Matrix Remodeling and Hyaluronan Production by Myofibroblasts and Cancer-Associated Fibroblasts in 3D Collagen Matrices

Abstract

The tumor microenvironment is a key modulator in cancer progression and has become a novel target in cancer therapy. An increase in hyaluronan (HA) accumulation and metabolism can be found in advancing tumor progression and are often associated with aggressive malignancy, drug resistance and poor prognosis. Wound-healing related myofibroblasts or activated cancer-associated fibroblasts (CAF) are assumed to be the major sources of HA. Both cell types are capable to synthesize new matrix components as well as reorganize the extracellular matrix. However, to which extent myofibroblasts and CAF perform these actions are still unclear. In this work, we investigated the matrix remodeling and HA production potential in normal human dermal fibroblasts (NHFB) and CAF in the absence and presence of transforming growth factor beta -1 (TGF-β1), with TGF-β1 being a major factor of regulating fibroblast differentiation. Three-dimensional (3D) collagen matrix was utilized to mimic the extracellular matrix of the tumor microenvironment. We found that CAF appeared to response insensitively towards TGF-β1 in terms of cell proliferation and matrix remodeling when compared to NHFB. In regards of HA production, we found that both cell types were capable to produce matrix bound HA, rather than a soluble counterpart, in response to TGF-β1. However, activated CAF demonstrated higher HA production when compared to myofibroblasts. The average molecular weight of produced HA was found in the range of 480 kDa for both cells. By analyzing gene expression of HA metabolizing enzymes, namely hyaluronan synthase (HAS1-3) and hyaluronidase (HYAL1-3) isoforms, we found expression of specific isoforms in dependence of TGF-β1 present in both cells. In addition, HAS2 and HYAL1 are highly expressed in CAF, which might contribute to a higher production and degradation of HA in CAF matrix. Overall, our results suggested a distinct behavior of NHFB and CAF in 3D collagen matrices in the presence of TGF-β1 in terms of matrix remodeling and HA production pointing to a specific impact on tumor modulation.

Keywords: cancer associated fibroblast; hyaluronan; matrix remodeling; myofibroblast; transforming growth factor beta 1; tumor microenvironment.

Figure 1

Effect of TGF-β1 on proliferation, aSMA expression, and matrix remodeling of NHFB and CAF. NHFB and CAF were cultured on 3D collagen matrices in the presence and absence of TGF-β1 for 7 days. Both cells were then characterized in terms of proliferation, aSMA expression, and matrix remodeling. (A) Quantitative analysis of cell number using commercial WST-1 assay. (B) Representative confocal images of NHFB and CAF. Cells were stained with DAPI and Phalloidin conjugated with Alexa Fluor 488 to visualize nucleus and actin, respectively. Collagen fibrils were imaged in the reflection mode. (C) The expression of aSMA was analyzed using qPCR and was normalized to NHFB cultured in the absence of TGF-β1. (D) Immunocytostaining of aSMA and (E) quantitative analysis of number of aSMA positive cells by manual count. Data are represented as mean ± SD; *—significance level of p < 0.05; n.s.—not significant. All quantitative experiments were performed at least in 3 replicates with fibroblasts and CAF from 3 different donors for each condition.

Figure 2

Matrix remodeling of NHFB and CAF in the presence and absence of TGF-β1. Collagen matrices were decellularized and analyzed regarding (A) matrix elastic modulus and (B) mean pore size. For both (A) and (B) the dashed line symbolizes empty collagen matrix pore size without cells. The relative gene expression of (C) alpha 1 chain of collagen type 1 (Coll1a1) and (D) fibronectin containing EDA-domain (EDA-FN) were analyzed using qPCR and were normalized to NHFB cultured in the absence of TGF-β1. Data are represented as mean ± SD; *—significance level of p < 0.05; n.s.—not significant. All quantitative experiments were performed at least in 3 replicates using fibroblasts and CAF from 3 different donors for each condition.

Figure 3

HA production and localization. HA amount of (A) cell culture supernatant and (B) decellularized matrices were analyzed using a commercial HA ELISA kit. Data are represented as mean ± SD; *—significance level of p < 0.05; n.s.—not significant. (C) Representative images of decellularized 3D collagen matrices of FB and CAF in dependency of TGF-β1. Matrices were stained with biotin-tagged HABP followed by streptavidin conjugated with Alexa Fluor 488 to visualize HA (dark blue). Collagen fibrils were visualized using reflection mode (grey-yellow). All quantitative experiments were performed at least in 3 replicates using fibroblasts and CAF from 3 different donors for each condition.

Figure 4

Analysis of HA molecular weight. (A) Representative images of electrophoresis gel of supernatant and soluble decellularized collagen matrices of FB and CAF in dependence of TGF-β1. (B) Representative density profile plot of molecular weight distribution of matrix bound HA of CAF treated with TGF-β1. (C) Analysis of the average HA molecular weight of decellularized matrices. Data are presence as mean ± SD; n.s.—not significant. All quantitative experiments were performed at least in 3 replicates with fibroblasts and CAF from 3 different donors for each condition.

Figure 5

Gene Expression of HA related metabolic enzymes. Quantitative analysis of gene expression of (A(i)) HAS1, (A(ii)) HAS2 and (A(iii)) HAS3, and (B(i)) HYAL1, (B(ii)) HYAL2 and (B(iii)) HYAL3 of FB and CAF in dependence of TGF-β1 presentation. Data are normalized to NHFB without TGF-β1 treatment. Data are represented as mean ± SD; *—significance level of p < 0.05; n.s.—not significant. For all quantitative experiments were performed at least 3 replicates with fibroblasts and CAF from 3 different donors for each condition.