Cancer-associated fibroblasts

Activated fibroblasts, that is, the myofibroblasts of the cancer microenvironment, have been classified as cancer-associated fibroblasts (CAFs) because the number of these cells increases significantly in various types of malignant neoplasms. Myofibroblasts have features similar to those of both smooth muscle fibers and fibroblasts, and significantly induce the growth and differentiation of cells during embryogenesis, wound healing, and other processes of tissue remodeling (79). Various cells can differentiate into myofibroblasts, not only fibroblasts, but also the smooth muscle fibers of vessels, pericytes, bone marrow precursor cells, and even cancer cells. PDGF secreted by the tumor stimulates fibroblast proliferation, while TGF-β released from macrophages chemoattracts fibroblasts in lower concentrations, while in higher concentrations it induces their transdifferentiation into myofibroblasts. Furthermore, tumor cells themselves express TGF-β (80). Myofibroblasts appear shortly before the cancer invasion, degrade the basal membrane and extracellular matrix through the secretion of serine proteases, matrix metalloproteinase, and urokinase activator of plasminogen. Myofibroblasts additionally express IGF and HGF/SF (hepatocyte growth factor/scatter factor), inducing the survival and migration of the cells and the expression of pro-angiogenic factors (FGF-2 and VEGF) and pro-inflammatory cytokines (IL-1, IL-6, and IL-8 i TNF-a). Myofibroblasts not only stimulate their own migration to the tumor site, they also induce the survival, proliferation, invasion of cancer cells, and angiogenesis, thus enhancing the ability of the tumor to grow and create metastases (81-83). Microenvironment fibroblasts participate in deciding whether the cancer cells proliferate, infiltrate the surrounding tissues, and metastasize.

In our previous studies, cancer-associated fibroblasts were identified in the cancer microenvironment of head and neck squamous cell carcinomas and adenocarcinomas; these cells expressed a strong level of vimentin (69-70). Statistically significantly higher vimentin immunoreactivity levels were observed in the tumor stroma in patients with advanced tumor size (T3 and T4) in head and neck squamous cell carcinoma. Cancer-associated fibroblasts  (CAFs) in the cancer microenvironment in both histological types of tumors of the head and neck were observed to express metallothionein. Metallothioneins are a family of low-molecular weight proteins (6 kDa) with a high affinity for divalent metals, such as zinc and copper, as well as toxic metals, such as cadmium and mercury (84-85). Human metallothioneins include four isoforms: MT-1 and MT-2, which are widely expressed in tissues, and MT-3 and MT-4, which are present exclusively in specialized cells (84). The ability to bind the metal ions is linked to the biological role of this protein, including protection against metal toxicity, reservoir of zinc and copper for metaloenzymes during the apoptosis process, the production of transcription factors, and protection against oxidative stress (84). MTs may also play an important role in the proliferation and differentiation of cells (86). It has been established that MT expression in the cytoplasm helps to protect against cytotoxicity, while its expression in the nucleus protects against genotoxicity (84, 86-87). Genotoxicity concerns the acquisition of cells of the malignant phenotype, as a result of mutations important in the carcinogenesis process. Cytotoxicity is important in the interaction of cancer cells with immune system cells. MT expression was observed in various types of malignant neoplasms and in cancer microenvironments as well as in healthy tissues adjacent to cancer nests. That the MT expression in healthy epithelia was localized to the basal part of the epithelium, which comprises intensively dividing cells responsible for its renewal, was demonstrated in various studies, while in the more superficial layers of the epithelium which were composed of well-differentiated cells, MT expression was not found (88-91) (Figure 5). MT expression was also observed in tumor-adjacent tissue, epithelium, and even in tumors without MT expression (88-93). Moreover, MT expression was found in healthy thyroid cartilage adjacent to laryngeal cancer, in healthy vessels, and glandular epithelium in the tumor vicinity. It has been suggested that migrating tumor cells may stimulate cancer microenvironment cells to respond (Figure 5) (88, 92-93). The cancer microenvironment

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expressed MT as well, and it has been shown that MT expression in the cancer microenvironment may be related to an increased degree of tumor invasiveness and local tumor s pread. Moreover, the level of expression was found to be significantly higher in patients with the presence of lymph node metastases in comparison to those patients without such metastases (88). MT expression in the cancer microenvironment may also be related to the increasing infiltration of immune system cells. The response of the tumor stroma with MT expression may be the result of increasing resistance to immune-induced apoptosis, as MT plays a protective role against apoptosis. Because of MT’s complex function, which includes anti-apoptotic, pro-proliferative, and immunomodulating properties, the local expression of MT may play a critical role in cancer invasion through CAFs. The immunomodulating role of MT expression has also been observed in nasal polyps, which are a symptom of chronic rhinosinusitis. Additionally, MT expression has been found in the stroma of nasal polyps, which is the area of excessive immune cell infiltration. There were differences in MT expression between the polyps depending on the type of the predominant immune cell infiltration. The presence of MT expression in the nasal polyp microenvironment seems to confirm its important immunoregulatory role as well as its participation in the maintenance of chronic inflammation (88, 94).

Cancer-associated fibroblasts have also been observed to express other important molecules that may determine the nature of their participation in creating the local suppressive microenvironment. For example, the presence of RCAS1 and B7-H4 positive CAFs was found in the cancer microenvironment of patients suffering from cervical cancer. The number of these cells in the cancer microenvironment did not, however, correlate with the stage of the disease, probably because these tumors were all in I and II stage (and hence operable). By contrast, in patients with ovarian cancer, the cancer microenvironment was characterized by the presence of RCAS1-positive carcinoma-associated fibroblasts, and this presence was more pronounced in the border part of the tumor (which is defined as a younger part having signs of dynamic growth) derived from those patients with the presence of  lymph node metastases in comparison to those without such metastases.

All these findings would seem to confirm that CAFs and TAMs expressing MT, RCAS1, and B7-H4 molecules participate in creating the suppressive profile of the cancer microenvironment thus enabling both local tumor spread and the creation of metastases.

Beginning with basal membrane disruption, the tumor stimulates the remodeling of its own microenvironment in order to enable local spread as well as the creation of distant metastases. The development of those abilities that enable cells to detach from the main tumor, such as cell adhesion, invasion, extracellular matrix degradation, and extravasation with homing of distant tissues and organs, results in the creation of metastases. The acquisition of this phenotype is related to the phenomenon of epithelial to mesenchymal transition (EMT)(94-96).