Tumor-targeting nanocomplex delivery of novel tumor suppressor RB94 chemosensitizes bladder carcinoma cells in vitro and in vivo

Abstract

Purpose: RB94, a truncated form of RB110, has enhanced tumor suppressor potency and activity against all tumor types tested to date including bladder carcinoma. However, efficient, systemic delivery of the gene encoding RB94 specifically to tumors, is an obstacle to clinical application as an anticancer therapeutic. We have developed a systemically given, nanosized liposome DNA delivery system that specifically targets primary and metastatic disease. The ability of RB94, delivered via this nanocomplex, to sensitize bladder carcinoma to chemotherapy in vitro and in vivo was assessed.

Experimental design: The nanocomplex is an RB94 plasmid encapsulated by a cationic liposome, the surface of which is decorated with a tumor-targeting moiety, either transferrin (Tf/Lip/RB94) or an antitransferrin receptor single-chain antibody fragment (TfRScFv/Lip/RB94). The ability of the complex to sensitize human bladder carcinoma HTB-9 cells to chemotherapeutics was assessed in vitro by XTT assay. In vivo tumor specificity and efficacy were tested in mice carrying HTB-9 tumors by PCR and tumor growth inhibition, respectively.

Results: Transfection with Tf/Lip/RB94 significantly sensitized HTB-9 cells to chemotherapeutic agents in vitro. Tumor specificity of the complex was shown in an orthotopic bladder tumor model by immunohistochemistry and PCR. Moreover, in mice bearing subcutaneous HTB-9 tumors, the combination of systemically given Tf/Lip/RB94 or TfRScFv/Lip/RB94 plus gemcitabine resulted in significant (P<0.0005) tumor growth inhibition/regression and induction of apoptosis.

Conclusions: Use of our tumor-targeting nanocomplex to specifically deliver the potent tumor suppressor RB94 efficiently to tumors has potential as a more effective treatment modality for genitourinary and other cancers.

Figures

Figure 1. RB94 expression in HTB-9 cells

Panel A: Diagram of pSCMV-RB94. Panel B: Western blot analysis of RB94 negative HTB-9 cells following transfection with various Tf/Lip encapsulated pSCMV-RB94 clones (X452-X456) and the parental plasmid construct containing a different CMV promoter. (X457). UT = untreated HTB-9 cells; Control = purified protein to confirm position of RB94. Panel C: RB94 staining in HTB-9 cells transfected with the Tf/Lip complex carrying either the original pCMV-RB94 (X457) or the pSCMV-RB94 (X455) construct 24 hr post- transfection. Top = transfection with pCMV-RB94; Bottom = Transfection with pSCMV. The magnification of both images is 400X. The broad arrows indicate strongly RB94 expressing cells; the thin arrow shows a non-transfected cell.

Figure 2. Chemosensitization of tumor cells by nanocomplex delivered RB94. XTT cell survival assays

Panel A: Evaluation of the degree of sensitization of bladder cancer cell line HTB-9 to gemcitabine by the Tf/LipD delivered RB94 gene or empty vector (0.05μg plasmid DNA). Tf/Lip only was used to assess the influence of non-specific cytotoxicity. Fold sensitization is a comparison of the IC50 values of Tf/Lip/Rb94 vs. Tf/Lip/empty vector. Panel B: Similar experiment as in Panel A in HTB-9 using the chemotherapeutic agent cisplatin (CDDP). The DNA dose was 0.1μg. Panel C: Assessment of the effect of nanocomplex Tf/LipD/RB94 (0.1μg) on the response of normal human endothelial cell line CRL1730 to gemcitabine. Note that for this normal cell line the gemcitabine concentration range is 1000 fold higher that that used with the tumor cell line.

Figure 3. In vivo expression of RB94 in nanocomplex transfected HTB-9 tumors

Panel A: Mice bearing subcutaneous RB94 negative HTB-9 human bladder cancer tumors were i.v. tail vein injected with TL/RB94 or the complex minus the Tf ligand (UL) as described in Materials and Methods. Sixteen hours post-injection, the tumor liver and lungs were excised, protein isolated and RB94 expression determined by Western Blot analysis using an anti-RB monoclonal antibody (QED Bioscience Inc., San Diego CA). Panel B: Mice bearing subcutaneous RB94 negative HTB-9 human bladder cancer tumors were i.v. tail vein injected with scL/RB94 or the complex minus the TfRscFv targeting moiety. Lipsome formulations A and D were both used in this experiment. Forty-eight hours post-injection the tumor and liver were excised, protein isolated and RB expression assessed as in Panel A. UT= untreated; Arrow indicates the position of the RB94 protein.

