Hypothesis Overview.

<p>An interaction complex involving discrete MP surface molecules and FERM domain proteins (including Ezrin) serve to selectively target and anchor P-gp to breast cancer cells upon MP binding. This leads to the selectivity in dissemination and acquisition of MDR trait in cells.</p

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

<p>20 µg total cell lysates of (<b>A</b>) drug sensitive cancer cells (leukaemia, CEM and breast cancer, MCF-7) and their co-cultures; (<b>C</b>) 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. (<b>A</b>) CD44 was only detected in the breast cancer DXMP but not in the malignant MCF-7 cell (<b>A</b>), or in any of the non-malignant cells (<b>B</b>) following DXMP co-culture. (<b>A</b>) CD44 was not detected in the leukaemic cell-derived VLBMP, the malignant cells or (<b>B</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. (<b>C</b>) <b>Detection of CD44 on breast cancer and leukaemic cells by flow cytometry</b>. MCF-7 (<b>a</b>–<b>b</b>) and DX (<b>c</b>–<b>d</b>) cells were harvested from culture flasks either by scraping (a and c) or by trypsinising (b and d). Leukaemic CEM (<b>e</b>) and VLB₁₀₀ (<b>f</b>) 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 (<b>a</b>) or with trypsin (<b>b</b>). 75% DX cells harvested without (<b>c</b>) or 46% DX cells harvested with trypsin (<b>d</b>), were detected positive for CD44 with respect to the secondary control by flow cytomtery. Almost equal percentage (3.1–3.5%) of CEM (<b>e</b>) and VLB₁₀₀ (<b>f</b>) cells were detected positive for CD44 with respect to the secondary control. Data are representative of a typical experiment.</p

Selective transfer of P-gp to MCF-7 breast cancer cells by DXMPs by Western Blot analysis.

<p>20 µg total cell lysates of non-malignant cells (breast epithelial cells (MBE), osteoblasts (HO-f) and urothelial (HUC) cells) and their co-cultures; and drug sensitive cancer cells (leukaemia, CEM and breast cancer, MCF-7) and their co-cultures with leukaemic cell-derived (<b>A</b>) VLBMP and (<b>B</b>) E₁₀₀₀MP overexpressing P-gp and MRP1, respectively, and breast cancer-derived (<b>C</b>) DXMP overexpressing P-gp, were analyzed by Western blot analysis. (<b>A</b>) P-gp and (<b>B</b>) MRP1 were readily detected in the drug resistant leukaemic donor cells, their MPs and also in both the recipient malignant and non-malignant cells after MP transfer using the anti-P-gp mAb, F4 and anti-MRP1 mAb, QCRL-1, respectively. (<b>C</b>) P-gp was also detected in the drug resistant breast cancer donor cell, its MPs (DXMP), recipient malignant drug sensitive cells but not in the recipient non-malignant cells after MP transfer. α-tubulin or β-actin were used as internal loading controls. (<b>D</b>) <b>Selective transfer of P-gp to MCF-7 breast cancer cells by malignant breast cancer-derived MPs by Confocal microscopy.</b> Malignant breast cells MCF-7 (<b>I</b>) or non-malignant osteoblasts, HO-f (<b>II</b>) cells were stained with fluorescent membrane probes PKH26 and the nucleus was stained with DAPI after 4 hours co-culture with GFP-MPs. (<b>I</b>) P-gp was detected in the breast MCF-7 cells only and not in (<b>II</b>) the osteoblasts. Panel A shows all channels captured, Panel B shows the DAPI in the 405 nm channel, Panel C shows the transferred GFP (or lack of) in the 488 nm channel, and Panel D shows the PKH26 in the 561 nm channel. Images were acquired using the Nikon A1 confocal microscope. Scale bar as indicated. Both Western blot and confocal microscopy experiments were repeated at least 2–3 times with similar results. Data are representative of a typical experiment.</p

P-gp localised and retained in tumour core, two weeks post MP administration.

<p>At 10 days after tumour cell implantation, tumours derived from female athymic mice bearing MCF-7 (negative control) and DX (positive control) or MCF-7 tumours with DXMP exposure (MCF-7+DXMP) were resected and fixed after 24 h and 14 days DXMP exposure. Five independent tumours (each from a different mouse) were tested in each mouse group. (<b>A</b>) Excised and sectioned tumour slides were subjected to haematoxylin and eosin (H&E) staining showing the presence of breast epithelial cells and blood vessels in the tumour sections at both 24 h and 14 days. Slides were examined at 40× followed by 100× magnification. (<b>B</b>) Tumour and (<b>C</b>) kidney specimens were subjected to immunohistochemical (IHC) staining with antibodies specific to P-glycoprotein (anti-P-gp mAb, F4) and its Ig G isotype control. (<b>B</b>) P-gp (indicated in black arrows) was found in MCF-7 tumours, 2 weeks post DXMP exposure. P-gp was observed in MCF-7 tumours (-ve control) and DX tumours (+ve control). (<b>C</b>) No P-gp was observed in kidney 14 days after DXMP administration. Slides were examined at 10× or 20× magnification. Images are of a representative animal.</p

Breast cancer-derived MPs transfer Ezrin to recipient cells.

