Metastatic Pancreatic Ductal Adenocarcinoma Research Articles

Abstract CT214: Paricalcitol addition to chemotherapy in patients with previously untreated metastatic pancreatic ductal adenocarcinoma (PINBALL)

Abstract Background Vitamin D agonism has been shown in preclinical studies to decrease the production of collagen, decrease myeloid derived suppressor cells, and decrease regulatory T cells in pancreatic ductal adenocarcinoma (PDAC). Paricalcitol is a vitamin D analog used for vitamin D replacement for those with secondary hyperparathyroidism. Two individuals with PDAC who had been developed resistance gemcitabine based combination therapy had shown clinical benefit with the addition of paricalcitol. This clinical study was done to study the effect of adding paricalcitol to individuals with metastatic PDAC receiving gemcitabine based combination therapy (NCT04054362, NCT03415854) . Methods Adults with a histologically- or cytologically-confirmed diagnosis of metastatic PDAC and who were previously untreated were eligible. Individuals received gemcitabine based combination- either gemcitabine plus nab-paclitaxel or gemcitabine plus nab-paclitaxel and cisplatin and had to either demonstrate progressive or stable disease. Paricalcitol was given at a fixed dose of 25 mcg IV three days a week. The combination continued until either disease progression or poor tolerance. The primary objective was to determine the clinical benefit of adding paricalcitol to gemcitabine based combination therapy for metastatic PDAC. Secondary objectives included assessing safety and pharmacodynamic through patient biopsies. Results A total of 34 patients were enrolled across two sites. 16 patients received paricalcitol. The overall response rate was 0% ( CR, PR). Disease control rate at 9 weeks was 21.4% (CR+PR+ SD). PFS upon addition of paricalcitol was 1.6 months. OS upon addition of paricalcitol was 4.8 months. Grade 3/4 AE's attributed to paricalcitol include 13% thrombocytopenia; 19% anemia; 6% transaminitis; 6% pleural effusion; 6% ascites- all grade 3. Conclusions The addition of paricalcitol demonstrated evidence of limited clinical benefit in individuals with metastatic PDAC receiving front-line gemcitabine based therapy. Paricalcitol was generally well tolerated. Tumor analysis is ongoing. Addition studies utilizing prospective treatment of paricalcitol for front line treatment of mPDAC are ongoing (NCT03520790). Trial funding provided by SU2C, CRUK, Lustgarten Foundation, and AACR. Citation Format: David Propper, Gayle Jameson, Denise Roe, Betsy Wertheim, Dan Von Hoff, Erkut H. Borazanci. Paricalcitol addition to chemotherapy in patients with previously untreated metastatic pancreatic ductal adenocarcinoma (PINBALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT214.

Abstract CT105: Early clinical experience with a novel first-in-class G-quadruplex experimental anti-cancer drug

Abstract The human genome contains G-rich regions that are over-represented in many cancer gene promoters. These can form higher-order G-quadruplex arrangements under the influence of an appropriate and selective small molecule compound. The resulting ligand-G-quadruplex complex may be resistant to polymerase read-through and thus results in transcriptional down-regulation of target genes. QN-302, based on a tetra-substituted naphthalene diimide chemotype, is a potent and selective G-quadruplex-binding compound that has been designed and initially developed at University College London. It displays single-digit cell growth inhibition in a panel of pancreatic ductal adenocarcinoma (PDAC) cell lines. RNA-seq analysis shows that it is a pan-quadruplex agent, down-regulating the expression of multiple G-quadruplex-promoter cancer genes and pathways in PDAC. It also shows in vivo anti-tumor activity in several animal models for PDAC, with low toxicity observed at therapeutic doses. QN-302 was licensed to Qualigen Therapeutics Inc in January 2022. It was granted Orphan Drug designation by the FDA in December 2022 for PDAC. This was followed by extensive regulatory toxicological and pharmacological studies together with successful scale-up synthesis, purification and formulation. QN-302 was granted IND clearance in August 2023 by the FDA for phase 1 clinical trials in advanced or metastatic solid tumors. This Phase 1a clinical trial is being conducted in two USA cancer centers (START Midwest, Grand Rapids, MI and Honor Health, Scottsdale, AZ) using a protocol based on 28-day once-weekly dose escalation and evaluation of three potential biomarkers, two of which have been suggested by transcriptomic analysis as being indicative of G-quadruplex-induced drug response and accompanying decrease of anti-tumor activity. So far 1 Cohort (3 patients) has been dosed at the lowest dose of 1.62 mg of QN-302 as part of this Phase 1a clinical trial. Patient #1, a male patient of metastatic pancreatic cancer, is currently in Cycle 4 of therapy showing Stable Disease and no AEs or SAEs reported. Patient #2, a female patient of metastatic colorectal cancer, completed Cycle 2 after which she showed progression upon CT Scan before entering Cycle 3 and was taken off study. Patient #3, a male patient of metastatic PDAC, is currently ending Cycle 2, about to enter Cycle 3, showing Stable Disease upon CT scan. All patients dosed showed no AEs, SAEs or side effects. They were also clinically observed to have improved QOL compared to prior SOC while on therapy with QN-302, This presentation will outline this unique approach and summarize safety, tolerability, PK/PD data and any early clinical data as described above including preliminary biomarker data now that clinical evaluation has commenced. The advantages and challenges of QN-302 will also be discussed. Citation Format: Tariq Arshad, Stephen Neidle, Sreenivasa Chandana, Erkut Borazanci. Early clinical experience with a novel first-in-class G-quadruplex experimental anti-cancer drug [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT105.

