Response Of Pancreatic Ductal Adenocarcinoma Research Articles

Abstract C039: Investigating ER stress responses in pancreatic ductal adenocarcinoma under lipid imbalance

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death, with a five-year survival rate of 12%. Dense desmoplasia results in poor intratumoral perfusion and elevated interstitial fluid pressure, creating regions with severe oxygen and nutrient deprivation. One consequence is a deficiency in unsaturated fatty acids (uFAs), as the de novo synthesis of uFAs requires oxygen. Our results indicate that PDAC cells experience endoplasmic reticulum (ER) stress and activate subsequent ER stress responses under uFA- limited conditions in vitro. ER stress responses are governed by three major stress sensors: inositol-requiring enzyme 1α (IRE1α), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). When IRE1α is activated, XBP1 mRNA is spliced, resulting in an activated transcription factor, XBP1s, which modulates lipid metabolism. XBP1s is upregulated in PDAC compared to PanIN and normal tissue in murine and human samples. PDAC cells have reduced viability under uFA-limited conditions. Moreover, ER stress is activated under uFA deficiency, and the stress response is diminished upon rescue by monounsaturated oleic acids. To study the role of ER stress responses, we genetically depleted PERK, ATF6, or XBP1 in PDAC cells. The depletion of these pathways rescued PDAC cell viability. Interestingly, B-I09, an inhibitor of XBP1 splicing, led to a mild tumor reduction in vivo. We have found that "basal-like" patient tumors are significantly enriched in the ER stress response pathways based on bulk patient RNA-seq datasets. Therefore, we hypothesize that the two PDAC subtypes “basal” and “classical” might have different responses or reliance on the ER stress pathways. We have created isogenic subtype models to represent basal and classical subtypes by modulating epithelial to mesenchymal (EMT) factors and deltaNP63. Signaling studies suggest that there are no differences between the subtypes’ response to uFA-limited conditions. We are currently assessing the role of XBP1s and IRE1α in vivo by implanting the gene-edited cells into subcutaneous and orthotopic tumor models. Furthermore, we are pursuing spatial mass spectrometry to study the lipid environment in PDAC tumors and investigate whether lipid imbalance is the main cause of ER stress activation in the tumor microenvironment (TME). Overall, our studies show that ER stress response is upregulated in PDAC tumors, and stress response promotes cell death under in vitro TME-like conditions. Subtype features such as EMT and deltaNP63 do not alter PDAC cells’ ER stress responses, and had minimal impact on their stress response reliance. Citation Format: Yanqing (Christine) Jiang, Xu Han, John Tobias, Carson Poltorack, Mai Wang, Pamela Burgess-Jones, Nathan Coffey, Brian Keith, Celeste Simon. Investigating ER stress responses in pancreatic ductal adenocarcinoma under lipid imbalance [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 C039.

Abstract IA-08: Syndecan 1 is a therapeutic target for KRAS-driven pancreatic cancer

Abstract KRAS inhibitors targeting G12C mutation have been approved by FDA for the treatment of non-small cell lung cancers and have shown promising clinical responses in pancreatic ductal adenocarcinoma (PDAC). RAS inhibitors targeting additional KRAS mutations or multiple RAS isoforms are under clinical development. Although targeting KRAS exhibits anti-tumor effect in PDAC, majority of tumors inevitably develop resistant disease, as is the case for most targeted therapies. In this new era of targeting KRAS driven cancer, understanding mechanisms driving this resistance to RAS targeted therapies and further devising combination strategies to overcome it have become a necessary, though challenging, task to achieve long term disease control. To tackle this problem, we employed various in vitro and in vivo preclinical model systems to explore the development of resistance to both genetic and pharmacological KRAS blockade. Our previous work identified the membrane proteoglycan Syndecan 1 (SDC1) as a KRAS downstream surrogate whose cell surface localization is tightly controlled by oncogenic KRAS signaling and is critical for KRAS-driven tumor development. Here, our analysis of KRAS driven genetically engineered PDAC mouse model and human pancreatic or colorectal cancer (CRC) cells with KRASG12C mutation discovered that, while cell surface SDC1 is reduced upon acute genetic or pharmacological inhibition of oncogenic KRAS, plasma membrane SDC1 is recovered in tumor cells that become resistant to KRAS inhibition (KRAS-bypass cells). Surface localization of SDC1 leads to the activation of multiple receptor tyrosine kinases (RTKs) and macropinocytosis, resulting in the resistance to KRAS blockade. We further identified that the recovery of plasma membrane SDC1 in KRAS-bypass cells is driven by YAP1 oncogene, which induces the downregulation of ARF6 GAPs, leading to the activation of ARF6, a small GTPase required for SDC1 membrane recycling. To therapeutically targeting SDC1, we have successfully developed a monoclonal antibody (22B mAb), which exhibits remarkable sensitivity and specificity for human SDC1. Our results indicate that 22B mAb exerts substantial inhibition of PDAC cell growth in vitro and desirable tumor inhibitory effects in vivo in murine PDAC tumors and human PDAC cell-derived xenograft tumors. Importantly, our 22B mAb synergizes with KRAS inhibitors, chemotherapy, or immunotherapy, resulting in significantly enhanced therapeutic effects. Taken together, our study identified SDC1 as a functional driver for the bypass of KRAS-dependence and therapeutically targeting SDC1 with a novel mAb will overcome the acquired resistance to KRAS targeted therapy. As such, our research yields important new insights in the fields of cancer biology regarding mechanisms of resistance employed by human cancers, and identifies new actionable therapeutic targets and approaches to overcome resistance to KRAS inhibition. Citation Format: Mitsunobu Takeda, Zecheng Yang, Madeline S Theardy, Alexey Sorokin, Oluwadara Coker, Shuaitong Chen, Long T Vien, Laura Bover, Haoqiang Ying, Scott Kopetz, Wantong Yao. Syndecan 1 is a therapeutic target for KRAS-driven 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 IA-08.

