Tumor-associated Macrophage Markers Research Articles

Water extract of ginseng and astragalus regulates macrophage polarization and synergistically enhances DDP's anticancer effect

Ethnopharmacological relevanceIn traditional Chinese medicine, supplementing Qi and strengthening body resistance are an important principle of anticancer treatment. Panax ginseng C.A.Mey. (ginseng) and Astragalus membranaceus Bunge (astragalus) are the representative herbs for this therapeutic principle. Aim of the studyThis study aims to explore the effect of the water extract of ginseng and astragalus (WEGA) on regulating macrophage polarization and mediating anticancer in the tumor microenvironment. Materials and methodsA549 cells were cultured in tumor-associated macrophage (TAM) supernatant with various concentrations of WEGA (0, 5, 10, 20 mg/mL). A549 cell proliferation was determined through methyl thiazole tetrazolium (MTT) assay and real-time cell analysis (RTCA), respectively. In vivo experiments were performed with a Lewis lung cancer (LLC) xenograft mouse model. Forty-eight mice were divided into six groups and treated with saline, WEGA, or cis-diamine dichloro platinum (DDP) with dosage of WEGA (0, 30, 60, 120 mg/kg body weight/day). The different groups were administered with drugs via oral or intraperitoneal injection once a day for 21 consecutive days. Tumor inhibition rate, spleen index, thymus index, cytokine, protein, and mRNA expression levels were detected in mice. ResultsIn a co-culture system, WEGA remarkably inhibited A549 cell proliferation, promoted the expression of M1 macrophage markers and inhibited M2 TAMs markers. Therefore, WEGA affected the biological behavior of cancer cells by regulating the expression of some markers relevant to macrophage polarization. In addition, the group of WEGA and DDP chemotherapy effectively inhibited the transplanted tumor growth in mice and improved weight loss and immunosuppressive with the cisplatin inducing. ConclusionsThis study provides mechanistic insights into the anticancer effect of WEGA through the regulation of macrophage polarization and highlights that WEGA could be a novel option for integrative cancer therapies.

Fucosyl-Agalactosyl IgG₁ Induces Cholangiocarcinoma Metastasis and Early Recurrence by Activating Tumor-Associated Macrophage.

Concern over roles of serum IgG agalactosylation in chronic inflammatory diseases has been mounting for years but less touched in cancers. The present study addressed the underlying role of agalactosylated IgG beyond tumorigenesis. Liquid-chromatography-tandem mass spectrometry was leveraged for the analysis of IgG1 and IgG2 N-glycomes. We found that a high percentage of serum fucosyl-agalactosyl IgG1 (IgG1-G0F) in patients with cholangiocarcinoma was associated with poor tumor differentiation and tumor metastasis. Results from Kaplan–Meier analyses and a stepwise Cox regression analysis showed that patients with serum IgG1-G0F ≥40% were highly correlated with poor recurrence-free survivals and overall survivals. Interestingly, patients with cholangiocarcinoma whose serum IgG1-G0F ≥40% had more CD163+ tumor-associated macrophages in cancerous tissues than adjacent non-cancerous counterparts. In vitro assays revealed that agalactosylated IgG upregulated tumor-associated macrophage markers CD163 and CD204 in human U-937 cells and peripheral macrophages. Moreover, a positive and a negative feedback loop of transforming growth factor-β1 and interferon-γ, respectively, on IgG agalactosylation was identified using hybridoma cells and verified in sera of the patients. In conclusion, agalactosylated IgG activates tumor-associated macrophages, thereby promoting tumor metastasis and recurrence of cholangiocarcinoma.

