Mouse Model Of Pancreatic Ductal Adenocarcinoma Research Articles

Abstract B108: Integrated in vivo dissection of metastatic phenotypes and molecular programs in pancreatic ductal adenocarcinoma

Abstract The ability of pancreatic ductal adenocarcinoma (PDAC) cells to form tissue destructive metastases remains a critical impediment to therapy. Metastasis is a complex multistep process that is poorly understood and the molecular events and cellular states that enable cancer cells to overcome each step of the metastatic cascade remain elusive. To investigate the regulators of PDAC metastasis, we accrued and characterized a panel of 31 cell lines generated from mouse models of PDAC. Initial experiments indicated that these cell lines have an ~40-fold difference in their ability to form lung metastases after transplantation into immunocompetent recipient mice. To more accurately quantify the relationship between metastatic seeding, growth, organ tropism, and immune evasion across all cell lines in parallel, we tagged each cell line with a barcode that provides both cell line-level and subclonal-level resolution. We pooled all 31 barcoded cell lines, transplanted them to seed metastases in different organs, and performed barcode sequencing on DNA from the metastasis-bearing tissues after short (seeding) and long (expansion) time periods. Neither seeding nor metastatic outgrowth correlated with growth in culture. Metastatic seeding did not directly correlate with overall metastatic burden, indicating that these processes depend on distinct biological processes. Transplantation into syngeneic immune-competent and immune-deficient recipient mice quantified the impact of immune responses towards each cell line. Bulk and single cell RNA-sequencing uncovered unexpected intra- and inter-cell line heterogeneity. Future experiments will integrate the data on metastatic phenotypes with molecular information to uncover drivers and inhibitors of each step of the metastatic cascade. Broad-scale molecular profiling coupled with quantitative in vivo analysis of metastatic phenotypes will uncover the relationships between metastatic phenotypes, create a resource for the community, and reveal novel pathways that govern PDAC metastasis, which could have implications for developing novel therapeutic strategies. Citation Format: Saswati Karmakar, Anastasia Lyulina, Xiaochen Xiong, Yuning J. Tang, Steven Lopez, Nicolas William Hughes, Laura Attardi, Dmitri A. Petrov, Monte M. Winslow. Integrated in vivo dissection of metastatic phenotypes and molecular programs in pancreatic ductal adenocarcinoma [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 B108.

Abstract B031: Targeting epigenetic machinery to revoke stromal pro-tumorigenicity and enhance efficacy in pancreatic cancer therapy

Abstract Oncogenic lesions in pancreatic ductal adenocarcinoma (PDAC) induce prominent desmoplasia in part by activating fibroblasts or their progenitors in the stroma. The resultant cancer-associated fibroblasts (CAFs) often switch on the pro-desmoplastic, pro-inflammatory and/or pro-tumorigenic transcriptional programs, contributing to tumor progression and therapeutic resistance. Identifying and targeting epigenetic machinery essential for stromal fibroblast activation provides potential strategies to revoke stromal pro-tumorigenicity and overcome therapeutic resistance in PDAC. Here we identify Class I histone deacetylases (HDACs) as key epigenetic regulators facilitating the induction of pro-desmoplastic and pro-tumorigenic transcriptional programs in stromal fibroblasts. Mechanistically, HDACs coordinate the establishment of chromatin architecture that enables the activation of pro-desmoplastic programs directed by the transcriptional factor axis of serum response factor (SRF) and forkhead box M1 (FOXM1). HDACs also facilitate the induction of pro-inflammatory program in stromal fibroblasts, supporting leukemia inhibitory factor (LIF)-mediated pro-tumorigenic crosstalk. Both HDAC depletion in CAFs and treatment of the HDAC inhibitor entinostat (Ent) in PDAC mouse models reduce stromal activation and curb tumor progression. Notably, HDAC inhibition (HDACi) enriches a novel lipogenic fibroblast subpopulation in CAFs, a potential precursor population for activated myofibroblasts in vivo. Our work pinpoints a critical role of HDACs in driving stromal pro-tumorigenicity and highlights the stromal targeting capacity of HDACi therapy. Furthermore, the clinical utility of HDACi therapy is often hindered by limited potency and nonnegligible systemic toxicity of the HDAC inhibitors. To facilitate the translation of HDACi-based therapy, we develop a polymer-based bottlebrush pro-drug (BPD) nanoparticle incorporating Ent. The Ent-BPD nanoparticle overcomes the limitations of conventional HDACi therapy by sustaining long-lasting release of HDACi activity specifically at the tumor sites. Administration of Ent-BPD in PDAC mouse models achieves superior intratumoral HDACi potency and enhanced therapeutic efficacy compared to the free drug Ent treatment; meanwhile, it avoids the systemic toxicity associated with the free drug treatment. Our work further demonstrates a promising strategy for the clinical adaptation of HDACi-based therapy. Citation Format: Gaoyang Liang, Jeremiah A. Johnson, Ronald M. Evans. Targeting epigenetic machinery to revoke stromal pro-tumorigenicity and enhance efficacy in pancreatic cancer therapy [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 B031.

Abstract IA20: Stromal-derived NRG1 enables oncogenic KRAS bypass in pancreas cancer

Abstract Oncogenic KRAS (KRAS*) is a signature mutation in pancreatic ductal adenocarcinoma (PDAC). While genetic extinction or pharmacological inhibition of KRAS* elicits anti-tumor responses in preclinical models and patients, we and others have identified cancer cell intrinsic (YAP, RTKs) and paracrine immune-mediated (TGFb) mechanisms enabling KRAS* bypass and tumor recurrence. Here, we explored the potential role of cancer associated fibroblasts (CAFs) as contributors to mechanisms driving KRAS* bypass and identified CAF-derived NRG1 activation of cancer cell ERBB2 and ERBB3 receptor tyrosine kinases as a mechanism by which KRAS* independent tumor regrowth is supported. We showed that genetic extinction or pharmacological inhibition of KRAS* results in upregulation of ERBB2 and ERBB3 expression in human and murine cancer cell models. We demonstrated that CAF-derived NRG1 supports KRAS*-dependent tumor growth. These insights prompted genetic depletion or pharmacological inhibitions studies targeting ERBB2/3 or NRG1, which showed abolition of KRAS* bypass and synergy with KRASG12D inhibitor in mouse and human PDAC models. Thus, CAFs via the NRG-ERBB2/3 paracrine axis provide yet another mechanism enabling KRAS* inhibitor therapy resistance, providing a highly actionable combination therapeutic strategy to improve the effectiveness of KRAS* inhibitors in PDAC patients. Citation Format: Jincheng Han, Jiaqian Xu, Yonghong Liu, Shaoheng Liang, Kyle A. LaBella, Deepavali Chakravarti, Denise J. Spring, Yan Xia, Ronald A. DePinho. Stromal-derived NRG1 enables oncogenic KRAS bypass in pancreas 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 IA20.