Abstract CT218: Phase II open label trial of Minnelide™ in patients with chemotherapy refractory metastatic pancreatic cancer

Abstract Background: Triptolide and its water-soluble prodrug, Minnelide™, have shown promising activity against pancreatic ductal adenocarcinoma (PDAC) in both in vitro and in vivo experiments. Triptolide induces apoptosis in pancreatic cancer cells by different pathways, including caspase-dependent apoptotic death and caspase -independent autophagic death. It inhibits the activity of the TFIIH general transcription factor complex and modulates the transcriptional landscape of both the stroma and cancer cell compartments. In the epithelial tumour compartment, triptolide down-regulates key transcription factors such as c-MYC. Preclinical investigations indicate significant activity against super enhancer targets. In the phase I clinical trial of Minnelide™ activity in highly refractory metastatic pancreatic carcinoma was observed. Hence, we undertook this study in patients with chemotherapy refractory metastatic PDAC. Our aims were to determine clinical anticancer effects, and to define effects of the drug on super enhancer expression and downstream effects. Methods: Eligibility criteria included stage IV PDAC progressing on standard chemotherapy, Karnofsky performance status ≥ 70%, and measurable disease by RECIST 1.1. Planned sample size was up to 35 patients with interim analysis after the first 18 evaluable patients, aiming for at least a 20% difference in DCR (10% v 30%). Minnelide™ 0.67 mg/m2 was administered intravenously over 30 minutes on days 1-21, followed by a 7-day rest period in a 28-day cycle. Exploratory objectives included: analyses of paired tumour biopsies for changes in immune cell infiltration, stromal activation, chromatin accessibility, proteomics and metabolism. Results: 17 patients were treated. There were no RECIST responses or stable disease. However, evidence of activity was observed in 4 of 15 evaluable patients. This activity included progression after 117 days treatment in 1 patient and reductions in CT tumour size or FDG-PET uptake and/or significant decreases in circulating CA19.9 levels in 3 others. Minnelide was generally well tolerated. Treatment related toxicities > 3 were: neutropenia 4 patients and cerebellar ataxia 1 patient.Exploratory analyses included ATAC-Seq on paired biopsies where gene ontology analyses showed downregulation of genes negatively regulating cell cycle and upregulation of genes controlling cell cycle checkpoints. Other exploratory analyses will be presented. Conclusions: The study identified a tolerable drug dose and evidence of activity in a highly chemotherapy-refractory population which provides encouraging proof for the principle of targeting TFIIH in human PDAC. Supported by SU2C, Cancer Research UK, Lustgarten Foundation Citation Format: David Propper, Erkut Borazanci, Peter J. O'Dwyer, Daniel Von Hoff, Joseph Hartlebury, Corina E. Antal, Gerard I. Evan, HaiYong Han, Michael Downes, Ronald Evans, Joshua Rabinowitz, Emmanuel Petricoin, Ashok Saluja, Mohana Velagapudi, Hitendra Patel. Phase II open label trial of Minnelide™ in patients with chemotherapy refractory metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT218.

LOAd703, an oncolytic virus-based immunostimulatory gene therapy, combined with chemotherapy for unresectable or metastatic pancreatic cancer (LOKON001): results from arm 1 of a non-randomised, single-centre, phase 1/2 study

Pancreatic ductal adenocarcinoma is characterised by low immunogenicity and an immunosuppressive tumour microenvironment. LOAd703, an oncolytic adenovirus with transgenes encoding TMZ-CD40L and 4-1BBL, lyses cancer cells selectively, activates cytotoxic T cells, and induces tumour regression in preclinical models. The aim of this study was to evaluate the safety and feasibility of combining LOAd703 with chemotherapy for advanced pancreatic ductal adenocarcinoma. LOKON001 was a non-randomised, phase 1/2 study conducted at the Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX, USA, and consisted of two arms conducted sequentially; the results of arm 1 are presented here. In arm 1, patients 18 years or older with previously treated or treatment-naive unresectable or metastatic pancreatic ductal adenocarcinoma were treated with standard 28-day cycles of intravenous nab-paclitaxel 125 mg/m2 plus gemcitabine 1000 mg/m2 (up to 12 cycles) and intratumoural injections of LOAd703 every 2 weeks. Patients were assigned using Bayesian optimal interval design to receive 500 μL of LOAd703 at 5 × 1010 (dose 1), 1 × 1011 (dose 2), or 5 × 1011 (dose 3) viral particles per injection, injected endoscopically or percutaneously into the pancreatic tumour or a metastasis for six injections. The primary endpoints were safety and treatment-emergent immune response in patients who received at least one dose of LOAd703, and antitumour activity was a secondary endpoint. This study was registered with ClinicalTrials.gov, NCT02705196, arm 2 is ongoing and open to new participants. Between Dec 2, 2016, and Oct 17, 2019, 23 patients were assessed for eligibility, leading to 22 patients being enrolled. One patient withdrew consent, resulting in 21 patients (13 [62%] men and eight [38%] women) assigned to a dose group (three to dose 1, four to dose 2, and 14 to dose 3). 21 patients were evaluable for safety. Median follow-up time was 6 months (IQR 4-10), and data cutoff was Jan 5, 2023. The most common treatment-emergent adverse events overall were anaemia (96 [8%] of 1237 events), lymphopenia (86 [7%] events), hyperglycaemia (70 [6%] events), leukopenia (63 [5%] events), hypertension (62 [5%] events), and hypoalbuminaemia (61 [5%] events). The most common adverse events attributed to LOAd703 were fever (14 [67%] of 21 patients), fatigue (eight [38%]), chills (seven [33%]), and elevated liver enzymes (alanine aminotransferase in five [24%], alkaline phosphatase in four [19%], and aspartate aminotransferase in four [19%]), all of which were grade 1-2, except for a transient grade 3 aminotransferase elevation occurring at dose 3. A maximum tolerated dose was not reached, thereby establishing dose 3 as the highest-evaluated safe dose when combined with nab-paclitaxel plus gemcitabine. Proportions of CD8+ effector memory cells and adenovirus-specific T cells increased after LOAd703 injections in 15 (94%) of 16 patients for whom T-cell assays could be performed. Eight (44%, 95% CI 25-66) of 18 patients evaluable for activity had an objective response. Combining LOAd703 with nab-paclitaxel plus gemcitabine in patients with advanced pancreatic ductal adenocarcinoma was feasible and safe. To build upon this novel chemoimmunotherapeutic approach, arm 2 of LOKON001, which combines LOAd703, nab-paclitaxel plus gemcitabine, and atezolizumab, is ongoing. Lokon Pharma, the Swedish Cancer Society, and the Swedish Research Council.

Systematic analysis of proteome turnover in an organoid model of pancreatic cancer by dSILO.

The role of protein turnover in pancreatic ductal adenocarcinoma (PDA) metastasis has not been previously investigated. We introduce dynamic stable-isotope labeling of organoids (dSILO): a dynamic SILAC derivative that combines a pulse of isotopically labeled amino acids with isobaric tandem mass-tag (TMT) labeling to measure proteome-wide protein turnover rates in organoids. We applied it to a PDA model and discovered that metastatic organoids exhibit an accelerated global proteome turnover compared to primary tumor organoids. Globally, most turnover changes are not reflected at the level of protein abundance. Interestingly, the group of proteins that show the highest turnover increase in metastatic PDA compared to tumor is involved in mitochondrial respiration. This indicates that metastatic PDA may adopt alternative respiratory chain functionality that is controlled by the rate at which proteins are turned over. Collectively, our analysis of proteome turnover in PDA organoids offers insights into the mechanisms underlying PDA metastasis.

