Influences Of Moderate Intermittent Negative Pressure On The Biological Characteristics Of MDA-MB-231 Triple Negative Breast Cancer Cells

Background:Along with the update on breast cancer-related treatment and also the increasing demand for breast reconstruction after receiving mastectomy or lumpectomy,breast reconstruction has been regarded as one part of the whole diagnosis and treatment process on breast cancers.And the relevant reconstructive surgical techniques have also been fully developed and applied.Among these techniques,autologous fat transferring was increasingly popular for its good biocompatibility,minimal invasiveness and nursing-friendliness.As the necessary assistance to the lipofilling-based reconstructive method,the application of negative pressure external.volume expansion during the reconstructive process optimized the clinical aesthetic outcome.Fat transferring has been widely applied on breast reconstruction recently.Its popularization led the attention on its oncological safety from the breast surgeons and patients.In 2009,American Society of Plastic Surgeons(ASPS)issued a statement regarding that the existed evidence could not prove its stimulation on breast cancer progression,fat transferring could still be considered as a safe and reliable approach to breast reconstruction.Meanwhile,as the assistant technique,it was considered significant to explore what kind of influences the negative pressure external volume expansion would exert on the cancer progression before its popularization.Objectives:1.Set up the in vitro moderate intermittent negative pressure cell culturing device and in vivo model of moderate intermittent negative pressure exerting on the local skin above the primary tumor xenografts.2.Comparing the influences of moderate intermittent negative pressure and also the normal pressure exert on the biological characteristics of MDA-MB-231 triple negative breast cancer cells:(1)Comparasion on their effects on MDA-MB-231 cell invasive ability.(2)Comparasion on their effects on MDA-MB-231 cell proliferation.(3)Comparasion on their effects on MDA-MB-231 cell death.Methods:1.Set up the in vitro and in vivo model(1)In vitro model:An enclosure chamber made from polypropylene was manufactured and put in the incubator for cell culturing.By keeping balance the inflow and outflow,the pressure in the chamber stayed at-25 mmHg which was in the range used by the clinical external volume expansion during working hours.After 12 working hours,gas in the box and incubator circulated without limit of the valve,and the pressure in the box returned to 0 mmHg.-25 mmHg group was cultured in the chamber,and 0 mmHg group was cultured in constant 0 mmHg environment.(2)In vivo model:MDA-MB-231 cell suspensions were injected subcutaneously into dorsal skin of nude mice bilaterally(0.15 ml/point).Tumors on one side of mice were randomly assigned to the non-EVE(0 mmHg)group,meanwhile tumors on the other side were regarded as samples in the EVE(-25 mmHg)group.The local skin of mice in-25 mmHg group was exposed to-25 mmHg every;3 hour per day.2.Comparative study of the influences the moderate intermittent negative pressure and the normal pressure exert on the biological characteristics of MDA-MB-231 triple negative breast cancer cells(1)Invasion:Transwell cell invasion assay and ELISA were used to evaluate cell invasive ability and the secretion of IL-8 in vitro,Realtime-PCR,Western blot and immunohistochemistry were applied to examine the epithelial-mesenchymal-transition(EMT)and angiogenesis between the two groups in in vivo experiments.(2)Proliferation:In vivo imaging,immunohistochemistry and Cell Counting Kit-8 assay(CCK-8)were used to evaluate cell proliferation;Immunohistochemistry,western blot analysis and ELISA were applied to trace the oxygen content,vascularization and inflammation.(3)Death:Annexin V and Dead Cell Assay and TUNEL assay were used to evaluate in vitro and in vivo cell death.ELISA was applied to examine the secretion of TNF-?,and Haematoxylin&Eosin staining was used to measure the ratio of necrosis area in the tumor tissues.Results:1.Moderate intermittent negative pressure increases invasiveness of MDA-MB-231 triple negative breast cancer cells MDA-MB-231 cells in the 0 mmHg group were more invasive and had higher expressions of IL-8(30.02 ± 10.44 pg/ml vs.18.82 ± 9.26 pg/ml,p<0.05)and TNF-a(20.59 ± 4.72 pg/ml vs.14.10 ± 3.36 pg/ml,p<0.05)than the-25 mmHg group.Grafted MDA-MB-231 tumors in-25 mmHg group showed a more obvious epithelial-mesenchymal transition at 2 week and better angiogenesis at 2 and 4 week,respectively.2.Moderate intermittent negative pressure does not significantly affect proliferation of MDA-MB-231 triple negative breast cancer cells MDA-MB-231 cells in-25 mmHg group did not show more significant increase on cell proliferation than the one in 0 mmHg group in both in vitro growth curve and in vivo 28-day follow-ups(p>0.05).The action of EVE decreased the real-time oxygen content of subcutaneous tissues but did not affect the tumor tissues beneath on postoperative day 14.Meanwhile,a higher CD31 expression was detected in-25 mmHg group at day 14 and day28,respectively.Besides,MDA-MB-231 cells from in vitro 0 mmHg group showed increasing production of IL-6(p<0.05).3.Moderate intermittent negative pressure induces apoptosis of MDA-MB-231 triple negative breast cancer cells on the early stage of tumor progression MDA-MB-231 cells in the-25 mmHg group had a higher percentage of apoptotic cell and a lower percentage of live cell(p<0.05).The supernatant of MDA-MB-231 cell culture medium in this group expressed a higher concentration of IL-8(p<0.05).Meanwhile,a higher Caspase-3 expression was detected at day 14,and larger percentage of necrotic area was measured at day28 in the grafts from the-25 mmHg group(p<0.05).Conclusions:1.Moderate intermittent negative pressure induces MDA-MB-231 cells to be more invasive.It also increases the cellular secretion of IL-8,blood vessel density and also the epithelial-mesenchymal-transition of the grafted tumor tissues.2.Moderate intermittent negative pressure suction is not associated with increasing MDA-MB-231 cell proliferation.However,it increases the amount of blood vessels in the grafted tumors and also the number of hypoxic cells in the layers of cuticle,derma and subcutaneous tissue.3.Moderate intermittent negative pressure increases the ratio of apoptotic MDA-MB-231 cells,meanwhile it decreases the ratio of live cells.And it also increases the secretion of TNF-a.Besides,the moderate intermittent negative pressure suction leads larger necrotic area of tumor tissues in the late period of tumor progression.