Mesenchymal stem cell transition to tumor-associated fibroblasts contributes to fibrovascular network expansion and tumor progression

Abstract

Background: Tumor associated fibroblasts (TAF), are essential for tumor progression providing both a functional and structural supportive environment. TAF, known as activated fibroblasts, have an established biological impact on tumorigenesis as matrix synthesizing or matrix degrading cells, contractile cells, and even blood vessel associated cells. The production of growth factors, cytokines, chemokines, matrix-degrading enzymes, and immunomodulatory mechanisms by these cells augment tumor progression by providing a suitable environment. There are several suggested origins of the TAF including tissue-resident, circulating, and epithelial-to-mesenchymal-transitioned cells.

Methodology/principal findings: We provide evidence that TAF are derived from mesenchymal stem cells (MSC) that acquire a TAF phenotype following exposure to or systemic recruitment into adenocarcinoma xenograft models including breast, pancreatic, and ovarian. We define the MSC derived TAF in a xenograft ovarian carcinoma model by the immunohistochemical presence of 1) fibroblast specific protein and fibroblast activated protein; 2) markers phenotypically associated with aggressiveness, including tenascin-c, thrombospondin-1, and stromelysin-1; 3) production of pro-tumorigenic growth factors including hepatocyte growth factor, epidermal growth factor, and interleukin-6; and 4) factors indicative of vascularization, including alpha-smooth muscle actin, desmin, and vascular endothelial growth factor. We demonstrate that under long-term tumor conditioning in vitro, MSC express TAF-like proteins. Additionally, human MSC but not murine MSC stimulated tumor growth primarily through the paracrine production of secreted IL6.

Conclusions/significance: Our results suggest the dependence of in vitro Skov-3 tumor cell proliferation is due to the presence of tumor-stimulated MSC secreted IL6. The subsequent TAF phenotype arises from the MSC which ultimately promotes tumor growth through the contribution of microvascularization, stromal networks, and the production of tumor-stimulating paracrine factors.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. In admixtures of tumor and MSC, MSC exhibit four attributes of TAF.

(A) Initially, MSC do not express all TAF markers. As shown by western, huMSC, prior to Skov-3 tumor exposure, express low levels of α-SMA, FAP and desmin and are negative for the expression of, FSP, TSP-1, and Tn-C. Following 16 days exposure to Skov-3-conditioned medium, the MSC express Tn-C, TSP-1, FSP and increase expression of α-SMA, FAP and desmsin. (B) The TAF is a fibroblastic cell that has four defining characteristics, which together, distinguish it from the normal fibroblast: The fibroblastic nature of the cell (red; Fibroblast activation protein and fibroblast specific protein); the aggressive/invasive nature of the cell defined by the secreted proteins (green; TSP-1, Tn-C and SL1); the vascularization potential of the cell (blue; α-SMA, desmin and VEGF); and the growth factors secreted by the cell to aid in tumor growth and development (yellow; EGF, HGF, IL-6 and bFGF).

Figure 2. Admixed Skov-3/MSC xenografts express fibroblast markers and invasive markers indicative of TAF.

IHC staining for markers that define fibroblast presence are visible in the Skov-3/MSC (1∶1) admixed xenografts but not in Skov-3-only xenografts. Fibroblast markers FAP (A) and FSP (B) are both present throughout the admixed tumors. The three tissue remodeling proteins Tn-C (C), TSP-1 (D) and SL-1 (E) are detectable within the admixed tumors. These two categories cover the first two characteristics of a TAF, and are observed only in tumors exposed to MSC. Human-specific, mouse non-cross-reactive antibodies allow identification of the human MSC-contributory components within the tumor microenvironment.

Figure 3. Admixed Skov-3/MSC xenografts express provascularization markers.

One characteristic of the TAF is their ability to form fibrovascular networks, which include vessels and the architectural foundation in which they lay. IHC staining reveals structural proteins α-SMA (A) and desmin (B) as well as factors suggestive of tumor vascularization, including VEGF (C) in the presence of admixed MSC but not in tumors that received no MSC (far right panel). HGF (D) is found within the stromal compartment of the tumor. A strong delineation separating the tumor and stromal compartments is formed when the admixed xenograft is stained for HGF. Likewise, EGF (E) is highly expressed in the stromal regions of the admixed Skov-3 tumor. (F) IL-6 staining is expressed in the Skov-3 alone tumors, however the expression is increased in the admixed tumors.

Figure 4. huMSC secreted growth factors promote growth of Skov-3 tumors.

The TAF phenotype includes secretion of pro-tumorigenic growth factors. (A) Supported by the secretion of growth factors by the huMSC. In vivo growth of xenograft Skov-3 tumors steadily progress but when mixed at a 1∶1 ratio with MSC rapid growth ensued after day 65. At day 91, the Skov-3/MSC 50/50 tumors were significantly larger than the Skov-3 alone (P<0.05). (B) Skov-3 tumor cell growth is species dependent. The Skov-3 ovarian tumor cells were growth in co-culture with huMSC, and several muMSC including cells isolated from balb/c and C57 mice, and a fibroblast cell line, 3T3. Growth is graphed as fold change relative to normal Skov-3 proliferation. The only cell line that produced adequate factors to induce cell growth is the huMSC (red circles). The huMSC induced Skov-3 growth significantly (P<0.01) better than the other cell lines. (C) Naïve MSC produce basal levels of IL-6, TGF-β, VEGF and HGF. All secreted factors with the exception of HGF are significantly increased upon stimulation with Skov-3 CM (P<0.001).

Figure 5. Growth factors are critical to Skov-3 tumor progression.

(A) An in vitro 3D tumor growth assay (TGA) at day 8. Briefly, RFP-labeled Skov-3 tumor cells were mixed with huMSC cells, huMSC conditioned media (CM) or various recombinant cytokines (FGF, HGF, TGF-β, VEGF, EGF, IL-6) in a 3D assays (as described in the materials and methods). EGF (P<0.05) and IL6 (P<0.01) significantly increased the proliferation of Skov-3 cells compared to the Skov-3 only, but less than the MSC (P<0.01) induced Skov-3 proliferation. (B) Immunoprecipitation of IL-6 from the Skov-3/MSC co-culture medium over an 8 day period reveals significantly reduced growth of the Skov-3 (P<0.01) cells as compared with the Skov-3/MSCco-culture. Cell growth is graphed as fold change relative to normal Skov-3 proliferation.