Pancreatic Ductal Adenocarcinoma Tumor Microenvironment Research Articles

Follicular Helper T Cells Remodel the Immune Microenvironment of Pancreatic Cancer via Secreting CXCL13 and IL-21.

Simple SummaryThe immunosuppressive microenvironment is closely related to the poor prognosis of patients with PDAC. Tfh cells play an anti-tumor function in various malignant solid tumors; however, the role of Tfh cells in PDAC has not been determined. In this study, we aimed to explore the function of Tfh cells in PDAC, and revealed a novel immunosuppressive mechanism mediated by Tfh cells. Tfh cells promoted the formation of an immunoactive tumor microenvironment by secreting CXCL13 and IL-21, and the high infiltration of Tfh cells correlated with better patient prognosis. However, the anti-tumor function of Tfh cells was inhibited by the PD-L1/PD-1 signaling pathway. Neoadjuvant chemotherapy could further reverse the function of Tfh cells. Our results provided new strategies to remodel the immunoactive tumor microenvironment of PDAC.Immunosuppression is an important factor for the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). Follicular helper T cells (Tfh cells) play an anti-tumor role in various malignant solid tumors and predict better patient prognosis. In the present study, we aimed to determine the immunosuppressive mechanism associated with Tfh cells and explore a new strategy to improve the tumor microenvironment of PDAC. Flow cytometry was used to detect the infiltration and proportion of Tfh cells in tumor tissues and peripheral blood from patients with PDAC. The spatial correlations of Tfh cells with related immune cells were evaluated using immunofluorescence. The function of Tfh cells was examined using in vitro and in vivo model systems. The high infiltration of Tfh cells predicted better prognosis in patients with PDAC. Tfh cells recruited CD8+ T cells and B cells by secreting C-X-C motif chemokine ligand 13 (CXCL13), and promoted the maturation of B cells into antibody-producing plasma cells by secreting interleukin 21 (IL-21), thereby promoting the formation of an immunoactive tumor microenvironment. The function of Tfh cells was inhibited by the programmed cell death 1 ligand 1 (PD-L1)/programmed cell death 1 (PD-1) signaling pathway in PDAC, which could be reversed using neoadjuvant chemotherapy. Treatment with recombinant CXCL13, IL-21 and Tfh cells alleviated tumor growth and enhanced the infiltration of CD8+ T cells and B cells, as well as B cell maturation in a PDAC mouse model. Our results revealed the important role of Tfh cells in mediating anti-tumor cellular immunity and humoral immunity in PDAC via secreting CXCL13 and IL-21 and determined a novel mechanism of immunosuppression in PDAC.

Abstract 2668: Pancreatic cancer cell extracellular vesicles drive the unfolded protein response in recipient normal pancreas cells

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related deaths by 2030. The 5-year survival rate remains low, hovering ~9%. A key driver of poor patient outcomes is late-stage diagnosis of disease. Currently, no diagnostics exist to identify indolent disease. A better understanding of the complex tumor microenvironment in PDAC is needed for discerning disease etiology and progression. Extracellular vesicles (EVs) are nanometer sized lipid-bilayer particles released from cells of all tissue types, which carry an array of biomolecules. EVs mediate a myriad of cancer progression events including tumor proliferation, angiogenesis, immune escape, and metastasis. Their role in PDAC tumorigenesis, however, is largely unknown. In this study we report on the effects of PDAC cell derived EVs on normal pancreatic epithelial cells. Results: We treated normal pancreas cells with pancreas cancer cell derived EVs (cEVs). Global gene expression analysis identified 376 differentially expressed genes (DEGs) enriched for genes in the aminoacyl-tRNA biosynthesis, tRNA aminoacylation, ATF4 and ATF6 signaling pathways. Gene ontology analysis identified enrichment in biological processes associated with endoplasmic reticulum (ER) stress, NF-kB signaling and DNA damage response. Lipid synthesis was severely altered, with decreased expression in several cholesterol, sterol, and acetyl-CoA pathways. Transcription factor analysis identified enrichment of CHOP/DDIT3 target motifs, as well as targets of XBP1, ATF6 and ATF4. Unfolded protein response (UPR) and ER stress genes in our dataset were significantly upregulated, including DDIT3, NFYB, ATF6 and ATF4. We validated upregulation of UPR/ER stress in these cells using qPCR and luciferase activity assays. Multi-omics characterization of EVs isolated from PDAC and normal cell lines showed distinct biochemical profiles. This analysis identified potential mediators of UPR/ER stress enriched in cEVs, including proteins which regulate protein trafficking and ubiquitination, lipid species esterified to palmitic acid and metabolites which regulate tRNA charging. Conclusions: We show that cEVs induce significant gene expression changes within recipient normal pancreatic cells and likely induce ER stress, leading to an UPR. Long-term UPR/ER stress can impact a variety of cancer hallmarks, including angiogenesis, genome stability, inflammation, metastasis, and drug resistance. ER stress has been shown to mediate progression of pancreatic intraepithelial neoplasia (PanIN) to PDAC. These results highlight a potential role of EVs in mediating cellular messages from pancreatic cancer cells to alter the function of recipient normal cells, aiding disease onset and progression. Further studies are ongoing to identify key mediators within cEVs for the induction of UPR/ER stress. Citation Format: Charles Phillip Hinzman, Shivani Bansal, Yaoxiang Li, Anton Iliuk, Jose Trevino, Partha Banerjee, Amrita Cheema. Pancreatic cancer cell extracellular vesicles drive the unfolded protein response in recipient normal pancreas cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2668.

