Cancer Associated Fibroblast-mediated Drug Resistance Of Lung Cancer A549 Cells By Up-regulating The PI3K/Akt And GRP78 Signaling On A Microfluidic Platform

Background Lung cancer is the leading cause of the cancer mortality worldwide with approximately 15% of survival rate in 5 years. Non-small cell lung cancer(NSCLC)accounts for 80%-85% of total lung cancers. Chemotherapy is still an important method to treat patients with advanced or metastatic NSCLC. However, the inevitable development of chemotherapy resistance is a major obstacle for the successful treatment of this disease. The activation of oncogene survival-promoting signaling pathways, such as phosphoinositide 3-kinase(PI3K)/Akt, has been demonstrated to be crucial for cancer cells to develop chemoresistance. Recent studies have also shown that tumor microenvironment provides a protective niche for cancer cells from drug treatment and eventually leads to chemoresistance. Cancer associated fibroblasts(CAF) are the most common stromal cells in the tumor microenvironment and are involved in the development of chemoresistance in cancer cells. CAF can produce growth factors to activate PI3K/Akt of adjacent tumor cells via a paracrine model.This suggests that PI3K-Akt signaling and its upstream molecules(or receptors) may be potential therapeutic targets against chemresistance in cancer. While dozens ofinhibitors targeting the pathways are currently under clinical studies, the efficacy of these inhibitors is clinically disappointing due to the poor specificity and severe adverse effect of these inhibitors and functional overlap of many kinases. This suggests that the combination of multiple drugs with different action mechanisms or that of targeting drugs with traditional cytotoxic agents could be potential strategies in NSCLC treatment. Alternatively, tumor microenvironment can trigger the stress response in cancer cell growth, leading to the accumulation of unfolded and/or misfolded proteins in the endoplasmic reticulum(ER) lumen, which subsequently provokes an evolutionarily conserved response, termed the unfolded protein response.Glucose-regulated protein 78(GRP78), is an UPR protein, and is a master regulator of the UPR. Up-regulation of GRP78 expression in tumors is associated with the development of chemoresistance. However, the underlying mechanisms remain largely unknown. Lately, it was reported that growth factor signaling could be coupled to ER chaperone expression, which suggested PI3K/AKT/m TOR pathway augmented the expression of GRP78 in mouse livers, but not via the canonical UPR. Thus, we hypothesize that HGF may regulate the GRP78 expression by activating the PI3K-Akt signaling and the GRP78 may be the key factor for development of chemoresistance in NSCLC.The investigations of targeting specific oncogenes or tumor-associated prosurvival pathways that can contribute to the progression of cancers and intrinsic or acquired drug resistance place significant demands on a systemic understanding of cancer biology. Microfluidic technology, also referred to as Lab-on-a-Chip(LOC)technology or micro-total analysis systems(μTAS), offers tremendous hope in the development of both point-of-care cancer biomarker measurements and personalized diagnostic strategies. Typically, the technology enables the actuation of fluids and manipulation of bioparticles at the microscale. Fluid flow in ultralow dimensions of micrometers is laminar and can be precisely controlled by adjusting the flow rate. The distinct property gives rise to more efficient and accurate mass delivery to cancer cells in controlled time and space. Also, unlike traditional tissue culture, microfluidic platforms have scalable sizes with most biological macromolecules, cells and bloodvessels, they provide unique functionality for the design and remodeling of three-dimensionally(3D) precise scaffolds, which mimick the physiological tumor microenvironment. Cancer cells have been found to present enhanced viability,invasion, and variable therapeutic response when embedded in microfluidic 3D culture systems. Most importantly, the microfluidic-based studies pave the way for integrated analyses involved in tumorigenesis as well as therapeutic resistance to anti-tumor drugs.In this study, we examined the impact of CAF and HGF on the Met/PIK3K/AKT phosphorylation and GRP78 expression and paclitaxel-induced apoptosis in human non-small cell lung cancer A549 cells cultured in the microfluidic 3D cell culture system: though non-contact co-culture of A549 cells and activated human fibroblasts(CAF), we firstly observed PI3K-AKT and GRP78 expression and then we investigated if CAF-mediated PI3K-AKT signaling pathway has a regulatory influence in the expression of GRP78.Method1. Microfluidic chip fabricationThe microfluidic device with two-layer structure was fabricated with polydimethylsiloxane by standard soft lithography method. The lower layer consisted of a combination of a linear concentration gradient generator(CGG) and four downstream parallel 3D cell culture units for A549 cell growth. The upper PDMS layer was used for 2D culture of CAFs, which possessed multiplexed perfusion channels making CAF-secreted growth factors as well as drugs flow to the cell chambers on the lower layer. The cell morphology was imaged by microscope at different time points.2. Determination of the Met-PI3k-Akt pathways in A549 cells via non-contact coculture with CAFsHFL1 cells were cultured in a mixture(1:1) of maintenance medium and the supernatants of cultured A549 cells in semiconfluent conditions for a minimum of 20 passages prior to matrix production. The CAF matrix-induced activation of the PI3k-Akt pathways was determined by immunofluorescence assay. The relative levelsof PI3Kp85, Akt1 phosphorylation in A549 cells were determined by western blot.The impact of modulation of the PI3K-Akt signaling on paclitaxel-induced A549 cell apoptosis was determined by a fluorescent assay. The apoptotic cells