<p>17.5 µg total cell lysates of breast epithelial cells, osteoblasts and urothelial cells and their co-cultures with (<b>A</b>) leukaemic cell-derived MPs namely VLBMP and (<b>B</b>) Breast cancer-derived MPs namely DXMP both overexpressing P-gp were analyzed by Western blot analysis followed by densitometric quantification after normalization to total protein loaded to the gel. (<b>A</b>) The presence of Ezrin in the leukaemic cell-derived VLBMP and in the recipient cells after MP transfer was readily detected using the anti-Ezrin mAb, 3C12. (<b>B</b>) Ezrin was also detected in the breast cancer DXMP and in all the recipient cells after MP transfer. β-actin was used as an internal loading control. The Western blot experiments were repeated at least three times with similar results. Data are representative of a typical experiment. Densitometric data represent the mean ± SEM of 3 independent experiments * <i>p</i><0.05.</p

Detection of PKH-26 labeled DxMP binding to normal cells by flow cytometry (FCM).

<p>After purification and staining, the co-incubation of PKH-26 DXMP labelled DXMPs (<b>A</b>) 96% of the breast epithelial (MBE) cells (black histogram) and (<b>B</b>) 48% of the osteoblasts (HO-f) showed PKH26 fluorescence (black histogram) with respect to the cells with no MP incubation (gray histograms). The experiment was repeated at least 2–3 times with similar results. Data are representative of a typical experiment.</p

Correlations between MP and some clinical/biological parameters.

<p>A to E: correlations between P-MP numbers and temperature, fever resolution time (FRT), Blantyre coma score (BCS), coma resolution time (CRT) and platelet counts, respectively. F: correlation between R-MP and hemoglobin levels (Hgb).</p

Flow cytometry analysis details and representative cytograms in a CM patient.

<p>MP were discriminated by their size and structures (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013415#pone-0013415-g001" target="_blank">Fig. <b>1A</b></a>). And events in MP gate were further analyzed to differentiate Annexin V⁺ MP from the background signal. An internal standard, constituted of fluorosphere beads of a known size and at a known concentration was used to help set the MP gate and calculate the MP level. Representative cytograms: <b>1B</b> to <b>1H</b> Illustrations of the differences in MP positive events in cytograms representing total MP and cell-derived MP in a CM patient. (<b>B</b>): T-MP:annexin V⁺; (<b>C</b>): P-MP:CD41⁺; (<b>D</b>): E-MP:CD105⁺; (<b>E</b>): R-MP:CD235a⁺; (<b>F</b>): E-MP:CD51⁺; (<b>G</b>): M-MP:CD11b⁺; (<b>H</b>): L-MP:CD3⁺.These MP positive events were used in the calculations of the number of MP/µl of plasma (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013415#s2" target="_blank">methods</a> section).</p

Total and cellular MP levels per µl of plasma at follow-up/discharge (7 days after admission:

<p><i>A</i>) versus levels on admission. (<b>A</b>): T-MP:annexin V⁺; (<b>B</b>): P-MP:CD41⁺; (<b>C</b>): E-MP:CD105⁺; (<b>D</b>): R-MP:CD235a⁺; (<b>E</b>): M-MP:CD11b⁺; (<b>F</b>): L-MP:CD3⁺. Dot plot representations showing how in most of the cases, MP levels decreases in patients with CM at discharge (<i>D</i>). Control group is represented to show that patient levels are returning to control levels. C: Controls; SA: severe malaria anemia patients on admission; CM: cerebral malaria patients on admission; CM+SA: patients with the combine symptoms of CM and SMA on admission; SA(D): severe malaria anemia patients at discharge; CM(D): cerebral malaria patients at discharge; CM+SA(D): patients with the combine symptoms of CM and SMA at discharge; MP levels are given in MP/µl of plasma. The values of the deceased patients are presented as black stars on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013415#pone-0013415-g003" target="_blank">Figure 3</a>. *: p<0.05, **: p<0.001, ***: p<0.0001.</p