Abstract 2740: Effect of pancreatic cancer cell derived extracellularvesicles on the tumor microenvironment

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one amongst the most lethal malignancies, with a 5 year survival rate of <10%.While the tumor microenvironment in PDAC is highly immunosuppressive, little is known about the mechanisms of immune invasion leading to rapid tumor progression. Hence, in-depth exploration of early cellular and molecular drivers of PDAC is desirable for early and precise diagnosis and to identify new biological targets for developing new therapies. Extracellular vesicles (EVs) are nanometer sized particles that are shed by most tissues/organs and are known to be important mediators of intercellular signaling and molecular transfer. We hypothesized that pancreatic cancer derived EVs may play an important role in mediating the interactions among the tumor and surrounding microenvironment (TME). Given that tumor-associated macrophages (TAMs) are integral components of the TME that can promote tumor progression, tumor invasion, and intravasation to other parts of the body, this study is focused on investigating the role of PDAC derived EVs in macrophages polarization, which in turn would impact tumor cell proliferation and invasion. Results: EVs were isolated from 6 pancreatic cancer (PaCa) and 2 normal established pancreatic epithelial cell lines. EVs were characterized using nanoparticle tracking (NTA) and immunoblot analysis. LC-MS/MS based shotgun proteomics analyses of EVs revealed that PaCa cell line derived EVs were significantly enriched in potentially immunomodulatory proteins such as S100 calcium-binding protein A4 (S100A4) and human leukocyte antigen-B (HLA-B). However, PaCa cell EVs had significantly less MHC-class-I-related chain A (MICA), a protein important for proper human leukocyte antigen (HLA) function, as compared to normal pancreatic epithelial cell derived EVs. These data suggest that PaCa derived EVs may influence immunomodulation and macrophage function within the tumor microenvironment thereby strengthening our hypothesis. Further experimental studies aimed at delineating the precise mechanism of action of EVs in macrophage polarization is ongoing. Conclusions: Our finding suggests significant difference among the molecular composition of PaCa derived cargos as compared to and normal cells derived EVs that could be useful in furthering our understanding about pancreatic cancer etiology. Furthermore, the novel insights obtained from these studies may augment the development of biomarkers that can be used for early detection of the disease and improved therapeutics to improve clinical outcomes. Citation Format: Baldev Singh, Charles Hinzman, Edina Wang, Anton Iliuk, Jose Trevino, Keith Unger, Partha Banerjee, Amrita K. Cheema. Effect of pancreatic cancer cell derived extracellularvesicles on the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2740.

Apoptosis in the Pancreatic Cancer Tumor Microenvironment-The Double-Edged Sword of Cancer-Associated Fibroblasts.

Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis. This is attributed to the disease already being advanced at presentation and having a particularly aggressive tumor biology. The PDAC tumor microenvironment (TME) is characterized by a dense desmoplastic stroma, dominated by cancer-associated fibroblasts (CAF), extracellular matrix (ECM) and immune cells displaying immunosuppressive phenotypes. Due to the advanced stage at diagnosis, the depletion of immune effector cells and lack of actionable genomic targets, the standard treatment is still apoptosis-inducing regimens such as chemotherapy. Paradoxically, it has emerged that the direct induction of apoptosis of cancer cells may fuel oncogenic processes in the TME, including education of CAF and immune cells towards pro-tumorigenic phenotypes. The direct effect of cytotoxic therapies on CAF may also enhance tumorigenesis. With the awareness that CAF are the predominant cell type in PDAC driving tumorigenesis with various tumor supportive functions, efforts have been made to try to target them. However, efforts to target CAF have, to date, shown disappointing results in clinical trials. With the help of sophisticated single cell analyses it is now appreciated that CAF in PDAC are a heterogenous population with both tumor supportive and tumor suppressive functions. Hence, there remains a debate whether targeting CAF in PDAC is a valid therapeutic strategy. In this review we discuss how cytotoxic therapies and the induction of apoptosis in PDAC fuels oncogenesis by the education of surrounding stromal cells, with a particular focus on the potential pro-tumorigenic outcomes arising from targeting CAF. In addition, we explore therapeutic avenues to potentially avoid the oncogenic effects of apoptosis in PDAC CAF.

