| BackgroundThe fasciaology originated from the Chinese Digital Human Research by Professor Yuanlin. This theory holds that people as an organic whole can be divided into two systems by fasciaology, which called Support and Reserve system and Function system. Creatures sustain a longer life cycle and maintain a stable internal environment of the body by Support and Reserve System. The Function system keeps a relatively stable structure and function by Support and Reserve system. And the Support and Reserve system will continue to provide a source of nutrients to cell source and cellular activities for cell renewal and activities of the Function system. Functional system is characterized by special features of functional cells. While the fascial scaffold differentiate into functional cells through various orientations of stem cells by the core of non-committed stem cells, which from Support and Reserve System. It also provides cell supplementary and a stable environment for various cells’ updating and metabolism for Function system, simultaneously.Tumor is an abnormal lesion of the result of carcinogenic factors in body that some cells lose control of its normal growth regulation at the genetic level in local tissue, which was the cause of abnormal clonal proliferation. The main factors of carcinogenicity were including: a chemical mutagen, X-ray, radiation, viral infection, long-term physical stimulation and so on. These factors could cause genetic mutations, which can promote uncontrolled growth and division of cells. From the perspective of fasciaology that the normal function will release cell differentiation factor to induce stem cells directly differentiate into functional cells during the process of damage, which will repair cell damage. And committed stem cells will release chemokines to recruited stem cells through the basement membrane to the parts of committed stem cells during their differentiation. The mutations would cause cell abnormal division during the process of the progression of tumor, as the committed stem cells differentiate into abnormal function cells. Then committed stem cells will generate stem cells chemokines, which will induce stem cells, concentrate to committed stem cells and complement the consumption of them through the basement membrane. This process will repeat more and more and lead to an increase of tumor. Moreover, if a large number of stem cells through the basement membrane will lead to the collapse of the basement membrane that the basement membrane between stem cells and committed stem cells will loss. The committed stem cells chemokines and directional barrier of stem cells will occur in the deep base film. The spread factor is partially reflected in this phenomenon.Breast cancer is considered the most commonly occurring cancers in women (approximately25%). Most patients suffering from cancer require mandatory radiotherapy after breast-conserving surgery or mastectomy, but the locoregional control and survival rate of breast cancer patients remains unsatisfactory. However, it has been reported that AMSCs might dramatically favor breast cancer recurrence. These results suggest that AMSCs are not safe for breast reconstruction or damage repair after cancer surgery in clinical applications.AMSCs are adult stem cells in the adipose tissue, which have multi-differentiation potential obtained from the fascial connective tissue. Zuk, the first one to isolate adipose tissue extracted cells (processed lipoaspirate cells, PLA cells) from human adipose tissues, which possess multipotent differentiation and can be steadily proliferating in vitro. Compared with other adult stem cells, the advantages of PLA cells are as follow: wide variety of sources, convenience of drawing, less trauma, no immunological rejection and so on. Experiments showed thatAMSCs have cross-mesoderm pluripotency.AMSCs not only can be used for functional cells complement as stem cells, but also can stimulate the body to secrete a variety of factors to promote the body’s repair. In the induction, it can differentiate into the germ layers cells, as fat cells, osteoblasts, chondrocytes, myocardial cells, neural cells derived from ectoderm, also endothelial cells and liver cells derived from endoderm.The studies have reported that AMSCs can be used for the treatment of tissue damage after breast cancer surgeries because of AMSCs have the above characteristics. But other studies have shown that AMSCs can promote breast cancer recurrence. So the safety of AMSCs for breast reconstruction and repair damage needed further study.ObjectiveThrough the observation of the effects of AMSCs in the sensitivity of ionizing radiations on breast cancer cells in vitro and the analysis of radiation resistance in vitro to explore the mechanisms and whether AMSCs can