Human Keratinocyte Carcinomas Have Distinct Differences in Their Tumor-Associated Macrophages

Despite both being keratinocyte carcinomas mainly caused by ultraviolet radiation, cutaneous squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs) have different clinical behaviors. Whether these distinct features are associated with tumor-associated macrophages (TAMs) is largely unknown. The main goal of this study was to conduct a comprehensive analysis of density and polarization states of TAMs in SCCs versus BCCs. The role of lactic acid in TAM polarization in SCC versus BCC was examined. We found that SCCs have a higher density of CD68 TAMs compared to BCCs. TAMs in SCCs express higher levels of TAM-associated markers (arginase-1, MMP9, CD40 and CD127) than those in BCCs. Interestingly, differential expression of TAM-associated markers between SCCs and BCCs was reproduced in human monocytic THP-1 cells stimulated with SCC- or BCC-conditioned media. Analysis of soluble factor(s) in these tumors further revealed that SCCs have a significantly higher concentration of lactic acid than BCCs, and lactic acid was sufficient to upregulate TAM markers. Our results demonstrate that TAMs in SCCs versus BCCs differ in density and polarization states, which can be determined by soluble factors including tumor-derived lactic acid. These differences in TAMs may contribute to the distinct clinical behaviors of SCCs versus BCCs. Funding Statement: This work was supported by grants from NIH (5K08CA172580), Doris Duke Charitable Foundation (15-007374), and NIH Medical Scientist Training Program (T32 GM007205). Declaration of Interests: Authors report no conflicts of interest. Ethics Approval Statement: De-identified, discarded tissue was obtained consistent with the general requirements of the protocol approved by the Yale Human Investigations Committee (HIC#200002048).

The relationship between chemokines CCL2, CCL3, and CCL4 with the tumor microenvironment and tumor-associated macrophage markers in colorectal cancer.

A complex network of chemokines can influence cancer progression with the recruitment and activation of hematopoietic cells, including macrophages to the supporting tumor stroma promoting carcinogenesis and metastasis. The aim of this study was to investigate the relation between tissue and plasma chemokine levels involved in macrophage recruitment with tumor-associated macrophage profile markers and clinicopathological features such as tumor-node-metastases stage, desmoplasia, tumor necrosis factor-α, and vascular endothelial growth factor plasma content. Plasma and tumor/healthy mucosa were obtained from Chilean patients undergoing colon cancer surgery. Chemokines were evaluated from tissue lysates (CCL2, CCL3, CCL4, CCL5, and CX3CL1) by Luminex. Statistical analysis was performed using Wilcoxon match-paired test ( p < 0.05). Macrophage markers (CD68, CD163, and iNOS) were evaluated by immunohistochemistry samples derived from colorectal cancer patients. Correlation analysis between chemokines and macrophage markers and clinicopathological features were performed using Spearman's test. Plasmatic levels of chemokines and inflammatory mediators' vascular endothelial growth factor and tumor necrosis factor-α were evaluated by Luminex. Tumor levels of CCL2 (mean ± standard deviation = 530.1 ± 613.9 pg/mg), CCL3 (102.7 ± 106.0 pg/mg), and CCL4 (64.98 ± 48.09 pg/mg) were higher than those found in healthy tissue (182.1 ± 116.5, 26.79 ± 22.40, and 27.06 ± 23.69 pg/mg, respectively p < 0.05). The tumor characterization allowed us to identify a positive correlation between CCL4 and the pro-tumor macrophages marker CD163 ( p = 0.0443), and a negative correlation of iNOS with desmoplastic reaction ( p = 0.0467). Moreover, we identified that tumors with immature desmoplasia have a higher CD163 density compared to those with a mature/intermediated stromal tissue ( p = 0.0288). Plasmatic CCL4 has shown a positive correlation with inflammatory mediators (tumor necrosis factor-α and vascular endothelial growth factor) that have previously been associated with poor prognosis in patients. In conclusion High expression of CCL4 in colon cancer could induce the infiltration of tumor-associated macrophages and specifically a pro-tumor macrophage profile (CD163+ cells). Moreover, plasmatic chemokines could be considered inflammatory mediators associated to CRC progression as well as tumor necrosis factor-α and vascular endothelial growth factor. These data reinforce the idea of chemokines as potential therapeutic targets or biomarker in CRC.