Abstract 2678: Tumoral CXCR4 and CXCL12 but not CXCR7 nor PD-L1 expression predicts disease free survival (DFS) and cancer specific survival (CSS) in resected pancreatic adenocarcinoma patients

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumor types and the 4th leading cause of tumor-related mortality in developed nations. PDAC is characterized by a highly fibrotic stroma that can physically exclude cytotoxic T cells from the vicinity of tumor cells. Targeting the axis CXCR4-CXCL12 improved T cell access to PDAC Tumor Microenvironment (TME) making tumors more susceptible to anti-PD-1/anti PD-L1 therapy. With the intent to evaluate the role of the entire CXCR4-CXCL12-CXCR7 axis in a context of PD-1/PD-L1 expression, 80 primary pancreatic cancers were evaluated in cancer cells and TME. Methods: 80 primary PDAC samples from patients undergone surgery collected at Ospedale P. Pederzoli Peschiera del Garda (Verona, Italy) were included in the study. Immunohistochemistry was conducted using primary antibodies: Anti-CXCR7/RDC-1 clone 11G8; Anti-CXCL12 Clone #79018; two commercial anti-CXCR4 clones #44716 and the UMB-2; two commercial anti-PD-L1, clone E1L3N and 22C3. Results: CXCR4 was expressed in 53 out of 73 tumors (72.6%). CXCL12 was predominantly identified into the membrane of cancer cells in 16 out of 75 (21.3%) tumors. CXCR7 staining was detectable in 44 out of 76 (57.8%) tumors. PD-L1 identified into the membrane of cancer cells in 35 out of 70 (50%) tumors. Patients with high CXCR4 tumor expression experienced worse outcome (DFS: 11.77 vs 22.85 months p=0.0071; CSS: 21.83 vs 35 months p>0.05, not significant) and predicted short DFS in R0 patients subgroup (n=48) (DFS: 11.20 months vs 33.40 months p=0.0140). CXCL12 expression predicted DFS (p=0.0002) and CSS (p=0.0046). Of note CXCL12 predicted short DFS (p=0.0015) and CSS (p=0.0213) in R0 patient's subgroup (n=48). Although stromal expression of CXCR4, CXCL12 and CXCR7 were not prognostic, CXCR4 positive inflammatory cells and tumoral CXCL12 significantly and directly correlated with the vascular invasion. Neither tumoral nor stromal expression of PD-L1 was prognostic but PD-L1 interestingly stained the perineural invasion and tumor-infiltrating inflammatory cells of body or tail tumors. In multivariate analysis, tumoral CXCR4 expression (p=0.0043), perineural invasion (p=0.0030) and AJCC lymph node status (p=0.0016) were independent prognostic factors for DFS. Tumoral CXCL12 expression (p=0.01467), AJCC Stage (p=0.0004) and perineural infiltration (p=0.03164) were independent prognostic factors for CSS. Conclusion: Tumoral CXCR4 and CXCL12 were prognostic factors at the univariate and multivariate analysis as for DFS and CSS in PDAC. Stromal PD-L1 stained perineural infiltration and stromal CXCR4 characterized inflammatory cells in the vascular invasion. Thus tumoral and stromal characterization for CXCR4 and CXCL12 in PDAC is worth to identify prognostic categories and to orient therapeutic approach. Citation Format: Crescenzo D'Alterio, Alessandro Giardino, Giosuè Scogmaliglio, Fabiana Tatangelo, Giovanni Butturini, Luigi Portella, Giuseppe Guardascione, Isabella Frigerio, Stefano Gobbo, Gerardo Botti, Stefania Scala. Tumoral CXCR4 and CXCL12 but not CXCR7 nor PD-L1 expression predicts disease free survival (DFS) and cancer specific survival (CSS) in resected pancreatic adenocarcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2678.

