High Tumor-associated Macrophages Research Articles

Tumor associated macrophages in breast cancer progression: implications and clinical relevance

Macrophages represent an immune cell population characterized by high plasticity and a range of properties and functions. Their activation status and specific phenotype are highly associated with their localization and the environmental cues they receive. The roles of macrophages in cancer development are diverse. Despite their antitumor effects at early stages of the disease, their presence in the tumor microenvironment (TME) has been linked to tumor promotion upon disease establishment. Tumor associated macrophages (TAMs) are key components of breast cancer TME and they have been associated with poor clinical outcomes. High TAM densities were found to correlate with tumor progression, increased metastatic potential and poor prognosis. Interestingly, considerably higher levels of TAMs were found in patients with triple negative breast cancer (TNBC)—the most aggressive type of breast cancer—compared to other types. The present review summarizes recent findings regarding the distinct TAM subsets in the TME and TAM involvement in breast cancer progression and metastasis. It highlights the constant interplay between TAMs and breast cancer cells and its major contribution to the progression of the disease, including such aspects as, polarization of macrophages toward a tumor promoting phenotype, induction of epithelial to mesenchymal transition (EMT) in cancer cells and enhancement of cancer stem cell properties. Further, we discuss the clinical relevance of these findings, focusing on how a better delineation of TAM involvement in breast cancer metastasis will facilitate the selection of more efficient treatment options.

Clinicopathologic and prognostic significance of tumor-associated macrophages in cervical cancer: a systematic review and meta-analysis.

The role of tumor-associated macrophages (TAMs) in cervical cancer (CC) remains controversial. Here, we report a meta-analysis of the association between TAMs infiltration and clinical outcomes. PubMed, Embase, Web of Science, and CNKI were searched systematically from inception until December 20, 2023. Studies involving TAMs and prognosis, clinical, or pathological features were included. Quality assessments of the selected studies were assessed. The fixed-effect or random-effects model, standard mean difference (SMD), odds ratios (OR), or hazard ratios (HR) with 95% confidence intervals (CIs) were used as the effect size estimate. 26 eligible studies with 2,295 patients were identified. Our meta-analysis revealed that TAMs were overexpressed in CC (OR = 12.93, 95% CI = 7.73-21.61 and SMD = 1.58, 95% CI = 0.95-2.21) and that elevated TAM levels were strongly associated with lymph node metastasis (LNM) (SMD = 0.51, 95% CI = 0.90-2.01) and FIGO stages (SMD = 0.46, 95% CI = 0.08-0.85). Subgroup analysis indicated a significant positive correlation between LNM and TAMs density in tumor stroma, but not in cancer nests (SMD = 0.58, 95% CI = 0.31-0.58). Furthermore, in early stage, a stronger correlation exists between LNM and TAM density (SMD = 1.21, 95% CI = 0.75-1.66). In addition, it revealed that patients with high TAMs expression had poorer overall survival (OS) (HR = 2.55 95% CI = 1.59-4.07) and recurrence-free survival (RFS) (HR = 2.17, 95% CI = 1.40-3.35). Our analyses suggest that a high density of TAMs predicts adverse outcomes in CC.

Is overexpression of CD163 and CD47 in tumour cells of breast carcinoma implicated in the recruitment of tumour-associated macrophages (TAMs) in tumour microenvironment? immunohistochemical prognostic study

ABSTRACT Background Now, targeted therapy and immunotherapy are promoted. tumour -Associated Macrophages (TAMs) are an essential component of immune-response in breast cancer(BC) with prognostic controversy. Additionally, their recruiting factors are still obscure. Purpose:This study aimed to evaluate the prognostic significance of CD163 and CD47 in BC of No Special Type (BC-NST) and to explore their suggested role in recruiting TAMs. Material and methods This immunohistochemical study was conducted on 91 archival specimens of breast cases. Immunoreactivity scores were correlated with TAMs density, clinicopathological data, and survival. Results Revealed the highest CD163 expression was detected in the pure DCIS group (p = 0.016), while the highest CD47 expression and high TAMs density were reported in the invasive group (p = 0.008, and p = 0.002 respectively) followed by the DCIS group. In IC-NSTs the CD163 and CD47 scores were associated with poor prognostic parameters like(high grade, advanced stage, distant metastasis, ER negativity,Ki67 index, post-surgical chemotherapy, poor NPI group, high mitotic count, dense infiltration of TAMs, shorter OS). Also, CD47 was associated with the dens infiltration of TAMs in DCIS (p = 0.001). There was a significant correlation between tumour cell expression of CD163 and CD47 in IC-NSTs and DCIS (p = 0.002 and p = 0.009 respectively). Conclusions High CD163 and CD47 expressions in both DCIS andIBC are intimately associated, significantly associated with poor prognosis and are important provoking factors of TAMs.