Specific clinical and immune features of CD68 in glioma via 1,024 samples.

BackgroundThere is a growing recognition that tumor-associated macrophages (TAMs) are recruited to the glioma environment, facilitating tumor proliferation and migration by creating an immunosuppressive microenvironment. CD68 has been widely reported as a specific marker of TAMs in cancer.PurposeTo clarify the role of CD68 in glioma, we investigated its function at the transcriptome level and relationship with clinical practice.Patients and methodsIn total, 325 RNA-seq data from Chinese Glioma Genome Atlas (CGGA) and 697 RNA-seq data from The Cancer Genome Atlas (TCGA) network were enrolled in this study. CD68-specific findings were further analyzed with R language, and the prognostic impacts were validated through analyzing the overall survival (OS).ResultsCD68 showed a positive correlation with the WHO grade of malignancy in glioma. Meanwhile, CD68 was predominantly expressed in IDH wide type and mesenchymal subtype. Gene ontology (GO) analysis revealed that CD68-related genes were closely related to inflammatory response and immune response. Moreover, seven cultures of metagenes further confirmed that CD68 was a specific marker for macrophages in inflammatory response and played an important role in suppressing T-cell-mediated immunity. The Pearson correlation test suggested that CD68 showed robust correlation with other markers of macrophages and immune checkpoints, including PD-1 and TIM-3. Clinically, a high expression level of CD68 in tumors exhibited a poor survival in glioma patients.ConclusionOur results demonstrated that CD68 acted as an immune suppressor and contributed to glioma progression in the tumor microenvironment. These findings may expand our understanding of CD68-specific clinical and immune features in glioma.

Correlation between the infiltration of tumor-associated macrophages in the tissues of breast carcinoma and the expressions of vascular endothelial growth factors

Objective To investigate the correlation between the infiltration of tumor-associated macrophages (TAM) in breast cancer and the expressions of vascular endothelial growth factors (VEGF). Methods The expressions of CD163 (TAM marker) and VEGF in 45 postoperative tissue specimens of primary breast cancer in Affiliated Hospital of Guilin Medical University from January 2014 to January 2018 were examined by using immunohistochemistry EnVision method. Then TAM was counted under light microscope and the expression of VEGF was determined by using semi quantitative integration method. Correlation between the expression of TAM and VEGF and their relationships with clinicopathological parameters were also analyzed. Results TAM infiltration (the number of TAM under per high power field) in breast cancer patients (≤ 51 years old) was significantly more than that in breast cancer patients (> 51 years old) [(78.1±11.9)/HP vs. (69.7±14.0)/HP, t=2.167, P=0.036]. TAM infiltration in breast cancer patients with lymph node metastasis was more than that in breast cancer patients without lymph node metastasis [(79.2±11.8)/HP vs. (70.2±13.6)/HP, t=2.362, P=0.023]. The positive rate of VEGF in breast cancer with lymph node metastasis was significantly higher than that in breast cancer without lymph node metastasis [100.00% (20/20) vs. 68.00% (17/25), χ2= 5.749, P=0.017]. Pearson correlation analysis showed that the expression of VEGF was positively correlated with TAM infiltration (r 2=0.800, P < 0.05). Conclusion TAM infiltration and the expression of VEGF can be used to predict the malignant degree of breast cancer, and can be used as a potential intervention target for adjuvant therapy and clinical prognosis of breast cancer. Key words: Breast neoplasms; Tumor-associated macrophages; Vascular endothelial growth factors; Immunohistochemistry

Colon cancer-derived conditioned medium induces differentiation of THP-1 monocytes into a mixed population of M1/M2 cells.

