High Tumor-associated Macrophages Research Articles

Tumor-Associated Macrophages as Multifaceted Regulators of Breast Tumor Growth.

Breast cancer is the most commonly occurring cancer in women of Western countries and is the leading cause of cancer-related mortality. The breast tumor microenvironment contains immune cells, fibroblasts, adipocytes, mesenchymal stem cells, and extracellular matrix. Among these cells, macrophages or tumor-associated macrophages (TAMs) are the major components of the breast cancer microenvironment. TAMs facilitate metastasis of the breast tumor and are responsible for poor clinical outcomes. High TAM density was also found liable for the poor prognosis of breast cancer. These observations make altering TAM function a potential therapeutic target to treat breast cancer. The present review summarizes the origin of TAMs, mechanisms of macrophage recruitment and polarization in the tumor, and the contributions of TAMs in tumor progression. We have also discussed our current knowledge about TAM-targeted therapies and the roles of miRNAs and exosomes in re-educating TAM function.

Infiltration by CXCL10 Secreting Macrophages Is Associated With Antitumor Immunity and Response to Therapy in Ovarian Cancer Subtypes.

Ovarian carcinomas (OCs) are poorly immunogenic and immune checkpoint inhibitors (ICIs) have offered a modest benefit. In this study, high CD3+ T-cells and CD163+ tumor-associated macrophages (TAMs) densities identify a subgroup of immune infiltrated high-grade serous carcinomas (HGSCs) with better outcomes and superior response to platinum-based therapies. On the contrary, in most clear cell carcinomas (CCCs) showing poor prognosis and refractory to platinum, a high TAM density is associated with low T cell frequency. Immune infiltrated HGSC are characterized by the 30-genes signature (OC-IS30) covering immune activation and IFNγ polarization and predicting good prognosis (n = 312, TCGA). Immune infiltrated HGSC contain CXCL10 producing M1-type TAM (IRF1+pSTAT1Y701+) in close proximity to T-cells. A fraction of these M1-type TAM also co-expresses TREM2. M1-polarized TAM were barely detectable in T-cell poor CCC, but identifiable across various immunogenic human cancers. Single cell RNA sequencing data confirm the existence of a tumor-infiltrating CXCL10+IRF1+STAT1+ M1-type TAM overexpressing antigen processing and presentation gene programs. Overall, this study highlights the clinical relevance of the CXCL10+IRF1+STAT1+ macrophage subset as biomarker for intratumoral T-cell activation and therefore offers a new tool to select patients more likely to respond to T-cell or macrophage-targeted immunotherapies.

A Nanoradiomics Approach for Differentiation of Tumors Based on Tumor-Associated Macrophage Burden.

Objective Tumor-associated macrophages (TAMs) within the tumor immune microenvironment (TiME) of solid tumors play an important role in treatment resistance and disease recurrence. The purpose of this study was to investigate if nanoradiomics (radiomic analysis of nanoparticle contrast-enhanced images) can differentiate tumors based on TAM burden. Materials and Methods In vivo studies were performed in transgenic mouse models of neuroblastoma with low (N = 11) and high (N = 10) tumor-associated macrophage (TAM) burden. Animals underwent delayed nanoparticle contrast-enhanced CT (n-CECT) imaging at 4 days after intravenous administration of liposomal-iodine agent (1.1 g/kg). CT imaging-derived conventional tumor metrics (tumor volume and CT attenuation) were computed for segmented tumor CT datasets. Nanoradiomic analysis was performed using a PyRadiomics workflow implemented in the quantitative image feature pipeline (QIFP) server containing 900 radiomic features (RFs). RF selection was performed under supervised machine learning using a nonparametric neighborhood component method. A 5-fold validation was performed using a set of linear and nonlinear classifiers for group separation. Statistical analysis was performed using the Kruskal–Wallis test. Results N-CECT imaging demonstrated heterogeneous patterns of signal enhancement in low and high TAM tumors. CT imaging-derived conventional tumor metrics showed no significant differences (p > 0.05) in tumor volume between low and high TAM tumors. Tumor CT attenuation was not significantly different (p > 0.05) between low and high TAM tumors. Machine learning-augmented nanoradiomic analysis revealed two RFs that differentiated (p < 0.002) low TAM and high TAM tumors. The RFs were used to build a linear classifier that demonstrated very high accuracy and further confirmed by 5-fold cross-validation. Conclusions Imaging-derived conventional tumor metrics were unable to differentiate tumors with varying TAM burden; however, nanoradiomic analysis revealed texture differences and enabled differentiation of low and high TAM tumors.

PD-1-Positive Tumor-Associated Macrophages Define Poor Clinical Outcomes in Patients With Muscle Invasive Bladder Cancer Through Potential CD68/PD-1 Complex Interactions.