Abstract B053: Physical activity regulates targetable transcriptomic changes in the adipose tissue of obese PDAC mice

Abstract Background: Obesity is a risk factor for pancreatic ductal adenocarcinoma (PDAC), a highly aggressive cancer with a low 5-year survival rate. The association between obesity and PDAC could be attributed to the inflammatory effects of obesity and its impact on the adipose microenvironment. Physical activity (PA) is a potential approach to counteract obesity and its associated inflammation. Moreover, a recent study revealed that exercise-induced interleukin (IL)-15 in tumors has an anti-tumor response. However, there is still limited understanding of the mechanisms underlying how PA impacts the adipose-tumor microenvironments in PDAC. In this study, we hypothesized that PA could upregulate anti-inflammatory signaling in the adipose tissue and that targeting some of these signaling pathways could be a novel therapy for obesity-associated PDAC. Methods: Transcriptomic analysis was performed on adipose tissue obtained from a genetically engineering mouse model of obesity-associated PDAC. The mice were fed an obesogenic high-fat diet and divided into groups with and without PA intervention for one month. An adipose-specific recombinant adeno-associated vector (rAAV) was generated and mouse IL-15 cDNA was subcloned into the vector. C57BL/6J mice were administered intraperitoneal injections of either the rAAV/empty or rAAV/IL-15 vectors. Three weeks after rAAV intraperitoneal injection, mice were orthotopically injected with mouse KRasG12D/Trp53−/−/PDX-1-CRE cells expressing firefly luciferase to monitor growth in vivo and expression of IL-15 in the adipose tissue was confirmed via qPCR. Results: PA intervention increased the expression of genes related to metabolic and inflammation-related pathways. The administration of a novel adipose-targeted rAAV resulted in the upregulation of IL-15 within the adipose tissue and not the tumor. Increasing IL-15 expression in the adipose tissue led to a notable delay in tumor growth in an orthotopic mouse model. Conclusions: Here, we show that PA modulates gene expression in the adipose tissue and the beneficial impact of adipose tissue molecular remodeling on anti-tumor effects. However, more studies are needed to characterize the full extent of the adipose-tumor interactions and how they can be effectively targeted for therapeutic interventions. Citation Format: Hsiang-Yin Hsueh, Valentina Pita-Grisanti, Ericka Vélez-Bonet, Lei Cao, Wei Huang, Zobeida Cruz-Monserrate. Physical activity regulates targetable transcriptomic changes in the adipose tissue of obese PDAC mice [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 B053.

Abstract B059: A novel pancreatic cancer mouse model with human immunity

Abstract The iRGD tumor-penetrating peptide delivers chemically linked drugs and even co-injected free drugs selectively to extravascular tumor tissue by targeting the αvβ5 integrin and neuropilin-1 (NRP1). A phase 1b clinical trial showed that iRGD appears to safely double the response rate of standard chemotherapy in patients with stage 4 pancreatic ductal adenocarcinoma (PDAC). We recently found that long-term iRGD treatment, even as a monotherapy, reduced regulatory T cells (Tregs) selectively in the PDAC tissue, which led to the expansion of CD8+ T cells and improved efficacy of immunotherapy in syngeneic PDAC mice. This finding led to the discovery of αvβ5 integrin being a novel targetable marker for tumor-resident Tregs in PDAC mice. Our recent data show that αvβ5 integrin+ Tregs are also present in human PDAC tissue. To study the significance of αvβ5 integrin+ human Tregs as a therapeutic target for iRGD, we developed a humanized PDAC (huPDAC) mouse model by transplanting human PDAC cells into human immune system (HIS) mice, which harbor fully matured human CD8+ and CD4+ T cells, natural killer T cells, B cells, and dendritic cells. The CD8+ T cells are human leukocyte antigen (HLA)-restricted and show a tumor antigen-specific response. We orthotopically implanted HLA-matched human PDAC cells into HLA-A2-positive HIS mice. Both an established human PDAC cell line (PANC-1) and patient-derived PDAC cells (0779E) successfully formed orthotopic tumors and liver metastases. The tumors were infiltrated by various human immune cells at a ratio similar to that found in patient-derived PDAC tissues. Of note, αvβ5 integrin+ human Tregs were also noted in the tumors but not in the spleen. iRGD monotherapy increased the ratio of CD8+ T cells per Tregs in the tumors, suggesting that iRGD will likely function as an immunomodulator in humans. The first patient was recently dosed in a new phase 1b/2a clinical trial (iLSTA Trial: ACTRN12623000223639), which assesses the safety and preliminary efficacy of iRGD in combination with standard-of-care chemotherapy and immunotherapy in locally advanced PDAC patients. Citation Format: Norio Miyamura, Kodai Suzuki, Richard A. Friedman, Aristidis Floratos, Yuki Kunisada, Kazuya Masuda, Andrew M. Lowy, Moriya Tsuji, Kazuki N. Sugahara. A novel pancreatic cancer mouse model with human immunity [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 B059.

Abstract B100: Netrin-1/Neogenin-1 interaction modulates pancreatic innervation to promote tumorigenesis and accelerates cancer progression