Macrophages play an important role in the immune response and in the maintenance of tissue homeostasis. It is well known that many tumors recruit monocytes from circulation and influence their differentiation, mainly into suppressive M2-like subsets. Since there are contradictory data concerning the importance of macrophages for colon cancer progression, we used in our experiments four colon cancer cell lines representing different stages of tumor development (HT29, LS180, SW948, SW620). An acute monocytic leukemia cell line THP-1 was used as a human model of monocytes. Our work revealed that conditioned medium from the tumor cell lines induced activation and differentiation of THP-1 cells. The changes involved increased expression of CD68, a macrophage differentiation marker. Moreover, we also observed increased expression of CD206 and CD163, which are widely considered as markers of tumor-associated macrophages. The tumor-derived conditioned medium decreased the proliferation of THP-1 cells and blocked their cell cycle at the G1 stage. The tumor-conditioned medium also upregulated the production of several cytokines and chemokines characteristic of both M1 and M2 subsets and induced the expression of important pro-angiogenic factors, vascular endothelial growth factor, and matrix metalloproteinase-9 in THP-1 cells. Moreover, the tumor-conditioned medium induced the expression of galectin-3, which is implicated in malignant transformation, and indoleamine 2,3-dioxygenase, that is, a key enzyme of the kynurenine pathway. Our data suggest that tumor cells can actively influence the phenotype of monocytes and switch their differentiation into a population of non-adherent mixed M1 and M2 cells. These preliminary studies suggest that colon cancer cells produce soluble factors that influence monocyte differentiation, most probably into suppressive subsets. These data provide a better understanding of the influence of colon cancer on polarization of monocytes.

IL-6 augments IL-4-induced polarization of primary human macrophages through synergy of STAT3, STAT6 and BATF transcription factors

ABSTRACTMacrophages in the tumor microenvironment respond to complex cytokine signals. How these responses shape the phenotype of tumor-associated macrophages (TAMs) is incompletely understood. Here we explored how cytokines of the tumor milieu, interleukin (IL)-6 and IL-4, interact to influence target gene expression in primary human monocyte-derived macrophages (hMDMs). We show that dual stimulation with IL-4 and IL-6 synergistically modified gene expression. Among the synergistically induced genes are several targets with known pro-tumorigenic properties, such as CC-chemokine ligand 18 (CCL18), transforming growth factor alpha (TGFA) or CD274 (programmed cell death 1 ligand 1 (PD-L1)). We found that transcription factors of the signal transducer and activator of transcription (STAT) family, STAT3 and STAT6 bind regulatory regions of synergistically induced genes in close vicinity. STAT3 and STAT6 co-binding further induces the basic leucine zipper ATF-like transcription factor (BATF), which participates in synergistic induction of target gene expression. Functional analyses revealed increased MCF-7 and MDA-MB 231 tumor cell motility in response to conditioned media from co-treated hMDMs compared to cells incubated with media from single cytokine-treated hMDMs. Flow cytometric analysis of T cell populations upon co-culture with hMDMs polarized by different cytokines indicated that dual stimulation promoted immunosuppressive properties of hMDMs in a PD-L1-dependent manner. Analysis of clinical data revealed increased expression of BATF together with TAM markers in tumor stroma of breast cancer patients as compared to normal breast tissue stroma. Collectively, our findings suggest that IL-4 and IL-6 cooperate to alter the human macrophage transcriptome, endowing hMDMs with pro-tumorigenic properties.

Positive correlation between the density of macrophages and T-cells in undifferentiated sarcoma.

Undifferentiated sarcoma (US) is a frequent soft tissue sarcoma. Although the 10-year survival rate is around 60%, advanced US is highly resistant to chemo/radiotherapy. The tumor microenvironment (TME) is closely associated with tumor progression. However, few studies of infiltrated immune cells in US have been published. In this study, we evaluated tumor-associated macrophages (TAMs) and CD8-positive cytotoxic T lymphocytes (CTLs) in 28 cases of US. Iba1, CD163, and CD204 were used as markers for TAMs. The density of CTLs was positively correlated with the density of TAMs. However, a negative correlation was seen between the density of CTLs and the percentage of CD204-positive TAMs. We found no significant association between the density of Iba1-/CD204-/CD8-positive cells and clinicopathological factors. No significant correlation between immune cell infiltration and clinical outcome was observed. Although we found no significant association between immune cells and clinicopathological factors, these findings may provide new insight into the characterization of immune cells in the TME of US.

