| Background:Breast cancer is one of the most common epithelial tumor,whose morbidity and mortality are high on female malignant tumor, which cause serious damage to women’s health andhas become a global health problem concerned. Over the past 20 years, the incidence of breast cancer continues to grow in most countries. According to the World Health Organization (World Health Organization, the WHO) statistics, there are about 1.384 million newly diagnosed cases and 459000 death cases of breast cancer women around the World. Breast cancer has become one of the biggest threats to the health of women.The harm of breast cancer and the development trend is influenced by many factors such as economic development, environmental and lifestyle changes and population aging. Breast cancer cell invasion and metastasis are the key factors affecting the prognosis of patients with breast cancer. The occurrence and metastasis of tumor and tumor cell microenvironment has a close relationship. Tumor microenvironment, a complex integrated system, the environment of the tumor development, consist of the tumor cells, mesenchymal cells and capillaries, tissue fluid, and a small amount of infiltrating cells together, which is different from the microenvironment formed by normal cell and its surrounding tissue.Hypoxia and acidosis, interstitial high pressure formation, plenty of growth factors and proteolytic enzymes and immune inflammatory reaction together form the biological characteristics of tumor tissue metabolism environment,and this feature is important for regulating the tumor occurrence and development, proliferation, invasion, migration, adhesion ability and the formation of new blood vessels. As one of the substantive tumor physical environment, hypoxia exerts a critical impact on the biological characteristics of tumor. When Substantial tumor growth rate surpass the ability of peripheral vascular including producing growth factors, nutrients, oxygen and taking away the metabolic waste products of the tumor itself, the imbalanceof oxygen supply and need will lead to hypoxia, low sugar and low pH level.Clinical and experimental evidence shows that hypoxic microenvironment is one of the important factors that increase the tumor metastasis ability. Tumor cells in the microenvironment hypoxia have a set of adaptation andsurvival mechanism, and the oxygen induced factor (hypoxia inducible factor, HIF) has played an important role. HIF-1 is a transcription activation factor in maintaining biological state of survival under anoxic condition.Also,research shows that HIF-1 regulates the process of energy metabolism, hypoxia angiogenesis, invasion and metastasis in tumor. It is found HIF have a big peony card family, including HIF-1, HIF-2and HIF-3. Especially, HIF-1 is one of the most studied and important members regulating tumor cell survival and malignant process.Epithelial mesenchymal transition refers to the phenomenon that epithelial cells transform to mesenchymal cells in the certain physiological and pathological circumstances, defined as a process thatthe epithelial cells with polarity and closely connection loses its polarity and cell morphology, after the cytoskeleton remodeling, and turns to the mesenchymal cells which are independent and have loose connections, and able to invade the extracellular matrix.Concept of EMT, as seen in developmental biology, mainly engaged in embryonic development, tissue reconstruction, wound healing, heart valves, and craniofacial structure and the formation of the musculoskeletal system, closely related to the pathogenesis with a variety of chronic diseases, such as renal fibrosis.In recent years, the study also found that the EMT process is also closely related with tumor cell growth, invasion and metastasis. The process of EMT signaling pathways involved in regulation of tumor cells and related gene research is becoming more and more attention, and has already become a hot spot in cancer research.Calcium connexins is one of the intercellular adhesion glycoprotein molecules across the membrane in epithelial tissue dependent on calcium ion, mainly involved in the connections between cells, and divided into E-cadherin, P-cadherin and N-cadherin.Especially E-cadherin and N-cadherin greatly influences the invasion and metastasis of tumor and also is the key molecules of EMT. E-cadherin, containing extracellular region, transmembrane region and cytoplasm area, is a transmembrane glycoprotein. Ca2+ area is located in the extracellular repeat unit of E-cadherin.The cytoplasmic areaof E-cadherin could forme complexesrespectively with alpha, beta, and gamma catenin. First of all, the newly synthetic E-cadherin combines with beta or gamma serial element, while from cytoplasmic to the cell membrane, E-cadherin undergoing the process of decoration and mature.As an intermediary, beta or gamma ring element can format least two kinds of E-cadherin/catenin compounds with alpha