Immune Cells In Tumor Microenvironment Research Articles

Genomic and Transcriptomic Landscape of RET Wild-Type Medullary Thyroid Cancer and Potential Use of Mitogen-Activated Protein Kinase-Targeted Therapy.

About 75% of medullary thyroid cancers (MTCs) are sporadic with 45-70% being driven by a RET mutation. Selpercatinib is an approved treatment for RET-mutated (mutRET) MTC, however, treatments are needed for wild-type RET MTC (wtRET). Genomic alterations and transcriptomic signatures of wtRET MTC may reveal new therapeutic insights. We did a retrospective analysis of MTC samples submitted for DNA/RNA sequencing and PD-L1 expression using IHC at a CLIA/CAP-certified lab. Tumor microenvironment immune cell fractions were estimated using RNA deconvolution (quanTIseq). Transcriptomic signatures of inflammation and MAP kinase pathway activation scores (MPAS) were calculated. Mann-Whitney U, chi-square, and Fisher exact tests were applied (p-values adjusted for multiple comparisons). The 160-patient cohort included 108 mutRET and 52 wtRET MTC samples. wtRET tumors frequently harbored MAPK pathway mutations, including HRAS (42.31%), KRAS (15.7%), NF1 (6.7%), and BRAF (2%) whereas only one MAPK pathway mutation (NF1) was identified among mutRET MTC. Recurrent mutations seen in wtRET MTC included MGA, VHL, APC, STK11, and NFE2L2. Increased transcriptional activation of the MAPK pathway was observed in wtRET patients harboring mutations in MAPK genes. While the frequency of PD-L1 expression was similar in wtRET and mutRET (10.2% vs 7.0%, p=0.531), wtRET tumors were more often TMB-high (7.7% vs 0.0%, p=0.011), and wtRET MTC exhibited higher expression of immune checkpoint genes. We identified molecular alterations and immune-related features that distinguish wtRET from mutRET MTC. While RET mutation drives MTC in the absence of other alterations, we showed that wtRET MTC frequently harbors MAPK pathway mutations. These findings may indicate a potential basis for MAPK-targeted therapy, possibly in combination with oncology immune-oncology agents for selected patients with wtRET MTC.

Interleukin-6-Positive Immune Cells as a Possible New Immunologic Marker Associated With the Colorectal Cancer Prognosis.

Chronic inflammation creates tumor microenvironment (TME) that facilitates colorectal cancer (CRC) cell proliferation, migration, metastasis, and tumor progression. Interleukin-6 (IL-6) is a proinflammatory cytokine with a pleiotropic effect on CRC development. We aimed to evaluate IL-6 expression in tumor cells and in immune cells in TME, to assess the serum level and IL6 -174 G/C genotype distribution and to correlate the results with selected morphologic and clinical parameters that may add useful information in understanding the mechanisms of human CRC progression. A total of 153 patients with CRC were recruited in the current study. We assessed the IL-6 serum concentration through the ELISA method, the expression of IL-6 in tumor and in immune cells by immunohistochemical and double immunofluorescence staining, the MSI status by immunоhistochemistry for 4 mismatch repair (MMR) proteins, and the genotype distributions for IL6 -174G/C (rs1800795) single-nucleotide polymorphism through PCR-RFLP method. Our results showed that serum IL-6 level were increased in CRC patients as compared with healthy controls (P<0.0001), and in patients with cancers with advanced histologic type (type IV). However, the higher concentration (above the median of 55.71pg/mL) was with borderline association with longer survival of the patients after surgical therapy (P=0.055, Log rank test). We also found that IL-6+ immune cells prevailed in the invasive front (IF) of tumors compared with the tumor stroma (TS) (P<0.0001). More IL-6+ cells were recruited in the tumors with less advanced histologic type (I+II), with stronger inflammatory infiltrate in the IF, in early pTNM stages (I+II), without lymph node and distant metastases and the higher levels of IL-6+ cells, especially in the IF, were associated with longer survival (P=0.012). The results of our study suggest that although the serum levels of IL-6 are higher in CRC, the increased IL-6+ cells in tumor microenvironment, both in the invasive front and in tumor stroma, as well as the higher serum levels are associated with good prognostic variables and longer survival of the patients mainly in the early stages of CRC.

The anti-colorectal cancer effect and metabolites of Agrimonia pilosa Ledeb

Ethnopharmacological relevanceAgrimonia pilosa Ledeb. (Rosaceae, A. pilosa) has been used in traditional medicine in China, Japan, Korea, and other Asian countries for treatment of acute and chronic enteritis and diarrhea. Secondary metabolites have been isolated and tested for biological activities. It remains unclear in terms of its potential components of anti-colorectal cancer properties. Aim of the studyThe study aimed to how extracts from A. pilosa and their components influenced tumor microenvironment and the colorectal tumor growth in vivo on AOM/DSS induced colorectal cancer mice, the metabolites of A. pilosa was also been studied. Materials and methodsDifferent methods have been used to extract different parts of A. pilosa. And the anti-proliferation effect of these extracts on colon cancer cells have been tested. The components of A. pilosa and its metabolites in vivo were analyzed by UPLC-QTOF-MS/MS. The anti-colorectal cancer (CRC) effects of A. pilosa and its components in vivo were studied on AOM/DSS induced CRC mice. The effects of constituents of A. pilosa on the composition of immune cells in tumor microenvironment (TME) were analyzed by flow cytometry. 16 S rDNA technology was used to analyze the effect of administration on the composition of intestinal microflora. Pathological section staining was used to compare the morphological changes and molecular expression of intestinal tissue in different groups. ResultsThe constituent exists in root of A. pilosa showed the strongest anti-proliferation ability on colon cancer cells in vitro. The extract from the root of A. pilosa could attenuate the occurrence of colorectal tumors induced by AOM/DSS in a concentration-dependent manner. Administration of the extract from the root of A. pilosa could affect the proportion of γδT cells, tumor associated macrophages and myeloid derived suppressor cells in TME, increasing the proportion of anti-tumor immune cells and decrease the immunosuppressive cells in the TME to promote the anti-tumor immune response. The administration of the extract adjusted the composition of gut microbiota and its components Agrimoniin and Agrimonolide-6-o-glucoside showed the strongest anti-CRC effect in vivo with adjusting the gut microbiota differently. ConclusionsThe extract from root of A. pilosa showed anti-colorectal cancer effects in vivo and in vitro, affecting the composition of gut microbiota and the anti-tumor immune response. Within all components of A. pilosa, Agrimoniin and Agrimonolide-6-o-glucoside showed remarkable anti-CRC efficiency in vivo and in vitro. Besides, the metabolites of extract from root of A. pilosa in gastrointestinal tract mainly composed of two parts: Agrimonolide-related metabolites and Urolithins. The extract from root of A. pilosa could contribute to potential drugs for assisting clinical anti-colon cancer therapy.