Expression of versican isoforms V0/V1 by pancreatic cancer associated fibroblasts increases fibroblast proliferation

BackgroundVersican is a large extracellular matrix (ECM) proteoglycan with four isoforms V0-3. Elevated V0/V1 levels in breast cancer and glioma regulate cell migration and proliferation, but the role of versican in pancreatic ductal adenocarcinoma (PDAC) remains unclear. MethodsIn this study, we evaluated the expression levels of versican isoforms, as well as their cellular source and interacting partners, in vivo, in human and mouse primary and metastatic PDAC tumours and in vitro, in pancreatic tumour cells and fibroblasts using immunostaining, confocal microscopy and qPCR techniques. We also investigated the effect of versican expression on fibroblast proliferation and migration using genetic and pharmacological approaches. ResultsWe found that versican V0/V1 is highly expressed by cancer-associated fibroblasts (CAFs) in mouse and human primary and metastatic PDAC tumours. Our data also show that exposing fibroblasts to tumour-conditioned media upregulates V0 and V1 expressions, while Verbascoside (a CD44 inhibitor) downregulates V0/V1 expression. Importantly, V0/V1 knockdown significantly inhibits fibroblast proliferation. Mechanistically, we found that inhibiting hyaluronan synthesis does not affect versican co-localisation with CD44 in fibroblasts. ConclusionCAFs express high levels of versican V0/V1 in primary and liver metastatic PDAC tumours and versican V0/V1 supports fibroblast proliferation.

Results of a Randomized, Double-Blind, Placebo-Controlled, Phase 1b/2 Trial of Nabpaclitaxel + Gemcitabine ± Olaratumab in Treatment-Naïve Participants with Metastatic Pancreatic Cancer.

The efficacy and safety of olaratumab plus nabpaclitaxel and gemcitabine in treatment-naïve participants with metastatic pancreatic ductal adenocarcinoma was evaluated. An initial phase 1b dose-escalation trial was conducted to determine the olaratumab dose for the phase 2 trial, a randomized, double-blind, placebo-controlled trial to compare overall survival (OS) in the olaratumab arm vs. placebo arms. In phase 1b, 22 participants received olaratumab at doses of 15 and 20 mg/kg with a fixed dose of nabpaclitaxel and gemcitabine. In phase 2, 159 participants were randomized to receive olaratumab 20 mg/kg in cycle 1 followed by 15 mg/kg in the subsequent cycles (n = 81) or the placebo (n = 78) on days 1, 8, and 15 of a 28-day cycle, plus nabpaclitaxel and gemcitabine. The primary objective of the trial was not met, with a median OS of 9.1 vs. 10.8 months (hazard ratio [HR] = 1.05; 95% confidence interval [CI]: 0.728, 1.527; p = 0.79) and the median progression-free survival (PFS) was 5.5 vs. 6.4 months (HR = 1.19; 95% CI: 0.806, 1.764; p = 0.38), in the olaratumab vs. placebo arms, respectively. The most common treatment-emergent adverse event of any grade across both arms was fatigue. Olaratumab plus chemotherapy failed to improve the OS or PFS in participants with metastatic PDAC. There were no new safety signals.

STIM1/SOX2 proteins are co-expressed in the tumor and microenvironmental stromal cells of pancreatic ductal adenocarcinoma and ampullary carcinoma

Pancreatic ductal adenocarcinoma (PDAC) and ampullary carcinoma (AAC) are lethal malignancies with modest benefits from surgery. SOX2 and STIM1 have been linked to anticancer activity in several human malignancies. This study included 94 tumor cases: 48 primary PDAC, 25 metastatic PDAC, and 21 primary AAC with corresponding non-tumor tissue. All cases were immunohistochemically stained for STIM1 and SOX2 and results were correlated with clinicopathologic data, patient survival, and BCL2 immunostaining results. Results revealed that STIM1 and SOX2 epithelial/stromal expressions were significantly higher in PDAC and AAC in comparison to the control groups. STIM1 and SOX2 expressions were positively correlated in the primary and metastatic PDAC (P = 0.016 and, P = 0.001, respectively). However, their expressions were not significantly associated with BCL2 expression. SOX2 epithelial/stromal expressions were positively correlated with the large tumor size in the primary AAC group (P = 0.052, P = 0.044, respectively). STIM1 stromal and SOX2 epithelial over-expressions had a bad prognostic impact on the overall survival of AAC (P = 0.002 and P = 0.001, respectively). Therefore, STIM1 and SOX2 co-expression in tumor cells and intra-tumoral stroma could contribute to the development of PDAC and AAC. STIM1/SOX2 expression is linked to a bad prognosis in AAC.