serial element, finally, tracted with actin which is combined with alpha serial element and reached to the adjacent cells connect, gathered into a fibrous.During the movement in the lipid bilayer, E-cadherin is linked together in cell junction. E-cadherin generally exists in all kinds of epithelial cells, mediating connection between the same type of cells,so as to maintaining normal epithelial cell morphology, cell polarity and structural integrity. In clinical pathological type of breast cancer, the incidence of infiltration and metastasis of breast cancer is high in the higher grade of infiltrating ductal carcinoma, three negative tumor, basal cell carcinoma, thus inducing poor prognosis. The cause is mainly that these types of cancer cells undergo the process of EMT, then gaining the strong vitality and invasive ability. Cells with such the ability often have some phenotypic change, and one of the most important landmark change is the reduction or loss of E-cadherin protein and increased expression of N-cadherin protein.It is showed that HIF may reduce E-cadherin levelby acting on Notch signaling pathways, thereby promoting tumor migration. However, there are few report about the molecular mechanism of tumor hypoxia whether breast cancer cells induced mesenchymal transformation of epithelial, in turn, affected the invasion and metastasis of breast cancer.The important principle of in vitro cell culture is to simulate in vivo cell growth environment, while the core of the most important factors in the simulation system is the interaction between cells and culture environment. Two-dimensional (2D)cell culture is the commonly used method in vitro growth, differentiation, invasion and metastasis of malignant tumor, apoptosis and screening and detection of antitumor drugs. Although some progress has been made in the study of tumor in vitro, but the system has the lack of 3D microenvironment of real tumor tissue, so as not to well simulate in vivo tumor microenvironment. When adherent growth cells lose the extracellular matrix, they will not display the original morphological characteristics and growth ability. When there is the lack of three-dimensional configuration, there will exist some serious limitations in studying the interaction between cells and cells, cells and microenvironment, especially in the study of tumor cellsstructure and metastasis in a three-dimensional environment. At the same time, there will easily appear to befalse positive in antitumor drug screening and detection, and the research results can not fully reflect the authenticity of solid tumors in vivo.So it requires that we need to find a better research methods instead of 2D culture system. Different from the traditional two-dimensional monolayer cell culture, three-dimensional cell culture technology (three-dimensional cell culture, TDCC) refers to the coculture in vitro concerning different material carrier of the three-dimensional structure and different kinds of cells,thus enable cells to migrate, grow, constitute a three-dimensional cell-carrier compounds carrier in three dimensional space structure.TDCC model is more suitable for studying gene function and biological organism regulation related pathways,promote the transformation of cancer biology research method, helps discover new tumor therapeutic targets. In recent years many three-dimensional (3D) in vitro tumor model is used to simulate entity tumor microenvironment in vivo to study the molecular mechanisms of cancer development and carry out in vitro tumor drug permeation study.In addition, the solid tumor tissues have different phenotypes of tumor cells, including proliferated cell, the non-proliferated cells and dead cells.The cells in three-dimensional multicellular tumor spheroid model can be divided into surface cells and deep cells, cells with abundant oxygen supply and cells lack of oxygen, etc, thus this model can better simulate in vivo tumor hypoxia microenvironment and the change of pH, closer to complete tumor tissue in the body. Therefore,3D structure provides more reliable choice forus to study in vitro tumor provides than 2D culture system. In the building process of 3D models, biological materials are one of the crucial factor. The biological materials can be divided into two major categories including natural and synthetic materials.Collagen, matrix, and the extracellular matrix, is the best natural biological material. With the rapid development of modern inorganic chemistry and polymer chemistry, artificial synthetic biomaterials also progressed rapidly, especially for polylactic acid、 poly(lactic-co-glycolic acid)、hydrogel and some synthetic peptides.In previous studies, the hydrogel,Indian silk protein, type I collagen and other biological materials promote the development of three-dimensional organization establishing. Natural materials such as collagen from sources in animal or human, possess excellent biological compatibility, but