Pancreatic Tumor Microenvironment Research Articles

Aryl hydrocarbon receptor knockout accelerates PanIN formation and fibro-inflammation in a mutant Kras-driven pancreatic cancer model.

The pathogenesis of pancreas cancer (PDAC) remains poorly understood, hindering efforts to develop a more effective therapy for PDAC. Recent discoveries show the aryl hydrocarbon receptor (AHR) plays a crucial role in the development of several cancers, and can be targeted for therapeutic effect. However, its involvement in the pathogenesis of PDAC remains unclear. To address this gap, we evaluated the role of AHR in the development of PDAC pre-cancerous lesions in vivo. We created a global AHR-null, mutant Kras-driven PDAC mouse model (A-/-KC) and evaluated the changes in PDAC precursor lesion formation (Pan-IN 1, 2, and 3) and associated fibro-inflammation between KC and A-/-KC at 5 months of age. We then examined the changes in the immune microenvironment followed by single-cell RNA-sequencing analysis to evaluate concomitant transcriptomic changes. We identified a significant increase in PanIN-1 lesion formation and PanIN-1 associated fibro-inflammatory infiltrate in A-/-KC vs KC mice. This was associated with significant changes in the adaptive immune system, particularly a decrease in the CD4+/CD8+ T-cell ratio, as well as a decrease in the T-regulatory/Th17 T-cell ratio suggesting unregulated inflammation. These findings show the loss of AHR results in heightened Kras-induced PanIN formation, through modulation of immune cells within the pancreatic tumor microenvironment.

CD200 is overexpressed in the pancreatic tumor microenvironment and predictive of overall survival

Pancreatic cancer is an aggressive disease with a 5 year survival rate of 13%. This poor survival is attributed, in part, to limited and ineffective treatments for patients with metastatic disease, highlighting a need to identify molecular drivers of pancreatic cancer to target for more effective treatment. CD200 is a glycoprotein that interacts with the receptor CD200R and elicits an immunosuppressive response. Overexpression of CD200 has been associated with differential outcomes, depending on the tumor type. In the context of pancreatic cancer, we have previously reported that CD200 is expressed in the pancreatic tumor microenvironment (TME), and that targeting CD200 in murine tumor models reduces tumor burden. We hypothesized that CD200 is overexpressed on tumor and stromal populations in the pancreatic TME and that circulating levels of soluble CD200 (sCD200) have prognostic value for overall survival. We discovered that CD200 was overexpressed on immune, stromal, and tumor populations in the pancreatic TME. Particularly, single-cell RNA-sequencing indicated that CD200 was upregulated on inflammatory cancer-associated fibroblasts. Cytometry by time of flight analysis of PBMCs indicated that CD200 was overexpressed on innate immune populations, including monocytes, dendritic cells, and monocytic myeloid-derived suppressor cells. High sCD200 levels in plasma correlated with significantly worse overall and progression-free survival. Additionally, sCD200 correlated with the ratio of circulating matrix metalloproteinase (MMP) 3: tissue inhibitor of metalloproteinase (TIMP) 3 and MMP11/TIMP3. This study highlights the importance of CD200 expression in pancreatic cancer and provides the rationale for designing novel therapeutic strategies that target this protein.

The nanobody targeting PD-L1 and CXCR4 counteracts pancreatic stellate cell-mediated tumour progression by disrupting tumour microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy worldwide owing to its complex tumour microenvironment and dense physical barriers. Stromal-derived factor-1 (SDF-1), which is abundantly secreted by tumour stromal cells, plays a pivotal role in promoting PDAC growth and metastasis. In this study, we investigated the impact and molecular mechanisms of the anti-PD-L1&CXCR4 bispecific nanobody on the TME and their consequent interference with PDAC progression. We found that blocking the SDF-1/CXCR4 signalling pathway delayed the epithelial-mesenchymal transition in pancreatic cancer cells. Anti-PD-L1&CXCR4 bispecific nanobody effectively suppress the secretion of SDF-1 by pancreatic stellate cells and downregulate the expression of smooth muscle actin alpha(α-SMA), thereby preventing the activation of cancer-associated fibroblasts by downregulating the PI3K/AKT signaling pathway. This improves the pancreatic tumour microenvironment, favouring the infiltration of T cells into the tumour tissue. In conclusion, our results suggest that the anti-PD-L1&CXCR4 bispecific nanobody exerts an antitumor immune response by changing the pancreatic tumour microenvironment. Hence, the anti-PD-L1&CXCR4 bispecific nanobody is a potential candidate for pancreatic cancer treatment.

