Increase in tumor-associated macrophages after antiangiogenic therapy is associated with poor survival among patients with recurrent glioblastoma

Antiangiogenic therapy is associated with increased radiographic responses in glioblastomas, but tumors invariably recur. Because tumor-associated macrophages have been shown to mediate escape from antiangiogenic therapy in preclinical models, we examined the role of macrophages in patients with recurrent glioblastoma. We compared autopsy brain specimens from 20 patients with recurrent glioblastoma who received antiangiogenic treatment and chemoradiation with 8 patients who received chemotherapy and/or radiotherapy without antiangiogenic therapy or no treatment. Tumor-associated macrophages were morphologically and phenotypically analyzed using flow cytometry and immunohistochemistry for CD68, CD14, CD163, and CD11b expression. Flow cytometry showed an increase in macrophages in the antiangiogenic-treated patients. Immunohistochemical analysis demonstrated an increase in CD68+ macrophages in the tumor bulk (P < .01) and infiltrative areas (P = .02) in antiangiogenic-treated patients. We also observed an increase in CD11b+ cells in the tumor bulk (P < .01) and an increase in CD163+ macrophages in infiltrative tumor (P = .02). Of note, an increased number of CD11b+ cells in bulk and infiltrative tumors (P = .05 and P = .05, respectively) correlated with poor overall survival among patients who first received antiangiogenic therapy at recurrence. In summary, recurrent glioblastomas showed an increased infiltration in myeloid populations in the tumor bulk and in the infiltrative regions after antiangiogenic therapy. Higher numbers of CD11b+ cells correlated with poor survival among these patients. These data suggest that tumor-associated macrophages may participate in escape from antiangiogenic therapy and may represent a potential biomarker of resistance and a potential therapeutic target in recurrent glioblastoma.     

Tumor-Associated Macrophages are Correlated with Tamoxifen Resistance in the Postmenopausal Breast Cancer Patients

Tumor-associated macrophages (TAMs) have been correlated with increased angiogenesis and poor prognosis in breast cancer. However, the precise role of TAMs in tamoxifen resistance remains unclear. We used immunohistochemical method to examine the expression of epidermal growth factor receptor (EGFR) and CD163+ macrophages in 100 breast cancer tissues. The clinical and biological features of 100 patients were estrogen receptor (ER)-positive and human epidermal growth factor receptor 2(Her-2)-negative tumors. The tamoxifen resistant tissues (n?=?48) were the surgical excision samples from patients who developed recurrence or metastasis at the time of adjuvant tamoxifen treatment. The tamoxifen resistant tissues were contrast to tamoxifen sensitive tissues (n?=?52). Positive staining for EGFR and CD163+ macrophages were observed in 21 samples (43.8 %) and in 26 samples (54.2 %) respectively in tamoxifen resistance group, which were higher than that of tamoxifen sensitive group (P?=?0.001 and P?=?0.000279 respectively). Significant positive correlations were found between the expression of EGFR and CD163+ macrophages (r?=?0.567, P?<?0.01). CD163+ macrophages were positively correlated with tumor size, lymph node metastasis and obesity. Obesity was also related to tamoxifen resistance (P?<?0.05). The patients with higher density of CD163+ macrophages infiltration suffered from shorter time to develop recurrence or metastasis (P?<?0.05). TAMs may be associated with tamoxifen resistance. Further studies are needed to investigate the potential mechanism between TAMs and tamoxifen resistance.     

High PTX3 expression is associated with a poor prognosis in diffuse large B‐cell lymphoma