Immune cell distribution and DNA methylation signatures differ between tumor and stroma enriched compartment in pancreatic ductal adenocarcinoma

The presence of abundant tumor stroma is a prominent characteristic of pancreatic ductal adenocarcinomas (PDAC) that potentially influences disease progression and therapy response. This study aims to investigate immune cell infiltration and epigenetic profiles in tumor cell enriched (“Tumor”) and stroma cell enriched (“Stroma”) regions within human PDAC tissue samples. By comparing those regions, we identified 25,410 differentially methylated positions (DMPs) distributed across 6,963 unique genes. Pathway enrichment analysis using the top 2,000 DMPs that were either hyper- or hypomethylated indicated that immune response pathways and the estrogen receptor pathway are epigenetically dysregulated in Tumor and Stroma regions, respectively. In terms of immune cell infiltration, we observed overall low levels of T cells in both regions. In Tumor regions however, occurrence of tumor-associated macrophages (TAMs) was higher than in Stroma regions (p = 0.02) concomitant with a dualistic distribution that stratifies PDAC patients into those with high and low TAM infiltration. By categorizing TAM levels into quartiles, our analysis revealed that PDAC patients with more than 1,515 TAMs per mm² exhibited significantly shorter overall survival (p = 0.036). Our data suggest that variations in inflammatory characteristics between the Tumor and Stroma defined compartments of PDAC may primarily stem from the presence of macrophages rather than lymphocytes. The abundance of TAMs within regions enriched with tumor cells correlates with patient survival, underscoring the potential significance of exploring therapeutic interventions targeting TAMs. Furthermore, directing attention towards the estrogen receptor pathway may represent a promising strategy to address the stroma cell component within the PDAC tumor microenvironment.

Mutational, immune microenvironment, and clinicopathological profiles of diffuse large B-cell lymphoma and follicular lymphoma with BCL6 rearrangement.

BCL6-rearrangement (BCL6-R) is associated with a favorable prognosis of follicular lymphoma (FL), but the mechanism is unknown. We analyzed the clinicopathological, immune microenvironment (immune checkpoint, immuno-oncology markers), and mutational profiles of 10 BCL6-R-positive FL, and 19 BCL6-R-positive diffuse large B-cell lymphoma (DLBCL) cases (both BCL2-R and MYC-R negative). A custom-made panel included 168 genes related to aggressive B-cell lymphomas and FL. FL cases were nodal, histological grade 3A in 70%, low Ki67; and had a favorable overall and progression-free survival. DLBCL cases were extranodal in 60%, IPI high in 63%, non-GCB in 60%, EBER-negative; and had a progression-free survival comparable to that of DLBCL NOS. The microenvironment had variable infiltration of M2-like tumor-associated macrophages (TAMs) that were CD163, CSF1R, LAIR1, PD-L1, and CD85A (LILRB3) positive; but had low IL10 and PTX3 expression. In comparison to FL, DLBCL had higher TAMs, IL10, and PTX3 expression. Both lymphoma subtypes shared a common mutational profile with mutations in relevant pathogenic genes such as KMT2D, OSBPL10, CREBBP, and HLA-B (related to chromatin remodeling, metabolism, epigenetic modification, and antigen presentation). FL cases were characterized by a higher frequency of mutations of ARID1B, ATM, CD36, RHOA, PLOD2, and PRPRD (p < 0.05). DLBCL cases were characterized by mutations of BTG2, and PIM1; and mutations of HIST1H1E and MFHAS1 to disease progression (p < 0.05). Interestingly, mutations of genes usually associated with poor prognosis, such as NOTCH1/2 and CDKN2A, were infrequent in both lymphoma subtypes. Some high-confidence variant calls were likely oncogenic, loss-of-function. MYD88 L265P gain-of-function was found in 32% of DLBCL. In conclusion, both BCL6-R-positive FL and BCL6-R-positive DLBCL had a common mutational profile; but also, differences. DLBCL cases had a higher density of microenvironment markers.

