Hyaluronidase and collagenase increase the transfection efficiency of gene electrotransfer in various murine tumors

Abstract

One of the applications of electroporation/electropulsation in biomedicine is gene electrotransfer, the wider use of which is hindered by low transfection efficiency in vivo compared with viral vectors. The aim of our study was to determine whether modulation of the extracellular matrix in solid tumors, using collagenase and hyaluronidase, could increase the transfection efficiency of gene electrotransfer in histologically different solid subcutaneous tumors in mice. Tumors were treated with enzymes before electrotransfer of plasmid DNA encoding either green fluorescent protein or luciferase. Transfection efficiency was determined 3, 9, and 15 days posttransfection. We demonstrated that pretreatment of tumors with a combination of enzymes significantly increased the transfection efficiency of electrotransfer in tumors with a high extracellular matrix area (LPB fibrosarcoma). In tumors with a smaller extracellular matrix area and less organized collagen lattice, the increase was not so pronounced (SA-1 fibrosarcoma and EAT carcinoma), whereas in B16 melanoma, in which only traces of collagen are present, pretreatment of tumors with hyaluronidase alone was more efficient than pretreatment with both enzymes. In conclusion, our results suggest that modification of the extracellular matrix could improve distribution of plasmid DNA in solid subcutaneous tumors, demonstrated by an increase in transfection efficiency, and thus have important clinical implications for electrogene therapy.

FIG. 1.

Green fluorescent protein (GFP) expression in LPB tumors 9 days postelectrotransfection. Shown are representative images of excised tumors cut into two halves, which were treated with plasmid DNA expressing GFP (A), pretreated with a combination of both enzymes followed by plasmid DNA injection alone (B) or pretreated with a combination of both enzymes followed by electrotransfer of plasmid DNA (C), and visualized under a digitalized fluorescence stereomicroscope. Original magnification, ×0.8. N, necrotic part; arrows, GFP fluorescent spots. (D) Percentage of transfected (GFP-positive) areas and (E) fluorescence intensity of transfected area. Data represent the mean±SEM of 3 or 4 tumors from 3 independent experiments (total, 9–12).*p<0.05 compared with all other groups. GFP, injection of plasmid DNA encoding GFP alone; Combination+GFP, injection of collagenase 24 hr and hyaluronidase 2 hr before injection of plasmid DNA; EPGFP, injection of plasmid DNA followed by application of EP; Collagenase+EPGFP, injection of collagenase 24 hr before electrotransfection of tumors; Hyaluronidase+EPGFP, injection of hyaluronidase 2 hr before electrotransfection of tumors; Combination+EPGFP, injection of collagenase 24 hr and hyaluronidase 2 hr before electrotransfection of tumors.

FIG. 2.

Time dependence of transfection efficiency in LPB tumors. (A) Percentage of transfected (GFP positive) areas and (B) fluorescence intensity of transfected area on days 2, 9, and 15. Data represent the mean±SEM of 3 or 4 tumors from 3 independent experiments (total, 9–12). *p<0.05 compared with all other groups. EPGFP, injection of plasmid DNA followed by application of EP; Collagenase+EPGFP, injection of collagenase 24 hr before electrotransfection of tumors; Hyaluronidase+EPGFP, injection of hyaluronidase 2 hr before electrotransfection of tumors; Combination+EPGFP, injection of collagenase 24 hr and hyaluronidase 2 hr before electrotransfection of tumors.

FIG. 3.

Collagen content (A) and glycosaminoglycan/proteoglycan content (B) in LPB tumors after collagenase and hyaluronidase treatment. Shown are representative histological images of LPB tumors stained for collagen (light blue) and glycosaminoglycans/proteoglycans (pink) in control tumors and after treatment with collagenase, hyaluronidase, or a combination of both enzymes. Extracellular matrix content: (C) glycosaminoglycans/proteoglycans; (D) collagen; and (E) cell density of LPB tumors after collagenase and/or hyaluronidase treatment. Data represent the mean±SEM of six tumors. *p<0.05 compared with the control group. ^#p<0.05 compared with treatment of tumors with hyaluronidase. NS, not significant.

FIG. 4.

Growth curves of LPB tumors after electrotransfection. Combination+GFP, injection of collagenase 24 hr and hyaluronidase 2 hr before injection of plasmid DNA; EPGFP, injection of plasmid DNA followed by application of EP; Collagenase+EPGFP, injection of collagenase 24 hr before electrotransfection of tumors; Hyaluronidase+EPGFP, injection of hyaluronidase 2 hr before electrotransfection of tumors; Combination+EPGFP, injection of collagenase 24 hr and hyaluronidase 2 hr before electrotransfection of tumors. Data represent the mean±SEM of six tumors.

FIG. 5.

Transfection efficiency in LPB (A), SA-1 (B), EAT (C), and B16 (D) tumors after pretreatment of tumors with collagenase and hyaluronidase. EP Luciferase, injection of plasmid DNA followed by application of EP; Collagenase+EP Luciferase, injection of collagenase 24 hr before electrotransfection of tumors; Hyaluronidase+EP Luciferase, injection of hyaluronidase 2 hr before electrotransfection of tumors; Combination+EP Luciferase, injection of collagenase 24 hr and hyaluronidase 2 hr before electrotransfection of tumors. Data represent the mean±SEM of 3 or 4 tumors from 3 independent experiments (total, 9–12). *p<0.05 compared with other treatment groups; ^#p<0.05 compared with the Hyaluronidase+EP Luciferase group.