Infiltration In Pancreatic Cancer Research Articles

Bioinformatic analysis of FOXN3 expression and prognostic value in pancreatic cancer.

In most cancers, forkhead box N3 (FOXN3) acts as a transcriptional inhibitor to suppress tumor proliferation, but in pancreatic cancer, the opposite effect is observed. To confirm and investigate this phenomenon, FOXN3 expression in various carcinomas was determined using GEPIA2 and was found to be highly expressed in pancreatic cancer. Kaplan-Meier plotter was then used for survival analysis, revealing that high FOXN3 expression in pancreatic cancer might be associated with a poor prognosis. Similarly, clinical samples collected for immunohistochemical staining and survival analysis showed consistent results. The RNA-seq data of pancreatic cancer patients from the TCGA were then downloaded, and the differential expression gene set was obtained using R for gene set enrichment analysis (GSEA). The intersection of the above gene sets and FOXN3-related genes was defined as related differentially expressed gene sets (DEGs), and enrichment analysis was performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Finally, we analyzed the relationship between FOXN3 and immune infiltration in pancreatic cancer. Collectively, our findings reveal that FOXN3 is involved in the occurrence and progression of pancreatic cancer and may be useful as a prognostic tool in pancreatic cancer immunotherapy.

Bioinformatics-Based Analysis: Noncoding RNA-Mediated COL10A1 Is Associated with Poor Prognosis and Immune Cell Infiltration in Pancreatic Cancer.

Background Collagen type X alpha 1 (COL10A1) is a structural component of the extracellular matrix that is aberrantly expressed in a variety of cancer tissues. However, its role in pancreatic cancer progression is not well understood. Methods The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Gene Expression Profiling Interaction Analysis (GEPIA) data were employed to explore the expression of COL10A1 in normal and tumor tissues and its prognostic value in pancreatic adenocarcinoma. The clinical data of pancreatic cancer in TCGA were used to explore the relationship between COL10A1 and clinical features. Genes coexpressed with COL10A1 were explored using multiple databases and analyzed for functional enrichment. In addition, the lncRNA/miRNA/COL10A1 axis that may be involved in COL10A1 regulation in pancreatic cancer was explored by constructing a competitive endogenous RNA (ceRNA) regulatory axis. Finally, COL10A1 was analyzed for correlation with immune cell infiltration and various immune checkpoint molecules in pancreatic cancer. Results It was found that the expression of COL10A1 was significantly increased in pancreatic cancer tissues. High expression of COL10A1 was related to the clinicopathological characteristics and the worse prognosis of pancreatic cancer patients. The TUG1/miR-144-3p/COL10A1 axis was identified as the most likely upstream noncoding RNA pathway for COL10A1 in pancreatic cancer. Besides, in pancreatic adenocarcinoma, the expression level of COL10A1 showed a significant positive correlation with tumor immune cell infiltration, biomarkers of immune cells, and expression of immune checkpoint molecules. Conclusion COL10A1 is an early diagnostic marker, and its high expression correlates with immune infiltration in pancreatic cancer. The TUG1/miR-144-3p/COL10A1 axis was identified as the most likely upstream noncoding RNA pathway for COL10A1 in pancreatic cancer.

Identification of MBOAT2 as an Unfavorable Biomarker Correlated with KRAS Activation and Reduced CD8+ T-Cell Infiltration in Pancreatic Cancer

Objectives Limited research on the role of membrane-bound O-acyltransferase domain–containing 2 (MBOAT2) in cancer biology exists. In particular, the underlying role of MBOAT2 and its potential mechanisms in pancreatic cancer have not yet been explored. Further study of MBOAT2 could provide new ideas about the carcinogenesis and treatment of pancreatic cancer (PC). Methods In the current study, the potential biological and clinical significances of MBOAT2 were explored by bioinformatics analysis. Real-time quantitative polymerase chain reaction and western blot analysis were performed to determine the level of MBOAT2 in pancreatic ductal adenocarcinoma (PDAC) cell lines. MTT, colony formation, and Transwell assays and flow cytometry of cell cycle were performed to analyze PDAC cell proliferation, migration, and cycle progression. The potential relationship between MBOAT2 level and tumor immunity was analyzed using the ESTIMATE algorithm, CIBERSORT algorithm, and single-sample gene set enrichment analysis. Results The level of MBOAT2 was remarkably upregulated in most tumors, especially pancreatic tumors, and was positively correlated with a greater rate of tumor recurrence, higher histologic grade, and worse overall survival. MBOAT2 overexpression was also closely correlated with the mutation status and expression level of driver genes, especially KRAS. Meanwhile, functional enrichment analysis demonstrated that MBOAT2 might be involved in cell–cell communication; cell cycling; the Ras signaling pathway; and immune-related biological functions such as the leukocyte activation involved in T-cell–receptor signaling pathway, the inflammatory response, and antigen processing and presentation. Furthermore, in vitro experiments demonstrated that MBOAT2 overexpression accelerated PC cell proliferation and migration. MBOAT2 overexpression also enhanced CDK2 and CCNA2 expression, leading to cell cycle progression from the G1 phase to the G2 phase. Lastly, MBOAT2 overexpression reduced the infiltration level of CD8+ T-cells, plasmacytoid dendritic cells, and activated dendritic cells but triggered a high type-2 T helper/type-1 T helper cell ration (Th2/Th1 ration) in PC. Conclusion Our findings suggest that MBOAT2 is a potential protooncogene in PDAC that predicts a poor prognosis and is related to KRAS activation and inferior infiltration of CD8+ T-cells in PC.