Abstract B059: Modulating the immunosuppressive pancreas tumor microenvironment through intratumoral delivery of cytokine-encoding mRNAs

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive cancer, with a mere 13% average 5-year survival rate. The hallmark desmoplastic stromal response in PDAC leads to poor vascularization, drug delivery challenges, and impaired cytotoxic immune cell infiltration, contributing to de novo therapy resistance. Our previous research has shown therapeutic induction of cellular senescence with RAS pathway targeting therapies can induce cytokine and chemokine production through the senescence-associated secretory phenotype (SASP) that can effectively activate anti-tumor T cell immunity and responses to anti-PD-1 immune checkpoint blockade. To build on these findings as well as overcome limitations associated with senescence-inducing therapy toxicity and induction of pro-tumorigenic SASP cytokines, here we leveraged mRNA technology to deliver specific SASP cytokines and chemokines we have found to stimulate immune responses directly into the suppressive PDAC TME as an immune oncology platform. We created an in vitro transcription pipeline for mRNA production and multiplexing, and demonstrate we can intratumorally deliver multiple mRNAs simultaneously into the TME of transplanted and autochthonous PDAC mouse models, stimulating the local production of cytokines normally absent in PDAC. We further characterized a novel combination of five cytokines and chemokines that can effectively recruit and activate both innate and adaptive immune responses against PDAC. Through repeated bi-weekly dosing, this mRNA combination also promotes tumor destruction and improves survival outcomes. Considering the recent success of off-the-shelf neoantigen mRNA vaccines in early human PDAC patient trials, we have now developed mRNAs that encode PDAC-specific antigens. By integrating our cytokine mRNA platform with antigen mRNAs, we observe a significant increase in intratumoral antigen-presenting dendritic cell populations and a substantially enhanced T-cell mediated anti- tumor immune response in orthotopic transplant models of PDAC. Overall, this study not only unveils pivotal insights into the cytokines absent in PDAC that are crucial for promoting anti- tumor immunity, but also pioneers an innovative immunotherapy approach. This platform has the potential to be integrated with existing immunotherapy modalities, addressing the longstanding challenge of therapy resistance in PDAC. Citation Format: Chaitanya Naimesh Parikh, Kelly De Marco, Katherine Murphy, Loretah Chibaya, Lin Zhou, Youwei Qiao, Griffin Kane, Li Li, Wen Xue, Marcus Ruscetti. Modulating the immunosuppressive pancreas tumor microenvironment through intratumoral delivery of cytokine-encoding mRNAs [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 B059.

Abstract B071: Antigen-presenting cancer-associated fibroblasts are required for ICI-sensitivity in pancreatic ductal adenocarcinoma

Abstract Cancer-associated fibroblasts (CAFs) are a hallmark of many cancers and are particularly prevalent in pancreatic ductal adenocarcinoma (PDAC). This highly fibrotic environment is thought to play a role in resistance to therapy; however, attempts to target CAFs have been unsuccessful in clinical trials. Deeper insights into CAF biology have revealed that these cells are heterogeneous in origin, gene expression, and function. RNA sequencing studies have identified a subset of CAFs, antigen-presenting CAFs (apCAFs), which are defined by MHC-II and interact with CD4 T cells. Given the importance of CD4 T cells in the antitumor response in PDAC, understanding apCAF biology could demonstrate ways to promote their differentiation and enhance T cell activity.Methods: Two clones differing in sensitivity to ICIs, sKPC (sensitive) and rKPC (resistant), derived from the autochthonous KPC murine PDAC model, were implanted orthotopically into the pancreas of syngeneic C57BL/6 mice. Mice were untreated, treated with dual checkpoint inhibition, or immunized with a KRAS peptide vaccine. Tumors were harvested for TME characterization, functional assays, single-cell RNA sequencing (scRNAseq), and imaging mass cytometry (IMC). apCAFs, CD11c+, and CD4+ T cells were isolated for functional assays. Peptide-pulsed apCAFs and CD11c+ cells were cultured with naïve OT-II CD4+ T cells or sKPC tumor infiltrating CD4+ T cells. apCAF KO mice were generated by crossing PDGFRαCre and MHC-IIflx mice.Results: Immunosensitive sKPC tumors are characterized by higher immune cell and apCAF infiltration than rKPC tumors. These apCAFs activate tumor-infiltrating CD4 T cells and induce regulatory T cell (Treg) differentiation, but not production of immunosuppressive cytokines. IMC analysis shows greater proximity of apCAFs to CD4 T cells in sKPC tumors compared with rKPC tumors. Remarkably, apCAF KO mice implanted with sKPC tumors lose sensitivity to ICI. scRNAseq has uncovered a unique transcriptome of apCAFs depending on tumor of origin and time following tumor implantation, revealing potential targets by which to quantitatively and qualitatively modulate apCAFs and enhance the antitumor response. Furthermore, scRNAseq of CD4 T cells cocultured with apCAFs has shown a unique pattern of CD4 T cell gene upregulation depending on the TME from which apCAFs were isolated, which could yield strategies to modulate not only apCAFs but also CD4 T cell activity.Conclusions: apCAFs are more abundant and more proximal to CD4 T cells in immunosensitive tumors, and apCAF KO sKPC tumor-bearing mice lose sensitivity to ICI. Together these data implicate apCAFs as essential mediators of the antitumor immune response. The unique transcriptome of apCAFs and cocultured CD4 T cells depending on tumor of origin suggests multiple strategies to modulate their interactions. Although a small proportion of the TME, modulating apCAF number or function could provide a solution to the currently unmet need of new and more effective treatments for PDAC patients. Citation Format: Saumya Maru, Meredith Wetzel, Jacob T Mitchell, Nicole Gross, Lalitya Andaloori, Kathryn Howe, Emma Kartalia, Guanglan Mo, James Leatherman, Won Jin Ho, Luciane Kagohara, Elana Fertig, Edward J Pearce, Elizabeth M Jaffee. Antigen-presenting cancer-associated fibroblasts are required for ICI-sensitivity in pancreatic ductal adenocarcinoma [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 B071.

