Pancreatic Ductal Adenocarcinoma Stroma Research Articles

Abstract A038: The hypoxic regulation of macrophage function in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is marked by an extensive fibroinflammatory stroma and a low-oxygen (hypoxic) microenvironment, which contribute to disease progression and therapeutic resistance. Within the PDAC stroma, macrophages and cancer-associated fibroblasts (CAFs) are the predominant cell types. Macrophages are a major immunosuppressive population in PDAC. These cells are highly plastic and, as a result, their environment plays an important role in regulating their function. Macrophages adapt to hypoxia primarily by stabilizing hypoxia-inducible factors (HIFs), which are oxygen-labile transcription factors. Although PDAC is profoundly hypoxic and both the tumor cells and stromal cells experience hypoxia, the effects of hypoxia and HIFs on macrophages and their interactions with other stromal cells in PDAC are not yet fully understood. We found that hypoxia promotes the acquisition of inflammatory cancer-associated fibroblasts (inflammatory CAFs), a fibroblast subtype that produces high levels of cytokines and chemokines. Utilizing a hypoxia marker in mouse models of PDAC, we have observed that inflammatory CAFs and macrophages are predominantly situated in hypoxic tumor regions compared with normoxic regions, while T cells are largely absent from these oxygen-poor regions. These preliminary data raise the possibility that inflammatory fibroblasts induced by hypoxia promote macrophage infiltration into hypoxic tumor regions and facilitate an immunosuppressive macrophage phenotype. We have also found that when macrophages are exposed to conditioned media derived from fibroblast cultures under either hypoxic or normoxic conditions, factors secreted from the hypoxia-exposed fibroblasts promote an immunosuppressive macrophage phenotype. We are currently identifying the factors released from hypoxic fibroblasts that drive the immunosuppressive macrophage phenotype. Future work will focus on evaluating the effects of macrophage-intrinsic responses to hypoxia through HIFs on pancreatic tumorigenesis in vivo. Citation Format: Sean Hannifin, Ashley M Mello, Tenzin Ngodup, Katelyn L Donahue, Marina Pasca di Magliano, Kyoung Eun Lee. The hypoxic regulation of macrophage function in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A038.

FAP-activated liposomes achieved specific macropinocytosis uptake by pancreatic stellate cells for efficient desmoplasia reversal

The desmoplastic stroma in pancreatic ductal adenocarcinoma (PDAC) severely compromises drug delivery and efficacy. To disrupt stromal barriers, we constructed an all-trans retinoic acid (ATRA)-loaded liposome, FrLip@R, that contained calcium phosphate (CaP) cores and arginine 12-mer peptide (r12) ligands shielded by fibroblast activation protein (FAP)-detachable polyglutamate. Shielding r12 protected FrLip@R from nonspecific uptake, e.g., eluding the mononuclear phagocyte system (MPS) uptake for extended blood retention. Then, FrLip@R could be specifically activated by FAP overexpressed on activated pancreatic stellate cells (PSCs) to expose r12, thereby promoting its PSC uptake via an efficient macropinocytosis pathway. Accordingly, FrLip@R exhibited 2.42- and 2.85-fold higher internalization by in vivo PSCs than r12-modified and unmodified liposomes, respectively. In particular, the FrLip amount in the FAP+ PSCs was 54.46-fold higher than that in the other intratumoral cells. In addition, CaP core dissolution in acidic endosomes promoted ATRA release in the cytoplasm. Through efficient and specific ATRA delivery, FrLip@R induced PSC quiescence and disrupted stromal barriers, enhancing intratumoral drug delivery. Finally, in a murine pancreatic cancer model, FrLip@R combined with gemcitabine exerted antitumor efficacy superior to a 1.5-fold dose of gemcitabine while avoiding its severe side effects. Furthermore, FrLip@R modulation created a microenvironment adverse to tumor growth. Therefore, FrLip@R represents a potent and safe stromal barrier-disrupting agent that can amplify chemotherapeutic efficacy in desmoplastic PDAC.

Increased Gremlin1 Expression in Pancreatic Ductal Adenocarcinoma Promotes a Fibrogenic Stromal Microenvironment.

