The present study revealed that distinct cellular morphology correlated with an altered gene and miRNA expression profile in mouse Lewis lung carcinoma LLC1 cells grown under lr-ECM 3D cell culture conditions as compared to 2D monolayer. Our results indicated that the ECM strongly affected the expression of particular genes associated with common biological pathways involved in cancer cell adaptation to 3D cell culture microenvironment and correlated with deregulated expression of miRNAs under these conditions. Furthermore, the present study also demonstrated that ECM-enriched cellular microenvironment induced a shift in gene and miRNA expression representative to expression levels in vivo. Hence, these results support the application of 3D cell culture to obtain more relevant results for the study of specific miRNAs involved in cell–ECM interaction and of ECM-mediated signaling networks in cancer.
Our findings demonstrated markedly altered gene expression signature of LLC1 cells grown under 2D and lr-ECM 3D cell culture conditions, as it was observed previously in other cell lines [17, 18]. In the present study differences in cell culture conditions resulted in 1884 differently expressed genes demonstrating the broad influence of ECM environment in gene expression regulation. In addition, we also found that the expression of selected hnf4a, infb1, klf8 and fgfr4 genes was significantly increased in LLC1 tumors likewise in LLC1 cells cultured under 3D cell culture conditions compared to gene expression levels in cells grown on plastic. Furthermore, we observed that metabolic, MAP kinase, cell adhesion and immune response functional pathway categories were most significantly altered in LLC1 cells between 2D and 3D culture conditions. The microarray data analysis identified differential expression of 73 genes related to metabolic pathways in LLC1 cells grown under lr-ECM 2D and 3D conditions. We found that the expression of genes involved in pyrimidine/purine, glycerophospholipid, unsaturated fatty acid, amino acid, monosaccharide and drug metabolism were markedly altered in an ECM dependent manner. This indicates that culturing LLC1 cells in 3D cell culture rearranges metabolic functions. In addition, changes in cellular metabolism are tightly connected to pH, nutrient and oxygen gradients leading to the formation of proliferation and hypoxia zones within the tumor microenvironment and 3D cell culture as well [19, 20]. However, genome-wide analyses of metabolic pathway rearrangement in cancer cells grown in an ECM 3D cell culture are limited. Our findings are supported by a previous report that indicated differential expression of genes involved in xenobiotic and lipid metabolism in HepG2 hepatoma cell spheroids suggesting that cells in a 3D culture could be more metabolically active compared to cells grown in monolayer [21]. In addition, Srisomsap et al. [22] revealed signatures of differentially expressed proteins associated with anaerobic glycolysis, mitochondrial and nucleotide metabolism in HepG2 cells grown in a collagen based 3D cell culture. Therefore, altogether these findings suggest that lr-ECM 3D cell culture significantly rearranges metabolic functions in LLC1 cells. Our findings are also in agreement with a previous report also indicating that cellular adaptation to a 3D culture environment significantly alters the expression of genes involved in ECM and cell adhesion [5, 23]. In addition, Luca et al. also observed significantly altered expression of genes involved in MAP kinase pathway [6]. Strikingly, the study also demonstrated altered EGFR protein levels and a switch between RAS-MAPK pathway activation between 2D and lr-ECM 3D environments implying that cellular behavior in different microenvironment could promote important mechanisms to acquire resistance during anticancer therapy. Hence, these findings suggest that the ECM strongly influences the expression of particular genes associated with common biological processes that are involved in cellular adaptation to 3D cell culture conditions. Therefore, results obtained in cells grown under 3D cell culture conditions might also be exploited for the development of targeted cancer therapy.
