Tumor-targeted delivery of sunitinib base enhances vaccine therapy for advanced melanoma by remodeling the tumor microenvironment

Abstract

Development of an effective treatment against advanced tumors remains a major challenge for cancer immunotherapy. We have previously developed a potent mannose-modified lipid calcium phosphate (LCP) nanoparticle (NP)-based Trp2 vaccine for melanoma therapy, but because this vaccine can induce a potent anti-tumor immune response only during the early stages of melanoma, poor tumor growth inhibition has been observed in more advanced melanoma models, likely due to the development of an immune-suppressive tumor microenvironment (TME). To effectively treat this aggressive tumor, a multi-target receptor tyrosine kinase inhibitor, sunitinib base, was efficiently encapsulated into a targeted polymeric micelle nano-delivery system (SUN_b-PM), working in a synergistic manner with vaccine therapy in an advanced mouse melanoma model. SUN_b-PM not only increased cytotoxic T-cell infiltration and decreased the number and percentage of MDSCs and Tregs in the TME, but also induced a shift in cytokine expression from Th2 to Th1 type while remodeling the tumor-associated fibroblasts, collagen, and blood vessels in the tumor. Additionally, inhibition of the Stat3 and AKT signaling pathways by SUN_b-PM may induce tumor cell apoptosis or decrease tumor immune evasion. Our findings indicated that targeted delivery of a tyrosine kinase inhibitor to tumors can be used in a novel synergistic way to enhance the therapeutic efficacy of existing immune-based therapies for advanced melanoma.

Keywords: Advanced melanoma; Peptide vaccine; Polymeric micelles; Sunitinib base; Tumor microenvironment.

Fig. 1

TEM images of (A) p-Trp2 LCP vaccine and (B) SUN_b-PM after negative staining. (C) In vitro stability of SUN_b-PM at 4°C. (D) In vitro release of sunitinib base from PLGA-PEG micelles at pH 5.8 and pH 7.4. (E) Cytotoxicity of SUN_OS and SUN_b-PM against B16F10 cells after 24 h. The error bars in the graphs represent standard derivations (n=3).

Fig. 2

(A) Tumor accumulation of ³H-labeled SUN_b-PM and SUN_OS in C57BL/6 mice bearing B16F10 tumors at t = 3, 6, 12 and 24 h after treatment. The dose of ³H-labeled drug was 50 μCi/kg. (B–E) Antitumor activity of PBS, VAC, SUN_OS, SUN_b-PM, V+SUN_OS and V+SUN_b-PM on B16F10 tumor-bearing mice. C57BL/6 mice were inoculated with 2×10⁵ B16F10 cells on day 0. VAC was injected on day 14 at a dose of 0.3 mg/kg; SUN_OS was given p.o. or SUN_b-PM was given i.v. on days 14, 16, and 18 at a dose of 30 mg/kg or 10 mg/kg, respectively. Tumor growth was measured every day until the 19^th day after inoculation. Mice were sacrificed on day 19 and tumors were harvested. (B) Tumor volumes of tumor-bearing mice as a function of time. The arrows (pink for VAC and blue for SUN) indicate the time points of drug administration. (C) Tumor inhibition ratio calculated based on the weight of tumor at the end of the tests. (D) H&E staining of tumor sections. The white scale bar represents 100 μm. (E) TUNEL-positive cells in tumor sections excised from the mice seven days after the first treatment. The yellow scale bar represents 50 μm. Three randomly selected microscopic fields were quantitatively analyzed using ImageJ. The results are displayed as mean ± S.D. (error bars). Statistical analyses were calculated by comparing with the untreated group, unless otherwise specified. *p < 0.05, **p < 0.01, ***p< 0.001, n = 5.

Fig. 3

Safety evaluations. (A) Body weight change of B16F10-bearing mice after treatment with VAC, SUN_OS, SUN_b-PM, V+SUN_OS, or V+SUN_b-PM. (B) Liver and (C) kidney function assays after treatment. Results were expressed as the mean ± S.D. (n = 5). The normal ranges for all the biochemical parameters are shown in the upper right corner of panels B and C.

