Tumor-associated Macrophages Count Research Articles

Tumor stroma ratio, tumor stroma maturity, tumor-infiltrating immune cells in relation to prognosis, and neoadjuvant therapy response in esophagogastric junction adenocarcinoma.

Accurate predictions on prognosis and neoadjuvant therapy response are crucial for esophagogastric junction adenocarcinoma (EGJA) patients. Therefore, we aimed to investigate the predictive abilities of several indicators, including tumor stroma ratio (TSR), tumor stroma maturity (TSM), and the density and spatial distribution of tumor-infiltrating immune cells (TIICs), such as T cells, B cells, and tumor-associated macrophages (TAMs). Resection and biopsy specimens of a total of 695 patients were included, obtained from the National Cancer Center (NCC) and The Cancer Genome Atlas (TCGA) cohorts. TSR and TSM were evaluated based on histological assessment. TIICs were quantified by QuPath following immunohistochemical (IHC) staining in resection specimens, while the Klintrup-Mäkinen (KM) grade was employed for evaluating TIIC in biopsy specimens. Patients with high stromal levels or immature stroma had relatively worse prognoses. Furthermore, high CD8+T cell count in the tumor periphery, as well as low CD68+ TAM count either in the tumor center or in the tumor periphery, was an independent favorable prognostic factor. Significantly, the combination model incorporating TSM and CD163+TAMs emerged as an independent prognostic factor in both two independent cohorts (HR 3.644, 95% CI 1.341-9.900, p = 0.011 and HR 1.891, 95% CI 1.195-2.99, p = 0.006, respectively). Additionally, high stromal levels in preoperative biopsies correlated with poor neoadjuvant therapy response (p < 0.05). In conclusion, our findings suggest that TSR, TSM, CD8+T cell, CD68+TAMs, and CD163+TAMs predict the prognosis to some extent in patients with EGJA. Notably, the combined model incorporating TSM and CD163+TAM can contribute significantly to prognostic stratification. Additionally, high stromal levels evaluated in preoperative biopsy specimens correlated with poor neoadjuvant therapy response.

High Numbers of CD163+ Tumor-Associated Macrophages Predict Poor Prognosis in HER2+ Breast Cancer

Tumor-associated macrophages (TAMs) are associated with a poor outcome in breast cancer (BC), but their prognostic value in different BC subtypes has remained somewhat unclear. Here, we investigated the prognostic value of M2-like TAMs (CD163+) and all TAMs (CD68+) in a patient cohort of 278 non-metastatic BC patients, half of whom were HER2+ (n = 139). The survival endpoints investigated were overall survival (OS), breast cancer-specific survival (BCSS) and disease-free survival (DFS). In the whole patient cohort (n = 278), a high CD163+ TAM count and a high CD68+ TAM count were associated with a worse outcome (p ≤ 0.023). In HER2+ BC, a high CD163+ TAM count was an independent factor for a poor prognosis across all the investigated survival endpoints (p &lt; 0.001). The prognostic effect was evident in both the HER2+/hormone receptor-positive (p &lt; 0.001) and HER2+/hormone receptor-negative (p ≤ 0.012) subgroups and regardless of the provision of adjuvant trastuzumab (p ≤ 0.002). In HER2-negative BC, the CD163+ TAM count was not significantly associated with survival. These results suggest that a high CD163+ TAM count predicts an inferior outcome, especially in HER2+ BC patients, and as adjuvant trastuzumab did not overcome the poor prognostic effect, combination treatments including therapies targeting the macrophage function could represent an effective therapeutic approach in HER2+ BC.

Myostatin expression in lung cancer induces sarcopenia and promotes cancer progression.

Many studies have demonstrated that sarcopenia among lung cancer predicts poor prognosis due to cancer progression. However, the cytokines that link sarcopenia and lung cancer progression remain unidentified. This study aimed to investigate whether lung cancer producing myostatin, which induces skeletal muscle atrophy, leads to sarcopenia and promotes cancer progression in patients with resected lung cancer. Tumor tissues were obtained from 148 patients who underwent curative resection for lung cancer. Tumor cells were stained with myostatin and tumor-associated macrophages (TAM) in the tumor microenvironment were stained with CD68. We assessed the association between myostatin expression and the clinicopathological features. High myostatin expression in lung cancer was significantly associated with low skeletal muscle mass. The 5-year overall survival and relapse-free survival were significantly worse among patients with high myostatin expression than those with low expression. A multivariate analysis showed that TAM count was positively correlated with high myostatin expression. Sarcopenia may be induced by myostatin secreted by lung cancer cells. Moreover, myostatin may promote TAM migration into the tumor microenvironment, leading to advance lung cancer. As a result, patients with high myostatin expression had poor prognosis.

