| Background Microfluidic-based systems, also refer to as‘lab on chip’, represents an intriguing approach and have shown inimitable advantages over conventional platform in performing analytical functions, including integration, miniaturization, automation and parallelization. Due to microfluidic chip channels can be fabricated at the micro-scale adapted to cells, faciliating cell culture and single cell operation or integrating multiple functional analysis, it has been employed as a desirable platform for cell-based research.Chemotherapy is a primary clinical strategy for the lung cancer treatment. However, drug resistance remains a major obstacle for successful chemotherapy in lung cancer cells. Tumor drug resistance mechanism is especially intricate, multiple factors and mechanisms have been implicated in the development of chemoresistance, among which, cancer-cell-specific factors might be major contributors. Investigators have paid more attention on several resistance related proteins involved in cancer-cell-specific issues. The most representative of them are P-glycoprotein (P-gp) and glutathione S-transferasesπ(GST-π). P-gp is an ATP-dependent plasma membrane transporter (Mr 170 000) encoded by the multidrug resistance (MDR1) gene that is responsible for innate and or acquired drug resistance in tumor cells due to its drug efflux function. GST-π, one of most investigated GSTs isoenzyme, was frequently overexpressed in many human tumors and was related to poor patient survival. In addition to cancer-cell-specific issues, cellular chemoresistance is closely associated with influence of tumor cell niches that occur naturally in solid tumors. The tumor microenvironment is featured by severe glucose depletion, anoxia, and acidosis, which can induced the increased glucose-regulated proteins (GRPs) synthesis. The GRPs serve multiple functions in maintaining cellular homeostasis. As molecular chaperones, they can protect cells against death, which is an adaptive mechanism for cell survival, whereas in tumors they could be potential contributor to drug resistance. The best-studied GRPs are GRP78 and GRP94. However, there is limited information on the functions of GRP78/GRP94 and Pgp/GST-πin human lung carcinoma chemoresistance. We have previously investigated the correlation between these concerned proteins and celullar chemoresistance in lung cancer with conventional experimental platform. But these methods were characterized by a relatively long time, high reagent consumption, poor sensitivity and complex operation procedures. In this study, we employed a novel microfluidic chip platform to investigated the chemoresistance of lung cancer cells as a feasible alternative to conventional platform by lowering reagent consumption, integration, flexible and improving sensitity and increased throughput. This study included four sections.Methods1. Fabrication of microfluidic chip platform.The microchip was fabricated in PDMS. A variety of microchannels and microchambers in different sizes were built and the microchannels surface were treated by rat tail collagen solution at different concentrations. Afterwards, the optimal condition was selected for further lung cancer cell culture. Of all the microchip with distinct combinatorial microchannels, the desirable chip units were designated to perform further experiment procedures. A couple of microfluidic systems with different cell culture condition were established to carry out research on drug resistance in lung cancer cell lines.2. Cell culture based on microfluidic chip platform and cell drug intervention..In different microfluidic chip systems, cell culture was performed under static medium and fluidic medium respectively. The morphology of cells in different growth stages was imaged sequentially by microscope at different time points. Cells in good condition of different systems were pretreated with respective drug stimulus, faciliating further parallel control experiment condition. Furthermore, chemotherapy agent was injected..3. Detection and analysis of drug resistance based on microfluidic chip platform in lung cancer cell lines.After cell culture and drug intervention, the detection of drug resistance related protein expression and apoptosis caused by VP-16 was carried out. Under static medium condition, Immunofluorescence assay and Western blotting was used to determine the protein expression of GRP78 and GRP94 and cell survival was determined by apoptosis assay with propidium (PI) staining. Under fluidic medium condition, the protein expression of P-gp and GST-πwas measured by immunofluorescence assay as well, while the cell survivel was determined by apoptosis assay with Hoechst33342 /PI stainning. According to cell survival results and the expression of GRP78/GRP94 and P-gp/GST-π, chemoresistance to VP-16 was analyed in lung cancer cells.4. Comparison on methods of microfluidic chip platform and conventional experimental platform in lung cancer drug resistance research. In order to display the characteristic of microfluidic chip platform, we compared it with conventional experimental platform in reagent consumption, operation procedure, time consuming and sensitivity etal. Similarly, the characteristic of the two microfluidic chip system would be described respectively.Results1.After screening of microchannels condition and chip design combination, two different microfluidic chip systems were developed to implement cellular chemoresistance analysis. 1) A microfluidic system with paralled combination of muti-channels was used to investigate GRP78/GRP94’s impacts on cellular drug resistance under static medium culture condition. 