Fig. 4

Tumor infiltrating immune cells after treatment. C57BL/6 mice were inoculated with 2×10⁵ B16F10 cells on day 0. VAC was injected on day 14 at a dose of 0.3 mg/kg; SUN_OS and SUN_b-PM were p.o. or i.v. administered on days 14, 16, and 18 at a dose of 30 mg/kg or 10 mg/kg, respectively. Mice were sacrificed on day 19 and tumors were harvested. Tumor tissues were assayed for CD8⁺ T cells, CD11b⁺/Gr1⁺ MDSC cells, and Foxp3⁺/CD4⁺ Treg cells with immunofluorescence staining, and representative immunofluorescence images were presented (A-C). Flow cytometric analysis was also used to quantify immune cells in the TME (D-F). The results are displayed as mean ± S.D. (error bars). Statistical analyses were calculated by comparing to the untreated group unless otherwise specified with markings. *p < 0.05, **p < 0.01, ***p< 0.001, n = 3.

Fig. 5

In vivo CTL response after vaccination under various conditions. C57BL/6 mice were subcutaneously injected with VAC on day 1, and treated with SUN_OS or SUN_b-PM on days 1, 3 and 5. Splenocytes from naive mice were pulsed with Ova or Trp2 peptide and stained with low (Ova) or high (Trp2) concentrations of CFSE, respectively. The cells were then mixed and injected i.v. into the vaccinated mice. After 18 h, splenocytes from the vaccinated mice were analyzed by flow cytometry and enumerated according to a published equation [20]. A representative graph from each group is shown. *p < 0.05, **p < 0.01, ***p< 0.001, n = 3.

Fig. 6

Tumor cytokine levels after vaccination. C57BL/6 mice were inoculated with 2×10⁵ B16F10 cells on day 0. VAC was given on day 14 and SUN treatments were given on days 14, 16, and 18. Mice were sacrificed on day 18, and tumors were collected for cytokine detection using RT-PCR. The results are displayed as mean ± S.D. (error bars). Statistical analyses were calculated by comparison with the untreated group. *p < 0.05, **p < 0.01, *** p< 0.001, n = 5.

Fig. 7

(A) Vessels in B16F10 tumors stained with CD31 antibody (red) to measure vasculature and DiI-labeled PLGA-PEG micelles (false color green) to measure tumor permeability. C57BL/6 mice were inoculated with 2×10⁵ B16F10 cells on day 0. VAC was injected on day 14 at a dose of 0.3 mg/kg; SUN_OS and SUN_b-PM were p.o. or i.v. administered on days 14, 16 and 18 at a dose of 30 mg/kg and 10 mg/kg, respectively. On day 19, the mice were i.v. injected with DiI-loaded PLGA-PEG micelles. Twenty four h after injection, the mice were sacrificed and tumors were harvested. The blood vessels were stained using an anti CD31 antibody; the cell nucleus was stained using DAPI (blue); the PLGA NPs were labeled with DiI. The white scale bar represents 100 μm. The white arrows indicate the thin and elongated vessels; the white arrowheads indicate the round and open vessels. (B) TAFs in tumors stained with α-SMA antibody (red), scale bar = 50 μm; (C) tumor sections stained with Masson’s Trichrome, scale bar = 50 μm. The tumor-bearing mice in (B) and (C) were treated according to the above schedule and sacrificed on day 19. In (C), the blue color represents collagen fibers (yellow arrows). To quantify the fields, three randomly selected microscopic fields were quantitatively analyzed using ImageJ (D, E, F). The results are displayed as mean ± S.D. (error bars). Statistical analyses were calculated by comparing to the untreated group unless otherwise specified. *p<0.05,**p < 0.01, ***p< 0.001, n = 3.

Fig. 8

Expression levels of p-Stat3, Stat3, p-AKT, AKT, and GAPDH in tumor samples after treatment. C57BL/6 mice were inoculated with 2×10⁵ B16F10 cells on day 0. VAC was injected on day 14 at a dose of 0.3 mg/kg; SUN_OS and SUN_b-PM were administered p.o. or i.v. on days 14, 16, and 18 at a dose of 30 mg/kg and 10 mg/kg, respectively. Mice were sacrificed on day 19 and tumors were collected for western blot analysis. The results are displayed as mean ± S.D. (error bars). Statistical analyses were calculated by comparing with the untreated group unless specified with markings. *p < 0.05, **p< 0.01, ***p < 0.001, n = 5.

Scheme 1

Schematic illustration of SUN_b-PM and LCP-Trp2 vaccine injected into the advanced melanoma-bearing mouse.