Tumor-associated macrophages drive glycolysis through the IL-8/STAT3/GLUT3 signaling pathway in pancreatic cancer progression

Glycolytic metabolism is a hallmark of pancreatic ductal adenocarcinoma (PDAC), and tumor-associated stromal cells play important roles in tumor metabolism. We previously reported that tumor-associated macrophages (TAMs) facilitate PDAC progression. However, little is known about whether TAMs are involved in regulating glycolysis in PDAC. Here, we found a positive correlation between CD68+ TAM infiltration and FDG maximal standardized uptake (FDG SUVmax) on PET-CT images of PDAC. We discovered that the glycolytic gene set was prominently enriched in the high TAM infiltration group through Gene Set Enrichment Analysis using The Cancer Genome Atlas database. Mechanistically, TAMs secreted IL-8 to promote GLUT3 expression in PDAC cells, enhancing tumor glycolysis both in vitro and in vivo, whereas this effect could be blocked by the IL-8 receptor inhibitor reparixin. Furthermore, IL-8 promoted the translocation of phosphorylated STAT3 into the nucleus to activate the GLUT3 promoter. Overall, we demonstrated that TAMs boosted PDAC cell glycolysis through the IL-8/STAT3/GLUT3 signaling pathway. Our cumulative findings suggest that the abrogation of TAM-induced tumor glycolysis by reparixin might exhibit an antitumor impact and offer a potential therapeutic target for PDAC.

Siglec10 Expression on Tumor-associated Macrophages Is an Independent Prognostic Factor in Stage I Lung Adenocarcinoma.

Prognostic indicators for postoperative lung adenocarcinoma are elusive. The interaction between CD24 on tumor cells and sialic-acid-binding Ig-like lectin 10 (Siglec10) on tumor-associated macrophages (TAMs) is implicated in immune evasion in distinct tumors. However, the therapeutic significance of phagocytic checkpoints in lung adenocarcinoma remains unknown. We aimed to investigate the clinical relevance and prognostic significance of phagocytosis checkpoints mediated by Siglec10 in TAMs of patients with lung adenocarcinoma who underwent curative resection. In this single-center retrospective study, we analyzed the data of 423 patients with stage I lung adenocarcinoma resected between 1999 and 2016. Tissue microarrays were constructed, and CD24, CD68, and Siglec10 immunohistochemistry was performed. Additionally, we assessed the clinical significance and prognostic associations of these markers. CD24 expression was higher in the Siglec10-high expression group than that in the -low expression group. Multivariate analysis showed that combined high Siglec10 and CD24 expression was an independent predictor of recurrence-free probability. The combined high Siglec10 and CD68 expression was a significant independent predictor of overall survival. Univariate analysis demonstrated that the 5-year probability of post-recurrence survival of patients with combined high Siglec10 and CD68 expression was lower than that of the other patients. High TAM Siglec10 expression and tumor CD24 expression are correlated, and the high Siglec10+CD24 combination is a major risk factor for recurrence. CD68+Siglec10 TAMs are important prognostic factors. Siglec10 expression on TAMs is essential for tumor microenvironment immunoregulation and offers a promising new immunotherapeutic approach for lung adenocarcinoma.

The prognostic effect of tumor-associated macrophages in stage I-III colorectal cancer depends on T cell infiltration.

Tumor-associated macrophages (TAMs) are associated with unfavorable patient prognosis in many cancer types. However, TAMs are a heterogeneous cell population and subsets have been shown to activate tumor-infiltrating T cells and confer a good patient prognosis. Data on the prognostic value of TAMs in colorectal cancer are conflicting. We investigated the prognostic effect of TAMs in relation to tumor-infiltrating T cells in colorectal cancers. The TAM markers CD68 and CD163 were analyzed by multiplex fluorescence immunohistochemistry and digital image analysis on tissue microarrays of 1720 primary colorectal cancers. TAM density in the tumor stroma was scored in relation to T cell density (stromal CD3+ and epithelial CD8+ cells) and analyzed in Cox proportional hazards models of 5-year relapse-free survival. Multivariable survival models included clinicopathological factors, MSI status and BRAFV600E mutation status. High TAM density was associated with a favorable 5-year relapse-free survival in a multivariable model of patients with stage I-III tumors (p = 0.004, hazard ratio 0.94, 95% confidence interval 0.90-0.98). However, the prognostic effect was dependent on tumoral T-cell density. High TAM density was associated with a good prognosis in patients who also had high T-cell levels in their tumors, while high TAM density was associated with poorer prognosis in patients with low T-cell levels (pinteraction = 0.0006). This prognostic heterogeneity was found for microsatellite stable tumors separately. This study supported a phenotypic heterogeneity of TAMs in colorectal cancer, and showed that combined tumor immunophenotyping of multiple immune cell types improved the prediction of patient prognosis.