Tumor‐associated macrophages (TAMs) are associated with a poor prognosis of diffuse large B‐cell lymphoma (DLBCL). As macrophages are heterogeneous, the immune polarization and their pathological role warrant further study. We characterized the microenvironment of DLBCL by immunohistochemistry in a training set of 132 cases, which included 10 Epstein–Barr virus‐encoded small RNA (EBER)‐positive and five high‐grade B‐cell lymphomas, with gene expression profiling in a representative subset of 37 cases. Diffuse large B‐cell lymphoma had a differential infiltration of TAMs. The high infiltration of CD68 (pan‐macrophages), CD16 (M1‐like), CD163, pentraxin 3 (PTX3), and interleukin (IL)‐10‐positive macrophages (M2c‐like) and low infiltration of FOXP3‐positive regulatory T lymphocytes (Tregs) correlated with poor survival. Activated B cell‐like DLBCL was associated with high CD16, CD163, PTX3, and IL‐10, and EBER‐positive DLBCL with high CD163 and PTX3. Programmed cell death‐ligand 1 positively correlated with CD16, CD163, IL‐10, and RGS1. In a multivariate analysis of overall survival, PTX3 and International Prognostic Index were identified as the most relevant variables. The gene expression analysis showed upregulation of genes involved in innate and adaptive immune responses and macrophage and Toll‐like receptor pathways in high PTX3 cases. The prognostic relevance of PTX3 was confirmed in a validation set of 159 cases. Finally, in a series from Europe and North America ({"type":"entrez-geo","attrs":{"text":"GSE10846","term_id":"10846"}}GSE10846, R‐CHOP‐like treatment, n = 233) high gene expression of PTX3 correlated with poor survival, and moderately with CSF1R, CD16, MITF, CD163, MYC, and RGS1. Therefore, the high infiltration of M2c‐like immune regulatory macrophages and low infiltration of FOXP3‐positive Tregs is associated with a poor prognosis in DLBCL, for which PTX3 is a new prognostic biomarker.     

Evaluation of the phenotype pattern of macrophages isolated from malignant and non-malignant pleural effusions

Macrophages are among the infiltrate components of most malignant tumors. Tumor-associated macrophages (TAMs) may secrete a variety of humoral factors, which promote or inhibit tumor growth. In general, depending on their activation pathway, macrophages exhibit two different patterns of phenotype, M1 or M2. It is assumed that TAMs comprise pattern M2. In the malignant pleural effusion, macrophages are a frequent component of cytological evaluation. In this microenvironment, TAMs could be involved in the development of immunity. The phenotype of macrophages represented in malignant and non-malignant pleural effusions is unknown. In this study, macrophages were isolated from 38 pleural effusions (15 malignant and 23 non-malignant) and the expression of a variety of immune mediators and their receptors was assessed to determine the type of activation (M1 vs. M2). The expression of mRNA was analyzed for IL-1β, IL-4, IL-6, IL-10, IL-11, IL-18, TNFα, TGFβ1, IL1R1, IL1RAP, TLR2, TLR4, VLA4, CD62L, MMP2, MMP9, VEGFA, PDGFA, and PDGFB. In immunohistochemical evaluation, the expressions of CD68, mesothelin, MAC387, IL-1β, IL-6, IL-10, IL-12, TNFα, and CD105 were assessed. The cytoplasmic expression of IFNγ, TNFα, IL-6, and IL-10 and the surface expression of CD11a, CD14, CD15, CD16, CD23, CD25, CD45, CD54, CD62L, CD69, VLA2, VLA3, VLA4, VLA6, TLR2, TLR4, and CCR7 were tested using flow cytometry. In supernatants from macrophages cultures, TNFα, IL-1β, IL-6, IL-8, IL-10, IL-12, MCP1, and VEGF were investigated by cytometric beads array method (CBA flex sets) and TGFβ1 by ELISA. Our results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other and suggest that macrophages isolated from non-malignant effusions show a pattern comparable to M1 while those isolated from malignant effusions express similarity to M2 phenotype, but they have not shown a classical M2 pattern.     

Human breast cancer cells educate macrophages toward the M2 activation status

Introduction

The immune system plays a major role in cancer progression. In solid tumors, 5-40 % of the tumor mass consists of tumor-associated macrophages (TAMs) and there is usually a correlation between the number of TAMs and poor prognosis, depending on the tumor type. TAMs usually resemble M2 macrophages. Unlike M1-macrophages which have pro-inflammatory and anti-cancer functions, M2-macrophages are immunosuppressive, contribute to the matrix-remodeling, and hence favor tumor growth. The role of TAMs is not fully understood in breast cancer progression.

Methods

Macrophage infiltration (CD68) and activation status (HLA-DRIIα, CD163) were evaluated in a large cohort of human primary breast tumors (562 tissue microarray samples), by immunohistochemistry and scored by automated image analysis algorithms. Survival between groups was compared using the Kaplan-Meier life-table method and a Cox multivariate proportional hazards model. Macrophage education by breast cancer cells was assessed by ex vivo differentiation of peripheral blood mononuclear cells (PBMCs) in the presence or absence of breast cancer cell conditioned media (MDA-MB231, MCF-7 or T47D cell lines) and M1 or M2 inducing cytokines (respectively IFN-γ, IL-4 and IL-10). Obtained macrophages were analyzed by flow cytometry (CD14, CD16, CD64, CD86, CD200R and CD163), ELISA (IL-6, IL-8, IL-10, monocyte colony stimulating factor M-CSF) and zymography (matrix metalloproteinase 9, MMP-9).