Furthermore, in our present data we also observed a strong modulation of inflammatory genes in LLC1 cells between 2D and 3D culture conditions. Our findings indicated an altered expression of 44 immune response related genes suggesting that ECM plays an important role in modulating tumor-immune system interactions. Surprisingly, interferon 1 beta (infb1) was the most significantly up-regulated inflammatory gene in LLC1 cells under 3D conditions. Interferons have been shown to promote anti-proliferative, anti-angiogenic and immunoregulatory effects on many tumor types [24, 25]. Nevertheless, we also observed increased ifnb1 levels in mouse LLC1 tumors suggesting that the primary primal role of elevated basal ifnb1 levels could be more associated with regulation of tumor immuno-surveillance, but not necessarily with tumor suppression. Our results also indicated increased expression of NFAT family fatc2 and fatc4 genes in LLC1 cells grown under lr-ECM 3D culture conditions as compared to 2D. As NFAT transcription factor family was originally identified to mediate the response of immune cells, recent studies have demonstrated that NFATs also perform important roles in formation of tumor microenvironment. Activation of NFAT signaling in cancer cells results in inflammatory chemokine production eventually leading to recruitment of inflammatory cells to the tumor [26]. Interestingly, recent report suggested that NFAT2 constitutive activation in transgenic mice also linked the microenvironment and the neighboring cells, as both tumor cells expressing NFAT2 and neighboring wild-type cells up-regulated c-Myc and STAT3 in spontaneous skin and ovary tumors [27]. On the other hand, previous reports also associated NFAT signaling axis to VEGF driven tumor angiogenesis regulation indicating complex nature of NFAT in metastatic niche formation [28]. In addition, our results also depicted the differential expression of cytokine receptors (il2ra, il12rb2, il21r and il22ra), chemokine receptors (ccr3, xrc1 and cxcr7) and tumor necrosis factor receptors (tnfrsf1b, 9, 11a and 25) supporting further modulation of cross-talk between cancer and their microenvironment in ECM dependent manner, which cannot be established in 2D cultures. These observations suggest that the investigation of the role of inflammatory genes under 3D cell culture conditions could be very important to understanding the basal influence of genes involved in tumor microenvironment – immune system interactions in vivo. Results obtained culturing cells under 3D cell conditions could be also strongly considered in preclinical targeted therapy research, since ECM environment could strongly influence the responsiveness of tumor cells to immunotherapy.
While it has been well observed that miRNAs regulate the expression of ECM molecules, emerging evidence shows that miRNA expression and function could be significantly affected by the ECM [29, 30]. Consistent with these observations, in the present study microarray data demonstrated a signature of significantly altered expression of 77 miRNAs in LLC1 cells grown under 2D and lr-ECM 3D cell culture conditions compared to cells cultured on plastic. Interestingly, our results showed that ECM strongly induced the up-regulation of miRNA in LLC1 cells grown under 3D culture conditions. This is in accordance with a previous report which suggested that global upregulation of miRNA expression may be linked with the changes in cellular density [31]. Furthermore, our results also indicated that the ECM induced upregulation of miR-466?~?467?~?669 (e.g. miR-466b,c,d), miR-376 (miR-376a, miR-376b, miR-376c), and miR-34 (miR-34b and miR-34c) clusters. The miR-466?~?467?~?669 cluster is known as one of the largest miRNA clusters in mouse genome containing 71 miRNAs. A previous report [32] suggested that members of this cluster are abundantly expressed during mouse embryo development and might regulate growth and survival of embryonic stem cells. On the other hand, it has been shown that miR-376 cluster miRNAs are associated with tumorigenesis. For example, elevated expression of miR-376a promoted tumor cell migration and invasion and also positively correlated with advanced tumor metastasis and shorter patient survival [33, 34]. In addition, overexpression of miR-376c increased ovarian cancer cell survival and was associated with poor response to chemotherapy [35]. Moreover, elevated levels of miR-376c were shown in plasma of early stage breast cancer patients [36]. By contrast, miR-34 cluster encodes miRNAs possessing tumor suppressive properties mediating apoptosis, cell cycle arrest and senescence [37]. Our miRNA microarray data were consistent with previous reports indicating that human cancer cells cultured on ECM 3D cell culture conditions have also exhibited a significantly altered miRNA expression profile compared to cells cultured on plastic [38–40]. ECM 3D cell culture associated miRNA profiles demonstrated altered expression of tumor suppressive and oncogenic miRNAs and also correlated with distinct cellular morphogenesis under 3D culture conditions highlighting the regulation of miRNA expression in the ECM dependent manner. Additionally, we also showed that the expression of selected miR-195a, miR-207, miR-376c and miR-466f miRNAs was also significantly increased in mouse LLC1 tumors as compared to miRNA expression levels in 2D indicating the potential role of these miRNAs in tumor progression in vivo. Altogether, these findings suggest that the 3D cell culture should be considered as a critical experimental approach for essential understanding of the miRNA biology associated with tumor microenvironment. Indeed, the gene expression signature of 3D culture of breast cancer cells has been found to define prognostic value for patients with breast cancer [41]. Understanding how ECM regulates miRNA expression will also further elucidate how miRNAs determine tumor development and reveal potential prognostic and therapeutic opportunities.
