The Postoperative Immunosuppressive Phenotypes of Peripheral T Helper Cells Are Associated with Poor Prognosis of Breast Cancer Patients.

PURPOSE: T helper cells play essential roles in anti-tumor immune response. However, the postoperative changes of peripheral T cell subsets and their clinical significance in breast cancer patients remain largely unknown. METHODS: We evaluated the perioperative changes of T lymphocyte subsets in invasive breast cancer (IBC) patients and breast fibroadenoma (BF) patients preoperatively (preop) and 6, 24, 72 hours postoperatively (POH6, POH24, and POH72). Proportions of CD3, CD4, CD8, T helper (Th) 1, Th2, Th17 cells, regulatory T cells (Treg), and CD4+/CD8+, Th1/Th2 ratio were detected by flow cytometry. Changes in T helper cell quantity were correlated to clinicopathological parameters. Furthermore, we explored the association between the perioperative variations of T cell subsets and disease-free survival (DFS) of IBC patients. RESULTS: In IBC patients, Th1 cells diminished while Tregs elevated in postoperative 72 hours in the peripheral blood. In contrast, no significant perioperative changes of T cell subsets were observed in BF patients. Postoperative lower Th1 cells at POH 72 of IBC patients were correlated with greater tumor burden, HER2 positive and Ki67 positive. The increased Tregs at POH 72 of IBC patients were correlated with larger tumor size and HER2 positive. Th1 cell decline and Treg increment were both associated with shorter DFS in IBC patients. CONCLUSIONS: The variations of peripheral T helper cell subsets showed postoperative immunosuppression and were associated with poor prognosis in IBC patients.

Cytokine pattern of the breast tumor supernatant.

Cytokine production was evaluated in supernatants of cultured tumor cells that were obtained by biopsy of the breast invasive ductal carcinoma (IDC) and breast fibroadenoma (FA) and grown in vitro. In the IDC supernatants, the concentrations of pro-inflammatory (pro-oncogenic) cytokines IL-17, IL-18, and IFNγ and of IL-1 receptor antagonist were significantly higher than in the FA cell supernatants. The concentrations of anti-inflammatory cytokine IL-10 and MCP-1 protein in supernatants of IDC cells were significantly lower than those determined in FA supernatants.

The crosstalk: Tumor-infiltrating lymphocytes rich in regulatory T cells suppressed cancer-associated fibroblasts.

BACKGROUND: The interactions between cancer-associated fibroblasts (CAFs) and cancer cells or tumor-infiltrating lymphocytes (TILs) and cancer cells play important roles in cancer progression and metastasis. However, studies related to the crosstalk between CAFs and TILs in tumor microenvironment (TME) are still lacking. In this study, we mainly investigated the interactions between CAFs and TILs. MATERIAL AND METHODS: The distribution of TILs rich in regulatory T cells (Tregs) in breast cancer tissues was evaluated using hematoxylin-eosin staining and immunohistochemistry with anti-CD3, anti-Foxp3, and anti-α-smooth muscle actin antibodies. Homologous CAFs/normal fibroblasts (NFs) and TILs cultured in vitro were identified and detected using immunocytochemistry and flow cytometry (FCM). The direct interaction among these cell types was studied via a factorial design in a co-cultured system. Their indirect interaction was assayed using Transwell plates. The cell cycle and apoptosis of CAFs/NFs co-cultured with TILs was analyzed using propidium iodide staining. RESULTS: Histochemistry demonstrated most of the TILs including Tregs, were distributed in the cancer stroma, adjoining to CAFs. This finding implies that both cell types interact closely in the TME. Identification of the cultured cells showed that CAFs maintained their activated phenotype within limited passages in vitro, and that the TILs population contained a high percentage of Tregs. Data analysis of the factorial design suggests significant interactions among CAFs, NFs, and TILs in both direct and indirect contact ways. The CAFs and NFs were suppressed signally by TILs, which are probably induced by the secretory cytokines derived from TILs or Tregs. Although apoptosis was not detected in CAFs/NFs, the cell cycle assay suggested that the CAFs/NFs were arrested in the G2/M phase by the TILs and their secretory cytokines. CONCLUSION: CAFs and NFs were dramatically suppressed by Tregs-rich TILs. This suggests the interaction between TILs and CAFs might modify the TME in an unknown manner.

CD44(+)/CD24(-) cells are transit progenitors and do not determine the molecular subtypes and clinical parameters in breast carcinomas.

