Cancer-associated Fibroblasts In Pancreatic Cancer Research Articles

Nano-Drug Delivery Systems Targeting CAFs: A Promising Treatment for Pancreatic Cancer.

Currently, pancreatic cancer (PC) is one of the most lethal malignant tumors. PC is typically diagnosed at a late stage, exhibits a poor response to conventional treatment, and has a bleak prognosis. Unfortunately, PC's survival rate has not significantly improved since the 1960s. Cancer-associated fibroblasts (CAFs) are a key component of the pancreatic tumor microenvironment (TME). They play a vital role in maintaining the extracellular matrix and facilitating the intricate communication between cancer cells and infiltrated immune cells. Exploring therapeutic approaches targeting CAFs may reverse the current landscape of PC therapy. In recent years, nano-drug delivery systems have evolved rapidly and have been able to accurately target and precisely release drugs with little or no toxicity to the whole body. In this review, we will comprehensively discuss the origin, heterogeneity, potential targets, and recent advances in the nano-drug delivery system of CAFs in PC. We will also propose a novel integrated treatment regimen that utilizes a nano-drug delivery system to target CAFs in PC, combined with radiotherapy and immunotherapy. Additionally, we will address the challenges that this regimen currently faces.

Abstract A020: Molecular basis of immune suppressive microenvironment specified by cancer-associated fibroblasts in pancreatic cancer

Abstract Despite constant efforts to improve treatment, the prognosis remains poor, with an overall survival rate of 6% (ranges from 2% to 9%) due to lack of efficient chemotherapy, radiotherapy, and targeted therapy. Immune checkpoint inhibitors (ICIs) has been reported to be effective in various solid cancers, such as melanoma, lung cancer, and renal cell carcinoma. However, pancreatic ductal adenocarcinoma (PDAC) is resistant against ICIs. The responsiveness of immune therapy is mainly determined by immune tumor microenvironment (TME) composed of tumor-infiltrating lymphocytes (TIL) and stromal cells. PDAC possesses unique TME, which is abundant of fibrosis, termed of desmoplasia, and the role of fibrosis in constructing immune suppressive TME remains unclear. First, in this study, to examine the immunological characteristics of TME, we constructed an integrated data base of TIL profiling, RNA-seq and whole exome seq using 31 fresh resected tissues of PDAC. An unsupervised clustering of based on numbers and percentages of 12 TIL types analyzed by flow cytometry showed that PDAC was divided into 2 types of cluster (myeloid cell-, T cell-dominant type), and the prognosis of myeloid type was significantly poorer than T cell-type. Myeloid type contained the high frequency of myeloid cell lineage including monocytes, macrophages and myeloid-derived suppressor cells (MDSC). Only MDSCs were significantly associated with poor prognosis among various immune cell types. Furthermore, gene enrichment analysis identified activated and suppressed pathways in each immune type, and the immune-related pathways were significantly down-regulated in myeloid cell-dominant type. Then, to understand the characteristics of MDSCs infiltrated into pancreatic cancer tissues, single cell RNA-seq with surgical specimens was performed, and the analysis showed that a specific subtype of MDSCs infiltrated into cancer tissues. In addition, activation status of cancer-associated fibroblasts (CAFs) was related with MDSC recruitment and activation, indicating that the interaction between MDSCs and CAFs is crucial to create MDSC-rich TME. Inhibition of interaction between MDSCs and CAFs is expected to overcome immunosuppressive TME can be a novel immunotherapy in PDAC. Citation Format: Kazunori Aoki, Hironori Fukuda, Eri Hashimoto, Kosuke Arai. Molecular basis of immune suppressive microenvironment specified by cancer-associated fibroblasts in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A020.

