N. Cherdyntseva

Intratumoral heterogeneity of macrophages and fibroblasts in breast cancer is associated with the morphological diversity of tumor cells and contributes to lymph node metastasis

Recent studies have highlighted the heterogeneity of the tumor microenvironment (ME) and the importance of its analysis to the understanding of its impact on clinical outcomes. In this study, we aimed to analyze the intratumoral distribution of macrophages and fibroblasts in breast cancer (BC) based on the morphological diversity of tumor cells (tubular, alveolar, solid, trabecular and discrete structures) and the clinicopathological parameters of the disease. Thirty-six patients with invasive breast carcinoma of no special type were included in the study. The distribution of macrophages and fibroblasts in the MEs of different morphological structures was assessed using laser microdissection-assisted quantitative RT-PCR analysis of marker genes and double immunofluorescence staining for the CD68, RS1, aSMA, and FAP proteins. Gene expression microarrays were used to determine the expression of genes involved in the regulation of macrophage and fibroblast phenotypes in different morphological structures. We found that different macrophage and fibroblast subpopulations were simultaneously observed in the MEs of morphologically distinct structures but that the frequency of their detection and number of cells detected varied significantly among these structures. In particular, macrophages and fibroblasts were more frequently detected in the ME of solid structures and were rarely observed in tubular structures. A high number of CD68+RS1+ macrophages in the ME of solid structures was found to be associated with an increased frequency of lymph node metastasis in luminal B HER2- BC. In contrast, in luminal B HER2+ BC, lymph node involvement was related to the high representation of aSMA+FAP+ fibroblasts around trabecular structures. Morphologically distinct structures differed in the mechanisms regulating the macrophage and fibroblast phenotypes. The highest number of overexpressed genes controlling macrophage and fibroblast functions was observed in discrete groups of tumor cells, and the lowest number was observed in alveolar and solid structures. Taken together, our findings indicate the heterogeneous distribution of macrophages and fibroblasts in breast tumors and its close relation to the intratumoral morphological diversity of BC and contribution to lymph node metastasis.

Tumor-associated macrophages in human breast cancer produce new monocyte attracting and pro-angiogenic factor YKL-39 indicative for increased metastasis after neoadjuvant chemotherapy

ABSTRACT In breast cancer, the tumor microenvironment plays a critical role in the tumor progression and responses to therapy. Tumor-associated macrophages (TAMs) are major innate immune cells in tumor microenvironment that regulate intratumoral immunity and angiogenesis by secretion of cytokines, growth factors as well as chitinase-like proteins (CLPs), that combine properties of cytokines and growth factors. YKL-39 is a chitinase-like protein found in human and absent in rodents, and its expression in TAMs and role in breast cancer progression was not studied to date. Here for the first time we demonstrate that YKL-39 is expressed on TAMs, predominantly positive for stabilin-1, but not by malignant cells or other stromal cells in human breast cancer. TGF-beta in combination with IL-4, but not IL-4 alone was responsible of the stimulation of the production of YKL-39 in human primary macrophages. Mechanistically, stabilin-1 directly interacted with YKL-39 and acted as sorting receptor for targeting YKL-39 into the secretory pathway. Functionally, purified YKL-39 acted as a strong chemotactic factor for primary human monocytes, and induced angiogenesis in vitro. Elevated levels of YKL-39 expression in tumors after neoadjuvant chemotherapy (NAC) were predictive for increased risk of distant metastasis and for poor response to NAC in patients with nonspecific invasive breast carcinoma. Our findings suggest YKL-39 as a novel therapeutic target, and blocking of its activity can be combined with NAC in order to reduce the risk of metastasis in breast cancer patients.

Genetic variability in the regulation of the expression cluster of MDR genes in patients with breast cancer

PurposeWe aimed to investigate the association between the polymorphism and expression patterns of multiple drug resistance genes (MDR) in breast cancer (BC).Materials and methodsThe MDR gene expression levels were measured in tumor tissues of 106 breast cancer patients using quantitative real-time PCR. Affymetrix CytoScan™ HD Array chips were used to assess genotypes. Pairwise correlation analysis for ABCB1, ABCC1, ABCC2 and ABCG2 gene expression levels was carried out to reveal co-expression clusters. Associations between SNPs of MDR genes and their preoperative expression levels were assessed using analysis of covariance adjusting for covariates.ResultsThe SNPs associated with the expression of the ABCB1, ABCC1, ABCC2 and ABCG2 genes before NAC were detected. In addition, 21 SNPs associated with the expression of four ABC-transporter genes and involved in the expression regulation were identified. Validation in an independent sample confirmed the association between the MDR cluster genes and 11 SNPs.ConclusionsFour MDR genes: ABCB1, ABCC1, ABCC2 and ABCG2 were shown to form the functional expression cluster in breast tumor. Further studies are required to discover precise mechanisms of the cluster regulation, thereby providing new approaches and targets to combat the development of the MDR phenotype during chemotherapy.

