Epithelial–Stromal Interaction in Squamous Cell Epithelium-derived Tumors: An Important New Player in the Control of Tumor Biological Properties

Epithelium–Matrix Interaction under Physiological Conditions

Squamous epithelium covers the mechanically stressed areas of external (skin) or internal (oral cavity, vocal cords, oesophagus etc.) surfaces of the human body. This epithelium is morphologically and functionally stratified, reflecting the functional properties of this tissue. The proliferating cells are located in the basal layer of epithelium under normal physiological conditions. These proliferating cells also include a pool of epidermal stem cells (SCs). In addition to the basal layer of glabrous areas, epidermal SCs are also present in hairy skin in the bulge region of the outer root sheath of the hair follicle (1, 2). Epidermal SCs are slowly cycling cells, and their division has an asymmetrical character. The arising daughter cells have either the character of epidermal SCs (responsible for self-renewal and long-lasting maintenance of the tissue) or so-called transit-amplifying cells (TACs), which start to differentiate. TACs proliferate quickly, but the number of mitotic divisions is restricted (3, 4).

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Figure 1.

Comparison of the appearance of advanced basal cell carcinoma of the nose 100 years ago (A) and today (B). The tumor is marked by an arrow. 100-year-old model (A) was prepared from a specimen of a patient in the Department of Dermatology of Faculty of Medicine, Charles University 100 years ago.

Proliferating cells, including stem cells require contact with the basement membrane, which is mediated predominantly by integrins forming contact of the hemidesmosomal type. Except type IV collagens, representatives of the laminin family and fibronectin, which are all directly recognized by integrins, oligo/polysaccharides including heparansulfate participate in the formation of the basement membrane. These carbohydrate components are recognized by the endogenous lectins, including the mannose-rich oligosaccharides of the laminin. They are also reactive for plant lectin – concavalin-A (Con-A) (Figure 2) (5). The carbohydrate–lectin interaction can modulate the protein-protein interaction performed by integrins and participates in the process of the control of the functional phenotype of proliferating cells (6). Detachment of epithelial cells induces, with several exceptions, a specific type of the programmed cell death, anoikis. The anoikis-resistant nucleated cells are (not surprisingly) leukocytes that are transported through the organism by blood/lymphatic vessels to the site of inflammation. Apart from these circulating cells of the immune system, adult (tissue) SCs including epidermal SCs (7, 8) can also be transported by the circulation and are anoikis resistant.

The basement membrane is clearly not only a simple passive scaffold of which the sole function is adherence. The basement membrane is reorganized during the life of the epithelium by activity of basal epithelial cells and underlying dermal/mucosal fibroblasts. The basement membrane represents an active fine-mesh screen that mediates adhesion and proliferation of epithelial cells and their support by nutritive substances, oxygen and signal molecules such hormones and growth factors (9, 10).

Epithelial–Mesenchymal Interaction in the Development of Adult Epithelium

Although as was mentioned above, the epithelial cells and fibroblasts are separated by the basement membrane, this barrier is not impermeable to oxygen, nutritive substances, metabolites and signal molecules. The important data regarding the cooperation of both cell types of squamous epithelium, i.e. epithelial cells and fibroblasts was obtained from the developing skin and mucosa and its appendages such as hair, teeth, mammary gland and lungs. Morphologically, the epithelium is thickened at the epithelial bud around which the fibroblasts are concentrated (Figure 2). The extensive cross-talk between the epithelial and mesenchymal cells across the basement membrane is mediated via the production of specific growth factors (distinct members of bone morphogenetic factors, Wnt or hedegehog proteins), which is necessary for successful development (11-15). The similar well-orchestrated signaling machinery is active repeatedly during the cycle of the hair follicle from the anagen to catagen during its life. Fibroblasts harvested from distinct body surface areas such as the palm/planta control the ectopic expression of specific keratins (K9) in keratinocytes obtained from the breast, originally K9-negative (16).

Normal and Cancer SCs

The majority of tissues including squamous cell epithelium contain SCs. As mentioned above, the epidermal SCs are located in the bulge region of the outer root sheath and in the basal layer of epithelium. Their membrane is equipped by special proteins responsible for the active efflux of xenobiotics out of the cytoplasm, so protecting the cells from the toxic damage (side cell population) (17). It should be noted here that the special microenvironment (so-called niche) is responsible for the maintenance of SC properties and preservation against their differentiation (18, 19).

An abundance of data indicate that leukemias and solid tumors also contain a pool of cells sharing SC properties. These cells were initially defined during transplantation experiments where only a small portion of cells of the tumor mass is responsible for the tumor successful transfer of tumor to the host (20, 21). Indirectly, the cells with phenotypic signature of tissue SCs were described in human and animal carcinomas, such as tumors of breast, skin, mucosa and prostate (4, 22-25). Moreover, both cell populations i.e. adult SCs and some of cancer cells are resistant to anoikis (7, 26). Because their other properties are also similar to that of normal adult SCs i.e. slow cell cycle, unlimited proliferation rate and membrane proteins responsible for exclusion of xenobiotics, their existence may be clinically very important. Their destruction by chemo/radiotherapy is thus not easy and their unique properties can cause clinically important phenomena such as minimal residual disease and multidrug resistance (4). These cells represent an ideal target for anticancer therapy. Unfortunately, no specific markers of these cells are known and, therefore, their specific targeting is not yet possible.

