Microarrays of 41 Human Tumor Cell Lines for the Characterization of New Molecular Targets: Expression Patterns of Cathepsin B and the Transferrin Receptor

Abstract

In recent years the use of the microarray technology has allowed the identification of numerous cancer-related genes and proteins. Today anticancer drug discovery is mainly target driven, which requires the characterization of molecular targets in existing cell lines or xenograft models. However, this analysis is time consuming and labor intensive. In order to ease this bottleneck, we have established tissue microarrays of 41 human tumor cell lines on glass slides. The purpose of our study was to (a) establish a simple and efficient method for cell line microarray construction, (b) apply the resulting array to the profiling of cathepsin B and transferrin receptor by immunohistochemistry and (c) verify the results in separate Western blot analyses. Ten to twenty million (1–2 × 10⁷) cells were harvested without trypsinization and fixed with Bouin’s solution containing 8% formalin. Cell pellets 2–5 mm in diameter were embedded in paraffin. Microarrays were assembled using a tissue arrayer. Pellet biopsies 0.6 cm in diameter were taken and arrayed in duplicate in a new recipient paraffin block. About 60 slides can be obtained from one block. Citrate buffer was used for antigen retrieval. Expression of cathepsin B was granular and located in the cytoplasm. High cathepsin B levels were detected in 2 melanomas (MEXF 514L and MEXF 276L) and in the renal cell line RXF 486L. Twenty-five cell lines showed only minimal positivity. Nine cell lines of leukemia and lymphoma, breast, ovarian, prostate and renal cancer origin were positive for the transferrin receptor, while 32 cell lines were negative. Western blotting confirmed the results obtained by immunohistochemistry. Using these cell line microarrays, cell lines overexpressing a target of interest can be selected for in vitro evaluation of specific inhibitors.

Role of Transferrin Receptor and the ABC Transporters ABCB6 and ABCB7 for Resistance and Differentiation of Tumor Cells towards Artesunate

“…Arrays of cell lines from the Oncotest Human Cell Line Collection were assembled using a tissue arrayer (Beecher Instruments, Sun Prairie, Wisc., USA) as recently described [36]. Pellet biopsies (0.6 mm in diameter) were taken from embedded cells and arrayed in duplicate in a new recipient paraffin block.…”

“…The arrays were stained with the primary antibody 71C03 against TfR (Neomarkers, Fremont, CA, USA). Staining was analyzed by light microscopy and semi-quantitatively evaluated as described [36].…”

“…This test was implemented into the WinSTAT Program (Kalmia, Cambridge, MA, U.S.A.). The TfR expression in the Oncotest panel of tumor cell lines has been determined by a tissue micro-array technique adapted for the examination of cell line and immunohistochemistry [36]. The results have been validated by conventional immunohistochemistry and Western blotting [36].…”

Enhanced expression of transferrin receptor 1 contributes to oncogenic signalling by sphingosine kinase 1

“…20 Notably, elevated TFR1 has been widely reported in a variety of human tumours, often correlating with tumour grade and patient outcome. [21][22][23][24][25][26] Furthermore, high TFR1 expression enhances cell growth and tumour formation in mice, 27,28 and inhibition of TFR1 by either genetic, pharmacological or antibody-neutralization approaches has been shown to reduce cancer cell proliferation, tumour growth and metastases in mice. [29][30][31][32] Together, these findings raised the possibility that TFR1 may have a role in SK1-mediated oncogenesis.…”

Automated ERCC1 immunochemistry on hybrid cytology/tissue microarray of malignant effusions: evaluation of antibodies 8F1 and D-10

“…Data from these cell block sections is highly comparable to corresponding sections of histologic tissue biopsies or surgical specimens due to the same cut thickness, in most laboratories 2 to 4 μm. Furthermore, paraffin cores of 0.6 mm diameter from sediment blocks or also cell line pellets can be assembled into a cytology microarray the same way than cores from histologic blocks into a tissue microarray [20-22]. A cell block may also be an effective quality assurance tool for cancer registries and national mortality statistics [23], since no further diagnostic procedures maybe performed if e.g.…”