Tumor-associated macrophages (TAMs) regulate tumor immunity. Previous studies have shown that the programmed cell death protein 1 (PD-1)-positive TAMs have an M2 macrophage phenotype. CD68 is a biomarker of TAMs and is considered to be a poor prognostic marker of several malignancies. Our results show that PD-1-positive TAMs can be a negative survival indicator in patients with muscle-invasive bladder cancer (MIBC), and that the mechanistic effects could result due to a combination of PD-1 and CD68 activity. We analyzed 22 immune cell types using data from 402 patients with MIBC from the TCGA database, and found that a high immune score and M2 TAMs were strongly associated with poor clinical outcomes in patients with MIBC. Further, we analyzed resected samples from 120 patients with MIBC and found that individuals with PD-1-positive TAMs showed a reduction in 5-year overall survival and disease-free survival. Additionally, PD-1-positive TAMs showed a significant association with higher programmed death-ligand 1 (PD-L1) expression, the Ki67 index, the pT stage and fewer CD8-positive T cells. Through the co-immunoprecipitation (co-IP) assay of THP-1 derived macrophages, we found that CD68 can bind to PD-1. The binding of CD68 and PD-1 can induce M2 polarization of THP-1 derived macrophages and promote cancer growth. The anti-CD68 treatment combined with peripheral blood mononuclear cells (PBMC) showed obvious synergy effects on inhibiting the proliferation of T24 cells. Together, these results indicate for the first time that CD68/PD-1 may be a novel target for the prognosis of patients with MIBC.

Prospective Analyses of Lifestyle Factors Related to Energy Balance and Ovarian Cancer Risk by Infiltration of Tumor-Associated Macrophages.

Lifestyle factors related to energy balance have been associated with ovarian cancer risk and influence the tumor immune microenvironment, including tumor-associated macrophages (TAM). However, no studies have assessed whether these factors differentially impact ovarian cancer risk by TAM densities. We conducted a prospective analysis in the Nurses' Health Studies to examine the associations of physical activity, sitting time, and a food-based empirical dietary inflammatory pattern (EDIP) score with invasive epithelial ovarian cancer risk by TAM density assessed by immunohistochemistry. We considered density of CD68 (marker of total TAMs) and CD163 (marker of pro-carcinogenic M2-type TAMs), and their ratios. We used multivariable Cox proportional hazards regression to calculate hazard ratios (HR) and 95% confidence intervals (CI) of exposures with risk of ovarian tumors with high versus low TAMs, including analyses stratified by body mass index. Analyses included 312 incident ovarian cancer cases with TAM measurements. Physical activity, sitting time, and EDIP score were not differentially associated with ovarian cancer risk by TAM densities (P heterogeneity > 0.05). Among overweight and obese women, higher EDIP score was associated with increased risk of CD163 low-density tumors (HR comparing extreme tertiles, 1.57; 95% CI, 0.88-2.80; P trend = 0.01), but not CD163 high-density tumors (comparable HR, 1.16; 95% CI, 0.73-1.86; P trend = 0.24), though this difference was not statistically significant (P heterogeneity = 0.22). We did not observe differential associations between lifestyle factors and ovarian cancer risk by TAM densities. Future investigations examining the interplay between other ovarian cancer risk factors and the tumor immune microenvironment may help provide insight into ovarian cancer etiology.

Tumor Cells Interleukin-22 Expression Associates with Elevated Tumor-Associated Macrophages Infiltrating and Poor Prognosis in Patients with Breast Cancer.

Interleukin-22 (IL-22), secreted by tumor infiltrated lymphocytes, is identified as a tumor-promoting factor in certain cancers, which was secreted by tumor infiltrated lymphocytes. However, the role of IL-22 in breast cancer remains conflicting. In this study, we assessed the expression of IL-22, IL-22 receptor 1 (IL-22R1), CD4, CD8, FOXP3, and CD68 in breast cancer by immunohistochemistry. IL-22 expression was exhibited in 105 (69.1%) cases in tumor cells (tIL-22), whereas only 24 (15.8%) samples displayed IL-22 expression in stromal cells. Multivariate analysis showed that tIL-22 expression was a poor prognostic factor for overall survival (OS) (p = 0.04). Meanwhile, IL-22R1 was predominantly presented in tumor cells (84.9%), which was associated with tIL-22 expression. The CD68-positive tumor-associated macrophages (TAMs) displayed the highest infiltration rate (50.7%) compared with CD4-, CD8-, and FOXP3-positive cells. Kaplan-Meier analysis confirmed patients with high TAM infiltration displayed significantly worse relapse-free survival (RFS) compared with low TAMs group (p = 0.017). TAM infiltration was also positively associated with tIL-22 and IL-22R1 expression. Furthermore, tIL-22 expression together with high TAM infiltration displayed the worst prognosis outcomes both in OS (p = 0.039) and RFS (p = 0.008). Instead of lymphocytes, our data indicated that tumor cells express IL-22 in breast cancer that is associated with IL-22R1, high TAM infiltrating, and poor prognosis.