Abstract 1107: Cell-surface vimentin-positive macrophages like CTCs as novel biomarkers of metastatic gastrointestinal stromal tumors

Abstract Background: Gastrointestinal stromal tumors (GISTs) are the most common gastrointestinal tract sarcomas and are heavily infiltrated with tumor-associated macrophages (TAMs). Biomarkers specifically capturing circulating tumor cells (CTCs) that can be used to noninvasively determine the disease status in GIST patients have been lacking. Patients and Methods: To fill this gap, we captured both regular CTCs and TAMs from GIST patients' blood and tumor samples using our proprietary cell-surface vimentin (CSV) antibody 84-1 and the TAM markers CD14 and CD68 based on our previous established CTC detection method from 104 localized or metastatic sarcoma patients and 10 healthy donors. Also, freshly procured metastatic GIST tissues were obtained from four patients. Results: We defined a rare population of CSV+ macrophage-like CTCs (ML-CTCs) in metastatic GIST patients' blood samples with expression of CD14 and CD68 but not CD45. Also, we demonstrated CSV+ ML-CTCs to be tumor microenvironment-derived. Metastatic GIST patients had markedly higher numbers of CSV+ ML-CTCs than localized GIST patients and healthy blood donors. However, regular CTC counts could not predict metastasis of GISTs but could for other types of sarcoma. Notably, CSV+ ML-CTCs were represented as M1-like macrophages in their phenotype and function. Conclusions: CSV+ ML-CTCs are novel biomarkers for prediction of metastatic risk and therapeutic response in GIST patients. Routinely monitoring these cells will be an important approach to liquid biopsy analysis of GISTs. Citation Format: Heming Li, Qing H. Meng, Hyangsoon Noh, Neeta Somaiah, Keila E. Torres, Xueqing Xia, Izhar Singh Batth, Cissimol P. Joseph, Zachary A. Mulder, Ruoyu Wang, Shulin Li. Cell-surface vimentin-positive macrophages like CTCs as novel biomarkers of metastatic gastrointestinal stromal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1107.

Expression of Adipocyte/Macrophage Fatty Acid-Binding Protein in Tumor-Associated Macrophages Promotes Breast Cancer Progression.

Tumor-associated macrophages (TAM) play a critical role in cancer development and progression. However, the heterogeneity of TAM presents a major challenge to identify clinically relevant markers for protumor TAM. Here, we report that expression of adipocyte/macrophage fatty acid-binding protein (A-FABP) in TAM promotes breast cancer progression. Although upregulation of A-FABP was inversely associated with breast cancer survival, deficiency of A-FABP significantly reduced mammary tumor growth and metastasis. Furthermore, the protumor effect of A-FABP was mediated by TAM, in particular, in a subset of TAM with a CD11b+F4/80+MHCII-Ly6C- phenotype. A-FABP expression in TAM facilitated protumor IL6/STAT3 signaling through regulation of the NFκB/miR-29b pathway. Collectively, our results suggest A-FABP as a new functional marker for protumor TAM.Significance: These findings identify A-FABP as a functional marker for protumor macrophages, thus offering a new target for tumor immunotherapy. Cancer Res; 78(9); 2343-55. ©2018 AACR.