Abstract C023: PTEN/STAT3 pathway in cancer-associated fibroblasts in Pancreatic Cancer

Abstract Pancreatic cancer is a devastating disease with a high mortality rate with a five-year survival rate of 12%. Recent studies have shed light on the crucial role of cancer-associated fibroblasts (CAFs) in shaping the tumor microenvironment. Our group demonstrated PTEN loss occurs in CAFs in ~25% of PDAC patient samples, which leads to increased activation of signaling pathways in CAFs, including the STAT3 pathway. Conditional deletion of STAT3 in CAFs in a dual flpO/cre recombinase PDAC model correlated with increased survival, decreased M2-like macrophages and increased T-cell infiltration. In the present study we aimed to test the hypothesis that direct interactions between PTEN and STAT3 in cancer CAFs contributes to an immuno-suppressive tumor microenvironment in pancreatic ductal carcinoma (PDAC). To address this hypothesis, we utilized CAFS isolated from the dual recombinase PDAC model. Proximity ligation assays with these CAFS demonstrated that PTEN-STAT3 are in a complex in the cell cytosol that translocate to the nucleus upon IL6 treatment. This finding was validated by co-immunoprecipitation experiments. ChIP-seq analysis of CAFs with PTEN and pSTAT3/STAT3 antibodies revealed a significant overlap in PTEN and pSTAT3/STAT3 occupation of gene promoters. Integrative analysis of PTEN-STAT3 bound genes with corresponding RNA-seq data revealed that the PTEN/STAT3 complex targets cytokines and ligands that directly affect T-cell function, contributing to an immune suppressive microenvironment. Analysis of human PDAC samples support the relationship between STAT3 activation and T-cell activity. Identifying PTEN/STAT3 targets in CAFs may provide new avenues for overcoming the anti-tumor immune responses in PDAC. Citation Format: Samaneh Saberi, Cameron Bumbleburg, Victoria Jordan, Julia E. Lefler, Sudarshana Sharma, Michael C. Ostrowski. PTEN/STAT3 pathway in cancer-associated fibroblasts in Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C023.

Medical Biology of Cancer-Associated Fibroblasts in Pancreatic Cancer.

Pancreatic cancer is one of the deadliest forms of cancer with one of the lowest 5-year survival rates of all cancer types. A defining characteristic of pancreatic cancer is the existence of dense desmoplastic stroma that, when exposed to stimuli such as cytokines, growth factors, and chemokines, generate a tumor-promoting environment. Cancer-associated fibroblasts (CAFs) are activated during the progression of pancreatic cancer and are a crucial component of the tumor microenvironment (TME). CAFs are primarily pro-tumorigenic in their activated state and function as promoters of cancer invasion, proliferation, metastasis, and immune modulation. Aided by many signaling pathways, cytokines, and chemokines in the tumor microenvironment, CAFs can originate from many cell types including resident fibroblasts, mesenchymal stem cells, pancreatic stellate cells, adipocytes, epithelial cells, endothelial cells, and other cell types. CAFs are a highly heterogeneous cell type expressing a variety of surface markers and performing a wide range of tumor promoting and inhibiting functions. Single-cell transcriptomic analyses have revealed a high degree of specialization among CAFs. Some examples of CAF subpopulations include myofibrotic CAFs (myCAFs), which exhibit a matrix-producing contractile phenotype; inflammatory CAFs (iCAF) that are classified by their immunomodulating, secretory phenotype; and antigen-presenting CAFs (apCAFs), which have antigen-presenting capabilities and express Major Histocompatibility Complex II (MHC II). Over the last several years, various attempts have been undertaken to describe the mechanisms of CAF-tumor cell interaction, as well as CAF-immune cell interaction, that contribute to tumor proliferation, invasion, and metastasis. Although our understanding of CAF biology in cancer has steadily increased, the extent of CAFs heterogeneity and their role in the pathobiology of pancreatic cancer remains elusive. In this regard, it becomes increasingly evident that further research on CAFs in pancreatic cancer is necessary.

Abstract 3643: PTEN/STAT3 pathway in cancer-associated fibroblasts in pancreatic cancer