were determined using the Hoechst33342/PI apoptosis assay kit.3. CAF-induced expression of GRP78 and its relevance to PI3k-Akt activationThe CAF matrix-induced expression of GRP78 was determined by immunofluorescence assay and western blot. To observe the influence of this survival signaling pathway on GRP78 expression, the expressions of GRP78 were interrogated as different inhibitors of PI3K-Akt signaling pathway were introduced. Similarly, the modulation of GRP78 on paclitaxel-induced A549 cell apoptosis was determined by a fluorescent assay.4. Determination of CAF derived cytokines based on the microfluidic platformThe supernatants from cultured CAFs were harvested at different time point and centrifuged at 2000×g, stored at-70°C and used as CAF culture medium after determination of HGF concentrations. Enzyme linked immunossorbent assay was used to detect the level of supernatant HGF secreted by CAFs. To identify the role of HGF in the chemoresistance of A549 cells, HGF was introduced into the microfluidic chip through the inputs in upper layer. Immunofluorescence assay was used to detect the expression of Met phosphorylation and relative downstream signaling molecules after cell 3D culture and drug intervention.Results1. Establishment of microfluidic cell culture platformBased on the characteristics of cell-cell, cell-drug and cell-medium interactions,fluid mechanics and control design principle, we fabricated a double layer, integrated and high through-put microfluidic chip platform.(1) Four parallel cell culture units on the upper layer were used for the growth of HFL-1, which produced cytokines and other soluble factors. These cytokines together with flowing medium can pour into the lower layer culture units for A549 cell growth.(2) The lower chip was composed of four parallel units, each of which included a 3D culture chamber for A549 cells and its upstream concentration gradient generator(CGG). The side flow perfusion moldwas adopted in 3D cell culture. Fresh medium of oxygen and nutrition was supplied with a syringe pump successively and automatically, which simulated physical blood flow and thus mimicked in vivo microenvironment. On top of CGG was drug input pores and four distinctive drug concentrations were generated through special design of CGG. We introduced a fluorescent molecule with similar molecular weight to drugs before study and got theoretical concentration in different branches of CGG.(3)The upper and lower chip were connected by the microchannels. It was demonstrated that cells could grow and spread well in the two different medium culture systems.2. The viability of A549 cells under 3D coculture condition was enhanced and the increased viability was associated with the upregulation of PI3K-AKT.Immunofluorescence assays showed that the expression of α-SMA in HFL-1cells cultured with NSCLC A549 cell supernants increased obviously, which has been demonstrated to be an important mark of CAF. Both HFL-1 cells in 2D culture condition on the upper layer and A549 cells in 3D culture condition on the lower layer grew well. In addition, cell survival analysis demonstrated that the cell apoptotic percentage for cocultured A549 cells decreased obviously than that of mono-cultured A549 cells(p<0.05). By the induction of CAF supernants, the expression of PI3 K and AKT in A549 cells were significantly up-regulated. PI3 K and AKT inhibitors could lead to the recovery of resistance in A549 cells to paclitaxel(p<0.05), suggesting that activation of prosurvival signaling pathways was relevant to the increased anti-apoptotic ability.3. GRP78-mediated chemotherapy resistance of A549 cells were associated with the upregulation of PI3K/AKT signaling.Immunofluorescence assay and western blot revealed that activated HFL-1 cells(CAFs) induced obvious upregulation of PI3 K, AKT and GRP78. Inhibition of the expression of PI3 K and AKT resulted in decreased level of GRP78 while inhibition of GRP78 could not affect the expression of PI3 K and AKT. The results suggested that GRP78 might be the downstream molecule of PI3 K and AKT signaling pathway.Similar to PI3K/AKT inhibitors, GRP78 inhibitor promoted A549 cell apoptosis,suggesting coculture induced GRP78 upregulation in A549 cells causedchemotherapy resistance via suppressing A549 cell apoptosis. In addition, the combination of PI3K/AKT inhibitor and GRP78 inhibitor had synergistic pro-apoptotic effects.4. Cell factor screening showed upregulation of GRP78 was dependent on CAF-derived HGF.The supernatant medium was collected at different time in the 2D cell culture chambers. Using ELISA method, we verified that the activated HFL-1 cells could secrete the cytokines HGF while A549 cells not(p<0.05). Similar to coculture with CAFs, exogenous introduction of HGF upregulated the expression of Met and PI3K/Akt as well as GRP78 in A549 cells. It could be postulated that the influence of CAFs on NSCLC A549 cells may at least partially, be mediated by HGF.Conclusions:1. The microfluidic-based studies pave the way for integrated cytological analyses to identify complicated signaling molecules potentially involved in tumorigenesis as well as therapeutic resistance to anti-tumor drugs owing to scalable sizes with most biological macromolecules, cells and blood vessels, which provide unique functionality for the design and remodeling of precise scaffolds, mimicking the physiological tumor microenvironment.2. Carcinoma associated fibroblasts can induce the upregulation of PI3K-AKT signaling pathway in NSCLC A549 cells and lead to chemotherapy insensitivity of tumor cells.3. GRP78 could be one of a downstream effector of PI3K-AKT pathway. This suggests potential crosstalk between prosurvival signaling pathway and endoplasmic reticulum stress pathway. PI3K/AKT inhibitor and GRP78 inhibitor promote synergistically the apoptosis of A549 cells.4. Activated HFL-1 cells can secrete HGF and thus induce the upregulation ofPI3K-AKT signalings and GRP78 overexpression.