Establishment, growth and in vivo differentiation of a new clonal human cell line, EM-G3, derived from breast cancer progenitors.

A new clonal cell line, EM-G3, was derived from a primary lesion of human infiltrating ductal breast carcinoma. The line consisted of cuboidal cells with occasional appearance of more differentiated branched cells apparently involved in cell-to-cell communication. The EM-G3 cells, population doubling time 34 h, are dependent on the epidermal growth factor. Multicolor fluorescence in situ hybridization (mFISH) analysis demonstrated a stable diploid genome with several genetic changes. Immunocytochemical analysis of EM-G3 in vitro revealed positivity for keratins (K) K5, K14, K18, nuclear protein p63, epithelial membrane antigen (EMA) and other proteins indicative of a pattern of mammary epithelium bipotent progenitors. Detection of integrins alpha-6, beta-1, and protein CD44 by cDNA array also pointed to the character of basal/stem cells. In contrast, dominant cells in the human original tumor showed the luminal character (K18+, K19+, K5-, K14-, and p63-). However, cells with the immunocytochemical profile similar to that of cultured EM-G3 cells were found in minor clusters in the patient's tumor sections. The EM-G3 cells formed limited tumors in nu/nu mice. The cells in mouse tumors were organized in primitive ductal-like structures consisting of 1-3 large central luminal-like cells (EMA+) surrounded by peripheral myoepithelial-like cells (p63+/EMA-). The large central cells gradually disintegrated, forming a pseudolumen. Apparently, EM-G3 cells are able to partially differentiate in vivo as well as in vitro. Our results indicate that EM-G3 cells were derived from a premalignant population of common progenitors of luminal and myoepithelial cells that were immortalized in an early stage of tumorigenesis.

KIT receptor is expressed in more than 50% of early-stage malignant melanoma: a retrospective study of 261 patients.

The c-kit gene encodes a transmembrane receptor (KIT) with tyrosine kinase activity which is a specific target for anti-cancer therapy. We investigated KIT expression in a group of patients with early-stage malignant melanoma. Primary tumour specimens obtained from 261 radically resected patients with stage I and II malignant melanoma were examined for KIT expression. Formalin-fixed, paraffin embedded tissues were stained with the polyclonal rabbit anti-human anti-KIT antibody (Dako Cytomation Inc., Carpenteria, California, USA). Patients were classified into four groups according to the level of expression (0%, <30%, 30-60% and >60%). Univariate and multivariate analyses examining the impact of KIT expression, Breslow thickness, Clark level and microscopic ulceration on disease-free survival were performed. Within the population of 261 patients with early-stage melanoma with 62 recurrences during a follow-up of 64 months, KIT expression was found in 144 cases (55%). KIT was expressed in more than 60% of cells in 20 patients (8%), in 30-60% of cells in 64 patients (24%) and in less than 30% of cells in 60 patients (23%). KIT expression was not found in 117 patients (45%). In univariate analyses, the influence of KIT expression on disease-free survival was not proven (P=0.4956; log-rank test). Increasing Breslow thickness, a higher Clark level, the presence of microscopic ulceration and a higher stage were significantly associated with a shorter disease-free survival (P<0.0001; log-rank test in all cases). In multivariate analysis, Breslow thickness, stage and KIT expression were significant negative prognostic factors for a shorter disease-free survival (P<0.0001, P=0.0028, P=0.0488, respectively; stepwise Cox regression model). It can be concluded that KIT is expressed in more than one-half of early-stage malignant melanoma. KIT may serve as an additive prognostic factor to Breslow thickness and stage within the tested population. The therapeutic impact of KIT expression in malignant melanoma is uncertain. Results of ongoing pilot phase II studies may validate the efficacy of imatinib mesylate in malignant melanoma expressing KIT.

Assessment of in vitro drug resistance of human breast cancer cells subcultured from biopsy specimens.

UNLABELLED: The low cellular yield of a breast cancer sample represents a limiting factor for in vitro chemosensitivity/chemoresistance testing. The use of in vitro serially cultured cells can help overcome this obstacle. MATERIALS AND METHODS: In vitro drug resistance of cells cultured from mammary carcinomas by the 3T3 feeder-layer technique was tested by the MTT assay. Out of the 33 tested cultures, 9 were derived from cells obtained from true-cut biopsies of primary tumours, with sample volume less than 0.03 cm3. The cultures were treated with 6 anticancer drugs at 6-8 concentrations. The chemoresistance of cultured cells was monitored by the surviving cell fraction as a function of the drug concentration. RESULTS: The average time to obtain a result was 28 days. The volume of an original sample had no effect on the in vitro resistance of a culture, suggesting minimal alteration of in vitro chemosensitivity of cells by their cultivation. Histopathological grade, estrogen receptor status or expression of the c-erb-B2 protein of the original tumours did not significantly correlate with the resistance of cultures. Individual drugs displayed distinct in vitro effectiveness. Paclitaxel and cisplatin were the most potent drugs. Gemcitabine, vinorelbine and mafosfamide were the least potent drugs. Doxorubicin and gemcitabine most frequently failed to completely metabolically inhibit 100% of cultured cells at any concentration. CONCLUSION: Combination of the optimised feeder-layer cultivation technique and the MTT test permits extensive drug resistance testing from very small breast cancer samples.