Melanoma Cell Reprogramming and Awakening of Antitumor Immunity as a Fingerprint of Hyper-Harmonized Hydroxylated Fullerene Water Complex (3HFWC) and Hyperpolarized Light Application In Vivo

Abstract

In our recent study, we showed that in vitro treatment of melanoma cells with hyperpolarized light (HPL) as well as with the second derivative of fullerene, hyper-harmonized hydroxylated fullerene water complex (3HFWC) reduced viability of cells by decreasing their proliferative capacity and inducing senescence and reprogramming towards a normal, melanocytic phenotype. Therefore, we wanted to determine whether these effects persisted in vivo in the syngeneic mouse melanoma model with a combined treatment of HPL irradiation and 3HFWC per os. Our results demonstrated the potent antitumor effects of 3HFWC nanosubstance assisted by HPL irradiation. These effects were primarily driven by the stimulation of melanoma cell growth arrest, the establishment of a senescent phenotype, and melanocytic differentiation on the one hand, and the awakening of the antitumor immune response on the other. In addition, the combined treatment reduced the protumorigenic activity of immune cells by depleting T regulatory cells, myeloid-derived suppressors, and M2 macrophages. The support of the 3HFWC substance by HPL irradiation may be the axis of the new approach design based on tumor cell reprogramming synchronized with the mobilization of the host's protective immune response.

Keywords: differentiation; hyperpolarized light; melanoma; second derivative of C60; senescence.

Figure 1

In vivo treatment with HPL, 3HFWC, or 3HFWC+HPL reduced B16 melanoma growth. Mouse melanoma B16 cells (2 × 10⁵) were inoculated s.c. into C57BL/6 mice (day 0). Five days post-implantation, mice were randomized into groups with the same average tumor volume (n = 6–7) and treated with 3HFWC, HPL, and with their combination. Controls were left untreated. Shown are (A) growth curves depicting average tumor volumes; (B) individual tumor volumes at the end (day 13) of a representative experiment; (C) mean body weight, and (D) average daily liquid (water of 3HFWC) uptake of each group. Data are presented as mean ± SEM (A,C,D). Statistical significance (p value) in comparison with the control group, Mann–Whitney test.

Figure 2

Inhibition of melanoma cells proliferation in vivo by HPL, 3HFWC, and HPL+3HFWC treatments, as demonstrated by (A) PCNA immunoexpression (green) in melanoma tissue, red signal—propidium iodide nuclear staining (scale bar and orig. magnification—25 μm, × 63); (B) PCNA nuclear immunopositivity levels (0/+ no/weak immunopositivity; ++/+++ medium/strong immunopositivity of nuclei); and (C) mitotic index in melanoma tissue. Mean values ± SEM; statistical significance in comparison to melanoma tissue of control animals (* p value) and between the treated groups.

Figure 3

Alterations of melanin pigmentation in melanoma tissue in vivo induced by HPL, 3HFWC or 3HFWC+HPL treatment. (A) Representative HE micrographs of melanoma tissue (scale bar and orig. magnification—50 μm, × 40); (B) quantification of melanin pigmentation of melanoma cells of untreated (control), HPL-, 3HFWC- and 3HFWC+HPL-treated animals; results presented as min to max values, with median; statistical significance in comparison to control (* p value) and between the treated groups.

Figure 4

Histological signs of cellular senescence in melanoma in vivo induced by HPL, 3HFWC, or 3HFWC+HPL treatment. (A) Representative SBB-stained micrographs of melanoma tissue with lipofuscin-loaded cells (*) (scale bar and orig. magnification—20 μm, × 100); (B) nuclear surface area of melanoma cells; (C) volume density of necrotic area in the tissue of untreated (control), HPL-, 3HFWC- and 3HFWC+HPL-treated animals, presented as mean ± SEM; statistical significance: in comparison to control (* p value) and between the treated groups.

Figure 5

Flow cytometric characterisation of immune cells infiltrating B16 tumors after HPL, 3HFWC, or 3HFWC+HPL co-treatment. Tumors and tumor-draining lymph (tdLN) nodes were harvested 13 days after the inoculation from B16 melanoma-bearing mice treated as in Figure 1. Tumor-infiltrating immune cells and immune cells from tdLN nodes isolated from the indicated groups of mice were subjected to flow cytometry analysis. Numbers in graphs represent the percentage of cells in the different gates: tumor-infiltrating CD8⁺ T cells (CD3⁺CD8⁺, (A), T_reg cells (CD4⁺CD25⁺FoxP3⁺; (B), MDSCs (Gr-1⁺CD11⁺; (C), M2 macrophages (CD206⁺; (D) and CD8⁺ T cells (E) and NK cells (NK1.1⁺CD3⁻; (F) from tdLN. Data are presented as mean ± SEM and compared with the control group, Mann-Whitney test.

Figure 6

Evaluation of prophylactic potential, and combined prophylactic and therapeutic potential of 3HFWC to suppress B16 growth in vivo. B16 mouse melanoma cells (2 × 10⁵) were s.c. inoculated into C57BL/6 mice on day 0. Mice were treated with 3HFWC from day −7 till day 0 (3HFWC prophylaxis), from day −7 till the end of the experiment (3HFWC prophylaxis + treatment) or were left untreated (control). (A) Growth curves depicting average tumor volumes; (B) individual tumor volumes at the end (day 21) of the representative experiment; (C) mean body weight, and (D) average daily liquid (water of aqueous 3HFWC solution) uptake of each group. Data are presented as mean ± SEM and compared with the control group, Mann–Whitney test.