Results

Clinically, we found that high numbers of CD163⁺ M2-macrophages were strongly associated with fast proliferation, poor differentiation, estrogen receptor negativity and histological ductal type (p<0.001) in the studied cohort of human primary breast tumors. We demonstrated ex vivo that breast cancer cell-secreted factors modulate macrophage differentiation toward the M2 phenotype. Furthermore, the more aggressive mesenchymal-like cell line MDA-MB231, which secretes high levels of M-CSF, skews macrophages toward the more immunosuppressive M2c subtype.

Conclusions

This study demonstrates that human breast cancer cells influence macrophage differentiation and that TAM differentiation status correlates with recurrence free survival, thus further emphasizing that TAMs can similarly affect therapy efficacy and patient outcome.

Electronic supplementary material

The online version of this article (doi:10.1186/s13058-015-0621-0) contains supplementary material, which is available to authorized users.     

Head and neck cancer relapse after chemoradiotherapy correlates with CD163+ macrophages in primary tumour and CD11b+ myeloid cells in recurrences

Background:

We investigated the prognostic role of tumour-associated macrophages (TAMs) in patients with head and neck squamous cell carcinoma (HNSCC) treated with definitive chemoradiotherapy (CRT).

Methods:

The expression of CD68+, CD163+ and CD11b+ cells was assessed using immunohistochemistry in n=106 pre-treatment tumour biopsy samples and was correlated with clinicopathological characteristics, including T-stage, N-stage, grading, tumour localisation, age and sex as well as local failure-free survival (LFFS), distant metastases-free survival (DMFS), progression-free (PFS), and overall survival (OS). Finally, TAMs expression and vessel density (CD31) were examined in n=12 available early local recurrence samples and compared with their matched primary tumours . The diagnostic images and radiotherapy plans of these 12 patients were also analysed. All local recurrences occurred in the high radiation dose region (⩾70 Gy).

Results:

With a median follow-up of 40 months, OS at 2 years was 60.5%. High CD163 expression in primary tumours was associated with decreased OS (P=0.010), PFS (P=0.033), LFFS (P=0.036) and DMFS (P=0.038) in multivariate analysis. CD163 demonstrated a strong prognostic value only in human papillomavirus (p16^INK4)-negative patients. Early local recurrence specimens demonstrated a significantly increased infiltration of CD11b+ myeloid cells (P=0.0097) but decreased CD31-positive vessel density (P=0.0004) compared with their matched primary samples.

Conclusions:

Altogether, baseline CD163 expression predicts for an unfavourable clinical outcome in HNSCC after definitive CRT. Early local recurrences showed increased infiltration by CD11b+ cells. These data provide important insight on the role of TAMs in mediating response to CRT in patients with HNSCC.     

Clinical significance of CD163+ tumor‐associated macrophages in patients with adult T‐cell leukemia/lymphoma

In several malignant tumors including lymphoma, macrophages that infiltrate tumor tissues are called tumor‐associated macrophages (TAMs). We discovered that TAMs, especially the CD163⁺ alternatively activated phenotype (M2), were closely involved with progression of adult T‐cell leukemia/lymphoma (ATLL). We used CD68 (a pan‐macrophage marker) and CD163 (an M2 marker) to immunostain 58 ATLL samples. Statistical analyses showed that a high number of CD68⁺ TAMs and an increased percentage of CD163⁺ cells among the TAMs were associated with a worse clinical prognosis; multivariate analysis indicated that the percentage of CD163⁺ cells was an independent prognostic factor. We also carried out in vitro coculture experiments with ATLL cell lines (ATN‐1 and TL‐Mor) and monocyte‐derived macrophages and found that direct coculture with M2 macrophages significantly increased BrdU incorporation into ATLL cell lines. A cytokine array analysis showed that macrophage‐derived soluble factors including C5a, tumor necrosis factor‐α, growth‐related oncogene‐α, CCL1/I‐309, and interleukin‐6 stimulated ATLL cell lines. CD163 expression in macrophages was strongly induced by direct contact with ATN‐1 cells, and downregulation of CD163 in macrophages significantly suppressed growth of cocultured ATN‐1 cells. These results suggest that interaction between M2 macrophages and lymphoma cells may be an appropriate target in treatment of patients with ATLL.     

