Tumor microbes are increasingly recognized for modulating tumor behavior and therapy responses. Intratumoral microbial burden (ITMB) analysis across cancers revealed regulation of immune pathways, and activated mast cells, mostly in colorectal (CRC) and gastric (STAD) cancers. High ITMB CRC leads to interferon regulation and is associated with improved outcomes in advanced disease. Single-cell sequencing revealed induction of interferon-related genes (IRGs) within microbes-containing human CRC. GI-luminal mismatch repair deficiency (MMRd) tumors had higher ITMB than proficient tumors (MMRp). In a rectal MMRd cohort with 100% remission after immune checkpoint blockade (ICB), tumor microbes and microbes-containing mast cells increased. In ICB-sensitive syngeneic murine MMRd tumor models, local tumor microbial depletion, impaired ICB efficacy while downregulating IFN signaling. Forced upregulation of IRGs in ADAR1-deficient cancer cells restored immunotherapy responses during microbial ablation. These data highlight dynamic interplay between ITMB, host defense, and immunogenicity which seems key to determine therapy responses.

Supplementary figure 4. Spatial transcriptomics deciphers the immune landscape underlying diabetic pancreatic cancer. a. The relative mRNA expression of CXCL8, CXCL6, CCL20, CCL13 and CXCL9 in the tumor region between DM and Non-DM groups. b. The spatial expressions of C3 and HIST1H4J which are associated with neutrophil extracellular traps formation pathway at spatial level. c. The number of MPO + CitH3+ cells in field were calculated between DM and Non-DM groups (PDAC, n = 5, PDAC with DM, n = 5). d. Bar chart showing the percentage of 22 immune subgroups in PDAC. e. Bar chart showing cell proportions of 22 immune subgroups between PDAC and PDAC with DM groups. f. Spatial resolved gene expression images and expression level of N2-meutrophils between DM and non-DM groups.

Abstract Pancreatic ductal adenocarcinoma (PDAC) has one of the lowest cancers related-survival rates with high resistance to therapies and no available preventive strategies. Even immunotherapy, which has transformed cancer treatment by providing survival benefits in many solid tumors, has shown limited efficacy in PDAC. Eating patterns can modulate the composition and metabolic activity of the gut microbiota, thereby affecting anti-tumor immune responses. Fasting, a common treatment for pancreatic inflammatory conditions, can affect the composition of gut microbiota, regulates immunity and host metabolic responses. Although fasting is known to offer therapeutic benefits in pancreatic disorders such as pancreatitis and diabetes, its potential role in the prevention or treatment of pancreatic cancer remains unclear. Intermittent fasting (IF) refers to dietary regimens that limit food intake to specific time windows while allowing ad libitum calorie consumption during those periods. Using multiple spontaneous genetically engineered premalignant and orthotopic murine cancer models, we show that IF delays pancreatic tumorigenesis and induces sensitivity to immunotherapy in PDAC. IF-mediated reversal of PD-1 blockade resistance is dependent on gut microbiota mediated CD8+ T cells activation and migration. IF modulates the relative abundance of gut microbiota as well as the serum metabolite levels, which activates intratumoral dendritic cells (DCs). Timeseries single cell transcriptomic analysis revealed that IF remodels the circadian clock of CD8+ T cells and promotes anti-tumor immune responses through shifting the oscillation of genes, including Kat2b, E2f4, Carns1 and Ccnc. Integration of these gene-expression profiles enables stratification of survival among PDAC patients. Spatial analysis confirmed that long-term fasting was associated with increased the proportion of CD8+ T cells which were in proximity to DCs compared to short-term fasting, suggesting clinical evidence supporting a role for fasting in the redistribution of immune cells within the tumor microenvironment of PDAC. Our findings demonstrate the efficacy of IF in PDAC prevention and reversal of therapy resistance by modulating the activation and egress of rhythmic CD8+ T cells, ultimately affecting the pancreas tumor ecosystem. Citation Format: Le Li, Vidhi Chandra, Fuduan Peng, Luca S. Santovito, Olivereen Le Roux, Rian M. Howell, Thais Bartelli, Virginia Tahan, Erika Y. Faraoni, Seyda Baydogan, Haiyan D. Miller, Kejing Song, Vasanta Putluri, Badrajee Piyarathna, Abu Hena Kamal, Nagireddy Putluri, Joseph Petrosino, Arun Sreekumar, Jay Kolls, James R. White, Zheng Sun, Lora V. Hooper, Linghua Wang, Faraz Bishehsari, Florencia McAllister. Intermittent fasting remodels gut ecosystem to influence initiation and therapy responses in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2087.