The pancreatic ductal adenocarcinoma (PDAC) microenvironment is primarily composed of cancer-associated fibroblasts (CAFs) and immune cells. Gremlin1 (Grem1) is a profibrogenic factor that promotes tumorigenesis in several cancers. However, the role of Grem1 in the PDAC microenvironment is not adequately defined. We correlated Grem1 levels with activated stroma and immune cells in human PDAC using The Cancer Genome Atlas (TCGA) RNA-sequencing data and characterized the expression of Grem1 transcripts and isoforms in pancreatic cell lines and PDAC tissues. We assessed the role of Grem1 in the microenvironment by in vitro studies. Grem1 expression is associated with an activated stroma and increased M1 and M2 macrophages. Only full length Grem1 variant 1 and isoform 1 were detectable in human pancreatic cells, and remarkably high levels of Grem1 were observed in pancreatic fibroblasts (P < 0.05). Immunohistochemistry detected Grem1 protein in PDAC tumor cells and stromal cells, which correlated with infiltrating macrophages in PDAC tumors. Grem1 knockdown in CAFs suppressed transforming growth factor (TGF)-β-induced extracellular matrix proteins (P < 0.05). Grem1 recombinant protein treatment in vitro increased M1 and M2 macrophages (P < 0.05). Grem1 acts as a profibrogenic factor in the PDAC microenvironment via modulation of fibroblasts and macrophages. Grem1 may have the potential to be developed as a therapeutic target for PDAC.

Pancreatic cancer-associated fibroblasts modulate macrophage differentiation via sialic acid-Siglec interactions

Despite recent advances in cancer immunotherapy, pancreatic ductal adenocarcinoma (PDAC) remains unresponsive due to an immunosuppressive tumor microenvironment, which is characterized by the abundance of cancer-associated fibroblasts (CAFs). Once identified, CAF-mediated immune inhibitory mechanisms could be exploited for cancer immunotherapy. Siglec receptors are increasingly recognized as immune checkpoints, and their ligands, sialic acids, are known to be overexpressed by cancer cells. Here, we unveil a previously unrecognized role of sialic acid-containing glycans on PDAC CAFs as crucial modulators of myeloid cells. Using multiplex immunohistochemistry and transcriptomics, we show that PDAC stroma is enriched in sialic acid-containing glycans compared to tumor cells and normal fibroblasts, and characterized by ST3GAL4 expression. We demonstrate that sialic acids on CAF cell lines serve as ligands for Siglec-7, -9, -10 and -15, distinct from the ligands on tumor cells, and that these receptors are found on myeloid cells in the stroma of PDAC biopsies. Furthermore, we show that CAFs drive the differentiation of monocytes to immunosuppressive tumor-associated macrophages in vitro, and that CAF sialylation plays a dominant role in this process compared to tumor cell sialylation. Collectively, our findings unravel sialic acids as a mechanism of CAF-mediated immunomodulation, which may provide targets for immunotherapy in PDAC.

Abstract 1595: Persistence of fetal gene signatures along the mesenchymal lineage trajectory defines heterogeneity and function of pancreatic cancer associated fibroblasts