Unveiling the immunosuppressive landscape of pancreatic ductal adenocarcinoma: implications for innovative immunotherapy strategies.

Pancreatic cancer, particularly pancreatic ductal adenocarcinoma (PDAC), stands as the fourth leading cause of cancer-related deaths in the United States, marked by challenging treatment and dismal prognoses. As immunotherapy emerges asapromising avenue for mitigating PDAC's malignant progression, a comprehensiveunderstanding of the tumor's immunosuppressive characteristics becomesimperative. This paper systematically delves into the intricate immunosuppressive network within PDAC, spotlighting the significant crosstalk between immunosuppressive cells and factors in the hypoxic acidic pancreatic tumor microenvironment. By elucidating these mechanisms, we aim to provide insights into potential immunotherapy strategies and treatment targets, laying the groundwork for future studies on PDAC immunosuppression. Recognizing the profound impact of immunosuppression on PDAC invasion and metastasis, this discussion aims to catalyze the development of more effective and targeted immunotherapies for PDAC patients.

Abstract 270: Dissecting the effects of p53 mutation and p53 loss on pancreatic tumor microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignant disease with a dismal prognosis. In the past decades, a plethora of genetically engineered mouse models (GEMMs) with autochthonous pancreatic tumor development have greatly facilitated the studies of pancreatic cancer. Commonly used GEMMs of PDAC often harbor the oncogenic KRAS driver mutation (KrasG12D), in combination with either p53 mutation by knock-in strategy (Trp53R172H) or p53 loss by conditional knockout (Trp53cKO) strategy, in pancreatic cell lineages. However, the systematic comparison of the tumor microenvironment between KrasG12D; Trp53R172H (KPmut) mouse models and KrasG12D; Trp53cKO (KPloss) mouse models is still lacking. In this study, we conducted cross-dataset single-cell RNA-sequencing (scRNA-seq) analyses to compare the pancreatic tumor microenvironment from KPmut mouse models and KPloss mouse models, especially focusing on the cell compositions and transcriptomic phenotypes of major cell types including cancer cells, B cells, T cells, granulocytes, myeloid cells, cancer-associated fibroblasts, and endothelial cells. We identified the similarities and differences between KPmut and KPloss mouse models, revealing the effects of p53 mutation and p53 loss on oncogenic KRAS-driven pancreatic tumor progression. Citation Format: Yang Chen. Dissecting the effects of p53 mutation and p53 loss on pancreatic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 270.

Abstract 5553: Glycan CA19-9 mediated immune modulation in the pancreatic tumor microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDA) is an intractable common malignancy with a pro-tumorigenic and immunosuppressive microenvironment (ME) that often limits treatment efficacy. Only 10-15% of patients are eligible for surgical resection, which is the only cure for PDA. The glycan CA19-9 is used to follow treatment response in PDA patients, but its functional role in PDA remained unknown until recently because rodents lack the ability to produce this carbohydrate. CA19-9 elevation in a KRAS-mutant background results in increased tumor proliferation and ME remodeling in mice. The tumor ME is encompassed by hypovascularized stroma consisting of cancer associated fibroblasts (CAFs) and tumor associated macrophages (TAMs) that contribute to desmoplasia and immunosuppression. All prior work investigating the tumor ME in PDA mouse models was performed in CA19-9 negative context, potentially missing a key element of PDA biology. Elevation of CA19-9 in mice caused expansion of both antigen-presenting (ap)CAFs and TAMs. CA19-9 induction in KRAS-mutant organoids increased gene expression of M-CSF, IL1a, and TGFb and led to increased apCAF and TAM differentiation. This project aims to uncover the mediators of CA19-9-mediated ME remodeling. CA19-9-modified macrophage colony-stimulating factor (M-CSF) was identified via immunoprecipitation and mass spectrometry from KRAS-mutant CA19-9-inducible pancreatic organoids. CA19-9-M-CSF induced increased immunosuppressive TAM differentiation in vivo and in vitro using a novel Macrophage, Organoid, and Fibroblast (MOrF) co-culture platform. This project will identify the mechanisms by which CA19-9 directly contributes to PDA ME immune modulation and unveil essential molecular underpinnings of PDA biology that will inform effective therapies in the future. Citation Format: Jasper Hsu, Tae Gyu Oh, Kristina L. Peck, Angelica E. Rock, Garret Bishop, Shira R. Okhovat, Susan M. Kaech, Dannielle D. Engle. Glycan CA19-9 mediated immune modulation in the pancreatic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5553.