Factors associated with favourable pathological tumour response after neoadjuvant chemotherapy in patients with pancreatic ductal adenocarcinoma

IntroductionPathological response of pancreatic ductal adenocarcinoma (PDAC) to neoadjuvant chemotherapy (NAT) has been associated with oncological outcomes. The aim of the study was to investigate factors associated with favourable tumour regression in patients undergoing pancreatic resection for PDAC. MethodsPatients who received NAT before undergoing PDAC resection at two institutions were reviewed. Tumour regression grading (TRG) was scored according to the College of American Pathologists (CAP) system. Interactions between chemotherapy, tumour and surgical factors with TRG were explored. Results54 patients were identified, with 12 (22%) displaying a favourable response to NAT. The type of chemotherapy agent received, the number of cycles or a dose reduction during NAT course was not significantly different between the groups. The time from diagnosis to chemotherapy and time from end of chemotherapy to surgery were also similar between the groups. A favourable TRG was associated with greater disease-free survival median 33.2 months vs. 10.3 months; p = 0.0) but not overall survival (median 43.8 months vs. 32.3 months; p = 0.200), which may be due to small sample size. ConclusionsChemotherapy factors were not significantly related to a favourable response to NAT. Future studies should seek to identify modifiable factors associated with a favourable TRG.

Impact of neoadjuvant chemoradiotherapy on pathologic response in pancreatic ductal adenocarcinoma: A systematic review and meta-analysis.

Impact of neoadjuvant chemoradiotherapy on pathologic response in pancreatic ductal adenocarcinoma: A systematic review and meta-analysis.

Dual drug-loaded tumor-targeted polymeric nanoparticles for enhancing therapeutic response in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease where standard-of-care chemotherapeutic drugs have limited efficacy due to the development of drug resistance and poor drug delivery caused by a highly desmoplastic tumor microenvironment. Combining multiple drugs in a tumor-targeting carrier would be a favorable approach to overcome these limitations. Hence, a tumor-targeted peptide (TTP) conjugated amphiphilic tri-block copolymer was developed to make targeted polymer nanoparticles (TTP-PNPs) serving as a vehicle for carrying gemcitabine (Gem), paclitaxel (PTX), and their combination (Gem + PTX). The TTP-PNPs in the form of empty polymer (P), single drug-loaded [P(Gem) and P(PTX)], and dual drug-loaded [P(Gem + PTX)] polymer nanoformulations exhibited stable and homogenous spherical shapes with 110–160 nm size. These nanoformulations demonstrated excellent stability under in vitro physiological conditions and led to an efficient release of the drugs in the presence of reduced glutathione (GSH). The efficacy of these nanoparticles was thoroughly evaluated in vitro and in vivo, demonstrating a notable capacity to selectively target and restrict PDAC cells (PANC-1 and KPC) growth. The cellular uptake and biodistribution study showed a significantly higher tumor-targeting ability of TTP-PNPs than PNPs without TTP. Notably, P(Gem + PTX) exhibited the lowest IC50 compared to all other controls and showed heightened synergistic effects in both cell lines. Furthermore, P(Gem + PTX) showed a significantly better tumor reduction and median overall survival in mouse models than single drug-loaded TTP-PNPs or a combination of free drugs (Gem + PTX). In summary, our TTP-PNP system shows great promise as a novel platform for delivering Gem + PTX specifically to pancreatic cancer (PC), maximizing the therapeutic benefits with lower concentrations of the drugs and potentially reducing toxic side effects.

Pathologic complete response following neoadjuvant chemotherapy in pancreatic ductal adenocarcinoma: Impact on survival and recurrence

BackgroundPathologic complete response after neoadjuvant treatment in pancreatic ductal adenocarcinoma is a rare occurrence. Similar to other malignancies, achieving a pathologic complete response in pancreatic ductal adenocarcinoma seems to correlate with improved survival. However, because of the rarity of such events, the true significance of pathologic complete response in pancreatic cancer remains unclear. The aim of the present study was to investigate the impact of pathologic complete response on survival and recurrence. MethodsIn a single-center retrospective study, pathologic complete response was defined as no evidence of viable tumor cells in resected specimen entirely sampled according to a rigorous protocol and in which a residual tumor bed was identified. Disease-specific survival and disease-free survival were measured from surgery. Independent predictors for disease-specific survival and disease-free survival were examined. ResultsOverall, 403 patients were included. Pathologic complete response was found in 15 patients (3.8%), after chemotherapy alone. After a median follow-up of 42 months (95% CI 38–45), 3-year disease-specific survival was 87% in pathologic complete response patients vs 43% in those without pathologic complete response (P = .014). The recurrence rate was 40% (n = 6/15) in the pathologic complete response group compared with 69.8% (n = 271/388) in those without pathologic complete response. Disease-free survival was longer in the pathologic complete response group, with higher 1- and 3-year rates compared with the no–pathologic complete response group (80% vs 60% and 48% vs 24%, respectively). Pathologic complete response was found to be an independent protective factor for disease-specific survival (P = .035) but not for disease-free survival (P = .052). ConclusionPathologic complete response in pancreatic ductal adenocarcinoma is not synonymous of cure but ensure a prolonged survival. Nevertheless, recurrence remains a significant concern, with high rates observed even among these exceptional responders.

