THE ROLE OF HIGH LET RADIATION TO ACTIVATE AN IMMUNOTHERAPEUTIC RESPONSE UNDER DIFFERENT OXYGEN STATUS

Purpose: Combining ionizing radiation with immune therapy has demonstrated some benefit in cancer treatment, where radiation applied to the primary tumor has resulted in the resolution of distant disease. Currently, the primary hypothesis is that radiation is activating the patient’s immune system to elicit anti-cancer response. However, this out-of-field response, or abscopal effect, very rarely occurs in patients, varies across and within cancer patient groups, and is difficult to reproduce. To date, the underlying biomechanisms remain largely unknown. In this study, we are investigating the potential biomechanisms regulating IFN-beta, an anti-cancer immune biomarker associated with immunological cell death and anit-cancer immune response (biological endpoint), and its subsequent dependency on dose, timing, LET, and oxygenation. In this study, the goal is to explore and compare the relative biological effectiveness of the stimulated immune response (IFN-beta) to low- and high-LET radiation across different polyomavirus-negative Merkel carcinoma cells (MCCs). In addition, changes in IFN-beta production in MCCs under hypoxic conditions are measured when treated with low-LET radiation to investigate how hypoxia affects immune response induction.

Methods: Merkel carcinoma cells MCC13, MCC26, UISO, and MCC14/2 were irradiated with x-rays (low-LET) or fast neutrons (high-LET) ranging from 0-24 Gy or 0-7.5 Gy, respectively. After which IFN-beta protein and gene levels were measured 48-,96- and 144-hours post-irradiation (RT-qPCR, ELISA). In addition, genes upstream of IFN-beta associated with STING-pathway (cGAS, STING, IFI16) and MAVS-pathway (MAVS, RIG-I, MDA5) and their potential modulators (TREX1, PRMT5) were evaluated, as well as micronuclei production (ICC/IF). These experiments were followed by immunofluorescent staining (ICC/IF) to assess the expression of key proteins (IFI16) and micronuclei (DAPI) and their spatial distribution, where these ICC images were accessed and analyzed Matlab-based codes developed in-house. For hypoxia experiments, cells were incubated in 0.1-0.3% oxygen prior to evaluation of their biomechanisms.

Results: The dose response of IFN-beta production per cell increased, peaked, and subsequently decreased with dose escalation, demonstrating a dose window. In MCC13, peak IFN-beta production occurred at 14 Gy for X-rays and 5.5 Gy for fast neutrons, consistent with RBE models of double-stranded break (DSB) induction. Even though the peak fast neutron dose of 5.5 Gy was equivalent to 14 Gy in physical dose, higher concentrations of IFN-beta secretion were observed, indicating that high LET can induce greater immune response. Changes in gene expression showed 100-fold and 15-fold increases in IFI16 and MDA5 for MCC13 at 14 Gy with IFI16 following a similar dose response as IFN-beta. Interestingly, IFI16 and IFN-beta were not upregulated 48 hours post-irradiation when exposed by either x-rays or fast neutrons but showed a dramatic increase at 96 hours with a subsequent decrease at 144 hours. ICC/IF measurements also revealed a shift in the ratio of nuclear-to-cytosol IFI16 protein expression with dose, consistent with IFN-beta and translocation of IFI16 from nucleus to cytosol. This held true under both low- and high-LET.

Unlike MCC13, MCC26 demonstrated a mild immune response after x-ray irradiation (peak IFN-beta production at 14 Gy) and no response was observed after fast neutron irradiation; andMCC14/2 did not demonstrate a measurable response (IFN-beta) to x-rays but did show a small increase at higher physical doses after fast neutron irradiation, suggesting that high-LET might restore immunogenicity. In both these cell lines, IFI16 gene and protein expression remained unchanged with dose. MCC26 exhibited a slight rise in cGAS and MDA5 gene expression at higher x-rays doses, with cGAS responding with a similar dose response as IFN-beta (14 Gy); MCC14/2 measured a slight rise in cGAS and IFI16 for fast neutrons but at a higher dose (7.5 Gy) compared to MCC13, consistent with IFN-beta secretion.

Lastly, we observed suppressed IFN-beta production in MCC13 while under chronic hypoxia. Compared to normoxia, IFN-beta secretions peaked at 15 Gy but production was significantly lower (~7X), with concomitant reduction in IFN-beta gene expression. Trex1 gene expression was also elevated under hypoxia compared to normoxia, which may be an additional factor contributing to lower IFN-beta secretions. Nevertheless, there might be more factors and regulators in the pathway resulting in the suppression from hypoxia.

Conclusion: We have identified an optimal dose for IFN-beta production in Merkel cell lines and compare the effect of high- and low-LET. Furthermore, the temporal response of IFN-beta is significantly impacted by IFI16 and its translocation. Together, these data point to both the dose and fractionation to be important factors in radiation induced immunogenicity and provide new information on how to better translate this novel technique into the clinic.