Intercellular transfer of P-glycoprotein mediates acquired multidrug resistance in tumor cells

Abstract

The overexpression of P-glycoprotein (P-gp) causes resistance to chemotherapy in many tumor types. Here, we report intercellular transfer of functional P-gp from P-gp-positive to P-gp-negative cells in vitro and in vivo. The expression of acquired P-gp is transient in isolated cells but persists in the presence of P-gp-positive cells or under the selective pressure of colchicine. The intercellular transfer of functional P-gp occurs between different tumor cell types and results in increased drug resistance both in vitro and in vivo. Most importantly, the acquired resistance permits tumor cells to survive potentially toxic drug concentrations long enough to develop intrinsic P-gp-mediated resistance. P-gp transfer also occurs to putative components of tumor stroma, such as fibroblasts, raising the possibility that multidrug resistance could be conferred by resistant tumor cells to critical stromal elements within the tumor mass. This is the first report, to our knowledge, that a protein transferred between cells retains its function and confers a complex biologic property upon the recipient cell. These findings have important implications for proteomic analyses in tumor samples and resistance to cancer therapy.

Fig. 1.

Transfer of P-gp expression between resistant and sensitive variants of the BE (2)-C human neuroblastoma cell line. (a) Evolution of P-gp transfer with graphs showing histograms of a 50/50 mixture of sensitive and BE (2)-C/CHC (0.2) cells measured at 0 (3 h), 2, 4, 6, and 8 days after coincubation. MRK-16 Ab was used in a sandwich assay with fluorescein labeled secondary Abs. (b) Dependence of the transferred P-gp expression on the P-gp levels in resistant cells. Scatter histograms were obtained by gating cells according to PE and GFP emission spectra. One day cocultures of sensitive cells with BE (2)-C/CHC(0.2) (Left Lower) and BE (2)-C/CHC (1) (Right Lower) are compared with controls of pure sensitive (Left Upper) and pure BE (2)-C/CHC (0.2) (Right Upper) cells. (c) Coincidence of the AqMDR population (the first peak in the mixed population histogram, gray; see text) with the population gated for GFP expression (black). In these and all other experiments, medium was free of colchicine. Coincubation was for 10 days with BE (2)-C/CHC(0.2) cells. A control showing mixture of BE (2)-C/CHC (1) and BE (2)-C with no coincubation is shown in Fig. 16.

Fig. 2.

Direct demonstration of P-gp transfer through imaging by confocal microscopy. Controls of pure BE (2)-C/CHC (1) (first row) and sensitive (second row) cells compared with dually labeled (AqMDR, 8 days 50:50 ratio coculture) cells (third and fourth rows). The arrows indicate a local “bright spot” of high P-gp-related membrane fluorescence. (Scale bars, 20 μm.)

Fig. 3.

Characterization of P-gp transfer. (a) Transferred P-gp is functional, as judged by the change in the Rhodamine 123 extrusion in coculture (gray line) versus BE (2)-C (rightmost black solid line showing concentration without mixing for reference) and BE (2)-CHC(0.2) (leftmost dotted line) cell lines (Left) and the same experiment in the presence of verapamil (Right). In Left, the most resistant subset has a very low peak of rhodamine, but even the most sensitive subset in the mixture shows a peak that has a definite shift toward less rhodamine retention, which is consistent with functioning P-gp transfer. The effect is virtually abolished by verapamil. (b) The extent of P-gp transfer depends on the relative proportions [sensitive/resistant ratio (S/R):80/20; 50/50; 20/80 ratios of BE (2)-C and BE (2)-C/CHC(10′) lines] and levels of P-gp expression in the resistant line [BE (2)-C/CHC(10′) less resistant versus BE (2)-C/CHC(0.2) more resistant]. The peak MDR expression in the AqMDR peak is higher when sensitive cells are incubated with more resistant cells, showing a kind of dose effect. The controls for these experiments are in Fig. 11.

Fig. 4.

Intercellular P-gp transfer occurs in vivo. Cells extracted from tumors grown in immunodeficient mice were isolated and analyzed by flow cytometry. (a) Results shown are for tumors grown from BE (2)-C (a Left), BE (2)-C/CHC (1) (a Right), and AqMDR cells from a 50/50 ratio after 2 days of coculture. (b Left) A negative control performed with same cells by using a nonspecific mAb with MRK-16 isotype. (b Right) More than 65% of the cells are in the right upper quadrant, indicating both GFP and P-gp, attesting to efficient transfer. The cells injected had scatter plot profiles qualitatively identical to Fig. 1b.

Fig. 5.

P-gp transfer is mediated by large microparticles or cell-to-cell contact. The control of sensitive cells labeled with P-gp nonspecific (SN) and P-gp specific (MRK-16, SP) Abs grown with no medium transfer as compared with sensitive cells labeled with specific Ab grown for 1 day in unfiltered or filtered (0.8 μm) medium transferred from BE (2)-C/CHC (1) cells. Virtual absence of resistant cells in the unfiltered medium is evident, as shown in Right Upper (in contrast to the scatter plot of the cell line from which the unfiltered medium was obtained, shown in Fig. 1b Right Upper).

Fig. 6.

Evolution of resistance transfer before and after FACS of AqMDR cells. (a) Percentage of BE (2)-C (♦), BE (2)-C/CHC (1) (▪) and AqMDR (•) cells as determined by flow cytometry. The sort occurred on the ninth day after coculture initiation as indicated. See Fig. 8 for simulation of this experiment. Note that because of the gap in time between the sort and the first subsequent measurement, a significant reduction in the P-gp content may have occurred. (b) An RT-PCR experiment showing mdr-1 levels in the indicated cell lines. The experiments with AqMDR were performed for 8 days (left lane) and 12 days (right lane) of coculture followed by FACS. (c and d) Transfer of resistance allows sensitive cells to survive cytotoxic drug concentrations and ultimately develop intrinsic resistance. BE (2)-C and BE (2)-C/CHC (1) cells were cocultured (in a 50:50 ratio) for 10 days and then dually labeled (AqMDR) cells were separated from the rest of the cells by FACS. Postsort AqMDR cells were then grown in colchicine-free medium for 7–10 days and treated with 5 ng/ml colchicine [close to an IC₅₀ of sensitive cells (4.5 ng/ml)] medium for 1–2 weeks. This sequence was repeated six times at the time intervals indicated. P-gp transfer measured by flow cytometry and colchicine resistance (c Inset) and RT-PCR data (d) are shown. In a, BE (2)-C (♦), BE (2)-C/CHC (1) (▪), and AqMDR (•) cells are shown. The lanes in d correspond to experiments performed after each indicated sort (a data point in c) and the controls of sensitive and resistant cells and irrelevant RNA from pAW109 plasmid.