Expression Of Tumor-associated Macrophages Research Articles

Abstract 2701: EREG reprograms M2-tumor-associated macrophage and suppresses colorectal cancer cell proliferation and migration

Abstract Colorectal cancer (CRC) represents a common malignancy within the digestive system, linked to significant morbidity and mortality rates. Tumors exert diverse effects on immune modulation, facilitating progression and metastasis. Amid the abundant immune cell populace within the tumor microenvironment (TME), tumor-associated macrophages (TAMs) play a pivotal role. M2-type TAMs actively foster tumor growth and engage in immune suppression by fostering angiogenesis and tissue restructuring. In a previous study, we analyzed single-cell RNA-sequencing (scRNA-seq) data derived from colorectal cancer patients. We observed an enrichment of the secreted phosphoprotein 1 (SPP1)+ macrophage fraction in both the tumor core and border when compared to normal tissues. Additionally, epiregulin (EREG), an epidermal growth factor receptor (EGFR) ligand, was found to be upregulated in SPP1+ macrophages, correlating with tumor growth and metastatic tendencies. Research on EREG is important because it has been found to play a significant role in cancer progression, including colorectal cancer. Understanding the mechanisms by which EREG contributes to tumor growth and metastasis can provide valuable insights into the development of targeted therapies for cancer treatment. Our hypothesis centered around the impact of EREG from TAMs on tumor progression. To identify TAM-specific EREG and its functional significance, we isolated tumor-associated macrophages from CT26 (mouse colorectal carcinoma)-bearing tumors and cultured them with conditioned and normal mediums. Subsequently, we examined EREG expression at the gene level using quantitative PCR. Our findings indicated significantly higher levels of EREG in the conditioned medium compared to the normal medium, suggesting an association between EREG and cancer cells. Remarkably, our investigation also confirmed that EREG has the capacity to reshape macrophage signatures. Notably, the surface marker CD206, characteristic of M2 macrophages, exhibited a significant decrease, while iNOS, a marker for M1 macrophages, showed an increase upon EREG depletion. We evaluated the interaction between CT26 cancer cells and TAMs using indirect co-culture methods. EREG-depleted TAMs were plated in the top chamber, while CT26 colorectal cancer cells were seeded at the bottom. Our results validated that knocking down EREG in TAMs not only hindered the proliferation of cancer cells but also affected the behavior of TAMs. Additionally, the reduced EREG expression in TAMs hindered the migration of cancer cells. These findings strongly suggest that targeting EREG could effectively restrain the growth and metastasis of colorectal cancer cells by reprogramming macrophage polarization in the TME. Consequently, this indicates its potential as a promising therapeutic target in colorectal cancer. Citation Format: Ah-Ran Yu, Seijong Kim, Yelin Jeong, Hye Kyung Hong, Yong Jae Shin, Yong Beom Cho. EREG reprograms M2-tumor-associated macrophage and suppresses colorectal cancer cell proliferation and migration [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 2701.

Abstract 4987: TREM2 is a radiation-induced target for immunomodulation of tumor-associated macrophages in the prostate cancer tumor microenvironment

Abstract Background: The prostate cancer (PCa) tumor microenvironment (TME) is characterized by a predominance of myeloid cells that may have a key role in immunosuppression and disease progression. Stereotactic Body Radiation Therapy (SBRT) is utilized for treatment of localized and metastatic PCa and promotes inflammatory remodeling of the TME. We analyzed human PCa specimens at acute post-SBRT timepoints to identify radiation-induced targets in the PCa TME and used in vitro and in vivo models to test and validate our findings. Methods: We performed 10X single-cell RNA sequencing (scRNA-seq) on prostatectomy specimens from men with high-risk localized PCa; 12,988 cells from 4 patients that underwent pre-operative SBRT and 47,314 cells from 6 patients that underwent prostatectomy alone. THP-1 monocytes polarized into macrophage (Mɸ) states [M0, M1, M2 and tumor-associated macrophages (TAM), were used to characterize TREM2 expression and immune responses to radiation or conditioned media (CM) derived from irradiated PC3 cells. In vivo studies with C57BL/6 mice inoculated with MC38 flank tumors or PTEN-/- SPOPmut CHD1del mouse prostate organoid line were used to analyze intratumoral myeloid populations in response to SBRT (37.5Gy in 5 fractions). Results: scRNA-seq analysis (Cell Ranger 7.1.0 pipelines; Seurat v5 R package) revealed enrichment of a cluster of myeloid cells defined by a damage-associated Mɸ gene signature (TREM2, APOE, SPP1, LGALS3, CD9) in response to SBRT compared with non-irradiated specimens (35.6% vs 5.7%). TREM2 expression was significantly upregulated in THP-1 cells polarized in M2 (p<0.001) and TAM (p<0.001) conditions, compared to M1 conditions. Irradiation (20Gy x1) of THP-1 cells significantly increased TREM2 expression in M0 (p<0.01), M1 (p<0.001), and TAM (p<0.01) conditions. Proliferation of PC3 cells exposed to CM from irradiated TAMs was significantly increased (p<0.0001) compared to CM from non-irradiated TAM. Alternatively, CM derived from irradiated PC3 cells significantly increased (p<0.05) TREM2 expression in M0-polarized Mɸ. In vivo studies, in MC38 and organoid models, noted a significant reduction (p<0.0001) in tumor volume at day 7 post-SBRT. Multi-parametric flow cytometry demonstrated robust myeloid infiltration in response to SBRT dominated by CD11b+F4/80+ TAMs in irradiated tumors. Among irradiated tumors, TREM2 expression was markedly elevated in TAMs (organoid and MC38, p<0.0001). Conclusion: Preliminary data in human irradiated PCa and validation studies in preclinical models suggest TREM2 expression on myeloid cells is a radiation-induced target. Further studies are underway to understand the functional and immunological implications of SBRT-induced TREM2 expression on TAMs and whether this can be therapeutically targeted to enhance anti-tumor immunity in PCa. Citation Format: Janny A. Villa-Pulgarin, Un In Chan, Francesca Khani, Fabio Socciarelli, Jiansheng Wu, Ryan Carelli, Jeffrey Kraynak, Silvia C. Formenti, Luigi Marchionni, Massimo Loda, Himanshu Nagar, Ariel E. Marciscano, Christopher E. Barbieri. TREM2 is a radiation-induced target for immunomodulation of tumor-associated macrophages in the prostate cancer 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 4987.

