CAFs on PDAC proliferation and chemotherapeutic response. These data demonstrate the feasibility of establishing a patient-derived matched coculture model and provide the backbone for expanding this model to include representative immune cells. Citation Format: Samantha Guinn, Joseph Tandurella, Jacquelyn Zimmerman, Richard Burkhart, Elizabeth M. Jaffee. Cancer associated fibroblasts are regulators of the tumor microenvironment in human 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 C036.