CD317 Signature in Head and Neck Cancer Indicates Poor Prognosis

Targeted therapy using monoclonal antibodies (mAbs) has emerged as a widely used form of immunotherapy in head and neck squamous cell carcinoma (HNSCC). Membrane-associated glycoprotein CD317 has been preferentially overexpressed by multiple myeloma cells, and its humanized mAb has been previously used in clinical trials. However, overexpression of CD317 in HNSCC and its correlation with tumor immunity is still uncertain. Here, the immunoreactivity of CD317 was detected in human HNSCC tissue microarrays, which contained 43 oral mucosa samples, 48 dysplasia samples, and 165 primary HNSCC. We found that CD317 expression was up-regulated in HNSCC tumor cells, and the CD317 expression level was independent of the histological grade, tumor size, and lymph node metastasis. Moreover, Kaplan–Meier survival curve analysis showed that patients with high expression of CD317 had a poor prognosis compared with patients with low expression. Furthermore, CD317 overexpression in HNSCC was correlated with immune checkpoint molecules PD-L1, B7-H3, and B7-H4 and tumor-associated macrophage markers (CD68 and CD163). We also observed that CD317 was overexpressed in immunocompetent mouse HNSCC tissue compared with normal tissue. Taken together, our findings demonstrate that CD317 overexpression indicates poor prognosis and is correlated with immune-related components in this patient cohort. CD317 may serve as a potential target for effective immunotherapy of HNSCC.

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.

Promotion of Sema4D expression by tumor-associated macrophages: Significance in gastric carcinoma.

AIMTo study the role of semaphorin 4D (Sema4D) expression promoted by tumor-associated macrophages (TAMs) in gastric carcinoma cells and its clinical significance in the invasion and metastasis of gastric carcinoma.METHODSCD68 and Sema4D expression was analyzed in gastric carcinoma and adjacent normal tissues from 290 patients using the immunohistochemical streptavidin-peroxidase method, and their relationships with clinicopathological features were evaluated. Human M2 macrophages were induced in vitro and co-cultured in non-contact with gastric carcinoma SGC-7901 cells. Changes in the secretory Sema4D level in the SGC-7901 cell supernatant were measured using an enzyme-linked immunosorbent assay. The effects of TAMs on SGC-7901 cell invasion and migration were assessed with invasion and migration assays, respectively.RESULTSCD68 and Sema4D protein expression was significantly higher in gastric carcinoma tissues than in adjacent normal tissues (71.7% vs 33.8% and 74.5% vs 42.8%, respectively; P < 0.01). CD68 and Sema4D protein expression was significantly associated with histological differentiation, TNM stage, and lymph node metastasis (P < 0.05), and their expression levels were positively correlated with one another (r = 0.467, P < 0.01). In the in vitro experiment, secretory Sema4D protein expression was significantly increased in the supernatant of SGC-7901 cells co-cultured with TAMs compared with the blank control (1224.13 ± 29.43 vs 637.15 ± 33.84, P < 0.01). Cell invasion and metastasis were enhanced in the Transwell invasion and migration assays (P < 0.01).CONCLUSIONTAMs promote the invasion and metastasis of gastric carcinoma cells possibly through upregulated secretory Sema4D protein expression. Combined detection of TAM markers, CD68 and Sema4D, in gastric carcinoma tissue shows potential to predict the trend of gastric carcinoma progression.

Blockage of the NLRP3 inflammasome by MCC950 improves anti-tumor immune responses in head and neck squamous cell carcinoma.