Autophagic Schwann cells promote perineural invasion mediated by the NGF/ATG7 paracrine pathway in pancreatic cancer

BackgroundPerineural invasion (PNI) and autophagy are two common features in the tumor microenvironment of pancreatic cancer (PanCa) and have a negative effect on prognosis. Potential mediator cells and the molecular mechanism underlying their relationships need to be fully elucidated.MethodsTo investigate the autophagy of Schwann cells (SCs) in PNI, we reproduced the microenvironment of PNI by collecting clinical PNI tissue, performing sciatic nerve injection of nude mice with cancer cells and establishing a Dorsal root ganglion (DRG) coculture system with cancer cell lines. Autophagy was detected by IHC, IF, transmission electron microscopy (TEM) and western blotting assays. Apoptosis was detected by IF, TEM and western blotting. NGF targeting molecular RO 08–2750(RO) and the autophagy inhibitor Chloroquine (CQ) were utilized to evaluate the effect on autophagy and apoptosis in SCs and PanCa cells in PNI samples.ResultsSC autophagy is activated in PNI by paracrine NGF from PanCa cells. Autophagy-activated Schwann cells promote PNI through a) enhanced migration and axon guidance toward PanCa cells and b) increased chemoattraction to PanCa cells. The NGF-targeting reagent RO and autophagy inhibitor CQ inhibited Schwann cell autophagic flux and induced Schwann cell apoptosis. Moreover, RO and CQ could induce PanCa cell apoptosis and showed good therapeutic effects in the PNI model.ConclusionsPanCa cells can induce autophagy in SCs through paracrine pathways such as the NGF/ATG7 pathway. Autophagic SCs exert a “nerve-repair like effect”, induce a high level of autophagy of cancer cells, provide a “beacon” for the invasion of cancer cells to nerve fibers, and induce directional growth of cancer cells. Targeting NGF and autophagy for PNI treatment can block nerve infiltration and is expected to provide new directions and an experimental basis for the research and treatment of nerve infiltration in pancreatic cancer.

CRMP4 Up-regulates M2 Macrophages and Myeloid-derived Suppressor Cells to Promote Pancreatic Cancer in Mice.

We previously observed higher prevalence of high-grade pancreatic intraepithelial neoplasia (PanIN) in LSL-KrasG12D/+; Pdx1Cre/+ (KC-Crmp4wild) mice than LSL-KrasG12D/+; Pdx1Cre/+; Crmp4-/- (KC-Crmp4-/-) mice. This study investigated the relationship between collapsin response mediator protein 4 (CRMP4) and immune cell infiltration in pancreatic cancer. PanIN was induced by intraperitoneal injection of caerulein into KC-Crmp4wild and KC-Crmp4-/- mice, and immune cells in PanIN lesions were compared. Subcutaneous tumors were created by injecting Pan02 cells, and tumor diameter was compared between Crmp4wild and Crmp4-/- mice every 7 days. Peritumoral immune cells were examined immunohisto chemically. High-grade PanIN in KC mice showed statistically significantly high expression of CD163 (p=0.031) and CD11b (p=0.027). Following subcutaneous injection of Pan02 cells, tumor diameter was greater in Crmp4wild mice than Crmp4-/- mice. Crmp4wild mice exhibited higher CD163 and CD11b expression than Crmp4-/- mice in tumors (p<0.001). CRMP4 might promote pancreatic cancer by up-regulating M2 macrophages and myeloid-derived suppressor cells.

High Expression of CHST11 Correlates with Poor Prognosis and Tumor Immune Infiltration of Pancreatic Cancer.