Supplementary Figure 7: Identification of IL-17-producing cell types

Supplementary figure 5. DM promotes colorectal and breast cancer progression in mice models. a. Representative images and quantification of Ki-67 positive cells in tumor tissues by IHC (Scale bar = 50 µm, n = 6). b. Comparison of N2 polarized neutrophils between tumors obtained from DM and control groups using mIHC, scale bar = 50 μm. c. Experimental outline of murine subcutaneous colorectal cancer model. d. Representative images of tumor size. Scale bar represents 5 mm. The tumor volume and weight were compared. e. Growth curves of subcutaneous tumors. f. Representative immunofluorescence images and quantification of CD45+ Ly6g+ and MPO + CitH3+ cells in MC38 tumors (Scale bar = 100 µm, n = 5). Green, CD45 and CitH3 positive cell; Red, Ly6g and Mpo positive cell. g. Experimental outline of murine subcutaneous breast model. h. Representative images of subcutaneous breast tumors. Scale bar represents 5 mm. i. The tumor volumes and weight were compared. j. Representative immunofluorescence images and quantification of MPO + CitH3+ cells in the breast tumors (Scale bar = 100 µm, n = 5). k. Representative images of oil red staining in the SW1990 cells stimulated by high concentration of glucose (HG) versus normal glucose (NG). The levels of cholesterol in cellular supernatant were detected (n = 3). L. Flow cytometry gating strategy.

BACKGROUND:Pancreatitis significantly alters the microbial composition of the oral and intestinal compartments, causing dysbiosis that may contribute to disease mechanisms and potentially serve as a basis for diagnosis or treatment. OBJECTIVE:To determine whether the oral or gut microbial signature can classify chronic pancreatitis (CP). METHODS:Stool samples (n=707) were collected from participants in the Prospective Evaluation of Chronic Pancreatitis for Epidemiologic and Translational Studies (PROCEED). Samples were distributed among 200 healthy (HC), 310 CP, 49 acute pancreatitis (AP), and 148 recurrent acute pancreatitis (RAP). In addition, saliva samples were collected for a subset of participants (n=156). Whole genome sequencing was performed to assess microbiome composition. Machine learning algorithms were utilized to identify a signature with microbial features predictive of CP. RESULTS:Gut alpha diversity was significantly decreased in AP, RAP, and CP compared with HC, with CP exhibiting the lowest diversity. In contrast, oral microbial diversity showed no significant variation across groups. Beta diversity analysis revealed distinct gut microbiome compositions between HC and pancreatitis subtypes, with CP showing the most pronounced differences. Random forest models using gut microbial species demonstrated robust predictive performance for CP using a minimum of 10 species (Area under the curve-AUC: 0.834; accuracy: 0.774). Despite similarities in gut microbiome composition across pancreatitis subtypes, a unique gut microbial signature for CP was identified highlighting the microbiome's potential in CP diagnosis. CONCLUSION:Our study reveals a gut microbial signature predictive of CP using machine learning models in a large US multi-institutional cohort.