CD44(+)/CD24(-) cells have been associated with breast cancer stem/progenitor cell features. However, the status of this phenotype cells in normal, benign and malignant breast tissues has not been studied, and the clinical correlation of this subpopulation in breast cancer is not fully understood. The present study sought to identify these cells in a series of normal, benign, and malignant breast tissues and explore their correlation to the molecular subtypes of breast carcinoma and conventional pathological features. Double-staining immunohistochemistry (DIHC) of CD44 and CD24 was performed on 30 normal breast tissues, 30 breast fibroadenomas (FA), 60 breast invasive ductal carcinomas (IDC), and 3 breast cancer cell lines (MCF-7, MDA-MB-468, and MDA-MB-231). In the normal breast tissues and FAs, three phenotypes were observed including CD44(+)/CD24(+), CD44(+)/CD24(-), and CD44(-)/CD24(-) cells. In the IDCs, CD44(-)/CD24(+) cells were detected, in addition to the three aforementioned phenotypes. The strong positive rate (+++, incidence >60%) of CD44(+)/CD24(-) was significantly increased from normal breast tissue, FAs to IDCs (0.0%-->6.7%-->21.7%). However, the CD44(+)/CD24(-) cells didn't correlate with ages of patients, lymph node metastasis, tumor size, molecular subtypes, and the expression of ER, PR, HER-2, PS2, Bcl-2, nm23. The proportion of CD44(+)/CD24(-) cells in MCF-7, MDA-MB-468, and MDA-MB-231 was about 1, 5, and 80%, respectively. The results indicate that the CD44(+)/CD24(-) cells are transit progenitors and have no association with the molecular subtypes and clinicopathological parameters in the IDCs.

Critical appraisal of stromal CD10 staining in fibroepithelial lesions of breast with a special emphasis on expression patterns and correlation with WHO grading.

BACKGROUND: Fibroepithelial lesions of the breast are classified into fibroadenoma and phyllodes tumor (PT). Although WHO has established a well-defined grading system for PT, dilemma of discriminating borderline from malignant PT still exists. Stromal CD10 is a known poor prognostic factor in invasive breast cancer, its expression in fibroepithelial lesions of the breast is not well-documented. Till date, only one study has correlated the CD10 staining score with tumor grade in PT. OBJECTIVE: To evaluate whether the differences in expression patterns of CD10 and staining intensity correlate with the degree of malignancy in fibroepithelial tumors of the breast. MATERIALS AND METHODS: This is a retrospective study in which stromal CD10 expression was studied in 75 cases of fibroepithelial lesions of the breast using immunohistochemistry. Statistical analysis was performed using Chi-square test. RESULTS: There was a statistically significant trend of increasing stromal expression of CD10 with increasing degree of malignancy (P = 0.001). CONCLUSIONS: CD10 staining pattern and its scoring can assist the pathologist to accurately grade the PT of the breast for adequate treatment and can also be used as a target for the development of novel therapies.

Stromal CD10 expression in mammary fibroadenomas and phyllodes tumours.

BACKGROUND/AIMS: CD10 (CALLA) has recently been reported to be expressed in spindle cell neoplasia, and has been used to differentiate endometrial stromal sarcoma from leiomyoma and leiomyosarcoma. In the breast, myoepithelial cells express CD10, but there are few studies of the expression of CD10 in mammary fibroepithelial lesions. METHODS: Stromal CD10 expression was studied in 181 mammary phyllodes tumours (102 benign, 51 borderline malignant, and 28 frankly malignant) and 33 fibroadenomas using immunohistochemistry, to evaluate whether differences in expression correlated with the degree of malignancy. RESULTS: There was a progressive increase in the patients' age and tumour size, from fibroadenoma to phyllodes tumours with an increasing degree of malignancy (p < 0.001). Stromal CD10 expression was positive in one of 33 fibroadenomas, six of 102 benign phyllodes tumours, 16 of 51 borderline malignant phyllodes tumours, and 14 of 28 frankly malignant phyllodes tumours. The difference was significant (p < 0.001) and an increasing trend was established. Strong staining was seen in subepithelial areas with higher stromal cellularity and activity. Stromal CD10 expression had a high specificity (95%) for differentiating between benign lesions (fibroadenomas and benign phyllodes tumours) and malignant (borderline and frankly malignant) phyllodes tumours. CONCLUSIONS: CD10 may be a useful adjunct in assessing malignancy in mammary fibroepithelial lesions.