Abstract Background: Nerves are a major component of the tumor microenvironment, with active crosstalk between cancer cells and nerves that may contribute to cancer progression. Netrin1 (Ntn1) is an axon guidance molecule that is important during early neural development. Recent studies suggest that Ntn1 may also play a role in tumorigenesis but its role in pancreatic cancer is not well understood. We hypothesized that Ntn1 plays a pro-tumorigenic role in pancreatic ductal adenocarcinoma (PDAC) progression and metastasis in part through modulation of nerves in the microenvironment. Methods: Expression of Ntn1 and its receptor Neogenin1(Neo1) were investigated in mouse models of spontaneous PDAC (LSL-Kras+/G12D/Pdx1-Cre; KC, LSL-Kras+/G12D/LSL-Trp53+/R172H/Pdx1-Cre; KPC). The influence of Ntn1 on pancreatic cancer progression and innervation was evaluated by KC mice with or without conditional knockout of Ntn1 (LSL-Kras+/G12D/Pdx1-Cre/Netrin1fl/fl; KCN). Murine PDAC cell lines and organoids were used to examine the functional role of Ntn1 and Neo1 through genetic and pharmacological modulations. The capacity of these cell lines to generate metastasis and the effect on neurite outgrowth was studied in a co-culture system with Dorsal Root Ganglia (DRG). Results: Expression of Ntn1 was not observed in normal acinar cells or ductal cells, but was found in both PanIN lesions and PDAC. Neo1 expression was similar to Ntn1 and there was a positive correlation between the expression of Ntn1 and Neo1. A lower rate of PanIN progression was observed in KCN mice compared to KC mice. In the metastatic model, overexpression of Ntn1 increased tumor burden and decreased survival while injections of a neutralizing Ntn1 antibody or genetic knockdown of Neo1 improved both endpoints. Ntn1 and Neo1 were upregulated in pancreatic organoids upon KRAS activation. Recombinant Ntn1 (rNTN1) promoted organoid forming capacity and upregulate Sox2 and Sox9 gene expression, which was inhibited by genetic knockdown of Neo1 or an anti-Neo1 antibody, supporting a direct autocrine role in modulating stemness. Nerve fibers were significantly increased at the site of PDAC liver metastasis, with Ntn1 showing the highest expression level among neurotrophins, as it was significantly increased in metastatic PDAC cells. Consistent with this observation, we observed less innervation in KCN mice, which correlated with reduced PanIN progression. Co-culture of DRGs with PDAC cells that overexpressed Ntn1 resulted in much greater axonal elongation consistent with a role of Ntn1 in promoting innervation. Conclusion: The Netrin1/Neogenin1 interaction is an important regulator of PDAC progression, acting both directly on PDAC cells to promote stemness, and indirectly via modulating tumor innervation. Blockade of Ntn1/Neo1 interaction reduces the capacity of PDAC to metastasize to the liver and improves survival. Overall, targeting the Ntn1/Neo1 interaction may represent a potential new therapeutic approach for the treatment of pancreatic cancer. Citation Format: Yosuke Ochiai, Sunagawa Masaki, Ermanno Malagola, Hiroki Kobayashi, Feijing Wu, Ruth A. White, Leah B. Zamechek, Timothy C. Wang. Netrin-1/Neogenin-1 interaction modulates pancreatic innervation to promote tumorigenesis and accelerates 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 B100.

Abstract A036: Quantitative molecular imaging of pancreatic tumor fibrosis for evaluation of response to neoadjuvant therapy: Pre-clinical and first-in-human application of collagen PET/MRI

Abstract Purpose: One of the major challenges in the management of pancreatic ductal adenocarcinoma (PDAC) is the lack of a reliable imaging tool to monitor tumor response to treatment. A high degree of fibrosis, mainly collagen type I, has been recognized as the hallmark of PDAC, which further increases in response to neoadjuvant chemoradiotherapy (CRT). This study introduces an image-guided paradigm for monitoring treatment response using the collagen type I specific PET imaging probe, 68Ga-CBP8 in mouse models and patients with PDAC. Methods: Subcutaneous mouse models of human PDAC were generated using FOLFIRNOX-sensitive (PANC1 and PDAC6) and FOLFIRINOX-resistant (SU8686) PDAC cells in nude mice (n=88). Mice were randomized into 2 groups of treatment with FOLFIRINOX or vehicle i.v. twice a week. Animals underwent PET/MRI with 68Ga-CBP8 (3.7-11.1 MBq) prior to, and at 7- and 15-days post-treatment (n=4-8/group). To assess the specificity of the probe, additional PANC1 tumor-bearing mice (n=4) were imaged with 68Ga-CBP8, and a collagen non-binding probe, 68Ga-CNBP (3.7-11 MBq). PET values were correlated with tumor growth over 21 days and quantification of fibrosis by Picrosirius red and IHC for collagen I, α-SMA, FAP-α, collagen rtPCR, and hydroxyproline assay. 8 patients (49-65 yrs.) with newly diagnosed PDAC underwent 68Ga-CBP8 PET (122-281 MBq) and standard MRI. Thus far, 3/8 subjects have undergone repeat PET/MRI after completion of neoadjuvant CRT. The pre-and post-CRT PET parameters for tumor and pancreas were compared and correlated with tumor size change, histology, and spatial analyses for PDAC and fibroblasts. Results: PET/MRI of PANC1 tumors showed significantly higher 68Ga-CBP8 uptake compared to control 68Ga-CNBP at each time point (p < 0.05), demonstrating the specificity of the probe in targeting collagen. PET values significantly increased over time in FOLFIRINOX-sensitive tumors (PANC1 %ID/cc: 0.8 ± 0.2 vs. 1.1 ± 0.3 vs. 1.9 ±0.2, p < 0.05 in day-0 vs. day-7 vs. day-15, respectively, p = 0.0005), whereas no significant change was seen in the probe uptake in the vehicle or resistant tumors (p > 0.05). There was a significant tumor growth reduction in responders compared to vehicle and resistant tumors. Histology showed a continuous increase of collagen in responding tumors (% collagen proportion area (CPA) in PANC1: 5.2 ± 3.8 vs. 16.2 ± 8.7 vs. 46.9 ± 6.0, p < 0.05, at Day-0 vs. Day-7 vs. Day-15, respectively, p < 0.0001), no change in resistant SU8686 (CPA: 33.0 ± 8.9 vs. 30.0 ± 9.7 vs. 28.1 ± 7.2, at Day-0 vs. Day-7 vs. Day-15, respectively, p = 0.66) and unchanged minimal collagen in pancreas. PET/MRI of patients showed a significantly higher SUVmean in the tumor compared to pancreas (2.4 ± 0.37 vs. 1.9 ± 0.31, p = 0.01), and a significantly higher uptake in the treated compared to untreated tumors (SUVmean: 2.8 ± 0.33 vs. 1.9 ± 0.56, p = 0.04). Response to treatment was confirmed on histology. Conclusion: Collagen PET could be used as a reliable non-invasive tool for monitoring response to neoadjuvant therapy in PDAC. Citation Format: Shadi A. Esfahani, Hua Ma, Shriya Krishna, Sergey Shuvaev, Arvin Hajmirzaian, Iris Zhou, Ciprian Catana, Mozhdeh Sojoodi, Eric Abston, David Ting, Pedram Heidari, Michael Lanuti, Kenneth Tanabe, Motaz Qadan, Carlos Fernandez de-Castillo, Aparna Parikh, Colin Weekes, Theodore Hong, Peter Caravan. Quantitative molecular imaging of pancreatic tumor fibrosis for evaluation of response to neoadjuvant therapy: Pre-clinical and first-in-human application of collagen PET/MRI [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 A036.