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Figure 2.

Developing hair follicle from skin of 14-day-old mouse embryo. The carbohydrates reactive for plant endogenous lectin-concavalin-A (ConA) exhibit a distinct signal. The high signal is present in the basement membrane separating the epithelium from underlayering mesenchyma (arrows) including the epithelim-thickening bud (asterisk). Mesenchyme cell accumulation around the epithelium bud (asterisk) is marked by a dashed line.

If we accept the existence of putative cancer SCs, it is possible to hypothesize the likely role of their niche on ‘successful’ growth of tumor and their metastasis.

Tumor Stroma as the Niche for Cancer SCs

Almost 100 years ago, the British surgeon Stephen Paget postulated the hypothesis that cancer cells need a special environment in a similar way to seeds needing fruitful soil (27). Pathologists know that the connective tissue surrounding the epithelial tumor cells buds, so-called tumor stroma, is usually different from the mesenchymal component of normal tissue. The tumor stroma is a complex tissue composed of fibroblasts (and extracellular matrix produced by these cells), capillaries (morphologically distinct) and different types of leukocytes (Figure 3A, B). The classical view of the tumor stroma predicted its role only for the adhesion, support and nutrition of cancer cells. However, the contemporary view suggests that the tumor stroma can influence the biological properties of tumor, mainly its local aggressivity and ability to metastasize (28-30). There are certain similarities concerning the arrangements of tumor stroma and connective tissue component of wounds (31). For example, we observed a remarkably strong expression of adhesion/growth regulatory lectin galectin-1 in the dermis of wounded skin, and stroma of basal (BCC), squamous cell carcinoma (SCC) and also in the dermis of psoriatic plaque (32-36). Another connecting feature of wound with tumor is the existence of a special population of fibroblasts, some of which exhibit smooth muscle actin (the myofibroblasts) (37). These cells originate predominantly from the local mesenchyme by the activity of transforming growth factor β (TGF-β) (Figure 3C, D) (38). Fibroblasts/myofibroblasts participate in defect closure by wound contraction (39). However, they are also known to be potent producers of extracellular matrix and of numerous cytokines/chemokines directly regulating the activation of epithelial cell proliferation and migration (Table I). The tumor stromal fibroblasts/myofibroblasts can participate in the control of the activation of cancer cells (including cancer SCs) (37, 40).

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Figure 3.

Advanced basal cell carcinoma (BCC) stained by hematoxilin and eosin (A) is characterized by the desmoplastic appearance of tumor stroma (asterisk). Cells of squamous cell carcinoma (SCC) of larynx (B) positive for pankeratin (kerat) (green signal) are surrounded by frequent stromal elements positive for vimentin (red signal). Dermal fibroblasts (DF) produce fibronectin (green signal) (C). The application of TGF-β1 significantly induces transformation of fibroblasts to myofibroblasts exhibiting α-smooth muscle actin (SMA) (red signal) (D). Production of fibronectin is preserved. Normal epidermal keratinocytes (K) were co-cultured with fibroblasts prepared from malignant fibrous histiocytoma (MFHF) (E) and from benign fibrous histiocytoma (dermatofibroma) (BFHF) (F) under identical conditions. The difference in expression of keratin 19 (red signal) is clearly visible. Nuclei are counterstained by 4, 6-diamidino-2-phenylindole (DAPI). Bar is 100 μm (A, B), 50 μm (C, D) and 70 μm (E, F) respectively.

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Figure 4.

Bidirectional mutual interaction between cancer epithelial cells and stromal fibroblasts influences formation of permissive cancer microenvironment. Blocking of molecules participating in this interaction, such as IGF-2 and BPM-4 (60) by antibodies significantly affects phenotype of epithelial cells to exhibit markers of advanced differentiation. Therapeutic potential of cancer microenvironment modulation is possible.

We have isolated cancer stromal fibroblasts from BCC of the skin and SCC of the tongue as previously published elsewhere (34, 41). Co-culturing these fibroblasts with normal human keratinocytes, we observed their strong impact on the phenotype of normal human keratinocytes acquiring a phenotype similar to that of tumor epithelium or the epidermal SC pool. BCC stromal fibroblasts induced expression of K19 in normal keratinocytes. This type of intermediate filament is expressed by the bulge epidermal SCs and more than 50% of BCCs exhibit cells positive for this keratin (8). This observation was supported by induction of the nuclear expression of nucleostemin and binding sites for galectin-1 that also indicate the low level of the differentiation of co-cultured keratinocytes. The observation of the expression of BCC marker (Ber-Ep4) in normal keratinocytes co-cultured with BCC fibroblasts underlines the role of stromal elements in normal epithelium.