Expression of M2 macrophage markers YKL-39 and CCL18 in breast cancer is associated with the effect of neoadjuvant chemotherapy

PurposeHigh activity of enzyme TOP2a in tumor cells is known to be associated with sensitivity to anthracycline chemotherapy, but 20% of such patients do not show clinical response. Tumor microenvironment, including tumor-associated macrophages (TAM), is an essential factor defining the efficiency of chemotherapy. In the present study, we analyzed the expression of M2 macrophage markers, YKL-39 and CCL18, in tumors of breast cancer patients received anthracycline-based NAC.MethodsPatients were divided into two groups according to the level of doxorubicin sensitivity marker TOP2a: DOX-Sense and DOX-Res groups. Expression levels of TOR2a, CD68, YKL-39 and CCL18 genes were analyzed by qPCR, the amplification of TOR2a gene locus was assessed by the microarray assay. Clinical and pathological responses to neoadjuvant chemotherapy were assessed.ResultsWe found that the average level of TOP2a expression in patients of DOX-Sense group was almost 10 times higher than in patients of DOX-Res group, and the expression of CD68 was 3 times higher in the DOX-Sense group compared to DOX-Res group. We demonstrated that expression levels of M2-derived cytokines but not the amount of TAM is indicative for clinical and pathological chemotherapy efficacy in breast cancer patients. Out of 8 patients from DOX-Sense group who did not respond to neoadjuvant chemotherapy (NAC), 7 patients had M2+ macrophage phenotype (YKL-39+CCL18− or YKL-39−CCL18+) and only one patient had M2− macrophage phenotype (YKL-39−CCL18−). In DOX-Res group, out of 14 patients who clinically responded to NAC 9 patients had M2− phenotype and only 5 patients had M2+ macrophage phenotype. Among pathological non-responders in DOX-Sense group, 19 (82%) patients had M2+ tumor phenotype and only 4 (18%) patients had M2− phenotype. In DOX-Res group, all 5 patients who pathologically responded to NAC had M2 phenotype (YKL-39−CCL18−). Unlike the clinical response to NAC, the differences in the frequency of M2+ and M2− phenotypes between pathologically responding and non-responding patients within DOX-Sense and DOX-Res groups were statistically significant.ConclusionsThus, we showed that in patients with breast cancer who received anthracycline-containing NAC the absence of clinical response is associated with the presence of M2+ macrophage phenotype (YKL-39-CCL18u2009+u2009or YKL-39u2009+u2009CCL18-) based on TOP2a overexpression data.

The effect of neoadjuvant chemotherapy on the correlation of tumor-associated macrophages with CD31 and LYVE-1

Angiogenesis and lymphangiogenesis play a crucial role in tumor growth, invasion and metastasis. Tumor-associated macrophages (TAM) induce both angiogenesis and lymphangiogenesis in mouse breast cancer models and positively correlate with these processes in human breast cancer patients. Neoadjuvant chemotherapy (NAC) is a widely used therapeutic option for cancer treatment. However, the effect of NAC on the distribution of TAM within intratumoral compartments and their correlation with angiogenesis and lymphangiogenesis remained unknown. In the present study we analyzed the effect of NAC on the distribution of CD68+ and stabilin-1+ TAM in five functionally distinct areas of human breast cancer and their correlations with microvessel density (MVD) and lymphatic microvessel density (LMVD), identified by CD31 and LYVE1, respectively. We found that NAC enhances blood vessel density in soft fibrous stroma and in coarse fibrous stroma. Without NAC the amount of CD68+ TAM in gaps of ductal tumor structures positively correlate with CD31+ microvessel density in soft fibrous stroma. NAC had enhancing effect on the amount of CD68+ TAM but not stabilin-1+ TAM in soft fibrous stroma. However, no correlation between TAM and CD31+ microvessel density was identified after NAC. NAC did not enhance the lymphatic microvessel density. But after NAC stabilin-1 expressing subpopulation of TAM positively correlated with expression of LYVE-1. We hypothesized that CD68+ TAM can support tumor angiogenesis primarily before NAC, while stabilin-1+ TAM can contribute to the maintenance of lymphatic microvessel density after NAC.

GLCE rs3865014 (Val597Ile) polymorphism is associated with breast cancer susceptibility and triple-negative breast cancer in Siberian population

d-Glucuronyl C5-epimerase (GLCE) is one of key enzymes in heparan sulfate biosynthesis and possesses tumour-suppressor function in breast carcinogenesis. Here, we investigated a potential involvement of GLCE polymorphism(s) in breast cancer development in Siberian women population. Comprehensive analysis of SNP databases revealed GLCE rs3865014 (Val597Ile) missense polymorphism as the main significantly present in human populations. According the TaqMan-based SNP assay, allele distributions for the rs3865014 (A>G) were similar in healthy Siberian women (n=136) and cancer patients (n=129) (A0,73:G0,27) and intermediate between the European and Asian populations, while genotype distributions were different, with the increase of AG rate in breast cancer patients (OR=1.76; 95% CI=1.04-1.90; P(Y)=0.035 χ2=4.44). Heterozygous AG genotype was associated with tumour size (OR=3.67, P(Y)=0.004), ER-negative tumours (OR=3.25, P(Y)=0.0028), triple-negative tumours (OR=4.94, P(Y)=0.015) but not menopausal status, PR and HER-2 status, local or distant metastasis. Homozygous GLCE genotypes (AA/GG) were more common for ER+PR+ luminal A breast cancer (OR=0.25, P(Y)=0.031). Loss-of-heterozigosity was identified in 5 of 51 breast tumours and the loss of G allele was associated with the decreased GLCE expression. Epidemiologic data for the GLCE SNP in different racial/ethnic groups demonstrated high AG genotype rates as a risk factor not for breast cancer incidence but for poor prognosis of the disease. The obtained data suggest an involvement of GLCE rs3865014 in breast cancer development. Heterozygous AG genotype might be a risk factor for breast cancer susceptibility in Siberian women and is associated with aggressive ER-negative and triple-negative cancer subtypes.