Pancreatic Ductal Adenocarcinoma Tumors Research Articles

A 15-Inflammation-Related Gene Signature Predicts the Prognosis of Patients With Pancreatic Ductal Adenocarcinoma

Chronic inflammation promotes the development of pancreatic ductal adenocarcinoma (PDAC) and PDAC-related inflammatory tumor microenvironment facilitates tumor growth and metastasis. Thus, we aimed to study the association between inflammatory response and prognosis in patients with PDAC. We conducted the whole transcriptomic sequencing using tissue samples collected from patients diagnosed with PDAC (n = 106) recruited from Shandong Cancer Hospital. We first constructed a prognostic signature using 15 inflammation-related genes in The Cancer Genome Atlas (TCGA) cohort (n = 177) and further validated it in an independent International Cancer Genome Consortium (ICGC) cohort (n = 90) and our in-house cohort. PDAC patients with a higher risk score had poorer overall survival (OS) (P < 0.001; HR, 3.02; 95% CI, 1.94-4.70). The association between the prognostic signature and OS remained significant in the multivariable Cox regression adjusting for age, sex, alcohol exposure, diabetes, and stage (P < 0.001; HR, 2.91; 95% CI, 1.73-4.89). This gene signature also robustly predicted prognosis in the ICGC cohort (P = 0.01; HR, 1.94; 95% CI, 1.14-3.30) and our cohort (P < 0.001; HR, 2.40; 95% CI, 1.45-3.97). Immune subtype C3 (inflammatory) was enriched and CD8+ T cells were higher in patients with a lower risk score (P < 0.05). Furthermore, PDAC patients with higher risk scores were more sensitive to chemotherapy, immunotherapy, and PARP inhibitors (P < 0.05). In sum, we identified a novel gene signature that was associated with inflammatory response for risk stratification, prognosis prediction, and therapy guidance in PDAC patients. Future studies are warranted to validate the clinical utility of the signature.

Evaluations of the combined use of blood- and tissue-based protein biomarkers for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a low 5-year survival rate. Biomarkers may be of value for the early diagnosis of pancreatic cancer. This study assessed blood- and tumour tissue-based biomarkers associated with pancreatic cancer. We studied 61 patients who underwent pancreatic resection. Of these 61 patients, 46 patients had PDAC, and 15 patients had inflammatory tumours. Blood and tumour tissue levels of VEGF, hypoxia-inducible factor 1α (HIF-1α) and glucose transporter 1 (GLUT1) were measured. Blood concentrations of VEGF (p < 0.000001) and HIF-1α (p = 0.000002) were significantly higher in the PDAC group than in the inflammatory tumour group. Tumour tissue concentrations of VEGF (p < 0.000001), HIF-1α (p = 0.000005) and GLUT1 (0.000002) were also significantly higher in the PDAC group. Univariate analyses revealed that age, BMI, and blood levels of CA19-9, VEGF, and HIF-1α were potential predictors of PDAC. Potential predictors of PDAC in tumour tissue were VEGF, HIF-1α and GLUT1. Multivariate analyses found that VEGF was the most powerful independent predictor of PDAC in blood (OR = 1.016; 95% CI: 1.007-1.025; 0.001) and tumour tissue (OR = 1.02; 95% CI: 1.008-1.032, p = 0.001). The cut-off point for blood VEGF was 134.56 pg/ml, with a sensitivity of 97.8%, specificity of 86.7%, PPV of 95.7%, and NPV of 92.9%. The cut-off point for tissue tumour VEGF in PDAC was 208.59 pg/mg, with a sensitivity, specificity, PPV and NPV of 97.7%, 92.9%, 97.7%, and 92.9%, respectively. There are significant differences in blood-based biomarkers for differentiating between PDAC and inflammatory tumours of the pancreas. VEGF was an independent predictor of PDAC independent of its addition to the routinely used tumour marker CA19-9 antigen.

Mesothelin CAR T-cells secreting anti-FAP/anti-CD3 molecules efficiently target pancreatic adenocarcinoma and its stroma.

Targeting solid tumors with CAR T-cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAFs), which may contribute to the limited efficacy of mesothelin-directed CAR T-cells in early-phase clinical trials. To provide a more favorable TME for CAR T-cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T-cells with an anti-mesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAFs through fibroblast activation protein (FAP) and engages T-cells through CD3 (termed mesoFAP CAR-TEAM cells). Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAFs, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAFs within the TME. We developed and used patient-derived ex vivo models including patient-derived organoids with patient-matched CAFs and patient-derived organotypic tumor spheroids (PDOTS). We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAFs compared to T cells engineered to target either antigen alone in our ex-vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors. CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.