The NLRP3 inflammasome is a critical innate immune pathway responsible for producing active interleukin (IL)-1β, which is associated with tumor development and immunity. However, the mechanisms regulating the inflammatory microenvironment, tumorigenesis and tumor immunity are unclear. Herein, we show that the NLRP3 inflammasome was over-expressed in human HNSCC tissues and that the IL-1β concentration was increased in the peripheral blood of HNSCC patients. Additionally, elevated NLRP3 inflammasome levels were detected in tumor tissues of Tgfbr1/Pten 2cKO HNSCC mice, and elevated IL-1β levels were detected in the peripheral blood serum, spleen, draining lymph nodes and tumor tissues. Blocking NLRP3 inflammasome activation using MCC950 remarkably reduced IL-1β production in an HNSCC mouse model and reduced the numbers of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and tumor-associated macrophages (TAMs). Moreover, inhibiting NLRP3 inflammasome activation increased the numbers of CD4+ and CD8+ T cells in HNSCC mice. Notably, the numbers of exhausted PD-1+ and Tim3+ T cells were significantly reduced. A human HNSCC tissue microarray showed that NLRP3 inflammasome expression was correlated with the expression of CD8 and CD4, the Treg marker Foxp3, the MDSC markers CD11b and CD33, and the TAM markers CD68 and CD163, PD-1 and Tim3. Overall, our results demonstrate that the NLRP3 inflammasome/IL-1β pathway promotes tumorigenesis in HNSCC and inactivation of this pathway delays tumor growth, accompanied by decreased immunosuppressive cell accumulation and an increased number of effector T cells. Thus, inhibition of the tumor microenvironment through the NLRP3 inflammasome/IL-1β pathway may provide a novel approach for HNSCC therapy.

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.

Effects of Obesity on the Regulation of Macrophage Population in the Prostate Tumor Microenvironment

ABSTRACTObesity is associated with a greater risk of prostate cancer mortality. However, the mechanisms connecting obesity to the progression of prostate cancer remain unknown. This study determined the impact of obesity on macrophage recruitment and tumor-associated macrophage (TAM) polarization in the prostate tumor microenvironment, since a high concentration of TAMs in tumors has been linked to progression in prostate cancer. We utilized an in vitro model in which pre-adipocytes, prostate cancer cells, and macrophages were exposed to sera from obese or nonobese men, or conditioned media generated under obese or nonobese conditions. Matrigel invasion chambers were used to assess macrophage recruitment in vitro, and immunohistochemical analysis evaluated recruitment in a PTEN knockout mouse model. qPCR was used to measure mRNA levels of CCL2, COX-2, IL-10, TGF-beta, VEGF-A, arginase-1, and MMP-9. PGE2 production was measured by ELISA. Obesity increased macrophage and TAM recruitment, and increased mRNA levels of TAM markers in macrophages. Similarly, obese conditions increased CCL2 and COX-2 expression, as well as PGE2 levels in prostate cancer cells. COX-2 inhibition resulted in lower expression of obesity-induced TAM markers. Our data suggest that obesity promotes macrophage infiltration into the prostate tumor microenvironment, and induces TAM polarization through the COX-2/PGE2 pathway.

Quantitative measurements of intercellular adhesion between a macrophage and cancer cells using a cup-attached AFM chip

Intercellular adhesion between a macrophage and cancer cells was quantitatively measured using atomic force microscopy (AFM). Cup-shaped metal hemispheres were fabricated using polystyrene particles as a template, and a cup was attached to the apex of the AFM cantilever. The cup-attached AFM chip (cup-chip) approached a murine macrophage cell (J774.2), the cell was captured on the inner concave of the cup, and picked up by withdrawing the cup-chip from the substrate. The cell-attached chip was advanced towards a murine breast cancer cell (FP10SC2), and intercellular adhesion between the two cells was quantitatively measured. To compare cell adhesion strength, the work required to separate two adhered cells (separation work) was used as a parameter. Separation work was almost 2-fold larger between a J774.2 cell and FP10SC2 cell than between J774.2 cell and three additional different cancer cells (4T1E, MAT-LyLu, and U-2OS), two FP10SC2 cells, or two J774.2 cells. FP10SC2 was established from 4T1E as a highly metastatic cell line, indicates separation work increased as the malignancy of cancer cells became higher. One possible explanation of the strong adhesion of macrophages to cancer cells observed in this study is that the measurement condition mimicked the microenvironment of tumor-associated macrophages (TAMs) in vivo, and J774.2 cells strongly expressed CD204, which is a marker of TAMs. The results of the present study, which were obtained by measuring cell adhesion strength quantitatively, indicate that the fabricated cup-chip is a useful tool for measuring intercellular adhesion easily and quantitatively.