Abstract B082: Extinction of oncogenic Kras in genetic mouse models eradicates pancreatic cancer by inducing Fas-dependent apoptosis by CD8+ T cells

Abstract Oncogenic KrasG12D (Kras*) is critical for the initiation and maintenance of pancreatic ductal adenocarcinoma (PDAC), and a known repressor of tumor immunity. Conditional extinction of Kras* in genetic mouse models of PDAC leads to reactivation of Fas, CD8+ T cell mediated apoptosis, and complete eradication of tumors. Kras* extinction recruits CD4+ and CD8+ T cells, promotes the activation of putative antigen presenting CD11b−CD11c+ dendritic cells, and suppresses CD11b+ myeloid cell infiltration. Our findings shows that Kras* drives T cell suppression in PDAC. Mechanistically, Kras* mediated immune evasion involves epigenetic regulation of the death receptor Fas in cancer cells, via repression of its promoter region mediated by functional recruitment of DNA methyltransferase 1 (DNMT1), trimethylation of histone 3 at lysine 27 (H3K27me3), the histone methyltransferase EZH2, and suppression of acetylation of histone 3 at lysine 27 (H3K27ac). Further, analysis of human RNA sequencing reveals that Kras* expression levels inversely correlate with FAS expression and PDAC tumors with high KRAS demonstrate low CD8+ T cell infiltration, which is associated with shorter overall survival in PDAC patients. Given that specific and effective targeting of Kras* results in the CD8+ T cell mediated, Fas dependent apoptosis of cancer cells and eradication of PDAC in mice, this study highlights the direct role of Kras* in suppression of tumor immunity and supports a role for immunotherapy in combination with Kras* targeting strategies to control PDAC. Citation Format: Valerie S. LeBleu, Krishnan K. Mahadevan, Elena V. Ramirez, Yang Chen, Bingrui Li, Amari M. Sockwell, Mihai Gagea, Hikaru Sugimoto, Desiree Tampe, Michael Zeisberg, Haoqiang Ying, Abhinav K. Jain, Ronald A. DePinho, Aniban Maitra, Kathleen M. McAndrews, Raghu Kalluri. Extinction of oncogenic Kras in genetic mouse models eradicates pancreatic cancer by inducing Fas-dependent apoptosis by CD8+ T cells [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 B082.

Abstract B026: αvβ5 Integrin serves as a tumor-specific marker for immunosuppressive regulatory T cells in pancreatic cancer

Abstract Regulatory T cells (Tregs) contribute to the immunosuppressive nature of pancreatic ductal adenocarcinoma (PDAC). Systemic depletion of Tregs can enhance anti-tumor T cell immunity, but can also cause unwanted autoimmune complications. A tumor-specific Treg-targeted therapy that effectively and safely enhances anti-tumor immunity for PDAC treatment remains an unmet need. We have recently found that αvβ5 integrin is expressed on Tregs that reside in the PDAC, but not those in the spleen, suggesting that the integrin can serve as a targetable marker for tumor-resident Tregs. Indeed, long-term treatment with the iRGD tumor-penetrating peptide, which effectively targets αvβ5+ cells in PDAC tissue, depleted Tregs in a tumor-specific manner leading to a significant expansion of CD8+ T cells in transgenic and syngeneic PDAC mouse models. αvβ5+ Tregs were induced from both naïve CD4+ T cells and naturally occurring Tregs. T cell receptor stimulation was found to be the common inducer of αvβ5+ Tregs. αvβ5+ Tregs formed a subpopulation of CCR8+ Tregs, which are preferentially found in the tumor tissue of various cancer types. Our most recent data show that the αvβ5 integrin marks a highly immunosuppressive fraction of the CCR8+ Tregs, suggesting that targeting αvβ5+ Tregs can lead to effective depletion of immunosuppressive Tregs in a tumor-specific manner. Citation Format: Yuki Kunisada, Kodai Suzuki, Norio Miyamura, Shingo Eikawa, Tatiana Hurtado de Mendoza, Evangeline S. Mose, Caisheng Lu, Yukihito Kuroda, Erkki Ruoslahti, Andrew M. Lowy, Kazuki N. Sugahara. αvβ5 Integrin serves as a tumor-specific marker for immunosuppressive regulatory T cells 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 B026.

Abstract A037: Unraveling the importance of pancreatic cancer extracellular signaling to endothelial cells within the tumor microenvironment

Abstract In many solid tumors, including pancreatic ductal adenocarcinoma (PDAC), the secretion of extracellular vesicles (EVs) has been shown to support tumor progression, chemotherapeutic resistance, and metastasis. Importantly, PDAC tumors are composed of up to 80% non-tumor cells and an extracellular matrix, yet no studies have been performed to determine whether PDAC EVs are preferentially imported by specific cells within the tumor microenvironment. Further, the RNA-binding protein HuR plays an important role in PDAC cells, supporting the stability and translation of transcripts that aid tumor cell stress responses. Our lab has recently found that EVs contain distinct mRNA and phosphorylated protein cargoes from donor cells. Further, HuR knockout (KO) cells produce EVs with significantly different mRNAs and phosphorylated proteins that EVs isolated from HuR wildtype (WT) cells. Considering the functional impact of EV cargoes on recipient cells, our study aims to identify the specific cell types in the tumor microenvironment that import PDAC EVs and investigate the role of PDAC cell HuR in this signaling process. We have generated human PDAC and mouse KrasG12D Trp53R172H driven (KPC) PDAC cell lines to express an EV reporter labeling the inner leaflet of cell membranes with GFP-nLuc. We utilized these cell lines in an immunocompetent mouse model of PDAC to determine which stromal cells import PDAC EVs in live mouse tumors. We found that endothelial cells have a significantly higher EV import than other stromal cells, and are now utilizing cyclic-immunofluorescence to confirm these findings and assess whether canonical endothelial cell tube formation proteins are increased with EV import. We will assess the functional importance of this signaling via single-cell RNA-sequencing of developed KPC EV reporter tumors. Future studies will interrogate the importance of HuR leveraging the EV reporter in KPC HuR WT vs. KO cells and through 3D endothelial cell tube formation assays treated with HuR WT vs. KO EVs. Collectively, our study sheds light on the preferential uptake of PDAC EVs by endothelial cells and highlights the potential role of tumor cell-intrinsic HuR in this signaling process. These findings expand our understanding of the interactions between PDAC and the tumor microenvironment, potentially paving the way for targeted therapeutic interventions in this devastating disease. Citation Format: Jennifer M. Finan, Yifei Guo, Kevin MacPherson, Valerie Calvert, Carl Pelz, Emanuel F. Petricoin, Julie A. Saugstad, Rosalie Sears, Jonathan R. Brody. Unraveling the importance of pancreatic cancer extracellular signaling to endothelial cells within the tumor microenvironment [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 A037.

