Macrophages In Pancreatic Adenocarcinoma Research Articles

Single-cell sequencing combined with spatial transcriptomics reveals that the IRF7 gene in M1 macrophages inhibits the occurrence of pancreatic cancer by regulating lipid metabolism-related mechanisms.

The main focus of this study is to explore the molecular mechanism of IRF7 regulation on RPS18 transcription in M1-type macrophages in pancreatic adenocarcinoma (PAAD) tissue, as well as the transfer of RPS18 by IRF7 via exosomes to PAAD cells and the regulation of ILF3 expression. By utilising single-cell RNA sequencing (scRNA-seq) data and spatial transcriptomics (ST) data from the Gene Expression Omnibus database, we identified distinct cell types with significant expression differences in PAAD tissue. Among these cell types, we identified those closely associated with lipid metabolism. The differentially expressed genes within these cell types were analysed, and target genes relevant to prognosis were identified. Flow cytometry was employed to assess the expression levels of target genes in M1 and M2 macrophages. Cell lines with target gene knockout were constructed using CRISPR/Cas9 editing technology, and cell lines with target gene knockdown and overexpression were established using lentiviral vectors. Additionally, a co-culture model of exosomes derived from M1 macrophages with PAAD cells was developed. The impact of M1 macrophage-derived exosomes on the lipid metabolism of PAAD cells in the model was evaluated through metabolomics analysis. The effects of M1 macrophage-derived exosomes on the viability, proliferation, division, migration and apoptosis of PAAD cells were assessed using MTT assay, flow cytometry, EdU assay, wound healing assay, Transwell assay and TUNEL staining. Furthermore, a mouse PAAD orthotopic implantation model was established, and bioluminescence imaging was utilised to assess the influence of M1 macrophage-derived exosomes on the intratumoural formation capacity of PAAD cells, as well as measuring tumour weight and volume. The expression of proliferation-associated proteins in tumour tissues was examined using immunohistochemistry. Through combined analysis of scRNA-seq and ST technologies, we discovered a close association between M1 macrophages in PAAD samples and lipid metabolism signals, as well as a negative correlation between M1 macrophages and cancer cells. The construction of a prognostic risk score model identified RPS18 and IRF7 as two prognostically relevant genes in M1 macrophages, exhibiting negative and positive correlations, respectively. Mechanistically, it was found that IRF7 in M1 macrophages can inhibit the transcription of RPS18, reducing the transfer of RPS18 to PAAD cells via exosomes, consequently affecting the expression of ILF3 in PAAD cells. IRF7/RPS18 in M1 macrophages can also suppress lipid metabolism, cell viability, proliferation, migration, invasion and intratumoural formation capacity of PAAD cells, while promoting cell apoptosis. Overexpression of IRF7 in M1 macrophages may inhibit RPS18 transcription, reduce the transfer of RPS18 from M1 macrophage-derived exosomes to PAAD cells, thereby suppressing ILF3 expression in PAAD cells, inhibiting the lipid metabolism pathway, and curtailing the viability, proliferation, migration, invasion of PAAD cells, as well as enhancing cell apoptosis, ultimately inhibiting tumour formation in PAAD cells in vivo. Targeting IRF7/RPS18 in M1 macrophages could represent a promising immunotherapeutic approach for PAAD in the future.

Prognostic Values and Clinical Significance of S100 Family Member's Individualized mRNA Expression in Pancreatic Adenocarcinoma.