promote breast cancer development. It provides some experimental basis for risks of clinical postoperative treatment and fat reengineering fill of breast cancer. Further it also provides some experimental evidences for fascia theories, which about the hypothesis of stem cell and cancer.1To observe whether AMSCs is present in non-specific connective tissue through the acquisition of the isolation and culture of fat tissue from liposuction, comparing with the stem cell lines.2To observe the proliferation of breast cancer cells by using AMSCs supernatant to culture them. And whether AMSCs can promote breast cancer cells, which is the premise of this experiment.3To observe whether the existence of chemotaxis and recruitment of breast cancer cells to AMSCs by using the transwell method to culture AMSCs and breast cancer cells.4To observe resistance of breast cancer cells to ionizing radiation through the colony formation assay and DNA damage repair experiments, which have been cultured by AMSCs supernatant. 5The expression of IGF-1R of AMSCs and breast cancer cells were detected by Western Blot and ELISA technique to explore the mechanism of AMSCs to promote radiation resistance.6To observe the resistance of ionizing radiation by transplanting AMSCs and breast cancer cells suspension into BALB/c-nu/nu mice.There was further confirmed that AMSCs can promote the resistance of breast cancer to ionizing radiation.Methods1The fat tissues were isolated by enzyme digestion and cultured adipose derived stem cells by liposuction and using morphology, functional learning and flow cytometry to identify whether have the characteristics of AMSCs, which to determine the existence of AMSCs in non-specific fascia connective tissue.2Cell Culture: There were MCF-7and BT474two breast cancer cells in this experiment. AMSCs and MCF-7were cultured with high glucose DMEM plus10%fetal bovine serum (100u/ml penicillin and streptomycin100u/ml) and BT474cells were cultured with RPMI1640plus10%fetal bovine serum (100u/ml penicillin and100u/ml streptomycin). All cells were placed in5%CO2,37℃incubator.3Cell proliferation was measured using the3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) dye reduction method. In brief, breast cancer cells (2×103/100μL) were plated into each well of96-well plates. After24h incubation, AMSCs culture supernatants were added into wells for24-72h and incubated an additional2h with50μL MTT solution (2mg/mL). The medium was subsequently removed, and the dark blue crystals were dissolved with100μL DMSO. The absorbance was measured using a microplate reader with reference wavelengths of570nm. The percent growth was determined relative to untreated controls. Each experiment was performed with triplicate samples and at least three times independently.4Cell migration assays were performed using the modified Boyden chamber method with an8-μm pore filter separating the top and bottom Transwell chambers (Corning, America). The AMSCs (104cells/200μL DMEM-HG) were added to the top chamber, and MCF-7cells (5×104cells/500μL DMEM-HG+10%FBS) were added to the bottom chambers under a humidified atmosphere of5% CO2at37℃. After48h incubation, the cells that failed to migrate from the top surface of the filters were removed with cotton swabs. The migrating cells on the surface of the filters were fixed with methanol and stained with crystal violet. The migration was quantitated by counting cells in six selected fields randomly on each filter under a microscope at a x200magnification, and the graphs were depicted as the mean of three independent experiments.5The testing of IGF-1, which were secreted from AMSCs and breast cancer cells. AMSCs, MCF-7or BT-474cells (5×106) were incubated for48h in5-mL culture medium. The supernatants were centrifuged and stored at-80℃IGF-1was quantitated by ELISA in accordance with the manufacturer’s procedure (Human IGF-I Quantikine ELISA Kit; R&D). The detection limit of IGF-1was0.1ng/mL. All samples were tested in triplicate independently.6Cells were trypsinized, harvested and counted. Cells were seeded in triplicate at varying concentrations in6-well plates according to the dose irradiated with or without AMSC supernatants or IGF-1. After24-h incubation, the cells were exposed at2,4,6or8Gy of6MV X-rays generated by linear accelerator (Varian2300EX, Varian, Palo Alto, CA) at a dose rate of5Gy/min. The cells were then incubated for10-14days at37℃to form colonies. After that, the colonies were fixed with100% methanol, and then stained with crystal violet. Colonies containing≥50cells were counted by microscopic inspection. The surviving fractions were calculated with following equation:(mean number of colonies)/(number of inoculated cellsxplating efficiency). Plating efficiency was defined as follows:(mean number of colonies)/(the number of inoculated control cells, which were not exposed to radiation). Survival curves were fitted using the classic multi-target single-hit model (SF=1-(1-e-D/D0)N) using GraphPad prism soft. Treatment with IGF-1R antagonist AG1024(Calbiochem, San Diego, CA, USA) was2h before radiation.The repair of DNA damage was detected by fluorescent microscope counting gamma-H2AX points, to reflect the resistance of radiation of breast cancer cells. 