Allysine-Targeted Molecular MRI Enables Early Prediction of Chemotherapy Response in Pancreatic Cancer.

Neoadjuvant therapy is routinely used in pancreatic ductal adenocarcinoma (PDAC), but not all tumors respond to this treatment. Current clinical imaging techniques are not able to precisely evaluate and predict the response to neoadjuvant therapies over several weeks. A strong fibrotic reaction is a hallmark of a positive response, and during fibrogenesis, allysine residues are formed on collagen proteins by the action of lysyl oxidases. Here, we report the application of an allysine-targeted molecular MRI probe, MnL3, to provide an early, noninvasive assessment of treatment response in PDAC. Allysine increased 2- to 3-fold after one dose of neoadjuvant therapy with FOLFIRINOX in sensitive human PDAC xenografts in mice. Molecular MRI with MnL3 could specifically detect and quantify fibrogenesis in PDAC xenografts. Comparing the MnL3 signal before and 3 days after one dose of FOLFIRINOX predicted subsequent treatment response. The MnL3 tumor signal increased by 70% from day 0 to day 3 in mice that responded to subsequent doses of FOLFIRINOX, whereas no signal increase was observed in FOLFIRINOX-resistant tumors. This study indicates the promise of allysine-targeted molecular MRI as a noninvasive tool to predict chemotherapy outcomes. Significance: Allysine-targeted molecular MRI can quantify fibrogenesis in pancreatic tumors and predict response to chemotherapy, which could guide rapid clinical management decisions by differentiating responders from nonresponders after treatment initiation.

A targetable secreted neural protein drives pancreatic cancer metastatic colonization and HIF1a nuclear retention.

Pancreatic ductal adenocarcinoma (PDAC) is an increasingly diagnosed cancer that kills 90% of afflicted patients, with most patients receiving palliative chemotherapy. We identified neuronal pentraxin 1 (NPTX1) as a cancer secreted protein that becomes over-expressed in human and murine PDAC cells during metastatic progression and identified adhesion molecule with Ig like domain 2 (AMIGO2) as its receptor. Molecular, genetic, biochemical and pharmacologic experiments revealed that secreted NPTX1 acts cell-autonomously on the AMIGO2 receptor to drive PDAC metastatic colonization of the liver-the primary site of PDAC metastasis. NPTX1-AMIGO2 signaling enhanced hypoxic growth and was critically required for hypoxia induced factor-1a (HIF1a) nuclear retention and function. NPTX1 is over-expressed in human PDAC tumors and upregulated in liver metastases. Therapeutic targeting of NPTX1 with a high-affinity monoclonal antibody substantially reduced PDAC liver metastatic colonization. We thus identify NPTX1-AMIGO2 as druggable critical upstream regulators of the HIF1a hypoxic response in PDAC.

Keratin 17 modulates the immune topography of pancreatic cancer

BackgroundThe immune microenvironment impacts tumor growth, invasion, metastasis, and patient survival and may provide opportunities for therapeutic intervention in pancreatic ductal adenocarcinoma (PDAC). Although never studied as a potential modulator of the immune response in most cancers, Keratin 17 (K17), a biomarker of the most aggressive (basal) molecular subtype of PDAC, is intimately involved in the histogenesis of the immune response in psoriasis, basal cell carcinoma, and cervical squamous cell carcinoma. Thus, we hypothesized that K17 expression could also impact the immune cell response in PDAC, and that uncovering this relationship could provide insight to guide the development of immunotherapeutic opportunities to extend patient survival.MethodsMultiplex immunohistochemistry (mIHC) and automated image analysis based on novel computational imaging technology were used to decipher the abundance and spatial distribution of T cells, macrophages, and tumor cells, relative to K17 expression in 235 PDACs.ResultsK17 expression had profound effects on the exclusion of intratumoral CD8+ T cells and was also associated with decreased numbers of peritumoral CD8+ T cells, CD16+ macrophages, and CD163+ macrophages (p < 0.0001). The differences in the intratumor and peritumoral CD8+ T cell abundance were not impacted by neoadjuvant therapy, tumor stage, grade, lymph node status, histologic subtype, nor KRAS, p53, SMAD4, or CDKN2A mutations.ConclusionsThus, K17 expression correlates with major differences in the immune microenvironment that are independent of any tested clinicopathologic or tumor intrinsic variables, suggesting that targeting K17-mediated immune effects on the immune system could restore the innate immunologic response to PDAC and might provide novel opportunities to restore immunotherapeutic approaches for this most deadly form of cancer.

