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

Abstract

Epithelial-mesenchymal transition (EMT) is an essential process in breast cancer metastasis. The aim of the present study was to determine the role of secretions of tumor-associated leukocytes (TALs) isolated from negative and positive lymph nodes (nLNs and pLNs, respectively) breast cancer patients in regulating EMT mechanism and the associated signaling pathways. We found an increased infiltration of TALs, which was associated with downregulation of E-cadherin and over-expression of vimentin in the breast carcinoma tissues of pLNs as compared to nLNs patients and normal breast tissues obtained from healthy volunteers during mammoplasty. Furthermore, TALs isolated from pLNs breast cancer patients secreted an elevated panel of cytokines by up to 2-5-fold when compared with those isolated from nLNs patients. Secretome of TALs of pLNs possessed higher TARC, IGF-1, IL-3, TNF-β, IL-5, G-CSF, IL-4, and IL-1α with more than a fivefold compared to those of nLNs. Using the human breast cancer cell lines MCF-7 and MDA-MB-231, we found that cytokines secreted by TALs isolated from nLNs and pLNs breast cancer patients promoted EMT via upregulation of TGF-β and vimentin and downregulation of E-cadherin at messenger RNA (mRNA) levels in both cell lines and at protein level in MCF-7. While TGF-β is over-expressed by MDA-MB-231 seeded in media conditioned by secretome of TALs isolated from nLNs and pLNs breast cancer patients. The downstream TGF-β signaling transcription factors, Snail, Slug, and Twist, known to be associated with EMT mechanism were over-expressed by MCF-7 and MDA-MB-231 seeded in media conditioned by secretome of TALs isolated from nLNs and pLNs breast cancer patients. Acquisition of EMT in MCF-7 cells is mechanistically attributed to the activation of EGFR and NF-κB/p65 signaling which are significantly highly expressed by MCF-7 cells seeded in media conditioned by secretome of TALs isolated from pLNs compared to nLNs patients. Overall, this study provides implications of secretome of TALs and activated EGFR and NF-κB/p65 in EMT process that may be considered a therapeutic strategy to inhibit lymph node metastasis in breast cancer patients.

Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3, Notch and EGFR signaling pathways

“…Immunohistochemical staining was performed on serial formalin-fixed and paraffin- embedded tissues sectioned at 4 μm-thickness as we previously described [40]. Tissue sections were deparaffinized by two consecutive incubations in xylene for 10 min each, followed by rehydration through two changes of absolute ethanol, graded decreasing concentrations of ethanol for 5 min each and finally in distilled water.…”

“…All steps needed to form 3D spheroids were analogously performed followed by starvation for 24 h. Media conditioned by the secretome of the cells were collected and subjected to profile 42 biological factors according to the manufacturer’s instructions. The signal intensity of each spot, which represents the secreted chemokine, cytokines, and growth factors was evaluated by subtracting from the background and normalized to positive controls using ImageJ software as we previously described [40]. …”

Characterization of The Surgical Leakage Collected After Breast Cancer Surgery and Studying Their Effect on Breast Cancer Cell Line

“…After overnight incubation, the membranes signals were detected by using the chemiluminescence detection reagent provided with the kit. Signal intensity values representing detected cytokines were subtracted from the background and normalized to positive controls on the same membrane using ImageJ software (National Institutes of Health, MD, USA) as described by the authors before (El-Ghonaimy et al, 2016). Cell Culture:…”

IL-10 correlates with the expression of carboxypeptidase B2 and lymphovascular invasion in inflammatory breast cancer: The potential role of tumor infiltrated macrophages

“…We studied the effect of recombinant human IL-10 on MDA-MB-231 (non-IBC) and SUM149 (IBC) cell lines (gift from Dr. Bonnie Sloane, Department of Pharmacology, Wayne State University, Detroit, MI, USA). Cell lines were cultured as previously described [36,45]. MDA-MB-231 and SUM-149 cells were seeded at the density of (3.5 × 10 5 /well) in six well plate and incubated at 37°C and 5% CO 2 for 24 h in appropriate complete media, Ham's F.12 containing 5% FBS and DMEM containing 10% FBS for SUM149 and MDA-MB-231, respectively [45,46].…”

“…Cell lines were cultured as previously described [36,45]. MDA-MB-231 and SUM-149 cells were seeded at the density of (3.5 × 10 5 /well) in six well plate and incubated at 37°C and 5% CO 2 for 24 h in appropriate complete media, Ham's F.12 containing 5% FBS and DMEM containing 10% FBS for SUM149 and MDA-MB-231, respectively [45,46]. Cultured media were removed and cells incubated in serum free media conditioned with recombinant IL-10 (R&D systems, Minneapolis, Minnesota, USA) at concentrations of 100, 200 and 400 ng/ml for 24 h [36].…”