Jiří Štork

Melanoma cells influence the differentiation pattern of human epidermal keratinocytes

BackgroundNodular melanoma is one of the most life threatening tumors with still poor therapeutic outcome. Similarly to other tumors, permissive microenvironment is essential for melanoma progression. Features of this microenvironment are arising from molecular crosstalk between the melanoma cells (MC) and the surrounding cell populations in the context of skin tissue. Here, we study the effect of melanoma cells on human primary keratinocytes (HPK). Presence of MC is as an important modulator of the tumor microenvironment and we compare it to the effect of nonmalignant lowly differentiated cells also originating from neural crest (NCSC).MethodsComparative morphometrical and immunohistochemical analysis of epidermis surrounding nodular melanoma (n = 100) was performed. Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively. Differentially expressed candidate genes were verified by RT-qPCR. Biological activity of candidate proteins was assessed on cultured HPK.ResultsEpidermis surrounding nodular melanoma exhibits hyperplastic features in 90% of cases. This hyperplastic region exhibits aberrant suprabasal expression of keratin 14 accompanied by loss of keratin 10. We observe that MC and NCSC are able to increase expression of keratins 8, 14, 19, and vimentin in the co-cultured HPK. This in vitro finding partially correlates with pseudoepitheliomatous hyperplasia observed in melanoma biopsies. We provide evidence of FGF-2, CXCL-1, IL-8, and VEGF-A participation in the activity of melanoma cells on keratinocytes.ConclusionWe conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro. This interaction further highlights the role of intercellular interactions in melanoma. The reciprocal role of activated keratinocytes on biology of melanoma cells shall be verified in the future.

Stromal fibroblasts from basal cell carcinoma affect phenotype of normal keratinocytes

Background  Epithelial–mesenchymal interactions are important not only to direct the course of prenatal development of skin and its appendages but also to influence the behaviour of transformed epithelial cells.

Comparative phenotypic characterization of keratinocytes originating from hair follicles.

The principal pool of epidermal stem cells is located in the bulge region of the hair follicle root sheath. In this research project, we have used a refined procedure to isolate porcine hair follicles including their root sheath and for comparison purposes also human cell material. These cells migrating from the hair follicles were then cytochemically characterized. A panel of antibodies and two labeled plant lectins were tested on cell material obtained under a range of assorted experimental conditions. Due to their role in growth regulation we also studied two endogenous lectins, specifically monitoring their expression and the presence of accessible ligands. These in vitro results were compared with findings on porcine and human hair follicles and human basal cell carcinomas in situ. The keratinocytes originating from hair follicles in the presence of feeder cells are rather undifferentiated and express galectin-1/galectin-1-binding sites but not galectin-3 in their nuclei associated with ΔNp63α positivity. Nuclear reactivity for galectin-1 was rarely observed in the bulge of the outer root sheath of the human hair follicle and of basal cell carcinomas and absent in porcine tissue samples. Exclusion of feeder cells from our cultivation system of porcine hair follicles led to the formation of spheroid bodies from these keratinocytes. Ki67 as a marker of proliferation was not present in the nuclei of cells forming these spheroids. One part of these bodies is positive for markers of post-mitotic differentiated cells, while the other spheroids are composed of poorly differentiated cells, which are able to adhere to feeder cells and form growing colonies. In summary, the detection of galectin-1 and also nuclear binding sites for this endogenous effector points to intracellular functionality of this lectin. It can be considered a potential marker of a distinct cell population, probably at the beginning of a differentiation cascade of keratinocytes.

Mouse 3T3 fibroblasts under the influence of fibroblasts isolated from stroma of human basal cell carcinoma acquire properties of multipotent stem cells

Background information. Multipotent mesenchymal stem cells can participate in the formation of a microenvironment stimulating the aggressive behaviour of cancer cells. Moreover, cells exhibiting pluripotent ESC (embryonic stem cell) markers (Nanog and Oct4) have been observed in many tumours. Here, we investigate the role of cancer‐associated fibroblasts in the formation of stem cell supporting properties of tumour stroma. We test the influence of fibroblasts isolated from basal cell carcinoma on mouse 3T3 fibroblasts, focusing on the expression of stem cell markers and plasticity in vitro by means of microarrays, qRT‐PCR (quantitative real‐time PCR) and immunohistochemistry.

Case reports. Tinea gladiatorum due to Trichophyton mentagrophytes

Summary. Two cases of tinea gladiatorum due to Trichophyton mentagrophytes var. quinckeanum are described. A pet rabbit was probably the primary source of infection, which was then spread further by man‐to‐man contact. Besides these two patients, four other members of the same wrestling team were affected by tinea corporis.

Granular cell basal cell carcinoma

A 69‐year‐old woman presented with a 2‐year history of a slowly growing tumour on the nose. The clinical diagnosis was basal cell carcinoma (BCC) and a complete excision was performed. Histologically, the tumour had the general features of a BCC but with sheets and nests of cells with granular cytoplasmic changes in the centre of the lesion. A pathological diagnosis of granular cell BCC was made. On immunohistochemical examination, the tumour cells were Ber‐EP4, cytokeratin AE1/AE3 and cytokeratin CAM 5.2 positive but S100 protein negative. Only the granular cells were CD68 antigen (monoclonal antibody KP1) positive.