the biggest drawback is biological performance differences between different batches of the product materials, thus affecting experimental repeatability. Morever, it has fast degradation speed and less mechanical strength. Hydrogels, as a kind of synthetic extracellular matrix, are very suitable for the delivery of therapeutic stem cells and regulatory factor. At the same time, you can use hydrogel and seed cells to construct various kinds of engineering tissue.The synthesized hydrogel has good physicochemical properties,good biocompatibility, and can be synthesized by the process of the regeneration of different damage tissues.The hydrogel can also be designed to be the extracellular matrix with special mechanical properties and specific molecular signals according to the special needs, through adjusting chemical composition, synthesis and crosslinking means. However, the hydrogel also has the shortcomings of poor biodegradability, poor cell adhesion, etc, limiting its application in the construction of the organization. In addition, the size of the biological materials and scale are also factors must be considered.In general, the diameter and pore size of natural materials and synthetic polymer materials are generally micron grade, which could not provide the real three-dimensional environment for the cells.Ideal three-dimensional scaffold materials should have the following characteristics:the structure size of the material should ensure cells in a real 3D environment; materials design; materials should be with material preparation process of economy, repeat production, biological safety, physiological conditions good compatibility. Widely used in recent years, the layers of self-assembly technology (LBL) is a good choice. Our laboratory has constructed successfully chitosan in vitro 3D model, on this basis, we use gelatin、alginate and chitosan as self-assembled materials to build the breast cancer in vitro three-dimensional cultivation model,discusses its simulation for tumor hypoxia state.In the second part, we transplant cell respectively from the traditional two-dimensional cultivation and three-dimensional model into nude mice in vivo,explore the advantages of the three-dimensional tumor model in comparison with 2D training model from the level of animal experiments, in order to provide a certain theoretical and experimental basis for further studying the biological characteristics of breast cancer and drug efficacy evaluation.Objective1 Establish an ideal gelatin-sodium alginate chitosan multilayer nano-filmon the basis of layers of self-assembly technique, cocultured with human breast cancer cells MDA-MB-231,testing its characterization. Simple simulate in vivo hypoxic microenvironment of tumor cell growth applying 3-Din vitro cultivation system to provide a better platform for the further research of the role of EMT mechanism in tumor development under the condition of hypoxia.2.Selectthe nude mice as experimental object, compare the state of tumor genesis and migration, preliminarily in nude mice of cells from between traditional two-dimensional culture conditions and three-dimensional model and explore the advantage of three-dimensional cultivation model, in order to provide certain theoretical and experimental basis for further carrying out research on the development and invasive metastasis of breast cancer.Methods1.Cell culture. Buy breast cancer cell line MDA-MB-231, after the conventional recovery, use RPMI-1640 medium containing 10% FBS,100 U/ml penicillin,100 μg/ml streptomycin for cell cultivation in 25 cm2 bottle,37℃ and 5% CO2 incubator.2-3 d passage, the cells after passage were divided into two groups, one group maintained in the traditional two-dimensional cultivation, another group mixed with self-assembled nano-membrane, building 3-D tumor tissue model.2.Breeding of nude mice. Buy nu BALB/c nude mice from southern medical university, careful breeding, observation of mental state in nude mice, activity, etc. All animal experiment should observe related animal institution of the animal experiment center, southern medical university.3.The construction of a 3-D model of tumor tissue. Human breast cancer cells MDA-MB-231 cells at 1×10/cm density were maintained in 75 cm2 culture bottle, 37℃ and 5% CO2 incubator. Change the liquid every other day. After cell being fusion to 80%-90%, wash twice using PBS, digest with 0.25% trypsin solution containing 0.02% EDTA, terminate digestion with a complete medium when the70% cells were rounded on the microscopic observation, gently blowing bottle cell, collecting cell suspension, centrifugate at 800 rpm 5min, discard the supernatant and then adjust the cell number to 107. Cells collected evenly disperse to 1% preheating gelatin solution (7ml), after ten minutes mixture of cells and material, centrifugate at 1000rpm 5min, remove excess gelatin solution. Then a layer of cationic polyelectrolytes deposited at the cell