Expression of a suicidal gene under control of the human secreted protein acidic and rich in cysteine (SPARC) promoter in tumor or stromal cells led to the inhibition of tumor cell growth

Abstract

The successful use of transcriptional targeting for cancer therapy depends on the activity of a given promoter inside the malignant cell. Because solid human tumors evolve as a "cross-talk" between the different cell types within the tumor, we hypothesized that targeting the entire tumor mass might have better therapeutic effect. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein overexpressed in different human cancers malignant melanomas both in the malignant cells compartment as in the stromal one (fibroblasts and endothelial cells). We have shown that expression of the herpes simplex virus-thymidine kinase (TK) gene driven by the SPARC promoter in combination with ganciclovir inhibited human melanoma cell growth in monolayer as well as in multicellular spheroids. This inhibitory effect was observed both in homotypic spheroids composed of melanoma cells alone as well as in spheroids made of melanoma cells and stromal cells. Expression of the TK gene was also efficient to inhibit the in vivo tumor growth of established melanomas when TK was expressed either by the malignant cells themselves or by coadministered endothelial cells. Our data suggest that the use of therapeutic genes driven by SPARC promoter could be a valuable strategy for cancer therapy aiming to target all the cellular components of the tumor mass.

Figure 1

Luciferase activity driven by different variants of SPARC promoter. A, activity of different variants of SPARC promoter in different cell lines B, activity of hSPPr and hSPPr(Δ10) in different cell types. Statistical analysis of hSPPr and hSPPr(Δ10) activity in the different cell lines was made in comparison to T-47D cells. ***, P < 0.001; **, P < 0.01; *, P < 0.05. Data include the activity in the different cell types of an SV40 promoter.

Figure 2

In vitro sensitivity to ganciclovir (GCV) of different cell types expressing TK. A, ganciclovir sensitivity of A375N-derived cell clones expressing TK driven by hSPPr and hSPPr(Δ10). Inset, TK and actin (ACTB) bands after semiquantitative reverse transcription-PCR. B, bystander effect induced by melanoma cells expressing TK driven by the two versions of SPARC promoter. C, ganciclovir sensitivity of a cell culture mix composed of Mel-(Δ10)-TK and either BAEC or WI-38 VA cells (1:1 cell ratio). ***, P < 0.001.

Figure 3

In vitro sensitivity to ganciclovir of homotypic and heterotypic spheroids. A, ganciclovir sensitivity of spheroids made of Mel-TK cells alone or mixed with Mel-EGFP cells. B, ganciclovir sensitivity of spheroids made of Mel-(Δ10)-TK alone or mixed with Mel-EGFP cells. C, ganciclovir sensitivity of spheroids made of melanoma cells expressing TK mixed with WI-38 fibroblasts. or BAEC.

Figure 4

In vitro sensitivity to ganciclovir of heterotypic cell cultures and spheroids containing stromal cells expressing TK. A, ganciclovir sensitivity of BAEC and WI-38 VA cells expressing TK. In all cases, ganciclovir was added for 5 d, the day after cells were seeded on the plates; data were obtained by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. B, ganciclovir sensitivity of cells monolayers containing BAEC-(Δ10)-TK or WI-38 VA-(Δ10)-TK cells mixed with A375N cells. C, ganciclovir sensitivity of heterotypic spheroids made of BAEC-(Δ10)-TK cells mixed with different malignant cell types. D, immunofluorescence of a heterotypic spheroid made of Mel-EGFP and BAEC-(Δ10)-TK cells showingthe presence of Mel-EGFP green fluorescent cells at the outer part. E, combined immunofluorescence of EGFP and proliferatingcell nuclear antigen on a spheroid made of Mel-EGFP and BAEC-(Δ10)-TK cells. A representative Mel-EGFP cell (arrow) showing a blue nucleus (corresponding to positive proliferating cell nuclear antigen staining) and green cytoplasm. F, similar to E but showinga potential BAEC-(Δ10)-TK cell (arrow) with a blue nucleus and no green fluorescence in the cytoplasm.

Figure 5

In vivo inhibition of established melanomas expressing TK and treated with ganciclovir. A, the different cell types were injected as described and treated with PBS (n = 4) or ganciclovir (n = 6). B, external visualization of the in vivo tumor growth (bright field and fluorescence). C, mice carrying Mel-(Δ10)-TK tumors were treated with ganciclovir (n = 6) or PBS (n = 4) as described. D, mice carryingchimeric BAEC-(Δ10)-TK:A375N tumors were treated with ganciclovir (n = 6) or PBS (n = 4) as described.