Abstract 6884: Evaluating tumor-infiltrating immune cells helps predicting prognosis in biliary tract cancer

Abstract Background: The tumor microenvironment (TME) is composed of not only cancer cells but also an extracellular matrix and many types of non-cancerous cells, including fibroblasts and immune cells. In recent years, infiltrating immune cells in TME are known as a predictor of chemotherapy and immunotherapy effectiveness. The purpose of this study is to evaluate infiltrations of lymphocytes and macrophages in biliary tract cancers (BTCs). Methods: Clinical data and tissues were obtained from 130 patients who underwent surgical treatment for BTCs (intrahepatic, perihilar, and distal bile duct cancer, gallbladder cancer, and ampullary cancer) at our institution between 2001 and 2017.The immunohistochemical evaluation was performed to evaluate the number of CD8+ T cells, CD4+ T cells and FOXP3+ T cells and CD68+ Macrophages around the tumor cells. We defined Immunoscore based on the status of infiltrating immune cells. With CD8-high, CD4-high, and FOXP3-high TILs and CD68-low TAMs as one factor, the number of factors was counted in each case, and Immunoscore was assigned a score of 5 stages, from 0 to 4. It was high for the score of 3-4 and low for the score of 0-2. Also, to evaluate the correlation with TME and systemic inflammatory reaction, we examined blood inflammatory biomarker from peripheral blood samples. Results: A total of 59 cases had high Immunoscore and 71 cases had low Immunoscore. Patients with high Immunoscore showed significantly superior overall survival (OS) and recurrence free survival (RFS) than those with low Immunoscore (median OS 60.8 vs. 26.4 months, p = 0.001; median RFS not reached vs. 17.2 months, p &amp;lt; 0.001). For OS, low Immunoscore, positive lymph node metastasis, presence of distant metastasis, and high serum CA19-9 level were independent poor prognostic factors (hazards ratio 2.05, 3.48, 1.7, and 2.05; p = 0.01, p = 0.005, p = 0.05, and p = 0.01, respectively). For RFS, low Immunoscore, T classification of pT3-4, and presence of distant metastasis were independent poor prognostic factors with hazards ratio of 2.41 (p = 0.001), 2.16 (p = 0.005), and 3.58 (p = 0.005), respectively. Patients with high preoperative Neutrophil-to-lymphocyte ratio (NLR) had a significantly lower average number of CD8+ T cells as compared to patients with low NLR (p = 0.02). Preoperative NLR was associated with infiltrating CD8+ T cells in TME. Conclusions: High Immunoscore group had significantly longer OS and RFS and was an independent good prognostic factor. Also, infiltrating CD8+ T cells in TME correlated preoperative NLR. Our findings indicated that in biliary tract cancer, the evaluation of infiltrating immune cells in TME was useful to predict patient prognosis, and preoperative NLR may predict the tumor infiltrating CD8+ T cells. Citation Format: Kousuke Hatta, Ryota Tanaka, Kenjiro Kimura, Shinpei Eguchi, Go Ohira, Hiroji Shinkawa, Kohei Nishio, Masahiko Kinoshita, Shigeaki Kurihara, Shuhei Kushiyama, Takahito Kawaguchi, Naoki Tani, Koichi Nakanishi, Mizuki Yoshida, Takeaki Ishizawa. Evaluating tumor-infiltrating immune cells helps predicting prognosis in biliary tract 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 6884.

Abstract 3851: Beta-lapachone promotes tumor associated neutrophils (TANs) polarized towards anti-tumor (N1) phenotype

Abstract NAD(P)H:quinone oxidoreductase1 (NQO1) is overexpressed in most solid cancers, emerging as a promising target for tumor selective killing. β-lapachone (β-lap), an NQO1bioactivatable drug, exhibits significant antitumor effects on NQO1+ human solid cancer cell lines. It also induces immunogenic cell death in the tumor microenvironment (TME)and synergizes with anti-PD-L1 immunotherapy. However, the interaction between β-lap-mediated anti-tumor immune response and neutrophils, a novel antigen presenting cells(APCs), remains unknown. This study found β-lap treatment increased intracellular ROS formation and DNA damage to kill NQO1+ murine tumor cell lines and dicoumarol spared the lethality. Furthermore, flow cytometric analysis of immune cells in TME of subcutaneous tumor models indicated that there is a significant infiltration of neutrophils(CD11b+ Gr1high cells) or Tumor Associated Neutrophils (TANs) following β-lap treatment. Depletion of neutrophils by co-addition of anti-Ly6G antibody abolished β-lap-induced anti-tumor efficacy in immune competent mice. Neutrophil mediated tumor cell killing determined ex vivo using bone marrow derived neutrophils and it was observed that in the presence of β-lap induced antigen, neutrophils were killing murine tumor cells. Moreover, exposure of these neutrophils to β-lap induced antigen medium induce antitumor N1-like properties, include increase in certain surface markers such as CD95(Fas) and CD54 (ICAM-1) as well as increase in intracellular cytokine IFN-β and decrease in TGF-β revealed by flow cytometric analysis. Corroborating to this, there is a significant increase of CD95 and IFN-β and a decrease in TGF-β were observed with TANs infiltrated into β-lap treated TME compared to vehicle treated tumors. Additionally, TLR/MyD88signaling deficiency decreased β-lap induced neutrophil tumor infiltration. BlockingHMGB1, depleting neutrophils, or TLR4/MyD88 deficiency lessened the antigen-specific T cell response. This study highlights that β-lap kills NQO1+ murine tumor cells through ROS production and DNA damage, and neutrophils play a pivotal role in its antitumor effects. Citation Format: Xiumei Huang, Soumya Tumbath, Liangxiang Jiang, Yang-Xin Fu. Beta-lapachone promotes tumor associated neutrophils (TANs) polarized towards anti-tumor (N1) phenotype [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 3851.