Cultivation-dependent plasticity of melanoma phenotype.

Malignant melanoma is a highly aggressive tumor with increasing incidence and high mortality. The importance of immunohistochemistry in diagnosis of the primary tumor and in early identification of metastases in lymphatic nodes is enormous; however melanoma phenotype is frequently variable and thus several markers must be employed simultaneously. The purposes of this study are to describe changes of phenotype of malignant melanoma in vitro and in vivo and to investigate whether changes of environmental factors mimicking natural conditions affect the phenotype of melanoma cells and can revert the typical in vitro loss of diagnostic markers. The influence of microenvironment was studied by means of immunocytochemistry on co-cultures of melanoma cells with melanoma-associated fibroblast and/or in conditioned media. The markers typical for melanoma (HMB45, Melan-A, Tyrosinase) were lost in malignant cells isolated from malignant effusion; however, tumor metastases shared identical phenotype with primary tumor (all markers positive). The melanoma cell lines also exerted reduced phenotype in vitro. The only constantly present diagnostic marker observed in our experiment was S100 protein and, in lesser extent, also Nestin. The phenotype loss was reverted under the influence of melanoma-associated fibroblast and/or both types of conditioned media. Loss of some markers of melanoma cell phenotype is not only of diagnostic significance, but it can presumably also contribute to biological behavior of melanoma. The presented study shows how the conditions of cultivation of melanoma cells can influence their phenotype. This observation can have some impact on considerations about the role of microenvironment in tumor biology.

Two Case Reports of Columnar Dyskeratosis, an Unusual Keratinisation Disorder

Keratinisation disorders with distinctive histopathological patterns are few in number. We describe two men with unusual dermatosis, characterised by a distinctive pattern of focal dyskeratosis. Both men suffered from generalised dermatosis formed by verrucous red-brown plaques. Repeated skin biopsies showed the same histopathological pattern with foci of vertically oriented dyskeratotic cells. The dyskeratotic cells on the level of the stratum spinosum and granulosum were positive for AE1/AE3, CK-HW, CK-LS and CK116 immunostaining. PCR for HPV was negative. The similar clinical appearance of skin lesions in both patients together with their identical histopathological pictures seems to represent a unique clinicopathological condition that we believe is best described by the term ‘columnar dyskeratosis’.

Phenotypic characterization of human keratinocytes in coculture reveals differential effects of fibroblasts from benign fibrous histiocytoma (dermatofibroma) as compared to cells from its malignant form and to normal fibroblasts.

BACKGROUND Benign and malignant fibrous histiocytoma present with a considerable difference concerning cellular organization in their vicinity. OBJECTIVE Normally appearing epithelium covers the malignant form in contrast to hyperplastic epidermis for benign tumors. It is an open question as to whether the tumor-associated fibroblasts are capable to affect phenotypic features of normal keratinocytes, prompting this comparative analysis. METHODS Fibroblasts were isolated from benign and malignant fibrous histiocytomas, respectively, and also from normal dermis. The resulting cell populations were thoroughly characterized immunocytochemically using a large panel of antibodies. The three fibroblast preparations were cocultured with normal interfollicular keratinocytes. Their phenotype was characterized for distinct properties including differentiation and proliferation. RESULTS Fibroblasts prepared from both tumor types were phenotypically practically identical with normal dermal fibroblasts. Their activities on keratinocytes were different. Cells prepared from benign fibrous histiocytoma were capable to effect strong expression of keratin 19 and production of a galectin-1-rich extracellular matrix. Fibroblasts isolated from malignant fibrous histiocytoma led to a phenotype very similar to that when keratinocytes were cocultured with normal dermal fibroblasts. CONCLUSION Fibroblasts prepared from benign fibrous histiocytoma were biologically active on keratinocytes in a particular manner. Our results on fibroblast activity are suggested to be relevant for morphologic differences observed in vivo between normal epidermis and epidermis adjacent to the studied tumor types.

Trichophyton rubrum suppurative tinea of the bald area of the scalp

An 83‐year‐old man presented with an approximately 1‐year history of an extensive inflammatory purulent crusted lesion in the bald area of the scalp diagnosed as tinea caused by Trichophyton rubrum. The scalp biopsy specimen showed suppurative folliculitis with perifollicular abscesses in upper dermis, and periodic acid‐Schiff‐positive fungal elements within the hair follicles and in the hyperkeratotic horny layer. The infection probably spread from diseased fingernails. A cure of the scalp lesion was achieved 2 months after starting daily oral treatment with 250 mg terbinafine. To our knowledge, the case presented is the first in which a suppurative abscess‐forming T. rubrum infection of the bald area of the scalp in an immunocompetent man has been described.