Key resources used in the conduct of experiments is provided

Supplementary Figure 1 provides additional details of orthotopic tumors treated with TCDD including histology, gross pictures and dose escalation. Data on additional cell lines including in vitro growth kinetics is provided.

Supplementary Figure 5 shows additional single cell sequencing data from orthotopic PDAC tumors in mice treated with TCDD or untreated.

Supplementary Figure 2 shows RNA and protein expression of interferon gamma and IL22. An image of the standard ELISA curve is provided.

Supplementary Figure 3 shows gating strategies used for flow analysis of CD4 cells from orthotopic tumors as well as serum and tissue IL22 measured by ELISA. IHC and multiplex IHC analysis of TCDD treated tumors in the absence of T regulatory cells is shown.

The tumor microenvironment (TME) is composed of tumor cells and surrounding stroma, including immune, mesenchymal, and vascular cells, as well as soluble factors. Traditional techniques to interrogate cellular interactions and infiltration are limited by the loss of tissue architecture or by labeling only a small number of antigens. Multiplex fluorescent immunohistochemistry (mfIHC) is a tissue staining technique that involves tyramide-based signal amplification to covalently bind a fluorophore to an antigen of interest, followed by removal of the primary and secondary antibodies, thereby permitting co-staining and localization of multiple antigens on a single sample. mfIHC retains spatial information, allowing for in-depth analysis of cellular phenotypes and their interactions within the TME. Described here is an overview of the mfIHC experimental design, staining, and image analysis in murine tissues utilizing a manual technique and the Akoya OPAL system.

Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to immune therapies. Limited biomarkers, such as mismatch repair proteins, have been used to identify those who may respond to immunotherapy. We identified a subset of aggressive PDACs (⁓25%) carrying mutations in the complex of proteins associated with SET1-like complex genes (CLCGs), which can be used as new biomarkers for targeted immunotherapy. In this study, we compared the immune microenvironment of PDACs harboring CLCG mutations with matched wild-type PDACs using multiplex fluorescent immunohistochemistry and computational imaging techniques. We observed that CLCG-mutant PDACs were infiltrated with fewer CD4+ T cells and antigen-presenting cells (APCs) but elevated immune checkpoint T cell immunoreceptor with Ig and ITIM domains (TIGIT) expression on CD4+ T cells and APCs. There was no difference in the expressions of other immune checkpoints, such as programmed death-1 receptor ligand and T-cell immunoglobulin and mucin domain-containing protein 3. More CD4+ T cells near epithelial cells (tumor cells) and APCs expressed TIGIT in CLCG-mutant PDACs. Additionally, CLCG-mutant PDACs displayed a malfunctional immune cell crosstalk. Single-cell RNA-sequencing data confirmed the elevated TIGIT expression on CD4+ T cells and increased exhausted CD4+ T cells in CLCG-low PDACs. These findings uncovered the unique underlying mechanisms of immune suppression in CLCG-deficient PDACs and identified CLCG as potential biomarkers to identify those who may benefit from TIGIT-targeting immunotherapies.

Supplementary Figure 4 shows histologic features of orthotic tumors placed in AhR knockout mice or those treated with an AhR inhibitor prior to TCDD administration.

Although smoking is a risk factor for pancreatic adenocarcinoma (PDAC), the underlying mechanisms promoting tumorigenesis and progression are unknown. In this study, we show that aryl hydrocarbon receptor (AHR) ligands found in cigarette smoke, like the carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin, promote pancreatic dysplasia and PDAC progression in a mouse model of this disease. This effect is mediated by AHR activation in CD4+ T cells, leading to their polarization to IL22-producing TH22 cells and regulatory T cell accumulation, ultimately driving a blunted CD8+ T-cell effector response. Analysis of human pancreata from organ donors revealed that smokers have increased AHR activation relative to nonsmokers. Similarly, PDAC tumors from patients with a history of cigarette smoking presented with increased regulatory T-cell accumulation compared with nonsmokers. These findings support a model whereby AHR ligands (AHRL) in cigarette smoke promote tumorigenesis and progression of PDAC through dysregulation of immune responses. SIGNIFICANCE:Our study investigates the mechanistic link between AHRL and pancreatic cancer. We determined that AHRLs polarize naïve T cells, resulting in increased production of IL22 and immunosuppression. Our findings identify a novel signaling axis linking environmental chemicals to pancreatic tumorigenesis via the immune system. See related commentary by Zhao and Hill, p. 13.