Further on we also investigated potential relations between 77 differently expressed miRNAs and their target genes to depict possible miRNA-mRNA interactions in LLC1 cells regulated by ECM microenvironment under 3D cell culture conditions. We found that 8629 unique target genes could be regulated by these differently expressed miRNAs. Pathway enrichment analysis also revealed that 69 KEGG pathways were enriched in target genes related to these miRNAs including pathways involved in tumor development. However, as it is known that miRNA targets multiple mRNAs, the ability to find the key pathways by computational approaches is highly dependent on size of miRNA profile. In addition, the statistical target analysis approach could be successful if the miRNA of interest has an effect on the abundance of expressed target gene, but not if expression of target gene is regulated only by translational inhibition. Hence, we focused on negative correlation analysis between differently expressed miRNA and genes associated with metabolic, MAPK, cell adhesion and immune response pathways in LLC1 cells grown under 2D and 3D cell culture conditions. Indeed, we found that differently expressed genes associated to these pathways could be potentially regulated by miRNAs differently expressed in LLC1 cells.
In the present study the miRNA target filter analysis identified miRNAs showing inverse correlations with metabolic genes indicating the role of miRNA in metabolic pathway regulation. For instance, the down-regulation of miR-9, miR-19a, miR-135a, miR-495 and miR-590 negatively correlated with the up-regulation of genes involved in polysaccharide synthesis including alpha-mannosidase man2a1, glycosyltransferase ext1 and beta-1,3-galactosyltransferase B3galt6. We also found that the down-regulation of genes involved lipid metabolism including pla2g2c, dhrs9, ppt1 and sc5d inversely correlated with the up-regulation of miR-297a, miR-346, miR-466b, miR-466d, miR-468 and miR-669b. In addition, our results also revealed that the differential expression of diacylglycerol kinase dgkb and inositol polyphosphate 5-phosphatase ocrl regulating lipid signaling and membrane trafficking inversely correlated with the expression of miR-9, miR-126, miR-590 and miR-466 g, respectively. These findings are supported by recent studies demonstrating important roles of miRNAs in metabolic rearrangement occurring in cancer cells [42, 43]. Furhtermore, our results indicated that the expression of kras, mknk1 and pak2 kinases involved in the MAP kinase pathway negatively correlated with the expression of miR-761, mir-195 and miR-297a, respectively. The target correlation analysis also depicted miR-34b, miR-34c, miR-466f and miR-500 miRNAs as potential negative regulators of sos2 gene expression. However, the evidence implicating miRNAs role in MAP kinase pathway is still emerging. Previous report suggested that miR-34c may suppress proliferation of lung cancer cells by inhibition of MAPK pathway [44]. In addition, previous data also associated regulation of miRNAs with MAP kinases in pancreatic cancer cells showing that expression of miR-34a inversely correlated with MAPK pathway activity [45]. Ichimura et al. also demonstrated that miR-34a suppressed the expression of MEK1 leading to repression of the MEK-ERK signalling axis [46]. In the present study we also observed a significant link between deregulated expression of miRNA and cell adhesion molecules. For example, our results indicated a negative correlation between expression of col1a1 and miR-135a, miR-137 and miR-590. In addition, decreased flna expression might be influenced by miR-328 and miR-761. These findings are consistent with a previous report indicating the presence of feedback mechanisms that promote ECM molecules, which are downstream targets of specific miRNA, to regulate expression of these miRNAs [40]. A similar target enrichment analysis also revealed that increased expression of miRNAs might be connected with the regulation of immune response pathway genes. For example, our results depicted a negative correlation between expression of chemokine receptor xcr1 and miR-669b. Additionally, we also noted that decreased expression of oas3 might be affected by numerous miRNAs. Thus, taken together these findings suggest that metabolic, MAP kinase, cell adhesion and immune response pathway genes might be regulated by miRNAs altered in ECM dependent manner. Therefore, the 3D cell culture model could be applied not only for further investigation of common cancer pathways altered in ECM dependent manner but also for the study of specific miRNAs involved in ECM-mediated cancer signaling networks. Further understanding of complex ECM dependent signaling networks in tumors could direct to novel cancer treatment strategies.