Figure 3. In vivo expression of RB94 in nanocomplex transfected HTB-9 tumors

Panel A: Mice bearing subcutaneous RB94 negative HTB-9 human bladder cancer tumors were i.v. tail vein injected with TL/RB94 or the complex minus the Tf ligand (UL) as described in Materials and Methods. Sixteen hours post-injection, the tumor liver and lungs were excised, protein isolated and RB94 expression determined by Western Blot analysis using an anti-RB monoclonal antibody (QED Bioscience Inc., San Diego CA). Panel B: Mice bearing subcutaneous RB94 negative HTB-9 human bladder cancer tumors were i.v. tail vein injected with scL/RB94 or the complex minus the TfRscFv targeting moiety. Lipsome formulations A and D were both used in this experiment. Forty-eight hours post-injection the tumor and liver were excised, protein isolated and RB expression assessed as in Panel A. UT= untreated; Arrow indicates the position of the RB94 protein.

Figure 4. In vivo expression of RB94 in tumor and liver at the single cell level by immunohistocemical analysis and PCR

In Panels A-D, HTB-9 tumor and liver cells from the mice shown in Figure 3B were immunohistochemically stained for RB94 expression. Panel A: Tumor from mouse injected with TfRscFv/LipA/RB94 in Figure 3B Panel B: Liver from the same mouse whose tumor is shown in Panel A. Panel C: Tumor from a mouse that received the untargeted LipA/RB94 complex in Figure 3B Panel D: Liver from the same mouse whose tumor is shown in Panel C Panel E: PCR analysis of tumor bearing bladder, and normal tissues from two individual mice bearing RB94 negative HTB-9 tumors which had been injected three times over 24 hours with the TfRscF/LipA/RB94 complex at 24ugDNA/mouse/injection. Lanes 1-4 are from mouse 1 while lane 6 is from a separate tumor bearing mouse. Lane 1 = liver; Lane 2 = large intestine; Lane 3 = kidney; Lanes 4 and 6 = bladder with tumor; Lane 5 = water blank. M = size markers (500bp and 1000bp hyperladder V and IV, respectively,) (Bioline Co., Randolph, MA).

Figure 5. Detection of cleaved caspase 3 in vivo after treatment with TfRscFv/LipD/RB94

Western blot analysis of the level of the 17kDa cleaved caspase 3 protein, a marker for apoptosis, in plasma for HTB-9 tumor bearing mice 16 hours after treatment with complete tumor targeting nanocomplex (TF/Lip/RB94 and TfRscFv/Lip/RB94), complex minus the targeting moiety (Unliganded) or the complex bearing a non- tumor specific ligand (CD2). The mice received three i.v. tail vein injections over 24 hours at 40μg DNA/mouse/injection. Protein was obtained and Western blot analysis performed as described in Materials and Methods.

Figure 6. Tumor response of the HTB-9 xenograft tumor model to ligand-targeted liposome delivery of RB94. HTB-9 tumors were induced in female athymic nude mice as described in Materials and Methods

Panel A: Mice bearing tumors of approximately 100mm3 were i.v. tail vein injected with Tf/LipA or TF/LipD complexed RB94 plasmid DNA (10μg DNA/mouse/injection) alone or in combination with gemcitabine (Gem). The last injection was on day 49. Points are the mean of 4-10 tumors/group ± S.E. Panel B: Mice bearing tumors of 50-100mm3 were i.v. tail vein injected with scL complexed RB94 plasmid DNA or empty vector (20μg DNA/mouse/Injection) in combination with gemcitabine. Unliganded Liposome/RB94 complex was also administered in combination with the chemotherapeutic agent. The last injection was on day 28. Points are the mean of 4-10 tumors/group ± S.E.

Figure 6. Tumor response of the HTB-9 xenograft tumor model to ligand-targeted liposome delivery of RB94. HTB-9 tumors were induced in female athymic nude mice as described in Materials and Methods

Panel A: Mice bearing tumors of approximately 100mm3 were i.v. tail vein injected with Tf/LipA or TF/LipD complexed RB94 plasmid DNA (10μg DNA/mouse/injection) alone or in combination with gemcitabine (Gem). The last injection was on day 49. Points are the mean of 4-10 tumors/group ± S.E. Panel B: Mice bearing tumors of 50-100mm3 were i.v. tail vein injected with scL complexed RB94 plasmid DNA or empty vector (20μg DNA/mouse/Injection) in combination with gemcitabine. Unliganded Liposome/RB94 complex was also administered in combination with the chemotherapeutic agent. The last injection was on day 28. Points are the mean of 4-10 tumors/group ± S.E.