To verify this observation, we employed another type of skin tumor. Dermatofibroma (benign fibrous histiocytoma) is a common fibrohistiocytic skin tumor of mesenchymal origin where the pool of tumor cells has properties of atypical histiocytes. Curiously, the epidermis overlying the tumor nodule (that is present in dermis) is hyperplastic and similar to BCC. The fibroblasts isolated from this type of tumor are very similar to the fibroblasts isolated from the BCC in relation to their biological activity, where they also induce expression of K19 in co-cultured normal keratinocytes. On the contrary, malignant fibrous histiocytoma, another soft tissue tumor of dermal origin is not associated with hyperplasia of overlying epidermis. In concordance with this experience from histopathology, no response in the normal human keratinocytes was observed during in vitro co-cultivation experiments (Figure 3E, F) (42, 43).

The phenotypic changes of normal keratinocytes under the influence of co-cultured fibroblasts prepared from the stroma of SCC were stronger in comparison with that of the fibroblasts from the BCC/dermatofibroma (41). In addition to the induction of expression of K19, we also detected cells exhibiting K8. This intermediate filament is never expressed by normal squamous epithelium postnatally, but it is present in SCC where it is an indicator of poor prognosis for the patient (44). Many normal keratinocytes co-cultured with SCC stromal fibroblasts also coexpressed keratin with vimentin. These cells also exhibited nuclear presence of transcription factor SNAIL that is known to participate in the control of epithelial-mesenchymal transition (EMT) (45). This observation suggests EMT in keratinocytes co-cultured with SCC stromal fibroblasts. This result seems to be functionally important because EMT participates in embryogenesis, wound healing and in the process of metastasis (46-48).

Mesenchymal bone marrow stromal cells are able to acquire properties of cancer-associated fibroblasts (49). Similarly, the cells formed by the EMT capture properties of mesenchymal SCs (50). Therefore it is probable that some stromal fibroblasts can originate directly in the cancer epithelium. This possibility was proposed for breast cancer (51). Mechanisms of how the cancer-associated fibroblasts originate are summarized in Table II.

The majority of SCCs of uterine cervix and many head and neck SCCs are induced by human Papillomavirus 16 (HPV16) (53). The genetically engineered epithelial cells transfected by protoncogenes E6 and E7 of HPV16 acquire morphological properties of mesenchymal cells (fibroblasts). Hence they can represent the mesenchymal cells arising from cancer epithelium by the EMT. These cells share the genetic alteration identical to that of the original tumor population, however their morphology and functional properties are strikingly different. We co-cultured these transfected cells of fibroblast-like morphology together with normal keratinocytes. Their influence on the normal keratinocytes was profound and very similar to the action of fibroblasts prepared from the stroma of SCC. This indicates that the tumor epithelium can be the source of bioactive stromal fibroblasts. This hypothesis needs verification by mutation analysis, because when the stromal fibroblasts should be originated in tumor stroma, their mutation should be the same as that, in cancer cells. Unfortunately, analyzing the tumors from clinical practice produces data that are controversial (54). Moreover, the inclusion of cancer cells migrating through the stroma in the course of metastasis can be a critical objection to this hypothesis because it is not possible to distinguish them from stromal elements formed by the epithelial-mesenchymal interaction.

When stromal fibroblasts are maintained in vitro for a long time their biological activity is preserved for at least 2 months, although these cells are isolated from the influence of tumor epithelium (34, 41). This property can be caused principally by two mechanisms. In addition to direct genetic alteration, epigenetic changes could also be responsible for this phenomenon. The data summarized by Polyak and co-workers indicate that epigenetics can play an important role in the biology of cancer stromal fibroblasts (55).

Although, the data demonstrated the effect of cancer stromal fibroblasts/myofibroblasts on the biology of epithelial cells, the unanswered question is whether the contact of cell populations i.e. stromal elements and the epithelium is necessary. In order to address this question, we co-cultured epithelial cells with stromal fibroblasts directly on coverslips and in a transwell system. Both cell types were separated by a microporous membrane, avoiding their direct contact, but enabling the free movement of cultivation medium containing soluble molecules including growth/regulatory factors. It should be noted here that the activity of fibroblasts relative to that of co-cultured epithelium was lower, but principally it was preserved (34, 41). This result indicates the important role of soluble proteins (cytokines/chemokines) produced by stromal fibroblasts/myofibroblasts (Table I) in mesenchymal-epithelial interaction in tumors, where the examples are shown here (56-60). The remarkable role of these regulatory factors is not so surprising because, as was shown above, both cell populations, i.e. epithelium and underlying mesenchyme, are separated by a permeable basement membrane under normal physiological conditions, where the epithelial cells are adapted to recognize these bioactive molecules. Malignant tumors are characterized by invasion of tumor cells across the basement membrane that characterizes the transition of the so-called carcinoma in situ to the invasive form. However, as was depicted in the case of head and neck SCC, the basement membrane, frequently incomplete, surrounds a substantial proportion of nodules of tumor epithelium, usually, except the site of invasive front of tumor with actively migrating cells (61). This data suggests that the epithelium is adapted for the signaling by active proteins/peptides across the permeable membrane even in cancer.

On the other hand, the opposite role of the niche as presented above was clearly demonstrated by experiments where the cells with a high tumorigenic potential were influenced by the embryonic microenvironment, minimizing their tumor-forming activity and malignant phenotype (62).