ADAM10 Evens Out the Double-Edged Sword of Radiotherapy in Pancreatic Cancer

Radiotherapy plays an important role in the management of pancreatic ductal adenocarcinoma (PDAC), especially when patients are not surgical candidates. Radiation-induced tumor death provokes an acute inflammation followed by a late-fibrotic response that parallels the fibroinflammatory tumor microenvironment of PDAC, inciting the question of whether radiation-induced fibrosis contributes to PDAC progression. The study published in this issue by Mueller and colleagues presents a potential mechanism linking radiation-induced fibrosis with expression of a disintegrin and metalloprotease 10 (ADAM10) and ephrinB2, which may also contribute to tumor progression. The authors show that ablation of ADAM10 decreases radiation-induced fibrosis and improves survival in preclinical models. These data suggest that targeting ADAM10 may help to improve clinical outcomes with radiotherapy, particularly if definitive radiation is not possible. A better understanding of the biology of radiotherapy in pancreatic cancer remains crucial, and Mueller and colleagues offer important insight in this regard.See related article by Mueller et al., p. 3255.

Suppression of tumor-associated neutrophils by lorlatinib attenuates pancreatic cancer growth and improves treatment with immune checkpoint blockade

Pancreatic ductal adenocarcinoma (PDAC) patients have a 5-year survival rate of only 8% largely due to late diagnosis and insufficient therapeutic options. Neutrophils are among the most abundant immune cell type within the PDAC tumor microenvironment (TME), and are associated with a poor clinical prognosis. However, despite recent advances in understanding neutrophil biology in cancer, therapies targeting tumor-associated neutrophils are lacking. Here, we demonstrate, using pre-clinical mouse models of PDAC, that lorlatinib attenuates PDAC progression by suppressing neutrophil development and mobilization, and by modulating tumor-promoting neutrophil functions within the TME. When combined, lorlatinib also improves the response to anti-PD-1 blockade resulting in more activated CD8 + T cells in PDAC tumors. In summary, this study identifies an effect of lorlatinib in modulating tumor-associated neutrophils, and demonstrates the potential of lorlatinib to treat PDAC.

Translating complexity and heterogeneity of pancreatic tumor: 3D in vitro to in vivo models

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive type of cancer with an overall survival rate of less than 7-8%, emphasizing the need for novel effective therapeutics against PDAC. However only a fraction of therapeutics which seemed promising in the laboratory environment will eventually reach the clinic. One of the main reasons behind this low success rate is the complex tumor microenvironment (TME) of PDAC, a highly fibrotic and dense stroma surrounding tumor cells, which supports tumor progression as well as increases the resistance against the treatment. In particular, the growing understanding of the PDAC TME points out a different challenge in the development of efficient therapeutics - a lack of biologically relevant in vitro and in vivo models that resemble the complexity and heterogeneity of PDAC observed in patients. The purpose and scope of this review is to provide an overview of the recent developments in different in vitro and in vivo models, which aim to recapitulate the complexity of PDAC in a laboratory environment, as well to describe how 3D in vitro models can be integrated into drug development pipelines that are already including sophisticated in vivo models. Hereby a special focus will be given on the complexity of in vivo models and the challenges in vitro models face to reach the same levels of complexity in a controllable manner. First, a brief introduction of novel developments in two dimensional (2D) models and ex vivo models is provided. Next, recent developments in three dimensional (3D) in vitro models are described ranging from spheroids, organoids, scaffold models, bioprinted models to organ-on-chip models including a discussion on advantages and limitations for each model. Furthermore, we will provide a detailed overview on the current PDAC in vivo models including chemically-induced models, syngeneic and xenogeneic models, highlighting hetero- and orthotopic, patient-derived tissues (PDX) models, and genetically engineered mouse models. Finally, we will provide a discussion on overall limitations of both, in vitro and in vivo models, and discuss necessary steps to overcome these limitations to reach an efficient drug development pipeline, as well as discuss possibilities to include novel in silico models in the process.

Prognostic significance and immune infiltration of microenvironment-related signatures in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is 1 of the highly fatal and most aggressive types of malignancies and accounts for the vast majority of Pancreatic Cancer. Numerous studies have reported that the tumor microenvironment (TME) was significantly correlated with the oncogenesis, progress, and prognosis of various malignancies. Therefore, mining of TME-related genes is reasonably important to improve the overall survival of patients with PDAC.The Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data algorithm was applied to identify differential expressed genes. Functional and pathway enrichment analyses, protein–protein interaction network construction and module analysis, overall survival analysis and tumor immune estimation resource database analysis were then performed on differential expressed genes.Data analysis indicated that higher immune scores were correlated with better overall survival (P = 0.033). Differential expression analysis obtained 90 intersection genes influencing both stromal and immune scores. Among these intersection genes, CA9, EBI3, SPOCK2, WDFY4, CD1D, and CCL22 were significantly correlated with overall survival in PDAC patients. Moreover, multivariate Cox analysis revealed that CA9, SPOCK2, and CD1D were the most significant prognostic genes, and were closely correlated with immune infiltration in TCGA cohort. Further analysis indicated that CD1D were significantly related with immune cell biomarkers for PDAC patients.In summary, our findings provide a more comprehensive insight into TME and show a list of prognostic immune associated genes in PDAC. However, further studies on these genes need to be performed to gain additional understanding of the association between TME and prognosis in PDAC.