OA10.05 EGFR Gene Mutations Affect Tumor-Infiltrating Stromal Cell Components in Early-Stage Lung Adenocarcinoma

Tumors are complex structures consisting of cancer cells surrounded by a tumor stroma that is now recognized to be critical for cancer progression. Although epidermal growth factor receptor (EGFR) mutations are frequently observed in non-small cell lung carcinoma, it remains poorly understood whether EGFR mutations in cancer cells affect the tumor stroma. In this study, we studied the status of EGFR mutations in early-stage lung adenocarcinoma and analyzed the relations of EGFR mutations to tumor-infiltrating stromal cell components. A total of 152 consecutive patients with clinical stage IA lung adenocarcinoma who underwent complete tumor resection in Keio University Hospital between 2010 and 2014 were studied. Genomic DNA was isolated from formalin-fixed, paraffin-embedded tumor sections and mutational analyses of EGFR gene exons 19, 20 and 21 were performed by a polymerase chain reaction-based method. Paraffin sections were also subjected to immunohistochemistry for CD3, FOXP3, CD163 or CD204. Numbers of CD3-, FOXP3-, CD163- or CD204-immunostained cells were counted by observing 5 different fields at x200 magnification. EGFR mutations were detected in 71 (47%) of the 152 patients with clinical stage IA lung adenocarcinoma and were found more frequently in women and non-smokers. These contained 38 patients with missense mutations in exon 21 (L858R) and 30 patients with deletions in exon 19. By immunohistochemistry, the number of stromal macrophages positive for CD163 or CD204, markers for tumor-associated macrophages (TAMs), was significantly decreased within tumors with EGFR mutations compared to within those with wild-type EGFR, whereas the number of CD3+ T cells or FOXP3+ regulatory T cells was comparable between these groups. Both tumors with missense mutations in exon 21 and deletions in exon 19 had a similar trend toward decreased TAMs in the tumor stroma. Our data suggest that EGFR mutations in early-stage lung adenocarcinoma are associated with decreased TAMs in the tumor stroma. EGFR mutation status might act on not only cancer cell behavior but tumor microenvironment.

CD68/macrosialin: not just a histochemical marker

CD68 is a heavily glycosylated glycoprotein that is highly expressed in macrophages and other mononuclear phagocytes. Traditionally, CD68 is exploited as a valuable cytochemical marker to immunostain monocyte/macrophages in the histochemical analysis of inflamed tissues, tumor tissues, and other immunohistopathological applications. CD68 alone or in combination with other cell markers of tumor-associated macrophages showed a good predictive value as a prognostic marker of survival in cancer patients. Lowression of CD68 was found in the lymphoid cells, non-hematopoietic cells (fibroblasts, endothelial cells, etc), and tumor cells. Cell-specific CD68 expression and differentiated expression levels are determined by the complex interplay between transcription factors, regulatory transcriptional elements, and epigenetic factors. Human CD68 and its mouse ortholog macrosialin belong to the family of LAMP proteins located in the lysosomal membrane and share many structural similarities such as the presence of the LAMP-like domain. Except for a second LAMP-like domain present in LAMPs, CD68/microsialin has a highly glycosylated mucin-like domain involved in ligand binding. CD68 has been shown to bind oxLDL, phosphatidylserine, apoptotic cells and serve as a receptor for malaria sporozoite in liver infection. CD68 is mainly located in the endosomal/lysosomal compartment but can rapidly shuttle to the cell surface. However, the role of CD68 as a scavenger receptor remains to be confirmed. It seems that CD68 is not involved in binding bacterial/viral pathogens, innate, inflammatory or humoral immune responses, although it may potentially be involved in antigen processing/presentation. CD68 could be functionally important in osteoclasts since its deletion leads to reduced bone resorption capacity. The role of CD68 in atherosclerosis is contradictory.