Abstract Introduction Pancreatic cancer is predicted to be the second leading cause of cancer deaths by 2030. Interleukin 17 (IL-17) signaling in pancreatic cancer cells can trigger neutrophil extracellular traps that impair immunotherapy efficacy. IL-17 signaling also maintains gut homeostasis. Intestinal epithelial cells interact with the gut microbiome, participate in injury repair, and are replenished by intestinal stem cells (ISCs). Our lab showed that deletion of IL-17 receptor A (IL-17RA) in the gut epithelium induces pro-tumorigenic IL-17 signaling due to the disruption of gut homeostasis, suggesting that gut IL-17/IL-17RA—mediated immune responses affect distant tumors. However, the role of IL-17 in specific epithelial compartments and its impact on gut homeostasis and cancer risk remain unclear. We hypothesized that disruption of IL-17/IL-17RA signaling within ISCs impacts tumor growth. Methods We utilized a lineage marker of ISCs, Lgr5, to drive Cre enzyme tissue specific expression to knock out Il17ra in gut ISCs (cKO mice). We used implantable pancreatic tumor models and performed immunoprofiling. Results We found that the pancreatic tumor growth was altered by the remote signal from gut ISCs in cKO mice. Furthermore, we identified that major transcriptomic changes were associated with innate and adaptive immune pathways, especially B cell activation. Conclusion We conclude that IL-17/IL-17RA signaling in gut ISCs changes intestinal immune responses locally that can affect distant tumor immunity. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Supplementary figure 6. DM promotes PDAC progression through SREBP2-induced NETs formation. a. Western blot assay compared the protein level of SREBP2 between control and Betulin-treated tumor cells. The mRNA level of SREBP2 between control and Betulin-treated tumor cells. Comparison of cholesterol level in the supernatant of tumor cells between control and Betulin-treated tumor cells. b. The mRNA level of CXCL1 in the Pan02 cells following the inhibition of SREBP2 or cholesterol biosynthesis and the level of CXCL1 in the cellular supernatant (n = 3). c. The expression of CXCL1 in the tumors of control, betulin and simvastatin groups (Scale bar = 100 µm, n = 6). d. Levels of cholesterol within tumor tissues in different treatment conditions. e. WT and Pad4-/- mice were injected with STZ to induce diabetes. Mice were then injected with Pan02 cells and sacrificed 3 weeks after tumor implantation. Representative images (duplicates) of pancreatic orthotopic tumors. Scale bar represents 5 mm. The tumor volume and weight at the endpoint were compared. f. Representative immunofluorescence images and quantification of CD45+ Ly6g + cells in the tumors (Scale bar = 100 µm, n = 5). Green, CD45 positive cell; Red, Ly6g positive cell. g. Confirmation of SREBP2 knockdown in Pan02 cells determined by western blotting. h. Representative images of western blot and quantification of total and phosphorylation of NF-κB and AKT proteins in the pancreatic cells (SW 1990) stimulated by Betulin. i. The serum cholesterol between shNC and shSREBP2 groups. j. The levels of CXCL1 in the cellular supernatant of shNC and shSREBP2 groups (n = 5). The level of cholesterol in the shNC and shSREBP2 PAN02 cells (n = 3). k. The relative mRNA expression of CXCL1 and the cellular supernatant level of CXCL1 in Pan02 (shNC and shSREBP2) cells stimulated by NF-κB inhibitor (DHMEQ). l. The relative mRNA expression of CXCL1 and the cellular supernatant level of CXCL1 in Pan02 (shNC and shSREBP2) cells stimulated by AKT inhibitor (PI3K/AKT-IN-1).

Abstract Androgen deprivation therapy (ADT) remains the standard treatment for advanced prostate cancer (PCa); however, most patients ultimately progress to lethal castration-resistant PCa (CRPC). Emerging evidence implicates RNA N⁶-methyladenosine (m⁶A) modification as a key regulator of cancer biology, yet its role in CRPC remains poorly understood. As a critical adaptor in the m⁶A methyltransferase complex, RNA-binding motif protein 15 (RBM15) directs m⁶A deposition to specific mRNA targets. Here, we identified RBM15 as the key methyltransferase member significantly upregulated in CRPC tissues and strongly correlated with poor patient survival. Functionally, RBM15 overexpression reduces PCa cell sensitivity to enzalutamide, whereas its knockdown suppresses tumor growth and invasion. Mechanistically, RBM15 is an androgen-responsive protein whose expression increases upon chronic androgen deprivation. It catalyzes m⁶A methylation at position A1384 of damaged DNA binding protein 1 (DDB1) mRNA, leading to YTHDF2-dependent transcript decay and reduced DDB1 protein levels. Lower DDB1 impairs K48-linked polyubiquitination of the androgen receptor (AR), thereby stabilizing AR and amplifying AR signaling. Importantly, AR transcriptionally activates RBM15, forming a feed-forward loop that drives CRPC progression. Collectively, our findings establish RBM15 as a central epitranscriptomic driver of CRPC and identify the RBM15–DDB1–AR axis as a promising therapeutic target. Dual inhibition of RBM15 and AR may offer a novel strategy to overcome treatment resistance in advanced PCa.

