A bioengineered microenvironment to quantitatively measure the tumorigenic properties of cancer-associated fibroblasts in human prostate cancer
Introduction
The cell microenvironment through multiple controlled signals directs fundamental cellular processes such as migration, proliferation, survival, and differentiation. Aberrant cues can result in diseases, such as cancer [1], [2]. Specifically, in prostate cancer, the stromal microenvironment is critical in determining epithelial cell differentiation and function [3]. In tumor stroma, carcinoma-associated fibroblasts (CAFs) have a distinct phenotype relative to normal prostate fibroblasts (NPFs) and confer tumorgenicity on non-tumorigenic prostatic epithelial (BPH-1) cells when xenografted in vivo [4], [5], [6], [7]. Based on these observations, CAFs are a therapeutic target.
At present, the only proven method of investigating CAF tumorigenicity is tissue recombination, where stroma and epithelia are co-grafted into immune-deficient mice and the outcome is whether or not a tumor is formed. We and others have widely used this bioassay, yet the techniques are lengthy and technically challenging; only recently we developed a method for unbiased semi-quantitative analyses [4]. To advance the field, there is a need to develop improved in vitro models to mimic the cancer cell interactions with the microenvironment and allow quantitative analyses of the outcome, in terms of their effect on the epithelium. In turn, this will enable the comparison of relative differences between different patient CAFs. Ultimately, this data is fundamental to identify mechanisms that underpin therapeutic targeting of tumor stroma.
In the past, in vitro models of cancer cells have largely depended on the use of two-dimensional (2D) tissue culture on plastic or glass surfaces. Although much has been learned from these studies about how paracrine mechanisms instruct or direct cell behavior, these approaches are limited by the fact that the cells on surfaces often experience artificial polar environments, atypical elastic properties of their environment, and non-physiological cell contacts and densities. Moreover, the flat surface of the tissue culture plate represents a poor topological approximation of the more complex three-dimensional (3D) architecture of the extracellular matrix (ECM) [8]. Therefore, more physiologically mimetic model systems to study both normal and abnormal functions of cells and tissues are desirable [9]. There is strong evidence exists that the highly porous nano- and microtopography that results from the 3D fibrillar associations of ECM proteins is essential for cell adherence, cytoskeletal organization, migration, signal transduction, morphogenesis and differentiation in cell culture [10], [11].
The hypothesis of this study was that production of ECM components in a cellularized co-culture method would allow in vitro validation of prostatic CAFs. Herein, a bioengineered approach was developed to compare the effects of human primary stromal fibroblasts on epithelial cell morphology and motility and test if there is a change with prostate cancer progression.
Section snippets
Isolation of primary prostatic stromal cells (CAFs and NPFs)
Human prostate specimens were obtained by the Australian Prostate Cancer BioResource with approval from the Cabrini Institute (03-14-04-08), Epworth Hospital (34306) and Monash University Human Research Ethics Committees (2004/145). Tissue was collected from radical prostatectomy specimens for CAFs and NPFs as previously described [4]. Transurethral resection of the prostate (TURP) tissue was used for benign prostate fibroblasts (BAFs) and CAFs from castrate resistant prostate cancer (CRPC).
Morphological and molecular characterization of prostatic stroma
For this study, primary fibroblasts were isolated from human radical prostatectomy specimens. Tissue pieces were selected from confirmed malignant areas and adjacent non-malignant areas, as assessed by a pathologist. Carcinoma-associated fibroblasts (CAFs) and normal prostatic fibroblasts (NPFs) were isolated from digested tissue and cultured in medium that selects for fibroblasts instead of prostate epithelial cells. As an additional control, benign-associated fibroblasts (BAFs) were isolated
Discussion
Xenograft models where epithelia are recombined with stroma to study tumorigenicity are based on complex cellular interactions that cannot be replicated in standard 2D cell cultures because cues from the cell matrix play a critical role in directing and maintaining cell fate in vivo. Misregulation within the extracellular space can cause cell death or other aberrant behaviors associated with developmental defects and diseases such as fibrosis and cancer. Thus, as one thinks about cell culture
Conclusions
This co-culture technique is a reliable in vitro model to quantify stromal–epithelial interactions in the tumor. The cellularized matrix, which includes production of ECM, more accurately mimics the microenvironment found in vivo. Using this method, quantitative comparisons were made between fibroblasts from individual men at different stages of prostate cancer progression. Our data demonstrate the ability of CAFs to induce phenotypic changes in BPH-1 cells, regardless of the tumor stage.
