Synchronous inhibition of mTOR and VEGF/NRP1 axis impedes tumor growth and metastasis in renal cancer

Abstract

Clear cell renal cell carcinoma (ccRCC) is known for its highly vascular phenotype which is associated with elevated expression of vascular endothelial growth factor A (VEGF), also known as vascular permeability factor (VPF). Accordingly, VEGF has been an attractive target for antiangiogenic therapies in ccRCC. Two major strategies have hitherto been utilized for VEGF-targeted antiangiogenic therapies: targeting VEGF by antibodies, ligand traps or aptamers, and targeting the VEGF receptor signaling via antibodies or small-molecule tyrosine-kinase inhibitors (TKIs). In the present article we utilized two entirely different approaches: targeting mammalian target of rapamycin (mTOR) pathway that is known to be involved in VEGF synthesis, and disruption of VEGF/Neuroplin-1 (NRP1) axis that is known to activate proangiogenic and pro-tumorigenic signaling in endothelial and tumor cells, respectively. Everolimus (E) and a small-molecule inhibitor EG00229 (G) were used for the inhibition of mTOR and the disruption of VEGF/NRP1 axis, respectively. We also exploited a liposomal formulation decorated with a proprietary tumor-targeting-peptide (TTP) to simultaneously deliver these two agents in a tumor-targeted manner. The TTP-liposomes encapsulating both Everolimus and EG00229 (EG-L) demonstrated higher in vitro and in vivo growth retardation than the single drug-loaded liposomes (E-L and G-L) in two different ccRCC models and led to a noticeable reduction in lung metastasis in vivo. In addition, EG-L displayed remarkable inhibition of tumor growth in a highly aggressive syngeneic immune-competent mouse model of ccRCC developed in Balb/c mice. Taken together, this study demonstrates an effective approach to achieve improved therapeutic outcome in ccRCC.

Keywords: Renal cell carcinoma; Targeted therapies.

Fig. 1

In vivo biodistribution of IR-780-dye-labeled liposomes in RCC xenografts. IVIS imaging showing higher tumor accumulation of IR-780 dye-labeled TTP-conjugated liposomes (TL) compared to control liposomes (CL) at 24 h (upper panel) and 48 h (lower panel) after IV administration into mice bearing subcutaneous 786-O (a) and A498 tumors (b). Ex vivo imaging of 786-O (c) and A498 (d) tumors and major organs, respectively, harvested at 48 h, demonstrated significant higher tumor uptake of TL compared to CL. Interestingly, significantly higher lung accumulation of CL was observed compared to TL. n = 1 mouse per treatment group.

Fig. 2

In vitro and in vivo efficacy of drug-loaded liposomes in RCC cell lines. 786-O (a) and A498 (b) cells were treated with various drug-loaded TTP-conjugated liposomes for 72 h. Then cell viability was determined with MTS assay. Dual-drug-loaded liposomes showed higher reduction in cell viability compared to single drug-loaded liposomes in all cell lines (n = 4 wells per dose). c 5 × 10⁶ 786-O cells were subcutaneously injected into the right flanks of 8-week-old male SCID mice. Tumors were allowed to grow until the average tumor size is ~400–500 mm³. Then mice were treated with drug-loaded liposomes (n = 1 mouse per treatment group) 3× per week for 3 weeks. Tumors were measured weekly and tumor volume was plotted to obtain the respective growth curves. In both cases dual-drug-loaded liposomes demonstrated stronger inhibition compared to single drug-loaded liposomes. Some of the mice were sacrificed before the completion of experiment due to ulceration of tumors. d Similar results were obtained in A498 xenografts (n = 1 mouse per treatment group).

Fig. 3

H&E and Ki67 staining of tumor sections obtained from the single mouse trial. a Representative images of the H&E and Ki67 stained tumor sections from different treatment groups displayed comparatively higher antiproliferative activity of EG-L. Bar length = 200 µm. b, c Quantification of Ki67-positive nuclei in 786-O and A498 tumor sections, respectively. Error bars in all graphical plots are given based on standard deviation. *, ** and *** denote p < 0.05, p < 0.01, and p < 0.001 compared to control, respectively (n = 5 spatially different regions from same tumor section).

Fig. 4

Validation of the result obtained from single mouse trial in cohorts of five mice. a 5 × 10⁶ 786-O cells were subcutaneously injected into the right flanks of 8-week-old male SCID mice. Tumors were allowed to grow until the average tumor size is ~300 mm³. Then mice were treated with vehicle or EG-L (n = 5 mice per treatment group) 3× per week for 4 weeks. Tumors were measured weekly and tumor volume was plotted to obtain the respective growth curves. EG-L demonstrated significant inhibition compared to the vehicle group. ** and *** denote p < 0.01 and p < 0.001 compared to control, respectively. b Images of the harvested tumors at the end of the experiment. c Representative images of H&E and Ki67 staining of the tumor tissue sections. Bar length = 200 µm. d Quantification of Ki67-positive nuclei. *** denotes p < 0.001 compared to control (n = 3 tumors per group, five spatially different regions from each tumor section).

Fig. 5

Antitumor efficacy of EG-L in an immune-competent mice model of RCC. a 1 × 10⁶ Renca cells were subcutaneously injected into the right flanks of 8 week-old-male Balb/c mice. Tumors were allowed to grow until the average tumor size is ~120 mm³. Then mice were treated with vehicle or EG-L (n = 5 mice per treatment group) 2× per week for 3 weeks. Tumors were measured weekly and tumor volume was plotted to obtain the respective growth curves. EG-L demonstrated significant inhibition compared to the vehicle group. ** and *** denote p < 0.01 and p < 0.001 compared to control, respectively. b Images of the harvested tumors at the end of the experiment. Two tumors from control groups were ruptured before harvest. c Representative images of H&E, Ki67, and YM1 staining of the tumor tissue sections. Bar length = 200 µm. d, e Quantification of Ki67 and YM1 staining respectively. *** denotes p < 0.001 compared to control (n = 3 tumors for control and 4 tumors for EG-L, five spatially different regions from each tumor section).

Fig. 6

EG-L downregulates the expression of C-X-C chemokines and TGF-β in an immune-competent mice model of RCC. Total RNA was isolated from tumors treated with vehicle or EG-L and subjected to real-time reverse transcription polymerase chain reaction (RT-PCR) for a CXCL9, b CXCL10, c CXCL11, and d TGFB1. EG-L significantly reduced the mRNA expression of the cytokines compared to the vehicle. * denotes p < 0.5 compared to control. (n = 3 tumors for control and 5 tumors for EG-L).

Fig. 7

Inhibition of lung metastasis in mice bearing 786-O xenografts. The dual-drug-loaded liposomes (EG-L) significantly inhibited lung metastasis in mice bearing 786-O subcutaneous tumors compared to Control, liposome only (L), or single-drug-loaded liposomes (E-L and G-L). Metastatic nodules are indicated by black arrows (upper panel). Respective higher magnification images are depicted in the lower panel (n = 1 mouse per treatment group).