Pancreatic cancer (PC) is the seventh leading cause of cancer death worldwide, and its prognosis is poor. It has been reported that carbohydrate sulfotransferase 11 (CHST11) is associated with tumor progression in various cancers but rarely reported in PC. The aim of this study was to comprehensively investigate the clinical value of CHST11 in PC. CHST11 gene expression analysis was conducted by The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), Gene Expression Omnibus (GEO), and Human Protein Atlas (HPA) databases. Survival analysis and receiver operating characteristic (ROC) curves were performed to evaluate the prognostic significance of CHST11 based on TCGA and International Cancer Genome Consortium (ICGC) databases. Additionally, functional enrichment analysis was also performed. Moreover, single-sample Gene Set Enrichment Analysis (ssGSEA) and ESTIMATE algorithm were used to assess immune infiltration. Finally, the relationship between CHST11 expression and immune checkpoint gene levels was estimated by Spearman's correlation analysis. CHST11 was highly expressed in PC tissues compared with normal tissues, and the expression of CHST11 was related to T stage, N stage, and histological grade. The survival time of PC patients with CHST11 low-expression was significantly better than those with CHST11 high-expression. The areas under the ROC curve corresponding to 1-, 3-, and 5-year survival in the TCGA dataset were 0.573, 0.671, and 0.740, respectively, and those in the ICGC dataset were 0.588, 0.602, and 0.626, respectively. Functional enrichment analysis showed that CHST11 may be involved in the regulation of immune function. Finally, the level of CHST11 was significantly correlated with 22 types of tumor-infiltrating immune cells, immune and stromal scores, and 7 immune checkpoint genes. High-expression of CHST11 was correlated with poor prognosis and tumor immune infiltration in PC. Moreover, CHST11 may act as a novel prognostic marker and potential therapeutic target of PC.

Collagen Organization Does Not Influence T-Cell Distribution in Stroma of Human Pancreatic Cancer.

Simple SummaryThe excessive desmoplasia is the hallmark of human pancreatic cancer that influences the local T-cell-based immune response. In the present work, the stromal collagen organization in normal and malignant pancreatic tissues as well as its relationsship to T-cell distribution in pancreatic cancer were studied. It was found that differences in collagen organization do not change the spatial orientation of T-cell migration and do not influence the availability of tumor cells for T-cells. The results of the study do not support the concept of use of stroma collagen organization for improvement of spatial T-cell distribution in the tumor. The dominant intrastromal T-cell infiltration in pancreatic cancer is mainly caused by the contact guidance through the excessive desmoplastic reaction and could represent one of the obstacles to an effective immune response in this tumor type. This study analyzed the collagen organization in normal and malignant pancreatic tissues as well as its influence on T-cell distribution in pancreatic cancer. Human pancreatic tissue was analyzed using immunofluorescence staining and multiphoton and SHG microscopy supported by multistep image processing. The influence of collagen alignment on activated T-cells was studied using 3D matrices and time-lapse microscopy. It was found that the stroma of malignant and normal pancreatic tissues was characterized by complex individual organization. T-cells were heterogeneously distributed in pancreatic cancer and there was no relationship between T-cell distribution and collagen organization. There was a difference in the angular orientation of collagen alignment in the peritumoral and tumor-cell-distant stroma regions in the pancreatic ductal adenocarcinoma tissue, but there was no correlation in the T-cell densities between these regions. The grade of collagen alignment did not influence the directionality of T-cell migration in the 3D collagen matrix. It can be concluded that differences in collagen organization do not change the spatial orientation of T-cell migration or influence stromal T-cell distribution in human pancreatic cancer. The results of the present study do not support the rationale of remodeling of stroma collagen organization for improvement of T-cell–tumor cell contact in pancreatic ductal adenocarcinoma.

YAP1 is a Prognostic Biomarker and Correlated with Immune Cell Infiltration in Pancreatic Cancer.

Yes-associated protein-1 (YAP1) is an important effector of the Hippo pathway and has crosstalk with other cancer signaling pathways. It induces an immunosuppressive tumor microenvironment by activating pathways in several cellular components. However, the mechanisms by which it drives immune infiltration in pancreatic cancer remain poorly understood. We analyzed the expression of YAP1 as well as its prognostic value and correlations with immune infiltrates in various cancers, with a focus on pancreatic cancer. In particular, using the Oncomine database and Gene Expression Profiling Interactive Analysis (GEPIA) database, we found that YAP1 is differentially expressed between tumor tissues and control tissues in a number of cancers and in particular, is elevated in pancreatic cancer. Using the Kaplan–Meier plotter, GEPIA, and Long-term Outcome and Gene Expression Profiling database of pan-cancers (LOGpc), we further established the prognostic value of YAP1. We found that YAP1 expression was significantly related to outcomes in multiple types of cancer based on data from The Cancer Genome Atlas, particularly in pancreatic cancer. Correlations between YAP1 and immune cell infiltration and immune cell marker expression were examined using Tumor Immune Estimation Resource and GEPIA. High expression levels of YAP1 were significantly associated with a variety of immune markers and immune cell subsets in pancreatic cancer. These results suggest that YAP1 is correlated with patient outcomes and tumor immune cell infiltration in multiple cancer types and is a valuable prognostic biomarker in pancreatic cancer.

