Abstract
The tumor microenvironment (TME) is composed of a heterogeneous biological ecosystem of cellular and non-cellular elements including transformed tumor cells, endothelial cells, immune cells, activated fibroblasts or myofibroblasts, stem and progenitor cells, as well as the cytokines and matrix that they produce. The constituents of the TME stroma are multiple and varied, however cancer associated fibroblasts (CAF) and their contribution to the TME are important in tumor progression. CAF are hypothesized to arise from multiple progenitor cell types, including mesenchymal stem cells. Currently, isolation of TME stroma from patients is complicated by issues such as limited availability of biopsy material and cell stress incurred during lengthy adaptation to atmospheric oxygen (20% O2) in cell culture, limiting pre-clinical studies of patient tumor stromal interactions. Here we describe a microenvironment mimetic in vitro cell culturing system that incorporates elements of the in vivo lung environment, including lung fibroblast derived extracellular matrix and physiological hypoxia (5% O2). Using this system, we easily isolated and rapidly expanded stromal progenitors from patient lung tumor resections without complex sorting methods or growth supplements. These progenitor populations retained expression of pluripotency markers, secreted factors associated with cancer progression, and enhanced tumor cell growth and metastasis. An understanding of the biology of these progenitor cell populations in a TME-like environment may advance our ability to target these cells and limit their effects on promoting cancer metastasis.
Highlights
The tumor microenvironment consists of a diverse milieu of transformed and non-transformed cells that coordinate to build and maintain a physical environment that supports tumor growth and potentiates escape and establishment at secondary systemic sites[1]
cancer associated fibroblasts (CAF) produce a modified extracellular matrix (ECM) which acts as a bioactive mechanical scaffold that can influence tumor cell survival and metastasis, as well as matrix metalloproteinases (MMP) that allow the dynamic regulation of the ECM architecture through degradation of local matrix proteins and activation of latent growth factors within the matrix[11]
In order to test the hypothesis that an in-vitro culturing system resembling the microenvironment of the human lung would facilitate the isolation and expansion of sensitive primary patient tumor cell populations, we developed a microenvironment mimetic culturing system that includes a fibroblast derived extracellular matrix (ECM) and an atmosphere that maintained the oxygen tension at a physiological level (Fig. 1A)
Summary
The tumor microenvironment consists of a diverse milieu of transformed and non-transformed cells that coordinate to build and maintain a physical environment that supports tumor growth and potentiates escape and establishment at secondary systemic sites[1]. CAF currently lack specific markers but display characteristics similar to activated fibroblasts such as expression of alpha-smooth muscle actin (α-SMA) and vimentin[8] They act in paracrine fashion by producing potent proliferative cytokines that elicit mitogenic signaling within tumor cells. CAF produce a modified extracellular matrix (ECM) which acts as a bioactive mechanical scaffold that can influence tumor cell survival and metastasis, as well as matrix metalloproteinases (MMP) that allow the dynamic regulation of the ECM architecture through degradation of local matrix proteins and activation of latent growth factors within the matrix[11] Despite these critical effects on tumor homeostasis and progression, the origin of the CAF remains elusive and may arise from a heterogeneous number of cell types. We have shown that fetal human lung fibroblasts produce a CDM that supports lung adenocarcinoma cells to regulate EMT processes and provide survival signaling to promote survival in conditions of serum starvation and hypoxia[16]
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