Immune heterogeneity in small-cell lung cancer and vulnerability to immune checkpoint blockade

Atezolizumab (anti-PD-L1), combined with carboplatin and etoposide (CE), is now a standard of care for extensive-stage small-cell lung cancer (ES-SCLC). A clearer understanding of therapeutically relevant SCLC subsets could identify rational combination strategies and improve outcomes. We conduct transcriptomic analyses and non-negative matrix factorization on 271 pre-treatment patient tumor samples from IMpower133 and identify four subsets with general concordance to previously reported SCLC subtypes (SCLC-A, -N, -P, and -I). Deeper investigation into the immune heterogeneity uncovers two subsets with differing neuroendocrine (NE) versus non-neuroendocrine (non-NE) phenotypes, demonstrating immune cell infiltration hallmarks. The NE tumors with low tumor-associated macrophage (TAM) but high T-effector signals demonstrate longer overall survival with PD-L1 blockade and CE versus CE alone than non-NE tumors with high TAM and high T-effector signal. Our study offers a clinically relevant approach to discriminate SCLC patients likely benefitting most from immunotherapies and highlights the complex mechanisms underlying immunotherapy responses.

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.

Tumour-associated macrophages as a potential target to improve natural killer cell-based immunotherapies.

Adoptive transfer of natural killer (NK) cells has been proposed as a novel immunotherapy for malignant tumours resistant to current therapeutic modalities. Several clinical studies have demonstrated that the NK cell-infusion is well tolerated without severe side effects and shows promising results in haematological malignancies. However, patients with malignant solid tumours do not show significant responses to this therapy. Such disappointing results largely arise from the inefficient delivery of infused NK cells and the impairment of their functions in the tumour microenvironment (TME). Tumour-associated macrophages (TAMs) are the most abundant stromal cells in the TME of most solid tumours, and a high TAM density correlates with poor prognosis of cancer patients. Although our knowledge of the interactions between TAMs and NK cells is limited, many studies have indicated that TAMs suppress NK cell cytotoxicity against cancer cells. Therefore, blockade of TAM functions can be an attractive strategy to improve NK cell-based immunotherapies. On the other hand, macrophages are reported to activate NK cells under certain circumstances. This essay presents our current knowledge about mechanisms by which macrophages regulate NK cell functions and discusses possible therapeutic approaches to block macrophage-mediated NK cell suppression.

Expression of CD68+ Tumor associated macrophages in relation to β-catenin in carcinoma stomach.

With no unified system for tumor associated macrophages (TAMs) density assessment, limited information is available on their relationship with β-catenin expression. To evaluate the density of CD68+ TAMs in gastric adenocarcinoma samples by immunohistochemistry and correlate it with grade, stage, invasion, and beta-catenin. Formalin fixed paraffin embedded (FFPE) blocks from gastrectomy specimens of proven gastric adenocarcinoma were prospectively and retrospectively were studied over a period of two years. Immunohistochemistry with CD68 and β-catenin was performed. TAM density was qualitatively compared in "tumor" versus "stroma" and "tumor" versus "non-tumor" regions. Quantitative CD68+ TAM density was assessed using different methods and compared. Cases were classified as high and low TAM based on the median value and correlated with histologic type, location, grade, stage and β-catenin expression pattern. Spearman's rank correlation test was used to compare the different methods of TAM density evaluation. The categorical variables were studied using Pearson's Chi-square or Fisher's exact test. CD68+ TAM density and β-catenin expression were correlated by analysis of variance. A P value ≤ 0.05 was taken as statistically significant. The CD68+ TAMs in the "tumor" versus "non-tumor" area (p = 0.34) and "tumor" versus "stroma distribution" (p = 0.81) did not show any statistical significance. All methods of TAM density were found to be comparable. High TAM group is significantly associated with lymphovascular invasion, tumor depth, lymph node metastasis, and abnormal β-catenin expression. TAMs density plays an important role in the tumor stage. Macrophages may possibly induce gastric cancer invasiveness by activating β-catenin pathway.