Correlations between tumor mutation burden and immune infiltrates and their prognostic value in pancreatic cancer by bioinformatic analysis

PurposeWe aimed to investigate the patterns and prognostic roles of tumor mutation burden and immune microenvironment in pancreatic cancer. MethodsThe somatic mutation data, transcriptome profiles and clinical information were downloaded from the Cancer Genome Atlas database. Gene expression difference, Gene ontology, KEGG, gene set enrichment analyses and “CIBERSORT” algorithm were performed to screen differentially expressed genes, enriched functions or pathways and immune infiltrates differences between high and low TMB groups. Single sample gene set enrichment and unsupervised consensus clustering analyses were used for immunity grouping. Immune cell infiltration and expressions of HLA and checkpoint genes were investigated. Finally, a nomogram model integrating TMB and immune infiltration was established. ResultsA total of 608 differentially expressed genes were identified between high and low TMB groups, KEGG base excision repair and DNA replication pathways were enriched in high TMB group. Infiltration levels of M0 macrophages were higher and dendritic resting cells were lower in high TMB group. The risk model based on TMB-related immune genes, FAM19A2 and SLC22A17 was established and high risk scores indicated poorer prognosis. The expressions of HLA genes and immune checkpoint genes were higher in high immunity group. The nomogram showed remarkable ability for individualized survival estimation with good AUC values (0.794 and 0.800, respectively) for 3- and 5-year survival rates prediction. ConclusionsThe characteristics of tumor mutation burden and immune infiltration in pancreatic cancer provide new insights into the tumor microenvironment, immunotherapies and a novel prognostic nomogram model for pancreatic cancer patients.

Composition, Spatial Characteristics, and Prognostic Significance of Myeloid Cell Infiltration in Pancreatic Cancer.

Although abundant myeloid cell populations in the pancreatic ductal adenocarcinoma (PDAC) microenvironment have been postulated to suppress antitumor immunity, the composition of these populations, their spatial locations, and how they relate to patient outcomes are poorly understood. To generate spatially resolved tumor and immune cell data at single-cell resolution, we developed two quantitative multiplex immunofluorescence assays to interrogate myeloid cells (CD15, CD14, ARG1, CD33, HLA-DR) and macrophages [CD68, CD163, CD86, IFN regulatory factor 5, MRC1 (CD206)] in the PDAC tumor microenvironment. Spatial point pattern analyses were conducted to assess the degree of colocalization between tumor cells and immune cells. Multivariable-adjusted Cox proportional hazards regression was used to assess associations with patient outcomes. In a multi-institutional cohort of 305 primary PDAC resection specimens, myeloid cells were abundant, enriched within stromal regions, highly heterogeneous across tumors, and differed by somatic genotype. High densities of CD15+ARG1+ immunosuppressive granulocytic cells and M2-polarized macrophages were associated with worse patient survival. Moreover, beyond cell density, closer proximity of M2-polarized macrophages to tumor cells was strongly associated with disease-free survival, revealing the clinical significance and biologic importance of immune cell localization within tumor areas. A diverse set of myeloid cells are present within the PDAC tumor microenvironment and are distributed heterogeneously across patient tumors. Not only the densities but also the spatial locations of myeloid immune cells are associated with patient outcomes, highlighting the potential role of spatially resolved myeloid cell subtypes as quantitative biomarkers for PDAC prognosis and therapy.

GTF2IRD1 overexpression promotes tumor progression and correlates with less CD8+ T cells infiltration in pancreatic cancer.

Background: General Transcription Factor II-I Repeat Domain-Containing Protein 1 (GTF2IRD1) is a member of the GTF21 gene family, which encodes a set of multifunctional transcription factors. However, the potential function of GTF2IRD1 in pancreatic cancer (PC) still remains unknown. Study on GTF2IRD1 might provide a new insight into the carcinogenesis and therapeutics of PC.Methods: In the current study, the clinical significance and potential biological of GTF2IRD1 were evaluated by bioinformatics analysis. The oncogenic role of GTF2IRD1 in PC was also determined using in vitro studies. Possible associations between GTF2IRD1 expression and tumor immunity were analyzed using ESTIMATE algorithm and single-sample Gene Set Enrichment Analysis (ssGSEA).Results: GTF2IRD1 expression was significantly up-regulated in tumor tissues, and positively associated with higher histologic grade, higher American Joint Committee on Cancer (AJCC) stage, and worse prognosis. Function enrichment analysis demonstrated that GTF2IRD1 may be involved in pancreatic adenocarcinoma pathway, TGF-β signaling pathway, and tumor-infiltrating lymphocyte (TIL) related biological functions, such as T-cell receptor signaling pathway, leukocyte transendothelial migration, resistin as a regulator of inflammation, and regulation of leukocyte-mediated cytotoxicity. Knockdown of GTF2IRD1 expression inhibited cancer cell proliferation, colony formation, and invasion in vitro. ESTIMATE algorithm and ssGSEA demonstrated that GTF2IRD1 expression negatively correlated with the infiltration and anti-tumor activity of TILs, especially for CD8+ T cells.Conclusion: The study demonstrates that GTF2IRD1 overexpression promotes tumor progression and correlates with less CD8+ T cells infiltration in PC.

