Dependence of Immunogenic Modulation of Tumor Cells by Proton Radiation on the Linear Energy Transfer

Abstract

Radiation therapy has been shown to induce immune-mediated tumor regression by multiple mechanisms. One of the mechanisms is to trigger an immunogenic phenotype characterized by upregulation of cell surface immune co-stimulators, antigen presenting machinery and checkpoint molecules that primes cells for subsequent elimination by cytotoxic T cells. The LET of the proton beam varies with the depth of dose deposition. Prior studies comparing proton radiation to photon radiation have failed to identify a stronger immunogenic phenotype with protons. However, the linear energy transfer (LET) was comparable for both modalities. We asked whether immunogenic phenotype induction would be different with higher LET, one that is typically noted towards the end of the Bragg peak of protons. Murine CT26 colorectal cancer cells were irradiated with 8Gy proton beams at 1.09keV/um (low), 2.58keV/um (medium) and 7.7keV/um (high) LET. 48hours after irradiation the cells were stained for surface expression of OX40L, ICOSL, CD40, CD70, 41BBL (immune co-stimulators), MHC-I, Calreticulin (antigen presenting machinery), ICAM-1 (cell adhesion molecule), PDL-1, CTLA-4 (immune checkpoint molecules) using a multiplexed flow staining panel. The treated cells were also plated on 8 well glass chamber slides and immunofluorescence (IF) staining for 41BBL, OX40L, CD40, MHC-1 and calreticulin was evaluated. Freshly extracted mouse splenocytes were used as biological positive control for both flow cytometry and immunofluorescence. We observed increase in percentage expression of OX40L, CD40, ICAM-1 and MHC-I, and drop in PDL-1 expression in the cells irradiated in the high LET region compared to low LET region of the proton beam as shown in the following table.Abstract 3444Treatment groupsMarker % expressionControl8Gy Low LET8Gy Medium LET8Gy High LETOX40L0.70.2360.6187.79CD4073.1383.292.5596.73MHC-I91.1195.1195.998.62ICAM-13.125.660.9627.42PDL-110.3256.4650.2138.36 Open table in a new tab This data is the average of independent experiments performed under same conditions. The other immune markers did not show statistically significant difference between the low and high LET proton radiation although the expression increased compared to the untreated controls. There was increased expression of 41BBL, OX40L, CD40, Calreticulin and MHC-I in the high LET group compared to low LET on immunofluorescence staining, which was assessed qualitatively. High LET proton radiation can be used to stimulate better immunogenic phenotype in tumor cells compared to low LET proton radiation. This preliminary data will form the foundation for future in vivo testing on animal models, to see if high LET proton therapy improves the local control and elicits a stronger systemic immune response compared to low LET radiation.