Although it is widely accepted that tumorigenesis is regulated by interactions between tumor cells and CAFs, the precise origin and function of CAFs has been unclear. We show that: (1) CAFs increase during chronic inflammation and gastric cancer progression, particularly during the transition to dysplasia; (2) CAFs are derived from αSMA+ MF present in the normal BM; and (3) αSMA+ MF are generated from MSC and contribute to the normal BM niche and MSC self-renewal. During chronic inflammation and carcinogenesis, these αSMA+ BM niche cells are (4) expanded in a TGF-β–dependent matter and recruited through CXCR4/SDF1α signaling together with Gremlin-1-expressing MSC to incipient tumors where they (5) contribute to a tumor niche that promotes and sustains tumor progression.
In inflammation-dependent models of gastric cancer, progression to dysplasia is associated with accumulation of αSMA-expressing CAFs, compared to collagen-α1-expressing fibroblasts. αSMA-expressing CAFs could in theory originate from local fibroblasts, endothelial cells, mesenchymal cells, local epithelial cells during the EMT, or recruited BMDC. Studies using RIPTag transgenic mice showed that BMDC can give rise to CAFs and fibroblasts (Direkze et al., 2004
), which was confirmed in other cancer models (Guo et al., 2008
). More importantly, studies in patients with gastric cancer that received gender-mismatched BM transplants have confirmed the BM origin of CAFs in gastric cancer (Worthley et al., 2009
). We demonstrate through BM reconstitution studies in mice that 20% of αSMA+ fibroblastic cells that accumulate in dysplasia are BM-derived, and that BM MSCs are the likely origin of these CAFs (). In contrast to previous reports (Simmons et al., 1987
; Yokota et al., 2006
), we show here that it is indeed possible to transplant BM MSCs in the mouse. CAFs recruited from the BM are functionally more active as promoters of tumor growth and invasion compared to normal or resident fibroblasts, and exhibit a previously reported NF-κB dependent inflammatory gene expression signature (Erez et al., 2010
), which accounts for higher levels of pro-inflammatory cytokines in CAFs (Karnoub et al., 2007
; Orimo et al., 2005
In mouse models of inflammation induced gastric dysplasia, αSMA+ CAF were recruited to the stomach after first accumulating in the peripheral blood and in the BM. Indeed, the accumulation of αSMA+ cells in the BM appeared to correlate with the development of dysplasia, and suggests that carcinogenesis involves an early stage of BM remodeling. In the normal BM, MSCs are able to give rise to αSMA+ cells that morphologically and functionally behave identically to CAFs isolated from the dysplastic stomach, and these αSMA+ MF cells appear to be expanded in cancer.
Previous studies have suggested that MSCs can be induced to differentiate into CAFs, particularly when exposed to tumor-conditioned media (Mishra et al., 2008
). However, we show that differentiation of MSC into αSMA+ cells is a normal pattern of differentiation in vitro
and in vivo
, compatible with asymmetric stem cell division in a broad definition, although more specification regarding an intrinsic or extrinsic mechanism would be needed (Morrison and Kimble, 2006
). Moreover, that αSMA+ myofibroblastic cells represent normal niche cells within the BM that maintain the self-renewal properties of MSCs. In culture, MSC lose their proliferative potential and ability to self-renew when αSMA+ cells are specifically eliminated, and these capabilities are restored if αSMA+ cells are added back to the MSC. The αSMA-RFP transgene has allowed us to sort the heterogeneous, adherent population of BM mesenchymal cells, revealing that the true MSC is αSMA- but can give rise to αSMA+ MF.
Most adult stem cells are extremely difficult to culture in vitro
in the absence of supportive niche cells or defined growth factors, and the fact that MSCs generate their own αSMA+ niche cells clarifies the self-renewal abilities of BM MSC cultures. The concept that a stem cell is able to generate its own niche cell has precedence (Mathur et al., 2010
; Snippert et al., 2010
). In addition, the presence of the αSMA+ cells in the BM raises interesting questions regarding the possible role of these cells beyond support of the MSC. The BM niche is thought to consist of osteoblasts (Lo Celso et al., 2009
), which only exist in the bone, but further studies are required to determine if CAF-like cells support the growth of stem cells beyond the MSC.