Abstract In pancreatic ductal adenocarcinoma (PDAC), cancer associated fibroblasts (CAFs) play critical and complex roles in the tumor microenvironment. CAFs are also a major cell type in the desmoplastic stroma in PDAC and may account for half of the entire tumor tissue. Here we aimed to investigate the origin, diversification, and function of CAFs. We constructed a dual-DNA-recombinase mouse genetic model carrying KrasG12D/+; Trp53frt/+; Pdx1Flpo/+; Isl1creER/+; R26Tomato/+ alleles, referred to as KPFIT. The DNA recombinase FlpO directs expression of an oncogene Kras (G12D mutation) and loss of a tumor suppressor p53 in pancreatic epithelial cells, while creER recombines the Tomato reporter in the splanchnic mesenchyme and its descendants. The splanchnic mesenchyme is a layer of fetal tissue surrounding the endoderm where the pancreatic epithelium arises. This approach capitalized on the independent functions of FlpO/Frt and CreER/LoxP systems, allowing us to lineage trace Tomato labeled splanchnic progenies (via IT) in a spontaneous pancreatic cancer model (via KPF) within the same mouse. Our study identified the splanchnic mesenchyme as the fetal origin of pancreatic fibroblasts during homeostasis and tumorigenesis. Notably, the other two postulated origins, bone marrow and epithelial cells, have minimal contributions to fibroblasts. Importantly, single cell transcriptomic analysis indicated persistent and dynamic gene expressions along the pancreatic mesenchymal trajectory during development, homeostasis, precancer lesion and cancer. Intriguingly, certain splanchnic factors are expressed in only subtypes of adult pancreatic fibroblasts in temporally and spatially distinct patterns. Furthermore, we constructed mouse genetic models to delete one of the splanchnic factors, Gata6, specifically in CAFs, which resulted in increased tumor burden in the pancreas. This suggests a non-cell autonomous function of GATA6 in CAFs to restrain pancreatic cancer progression. In summary, this study delineated a continuous cell trajectory of the mesenchymal lineage in the pancreas across different life stages. Moreover, persistent gene expressions along the mesenchymal trajectory contributes to pancreatic CAF heterogeneity. Importantly, such persistence may constitute an inherent mechanism to suppress pancreatic cancer. The enhancement of this mechanism could be explored further for therapeutic benefits. Citation Format: Lu Han, Tom Walter, Joseph Beaudet, Caroline Everett, Kun Fang, Michael Zimmermann, Angela Mathison, Raul Urrutia, Victor Jin, Gustavo Leone, Michael Ostrowski. Persistence of fetal gene signatures along the mesenchymal lineage trajectory defines heterogeneity and function of 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 1595.

Unraveling the impact of cancer-associated fibroblasts on hypovascular pancreatic neuroendocrine tumors

BackgroundPancreatic neuroendocrine tumors (PNETs) with low microvessel density and fibrosis often exhibit clinical aggressiveness. Given the contribution of cancer-associated fibroblasts (CAFs) to the hypovascular fibrotic stroma in pancreatic ductal adenocarcinoma, investigating whether CAFs play a similar role in PNETs becomes imperative. In this study, we investigated the involvement of CAFs in PNETs and their effects on clinical outcomes.MethodsWe examined 79 clinical PNET specimens to evaluate the number and spatial distribution of α-smooth muscle actin (SMA)–positive cells, which are indicative of CAFs. Then, the findings were correlated with clinical outcomes. In vitro and in vivo experiments were conducted to assess the effects of CAFs (isolated from clinical specimens) on PNET metastasis and growth. Additionally, the role of the stromal-cell-derived factor 1 (SDF1)–AGR2 axis in mediating communication between CAFs and PNET cells was investigated.ResultsαSMA-positive and platelet-derived growth factor-α–positive CAFs were detected in the hypovascular stroma of PNET specimens. A higher abundance of α-SMA-positive CAFs within the PNET stroma was significantly associated with a higher level of clinical aggressiveness. Notably, conditioned medium from PNET cells induced an inflammatory phenotype in isolated CAFs. These CAFs promoted PNET growth and metastasis. Mechanistically, PNET cells secreted interleukin-1, which induced the secretion of SDF1 from CAFs. This cascade subsequently elevated AGR2 expression in PNETs, thereby promoting tumor growth and metastasis. The downregulation of AGR2 in PNET cells effectively suppressed the CAF-mediated promotion of PNET growth and metastasis.ConclusionCAFs drive the growth and metastasis of aggressive PNETs. The CXCR4–SDF1 axis may be a target for antistromal therapy in the treatment of PNET. This study clarifies mechanisms underlying PNET aggressiveness and may guide future therapeutic interventions targeting the tumor microenvironment.