Transcriptional and immunohistological assessment of immune infiltration in pancreatic cancer.

Pancreatic adenocarcinoma is characterized by a complex tumor environment with a wide diversity of infiltrating stromal and immune cell types that impact the tumor response to conventional treatments. However, even in this poorly responsive tumor the extent of T cell infiltration as determined by quantitative immunohistology is a candidate prognostic factor for patient outcome. As such, even more comprehensive immunophenotyping of the tumor environment, such as immune cell type deconvolution via inference models based on gene expression profiling, holds significant promise. We hypothesized that RNA-Seq can provide a comprehensive alternative to quantitative immunohistology for immunophenotyping pancreatic cancer. We performed RNA-Seq on a prospective cohort of pancreatic tumor specimens and compared multiple approaches for gene expression-based immunophenotyping analysis compared to quantitative immunohistology. Our analyses demonstrated that while gene expression analyses provide additional information on the complexity of the tumor immune environment, they are limited in sensitivity by the low overall immune infiltrate in pancreatic cancer. As an alternative approach, we identified a set of genes that were enriched in highly T cell infiltrated pancreatic tumors, and demonstrate that these can identify patients with improved outcome in a reference population. These data demonstrate that the poor immune infiltrate in pancreatic cancer can present problems for analyses that use gene expression-based tools; however, there remains enormous potential in using these approaches to understand the relationships between diverse patterns of infiltrating cells and their impact on patient treatment outcomes.

FAM83H overexpression predicts worse prognosis and correlates with less CD8+ T cells infiltration and Ras-PI3K-Akt-mTOR signaling pathway in pancreatic cancer.

Family with sequence similarity 83 members H (FAM83H) is one member of Family with sequence similarity 83 (FAM83) family, which possess oncogenic properties in several types of cancer. However, the potential function of FAM83H in pancreatic cancer (PC) still remain unknown. This study aims to explore the role of FAM83H during pancreatic carcinogenesis and the regulation of immune infiltration in PC. In the current study, the clinical significance and potential biological of FAM83H were evaluated by bioinformatics analysis. Possible associations between FAM83H expression and tumor immunity were analyzed using ESTIMATE algorithm and single-sample gene set enrichment analysis (ssGSEA). FAM83H expression was significantly upregulated in tumor tissues, and positively associated with higher histologic grade, tumor recurrence, and worse prognosis. FAM83H overexpression is notably associated with KRAS activation. And functional enrichment analysis demonstrated that FAM83H may be involved in positive regulation of cell proliferation and migration, Ras protein signal transduction, regulation of cell-matrix adhesion, epithelial to mesenchymal transition (EMT), TGF-β receptor signaling in EMT, and activated NOTCH transmits signal to the nucleus. ESTIMATE algorithm and ssGSEA demonstrated that FAM83H overexpression suppressed the infiltration and antitumor activity of tumor-infiltrating lymphocytes (TILs), especially for CD8+ T cells. Besides, FAM83H overexpression significantly correlated with low expression of TIL-related gene markers (e.g. CD8A, CD8B, CD2, CD3D, and CD3E). The study suggests that FAM83H overexpression predicts poor prognosis and correlates with less CD8+ T cells infiltration and Ras-PI3K-Akt-mTOR signaling pathway in PC.

Prognostic alternative splicing signature reveals the landscape of immune infiltration in Pancreatic Cancer.

Background: Pancreatic cancer (PC) is an aggressive cancer with worse survival in the world. Emerging evidence suggested that the imbalance of alternative splicing (AS) is a hallmark of cancer and indicated poor prognosis of patients. Genes-derived splicing events can produce neoepitopes for immunotherapy. However, the profound study of splicing profiling in PC is still elusive. We aimed to identification of novel prognostic signature across a comprehensive splicing landscape and reveal their relationship with tumor-infiltrating immune cells in pancreatic cancer microenvironment.Methods: Based on integrated analysis of splicing profiling and clinical data, differentially splicing events were filtered out. Then, stepwise Cox regression analysis was applied to identify survival-related splicing events and construct prognostic signature. Functional enrichment analysis was performed to explore biology function. Kaplan-Meier curves and receiver operating characteristic (ROC) curves were performed to validate the predictive effect of predictive signature. We also verified the clinical value of prognostic signature under the influence of different clinical parameters. For deeper analysis, we evaluated the correlation between prognostic signature and infiltrating immune cells by CIBERSORT.Results: According to systematic analyzing, a final six splicing events were identified and validated the good prognostic capability in entire TCGA dataset, validation set 1 and validation set 2 by Kaplan-Meier curves (P < 0.0001). The area under the curve (AUC) of ROC curves were also confirmed the high predictive efficiency of the prognostic signature in these three cohorts (AUC = 0.857, 0.895 and 0.788). In order to validate whether prognostic signature highlights a correlation between AS and immune contexture, CIBERSORT was performed to analyze the proportion of tumor-infiltrating immune cells in PC. Based on prognostic signature, we identified survival-related immune cells including CD8 T cells (P = 0.0111), activated CD4 memory T cells (P = 0.0329) and resting mast cells (P = 0.0352).Conclusion: In conclusion, our study contribute to provide a promising prognostic signature based on six splicing events and revealed prognosis-related immune cells which indeed represented novel tumor drivers and provide potential targets for personalized therapeutic.