Abstract This study aims to test the hypothesis that direct interactions and auto-regulatory loop between PTEN and STAT3 in cancer-associated fibroblasts (CAFs) contribute to an immunosuppressive tumor microenvironment in pancreatic ductal carcinoma (PDAC). Pancreatic cancer accounts for almost as many deaths as cases because of its poor prognosis. Many studies have led to the paradigm that stromal elements contribute to pancreatic tumor growth. Cancer-associated fibroblasts (CAFs) are a primary cell type in the desmoplastic stroma in PDAC, and targeting them to improve PDAC treatment is thought to be a promising strategy, however; studies have shown both tumor-promoting and tumor-limiting activities associated with PDAC CAFs. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is one of the most well-described tumor suppressor genes and can regulate essential transcription factors in stromal fibroblasts. On the other hand, STAT3 signaling in pancreatic CAFs contributes to the immunosuppressive and fibrotic stroma observed with disease progression. In this study, we used novel, dual recombinase mouse models of PDAC, with Kras and p53 mutations engineered in the pancreatic epithelium (Flp-based) and Stat3 deleted in a subpopulation of CAFS (Cre). CAFs isolated from these mice were used to probe PTEN-STAT3 interaction in vitro using the Proximity Ligation Assay (PLA) and by ChIP-seq using an RNA seq. Mouse and Human pancreatic tumor samples were analyzed and quantified using multiplex-immunofluorescence and multispectral imaging. In the mouse genetic model, we found PTEN downregulated in ~80% of CAFs, consistent with previous work from our group that demonstrated PTEN loss in 25% of PDAC patient samples. At the same time, pY705-STAT3 was activated in CAFs, and deletion of STAT3 in CAFs correlated with increased survival, decreased M2-like macrophages, and increased T-cell infiltration. STAT3 ablation in CAFS resulted in the re-expression of PTEN in CAFS, implying an autoregulatory loop. PLA on CAFs isolated revealed that IL6 treatment resulted in the transfer of a STAT3-PTEN protein complex into the nucleus ChIP-seq on CAFs with PTEN STAT3 antibodies revealed a significant overlap in PTEN and STAT3 occupation of gene promoters. This result is consistent with in silico analysis on the PTEN and STAT3 ChIP-Seq data in human cell lines. ChIP-Seq data is being interacted with RNA data to identify essential target genes. Human PDAC tissue microarrays are stained with pSTAT3, PTEN, CD3, and CD8.Our study suggests an autoregulatory look between PTEN and STAT3 in PDAC CAFs. PTEN may suppress specific STAT3 target genes through direct interactions with chromatin. Alternatively, PTEN/STAT3 complexes may target CAF genes that suppress tumor growth or activate anti-tumor immune responses. These investigations could identify potential targets in CAFs for restoring the anti-tumor immune responses in PDAC. Citation Format: Samaneh Saberi, Sudarshana Sharma, Cameron Bumbleburg, Julia E. Lefler, Michael C. Ostrowski. PTEN/STAT3 pathway in cancer-associated fibroblasts in pancreatic cancer. [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 3643.

COL12A1 Acts as a Novel Prognosis Biomarker and Activates Cancer-Associated Fibroblasts in Pancreatic Cancer through Bioinformatics and Experimental Validation

Pancreatic cancer remains one of the most challenging malignancies to date and is associated with poor survival. Cancer-associated fibroblasts (CAFs) are key stromal cells in the tumor microenvironment (TME) that play a crucial role in tumor progression in pancreatic cancer. Thus, uncovering the key genes involved in CAF progression and determining their prognostic value is critically important. Herein, we report our discoveries in this research area. Analysis of The Cancer Genome Atlas (TCGA) dataset and investigation of our clinical tissue samples indicated that COL12A1 expression was aberrantly highly expressed in pancreatic cancer. Survival and COX regression analyses revealed the significant clinical prognostic value of COL12A1 expression in pancreatic cancer. COL12A1 was mainly expressed in CAFs but not in tumor cells. This was verified with our PCR analysis in cancer cells and CAFs. The knocking down of COL12A1 decreased the proliferation and migration of CAFs and down-regulated the expression of CAF activation markers actin alpha 2 (ACTA2), fibroblast activation protein (FAP), and fibroblast-specific protein 1 (FSP1). Meanwhile, the interleukin 6 (IL6), CXC chemokine Ligand-5 (CXCL5), and CXC chemokine Ligand-10 (CXCL10) expressions were inhibited, and the cancer-promoting effect was reversed by COL12A1 knockdown. Therefore, we demonstrated the potential prognostic and target therapy value of COL12A1 expression in pancreatic cancer and elucidated the molecular mechanism underlying its role in CAFs. The findings of this study might provide new opportunities for TME-targeted therapies in pancreatic cancer.