Abstract C025: Fibroblast-specific IL1R1-p38 MAPK signaling sustains stromal inflammation and contributes to therapeutic resistance in pancreatic cancer

Abstract Introduction: The desmoplastic stroma of pancreatic ductal adenocarcinoma (PDAC) perpetuates therapeutic resistance by impairing drug delivery and effector immune cell infiltration and activation. We have identified tumor cell-derived interleukin-1α (IL1α) as a critical activator of the stroma, comprised of cancer-associated fibroblasts (CAF), towards a pro-inflammatory phenotype via the IL1 receptor, IL1R1, and subsequent activation of p38 MAPK. Our hypothesis is that disruption of the CAF-specific IL1α/p38 MAPK signaling axis can improve chemotherapeutic resistance by remodeling the fibrotic stromal landscape and the overall immune microenvironment in PDAC. Methods: p38 MAPK was pharmacologically inhibited with pexmetinib and genetically with an shRNA lentiviral system. Bulk RNA and ATAC sequencing (RNAseq and ATACseq) were performed on human pancreatic stellate cells (hPSC). Gene set enrichment analyses and motif enrichment analyses were performed on the RNAseq and ATACseq datasets, respectively. Tumor cells (KPC-6694), derived from LSL-KrasG12D/+; LSL-Tp53R172H/+; Pdx1Cre/+ (KPC) murine tumors, were injected orthotopically into Col1a2Cre/+; Mapk14f/f and Col1a2Cre/+; Il1r1f/f mice, and tumors were harvested for downstream analysis. Ptf1aCre/+; LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice were treated with pexmetinib (PEX, 30mg/kg, daily PO), anakinra (AKA, 50mg/kg IP, BID) or vehicle control for 2.5 weeks prior to sacrifice for downstream analyses and single-cell RNA sequencing (scRNA) on tumors. Results: In vitro inhibition of p38 MAPK prevented CAF activation into an inflammatory fibroblast, when stimulated with IL1α, demonstrated through bulk RNAseq and ATACseq. Bulk ATACseq revealed epigenetic regulation of the inflammatory CAF phenotype by IL1-mediated activation of p38 MAPK. Stromal-specific deletion of Il1r1 or Mapk14 resulted in signification reduction of tumor weight and intratumoral myeloid-derived suppressor cells. Additionally, pharmacologic blockade of both IL1R1 (AKA) and p38 MAPK inhibition (PEX) altered intratumoral immune cell and CAF populations, by flow cytometry and scRNA. Lastly, p38 inhibition or IL1R1 blockade combined with chemotherapy significantly reduced tumor burden and favorably altered the immune landscape in a PDAC genetically engineered mouse model. Conclusions: These findings provide important mechanistic data in preclinical PDAC models to explore p38 MAPK inhibition in combination therapy to target the fibrotic stroma and immunosuppressive tumor microenvironment. Citation Format: Samara P. Singh, Austin R. Dosch, Siddharth Mehra, Iago de Castro Silva, Vanessa Tonin Garrido, Anna Bianchi, Haleh Amirian, Edmond W. Box, Jashodeep Datta, Nagaraj S. Nagathihalli, Nipun B. Merchant. Fibroblast-specific IL1R1-p38 MAPK signaling sustains stromal inflammation and contributes to therapeutic resistance 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 C025.

Abstract A045: Persistence of the splanchnic gene signature along the mesenchymal cell trajectory during pancreatic cancer progression