Abstract 1194: Understanding radiation and immunotherapy induced cell state shifts in the pancreatic tumor microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with few effective treatment options. It is characterized by a highly desmoplastic tumor microenvironment and a paucity of dendritic cells, leading to low immune cell infiltration and poor outcomes with immunotherapy treatments that are highly effective in other cancers. In a recent pilot study, metastatic PDAC patients who had progressed on chemotherapy were treated with a combination of dual immune checkpoint blockade (ICB) therapy and radiation which resulted in an 18% overall response rate (ORR) and 29% disease control rate (DCR). This led to a phase 2 study in order to further test this combination with the goal of deep correlative analysis to understand the determinants of immunotherapy response and resistance. In this follow up there was a 3.6% ORR, 10.7% DCR, progression free survival (PFS) of 2.3 months, and a single patient with complete response. We collected 36 samples from 23 patients including primary pancreas tumor tissue and liver metastases, and 13 pairs of pre and on treatment biopsies. We performed single-nucleus and paired single-cell RNA and TCR sequencing on these samples to provide comprehensive insight into the cell types and biology present in this unique tumor microenvironment. Using supervised non-negative matrix factorization we have identified both previously established and novel gene programs that co-vary between cell types. Within the various tissue sites we have identified differing proportions of basal to classical tumor cell types and a distinct shift towards the classical state post-radiation. Of particular interest is a group of interferon related gene programs present within epithelial cell subtypes, C1QC+ and MHCII+ macrophages, and CD14+/CD16+ monocytes that indicate cell type specific responses to treatment. Focusing on the T cells, we found a distinct shift towards exhausted or proliferating states on treatment and using TCR sequencing data we have identified novel and expanding clones on treatment, largely consisting of those same cell subtypes. We also collected spatial transcriptomics data on a subset of patient samples, and analysis is ongoing to further confirm the cell type relationships identified in single-cell resolution data, along with spatial localization of basal to classical tumor states. This vast multimodal dataset allows us novel insight into the radiation and dual ICB induced cellular state shifts in the tumor microenvironment. Citation Format: Milan Parikh, Ryan Park, Aparna R. Parikh, Julie L. Koenig, Leon Pappas, Moshe Sade-Feldman, Lynn Bi, Nicole Carzo, Tarin M. Grillo, Islam Baiev, Olanike Asupoto, Irena Gushterova, Tom LaSalle, Anna Gonye, Emily Blaum, David P. Ryan, David T. Ting, Theodore S. Hong, Dana Pe'er, Nir Hacohen, Arnav Mehta. Understanding radiation and immunotherapy induced cell state shifts in the pancreatic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1194.

Abstract 4043: Nanoparticle-mediated combination therapy to synergistically harness Type I interferons and senescence in the pancreatic tumor microenvironment

Abstract We seek to combine a unique innate immunomodulatory nanoparticle (NP) system with RAS inhibitors for a multi-faceted therapy that mitigates the formidable drug delivery challenges in pancreatic ductal adenocarcinoma (PDAC). PDAC has quickly risen to the third most deadly cancer largely due to its fibrotic, desmoplastic, and immunosuppressive tumor microenvironment (TME) that limits delivery and efficacy of chemo- and immunotherapeutics. We have engineered unique lipid-based NPs that co-encapsulate agonists of the Stimulator of Interferon Genes (STING) and Toll-like Receptor 4 (TLR4) pathways. NPs can be safely delivered in the systemic blood circulation to achieve TME deposition, uptake by innate antigen-presenting cells (APCs), and robust, synergistic production of Type I interferons by dual STING/TLR4 activation. Here, we hypothesize that combination therapy of proinflammatory NPs with tumor senescence-inducing RAS inhibitors, tremetinib and palbociclib, will not only augment CD8+ T cell recruitment to the TME but enhance their sustained activation by mitigating local immunosuppression. In mice bearing orthotopic transplant KPC tumors or KPC GEMMs, combination NP/inhibitor treatment promoted significant NP delivery to tumors, APC and natural killer (NK) cell activation, and CD8+ T cell-mediated clearance, compared to monotherapies and untreated controls. Unexpectedly and strikingly, these studies also showed that, besides innate immune cells, combination therapy also promoted Type I interferon and other proinflammatory cytokine production by PDAC tumor cells, which are otherwise notoriously unresponsive to current state-of-the-art treatments. Further, in KPC GEMMs, 25% of mice exhibited apparent complete responses following combination treatment, which, to our knowledge, is challenging to achieve in this model. Current studies include the mechanistic elucidation of immune- and tumor-intrinsic factors that govern efficacy of our combination therapy. In conclusion, these findings strongly corroborate the use of this NP-based system as a platform therapy for similar drugs and across other aggressive cancers. They also strongly make the case for the rational design of combination therapies to achieve synergistic therapeutic outcomes with minimal systemic toxicities. Citation Format: Prabhani Atukorale, Marcus Ruscetti, Loretah Chibaya, Christina Lusi, Kelly DeMarco, Griffin Kane, Meghan Brassil, Chaitanya Parikh, Katherine Murphy. Nanoparticle-mediated combination therapy to synergistically harness Type I interferons and senescence in the pancreatic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4043.