Abstract A19: Aspirin use and ovarian cancer risk by lymphocyte infiltration

Abstract Introduction: There is growing evidence to support a modest inverse association between daily aspirin use and ovarian cancer risk. We previously hypothesized that this association may differ by immune cell infiltration. In our prior study evaluating heterogeneity by tumor-associated macrophage (TAM) infiltration, aspirin use was associated with lower risk of ovarian cancer with high protumorigenic M2-type TAM infiltration, but not low M2-type TAM infiltration. The goal of the present study was to evaluate the hypothesis that the association between aspirin use and ovarian cancer risk also differs by infiltration with tumor-infiltrating lymphocytes (TILs). Methods: We included cases and matched controls from the Nurses’ Health Study (NHS), NHSII, and New England Case Control Study. Study participants self-reported aspirin use including regular use (≥1 x per week), duration of use, and number of tablets. A gynecologic pathologist (JLH), who was blinded to exposure status, reviewed hematoxylin and eosin stained tumor slides to quantify TILs for all cases (n=547). We used polytomous logistic regression, adjusted for ovarian cancer risk factors, to estimate odds ratios (OR) for aspirin use and ovarian cancer risk by TIL infiltration. Results: Overall, recent aspirin use was associated with lower risk of ovarian cancer compared with no regular aspirin use (OR=0.65, 95%CI=0.47-0.89). Recent aspirin use was associated with a lower risk of ovarian cancer exhibiting TIL infiltration (OR=0.38, 95%CI=0.26-0.56) but not with TIL-negative cancers (OR=1.00, 95%CI=0.71-1.41; p-heterogeneity &amp;lt;0.001). We observed similar results for aspirin duration and tablets. For duration, comparing ≥10 years of aspirin use to &amp;lt;1 year we observed a significantly lower risk of ovarian cancer with TILs (OR=0.47, 95%CI=0.41-0.71; p-trend=0.004), but a nonsignificant reduction for ovarian cancer without TILs (OR=0.86, 95%CI=0.59-1.26; p-trend=0.72; p-heterogeneity=0.03). Comparing ≥6 tablets to &amp;lt;1 tablet per week, there was evidence of a lower risk of ovarian cancer with TILs (OR=0.47, 95%CI=0.30-0.75; p-trend=0.001) but not without TILs (OR=1.02, 95%CI=0.70-1.48; p-trend=0.84; p-heterogeneity=0.003). Conclusions: Our results suggest that prediagnosis aspirin use may be an important modulator of immune cell infiltration in ovarian tumors, supporting an immunogenic mechanism for aspirin in ovarian carcinogenesis. Further work is under way to evaluate nonaspirin nonsteroidal anti-inflammatory drugs, and to consider which subsets of TAMs and TILs may be driving heterogeneity in the associations between anti-inflammatory drugs and ovarian cancer risk. Citation Format: Mollie E. Barnard, Britton Trabert, Jonathan L. Hecht, Ellen L. Goode, Naoko Sasamoto, Kathryn L. Terry, Shelley S. Tworoger. Aspirin use and ovarian cancer risk by lymphocyte infiltration [abstract]. In: Proceedings of the AACR Special Conference on Modernizing Population Sciences in the Digital Age; 2019 Feb 19-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(9 Suppl):Abstract nr A19.

Prognosis of Macrophage Density in the Absence of Neutrophils in Differentiated Thyroid Cancer

BackgroundDespite the advances in treatment of differentiated thyroid cancer (DTC), predicting prognosis remains a challenge. Immune cells in the tumor microenvironment may provide an insight to predicting recurrence. Therefore, the objective of this study was to investigate the association of tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) with recurrence in DTC and to identify serum cytokines that correlate with the presence of these immune cells in the tumor. Materials and methodsForty-two DTC tissues from our institutional neoplasia repository were stained for immunohistochemistry markers for TAMs and TANs. In addition, cytokine levels were analyzed from these patients from preoperative blood samples. TAM and TAN staining were compared with clinical data and serum cytokine levels. ResultsNeither TAM nor TAN scores alone correlated with tumor size, the presence of lymph node metastases, multifocal tumors, lymphovascular or capsular invasion, or the presence of BRAFV600E mutation (all P > 0.05). There was no association with recurrence-free survival (RFS) in TAN density (mean RFS, 169.1 versus 148.1 mo, P = 0.23) or TAM density alone (mean RFS, 121.3 versus 205.2 mo, P = 0.54). However, when scoring from both markers were combined, patients with high TAM density and TAN negative scores had significantly lower RFS (mean RFS, 50.7 versus 187.3 mo, P = 0.04) compared with the remaining cohort. Patients with high TAM/negative TAN tumors had significantly lower serum levels of interleukin 12p70, interleukin 8, tumor necrosis factor alpha, and tumor necrosis factor beta. ConclusionsIn DTCs, high density of TAMs in the absence of TANs is associated with worse outcome. Assessment of multiple immune cell types and serum cytokines may predict outcomes in DTC.

Tumor-Associated Macrophage Status in Cancer Treatment.