Abstract In pancreatic ductal adenocarcinoma (PDAC), cancer associated fibroblasts (CAFs) play critical and complex roles in the tumor microenvironment. While fibroblasts are sparse in the normal adult pancreas, CAFs are a major component in the desmoplastic stroma of PDAC and may account for half of the entire tumor tissue. Here we aimed to investigate the origin, diversification, and function of CAFs. We constructed a dual-DNA-recombinase mouse genetic model, which allowed for genetic alternations in pancreatic epithelial cells and fibroblasts independently and simultaneously. The splanchnic mesenchyme is a layer of tissue adjacent to the pancreatic endoderm during fetal development. Our lineage tracing studies demonstrated that the splanchnic mesenchyme is the fetal origin of adult pancreatic fibroblasts during homeostasis and tumorigenesis. Notably, the other two postulated CAF sources, bone marrow and epithelial cells, have minimal contributions to fibroblasts. We further compared mice carrying no genetic mutations, only Kras mutation, and Kras/p53 dual mutations in pancreatic epithelial cells. We found that fibroblasts in all three conditions are derived from the splanchnic mesenchyme, suggesting a continuous mesenchymal trajectory in coordination with the pancreatic epithelium. Additionally, single cell transcriptomic analysis indicated persistent and dynamic gene expressions along the pancreatic mesenchymal trajectory during development, homeostasis, precancer lesion and cancer. Intriguingly, certain splanchnic factors are expressed in adult pancreatic fibroblasts in temporally and spatially distinct patterns. To determine the function of splanchnic factors in fibroblasts, we constructed mouse genetic models to delete GATA6 specifically in CAFs, which resulted in increased tumor burden in the pancreas. This suggests a non-cell autonomous function of GATA6 in CAFs to restrain pancreatic cancer progression. In summary, this study delineated a continuous cell trajectory of the mesenchymal lineage in the pancreas across different life stages. Furthermore, persistent gene expressions along the mesenchymal trajectory contributes to pancreatic CAF heterogeneity. Importantly, such persistence may constitute an inherent mechanism to restrain pancreatic cancer. Citation Format: Lu Han, Tom Walter, Joseph Beaudet, Caroline Everett, Julia Piermattei, Alex Adams, Kun Fang, Michael Zimmermann, Angela Mathison, Raul Urrutia, Victor Jin, Gustavo Leone, Michael Ostrowski. Persistence of the splanchnic gene signature along the mesenchymal cell trajectory during pancreatic cancer progression [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 A045.

The Degree of Fibrous Stroma in Pancreatic Ductal Adenocarcinoma Does Not Serve as A Reliable Marker for Survival: A Case Study

The Degree of Fibrous Stroma in Pancreatic Ductal Adenocarcinoma Does Not Serve as A Reliable Marker for Survival: A Case Study

Modelling and breaking down the biophysical barriers to drug delivery in pancreatic cancer.

The pancreatic ductal adenocarcinoma (PDAC) stroma and its inherent biophysical barriers to drug delivery are central to therapeutic resistance. This makes PDAC the most prevalent pancreatic cancer with poor prognosis. The chemotherapeutic drug gemcitabine is used against various solid tumours, including pancreatic cancer, but with only a modest effect on patient survival. The growing PDAC tumour mass with high densities of cells and extracellular matrix (ECM) proteins, i.e., collagen, results in high interstitial pressure, leading to vasculature collapse and a dense, hypoxic, mechanically stiff stroma with reduced interstitial flow, critical to drug delivery to cells. Despite this, most drug studies are performed on cellular models that neglect these biophysical barriers to drug delivery. Microfluidic technology offers a promising platform to emulate tumour biophysical characteristics with appropriate flow conditions and transport dynamics. We present a microfluidic PDAC culture model, encompassing the disease's biophysical barriers to therapeutics, to evaluate the use of the angiotensin II receptor blocker losartan, which has been found to have matrix-depleting properties, on improving gemcitabine efficacy. PDAC cells were seeded into our 5-channel microfluidic device for a 21-day culture to mimic the rigid, collagenous PDAC stroma with reduced interstitial flow, which is critical to drug delivery to the cancer cells, and for assessment with gemcitabine and losartan treatment. With losartan, our culture matrix was more porous with less collagen, resulting in increased hydraulic conductivity of the culture interstitial space and improved gemcitabine effect. We demonstrate the importance of modelling tumour biophysical barriers to successfully assess new drugs and delivery methods.

Inhibiting stromal Class I HDACs curbs pancreatic cancer progression

Oncogenic lesions in pancreatic ductal adenocarcinoma (PDAC) hijack the epigenetic machinery in stromal components to establish a desmoplastic and therapeutic resistant tumor microenvironment (TME). Here we identify Class I histone deacetylases (HDACs) as key epigenetic factors facilitating the induction of pro-desmoplastic and pro-tumorigenic transcriptional programs in pancreatic stromal fibroblasts. Mechanistically, HDAC-mediated changes in chromatin architecture enable the activation of pro-desmoplastic programs directed by serum response factor (SRF) and forkhead box M1 (FOXM1). HDACs also coordinate fibroblast pro-inflammatory programs inducing leukemia inhibitory factor (LIF) expression, supporting paracrine pro-tumorigenic crosstalk. HDAC depletion in cancer-associated fibroblasts (CAFs) and treatment with the HDAC inhibitor entinostat (Ent) in PDAC mouse models reduce stromal activation and curb tumor progression. Notably, HDAC inhibition (HDACi) enriches a lipogenic fibroblast subpopulation, a potential precursor for myofibroblasts in the PDAC stroma. Overall, our study reveals the stromal targeting potential of HDACi, highlighting the utility of this epigenetic modulating approach in PDAC therapeutics.