Distribution of tumor-associated macrophages and M1/M2 polarization in different types and grades of ovarian tumors

Ovarian cancer represents one of the major causes of gynaecologic cancer mortality worldwide. Tumor-associated macrophages (TAM) are associated with the development and progression of ovarian cancer. TAMs are mainly represented by two types M1 and M2. We investigated the distribution of total, M1, M2 macrophages, tumor cell/macrophage ratio and M1/M2 ratio in mucinous and serous ovarian tumors. The study results showed that total TAM count is significantly higher in serous ovarian tumors, compared to mucinous tumors and the highest infiltration rate is detected in high-grade serous ovarian carcinomas. In addition, the number of M2 macrophages is significantly increased in higher-grade serous ovarian carcinomas. The evaluation of tumor/cell macrophage ratio could be used as an objective measure of macrophage infiltration in ovarian tumors.

Evaluation of the immunomodulatory activity of thalidomide on tumor-associated macrophages in the 4T1 murine metastatic breast cancer model

ABSTRACT The present work evaluated the immunomodulatory effect of thalidomide (Thal) at different doses on tumor-associated macrophages (TAMs) using a mouse model of human breast cancer. Mice were inoculated with 4T1 cells in the left flank and treated with Thal once a day at concentrations of 50, 100, and 150mg/kg body weight from the 5th day until the 28th day of tumor inoculation. The tumors were sized, proliferation index and TAMs count were evaluated in primary tumors and metastatic lungs. In addition, the metastasis rate was evaluated in the lungs. Thal at 150mg/kg significantly decreased tumor growth, proliferation index, and TAMs infiltration in primary tumors. Conversely, a higher number of TAMs and lower proliferation index were observed in metastatic lungs in mice treated with 150mg/kg of Thal. Furthermore, Thal at 150mg/kg significantly decreased the metastatic nodules in the lungs. Our findings demonstrated that Thal treatment considerably decreased the primary tumor and lung metastasis in mice associated with different TAM infiltration effects in these sites.

Potential role of tumor-associated macrophages and CD163/CD68 ratio in mycosis fungoides and Sézary syndrome in correlation with serum sCD163 and CCL22.

Currently, there are no curative treatment options for mycosis fungoides (MF) and Sézary syndrome (SS) other than stem cell transplant. Understanding the interplay between tumor cells and tumor microenvironment could aid in the development of new therapies. Tumor-associated macrophages (TAMs) mostly have M2 phenotype that promotes tumor progression. This study investigated CD68+ and CD163+ TAMs as well as CD163/CD68 ratio in skin lesions from different stages of MF, large-plaque parapsoriasis, and SS. Moreover, we analyzed serum levels of sCD163 and CCL22 in correlation with TAMs count and CD163/CD68 ratio. CD68+ and CD163+ TAMs count significantly increased as the disease progressed. CD163/CD68 ratio was highest at MF tumor stage and SS indicating M2 polarization with disease progression. Significant positive correlations were detected between serum levels of sCD163 and CCL22 and CD68+ and CD163+ TAMs count and CD163/CD68 ratio. We concluded that TAMs play an important role in MF progression. High CD163/CD68 ratio in tumor stage MF and SS indicates M2 polarization of TAMs with tumor progression. CD163/CD68 ratio should be considered in assessing TAMs rather than total TAMs count. Also, sCD163 and CCL22 serum levels reflect M2 load and thus could be used as markers to assess disease progression.

Clinicopathologic and Prognostic Significance of CD47 Expression and Tumor-associated Macrophages in Endometrial Carcinoma.