CLEC4E upregulation in gastric cancer: A potential therapeutic target correlating with tumor-associated macrophages

BackgroundCLEC4E has been reported to promote lung cancer progression. Tumor-associated macrophages (TAMs) play an important role in tumorigenesis. Whether the expression of CLEC4E in TAMs is associated with gastric carcinogenesis remains unclear. MethodsThe TIMER, UALCAN, UCSC Xena, and KM plotter databases are used to examine the expression of CLEC4E and its prognostic significance in gastric cancer (GC). Additionally, GO, KEGG, and GSEA analysis were conducted, and single-cell RNA-seq (scRNA-seq) datasets were utilized. The Coremine medical database was used to predict therapeutic drugs, and molecular docking was performed. Human GC samples were obtained, and co-culture models were constructed to evaluate the effects of CLEC4E in TAMs on tumor growth, migration, and invasion in vitro. ResultsCLEC4E was significantly upregulated in GC, and high CLEC4E expression was associated with poor prognosis. Western blotting and immunostaining showed increased protein levels of CLEC4E in GC. GO, KEGG, and GSEA results indicated that CLEC4E is involved in immune response. Immune infiltration analysis demonstrated that CLEC4E expression positively correlated with multiple immune cell types. scRNA-seq analyses revealed that CLEC4E was predominantly expressed in myeloid cells specifically TAMs, in GC. In vitro experiments confirmed that MFC induced CLEC4E expression in TAMs to mediate tumor progression. Specifically targeting CLEC4E by si-CLEC4E or stigmasterol inhibited cancer cell migration and invasion. ConclusionCLEC4E is a potential prognostic biomarker and new therapeutic target for GC that can be specifically targeted by stigmasterol.

Programmed Cell Death Ligand 1 Expression in CD163 + Tumor-associated Macrophages in Cancer Gland Rupture Microenvironment.

In this study, we aimed to examine the relationship among cancer gland rupture microenvironment, programmed cell death ligand 1 (PD-L1) expression in CD163 + tumor-associated macrophages (TAMs), and prognosis in colon adenocarcinoma. A total of 122 patients were diagnosed with colon adenocarcinoma between 2010 and 2019. PD-L1 + (clone 22C3) "macrophage scores" in the microenvironment of cancer gland rupture were calculated. The effects of these variables on prognosis were statistically analyzed. CD163 + TAMs were denser in the cancer gland rupture microenvironment. PD-L1 + TAMs were observed in the tumor periphery, and there was a significant difference between the rates of PD-L1 expression in TAMs and survival time (log-rank = 10.46, P = 0.015), clinical stage 2 ( P = 0.038), and primary tumor 3 and primary tumor 4 cases ( P = 0.004, P = 0.013). The risk of mortality was 4.070 times higher in patients with a PD-L1 expression rate of ≥1% in CD163 + TAMs. High PD-L1 expression in CD163 + TAMs is associated with poor overall survival. Therefore, blocking PD-L1 in CD163 + TAMs can be used as a target for immunotherapy.

IRG1 restrains M2 macrophage polarization and suppresses intrahepatic cholangiocarcinoma progression via the CCL18/STAT3 pathway.

Intrahepatic cholangiocarcinoma (ICC) is a highly malignant and aggressive cancer whose incidence and mortality continue to increase, whereas its prognosis remains dismal. Tumor-associated macrophages (TAMs) promote malignant progression and immune microenvironment remodeling through direct contact and secreted mediators. Targeting TAMs has emerged as a promising strategy for ICC treatment. Here, we revealed the potential regulatory function of immune responsive gene 1 (IRG1) in macrophage polarization. We found that IRG1 expression remained at a low level in M2 macrophages. IRG1 overexpression can restrain macrophages from polarizing to the M2 type, which results in inhibition of the proliferation, invasion, and migration of ICC, whereas IRG1 knockdown exerts the opposite effects. Mechanistically, IRG1 inhibited the tumor-promoting chemokine CCL18 and thus suppressed ICC progression by regulating STAT3 phosphorylation. The intervention of IRG1 expression in TAMs may serve as a potential therapeutic target for delaying ICC progression.