Abstract 6433: Anti-CD36 monoclonal antibody can sensitize the neoadjuvant chemotherapy response in PDAC by eliminating the senescent tertiary lymphatic structure

Abstract Objective: Pancreatic ductal adenocarcinoma (PDAC) is deadly and aggressive. Neoadjuvant chemotherapy (NAC) may reduce tumor volume and alter the resectability of the tumor. The efficacy of neoadjuvant chemotherapy in patients is still limited. Tertiary lymphoid structures (TLSs) develop in peripheral non-lymphoid tissues and are essential for the immune response and correlate with chemotherapy in various tumor types. However, more research is needed to determine how TLSs affect neoadjuvant chemotherapy in PDAC. We aim to determine how neoadjuvant chemotherapy affects tumor-infiltrating lymphocytes (TLSs) in PDAC patients. We also want to find a biomarker to boost PDAC neoadjuvant chemotherapy response. Methods: We retrospectively collected formalin-fixed paraffin embedded (FFPE) tissues from 115 PDAC patients without preoperative treatment and 72 patients pretreated with neoadjuvant chemotherapy. Multispectral images were processed using AI-powered image analysis software (Tissue Gnostics; Zeiss, Germany). The 10X Genomics platform was used to sequence single cells in tissue samples from five patients who received neoadjuvant chemotherapy (NAC) and five patients who had not received any treatment before analysis (pretreatment naïve, PN). TLS was separated using Laser Microdissection. The mouse model was created by orthotopically injecting tumor cells into the pancreas. Results: Patients who received NAC have a senescent microenvironment. High-throughput single-cell sequencing showed that the senescent gene signature score was enriched in the NAC group, especially in immune cells. The percentage of p16+ CD20+ B cells and P16+ CD3+ T cells in TLSs also increased in the NAC group. Spatial transcriptomics analysis exhibited that CD36 is highly expressed in senescent TLSs, which suggested that CD36 may be a marker for senescent TLSs. Moreover, CD36+ TLSs induced the infiltration of MDSCs and regulatory T cells. CD36+ TLSs were an independent prognostic factor for poor overall survival (OS) (HR=0.67, p=0.002) and progression-free survival (PFS) (HR=0.71, p=0.003). A mice orthotopic xenograft model showed that an anti-CD36 monoclonal antibody eliminated senescent TLSs in pancreatic tissue. MDSCs and Tregs in the surrounding tissue also decreased. Additionally, in vivo KPC mice model showed that the combination of an anti-CD36 and chemotherapy reduced tumor volume and increased survival compared to chemotherapy alone. Conclusion: NAC has the potential to induce the senescence of tumor-associated TLSs, a phenomenon that has been linked to unfavorable prognosis in PDAC. CD36 serves as a viable marker for senescent TLSs that can be targeted. The administration of an anti-CD36 monoclonal antibody can potentially enhance the efficacy of chemotherapy in PDAC by selectively eliminating CD36+ senescent TLSs in mouse models. Citation Format: Jingrui Yan, Tianxing Zhou, Chao Yang, Yongjie Xie, Jun Yu, Jihui Hao. Anti-CD36 monoclonal antibody can sensitize the neoadjuvant chemotherapy response in PDAC by eliminating the senescent tertiary lymphatic structure [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 6433.

Abstract 6649: Tumor treating fields (TTFields) induce an anti-tumor immune response in a pancreatic cancer mouse model

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with few therapeutic options. Tumor Treating Fields (TTFields) are low-intensity electric fields that disrupt cellular processes critical for cancer cell division and tumor progression and are approved for the treatment of glioblastoma and pleural mesothelioma. A phase 3 clinical trial is in progress to test the efficacy and safety of TTFields concomitant with standard-of-care gemcitabine and nab-paclitaxel (GnP) as front-line treatment of locally advanced PDAC. TTFields have been shown to induce immunogenic cell death (ICD) in cancer cells and elicit a systemic anti-cancer immune response. Accordingly, the benefit of applying TTFields together with immune checkpoint inhibitors (ICI) was demonstrated in a phase 3 study in patients with non-small cell lung carcinoma. This preclinical study investigated TTFields-induced immune responses in PDAC. Methods: An orthotopic PDAC tumor model was established by inoculating 4 × 105 Panc02 pancreatic cancer cells with stable luciferase expression into the pancreas tail of immune-competent C57BL/6 mice. The mice were randomized into four groups following bioluminescence detection of tumor volume: control, TTFields (150 kHz, 10 days), GnP (50 mg/kg for each drug, i.p. injected once a week for a total of two administrations), and GnP + TTFields. At the end of treatment, tumor volume was measured using caliper, and blood was collected for spectral flow cytometry-based immunophenotyping and ELISA testing for the ICD marker HMGB-1. In vitro, Panc02 cells were treated for 72h with TTFields (150 kHz, 1.62 V/cm RMS) and examined by flow cytometry for apoptosis and ATP secretion (using quinacrine staining) and surface exposure of calreticulin. Results: TTFields markedly reduced the tumor size in Panc02 tumor-bearing mice compared to control mice. Analysis of peripheral blood effector (CD62L-CD44+) and central (CD62L+CD44+) memory T cells demonstrated the induction of circulating effector-memory cytotoxic CD8+ T cells. Additionally, serum HMGB-1 concentrations were significantly elevated in mice treated with TTFields compared to control mice, implying the involvement of ICD in the response to TTFields. In vitro, TTFields application induced apoptosis of Panc02 cells and increased ICD, as indicated by increased surface exposure of calreticulin and ATP depletion. When TTFields were used in Panc02 tumor bearing mice concomitantly with GnP, a substantial reduction in tumor size was detected, and a complete tumor regression was observed in a number of mice. Conclusions: Our results suggest that TTFields may enhance the effects of standard-of-care chemotherapy for the treatment of PDAC. Furthermore, TTFields were shown to induce ICD and activate an antitumor immune response in PDAC, supporting future studies evaluating the efficacy of TTFields together with ICI in PDAC. Citation Format: Tal Kan, Tharwat Haj Khalil, Yiftah Barsheshet, Tali Voloshin, Avital Vorontsov, Boris Brant, Lilach Koren, Roni Blatt, Shay Cahal, Efrat Zemer-Tov, Rom Paz, Anat Klein-Goldberg, Catherine Tempel Brami, Sara Jacobovitch, Alexandra Volodin, Bella Koltun, Cfir David, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Tumor treating fields (TTFields) induce an anti-tumor immune response in a pancreatic cancer mouse model [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 6649.