2)A chip system with fluidic medium culture condition was employed to mimick physiological microenvironment in vivo and to investigate P-gp/GST-π’s effects on cellular drug resistance. In this system, a simple external small clip was served as microvalve to control the fluidic flow so that parallel control experiment could be carried out simultaneously and a syring pump, which supplied the cells with fresh medium of oxygen and nutrition successively and automatically.2. Cell culture with microfluidic chip and cell drug intervention.On basis of conventional cell culture by common culture flask, cell culture in microfluidic platform with static and fluidic medium chip system was successfully achieved. The results demonstrated that the lung cancer cells could grow and spread well in the two different medium chip systems. By contrast, the described chip system with fluidic medium condition could provide a more suitable and better microenvironment for cells growth and proliferation. After that,the cells were divided into experimental group and control group. For the chip system with static medium condition, inducer A23187 was introduced into respective experiment microchambers of SPCA1 and NCI-H-460 cells. For the chip system with fluidic medium condition, the cells in experimental groups were exposed to culture medium containing corresponding inhibitors of P-gp/GST-π, wherea the cells in control groups were subjected to free medium (absent of inhibitors). For both of the two systems, VP-16 solution was injected into each microchamber, preparing for further analysis.3. Detection and analysis of drug resistance in lung cancer cell lines. For the chip system with static medium, the results of immunofluorescence method and Western boltting were similar, there was a significant elevation of GRP78 and GRP94 expression in SPCA1 and NCI-H-460 experimental cells compared with their respective control cells. Cell survival analysis showed the apoptotic percentage for the experimental cells (A23187 pretreatment) of SPCA1 and NCI-H-460 obviously decreased than that of the control group (A23187 pretreatment), indicating that the up-regulation of GRP78 and GRP94 could confer survival advantage to SPCA1 and NCI-H-460 cells when exposed to VP-16, which led to chemotherapy resistance. For the chip system with fluidic medium, immunofluorescence assays showed that the expression of P-gp and GST-πin experimental cells decreased obviously compared with their respective control cells. Cell survival analysis demonstrated that the cell apoptotic percentage for verapamil-pretreated group cells increased around 2.9-fold than that of the non-verapamil pretreated cells (23.7±2.6% versus 8.1±3.0%, p<0.05). Different from verapamil, EA–pretreatment didn’t caused an apparent alteration of apoptotic rate in experimental cells compared with the control cells (10.9±3.7% versus 9.0±3.1% Figure 9), suggesting that P-gp (rather than GST-π) was associated with the chemeoresistance to VP-16 in SPCA1 cells.4. Comparison on methods of microfluidic chip platform and conventional experimental platform in lung cancer drug resistance research. The analysis of chemoresistance to VP-16 could be achieved with both the microfluidic chip platform and the results of microfluidic chip platform were similar to those of the conventional exprimental platform. However microfluidic method displayed unique advantage over conventional method, such as online situ monitoring with micro spatial, more sensitive, simple opreration, low lost and low reagent consumption. Furthermore, for the microfluidic chip platform, there was also difference between the static medium chip system and the fluidic medium chip system. Compared to the static medium chip system with increasing throughput, the fluidic medium chip system could supply cells with fluidic fresh medium that similar to physiological microenvironmental condition. In addtion, cell culture and functional assay revealed further integration due to the use of simple microvalve. Conclusions1. In this study, two microfluidic chip systems were successfully established for cell culture and cellular chemoresistance research under different medium condition. The parallel muti-channels microfluidic chip was used for cell culture under static medium condition, detecion of GRP78 and GRP94 expression and cell survival to chemoterapy, and the microfluidic chip with microvavle was used for cell culture under fluidic medium condition, detection of P-gp and GST-πexpression and cell survival to chemotherapy.2. In this section, there were two conclusions: 1)Cell culture could be performed under different medium condition with the two microfluidic chip systems and the cells in both system could maintain in good condition. By comparison with the cells under static medium, to some extent, the described microfluidic device with vavle and pump could provide a suitable even better fluidic microenvironment for cells growth and proliferation. 2) On basis of cell culture under different condition, the online.drug intervene was impleimented.3. The detection of drug resistance related protein expression and cell survial to VP-16 was successfully achieved with the two microfluidic chip systems, showing its advantages of high sensitivity, low cost and online Situ Monitoring.4. The results on drug resistance anaylsis in lung cancer cells by the microfluidic chip platform was similar to those by the conventional experimental platformm, that is 1 ) Up-regulation of GRP78 and GRP94 by A23187 was associated with chemoresistance to VP-16 in lung cancer cell SPCA1 and NCI-H-460. 2)P-gp and GST-πexpression can be suppressed by respective inhibitor, however, P-gp rather than GST-π, was associated with the chemoresistance to VP-16 in SPCA cells. However, the microfluidic chip platform has shown its unique advantages over conventional platform such as integration, high throughput, low reagent consumption and increasing sensitivity. The muti-channnels chip was characteristic by high throughput, wherea, the chip equiped with vavle and pump with fluidic medium culture condition showed further integration and automation advantages. Taken together, the two prescribed microchip systems are capable of integrating parallel operation on cell culture and functional assays, offering an easy and flexible platform for cellular chemotheray resistance research. |
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