Multifaceted Tumor Stromal Fibroblasts

Inflammation and Fibroblast Activation

Chronic inflammation is a hallmark of cancer [22], a lot of factors in cancer-associated inflammation are reported to induce the activation of fibroblasts. Among them transforming growth factor beta (TGF-β) and platelet-derived growth factor (PDGF) are the most well investigated ones to activate fibroblasts. TGF-β has been reported to be capable of inducing fibroblasts to differentiate into activated smooth-muscle reactive fibroblasts, termed myofibroblasts; while PDGF is in favor of the proliferation of myofibroblasts [2, 23, 24]. Erez et al. have found that Interleukin (IL)-1 treatment on fibroblasts activated the nuclear factor (NF)-κB signaling pathway and induced the expression of pro-inflammatory genes [25]. Similarly, hepatic stellate cells (HSCs), the main kind of fibroblasts in liver, were found sensitive to LPS derived from the intestinal tract, which resulted in the increased activation and survival rate of HSCs, and contributed to liver fibrosis and hepatocellular carcinoma [26, 27]. Surprisingly, cytokine signaling through JAK kinases is involved in generating actomyosin contractility in CAFs [28]. Except for cytokines, mitochondrial oxidative stress can also facilitate cancer-associated fibroblasts to produce lactate and promote the tumor metabolism [29, 30].

Mechanical Forces and Fibroblast Activation

It was previously reported that primary cultured fibroblasts on petri dishes were activated “automatically” [31], and this phenomenon implies mechanical stimuli may acting as a driving force for myofibroblast differentiation. Recent studies have shown that mechanical force induces latent TGF-β activation and myofibroblast differentiation [20]. Fibroblasts were also reported to secrete higher levels of tumor necrosis factor (TNF)-α in response to compression [19]. Integrin–mediated activation of fibroblasts triggered by mechanical stress is also viewed as the major mechanism by several groups [20, 32]. It has been clearly reported that stiffened ECM, accompanied by collagen cross linking, facilitate epithelium invasion by promoting its focal adhesions and enhanced PI3 kinase (PI3K) activity [33]. However, it is still elusive about the exact mechanism for mechanically induced fibroblast activation in tumor microenvironment.

As an Inflammation Regulator

Fibroblasts are proved to promote tumor initiation, which relies on facilitating tumor-associated chronic inflammation. Our previous work has demonstrated that fibroblasts promoted skin carcinogenesis via maintaining chemokine (C-C motif) ligand (CCL)-2-mediated macrophage infiltration and chronic inflammation. The inflammatory reaction was impaired after the selective depletion of proliferating fibroblasts in the skin of FSP-TK transgenic mice. This led to reduced local CCL-2 concentration and impaired macrophage infiltration. As a result of down-regulated inflammation, the incidence of skin tumor was reduced [37]. In a spontaneous tumor model of K14-HPV16 mice, the inflammatory signature was already activated in CAFs isolated from the initial hyperplastic stage in multistep skin tumorigenesis. CAFs from this pathway promoted macrophage recruitment, neovascularization, and tumor growth in a NF-κB signaling-dependent way [38]. It was also reported that overexpression of TGF-β and/or hepatocyte growth factor in mouse fibroblasts induced the initiation of breast cancer within the normal human epithelium [39].

CAFs helped tumor cells to escape from immune-mediated rejection by recruiting myeloid-derived suppressor cells (MDSCs), T regulatory cells (Tregs) and promoting Th2 polarization of the tumor microenvironment [40]. Haniffa et al. showed that IFN-γ–stimulated fibroblasts can also suppress allogeneic T cell proliferation by upregulated indoleamine 2,3-dioxygenase and accelerated tryptophan metabolism in fibroblasts. Kraman et al. found that ablation of fibroblasts in the established tumor aroused adaptive tumor immunity involving IFN-γ and TNF-α [41]. These indicate that fibroblasts may have the immunosuppressive properties [42].

As a Damage Healer

Lots of tumor inducing agents, such as microorganisms or many kinds of carcinogens as well as growing tumors will produce tissue damage. Back to 80 years ago, G. Friedrich [43] and R. Rössle [44] found in a series of patients (frequently smokers) that lung carcinomas grew at sites of scars. They suggested that scars predisposed to cancer be termed Narbenkrebs (scar cancer). Scars within the carcinomas had an immature phenotype (increased collagen type 3 content) indicative for an early stage of fibrotic process, whereas scars at some distance from the neoplasm revealed a mature, late stage of the fibrotic process (decreased type 3 and increased type 1 and 4 collagen) [45].