The Co-Localization of NLRP3 and ASC Specks Does Not Automatically Entail NLRP3 Inflammasome Functionality in PDAC Cell Lines

The NLRP3 inflammasome is an important mediator of the host inflammatory response, and downregulation of inflammation is important in cancer treatment. Here, we investigated four different pancreatic ductal adenocarcinoma (PDAC) cell lines, AsPC-1, BxPC-3, CFPAC-1 and Panc-1, with regards to NLRP3 inflammasome formation and cytokine secretion. ASC specks were observed in all the cell lines investigated, but AsPC-1 was the only cell-line with the co-localization of anti-ASC and anti-NLRP3 and spontaneously formed multiple NLRP3 inflammasomes per cell. The co-localization of NLRP3 and ASC was not accompanied by IL-1β release nor significant IL-18 release. BxPC-3 displayed relatively high expression of the inflammasome-related genes IL1B and CASP1 and had the highest levels of IL1β and IL18 secretion and the highest amount of ASC. The inflammasome-associated genes IL18 and PYCARD were up-regulated in the PDAC primary tumors compared to normal tissue, and high PDAC tumor expression of IL18, CASP1 and PYCARD correlated with low patient survival. We have shown that PDAC cell lines display significant variations in their inflammasome-related gene expression and readouts. We conclude that spontaneous ASC speck formation is possible in PDAC cells and that multiple NLRP3 inflammasomes are formed spontaneously in AsPC-1 cells but that the co-localization of NLRP3 and ASC specks does not automatically entail inflammasome function.

Inoculation of Pan02 cells produces tumor nodules in mouse pancreas: Characterization of a novel orthotopic pancreatic ductal adenocarcinoma tumor model for interventional studies.

Preclinical models of cancer are vital for assessing and predicting efficacies and toxicities of novel treatments prior to testing in human subjects. Current pancreatic tumor models exhibit variable growth rates, unpredictable tumor size after implantation in non-native tissues, or require surgical implantation. Surgical implantation in the pancreas may produce not only unpredictable tumor uptake but could also elicit additional inflammatory responses. In searching for a pancreatic carcinoma cell that can be introduced into a mouse via simple injection, we found that Pan02, a murine ductal pancreatic adenocarcinoma derived from a pancreatic lesion of a C57BL/6 mouse, inoculated peritoneally can consistently produce pancreatic tumors. This intraperitoneal, but not intravenous, introduction of Pan02 cells leads to the attachment and growth of Pan02 in the pancreas before spreading to other tissues. Time-course tissue analysis indicates that the Pan02 cells first find, infiltrate, and grow within the pancreas, producing a pancreatic tumor model. This model appears to mimic pancreatic cancer development in humans and is the first reported use of Pan02 cells to produce orthotopic pancreatic and metastatic neoplasms in a mouse model without the need for tumor implantation within matrices or survival surgeries. This orthotopic pancreatic tumor model, with consistent tumor uptake, synchronized tumor development and survival, and predictable outcomes may enable and accelerate the preclinical evaluation of treatment candidates for pancreatic cancer.

Collagen Lattice Model, Populated with Heterogeneous Cancer-Associated Fibroblasts, Facilitates Advanced Reconstruction of Pancreatic Cancer Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is a solid-tumor malignancy. To enhance the treatment landscape of PDAC, a 3D model optimized for rigorous drug screening is essential. Within the PDAC tumor microenvironment, a dense stroma comprising a large extracellular matrix and cancer-associated fibroblasts (CAFs) is well-known for its vital role in modulating tumor growth, cellular heterogeneity, bidirectional paracrine signaling, and chemoresistance. In this study, we employed a fibroblast-populated collagen lattice (FPCL) modeling approach that has the ability to replicate fibroblast contractility in the collagenous matrix to build dense stroma. This FPCL model allows CAF differentiation by facilitating multifaceted cell-cell interactions between cancer cells and CAFs, with the differentiation further influenced by mechanical forces and hypoxia carried within the 3D structure. Our FPCL models displayed hallmark features, including ductal gland structures and differentiated CAFs with spindle shapes. Through morphological explorations alongside in-depth transcriptomic and metabolomic profiling, we identified substantial molecular shifts from the nascent to mature model stages and potential metabolic biomarkers, such as proline. The initial pharmacological assays highlighted the effectiveness of our FPCL model in screening for improved therapeutic strategies. In conclusion, our PDAC modeling platform mirrors complex tumor microenvironmental dynamics and offers an unparalleled perspective for therapeutic exploration.

Abstract 7217: Orthogonal targeting of NAD metabolism and ErbB2/ErbB3 signaling in pancreatic adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is now the third leading cause for cancer related deaths in the US with a five-year survival of ~10% that has improved little over the past 20 years. Thus, there is a critical need to fully understand mechanisms of PDAC tumorigenesis in order to develop novel and effective therapies. Nicotinamide Adenine Dinucleotide (NAD), a critical co-factor and energy source required for cellular processes such as DNA repair and epigenetic regulation is frequently upregulated in PDAC. We recently reported a novel oncogenic axis connecting the NAD-dependent epigenetic regulator and oncogene C-terminal Binding Protein (CtBP) with the EGFR family growth factor receptors ErbB2/ErbB3 (ErbB2/3) in PDAC cells and tumors. Indeed, previous reports have shown ~30% of human PDAC tumors overexpress ErbB3, and our work has shown that PDAC tumor cell growth relies on maintenance of ErbB3 expression. Moreover, we show that the majority of PDAC cells with high levels of ErbB3 expression are uniquely sensitive to the ErbB2-targeted tyrosine kinase inhibitor lapatinib. In addition, we have recently shown that co-targeting NAD and CtBP with small molecule inhibitors is an effective and synergistic therapeutic strategy in PDAC model systems. Methods and Results: In the current study, we demonstrate NAD-dependent regulation of ErbB2/3 signaling in PDAC cells in vitro. Upon depletion of NAD using the NAD salvage pathway inhibitor GMX1778 in human PDAC cells, we observed a striking coordinate decrease in the levels of both ErbB2 and ErbB3. Furthermore, stable knockdown of the limiting enzyme of the complementing Preiss-Handler NAD synthetic pathway, Nicotinamide Adenine Phosphoribosyl Transferase, also resulted in robust downregulation of ErbB2/3 protein levels, suggesting a direct linkage in PDAC cells between the status of NAD metabolism and synthesis and activity of the key growth factor receptors ErbB2/3. Conclusions: Our findings highlight the novel connection between NAD and ErbB2/3 in PDAC. Furthermore, our data suggests combining chemical or biologic inhibitors of ErbB2/3 with an inhibitor of NAD synthesis may result in highly synergistic cytotoxic effects in PDAC cells and PDAC preclinical models. Such data could support the future development of a human PDAC clinical trial repurposing agents targeting NAD synthesis and ErbB2/3 that are already FDA-approved or currently in clinical testing. Citation Format: Kranthi Kumar Chougoni, Nari Kim, Martin M. Dcona, Diana T. Dcona, Gautam K. Malhotra, Steven R. Grossman. Orthogonal targeting of NAD metabolism and ErbB2/ErbB3 signaling in pancreatic 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 7217.