High-fat diet-fed ovariectomized mice are susceptible to accelerated subcutaneous tumor growth potentially through adipose tissue inflammation,local insulin-like growth factor release,and tumor associated macrophages

Background: The association between obesity and colorectal cancer (CRC) risk has been well established. This relationship appears to be more significant in men than in women, which may be attributable to sex hormones. However, controlled animal studies to substantiate these claims and the mechanisms involved are lacking.Materials and Methods: MC38 murine colon adenocarcinoma cells were injected subcutaneously into high-fat diet (HFD) fed male, female and ovariectomized (OVX) female C57BL/6 mice.Results: HFD increased tumor growth (main effect) that was consistent with metabolic perturbations (P < 0.01). HFD OVX mice exhibited the most significant tumor growth compared to HFD male and female mice (p < 0.05) and this was associated with increased subcutaneous adipose tissue (p < 0.05). Further, the subcutaneous adipose tissue depots within HFD OVX mice exhibited more severe macrophage associated inflammation compared to female (P < 0.01), but not male mice. Conditioned media from subcutaneous adipose tissue of HFD OVX contained higher IGF-1 levels compared to male (P < 0.01), but not female mice. Finally, HFD OVX mice had increased M2-like gene expression in their tumor-associated macrophages (TAMs) compared to female mice (P < 0.01).Conclusions: This work provides evidences suggesting adiposity, adipose specific IGF-1, macrophage associated adipose inflammation, and TAMs as potential mechanisms driving obesity-enhanced CRC in females lacking ovarian hormones.     

Tumour-associated CD66b+ neutrophil count is an independent prognostic factor for recurrence in localised cervical cancer

Background:

The prognostic impact of tumour-promoting immune cells in cervical cancer is unclear.

Methods:

Federation of Gynaecology and Obstetrics (FIGO) stage IB and IIA cervical cancer patients (N=101) were assessed for tumour-associated CD66b⁺ neutrophils and CD163⁺ macrophages by immunohistochemistry in whole tissue sections using stereology. Results were correlated with previous results on tumour-infiltrating CD3⁺, CD4⁺, and CD8⁺ lymphocytes in the same cohort with recurrence-free survival (RFS) as end point.

Results:

The highest densities of CD66b⁺ neutrophils and CD163⁺ macrophages were observed in the peritumoural compartment (median 53.1 cells mm⁻² and 1.3% area fraction, respectively). Above median peritumoural and stromal CD66b⁺ neutrophils and peritumoural CD163⁺ macrophages were significantly associated with short RFS. Multivariate analysis identified high peritumoural neutrophils (HR 2.27; 95% CI 1.09–4.75; P=0.03), low peritumoural CD8⁺ lymphocytes (HR 3.67; 95% CI 1.63–8.25; P=0.002), and lymph node metastases (HR 2.70; 95% CI 1.26–5.76; P=0.01) as independent prognostic factors for short RFS, whereas CD163⁺ macrophages were not significant. An index of combined intratumoral and peritumoral CD66b⁺ neutrophils to CD8⁺ lymphocytes had good discriminatory power for each quartile with 5-year RFS of 92%, 80%, 62%, and 44% (P=0.001).

Conclusion:

Tumour-associated neutrophil count is an independent prognostic factor for short RFS in localised cervical cancer. Combining CD66b and CD8 may further improve prognostic stratification. These findings require prospective validation.     

Impaired T cell function in malignant pleural effusion is caused by TGF‐β derived predominantly from macrophages

Malignant pleural effusion (MPE) is an indication of advanced cancer. Immune dysfunction often occurs in MPE. We aimed to identify the reason for impaired T cell activity in MPE from lung cancer patients and to provide clues toward potential immune therapies for MPE. The surface inhibitory molecules and cytotoxic activity of T cells in MPE and peripheral blood (PB) were analyzed using flow cytometry. Levels of inflammatory cytokines in MPE and PB were tested using ELISA. TGF‐β expression in tumor‐associated macrophages (TAMs) was also analyzed. The effect of TAMs on T cells was verified in vitro. Lastly, changes in T cells were evaluated following treatment with anti‐TGF‐β antibody. We found that expression levels of Tim‐3, PD‐1 and CTLA‐4 in T cells from MPE were upregulated compared with those from PB, but levels of IFN‐γ and Granzyme B were downregulated (p < 0.05). The amount of TGF‐β was significantly higher in MPE than in PB (p < 0.05). TGF‐β was mainly produced by TAMs in MPE. When T cells were co‐cultured with TAMs, expression levels of Tim‐3, PD‐1 and CTLA‐4 were significantly higher than controls, whereas levels of IFN‐γ and Granzyme B were significantly decreased, in a dose‐dependent manner (p < 0.05). In vitro treatment with anti‐TGF‐β antibody restored the impaired T cell cytotoxic activity in MPE. Our results indicate that macrophage‐derived TGF‐β plays an important role in impaired T cell cytotoxicity. It will therefore be valuable to develop therapeutic strategies against TGF‐β pathway for MPE therapy of lung cancer.     