Abstract 1582: Cabozantinib changes the tumor microenvironment to increase the efficacy of immunotherapy in refractory metastatic pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. Tumors evade immune surveillance by expressing PD-L1 that interacts with the T cell protein PD-1, subsequently inhibiting CD8+ cytotoxic T lymphocyte proliferation and effector function. A plausible reason for this observation may be that other, redundant, immune suppressive mechanisms are at play. Our research team has shown that elevated infiltration of polymorphonuclear (PMN)-MDSCs within the PDAC tumor microenvironment (TME) inhibit T cell effector function, regardless of PD-1/PD-L1 inhibition (Holokai et al., 2020). Objective: A Phase II clinical trial was developed to identify the potential therapeutic benefit of combinatorial anit-PD-L1/PD-1 and cabozantinib (cabo) treatment in therapy-resistant (refractory and metastatic) PDAC patients. Methods: NanoString CosMx™ spatial molecular transcriptomic imaging (CosMx™ SMI) was performed using FFPE sections collected from a liver or lung metastatic target lesions at visit 1 (pretreatment) and visit 2 (9 weeks on combinatorial treatment) from individual patients. FFPE sections were prepared from the same target lesion, and fields of view were chosen on the H&E in consultation with GI pathologist. Seurat V 4.0 was used for the analysis and visualization by spatial mapping of each FOV showing identified cell population indicated by color coding in visit 1 and visit 2 within the same patient. Multiplex immunofluorescence (MxIF) of patient FFPE tissues and single cell RNA sequencing (scRNAseq) of PDAC organoids (PDOs) generated from the core biopsies collected at visit 1 and matched visit 2 from each patient were used to validate the CosMx™ SMI data. Results: A uniform manifold approximation and projection showed loss of tumor cell populations within the PDAC TME in response to combinatorial treatment compared to pretreatment visit 1. Comparison between spatial maps of visit 1 to visit 2 demonstrated a shift in cell populations within defined NICHES (local cellular TME) whereby there was 1) a loss of tumor cell populations, 2) increase in CTL infiltration, 3) increase in endothelial cells, 4) decrease immunosuppressive MDSCs, 5) decrease in antigen presenting CAFs, and 6) change in myCAFs to an inflammatory CAF phenotype. Within the iCAF population IL-6, CXCL1, LIFR, ACTA2 and FAK were expressed; correlating with an increase in CD8 perforin and granzyme B positive cells in response to cabo and checkpoint inhibition. MxIF, spectral flow cytometry and scRNAseq analyses of the matched in vitro PDO model showed tumor cell death that correlated with decreased MDSC viability, increased CTL proliferation and a shift in CAF phenotypes. Conclusion: These findings suggest that cabo induces a TME conducive to increased response to immunotherapy in PDAC patients with refractory metastatic disease. Citation Format: Jayati Chakrabarti, Pritha Adhikary, Jiang Wang, Davendra Sohal, Syed A. Ahmad, Junaid Arshad, Aaron J. Scott, Rachna T. Shroff, Yana Zavros. Cabozantinib changes the tumor microenvironment to increase the efficacy of immunotherapy in refractory metastatic pancreatic ductal adenocarcinoma [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 1582.

Abstract 4737: The combination of the BET inhibitor JQ1 and gemcitabine induces regressions in gemcitabine resistant patient-derived xenograft models of pancreatic cancer

Abstract Efforts to develop targeted therapies for drug resistant tumors have been relatively unsuccessful. The 5-year survival for patients with advanced pancreatic cancer is ~3%. Gemcitabine is usually combined with nab-paclitaxel as a standard of care for treatment of locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). However, no chemotherapeutic regimens thus far evaluated have been curative. We developed the first PDAC patient-derived xenograft (PDX) models in which resistance against gemcitabine was acquired in vivo. We used RNA-seq and ingenuity pathway analysis to compare expression profiles of paired parent vs gemcitabine resistant PDX models. This comparison suggested a previously unreported mechanism for gemcitabine resistance, involving aberrations of cholesterol biosynthesis and lipid metabolism pathways. We also made the unexpected observation that gemcitabine-resistant PDAC models derived from primary tumors have high levels of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), an enzyme essential to cholesterol biosynthesis and rate limiting in ketogenesis. Notably, the BET bromodomain inhibitor JQ1 decreases HMGCS2 levels; and BET inhibitors + gemcitabine exert synergistic cytotoxicity in gemcitabine-resistant PDAC cells. We determined that the BET bromodomain inhibitor JQ1 + gemcitabine induces regressions of two in vivo gemcitabine-resistant tumor models which we developed directly from primary human tumor tissue. These mechanism-based studies suggest that inhibiting HMGCS2 represents a novel approach to therapy for PDAC. We propose that combining gemcitabine with agents that decrease levels of HMGCS2 comprises effective treatment for patients with drug resistant PDAC. Citation Format: Aubrey L. Miller, Samuel C. Fehling, Eric Josh Brown, Eric O. Heard, Tracy L. Gamblin, Rebecca B. Vance, Karina J. Yoon. The combination of the BET inhibitor JQ1 and gemcitabine induces regressions in gemcitabine resistant patient-derived xenograft models of pancreatic cancer [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 4737.

Abstract 934: RNA-based precision medicine predicts sensitivity to selinexor in select pancreatic ductal adenocarcinoma patients

Abstract Sensitivity to therapeutic agents is impacted both by genetic heterogeneity between patients or among clones within a tumor and by heterogeneity of epigenetic cell states. Prior work demonstrates that human pancreatic tumors comprise malignant cells in a mixture of multiple cell states with distinct genetic dependencies, leading to a reservoir of chemoresistance across the tumor; targeting just one or two malignant cell states is insufficient to achieve durable therapeutic responses. One approach to overcome this challenge is to identify agents with efficacy against malignant cells in multiple cell states. However, in the absence of genetic distinctions between cell states, identifying therapeutic vulnerabilities for each malignant cell state present in a patient’s tumor in real time remains a challenge. OncoTreat is a CLIA certified algorithm that matches drugs to patients based on tumor gene expression profiles. The approach utilizes regulatory network analysis, a systems biology framework that transforms gene expression data into regulatory protein activity profiles, in a manner that facilitates the identification of master transcriptional regulators of cell state. We carried out a Phase 1 clinical trial of the OncoTreat framework to examine feasibility for implementing RNA-based precision medicine in patients with metastatic pancreatic ductal adenocarcinoma (2nd line and beyond). Patient-derived xenografts were established from subjects prior to 1st line therapy, and selected agents matched by OncoTreat were assessed co-clinically while subjects received standard of care regimens, with the potential for subsequent treatment with successful agents upon progression. Two subjects in the study matched to selenexor, an XPO1 inhibitor that is FDA approved for multiple myeloma. Co-clinical studies of selinexor in patient-derived xenograft models generated from the matched subjects showed prolonged survival compared to control regimens. Single-cell RNA sequencing analysis of these tumors found that the activity of master regulators confirmed that the activity of selinexor-responsive master regulators were inverted in response to treatment in vivo, reducing the fraction of malignant cells in the tumors. Strikingly, selenexor treatment was associated with significant inhibition of RAS/MAPK signaling, suggesting a potential novel role for selinexor targeting Ras. Together, these data indicate a potential therapeutic vulnerability for a subset of PDAC patients that can be predicted by a CLIA-certified RNA-based precision medicine platform. Citation Format: Alvaro Curiel-Garcia, Lorenzo Tomassoni, Tanner C. Dalton, Amanda R. Decker-Farrell, Carmine F. Palermo, Daniel R. Ross, Stephen A. Sastra, Urszula N. Wasko, Isabel M. Goncalves, Prabhot Mundi, Basil S. Bakir, Rachael A. Safyan, Hanina Hibshoosh, Gulam A. Manji, Andrea Califano, Kenneth P. Olive. RNA-based precision medicine predicts sensitivity to selinexor in select pancreatic ductal adenocarcinoma patients [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 934.