Abstract 2895: ILRAP blockade mediates anti-fibrotic effects in pancreatic cancer-associated fibroblasts

Abstract Introduction: Pancreatic cancer (PDAC) patients have poor prognosis partly due to excessive activity of cancer-associated fibroblasts (CAFs). CAFs drive the fibrosis that causes excessive type III collagen and extracellular matrix deposition that in turn reduces drug response resulting in poor survival. In support, high levels of the type III collagen serum biomarker PRO-C3 correlates with poor survival in PDAC. TGF-β is thought to be the main driver of PRO-C3 and tumor fibrosis. Cytokines such as Interleukin 1 (IL-1) play a key role in the pancreatic tumor microenvironment and may play a role in tumor fibrosis as well. In this study, we first investigated the potential of IL-1 in activating fibroblasts to drive fibrosis and produce PRO-C3. Subsequently, we established a co-culture of pancreatic cancer cells and pancreatic CAFs to investigate the anti-fibrotic properties of nadunolimab, an antibody that blocks IL-1 signaling by targeting IL-1 Receptor Accessory Protein (IL1RAP). Nadunolimab is currently in phase I/IIa clinical development for treatment of pancreatic cancer (CANFOUR, NCT03267316). Methods: Human primary pancreatic cancer-associated fibroblasts (CAFs) were cultured in Ficoll-media (Scar-in-a-jar, SiaJ) supplemented with TGF-β, IL-1α or IL-1β. The fibrotic activity of the fibroblasts was investigated by measuring the formation of type III collagen (PRO-C3) at days 3, 6, 9 and 12. Then, pancreatic cancer cells (BxPc3) and pancreatic CAFs were cultured either alone or in a co-culture. Nadunolimab or isotype control were added at the start of these cultures and supernatants were collected after three days. The level of PRO-C3 was measured by ELISA. Results: Both IL-1α or IL-1β were equipotent to TGF-β in inducing PRO-C3 in the SiaJ monoculture, indicating that IL-1 is pro-fibrotic. In addition, when cancer cells and CAFs were co-cultured, PRO-C3 levels increased compared to single-cell cultures (1.5-6-fold). When co-cultures were treated with nadunolimab, the induction of PRO-C3 was blocked to levels similar to monocultures, whereas the isotype control had no effect on PRO-C3 levels. Conclusion: IL-1 activated fibroblasts and induced type III collagen formation (PRO-C3), suggesting that IL-1 is a driver of tumor fibrosis. In support, pancreatic tumor cells induced collagen type III formation (PRO-C3) in pancreatic CAFs and blockade of IL1RAP with nadunolimab inhibited this collagen formation. Thus, nadunolimab may have anti-fibrotic properties and PRO-C3 could potentially be used for prognostic/predictive enrichment and as a pharmacodynamic marker in future studies evaluating anti-IL-1 modalities in PDAC. Citation Format: Neel I. Nissen, Nils Hansen, Elin Jaensson Gyllenbäck, Camilla R. Millrud, Marcus Järås, David Liberg, Morten A. Karsdal, Nicholas Willumsen. ILRAP blockade mediates anti-fibrotic effects in pancreatic cancer-associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2895.