Tumor-associated macrophages (TAMs) represent the most abundant innate immune cells in tumors. TAMs, exhibiting anti-inflammatory phenotype, are key players in cancer progression, metastasis and resistance to therapy. A high TAM infiltration is generally associated with poor prognosis, but macrophages are highly plastic cells that can adopt either proinflammatory/antitumor or anti-inflammatory/protumor features in response to tumor microenvironment stimuli. In the context of cancer therapy, many anticancer therapeutics, apart from their direct effect on tumor cells, display different effects on TAM activation status and density. In this review, we aim to evaluate the indirect effects of anticancer therapies in the modulation of TAM phenotypes and pro/antitumor activity.

Abstract D063: Intratumoral macrophage analysis and characterization of HER2+ pretreatment breast tumor biopsy using multiplex immunofluorescence

Abstract Background: Latinas in the US experience overall worse breast cancer (BC) survival than non-Latinas. Higher rates of aggressive BC subtypes including estrogen receptor negative(ER-) and HER2+ in Latinas are partly suspected. High intra-tumoral immune cells including myeloid suppressive M2 type macrophages (MAC) that suppress tumor immune responses are positively associated with poor outcomes and low response to therapy. Unclear is if tumor associated immune cells differ by race/ethnicity. Objective: To develop and evaluate the performance of a multiplex immunofluorescence (IF) methods to classify tumor-associated macrophages (TAM) as M1 and M2 in HER2+ breast tumors. Methods: Two panels of three antibodies were studied in HER2+ breast cancer biopsy specimens (n=19 patients). Two myeloid panels targeting CD80, CD68, CD163, CD206 with a nuclear (DAPI) and tumor cell (cytokeratin 8/18) was investigated. Banked breast tumor tissues were deparaffinized, and stained using indirect multiplex IF. Each tissue underwent multiple staining for CD206, CD68, and CD163 with stripping. A tyramide signal amplification IF (Perkin Elmer) was used in fluorescent labeling. Whole slide analysis image analysis was performed using HALO image analysis software. Results: All biopsy tissues yielded high quality IF profiles. Analyses per patient tumor (i.e., 5 regions of high tumor cell density containing a minimum of 5000 nucleated cells) revealed a fairly homogeneous MAC profile. In contrast, significant between patient heterogeneity in intratumoral M2 MAC density and differences by ER status was observed. In this sample, Her2+ BCs contained a high intratumoral TAM content – i.e., mean 27% (range &amp;lt;1% to 70%) of cells in tumor dense regions were positive for the pan MAC marker CD68+. Further, intratumoral CD68+ cells (all MAC) were slightly higher in abundance in ER-/HER2+ tumors at 31% compared to 20% in ER+/HER2+ tumors. Further, the relative abundance of CD68+/CD206+ cells (M2 type) in tumor rich regions showed a high degree of variability between cases with a mean 7.1% of total cells (range 0 to 48%). Notable, the relative abundance of double CD68/CD206 cells was higher in the ER+ tumors at 8.8% (range 0 to 48%) compared to 3.3% in ER- that showed a narrow range of 0 to 8%. A similar pattern was observed for the CD68/CD163 double positive M2 type that included large heterogeneity between patients and higher abundance in ER+/HER2+ tumors. In contrast, while exhibiting high heterogeneity between patients, the triple positive CD68, CD163, CD206 cells population appeared to be proportionally higher in ER-/HER2 tumors. Ongoing work includes characterization of the M1 type MAC and their relative abundance in HER2+ tumors by ER status. Conclusion: These results demonstrate the feasibility of multiplex IF to characterize TAM subsets in patient derived BC tissues. Future efforts will include relating TAM to HER2 targeted therapy response rates and examination of differences in TAM profiles in patient populations by race/ethnicity. Citation Format: Emmaly D Gutierrez, Christina Preece, Alison Stopeck, Patricia Thompson. Intratumoral macrophage analysis and characterization of HER2+ pretreatment breast tumor biopsy using multiplex immunofluorescence [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D063.

A novel CSF-1R neutralizing antibody, DCB-AB21, as a potential therapeutics for tumors.

e15006 Background: Tumor-associated macrophages (TAMs) represent the major cell components of immune infiltrate in solid tumors, and it has been reported that high TAMs density is associated with poor clinical prognosis. Within the tumor microenvironment, TAMs rely on signaling through colony stimulating factor 1 receptor (CSF-1R)/CSF-1 axis for various tumor promoting processes such as tumor growth, drug resistance, angiogenesis, and metastasis. Therefore, decrease of TAMs density in tumors through CSF-1R inhibition is considered an attractive therapeutic target for drug development. Methods: DCB-AB21 was screened from immunized mouse hybridoma clones. ELISA-based and flow cytometry-based affinity screening is performed and then the growth inhibition test by cell-based CSF-1-dependent cell line were further used as antibody screening assay. Results: A novel anti-CSF-1R monoclonal antibody DCB-AB21 which specifically binds to CSF-1R and inhibits CSF-1R downstream signaling. Our results showed DCB-AB21 can block the CSF-1 and IL-34-induced cell proliferation. Potent inhibition of CSF-1R-dependent signaling was observed in the Ba/F3 CSF-1R driven model cells, and in human THP-1 leukemia monocytic cell lines. Humanization engineering further improves this antibody’s affinity and anti-proliferative efficacy. The discovery of novel binding epitope of DCB-AB21 differentiates itself from the current CSF-1R targeting antibodies testing in clinical trials. In vivo efficacy of DCB-AB21 showed AB21 alone has moderate anti-cancer activity and it further potentiate anti-PD-1’s efficacy. Conclusions: These data demonstrate that DCB-AB21 can potently inhibit CSF-1R in cellular contexts and in vivo model, and has the potential to induce a phenotypic effect on macrophages. These data suggest that DCB-AB21 is a promising new agent with possibility to combine with immune checkpoint inhibitors to relief macrophage-dependent immune suppression and would yield clinical benefit.

