Retroviral gene transfer is inhibited by chondroitin sulfate proteoglycans/glycosaminoglycans in malignant pleural effusions

Abstract

Gene therapy may be an important adjuvant for treating cancer in the pleural space. The initial results of retroviral gene transfer to cancer cells in malignant pleural effusions revealed that transduction was markedly inhibited, and studies to characterize the inhibitory factor(s) were performed. The inhibition was contained within the soluble, rather than cellular, components of the effusions and was demonstrated with amphotropic, gibbon ape leukemia virus, and vesicular stomatitis virus-glycoprotein pseudotyped retroviral vectors. After excluding complement proteins, a series of studies identified chondroitin sulfates (CSs) as the inhibitory substances. First, treatment of the effusions with mammalian hyaluronidase or chondroitinases, but not Streptomyces hyaluronidase, abolished the inhibitory activity. Second, addition of exogenous CS glycosaminoglycans mimicked the inhibition observed with pleural effusions. Third, immunoassays and biochemical analyses of malignant pleural effusion specimens revealed CS in relevant concentrations within pleural fluid. Fourth, proteoglycans/glycosaminoglycans isolated from the effusions inhibited retroviral gene transfer. Analyses of the mechanism of inhibition indicate that the chondroitin sulfates interact with vector in solution rather than at the target cell surface. These results suggest that drainage of the malignant pleural effusion, and perhaps enzymatic pretreatment of the pleural cavity, will be necessary for efficient retroviral vector mediated gene delivery to pleural metastases.

Fig. 1. Comparison of the inhibition of amphotropic retroviral transduction efficiency in the presence of whole (unfractionated) pleural effusions (A), RBCs (B), and supernatants (C) fractionated from whole pleural effusions

Mv1Lu cells were transduced with the LNPOZ amphotropic retroviral vector in the presence or absence (control) of freshly harvested malignant pleural effusions (n = 5), RBCs (n = 9, three separate experiments using RBCs from three subjects), or effusion supernatants (n = 16). The efficiency of reporter lacZ gene expression was quantified as the percentage of cells transduced by flow cytometry. Shown is the inhibition to transduction (compared with control) by the vector in the presence of increasing volume percentages of the effusions or hematocrits in LNPOZ-containing medium. Data are presented as means = S.E. *, p = 0.05; **, p = 0.001, for a statistically significant difference from control by the Student-New-man-Keuls pairwise multiple comparison analysis.

Fig. 2. CFT1 transduction in the presence or absence of effusions over a range of amphotropic RV vector concentration (A) or using VSV-G (B) or GALV (C) pseudotyped retroviral vectors

CFT1 cells were transduced with various vectors admixed (50%, v/v) with effusion supernatants or medium. 48 h after transduction, the efficiency of gene transfer was quantitated using the 5-bromo-4-chloro-3-indolyl β-D-galactoside histochemical assay and by microscopically counting the percentage of blue cells. Each data point in A represents the mean transduction efficiency of two groups of cells in the absence (□) or presence (○,▵) of two different effusions. B and C show the mean ± S.E. transduction efficiency of three groups of cells exposed to the VSV-G or GALV pseudotyped vectors in the absence (C) or presence (E) of effusions. *, a statistically significant (p < 0.01) difference between the groups by a paired t test analysis.

Fig. 3. Comparison of the inhibition of amphotropic retroviral transduction by untreated pleural effusion supernatants, supernatants heated (60 °C for 1 h) to inactivate complement, or supernatants treated with bovine testicular hyaluronidase (250 units/ml BTH)

Shown is the inhibition to transduction of Mv1Lu cells by the LNPOZ amphotropic retroviral vector admixed (50%, v/v) with untreated effusion supernatants, heated effusion supernatants, or effusion supernatants treated with BTH. The reporter lacZ gene expression is quantified by flow cytometry. n =16 for each group, data presented as means ± S.E., *, the two-tailed p value from a paired t test analysis where p < 0.001 for a statistically significant difference between the untreated and BTH-treated group.

Fig. 4. Comparison of the inhibition of amphotropic retroviral transduction in the presence of untreated effusion supernatants or in the presence of effusion supernatants treated with the mammalian BTH or the bacterial S-Hya

Mv1Lu cells were transduced with the LNPOZ amphotropic retroviral vector in the presence (50%, v/v) of four effusion supernatants treated with BTH or S-Hya at equivalent unit concentrations, and the reporter lacZ gene expression was quantified. n = 4 effusion specimens, data presented as means ± S.E. *, p < 0.05 for a statistically significant difference from control by the Bonferroni pairwise multiple comparison analysis.

