Figure 4. Breast cancer MPs carry but do not transfer CD44 to recipient cells.

20 µg total cell lysates of (A) drug sensitive cancer cells (leukaemia, CEM and breast cancer, MCF-7) and their co-cultures; (C) non-malignant cells (breast epithelial cells (MBE), osteoblasts (HO-f) and urothelial (HUC) cells) and their co-cultures; with leukaemic cell-derived VLBMP and breast cancer-derived DXMP both overexpressing P-gp were analyzed by Western blot analysis. (A) CD44 was only detected in the breast cancer DXMP but not in the malignant MCF-7 cell (A), or in any of the non-malignant cells (B) following DXMP co-culture. (A) CD44 was not detected in the leukaemic cell-derived VLBMP, the malignant cells or (B) the non-malignant cells follwoing VLBMP co-culture using the anti-CD44 mAb, clone EPR1013Y. β-actin or α-tubulin or were used as an internal loading control. The Western Blot experiments were repeated at least three times with similar results. (C) Detection of CD44 on breast cancer and leukaemic cells by flow cytometry. MCF-7 (a–b) and DX (c–d) cells were harvested from culture flasks either by scraping (a and c) or by trypsinising (b and d). Leukaemic CEM (e) and VLB₁₀₀ (f) cells were harvested by centrifuging the cell suspension. Cells were surface labelled with anti-CD44 (1∶30) (mAb, clone EPR1013Y) followed by Alexa Fluor 647-conjugated goat anti-rabbit IgG (1∶400). No CD44 was detected on the MCF-7 cells harvested without (a) or with trypsin (b). 75% DX cells harvested without (c) or 46% DX cells harvested with trypsin (d), were detected positive for CD44 with respect to the secondary control by flow cytomtery. Almost equal percentage (3.1–3.5%) of CEM (e) and VLB₁₀₀ (f) cells were detected positive for CD44 with respect to the secondary control. Data are representative of a typical experiment.