Polycyclic aromatic hydrocarbons (PAH) are a group of environmental pollutants some of which (e.g. Benzo(a)pyrene) have been shown to cause human cancers [46]. Methylcholanthrene (MCA), another PAH molecule, has been widely used in mice to study chemical induced carcinogenesis [47–49]. Injection of MCA/oil induced a series of local reactions against the carcinogen emulsion [50], including the infiltration of inflammatory cells, the recruitment and proliferation of fibroblasts and finally the encapsulation of MCA by ECM which is called “foreign body reaction” [51]. The “foreign body reaction” is characterized by encapsulation of foreign materials. It is phylogenetically one of the oldest defense mechanisms predating adaptive immunity, a major protective mechanism in invertebrates and usually observed as a pathological reaction in humans [14]. Further investigation shows that treatment with collagenase led to destruction of the MCA encapsulation and then a rapid tumor development in the long term “tumor free” mice. Fibroblasts protected epithelial cells from DNA damage, epithelial malignancy occurred in the absence of local activating fibroblasts (unpublished data). Besides chemical carcinogen, it appears that fibroblasts-derived fibrotic capsule is also able to enclose neoplasm. In clinical cases, mainly in hepatocellular carcinomas and mammary carcinomas, the presence of a capsule around neoplastic cells is recognized as an indication of good prognosis [52–54]. Encapsulated tumors have low growth rate, or none at all. Once the capsule is disrupted, growth of tumor resumes [55, 56].

Our results indicate that inflammation and scarring, both suspected to contribute to malignancy, prevent cancer in certain situations [14]. Whether scar cancer results from inefficient encapsulation of carcinogen is not yet known, however, benzo(a)pyrene was detected in substantial amounts in lung tissues of smokers [45, 57] and former smokers retain a substantial risk of developing lung cancer [58].

As an Inflammation Regulator

CAFs promote tumor progression through producing a cancer cell-favorable inflammatory microenvironment. Fibroblast-derived cytokines such as IL-1 and CXCL-14 also have been shown to play vital roles as immune modulators [63, 64]. CAF-derived CXCL-12 was shown to be responsible for recruiting endothelial progenitor cells to breast tumors, which stimulated tumor blood vessel growth [62]. Vascular endothelial growth factor (VEGF) has been reported as an important tumor angiogenesis factor in many studies, which can be secreted by tumor cells, macrophages, mast cells and fibroblasts [69]. Studies by Fukumura et al. have shown that VEGF promoter activity is high in stromal fibroblasts in the transplant and spontaneous mammary tumor models [70], indicating that fibroblasts may be the major producer of VEGF and therefore be crucial for tumor angiogenesis in specific tumor models. Consistent with that, our study also shows that stromal fibroblasts express VEGF at both the RNA and protein levels. Further studies showed that fibroblasts promoted tumor growth when coinjected with tumor cells. However, the presence of IFN-γ, well known to promote immunologically induced rejection of transplanted tumor cells [71], significantly impaired the ability of fibroblasts to promote tumor growth due to a reduced angiogenesis. The mechanism relied mainly on the IFN-γ-mediated down-regulation of VEGF production by fibroblasts [13]. Importantly, PDGF-C from fibroblast may rescue angiogenesis in some anti-VEGF resistant tumors [72]. Several factors secreted by fibroblasts, such as epidermal growth factor, hepatocyte growth factor, insulin-like growth factor and IL-6, can stimulate cancer cell proliferation, maintain their survival and enhance their invasive properties [24, 65].

As a Damage Healer

Fibroblasts decorate the tumor microenvironment through direct cell-to-cell contacts and secretion of MMPs, tissue inhibitors of metalloproteinase (TIMPs), and ECM components, such as collagen and hyaluronan [73]. It was reported that tumor cells can get signals from the former enzymes and their targets, be stimulated to get epithelial–mesenchymal transition, migrate along tracks made of ECM collagen fibers, and then cross tissue boundaries and escape the primary tumor site, and finally achieve the metastasis with the help of fibroblasts to creating a niche [28, 74–78]. Actually, CAF derived MMP-1, 2, 3, 9, 11, 13, 14, 19, ADAMT-5 and TIMP-1, 2, 3 are reported involving in angiogenesis and the proliferation, adhesion, migration, differentiation and apoptosis of tumor cells or stromal cells [75].

The biomechanical remodeling of the microenvironment by fibroblasts is also important for the progression of tumor. Goetz et al. found Caveolin-1 positive fibroblasts are enriched in stroma of human breast, kidney and colon carcinoma and melanoma metastasis. Due to decreased CAF contractility, Caveolin-1-deficient mice got disorganized stromal tissue architecture [18]. It was also reported that cytokine signaling through the receptor subunit GP130-IL6ST generated contractile force in stromal fibroblasts to remodel the ECM and promote tumor invasion [28]. Similar to “foreign body reaction”, fibroblasts repressed the early stages of tumor progression by facilitating the formation of gap junctions between activated fibroblasts and thereby exerting contact inhibition on cancer cells [79, 80].