Arachidonate 15-lipoxygenase-mediated production of Resolvin D5n-3 DPA abrogates pancreatic stellate cell-induced cancer cell invasion.

Activation of pancreatic stellate cells (PSCs) to cancer-associated fibroblasts (CAFs) is responsible for the extensive desmoplastic reaction observed in PDAC stroma: a key driver of pancreatic ductal adenocarcinoma (PDAC) chemoresistance leading to poor prognosis. Specialized pro-resolving mediators (SPMs) are prime modulators of inflammation and its resolution, traditionally thought to be produced by immune cells. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipid mediator profiling PSCs as well as primary human CAFs express enzymes and receptors to produce and respond to SPMs. Human PSC/CAF SPM secretion profile can be modulated by rendering these cells activated [transforming growth factor beta (TGF-β)] or quiescent [all-trans retinoic acid (ATRA)]. ATRA-induced nuclear translocation of arachidonate-15-lipoxygenase (ALOX15) was linked to increased production of n-3 docosapentaenoic acid-derived Resolvin D5 (RvD5n-3 DPA), among other SPMs. Inhibition of RvD5n-3 DPA formation increases cancer cell invasion, whereas addback of this molecule reduced activated PSC-mediated cancer cell invasion. We also observed that circulating concentrations of RvD5n-3 DPA levels were decreased in peripheral blood of metastatic PDAC patients when compared with those measured in plasma of non-metastatic PDAC patients. Together, these findings indicate that RvD5n-3 DPA may regulate cancer-stroma cross-talk and invasion.

The tumor stroma influences immune cell distribution and recruitment in a PDAC-on-a-chip model.

The dense tumor stroma of pancreatic ductal adenocarcinoma (PDAC) and its secreted immune active molecules provide a barrier for chemotherapy treatment as well as for immune cell infiltration to the tumor core, providing a challenge for immunotherapeutic strategies. Consequently, the investigation of processes underlying the interaction between the tumor stroma, particularly activated pancreatic stellate cells (PSCs), and immune cells may offer new therapeutic approaches for PDAC treatment. In this study, we established a 3D PDAC model cultured under flow, consisting of an endothelial tube, PSCs and PDAC organoids. This was applied to study the role of the tumor microenvironment (TME) on immune cell recruitment and its effect on partly preventing their interaction with pancreatic cancer cells. We observed that stromal cells form a physical barrier, partly shielding the cancer cells from migrating immune cells, as well as a biochemical microenvironment, that seems to attract and influence immune cell distribution. In addition, stromal targeting by Halofuginone led to an increase in immune cell infiltration. We propose that the here developed model setups will support the understanding of the cellular interplay influencing the recruitment and distribution of immune cells, and contribute to the identification of key players in the PDAC immunosuppressive TME as well as support the discovery of new strategies to treat this immune unresponsive tumor.

Visual Investigation of Tumor-Promoting Fibronectin Potentiated by Obesity in Pancreatic Ductal Adenocarcinoma Using an MR/NIRF Dual-Modality Dendrimer Nanoprobe.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease characterized by dense stroma. Obesity is an important metabolic factor that greatly increases PDAC risk and mortality, worsens progression and leads to poor chemotherapeutic outcomes. With omics analysis, magnetic resonance and near-infrared fluorescence (MR/NIRF) dual-modality imaging and molecular functional verification, obesity as an important risk factor is proved to modulate the extracellular matrix (ECM) components and enhance Fibronectin (FN) infiltration in the PDAC stroma, that promotes tumor progression and worsens response to chemotherapy by reducing drug delivery. In the study, to visually evaluate FN in vivo and guide PDAC therapy, an FN-targeted nanoprobe, NP-CREKA, is synthesized by conjugating gadolinium chelates, NIR797 and fluorescein isothiocyanate to a polyamidoamine dendrimer functionalized with targeting peptides. A dual-modality strategy combining MR and NIRF imaging is applied, allowing effective visualization of FN in orthotopic PDAC with high spatial resolution, ideal sensitivity and excellent penetrability, especially in obese mice. In conclusion, the findings provide new insights into the potential of FN as an ideal target for therapeutic evaluation and improving treatment efficacy in PDAC, hopefully improving the specific management of PDAC in lean and obese hosts.