Abstract A018: Exercise stimulates anti-tumoral immunity in metastatic PDAC

Abstract The majority of pancreatic ductal adenocarcinoma (PDAC) patients are metastatic at presentation with limited treatment options and a poor overall 5-year survival of 3%. The liver is the predominant site of distant metastasis in PDAC. Our group has previously shown that aerobic exercise can inhibit tumorigenesis in a primary PDAC mouse model by promoting anti-tumor immunity, however, the effects of exercise in the metastatic setting have not been explored. As such, we developed a model of PDAC liver metastasis by isolating primary pancreatic and liver metastatic cell lines from 18–20-week-old LSL-KRasG12D/+; LSL-Trp53R172H/+; p48Cre; Rosa26LSL-tdTomato/+ (KPCTpancreas or KPCTliver, respectively) mice. KPCTpancreas cells were surgically implanted into the pancreata of syngeneic wild-type mice. One week after pancreatic implantation, KPCTliver cells were implanted into the liver either by direct injection into the parenchyma or via the portal vein. The portal vein method enables us to account for the extravasation step of the metastatic cascade, while the direct parenchymal injection offers a less invasive surgery. Both models preserve the spleen, which is the largest secondary lymphoid organ and essential to the immune response. Treadmill running was initiated in the aerobic exercise cohort 5 days per week for 2 weeks or mice were allowed to rest. Endpoint analysis revealed that aerobic exercise treatment decreased tumor burden in both primary tumors and liver metastasis as measured by tumor weights and tdTomato fluorescence imaging when compared to the rested controls. Immune profiling of the liver metastases revealed elevated IL-15Rɑ+ CD8+ T cells as well as CD19+ B cells in the exercise cohort. While identification of IL-15Rɑ+ CD8+ T cells is consistent with the immune changes we have previously reported in the primary pancreatic tumor of exercised mice, the latter changes reveal organ specific immune cell recruitment not observed in the primary pancreatic tumor of exercised mice. The combined immune profiling and phenotypic changes suggest that aerobic exercise reduces PDAC metastatic growth by modulating systemic and intra-tumoral immunity. The induction of an anti-tumorigenic immune response by aerobic exercise could open avenues for potential pharmacological intervention in downstream pathways to aid in the treatment of metastatic PDAC. Citation Format: Mansour E. Riachi, Carolina G. Alcantara Hirsch, Erica Ma, Beny Shapiro, Ammar Javed, Christopher L. Wolfgang, Dafna Bar-Sagi. Exercise stimulates anti-tumoral immunity in metastatic PDAC [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 A018.

Abstract C101: Calcium signaling rewiring activates epithelial-to-mesenchymal transition

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States and is often not diagnosed until it has already metastasized. Calcium and downstream signaling cascades contribute to epithelial-to-mesenchymal transition (EMT) and facilitate metastasis formation in PDAC. Ca2+/calmodulin-dependent kinase-II (CaMK2), a downstream effector kinase activated by Ca2+, has previously been shown in vitro to be an important mediator of a partial/hybrid EMT program that gives tumor cells high metastatic proclivity. We hypothesize that CaMK2 is a critical node in the partial EMT state and thus deleted Camk2b in vivo to study the effect of its loss on tumor progression and metastasis. Methods: We generated a genetically engineered mouse model where Camk2b was knocked out in the KPCY autochthonous PDAC mouse model (herein KPCY-Camk2bcKO). Isolated cell lines were treated with the EMT-inducing ligand TGF-b to study the effect of Camk2b on tumor cell plasticity. Results: Prior in vitro work indicated that CAMK2 is required for hybrid EMT and thus we expected KPCY-Camk2bcKO mice to have fewer metastases and live longer. Surprisingly, the genetic deletion of Camk2b accelerated tumor formation and reduced overall survival relative to KPCY controls. Despite dying sooner, there was no difference in overall tumor burden in KPCY-Camk2bcKO mice. Pathological and immunofluorescence analysis of KPCY-Camk2bcKO tumors showed an increased number of poorly differentiated regions and EMT as indicated by loss of the epithelial cell marker ECADHERIN (E-CAD) in YFP lineage-labeled tumor cells. In stark contrast to prior work, the loss of Camk2b increased tumor cell plasticity, potentially highlighting a rewiring of calcium signaling circuits in the Camk2b-null state. To this end, KPCY-Camk2bcKO tumor cell lines displayed a baseline mesenchymal morphology in vitro and had lower expression of Cdh1 (the gene encoding E-CAD) mRNA. Using a flow cytometry-based approach, we also found baseline E-CAD protein levels to be diminished relative to KPCY lines. Additionally, KPCY-Camk2bcKO tumor cells were more responsive to the EMT-inducing ligand TGF-b and downregulated their E-CAD levels more than KPCY cells. Notably, treatment with low dose TGF-b also dramatically reduced E-CAD expression in KPCY-Camk2bcKO but not KPCY cells, indicating that the knockout cells are easily pushed towards a mesenchymal state. Conclusions: While CAMK2 has been studied previously as a facilitator of the EMT program, this work paradoxically demonstrates that Camk2b loss enhances cellular plasticity within PDAC. In this context, we speculate that loss of Camk2b rewires calcium signaling to drive EMT. In future studies, we will investigate the effect of Camk2b loss on the metastatic cascade and uncover mechanisms through which Camk2b loss makes PDAC tumor cells more plastic. We will also begin to characterize the non-cell autonomous roles of CAMK2B in crosstalk with the tumor microenvironment. Citation Format: Jessica Peura, Yamini Ogoti, Jason Pitarresi. Calcium signaling rewiring activates epithelial-to-mesenchymal transition [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 C101.