Accumulation of FOXP3+T-cells in the tumor microenvironment is associated with an epithelial-mesenchymal-transition-type tumor budding phenotype and is an independent prognostic factor in surgically resected pancreatic ductal adenocarcinoma

Here we explore the role of the interplay between host immune response and epithelial-mesenchymal-transition (EMT)-Type tumor-budding on the outcome of pancreatic adenocarcinoma (PDAC).CD4+, CD8+, and FOXP3+T-cells as well as iNOS+ (M1) and CD163+-macrophages (M2) were assessed on multipunch tissue-microarrays containing 120 well-characterized PDACs, precursor lesions (PanINs) and corresponding normal tissue. Counts were normalized for the percentage of tumor/spot and associated with the clinico-pathological features, including peritumoral (PTB) and intratumoral (ITB) EMT-Type tumor-budding and outcome.Increased FOXP3+T-cell-counts and CD163-macrophages and decreased CD8+T-cell-counts were observed in PDACs compared with normal tissues and PanINs (p < 0.0001). Increased peritumoral FOXP3+T-cell-counts correlated significantly with venous invasion, distant metastasis, R1-status, high-grade ITB, PTB and independently with reduced survival. Increased intratumoral FOXP3+T-cells correlated with lymphatic invasion, N1-stage, PTB and marginally with adverse outcome. High peritumoral CD163-counts correlated with venous invasion, PTB and ITB. High intratumoral CD163-counts correlated with higher T-stage and PTB.PDAC-microenvironment displays a tumor-favoring immune-cell composition especially in the immediate environment of the tumor-buds that promotes further growth and indicates a close interaction of the immune response with the EMT-process. Increased peritumoral FOXP3+T-cell density is identified as an independent adverse prognostic factor in PDAC. Patients with phenotypically aggressive PDACs may profit from targeted immunotherapy against FOXP3.     

CD45RO+ Memory T Lymphocytes — a Candidate Marker for TNM-Immunoscore in Squamous Non–Small Cell Lung Cancer

Tumor-infiltrating lymphocytes (TILs) are vital in limiting cancer progression and may supplement the TNM classification. CD45RO⁺ memory TILs show major prognostic impact in various malignancies but have not been extensively explored in non–small cell lung cancer (NSCLC). In this study, we aimed to evaluate their potential in a NSCLC TNM-Immunoscore. Tissue microarrays were constructed from tumor tissue samples from two cohorts including in total 536 patients (University Hospital of North Norway, n = 285; Nordland Hospital, n = 251) with primary resected stage I to IIIA NSCLC. The density of CD45RO⁺ and CD8⁺ TILs in tumor epithelial and stromal compartments of the tumors was evaluated by immunohistochemistry. In univariate analyses, intraepithelial CD45RO⁺ TIL density (T-CD45RO) was a significant prognostic factor for disease-specific survival (P = .007), limited to the squamous cell carcinoma (SCC) histology subgroup (P < .001), where it was significant in both cohorts (University Hospital of North Norway, P = .003; Nordland Hospital, P = .022). Combining T-CD45RO and stromal CD8⁺ TIL density (S-CD8) increased the prognostic impact in SCC (P < .001) and showed a significant impact within all pathological stages (I, P = .025; II, P < .001; III, P = .001). In the multivariate analysis, T-CD45RO was an independent positive prognostic factor for SCC (hazard ratio 2.65, 95% confidence interval 1.64-4.28, P < .001), and in combination with S-CD8, the prognostic impact increased vastly (high + high versus low + low: hazard ratio 6.50, 95% confidence interval 3.54-11.91, P < .001). In conclusion, T-CD45RO was an independent prognostic factor for SCC NSCLC. When combined with S-CD8, the prognostic impact increased and was significant within each pathological stage. We propose CD45RO as a candidate marker for TNM-Immunoscore in SCC NSCLC.     