The complement regulatory proteins CD46 and CD59, but not CD55, are highly expressed by glandular epithelium of human breast and colorectal tumour tissues.

Three of the proteins protecting cells from autologous lysis by complement are: membrane cofactor protein (MCP; CD46), an inhibitor of the membrane attack complex formation (CD59), and decay accelerating factor (DAF; CD55). We have investigated the expression of these proteins in breast and colorectal carcinoma by immunohistochemistry and immunoblotting of breast tissue for CD46. CD46 was consistently and strongly expressed in the epithelial compartment in 26/28 ductal carcinomas of the breast, 9/9 fibroadenomas, and 9/11 cases of control non-neoplastic breast tissue. CD59 showed a similar degree of expression in the fibroadenomas (9/9), but was less strongly expressed in carcinomatous (22/28) and control (5/11) tissues. In marked contrast, no CD55 expression was detected in tissue from 15 ductal carcinomas. Immunoblotting of breast tissue for CD46 showed the same size of the molecule as for lymphocytes. It had however considerably stronger expression in tumour tissue than in non-neoplastic tissue. CD46 and CD59 were either lacking or only weakly expressed in the epithelial component of control colorectal mucosa: 2/15 and 5/15, respectively. In contrast, tissue samples from colorectal adenocarcinomas showed clear staining for both CD59 (10/18) and, more markedly, CD46 (15/18). There was no association between the pattern or intensity of CD46 and CD59 expression and tumour differentiation. As the complement regulatory proteins CD46 and CD59 are also strongly expressed by trophoblast at the feto-maternal tissue interface, these results support the concept that similar mechanisms are employed both by the genetically dissimilar fetus and certain tumours to evade immune attack by their host.

The role of lymphocytes and macrophages in human breast tumorigenesis: an immunohistochemical and morphometric study.

The objective of the present study was to analyze the function of lymphoid elements in the tumorigenesis of human breast cancer, similar to their elucidation in human ovarian cancer in our previous work. The lymphocytic and macrophageal content of lymphocytes and macrophages was analyzed immunohistochemically and morphometrically in 49 human breast tumors of different types. The following types of tumors were studied: 1) fibrocystic disease, 2) fibroadenoma, 3) carcinoma in situ, 4) infiltrating ductal and lobular carcinoma with high lymphoid infiltration, and 5) infiltrating ductal and lobular carcinoma with lymphoid depletion. The first two had little lymphoid infiltration and few lymphocytes (mainly T cells), while carcinoma in situ had extensive lymphoid infiltration and increased lymphocytic density, the consequence of a sharp rise in total lymphocytes reflecting the intensified immune response. In ductal and lobular infiltrating carcinoma with high infiltration, T cells were in large excess of B cells (81% and 87% vs. 11%) and CD8+ lymphocytes were the predominant type of T cells (up to 90%), in both tumoral parenchyma and stroma. In infiltrating carcinoma with lymphoid depletion, the total lymphocyte and macrophage count and areas of lymphoid infiltrates decreased, relative to highly infiltrated carcinomas, as signs of deep subcompensation of the lymphoid system. The host's reaction to disease was reflected in high correlations between the densities of the lymphoid cellular elements as tumorigenesis evolved. We suggest that the stromal immunocompetent cells are a reservoir of T killers that eventually cross into the parenchyma and join T helpers and B lymphocytes in the immune antitumor response. In later stages of cancer the response is exhausted, as manifested in lymphoid subcompensation.

Different transendothelial migration behaviour pattern of blood monocytes derived from patients with benign and malignant diseases of the breast.

BACKGROUND: In this study we compared the expression of selected monocyte surface antigens with the potential to transmigrate through an endothelial layer before and after surgery from breast cancer patients (CA) and patients with benign disease of the breast (BE). MATERIALS AND METHODS: Transmigration capacity of mononuclear cells was determined after isolation by Ficoll density gradient, layered over human umbilical vein endothelial cells and cultured in a two chamber plate added with fMLP as a chemotactic stimulus. We determined monocyte phenotye (HLA-DR, FcgRI/CD64, CR1/CD11b and LFA-1/CD11a) and the phagocytosis of E. coli by flow cytometry. RESULTS: Before surgery blood monocytes had an equal expression of the measured surface antigens, but were different in regard to their interaction with endothelial cells. Monocytes derived from CA had a higher transmigration potency than those of BE. Moreover, the migration through the endothelial cell layer created different populations of monocytes. Surgical stress modified transmigrated monocytes of BE into the direction of monocytes from CA. Phagocytic capacity of peripheral blood monocytes from CA was significantly diminished and was further reduced after surgery when measured in transmigrated cells. CONCLUSION: Our study shows that monocytes from CA and BE can be discriminated in regard to their interaction with endothelial cells.