Abstract 2376: Mitazalimab, a potent CD40 agonist in combination with FOLFIRINOX demonstrates changes consistent with increased immune activation in TME and peripheral blood in a preclinical pancreatic cancer tumor model

Abstract Mitazalimab is a human CD40 FcγR-conditional agonistic IgG1 antibody developed for cancer immunotherapy. Immune-excluded tumors, such as pancreatic ductal adenocarcinoma (PDAC), are defined by low infiltration of immune cells. In PDAC, effector CD8+ T cells are excluded from the tumor by its desmoplastic stroma which surrounds cancer cell islets and hosts immunosuppressive macrophages. CD40 is expressed on key myeloid cell populations and targeting this receptor has a two-fold mode of action. First, mitazalimab activates macrophages, skewing them towards a tumoricidal phenotype that can potentially reverse the suppressive tumor microenvironment found in PDAC and sensitizes the tumor for chemotherapy by stromal degradation. Second, mitazalimab activates antigen-presenting cells, resulting in priming and expansion of tumor-specific T cells with the potential to generate an effective immune response that could provide immunological memory resulting in long term benefits to patients. Mitazalimab is currently being investigated in a phase 2 study in patients with metastatic PDAC (OPTIMIZE-1) in combination with modified FOLFIRINOX (NCT04888312). The treatment starts with a dose of mitazalimab administered one week prior to chemotherapy in the first cycle to leverage the mode of action and increase the efficacy of the combination therapy. Interim results of the study demonstrated encouraging anti-tumor activity with good durability of responses. To study the changes induced by mitazalimab and FOLFIRINOX in the tumor microenvironment, we evaluated the combination in human CD40 transgenic mice using the KPCY pancreatic tumor model. Tumor, tumor draining lymph nodes, blood and spleen were analyzed for changes in the immune compartment using multiplex assays at different time points. The results showed distinct immunophenotypical changes induced by mitazalimab, FOLFIRINOX and the combination. The combination of mitazalimab and FOLFIRINOX resulted in an increased tumoricidal immune profile, including a marked increase in effector CD8+ T cells compared to the individual therapies. Overall, these preclinical data support the chosen dosing regimen of mitazalimab and modified FOLFIRINOX in the OPTIMIZE-1study and underline the synergistic potential of this combination for an improved antitumor response. Citation Format: Karin Enell Smith, David Gomez Jimenez, Mia Thagesson, Anneli Nilsson, Yago Pico de Coaña, Sumeet Ambarkhane, Peter Ellmark. Mitazalimab, a potent CD40 agonist in combination with FOLFIRINOX demonstrates changes consistent with increased immune activation in TME and peripheral blood in a preclinical pancreatic cancer tumor 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 2376.

Abstract 5: Cytosine base editor ameliorates the safety profile of TCR edited T cells for the adoptive cell therapy of gastrointestinal tumors

Abstract Among gastrointestinal tumors, colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) are major unmet medical needs, with a high mortality rate mainly related to the onset of metastases, hindering the therapeutic efficacy of conventional treatments. Adoptive Cell Therapy (ACT) with TCR-edited T cells is a promising therapeutic strategy for patients with late-stage tumors, but its applicability is limited by the paucity of TCRs targeting relevant tumor antigens and the presence of an immunosuppressive tumor microenvironment. Moreover, the safety of the therapeutic cellular products, generated by genome editing tools, is fundamental for an effective and rapid clinical translation. With the aim of improving the safety profile of T-cell based therapeutic products manufactured by genome editing, we evaluated the efficacy and safety of cytosine base editor (CBE). We disrupted the endogenous TCR to redirect T cells specificity toward tumor cells, and TIGIT, a major inhibitory molecule in gastrointestinal tumors to enhancing T cell ability to counteract immunosuppression. Beyond the high efficiency of CBE in simultaneously disrupting the target genes, we observed only marginal sgRNA-dependent and -independent off-target events as measured by targeted sequencing of the top predicted off-target loci and by ultra-deep whole exome sequencing, respectively. In addition, unlike CRISPR/Cas9-treated cells, we could not detect translocations in CBE edited T cells. To select the antigens to target by engineered T cells, we investigated published datasets and identified 19 relevant tumor-associated antigens. By clonal tracking of the TCR repertoire of T cells stimulated with autologous APCs loaded with the selected antigens, we isolated 5 different TCRs specificities, including mesothelin. Importantly, these antigens are expressed in our cohort of primary and metastatic PDAC and CRC patients. TCR-edited T cells displayed anti-tumor activity against PDAC cell lines, CRC and PDAC patient-derived organoids while sparing HLA-unrelated and antigen-negative controls. To boost the efficacy of anti-tumor T cells, we coupled TIGIT disruption with TCR editing. By challenging TCR-edited TIGITKO T cells in vitro and in an orthotopic model of CRC liver metastases, we observed that TIGIT disruption can ameliorate the ability of tumor-specific T cells to control tumor growth. Our findings suggest that base editors can generate cellular products with a better safety profile and a promising therapeutic efficacy in gastrointestinal tumors. Citation Format: Martina Spiga, Alessia Potenza, Stefano Beretta, Alessia Airaghi, Chiara Iozzi, Martina Fiumara, Samuele Ferrari, Neda Mohammadi, Oronza Botrugno, Maximilian Reichert, Maria Pia Protti, Stefano Crippa, Massimo Falconi, Luigi Naldini, Eliana Ruggiero, Chiara Bonini. Cytosine base editor ameliorates the safety profile of TCR edited T cells for the adoptive cell therapy of gastrointestinal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5.