Abstract 1149: Spatial transcriptomic analysis of tumor microenvironment in patients with triple negative breast cancer with residual disease after neoadjuvant chemotherapy with immune checkpoint inhibitor

Abstract Recent advance of treatment strategy for triple negative breast cancer (TNBC) including immune checkpoint inhibitor improved survival outcome. Especially, adding pembrolizumab on neoadjuvant chemotherapy (NAC) increased pathologic complete response (pCR) rate at the time of curative surgery. Previous studies suggested that PD-L1 expression in both tumor and immune cells was associated with the response of pembrolizumab in TNBC, but PD-L1 expression state were not associated with pembrolizumab response in NAC setting. In addition, neoadjuvant pembrolizumab increased event-free survival (DFS) rate after NAC even though they had not achieved pCR. However, there were no biomarker for EFS increasement of using pembrolizumab. We designed prospective translational research for evaluating tumor microenvironment (TME) of residual TNBC which had treated with NAC followed by curative surgery. We collected formalin fixed paraffin embedded (FFPE) surgical specimens after NAC with or without pembrolizumab. TNBC samples were used for spatial transcriptomics (ST) analysis, with H&amp;E staining and inspection by an experienced pathologist. Libraries were prepared using Visium Spatial Gene Expression Reagent Kit protocol for FFPE (10x Genomics). Sequencing was conducted on the NovaSeq 6000 System (Illumina). From February 2023 to October 2023, 23 TNBC specimens from 22 patients were collected. Twenty specimens were breast tissues and three were lymph nodes, 18 TNBCs had been treated with NAC with pembrolizumab and four with only NAC, and five had residual cancer burden(RCB) class I, 11 of RCB class II and one of RCB class III. We collected BC and LN samples from BC with class III. Of 23 samples, we performed spatial transcriptomic analysis in 12 samples. In intrinsic subtyping, eight specimens were categorized into normal like, two of basal like and two of luminal A BC subtypes. In TNBC subtyping, mesenchymal stem cell like (MSL) were in six specimens, three of stem cell like (SL), two of luminal androgen receptor (LAR) and one of immunomodulatory (IM). We also performed cell type annotation with unsupervised method and categorized into 11 cell types; three cancer cell types, two macrophages, B and T cells, adipocytes, normal epithelium and perivascular-like and cancer associated fibroblasts. Among these 11 cell types, increasement of immune cells including B cells, T cells and macrophage proportions was associated with pembrolizumab treatment in NAC setting. In our study, we evaluated TME state of TNBC after NAC with or without pembrolizumab. We suggested that MSL or SL TNBC subtypes were associated with residual disease after NAC regardless of pembrolizumab use and pembrolizumab may recruit more immune cells in TME of TNBC. For concreating our suggestion, further analyses would be warranted. Citation Format: Ji-Yeon Kim, Kyunghee Park, Yoon La Choi, Jeong Eon Lee, Byung Joo Chae, Jonghan Yu, Seok Won Kim, Seok Jin Nam, Jin Seok Ahn, Woong-Yang Park, Yeon Hee Park. Spatial transcriptomic analysis of tumor microenvironment in patients with triple negative breast cancer with residual disease after neoadjuvant chemotherapy with immune checkpoint inhibitor [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 1149.

Abstract 3644: Uncover spatial signatures of tumor microenvironment and oncogenic pathways using 6,000-plex single-cell spatial molecular imaging on FFPE skin squamous cell carcinoma

Abstract Background: Tumor progression and therapeutic response are regulated by the tumor microenvironment. Understanding the spatial association and architecture of molecular characteristics and composition of tumor immune microenvironment at single-cell and subcellular resolution encourages improvements in clinical prognosis and immunotherapy benefits. Single-cell Spatial profiling technologies permit the study of transcriptional activity at the spatial, single-cell level and provide abundant, high-resolution information required for the identification of clinical-related features in immuno-oncology. Methods: We performed an ultra-high-plex RNA assay to detect 6,000 targets simultaneously in situ on FFPE human skin squamous cell carcinoma using the CosMx™ Spatial Molecular Imager (SMI). For the selection of regions of interest, four protein markers and DAPI were co-detected on the same tissue slide. Tertiary analysis algorithms were developed for cell typing, co-localization of genes and ligands, cell-cell interaction, and pathway analysis. Results: Thousands of transcripts were simultaneously detected with high sensitivity and specificity on the FFPE skin squamous cell carcinoma tissue section at single-cell subcellular resolution. We investigated the tumor microenvironment including cell types, their spatial distribution and proportion of diversified immune cells in the tumor compartment. The cell typing results revealed many cancerous subpopulations, which resolved spatially from cells that lacked any defining marker genes. We also assessed distances between immune cells and their nearest functional-related neighbors. Moreover, we revealed the ligands co-localization, as well as spatial patterns of direct cell-cell interactions and signaling pathways. We found that some genes have elevated expression in macrophages which are near cancer compared to those are far from cancer cells, empowering the molecular investigation of novel signaling between immune and cancer cells in tumor immune microenvironment. Conclusions: Single-cell spatial measurements of 6,000 RNA and four proteins on the same tissue section, along with a large viewing area on archival FFPE tissue, provide a novel tool to reveal the spatial signature of tumor microenvironment and oncogenic pathways, facilitating the next level of cancer and therapeutic research. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Michael Patrick, Shanshan He, Saskia Ilcisin, David Kroeppler, Patrick Danaher, Jason Reeves, Mark Gregory, Haiyan Zhai, Michael Rhodes, Joseph Beechem. Uncover spatial signatures of tumor microenvironment and oncogenic pathways using 6,000-plex single-cell spatial molecular imaging on FFPE skin squamous cell carcinoma [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 3644.