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.

Abstract 2118: SGN-CEACAM5C/SAR445953, a novel topoisomerase I inhibitor antibody-drug conjugate targeting CEACAM5, has potent anti-tumor activity in CRC, PDAC, GC and lung cancer tumor models

Abstract Carcinoembryonic antigen cell adhesion molecule 5, CEACAM5, is a glycosylphosphatidylinositol-anchored glycoprotein expressed on the cell surface of most of the colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) and of more than 2/3 of gastric cancer (GC) and non-small cell lung cancer (NSCLC) while normal tissue expression is limited. The high prevalence of CEACAM5 expression prompted us to develop a novel CEACAM5 topoisomerase I inhibitor (Topo1i) antibody drug conjugate (ADC) with a DAR of 8, SGN-CEACAM5C/SAR445953. The anti-CEACAM5 antibody was chosen based on its high selectivity for CEACAM5 and its potential to direct cytotoxic payloads to tumor. The Topo1i payload was optimized for potency and enhanced bystander activity. SGN-CEACAM5C/SAR445953 is rapidly internalized and demonstrates in vitro cytotoxicity with EC50 values in the sub-nM range while it induces no toxicity on CEACAM5-negative cells. The potent anti-tumor activity is mediated by direct cytotoxicity on CEACAM5-expressing tumor cells and by a strong bystander effect due to the diffusion of the payload to the neighboring CEACAM5-negative tumor cells. Accordingly, the ADC incubated in a co-culture of CEACAM5-positive and -negative cells at a ratio of 1/1 induces a ~50% growth inhibition of CEACAM5-negative cells. Interestingly, with incubated ADC, only 1% of CEACAM5-positive cells in co-culture are sufficient to provide 11% growth inhibition of CEACAM5-negative cells. In vivo, SGN-CEACAM5C/SAR445953 is stable in circulation in SCID mice with a t1/2 close to 15 days. In vivo efficacy at 1, 3 and 10 mg/kg (single administration) was evaluated in panels of 4 CRC, 3 PDAC, 4 NSCLC and 3 GC patient-derived xenograft (PDX) models. At 10 mg/kg, the ADC elicits antitumor regression in 14/14 models. At 3 mg/kg, tumor regression occurs in 12/14 models. This potent, specific and dose dependent anti-tumor activity was further confirmed in Single Mouse Trials (SMT) of 20 CRC PDX models, 31 lung cancer PDX models and 19 gastric cancer PDX models. SMT consists in use of one animal per PDX model per treatment arm and for which the evaluation of efficacy is based on RECIST (Response Evaluation Criteria In Solid Tumors) criteria used in clinic. In CRC, gastric and lung cancer SMT, disease control rates are 95%, 84% and 87%, respectively with overall response rates of 55%, 68% and 71% including 15%, 10% and 26% of complete responses, respectively. The high anti-tumor activity across panels of PDX models of several CEACAM5 positive indications supports further evaluation of SGN-CEACAM5C/SAR445953 in patients with CRC, PDAC, GC and lung cancers (NCT06131840). Citation Format: Yves Baudat, Celine Nicolazzi, Johann Petur Sigurjonsson, Celine Amara, Astrid Clarke, Ryan lyski, Dave Meyer, Valeria Fantin, Marielle Chiron, Stephanie Decary. SGN-CEACAM5C/SAR445953, a novel topoisomerase I inhibitor antibody-drug conjugate targeting CEACAM5, has potent anti-tumor activity in CRC, PDAC, GC and lung cancer tumor models [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 2118.