Glycolysis Induced by METTL14 Is Essential for Macrophage Phagocytosis and Phenotype in Cervical Cancer.

N 6-methyladenosine (m6A) is the most abundant mRNA modification in mammals and it plays a vital role in various biological processes. However, the roles of m6A on cervical cancer tumorigenesis, especially macrophages infiltrated in the tumor microenvironment of cervical cancer, are still unclear. We analyzed the abnormal m6A methylation in cervical cancer, using CaSki and THP-1 cell lines, that might influence macrophage polarization and/or function in the tumor microenvironment. In addition, C57BL/6J and BALB/c nude mice were used for validation invivo. In this study, m6A methylated RNA immunoprecipitation sequencing analysis revealed the m6A profiles in cervical cancer. Then, we discovered that the high expression of METTL14 (methyltransferase 14, N6-adenosine-methyltransferase subunit) in cervical cancer tissues can promote the proportion of programmed cell death protein 1 (PD-1)-positive tumor-associated macrophages, which have an obstacle to devour tumor cells. Functionally, changes of METTL14 in cervical cancer inhibit the recognition and phagocytosis of macrophages to tumor cells. Mechanistically, the abnormality of METTL14 could target the glycolysis of tumors invivo and vitro. Moreover, lactate acid produced by tumor glycolysis has an important role in the PD-1 expression of tumor-associated macrophages as a proinflammatory and immunosuppressive mediator. In this study, we revealed the effect of glycolysis regulated by METTL14 on the expression of PD-1 and phagocytosis of macrophages, which showed that METTL14 was a potential therapeutic target for treating advanced human cancers.

PU.1 induces tumor-associated macrophages promoting glioma progression through BTK-mediated Akt/mTOR pathway activation.

Glioma, the most common primary malignant brain tumor, is characterized by infiltrating immune cells that contribute to tumor progression and therapeutic resistance. Tumor-associated macrophages (TAMs) constitute a significant proportion of these infiltrating immune cells and have been implicated in glioma progression. However, the underlying molecular mechanisms by which TAMs promote glioma progression remain elusive. In this study, we investigated the role of PU.1, a crucial transcription factor involved in myeloid cell development, in glioma-associated macrophage polarization and activation. First, bioinformatics and analysis of clinical glioma samples demonstrated a positive correlation between PU.1 expression in TAMs and disease severity. Further experiments using in vitro coculture systems revealed that the expression of PU.1 is increased in glioma cells vs. control cells. Importantly, PU.1-overexpressing macrophages exhibited a protumorigenic phenotype characterized by enhanced migration, invasion, and proliferation. Mechanistically, we found that PU.1-induced activation of the Bruton tyrosine kinase (BTK) signaling pathway led to Akt/mTOR pathway activation in macrophages, which further enhanced their protumorigenic functions. Furthermore, pharmacological inhibition of the BTK or Akt/mTOR pathway reversed the protumorigenic effects of macrophages in vitro and impaired their ability to promote glioma progression in vivo. In conclusion, our study elucidates a novel mechanism by which PU.1 induces the polarization and activation of TAMs in the glioma microenvironment. We highlight the significance of BTK-mediated Akt/mTOR pathway activation in driving the protumorigenic functions of TAMs. Targeting PU.1 and its downstream signaling pathways in TAMs may provide a promising therapeutic strategy to suppress glioma progression and improve patient outcomes.

Tumor cell senescence-induced macrophage CD73 expression is a critical metabolic immune checkpoint in the aging tumor microenvironment.

Background: The role of senescent cells in the tumor microenvironment (TME) is usually bilateral, and diverse therapeutic approaches, such as radiotherapy and chemotherapy, can induce cellular senescence. Cellular interactions are widespread in the TME, and tumor cells reprogram immune cells metabolically by producing metabolites. However, how senescent cells remodel the metabolism of TME remains unclear. This study aimed to explore precise targets to enhance senescent cells-induced anti-tumor immunity from a metabolic perspective. Methods: The in vivo senescence model was induced by 8 Gy×3 radiotherapy or cisplatin chemotherapy, and the in vitro model was induced by 10 Gy-irradiation or cisplatin treatment. Metabonomic analysis and ELISA assay on tumor interstitial fluid were performed for metabolites screening. Marker expression and immune cell infiltration in the TME were analyzed by flow cytometry. Cell co-culture system and senescence-conditioned medium were used for crosstalk validation in vitro. RNA sequencing and rescue experiments were conducted for mechanism excavation. Immunofluorescence staining and single-cell transcriptome profiling analysis were performed for clinical validation. Results: We innovatively reveal the metabolic landscape of the senescent TME, characterized with the elevation of adenosine. It is attributed to the senescent tumor cell-induced CD73 upregulation of tumor-associated macrophages (TAMs). CD73 expression in TAMs is evoked by SASP-related pro-inflammatory cytokines, especially IL-6, and regulated by JAK/STAT3 pathway. Consistently, a positive correlation between tumor cells senescence and TAMs CD73 expression is identified in lung cancer clinical specimens and databases. Lastly, blocking CD73 in a senescent background suppresses tumors and activates CD8+ T cell-mediated antitumor immunity. Conclusions: TAMs expressed CD73 contributes significantly to the adenosine accumulation in the senescent TME, suggesting targeting CD73 is a novel synergistic anti-tumor strategy in the aging microenvironment.

