Fibroblasts Influence Survival and Therapeutic Response in a 3D Co-Culture Model

RESEARCH ARTICLE Fibroblasts Influence Survival and Therapeutic Response in a 3D Co-Culture Model Meher Majety*, Leon P. Pradel, Manuela Gies, Carola H. Ries Discovery Oncology, Roche Innovation Center Penzberg, Pharmaceutical Research and Early Development, Penzberg, Germany * Abstract OPEN ACCESS Citation: Majety M, Pradel LP, Gies M, Ries CH (2015) Fibroblasts Influence Survival and Therapeutic Response in a 3D Co-Culture Model. PLoS ONE 10(6): e0127948. doi:10.1371/journal.pone.0127948 Academic Editor: Salvatore V Pizzo, Duke University Medical Center, UNITED STATES Received: October 1, 2013 Accepted: March 3, 2015 Published: June 8, 2015 Copyright: © 2015 Majety et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded internally by Roche Diagnostics GmbH. The funder provided support in the form of salaries for all authors, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the author contributions section. Competing Interests: The authors have the following interests: This study was funded internally by Roche Diagnostics GmbH, the employer of all authors. The anti-IGF1R antibody (R1507) and the anti-cMet antibody (Onartuzumab) are described in the patents US7572897 titled “Antibodies against Insulin-like growth factor I receptor and uses thereof” In recent years, evidence has indicated that the tumor microenvironment (TME) plays a significant role in tumor progression. Fibroblasts represent an abundant cell population in the TME and produce several growth factors and cytokines. Fibroblasts generate a suitable niche for tumor cell survival and metastasis under the influence of interactions between fibroblasts and tumor cells. Investigating these interactions requires suitable experimental systems to understand the cross-talk involved. Most in vitro experimental systems use 2D cell culture and trans-well assays to study these interactions even though these paradigms poorly represent the tumor, in which direct cell-cell contacts in 3D spaces naturally occur. Investigating these interactions in vivo is of limited value due to problems regarding the challenges caused by the species-specificity of many molecules. Thus, it is essential to use in vitro models in which human fibroblasts are co-cultured with tumor cells to understand their interactions. Here, we developed a 3D co-culture model that enables direct cell-cell contacts between pancreatic, breast and or lung tumor cells and human fibroblasts/ or tumorassociated fibroblasts (TAFs). We found that co-culturing with fibroblasts/TAFs increases the proliferation in of several types of cancer cells. We also observed that co-culture induces differential expression of soluble factors in a cancer type-specific manner. Treatment with blocking antibodies against selected factors or their receptors resulted in the inhibition of cancer cell proliferation in the co-cultures. Using our co-culture model, we further revealed that TAFs can influence the response to therapeutic agents in vitro. We suggest that this model can be reliably used as a tool to investigate the interactions between a tumor and the TME. Introduction The tumor-stroma interaction has been identified as a hallmark of cancer[1]. The role of stromal cells in cancer progression has partially been elucidated, and several processes from growth factor secretion to evading immune response have been attributed to the stromal cells. The PLOS ONE | DOI:10.1371/journal.pone.0127948 June 8, 2015 1 / 18 Influence of Fibroblasts on Tumor Cell Growth and US7476724 titled “Humanized anti-cMet antibodies” respectively. There are no products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. ratio of tumor stroma has been shown to serve as an independent prognostic factor for breast cancer patients that indicates a three-fold increased risk of relapse for stroma-rich tumors [2]. Further, stroma-related molecular signatures can be used to predict the resistance of breast cancer to neo-adjuvant chemotherapy [3]. A desmoplastic reaction involving a variety of stromal cell types is often described as a distinct unique characteristic of pancreatic cancer [4]. Similarly, stromal cells have also been implicated in cancer progression and prognosis of lung cancer [5]. Fibroblasts constitute one of the most abundant cell types in the tumor stroma [6]. In normal tissues, fibroblasts play an important role in maintaining homeostasis and in wound healing by producing an array of factors that constitute the extracellular matrix (ECM) and other growth factors and cytokines that are essential for healing [7]. The cross-talk between the tumor cells and stromal fibroblasts in the TME influences to the secretion of an array of growth factors and cytokine/chemokines that, in turn, support tumor cell growth or survival, induce neo-vascularization and generate an immuno-suppressive TME in several cancers [8, 9]. Currently, TAFs appear to play a key role in tumor progression, and provide significant predictive or prognostic value, as well as serve as potential therapeutic targets [10]. To understand the mechanisms underlying the cross-talk between tumor cells and TAFs in vitro, a co-culture system in which tumor cells can interact with fibroblasts, similar to the TME in situ, is required. Conventionally, trans-well chambers (Boyden chambers) are used for this purpose. Using this approach, cells are separated by a porous membrane through which soluble factors are able to diffuse freely but direct cell-cell interaction is absent. The importance of direct cell-cell contact in this context has been demonstrated by experiments showing that the collagenbased co-culture of breast cancer cells with serum-activated fibroblasts induced clonogenic growth in vitro [11]. Recently, it has been shown that the direct interaction between luminal-/ basal-like breast cancer cells and fibroblasts invokes distinct phenotypic and gene expression changes that differ from trans-well co-cultures [12]. In addition, Fujita et al., showed that pancreatic cancer cell proliferation was enhanced by directly co-culturing these cells with pancreatic stromal cells, allowing the two cell types to directly interact in the culture dishes [13]. However, these studies were performed by culturing either one of the cell types on a flat 2D surface, which hardly represents the complex TME in vivo. It has been clearly demonstrated that the 2D culture system, although convenient for most applications, is a poor environment to study d (...truncated)