Macrophage polarisation changes within the time between diagnostic biopsy and tumour resection in oral squamous cell carcinomas—an immunohistochemical study

Background:

The prognosis of solid malignancies has been shown to depend on immunological parameters, such as macrophage polarisation (M1/M2). Recently, it was reported that preoperative oral surgery leads to a worsening of oral squamous cell carcinomas (OSCC) prognosis. Diagnostic incision biopsies are oral surgery procedures that might lead to healing-associated M2 macrophage polarisation with a potential negative influence on tumour biology. No studies have compared macrophage polarisation in OSCC biopsies and tumour specimens.

Methods:

Preoperative diagnostic incision biopsies (n=25) and tumour resection specimens (n=34) of T1/T2 OSCC were processed for immunohistochemistry to detect CD68-, CD11c-, CD163- and MRC1-positive cells. Samples were digitised using whole-slide imaging, and the expression of macrophage markers was quantitatively analysed.

Results:

Carcinoma tissues obtained during OSCC tumour resections showed a significantly (P<0.05) increased CD163 cell count (M2 macrophages) compared with tissues obtained during preoperative incision biopsies. Additionally, the CD163/CD68 ratio (an indicator of M2 polarisation) was significantly (P<0.05) higher in tumour resection specimens than in biopsies.

Conclusions:

This study revealed for the first time an increase in M2 polarisation in samples obtained during OSCC tumour resection surgery compared with preoperative incision biopsies. The biopsy-induced tissue trauma might explain the observed shift in macrophage polarisation towards the tumour-promoting M2 type and could lead to accelerated tumour progression.     

Apolipoprotein E expression promotes lung adenocarcinoma proliferation and migration and as a potential survival marker in lung cancer

Many human lung cancer cell lines express apolipoprotein E (ApoE), especially cells derived from malignant pleural effusions (MPE) in patients with lung adenocarcinoma. This study aimed to investigate the influence of ApoE expression on lung cancer. In lung cancer tissues, ApoE expression was more frequently found in malignant pleural effusions (MPE)-associated lung adenocarcinoma than in lung adenocarcinoma or squamous cell carcinoma without MPE (P < 0.05), indicating that ApoE is associated with the pathogenesis of MPE in patients with lung adenocarcinoma. Next, we examined the roles of ApoE in an MPE-derived lung adenocarcinoma cell line that endogenously over-expresses ApoE, PC14PE6/AS2 (AS2). In that experiment we inhibited ApoE expression by transfection of a plasmid carrying ApoE siRNAs into AS2 cells to generate AS-S2 and AS-S3 cells. Compared to vector-control cells and parental AS2 cells, AS2-S2 and AS2-S3 cells grew slower (P < 0.05), were more sensitive to cisplatin, and had significantly impaired cellular migration (P < 0.05). Furthermore, over-expression of ApoE was independently associated with poor survival in lung adenocarcinoma patients who had MPE at the time of diagnosis (P < 0.001). Conclusively, ApoE over-expression promotes cancer proliferation and migration and contributes to an aggressive clinical course in patients with lung adenocarcinoma and MPE.     

18F‐fluorodeoxyglucose uptake in PET is associated with the tumor microenvironment in metastatic lymph nodes and prognosis in N2 lung adenocarcinoma

Positron emission tomography is a useful technique for diagnosing lymph node (LN) metastasis. This study aimed to elucidate the association between fluorodeoxyglucose accumulation and the microenvironment in metastatic LNs in lung adenocarcinoma. We retrospectively analyzed 62 patients with surgically resected pathological N2 lung adenocarcinoma who underwent preoperative PET. The maximum standardized uptake value (SUV_max) in the metastatic LNs was measured. Lymph node specimens were immunohistochemically analyzed for CD8⁺, FoxP3⁺, and CD79a⁺ lymphocytes, CD204⁺ tumor‐associated macrophages (TAMs), and alpha‐smooth muscle actin‐positive cancer‐associated fibroblasts (αSMA⁺ CAFs). We compared the clinicopathologic and immunohistochemical characteristics between two groups with high and low LN SUV_max. Using novel 3D hybrid spheroid models, we investigated the change in invasiveness of cancer cells in the presence of CAFs. In the multivariate analyses, LN SUV_max was an independent prognostic factor. The overall survival in the LN SUV_max high group was significantly worse than in the low group (P = .034). In the LN SUV_max high group, metastatic cancer cell invasion of extranodal tissue was more frequent (P = .005) and the number of CD204⁺ TAMs and αSMA⁺ CAFs in metastatic LNs was significantly higher than in the low group (P < .001 and P = .002, respectively). Hybrid spheroid models revealed that cancer cells coexisting with CAFs were more invasive than those without CAFs. Our results indicated a strong association between LN SUV_max and poor prognosis in patients with N2 lung adenocarcinoma. Moreover, LN SUV_max was suggested to be associated with the presence of tumor‐promoting stromal cells in metastatic LNs.     