LncRNA-HOXC-AS2 regulates tumor-associated macrophage polarization through the STAT1/SOCS1 and STAT1/CIITA pathways to promote the progression of non-small cell lung cancer

Tumor-associated macrophages (TAMs) mainly exhibit the characteristics of M2-type macrophages, and the regulation of TAM polarization is a new target for cancer therapy, among which lncRNAs are key regulatory molecules. This study aimed to explore the effects of lncRNA-HOXC-AS2 on non-small cell lung cancer (NSCLC) by regulating TAM polarization. THP-1 cells were used to differentiate into macrophages, and TAMs were obtained by coculture with A549 cells. The M1/M2 cell phenotype and HOXC-AS2 expression were detected, and A549-derived exosomes (A549-exo) were used to elucidate the effects of A549 on macrophage polarization and HOXC-AS2 expression. Then, by interfering with HOXC-AS2 or STAT1, the effects of HOXC-AS2 regulation of STAT1 on the TAM phenotype and STAT1/SOCS1 and STAT1/CIITA pathways were analyzed, and the proliferation and metastasis of NSCLC cells in the coculture system were also detected. Results showed that HOXC-AS2 expression in M2 macrophages and TAMs was significantly higher than that in M1 macrophages, and A549-exo promoted HOXC-AS2 expression and M2 polarization. Intervention HOXC-AS2 resulted in increased M1 marker expression, decreased M2 marker expression, and activation of STAT1/SOCS1 and STAT1/CIITA pathways in TAMs. In addition, HOXC-AS2 was mainly expressed in the cytoplasm of TAMs and could bind to STAT1. Further experiments confirmed that intervention HOXC-AS2 promoted the M1 polarization of TAMs by targeting STAT1 and weakened the promoting effects of TAMs on the proliferation and metastasis of NSCLC. In conclusion, HOXC-AS2 inhibited the activation of STAT1/SOCS1 and STAT1/CIITA pathways and promoted M2 polarization of TAMs by binding with STAT1, thus promoting NSCLC.

RNA-based modulation of macrophage-mediated efferocytosis potentiates antitumor immunity in colorectal cancer

Tumor-associated macrophages play pivotal roles in tumor progression and metastasis. Macrophage-mediated clearance of apoptotic cells (efferocytosis) supports inflammation resolution, contributing to immune evasion in colorectal cancers. To reverse this immunosuppressive process, we propose a readily translatable RNA therapy to selectively inhibit macrophage-mediated efferocytosis in tumor microenvironment. A clinically approved lipid nanoparticle platform (LNP) is employed to encapsulate siRNA for the phagocytic receptor MerTK (siMerTK), enabling selective MerTK inhibition in the diseased organ. Decreased MerTK expression in tumor-associated macrophages results in apoptotic cell accumulation and immune activation in tumor microenvironment, leading to suppressed tumor growth and better survival in both liver and peritoneal metastasis models of colorectal cancers. siMerTK delivery combined with PD-1 blockade further produces enhanced antimetastatic efficacy with reactivated intratumoral immune milieu. Collectively, LNP-based siMerTK delivery combined with immune checkpoint therapy may present a feasible modality for metastatic colorectal cancer therapy.

Prognostic significance of annexin A2 and tumor associated macrophages (TAMs) in metastatic renal cell carcinoma and their relation to Sunitinib resistance

ABSTRACT Sunitinib, an antiangiogenic tyrosine kinase inhibitor, is the main treatment for metastatic renal cell carcinoma (mRCC). Development of resistance is a major obstacle against therapy success. The aim of this study was to assess annexin A2 and CD163+ tumor associated macrophages (TAMs) immunohistochemical expression in 50 mRCC cases as regard to patients’ prognosis and Sunitinib response. Also, to assess the correlation between annexin A2 and TAMs expression. High annexin A2 expression and TAMs density were associated with serum calcium level (P = 0.024 and 0.037, respectively), larger tumor size (P < 0.001), high tumor grade (P = 0.014 and <0.001, respectively), and the presence of tumor necrosis (P < 0.001). High annexin A2 and TAMs expressions were related to shorter patients’ overall survival (P = 0.009 and 0.001, respectively) and progression-free survival (P = 0.003 and 0.001, respectively). Annexin A2 was correlated with TAMs density (r = 0.890). Annexin A2 and TAMs are associated with poor prognostic parameters in mRCC patients, including high nuclear grade, increased tumor size, and the presence of tumor necrosis, together with shorter patients’ survivals and poor response to Sunitinib. Annexin A2 expression is correlated with TAMs density suggesting immunomodulatory role of annexin A2.