Abstract 2688: Age-associated changes in macrophage function and phenotype affects cancer development, and the efficacy of macrophage targeted anticancer therapies in murine melanoma model

Abstract Introduction: Aging of the immune system is a critical risk factor for chronic diseases, including cancer. Globally, cancer incidence is higher between 45 to 75 years of age and declines thereafter. However, the implications of aging in cancer are understudied, and aged populations are underrepresented, both in preclinical and clinical studies. The role of macrophages in cancer progression has gained attention as a tool and target for anticancer therapies. Tumor-associated macrophages (TAMs) mainly exhibit an anti-inflammatory M2 phenotype, promote an immunosuppressive tumor microenvironment (TME), and contribute to developing resistance against immunotherapies. Therefore, several strategies targeting the phenotype and function of TAMs are being explored, including inhibitors of histone deacetylases (HDACis), adoptive macrophage cell therapy, and CAR macrophages. Studies from our group have shown that ultra-specific HDAC6is can switch macrophages from anti-inflammatory, M2, to pro-inflammatory and antitumoral M1 phenotype with minimal cytotoxicity to cancer cells. However, it is unknown if age-related changes in macrophages affect such therapeutic strategies and the TME. Objective: To characterize the changes in macrophage function and phenotype with aging and study its implications on the TME and the efficacy of macrophage-based anticancer therapies. Methods: Wildtype mice of ages 6-8 WKS, 3-6 MO, 10-12 MO, and 20-24 MO were injected with SM1 melanoma cells to study tumor growth kinetics. Bone marrow-derived macrophages (BMDMs) isolated from tumor-bearing and healthy mice were polarized to M1 and M2 phenotype treated with HDAC6i to study age-associated changes in phagocytic activity, antigen presentation, and gene expression. Pathways associated with macrophage polarization were examined by flow cytometry, qRT-PCR, western blot, and single-cell proteomics. Immune cells in TME were characterized in flow cytometry and single-cell RNA and secretome analyses. Results: Tumor growth in SM1 murine melanoma model was significantly slower in older mice when compared to younger mice. Aged macrophages demonstrated significant defect in polarization to M1 and M2 phenotypes that was more pronounced in BMDMs from tumor-bearing mice. Tumor-associated macrophages showed inefficient monocyte-to-macrophage transition, as also reflected in the single-cell secretome analysis. The ability of HDAC6i to enhance macrophage function was reduced among older mice. Conclusions: The functional and phenotypic characteristics of macrophages change with age and can significantly affect tumor growth and the effectiveness of macrophage-targeted anticancer therapies. Our study highlights the importance of studying aging in macrophages and better representation of aged populations in preclinical and clinical studies. Citation Format: Manasa Suresh, Xintang Li, Satish Noonepalle, Nithya Gajendran, Marie Durr, David Quiceno-Torres, Karen Tan, Alejandro Villagra. Age-associated changes in macrophage function and phenotype affects cancer development, and the efficacy of macrophage targeted anticancer therapies in murine melanoma model [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 2688.

Abstract 1790: Pyruvate carboxylase regulates tumor progression through central carbon metabolism and immunosuppression

Abstract The tumor microenvironment (TME) is characterized by metabolic reprogramming and immune evasion, essential features in tumor progression. Tumor metabolic dysregulation, particularly the high production of lactate, plays a pivotal role in suppressing antitumor immunity within the TME. Pyruvate carboxylase (PC), responsible for converting pyruvate to oxaloacetate, is a critical determinant for lung metastasis in triple-negative breast cancer (TNBC). Herein we test the hypothesis that PC-mediated regulation of central carbon metabolism controls TME immunosurrveilance. Wild-type C57BL6/J and NSG mice received orthotopic injections of E0771 or M-Wnt tumor cells with shRNA targeting PC or a scramble control. RNA from tumors underwent transcriptomic profiling using Affymetrix Clariom D microarray (n=3-6/group per experiment). Gene set enrichment analysis identified pathways enriched in each condition, including a PC-associated hypoxia signature. Immunohistochemistry with anti-CD3 antibody and full spectrum flow cytometry assessed T cell infiltration and TME immune cell composition. Untargeted metabolomics, extracellular flux analysis, and high-resolution respirometry determined how loss of PC altered cellular metabolism. We found that suppressing PC expression increased primary tumor growth (~2 fold), potently drove immunosuppression, and remodeled cellular metabolism towards lactate production. Injection of PC-suppressed cells increased regulatory T cell levels in wild type mice. Importantly, PC-suppressed cells injected into NSG mice, which lack T, B, and NK cells, demonstrated slower, rather than accelerated, tumor growth. PC-suppression reduced oxygen consumption rate (OCR), but supply of anaplerotic carbon through exogenous malate or oxaloacetate restored OCR. Conversely, both the rate of and reliance on lactate production was increased by PC suppression. Notably, loss of immunosurveillance following downregulation of PC was reversed by inhibiting lactate transport. Additionally, lower PC expression was associated with increased hypoxia in tumors, supporting in vitro evidence of PC-repression by hypoxia. In summary, hypoxia-induced PC suppression emerges as a crucial mechanism through which primary tumors hinder antitumor immunity, partly via increased lactate production. These findings underscore the significance of PC-directed tumor metabolism as an understudied nexus in the interplay between tumor progression and antitumor immunity. Citation Format: Michael F. Coleman, Eylem Kulkoyluoglu Cotul, Alexander J. Pfeil, Alyssa N. Ho, Emily N. Devericks, Muhammad H. Safdar, Hao Chen, Numair Attaar, Violet A. Kiesel, Dorothy Teegarden, Stephen D. Hursting, Michael K. Wendt. Pyruvate carboxylase regulates tumor progression through central carbon metabolism and 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 1790.