Acknowledgments
This research was funded by the Prostate Cancer Foundation of Australia (Project Grant NCG4712, Movember Young Investigator Grants to R.A.T., S.J.E. and M.G.L.), Australian Prostate Cancer Research Centre-Queensland, Peter and Lyndy White Foundation, Australian Research Council (Discovery Project – DP110103890), Cancer Australia (1044458), National Health and Medical Research Council (Fellowships to G.P.R. and M.G.L.; 1035721) and Victorian Prostate Cancer Research Consortium (Scholarship to
References (25)
Fibroblast biology in three-dimensional collagen matrices
Trends Cell Biol
(2003)- et al.
Three-dimensional cancer-bone metastasis model using ex-vivo co-cultures of live calvarial bones and cancer cells
Biomaterials
(2012) - et al.
Mimicking normal tissue architecture and perturbation in cancer with engineered micro-epidermis
Biomaterials
(2012) - et al.
Modeling tissue morphogenesis and cancer in 3D
Cell
(2007) Can cancer be reversed by engineering the tumor microenvironment?
Semin Cancer Biol
(2008)- et al.
The locomotion of mouse fibroblasts in tissue culture
Biophys J
(1970) - et al.
Prostate cancer cell death produced by the co-delivery of Bcl-xL shRNA and doxorubicin using an aptamer-conjugated polyplex
Biomaterials
(2010) - et al.
Cell–matrix adhesion
J Cell Physiol
(2007) - et al.
Prostatic tumor stroma: a key player in cancer progression
Curr Cancer Drug Targets
(2008) - et al.
Human epithelial basal cells are cells of origin of prostate cancer, independent of CD133 status
Stem Cells
(2012)
Cross-talk between paracrine-acting cytokine and chemokine pathways promotes malignancy in benign human prostatic epithelium
Cancer Res
Identification of SFRP1 as a candidate mediator of stromal-to-epithelial signaling in prostate cancer
Cancer Res
Cited by (53)
The functional cross talk between cancer cells and cancer associated fibroblasts from a cancer mechanics perspective
2021, Biochimica et Biophysica Acta - Molecular Cell ResearchCitation Excerpt :CAFs provide nutrients for cancer cells by secretion of exosomes [152], and coordinate some cancer cell behaviors such as proliferation, angiogenesis and invasion to other tissues [153–155]. In an in vitro study using 3D matrix, the key role of CAFs in increased motility and the subsequent invasive behavior of prostate cancer cells were confirmed [156]. It has been observed that in some occasions during invasion to other tissues, CAFs accompany cancer cells [157].
Cancer-associated fibroblasts of the prostate promote a compliant and more invasive phenotype in benign prostate epithelial cells
2020, Materials Today BioCitation Excerpt :In this study, we compared the mechanical properties of primary CAFs and non-malignant prostate tissue fibroblasts (NPFs) isolated from malignant or benign regions of prostate tissue from the same patient [36]. Previous works have identified distinct proteomic and epigenetic profiles in patient-matched pairs of stromal cells, altered interactions with epithelial and immune cells, as well as aberrant ECM deposition, compared with normal fibroblasts from the prostate [17,37–41]. Here, we compare the mechanical characteristics of patient-matched CAFs and NPFs as well as their co-cultures with benign prostatic epithelial cells using AFM and RT-FDC.
In vitro three-dimensional modeling for prostate cancer
2020, Biomaterials for 3D Tumor ModelingTissue engineered human prostate microtissues reveal key role of mast cell-derived tryptase in potentiating cancer-associated fibroblast (CAF)-induced morphometric transition in vitro
2019, BiomaterialsCitation Excerpt :Prostatic cancer-associated fibroblasts (CAFs) and their non-malignant counterparts (non-malignant prostatic fibroblasts; NPFs) exhibit biochemical, proteomic and epigenetic differences [3–6]. Functionally, these differences enable CAFs to initiate and potentiate tumourigenicity in adjacent epithelia [3,4,7,8]. In particular, CAFs alter the physical environment of the epithelia by depositing an aberrant extracellular matrix (ECM) [9].
Proteomic profiling of human prostate cancer-associated fibroblasts (CAF) reveals LOXL2-dependent regulation of the tumor microenvironment
2019, Molecular and Cellular Proteomics
- 1
Authors contributed equally.