Metabolic reprogramming of cancer-associated fibroblasts in pancreatic cancer contributes to the intratumor heterogeneity of PET-CT

Intratumor heterogeneity of positron emission tomography-computed tomography (PET-CT) is reflected by variable 18F-fluorodeoxyglucose (FDG) uptake. Increasing evidence has shown that neoplastic and non-neoplastic components can affect the total 18F-FDG uptake in tumors. Cancer-associated fibroblasts (CAFs) is considered as the main non-neoplastic components in tumor microenvironment (TME) of pancreatic cancer. Our study aims to explore the impact of metabolic changes in CAFs on heterogeneity of PET-CT. A total of 126 patients with pancreatic cancer underwent PET-CT and endoscopic ultrasound elastography (EUS-EG) before treatment. High maximum standardized uptake value (SUVmax) from the PET-CT was positively correlated with the EUS-derived strain ratio (SR) and indicated poor prognosis of patients. In addition, single-cell RNA analysis showed that CAV1 affected glycolytic activity and correlated with glycolytic enzyme expression in fibroblasts in pancreatic cancer. We also observed the negative correlation between CAV1 and glycolytic enzyme expression in the tumor stroma by using immunohistochemistry (IHC) assay in the SUVmax-high and SUVmax-low groups of pancreatic cancer patients. Additionally, CAFs with high glycolytic activity contributed to pancreatic cancer cell migration, and blocking CAF glycolysis reversed this process, suggesting that glycolytic CAFs promote malignant biological behavior in pancreatic cancer. In summary, our research demonstrated that the metabolic reprogramming of CAFs affects total 18F-FDG uptake in tumors. Thus, an increase in glycolytic CAFs with decreased CAV1 expression promotes tumor progression, and high SUVmax may be a marker for therapy targeting the neoplastic stroma. Further studies should clarify the underlying mechanisms.

Exploring the Biology of Cancer-Associated Fibroblasts in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy characterised by a stubbornly low 5-year survival which is essentially unchanged in the past 5 decades. Despite recent advances in chemotherapy and surgical outcomes, progress continues to lag behind that of other cancers. The PDAC microenvironment is characterised by a dense, fibrotic stroma of which cancer-associated fibroblasts (CAFs) are key players. CAFs and fibrosis were initially thought to be uniformly tumour-promoting, however this doctrine is now being challenged by a wealth of evidence demonstrating CAF phenotypic and functional heterogeneity. Recent technological advances have allowed for the molecular profiling of the PDAC tumour microenvironment at exceptional detail, and these technologies are being leveraged at pace to improve our understanding of this previously elusive cell population. In this review we discuss CAF heterogeneity and recent developments in CAF biology. We explore the complex relationship between CAFs and other cell types within the PDAC microenvironment. We discuss the potential for therapeutic targeting of CAFs, and we finally provide an overview of future directions for the field and the possibility of improving outcomes for patients with this devastating disease.

PD-L1-directed PlGF/VEGF blockade synergizes with chemotherapy by targeting CD141+ cancer-associated fibroblasts in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year overall survival rate. Patients with PDAC display limited benefits after undergoing chemotherapy or immunotherapy modalities. Herein, we reveal that chemotherapy upregulates placental growth factor (PlGF), which directly activates cancer-associated fibroblasts (CAFs) to induce fibrosis-associated collagen deposition in PDAC. Patients with poor prognosis have high PIGF/VEGF expression and an increased number of PIGF/VEGF receptor-expressing CAFs, associated with enhanced collagen deposition. We also develop a multi-paratopic VEGF decoy receptor (Ate-Grab) by fusing the single-chain Fv of atezolizumab (anti-PD-L1) to VEGF-Grab to target PD-L1-expressing CAFs. Ate-Grab exerts anti-tumor and anti-fibrotic effects in PDAC models via the PD-L1-directed PlGF/VEGF blockade. Furthermore, Ate-Grab synergizes with gemcitabine by relieving desmoplasia. Single-cell RNA sequencing identifies that a CD141+ CAF population is reduced upon Ate-Grab and gemcitabine combination treatment. Overall, our results elucidate the mechanism underlying chemotherapy-induced fibrosis in PDAC and highlight a combinatorial therapeutic strategy for desmoplastic cancers.

