| BackgroundMicro total analysis systems, also called ‘‘lab-on-a-chip’’, integrate analyticalprocesses for sequential operations like sampling, sample pre-treatment, analyticalseparation, chemical reaction, analyst detection, and data analysis in a singlemicrofluidic device. Compared with conventional platform, the microfluidic chip-basedsystems have shown inimitable advantages featured by low reagent and powerconsumption, short reaction time, portability for in situ use, low cost, versatility indesign, and potentials for parallel operation and for integration with other miniaturizeddevices. Microfluidic technology, which is being developed fast currently, is widelyused in the cell research field, due to its micro-scale channels and flexible design.Lung cancer is the leading cause of the cancer mortality worldwide. Although thetreatment progress, due to lung cancer recurrence， metastasis and resistance tochemotherapy drugs, mortality remains high. Despite the fact that many intrinsicmechanisms associated with chemoresistance have been studied and identified, factorspromoting drug resistance remain poorly defined. In addition to cancer-cell-specificissues, cellular chemoresistance is closely associated with influence of tumor cell nichesthat occur naturally in solid tumors. The tumor microenvironment is featured by severeglucose depletion, anoxia, and acidosis, which can induce the increasedglucose-regulated proteins (GRPs) synthesis. They may be potential factors ofchemoresistance and the best-studied GRPs are GRP78. We have previouslyinvestigated the correlation between these concerned proteins and cellularchemoresistance in lung cancer with conventional experimental platform. However,there is limited information on the functions of GRP78in human stromal fibroblastschemoresistance. These conventional methods were characterized by a relatively long time, high reagent consumption, poor sensitivity and complex operation procedures. Inthis study, we employed a novel microfluidic chip platform to investigate thechemoresistance of lung cancer cells and focused on the determination of molecularstructure and contents of the lung cancer cell secretion and signaling factor. Theinteraction and signaling function between cells was also studied. With this newplatform, we are able to analyze the function of the myofibroblasts in thechemoresistance to anticancer drug and its possible mechanism associated with GRP78.This study included four sections.Methods1. Fabrication of microfluidic co-culture chip platform.The microchip was fabricated in PDMS. A variety of microchannels andmicrochambers in different sizes were built in the upstream of the device and theoptimal condition was selected for further two dimensional (2D) lung cancer cellcultures. A variety of medium perfusion model was designed for the downstream3Dcell culture chambers and the optimal condition was selected. Between the upstream ofthe2D culture chamber and the3D culture chambers, a series of micro-pillars with themicro-gaps less than cells were placed to block the cells cross from the upstream and asa result, only the soluble secreted from the cells could move through to the downstreamof3D cell culture chambers.2. Cell co-culture based on microfluidic chip platform and cell drug intervention.In different microfluidic cell culture system, cells were performed under2D cellculture,3D cell culture, and indirect contact cell co-culture respectively. Themorphology of cells in different growth molds was imaged sequentially by microscopeat different time points. Cells in good condition of different systems were pretreatedwith respective drug stimulus, facilitating further parallel control experiment condition.Furthermore, chemotherapy agent was injected.3. Analysis of correlation between over-expression of GRP78and drug resistancebased on microfluidic chip platform.After cell culture and drug intervention, the detection of drug resistance relatedprotein expression and apoptosis caused by VP-16was carried out. Under2D cellculture system, Immunofluorescence assay and Western blotting was used to determinethe protein expression of GRP78and cell survival was determined by apoptosis assaywith Hoechst33342staining. Under cell co-culture condition, the protein expression ofα-SMA and GRP78in myofibroblasts induced by lung cancer cells was measured by immunofluorescence assay as well, while the cell survivals was determined byapoptosis assay with Hoechst33342/PI staining. According to cell survival results andthe expression of GRP78, chemoresistance to VP-16was analyzed in lung cancer cellsand stromal fibroblasts.4. Cell factor screening and validation based on the microfluidic platformNCI-H460cells were allowed to attach and secrete cytokine for12h, then, mediumwas collected at the outlet at different time in the2D cell culture system. Enzyme linkedimmunossorbent assay (ELISA) was used to detect the supernatant cytokine levelsecreted by lung cancer cells. To identify the role of cytokines TGF-β1secreted by lungcancer cells in the transformation and chemoresistance in stromal fibroblasts,Immunofluorescence assay was used to detect the expression of α-SMA and GRP78after cell3D culture and drug intervention.Results1. After screening of microchannels condition and chip design combination, twodifferent microfluidic chip