Macrophage CD5L is a target for cancer immunotherapy

Reprogramming of immunosuppressive tumor-associated macrophages (TAMs) presents an attractive therapeutic strategy in cancer. The aim of this study was to explore the role of macrophage CD5L protein in TAM activity and assess its potential as a therapeutic target. Monoclonal antibodies (mAbs) against recombinant CD5L were raised by subcutaneous immunization of BALB/c mice. Peripheral blood monocytes were isolated from healthy donors and stimulated with IFN/LPS, IL4, IL10, and conditioned medium (CM) from different cancer cell lines in the presence of anti-CD5L mAb or controls. Subsequently, phenotypic markers, including CD5L, were quantified by flow cytometry, IF and RT-qPCR. Macrophage CD5L protein expression was studied in 55 human papillary lung adenocarcinoma (PAC) samples by IHC and IF. Anti-CD5L mAb and isotype control were administered intraperitoneally into a syngeneic Lewis Lung Carcinoma mouse model and tumor growth was measured. Tumor microenvironment (TME) changes were determined by flow cytometry, IHC, IF, Luminex, RNAseq and RT-qPCR. Cancer cell lines CM induced an immunosuppressive phenotype (increase in CD163, CD206, MERTK, VEGF and CD5L) in cultured macrophages. Accordingly, high TAM expression of CD5L in PAC was associated with poor patient outcome (Log-rank (Mantel-Cox) test p=0.02). We raised a new anti-CD5L mAb that blocked the immunosuppressive phenotype of macrophages invitro. Its administration invivo inhibited tumor progression of lung cancer by altering the intratumoral myeloid cell population profile and CD4+ T-cell exhaustion phenotype, thereby significantly modifying the TME and increasing the inflammatory milieu. CD5L protein plays a key function in modulating the activity of macrophages and their interactions within the TME, which supports its role as a therapeutic target in cancer immunotherapy. For a full list of funding bodies, please see the Acknowledgements.

Reversal of Lactate and PD-1-mediated Macrophage Immunosuppression Controls Growth of PTEN/p53-deficient Prostate Cancer.

Phosphatase and tensin homolog (PTEN) loss of function occurs in approximately 50% of patients with metastatic castrate-resistant prostate cancer (mCRPC), and is associated with poor prognosis and responsiveness to standard-of-care therapies and immune checkpoint inhibitors. While PTEN loss of function hyperactivates PI3K signaling, combinatorial PI3K/AKT pathway and androgen deprivation therapy (ADT) has demonstrated limited anticancer efficacy in clinical trials. Here, we aimed to elucidate mechanism(s) of resistance to ADT/PI3K-AKT axis blockade, and to develop rational combinatorial strategies to effectively treat this molecular subset of mCRPC. Prostate-specific PTEN/p53-deficient genetically engineered mice (GEM) with established 150-200 mm3 tumors, as assessed by ultrasound, were treated with either ADT (degarelix), PI3K inhibitor (copanlisib), or anti-PD-1 antibody (aPD-1), as single agents or their combinations, and tumors were monitored by MRI and harvested for immune, transcriptomic, and proteomic profiling, or ex vivo co-culture studies. Single-cell RNA sequencing on human mCRPC samples was performed using 10X Genomics platform. Coclinical trials in PTEN/p53-deficient GEM revealed that recruitment of PD-1-expressing tumor-associated macrophages (TAM) thwarts ADT/PI3Ki combination-induced tumor control. The addition of aPD-1 to ADT/PI3Ki combination led to TAM-dependent approximately 3-fold increase in anticancer responses. Mechanistically, decreased lactate production from PI3Ki-treated tumor cells suppressed histone lactylation within TAM, resulting in their anticancer phagocytic activation, which was augmented by ADT/aPD-1 treatment and abrogated by feedback activation of Wnt/β-catenin pathway. Single-cell RNA-sequencing analysis in mCRPC patient biopsy samples revealed a direct correlation between high glycolytic activity and TAM phagocytosis suppression. Immunometabolic strategies that reverse lactate and PD-1-mediated TAM immunosuppression, in combination with ADT, warrant further investigation in patients with PTEN-deficient mCRPC.

Osteoclast-like stromal giant cells in breast cancer likely belong to the spectrum of immunosuppressive tumor-associated macrophages.