surface membrane. Then use 0.1% preheating sodium alginate solution (7ml) reaction with polymer electrolyte membrane for 10 minutes,1000rpm 5min, remove excess sodium alginate solution, and an anionic polyelectrolyte thin film deposited. The above two steps were repeated three times, finally polyanion multi-layer nanometer film with a large number of breast cancer cells could be obtained. Mix 0.2% chitosan solution (7ml) and the same volume ofRPMI-1640 medium, adjust pH value to 6.7 or so, and react with polyanion multilayer nano-membrane for 10 minutes,1000 rpm 5min, removing redundant chitosan solution, thus forming polycation nano-membrane. The nano-film was gently suspended with medium, put in six orifice plate,37℃ and 5% CO2 cell culture incubator, change liquid in a day. Cultivate a day later, the nano-membrane was transfered to another hole to continue to develop. Normal two-dimensional culture cells as a control. Take out the polyelectrolyte nano-membrane containg MDA-MB-231 breast cancer cells, cleaning three times with PBS, observing cell survival condition on the surface of nano-membrane applying the living dead staining kits. After 2.5% glutaraldehyde fixed, observe the growth condition of the cells on and within the nano-membrane using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) respectively. Fixed with 4% paraformaldehyde at room temperature and frozen section, test the activity of cells in the interior of nano-membrane utilizing living dead staining kits and observe HIF expression of tumor cells with immuno-fluorescence staining.4.Nude mice tumor inoculation. Two-dimensional cells (cells in 2D culture), and cells migrated from the nano membrane of cells (cells migrated from 3D culture) for 4 days were digested with 0.25% trypsin containing 0.02% EDTA, and mixed with same volume of PBS and Matrigel rubber suspension after collecting samples, thoroughly incorporated.100 ul cell suspension was extracted (total number of cells:5 ×106)with 1 ml syringe, and injected into nude mice right help department skin with right hand, meanwhile left thumb forefinger head fixing nude mice, ring finger and little finger fixing tail. Finally, nude mice transplanted with two-dimensional culture cell of nude mice (2D,3 only) and transplanted with cells migrated from nano-membrane (3Dm,3 only) were obtained. A small incision about 1.0 cm was made in the right side of the chest wall skin of nude mice, fat pad exposed, and then nano-membrane washing by PBS for 3 times was transplanted into the subcutaneous space and the incision was sutured, thus getting transplanted with 3D tumor in nude mice (3Di,5 only). Observe the survival situation, state of mental activity, weight detection very day. Observe its tumor status every day. After tumor grows, observe the tumor shape every other day, measure major length of tumor in nude mouse minor length, calculate its volume and draw tumor growth curve. Mice were euthanized at 50 days later, weighing tumor tissue5. The detection of cells undergoing the process of epithelial mesenchymal transition in nude mice tumor. Sentence nude mice to death using the method of cervical spinal broken, stripped nude mice tumor tissue, keep its envelope, put it in 4% paraformaldehyde fixed for 24h, freezing slice to make N-cadherin, E-cadherin protein immuno-fluorescence test.6.Statistical analysis. Complete statistical analysis using SPSS 13.0 statistical software. The results presented as the mean and standard error. The independent sample t-test analysis was applied to two independent sample, adopting the Equality of Variances method or Satterthwaite approximate t-test method, p<0.05 represents the difference was statistically significant. The comparison between groups could be used single factor analysis of variance (One-way ANOVA), and applying the Tukey method in groups with homogeneity of variance, while the variance not neat, using Dunnett’s T3 method, p< 0.05 think the difference was statistically significant.Results1. Multi-layer nano-membrane through LBL method displayed a white transparent structure containing a large number of cell, and possessed good biocompatibility. Tumor cells continuously climb out from the nano-membrane as the incubation time extension.2. Scanning electron microscopy results showed that the3D multicellular tumor spheroid formed by nano-membrane exhibited porous three-dimensional network structure, with small network clearance which is good for moisture, nutrients and oxygen permeability; the cells on the surface of tumor spheroid grew well. There was the interaction between cells and cell, cells and extracellular matrix, thus leading to the formation of 3D environment facilitating the tumor cell growth. The tumor cells well adhered on the surface of the 3D material scaffold.3. The living dead dyeing results showed a small number of death cells (red)on the surface of tumor ball. Tumor spheroid exhibited a three-dimensional