Abstract 6313: Enhancing CAR T cell-based immunotherapy outcomes in PDAC: Evaluating iC9.B7-H3 CAR T cells in adjuvant, neoadjuvant, and monotherapy settings

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is among the most aggressive and lethal cancers worldwide. Despite negative margin resections and subsequent systemic therapy, PDAC often recurs. Additionally, most patients present with locally advanced and unresectable tumors at the time of diagnosis. Cancer-associated fibroblasts (CAFs) contribute to the highly desmoplastic tumor microenvironment (TME) and form a biophysical barrier, limiting immune cell infiltration. Moreover, CAF-secreted factors suppress the anti-tumor activity of T cells and shift cancer cells toward invasive proliferative and mesenchymal phenotypes. Therefore, targeting CAFs can enhance T-cell infiltration and improve the efficacy of T-cell based immunotherapeutic strategies. B7-H3 is an immune checkpoint molecule highly expressed on PDAC cells and CAFs. In this study, we investigate the use of Chimeric Antigen Receptor (CAR) T cells targeting B7-H3 to eliminate metastatic PDAC in a preclinical mouse model in three different settings: I. Adjuvant II. Neoadjuvant III. CAR T cell monotherapy. Our CART-B7-H3 construct includes an inducible caspase 9 (iC9) suicide gene, which allows for the controlled elimination of the CAR T cells in the event of overwhelming cytokine release syndrome. Methods: To validate the therapeutic potential of iC9.B7-H3 CAR T cells in vivo, NSG mice were orthotopically engrafted with 1x105 patient-derived PDAC6 cells and 9x105 hCAF1 cells at a ratio of 1:9, reflecting a clinically relevant model. In the adjuvant group, mice underwent surgical resection of the primary tumor (distal pancreatectomy and splenectomy) after 25 days. On days 30 and 60, they received systemic iC9.B7-H3 CAR T cell treatment (5x106). In the neoadjuvant group, mice were systemically treated with iC9.B7-H3 CAR T cells on day 19 and underwent surgical resection of the primary tumor on day 25, following another systemic CAR T cell dose on day 60. In the CAR T monotherapy group, mice received systemic iC9.B7-H3 CAR T cells on days 19 and 60. Results: In vivo, iC9.B7-H3 CAR T cells in the neoadjuvant group effectively eradicated both local and distant PDAC metastases for the length of the experiment (180 days) and significantly increased the survival (P <0.0001), indicating long-term efficacy in the treatment of PDAC metastases with no adverse side effects. In the adjuvant and CAR T monotherapy groups, iC9.B7-H3 CAR T cells effectively control tumor growth, eliminate metastases, and significantly increase survival (P=0.0018 and P=0.0007, respectively). Conclusion: These data suggest that the neoadjuvant application of iC9.B7-H3 CAR T cells may demonstrate persistent efficacy in eradicating both local and distant PDAC metastases. iC9.B7-H3 CAR T cells adjuvantly and as monotherapy achieved significant tumor control. Citation Format: Shahrzad Arya, Seyed Amir Sanatkar, Marco Ventin, Giulia Cattaneo, Cristina Martin, Gabriella Lionetto, Jingyu Jia, Feng Chen, David T. Ting, Xinhui Wang, Sandra Ryeom, Cristina R. Ferrone, Soldano Ferrone. Enhancing CAR T cell-based immunotherapy outcomes in PDAC: Evaluating iC9.B7-H3 CAR T cells in adjuvant, neoadjuvant, and monotherapy settings [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 6313.

Abstract 1392: Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling

Abstract Neutrophil extracellular traps (NETs) are an important contributor to the immunosuppressive tumor microenvironment and facilitate metastasis of solid cancers. NETs have been shown to stimulate breast and colon cancer cell metastasis through a mechanism potentially involving an interaction between integrin-linked kinase (ILK) and the cell surface receptor coiled-coil domain containing protein 25 (CCDC25). Here, we have interrogated whether ILK is required for NET-induced metastasis in pancreatic ductal adenocarcinoma (PDAC), an inflammation-driven tumor that is difficult to treat. Evaluation of the circulating neutrophil to lymphocyte ratio (NLR) in the blood samples of patients presenting with metastatic PDAC showed that a higher baseline NLR is associated with significantly shorter overall patient survival. Western blot and immunofluorescence analyses demonstrated that human PDAC cell lines, patient-derived xenograft cells, cells derived from the KPCY genetic model of PDAC and patient-derived organoids co-express CCDC25 and ILK. Co-immunoprecipitation analyses showed that CCDC25 and ILK associate in both mouse and human PDAC cells, and that exposure of the cells to NETs results in an increased amount of ILK associated with CCDC25. Exposure of human and KPCY mouse PDAC cells to NETs increases phosphorylation of GSK3-β and ERK, increases activation of Rac1 and Cdc42 and upregulates markers of epithelial-to-mesenchymal transition (EMT), including ZEB1 and Snail, in a time and concentration-dependent manner. Furthermore, time-lapse imaging using Incucyte-based wounding assays showed that exposure to NETs induces pseudopodia-like protrusions, migration and invasion by these cells. RNAi-mediated suppression of ILK expression or inhibition of ILK activity using a highly selective inhibitor of ILK, QLT-0267, significantly inhibits the NET-induced increase in GSK3-β phosphorylation, Rac1/Cdc42 activation, migration and invasion of PDAC cells. In vivo, co-localization of CCDC25 and ILK is observed in tumors and metastases from KPCY mouse and MIA PaCa-2 human PDAC xenograft models stained by multispectral immunofluorescence and analyzed by confocal microscopy. Tumors and metastases show the presence of infiltrating neutrophils and NETs as indicated by staining for myeloperoxidase and citrullinated histone H3. Importantly, doxycycline-inducible shRNA-mediated suppression of ILK expression by luciferase-positive PDAC cells following injection of these cells through the tail vein of mice administered LPS intranasally results in a significant reduction in metastasis as measured by bioluminescence-based imaging, compared to control animals. Collectively, these data identify NET-induced ILK signaling as an important contributor to PDAC progression and suggest the potential for targeting this axis to prevent PDAC progression and metastasis. Citation Format: Paul C. McDonald, Shannon J. Awrey, Hossein Tavakoli, Rebekah Carroll, Zachary J. Gerbec, Joanna M. Karasinska, David F. Schaeffer, Daniel J. Renouf, Shoukat Dedhar. Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling [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 1392.