Tumor-associated macrophages (TAMs) influence cancer progression. CD47 is an antiphagocytic molecule aiding tumor resistance against host immune surveillance. The relationship between CD47 expression and TAM-related microenvironment in endometrial carcinoma (EC) is poorly understood. The expression and prognostic significance of CD47 and CD163-labeled TAMs in 165 EC cases was assessed with CD47 and CD163 immunohistochemical studies. CD47 expression was found in 156/165 (94.6%) cases. CD47 expression was significantly higher in nonendometrioid carcinomas. CD47 overexpression was associated with histologic grade. High epithelial and stromal TAMs counts were also associated with high tumoral CD47 expression. High epithelial, stromal, and margin TAMs counts were associated with higher histologic grade and lymphovascular invasion. Epithelial TAMs counts were higher in patients with nonendometrioid carcinomas (P=0.0001) and cases with recurrence (P=0.018). High stromal TAMs counts were associated with deeper myometrial invasion (P=0.017) and the presence of distant metastasis (P=0.024). The counts of margin TAMs was significantly correlated with the depth of myometrial invasion, lymphovascular invasion, FIGO stage, lymph node metastases, distant metastasis, and recurrence (P=0.0001, 0.0001, 0.004, 0.005, 0.014, and 0.04, respectively). CD47 expression was not associated with overall survival (OS) and progression-free survival. However, high epithelial and stromal TAM counts were associated with shorter OS. Besides, high epithelial and margin TAM counts were associated with shorter progression-free survival. Furthermore, increased stromal and margin TAM counts were determined to be an independent prognostic marker of reduced OS. TAM count is, therefore, a significant prognostic factor in EC and the CD47 assessment has potential benefit for future clinical use.

Role of tumor-associated macrophages in relation to angiogenesis in urothelial bladder carcinoma

Background: Urothelial Bladder carcinoma is an aggressive tumor with male predominance. The tumor microenvironment is found to affect tumor progression and biology. One of its main constituents is tumor-associated- macrophages (TAMs). Angiogenesis is a powerful factor in the development of many tumors and is supposed to be induced by tumor cells and some stromal components that regulate tumor behavior. Aim: To assess the correlation of TAMs and angiogenesis in the stroma of urothelial carcinoma and its correlation with the tumor grade and stage and nodal metastasis. Material and Methods: This study was carried out on 30 paraffin blocks of bladder urothelial carcinoma. Hematoxylin and eosin staining was done for confirmation of the histopathological diagnosis. Immunohistochemical staining by CD68 antibody was done for counting of TAMs and CD34 antibodies for tumor-associated angiogenesis (microvessel count). Results: Significant higher TAMs count was detected in high-grade and invasive bladder cancers compared to low-grade and non-invasive types and in patients with nodal metastasis. Moreover, microvessel counts (MVCs) were significantly higher in higher grade, invasive bladder cancers and in patients with nodal metastasis. There was a positive correlation between the predominance of CD68+ TAMs and CD34+ MVCs in urothelial carcinoma. Conclusion: There is a positive correlation between TAMs and angiogenesis in the stroma of urothelial carcinoma and both are positively correlated with tumor grade, stage, and nodal metastasis. Our data indicated that both TAMs and MVCs could serve as prognostic markers for urothelial bladder cancer.

CD68+ Tumor Associated Macrophages Have Significant Impact on Therapeutic Outcome in Newly Diagnosed Multiple Myeloma (MM)