Brain metastasis in renal cancer patients: metastatic pattern,tumour-associated macrophages and chemokine/chemoreceptor expression

Background:

The mechanisms of brain metastasis in renal cell cancer (RCC) patients are poorly understood. Chemokine and chemokine receptor expression may contribute to the predilection of RCC for brain metastasis by recruitment of monocytes/macrophages and by control or induction of vascular permeability of the blood–brain barrier.

Methods:

Frequency and patterns of brain metastasis were determined in 246 patients with metastatic RCC at autopsy. Expression of CXCR4, CCL7 (MCP-3), CCR2 and CD68⁺ tumour-associated macrophages (TAMs) were analysed in a separate series of 333 primary RCC and in 48 brain metastases using immunohistochemistry.

Results:

Fifteen percent of 246 patients with metastasising RCC had brain metastasis. High CXCR4 expression levels were found in primary RCC and brain metastases (85.7% and 91.7%, respectively). CCR2 (52.1%) and CCL7 expression (75%) in cancer cells of brain metastases was more frequent compared with primary tumours (15.5% and 16.7%, respectively; P<0.0001 each). The density of CD68⁺ TAMs was similar in primary RCC and brain metastases. However, TAMs were more frequently CCR2-positive in brain metastases than in primary RCC (P<0.001).

Conclusion:

Our data demonstrate that the monocyte-specific chemokine CCL7 and its receptor CCR2 are expressed in tumour cells of RCC. We conclude that monocyte recruitment by CCR2 contributes to brain metastasis of RCC.     

The relationship between lymphocyte subsets and clinico-pathological determinants of survival in patients with primary operable invasive ductal breast cancer

Background:

The importance of lymphocyte subtypes in determining outcome in primary operable ductal invasive breast cancer remains unclear. The aim of present study was to examine the relationship between tumour lymphocyte subsets infiltrate and standard clinico-pathological factors and survival in patients with primary operable invasive ductal breast cancer.

Methods:

The analysis of the inflammatory cell infiltrate, including lymphocyte subtypes, was undertaken using immunohistochemical techniques and visual quantitative and semi-quantitative techniques in 338 patients with ductal breast cancer.

Results:

The majority (91%) of patients had high grade inflammatory cell infiltrate. The median follow-up of the survivors was 164 months. During this period, 65 died of their cancer. On univariate analysis, tumour inflammatory cell infiltrate, macrophages infiltrate (P<0.05), lymphocytic infiltrate (P<0.001) and CD8+ T-lymphocytic infiltrate (P<0.01) were associated with improved cancer-specific survival, whereas neutrophil (P<0.05) and CD138+ B-lymphocytic infiltrate (P<0.001) were associated with poorer cancer-specific survival. On multivariate analysis, tumour lymphocytic infiltrate (P<0.001), macrophage infiltrate (P<0.05), CD8+ T-lymphocytic infiltrate (P<0.01) and CD138+ B-lymphocytic infiltrate (P<0.001) were independently associated with cancer survival. When the significant inflammatory cell types were included with tumour-based factors in multivariate analysis only tumour size (Hazard ratios (HR): 2.55, 95% confidence interval (CI): 1.53–4.27, P<0.001), Ki-67 index (HR: 2.08, 95% CI: 1.08–4.00, P<0.05), lymphovascular invasion (HR: 4.40, 95% CI: 2.07–9.35, P<0.001), macrophage infiltrate (HR: 0.49, 95% CI: 0.33–0.73, P<0.001), lymphocytic infiltrate (HR: 0.11, 95% CI: 0.05–0.23, P<0.001), CD8+ T-lymphocytic infiltrate (HR: 0.57, 95% CI: 0.38–0.87, P<0.001) and CD138+ B-lymphocytic infiltrate (HR: 2.86, 95% CI: 1.79–4.56, P<0.001) were independently associated with cancer survival.

Conclusion:

The majority of patients with invasive ductal breast cancer had high-grade inflammatory cell infiltrate. In these patients, inflammatory cells including macrophage and lymphocytic infiltrate, and subsets CD8+ T-lymphocytic infiltrate and CD138+ B-lymphocytic infiltrate had superior prognostic value, compared with hormone status and lymph node involvement in patients with primary operable invasive ductal breast cancer.     