PD-1 Expression By Dendritic Cells Is a Key Regulator of T-Cell Immunity in Cancer

Programmed cell death 1 (PD-1) is an inhibitory receptor expressed on a variety of immune cells and a therapeutic target of checkpoint immunotherapy for cancer patients. PD-1 engagement by PD-L1 or PD-L2 can inhibit activation and expansion of tumor antigen-specific T cells. PD-1 expression in tumor-associated macrophages (TAM) correlates with impaired phagocytosis and antigen presenting function and enhanced immunosuppression. PD-1 is also expressed in dendritic cells (DC) and has been shown to correlate negatively with the outcome of DC-based cancer vaccines. On DC, PD-1 can interact with PD-L1 in cis when both of these receptors are expressed on the same cell surface. DCs are the major antigen presenting cells for cross-presenting tumor antigens to T cells, and PD-L1 upregulation protects them from killing by cytotoxic T lymphocytes, yet dampens the antitumor responses. Very little is known about the specific role of PD-1 on DCs function. In the present study, we sought to understand the role of PD-1 on DCs in the context of cancer. We generated mice with conditional targeting of the Pdcd1 gene (encoding for PD-1) and crossed them with CD11cCre ( Pdcd1 f/fCD11cCre) or Clec9Cre ( Pdcd1 f/fClec9Cre) resulting in specific deletion of PD-1 in DC cells. We used the MC17 fibrosarcoma syngeneic mouse model to investigate the tentative implications of DC-specific PD-1 ablation on anti-tumor responses. We found larger tumor sizes and weights in tumor-bearing Pdcd1 f/fCD11cCre mice compared to their Pdcd1 wt/wtCD11cCre control counterparts. Pdcd1 f/fCD11cCre tumor-bearing mice had significantly higher fractions of DCs in spleens and tumor-draining lymph nodes (tdLNs) but only minimal differences in their activation state and checkpoint marker expression. Similarly, CD4 + and CD8 + T cells from spleens and tdLNs were comparable in percentages and activation states. However, characterization of the immune cells at the tumor site, indicated that Pdcd1 f/fCD11cCre tumor-bearing mice had a more immunosuppressive tumor microenvironment with increased fractions of CD11b +Ly6C hiLy6G -monocytic myeloid derived suppressor cells (M-MDSCs) compared to control tumor-bearing mice. Moreover, flow cytometric analysis revealed that intratumoral M-MDSC in Pdcd1 f/fCD11cCre mice displayed enhanced expression of PD-L1 and CD38, consistent with a more activated and more immunosuppressive phenotype. Characterization of tumor infiltrating CD4 + and CD8 + T cells also revealed vast differences in composition and activation states. Specifically, Pdcd1 f/fCD11cCre mice had larger populations of intratumoral CD3 + T cells, with a greater CD4/CD8 T cell ratio, and higher percentages of CD4 + T regulatory cells. Conversely, intratumoral CD8 + T cells were reduced, and were characterized by diminished expansion of CD8 + T effector cells, and a less activated phenotype as determined by lower expression of CD25, CD69, and GITR. In syngeneic tumor experiments with B16 melanoma expressing ovalbumin (B16-Ova), Pdcd1 f/fCD11cCre mice and Pdcd1 f/fClec9Cre mice similarly had greater tumor sizes and weights compared to their control counterparts. These results indicate that selective ablation of PD-1 in DCs cells confers a more immunosuppressive tumor microenvironment by promoting T regulatory cell expansion and diminishing CD8 + T effector cell activation compromising anti-tumor responses.

CTHRC1 promotes colorectal cancer progression by recruiting tumor-associated macrophages via up-regulation of CCL15.

Tumor-associated macrophages (TAMs) represent a key factor in the tumor immune microenvironment (TME), exerting significant influence over tumor migration, invasion, immunosuppressive features, and drug resistance. Collagen triple helix repeat containing 1 (CTHRC1), a 30 KDa protein which was secreted during the tissue-repair process, is highly expressed in several malignant tumors, including colorectal cancer (CRC). Previous studies demonstrated that CTHRC1 expression in TAMs was positively correlated to M2 macrophage polarization and liver metastasis, while our discovery suggesting a novel mechanism that CTHRC1 secreted from cancer cell could indirectly interplay with TAMs. In this study, the high expression level of CTHRC1 was evaluated in CRC based on GEO and TCGA databases. Further, CTHRC1 was detected high in all stages of CRC patients by ELISA and was correlated to poor prognosis. Multispectral imaging of IHC demonstrated that M2 macrophage infiltration was increased accompanied with CTHRC1 enrichment, suggesting that CTHRC1 may have chemotactic effect on macrophages. In vitro, CTHRC1 could have chemotactic ability of macrophage in the presence of HT-29 cell line. Cytokine microarray revealed that CTHRC1 could up-regulate the CCL15 level of HT-29, pathway analysis demonstrated that CTHRC1 could regulate CCL15 by controlling the TGFβ activation and Smad phosphorylation level. In vivo, knocking down of CTHRC1 from CT-26 also inhibits tumor formation. In conclusion, CTHRC1 could promote the chemotactic ability of macrophages by up-regulating CCL15 via TGFβ/Smad pathway; additionally, a high level of CTHRC1 could promote macrophage's M2 polarization. This discovery may be related to tumor immune tolerance and tumor immunotherapy resistance in CRC. KEY MESSAGES: CTHRC1 promotes CRC progression by up-regulating CCL15 via TGF-β/Smad pathways to further recruit tumor-associated macrophages. By the means of autocrine or paracrine, CTHRC1 can indeed promote macrophage chemotaxis and enhance the infiltration of macrophages in tumor tissues but in the presence of tumor cells. CAFs were another source of CTHRC1, indicating CTHRC1 can infiltrate tumor islet as well as the stomal and be secreted from both tumor cells and CAFs. This study validated CTHRC1 as a potential immune therapy target CRC.