Background: The tumor microenvironment (TME) of MM is characterized by an immunosuppressive milieu fostering MM growth. The impact of tumor associated macrophages (TAM) has not been well-studied in the MM TME. We evaluated the bone marrow biopsies (BMBx) obtained at the initial diagnosis of MM and evaluated the correlation between the number of CD68+ macrophages and clinical parameters and outcomes. Methods: We evaluated the BMBx at initial diagnosis of 34 consecutive MM patients (pts) diagnosed between 2007-2016 at Mayo Clinic Florida. Immunohistochemistry (IHC) was performed with an anti-CD68 (KP1) antibody to identify and count CD68+ TAMs. Survival was analyzed with the Kaplan-Meier method. Chi-square tests were performed to study the correlation between the number of CD68+ TAMs and various clinical parameters. Results: Nineteen (56%) pts were male, 29 (85%) were caucasian. Median age at MM diagnosis was 58.5 years (95% CI 57-63), 18 (53%) pts has ISS I/II disease, 13 (38%) pts had ISS III disease, 3 (9%) has unknown ISS. Eight (24%) had high risk cytogenetics; 4(12%) had del17p, 2 (6%) had t(14;16), 1(3%) had 1q amplification, 3 (9%) had t(4;14). Eight (24%) had t(11;14). Twenty nine (85%) pts underwent autologous transplantation, 11(32%) received maintenance therapy. Overall response rate (≥partial response) to induction therapy was 100%. The ≥ very good partial response (VGPR) rate was 73.5%. Median number of induction cycles was 4.5 (95% CI 4.8-8.3). Twelve (35%) pts received lenalidomide (R)-dexamethasone (D) induction (RD), 9 (26%) received bortezomib (V)-cyclophosphamide(C)-D (VCD) induction, 7 (21%) received VRD induction, 5(15%) received VD induction and 1(3%) received CRD induction. The median follow-up was 92 months (m) (95% CI 85-99) The median progression free survival (mPFS) of the pt population was 40m (95% CI 27-68) and the median overall survival (mOS) was 96m (95% CI 81-126). Mean CD68+ TAM count was 12.5% (95% CI 8.1-16). There was a trend towards improved PFS for patients with ≥5% vs. &amp;lt;5% CD68+ TAM (47m vs. 36m, p=0.055) and there was improved OS for patients with ≥5% vs. &amp;lt;5% CD68+ TAM (101m vs. 73m, p=0.042, Figure 1) in their initial diagnostic BMBx. Pts with high risk cytogenetics did not have a significantly different CD68+ TAM (8.6%vs.12.8%, p=0.363) compared to those with standard risk cytogenetics. Pts with ISS I/II disease were more likely to have ≥5% CD68+ TAM in their diagnosis BMBx compared to patients with ISS III disease, χ²=8.58, p=0.003. Pts who achieved ≥VGPR to induction therapy were more likely to have ≥5% CD68+ TAM in their diagnosis BMBx compared to those who achieved &amp;lt;VGPR; χ²=5.35, p=0.020. Pts who received R-based induction and had ≥5% CD68+ TAM in their diagnosis BMBx had superior OS (96m vs. 62m, p=0.044) compared to those with &amp;lt;5%.This finding was not seen in patients who received V-based induction (p=0.33). Conclusion: Pts with ≥5% CD68+ TAM in their initial diagnostic BMBx are more likely to have deeper responses to induction therapy as well as superior OS. In pts induced with an R-based regimen, increased CD68+ TAM was associated with significantly better OS raising the possibility of a therapeutic and mechanistic role of TAM in R-related therapeutic effect. ≥5% CD68+ TAM were noted in patients with lower risk disease as defined by ISS stage but not by cytogenetics. Further exploration of the role of TAM in the biology of MM and its impact on clinical and therapeutic outcomes is warranted. Disclosures Alhaj Moustafa: Acrotech: Consultancy. Tun:DTRM Biopharma: Research Funding; Acrotech: Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Research Funding; Mundipharma: Research Funding; Curis: Research Funding; TG Therapeutics: Research Funding.

Clinical significance and prognostic role of tumor-associated macrophages infiltration according to histologic location in canine mammary carcinomas

Tumor-associated macrophages (TAMs) have been involved in growth and metastases of human and canine mammary tumors. However, the prognostic importance of TAM specific location in canine mammary tumors (CMT) was not evaluated. In this study, we evaluated the potential role of TAMs in specific histologic locations – intratumoral (iTAM) and stromal (sTAM), as well as total macrophage (tTAM) counts – as prognostic indicators in CMT. Clinicopathologic data from 66 animals with mammary carcinoma and their tumors were used in this study. Samples were stained with anti-macrophage antibody for subsequent TAM count. High levels of iTAM, sTAM, and tTAM were related with advanced clinical stage and vascular invasion. Additionally, tTAM revealed a relation with larger tumor size, while high levels of sTAM and tTAM were also correlated with node metastasis and a poor prognosis based on survival analysis. CMT with aggressive features can reveal higher TAM counts. TAMs are associated with vascular invasion and nodal metastasis, and sTAM and tTAM counts are correlated with overall survival, suggesting they could be used as prognostic indicators in canine mammary carcinomas.

Tumor microenvironment changes tumor cell sensitivity to action of energy metabolism modifiers.

Taking into account differences in the bioenergetics between malignant and normal cells a search of antitumor drugs among the modifiers of tumor metabolism has a reasonable excuse. Earlier it was found that the cytotoxic/cytostatic action of sodium dichloroacetate (DCA) against Lewis lung carcinoma (LLC) cells in vitro was enhanced in the case of its combination with metformin (MTF). To study the antitumor action of DCA in combination with MTF against LLC in vivo. LLC/R9, a low metastatic variant of LLC cells, was used. LLC/R9bearing mice were treated with MTF (at a total dose 0.15g/kg b.w.) alone or in combination with DCA (at a total dose of 0.75g/kg b.w.). LLC/R9growth kinetics and the primary tumor growth and metastasis indices on the 23rd day after tumor cell inoculation were evaluated by routine procedures. The state of the electron transport chain of mitochondria in tumor cells was studied using electron paramagnetic resonance. The content of lactate and glucose in blood plasma from mice was measured by enzymatic methods using biochemical analyzer. The number of tumor-associated macrophages (TAMs) and their distribution by M1/M2phenotype were estimated by flow cytometry using antibodies against CD68and CD206. In LLC/R9-bearing mice treated with DCA in combination with MTF, tumor growth and metastasis indices, as well as circulating glucose and lactate levels were not significantly different from those in the control group. The level of nitrosylation of non-heme and heme proteins and the content of iron-sulfur centers in the mitochondria of tumor cells in LLC/R9-bearing mice administered with DCA in combination with MTF did not also differ from the corresponding indices in control. Instead, in tumors treated with MTF alone and in combination with DCA the total CD68+ TAMs count was almost 27% (p< 0.05) and 43% lower (p< 0.05) correspondingly than that in control, but this decrease was not accompanied by redistribution of CD68+/CD206+ and CD68+/D206- subsets. DCA in combination with MTF, at least in doses applied, did not affect LLC/R9growth and metastasis in vivo. The complete absence of an antitumor effect of DCA in combination with MTF was simultaneously associated with the absence of significant changes in the functional state of electron transport chain of mitochondria in tumor cells, circulating glucose and lactate levels, and the decrease of the TAMs amount in tumors. It suggests that the antitumor activity of DCA and MTF could be determined by both their local effects within a tumor and their multiple systemic impacts.