Abstract 5617: Combined inhibition of ATR and ATM with tuvusertib and lartesertib (M4076) impacts the tumor microenvironment

Abstract Ataxia telangiectasia and Rad3-related protein (ATR) and ataxia telangiectasia mutated (ATM) are two of the main kinases of the DNA damage response (DDR). Recent work has demonstrated that the addition of ATM inhibitors enhances the cytotoxicity and in vivo anti-tumor efficacy of ATR inhibitors by abrogating the ATR inhibitor mediated G1 cell cycle arrest and enhancing chromosomal damage.1 Prior to causing cell death, damaged chromosome fragments can end up in the cytosol where they are detected by cytosolic nucleic acid sensors such as cGAS, resulting in the induction of inflammatory signaling pathways and an anti-tumor immune response. This suggests that the mode of action of DDR inhibitors may involve the tumor microenvironment (TME). We therefore aimed to assess the influence of combined pharmacological ATR and ATM inhibition on the TME to better understand how this could be exploited therapeutically. In line with published results in human tumor models, the treatment of murine colon cancer MC-38 cells in vitro with a combination of ATR inhibitor tuvusertib and ATM inhibitor lartesertib led to cell death. Further analysis revealed an activation of the cGAS-STING signaling pathway, an upregulation of PD-L1 and the release of inflammatory cytokines. These results confirmed the activation of immune signaling pathways upon combined inhibition of ATR and ATM. To analyze the stimulation of an anti-tumor immune response upon tuvusertib + lartesertib treatment in vivo, MC-38 cells were transplanted into immunocompetent C57BL/6J mice. Immunoprofiling by flow cytometry and immunohistochemistry staining confirmed that treatment with tuvusertib + lartesertib indeed altered the TME in this setting. In line with the in vitro findings, a significant upregulation of PD-L1 on immune and tumor cells could be observed after 24, 48, and 72 h of treatment even though MC-38 cells expressed high levels of PD-L1 already. Immunoprofiling did not reveal any changes in the TME immune cell content at these early time points. Interestingly, PD-L1 expression as well as tumor immune population changed after prolonged (23 days) treatment. PD-L1 was still expressed on tumor cells but to a significantly lesser extent and a depletion of CD8+ T-cells was observed. However, a significant increase in NK cells could be detected. In summary, we show that inhibition of the DDR through a combination of ATR and ATM inhibitors influences the TME in the MC-38 tumor model. These findings suggest an additional mode of action beyond direct cancer cell cytotoxicity. Further studies on how this may be leveraged therapeutically, for example through a combination with immune checkpoint inhibitors, are warranted. 1. Turchick A, Zimmermann A, et al. Mol Cancer Ther 2023;22(7):859-872. Citation Format: Karin Laaber, Audrey Turchick, Julia Jabs, Brian Elenbaas, Lyubomir T. Vassilev, Astrid Zimmermann. Combined inhibition of ATR and ATM with tuvusertib and lartesertib (M4076) impacts 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 5617.

Spatial relationships in the urothelial and head and neck tumor microenvironment predict response to combination immune checkpoint inhibitors

Immune checkpoint inhibitors (ICI) can achieve remarkable responses in urothelial cancer (UC), which may depend on tumor microenvironment (TME) characteristics. However, the relationship between the TME, usually characterized by immune cell density, and response to ICI is unclear. Here, we quantify the TME immune cell densities and spatial relationships (SRs) of 24 baseline UC samples, obtained before pre-operative combination ICI treatment, using multiplex immunofluorescence. We describe SRs by approximating the first nearest-neighbor distance distribution with a Weibull distribution and evaluate the association between TME metrics and ipilimumab+nivolumab response. Immune cell density does not discriminate between response groups. However, the Weibull SR metrics of CD8+ T cells or macrophages to their closest cancer cell positively associate with response. CD8+ T cells close to B cells are characteristic of non-response. We validate our SR response associations in a combination ICI cohort of head and neck tumors. Our data confirm that SRs, in contrast to density metrics, are strong biomarkers of response to pre-operative combination ICIs.

PDE5 inhibitors against cancer via mediating immune cells in tumor microenvironment: AI‐based approach for future drug repurposing exploration

AbstractPhosphodiesterase‐5 (PDE5) inhibitors are used clinically for the treatment of erectile dysfunction, pulmonary arterial hypertension, and other urological diseases. Emerging evidences have suggested the therapeutic capacity of PDE5 inhibitors as the repurposed drugs in oncology. However, the essential immune function of PDE5 inhibitors against cancer in tumor microenvironment (TME) remains unclear. This review aimed to summarize the recent advances regarding the repurposing of PDE5 inhibitors as anti‐cancer agents for cancer management to enhance the anti‐tumor immune response by mediating various immune cells, which included the myeloid‐derived suppressor cells, macrophages, T cells, fibroblasts, and natural killer cells in TME. Moreover, artificial intelligence (AI), as a new approach, is composed of traditional machine learning and deep learning methods and could be potentially used to identify the targets of immune cells in TME and predict the efficacy for repurposed drug toward malignancies. In summary, these endeavors provide novel insights into the comprehensive strategies for PDE5 inhibitors mediating immune cells against cancer and AI‐based approach for future drug repurposing exploration.

PD-L1 Expression and Tumour Microenvironment Patterns in Resected Non-Small-Cell Lung Cancer.