Abstract 2495: CCL3 predicts an exceptional response to TGFβ inhibition in pancreatic ductal adenocarcinoma by sustaining a basal-like ecotype enriched in LIF-producing macrophages

Abstract The TGFβ receptor inhibitor galunisertib (GAL) exhibited promising efficacy in patients with pancreatic ductal adenocarcinoma (PDAC) in the randomized phase 2 H9H-MC-JBAJ study. However, the identification of predictive biomarkers remains of unique importance. We used a 279 multi-analyte panel and confirmed CCL3 as the most significant plasma protein for chemoresistance in patients receiving gemcitabine (GEM) [overall survival (mOS) (95%CI), high vs low, 3.6 (2.5-4.0) vs 10.1 (7.2-17.0) months, HR (95%CI)= 3.92 (2.14-7.20); p&amp;lt;0.001]. GAL dramatically reverted this resistance and CCL3 was the most significant predictive marker for the combination of this agent plus GEM [mOS (95%CI), GAL + GEM vs. placebo + GEM, 9.0 (5.5-12.1) vs 3.6 (2.5-4.0) months, HR (95%CI)= 0.39 (0.23-0.68); p&amp;lt;0.001]. We delved into the mechanisms behind this exceptional response in PDAC with this prognostically negative ecotype sustained by CCL3 in 6 different murine PDAC models. Mice bearing high-Ccl3 tumors had a significantly shorter mOS, a higher infiltration of M2-macrophages (TAMs), and a more mesenchymal/basal-like phenotype than did models with undetectable Ccl3.Ccl3 was stably expressed in DT4313 and PAN610, and silenced in RC416 cells. In co-culture models, DT4313Ccl3 and PAN610Ccl3 had a more mesenchymal phenotype and induced a M2 polarization of RAW264.7 murine TAMs than did controls. Mice bearing DT4313Ccl3 or PAN610Ccl3 tumors had a significantly shorter mOS, an higher infiltration of M2-polarized TAMs and a more mesenchymal/basal-like phenotype than controls. The expression of Tgfβ1 ligand and the phosphorylation of Smad2 were triggered only in TAMs if co-cultured with Ccl3-high RC416scr, DT4313Ccl3 or PAN610Ccl3 cells. This activation of Tgfβ pathway was blocked by maraviroc, a selective inhibitor of Ccl3-receptor Ccr5. In co-culture, GAL reverted the mesenchymal phenotype of PDAC cells and the M2 polarization of TAMs. By using an 111 different cytokine immunoassay, Leukemia Inhibitory Factor (Lif) was the most significantly factor overexpressed by TAMs if stimulated by Ccl3. GAL suppressed this Lif expression. In mice bearing DT4313Ccl3 tumors GEM was ineffective, whereas it prolonged mOS in those with DT4313NTC tumors. Conversely, GAL plus GEM doubled mOS duration vs. GEM, modulated M2- TAMs infiltration, the mesenchymal/basal-like phenotype and plasma Lif only in mice bearing DT4313Ccl3 tumors, but not in those with DT4313NTC controls. In summary, we demonstrated that tumor-derived Ccl3 activates an autocrine Tgfβ signaling in TAMs, triggering their M2-phenotype and secretion of Lif. This Tgfβ-Lif axis sustains paracrinally a mesenchymal/basal-like PDAC ecotype resistant to GEM. Inhibition of Tgfβ signaling modulates this poor prognosis ecotype, and explains the exceptional response to GAL in patients with high-CCL3 PDAC. Citation Format: Silvia Pietrobono, Monica Bertolini, Veronica De Vita, Enza Scarlato, Federica Fazzini, Fabio Sabbadini, Domenico Mangiameli, Alberto Quinzii, Simona Casalino, Davide Melisi. CCL3 predicts an exceptional response to TGFβ inhibition in pancreatic ductal adenocarcinoma by sustaining a basal-like ecotype enriched in LIF-producing macrophages [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 2495.

Abstract 6631: Aging influences the pancreatic ductal adenocarcinoma tumor immune microenvironment to promote features of immunosuppression

Abstract Advancing age is the strongest independent risk factor for the development of pancreatic ductal adenocarcinoma (PDAC) and a negative prognostic factor. PDACs are typically immunologically “cold” tumors and do not often respond to currently approved immunotherapy treatment approaches. How aging may alter non-malignant cells, such as fibroblasts and immune cell populations, in the PDAC tumor microenvironment (TME) to decrease anti-tumor immune responses is not understood. We identified that aged microenvironments significantly alter the growth and progression of PDAC tumors. Orthotopic KPC and Panc02 mouse PDAC cell line injections into the pancreata of aged (&amp;gt;64-week-old) compared to young (6-8-week-old) C57BL/6 mice result in more rapid tumor growth and increased frequency of liver metastases in vivo. Tumors from aged mice have high expression of the TGFβ family member GDF-15 and hyperactivation of AKT signaling driven by the release of secreted factors from aged pancreatic fibroblasts. We found that in aged microenvironments there is a marked reduction in the number of CD8+ T cells and an increase in FoxP3+ regulatory T cells in primary orthotopic pancreatic tumors using the KPC and Panc02 models. In addition, we performed portal vein injections to establish liver metastases to study the differences in immune cell infiltration in metastases from young vs. aged microenvironments. Aged mice form more and larger liver metastases compared to young mice, and metastatic lesions in aged mice have decreased CD8+ T cell infiltration when compared to young mice. Since tumors in aged microenvironments have increased AKT activation compared to young microenvironments, we next tested if AKT signaling was sufficient to alter immune cell infiltration in PDAC tumors. In young mice, treatment with recombinant GDF-15, which activates AKT signaling, reduced CD8+ T cell infiltration into tumors. Conversely, in KPC tumors grown in aged mice treated with the AKT inhibitor MK-2206, there was a trend toward increased CD8+ T cell infiltration and a significant reduction in immunosuppressive FoxP3+ Tregs. Together, these data highlight age-dependent differences in the immune infiltration in PDAC tumors. They suggest that activation of AKT signaling can drive features of immunosuppression in PDAC, providing the rationale for targeting AKT signaling to improve antitumor immune responses in PDAC. Citation Format: Emma Kartalia, James M. Leatherman, Jae W. Lee, Elizabeth M. Jaffee, Ashani T. Weeraratna, Daniel J. Zabransky. Aging influences the pancreatic ductal adenocarcinoma tumor immune microenvironment to promote features of immunosuppression [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 6631.