THE ROLE OF TUMOR-ASSOCIATED MACROPHAGES IN THE PATHOGENESIS OF ORAL SQUAMOUS CELL CARCINOMA CORRELATED WITH THE CLINICOPATHOLOGICAL PARAMETERS

Introduction: Oral cancer is a major health problem, causing high morbidity and mortality rates. Oral squamous cell carcinoma (OSCC) accounts for 90-95% of all oral malignancies. During the last decade, significant evidence has suggested that inflammatory tumor microenvironment (TME) plays an important role in tumorigenesis. The inflammatory cells and their signals are indispensable participants in the neoplastic process, fostering proliferation, survival, and migration of cancer cells. The macrophages are the most abundant and important stromal cells in the TME, which orchestrate the inflammatory response. They control the cellular proliferation and survival by stimulating the immune cells and by promoting integrated processes of inflammation and tissue repair. Therefore, the tumor associated macrophages (TAMs) may be of great prognostic significance in different types of tumors. Objectives: This study was conducted to assess the TAMs density in OSCC using CD163 in metastasizing and non-metastasizing OSCC. Moreover, the correlation of TAM density with the lymph node status and the different histopathological grades of OSCC was assessed. Materials and methods: The density of TAM was calculated in 30 surgical specimens taken from OSCC patients. Biopsies were taken from the primary tumor of 15 cases with lymph node metastasis and 15 cases with no lymph node metastasis. Fifteen normal mucosal tissues were taken from healthy individuals indicated for alveoloplasty as a control group. Immunohistochemical staining using the CD163 antibody was performed, using Labeled Strept-Avidin Biotin complex method. Positive cells were counted using the Image J free software package. Results:CD163 was expressed in human OSCC and the TAMs count was significantly correlated with lymph node metastasis and with the tumor differentiation. Higher density was detected in metastatic tumors and in the poorly differentiated OSCC than in the well and moderately differentiated cases. Conclusions: CD163 positive TAMs could be a prognostic factor in OSCC cases as TAM density was significantly correlated with the lymph node status and the grade of differentiation of OSCC.

Abstract P1-05-03: JNK signaling regulates tumor cell–tumor-associated macrophage cross-talk in triple-negative breast cancer