Supplementary Figure 10: IL-17 directly stabilizes B7-H4 mRNA

Supplementary Figure 6: Stemness signature in pancreatic epithelial cells lacking IL-17RA.

Patients with pancreatic ductal adenocarcinoma (PDAC) with diabetes mellitus (DM) exhibit poor clinical outcomes. Metabolic reprogramming of both cancer cells and immune compartments plays a crucial role in shaping the antitumor immune response in PDAC. DM-induced metabolic alteration may disrupt the intricate cross-talk between immune cells and tumor-associated immune factors, profoundly influencing PDAC progression. In this study, we performed an integrated, spatially resolved multiomics study to investigate DM-associated, cell-specific metabolic remodeling within the PDAC tumor microenvironment. DM influenced interactions between tumor cells and immune cells, which accelerated PDAC growth in both humans and mice. Patients with PDAC with DM exhibited higher tumor stage, poorer differentiation, and worse outcomes. Spatial metabolic and transcriptional profiling revealed that SREBP2-dependent cholesterol biosynthesis exacerbated PDAC progression. Increased cholesterol biosynthesis promoted neutrophil recruitment and accelerated the formation of neutrophil extracellular traps (NET) by stimulating the CXCL1/CXCR1/CXCR2 signaling axis, ultimately promoting PDAC growth. Inhibition of SREBP2, pharmacologic blockade of CXCL1, or perturbation of NETs markedly reduced PDAC growth in diabetic mouse models. Together, these multiomics analyses and follow-up mechanistic studies constitute an integrated approach that elucidates a metabolic mechanism by which diabetes promotes PDAC development by remodeling the tumor-immune microenvironment and highlights a potential therapeutic strategy for PDAC with DM. Significance: Diabetes induces metabolic reprogramming that promotes neutrophil recruitment and neutrophil extracellular trap formation to drive pancreatic cancer progression, providing a targetable metabolism-immune axis to improve the outcomes of diabetic pancreatic cancer patients.

Supplementary figure 7. Single cell sequencing revealed that DM promotes PDAC progression by increasing neutrophil infiltration. a. Schematic diagram showed the experimental design of multi-omics in diabetic PDAC progression. b. Heatmap plotting markers for all clusters. c. Pathway analysis from KEGG analysis showing metabolism-related pathways enriched by different genes in cancer cells between DM and Non-DM groups. d. The expression of CXCL1 in cancer cells. e. Functional enrichment of up-regulated genes in the cancer cells between DM and Non-DM groups. f. KEGG analysis showing the pathways enriched by up-regulated genes in neutrophils between DM and Non-DM groups.

Supplementary figure 3. The cluster analysis for spatial transcriptomics. a. Mapping of cell clusters to spatial locations in all samples. b. UMAP visualizing 15 clusters in all samples. Comparison of 15 spatial clusters between two groups using UMAP. c. Heatmap plotting markers for all clusters.

Supplementary Table 1. Characteristics of primary PDAC samples (HTAN) with diabetes status.

Abstract Tumor resident microbes are a well-recognized component of the tumor microenvironment. Microbial subcellular location across tumors along with their functionality remains to be determined. Bulk microbial profiling techniques lack subcellular and spatial resolution and ultimately cannot distinguish between microbial signals or live microbial presence. To address these limitations, we performed orthogonal methods for functional microbial-host characterization. We first developed advanced quantitative fluorescent imaging methodologies that allows visualization of microbial cellular compartmentalization across three different tumor types (total n=30). Using this methodology, we performed spatial microbial transcriptomics at the regional and single cell levels to determine microbial distribution and to interrogate microbial regulation of tumor cell signaling in human pancreatic tumors (n=55). To confirm presence of viable microbes, we performed multiplexed culturomics of patient tumors and normal adjacent tissue specimens (n=80), followed by Whole Genomic Sequencing (WGS) analysis. We tested the effect of the isolated clinical strains on tumor cell signaling pathways with in vitro co-culture assays, and upon genetic fluorescent labelling we defined their role on in vivo tumor growth in murine models. These experiments confirmed their role in promoting tumors, driving resistance to therapeutics and modulation of host signaling mechanisms. Overall, our results identified several pathways under microbial regulation within cancer cells that can drive immune evasion through impaired antigen presentation. In summary, using multiple complimentary novel methodologies we characterize the microbial niche of tumors (MiNT) that uncover microbial regulation of host cell signaling and patient outcomes. Microbial modulatory approaches may be needed to reverse resistance to therapies in pancreatic cancer. Citation Format: Vidhi Chandra, Le Li, Seyda Baydogan, Fuduan Peng, Thais Bartelli, Haoyue Liu, Fernando Jimenez-Arancon, David Romanin, Javier A. Gomez, Steven Maron, Erick M. Riquelme, Mark Hurd, Anirban Maitra, Luis A. Diaz, Ismet Sahin, Adriana Paulucci-Holthauzen, Jared K. Burks, Huamin Wang, Jay Kolls, James R. White, Linghua Wang, Michael P. Kim, Florencia McAllister. Functional interrogation of pancreatic cancer resident microbes reveals their role in host modulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4908.