Immunocytochemical detection of carcinoembryonic antigen in fine-needle aspirates from patients with diverse breast diseases.

Fine-needle aspirates and paraffin-embedded tissue sections from 91 cases with diverse breast diseases were immunostained with carcinoembryonic antigen (CEA) monoclonal antibody using a BioGenex StrAviGen kit based on the biotin-streptavidin amplified methodology. The results were compared with histopathologic tumor type and tumor stage. CEA was not expressed in fibrocystic changes with mild or florid epithelial hyperplasia (n = 23) and fibroadenomas (n = 8). On the other hand, 90% (56/60) of primary breast carcinomas showed positive cytoplasmic staining for CEA. No correlation was found between CEA expression, histopathologic tumor type, and tumor stage. We suggest that CEA immunocytochemistry will help in the accurate diagnosis of primary breast carcinoma in fine-needle aspirates in addition to the usual cytological criteria.

Localization of p53 and proliferating cell nuclear antigen in fine-needle aspirates of benign and primary malignant tumors of the human breast: an immunocytochemical study using supersensitive monoclonal antibodies and the biotin-streptavidin-amplified method.

p53 and proliferating cell nuclear antigen (PCNA) status was determined in fine-needle aspirates (FNAs) and methacarn-fixed paraffin-embedded tissue sections of six fibroadenomas and 50 primary breast carcinomas using supersensitive monoclonal antibodies and the biotin-streptavidin-amplified method. Nuclear accumulation of p53 was identified in 28% of carcinomas, while a heterogeneous immunostaining for PCNA was seen in all benign and malignant tumors examined. p53 expression in relation to nuclear plemorphism and lymph-node status showed weak correlation only as to nuclear grade (r = 0.28; P < 0.01). No direct or inverse correlation was found to exist between PCNA score and the evaluated prognostic parameters. In conclusion, although the identification of p53 in FNAs of breast tumors may assist in the diagnosis of malignancy, its application in the laboratory practice of cytopathology appears to be limited, since only 28% of primary breast carcinomas accumulate p53. Moreover, PCNA immunocytochemistry can be used as an alternative to traditional methods of evaluating the proliferative rate of tumors in FNAs.

Immunostaining of estrogen receptor, progesterone receptor, MIB1 antigen, and c-erbB-2 oncoprotein in cytologic specimens: a simplified method with formalin fixation.

An effective but simple fixation protocol for the immunocytochemical staining of cytologic smears for estrogen and progesterone receptors, the Ki-67 antigen (using MIB1 antibody), and c-erbB-2 protein is described. One hundred twenty-seven smears from a variety of malignant and benign breast lesions showed good preservation of antigenicity when subjected to the following fixation protocol: Freshly made smears were air-dried for 20 min to 14 h at 22 degrees C before immersing in 10% buffered formalin for 2-14 h. Immunostaining followed microwave-stimulated epitope retrieval. There was strong concordance of staining with corresponding tissue sections in 15 cases of malignant tumors (ER: r = 0.7381; PR: r = 0.6684; MIB1: r = 0.7234). Immunostaining staining, when delayed for 5-10 days in about half the smears, showed no noticeable difference in reactivity, attesting to effective storage of the formalin-fixed smears at room temperature.

[Expression of a plant associated human cancer antigen in breast cancer].

OBJECTIVE: To study the expression of a glycoprotein of plant origin in normal, benign and malignant breast tissues. METHODS: Expression of a plant glycoprotein was examined in 5 samples of normal breast tissues, 20 fibro-adenoma and 136 breast cancer by SABC immunohistochemical staining and the results were analyzed by SPSS statistics software. RESULTS: No positive staining was detected in normal breast tissues (0/5). Weak staining was observed in 4 of 20 (20.0%) breast fibro-adenoma. Positive staining was demonstrated in 116 out of 136 (85.3%) breast cancer specimens. The differences were statistically significant. The expression of plant-associated human cancer antigen was related to pathological grade (P < 0.05), tissue invasiveness (P < 0.01) and recurrence (P < 0.05), but not to patients' age, tumor size and c-erbB-2 expression. CONCLUSION: The plant glycoprotein studied may be a human cancer-associated antigen which might be a potential marker of breast cancer.