Hydrogel Models with Stiffness Gradients for Interrogating Pancreatic Cancer Cell Fate.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer and has seen only modest improvements in patient survival rate over the past few decades. PDAC is highly aggressive and resistant to chemotherapy, owing to the presence of a dense and hypovascularized fibrotic tissue, which is composed of stromal cells and extracellular matrices. Increase deposition and crosslinking of matrices by stromal cells lead to a heterogeneous microenvironment that aids in PDAC development. In the past decade, various hydrogel-based, in vitro tumor models have been developed to mimic and recapitulate aspects of the tumor microenvironment in PDAC. Advances in hydrogel chemistry and engineering should provide a venue for discovering new insights regarding how matrix properties govern PDAC cell growth, migration, invasion, and drug resistance. These engineered hydrogels are ideal for understanding how variation in matrix properties contributes to the progressiveness of cancer cells, including durotaxis, the directional migration of cells in response to a stiffness gradient. This review surveys the various hydrogel-based, in vitro tumor models and the methods to generate gradient stiffness for studying migration and other cancer cell fate processes in PDAC.

Deciphering the Prognostic Implications of the Components and Signatures in the Immune Microenvironment of Pancreatic Ductal Adenocarcinoma

Background: The treatment modalities for pancreatic ductal adenocarcinoma (PDAC) are limited and unsatisfactory. Although many novel drugs targeting the tumor microenvironment, such as immune checkpoint inhibitors, have shown promising efficacy for some tumors, few of them significantly prolong the survival of patients with PDAC due to insufficient knowledge on the tumor microenvironment.Methods: A single-cell RNA sequencing (scRNA-seq) dataset and seven PDAC cohorts with complete clinical and bulk sequencing data were collected for bioinformatics analysis. The relative proportions of each cell type were estimated using the gene set variation analysis (GSVA) algorithm based on the signatures identified by scRNA-seq or previous literature.Results: A meta-analysis of 883 PDAC patients showed that neutrophils are associated with worse overall survival (OS) for PDAC, while CD8+ T cells, CD4+ T cells, and B cells are related to prolonged OS for PDAC, with marginal statistical significance. Seventeen cell categories were identified by clustering analysis based on single-cell sequencing. Among them, CD8+ T cells and NKT cells were universally exhausted by expressing exhaustion-associated molecular markers. Interestingly, signatures of CD8+ T cells and NKT cells predicted prolonged OS for PDAC only in the presence of “targets” for pyroptosis and ferroptosis induction. Moreover, a specific state of T cells with overexpression of ribosome-related proteins was associated with a good prognosis. In addition, the hematopoietic stem cell (HSC)-like signature predicted prolonged OS in PDAC. Weighted gene co-expression network analysis identified 5 hub genes whose downregulation may mediate the observed survival benefits of the HSC-like signature. Moreover, trajectory analysis revealed that myeloid cells evolutionarily consisted of 7 states, and antigen-presenting molecules and complement-associated genes were lost along the pseudotime flow. Consensus clustering based on the differentially expressed genes between two states harboring the longest pseudotime span identified two PDAC groups with prognostic differences, and more infiltrated immune cells and activated immune signatures may account for the survival benefits.Conclusion: This study systematically investigated the prognostic implications of the components of the PDAC tumor microenvironment by integrating single-cell sequencing and bulk sequencing, and future studies are expected to develop novel targeted agents for PDAC treatment.

Opportunities and delusions regarding drug delivery targeting pancreatic cancer-associated fibroblasts.

A dense desmoplastic stroma formed by abundant extracellular matrix and stromal cells, including cancer-associated fibroblasts (CAFs) and immune cells, is a feature of pancreatic ductal adenocarcinoma (PDAC), one of the most lethal cancer types. As the dominant cellular component of the PDAC stroma, CAFs orchestrate intensive and biologically diverse crosstalk with pancreatic cancer cells and immune cells and contribute to a unique PDAC tumor microenvironment promoting cancer proliferation, metastasis, and resistance against both chemo- and immunotherapies. Therefore, CAFs and CAF-related mechanisms have emerged as promising targets for PDAC therapy. However, several clinical setbacks and accumulating knowledge of the PDAC stroma have revealed the heterogeneity and multifaceted biological roles of CAFs, and concerns regarding "what to deliver" and "how to deliver" have arisen when designing CAF-targeted drug delivery systems to specifically inhibit tumor-supporting CAFs without impairing tumor-restricting CAFs. In this review, we will discuss the complexity of CAFs in the PDAC stroma as well as the potential opportunities and common misconceptions regarding drug delivery efforts targeting PDAC CAFs.