Neutrophil Attenuation Improves Chemosensitivity by Mitigating Inflammatory Polarization of Cancer-Associated Fibroblasts in Pancreatic Cancer

Neutrophil Attenuation Improves Chemosensitivity by Mitigating Inflammatory Polarization of Cancer-Associated Fibroblasts in Pancreatic Cancer

Prognostic Value of Cancer-Associated Fibroblasts in Pancreatic Cancer: A Meta-Analysis

Prognostic Value of Cancer-Associated Fibroblasts in Pancreatic Cancer: A Meta-Analysis

Abstract 1565: Targeting stromal-specific p38 MAPK signaling to stifle inflammatory reprogramming of cancer-associated fibroblasts in pancreatic cancer

Abstract Introduction: Major contributors to therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC) are the desmoplastic stroma that acts as a barrier to drug delivery and effector immune cell infiltration. In our efforts to identify mediators of therapeutic resistance, we identified the pro-inflammatory cytokine interleukin-1α (IL-1) to be critical in the activation and reprogramming of neighboring cancer-associated fibroblasts (CAFs) within the stroma to a pro-inflammatory phenotype to promote myeloid cell chemotaxis. Our mechanistic studies have identified the p38 MAPK pathway as a novel mediator of IL-1-induced CAF activation from quiescent stellate cells into the inflammatory fibroblast. Our central hypothesis is that disruption of the IL-1/p38 MAPK signaling cascade in pancreatic stellate cells (PSCs) and CAFs can improve therapeutic resistance by remodeling the fibrotic stromal landscape and the overall immune microenvironment in PDAC tumors. Methods: Inhibition of phosphorylated p38 MAPK was achieved pharmacologically with Pexmetinib and genetically with an shRNA lentiviral system in CAF and PSC cell lines. Inflammatory PSC/CAF activation was determined by qPCR, immunofluorescence, and vitamin A assay. Ptf1acre/+;LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice were treated with the p38 inhibitor, pexmetinib (30mg/kg, daily PO), or vehicle control for 2.5 weeks prior to sacrifice. For survival studies, PKT mice were treated with vehicle, gemcitabine (20 μg/twice weekly), pexmetinib, or combination until moribund. Results: Inhibition of p38 MAPK in PSCs prevented activation into an inflammatory fibroblast in vitro when stimulated with IL-1 or tumor cell cocultures. p38 MAPK inhibition in vivo resulted in signification reduction of PDGFR+ CAFs in a PDAC GEMM. Additionally, p38 MAPK inhibition resulted in a significant reduction of circulating myeloid cells (CD11b+) and intratumoral monocytic MDSCs (Ly6C+). p38 inhibition, in combination with chemotherapy, significantly improves overall survival in a PDAC GEMM. Conclusions: These findings provide important mechanistic data to explore p38 MAPK inhibition to target the fibrotic stroma and reduce immunosuppressive myeloid levels in tumors and provide compelling preclinical evidence to combine pexmetinib with chemotherapy to improve overall survival in PDAC. Citation Format: Samara Singh, Austin R. Dosch, Siddharth Mehra, Iago de Castro Silva, Anna Bianchi, Vanessa Tonin Garrido, Nilesh Deshpande, Zhiqun Zhou, Jashodeep Datta, Nagaraj Nagathihalli, Nipun Merchant. Targeting stromal-specific p38 MAPK signaling to stifle inflammatory reprogramming of cancer-associated fibroblasts in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1565.

Targeting Endoglin Expressing Cells in the Tumor Microenvironment Does Not Inhibit Tumor Growth in a Pancreatic Cancer Mouse Model.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer and is known to have low immunogenicity and an immunosuppressive microenvironment. It is also characterized by high accumulation of dense stroma, composed of mostly cancer-associated fibroblasts (CAFs). Multiple subsets of CAFs are described, with one of them expressing the transforming growth factor (TGF)-β co-receptor endoglin. In previous work, we and others have shown that endoglin-expressing CAFs stimulate tumor progression and metastasis. Therefore, in this study, we set out to investigate the role of endoglin-expressing CAFs in pancreatic cancer progression.MethodsFirst, we investigated the expression of endoglin on CAFs in both human tissues as well as a mouse model for PDAC. Since CAF-specific endoglin expression was high, we targeted endoglin by using the endoglin neutralizing antibody TRC105 in the murine KPC model for PDAC.ResultsAlthough some signs of immune activation were observed, TRC105 did not affect tumor growth. Since 90% of the CD8+ T-cells expressed the immune checkpoint PD-1, we investigated the combination with a PD1 checkpoint inhibitor, which did not enhance therapeutic responses. Finally, genetic deletion of endoglin from collagen 1a1 expressing cells also did not affect the growth of the mouse KPC tumors.ConclusionOur results show that although endoglin is highly expressed on PDAC-CAFs and signaling is efficiently inhibited by TRC105, this does not result in decreased tumor growth in the KPC model for pancreatic cancer.

