Acute remote ischemic conditioning enhances (CD3+)- but not (FoxP3+)-T-cell invasion in the tumor center and increases IL 17 and TNF-alpha expression in a murine melanoma model

Abstract

Introduction: Hypoxia can drive tumor progression, suppress anti-tumor immunity, and reduce the effectiveness of radiotherapy and chemotherapy. This study aimed to assess the impact of remote ischemic conditioning (RIC) on tumor oxygenation (sO2) and the anti-tumor immune response.

Material and methods: Fourteen B16-Ova tumor-bearing C57BL/6N mice received six 5-minute RIC cycles, while another fourteen underwent anesthesia only. Pimonidazole was administered 1.5 hours before sacrifice. Blood flow, sO2, and hemoglobin levels were measured in the non-ischemic hind limb and tumor. Tumor hypoxia was assessed using pimonidazole and CA IX immunohistochemistry, and T cell infiltration by CD3 and FoxP3 staining. Serum levels of 23 cytokines were analyzed using a multiplex immunoassay.

Results: Isoflurane anesthesia caused a slight intraindividual increase in blood flow (p = 0.07) and sO₂ (p = 0.06) of the hind limb and a sole increase in tumor sO₂ (p = 0.035), whereas RIC improved sO₂ of the tumor in relation to the hind limb (p=0.03). The median of the tumor oxygen saturation reached 51.4% in the control group and 62.7% in the RIC group (p = 0.09), exhibiting a slight tendency towards better oxygenation in the RIC group. Pimonidazole (p=0.24) and CA IX hypoxia score (p=0.48) did not reveal statistically significant differences between the two groups. In RIC-treated tumors, the number of CD3 (p=0.006), but not FoxP3- positive cells (p = 0.84), in the tumor core was significantly higher compared to the control group. In the RIC group, the mean fluorescence intensity (MFI) of IL-17 was significantly higher (p=0.035), and TNF-α was trend-wise higher (p=0.063) compared to the control group.

Conclusion: Both isoflurane anesthesia and RIC have an impact on microcirculation. The application of RIC counteracted some of the effects of isoflurane, primarily in healthy tissue, and led to a significant improvement in relative tumor tissue oxygenation compared to the non-ischemic hind limb. RIC selectively enhanced immune infiltration within the tumor center, probably by previously activated tumor infiltrating T cells, while having no significant impact on T-regulatory cells. RIC appears to impact the cytokine profile, as indicated by elevated MFIs of TNF-α and IL-17.

Keywords: hypoxia; immunomodulation; melanoma (B16); remote ischemic conditioning (RIC); tumor perfusion.