systems were developed to implement cellularchemo-resistance analysis.1) The circular cell culture chamber was designed for the2Dcell culture.2) The micro-pillars can effectively intercept the lung caner cells in the2Dcell culture chamber, while the cytokines secreted by lung cancer cells can flow to thedownstream. Through the indirect contact co-culture, fibroblasts were thus induced totransform into myofibroblasts via indirect contact.3) The side flow perfusion mold wasadopted in3D cell culture. A syringe pump was used which could supply the cells withfresh medium of oxygen and nutrition successively and automatically, mimicking invivo micro-physiological microenvironment.2. Cell co-culture with microfluidic chip and cell drug intervention.Cell culture in microfluidic platform with2D and3D fluidic system wassuccessfully achieved. The results demonstrated that cells could grow and spread well inthe two different medium culture systems. After that，the cells were divided intoexperimental group and control group. For the chip system with2D cell culturecondition, inducer A23187was introduced into respective experiment microchambersof NCI-H-460cells. For the chip system with3D cell co-culture condition, the lungcancer cells and the fibroblasts in the experimental group were co-cultured in2D and3D mode, respectively; in the control group, only the fibroblasts were cultured in3Dmode with the medium supply from the upstream2D cell culture chamber. For both ofthe two systems, VP-16solution 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 with2D cell culture mold, the results of immunofluorescencemethod and Western blotting were similar; there was a significant elevation of GRP78and expression in NCI-H-460experimental cells compared with their respective controlcells. Cell survival analysis showed the apoptotic percentage for the experimental cells(A23187pretreatment) of NCI-H-460obviously decreased than that of the controlgroup (A23187pretreatment). EGCG could suppress the up-regulation of GRP78induced by A23187which could lead to the recovery of chemotherapy resistance toVP-16in NCI-H460cells. For the chip system co-culture mold, immunofluorescenceassays showed that the expression of α-SMA and GRP78in HFL1cells induced by lungcancer cells increased obviously compared with their respective control cells. Cellsurvival analysis demonstrated that the cell apoptotic percentage for NCI-H460inducergroup cells increased greatly than that of the non-induced cells (p<0.05). EGCG couldsuppress the function of GRP78in myofibroblsts which could lead to the recovery ofchemotherapy resistance to VP-16(p<0.05).4. Cell factor screening and validation based on the microfluidic platformThe supernatant medium was collected at the outlet at different time in the2D cellculture chambers. Using the Enzyme linked immunossorbent assay (ELISA) method,we verified that the NCI-H460cells could secrete the cytokines TGF-β1(p<0.05). Afterstimulated by exogenous TGF-β1, the expression of α-SMA and GRP78in HFL1cellswas abundantly up-regulated. All these means that the influence of NCI-H460lungcancer cells on fibroblasts may be mediated by TGF-β1.Conclusions1. In this study, a novel microfluidic co-culture device was successfully established,based on which, different cell culture mold can be achieved, such as2D cell culture,3Dcell culture, and indirect contact cell co-culture. The upstream2D culture system can beused for lung cancer cell culture, detection of GRP78expression and cell survival tochemotherapy. The cell co-culture system can be used for or the investigation of theresponse of myofibroblasts induced by lung cancer cells to anticancer drug VP-16. Thedownstream3D culture system can be applied to cell soluble factor screening.2. In this section, there were two conclusions:1) Cell culture could be performedunder different culture mold with the microfluidic chip the cells in both systems couldmaintain in good condition. No matter in2D cell culture and3D cell culture mold, the cells could growth and proliferation well.2) The detection of drug resistance relatedprotein expression and cell survival to VP-16was successfully achieved under differentcondition the online with the microfluidic chip, showing its advantages of highsensitivity, low cost and online Situ Monitoring.3. In this section, there were three conclusions:1) Up-regulation of GRP78byA23187was associated with chemoresistance to VP-16in lung cancer cell NCI-H-460.EGCG could suppress the up-regulation of GRP78induced by A23187which couldlead to the recovery of chemotherapy resistance to VP-16in NCI-H460cells.2）Thecytokines from lung cancer cells effectively transformed the co-cultured fibroblasts intomyofibroblasts. The expression of GRP78in myofibroblasts could be elevated by theinduction of lung cancer cells, and the up-regulation of GRP78could protect the cellsfrom apoptosis induced by VP-16.3）The novel microchip systems are capable ofintegrating parallel operation on cell culture and functional assays, offering an easy andflexible platform for cellular chemotherapy resistance research.4. NCI-H460lung cancer cells can secrete TGF-β1, and the secretion of TGF-β1was associated with the trans-differentiation of lung fibroblasts and up-regulation ofGRP78in fibroblasts. |
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