TMIC-17. THE ROLE OF ICAM1 IN GLIOBLASTOMA TUMORIGENESIS UNDER HYPOXIC CONDITIONS

Abstract Glioblastoma (GBM) is a deadly brain cancer in adults with limited treatment options. Cell adhesion molecules (CAMs) mediate cell-cell interactions and migration. Intercellular adhesion molecule 1 (ICAM-1) is a CAM expressed by various cell types in solid tumors, including macrophages, tumor and endothelial cells. We previously demonstrated that ICAM-1 expression is linked to shorter overall survival in GBM patients. We observed elevated ICAM-1 levels in hypoxic regions of GBMs. Considering the role of tumor-associated macrophages (TAMs) in GBM tumorigenicity, our objective was to investigate how ICAM-1 expression in TAMs contributes to GBM growth within the hypoxic tumor microenvironment (TME). We employed a comprehensive approach utilizing in vitro and in vivo models, as well as genomic analyses at both bulk and single-cell levels, to elucidate the association between ICAM-1 and TAMs in tumorigenicity. Our findings demonstrated that injecting GBM cells into ICAM-1 knockout mice led to prolonged survival, smaller tumors, reduced macrophage infiltration, and enhanced M1-like polarization of TAMs. Additionally, bone marrow reconstitution experiments confirmed the tumor-promoting role of ICAM-1-expressing TAMs. Cell deconvolution, immunohistochemical, and flow cytometry analyses of mouse tumors supported these findings. Gene set enrichment analysis of differentially expressed genes in xenografted tumors revealed that ICAM-1 presence upregulated key tumorigenic pathways, including epithelial-mesenchymal transition, IL2-STAT5, TNF-α via NFκB, IL6-JAK-STAT3, and RAS/MAPK. In vitro analysis indicated that ICAM-1 promoted macrophage migration and polarization towards M2-like state, which was further augmented by hypoxia-induced ICAM1 expression. Flow cytometry, cytometry by time of flight (cyToF), and single-cell RNA sequencing analysis of human and mouse GBMs revealed elevated ICAM-1 expression in hypoxic TAMs, suggesting the hypoxic microenvironment promotes the tumorigenic functions of TAMs. In conclusion, ICAM1 likely enhances GBM tumorigenesis by promoting TAM migration into the TME. Furthermore, environmental factors such as hypoxia contribute to increased ICAM-1 expression in TAMs, further exacerbating GBM tumorigenicity.

AIM2 fosters lung adenocarcinoma immune escape by modulating PD-L1 expression in tumor-associated macrophages via JAK/STAT3

ABSTRACT This work was devised to discuss the effect of AIM2 on the immunosuppression of LUAD tumors, as well as its molecular mechanism. An allograft mouse model was built. Mouse macrophages were isolated and collected. The infiltration level of Mø and expression of M1 Mø, M2 Mø markers, and PD-L1 were assayed by IHC and flow cytometry. Expression levels of M1 Mø and M2 Mø marker genes and PD-L1 were detected by qPCR. The expression of proteins linked with JAK/STAT3 was tested by western blot. CD8+T cells and NK cells were activated in vitro and co-cultured with mouse macrophages, and their cytotoxicity was detected by LDH method. The proportion of CD206+PD-L1+ cells and the activation and proliferation of CD8+T cells were assayed by flow cytometry. Multicolor immunofluorescence was utilized to assay the co-localization of proteins. AIM2 demonstrated a high expression in LUAD, exhibiting a conspicuous positive correlation with the expression of the M2 Mø markers as well as PD-L1. Expression of M1 markers was upregulated after knockdown of AIM2, while M2 markers expression and PD-L1 were downregulated, and the colocalization of proteins linked with PD-L1 and M2 Mø was decreased. The infiltration and cytotoxicity of CD8+T cells and NK cells increased after silencing AIM2. After the knockdown of AIM2, which was enriched in the JAK/STAT3 pathway, the phosphorylation levels of JAK1, JAK2, and STAT3 were reduced, the immune infiltration level of CD8+T cells increased, and the co-localization level of PD-L1 and PD-1 dropped. The activity and proliferation level of CD8+T cells were increased with the reduced PD-1 expression. AIM2 fosters M2 Mø polarization and PD-L1 expression via the JAK/STAT3 pathway. Moreover, AIM2 promotes the immune escape of LUAD via the PD-1/PD-L1 axis. Our work may blaze a trail for the clinical treatment of LUAD.