Abstract A087: Loss of KDM5A supports KRAS-driven pancreatic cancer

Abstract Objective: Our objective is to investigate the potential link between the Kras and hypoxia in pancreatic ductal adenocarcinoma (PDAC). PDAC is notorious for its resistance to chemotherapy, radiotherapy, and immunotherapy, owing to its dense stroma and high intratumoral hypoxia. Here, we explore how loss of Kdm5a reprograms the epigenome and contributes to Kras-driven pathology in PDAC. We believe that loss of Kdm5a plays an essential role in enabling cancer cells to survive under hypoxic conditions. Methods: We used cell lines (iKPCs) derived from a genetically engineered mouse model of PDAC with doxycycline-inducible KrasG12D. To induce KrasG12D expression, cells were cultured in media containing 1 mcg/ml doxycycline (Kras-on). To turn off KrasG12D expression, cells were cultured in tetracycline-free media (Kras-off). Pharmaceutical inhibition of KrasG12D, MEK, and ERK was performed by treating iKPCs with MRTX-1133, mirdametinib, and ravoxertinib, respectively. MG-132 was used to inhibit the proteasome pathway. Kdm5a was overexpressed in iKPCs by lentiviral infection. Profiling of histone modifications was performed by epiproteomic mass spectrometry of iKPCs under three conditions: Kras-off, Kras-on, and Kras-on overexpressing Kdm5a. Results: The induction of oncogenic Kras led to a decrease in Kdm5a protein levels, which was restored by reduced oncogenic Kras expression induced by doxycycline withdrawal or Kras-specific inhibition with MRTX-1133. Proteasomal inhibition using MG-132 also stabilized Kdm5a. This suggests that phosphorylation of Kdm5a by Erk may lead to activation of a phosphodegron, ubiquitination, and subsequent proteasomal degradation. Immunoprecipitation of Kdm5a confirmed elevated phosphorylated-Ser/Thr and ubiquitin in the Kras-on condition. Although Kdm5a is a H3K4me3 demethylase, we observed no differences in the total abundance of H3K4me3 in the conditions we profiled. Conversely, acetylation of several histone marks, including H3K14ac and H4K16ac, were elevated in Kras-off compared to Kras-on iKPCs. The overexpression of Kdm5a in Kras-on iKPCs was found to rescue the reduction in histone acetylation. Conclusions: We present compelling evidence that oncogenic Kras induces the proteasomal degradation of Kdm5a in PDAC. Restoring Kdm5a in PDAC mitigates some epigenetic alterations induced by Kras. Our research unearths a hitherto unexplored pathway through which Kras can reprogram the epigenome. Although mass spectrometry studies of Kdm5a have previously found that it could interact with subunits of histone acetyltransferase (HAT) complexes, including Tip60 and Mrg15, direct evidence that Kdm5a facilitates histone acetylation has not been described. Thus, our findings could broaden understanding for predicting and identifying potential escape mechanisms that could arise upon Kras inhibition before they manifest in patients, providing a more proactive approach to managing PDAC. Citation Format: Jasper Chen, Jincheng Han, Ronald A. DePinho, Cullen M. Taniguchi. Loss of KDM5A supports KRAS-driven 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 A087.

Abstract C003: RAMP 205: A phase 1b/2a study of gemcitabine, nab-paclitaxel, avutometinib, and defactinib in untreated metastatic pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) accounts for ~90% of pancreatic cancer. Surgery is the only curative treatment. Unfortunately, most patients are diagnosed with advanced disease and are not candidates for resection. Combination chemotherapy approaches have provided incremental improvements to PDAC outcomes. However, impact on survival for patients with inoperable disease remains modest, with a 2-year survival rate of less than 7%. Activating mutations in the KRAS oncogene are a key initiating event in ~80-90% of PDAC. Permanent activation of KRAS drives tumor cell proliferation and survival, tumor microenvironment modulation, and metabolic alterations in PDAC. Patients with KRAS mutant PDAC have worse outcomes compared to those with KRAS wild-type PDAC. In addition, hyperactivation of focal adhesion kinase (FAK) has been correlated with high stromal density, resulting in therapeutic resistance and poor survival. Avutometinib is a first-in-class oral RAF/MEK clamp that potently inhibits MEK kinase activity while also blocking the compensatory reactivation of MEK by upstream RAF. Defactinib is a selective inhibitor of focal adhesion kinase (FAK), which has been shown to mediate resistance to multiple anticancer agents. Preclinical data have shown that the combination of avutometinib + FAK inhibition (FAKi) + chemotherapy (gemcitabine + paclitaxel as a surrogate for nab-paclitaxel) induced tumor regression in a autochthonous KRAS/TP53 mutant PDAC mouse model. Prior clinical experience of defactinib + pembrolizumab + chemotherapy was safe, showed responses, decreased stromal density, and increased CD8+ T cell infiltration in patients with PDAC (NCT02546531). Methods: RAMP 205 is a multi-center, open-label, single arm Phase 1b/2a study of gemcitabine and nab-paclitaxel in combination with avutometinib and defactinib in patients with previously untreated metastatic PDAC (NCT05669482). The study is being conducted in 2 parts: Parts A (dose evaluation) and Part B (dose expansion). The starting dose is 2.4 mg avutometinib orally (PO) twice weekly + 200 mg defactinib PO twice per day, in combination with standard once weekly gemcitabine/nab-paclitaxel (QW IV). All study drugs are dosed 3 weeks on, 1 week off. Following determination of the Recommended Phase 2 Dose (RP2D) in Part A, Part B will evaluate efficacy of the RP2D via objective response rate (primary endpoint). Mandatory tumor biopsies and blood samples will be collected for correlative studies, including circulating tumor DNA, molecular profiling and pharmacodynamic markers. Eligible patients must have histologic or cytologic evidence of metastatic PDAC and measurable disease according to RECIST v1.1. Patients with prior or concomitant treatment for metastatic PDAC or prior treatment with RAS/MAPK or FAK inhibitors are not eligible. The study will enroll ~35 patients with 6-12 patients in Part A, and 23 patients in Part B. Citation Format: Kian-Haut Lim, Manuel Hidalgo, Mark H. O'Hara, Kristen R. Spencer, Ignacio Garrido-Laguna, David G. DeNardo, Vijeta Bhambhani, Gloria Patrick, Yaofeng Cheng, Silvia Coma, Jonathan A. Pachter, Louis J. Denis. RAMP 205: A phase 1b/2a study of gemcitabine, nab-paclitaxel, avutometinib, and defactinib in untreated metastatic pancreatic ductal adenocarcinoma [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 C003.