7After incubated, cells were lysed in cell lysis buffer with a phosphatase inhibitor cocktail and proteinase inhibitor cocktail (Sigma), and the protein concentrations were quantified with a bicinchoninic acid protein assay kit (Pierce Biotechnology). Cell lysates were harvested, and western blot analysis was performed. Proteins were resolved by SDS-polyacrylamide gel (Bio-Rad) electrophoresis and transferred onto polyvinylidene difluoride membranes (Bio-Rad). Afterward, the membranes were blocked with nonfat milk for1hour at room temperature and followed by incubation with primary antibodies: rabbit anti-IGF-1R (Abcam) or mouse anti-β-actin (ProteinTech) overnight at4℃. After washing thrice, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for1h at room temperature. Immunoreactive bands were visualized with ECL western blotting substrate (Pierce, Rockford, IL, USA). Each experiment was performed at least three times independently.8Suspensions of5x106MCF-7cells with or without AMSCs in a2:1ratio were injected into the subcutaneous tissue of the right hindlimbs of4-to6-week-old female BALB/c-nu/nu nude mice. After tumor cells implantation,33micrograms of17β-estradiol (Sigma) was administered by intramuscular injection weekly. When tumor volumes reached about150mm3, mice with MCF-7cells and mice with MCF-7cells and AMSCs were randomly assigned to control and treated groups (5mice per group), respectively. Mice in the treated groups were irradiated at a single dose of8Gy with a linear accelerator (Varian2300EX, USA) with a dose rate of500cGy/min with copper shielding. The source-to-target distance was100cm. Tumor volumes were calculated every5days using the formula (lengthxwidth2)/2. The length and width of the tumors were measured with calipers. When tumor volumes reached about2000mm3, the mice were killed, and tumor tissues were collected for immunohistochemistry (IHC).9Results represent the mean of at least3independent experiments. Student’s t-test was used for comparison of the difference between two groups. One-way ANOVA was used for comparison of the difference between more than two groups. Two-way ANOVA were carried out for factorial analysis. All p values lower than0.05 were considered statistically significant.Results1AMSCs exhibited long spindle and polygonal morphology and cell colonies. The two passage cells were adherent and had a spindle-shaped morphology; the detection of cell surface markers showed that the cells with ability of multiplication of mesenchym in vitro with a mesenchymal stem cell properties. The comparative results of the yield of stromal vascular fraction cells between obese-derived fat tissue and breast cancer-derived fat tissue was significantly, t=l7.725, P<0.001. The comparative results of the yield of adherent cells between obese-derived fat tissue and breast cancer-derived fat tissue was significantly, t=15.357, P<0.001.2AMSC supernatants promoted proliferation of human breast cancer cells. To investigate whether the susceptibility of breast cancer cells was affected by AMSCs, we cultured MCF-7and BT474cells with AMSC supernatants. We observed that the proliferation of MCF-7and BT474cells was increased in the presence of supernatants of AMSCs compared with the control group F=53.636, F=50.164, all P<0.05. These results suggested that there seem to be some cytokines secreted by AMSCs promote proliferation of breast cancer cells. This study provided an experimental premise for our subsequent experiments.3Human breast cancer cells induce recruitment of AMSCs. AMSCs have been shown to possess the ability to be recruited. These cells originate from adipose tissue throughout the whole body, which can produce secretions to promote the migration of these cells to the target damaged organs. We speculated that breast cancer cells might affect the behavior of AMSCs, particularly their recruitment. We therefore assessed the effect of breast cancer cells on AMSCs migration. In the presence of medium alone, only a few AMSCs migrated through the filters. In contrast, MCF-7cells dramatically induced AMSCs migration, especially after radiation, F=218.491, P<0.001. These data suggested that breast cancer cells might recruit AMSCs to increase their radiation resistance.4AMSC supernatants induced radioresistance in breast cancer cells. To confirm the effect of the supernatants of AMSCs on the