Abstract 5943: Development of a SMART theranostic agent for precision pancreatic cancer therapeutics

Abstract Pancreatic cancer is predicted to emerge as the second leading cause of cancer-related deaths in the United States by 2030. Pancreatic ductal adenocarcinoma (PDAC) - the most common form of pancreatic cancer - is often diagnosed as metastatic disease and has demonstrated the least improvement in overall survival over the last three decades with incidence rising by 0.5% to 1.0% per year due in part to lack of effective and tumor-selective treatment strategies. One approach to reduce host toxicity is the development of novel molecules and innovative therapeutic approaches that are nontoxic to normal cells but can be activated only under tumor-specific conditions for lethality, which we coined as SMART (Selective Method of Activation for Response in Tumors) therapeutic agents. We utilized explicit properties and biomarkers present in PDAC tumors but not in normal tissues to develop a SMART DNA-damaging anti-cancer agent with both therapeutic and diagnostic (theranostic) activities. We evaluated the tumoricidal effect of our SMART agent using long-term survival assays in PDAC cell lines and 3D co-culture spheroids, as well as in tumor xenograft model studies in mice. We also tested the ability of our SMART agent to treat, detect/diagnose (via click chemistry), and identify critical resistance mechanisms (via proteomics) as a single agent or as a chemosensitizer with predictive tumor biomarkers for tumor-selective therapeutic response. We found that our SMART small molecule induces tumor-selective lethal effects as a monotherapy or in combination with DNA repair inhibitors or small molecules known to generate hydrogen peroxide (H2O2) in a tumor-selective manner for activation. Using “click” chemistry, we detected and quantified the incorporation of our SMART small molecule as a DNA-damaging agent via flow cytometry that correlated with therapeutic response, which is a feature that could facilitate the stratification, treatment monitoring, and management of patients with pancreatic cancers. We also found that our SMART agent is less toxic and more effective as a monotherapy than cisplatin in PDAC cell line-derived xenograft studies in mice. Overall, our novel SMART theranostic agent could provide a significant advancement as they are preferentially activated under tumor-specific conditions to induce tumor-selective therapeutic response. Furthermore, the diagnostic component our SMART agent could provide oncologists with essential information to make rapid clinical decisions on treatment strategies. Our SMART agent is also remarkably versatile as it can be utilized as a chemosensitizer or as a chemical probe to identify targetable vulnerabilities to potentiate its therapeutic effects at lower doses. Citation Format: Edward A. Motea, Jarrett J. Smith, Oluwasijibomi Ketiku. Development of a SMART theranostic agent for precision pancreatic cancer therapeutics [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 5943.

Abstract 3155: Metabolic stress induces an intrinsic inflammatory CAF phenotype that regulates stromal architecture in pancreatic cancer

Abstract Macropinocytosis is a critical amino acid supply pathway that fuels the metabolism of tumor cells and cancer-associated fibroblasts (CAFs) in pancreatic ductal adenocarcinoma (PDAC). PDAC tumors are deficient in glutamine, a critical amino acid that tumor cells and CAFs use to sustain their cellular fitness. We have previously shown that both PDAC cells and CAFs stimulate macropinocytosis as an adaptive response to glutamine depletion. How these linked metabolic mechanisms might co-operate to control stromal architecture remains elusive. In this study, we employed glutamine starvation and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), a selective inhibitor of macropinocytosis, to investigate how metabolic stress impacts the tumor microenvironment. We find that concomitant suppression of glutamine metabolism and macropinocytosis synergizes to drive an intrinsic inflammatory CAF phenotype that requires metabolic stress-induced ERK/MAPK signaling. Utilizing immunocompetent syngeneic mouse models of PDAC, we find that blocking macropinocytosis in these glutamine-depleted tumors diminishes tumor growth and reorganizes the tumor stroma by decreasing collagen deposition, increasing vasculature expansion and enhancing the infiltration of both CD8+ and CD4+ T cells. Alterations in the stromal landscape under these conditions are linked to the enhancement of CAFs that display an inflammatory signature while simultaneously suppressing more myofibroblastic identities within the tumor. Interestingly, we find that these effects on the tumor microenvironment can be harnessed to improve therapeutic responses. We determine that combining macropinocytosis inhibition with immune checkpoint blockade reduces PDAC tumor burden and significantly inhibits metastasis. We also find that macropinocytosis inhibition enhances drug delivery, thereby improving the efficacy of gemcitabine chemotherapy. Our findings demonstrate that intratumoral metabolic stress plays a critical part in preserving and modulating the tumor stroma. Citation Format: Yijuan Zhang, Swetha Maganti, Jennifer Hope, Li Lin, Cheska Marie Galapate, Florent Carrette, Linda Bradley, Cosimo Commisso. Metabolic stress induces an intrinsic inflammatory CAF phenotype that regulates stromal architecture in 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 3155.

Abstract 1578: RAS-GTP inhibition modulates Hedgehog signaling, suppressing myCAFs and promoting iCAFs in pancreatic ductal adenocarcinoma