Tumor-Infiltrating Neutrophils Predict Poor Survival of Non-Functional Pancreatic Neuroendocrine Tumor.

This study retrospectively characterized the immune infiltrating profile in nonfunctional pancreatic neuroendocrine tumors (NF-PanNETs). Tumor tissues from the 109-patient Fudan cohort and a 73-patient external validation set were evaluated by immunohistochemistry for 9 immune cell types: tumor-infiltrating neutrophils (TINs), tumor-associated macrophages (TAMs), CD11c+ dendritic cells, anti-NCR1+ natural killer (NK) cells, CD4+ and CD8+ T cells, CD45RO+ memory T cells, FOXP3+ regulatory T cells (Tregs), and CD20+ B cells. TINs were primarily distributed in the intratumoral area, dendritic cells and NK cells were scattered evenly in intratumoral and stromal areas, and Tregs were rarely detected. The remaining 5 cell types were primarily present in peritumoral stroma. Total TINs (P < .001) and TAMs (P = .002) increased as NF-PanNET grade rose. Kaplan-Meier analyses showed that high intratumoral TINs, total TAMs, and stromal CD4+ T-cell infiltration correlated with shorter recurrence-free survival (RFS, P = .010, P = .027, and P = .035, respectively) and overall survival (OS, P = .017, P = .029, and P = .045, respectively). Additionally, high intratumoral CD8+ T cell infiltration correlated with prolonged RFS (P = .039). Multivariate Cox regression demonstrated that intratumoral TINs, World Health Organization (WHO) classification, and eighth edition of the American Joint Committee on Cancer tumor-node-metastasis staging system (AJCC8th TNM) were independent factors for RFS (P = .043, P = .023, and P = .029, respectively), whereas intratumoral TINs and WHO classification were independent factors for OS (P = .010 and P = .007, respectively). Furthermore, the combination of TINs, WHO classification, and AJCC8th TNM remarkably improved prognostic accuracy for RFS. These results have been verified in the external validation set. Intratumoral TINs are an independent and unfavorable predictor of postoperative NF-PanNETs. A combination of TINs, WHO classification, and AJCC8th TNM could improve prognostic accuracy for RFS.

Abstract B53: Immunomodulatory effect of prostaglandin dehydrogenase in metastatic colon cancer

Abstract Cyclooxygenase-2, the key player in the biosynthesis of prostaglandin-E2 (PGE2) is often upregulated in colon cancer (CC). TNF-α, the main regulator of COX-2 and mPGES-1, contributes to the increased synthesis of PGE2, which is inhibited by overexpressed prostaglandin dehydrogenase (15-PGDH), a tumor suppressor playing major role in PGE2 catabolism but often downregulated in CC. Interestingly, CC patients expressing high levels of cysteinyl leukotriene receptor-2 (CysLT2R) have good prognosis and therefore, we investigated a potential link between CysLT2R-signaling and 15-PGDH in metastatic CC. Elevation of 15-PGDH expression by LTC4, a CysLT2R ligand, exhibited anti-tumor activity in colon cancer cells with significant phosphorylation of β-catenin with concurrent activation of CASPASE-3 and dramatic down-regulation of TNF-α mRNA expression in two different colon cancer cells. Colony-stimulating factor 1 (CSF-1) is a key regulator of macrophage differentiation that supports the protumorigenic role of tumor-associated macrophages (TAMs). We observed high CSF-1 in metastatic colon cancer with low CysLT2R expression, which is correlated with high level of CD-8+ T-cells and CD-163+ TAMs. CC cell lines produced high amount of CSF-1 when co-cultured with CC-specific CD-8+ T-cell or exposed to T-cell derived cytokines in vitro and exhibited significant reduction with LTC4 stimulation or 15-PGDH overexpression. Furthermore, longer use of 15-PGDH led to downregulation in the PD-L1 in a time-dependent manner. Combination of anti-CSF-1, anti-PD-1 and LTC4 exhibited significant reduction in CC tumor rich in macrophages. Blockade of PD-1/PD-L1 axis also facilitated upregulation of IFN-γ in γδ-T cells. Hence restoration of 15-PGDH expression through CysLT2R-signaling promotes immunomodulatory effect, indicating a promising therapeutic alternative for metastatic CC with high TAM infiltration. Citation Format: Shakti R. Satapathy, Anita Sjolander. Immunomodulatory effect of prostaglandin dehydrogenase in metastatic colon cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B53.