radioresistance of breast cancer cells, we performed a clonogenic survival assay with MCF-7and BT474cells. Survival fraction curves indicated that pretreatment with supernatants promoted the clonogenic survival of both MCF-7and BT474cells after varying doses of radiation (SER10=0.802,0.756). To further evaluate the effect of supernatants of AMSCs on the radioresistance of MCF-7and BT474cells, we investigate the DNA damage response by measuring the number of y-H2AX foci after irradiation, which is a well-known marker of DNA double-strand breakage and repair. We observed that pretreatment with supernatants in combination with IR (6Gy) led to a dramatic reduction in the number of y-H2AX foci24h post-IR administration compared with exposure to IR alone(BT474: t=13.50, p<0.001; MCF-7:t=11.88, p<0.001). These results indicated that AMSCs supernatants induced radiation resistance in breast cancer cells.5IGF-1derived from AMSCs was correlated with ion radiation (IR) resistance in human breast cancer cells. The breast cancer cell lines MCF-7and BT474secrete lower levels of IGF-1into their culture supernatants as compared with AMSCs. However, the expression of IGF-1R in MCF-7and BT474cells were higher than that in AMSCs (F=382.431, p<0.001). Twenty-four hours after various dose of radiation, IGF-1R expression in MCF-7and BT474cells were gradually increased (F=87.069, p<0.001). While the secreted level of IGF-1in the culture supernatants of AMSCs after radiation was gradually increased in a time-dependent manner.To further confirm the effect of IGF-1derived from AMSCs on radioresistance in human breast cancer cells, we performed a clonogenic survival assay with MCF-7and BT474cells with or without IGF-1, anti-IGF-1R, or culture supernatants of AMSCs. After8Gy radiation, cultured with the supernatants of AMSCs or IGF-1, the survival fraction of both MCF-7and BT474cells were significantly increased (p<0.05). Under these experimental conditions, both MCF-7and BT474cells became highly resistant to radiation in the presence of culture supernatants of AMSCs (p<0.05), but this effect was inhibited by treatment with anti-IGF-1R neutralizing antibody (p<0.05). Similar results were obtained in the presence of IGF-1(p<0.05).6AMSCs induced radiation resistance of breast cancer cells in vivo.To investigate whether the radiation resistance of breast cancer cells could be affected by AMSCs in vivo, we co-injected MCF-7cells with or without AMSCs into BALB/c-nu/nu nude mice subcutaneously. Without ion radiation (IR), the growth curves indicated that the tumors in mice injected with MCF-7plus AMSCs grew significantly faster than the tumors in mice injected with MCF-7cells alone. Similarly, in mice subjected to ion radiation (IR), the tumors with MCF-7plus AMSCs grew significantly faster than the tumors with MCF-7cells alone. Two-way ANOVA were carried out for factorial analysis that indicated the crosstalk between AMSCs and the radioresistance of breast cancer, and AMSCs enhance the radiation resistance of breast cancers. Subsequently, tumor growth delay of the tumors with MCF-7plus AMSCs were significantly shorter than that of tumors with MCF-7alone (t=3.753, P=0.006). We further investigated the levels of IGF-1R of MCF-7tumor xenografts by immunohistochemical Staining. With or without IR, the tumors in mice injected with MCF-7cells alone expressed low detectable levels of IGF-1R, whereas the tumors in mice injected with MCF-7cells and AMSCs produced higher levels of IGF-1R. In irradiated mice, although the levels of IGF-1R of MCF-7tumors with or without AMSCs were significantly lower than in without IR group, the tumors injected with MCF-7and AMSCs cells produced higher levels of IGF-1R than the tumors injected with MCF-7cells alone. These results indicated that AMSCs in the tumor microenvironment might enhance radiation resistance in breast cancer cells in vivo.Conclusions1In our study, the isolated and cultured AMSCs showed similar cell morphology that was homogeneous, polygonal or fusiform shape as reported. The induction and differentiation have showed the ability to differentiate into adipogenic and osteogenic. Phenotypic identification was as reported. There were undifferentiated mesenchymal stem cells in human adipose tissue.2The comparative results of the yield of stromal vascular fraction cells and adherent cellsbetween obese-derived fat tissue and breast cancer-derived fat tissue was significantly, but the cell morphology, induction and differentiation, phenotypic identification were not significantly.3The AMSCs can promote the resistance of breast cancer cells to ionizing radiation, in vitro.4, The IGF-1could promote the resistance of breast cancer cells to ionizing radiation, which were derived from AMSCs, in vivo. |
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