structure and possessed good biocompatibility.4. The living dead dyeing results on nano-membrane after frozen section showed lots of living cells (green) on the surface of tumor spheroid, while inside the tumor, lots of dead cells (red) could be saw. Immuno-fluorescence test displayd the expression of HIF proteins (568-nm stimulate red light), expression of CD47 proteins (488-nm stimulate green light). DAPI staining (blue) revealed that large number of nucleus fragments, unclear edge and apoptotic cell. HIF expression could not be shown in normal two-dimensional culture group, and DAPI (blue) staining revealed clear nuclear. TEM results of TEM showed that cells inside the tumor happened to necrosis and lipid droplets.5. Tumor was found in nude mice of 2D and 3 Dm group 2D after 10-day inoculation, volume were 277.86±50.00 mm3,72.97±88 mm3. The tumor volume increased in 2D and 3 Dm group as the growth of the time. Tumor size in 2D group significantly greater than that in 3 Dm group, and the tumor growth rate was faster than that of 3Dm group. In 21 days after tumor inoculation, tumor size in2Dgroup and 3Dm group was 1685.87±258.09 mm3 and 486.33±373.98 mm3 respectively, tumor volume being significant different between 3 Dm group and 2D group (p< 0.05). In 29 days after tumor transplantation, tumor size in 2Dgroup and 3Dm group was 2160.62±17.19 mm3 and 695.90±525.75 mm3 respectively. Tumor volume in 2D group was significantly higher than the tumor size in 3Dm group, and the difference was statistically significant (p< 0.01).In 40 days after inoculation, tumor size was 3561.47 ±617.42 mm3in 2Dgroup, and 939.15±706.74 mm3 in 3Dm group. Tumor volume in 3Dm group was obviously less than that in 2Dgroup with statistical significance (p<0.01). In 26 days after inoculation, tumor was found in axillary of nude mice from 3Di group. The tumor is small and not easy to measure, with slow growth. The tumor volume in 2Dgroup increased faster and in 8 days, ulceration necrosis appeared on the surface of tumor. Ulceration was found in one mouse in 3Dm group, while there is no ulceration appeared in 3Di group. In 50 days after inoculation, one in mice from 2Dgroup was dead, experiment terminated.6. After tumor inoculation, mental status and activities of the mice in three groups have been very good, no obvious abnormal activity. Due to the rapid growth of tumor volume in 2D group, the late state gradually became weak. In 50 days after tumor inoculation, one in mice of 2D group was dead, experiment terminated. After drawing materials, tumor appeared in three nude mice from 2D and 3Dm group, forming a complete capsule tumor, tumor weight being 0.456±0.052,0.102±0.822 g. However, tumor was found in only one mouse from 3Dm group, whose weight was0.031±0.010g. Tumor weight in 3Dm group and 3Di tumor tumor had no significant difference (p> 0.05), while there was significant difference in both 2Dgroup and 3Dm group (p< 0.05). Tumor weight in 3Di group was also significantly less than that in 2Dgroup (p< 0.01). In addition, tumor-like white tissue appeared in the other two mice in 3Di group, not forming a complete capsule.7. Immuno-fluorescence analysis was using to detect expression of N-cadherin protein (568-nm stimulate green) and E-cadherin protein (488-nm stimulate green light) in tumor tissue from 2D,3Dm group and 3Di group. The results indicated that here was strong expression of N-cadherin protein (red) in 2D group. Nuclear stained by DAPI (blue), obvious cellular pleomorphism, vacuolated nuclear and nucleus fragments could be found. High expression of E-cadherin protein (green) appeared in 3 Dm group with clear nuclei by DAPI staining. There was positive expression of N-cadherin protein (red) and E-cadherin protein (green) in tumor tissue from 3Di group, without abnormal DAPI staining nuclei (blue).High E-cadherin protein expression (green) was obvious in tumor-like white tissue with clear DAPI (blue) staining nuclei.Conclusion1.3Din vitro tumor model based on self-assembly technology using gelatin, sodium alginate and chitosan has good biocompatibility, representing the interaction between cells and cells, cells and matrix, providing three-dimensional space structure similar to in vitro microenvironment. Morever, the 3D in vitro tumor model could simulate the anoxic condition in vivo tumor microenvironment, closer to complete tumor tissue in the body.2. In the animal level,3D tumor model not only simulated the process of epithelial mesenchymal transformation mediated by hypoxia which could promote tumor invasion ability, but also build the dormant microenvironment which could restricts the rapid migration of tumor cell, functioned as sleep barrier, and simulate the incubation stage of the tumor growth, thus recreating the process of tumor growth in vivo, which is more conducive to our research on breast cancer occurrence, development and metastasis. |
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