Transcytosis Mediated Deep Tumor Penetration for Enhanced Chemotherapy and Immune Activation of Pancreatic Cancer

AbstractThe hyperproliferative tumor stroma of pancreatic ductal adenocarcinoma (PDAC) severely limits drug permeation and constructs an immunosuppressive microenvironment, causing resistance to chemotherapy and immunotherapy. Traditional nanomedicine mainly focuses on manipulating nanoparticles’ particle size or electrical characteristics to penetrate deep PDAC through the paracellular pathway, but the transcellular pathway is often ignored. Therefore, a versatile drug‐polymer conjugate PODEA‐Gem‐HMI is prepared and assembled into nanoparticles for the codelivery of chemotherapy drug gemcitabine and focal adhesion kinase (FAK) inhibitor defactinib. While sensing the mild acidity in the tumor microenvironment, the nanoparticle will disintegrate and release defactinib to modulate the tumor stroma. The PODEA block of the conjugate can bind with cell membranes reversibly and trigger adsorption‐mediated transcytosis (AMT) for promoted tumor penetration and cellular uptake. The internalized conjugates will release gemcitabine responding to the overexpressed glutathione (GSH) for enhanced chemotherapy, and PHMI can condensate the STING monomers for prolonged spontaneous immune stimulation.

Abstract 5847: The splanchnic mesenchyme is the main tissue origin of fibroblasts in the pancreas during homeostasis and tumorigenesis

Abstract In pancreatic ductal adenocarcinoma (PDAC), cancer associated fibroblasts (CAFs) play critical and complex roles in the tumor microenvironment. CAFs are also a major cell type in the desmoplastic stroma in PDAC and may account for half of the entire tumor tissue. Multiple subtypes of CAFs have been suggested, but the tissue origin(s) of CAF subtypes are unknown and genetic tools to robustly target them in vivo are lacking. Here we aimed to examine three potential tissue sources of CAFs: the pancreatic epithelium (through epithelium-to-mesenchyme transition), the bone marrow (through circulation), and the pancreatic mesenchyme or tissue resident fibroblasts (TRFs) in the normal pancreas (through proliferation). We utilized a genetically engineered mouse model of PDAC, where Kras and p53 mutations were engineered in the pancreatic epithelium using an Flp-Frt system. To determine whether the pancreatic epithelium gives rise to CAFs, we permanently labeled the pancreatic epithelium with a GFP reporter and traced their cell descendants by GFP expression. Despite robust GFP labeling of the epithelium, GFP expression was rarely identified in CAFs. To determine whether the bone marrow gives rise to CAFs, we transplanted donor bone marrow carrying a ubiquitously expressed GFP reporter to GFP-negative recipient mice. We found that minimal proportion of pancreatic CAFs were tagged with GFP. Lastly, to determine whether pancreatic TRFs give rise to CAFs, we used an inducible CreER-LoxP system to allow for permanent Tomato labeling in TRF progenitors, the splanchnic mesenchyme, during mid-gestation. Lineage tracing in PDAC showed that the vast majority of CAFs were labeled with Tomato expression, suggesting their splanchnic origin. Furthermore, certain splanchnic gene expression signatures persisted in subsets of CAFs in both the PDAC mouse model and human patient samples. Deletion of one of the splanchnic genes, Gata6, in CAFs resulted in increased tumor burden in the pancreas, suggesting a tumor-restraining role of Gata6 in CAFs. In summary, we found that the pancreatic epithelium and bone marrow contributes to a minimal proportion of CAFs in PDAC. Meanwhile, pancreatic TRFs are derived from the splanchnic mesenchyme during fetal development and they expand to contribute to the vast majority of CAFs in PDAC. Moreover, the persistence of splanchnic signature defines subtypes of CAFs, with a potential tumor-suppressing function. This study provides genetic approaches to robustly target CAFs in vivo, and novel insights into CAF origin, heterogeneity and function in PDAC. Citation Format: Lu Han, Yongxia Wu, Kun Fang, Sean Sweeney, Ulyss Roesner, Melodie Parrish, Khushbu Patel, Tom Walter, Julia Piermattei, Anthony Trimboli, Julia Lefler, Cynthia Timmers, Xue-Zhong Yu, Victor Jin, Michael Zimmermann, Angela Mathison, Raul Urrutia, Michael Ostrowski, Gustavo Leone. The splanchnic mesenchyme is the main tissue origin of fibroblasts in the pancreas during homeostasis and tumorigenesis [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 5847.