Abstract A52: Modulation of Hedgehog signaling alters immune infiltration in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDA) has a dismal 5-year survival rate of 9%, making this disease one of the deadliest human malignancies. Primary barriers to the treatment of pancreatic cancer include extensive stromal interactions and sustained immune suppression. Aberrant Hedgehog (HH) pathway activity is a hallmark of pancreatic tumorigenesis. Tumor-derived HH ligands signal in a paracrine fashion to the surrounding stroma to influence tumor growth. Expression of HH ligands increases during PDA progression, and previous work has shown that genetic deletion of Sonic HH from the epithelium of mice with pancreatic tumors results in increased Indian HH (Ihh) expression. Methods: Ihh was deleted in tumor cells lines (IhhKO) derived from a genetically engineered mouse model of pancreatic cancer (KrasG12D;Trp;P48-Cre), using CRISPR/Cas-9 gene editing to assess the role of Ihh in the tumor microenvironment. The level of HH signaling was determined using tumor cell co-cultures with Gli1lacZ fibroblasts (derived from mice with a lacZ reporter allele knocked into the Gli1 locus), in which beta galactosidase activity serves as a readout for HH signaling. WT and IhhKO tumor cells were orthotopically transplanted into the pancreas of syngeneic C57BL/6 mice. Human pancreas samples were obtained from surgical resection of pancreatic adenocarcinoma or fine-needle biopsy procedure (FNB). Immune profiling of mouse and human pancreatic tumors was performed using Cytometry Time-of-Flight analysis, and tumor composition was analyzed by single-cell RNA sequencing. In vitro cultures with pancreatic fibroblasts treated with either WT or IhhKO tumor cell conditioned media (CM) were cultured to assess tumor crosstalk. Results: Tumor cells lacking Ihh were generated through CRISPR/Cas-9 deletion, and this was confirmed by qRT-PCR. Co-culture of IhhKO tumor cells with Gli1lacZ fibroblasts results in decreased Gli1 expression both in vitro and in vivo. Immune profiling revealed that tumors lacking Ihh have significantly fewer macrophages (CD11b+/F4/80+), resulting in decreased presence of immunosuppressive factors such as arginase 1 and PDL1. Immune profiling of human PDA revealed similar populations of immunosuppressive myeloid cells present in tumors. In vitro co-cultures demonstrated CCL2 expression was reduced in pancreatic fibroblasts cultured with IhhKO-CM, providing mechanistic insight into the in vivo phenotype observed. Further, scRNA seq analysis suggests that modulation of HH signaling in the tumor microenvironment alters chemokine and immunomodulatory signaling pathways driven by fibroblasts in the pancreatic tumor microenvironment. Significance of Impact: HH signaling in pancreatic fibroblasts contributes to the establishment of an immune-suppressive environment in pancreatic cancer. Combining methods to target HH signaling and immune checkpoint therapy has translational potential in treating pancreatic cancer patients. Citation Format: Nina Steele, Samantha Kemp, Valerie Irizarry-Negron, Veerin Sirihorachai, Ahmed Abbas, Eileen Carpenter, Christopher Halbrook, Carlos Espinoza, Costas Lyssiotis, Howard Crawford, Timothy Frankel, Filip Bednar, Ben Allen, Marina Pasca di Magliano. Modulation of Hedgehog signaling alters immune infiltration in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A52.

Cancer‑associated adipocytes exhibit distinct phenotypes and facilitate tumor progression in pancreatic cancer.