Background and Objectives: Although perioperative immunotherapy is implemented as a standard of care for resected non-small cell lung cancer (NSCLC), there is unmet need for predictive biomarkers as programmed death-ligand 1 (PD-L1) is not the perfect one. The functionality of tumour-infiltrating immune cells in the tumour microenvironment (TME) and the involvement in immune system response is one of the crucial factors that lead to pro- or anti-tumourigenic role and could predict response to PD-1 and PD-L1 inhibitors. So, the investigation of PD-L1 expression in the context of TME in early stages of resected NSCLC is urgent required. Materials and Methods: PD-L1 expression by three scoring methods: tumour proportion score (TPS), immune cell score (IC), and combined proportion score (CPS) was assessed in 72 archival tumour tissue specimens from stage I-III surgically resected NSCLC patients and associations with immune cells in TME were explored. Results: PD-L1 expression ≥1% evaluated by TPS, IC, and CPS was detected in 28%, 36%, and 39% of cases and moderate, substantial, and strong agreement between TPS and IC, TPS and CPS, CPS and IC was detected (Cohen's κ coefficient 0.556, 0.63, and 0.941, respectively). PD-L1 TPS, IC, and CPS correlated with smoking intensity defined as pack-years (r = 0.0305, p = 0.012; r = 0.305, p = 0.013, and r = 0.378, p = 0.002, respectively). Only PD-L1 TPS was associated with squamous cell carcinoma (p = 0.028). PD-L1 IC ≥1% was more often seen in tumours with high CD4+ T cells infiltration (p = 0.02), while PD-L1 CPS ≥1%-in tumours with high CD4+ and CD8+ T cells infiltration (p = 0.021 and p = 0.048, respectively). PD-L1 IC and CPS ≥10% was more often detected in tumours with greater number of tumour-infiltrating CD4+Foxp3+ T cells (p = 0.01 and p = 0.025, respectively). PD-L1 TPS ≥50% was associated with higher probability to detect greater number of tumour-infiltrating M2 macrophages (p = 0.021). No association was found between PD-L1 alone or in combination with tumour-infiltrating lymphocytes, macrophages, and disease-free or overall survival. Conclusions: This study results revealed that rates of PD-L1 expression correlated among three scoring methods (TPS, IC, and CPS). Moreover, PD-L1 expression was significantly associated with smoking intensity, squamous histology, and tumour-infiltrating immune cells.

COL10A1 as a Prognostic Biomarker in Association with Immune Infiltration in Prostate Cancer.

The collagen type X alpha 1 (COL10A1) has recently been found to play an important role in the development and progression of cancer. However, the link between COL10A1 and the tumor immune microenvironment remains understood scantily. In the current study, the pan-cancer data of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were used to investigate the expression mode, the clinical prognostic and diagnostic value of COL10A1 in different tumors. We used TCGA data to assess the correlations between COL10A1 and clinical symptoms of prostate cancer. The R packages "edgR" and "clusterProfiler" were used for differential expression gene and enrichment analysis of COL10A1. Immunohistochemistry was further employed to corroborate the expression of COL10A1 gene in prostate cancer. After that, we used TIMER to evaluate the pertinence of COL10A1 expression to immune infiltration level in prostate cancer. On the whole, COL10A1 was expressed at significantly higher levels in a variety of tumor tissues than in the corresponding normal tissues. Besides, significant correlations with tumor prognosis and relative exactitude in predicting tumors show that COL10A1 may be a probable prognostic and diagnostic biomarker of prostate cancer. In addition, the evidence indicates a significant correlation between COL10A1 and clinical symptoms of prostate cancer. Furthermore, the main molecular functions of COL10A1 included humoral immune response, complement activation, immunoglobulin, regulation of complement activation, and regulation of humoral immune response. Finally, we found that COL10A1 expression is positively correlated with enhanced macrophage and M2 macrophage infiltration in prostate cancer. The study indicates that COL10A1 might participate in M2 macrophage polarization in prostate cancer. COL10A1 might be an innovative biomarker to evaluate tumor microenvironment immune cell infiltration and prognosis in prostate cancer.

Tumorous IRE1α facilitates CD8+T cells-dependent anti-tumor immunity and improves immunotherapy efficacy in melanoma

BackgroundTumor cells frequently suffer from endoplasmic reticulum (ER) stress. Previous studies have extensively elucidated the role of tumorous unfolded protein response in melanoma cells, whereas the effect on tumor immunology and the underlying mechanism remain elusive.MethodsBioinformatics, biochemical assays and pre-clinical mice model were employed to demonstrate the role of tumorous inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α) in anti-tumor immunity and the underlying mechanism.ResultsWe firstly found that IRE1α signaling activation was positively associated with the feature of tumor-infiltrating lymphocytes. Then, pharmacological ER stress induction by HA15 exerted prominent anti-tumor effect in immunocompetent mice and was highly dependent on CD8+T cells, paralleled with the reshape of immune cells in tumor microenvironment via tumorous IRE1α-XBP1 signal. Subsequently, tumorous IRE1α facilitated the expression and secretion of multiple chemokines and cytokines via XBP1-NF-κB axis, leading to increased infiltration and anti-tumor capacity of CD8+T cells. Ultimately, pharmacological induction of tumorous ER stress by HA15 brought potentiated therapeutic effect along with anti-PD-1 antibody on melanoma in vivo.ConclusionsTumorous IRE1α facilitates CD8+T cells-dependent anti-tumor immunity and improves immunotherapy efficacy by regulating chemokines and cytokines via XBP1-NF-κB axis. The combination of ER stress inducer and anti-PD-1 antibody could be promising for increasing the efficacy of melanoma immunotherapy.

LMTK3 gene expression and the molecular landscape of colorectal cancer (CRC).

