Capturing and Characterizing Immune Cells from Breast Tumor Microenvironment: An Innovative Surgical Approach

Abstract

Background In breast cancer patients, venous drainage of the breast may contain cells of immunological importance, tumor cells undergoing dissemination, and other biological factors derived from the tumor microenvironment. Collecting axillary venous blood during modified radical mastectomy and thus before dilution in the circulation may allow us to define biological properties of the tumor microenvironment. Aims were to (1) develop a surgical approach to collect blood from the breast tumor microenvironment through tributaries of the axillary vein and (2) characterize and compare immune cells collected from the axillary vein with those in peripheral blood of breast cancer patients. Materials and Methods We enrolled 17 women aged 30–50 years and diagnosed with breast cancer by mammography, ultrasound, and biopsy (stages II–III). All patients were, preoperatively, treatment-naive. During routine surgical dissection, blood was collected in heparin tubes, 10 mL from tributaries of the axillary vein and 10 mL from peripheral blood. Mononuclear cells were separated, and percentages of different leukocyte populations were determined by flow cytometry. Results We detected a significant increase in the percentage of total T lymphocytes and T helper cells collected from axillary tributaries, but not in the percentages of cytotoxic T cells, monocytes, natural killer, or B cells compared with peripheral blood. Conclusions The present study validated using an intra-operative surgical approach to collect leukocytes drained from the tumor microenvironment through axillary tributaries. Our results showed an increase in the infiltration of total T-lymphocytes and T helper cells in the tumor microenvironment, suggesting that they may contribute to tumor pathogenesis.

Cytokines secreted by macrophages isolated from tumor microenvironment of inflammatory breast cancer patients possess chemotactic properties

“…Collected blood was transferred directly to the laboratory for isolation of leukocytes as we have described (El-Shinawi et al, 2010). Briefly, blood was diluted with an equal amount of PBS, pH 7.2, at room temperature.…”

“…Cells were suspended in RPMI 1640 medium containing 5% heat inactivated FBS at density of 5 × 10 6 cells/ml. To determine the percentage of TAMs in the total isolated leukocytes, 1 × 10 5 cells/ml were double stained with fluorochrome-labeled monoclonal antibodies (APC-CD14 and PerCP-CD3) and the percentage of cells staining for CD14+/CD3- in the isolated leukocytes was assessed using FACS Calibur flow cytometer as we described previously (El-Shinawi et al, 2010). We purified TAMs (i.e., CD14+ cells) from the mononuclear cells using “Human Monocyte Negative Selection Enrichment kit without CD16 Depletion” (StemCell Technologies, Vancouver, Canada).…”

Secretome of tumor-associated leukocytes augment epithelial-mesenchymal transition in positive lymph node breast cancer patients via activation of EGFR/Tyr845 and NF-κB/p65 signaling pathway

“…Collected blood containing mononuclear cells was transferred immediately to the laboratory for isolation of TALs using density-gradient centrifugation, as we described before [17]. For each patient sample, the buffy coat layer containing TALs cells was separated and washed twice in PBS.…”

“…For each patient sample, the buffy coat layer containing TALs cells was separated and washed twice in PBS. Cell viability was assessed using trypan blue exclusion assay [17]. Cells were suspended in RPMI 1640 medium containing 1 % heat-inactivated FBS at density of 1 × 10 6 cells/ml in 37°C in a 5 % CO 2 humidified incubator.…”

Anticancer activity of tuftsin-derived T peptide in postoperative residual tumors

“…In clinical settings, surgical resection of the tumor may not always be beneficialalthough the procedure favorably removes tumor burden, it can hasten the metastatic process [7]. However, postoperative adjuvant therapies -which are highlighted by many clinicians -have been adopted to control the potential risks associated with tumor resection, such as dissemination of tumor cells into blood, production of negative cytokines, and immunocompromise [8][9][10].…”