Adipocyte infiltration in pancreatic cancer (PC) has been demonstrated to be independently associated with PC risk and an active contributor to tumor progression. However, to date, little is known about these unique pancreatic tumor-surrounding adipocytes, or their response to cancer cells. The present study utilized an in vitro indirect coculture model in which the phenotypic changes of adipocytes following exposure to PC cells were directly observed. RNA-sequencing was performed on 3T3-L1 adipocytes cultured with or without Panc-1 cancer cells, and significant changes were identified at the transcriptional level. In terms of delipidation and the impaired function of glucose and lipid metabolism, coculture with tumor cells resulted in an altered metabolic phenotype in mature adipocytes. In co-cultured adipocytes, the appearance of fibroblast-like cells was observed, and the mesenchymal cell differentiation pathway was enriched following the integrated analysis into the transcriptome. In addition, reverse transcription-quantitative PCR analyses of co-cultured adipocytes revealed a loss in gene expression of mature adipocyte markers, and a gain in gene expression of fibroblast-specific markers. It was also confirmed that newly generated cancer-associated adipocytes could facilitate the invasive capacities of the tumor, and may contribute to PC stromal remodeling. The present study supports a novel concept that reprogramming of stromal adipocytes orchestrated by PC cells may generate cancer-associated adipocytes with activated phenotypes, which may ultimately drive pancreatic tumor progression.

Abstract 1500: Mediators of CD8+ cytotoxic T lymphocyte infiltration in pancreatic cancer

Abstract Pancreatic cancer continues to have the highest mortality rate of all solid cancers, with a 5-year overall survival of approximately 8%. Despite recent progress in other malignancies, immunotherapy remains ineffective against pancreatic cancer. Previous work from our group and others has shown that tumors with mismatch repair (MMR) and homologous repair (HR) deficiency have increased immune activity, likely attributable to the inherently increased mutational and neoantigen load in these patients. However, there remains a subset of pancreatic cancer patients with increased CD8+ T cell infiltration, despite lacking these specific mutational signatures. Furthermore, an increasing body of evidence has shown that mutational burden and neoantigen load only partially explain the T cell-inflamed phenotype seen in tumors, implicating the presence of additional mechanisms that drive T cell infiltration in cancer. Several recent studies have highlighted the importance of proper CD8+ T cell priming by antigen presenting cells (APCs), particularly Batf3+ dendritic cells (DCs), and subsequent tumor infiltration via chemokines for immunotherapy to be effective. In this study, we investigated the involvement of chemokines in cytotoxic T lymphocyte infiltration in pancreatic cancer. Using a bioinformatics-driven approach, we analyzed 78 treatment-naïve, primary pancreas cancer resections for associations between CD8+ tumour infiltrating lymphocytes (TILs) and chemokine expression by RNAseq. A panel of chemokines, including CCL4, CCL5, CXCL9, CXCL10, and CXCL11, was highly associated with CD8+ TILs (p &amp;lt; 0.001). Segregating 173 tumour-enriched patient RNAseq samples based on expression of CXCL9 and CXCL10, we found those with higher expression of these chemokines had increased immune activation signatures. Importantly, this included increased MHC I presentation, presence of Batf3+ DCs, and T cell/APC co-stimulation (p &amp;lt; 0.001), while we observed no differences in conventional predictors of CD8+ T cell infiltration such as SNV counts or neoantigens between groups. These results were consistent across ICGC and TCGA data sets. Moreover, these results were also recapitulated in 72 tumor-enriched liver metastases, suggesting an underlying immunobiology that may occur in both primary and metastatic sites. The cellular sources of these chemokines, as determined by immunohistochemical analysis, and the role of tumor-sensing innate immune pathways leading to CD8+ T cell priming, by pathway and differential gene expression analysis, are currently being investigated. Taken together, these results demonstrate a potential role for these chemokines in recruiting CD8+ T cells in pancreatic cancer. Understanding mediators driving cytotoxic T cell infiltration will help identify mechanisms leading to proper CD8+ T cell priming and homing into tumors, to stratify patients amenable for known and novel immunotherapies. Citation Format: Joan Miguel Romero, Barbara Grünwald, Ashton Connor, Gun Ho Jang, Prashant Bavi, Aaditeya Jhaveri, Mehdi Masoomian, Sandra Fischer, Amy Zhang, Robert E. Denroche, Tracy McGaha, Faiyaz Notta, Pamela Ohashi, Grainne O'Kane, Julie Wilson, Jennifer Knox, Steven Gallinger. Mediators of CD8+ cytotoxic T lymphocyte infiltration in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1500.