LIPOXINS SELECTIVELY CHANGE M2‐LIKE PHENOTYPE OF TUMORASSOCIATED MACROPHAGES BY CHANGING THEIR UNIQUE METABOLIC PROFILE AND IMPAIRING TUMOR PROGRESSION

In tumor microenvironments, most tumor‐associated macrophages (TAM) exhibit a characteristic M2 phenotype that favors tumor development, with deficient production of NO and ROS, key features of pro‐inflammatory M1 cytotoxic macrophages. Evidences have shown a positive correlation between high TAM density and poor prognosis. The balance between the different tumor derived mediators may modulate the outcome of the Mϕ response and consequent tumor development. Lipoxins (LX) are specialized pro‐resolving lipid mediators, able to shift inflammatory monocytes and macrophages towards M2‐like profile, but their effects on TAMs remained non‐elucidated. We have shown that, in vitro, LX selectively induce a switch of TAM M2‐like phenotype towards an M1‐like profile, increasing ROS and NO production, and enabling these cells to promote tumor cell apoptosis. LX did not affected the M2 profile of IL‐4‐stimulated macrophages. In vivo, the treatment of tumor‐bearing mice with LX, diminished the population of LY6Chi monocytes (TAM precursors) in spleen, blood and bone marrow, decreasing macrophage infiltration into the tumor, reducing the M2 markers expression on TAMs, and impairing tumor progression, increasing mice survival. The differential activation of macrophages is supported by profound intracellular metabolic changes, being well accepted that the pro‐inflammatory M1/M(LPS+IFN‐γ) phenotype rely on aerobic glycolysis, while anti‐inflammatory M2/M(IL‐4) macrophages depend on oxidative metabolism. However, little is known about TAM metabolism. We investigated the metabolic profile and requirements of TAMs, analyzing the lipoxin effects on their metabolic pathways. We show for the first time that despite TAMs present high expression of M2 markers, similarly to the M1, but distinct from M2 cells, have high glycolytic activity, with high lactate secretion, mTOR/Akt activity and increased GLUT‐1 expression. Inhibition of glycolysis, but not the oxidative phosphorylation or PPP, decreased M2 markers expression in TAMs. The inhibition of PPP impairs lipoxin to shift the M2‐profile and restore cytotoxic properties in TAMs. In conclusion, TAMs although phenotyped as M2, are metabolically distinct from these cells, being rather similar to M1, depending on the glycolytic metabolism to support their profile and functions, that lipoxin effects rely on PPP activation.Support or Funding Informationby: FAPERJ # E‐26/202.782/2017; CAPES: Finantial code # 001; CAPES/PROEX # 0574/2018; CNPq # 302413/2017‐0; 118566/2017‐2, Brazil.

Cytotoxic and immunosuppressive inflammatory cells predict regression and prognosis following neoadjuvant radiochemotherapy of oesophageal adenocarcinoma

Background and purposeTumour infiltrating lymphocytes (TIL) and tumour associated macrophages (TAM) play a key role in anticancer immunosurveillance. We studied their influence on response to neoadjuvant radiochemotherapy (RCT) and prognosis in patients with oesophageal adenocarcinoma (OAC). Materials and methodsBetween 10/2004 and 06/2018, pre-RCT biopsy-specimens were available from 76 patients with locally advanced, non-metastatic OAC scheduled for trimodality therapy. We evaluated intra- and peritumoural expression of FoxP3+-, CD8+-TIL and CD68+-, CD163+-TAM, contemplating cell density, cell ratios and cell-to-cell distances to determine a possible influence on tumour regression grade (TRG) and survival. Median follow-up time for all patients was 18 months (IQR 9–43), and 54 months (25–97) for surviving patients. Data were analysed using risk analysis, logrank test and Cox regression. ResultsPoor tumour regression was detected for cN+ (RR 0.77 [95% CI 0.66–0.90], p = 0.001), low intratumoural FoxP3+/CD8+ ratio (RR 0.75 [0.60–0.96], p = 0.020), high peritumoural CD163+/CD68+ ratio (RR 0.77 [0.60–0.99], p = 0.045) and high intratumoural TAM density (RD −0.44 [−0.82 to −0.06], p = 0.023).Apart from poor resection quality and TRG, pretherapeutic high peritumoural CD8+ infiltration (HR 2.36 [1.21–4.61], p = 0.012) and short intratumoural FoxP3+ to CD8+ cell-to-cell distances in middle ranged CD8+ density (HR 2.55 [1.00–6.52], p = 0.050) were significant unfavourable prognostic factors in multivariate analysis. ConclusionsImmunologic parameters, such as CD8+-, FoxP3+-TIL and CD68+-, CD163+-TAM, were identified to be of independent predictive and prognostic value in patients with OAC. Further and independent validation of these biomarkers by a large size dataset may urgently be contemplated.