Tumor associated macrophage expressing CD204 is associated with tumor aggressiveness of esophageal squamous cell carcinoma

Tumor associated macrophages (TAMs) are the most abundant cancer stromal cells educated by tumor microenvironment to acquire trophic functions facilitating angiogenesis, matrix breakdown and cancer cell motility. Tumor associated macrophages have anti‐inflammatory properties or “alternatively” activated (M2) phenotype expressing CD204 and/or CD163. To know the role of TAMs in the growth and progression of esophageal squamous cell carcinomas (ESCCs), we calculated intratumoral CD204, CD163 or CD68 expressing macrophage count (M?C) and CD34‐positive microvessel density (MVD) by immunohistochemistry in 70 cases of surgically resected ESCCs and compared them with the clinicopathological factors and prognosis of patients. M?C had positive linear association with MVD. High CD204⁺ M?C were significantly correlated with more malignant phenotypes including depth of tumor invasion, lymph and blood vessel invasion, lymph node metastasis as well as clinical stages. On the other hand, CD163⁺ M?C did not associate with these clinicopathological factors with the exception of depth of tumor invasion and blood vessel invasion. Patients with high CD204⁺ M?C ESCCs showed poor disease‐free survival (P = 0.021). Conditioned media of five ESCC cell lines (TE‐8, ‐9, ‐10, ‐11 and ‐15) induced mRNA as well as protein expression of CD204 but not of CD163 with upregulation of vascular endothelial growth factor‐A mRNA in TPA treated human acute monocytic leukemia cell line THP‐1. These results overall indicate that CD204 is a useful marker for TAMs contributing to the angiogenesis, progression and prognosis of ESCCs whose specific tumor microenvironment may educate macrophages to be CD204⁺ M2 TAMs.     

Melanoma-initiating cells exploit M2 macrophage TGFβ and arginase pathway for survival and proliferation

M2 macrophages promote tumor growth and metastasis, but their interactions with specific tumor cell populations are poorly characterized. Using a mouse model of spontaneous melanoma, we showed that CD34⁻ but not CD34⁺ tumor-initiating cells (TICs) depend on M2 macrophages for survival and proliferation. Tumor-associated macrophages (TAMs) and macrophage-conditioned media protected CD34⁻ TICs from chemotherapy in vitro. In vivo, while inhibition of CD115 suppressed the macrophage-dependent CD34⁻ TIC population, chemotherapy accelerated its development. The ability of TICs to respond to TAMs was acquired during melanoma progression and immediately preceded a surge in metastatic outgrowth. TAM-derived transforming growth factor-β (TGFβ) and polyamines produced via the Arginase pathway were critical for stimulation of TICs and synergized to promote their growth.     

Prognostic impact of count of extratumoral lymphatic permeation in lung adenocarcinoma and its relation to the immune microenvironment

Extratumoral lymphatic permeation (ly‐ext) has been reported as an independent poor prognostic factor for lung adenocarcinoma, but whether or not the number of ly‐ext foci is associated with prognosis and its relationship to the immune microenvironment is unclear. We counted the number of ly‐ext foci on pathological slides from patients with completely resected lung adenocarcinoma with ly‐ext, and divided them into two groups: a group with a high number of ly‐ext foci (ly‐ext high) and one with a low number of ly‐ext foci (ly‐ext low). Among the patients with ly‐ext, only a high number of ly‐ext foci was an independent poor prognostic factor. The 3‐year recurrence‐free survival (RFS) rate of the ly‐ext high group was significantly lower than that of the ly‐ext low group (14.7% vs. 50.0%, P < 0.01). Then, we analyzed the immune microenvironment of pT1 lung adenocarcinoma with ly‐ext (13 cases of ly‐ext high and 11 cases of ly‐ext low tumor) by immunohistochemistry using antibodies for stem cell markers (aldehyde dehydrogenase 1 A1 and CD44), tumor‐promoting mucin (MUC1), tumor‐infiltrating lymphocytes (CD4, CD8, FOXP3, and CD79a), and tumor‐associated macrophages (CD204). The number of CD8+ TILs within the primary lesion was significantly lower and the number of FOXP3+ TILs within the primary lesion was significantly higher in the ly‐ext high group (P < 0.05 and P < 0.01, respectively). Our results indicated that a high number of ly‐ext foci was an independent poor prognostic factor. Moreover, tumors with high numbers of ly‐ext foci had a more immunosuppressive microenvironment.