Background: Breast cancer with osteoclast-like stromal giant cells (OSGC) is an exceedingly rare morphological pattern of invasive breast carcinoma. The tumor immune microenvironment (TIME) of these tumors is populated by OSGC, which resemble osteoclasts and show a histiocytic-like immunophenotype. Their role in breast cancer is unknown. The osteoclast maturation in the bone is regulated by the expression of cytokines that are also present in the TIME of tumors and in breast cancer tumor-associated macrophages (TAMs). TAMs-mediated anti-tumor immune pathways are regulated by miRNAs akin to osteoclast homeostasis. Here, we sought to characterize the different cellular compartments of breast cancers with OSGC and investigate the similarities of OSGC with tumor and TIME in terms of morphology, protein, and miRNA expression, specifically emphasizing on monocytic signatures. Methods and Results: Six breast cancers with OSGC were included. Tumor-infiltrating lymphocytes (TILs) and TAMs were separately quantified. The different cellular populations (i.e., normal epithelium, cancer cells, and OSGC) were isolated from tissue sections by laser-assisted microdissection. After RNA purification, 752 miRNAs were analyzed using a TaqMan Advanced miRNA Low-Density Array for all samples. Differentially expressed miRNAs were identified by computing the fold change (log2Ratio) using the Kolmogorov-Smirnov test and p values were corrected for multiple comparisons using the false discovery rate (FDR) approach. As a similarity analysis among samples, we used the Pearson test. The association between pairs of variables was investigated using Fisher exact test. Classical and non-classical monocyte miRNA signatures were finally applied. All OSGC displayed CD68 expression, TILs (range, 45–85%) and high TAMs (range, 35–75%). Regarding the global miRNAs profile, OSGC was more similar to cancer cells than to non-neoplastic ones. Shared deregulation of miR-143-3p, miR-195-5p, miR-181a-5p, and miR-181b-5p was observed between OSGC and cancer cells. The monocyte-associated miR-29a-3p and miR-21-3p were dysregulated in OSGCs compared with non-neoplastic or breast cancer tissues. Conclusion: Breast cancers with OSGC have an activated TIME. Shared epigenetic events occur during the ontogenesis of breast cancer cells and OSGC but the innumophenotype and miRNA profiles of the different cellular compartmens suggest that OSGC likely belong to the spectrum of M2 TAMs.

Tumor-associated macrophages related signature in glioma

Background: Glioma is the most common malignant primary tumor with a poor prognosis. Infiltration of tumor-associated macrophages (TAMs) is a hallmark of glioma. However, the regulatory mechanism of TAMs and the prognostic value of related signature in glioma remain unclear.Methods: TAMs were analyzed by EPIC, MCPCOUNTER and XCELL methods in multiple cohorts, including the TCGA merged GBMLGG, CGGA mRNAseq-325, and CGGA mRNAseq-693. Weighted correlation network analysis (WGCNA) were performed to identify candidate hub genes that might be related to TAMs. The prognostic genes were selected by Univariate Cox regression, Kaplan-Meier analysis and the least absolute shrinkage and selection operator (LASSO) multivariate Cox regression algorithm, and were used to construct a high efficacy prediction model.Results: Compared with LGG, TAMs of GBM in the TCGA merged GBMLGG, CGGA mRNAseq-693, and CGGA mRNAseq-325 cohorts were increased, and high TAMs levels predicted poorer overall survival for gliomas. The prediction model constructed by nine prognostic genes was highly efficient. The TAMs related risk-score was an independent risk factor for glioma. Moreover, high risk score was correlated with an increased population of TAMs in glioma, as well as the high immune scores, stromal scores and ESTIMATE scores.Conclusions: Increased TAMs might be an immune evasion mechanism of glioma. In addition, our findings suggested that TAMs-related signature was a valuable prognostic biomarker in glioma and provided therapeutic targets for glioma.

Abstract P1-04-07: Xiap expression is associated with infiltration of cd163+ tumor-associated macrophages in the tumor micro-environment of inflammatory breast cancer