Abstract 2795: An investigation to identify the phylogeny of bacterial communities in the pancreatic cancer microbiome by 16SrDNA sequencing and bioinformatic analysis

Abstract The microbiome refers to the diverse community of microorganisms, including bacteria, viruses, fungi, and other microbes, that inhabit various parts of the human body, such as the skin, mouth, gut, and other mucosal surfaces. The role of the microbiome in cancer is a complex and evolving area of research. While much is still being learned, there is evidence suggesting that the microbiome can influence cancer development and progression in several ways. Some bacteria within the microbiome can produce substances that may be either carcinogenic or protective against cancer. For instance, the conversion of certain dietary components by gut bacteria can produce metabolites that may either promote or inhibit cancer development. Different cancers may be associated with specific alterations in the microbiome, for example, the gut microbiome has been extensively studied in relation to colorectal cancer, with certain bacterial species potentially contributing to the development of this cancer. However, the role of microbiome in Pancreatic cancer is still not extensively studied, henceforth, we did a preliminary study to identify novel bacterial communities around pancreatic cancer microbiome by 16SrDNA sequencing in human pancreatic cancer xenograft model mice. Isolating total genomic DNA from the pancreatic model mice feces, 16SrDNA PCR was done using specific primers and the amplicon obtained as PCR product was confirmed by standard DNA gel electrophoresis with a 1 kb standard ladder to be 1.5kb in size universal for all bacterial 16SrDNA. The amplicon was then sent for Next Generation Sequencing at Zymo Research Corporation (CA). The resultant DNA sequences were analyzed by bioinformatics studies and their phylogeny identified by comparing with 16SrDNA data base. Differential abundance of the bacteria’s of the genera Lachnoclostridium, Robinsoniella, Alistipes, Akkermansia and Lactobacillus were observed analyzing the data along with other bacterial communities. We are currently continuing the project to identify more bacterial communities of significance in the pancreatic cancer microbiome and investigating the role and significance of these bacterial communities in pancreatic cancer tumor microenvironment. Acknowledgement This study was done by a joint collaboration between scientists and students of Elizabeth City State University (ECSU) campus of the University of North Carolina Systems and Mayo Clinic, Jacksonville. This research is supported by the NIH-NIGMS grant#T34GM100831-10 to Dr. H. Banerjee and a University of North Carolina General Administration Grant to Dr. Colby Hunter at ECSU. Citation Format: Hirendra N. Banerjee, Colby Hunter, Narendra Banerjee, Karrington Perry, Khadimou Dia, Anasua Banerjee, Santanu Bhattacharya, Hari Krishnareddy Rachamala, Enfang Wang, Shamit Dutta. An investigation to identify the phylogeny of bacterial communities in the pancreatic cancer microbiome by 16SrDNA sequencing and bioinformatic analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2795.

Abstract 4163: Improving the dynamic range of pH low insertion peptides for targeted imaging of pancreatic tumors

Abstract Objective: Current intraoperative imaging modalities such as near-infrared fluorescent imaging (NIR) or ultrasound are limited due to operator experience and depth, and targeted imaging of pancreatic tumors would be beneficial. However, specific pancreatic tumor biomarkers available for targeting in a novel imaging protocol are lacking. As such, we focus on developing a novel targeted imaging paradigm utilizing multispectral optoacoustic imaging (MSOT) to target the acidic microenvironment of pancreatic tumors. Peptides know as pH low insertion peptides (pHLIPs) when bound to dye allow for contrasted imaging selectively based on pH using MSOT. We have identified the need for a pHLIP with improved dynamic range to improve imaging accuracy. Here, we synthesize the novel pHLIP V7FS for comparison to V3 with aims of improving the dynamic range of imaging with MSOT in pancreatic cancer. Methods: Using microwave chemistry, V7FS and V3 were synthesized with >90% purity. Peptides were conjugated to HiLyte 750 dye, and peptide-dye conjugation confirmed via spectroscopy. Intraoperative measurements of human pancreatic tumor and surrounding uninvolved tissue were obtained using a microsensor. Near-infrared fluorescent imaging was used to measure probe uptake in S2VP10 and S2013 pancreatic cancer cells lines grown in pH specific media (7.4, 6.8, 6.6) in replicates. MSOT was then used to image probes in phantoms mimicking tissue and then in mice with pancreatic tumors from orthotopically implanted S2VP10. Statistical analysis was performed using ANOVA. Results: Compared to surrounding uninvolved pancreatic tissue, human pancreatic tumor was more acidic with pH 6.2-6.7 vs 7.2-7.3 (p<0.01). Signal at pH 7.4, 6.8 and 6.6 was higher for V7FS-750 compared to V3-750 (p=0.0105). Signal for V7FS-750 was centered around pH 6.6 and signal for V3-750 centered around pH 6.8. Interaction between probe and pH did not reach significance (p=0.745). Peak uptake in orthotopic murine models was observed at 3 hours following intravenous injection. Conclusions: V7FS-750 was shown to have higher signal accumulation compared to V3-750 favoring a more acidic environment (pH 6.6 vs pH 6.8). This novel pHLIP was demonstrated to be viable for use in an imaging paradigm utilizing MSOT in an orthotopic murine model. Continued improvement in the dynamic range of these probes is promising for the use of pHLIPS in imaging of pancreatic cancer with MSOT. Citation Format: Ryan Bynum, Emma Sanderson, Barish H. Edil, Ajay Jain, Lacey R. McNally. Improving the dynamic range of pH low insertion peptides for targeted imaging of pancreatic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4163.