[Expression of the adhesion molecule CD44v6 in infiltrating ductal carcinomas of the breast is associated with hormone dependence. Our experience with 168 cases]. / La expresión de la molécula de adhesión CD44v6 en carcinomas ductales infiltrantes de mama se asocia con la hormonodependencia. Nuestra experiencia con 168 casos.

In order to investigate the possible hormone-dependence of CD44v6 in human breast cancer, we assayed the concentrations of this isoform in the membrane fraction of 168 invasive ductal carcinomas (IDC) and in 26 normal breast tissue samples, 18 fibradenomas (FAD), 3 fibrocystic disease specimens (FD), 7 mucinous carcinomas and 4 medullary carcinomas using the ELISA method. The results were compared with those of the estrogen (ER) and progesterone (PR) receptors, pS2, tissue type plasminogen activator (t-PA), cathepsin D, epidermal growth factor receptor (EGFR) and c-erbB2/neu oncoprotein concentrations. Menopausal status, size of the tumor in the cases of cancers, axillary lymph node involvement, histologic grade, ploidy, cellular synthesis phase, multifocality and multicentricity were also considered as variables. The cut-off value for CD44v6-positivity was set at 5 ng/mg prt. membrane protein content. 64/138 (38.1%) infiltrating ductal carcinomas scored positive. This was significantly higher than for the normal breast tissue (0/26; p: 0.0001), similar to that seen in the FAD (3/18), fibrocystic disease (0/3), infiltrating mucinous carcinomas (4/7) and lobular (3/15) and significantly lower than for the infiltrating medullary carcinomas (4/4; p: 0.027). There were no significant differences with the other groups of tissues studied. Furthermore, CD44v6-positive IDC showed significantly higher concentrations of ER, PR and cathepsin D and lower (p: 0.051) concentrations of EGFR when compared to their CD44v6-negative counterparts. The significant coexpression of ER, PR and cathepsin D seems to indicate a possible role for hormonal regulation of CD44v6 expression while the role of pS2 and t-PA, estrogen related proteins, was very reduced.

Development and identification of monoclonal antibodies against c-erbB-2 p185 intracellular domain.

OBJECTIVE: To develop and identify the monoclonal antibodies (mAb) against c-erbB-2 p185 intracellular domain for detection of c-erbB-2 protein overexpression in breast tumor cells. METHODS: BALB/C mice were immunized with a synthesized p185 peptide of intracellular domain. The biological characteristics and immunoactivities were identified by different techniques. RESULTS: Three hybridoma cell strains secreting mAbs to c-erbB-2 protein were established and one of the mAbs, No. 035-E61 was tested for c-erbB-2 protein immunostaining on 39 breast carcinoma sections, 30 breast fibroadenoma sections and 16 sections from various normal organs. The results showed that the positive detection frequency of protein expression was 26% (10/39) in breast cancer, none (0/30) in fibroadenoma and 3 sections from normal organs were positively stained. The consistency between the 035-E61 and the DAKO reagent approved by FDA was 74%. CONCLUSIONS: No. 035-E61 mAb can specifically recognize the p185 intracellular domain and may be useful in guiding clinical Herceptin treatment.

[Morphogenetic potential of the communication systems in breast dysplasia and fibroadenoma]. / Morfogeneticheskia potentsii kommunikatsionnykh sistem pri displaziiakh i fibroadenomakh molochnoi zhelezy.

The results of this study indicate participation of the adrenergic vegetative neural terminals (VNT) in the stroma formation due to indirect influence on the vascular structures. The involvement of the immunocompetent cells in these processes is not denied. Moreover, the determining role of VNT of the adrenergic part in the development of dysplasia can be suggested. The peculiar direction of the stroma formation in fibroadenoma and "autonomization" of this process from VNT and immunocompetent cells influence is revealed.

[The content of DNA in the oral mucosal epitheliocytes and the expression of oncofetal antigens on the peripheral blood T-lymphocytes of women with breast dysplasias]. / Soderzhanie DNK v épiteliotsitakh slizistoi obolochki polosta rta i ékspressiia onkofetal'nykh antigenov na T-limfotsitakh perifericheskoi krovi u zhenshchin pri displaziiakh molochnykh zhelez.