Objective: Pancreatic adenocarcinoma (PAAD) is a common malignant tumor worldwide. S100 family (S100s) is wildly involved in regulating the occurrence, development, invasion, metastasis, apoptosis, and drug resistance of many malignant tumors. However, the expression pattern, prognostic value, and oncological role of individual S100s members in PAAD need to be elucidated. Methods: The transcriptional expression levels of S100s were analyzed through the Oncomine and GEPIA, respectively. The protein levels of S100s members in PAAD were studied by Human Protein Atlas. The correlation between S100 mRNA expression and overall survival and tumor stage in PAAD patients was studied by GEPIA. The transcriptional expression correlation and gene mutation rate of S100s members in PAAD patients were explored by cBioPortal. The co-expression networks of S100s are identified using STRING and Gene MANIA to predict their potential functions. The correlation of S100s expression and tumor-infiltrating immune cells was tested by TIMER. Pathway activity and drug target analyzed by GSCALite. Results: 13 S100s members were upregulated in PAAD tissues. 15 S100s members were associated with TP53 mutation. Expression levels of S100A3/A5/A6/A10/A11/A14/A16/B/P/Z were significantly correlated with the pathological stage. Prognosis analysis demonstrated that PAAD patients with low mRNA levels of S100A1/B/Z or high levels of S100A2/A3/A5/A10/A11/A14/A16 had a poor prognosis. Immuno-infiltration analysis showed that the mRNA levels of S100A10/A11/A14/A16 were correlated with the infiltration degree of macrophages in PAAD. Drug sensitivity analysis showed that PAAD expressing high levels of S100A2/A6/A10/A11/A13/A14/A16 maybe resistant to small molecule drugs. Conclusion: This study identifies the clinical significance and biological functions of the S100s in PAAD, which may provide novel insights for the selection of prognostic biomarkers.

Abstract A58: Notch signaling is a key regulator for immune-suppressive function of tumor-associated macrophages in pancreatic adenocarcinoma

Abstract Objective: Pancreatic ductal adenocarcinoma (PDA) has the most dismal prognosis among all human cancers. The poor survival rate has been attributed to high rate of metastasis and low efficacy of current therapies, which may result from the complex immunosuppressive tumor microenvironment. We explored the role of Notch signaling, a key regulator of immune responses, within the PDA microenvironment. Our goal is to better define these immunosuppressive pathways and to develop effective therapies for patients with PDA. Methods: Two primary mouse PDA cell lines (7940B and mT3-2D) were used for implantation into the pancreata of transgenic CBF:H2B-Venus mice; this enabled single-cell reporting of Notch signaling within the PDA microenvironment. Flow cytometry was used to assess Venus expression in different cell populations. In addition, tumor-associated macrophages (TAMs) were sorted by Notch activity [Venus (+) vs. Venus (-)] to analyze expression of Notch targets and macrophage polarization markers by qPCR. Next, a bone marrow-derived macrophage (BMDM) culture system was developed to investigate Notch activation in vitro. Lastly, we evaluated the therapeutic effect of Notch pathway inhibition as monotherapy and in combination with immune checkpoint programmed death receptor-1 (PD-1) blockade in an orthotopic PDA model. Results: Flow cytometry revealed that TAMs were the most abundant immune cell population in the orthotopic PDA microenvironment. About 50% of TAMs were Venus (+), which had significant enrichment in M2 polarization markers such as Arginase 1 (Arg1) and Notch targets (such as Hes1) when compared to Venus (-) TAMs by qPCR. Immunofluorescence showed co-localization of Venus and Hes1 in F4/80+ TAMs. In vitro experiments identified that Notch activation was only present in BMDMs from Venus mice when co-cultured directly with 7940B PDA cells and that Arg1 expression was only found in Venus (+) macrophages. Notch inhibitor LY3039478 led to decreased expression of both M2 polarization markers such as Arg1, Mrc1, Ym1, and immunosuppressive cytokine TGFβ in co-cultured BMDM. In the orthotopic PDA mouse model, neither LY3039478 nor anti-PD1 was effective in treating PDA in vivo, whereas combined inhibition of the Notch pathway and immune checkpoint resulted in significant reduction in tumor growth. Conclusion: Our data demonstrate that Notch activation within TAMs from murine PDAs is associated with an immunosuppressive (M2-like) phenotype, thereby suggesting that Notch signaling plays a role in macrophage polarization within the PDA microenvironment. While pharmacologic inhibition of the Notch pathway was ineffective in treating orthotopic murine PDA in vivo, the combination of Notch inhibition with PD-1 blockade resulted in a therapeutic effect. Targeting the Notch signaling pathway may lessen the immunosuppressive phenotype of TAMs within the PDA microenvironment and improve the efficacy of immune-based therapies for patients with PDA. Citation Format: Wei Yan, Yaqing Zhang, Rosa Menjivar, Filip Bednar, Marina Pasca di Magliano, William Reece Burns. Notch signaling is a key regulator for immune-suppressive function of tumor-associated macrophages in pancreatic adenocarcinoma [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 A58.