Gene and miRNA expression signature of Lewis lung carcinoma LLC1 cells in extracellular matrix enriched microenvironment

Stankevicius et al. BMC Cancer (2016) 16:789 DOI 10.1186/s12885-016-2825-9 RESEARCH ARTICLE Open Access Gene and miRNA expression signature of Lewis lung carcinoma LLC1 cells in extracellular matrix enriched microenvironment Vaidotas Stankevicius1,2, Gintautas Vasauskas1,2, Danute Bulotiene1, Stase Butkyte3, Sonata Jarmalaite1,4, Ricardas Rotomskis1,5 and Kestutis Suziedelis1,2,6* Abstract Background: The extracellular matrix (ECM), one of the key components of tumor microenvironment, has a tremendous impact on cancer development and highly influences tumor cell features. ECM affects vital cellular functions such as cell differentiation, migration, survival and proliferation. Gene and protein expression levels are regulated in cell-ECM interaction dependent manner as well. The rate of unsuccessful clinical trials, based on cell culture research models lacking the ECM microenvironment, indicates the need for alternative models and determines the shift to three-dimensional (3D) laminin rich ECM models, better simulating tissue organization. Recognized advantages of 3D models suggest the development of new anticancer treatment strategies. This is among the most promising directions of 3D cell cultures application. However, detailed analysis at the molecular level of 2D/3D cell cultures and tumors in vivo is still needed to elucidate cellular pathways most promising for the development of targeted therapies. In order to elucidate which biological pathways are altered during microenvironmental shift we have analyzed whole genome mRNA and miRNA expression differences in LLC1 cells cultured in 2D or 3D culture conditions. Methods: In our study we used DNA microarrays for whole genome analysis of mRNA and miRNA expression differences in LLC1 cells cultivated in 2D or 3D culture conditions. Next, we indicated the most common enriched functional categories using KEGG pathway enrichment analysis. Finally, we validated the microarray data by quantitative PCR in LLC1 cells cultured under 2D or 3D conditions or LLC1 tumors implanted in experimental animals. Results: Microarray gene expression analysis revealed that 1884 genes and 77 miRNAs were significantly altered in LLC1 cells after 48 h cell growth under 2D and ECM based 3D cell growth conditions. Pathway enrichment results indicated metabolic pathway, MAP kinase, cell adhesion and immune response as the most significantly altered functional categories in LLC1 cells due to the microenvironmental shift from 2D to 3D. Comparison of the expression levels of selected genes and miRNA between LLC1 cells grown in 3D cell culture and LLC1 tumors implanted in the mouse model indicated correspondence between both model systems. (Continued on next page) * Correspondence: 1 National Cancer Institute, Vilnius, Lithuania 2 Department of Biochemistry and Molecular Biology, Faculty of Natural Sciences, Joint Life Sciences Center, Vilnius University, Vilnius, Lithuania Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Stankevicius et al. BMC Cancer (2016) 16:789 Page 2 of 13 (Continued from previous page) Conclusions: Global gene and miRNA expression analysis in LLC1 cells under ECM microenvironment indicated altered immune response, adhesion and MAP kinase pathways. All these processes are related to tumor development, progression and treatment response, suggesting the most promising directions for the development of targeted therapies using the 3D cell culture models. Keywords: 3D cell culture, ECM, Gene and miRNA expression signature, MAPK signaling pathway, Cell adhesion, Inflammatory response Background The extracellular matrix (ECM), as one of the key components of tumor microenvironment, has a significant impact on cancer development and highly influences tumor cell features and therefore the response to treatment [1]. ECM contributes not only structural support of growing tumor cells, but also affects other cellular functions such as cell differentiation, migration, survival or proliferation [2–4]. Moreover, gene and protein expression levels are regulated in cell-ECM interaction dependent manner [5, 6]. Not surprisingly, clinical trials based on preclinical two-dimensional (2D) monolayer cell culture models which lack representation of ECM dependent molecular processes occurring in tumors currently have a failure rate of up to 95 %. Cancer cell growth under three-dimensional (3D) culture conditions simulating ECM microenvironment better resembles tumor cell properties in vivo [7]. Thus, investigations using such 3D cell culture models are expected to result in more successful clinical trials. Vast amount of evidence indicates the superiority of 3D cell cultures compared to 2D models for investigating cancer tumor microenvironment dependent cancer cell properties [8, 9]. Obvious advantages of 3D cell culture models are the cellular-ECM interactions and cell-cell contacts, the formation of active proliferation, quiescent viable cell and necrotic cell zones, as well as the formation of nutritional, oxygen and drug gradients better reflecting cellular organization and the microenvironment in tumor tissue [10]. Nevertheless, the 3D cell cultures do not resemble the full complexity of tumor tissue environment in vivo. Few obvious limitations of 3D cell cultures as a cancer research model are the lack of vasculature, host immune response and other cell-cell interactions that occur between cancer and stromal cells in tumors [11]. Recognized advantages and limitations of 3D cell culture models suggest that the most successful directions of 3D model application include the development of new anticancer treatment strategies. Hence, detailed analysis at the molecular level of 2D/3D cell cultures and tumors in vivo are still needed to unlock the power of 3D cell culture models in translational research. In order to elucidate which biological pathways are altered during microenvironmental shift, we have analyzed whole genome mRNA and miRNA expression changes in murine Lewis lung cancer LLC1 cells cultured in 2D or laminin rich ECM (lr-ECM) 3D conditions. LLC1 cell line was established from the lung of a C57BL mouse bearing a tumor of primary Lewis lung carcinoma. This cell line is highly tumorigenic and the implanted cells are immunologically compatible with the murine immune system, unlike the widely used human cancer (...truncated)