Abstract 2499: Window-of-opportunity trial of metastatic pancreatic cancer reveals potential mechanisms of response to targeting RAS signaling

Abstract KRAS is mutated in ~85% of pancreatic ductal adenocarcinoma (PDAC) patient's tumors, suggesting that it is a key node of PDAC targeted therapy. RAF/MEK/ERK signaling is downstream of KRAS signaling. Preclinical studies targeting MEK showed promises but clinical trials failed to show the benefit compared to Standard-of-Care treatment chemotherapy. To elucidate the mechanism of action and resistance to several targeted therapies including MEK inhibition in human PDAC, we conducted a Window-Of-Opportunity trial for Metastatic pancreatic cancer (WOOM trial). In this trial, metastatic PDAC patients received 10 days of Cobimetinib, a FDA approved MEK inhibitor, therapy and specimens of pre- and post- treatment were obtained by biopsy to evaluate the response to targeted treatment using deep multi-omic analytics including DNA-seq, RNA-seq, multiplex IHC, cyclic IF and Digital Spatial Profiling (DSP). The clinical grade KI67 staining positivity was used as a readout of tumor response. We found three of 15 patient’s tumors had significantly decreased KI67 staining positivity after Cobimetinib treatment and defined them as Responsive tumors. Genomic sequence showed that KRAS allelic imbalance and KrasG12R mutations were seen significantly higher frequency in Responsive tumors compared to Non-responsive tumors. RNA-seq analysis showed that these Responsive tumors were more likely to be classical-like subtype compared to Non-responsive tumors. DSP analysis confirmed the downregulation of ERK phosphorylation after treatment in all examined cases consistent with effective target inhibition. Interestingly, MEK phosphorylation was upregulated after treatment paradoxically. This suggests that paradoxical upstream pathway activation including RAF activation could be potential resistance mechanism. MYC protein expression change was significantly correlated with KI67 protein expression change, which suggests MYC downregulation could contribute to action of Cobimetinib. We also found that Cobimetinib treatment increased the immunosuppressive TREM2+ Macrophage population from single cell RNA-seq data. Furthermore, PDX from biopsy recapitulated patient's response to therapy and could be a great tool for testing new therapeutic strategies. Here, we summarize the multi-omics data and discuss potential mechanisms of response and resistance to targeting RAS signaling and related microenvironmental changes. Citation Format: Motoyuki Tsuda, Colin Daniel, Xiaoyan Wang, Carl Pelz, Hayley Zimny, Alexander Smith, John Muschler, Xi Li, Tugba Yildiran Ozmen, Furkan Ozmen, Dove Keith, Christopher Corless, Koei Chin, Jonathan Brody, Charles Lopez, Gordon Mills, Rosalie Sears. Window-of-opportunity trial of metastatic pancreatic cancer reveals potential mechanisms of response to targeting RAS signaling [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 2499.

Abstract 445: Determining the molecular and biologic effects of HER3 and IDH1 antagonism on liver endothelium-PDAC crosstalk

Abstract INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy due to the lack of early diagnosis and limited response to treatments. Austere and nutrient-deprived conditions typically present in the PDAC tumor microenvironment (TME) induce chemotherapy resistance. Therefore, novel therapeutic strategies are needed. We have previously demonstrated the primary tumor expresses IDH1 which supports the pro-survival oxidative phosphorylation (OXPHOS) metabolism, and has been successfully targeted by ivosidenib, an IDH1 inhibitor, in preclinical studies. Meanwhile, endothelial cells (ECs) from the liver, the most common site of PDAC metastases, secrete soluble factors in the conditioned medium (CM) that activate HER3, a receptor tyrosine kinase, involved in cell proliferation and metabolism. OBJECTIVES: We sought to (1) uncover the mechanisms involved in driving the metabolic differences between primary and metastatic tumors through the utilization of ECs from the liver (2) conduct metabolic analyses to determine metabolic effects of HER3 activation, and (3) examine potential synergism of combination therapy against HER3 and IDH1 in primary and metastatic niches. METHODS: The effect of CM on PDAC cell growth was determined by PICO Green Assay. The effects of ECs HER3/IDH1 inhibitions on cellular metabolism were measured by multiple metabolic assays including Seahorse, CellTiter-Glo, lactate ELISA, and TMRE assays. HER3-specific antibody Seribantumab, HER3 inhibitor Sapitanib, and IDH1 inhibitor ivosidenib were used to block HER3 and/or IDH1. Moreover. A cell viability-based synergy study was performed to assess the effects of HER3-IDH1 combination. Effects of HER3 and IDH1 inhibitions on PDAC tumor growth and mouse survival in vivo were determined in syngeneic, orthotopic models with PDAC primary tumors and liver metastases. RESULTS: CM from liver ECs significantly increased cell growth in PDAC cells in vitro and increased glycolytic tropism in multiple HER3 positive PDAC cell lines measured by metabolic assays mentioned above. HER3 antibody and HER3 inhibitor both increased oxidative consumption rate (OCR), an indirect measure of OXPHOS, and decreased extracellular acidification rate (ECAR), a measure of glycolysis measured by Seahorse. Combination therapy utilizing HER3 and IDH1 inhibitors revealed synergy. In our in vivo tumor model, mice treated with combination therapy had significantly longer overall survival than those given either monotherapy or no treatment. CONCLUSIONS: Our results demonstrated a paracrine role of liver ECs in promoting cell growth via activating HER3 in PDAC cells as well as reprograming cellular metabolism from OXPHOS towards glycolysis. Identification of the metabolic shift that takes place in metastatic PDAC to the liver due to HER3 activation gives way for the development of a potentially lethal drug combination. Citation Format: Christina S. Boutros, Alexander W. Loftus, Moeez Rathore, Mehrdad Zarei, Kimberly Curry, Jordan M. Winter, Rui Wang. Determining the molecular and biologic effects of HER3 and IDH1 antagonism on liver endothelium-PDAC crosstalk [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 445.

Abstract 1384: Highly metastatic PDAC adopts mesenchymal morphology and spatially coordinates endothelial cells with macrophages