Abstract PR002: STAT3 in cancer-associated fibroblasts promotes an immunosuppressive tumor microenvironment in PDAC

Abstract One of the defining characteristics of pancreatic ductal adenocarcinoma (PDAC) is the formation of a dense stroma comprised of cancer associated fibroblasts (CAFs) and immune cell populations. This stroma is immunosuppressive and can act as a physical barrier against common therapeutic treatments. Attempts to therapeutically target the PDAC stroma have yielded contradictory results, suggesting both tumor promoting and tumor limiting roles for CAFs. These studies emphasize the need to understand important transsignaling pathways between CAFs, tumor cells, and the immune microenvironment. IL-6 is a pleiotropic cytokine involved in several physiological functions and its increased expression is strongly associated with poor survival rates in PDAC patients. STAT3 is a major downstream target of IL-6, and its aberrant activation has been implicated in PDAC tumor progression and immune evasion. IL-6 expression and the IL-6/STAT3 signaling axis in PDAC has been characterized in epithelial tumor cells, however its stromal-specific function on PDAC has yet to be elucidated. We hypothesized that the STAT3 signaling axis in pancreatic CAFs contributes to the immunosuppressive and fibrotic phenotype seen with disease progression. Employing CreLoxP technology, the fibroblast specific protein-1 (Fsp-Cre) transgene was used to conditionally delete STAT3 in fibroblasts in the PdxFlp; KrasG12D; p53frt/frt (KPF) PDAC mouse model developed by our lab. Deletion of STAT3 in fibroblasts significantly increased the survival in a cohort of KPF mice compared to those with intact STAT3. Loss of fibroblast STAT3 also resulted in a significant decrease in ECM deposition in the pancreas. In preliminary investigations into the immune microenvironment, we found an increase in overall T cell infiltration and decrease in regulatory T cell populations in the STAT3 deleted cohort. Further, STAT3 deletion resulted in an increase in the ratio of M1 to M2 macrophages within the PDAC stroma, suggesting CAF STAT3 signaling is important to maintaining an immune microenvironment conducive to tumor progression. Ultimately, these preliminary results suggest STAT3 signaling in PDAC CAFs contributes to an overall survival disadvantage, an immunosuppressive tumor microenvironment, and an increase in ECM deposition. Citation Format: Julia E. Lefler, Katie MarElia, Michael C. Ostrowski. STAT3 in cancer-associated fibroblasts promotes an immunosuppressive tumor microenvironment in PDAC [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PR002.

The Role of Exosomes in Pancreatic Cancer From Bench to Clinical Application: An Updated Review.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most dismal gastrointestinal malignancies with an overall 5-year survival rate of 8%–9%. The intra-tumor heterogeneity and special tumor microenvironment in PDAC make it challenging to develop effective treatment strategies. Exosomes are extracellular vesicles that originate from the endosomes and have a diameter of 40–160 nm. A growing body of evidence has shown that exosomes play vital roles in tumor initiation and development. Recently, extensive application of exosomes as biomarkers and drug carriers has rendered them attractive in the field of PDAC. This review summarizes the latest progress in the methodologies for isolation, modification, and tracking of exosomes, exosome-mediated cell-to-cell communication, clinical applications of exosome as minimally invasive liquid biopsy and drugs carriers, as well as their involvement in the angiogenic regulation in PDAC. In spite of these advancements, some obstacles are still required to be overcome to use the exosome-based technologies for early diagnosis or improvement of prognosis of patients with PDAC.

KLHDC7B-DT aggravates pancreatic ductal adenocarcinoma development via inducing cross-talk between cancer cells and macrophages.

Tumor microenvironment (TME) exerts key roles in pancreatic ductal adenocarcinoma (PDAC) development. However, the factors regulating the cross-talk between PDAC cells and TME are largely unknown. In the present study, we identified a long noncoding RNA (lncRNA) KLHDC7B divergent transcript (KLHDC7B-DT), which was up-regulated in PDAC and correlated with poor survival of PDAC patients. Functional assays demonstrated that KLHDC7B-DT enhanced PDAC cell proliferation, migration, and invasion. Mechanistically, KLHDC7B-DT was found to directly bind IL-6 promoter, induce open chromatin structure at IL-6 promoter region, activate IL-6 transcription, and up-regulate IL-6 expression and secretion. The expression of KLHDC7B-DT was positively correlated with IL-6 in PDAC tissues. Via inducing IL-6 secretion, KLHDC7B-DT activated STAT3 signaling in PDAC cells in an autocrine manner. Furthermore, KLHDC7B-DT also activated STAT3 signaling in macrophages in a paracrine manner, which induced macrophage M2 polarization. KLHDC7B-DT overexpressed PDAC cells-primed macrophages promoted PDAC cell proliferation, migration, and invasion. Blocking IL-6/STAT3 signaling reversed the effects of KLHDC7B-DT on macrophage M2 polarization and PDAC cell proliferation, migration, and invasion. In conclusion, KLHDC7B-DT enhanced malignant behaviors of PDAC cells via IL-6-induced macrophage M2 polarization and IL-6-activated STAT3 signaling in PDAC cells. The cross-talk between PDAC cells and macrophages induced by KLHDC7B-DT represents potential therapeutic target for PDAC.