Abstract More than 90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS. RMC-7977 is a potent inhibitor of GTP-bound RAS proteins (RAS(ON), including both wild type and mutant variants of KRAS, NRAS, and HRAS. The related investigational agent, RMC-6236, is a first-in-class, potent, orally bioavailable, RASMULTI(ON) inhibitor currently in Phase 1/1b clinical trials (NCT05379985). We performed preclinical studies in a range of models of PDAC, including the highly chemoresistant K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) genetically engineered mouse. RMC-7977 exhibited broad anti-tumor activity at tolerable doses, provoking radiographic responses in KPC pancreatic tumors and extending overall survival by 3-fold, the largest response observed yet in this model. RMC-7977 dosing produced a metronomic effect on RAS signaling in tumor and normal tissues. This yielded tumor-selective effects on apoptosis and proliferation, consistent with RAS oncogene addition in PDAC. PDAC tumors are characterized by an expansive stroma that harbors multiple subtypes of cancer associated fibroblasts (CAFs) that manufacture a fibrotic, inflammatory extracellular matrix. CAF proliferation is driven by signals emanating from the malignant epithelial cells, such as Sonic Hedgehog (SHH), a secreted ligand that drives downstream Hedgehog pathway signaling in myofibroblastic CAFs (myCAFs). To understand the impact of RAS-GTP inhibition on paracrine signaling to CAFs in PDAC, To understand the dynamic responses of malignant epithelial cells following GTP-RAS inhibition, we performed single cell RNA sequencing (scRNAseq) on KPC pancreatic tumors treated with RMC-7977 or vehicle at multiple timepoints. Using the ARACNe and VIPER algorithms, we performed single cell regulatory network analysis on the expression profiles of &amp;gt;210,000 cells to calculate the activities of thousands of transcriptional regulatory protein in each cell. We found that the RAS-GTP inhibition blocked SHH expression in malignant epithelial cells, resulting in loss of downstream Hh pathway activity in CAFs within 24 hours of treatment. By one week of treatment, myCAFs were significantly depleted whereas inflammatory CAFs (iCAFs) were significantly accumulated. Strikingly, CAF heterogeneity was restored in KPC pancreatic tumors that acquired resistance to RMC-7977, despite continued suppression of Hedgehog pathway signaling. Consistent with prior studies of Smoothened inhibitors, the depletion of myCAFs from RAS-GTP inhibition was associated with the rapid opening and hypersprouting of intra-tumoral blood vessels. Ongoing studies using the mutant-selective KRASG12D inhibitor RM-044 should discriminate between direct effects of wild type RAS inhibition in CAFs versus the paracrine consequences of mutant RAS inhibition in malignant epithelial cells. Citation Format: Marie Hasselluhn, Lorenzo Tomassoni, Urszula Wasko, Alvaro Curiel-Garcia, Tanner Dalton, Steve A. Sastra, Carmine Palermo, Andrea Califano, Kenneth P. Olive. RAS-GTP inhibition modulates Hedgehog signaling, suppressing myCAFs and promoting iCAFs in 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 1578.

Abstract 1577: Cancer associated fibroblast - tumor cell crosstalk enhances epithelial to mesenchymal transition and promotes a classical to basal switch in human pancreatic ductal adenocarcinoma

Abstract Introduction: Cancer associated fibroblasts (CAF) drive a complex tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) through incompletely understood interactions. Two major CAF subtypes, myofibroblastic (myCAF) and inflammatory (iCAF), dominate the tumor landscape and have been defined by gene expression profiles. There is significant plasticity between subtypes and alterations in CAF behavior likely drive changes in tumorigenicity and immune evasion. Additionally, PDAC tumor cells usually present with a predominant classical or basal phenotype that is associated with clinical outcomes. We hypothesized that CAF phenotype impacts tumor cell gene expression and alters the clinically-relevant tumor subtyping. Methods: We interrogated cellular crosstalk in the PDAC TME using a novel three-dimensional, patient-matched coculture system of patient-derived organoids (PDO) and CAFs, established from 12 patients undergoing surgical resection. Molecular characterization included bulk RNA sequencing, proteome profiling (107 factor), flow cytometry, multiplex RNA Scope, imaging mass cytometry (IMC), and RT-qPCR. Results: PDO gene expression analysis from bulk RNAseq demonstrated CAF coculture induced phenotypic shifts in the Moffitt subtype from a classical to a basal phenotype. Further, CAF coculture enhanced epithelial to mesenchymal transition (EMT) across all patients, even those with a baseline basal phenotype. We hypothesized this could be due to both cell-cell crosstalk and secreted factors. We therefore treated PDO with CAF-conditioned media (CM) and demonstrated that CM is sufficient to drive the observed classical-to-basal shift by RT-qPCR. Secretome analysis of CM derived from 3 CAF lines identified HGF, FGF19, and IL-8 as factors contributing to this transcriptional change. Next, we used spatial - omics (transcriptomics and proteomics) to examine the validity of these findings in source patient tissue. Using RNA Scope, we discovered distinct tumor neighborhoods expressing basal mRNAs KRT17 and KRT6a surrounded by CAFs compared to those expressing classical genes TFF1 and GATA6. We then used IMC to further explore CAF subtypes in patient tumors and found FAP+ myCAF regions corresponded to basal tumor regions, while CXCL12+ iCAFs were found in classical regions. Conclusions: PDAC tumor biology is likely driven by interactions between cancer cells and CAFs. Potential mediators of these interactions include both cell-cell contact and secreted factors. We introduce a novel approach combining in vitro coculture with tissue-based assays, demonstrating that modern culture techniques preserve cell type heterogeneity and plasticity. Together, these data demonstrate that a CAF-rich TME drives tumor cell plasticity not only towards EMT but also a more basal disease phenotype. Citation Format: Samantha Guinn, Brayan Perez, Jignasha Patel, Jae Lee, Daniel Zabransky, Won Ho, Elana J. Fertig, Emma Kartalia, Joseph Tandurella, Richard Burkhart, Jacquelyn W. Zimmerman, Elizabeth Jaffee. Cancer associated fibroblast - tumor cell crosstalk enhances epithelial to mesenchymal transition and promotes a classical to basal switch in human 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 1577.

