Acquisition of multidrug resistance by L1210 leukemia cells decreases their tumorigenicity and enhances their susceptibility to the host immune response

Abstract

The use of antineoplastic drugs for cancer treatment is frequently associated with the acquisition of a multidrug-resistant (MDR) phenotype that renders tumoural cells insensitive to antineoplastics. It remains elusive whether the acquisition of the MDR phenotype alters immunological parameters that could influence the cell sensitivity to an eventual host immune response. We report that immunisation of syngeneic mice with gamma-irradiated L1210S (parental line) and L1210R (MDR phenotype) cells results in a significant rejection of subsequently implanted L1210R-based tumours, but not of the L1210S ones. Notably, L1210R tumours display a twofold reduction in vivo proliferative capacity and are less aggressive in terms of mouse survival than their sensitive counterparts. Also, analysis of surface expression of molecules involved in antigen presentation and cytokine activity revealed a slight increase in IFN-gamma receptor expression, a decrease of Fas molecule, and a fourfold up-regulation of MHC class I molecules in L1210R cells. Nonetheless, both cell lines were able to induce a cytotoxic response in syngeneic mice and were equally susceptible to cytotoxicity by splenic cells. Together, these findings indicate that acquisition of drug resistance by L1210 cells is accompanied by pleiotropic changes that result in reduced tumour proliferative capacity and tumorigenicity in syngeneic mice. Hence, immunological studies of MDR tumours may assist in the design of specific therapeutic strategies that complement current chemotherapy treatments.

Fig. 1

In vitro proliferative capacity of L1210S and L1210R cell lines. Cells (2×10⁴ cells/well) were plated on a P-24 flat-bottom plate for 5 days, and the number of viable cells was counted by using trypan blue staining. Data are mean ± SEM of three separate experiments.

Fig. 2

Tumorigenicity of L1210S and L1210R cells in syngeneic DBA/2 mice. Mice (eight per group) were inoculated (s.c.) in the right flank with 0.5×10³ L1210S (grey circles) or L1210R (black circles) cells. Growth rate of tumours (a) and percentage of mouse survival (b) were monitorised from the day of cell inoculation (day 0).

Fig. 3

Tumorigenicity and immunological response of L1210S and L1210R cells in syngeneic DBA/2 mice. Mice (eight per group) were inoculated (s.c.) in the right flank with 5×10⁵ γ-irradiated cells L1210S (Irrad-L1210S, grey circles) or L1210R (Irrad-L1210R, black circles). After 15 days, these mice were injected s.c. on the left flank with 0.5×10³ L1210S (a, c, e) or L1210R (b, d, f) cells. As control (white circles) non-preimmunised mice were inoculated with 0.5×10³ L1210S or L1210R cells. Percentage of tumour-free mice (a, b), growth rate of tumours (c, d) and mouse survival (e, f) were monitored from the day of viable cell inoculation (day 0).

Fig. 4

a–c Differential expression of surface molecules on L1210S and L1210R cells. The expression levels were measured by flow cytometry using specific antibodies (black bars). As NC, a non-related antibody with the same isotype as the experimental antibodies (data not shown). A different scale has been used to represent MHC class I expression. Data are mean ± SEM of eight separate experiments. *p<0.05; **p<0.01, as compared to expression levels on L1210S parental cell line (Student’s t test).

Fig. 5

Cell death induced on L1210S and L1210R cells by an antibody specific for mouse Fas. Cells were incubated with protein G (2 μg/ml) plus anti-Fas antibody (10 μg/ml) for 24 h. As NC, protein G (2 μg/ml) plus 10 μg/ml of a non-related antibody from the same isotype as the anti-Fas antibody (hamster IgG2, λ) was used. Apoptosis was monitored with the annexin V assay. a Flow cytometry histograms of L1210S, L1210R cells and thymocytes treated with protein G plus isotype control (NC) or protein G plus anti-Fas antibody. b Bar histograms of annexin V-FITC assay results showing background death (white bars) and Fas-dependent cell death (black bars). Thymocytes were used as positive control for Fas-induced apoptotic death. Data are mean ± SEM of three separate experiments. *p<0.05; **p<0.01, as compared to NC (Student’s t test).

Fig. 6

Cytotoxic activity of L1210S- or L1210R-immunised mice. a Specific lysis of L1210S cells (target cells) by splenic cells from control, L1210S- or L1210R-immunised mice. b Specific lysis of L1210R cells (target cells) by CTLs from control, L1210S- or L1210R-immunised mice. Data are mean ± SEM of four separate experiments.