Expression of miR-141, TAMs, and CD147 in Bladder Cancer Cells: Implications for Tumor Grade and Prognosis

This study aimed to investigate the significance of miR-141 expression, tumor-associated macrophages (TAMs), and CD147 expression in bladder cancer (BCa) cells, and their relationship with tumor grade. A total of 80 BCa patients were selected, and they were categorized into two groups: Group G1 (56 cases) with poorly differentiated tumors, and Group G2 (24 cases) with moderately to well-differentiated tumors according to the WHO classification criteria for urothelial carcinoma. We analyzed the differences in miR-141, TAMs, and CD147 expression levels among different tumor grades. The miR-141 level in BCa tissues of patients in Group G2 showed a significant decrease (1.88±0.4) compared to Group G1 (P &lt;0.05). As the tumor grade increased, TAMs levels in tumor tissues also increased (P &lt;0.05). Notably, 66.7% of CD147-positive patients in Group G2 exhibited a clear increase compared to Group G1 (P &lt;0.05). Importantly, significant correlations were observed between miR-141, TAMs, CD147 levels, and tumor grade (P &lt;0.05). These findings suggest that as BCa grades increase, miR-141 levels decrease while TAMs and CD147 levels increase. The levels of miR-141, TAMs, and CD147 in BCa tissue cells can serve as indicators for evaluating the prognosis of BCa patients.

C5aR1 blockade reshapes immunosuppressive tumor microenvironment and synergizes with immune checkpoint blockade therapy in high-grade serous ovarian cancer

ABSTRACT High-grade serous ovarian cancer (HGSC), with a modest response to immune checkpoint blockade (ICB) targeting PD-1/PD-L1 monotherapy, is densely infiltrated by M2-polarized tumor-associated macrophages (TAMs) and regulatory T (Treg) cells. The complement C5a/C5aR1 axis contributes to the programming of the immunosuppressive phenotype of TAMs in solid tumors and represents a promising immunomodulatory target for treating HGSCs. Here, we aimed to identify the relevance of C5aR1 in prognosis, immune microenvironment, and immunotherapy response in HGSCs. The expression and relationship of C5aR1 with tumor-infiltrating immune cells were assessed by immunohistochemistry and flow cytometry in the training cohort (n = 120) and fresh HGSC tissues (n = 36). Transcriptomic analyses of the xenografts delineated the mechanisms driving the immunomodulatory activity of PMX53, an orally bioavailable C5aR1 inhibitor. Therapeutic relevance was confirmed in ex vivo tumor cultures and The Cancer Genome Atlas (TCGA) datasets. C5aR1 expression independently predicted dismal prognosis and was linked to the immunoevasive subtype of HGSC, characterized by increased infiltration of pro-tumor cells (Treg cells, M2-polarized macrophages, and neutrophils) and impaired CD8+T functions. PMX53 antagonized subcutaneous tumor growth, modulated immunosuppressive mechanisms and synergized with aPD-1 in several tumor types. Single-cell RNA-seq analysis revealed predominant C5aR1 expression in TAMs, with an immunosuppressive-related expression signature in C5aR1+TAMs. Furthermore, the combination of C5aR1 and PD-L1 was associated with specific molecular characteristics and matched clinical response annotations. Therefore, the abundance of C5aR1 could predict an inferior prognosis in HGSCs, and incorporating PD-L1 may serve as a novel predictive biomarker to guide therapeutic options.

ChemR23 activation reprograms macrophages toward a less inflammatory phenotype and dampens carcinoma progression.

Tumor Associated Macrophages (TAM) are a major component of the tumor environment and their accumulation often correlates with poor prognosis by contributing to local inflammation, inhibition of anti-tumor immune response and resistance to anticancer treatments. In this study, we thus investigated the anti-cancer therapeutic interest to target ChemR23, a receptor of the resolution of inflammation expressed by macrophages, using an agonist monoclonal antibody, αChemR23. Human GM-CSF, M-CSF and Tumor Associated Macrophage (TAM)-like macrophages were obtained by incubation of monocytes from healthy donors with GM-CSF, M-CSF or tumor cell supernatants (Breast cancer (BC) or malignant pleural mesothelioma (MPM) cells). The effects of αChemR23 on macrophages were studied at the transcriptomic, protein and functional level. Datasets from The Cancer Genome Atlas (TCGA) were used to study CMKLR1 expression, coding for ChemR23, in BC and MPM tumors. In vivo, αChemR23 was evaluated on overall survival, metastasis development and transcriptomic modification of the metastatic niche using a model of resected triple negative breast cancer. We show that ChemR23 is expressed at higher levels in M-CSF and tumor cell supernatant differentiated macrophages (TAM-like) than in GM-CSF-differentiated macrophages. ChemR23 activation triggered by αChemR23 deeply modulates M-CSF and TAM-like macrophages including profile of cell surface markers, cytokine secretion, gene mRNA expression and immune functions. The expression of ChemR23 coding gene (CMKLR1) strongly correlates to TAM markers in human BC tumors and MPM and its histological detection in these tumors mainly corresponds to TAM expression. In vivo, treatment with αChemR23 agonist increased mouse survival and decreased metastasis occurrence in a model of triple-negative BC in correlation with modulation of TAM phenotype in the metastatic niche. These results open an attractive opportunity to target TAM and the resolution of inflammation pathways through ChemR23 to circumvent TAM pro-tumoral effects.