Supplementary Figure 6 shows the dysplasia and AhR expression in a spontaneous murine model of PDAC. Additionally expression in human tissues is shown.

Heat map of FlowSOM CD3+ T cell meta-clusters.

CD69+CD103+ CD8+ resident memory (TRM) cells are identified by mIHC.

Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to immune therapies. There are few biomarkers to guide the selection of patients likely to benefit from available immunotherapyies. Recent whole genome sequencing reveal that KMT2D, a histone-modifying enzyme, is mutated in up to 5% of PDAC cases. Our prior work established a tumor-suppressive role for KMT2D in regulating pancreatic cancer cell plasticity, especially showing that TGF-β-driven microRNA-147b silences KMT2D post-transcriptionally, and that KMT2D loss induces activin A secretion, triggering a noncanonical p38 MAPK pathway and promoting a mesenchymal phenotype. However, the impact of KMT2D deficiency on the tumor microenvironment (TME) remains unexplored. A comprehensive analysis comparing immune composition, immune checkpoint expression, and tumor-immune cell interactions in KMT2D-deficient versus wild-type PDAC has not been performed. We profiled the immune landscape in human PDAC tissues (n=5 KMT2D-mutant, n=8 wild-type [WT]) using tyramide signal amplification multiplex fluorescent immunohistochemistry (mfIHC) with two distinct antibody panels (Panel 1: PanCK, CD163, PD-L1, CD3, CD8, FoxP3; Panel 2: PanCK, CD163, CD3, CD8, TIGIT, TIM3). Eighty-six tumor-enriched regions were analyzed using InForm Cell Analysis software. For transcriptomic profiling, single-cell RNA sequencing (scRNA-seq) data from 30 PDAC patients were analyzed using the Seurat pipeline. All statistical analyses were conducted in R. KMT2D-mutant PDACs exhibited increased expression of the immune checkpoint TIGIT on CD4 T cells. Cellular engagement analysis demonstrated more CD4 T cells in proximity to epithelial cells (tumor cells) expressed TIGIT in KMT2D-mutant PDACs compared to WT PDACs. Additionally, disrupted immune cell crosstalk, as evidenced by altered spatial correlations in the proximity of antigen presenting cells (APC) to CD4 T cells, and in the distance between tumor cells and CD8 T cells in the mutant tumors. scRNA-seq analyses corroborated the enhanced TIGIT and CTLA4 expression, as well as the upregulation of the exhaustion-associated transcription factor PRDM1 in CD4 T cells from KMT2D-low PDACs, supporting the emergence of a dysfunctional, immunosuppressive CD4 T cell phenotype. Our findings reveal that KMT2D mutations are associated with an immunosuppressive tumor microenvironment in PDAC, marked by CD4 T cell dysfunction and upregulation of the immune checkpoint TIGIT. These results nominate TIGIT as a promising therapeutic target for this subset of PDAC patients and provide new insight into the mechanisms of immunotherapy resistance in KMT2D-mutant tumors. Shungang Zhang, Elaina Daniels, Jake McGue, Hongsun C. Kim, Ranga Sudharshan, Dafydd Thomas, Timothy Frankel, Jiaqi Shi. TIGIT-mediated immune suppression in KMT2D-mutant pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A061.

ACPP DDLPS transcriptomic gene signature successfully clusters with human DDLPS.