Abstract Background: Patients with pancreatic ductal adenocarcinoma (PDAC) often have metastatic disease at diagnosis and are highly resistant to cytotoxic and immunotherapies. Increased presence of myeloid cells, specifically high myeloid-to-T cell ratios, correlates with worse prognoses. However, mechanisms by which myeloid cells regulate cancer invasion and metastasis remain poorly understood. To study the role of immunosuppressive myeloid cells in driving metastasis, we employed murine PDAC models in conjunction with organoid systems and imaging mass cytometry (IMC). Methods: We used two murine cell lines driven by KrasG12D and Trp53R172H under Pdx1-Cre, KPCY 2838c3 and KPCY 6419c5, which exhibit low-metastatic and high-metastatic propensities, respectively. To characterize differences in the metastatic cascade between these cell lines, flow cytometry, histological analysis, and 3D organoid modeling were used. IMC was also used to interrogate the tumor microenvironment and identify spatial relationships. Results: These cell lines were orthotopically injected into syngeneic C57BL/6J mice to generate tumors that naturally metastasize to the liver. Flow cytometry analysis of livers from mice bearing 2838c3 or 6419c5 pancreatic tumors validated the higher propensity for metastasis with the 6419c5 cells (p=0.0484). Furthermore, the steps along the metastatic cascade contrasted significantly between 6419c5 and 2838c3. Highly metastatic PDAC adopted mesenchymal rather than collective invasion, increased intravasation, and more robust metastatic outgrowth. Notably, IMC revealed that highly metastatic PDAC had increased endothelial cell abundance (***p ≤ 0.001), lower cytotoxic CD8+ T cell to CD4+ regulatory T cell ratios (***p ≤ 0.001), and lower M1-like to M2-like tumor associated macrophage (TAM) ratios (*p ≤ 0.05). Furthermore, spatial analysis demonstrated that highly metastatic PDAC excluded lymphocytes from the tumor and situated Arginase-1+ TAMs near Ki67+ tumor cells and CD31+ endothelial cells. In contrast, low-metastatic PDAC had closer lymphocyte proximities to tumor and greater distances between Arginase-1+ TAMs, tumor, and endothelial cells. Conclusions: The highly metastatic PDAC tumor line utilizes mesenchymal invasion, has increased intravasation, and robust metastatic outgrowth compared to the low-metastatic PDAC cells. The TME of the highly metastatic model had disproportionate abundances of T regulatory cells and M2-like macrophages, poor lymphocyte infiltration, and enhanced vascularization. These overabundant M2-like macrophages (Arginase-1+) were also spatially coordinated with proliferating tumor cells and vasculature, suggesting a potential role for macrophages in tumor cell invasion and/or angiogenesis. Citation Format: Nicole E. Gross, Soren Charmsaz, Jae W. Lee, Cheng-Chieh Huang, Alexei G. Hernandez, Sarah M. Shin, Erin M. Coyne, Courtney Cannon, Xuan Yuan, Laura D. Wood, Elizabeth M. Jaffee, Won Jin Ho. Highly metastatic PDAC adopts mesenchymal morphology and spatially coordinates endothelial cells with 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 1384.

Abstract 1579: Macrophage infiltration and polarization in pancreatic ductal adenocarcinoma following stereotactic body radiation therapy

Abstract Introduction: Stereotactic body radiation therapy (SBRT) has emerged as a promising treatment modality for pancreatic ductal adenocarcinoma (PDAC) due to its precise delivery and potential to improve local control. Beyond its direct impact on tumor cells, accumulating evidence suggests that SBRT may also modulate the tumor microenvironment (TME), influencing immune responses. Macrophages play a crucial role in the TME with their polarization into pro-inflammatory (M1) or pro-tumoral (M2) phenotypes significantly impacting cancer progression. Studying the TME and developing immune therapies may be helpful for metastatic PDAC, which currently only has non-curative treatment. The TME, in particular the polarization of macrophages, has not been well studied following radiation treatment. Therefore, our work aims to study how SBRT affects the infiltration and polarization of macrophages in the PDAC TME in a time-dependent manner. Methods: To establish and study PDAC C57BL/6J mice were orthotopically injected with a luciferase-labeled KPC tumor cell line. SBRT was administered to tumor-bearing mice following a schedule of 10Gy radiation per fraction in total of 5 fractions on days 10-14 post tumor implantation. Mice were euthanized on day 1, 3 and 7 after irradiation. Tumor tissues were collected for immunohistochemistry (IHC) staining and flow cytometry with antibodies targeting CD68, CD11b, CD86 (M1 marker), and CD206 (M2 marker). Images were analyzed and quantified using ImageJ and GraphPad Prism. Results: Via the use of IHC staining, we found no significant difference in the number of cells stained with the CD68 antibody (macrophage marker) when compared throughout groups. Additionally, we found no significant difference in the number of macrophages stained with the M2 marker. However, we did observe a significant increase in the number of macrophages stained with the M1 marker in the group receiving SBRT. Through flow cytometry of the tumor tissues we observed an increasing trend, as time passed, in the number of macrophages expressing the M1 marker. The same trend was observed in macrophages expressing both the M1 and M2 markers. However, a change in the total number of macrophages or those expressing only the M2 marker was not observed. Conclusions: SBRT does not affect the total number of infiltrated macrophages in the TME or the amount of M2 macrophages. However, we did observe that the percentage of M1 macrophage increases significantly as time passes after SBRT. We theorize that the increase in M1 macrophages may be due to an M2 to M1 transition in the M2 population. Our study provides preliminary rationale to use macrophage-targeted immune therapy to potentiate the efficacy of SBRT for PDAC treatment, though we understand that further follow-up studies with a larger sample size and longer time course are required to obtain significant and meaningful results. Citation Format: Emil D. Gonzalez Marrero, Liang Wang, Benjamin R. Schrank, Albert C. Koong. Macrophage infiltration and polarization in pancreatic ductal adenocarcinoma following stereotactic body radiation therapy [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 1579.

Ectopic CXCR2 expression cells improve the anti-tumor efficiency of CAR-T cells and remodel the immune microenvironment of pancreatic ductal adenocarcinoma

BackgroundRecent progressions in CAR-T cell therapy against pancreatic ductal adenocarcinoma (PDAC) remain disappointing, which are partially attributed to the immunosuppressive microenvironment including macrophage-mediated T cell repletion.MethodsWe first characterized the expression patterns of macrophage-relevant chemokines and identified CXCR2 as the key factor regulating T cell trafficking and tumor-specific accumulation in PDAC microenvironment. After that, we synthesized and introduced a CXCR2 expression cascade into Claudin18.2 CAR-T cells and compared the behaviors of CAR-T cells in vitro and in vivo. The therapeutic potential of CXCR2 CAR-T was evaluated in two different allogeneic models: subcutaneous allografts and metastatic PDAC models.ResultsThe results showed that CXCR2 CAR-T not only reduced the size of allografted PDAC tumors, but also completely eliminated the formation of metastases. Lastly, we investigated the tumor tissues and found that expression of ectopic CXCR2 significantly improved tumor-targeted infiltration and residence of T cells and reduced the presence of MDSCs and CXCR2 + macrophages in PDAC microenvironment.ConclusionOur studies suggested that ectopic CXCR2 played a significant and promising role in improving the efficiency of CAR-T therapy against primary and metastatic PDAC and partially reversed the immune-suppressive microenvironment.