3373 Modulation of Hedgehog Signaling Alters Immune Infiltration in Pancreatic Cancer

OBJECTIVES/SPECIFIC AIMS: Pancreatic ductal adenocarcinoma (PDA) has a dismal 5-year survival rate of 9%, making this disease one of the deadliest human malignancies (https://seer.cancer.gov/). Primary barriers to the treatment of pancreatic cancer include extensive stromal interactions and sustained immune suppression. Aberrant Hedgehog (HH) pathway activity is a hallmark of pancreatic tumorigenesis. Tumor-derived HH ligands signal in a paracrine fashion to the surrounding stroma to influence tumor growth. Expression of HH ligands increases during PDA progression, and previous work has shown that genetic deletion of Sonic HH (Shh) from the epithelium of mice with pancreatic tumors results in increased Indian HH (Ihh) expression. This research aims to investigate the translational impact of changes in immune infiltration following deletion of IHH in a preclinical mouse model of pancreatic cancer. METHODS/STUDY POPULATION: Ihh was deleted in tumor cells lines (IhhKO) derived from a genetically engineered mouse model of pancreatic cancer (LSL-KrasG12D/+;LSL-TrpR270H;P48-Cre), using CRISPR/Cas-9 gene editing to assess the role of Ihh in the tumor microenvironment. The level of HH signaling was determined using tumor cell co-cultures with Gli1lacZ fibroblasts (derived from mice with a lacZ reporter allele knocked into the Gli1 locus), in which Beta Galactosidase activity serves as a readout for HH signaling. WT and IhhKO tumor cells were orthotopically transplanted into the pancreas of syngeneic C57BL/6 mice. Human pancreas samples were obtained from surgical resection of pancreatic adenocarcinoma, or fine needle biopsy procedure (FNB). Immune profiling of mouse and human pancreatic tumors was performed using Cytometry Time-of-Flight analysis (CyTOF), and tumor composition was analyzed by single-cell RNA sequencing (scRNA seq). In vitro cultures with pancreatic fibroblasts treated with either WT or IhhKO tumor cell conditioned media (CM) were cultured with bone-marrow derived macrophages to assess tumor crosstalk. RESULTS/ANTICIPATED RESULTS: Tumor cells lacking Ihh were generated through CRISPR/Cas-9 deletion, and this was confirmed by qRT-PCR. Co-culture of IhhKO tumor cells with Gli1lacZ fibroblasts results in decreased Gli1 expression both in vitro and in vivo. Immune profiling revealed that tumors lacking Ihh have significantly fewer tumor associated macrophages (CD11b+/F4/80+/CD206+), resulting in decreased presence of immunosuppressive factors such as arginase 1 and PDL1. Immune phenotyping of human pancreatic tissues revealed similar populations of immunosuppressive myeloid cells present in tumors. In vitro co-cultures demonstrated that, in the presence of bone-marrow derived macrophages, immunosuppressive IL-6 production was reduced in pancreatic fibroblasts cultured with IhhKO-CM, as compared to fibroblasts cultured with WT-CM, providing mechanistic insight into the in vivo phenotype observed. Further, scRNA seq analysis suggests that modulation of HH signaling in the tumor microenvironment alters chemokine and immunomodulatory signaling pathways driven by fibroblasts in the pancreatic tumor microenvironment. DISCUSSION/SIGNIFICANCE OF IMPACT: HH signaling in pancreatic fibroblasts contributes to the establishment of an immune suppressive environment in pancreatic cancer. Combining methods to target HH signaling and immune checkpoint therapy has translational potential in treating pancreatic cancer patients.

Relationship between desmoplastic pattern and lymphocytes infiltration in pancreatic cancer.

Relationship between desmoplastic pattern and lymphocytes infiltration in pancreatic cancer.

Relationship between desmoplastic pattern and lymphocytes infiltration in pancreatic cancer.

317 Background: The proposed histologic desmoplastic reaction (DR) categorization and lymphocyte infiltration (LI) directly reflects tumor behavior in a modulating stromal environment and could provide valuable prognostic information for colorectal cancer patients, but the prognostic role of DR and LI has not been investigated in pancreatic cancer patients. Methods: A total of 87 pancreatic cancer patients who underwent potentially curative surgery for their tumors in our hospital (2008 to 2013) were included. DR was classified as mature, intermediate, or immature on the basis of the existence of keloid-like collagen and myxoid stroma-distinctive histologic products of extracellular matrix remodeling. LI was classified as Pattern I; circumferential lymphocytes zone without disruptions around tumor, Pattern II; lymphocytes zone with partial disruptions and thinning around tumor, Pattern III; focal lymphocytes foci around tumor at most invasive border without lymphocyte zone, and Pattern IV; no lymphocyte foci around tumor. Results: With regard to DR, 0, 23, and 64 patients were classified as mature, intermediate, and immature, respectively. Five-year overall survival rates (#%) were higher in immature group (p=0.0040) than those of intermediate group (#%). Multivariate analysis revealed that DR was an independent prognostic factor along with residual tumor (p=0.0300). With regard to LI, 0, 6, 70, and 11 patients were classified as Pattern I, Pattern II, Pattern III, and Pattern IV, respectively. There was no relationship between DR and LI. Conclusions: The DR categorization seemed to reflect tumor behavior and could provide valuable prognostic information for pancreatic cancer patients. We did not show any relationship between DR and LI in pancreatic cancer.