Abstract B104: Immunomodulatory effect of 15-hydroxy prostaglandin dehydrogenase in colon cancer

Abstract Cyclooxygenase-2, the key player in the biosynthesis of prostaglandin-E2 (PGE2) is often upregulated in colon cancer. In addition, TNF-α, the prime regulator of COX-2 and mPGES-1, also contributes to increased synthesis of PGE2. However, 15-hydroxy prostaglandin dehydrogenase, a tumor suppressor playing major role in PGE2 catabolism, is often downregulated in colon cancer. Interestingly, colon cancer patients expressing high levels of cysteinyl leukotriene receptor-2 (CysLT2R) show good prognosis and therefore, we investigated a potential link between CysLT2R-signaling and 15-PGDH in metastatic colon cancer. Elevation of 15-PGDH expression by LTC4, a CysLT2R ligand, exhibited antitumor activity in colon cancer cells with significant phosphorylation of β-catenin with concurrent activation of CASPASE-3 and dramatic downregulation of TNF-α as well as NFκβ1 mRNA expression in HT-29 and Caco-2 colon cancer cells. The interaction between programmed death ligand 1 (PD-L1) and programmed death receptor 1 (PD-1) of cancer cells and T cells, respectively, leads to T-cell inactivation and tumor escape. We observed that recombinant TNF-α stimulated colon cancer cells expressed high PD-L1 compared to the unstimulated counterparts, which was downregulated by LTC4 treatment. Protumorigenic role and association of tumor-associated macrophage (TAMs) with PD-L1 was also evident with increase in CD-47 mRNA expression. Human (SW480, SW620) and mouse (MC-38) colon cancer cells exposed to conditioned media of chemically differentiated THP-1 and RAW 264.7 cells, respectively, showed abundant expression of PD-L1 in both mRNA and protein level along with CD-47 in mRNA level, which was inhibited by LTC4 stimulation via activation of CysLT2R in a 15-PGDH dependent manner. Hence restoration of 15-PGDH expression through CysLT2R-signaling promotes immunomodulatory effect, indicating a promising therapeutic alternative for metastatic colon cancer with high TAM infiltration. Citation Format: Shakti R. Satapathy, Anita Sjolander. Immunomodulatory effect of 15-hydroxy prostaglandin dehydrogenase in colon cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B104.

Increased tumor-associated macrophages in the prostate cancer microenvironment predicted patients' survival and responses to androgen deprivation therapies in Indonesian patients cohort.

BackgroundTumor-associated macrophages (TAMs) and microvessel density (MVD) play an essential role for tumor progression in prostate cancer (PCa). In this study, we evaluated the association between TAMs, the infiltration with tumor angiogenesis and the response to androgen deprivation therapies (ADTs) in PCa to evaluate TAM infiltration as a predictive factor for PCa survival.Materials and methodsFifty-four specimens were collected and stained with CD 68 antibody to investigated TAM infiltration in tumor. Von Willebrand factor was stained to evaluate MVD around the cancer foci. We assessed the association between patient's age, preoperative serum prostate-specific antigen, pathologic Gleason sum (GS), TAM infiltration, MVD, and the response to ADT for 5 years after PCa diagnosis.ResultsThe median TAM was observed in 28 (6-76)/high power field (x400). Increasing TAM correlated with increasing tumor angiogenesis (P < 0.001, r = 0.61), and the response to ADT was significantly better in patients with fewer TAMs (<28) than in patients with higher TAMs (>28) (P = 0.003). TAM infiltration was significantly higher in those with higher serum prostate-specific antigen, higher GS, and metastasis. Multivariate analysis showed that GS, ADT type, and MVD number were significant prognostic factors for response to ADT in PCa (P < 0.0001). An increased infiltration of TAM [hazards ratio (HR) = 4.47; 95% confidence interval (CI): 1.97–10.15], MVD (HR = 2.66; 95% CI: 1.27–5.61), metastatic status (HR = 2.29; 95% CI: 0.14-0.60), and prostate volume (HR = 2.19; 95% CI: 1.27–3.12) significantly correlated with shorter survival in PCa patients by univariate analysis (P < 0.05). Multivariate analyses revealed that TAM and metastatic status significantly correlated with poor overall survival.ConclusionsTAM infiltration is associated with response to ADT and increased tumor angiogenesis in PCa. GS, ADT type, and MVD in PCa specimens are useful predictive factors for poor response to ADT. Increasing TAM and positive metastatic status were prognostic factors for a poorer survival in PCa patients.