Abstract Despite advances in our understanding of the molecular mechanisms underlying the aggressiveness of triple-negative breast cancer (TNBC), the contribution of tumor-associated macrophages (TAMs) to TNBC pathogenesis has not been therapeutically exploited. TAMs are the most abundant cell types in the tumor microenvironment (TME) and the key contributor to tumor progression and invasion. We have found that c-Jun NH2-terminal kinase (JNK), a member of the MAPK family and a major regulator of inflammation, contributes to TNBC tumorigenesis by promoting the cancer stem-like cell phenotype. However, whether the JNK pathway regulates TAMs and their cross-talk with tumor cells in TNBC remains unknown. Here, we tested the hypothesis that JNK signaling contributes to TNBC aggressiveness by promoting the tumor cell–TAM cross-talk that facilitates TNBC cell invasiveness. We found that, among 80 patients with primary inflammatory breast cancer (IBC), TNBC tumors (n=18) had 2-fold more TAMs than non-TNBC tumors (n=62, P=0.05) and that high TAM counts were correlated with shorter disease-free survival of patients with IBC (P=0.05). Both JNK1 and c-Jun were highly activated in TAMs, and JNK-IN-8, a pan-inhibitor of JNK, suppressed c-Jun activation. JNK-IN-8 also increased expression of M1 macrophage markers (CD80 and HLA-DR) but reduced expression of TAM markers (CD163 and CD206), suggesting that JNK suppresses M1 macrophage differentiation but promotes TAM differentiation. Co-culture with TAMs significantly enhanced migration and invasion of HCC70 and MDA-MB-468 human and 4T1 murine TNBC cells. Similarly, an enhancement in TNBC cells migration and invasion was observed following culture with TAM-conditioned medium, suggesting that TAMs enhance TNBC cellular activities through paracrine signaling. In addition, inhibition of JNK signaling in TNBC cells or in TAMs by JNK-IN-8 significantly suppressed TAM-promoted enhancement of TNBC cell migration and invasion. These studies strongly suggest that JNK regulates M1/TAM differentiation and TNBC cell–TAM cross-talk. Furthermore, cytokine/chemokine profiling analysis showed that, of the identified molecules, MCP-1 (secreted by TAMs) and VEGF (secreted by TNBC cells) had the highest expression levels and that their expression was dramatically reduced following JNK-IN-8 treatment. Stimulation with recombinant VEGF increased proliferation of MDA-MB-468 cells, and stimulation with recombinant MCP-1 enhanced migration of the cells. These findings suggest that VEGF and MCP-1 are involved in JNK-mediated TNBC cell–TAM cross-talk. Together, our results suggest that JNK signaling regulates tumor cell–TAM cross-talk through MCP-1– and/or VEGF-mediated paracrine signaling and that JNK is an important therapeutic target in TNBC. Further animal studies using JNK-knockout TNBC cells co-injected with TAMs are needed to confirm our in vitro findings. Citation Format: Xie X, Otsuka S, Chu K, Lu AY, Tripathy D, Dalby KN, Hittelman WN, Van Laere S, Bartholomeusz C, Ueno NT. JNK signaling regulates tumor cell–tumor-associated macrophage cross-talk in triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-05-03.

CCL26 Participates in the PRL-3-Induced Promotion of Colorectal Cancer Invasion by Stimulating Tumor-Associated Macrophage Infiltration.

Both phosphatase of regenerating liver-3 (PRL-3) and tumor-associated macrophages (TAM) influence cancer progression. Whether PRL-3 plays a critical role in colorectal cancer invasion and metastasis by inducing TAM infiltration remains unclear. In the current study, we investigated the effects of chemokine ligand 26 (CCL26) on TAM infiltration and colorectal cancer invasion and the underlying mechanism in colorectal cancer cells by overexpressing or silencing PRL-3. We found that PRL-3 upregulated CCL26 expression correlatively and participated in cell migration, according to the results of gene ontology analysis. In addition, IHC analysis results indicated that the PRL-3 and CCL26 levels were positively correlated and elevated in stage III and IV colorectal cancer tissues and were associated with a worse prognosis in colorectal cancer patients. Furthermore, we demonstrated that CCL26 induced TAM infiltration by CCL26 binding to the CCR3 receptor. When LoVo-P and HT29-C cells were cocultured with TAMs, CCL26 binding to the CCR3 receptor enhanced the invasiveness of LoVo-P and HT29-C cells by mobilizing intracellular Ca2+of TAMs to increase the expression of IL6 and IL8. In addition, IHC results indicated that protein levels of CCR3 and TAMs counts were higher in stage III and IV colorectal cancer tissues and correlated with CCL26. Moreover, similar results were observed in vivo using mice injected with LoVo-P and HT29-C cells. These data indicate that PRL-3 may represent a potential prognostic marker that promotes colorectal cancer invasion and metastasis by upregulating CCL26 to induce TAM infiltration. Mol Cancer Ther; 17(1); 276-89. ©2017 AACR.

Regulatory T Cells and Tumor-Associated Macrophages in the Tumor Microenvironment in Non-Muscle Invasive Bladder Cancer Treated with Intravesical Bacille Calmette-Guérin: A Long-Term Follow-Up Study of a Japanese Cohort

The clinical significance of regulatory T cells (Treg) and tumor-associated macrophages (TAM) in the tumor microenvironment of human bladder cancer remains unclear. The aim of this study is to explore their relevance to oncological features in non-muscle invasive bladder cancer (NMIBC). We carried out immunohistochemical analysis of forkhead box P3 (FOXP3, Treg maker), CD204 (TAM marker), and interleukin-6 (IL6) using surgical specimens obtained from 154 NMIBC patients. The Treg and TAM counts surrounding the cancer lesion and IL6-positive cancer cell counts were evaluated against clinicopathological variables. We focused on the ability of the Treg and TAM counts around the cancer lesion to predict outcomes after adjuvant intravesical Bacille Calmette–Guérin (BCG) treatment. High Treg counts were associated with female patients, older age, T1 category, and high tumor grade. TAM count was significantly correlated with Treg count and with IL6-positive cancer cell count. In our analysis of 71 patients treated with BCG, high counts of Treg and TAM were associated with shorter recurrence-free survival, and the former was an independent predictor of recurrence. Poor response to intravesical BCG was associated with Treg and TAM in the tumor microenvironment. Disrupting the immune network can be a supplementary therapeutic approach for NMIBC patients receiving intravesical BCG.