The DNA content in epitheliocytes of the mucous membrane of the oral cavity and expression of carcinoembryonic antigen and trophoblast-specific beta-1 glycoprotein on peripheral blood lymphocytes, peripheral blood T-lymphocytes subsets into three groups of patients: 1--diffusion mastopathy; 2--diffusion mastopathy with fibromyoma uteri and 3--fibroadenoma were studied. It was indicated that DNA content was higher in all groups in comparison with healthy women and it was shown that in contrast to healthy women, 45% of patients studied had abnormal rate of T-cells subsets, about 30% of them had abnormal expression of CEA and TSG on peripheral blood lymphocytes. The data obtained show the significant disbalance in the mechanisms of defence and compensation taking place in the organisms of patients with nonmalignant mammary diseases.

[Use of the peroxidase-antiperoxidase method in the determination of cellular antigens on cryostat sections using IKO series monoclonal antibodies]. / Ispol'zovanie PAP-metoda dlia opredeleniia kletochnykh antigenov na kriostatnykh srezakh s pomoshch'iu monoklonal'nykh antitel serii IKO.

Presents the peroxidase-antiperoxidase method for the detection of cellular antigens on cryostate sections of various tissues, making use of the IKO monoclonal antibodies. Analyzes the possible errors and causes of potential artefacts. Describes the results obtained with the use of this method in examinations of the sections of malignant and benign tumors of the mammary gland and regional lymph nodes.

Active immunotherapy for mouse breast cancer with irradiated whole-cell vaccine expressing VEGFR2.

As tumor-associated antigens are not well characterized for the majority of human tumors, polyvalent vaccines prepared with whole-tumor antigens are an attractive approach for tumor vaccination. Vascular endothelial growth factor receptor-2 (VEGFR2), as a model antigen with which to explore the feasibility of immunotherapy, has shown great promise as a tumor vaccine. However, the efficacy of immunotherapy is often not ideal when used alone. In this study, we explored the therapeutic efficacy of an irradiated AdVEGFR2-infected cell vaccine-based immunotherapy in the weakly immunogenic and highly metastatic 4T1 murine mammary cancer model. An adenovirus encoding the VEGFR2 gene (AdVEGFR2) was constructed. Lethally irradiated, virus-infected 4T1 cells were used as vaccines. Vaccination with lethally irradiated AdVEGFR2-infected 4T1 cells inhibited subsequent tumor growth and pulmonary metastasis compared with challenge inoculations. Angiogenesis was inhibited, and the number of CD8+ T lymphocytes was increased within the tumors. Antitumor activity was also caused by the adoptive transfer of isolated spleen lymphocytes. In vitro, the expression of HMGB1 and HSP70 in the AdVEGFR2­infected 4T1 cells was increased, and was involved in the activation of tumor antigen-specific T-cell immunity. Our results indicate that the immunotherapy based on irradiated AdVEGFR2-infected whole-cancer cell vaccines may be a potentially effective strategy for 4T1 cancer treatment.

Autoantibodies against muscarinic receptors in breast cancer: their role in tumor angiogenesis.

The presence of autoantibodies in cancer has become relevant in recent years. We demonstrated that autoantibodies purified from the sera of breast cancer patients activate muscarinic acetylcholine receptors in tumor cells. Immunoglobulin G (IgG) from breast cancer patients in T1N0Mx stage (tumor size≤2 cm, without lymph node metastasis) mimics the action of the muscarinic agonist carbachol stimulating MCF-7 cell proliferation, migration and invasion. Angiogenesis is a central step in tumor progression because it promotes tumor invasion and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) is the main angiogenic mediator, and its levels have been correlated with poor prognosis in cancer. The aim of the present work was to investigate the effect of T1N0Mx-IgG on the expression of VEGF-A, and the in vivo neovascular response triggered by MCF-7 cells, via muscarinic receptor activation. We demonstrated that T1N0Mx-IgG (10(-8) M) and carbachol (10(-9) M) increased the constitutive expression of VEGF-A in tumor cells, effect that was reverted by the muscarinic antagonist atropine. We also observed that T1N0Mx-IgG and carbachol enhanced the neovascular response produced by MCF-7 cells in the skin of NUDE mice. The action of IgG or carbachol was reduced in the presence of atropine. In conclusion, T1N0Mx-IgG and carbachol may promote VEGF-A production and neovascularization induced by breast tumor cells via muscarinic receptors activation. These effects may be accelerating breast tumor progression.