Background Undifferentiated carcinoma (UC) is a rare subtype of pancreatic cancer distinguished from UC with osteoclast-like giant cells (UC-OGC) in 2019, affecting interpretation of literature that does not distinguish these subtypes. We sought to identify translationally relevant differences between these 2 variants and compared with pancreatic ductal adenocarcinoma.Methods We characterized clinical and multiomic differences between UC (n = 32) and UC-OGC (n = 15) using DNA sequencing, RNA sequencing, and multiplex immunofluorescence and compared these findings with pancreatic ductal adenocarcinoma.Results Characteristics at diagnosis were similar between UC and UC-OGC, though the latter was more resectable (P = .009). Across all stages, median overall survival was shorter for UC than for UC-OGC (0.4 years vs 10.8 years, respectively; P = .003). This shorter survival was retained after stratification by resection, albeit without statistical significance (1.8 years vs 11.9 years, respectively; P = .08). In a subset of patients with available tissue, the genomic landscape was similar among UC (n = 9), UC-OGC (n = 5), and pancreatic ductal adenocarcinoma (n = 159). Bulk RNA sequencing was deconvoluted and, along with multiplex immunofluorescence in UC (n = 13), UC-OGC (n = 5), and pancreatic ductal adenocarcinoma (n = 16), demonstrated statistically significantly increased antigen-presenting cells, including M2 macrophages and natural killer cells, and decreased cytotoxic and regulatory T cells in UC and UC-OGC vs pancreatic ductal adenocarcinoma. Findings were similar between UC and UC-OGC , except for decreased regulatory T cells in UC-OGC (P = .04).Conclusions In this series, UC was more aggressive than UC-OGC, with these variants having more antigen-presenting cells and fewer regulatory T cells than pancreatic ductal adenocarcinoma, suggesting potential for immune-modulating therapies in the treatment of these pancreatic cancer subtypes.

The fundamental biology of pancreatic ductal adenocarcinoma has been greatly impacted by the characterization of genetically modified mouse models that allow temporal and spatial activation of oncogenic KRAS (KRAS G12D ). The most commonly used model involves targeted insertion of a cre recombinase into the Ptf1a gene. However, this approach disrupts the Ptf1a gene, resulting in haploinsufficiency that likely affects sensitivity to oncogenic KRAS ( KRAS G12D ). The goal of this study was to determine if Ptf1a haploinsufficiency affected the acinar cell response to KRAS G12D before and after induction of pancreatic injury. We performed morphological and molecular analysis of three mouse lines that express a tamoxifen-inducible cre recombinase to activate KRAS G12D in acinar cells of the pancreas. The cre-recombinase was targeted to the acinar-specific transcription factor genes, Ptf1a and Mist1/Bhlha15 , or expressed within a BAC-derived Elastase transgene. Up to two months after tamoxifen induction of KRAS G12D , morphological changes were negligible. However, induction of pancreatic injury by cerulein resulted in stark differences in tissue morphology between lines within seven days, which were maintained for at least five weeks after injury. Ptf1a creERT pancreata showed widespread PanIN lesions and fibrosis, while the Mist1 creERT and Ela-creERT models showed reduced amounts of pre-neoplastic lesions. RNA-seq analysis prior to inducing injury suggested Ptf1a creERT and Mist1 creERT lines have unique profiles of gene expression that predict a differential response to injury. Multiplex analysis of pancreatic tissue confirmed different inflammatory responses between the lines. These findings suggest understanding the mechanisms underlying the differential response to KRAS G12D will help in further defining the intrinsic KRAS-driven mechanisms of neoplasia initiation.

Spectral flow cytometry analysis of immune populations in syngeneic orthotopic allografts.

Supplemental Table 1: Demographics and clinical data for donor Gife of Life Samples Supplemental Table 2: Putative Ligand-Receptor pairs comparing all cell types from single cell sequencing of tumor samples compared to healthy samples. Supplemental Table 3: Differential gene expression using linear mixed model analysis of cell types from spatial transcriptomic ROIs. Supplemental Table 4: Kras mutational profiling on select donor samples Supplemental Table 5: Antibodies used for multiplex immunofluoresence. Supplemental Table 6: Antibodies and RNAScope probe used for co-IF/ISH.

Human LPS tumors have varied TIMEs.