Defeating Melanoma Through a Nano-Enabled Revision of Hypoxic and Immunosuppressive Tumor Microenvironment

Abstract

Rationale: Reversing the hypoxic and immunosuppressive tumor microenvironment (TME) is crucial for treating malignant melanoma. Seeking a robust platform for the effective reversion of hypoxic and immunosuppressive TME may be an excellent solution to revolutionizing the current landscape of malignant melanoma treatment. Here, we demonstrated a transdermal and intravenous dual-administration paradigm. A tailor-made Ato/cabo@PEG-TK-PLGA NPs were administrated transdermally to melanoma with the help of a gel spray containing a skin-penetrating material borneol. Nanoparticles encased Ato and cabo were released and thereby reversed the hypoxic and immunosuppressive tumor microenvironment (TME).

Methods: Ato/cabo@PEG-TK-PLGA NPs were synthesized through a self-assembly emulsion process, and the transdermal ability was assessed using Franz diffusion cell assembly. The inhibition effect on cell respiration was measured by OCR, ATP, and pO₂ detection and in vivo photoacoustic (PA) imaging. The reversing of the immunosuppressive was detected through flow cytometry analysis of MDSCs and T cells. At last, the in vivo anti-tumor efficacy and histopathology, immunohistochemical analysis and safety detection were performed using tumor-bearing mice.

Results: The transdermally administrated Ato/cabo@PEG-TK-PLGA NPs successfully spread to the skin surface of melanoma and then entered deep inside the tumor with the help of a gel spray and a skin puncturing material borneol. Atovaquone (Ato, a mitochondrial-respiration inhibitor) and cabozantinib (cabo, a MDSCs eliminator) were concurrently released in response to the intratumorally overexpressed H₂O₂. The released Ato and cabo respectively reversed the hypoxic and immunosuppressive TME. The reversed hypoxic TME offered sufficient O₂ for the intravenously administrated indocyanine green (ICG, an FDA-approved photosensitizer) to produce adequate amount of ROS. In contrast, the reversed immunosuppressive TME conferred amplified systemic immune responses.

Conclusion: Taken together, we developed a transdermal and intravenous dual-administration paradigm, which effectively reversed the hypoxic and immunosuppressive tumor microenvironment in the treatment of the malignant melanoma. We believe our study will open a new path for the effective elimination of the primary tumors and the real-time control of tumor metastasis.

Keywords: hypoxia; immunosuppression; melanoma; transdermal administration; tumor microenvironment.

Scheme 1

A scheme illustrating the action mechanism of reversing the hypoxic and immunosuppressive TME with a transdermal and intravenous dual-administration paradigm for treating malignant melanoma. (A) Schematic showing the gel sprayed on the skin which containing Ato/cabo@PEG-TK-PLGA nanoparticles. ROS-responsible linkage triggered the release of Ato and cabo, which inhibit mitochondria-associated OXPHOS and MDSCs’ effect on T cells. (B) Schematic showing the PDT effect could be further enhanced by increasing tumor oxygen content and blocking immunosuppressive after transdermal absorption. What’s more, T cells were increased, which developed an effective immunogenic treatment against lung metastasis.

Figure 1

Characteristics of Ato/cabo@PEG-TK-PLGA NPs. TEM images of (A) PEG-TK-PLGA NPs and (B) Ato/cabo@PEG-TK-PLGA NPs. (C) DLS of PEG-TK-PLGA NPs and Ato/cabo@PEG-TK-PLGA NPs. (D) UV-VIS-NIR of PEG-TK-PLGA, Ato, cabo, and Ato/cabo@PEG-TK-PLGA NPs. Solvent: dichloromethane for Ato group and cabo; H₂O₂ for others. Release behaviors of (E) Ato and (F) cabo from Ato/cabo@PEG-TK-PLGA NPs treated with or without H₂O₂.

Figure 2

Transdermal administration of NPs. (A) Transdermal ability of nanoparticles test by Franz diffusion cell. (B) Laser confocal microscope fluorescence image and (C) Nile red transdermal curve of permeate fluorescence labeled nanoparticles. Scale bars: 100 μm. (**P < 0.01, ****P < 0.0001, n = 3).

Figure 3

The revision of hypoxic TME. (A-C) Cell respiratory depression was evaluated via the relative OCR (A); the relative ATP content (B); pO₂ (C) of B16F10 cells with indicated treatments. (D) Photoacoustic images were showing the ratio of oxygenated hemoglobin to deoxygenated hemoglobin in tumors after indicated treatments. (E) HIF-staining images showing the hypoxia degree in tumors after indicated treatments. Scale bars: 50 μm. (****P < 0.0001 compared with control group, n = 6) (^####P < 0.0001, compared with other groups, n = 6).

Figure 4

The revision of MDSC-mediated T cell suppression in tumor-bearing mice. Amount of MDSCs with different treatment in blood (A) and spleen (B). Differentiation markers of MDSCs in blood (C-F) and spleen (G-J). The two subsets of T cell in blood (K and L) and spleen (M and N) during treatment. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with control group, n = 6) (^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001, compared with other groups, n = 6).

Figure 5

In vivo tumor inhibition of different treatment against melanoma. (A) Schematic illustration of treatment plan (Tumor model: B16F10 right flank inoculation only; Lung metastatic model: B16F10 inoculated on right flank and also tail vein injection). (B) Photographs of tumors obtained after treatment. (C) Variation of tumor during different treatment. (D) Relative average body weight of mice after treatment. (E) Tumor weight of different treated group during treatment. Representative TUNEL (F) and Ki67 (G) immunohistochemical test of tumor slices after different treatment. Scale bars: 50 μm. (*P < 0.05, **P < 0.01, ****P < 0.0001 compared with control group, n = 6) (^#P < 0.05, ^###P < 0.001 compared with other groups, n = 6).

Figure 6

The efficiency of immunogenic treatment against lung metastasis. (A) Photographs of lungs obtained after treatment. (B)The number of melanoma spots on lungs during different treatments. Representative histology H&E (C) and PCNA staining (D) images of lung slices after different treatment. Scale bars: 100 μm. (****P < 0.0001 compared with control group, n = 4) (^###P < 0.001 compared with other groups, n = 4).

Figure 7

Long-term in-vivo safety (A) H&E staining of major organs after different treatments (Scale bars: 100 μm). (B) The variation of liver and kidney function showed safety after indicated treatment.