Abstract 2802: Enterococcus faecalis-derived X facilitates pancreatic tumor by regulating immune microenvironment

Abstract Previous research in human samples has found a link between the intratumoral microbiome and the development and progression of pancreatic cancer. Nevertheless, the bacterial strains that may have an effect, and the underlying mechanisms, are largely unknown. To identify potential bacterial species, microbiome sequencing was performed on fecal and tumor samples obtained from KPC mice and patients with pancreatic tumors in this study. To identify metabolites associated with bacteria, untargeted metabolomics were analyzed on serum and tumor samples. Furthermore, advanced and high-throughput techniques, such as single-cell RNA sequencing, tumor organoids, engineered bacteria and germ-free mouse models, were used to elucidate the specific influence on the immune microenvironment of pancreatic tumors. Our findings reveal an increased abundance of Enterococcus faecalis (E. faecalis) in the gut during the development of pancreatic cancer, accompanied by its accumulation in the tumor. Patient-derived E. faecalis strain and its metabolite X, facilitated pancreatic tumor growth, reduced the recruitment of CD8+T cells, and caused their dysfunction in the tumor, relying heavily on E. faecalis intratumoral colonization. We found that X inhibits the production of anti-tumor chemokines like CCL21 and CCL8 in tumor cells, reducing peripheral CD8+ T cell infiltration into the tumor. Furthermore, X was found to inhibit glycolysis and compromise the function of tumor-infiltrating CD8+ T cells by decreasing the activity of lactate dehydrogenase (LDH) in CD8+ T cells. Additionally, oral doxycycline, an antibiotic that targets intratumoral E. faecalis, effectively enhanced the therapeutic effect of gemcitabine in pancreatic cancer. Finally, we present evidence that X may predicts efficacy and survival in pancreatic cancer patients. Together, our findings provide new insights into the role of E. faecalis in mediating the immune environment of pancreatic tumors, highlighting E. faecalis and X as potential therapeutic targets for pancreatic cancer patients. Citation Format: Bin Wang, Chao Yang, Yongjie Xie, Tianxing Zhou, Jun Yu, Jihui Hao. Enterococcus faecalis-derived X facilitates pancreatic tumor by regulating immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2802.

Abstract 233: Cabozantinib changes the landscape of the pancreatic tumor microenvironment and improves the efficacy of pembrolizumab

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with an approximate 10% 5-year survival rate. PDAC patients rarely benefit from treatment with immune checkpoint inhibitors (ICIs) due to a tumor microenvironment (TME) associated with constrained effector T cell infiltration, and the presence of a suppressive myeloid cell population. Objective: To identify a combinatorial therapeutic strategy targeting the myeloid derived suppressor cells (MDSCs) and cancer associated fibroblasts (CAFs) within the PDAC TME to increase the efficacy of ICIs. Methods: C57BL/6 mice were orthotopically transplanted with syngeneic 7940b PDAC cells derived from transgenic KPC mouse. 7 days post-transplantation mice were treated with InVivoPlus anti-mouse PD-1 (CD279), cabozantinib (cabo, tyrosine kinase inhibitor) or a combination of drugs for a further 7 days. NanoString CosMx™ spatial molecular imaging (CosMx™ SMI) was performed using FFPE sections collected from core biopsies and surgically resected tissues. PDAC patient derived organoids (PDOs) were generated from the core biopsies or surgical biospecimens. Cancer-associated fibroblasts (CAFs) were isolated from the organoid cultures. Autologous immune cells (IMM) were cultured from patient peripheral blood mononuclear cells. PDO/CAF/IMM co-cultures were used to predict the patient response to cabo plus pembrolizumab combinatorial therapy. Results: Mice treated with a combination of anti-PD-1 and cabo exhibited significant EpCAM/PD-L1-expressing cell death that correlated with increased CTL proliferation and decreased PMN-MDSC death. Mice that were depleted of CD8+ CTLs using anti-CD8 antibody in vivo did not exhibit tumor cell death in response to combinatorial therapy and thus supporting the role of CTLs in driving effective immunotherapy in combination with cabozantinib treatment. Unexpectedly, a significant decrease in extracellular matrix protein fibronectin was observed. Human PDOs orthotopically transplanted into NSG mice developed pathology consistent with human PDAC with strong expression of CD44v9, IL6, vimentin and αSMA. Using PDAC PDO/CAFs/IMM co-cultures, pembro significantly induced tumor cell death in the presence CTLs, MDSCs significantly inhibited this response. Depletion of MDSCs using cabo in the PDO/CAFs/IMM co-cultures resulted in a significant increase in tumor cell death with a concomitant increase in CTL proliferation. Compared to core biopsies, surgical biospecimens collected post-standard of-care demonstrated a shift in cell populations within defined NICHES (local cellular TME) whereby there was 1) increases MDSCs, 2) change in CAFs phenotype and 3) increased expression of CD44v9. Conclusions: Cabozantinib depletes the PDAC TME of MDCSs leading to sensitization to ICI and tumor cell death and thus offers a potential combinatorial therapeutic approach. Citation Format: Pritha Adhikary, Jayati Chakrabarti, Meenu Priya Resmi, Jiang Wang, Davendra Sohal, Rachna T. Shroff, Syed A. Ahmad, Yana Zavros. Cabozantinib changes the landscape of the pancreatic tumor microenvironment and improves the efficacy of pembrolizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 233.