Abstract BACKGROUND: Inflammatory breast cancer (IBC) is a rare, but aggressive type of locally advanced breast cancer for which a specific immune response seems to be an important driver (Van Laere et al., CCR 2013). The tumor immune micro-environment (TIME) of IBC is characterized by an influx of tumor-associated macrophages (TAMs) and patients with spatial colocalization of TAMs with tumor cells seem to have a worse prognosis (Van Berckelaer et al., SABCS 2019). In contrast with TAMs, the presence of CD8+ T cells was associated with a better prognosis in IBC. Cytotoxic T cells mediate killing of the tumor by inducing apoptosis after activation of effector caspases-3/-7, regulated by the X-Linked Inhibitor of Apoptosis protein (XIAP). Recently, we reported that XIAP in breast cancer is associated with shorter survival and resistance to chemotherapy (Devi et al, Cancers 2021). In IBC, XIAP is translationally upregulated during cellular stress and induces, via NFkB, an immunosuppressive signaling (Evan et al, Cancer Res 2018). Therefore, we investigated the interplay between tumor cell driven XIAP expression and the TIME. METHODOLOGY: IBC patient clinicopathological variables (n=75) along with Affymetrix data (n=30/75) were collected. Immunostainings for XIAP, CD8 and CD163 (Hematoxylin-DAB) were done according to validated protocols. All slides were digitized and evaluated in Visiopharm to quantify the number of DAB+ immune cells and the relative marker area (RMA: the DAB+ area/total area of interest). Spatial interactions were examined using an effector index (EI) that counts the number of immune cells in a circle with a radius of 30 mcm around a CD8+ T cell or a tumor cell. We evaluated correlations between XIAP protein and mRNA expression, immune cell signatures (using CIBERSORT) &amp; gene-expression pathways as a validation of our IHC findings. RESULTS: High XIAP expression was detected in 45.3% of the IBC samples (n= 34/75). Although, XIAP expression was not associated with any clinicopathological parameters like grade, molecular subtype, or disease stage. High XIAP expressing tumors had more sTIL infiltration (P= 0.008), PDL1 expression (P= 0.012) and infiltration with TAMs (RMA: 1.03% (0.03% – 5.97%) vs. 2.73% (0.01% – 11.3%), P= 0.003). Furthermore, in these XIAP high samples TAMs were also located closer to the tumor cells (EI: 0.95 cells (0.06 – 3.48) vs. 2.04 cells (0.28 – 5.20), P&amp;lt; 0.001) and the CD8+ T cells (EI: 2.85 cells (0.26 – 8.85) vs. 5.13 cells (0.87 – 10.16), P&amp;lt; 0.001), while there was no correlation between XIAP and the total number of CD8+ cells. Although gene expression data analysis did not reveal any correlation between protein and mRNA expression of XIAP, there was a significant correlation between XIAP activation signature and protein expression (P&amp;lt;0.001). Furthermore, we discovered a strong association between XIAP protein and cellular stress response- &amp; inflammation-related gene sets. Finally, in the CIBERSORT data, we observed the strongest correlation between XIAP expression and M2 macrophages &amp; activated dendritic cells. CONCLUSION: This is the first study showing high XIAP expressing IBC tumors have increased infiltration of immunosuppressive TAMs, which co-localized near tumor and cytotoxic T cells. We also demonstrated an association between XIAP protein expression and inflammation related &amp; cellular stress response gene signatures. This is relevant as XIAP function depends on translational upregulation during cellular stress which leads to immunosuppressive signaling and an aggressive breast cancer phenotype. XIAP antagonists are in clinical trials and these data suggest potential benefit in targeting XIAP to enhance immunotherapeutic efficacy in IBC therapy. FUNDING: DoDs-W81XWH-17-1-0297,W81XWH-20-1-0153, ACS Mission Boost Grants(GRD) Citation Format: Christophe Van Berckelaer, Steven Van Laere, Joseph Gerardts, Luc Dirix, Michael Morse, Mark Kockx, François Bertucci, Peter Van Dam, Gayathri R Devi. Xiap expression is associated with infiltration of cd163+ tumor-associated macrophages in the tumor micro-environment of inflammatory breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-07.

“THE EFFECT OF GROWTH DIFFERENTIATION FACTOR 11 (GDF11) ON THE RESPONSE OF TUMOR-ASSOCIATED MACROPHAGES IN HEPATOCELLULAR CARCINOMA DERIVED CELLS”