PERK-eIF2α-ERK1/2 axis drives mesenchymal-endothelial transition of cancer-associated fibroblasts in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by remarkable desmoplasia, usually driven by cancer-associated fibroblasts (CAFs), influencing patient prognosis. CAFs are a group of plastic cells responsible for tumor growth and metastasis. Fibroblasts have been reported to directly contribute to angiogenesis by undergoing mesenchymal-endothelial transition (MEndoT) after ischemic injury in the heart, brain, and hindlimbs. However, whether CAFs can undergo similar transdifferentiation in the hostile tumor microenvironment and directly contribute to tumor angiogenesis remains unclear. Herein, we provide evidence that CAFs can adopt an endothelial cell-like phenotype and directly contribute to tumor angiogenesis in vitro and in vivo. Furthermore, this program is regulated by the PERK-eIF2α-ERK1/2 axis. Pharmacological inhibition of PERK with GSK2606414 limited the phenotypic transition of CAFs. In conclusion, our results suggest that CAFs contribute to tumor angiogenesis by undergoing the MEndoT, thus representing therapeutic targets for improving PDAC prognosis.

Distinct Stromal and Immune Features Collectively Contribute to Long-Term Survival in Pancreatic Cancer

Background: The aggressive biology and treatment refractory nature of pancreatic ductal adenocarcinoma (PDAC) significantly limits long-term survival. Examining the tumor microenvironment (TME) of long-term survivors (LTS) of PDAC offers the potential of unveiling novel biological insights and therapeutic targets.Methods: We performed an integrated approach involving immunophenotyping, stromal scoring and histomorphological profiling of a cohort of 112 PDAC-cases, including 25 long-term survivors (LTSs, OS ≥ 60 months). Mutational frequencies were assessed using targeted next generation sequencing. Finally, we validated our findings in silico using an external cohort of microarray data from PDAC patients.Results: LTS cases exhibit a largely quiescent population of cancer-associated fibroblasts (CAFs). Immune profiling revealed key differences between LTS and NON-LTS cases in the intratumoral and stromal compartments. In both compartments, LTS cases exhibit a T cell inflamed profile with higher density of CD3+ T cells, CD4+ T cells, iNOS+ leukocytes and strikingly diminished numbers of CD68+ total macrophages, CD163+ (M2) macrophages and FOXP3+ Tregs. A large proportion of LTS cases exhibited tertiary lymphoid tissue (TLT) formation, which has been observed to be a positive prognostic marker in a number of tumor types. Using a Random-Forest variable selection approach, we identified the density of stromal iNOS+ cells and CD68+ cells as strong positive and negative prognostic variables, respectively. In an external cohort, computational cell-type deconvolution revealed a higher abundance of T cells, B lymphocytes and dendritic cells (DCs) in patients with long-term OS compared to short-term survivors. Thus, in silico profiling of long-term survivors in an external cohort, strongly corroborated the T cell-inflamed TME observed in our LTS group.Conclusions: Collectively, our findings highlight the prognostic importance of TME profiles in PDAC, underlining the crucial role of tumor associated macrophages (TAMs) and the potential interdependence between immunosuppressive TAMs and activated CAFs in pancreatic cancer. Additionally, our data has potential for precision medicine and patient stratification. Patients with a T cell inflamed TME might derive benefit from agonistic T cell antibodies (e.g., OX40 or CD137 agonists). Alternately, patients with activated CAFs and high infiltration of immunosuppressive TAMs are highly likely to exhibit therapeutic responses to macrophage targeted drugs (e.g., anti-CSF1R) and anti-CAF agents.