Cancer associated fibroblasts: the major player in pancreatic cancer

Tumor cells are not alone in the tumor mass; in fact they are surrounded by a complex and active microenvironment, composed by fibroblasts and their extracellular matrix (ECM), immune cells, nerves, blood vessels, and secreted factors. It is now well recognized that the microenvironment plays an important role in tumor development and thus it is imperative for the comprehensive understanding of the interplay between cancer and its microenviroment for the development of better preventative and therapeutic strategies. Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a 5 year survival rate of only 10% after diagnosis[1]. One of the main reasons for this outcome is the poor understanding of the unique microenvironment of PDAC, where up to 90% of the tumor mass can be composed by the stroma, with most of it being the expansion of activated fibroblasts and their ECM[2]. Even though CAFs represent an important component of PDAC (and other types of cancers), they are still incompletely understood. This can be partially explained by the fact that there are no specific markers to discriminate CAFs, and researchers need to rely on negative selections, absence of mutations that characterize the transformed epithelial cells plus the presence of mesenchymal markers, all together with the assessment of fibroblastic function2. There are still controversies about the pro- and anti-tumor effects of CAFs and their origin. Recently, a consensus was published2, where authors suggest that CAFs are mostly originated by the local activation and proliferation of resident fibroblasts, stimulated by tissue injury, reactive oxidative species, growth factors and more. These CAFs are characterized by their plasticity, as they can interchange between functions according to the signals of the environment. Many are the functions attributed to these cells; CAFs produce a very dense ECM, which can lead to the collapse of blood vessels, thus affecting nutrient supply and the delivery of therapies to this environment[3]. Moreover, CAFs can be immunusuppressive, producing a millieu of cytokines and chemokines that can turn off anti-tumor immune cells and recruit pro-tumor ones[4]. Interestingly, CAFs can also contribute to the metabolic signature of the environment, as they produce and modify a large range of metabolites, often supporting cancer cell survival[5]. Recently, our research group uncovered the pro-tumor roles of the synaptic protein Netrin G1 in CAFs[6]. The expression of Netrin G1 in CAFs from PDAC patients inversely correlated with overall survival. Moreover, the loss of Netrin G1 in CAFs led to decreased production of immunosuppressive factors, allowing NK cells to kill PDAC cells in vitro. Netrin G1 expression was also important for the production of metabolites (mainly glutamine and glutamate) by CAFs and for the support of nutrient deprived PDAC cells. It is important to mention that the Netrin G1 related studies were perfomed using 3D co-cultures, and that its expression can only be detected in CAFs growing in their 3D environment, further reinforcing the need for better strategies to study and understand the tumor microenvironment in PDAC. Therefore, CAFs can be seen as the major intermediaries of PDAC microenvironment and to target these cells in an attempt to normalize them rather than eliminate them, might be an effective strategy for PDAC therapy.

Composition, Spatial Characteristics, and Prognostic Significance of Myeloid Cell Infiltration in Pancreatic Cancer.

Although abundant myeloid cell populations in the pancreatic ductal adenocarcinoma (PDAC) microenvironment have been postulated to suppress antitumor immunity, the composition of these populations, their spatial locations, and how they relate to patient outcomes are poorly understood. To generate spatially resolved tumor and immune cell data at single-cell resolution, we developed two quantitative multiplex immunofluorescence assays to interrogate myeloid cells (CD15, CD14, ARG1, CD33, HLA-DR) and macrophages [CD68, CD163, CD86, IFN regulatory factor 5, MRC1 (CD206)] in the PDAC tumor microenvironment. Spatial point pattern analyses were conducted to assess the degree of colocalization between tumor cells and immune cells. Multivariable-adjusted Cox proportional hazards regression was used to assess associations with patient outcomes. In a multi-institutional cohort of 305 primary PDAC resection specimens, myeloid cells were abundant, enriched within stromal regions, highly heterogeneous across tumors, and differed by somatic genotype. High densities of CD15+ARG1+ immunosuppressive granulocytic cells and M2-polarized macrophages were associated with worse patient survival. Moreover, beyond cell density, closer proximity of M2-polarized macrophages to tumor cells was strongly associated with disease-free survival, revealing the clinical significance and biologic importance of immune cell localization within tumor areas. A diverse set of myeloid cells are present within the PDAC tumor microenvironment and are distributed heterogeneously across patient tumors. Not only the densities but also the spatial locations of myeloid immune cells are associated with patient outcomes, highlighting the potential role of spatially resolved myeloid cell subtypes as quantitative biomarkers for PDAC prognosis and therapy.