Association of Macrophages With Angiogenesis in Oral Epithelial Dysplasia, Oral Verrucous Carcinoma, and Oral Squamous Cell Carcinoma: An Immunohistochemical Study.

The present study aimed to elucidate the role of tumor-associated macrophages (TAMs) and angiogenesis in tumor progression by assessing their immunohistochemical expression in oral epithelial dysplasia (OED), oral verrucous carcinoma (OVC), and oral squamous cell carcinoma (OSCC). About 20 histopathologically confirmed cases of OED, OVC, and OSCC each and 10 cases of normal oral mucosa taken as controls were stained immunohistochemically using CD68 and CD31 antibodies. The average TAM count and the microvessel density (MVD) were calculated for each group and expressed as mean±SD and compared using the Mann-Whitney U Test. Pearson correlation was applied to assess the correlation between TAM and MVD in different groups. The CD68 count was found to be significantly higher in all the groups as compared with controls, with the highest counts in OSCC, followed by OED and OVC. MVD was significantly higher in all the test groups as compared with controls. The increase in MVD was highly significant in OSCC as compared with OVC, and in OVC as compared with OED. No association could be determined between TAM and MVD. There does not seem to be any direct influence of macrophages on angiogenesis in the microenvironment of OED, OVC, and OSCC.

High-mobility group protein box1 expression correlates with peritumoral macrophage infiltration and unfavorable prognosis in patients with hepatocellular carcinoma and cirrhosis.

BackgroundHigh-mobility group protein box1 (HMGB1) is a pivotal factor in the development and progression of many types of tumor. Its role in hepatocellular carcinoma (HCC), and especially its correlation with intratumoral and peritumoral macrophage infiltration, remains obscure. We analyzed the potential roles and prognostic value of HMGB1 and explored the correlation between HMGB1 and macrophage infiltration in HCC using clinical samples.MethodsWe reviewed clinicopathological and follow-up data on a cohort of 149 patients with HCC complicated with Hepatitis B-related cirrhosis. We measured the expression of HMGB1 and CD68 in tumoral and peritumoral liver tissues after curative resection and assessed the impacts of the tumor-associated macrophage (TAM) count and HMGB1 expression on clinicopathologic characteristics, overall survival (OS), and recurrence-free survival (RFS).ResultsNinety-four of the patients had elevated tumoral HMGB1 expression and 59 of the patients had elevated peritumoral HMGB1 expression, compared to only 4 patients with elevated peritumoral HMGB1 expression in 36 pateints with Hepatitis B virus (HBV)-negative HCC without liver cirrhosis (p < 0.001). The peritumoral HMGB1 expression levels were correlated with tumor invasiveness, BCLC stage, and recurrence. The degree of TAM infiltration was higher in peritumoral tissues with high HMGB1 expression than in peritumoral tissues with low HMGB1 expression (p < 0.001). There was no significant difference in TAM infiltration between tumoral tissues with high and low HMGB1 expression. Kaplan-Meier analysis showed that intratumoral HMGB1 overexpression was associated with poor OS, but not with RFS. High peritumoral HMGB1expression and TAM count, which correlated positively with tumor size and BCLC stage, were independent prognostic factors for OS (p < 0.001 and p = 0.017, respectively) and RFS (p = 0.002 and p = 0.024, respectively). Multivariate analyses indicated peritumoral HMGB1 expression (p = 0.014) and TAM count (p = 0.037), as well as tumor differentiation (p = 0.026), to be independent significant prognostic factors for RFS.ConclusionsHigh HMGB1 expression in peritumoral liver tissues correlated with peritumoral macrophage infiltration and had prognostic value in HCC, suggesting that peritumoral HMGB1 might show promise as a new biomarker to predict HCC progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2883-z) contains supplementary material, which is available to authorized users.

Predominance of M2-polarized macrophages in bladder cancer affects angiogenesis, tumor grade and invasiveness.