Cellular Heterogeneity of Pancreatic Stellate Cells, Mesenchymal Stem Cells, and Cancer-Associated Fibroblasts in Pancreatic Cancer.

Simple SummaryCancer-associated fibroblasts, which are derived from several cell types such as pancreatic stellate cells or mesenchymal stem cells, play a major role in the progression and drug resistance of pancreatic cancer. Targeting cancer-associated fibroblasts can, therefore, be a promising therapeutic strategy. However, several studies show that deletion of cancer-associated fibroblasts may also have tumor-promoting effects. These contrasting observations suggest that cancer-associated fibroblasts exhibit functional heterogeneity in pancreatic cancer.Pancreatic cancer is projected to become the second deadliest cancer by 2030 in the United States, and the overall five-year survival rate stands still at around 9%. The stroma compartment can make up more than 90% of the pancreatic tumor mass, contributing to the hypoxic tumor microenvironment. The dense stroma with extracellular matrix proteins can be a physical and metabolic barrier reducing therapeutic efficacy. Cancer-associated fibroblasts are a source of extracellular matrix proteins. Therefore, targeting these cells, or extracellular matrix proteins, have been considered as therapeutic strategies. However, several studies show that deletion of cancer-associated fibroblasts may have tumor-promoting effects. Cancer-associated fibroblasts are derived from a variety of different cell types, such as pancreatic stellate cells and mesenchymal stem cells, and constitute a diverse cell population consisting of several functionally heterogeneous subtypes. Several subtypes of cancer-associated fibroblasts exhibit a tumor-restraining function. This review article summarizes recent findings regarding origin and functional heterogeneity of tumor-promoting as well as tumor-restraining cancer-associated fibroblasts. A better understanding of cancer-associated fibroblast heterogeneity could provide more specific and personalized therapies for pancreatic cancer patients in the future.

Significance of meflin-positive cancer-associated fibroblasts in predicting response to immune checkpoint inhibitors in non-small cell lung cancer.

3118 Background: Tumor immunity is regulated by complex interactions between cancer and immune cells, which also involves other components of the tumor microenvironment (TME). Recently, cancer-associated fibroblasts (CAFs), a major constituent of the TME, have emerged as important regulators of tumor immunity. Specifically, for example, α-smooth muscle actin or leucine-rich repeat containing 15-positive CAFs have been shown to be crucial for the suppression of tumor immunity. However, a comprehensive picture of how other CAF subset(s) are involved in tumor immunity is still lacking. Here, we show the involvement of a CAF subset highly expressing Meflin, which was recently identified as a marker of cancer-restraining CAFs in pancreatic cancer (Mizutani et al., Cancer Res, 2019), in the response of non-small cell lung cancer (NSCLC) patients to immune checkpoint inhibitors (ICIs). Methods: A sample cohort of 122 subjects with NSCLC who had received ICI monotherapy with nivolumab, pembrolizumab, or atezolizumab was identified at the Department of Respiratory Medicine at Nagoya University Hospital. We selected 92 eligible patients, collected formalin-fixed paraffin-embedded tumor tissues, and prepared 4–µm-thick slides for the analysis of Meflin expression by RNA-in situ hybridization assay, followed by the evaluation of treatment response of 88 patients using the iRECIST criteria. We assessed the number of Meflin-positive CAFs and divided the patients into Meflin-High (20% and more CAFs express Meflin) and -Low groups. The cut-off value was obtained by the ROC analysis. Primarily, objective response rate (ORR) was compared between Meflin-High and –Low groups. Overall survival (OS), and progression free survival (PFS) were also assessed. Results: Patients who started to receive ICIs till the end of March 2019 were enrolled and followed-up until the end of 2019. Analysis of the tumor tissues revealed that 24 (40.7%) of 59 Meflin-High patients responded to the ICI monotherapy. In contrast, none (0%) of 29 Meflin-Low patients showed any significant response (p-value: 0.0000174). Meflin-High groups showed statistically significant prolongations in both OS and PFS with the hazard ratios of 0.3114 [0.1591-0.6094] and 0.3997 [0.2290-0.6976], respectively. Conclusions: This retrospective observation indicated that the high infiltration of Meflin-positive CAFs may shape tumor-suppressive immune response and increase the sensitivity to ICIs, which differs from those of other CAF subsets.