Fig. 5. Inhibition of amphotropic retroviral transduction in the presence (50%, v/v) of untreated effusion supernatants (U) compared with effusion supernatants treated with varying concentrations of chondroitinase AC (CAC), chondroitinase ABC (CABC), or BTH

Mv1Lu cells were transduced with the LNPOZ amphotropic retroviral vector in the presence (50%, v/v) of five separate effusion supernatants or of the same effusion supernatants treated with chondroitinase AC or CABC or BTH. The reporter lacZ gene expression is quantified as the percentage of cells transduced by flow cytometry. Shown is the relative inhibition to trans-duction by the vector in the presence of untreated versus treated effusion supernatants in LNPOZ-containing medium. n = 5 effusion specimens. Data are presented as means ± S.E. *, p < 0.05 for a statistically significant difference from control by the Bonferroni pairwise multiple comparison analysis.

Fig. 6. Inhibition of amphotropic retroviral transduction in the presence of exogenous glycosaminoglycans (GAG; μg/ml) admixed with vector containing medium

Mv1Lu cells were transduced with the LNPOZ amphotropic retroviral vector in the presence of hyaluronic acid (▪), chondroitin sulfate A (•), chondroitin sulfate B (▴), or chondroitin sulfate C (⋆) at concentrations ranging from 0 to 100 μg/ml GAG. The reporter lacZ gene expression is quantified as the percentage of cells transduced by flow cytometry. Shown is the relative inhibition of retroviral transduction (compared with control) as a function of increasing concentrations of exogenous GAGs in LNPOZ-containing medium (n = 3 separate experiments; data are presented as means ± S.E.).

Fig. 7. Dot blot immunoassays testing for the presence of chondroitin 4-sulfate (using the 2B6 antibody), chondroitin 6-sulfate (using the 3B3 antibody), and native chondroitin sulfate epitopes (using the 7D4 antibody) in malignant pleural effusion supernatants

100 μl of seven (–7) serially diluted effusion specimens (1:1000, 1:5000, 1:10,000, and 1:50,000 in Tris-salt buffer) were assayed for the presence of chondroitin 4-sulfate and chondroitin 6-sulfate using bovine nasal cartilage core (at concentrations of 50, 10, 5, and 1 ng/ml) as the control (C) antigen. For these analyses, samples were applied to the nitrocellulose, blocked, then pretreated with chondroitinase ABC to generate epitopes recognized by antibodies 2-B-6 and 3-B-3 (, , 47). Similarly, 100 μl of six serially diluted effusion specimens (1:1000, 1:2500, 1:10,000, and 1:25,000) were assayed for the presence of native chondroitin sulfate epitope (7D4 antibody) without chondroitinase pretreatment and using digested shark cartilage (50, 10, 5, and 1 ng/ml) as the control (C) epitope. The primary murine antibodies (diluted 1:1000 in 1% BSA) were recognized by a secondary (diluted 1:7500 in 1% BSA) anti-mouse IgG conjugated to AP and detected following exposure to the AP substrate.

Fig. 8. Comparison of the inhibition of amphotropic retroviral transduction by untreated effusion supernatants, PG/GAGs isolated from effusions, or PG/GAGs isolated from the effusions that have been enzymatically treated with a variety of specific enzymes

Shown is the relative inhibition to transduction conferred by supernatants from three malignant effusions, the PG/GAGs isolated with the D1 fraction (specific gravity 1.6 g/ml) from those effusions, or the PG/GAGs isolated with the D1 fraction that have been treated with a panel of specific degradative enzymes. A, 50% (v/v with LNPOZ vector stock) effusion supernatant; B, 50% D1 fraction; C, 50% D1 fraction plus Streptomyces hyaluronidase (10 units/ml); D, 50% D1 fraction plus bovine testicular hyaluronidase (250 units/ml); E, 50% D1 fraction plus chondroitinase AC (0.5 units/ml); F, D1 fraction plus chondroitinase ABC (0.5 units/ml); G, 50% D1 fraction plus heparinase (5 units/ml); H, 50% D1 fraction plus heparitinase (5 units/ml); I, 50% D1 fraction plus keratanase (1 unit/ml). n = 3 effusion specimens; data are presented as means ± S.E. *, p<0.001 for a statistically significant difference from control by the Student-Newman-Keuls pairwise multiple comparison analysis.

Fig. 9. Transduction efficiency of LNPOZ into three cell lines (H28 (human malignant mesothelioma,▪), H226 (human lung squamous cell carcinoma,•), and Mv1Lu (mink lung epithelium,▴)) pretreated in culture with BTH (units/ml in maintenance medium)

Cells were transduced with the LNPOZ amphotropic retroviral vector following pretreatment of the cells with BTH and analyzed for lacZ expression by flow cytometry. (Each data point represents the mean efficiency of transduction from two measurements).