The XOR-IDH3α axis controls macrophage polarization in hepatocellular carcinoma

Tumor-associated macrophages (TAMs) are indispensable in the hepatocellular carcinoma (HCC) tumor microenvironment. Xanthine oxidoreductase (XOR), also known as xanthine dehydrogenase (XDH), participates in purine metabolism, uric acid production, and macrophage polarization to a pro-inflammatory phenotype. However, the role of XOR in HCC-associated TAMs is unclear. We evaluated the XOR level in macrophages isolated from HCC tissues and paired adjacent tissues. We established DEN/carbon tetrachloride (CCl4)-induced and orthotopically implanted HCC mouse models using mice with Xdh-specific depletion in the myeloid cell lineage (Xdhf/fLyz2cre) or Kupffer cells (Xdhf/fClec4fcre). We determined metabolic differences using specific methodologies, including metabolomics and metabolic flux. We found that XOR expression was downregulated in HCC TAMs and positively correlated with patient survival, which was strongly related to the characteristics of the tumor microenvironment (TME), especially hypoxia. Using HCC-inflicted mice (Xdhf/fLyz2cre and Xdhf/fClec4fcre), we revealed that XOR loss in monocyte-derived TAMs rather than Kupffer cells promoted their M2 polarization and CD8+ T-cell exhaustion, which exacerbated HCC progression. In addition, the tricarboxylic acid cycle was disturbed, and the generation of α-ketoglutarate (α-KG) was enhanced within XOR-depleted macrophages. XOR inhibited α-KG production by interacting with IDH3α catalytic sites (K142 and Q139). The increased IDH3α activity caused increased adenosine and kynurenic acid production in the TAMs, which enhanced the immunosuppressive effects of the TAMs and CD8+ T cells. The XOR-IDH3α axis mediates TAM polarization and HCC progression and may be a small-molecule therapeutic or immunotherapeutic target against suppressive HCC TAMs. HCC is one of the cancers with the highest morbidity in the world, and its 5-year survival rate is low. Currently, lots of immunotherapies have been applied to the treatment of HCC, but the curative effects are not satisfactory. TME of HCC is full of different infiltrating immune cells. TAMs are vital components in TME and involved in HCC progression. Herein, we confirm the downregulation of XOR expression in TAMs isolated from human HCC tissues. The loss of XOR in monocyte-derived macrophages increases IDH3 activity and results in an increase in α-KG production, which can promote M2-like polarization. Additionally, XOR-null TAMs derived from monocytes promote CD8+ T-cell exhaustion via the upregulation of immunosuppressive metabolites, including adenosine and KYNA. Given the prevalence and high rate of incidence of HCC and the need for improved therapeutic options for patients, our findings identify potential therapeutic targets that may be further studied to develop improved therapies.

Tumor-derived small extracellular vesicles promote breast cancer progression by upregulating PD-L1 expression in macrophages

BackgroundThe metastasis of breast cancer (BC) is a complex multi-step pathological process, strictly dependent on the intrinsic characteristics of BC cells and promoted by a predisposing microenvironment. Although immunotherapy has made important progress in metastasis BC, the heterogeneity of PD-L1 in tumor associated macrophages (TAMs) in BC and the underlying mechanisms in the metastasis development of BC are still not completely elucidated. Small extracellular vesicles (sEVs) represent essential interaction mediators between BC cells and TAMs. It is worth noting to explore the underlying mechanisms typical of sEVs and their role in the metastasis development of BC.MethodsThe structure of sEVs was identified by TEM, while the particle size and amounts of sEVs were detected by BCA and NTA analysis. The specific PD-L1 + CD163 + TAM subpopulation in metastasis BC was identified by scRNA-seq data of GEO datasets and verified by IHC and IF. The function of TAMs and sEVs in metastasis BC was explored by RT-qPCR, WB, IF, flow cytometry and in vivo experiment. The expression profiles of plasma sEVs-miRNA in relation to BC metastasis was analyzed using next-generation sequencing. Further detailed mechanisms of sEVs in the metastasis development of BC were explored by bioinformatics analysis, RT-qPCR, WB and luciferase reporter assay.ResultsIn this study, we identified that the immunosuppressive molecule PD-L1 was more abundant in TAMs than in BC cells, and a specific PD-L1 + CD163 + TAM subpopulation was found to be associated with metastasis BC. Additionally, we found that BC cells-derived sEVs can upregulate the PD-L1 expression and induce the M2 polarization, enhancing the metastasis development both in vitro and in vivo. Also, Clinical data showed that sEV-miR-106b-5p and sEV-miR-18a-5p was in relation to BC metastasis development and poor prognosis of BC patients. Further mechanistic experiments revealed that BC-derived sEV-miR-106b-5p and sEV-miR-18a-5p could synergistically promoted the PD-L1 expression in M2 TAMs by modulating the PTEN/AKT and PIAS3/STAT3 pathways, resulting in the enhancement of the BC cells invasion and metastasis.ConclusionsOur study demonstrated that BC-derived sEVs can induce metastasis in BC through miR-106b-5p/PTEN/AKT/PD-L1 and miR-18a-5p/PIAS3/STAT3/PD-L1 pathways in TAMs. Therefore, the inhibition of these specific interactions of signaling pathways would represent a promising target for future therapeutic strategies for treatment of BC.