172 Background: Lemur tail kinase 3 (LMTK3) plays a critical role in multiple cellular pathways such as Wnt signaling, KIT modulation, and the estrogen receptor pathway. We previously reported that LMTK3 gene polymorphisms are associated with clinical outcome in patients with CRC, and that LMTK3 and estrogen-mediated signaling play a crucial role in CRC tumorigenesis in vitro. Here, we aimed to characterize the molecular features associated with LMTK3 gene expression in CRC. Methods: 20,219 CRC were tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (592 genes or WES) and RNA (WTS). Top quartile transcripts per million (TPM) for LMTK3 expression (Q4) were considered high while bottom quartile (Q1) low. Consensus molecular subtypes (CMS) were assessed using RNAseq. Cell infiltration (CI) in the tumor microenvironment (TME) was estimated by QuantiSEQ. X2 and Fisher-Exact tests were used, and significance was determined as p-value adjusted for multiple comparisons ( q &lt; 0.05). Gene expression profiles were analyzed for transcriptional signatures predictive of response to immunotherapy (interferon gamma [IFNγ] score; T-cell inflamed signature [TIS] score). Results: LMTK3 expression was highest in left-sided (0.98 median TPM) followed by right-sided (0.96) and lowest in rectal tumors (0.92). No difference in patient sex distribution was observed between Q1 and Q4 cohorts. Overall, LMTK3 Q1 was associated with TMB-high (≥ 10 Mut/Mb, 11.4 vs 8.7%) and dMMR/MSI-H (8.1 vs 4.6%) ( q &lt; 0.0001); however, these associations were no longer significant in pMMR/MSS tumors. In the pMMR/MSS cohort, LMTK3 expression was highest in CMS4 (1.09 median TPM) followed by CMS2 (0.98), CMS1 (0.88), and lowest in CMS3 (0.70, all intergroup q &lt; 0.05). LMTK3 high showed differential rates of mutations and copy number alterations (CNA) in several genes, including higher mutation rates of APC, TP53, and CDX2 CNA, while lower mutation rates of KMT2A/D, ASXL1, ATM, SMAD4, RNF43 and AMER1 in the overall cohort ( q &lt; 0.05). In the pMMR/MSS cohort, the associations with APC, TP53, SMAD4 and ATM mutations and CDX2 CNA still held true ( q &lt; 0.05). High LMTK3 was associated with higher immune CI in the TME, including B cells, neutrophils, NK cells, CD4+ T cells, monocytes and dendritic cells, while macrophages and Tregs were lower regardless of tumor MSI status (fold change: 0.83-2.4, all q &lt; 0.0001). The IFNγ score was significantly lower in LMTK3 Q4 while the TIS score was significantly higher in LMTK3 Q4 ( q &lt; 0.0001). Conclusions: Our data show a strong association between LMTK3 gene expression and distinct molecular features and TME immune cell infiltration in CRC. These findings suggest that LMTK3 may be an important molecular factor that plays a role in determining the composition of the TME, thus targeting LMTK3 could represent a novel strategy in selected CRC subgroups.

Characterization of the cachexia pathway in pancreatic ductal adenocarcinoma.

699 Background: Cancer cachexia is characterized by progressive weight loss and skeletal muscle degradation, contributing to 33% of pancreatic ductal adenocarcinoma (PDAC) deaths. Targeting myostatin in the myostatin-activin pathway has been shown to reverse cachexia. Here, we present comprehensive clinical and molecular characterization of the myostatin-activin pathway in PDAC. Methods: 9,607 samples of PDAC tested at Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) were analyzed. Cachexia gene scores (GS) were calculated by averaging the positive z scores of activators and negative z scores of repressors in the myostatin-activin pathway. Activators were ACVR1B, ACVR1C, ACVR2A, ACVR2B, SMAD2, SMAD3, SMAD4, and TGFBR1, while repressors were SMAD1, SMAD5, SMAD6, SMAD7, SMURF1, and SMURF2. The top quartile (Q4) and bottom quartile (Q1) of GS were compared using chi-squared and Fisher-Exact tests. RNA deconvolution analysis with QuantiSEQ estimated cell infiltration in the tumor microenvironment (TME). Differences in overall survival (OS) were analyzed from insurance claims data and calculated from time of tissue collection using Kaplan-Meier estimates. Statistical significance was determined as a P-value adjusted for multiple comparisons ( q&lt;0.05). Results: GS were higher in primary tumors compared to metastases (median: -0.71 vs -0.86, q&lt;0.05). GS was associated with increased PD-L1 IHC expression (Q1 21.2% vs Q4 10.3%, q&lt;0.001), but not TMB-high (1.9% vs 2.1%, q=1) or MSI-H status (1.1% vs 1.3%, q=1). GS correlated with increased expression of immune related genes ( CD274, CD80, IDO1, CD86, PDCD1, LAG3, CTLA4, HAVCR2, and IFNG, q&lt;0.05). However, TME immune cell infiltration did not vary based on cachexia GS. Mutation rates of TP53 (Q1 79.4% vs Q4 73.7%), ARID1A (Q1 11.8% vs Q4 6.9%) and KRAS (Q1 92.7% vs Q4 86.4%) were associated with Q1-GS (all q&lt;0.01), while STK11 mutations (1.1% vs 3.0%, q=0.001) were associated with Q4-GS. Spearman correlation linked cachexia GS with the lipid metabolizing genes UCP2 (rho=0.49) and UCP3 (rho=0.30), as well as the inflammatory markers CCL2/MCP-1(rho=0.34) and IL1B (rho=0.33). Decreased OS was seen with higher tumor expression of the myostatin-activin pathway activator, SMAD3 (6.6 vs 8.5 mo, HR=1.22, CI 1.12-1.33, P&lt;0.001), and lower expression of the repressor SMAD7 (6.9 vs 8.9 mo, HR=0.91, CI 0.79-0.90, P=0.034) in metastatic PDAC. Conclusions: This is the largest molecular and clinical characterization of the myostatin-activin cachexia pathway in PDAC. Our data show that increased activation of the myostatin-activin pathway is associated with immune mediators, lipid metabolism, and inflammatory gene activation. Activators and repressors are significant predictors of survival in PDAC, suggesting possible novel therapeutic targets.

Systemic Delivery of Paclitaxel by Find-Me Nanoparticles Activates Antitumor Immunity and Eliminates Tumors.

Local delivery of immune-activating agents has shown promise in overcoming an immunosuppressive tumor microenvironment (TME) and stimulating antitumor immune responses in tumors. However, systemic therapy is ultimately needed to treat tumors that are not readily locatable or accessible. To enable systemic delivery of immune-activating agents, we employ poly(lactic-co-glycolide) (PLGA) nanoparticles (NPs) with a track record in systemic application. The surface of PLGA NPs is decorated with adenosine triphosphate (ATP), a damage-associated molecular pattern to recruit antigen-presenting cells (APCs). The ATP-conjugated PLGA NPs (NPpD-ATP) are loaded with paclitaxel (PTX), a chemotherapeutic agent inducing immunogenic cell death to generate tumor antigens in situ. We show that the NPpD-ATP retains ATP activity in hostile TME and provides a stable "find-me" signal to recruit APCs. Therefore, the PTX-loaded NPpD-ATP helps populate antitumor immune cells in TME and attenuate the growth of CT26 and B16F10 tumors better than a mixture of PTX-loaded NPpD and ATP. Combined with anti-PD-1 antibody, PTX-loaded NPpD-ATP achieves complete regression of CT26 tumors followed by antitumor immune memory. This study demonstrates the feasibility of systemic immunotherapy using a PLGA NP formulation that delivers ICD-inducing chemotherapy and an immunostimulatory signal.