Group A streptococcal collagen-like protein 1 restricts tumor growth in murine pancreatic adenocarcinoma and inhibits cancer-promoting neutrophil extracellular traps.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer associated with an immunosuppressive environment. Neutrophil extracellular traps (NETs) were initially described in the context of infection but have more recently been implicated in contributing to the tolerogenic immune response in PDAC. Thus, NETs are an attractive target for new therapeutic strategies. Group A Streptococcus (GAS) has developed defensive strategies to inhibit NETs. In the present work, we propose utilizing intra-tumoral GAS injection to stimulate anti-tumor activity by inhibiting cancer-promoting NETs. Mice harboring Panc02 or KPC subcutaneous tumors injected with three different M-type GAS strains. Tumors and spleens were harvested at the endpoint of the experiments to assess bacterial colonization and systemic spread, while sera were analyzed for humoral responses toward the streptococcal antigens, especially the M1 and Scl1 proteins. Role of the streptococcal collagen-like protein 1 (Scl1) in anti-PDAC activity was assessed in vivo after intratumoral injection with M1 GAS wild-type, an isogenic mutant strain devoid of Scl1, or a complemented mutant strain with restored scl1 expression. In addition, recombinant Scl1 proteins were tested for NET inhibition using in vitro and ex vivo assays assessing NET production and myeloperoxidase activity. Injection of three different M-type GAS strains reduced subcutaneous pancreatic tumor volume compared to control in two different murine PDAC models. Limitation of tumor growth was dependent on Scl1, as isogenic mutant strain devoid of Scl1 did not reduce tumor size. We further show that Scl1 plays a role in localizing GAS to the tumor site, thereby limiting the systemic spread of bacteria and off-target effects. While mice did elicit a humoral immune response to GAS antigens, tested sera were weakly immunogenic toward Scl1 antigen following intra-tumoral treatment with Scl1-expressing GAS. M1 GAS inhibited NET formation when co-cultured with neutrophils while Scl1-devoid mutant strain did not. Recombinant Scl1 protein inhibited NETs ex vivo in a dose-dependent manner by suppressing myeloperoxidase activity. Altogether, we demonstrate that intra-tumoral GAS injections reduce PDAC growth, which is facilitated by Scl1, in part through inhibition of cancer promoting NETs. This work offers a novel strategy by which NETs can be targeted through Scl1 protein and potentiates its use as a cancer therapeutic.

Microscopic tumor mapping of post-neoadjuvant therapy pancreatic cancer specimens to predict post-surgical recurrence: A prospective cohort study

BackgroundAlthough various pathological grading systems are available for evaluating the response of pancreatic ductal adenocarcinoma (PDAC) to neoadjuvant therapy (NAT), their prognostic value has not been thoroughly validated. This study examined whether microscopic tumor mapping of post-NAT specimens could predict tumor recurrence. MethodsThis prospective study enrolled 52 patients who underwent pancreaticoduodenectomy after NAT for PDAC between 2019 and 2021. Microscopic mapping was performed to identify residual tumor loci within the tumor bed using 4 mm2 pixels. Patients were divided into small extent (SE; n = 26) and large extent (LE; n = 26) groups using a cutoff value of 226 mm2. The diagnostic performance for predicting tumor recurrence was evaluated using receiver operating characteristic (ROC) curves. ResultsCarbohydrate antigen 19-9 levels were normalised after NAT in more patients in the SE group (SE 21 [80.8%] vs. LE 13 [50.0%]; P = 0.041). Tumor size (P < 0.001), T stage (P < 0.001), positive lymph node yield (P = 0.024), and perineural invasion rate (P = 0.018) were significantly greater in the LE group. The 3-year disease-free survival rate was significantly lower in the LE group (SE 83.3% vs. LE 50.0%, P = 0.004). The area under the ROC curve for mapping extent was 0.743, which was greater than that of the other tumor response scoring systems. ConclusionsMicroscopic tumor mapping of the residual tumor in post-NAT specimens is a significant predictor of post-surgical recurrence, and offers better prognostic performance than the current grading systems.

Abstract 1190 Investigating chemoresistance mechanisms and collateral responses in pancreatic ductal adenocarcinoma

Abstract 1190 Investigating chemoresistance mechanisms and collateral responses in pancreatic ductal adenocarcinoma

Inter-reader variability of imaging biomarkers for therapeutic response in pancreatic ductal adenocarcinoma.

