NBTXR3 Nanoparticle With Immunoradiation Might Reshape Metastatic Tumor-Infiltrating T Cell Repertoire in Murine Lung Cancer Model.

Abstract

PURPOSE/OBJECTIVE(S)\nRadiation therapy (XRT) induces lymphocyte infiltration of primary tumors that is increased when using NBTXR3 radioenhancer nanoparticle, leading to improved response to combination immunotherapy (IT). However, treatment effects on the metastatic tumor microenvironment are poorly understood. We studied the effects of NBTXR3 injection into primary tumors plus XRT plus dual-agent immunotherapy on secondary tumor-infiltrating T cells in a mouse model of lung cancer. The hypothesis is that intratumoral primary tumor treatment with NBTXR3 nanoparticle enriches conserved T cell receptor repertoires among metastatic tumor-infiltrating lymphocytes.\n\n\nMATERIALS/METHODS\nFour groups of 4 mice each were inoculated subcutaneously with anti-PD1-resistant 344SQR murine lung cancer cells in each hind leg, 3 days apart, to establish primary (right) and secondary (left, which represented metastasis) tumors. All mice received intraperitoneal anti-PD1 and anti-CTLA-4 (IT) on days 4, 7, 10, and 13, and 12-Gy high-dose XRT (HD) to the primary tumors on days 7, 8 and 9. Primary tumors in groups 2 and 4 also received intratumoral NBTXR3 on day 6. Secondary tumors in groups 3 and 4 were irradiated with 1-Gy low-dose XRT (LD) on days 12 and 13. Treatment groups were designated as 1\u202f=\u202fHD+IT, 2\u202f=\u202fNBTXR3+HD+IT, 3\u202f=\u202fHD+LD+IT, and 4\u202f=\u202fNBTXR3+HD+LD+IT. All secondary tumors were harvested on day 19 for tumor-infiltrating T-cell RNA extraction and sequencing on an Illumina MiSeq. T-cell receptor (TCR) beta complementarity-determining region 3 (CDR3b) repertoires were analyzed with MiXCR v3.0.12 software, and CDR3b analyses and Circos plots were generated using software. P < 0.05 from Mann-Whitney U tests were considered statistically significant.\n\n\nRESULTS\nNBTXR3 significantly increased the proportion of conserved CDR3b clonotypes within the secondary tumors among mice that received only HD XRT to the primary tumors (group 2 vs 1, P\u202f=\u202f0.0476). When the secondary tumors were exposed to LD XRT, NBTXR3 still increased the frequency of overlapping clonotypes although not significantly (group 4 vs 3, P\u202f=\u202f0.3095). Most interestingly, there were significantly more conserved CDR3b repertoires from the secondary tumors that were shared between groups 2 and 4 that received NBTXR3 compared to those shared between groups 1 and 3 (P < 0.0001). Comparisons of CDR3b normalized Shannon clonality indices and Circos plots did not reveal any difference in repertoire diversity between NBTXR3-treated and non-NBTXR3-treated groups.\n\n\nCONCLUSION\nNBTXR3 radioenhancer nanoparticle, given with immunoradiation, might reshape the TCR repertoire of metastatic lung cancer tumor-infiltrating T cells by driving the selection of conserved CDR3b clonotypes.