Recent advances in understanding cancer-associated fibroblasts in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is a devastating disease with a poor survival rate. It is resistant to therapy in part due to its unique tumor microenvironment, characterized by a desmoplastic reaction resulting in a dense stroma that constitutes a large fraction of the tumor volume. A major contributor to the desmoplastic reaction are cancer-associated fibroblasts (CAFs). CAFs actively interact with cancer cells and promote tumor progression by different mechanisms, including extracellular matrix deposition, remodeling, and secretion of tumor promoting factors, making CAFs an attractive target for PDA. However, emerging evidences indicate significant tumor-suppressive functions of CAFs, highlighting the complexity of CAF biology. CAFs were once considered as a uniform cell type within the cancer stroma. Recently, the existence of CAF heterogeneity in PDA has become appreciated. Due to advances in single cell technology, distinct subtypes of CAFs have been identified in PDA. Here we review recent updates in CAF biology in PDA, which may help develop effective CAF-targeted therapies in the future.

Targeting genetically-tuned CAFs in pancreatic cancer via perlecan manipulation

ABSTRACTIntroduction: Pancreatic cancer (PC) is responsible for significant worldwide cancer-associated mortality and has one of the lowest five-year survival rate post-diagnosis of all epithelial cancers. A major contributor to this dismal outcome is the extensive stromal reaction that occurs during PC progression. As such, targeting key components of the pancreatic tumor stroma in combination with standard-of-care chemotherapy has been a recent focus in both the pre-clinical and clinical settings.Areas Covered: In this commentary, we highlight how perlecan was identified as a new potential target for this disease.Expert Opinion: Perlecan is deposited by cancer-associated fibroblasts (CAFs) in the pancreatic tumor stroma, and work from our laboratory group recently demonstrated that depleting perlecan reduces metastatic spread, while also improving chemotherapy efficacy in pancreatic tumors harboring a gain-of-function p53 mutation. We also discuss potential strategies to therapeutically target perlecan which could be tested in pre-clinical models prior to translation into the clinic.

The IL-1/IL-1 receptor axis and tumor cell released inflammasome adaptor ASC are key regulators of TSLP secretion by cancer associated fibroblasts in pancreatic cancer

BackgroundThe thymic stromal lymphopoietin (TSLP), a key cytokine for development of Th2 immunity, is produced by cancer associated fibroblasts (CAFs) in pancreatic cancer where predominant tumor infiltrating Th2 over Th1 cells correlates with reduced patients’ survival. Which cells and molecules are mostly relevant in driving TSLP secretion by CAFs in pancreatic cancer is not defined.MethodsWe performed in vitro, in vivo and ex-vivo analyses. For in vitro studies we used pancreatic cancer cell lines, primary CAFs cultures, and THP1 cells. TSLP secretion by CAFs was used as a read-out system to identify in vitro relevant tumor-derived inflammatory cytokines and molecules. For in vivo studies human pancreatic cancer cells and CAFs were orthotopically injected in immunodeficient mice. For ex-vivo studies immunohistochemistry was performed to detect ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) expression in surgical samples. Bioinformatics was applied to interrogate published data sets.ResultsWe show in vitro that IL-1α and IL-1β released by pancreatic cancer cells and tumor cell-conditioned macrophages are crucial for TSLP secretion by CAFs. Treatment of immunodeficient mice orthotopically injected with human IL-1 positive pancreatic cancer cells plus CAFs using the IL-1R antagonist anakinra significantly reduced TSLP expression in the tumor. Importantly, we found that pancreatic cancer cells release alarmins, among which ASC, able to induce IL-1β secretion in macrophages. The relevance of ASC was confirmed ex-vivo by its expression in both tumor cells and tumor associated macrophages in pancreatic cancer surgical samples and survival data analyses showing statistically significant inverse correlation between ASC expression and survival in pancreatic cancer patients.ConclusionsOur findings indicate that tumor released IL-1α and IL-1β and ASC are key regulators of TSLP secretion by CAFs and their targeting should ultimately dampen Th2 inflammation and improve overall survival in pancreatic cancer.