700 Background: Indeterminate borders of pancreatic ductal adenocarcinoma (PDAC) can impair reliability of lesion annotation in imaging assessment of therapeutic response (TR) after neoadjuvant chemotherapy (NAT). Previous work showed that longitudinal image registration algorithms can track treatment-related changes and produce quantitative imaging biomarkers (QIB) for TR that predict overall (OS) and recurrence-free survival (RFS). We compare inter-reader agreements of these biomarkers against conventional RECIST and tumor volume QIB. Methods: N=30 patients enrolled in a Phase II clinical trial comparing outcomes of a NAT regimen were retrospectively analyzed. In baseline and restaging CT, whole pancreas and tumor segmentations and tumor diameters were annotated by 2 expert attending radiologists and 2 novice radiology fellows. For each reader, percent change in tumor diameter (RECIST score), percent change in tumor volume (ΔV), and difference in ratio of tumor burden to pancreas volume (ΔB) were computed as QIB for TR across the NAT interval via two approaches: image registration of the baseline tumor region of interest, and direct segmentation of the PDAC lesion in baseline and restaging images. Inter-reader agreements of RECIST, ΔV, and ΔB QIB from both approaches were compared via concordance correlation coefficient (CCC). Associations with OS and RFS were compared over 5-year follow-up via Harrell’s C-index. Results: Among expert radiologists, RECIST score CCC was 0.77 [95% CI: 0.59-0.88], whereas image registration QIB CCC values were significantly higher (ΔV: 0.95 [0.90-0.98], p=0.002; ΔB: 0.93 [0.86-0.97], p=0.014). ΔV and ΔB QIB measured by direct tumor segmentation had significantly lower CCC than by image registration (p&lt;0.001). Among novice readers, image registration QIB (ΔV: 0.83 [0.67-0.91], ΔB: 0.70 [0.48-0.84]) produced higher agreement than RECIST (0.26 [-0.17-0.61]; p=0.002 and p=0.035), with CCC more similar to expert readers. While image registration QIB did not differ from RECIST in association with RFS, registration-based QIB predicted OS with significantly higher C-index (ΔV: 0.58 [0.56-0.60], p&lt;0.001; ΔB: 0.64 [0.62-0.66], p&lt;0.001) than RECIST (0.50 [0.45-0.55]) among experts. Conclusions: Longitudinal registration of the pancreas yields more consistent QIB for PDAC TR with stronger association to OS outcomes than RECIST. These registration-based QIB aim to advance PDAC management by improving patient-specific discrimination of TR after NAT. [Table: see text]

Abstract B054: Investigating ER stress responses in pancreatic ductal adenocarcinoma under lipid imbalance

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death with a five-year survival rate of 10%. Dense desmoplasia results in poor intratumoral perfusion and elevated interstitial fluid pressure, creating regions with severe oxygen and nutrient deprivation. One result is a deficiency in unsaturated fatty acids (uFAs), as the de novo synthesis of uFAs requires oxygen. Our results indicate that PDAC cells experience endoplasmic reticulum (ER) stress and trigger subsequent ER stress responses under uFA limited conditions in vitro. ER stress responses are governed by three major stress sensors: Inositol-requiring enzyme 1𝛼 (IRE1𝛼), PKR-like ER kinase (PERK) and activating transcription factor 6 (ATF6). ER stress responses are also activated in PDAC murine and patient samples. Interestingly, the expression of ER stress markers is much higher in malignant lesions than benign pancreatic intraepithelial neoplasia. In contrast to tumor promoting, we found that PERK, ATF6 and IRE1𝛼 - XBP1 are cytotoxic to PDAC cells in vitro, as their knockout improved viability under stress conditions. Surprisingly, pharmacological inhibitor of XBP1 splicing, B-I09, didn’t recapitulate the results of the genetic approach. While B-I09 has cytoprotective effects in some PDAC lines, it is cytotoxic in others. In addition to inhibiting XBP1 splicing, B-I09 also inhibits regulated IRE1𝛼 dependent decay (RIDD). We speculate that RIDD might have opposite roles to XBP1s, leading to the discrepancy in the cellular phenotypes we observed. Most B-I09 responders are reported to be the basal-like transcriptional subtype. Therefore, we hypothesize that the basal-like subtype is more susceptible to B-I09. So far, we have shown that basal-like patient tumors are significantly enriched in the ER stress response pathways from bulk patient RNA-seq datasets, and we are currently validating this result in the publicly available scRNA-seq datasets from patients which contain basal-like and classical subtypes. We have generated isogenic models with basal-like features, such as epithelial to mesenchymal transition and deltaNP63 overexpression. However, our results suggest that these basal-like features did not sensitize classical PDAC cells to B-I09. Our lab has previously shown that B-I09 is selectively lethal to the MYC-transformed tumors. We are currently testing whether MYC overexpression would sensitize the non-responders to B-I09. It is exciting to see that some of the PDAC lines such as PANC1 and KPC murine line 4662 have reduced viability under B-I09 treatment. However, in our subcutaneous xenograft and allograft experiments, B-I09 only leads to a mild tumor growth suppression, which is unlikely to be therapeutically beneficial by itself. We are currently testing whether XBP1 inhibition could result in an enhanced tumor suppression using OVA antigen stimulated system, as XBP1 inhibition in tumor cells was shown to have tumor-repressing roles via immune system. Citation Format: Yanqing (Christine) Jiang, Xu Han, Nathan Coffey, Mai Wang, John Tobias, Raymond Ng, Carson Poltorack, M. Celeste Simon. Investigating ER stress responses in pancreatic ductal adenocarcinoma under lipid imbalance [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 B054.