Hepatocellular carcinoma (HCC) is the most common and aggressive form of liver cancer. Until 2020, worldwide, it has been ranked 5 th and 3 rd in terms of incidence and mortality, respectively. HCC has various etiologies, but tumors formed by high cholesterol intake induce an aggressive phenotype and high tumor-associated macrophages (TAM) infiltration. TAM acquires an alternative polarity (M2) with immunosuppressive and pro-tumoral activities. HCC patients with high TAM recruitment have a poor prognosis with a low free-survival rate. The design of therapies represents a challenge. The growth differentiation factor 11 (GDF11) has been proposed as a promising molecule for treating HCC, favors a reduction in proliferation, invasion, and lipid metabolism, and therefore reduces the aggressive phenotype. In our research group, we have found that GDF11 also has various effects on cells belonging to the tumor microenvironment, particularly in TAM. To evaluate TAM polarity and their intercommunication with HCC-derived cells during treatment with GDF11. THP-1-macrophages cell line was used; lipopolysaccharides were used for the M1 polarity acquisition and IL-4/13 or conditioned medium (CM) derived from Huh-7 cells for M2 polarity acquisition, flow cytometry (FCM) was used. GDF11 (50 ng/mL) was applied every 24 h up to 3 days. We used anti-Smad2 and 3 antibodies in Western blot (WB). Cell viability and proliferation studies were performed using crystal violet. The quantification of cholesterol was performed using o-Phthaldialdehyde and free radicals’ detection was performed using dihydroethidium. Data were presented as mean ± standard error of the mean (SEM). The analysis of variance (ANOVA) test was used to compare the mean values ​​between groups. Each result has at least 3 independent experiments. Statistical significance was indicated with an asterisk (*P<0.05). GDF11 treatments induced Smad proteins activation by specific phosphorylation, indicating that THP-1 macrophages respond to treatment in times from 5 to 60 min, but maintaining their response up to 72 h. This could suggest that the difference in the subsequent response may be involved in the binding or activation of additional adapter proteins of this signaling pathway. Furthermore, it was observed that GDF11 does not affect the cell viability, proliferation, and morphology studied in their different polarities. Macrophage re-education or re-polarization assays were carried out by adding GDF11 for an additional 72h. The data obtained show that TAM populations lost CD206 marker, going from 90% to 40% of the population, suggesting a loss of this pro-tumor polarity and a re-education towards anti-tumor macrophages responses. However, experiments show a dual role of GDF11 in macrophages due to the increase of specific markers in non-activated cells, indicating that it has a different effect depending on the activation state. Since GDF11 compromises cholesterol metabolism, which is observed in a decrease in total cholesterol levels in non-activated macrophages, which is observed in other studies using HCC cell lines. Also, it was possible to corroborate that GDF11 is behaving like a statin used as a positive control in macrophages and Huh-7 cells. Finally, GDF11 increases ROS levels, specifically superoxide anion, characteristic of phagocytic M1 macrophages. To evaluate response on HCC cells, wound-healing assays indicated that macrophage secretion is a key factor for cell displacement rather than viability and proliferation. In the present study, we found that GDF11 has immunomodulatory effects in TAM, decreasing pro-tumoral markers and lipid content that could be important to design HCC therapies. The authors declare that there is no conflict of interest.

DMF Activates NRF2 to Inhibit the Pro-Invasion Ability of TAMs in Breast Cancer.

Tumor-associated macrophages (TAMs) account for more than 50% of the cells in the tumor immune microenvironment of patients with breast cancer. A high TAM density is associated with a poor clinical prognosis. Targeting TAMs is a promising therapeutic strategy because they promote tumor growth, development, and metastasis. In this study, we found that dimethyl formamide (DMF) significantly inhibited the tumor invasion-promoting ability of TAMs in the co-culture system and further showed that DMF functioned by reducing reactive oxygen species (ROS) production in TAMs. The orthotopic 4T1 cell inoculation model and the spontaneous mouse mammary tumor virus-polyoma middle tumor-antigen tumor model were used to evaluate the antitumor effect of DMF. The results showed that DMF significantly inhibited tumor metastasis and increased T-cell infiltration into the tumor microenvironment. Mechanistically, NRF2 activation was necessary for DMF to exert its function, and DMF can play a role in breast cancer as an anticancer drug targeting TAMs.

TGR5 deficiency activates antitumor immunity in non-small cell lung cancer via restraining M2 macrophage polarization

The bile acid-responsive G-protein-coupled receptor TGR5 is expressed in monocytes and macrophages, and plays a critical role in regulating inflammatory response. Our previous work has shown its role in promoting the progression of non-small cell lung cancer (NSCLC), yet the mechanism remains unclear. Here, using Tgr5-knockout mice, we show that TGR5 is required for M2 polarization of tumor-associated macrophages (TAMs) and suppresses antitumor immunity in NSCLC via involving TAMs-mediated CD8+ T cell suppression. Mechanistically, we demonstrate that TGR5 promotes TAMs into protumorigenic M2-like phenotypes via activating cAMP-STAT3/STAT6 signaling. Induction of cAMP production restores M2-like phenotypes in TGR5-deficient macrophages. In NSCLC tissues from human patients, the expression of TGR5 is associated with the infiltration of TAMs. The co-expression of TGR5 and high TAMs infiltration are associated with the prognosis and overall survival of NSCLC patients. Together, this study provides molecular mechanisms for the protumor function of TGR5 in NSCLC, highlighting its potential as a target for TAMs-centric immunotherapy in NSCLC.