The development of dysplasia and tumors in the stomachs of mice was associated temporally with an expansion of αSMA+ cells in the BM, which was reproduced by co-culture of MSCs with gastric cancer cells, indicating the effect of a soluble, secreted factor. Previous studies have shown that co-culture of MSCs with cancer cells could result in differentiation into CAFs (Mishra et al., 2008
), and that DNA hypomethylation induced by 5-aza-dC promoted the differentiation of pooled human MSC cultures into CAFs. We had shown that CAFs become markedly hypomethylated during early stages of human gastric carcinogenesis (Jiang et al., 2008
). Here, we confirm that hypomethylation is sufficient to induce differentiation of CAFs and that the effect is specific for the RFP- MSCs. However, we also demonstrate that a soluble growth factor often secreted by tumors, TGF-β, may contribute to the development of CAFs from MSCs during tumorigenesis. Incubation of RFP- BM-derived MSCs with TGF-β induced DNA hypomethylation and accelerated their differentiation into αSMA+ CAFs. The promotion by TGF-β of myofibroblastic differentiation occurs in part through an SDF-1α dependent pathway, since inhibition of CXCR4 blocked this differentiation process. Thus, although global hypomethylation is a well-known feature of malignant tumors, we demonstrate that TGF-β can induce both fibroblastic differentiation and global DNA hypomethylation rapidly in vitro
The αSMA+ MF niche cells express a number of factors that likely contribute to the stem cell niche and also maintain the self-renewal properties of incipient tumors (). Factors such as Wnt5a, BMP4 and IL-6 were highly expressed by RFP+ cells, as well as BM-derived CAFs isolated from gastric dysplasia. Wnt signaling has been shown in the gut to be important both for the maintenance of tissue stem cells and the generation of cancer stem cells (CSC) (Brabletz et al., 2009
) and recently was associated with maintaining or inducing stemness in CSC (Vermeulen et al.). BMPs have also been linked to intestinal and hematopoietic stem cell maintenance, controlling the stem cell number through regulation of the niche size (Zhang and Li, 2005
). IL-6 appears to be required for the survival of intestinal epithelial cells and the development of inflammation-associated cancer of the gut, probably through activation of STAT-3 pathways in intestinal progenitors (Grivennikov et al., 2009
). However, although the RFP+ CAFs promoted in vitro
proliferation of cancer cells in organotypic models, the RFP+/− cells, which contained MSCs and CAFs, appeared to be very important in promoting in vivo
tumor growth, particularly when injected at a distance from the tumor site. These findings indicate that MSCs are required for the production or migration of CAFs over time. Our results suggest that SDF-1α is likely produced by the MSC-containing RFP- population, rather than the RFP+ CAFs; SDF-1α seems to function in an autocrine function to stimulate more niche cells, and a paracrine function to attract and maintain CAFs in close proximity to tumors. Thus, CXCR4 antagonism - in our xenograft studies and our gastric carcinogenesis studies - appeared to inhibit stromal cell recruitment to the tumors and also block the production of CAFs in the BM of mice with cancer.
The two cell types (RFP- MSC and RFP+ MF) function together as stem cell and niche cell and communicate with each other. A cross-talk between the MSC and MF results in a unique pattern of gene expression characteristic of the niche, compared to those of each individual cell population. For example, DKK1 and Shh are significantly upregulated in heterogeneous RFP+/− cultures but not in sorted RFP- and RFP+ cells, indicating that these factors can only be expressed in a functioning stem cell niche. Gremlin-1 was expressed on RFP- cells, presumably the true MSC, but mainly under the conditions when the MSCs were cultured together in a mixed RFP+/RFP- population. Interestingly, Gremlin-1, an antagonist of the BMP pathway, is not expressed by normal adult fibroblasts or MF from the skin or most solid organs; expression of Gremlin-1 has been reported to be unique to stromal cells in the setting of cancer (Sneddon et al., 2006
) and was consistently upregulated in our model of gastric carcinogenesis. Since Gremlin-1 expression was observed only in cultures that contain MSCs and MFs/CAFs, it is possible that MSC expression of Gremlin-1 occurs in response to BMP signals from the αSMA+ myofibroblastic cells. Consistently, high levels of Gremlin-1 have been observed in mouse embryonic fibroblast cells that are capable of maintaining human embryonic stem cells in culture (Pera et al., 2004
In summary, we show that chronic inflammation and epithelial dysplasia lead to remodeling of the BM and expansion of αSMA+ MFs in a manner that promotes cancer growth and progression. The CAF-like MFs contribute to the niche for the MSCs, and it is this fully intact MSC niche that is recruited to the tumor site and that stimulates malignant progression. The recruitment of the niche to the tumor site can be blocked by CXCR4 inhibition and the differentiation of MSCs can be abrogated by TGF-β inhibition. We propose a model in which during the earliest stages of inflammation-induced tumor development, the BM undergoes remodeling, mediated in part by TGF-β, and then the MSC-CAF stem cell niche promotes tumor progression through SDF-1α signaling (). These observations have implications for the early diagnosis and treatment of cancer.