Dual Drug Loaded Nanotheranostic Platforms as a Novel Synergistic Approach to Improve Pancreatic Cancer Treatment

AbstractThis study focuses on the development of theranostic, dual drug‐loaded nanocarriers to propose a proof‐of‐principle therapeutic approach in the treatment of pancreatic ductal adenocarcinoma (PDAC). The nanoconstructs consist of a core of zinc oxide nanocrystals doped with gadolinium, useful as a potential contrast agent in magnetic resonance imaging applications. After functionalizing their surface with amino‐propyl groups, the physical adsorption of two hydrophobic drugs is performed: Vismodegib and Sorafenib. Their synergistic use might improve PDAC treatment and stroma depletion when co‐delivered in the tumor microenvironment for future in vivo applications. To enhance the nanoconstructs’ biostability, the ensemble is coated by a lipid bilayer and a tumor targeting peptide is incorporated on the outer shell surface. As a first proof of concept, the resulting nanoconstructs are tested against two pancreatic cancer cell lines, showing a modest increase in treatment efficacy compared to the free drug counterparts and proving to spare healthy pancreatic cells. In a second testing set, the dual‐drug loaded nanoconstructs are tested on both cell lines previously sensitized to a first‐line chemotherapeutic drug, Gemcitabine, showing an improved treatment response. From these preliminary results, the nanotheranostic platforms might constitute a good starting point for future PDAC therapy and diagnosis studies.

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

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

Stroma-targeting strategies in pancreatic cancer: a double-edged sword.

Pancreatic ductal adenocarcinoma (PDAC) is a type of cancer with limited treatment options and terrible long-term survival, and it is expected to become the second leading cause of cancer-related death by 2030. One reason why this cancer is so aggressive and resistant is the formation of dense stroma that surrounds the neoplastic epithelium, which promotes tumor progression, invasion, metastasis, and resistance. The three major components of PDAC stroma are extracellular matrix (ECM), cancer-associated fibroblasts (CAFs), and vasculature. The dense ECM acts as a natural physical barrier, impeding drug penetration to PDAC tumor cells. Consequently, the method that combines stroma-targeting with anticancer therapy may be a viable alternative for increasing drug penetration. Additionally, blood vessels are key entities of the tumor stroma, serving as a pathway for nutrition as well as the only way for chemical medicines and immune cells to act. Finally, PDAC CAFs and tumor cells have crosstalk effects in the tumor microenvironment, where they are responsible for enhanced matrix deposition. In this review, we aim to provide an overview of our current comprehension of the three key components of PDAC stroma and the new promising therapeutic targets for PDAC.

236P A novel microfluidic platform for understanding the role of PDAC stroma in immune response

Pancreatic Ductal Adenocarcinoma (PDAC), the most common pancreatic cancer type is estimated to become the second leading cause of cancer-related deaths by 2030 with mortality rates of up to 93%. PDAC is characterized by high-density stroma and an immunosuppressive tumor microenvironment that lead to a lack of efficacy of immunotherapeutic strategies. Those facts highlight the urgent need for more relevant PDAC in vitro models that support the discovery of novel (immuno-)therapies. Here, we describe the establishment of a multiple cell type culture in a microfluidic platform that will be applied for modeling immune and stroma cell interplay in PDAC.