Unraveling the Connection: Pancreatic Cancer Cells and Schwann Cells.

Pancreatic ductal adenocarcinoma is one of the most lethal solid malignancies, characterized by its aggressiveness and metastatic potential, with a 5-year survival rate of only 13%. Progress in the management of metastatic disease has been modest. A robust connection between nervous system and tumor progression exists, with prominent neural alterations having been observed during pancreatic cancer's progression, including neural hypertrophy, neural density, and neural remodeling. The pancreatic tumor microenvironment includes s set of cells and structures that constantly dialogue with cancer cells, influencing its growth and behavior. The microglia is key cellular components of the tumor microenvironment, and Schwann cells are the principal glial cells in the peripheral neural system. Schwann cells can regulate changes in the tumor microenvironment and immune responses by secreting a variety of factors and can support a tumor's invasion of nerves and distant metastasis, with further pain exacerbation. Schwann cells secrete various pain-related molecules, such as the neural growth factor, to mediate the activation of primary sensory neurons, leading to pain induction. The binding of the neural growth factor to tropomyosin receptor kinase A is an important signaling mechanism for pain perception in humans. Consequently, directing efforts towards targeting neural invasion may provide an alternative strategy to improve the prognosis of and alleviate pain in patients with pancreatic cancer.

Synthesis and biological activity of 11-Oxygenated and heterocyclic estrone analogs in pancreatic cancer monolayers and 3D spheroids

Pancreatic Ductal Adenocarcinoma (PDAC), representing over 90 % of pancreatic cancer diagnoses, is an aggressive disease with survivability among the worst of all cancers due to its difficulty in detection and its high metastatic properties. Current therapies for PDAC show limited success at extending life expectancies, primarily due to cancer resistance and lack of patient-specific targeted therapies. This work highlights the design and evaluation of estrone-derived analogs with both heterocyclic side-chain functionality and 11-oxygenated functionality for use in pancreatic cancer. First-round heterocyclic analogs show preliminary promise in AsPC-1 and Panc-1 cell lines, with IC50 values as low as 10.16 ± 0.83 µM. Their success, coupled with design choices from other studies, led to the synthesis of novel 11-hydroxyl and 11-keto estrone analogs that show potent in-vitro toxicity against various pancreatic cancer models. The three most cytotoxic analogs, KA1, KA2, and KA9 demonstrated low micromolar activities in both MTT and CellTiter assays in three pancreatic cancer cell lines: AsPC-1, Panc-1, and BxPC-3, as well as in a co-culture of Panc-1 and pancreatic stellate cells. IC50 values for KA9 (4.17 ± 0.90, 5.28 ± 1.87, and 5.70 ± 0.65 µM respectively) shows consistency in all cell lines tested. KA9 is also able to cause an increase in caspases 3 and 7 activity, key markers for apoptosis, at non-cytotoxic concentrations. Additional work was performed by generating 3D pancreatic cancer spheroids to better modulate the pancreatic tumor microenvironment, and KA9 continued to show the best IC50 values (21.0 and 24.3 µM) in both cell types tested. KA9 was also able to prevent the growth of spheroids whereas the standard chemotherapy, Gemcitabine, could not, suggesting that it may be a potent analog for future development of treatments. Molecular dynamic simulations were also performed to confirm biological findings and uncovered that KA9′s preferential binding location is in the active site pocket of key proteins involved in cytotoxicity.