Abstract 6485: Profile guided low dose drug combination strategies and kinase activities with prognostic and therapeutic avenues in pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease, commonly characterized by multiple aberrant signaling. Phosphoproteomics provides a direct read-out of these complex signaling networks and the resultant clinically relevant phenotype, as well as a functional scaffold to identify new targets. In the absence of an oncogenic driver, low dose (LD) kinase inhibitor (KI) combinations against multiple (parallel) activated kinases might provide higher efficacy and reduce toxicity as compared to single drug treatment. Aims: 1) To identify targets and test combinations of multiple KIs at LDs for potential synergism in preclinical models. 2) To chart the phosphoproteome of 42 PDAC tumors to reveal signalling pathways that may be involved in PDAC progression. Methods: By using a two step phosphopeptide enrichment with phosphotyrosine immunoprecipitation and immobilized metal affinity chromatography, followed by label free MS analysis, we analyzed phosphoproteome data of 7 immortalized and 2 primary PDAC cell lines, and of 42 PDAC tumors. We used integrative inferred kinase activity (INKA) scoring of the maxquant output to identify hyperactive kinases. For the cell line panel, five KIs were selected based on targeting coverage of the INKA profiles. LD were set as IC20s for 2,3,4 drug combinations. Cell growth inhibition was assessed by SRB assay. Median-effect analysis was used to assess synergy. Functional testing was performed in immortalized 2D, xenoderived 2D and 3D cultures. Effective low dose 3 drug combinations were further validated using patient-derived xenografts. Results: High INKA scoring of multiple activities per cell line without clear outliers of single kinases underscores the need for combination therapies. Multiple LD combinations showed effective growth inhibition (70 to 92%) and synergism, mostly 3 drug combinations, which required targeting of several RTKs and downstream signaling. Different responses were further observed between epithelial and mesenchymal cell lines. These top performing combinations were further validated in PDAC organoids and in vivo. Clinical utility of these kinase targets was then confirmed in 42 tumor phosphoprofiles, which were characterized in different subtypes with distinct therapeutic options. Phosphoproteome signals and kinases activities with potential prognostic value and mutational associations were further described. Conclusion: Our INKA pipeline, which can rank kinase activities in individual tumors, is optimally suited to specifically prioritize actionable kinases with targeting purposes. Tailored LD combination strategies exhibited promising efficacy in preclinical models and may ultimately improve treatment outcomes. Next Steps: Multicellular patient-derived models will be used to further study and target the TME in PDAC tumors. Citation Format: Andrea Vallés Martí, Giulia Mantini, Cynthia Waasdorp, Richard R. de Goeij- de Haas, Alex A. Henneman, Sander R. Piersma, Thang V. Pham, Jaco C. Knol, Joanne Verheij, Frederike Dijk, Hans Halfwerk, Elisa Giovannetti, Connie R. Jiménez, Maarten F. Bijlsma. Profile guided low dose drug combination strategies and kinase activities with prognostic and therapeutic avenues in 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 6485.

Abstract 6674: Fungi utilizes exocytosis pathway to facilitate the release of DAMPs from pancreatic cancer cells

Abstract Recent studies overwhelmingly show that microbiome is pervasive to tumor and play key role in immunotherapy response and reprograming tumor immune microenvironment (TIME). However, it remains unknown how microbiome facilitates the host-immune cell crosstalk and promote tumorigenesis. Our recent study in pancreatic ductal adenocarcinoma (PDAC) show that intratumor fungal microbiome facilitate the release of a damage associated molecular pattern (DAMP) molecule IL-33 from cancer cells. The secreted IL33 from cancer cells in turns recruits ILC2 and TH2 cells which promotes PDAC tumorigenesis. However, the mechanism of fungal mediated IL-33 release from PDAC cells remains unknown. Our follow up studies using high throughput integrated proteomic profiling and immunoprecipitation revealed a group of exocyst complex proteins interacting with IL-33 in the presence of fungus. The exocyst complex consists of eight sub-unit proteins that tethers the secretory vesicles to the plasma membrane and implicated in cellular exocytosis. We found EXOC6B a component of exocyst complex, modulates IL-33 release via exocytosis pathway from the PDAC cells. Genetic depletion of EXOC6B resulted in shunted IL-33 release and significantly abrogated PDAC tumor progression. Mechanistically, we found that mycobiome activates a non-canonical TLR2-CARD9 pathway, thereby driving IL-33 nucleocytoplasmic transport and exocytosis. This study identifies a unique mechanism of EXOC6B mediated release of IL-33 that expands our knowledge on how host-mycobiome interaction shapes PDAC tumor microenvironment by intricate molecular mechanism. Our study identifies EXOC6B as a new therapeutics target involved in the mycobiome driven IL-33 secretion in pancreatic cancer. Citation Format: Aftab Alam, Shyamananda Singh Mayengbam, Sharon Senchanthisai, Bektas Irmak, Prasenjit Dey. Fungi utilizes exocytosis pathway to facilitate the release of DAMPs from pancreatic cancer cells [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 6674.