Abstract C044: Macropinocytic stress reprograms cancer-associated fibroblasts and enhances therapeutic responses in pancreatic cancer

Abstract Macropinocytosis is a fundamental metabolic pathway that has been demonstrated to support the survival and function of tumor cells and cancer-associated fibroblasts (CAFs) in pancreatic ductal adenocarcinoma (PDAC) through in vitro studies and xenograft mouse models. However, the impact of macropinocytosis on the PDAC tumor microenvironment in an immune-competent setting remains poorly understood. In this study, we utilized the selective inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) to block macropinocytosis in both tumor cells and CAFs in syngeneic PDAC mouse models. We observed that EIPA treatment inhibited PDAC tumor growth, which was accompanied by reduced tumor proliferation and increased cell apoptosis. Moreover, EIPA treatment decreased the presence of myofibroblastic CAFs and collagen deposition within PDAC tumors, while also promoting the expansion of tumor vasculature. By modifying the tumor stroma, EIPA priming enhanced drug delivery and improved the effectiveness of PDAC chemotherapy. Interestingly, immune profiling of PDAC tumors revealed that EIPA administration specifically increased the infiltration of CD8+ and CD4+ T cells. We discovered that EIPA induced changes in the cytokine/chemokine landscape within tumors by triggering an inflammatory CAF (iCAF)-like phenotype. Mechanistically, we discerned that inhibition of macropinocytosis triggers metabolic stress that in turn activates stress-responsive transcription factors and promotes the expression of early-phase proinflammatory cytokines and chemokines in mouse CAFs. Notably, combination treatment with EIPA and immune checkpoint blockade (ICB) reduced PDAC tumor burden and significantly inhibited tumor metastasis. Overall, our findings establish that macropinocytosis functions to support the barrier function of the stroma by maintaining fibroblastic identity and provide further evidence that inhibition of macropinocytosis represents a promising therapeutic modality in PDAC. Citation Format: Yijuan Zhang, Swetha Maganti, Jennifer Hope, Li Ling, Cheska Marie Galapate, Florent Carrette, Linda Bradley, Cosimo Commisso. Macropinocytic stress reprograms cancer-associated fibroblasts and enhances therapeutic responses 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 C044.

Abstract A089: Quantitative MRI metrics and single cell transcriptome reveal effects of stroma-directed drug and chemotherapy in a GEM model of pancreatic cancer

Abstract Hypothesis: The dense stroma present in pancreatic ductal adenocarcinoma (PDA) harbors a unique tumor microenvironment (TME) that is immune suppressive and underlies the chemo-resistance of PDA. We have evaluated synthetic vitamin D combined with chemoimmunotherapy in a pilot clinical study (NCT03519308). To elucidate the underlying mechanisms of this treatment, we designed a matched study in a genetically engineered mouse model of PDA (KPC mice) to test the hypothesis that synthetic vitamin D enhances the effect of chemotherapy with or without immune checkpoint inhibition. To detect changes in TME, we have integrated diffusion weighted (DW) and dynamic contrast enhanced (DCE) MRI with immunohistochemistry (IHC) and single cell RNA sequencing (scRNAseq). Methods: KPC mice were randomly assigned to one of four groups: 1) Control (untreated); 2) Nab-paclitaxel + gemcitabine + cisplatin (Chemo); 3) Chemo + synthetic vitamin D (calcipotriol, Cal); 4) Chemo + Cal + PD-L1. The treatment lasted for 14 days followed by euthanasia and tumor collection for IHC and scRNAseq. Respiratory motion-robust DW- and DCE-MRI were performed on treatment days 0, 7 and 14. Results: Compared to consistent tumor growth (measured by MRI) in the Control group, both progression and regression were observed in groups 2) - 4), suggesting that the KPC model captured the varied treatment responses observed in the clinic. Capillary perfusion/permeability of the tumor measured by Ktrans (a DCE-MRI metric) was reduced at day 7 in the Chemo group (P<0.05), whereas Chemo+Cal reversed the reduction of Ktrans. Chemo-induced Ktrans reduction was corroborated with a significant reduction of microvascular density by IHC of CD31 compared to the Control. Addition of PD-L1 did not appear to enhance the tumor growth delay by Chemo+Cal. scRNAseq analysis revealed that while Chemo significantly delayed the tumor growth compared to the Control, it increased the portion of mesenchymal cells in the tumor and upregulated epithelial-to-mesenchymal transition (EMT) genes known to resist chemotherapy. In contrast, Chemo+Cal increased the fraction of epithelial cells and downregulated EMT genes compared to Chemo. Trasncriptome analysis of the tumor cells demonstrated that Chemo treatment increased the expression of Fibronectin 1 (FN1), a gene associated with tumor invasiveness, EMT, and metastasis, while lowering the expression of Trefoil Factor Family1 (Tff1), which is related to tumor-suppression and chemosensitivity. Chemo+Cal treatment reversed the expression of both FN1 and Tff1 gene compared to Chemo treatment. Conclusion: Single cell transcriptome analysis provided valuable insights into treatment-induced molecular subtype changes, which may predict future resistance and invasiveness. Our study revealed important mechanisms of calcipotriol that enhance the efficacy of chemotherapy by improving microvascular perfusion and permeability and by reducing chemo-resistance by enriching epithelial cells in PDA tumors. Citation Format: Hoon Choi, Mamta Gupta, Thomas Karasic, Miguel Joaquim, Emma Furth, Stephen Pickup, Cynthia Clendenin, Hee Kwon Song, Yong Fan, Jeffrey Duda, James Gee, Mark Rosen, Peter O’Dwyer, Rong Zhou. Quantitative MRI metrics and single cell transcriptome reveal effects of stroma-directed drug and chemotherapy in a GEM model of 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 A089.

NDRG1 overcomes resistance to immunotherapy of pancreatic ductal adenocarcinoma through inhibiting ATG9A-dependent degradation of MHC-1

AimsPancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is resistant to immune checkpoint blockade (ICB) therapies. Emerging evidence suggests that NDRG1 may be an important target for the development of new therapies for PDAC. Herein, we investigated the novel roles of NDRG1 and Combretastatin A-4 (CA-4) in the treatment of PDAC ICB resistance. MethodsEnrichment of MHC class I was detected by RNA sequence and verified by RT-qPCR and immunoblotting in NDRG1-knockdown human pancreatic cancer cell lines. The protein degradation mode was found by stimulation with various inhibitors, and the autophagy degradation pathway was found by immunoprecipitation and immunolocalization. The roles of NDRG1 and MHC-I in immunotherapy were investigated by orthotopic solid tumors, histology, immunohistochemistry, multiplex immunofluorescence staining and flow cytometry. ResultsHere, we identified a previously undescribed role of NDRG1 in activating major histocompatibility complex class 1 (MHC-1) expression in pancreatic ductal adenocarcinoma (PDAC) cells through lysosomal-autophagy-dependent degradation. In mouse models of PDAC, either tumor cell overexpression or pharmacologic activation of NDRG1 leads to MHC-1 upregulation in tumor cells, which in turn promotes the infiltration and activity of CD8 + T cells, enhances anti-tumor immunity, and overcomes resistance to ICB therapy. Moreover, combination therapy of CA-4 and ICB overcomes the drug resistance of pancreatic cancer to ICB therapy. In PDAC patients, NDRG1 expression correlates with high MHC-1 expression and better survival. ConclusionOur results reveal NDRG1 in PDAC cancer cells as a tumor suppressor and suggest that pharmaceutically targeting NDRG1 is a promising way to overcome pancreatic cancer resistance to immunotherapy and provides a potential therapeutic strategy for the treatment of pancreatic cancer patients.