Tumor-associated macrophages (TAMs) often assume an immunoregulatory M2 phenotype. Thus, the aim of the present study was to clarify the correlation of vascularity and TAMs, in particular the M2 phenotype in the stroma and tumor areas, with the clinical and pathological outcomes of patients with bladder cancer. The TAM counts and microvessel counts (MVCs) were determined immunohistochemically in 21 patients with bladder cancer. The number of infiltrating TAMs was measured using immunohistochemistry with anti-cluster of differentiation (CD)68 and anti-CD163 antibodies, to identify a macrophage lineage marker and an M2-polarized-specific cell surface receptor, respectively. CD68+ and CD163+ macrophages were evaluated in the stroma and tumor areas, and areas with a high density of infiltrating cell spots were counted. MVCs were determined using immunohistochemistry with anti-CD34 antibodies. The results revealed that the higher ratio of CD163+/CD68+ macrophages in the stroma, tumor and total tumor tissues were correlated with a higher stage and grade (P<0.05). In addition, the low ratio of CD68+/CD34+ microvessels was correlated with a higher stage (P<0.05). There was also a positive correlation between TAMs and MVC (r2=0.25; P<0.05). These results suggest that the TAM polarized M2 phenotype affects microvessels, pathological outcome, tumor grade and invasiveness.

Correlation analysis between tumor-associated macrophages and epithelial-mesenchymal transition in cutaneous malignant melanoma

Objective To investigate the relationship of tumor-associated macrophage (TAM) infiltration in lesions with clinicopathologic features of cutaneous malignant melanoma (CMM) and epithelial-mesenchymal transition (EMT) -related indicators. Methods Fifty-four CMM and 15 benign melanocytic nevus paraffin-embedded tissue samples were collected. An immunohistochemical study was performed to determine the expression pattern and intensity of the TAM marker CD163 as well as EMT indicators epithelial cadherin (E-cadherin) , neurologic cadherin (N-cadherin) and vimentin in these tissue samples. Count data were expressed as mean ± standard deviation. Statistical analysis was carried out using t test, one-way analysis of variance (ANOVA) and multivariate linear regression analysis with the software SPSS 17.0. Results CD163-positive TAMs were detected in CMM samples, but not in benign melanocytic nevus samples. The mean TAM count per high power field in tissue samples was significantly different between invasive and in situ CMM (35.08 ± 13.78 vs. 16.97 ± 8.74, P 0.05) . No significant difference was observed in TAM count between patients of different gender or age groups, between acral and non-acral melanoma, or between ulcerated and non-ulcerated melanoma (all P ≥ 0.05) . Multivariate linear regression analysis showed that TAM count in CMM was associated with five factors: Clark level, ulceration, tumor progression, N-cadherin and vimentin. Conclusion TAMs may accelerate tumor progression and increase tumor malignancy by promoting EMT in CMM. Key words: Melanoma; Nevi and melanomas; Macrophages; Epithelial-mesenchymal transition; Pathology

Infiltration of CD3+ and CD68+ cells in bladder cancer is subtype specific and affects the outcome of patients with muscle-invasive tumors

ObjectivesUrothelial carcinoma (UC) aggressiveness is determined by tumor inherent molecular characteristics, such as molecular subtypes, as well as by host reactions directed toward the tumor. Cell types responsible for the host׳s response include tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs). The aim of the present investigation was to explore the immunological response in relation to UC molecular subtypes and to evaluate the prognostic effect of TIL and TAM counts in tissue sections from muscle-invasive (MI) tumors. Methods and materialsTissue microarrays with 296 tumors spanning all pathological stages and grades were analyzed with antibodies for CD3, CD8, FOXP3, CD68, and CD163. Cases were classified into the following molecular subtypes: urobasal, genomically unstable, and squamous cell carcinoma–like using a combination of immunohistochemistry and histology. The Cox regression and Kaplan-Meier analyses were performed with progression-free survival and disease-specific survival as end points. ResultsUC molecular subtypes demonstrate different degrees of immunological responses; the urobasal subtype induces a weak response, the genomically unstable subtype induces an intermediate response, and the squamous cell carcinoma–like subtype induces a strong response. These subtype specific responses are independent of tumor stage and include both TILs and TAMs. The presence of infiltrating CD3+ TILs was significantly associated with good prognosis in the MI cases (P<0.01). This positive association was modulated by the presence of CD68+ TAMs. The strongest association with poor survival was observed for a high ratio between CD68 and CD3 (P = 7×10−5). ConclusionUC molecular subtypes induce immunological responses at different levels. A high CD68/CD3 ratio identifies a bad prognosis group among MI UC cases.