Abstract 1590: The role of mucosal associated invariant T (MAIT) cells in pancreatic cancer liver metastasis

Abstract Pancreatic cancer liver metastasis (PCLM) is present in 50% of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) and leads to a median survival of &amp;lt;6 months. Here, we sought to identify urgently needed therapeutic options for treating PCLM by targeting mucosal associated invariant T (MAIT) cells, an invariant T cell restricted to MHCI related protein (MR1). MAIT cells are a unique subset of T cells that are classified as “protumor” MAIT17 and “antitumor” MAIT1 cells. We hypothesize that the PCLM tumor microenvironment (TME) induces direct MAIT cell activation via MR1 which primes MAIT cells towards a “protumor” phenotype. Using publicly available TCGA data, we found that PDAC tumors were enriched for MR1 gene expression compared to healthy tissue and patients with lower tumor MR1 expression had better overall survival. Next, immunostaining of MR1 on tumor samples from primary PDAC and PCLM revealed that that MR1 protein expression is highly enriched in the TME and localized to the surface of tumor cells. This suggests that MAIT cells in the PCLM TME are being activated directly via an MR1 dependent pathway. Next, we performed hemi-spleen injection of mouse pancreatic cancer cells into WT and MR1 KO (lacks MAIT cells) mice to model PCLM. Interestingly, MAIT cells in the WT PCLM mice were shifted towards the “pro-tumor” MAIT17 phenotype. Furthermore, the lack of MAIT cells in MR1 KO mice significantly reduced tumor burden compared to WT mice. Lastly, we found that MR1 KO mice had a reduction in exhausted NK and NKT cells which likely promoted anti-tumor immunity. Overall, we find that the PCLM TME promotes “pro-tumor” MAIT17 cells via MR1 mediated activation and these MAIT17 cells inhibit anti-tumor cells such as NK and NKT cells. This work identifies targeting MAIT cells as a novel immunotherapy approach for PCLM. Citation Format: Jugmohit Toor, Peter Dimitrion, Rachel Krevh, Kalpana Subedi, Bobby Zuniga, Jie Wang, Li Zhou, Qing-Sheng Mi. The role of mucosal associated invariant T (MAIT) cells in pancreatic cancer liver metastasis [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 1590.

Abstract 1602: Leukemia inhibitory factor (LIF)-factor XIIIA mediated macrophage-stromal crosstalk in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy marked by a highly immunosuppressive tumor microenvironment (TME). Macrophages are known to promote immunosuppression and exhibit a profibrotic transcriptional profile with potential involvement in extracellular matrix (ECM) remodeling. Previously, we identified hyperactivation of cancer cell intrinsic cyclic AMP response element binding protein 1 (CREB) regulates leukemia inhibitory factor (LIF) to mediate macrophage infiltration and polarization within the TME of PDAC. Subsequently, we observed that disrupting LIF-LIF receptor (LIFR) signaling therapeutically not only diminishes the infiltration of tumor-associated macrophages (TAMs) but also results in a decrease in ECM deposition. This study delves into the potential regulatory pathway through which LIF induces remodeling of the ECM within the PDAC TME. Methods: We targeted LIF-induced macrophage-stromal crosstalk using the LIFR antagonist (EC359) in an orthotopically tumor implanted LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice model of PDAC. Sirius red staining in tumor sections was performed to assess ECM (collagen) deposition. Moreover, we performed RNA transcriptomics-based analysis of recombinant (rLIF) induced bone marrow derived macrophages harvested from syngeneic C57BL/6 mice. The Cancer Genome Atlas (TCGA) pancreatic cancer (PAAD) data set along with single-cell RNA sequencing (scRNA seq) were employed to assess the expression of major downstream targets of LIF. To validate the RNA-seq results, we conducted immunohistochemical analysis (IHC) on mouse PDAC tissue sections and quantitative polymerase (qPCR) analysis on RAW 264 macrophage cell line treated with rLIF or KPC CREBWT PDAC tumor cells conditioned media. Results: We observed a drastic decrease in both TAM infiltration and, of note, ECM deposition in orthotopically implanted KPC pancreas tumor mice tissue treated with EC359 as compared to vehicle. Mechanistically, our RNA-seq analysis unveiled a significant upregulation of Factor XIIIA (FXIIIa), a major downstream target of LIF. Notably, TCGA data revealed that FXIIIa mRNA expression is upregulated across PDAC tumors marked by poor survival outcomes. Moreover, scRNA seq and IHC established that TAMs highly express FXIIIa in the PDAC TME. In exploring the effect of LIF-LIFR signaling in the ECM, mouse RAW 264.7 macrophage lines incubated in high LIF conditions confirmed transcriptional upregulation of ECM-associated proteins such as FXIIIa as well as fibronectin. Conclusion: Our investigation provides valuable insights into the possible mechanism by which LIF triggers ECM remodeling, thereby contributing to the desmoplastic stroma. Significantly, our findings propose that LIF-regulated FXIIIa signaling in macrophages serves as a plausible mediator of the LIF-induced ECM remodeling in PDAC. Citation Format: Nishah Jaferi, Siddharth Mehra, Varun Krishnamoorthy, Sudhakar Jinka, Anna Bianchi, Vanessa T. Garrido, Luis A. Nivelo, Ban Yuguang, Nagaraj S. Nagathihalli. Leukemia inhibitory factor (LIF)-factor XIIIA mediated macrophage-stromal crosstalk in 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 1602.