Abstract B019: Tumor-derived PTHrP molds an immunosuppressive microenvironment

Abstract Purpose[JP1] : Molecular phenotyping of PDAC [PJ2] has revealed an aggressive squamous/basal/quasi-mesenchymal[JP3] subtype, clinically presenting as highly metastatic and harboring genetic amplifications of KRAS. Our previous work indicated that a squamous identity gene, Parathyroid Hormone Like Hormone (PTHLH; encoding for the PTHrP protein), is frequently co-amplified with KRAS and skews PDAC towards a squamous identity. Further, PTHrP is secreted by tumor cells and promotes tumor growth and metastasis through autocrine signaling. However, pathological analysis of PTHrP-null tumors indicated that the tumor microenvironment (TME) was re-organized upon loss of PTHrP. Thus, we hypothesized that PTHrP is a tumor-derived factor that shapes the TME through its systemic secretion and mobilization of immunosuppressive myeloid cells. Methods: In vivo experiments used KrasG12D; p53R172H; Pdx1-Cre; YFP (KPCY) and KPCY-PthlhloxP/WT (KPCY-Pthlhhet) for spontaneous tumor formation in immune competent mice. Orthotopic implantation experiments used Pthlh overexpressing KPCY tumor cells (herein KPCY-PthlhOE) or empty vector control cells (KPCY-PthlhEV). Tumor immunophenotyping was done using flow cytometry. Cytokine expression was performed using qPCR and ELISA. Results: In vivo investigation of tumors from KPCY-Pthlhhet mice revealed less MDSC and greater CD8+ T cell infiltration than KPCY controls, demonstrating the necessity of PTHrP for immunosuppression[PJ4]. Orthotopic implantation of KPCY-PthlhOE cell lines reduced overall TME immune cell recruitment, while simultaneously increasing the proportion of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) infiltration. A concomitant decrease in the frequency of T cells in the KPCY-PthlhOE tumors was also observed, showing the sufficiency of PTHrP to organize an immunosuppressive TME[PJ5]. Additionally, a reduction of PD-1+ and CD44high CD8+ T cells revealed they were less activated. Interestingly, recombinant human PTHrP was sufficient to induce MDSC mobilization in vivo.[JP6] To interrogate the mechanism of MDSC trafficking into the TME, cytokine production by was analyzed in vitro. KPCY-PthlhOE exhibited a pronounced increase and decrease of Cxcl1 and Cxcl10 expression, respectively, relative to the KPCY-EV control. Conclusions: The work herein describes a new mechanism for the mobilization and infiltration of immune suppressive MDSC in the tumor microenvironment, initiated by PTHrP. The PTHrP-dependent immunosuppressive TME excludes and inhibits effector immune cells, such as CD8+ T cells. This phenomenon has been described in squamous-like PDAC but may also apply to other squamous adenocarcinomas. [PJ7] Further elucidation of the mechanism by which PTHrP is the genesis of the immune suppressive TME may spur the development of novel treatments for these more clinically severe cancer subtypes. Citation Format: Calvin P. Johnson, Boyang Ma, Jason Pitarresi. Tumor-derived PTHrP molds an immunosuppressive microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B019.

TRIM65 knockout inhibits the development of HCC by polarization tumor-associated macrophages towards M1 phenotype via JAK1/STAT1 signaling pathway

Background & aimsTumor-associated macrophages (TAMs) are main components of immune cells in tumor microenvironment (TME), and play a crucial role in tumor progression. Tripartite motif-containing protein 65 (TRIM65) has been associated with tumor progression. However, whether TRIM65 regulate the interaction of tumor cell and TAMs in HCC and the underlying mechanisms remain unknown. In this study, we investigated the role of TRIM65 in TME of HCC and explored its underlying mechanisms. MethodsThe relation of TRIM65 expression level with tumor grades, TNM stages, and worse prognosis of HCC patients was evaluated by bioinformatics analysis, as well as immune infiltration level of macrophages. TRIM65 shRNA was transfected into HepG2 cells, and TRIM65 overexpression plasmid was transfected into Huh7 cells, and the effect of TRIM65 on cell growth was examined by EdU assay. The mouse subcutaneous Hep1-6 tumor-bearing model with WT and TRIM65-/- mice was established to study the role of TRIM65 in HCC. Immunohistochemistry staining, Immunofluorescence staining, qRT-PCR and western blot were performed to evaluate the effect of TRIM65 on TAM infiltration, TAM polarization and JAK1/STAT1 signaling pathway. ResultsBioinformatics analysis revealed that TRIM65 was upregulated in 16 types of cancer especially in HCC, and high level of TRIM65 was strongly correlated with higher tumor grades, TNM stages, and worse prognosis of patients with HCC as well as immune infiltration level of macrophages (M0, M1, and M2). Moreover, we observed that TRIM65 shRNA-mediated TRIM65 knockdown significantly inhibited the HepG2 cells growth while TRIM65 overexpression highly increased the Huh7 cells growth in vitro. TRIM65 knockout significantly inhibited the tumor growth as well as macrophages polarization towards M2 but promoted macrophages polarization towards M1 in vivo. Mechanistically, the results demonstrate that TRIM65 knockout promoted macrophage M1 polarization in conditioned medium-stimulated peritoneal macrophages and in tumor tissues by activating JAK1/STAT1 signaling pathway. ConclusionsTaken together, our study suggests that tumor cells utilize TRIM65-JAK1/STAT1 axis to inhibit macrophage M1 polarization and promote tumor growth, reveals the role of TRIM65 in TAM-targeting tumor immunotherapy.