Supplementary Figure 2. IL-17RA in the pancreatic immune compartment is not required for tumorigenesis.

Supplementary figure 1. The prognostic implication of DM for pancreatic cancer. a. Forest plot showing the association between DM and the incidence of PDAC. b. Logistics analysis of potential predictors of development of PDAC.

Purpose: DNA repair and DNA damage responses in cancer cells are regulated by metabolic reprogramming, which is increasingly recognized as a key factor contributing to PARP inhibitor (PARPi) treatment failure. This study aims to explore the metabolic mechanisms underlying PARPi resistance in PTEN-deficient prostate cancer and identify clinically viable metabolic interventions to overcome therapy failure. Experimental Design: A multicenter retrospective cohort was analyzed to evaluate the efficacy of combined metformin-PARPi therapy. Mechanistic studies utilized molecular assays to elucidate PARPi resistance and its critical determinants. Machine learning models predicting PARPi response were developed using clinical datasets and interpreted via SHAP analysis. Results: In PTEN-deficient cancer cells, lactate accumulation activated the NHE1/PKC/NOX1 axis, sustaining elevated NADP+ levels. NADP+ competitively inhibited the formation of PARPi-PARP-DNA complexes, leading to PARPi resistance. However, metformin administration significantly elevated NADP+ levels, inducing allosteric effects on PARP structures and enhancing PARPi efficacy. Based on these findings, we developed and validated a predictive machine learning model for PARPi response, which was interpreted using SHAP and deployed on a web platform. Conclusions: Metformin modulates NADP+ levels to influence PARPi sensitivity in PTEN-deficient prostate cancer. Additionally, we developed a machine learning model to provide clinicians with personalized predictions for PARPi response.

Introduction: Artemisia argyi (AA) demonstrates anti-cancer potential. However, its mechanisms against pancreatic ductal adenocarcinoma (PDAC) remain unclear. This study investigates the anti-tumor mechanisms of AA in PDAC. Methods: The Traditional Chinese Medicine Systems Pharmacology (TCMSP) database was used to identify potential bioactive compounds. The Analyze Network tool revealed that stigmasterol (SS) was a key bioactive compound of AA. Then, the corresponding targets of SS were predicted through SwissTargetPrediction and SuperPred. A compound-target interaction network was subsequently constructed with Cytoscape, followed by the generation of a protein-protein interaction (PPI) network using the STRING database. Molecular docking was performed to evaluate the binding affinity between SS and the targets. Finally, the specific mechanism of SS on PDAC was experimentally investigated. Results: SS was identified as a major bioactive component of AA. A total of 107 overlapping targets between SS and PDAC were collected. Molecular docking demonstrated favorable binding interactions between SS and its candidate protein targets. The experimental results suggested that SS could promote PDAC cell apoptosis through the AMPK/mTOR pathway. Discussion: Our findings demonstrate that SS, a primary bioactive component of AA, exerts significant antitumor effects in PDAC by inducing tumor cell apoptosis through the activation of the AMPK/mTOR signaling pathway. Furthermore, we reveal a novel immunomodulatory dimension whereby SS remodels the immunosuppressive PDAC microenvironment. This dual action on both tumor cells and the immune landscape positions SS as a promising multi-faceted therapeutic candidate for PDAC. Conclusion: SS was characterized as the key bioactive component of AA, supporting its potential role as a promising therapeutic agent for PDAC.