Collagen type I PET/MRI enables evaluation of treatment response in pancreatic cancer in pre-clinical and first-in-human translational studies.

Pancreatic ductal adenocarcinoma (PDAC) is an invasive and rapidly progressive malignancy. A major challenge in patient management is the lack of a reliable imaging tool to monitor tumor response to treatment. Tumor-associated fibrosis characterized by high type I collagen is a hallmark of PDAC, and fibrosis further increases in response to neoadjuvant chemoradiotherapy (CRT). We hypothesized that molecular positron emission tomography (PET) using a type I collagen-specific imaging probe, 68Ga-CBP8 can detect and measure changes in tumor fibrosis in response to standard treatment in mouse models and patients with PDAC. Methods: We evaluated the specificity of 68Ga-CBP8 PET to tumor collagen and its ability to differentiate responders from non-responders based on the dynamic changes of fibrosis in nude mouse models of human PDAC including FOLFIRNOX-sensitive (PANC-1 and PDAC6) and FOLFIRINOX-resistant (SU.86.86). Next, we demonstrated the specificity and sensitivity of 68Ga-CBP8 to the deposited collagen in resected human PDAC and pancreas tissues. Eight male participant (49-65 y) with newly diagnosed PDAC underwent dynamic 68Ga-CBP8 PET/MRI, and five underwent follow up 68Ga-CBP8 PET/MRI after completing standard CRT. PET parameters were correlated with tumor collagen content and markers of response on histology. Results: 68Ga-CBP8 showed specific binding to PDAC compared to non-binding 68Ga-CNBP probe in two mouse models of PDAC using PET imaging and to resected human PDAC using autoradiography (P < 0.05 for all comparisons). 68Ga-CBP8 PET showed 2-fold higher tumor signal in mouse models following FOLFIRINOX treatment in PANC-1 and PDAC6 models (P < 0.01), but no significant increase after treatment in FOLFIRINOX resistant SU.86.86 model. 68Ga-CBP8 binding to resected human PDAC was significantly higher (P < 0.0001) in treated versus untreated tissue. PET/MRI of PDAC patients prior to CRT showed significantly higher 68Ga-CBP8 uptake in tumor compared to pancreas (SUVmean: 2.35 ± 0.36 vs. 1.99 ± 0.25, P = 0.036, n = 8). PET tumor values significantly increased following CRT compared to untreated tumors (SUVmean: 2.83 ± 0.30 vs. 2.25 ± 0.41, P = 0.01, n = 5). Collagen deposition significantly increased in response to CRT (59 ± 9% vs. 30 ± 9%, P=0.0005 in treated vs. untreated tumors). Tumor and pancreas collagen content showed a positive direct correlation with SUVmean (R2 = 0.54, P = 0.0007). Conclusions: This study demonstrates the specificity of 68Ga-CBP8 PET to tumor type I collagen and its ability to differentiate responders from non-responders based on the dynamic changes of fibrosis in PDAC. The results highlight the potential use of collagen PET as a non-invasive tool for monitoring response to treatment in patients with PDAC.

EGFR-activated myofibroblasts promote metastasis of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. Cancer-associated fibroblasts (CAFs) are recognized potential therapeutic targets, but poor understanding of these heterogeneous cell populations has limited the development of effective treatment strategies. We previously identified transforming growth factor beta (TGF-β) as a main driver of myofibroblastic CAFs (myCAFs). Here, we show that epidermal growth factor receptor/Erb-B2 receptor (EGFR/ERBB2) signaling is induced by TGF-β in myCAFs through an autocrine process mediated by amphiregulin. Inhibition of this EGFR/ERBB2-signaling network in PDAC organoid-derived cultures and mouse models differentially impacts distinct CAF subtypes, providing insights into mechanisms underpinning their heterogeneity. Remarkably, EGFR-activated myCAFs promote PDAC metastasis in mice, unmasking functional significance in myCAF heterogeneity. Finally, analyses of other cancer datasets suggest that these processes might operate in other malignancies. These data provide functional relevance to myCAF heterogeneity and identify a candidate target for preventing tumor invasion in PDAC.

Down-regulation of microRNA-23a promotes pancreatic ductal adenocarcinoma initiation and progression by up-regulation of FOXM1 expression

Transcriptional factor Forkhead box M1 (FOXM1) plays an important role in pancreatic ductal adenocarcinoma (PDAC) development and progression. The molecular mechanisms underlying its dysregulation remain unclear. We identified and functionally validated the microRNAs (miRNAs) that critically regulate FOXM1 expression in PDAC. The expression levels of miRNA-23a (miR-23a-3p and -5p) were altered in PDAC cell lines and their effects on FOXM1 signaling and cell proliferation and migration and tumorigenesis were examined in vitro and in vivo using mouse PDAC models. Compared with non-tumor pancreatic tissues, PDAC tissues and cell lines exhibited significantly reduced levels of miR-23a expression. Reduced miR-23a expression and concomitant increase in FOXM1 expression were also observed in acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasia, the major premalignant lesions of PDAC. Transgenic expression of miR-23a reduced the expression of FOXM1 and suppressed cell proliferation and migration in PDAC cells, whereas the inhibitors of miR-23a did the opposite. Loss or reduced levels of miR-23a increased the levels of FOXM1 expression, while increased expression of FOXM1 down-regulated miR-23a expression, suggesting that miR-23a and FOXM1 were mutual negative regulators of their expression in PDAC cells. Therefore, the miR-23a/FOXM1 signaling axis is important in PDAC initiation and progression and could serve as an interventional or therapeutic target for patients with early or late stages of PDAC.