Abstract 1586: Unraveling the importance of pancreatic cancer extracellular signaling to endothelial cells within the tumor microenvironment

Abstract In many solid tumors, including pancreatic ductal adenocarcinoma (PDAC), the secretion of extracellular vesicles (EVs) has been shown to support tumor progression, chemotherapeutic resistance, and metastasis. Importantly, PDAC tumors are composed of up to 80% non-tumor cells and an extracellular matrix, yet no studies have been performed to determine which cells within PDAC tumors internalized PDAC EVs. Further, the RNA-binding protein HuR plays an important role in PDAC cells, supporting the stability and translation of transcripts that aid tumor cell stress responses. Our lab has recently found that EVs harbor distinct mRNA and phosphorylated protein cargoes from donor cells. Further, HuR knockout (KO) cells produce EVs with significantly different mRNAs and phosphorylated proteins that EVs isolated from HuR wildtype (WT) cells. Specifically, proteins relating to endothelial function and angiogenesis were significantly differentially phosphorylated. Considering the functional impact of EV cargoes on recipient cells, our study aims to identify the specific cell types in the tumor microenvironment that import PDAC EVs and investigate the role of PDAC cell HuR in this signaling process. We have generated human PDAC and mouse KrasG12D Trp53R172H driven (KPC) PDAC cell lines to express an EV reporter, PalmGRET. Utilizing these cells lines in an immunocompetent mouse model of PDAC, we found that endothelial cells are major importers of PDAC EVs. We next performed bulk RNA-sequencing on endothelial cells sorted from these tumors that have (GFP+) versus have not (GFP-) imported PDAC EVs to assess the functional impact of PDAC EVs. Subsequent studies will focus on delineating the importance of this signaling axis by ablating EV secretion in addition to assessing endothelial cell function when treated with EVs from HuR WT vs. KO cells. Collectively, our study sheds light on the preferential uptake of PDAC EVs by endothelial cells and highlights the potential role of tumor cell-intrinsic HuR in this signaling process. These findings expand our understanding of the interactions between PDAC and the tumor microenvironment, potentially paving the way for targeted therapeutic interventions in this devastating disease. Citation Format: Jennifer M. Finan, Yifei Guo, Alexandra Q. Bartlett, Kevin MacPherson, Valerie Calvert, Madeline D. Hedberg, Emanuel Petricoin, Rosalie Sears, Jonathan R. Brody. Unraveling the importance of pancreatic cancer extracellular signaling to endothelial cells within the tumor microenvironment [abstract]. In: Proceedings of the 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 1586.

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 6627: Boosting macrophage-specific BCAA oxidation enhances immune activation within the tumor microenvironment and diminishes tumor growth in pancreatic cancer

Abstract Treating pancreatic ductal adenocarcinoma (PDAC) poses a significant challenge, mainly due to therapy resistance linked to the immunosuppressive tumor microenvironment (TME) within PDAC tumors. This TME is characterized by a fibrotic stroma and an abundance of immunosuppressive cells, notably tumor-associated macrophages (TAMs). TAMs crucially hinder effector T cell activity and contribute to T cell exhaustion. Redirecting TAMs toward an immunostimulatory state has emerged as a promising approach in PDAC treatment. Recent investigations, including our own studies, reveal a profound influence of metabolic pathways on the immune phenotype of TAMs. Specifically, dysregulation in branched-chain amino acid (BCAA) metabolism has been observed in PDAC patients, manifesting as elevated circulating BCAA levels correlating with a doubled risk of PDAC development. However, the extent to which BCAA metabolism influences TAM phenotype and anti-tumor immune responses remains a largely unexplored territory, presenting a challenge for PDAC treatment. Our focus revolves around understanding how heightened BCAA levels in PDAC affect TAM phenotype, the immunosuppressive tumor environment, and tumor progression. Employing an orthotopic PDAC model and genetically modified mice, specifically BCKDKfl/fl BCKDK LysMcre/+ (mimicking increased BCAA oxidation), alongside a BCKDK-targeting pharmacological inhibitor (BT-2), multicolor flow cytometry, and in vitro models, our research has revealed crucial insights. We found a diminished expression of BCAA oxidation genes in TAMs compared to normal pancreatic macrophages. Interestingly, M1 and M2 macrophages seemed to utilize BCAAs differently, reflected in varying levels of TCA cycle intermediates derived from BCAA oxidation. Notably, boosting BCAA oxidation specifically in TAMs through genetic modifications resulted in reduced PDAC growth associated with an enhanced immunostimulatory TAM phenotype and activation of CD8+ T cells in the TME. Conversely, administering BCAAs induced an immunosuppressive TAM phenotype, fostering PDAC growth, suggesting a direct influence of BCAAs on TAM phenotype. Pharmacologically enhancing BCAA oxidation via BT-2 bolstered immune activation within the tumor environment and reduced PDAC growth. Intriguingly, our analysis of publicly available data unveiled a significant correlation between high BCAA oxidation and favorable responses to anti-PD1 therapy in cancer patients. Our findings emphasize the pivotal role of BCAA metabolism in shaping the immune landscape of PDAC, offering potential avenues for therapeutic interventions targeting TAMs and mitigating TME immunosuppression. Citation Format: Gauri Mirji, Sajad Ahmad Bhat, Zachary Schug, Ben Z. Stanger, Zoltan Arany, Rahul S. Shinde. Boosting macrophage-specific BCAA oxidation enhances immune activation within the tumor microenvironment and diminishes tumor growth in 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 6627.