Prognostic significance of combined Lymphocyte-monocyte Ratio and Tumor-associated Macrophages in Gastric Cancer Patients after Radical Resection

Background: Immune function is recognized as an important prognostic indicator in gastric cancer (GC). The relationship between the lymphocyte-monocyte ratio (LMR) and tumor-associated macrophage (TAM) has received far less attention.Methods: A total of 401 patients from a prospective trial (NCT02327481) were enrolled in this study. The relationships between the LMR, TAM, and clinicopathologic variables were analyzed using a Kaplan-Meier log-rank survival analysis, and multivariate Cox regression models were used to identify associations with recurrence-free survival (RFS) and overall survival (OS). The discriminatory power of the prognostic models for both RFS and OS were compared. The decision curve analysis was performed to compare the clinical utility of the prognostic models.Results: High LMR was observed in 81.5% of the 401 GC patients, and high TAM infiltration was observed in 45.9% of the patients. In a multivariate Cox analysis of all patients, LMR and TAM were both independent prognostic factors for RFS and OS. Patients with high TAM expression had similar mean LMR levels than patients with low TAM expression. Moreover, LMR appeared to lose its prognostic significance in patients with high TAM expression levels. Finally, the model that included the TAM had better predictive capability and clinical utility for both RFS and OS.Conclusions: Although LMR and TAM are both independent predictors of RFS and OS in resectable GC patients, LMR seem to attenuate its prognostic significance in patients with high TAM expression. This information may be helpful in the clinical management of patients with GC. Further external studies are warranted to confirm this hypothesis.

Integration of intratumoral RASSF10 expression and tumor-associated macrophages into the established clinical indicators better predicts the prognosis of clear cell renal cell carcinoma patients

ABSTRACT A helpful evaluation system is crucial for the postoperative prognosis prediction of clear cell renal cell carcinoma (ccRCC) patients. This study determined the prognostic value of combining intratumoral RASSF10 expression and tumor-associated macrophages (TAMs) with the established clinicopathological indicators in ccRCC patients. RASSF10 expression was analyzed in ccRCC patient data from online databases and ccRCC cell lines. Two independent ccRCC patient cohorts were employed to examine the prognostic value of RASSF10 and other markers by immunohistochemistry (IHC) and statistical analyses. We found that RASSF10 expression was downregulated in ccRCC specimens from the TCGA datasets and three independent institutions. RASSF10 expression was negatively correlated with disease progression and TAM infiltration in ccRCC. In addition, low RASSF10 expression and high TAM infiltration predicted a high TNM stage, SSIGN score, WHO/ISUP grading, and a poor prognosis in two independent ccRCC patient cohorts. Moreover, RASSF10, CD68 or CD163, TNM stage, and SSIGN score were identified as independent risk factors in predicting ccRCC patients’ prognosis. Time-dependent c-index analyses revealed that the combination of RASSF10 and TAMs resulted in a higher index than that resulting from each alone in the postoperative prognosis of ccRCC patients, and the integration of RASSF10 and TAMs with the TNM stage or SSIGN score achieved the best accuracy in assessing the prognosis of ccRCC patients. These findings were validated in the randomized training, validation, and combined cohorts. Taken together, the combination of the RASSF10-TAM classifier and current clinical parameters yields superior accuracy in predicting the ccRCC patients’ postoperative outcome.

MicroRNA-148b-colony-stimulating factor-1 signaling-induced tumor-associated macrophage infiltration promotes hepatocellular carcinoma metastasis

BackgroundMicroRNAs (miRNAs) are small non-coding molecules that exhibit important regulatory roles in various biological or cellular functions, including tumor metastasis. However, the detailed mechanisms of the expression and functions of miRNAs in hepatocellular carcinoma (HCC) have not yet been completely investigated. MethodsIn this study, the levels of miR-148b in HCC cells and patient specimens were determined using qPCR assays. MiR-148b-overexpressing HCC cells were used to investigate the effect of miR-148b in vitro and in vivo. The relationship between the expression of miR-148b and colony stimulating factor-1 (CSF1) in HCC patients and the infiltration of macrophages into the tumor microenvironment were assessed by immunohistochemical staining. ResultsMiR-148b expression was decreased in metastatic HCC cells. A positive association between downregulated miR-148b expression and several clinical parameters, including recurrence, metastasis, and poor prognosis, was observed in patients with HCC. The results of bio-functional experiments indicated that the biological characteristics of HCC cells were not affected by miR-148b deficiency in vitro. However, miR-148b deficiency significantly enhanced the progression and metastasis of HCC in nude mice. By analyzing the gene expression profiles, we demonstrated that CSF1 was regulated by miR-148b and that miR-148b deficiency promoted HCC growth and metastasis through CSF1/CSF1 receptor (CSF1R)-mediated tumor-associated macrophage (TAM) infiltration. These results were supported by the negative relationship between miR-148b and CSF1 expression and TAM infiltration in HCC patients. Moreover, HCC patients with low miR-148b levels and high TAM infiltration were associated with poorer prognosis. ConclusionMiR-148b-CSF1 signaling-induced TAM infiltration promotes HCC metastasis. Therefore, miR-148b plays a suppressor role in HCC and might be a potential prognostic factor and therapeutic candidate for HCC.