Tumor immune escape by autotaxin: keeping eosinophils at bay

Secreted autotaxin (ATX) promotes tumor progression by producing the pleiotropic lipid mediator lysophosphatidic acid (LPA). In a recent Nature Cancer paper, Bhattacharyya et al. show that ATX/LPA signaling suppresses CCL11-driven infiltration of eosinophils into the pancreatic tumor microenvironment to facilitate tumor progression, thus revealing a new ATX-mediated immune escape mechanism and highlighting the antitumor potential of eosinophils.

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.

Schwann cells in pancreatic cancer: Unraveling their multifaceted roles in tumorigenesis and neural interactions

Pancreatic ductal adenocarcinoma (PDAC), characterized by heightened neural density, presents a challenging prognosis primarily due to perineural invasion. Recognized for their crucial roles in neural support and myelination, Schwann cells (SCs) significantly influence the process of tumorigenesis. This review succinctly outlines the interplay between PDAC and neural systems, positioning SCs as a nexus in the tumor-neural interface. Subsequently, it delves into the cellular origin and influencers of SCs within the pancreatic tumor microenvironment, emphasizing their multifaceted roles in tumor initiation, progression, and modulation of the neural and immune microenvironment. The discussion encompasses potential therapeutic interventions targeting SCs. Lastly, the review underscores pressing issues, advocating for sustained exploration into the diverse contributions of SCs within the intricate landscape of PDAC, with the aim of enhancing our understanding of their involvement in this complex malignancy.

Pancreatic cancer tumor microenvironment is a major therapeutic barrier and target.

Pancreatic Ductal Adenocarcinoma (PDAC) is projected to become the 2nd leading cause of cancer-related deaths in the United States. Limitations in early detection and treatment barriers contribute to the lack of substantial success in the treatment of this challenging-to-treat malignancy. Desmoplasia is the hallmark of PDAC microenvironment that creates a physical and immunologic barrier. Stromal support cells and immunomodulatory cells face aberrant signaling by pancreatic cancer cells that shifts the complex balance of proper repair mechanisms into a state of dysregulation. The product of this dysregulation is the desmoplastic environment that encases the malignant cells leading to a dense, hypoxic environment that promotes further tumorigenesis, provides innate systemic resistance, and suppresses anti-tumor immune invasion. This desmoplastic environment combined with the immunoregulatory events that allow it to persist serve as the primary focus of this review. The physical barrier and immune counterbalance in the tumor microenvironment (TME) make PDAC an immunologically cold tumor. To convert PDAC into an immunologically hot tumor, tumor microenvironment could be considered alongside the tumor cells. We discuss the complex network of microenvironment molecular and cellular composition and explore how they can be targeted to overcome immuno-therapeutic challenges.

Integrative analysis of spatial and single-cell transcriptome data from human pancreatic cancer reveals an intermediate cancer cell population associated with poor prognosis

BackgroundRecent studies using single-cell transcriptomic analysis have reported several distinct clusters of neoplastic epithelial cells and cancer-associated fibroblasts in the pancreatic cancer tumor microenvironment. However, their molecular characteristics and biological significance have not been clearly elucidated due to intra- and inter-tumoral heterogeneity.MethodsWe performed single-cell RNA sequencing using enriched non-immune cell populations from 17 pancreatic tumor tissues (16 pancreatic cancer and one high-grade dysplasia) and generated paired spatial transcriptomic data from seven patient samples.ResultsWe identified five distinct functional subclusters of pancreatic cancer cells and six distinct cancer-associated fibroblast subclusters. We deeply profiled their characteristics, and we found that these subclusters successfully deconvoluted most of the features suggested in bulk transcriptome analysis of pancreatic cancer. Among those subclusters, we identified a novel cancer cell subcluster, Ep_VGLL1, showing intermediate characteristics between the extremities of basal-like and classical dichotomy, despite its prognostic value. Molecular features of Ep_VGLL1 suggest its transitional properties between basal-like and classical subtypes, which is supported by spatial transcriptomic data.ConclusionsThis integrative analysis not only provides a comprehensive landscape of pancreatic cancer and fibroblast population, but also suggests a novel insight to the dynamic states of pancreatic cancer cells and unveils potential therapeutic targets.Graphical