The Role of Stellate Cells in Pancreatic Ductal Adenocarcinoma: Targeting Perspectives.

Pancreatic ductal adenocarcinoma (PDAC) is a gastrointestinal malignancy with a dismal clinical outcome. Accumulating evidence suggests that activated pancreatic stellate cells (PSCs), the major producers of extracellular matrix (ECM), drive the severe stromal/desmoplastic reaction in PDAC. Furthermore, the crosstalk among PSCs, pancreatic cancer cells (PCCs) as well as other stroma cells can establish a growth-supportive tumor microenvironment (TME) of PDAC, thereby enhancing tumor growth, metastasis, and chemoresistance via various pathways. Recently, targeting stroma has emerged as a promising strategy for PDAC therapy, and several novel strategies have been proposed. The aim of our study is to give a profound review of the role of PSCs in PDAC progression and recent advances in stroma-targeting strategies.

Irreversible electroporation ablation overcomes tumor-associated immunosuppression to improve the efficacy of DC vaccination in a mice model of pancreatic cancer

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is associated with highly immunosuppressive tumor microenvironment (TME) that can limit the efficacy of dendritic cell (DC) vaccine immunotherapy. Irreversible electroporation (IRE) is a local ablation approach. Herein, we test the hypothesis that IRE ablation can overcome TME immunosuppression to improve the efficacy of DC vaccination using KrasLSL-G12D-p53LSL-R172H-Pdx-1-Cre (KPC) orthotopic mouse model of PDAC. The median survival for mice treated with the combined IRE and DC vaccination was 77 days compared with sham control (35 days), DC vaccination (49 days), and IRE (44 days) groups (P = .006). Thirty-six percent of the mice treated with combination IRE and DC vaccination were still survival at the end of the study period (90 days) without visible tumor. The changes of tumor apparent diffusion coefficient (ΔADC) were higher in mice treated with combination IRE and DC vaccination than that of other groups (all P < .001); tumor ΔADC value positively correlated with tumor fibrosis fraction (R = 0.707, P < .001). IRE induced immunogenic cell death and alleviation of immunosuppressive components in PDAC TME when combined with DC vaccination, including increased tumor infiltration of CD8+ T cells and Granzyme B+ cells (P = .001, and P = .007, respectively). Our data show that IRE ablation can overcome TME immunosuppression to improve the efficacy of DC vaccination in PDAC. Combination IRE ablation and DC vaccination may enhance therapeutic efficacy for PDAC.

Inhibition of focal adhesion kinase enhances antitumor response of radiation therapy in pancreatic cancer through CD8+ T cells.

Objective:Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy, due in large part to its resistance to conventional therapies, including radiotherapy (RT). Despite RT exerting a modest antitumor response, it has also been shown to promote an immunosuppressive tumor microenvironment. Previous studies demonstrated that focal adhesion kinase inhibitors (FAKi) in clinical development inhibit the infiltration of suppressive myeloid cells and T regulatory (T regs) cells, and subsequently enhance effector T cell infiltration. FAK inhibitors in clinical development have not been investigated in combination with RT in preclinical murine models or clinical studies. Thus, we investigated the impact of FAK inhibition on RT, its potential as an RT sensitizer and immunomodulator in a murine model of PDAC.Methods:We used a syngeneic orthotopic murine model to study the effect of FAKi on hypofractionated RT.Results:In this study we showed that IN10018, a small molecular FAKi, enhanced antitumor response to RT. Antitumor activity of the combination of FAKi and RT is T cell dependent. FAKi in combination with RT enhanced CD8+ T cell infiltration significantly in comparison to the radiation or FAKi treatment alone (P < 0.05). FAKi in combination with radiation inhibited the infiltration of granulocytes but enhanced the infiltration of macrophages and T regs in comparison with the radiation or FAKi treatment alone (P < 0.01).Conclusions:These results support the clinical development of FAKi as a radiosensitizer for PDAC and combining FAKi with RT to prime the tumor microenvironment of PDAC for immunotherapy.