Illumina NovaSeq 6000 paired end sequencing; Evading immune evasion via reprograming of the tumor microenvironment by radiotherapy and simultaneous targeting of TGF-β/PD-L1
Evading immune evasion via reprograming of the tumor microenvironment by radiotherapy and simultaneous targeting of TGF-β/PD-L1
C57BL/6J mice were used in this experiment. Dissociation according to Miltenyi mouse lung dissociation kit (https://www.miltenyibiotec.com/DE-en/products/lung-dissociation-kit-mouse.html#gref). The Miltenyi proprietary protocol of the kit was strictly followed. Debris were removed using the debris removal solution by Miltenyi, following strictly the Miltenyi protocol (https://www.miltenyibiotec.com/DE-en/products/debris-removal-solution.html#gref). No particular cell types were enriched or filtered out. There were 2-3 mice per experimental group, which were pooled for each sample. For all studies, mice were randomized into treatment groups according to tumor volume on the day of treatment initiation (day 0). Bintrafusp alfa, anti–PD-L1, TGF-β trap, and isotype control  Bintrafusp alfa (BA, previously reported as M7824) is a bifunctional fusion protein composed of the extracellular domain of the TGF-βRII to function as a TGF-β “trap” fused to a human IgG1 antibody blocking PD-L1. The isotype control is a mutated version of anti–PD-L1, which completely lacks PD-L1 binding.  In tumor-bearing mice, BA (492 μg) or isotype control (400 μg) were administered with an intravenous injection (i.v.) in 0.2 mL PBS. Mice were treated with 15 Gy RT and, 16 weeks later, treated with Bintrafusp (492 μg) or isotype control (400 μg) on day 0, 3, and 6.  Radiation (RT) To assess the combination of radiation with Bintrafusp (BA), mice were randomized into the following treatment groups: isotype control (400 μg), RT (exact dose and schedule provided in the figure legends) + isotype control, BA (492 μg), or BA + RT (BART). Mice were treated with 15 Gy RT 16 weeks prior to BA or isotype control. Whole thoracic irradiation was administered. Briefly, a photon beam was delivered by a 6 MV Artiste Linac (Siemens) at a dose rate of 3 Gy/min in anesthetized mice. The LINAC generates a (6 MV) x-ray photon beam with a dose rate of 3 Gy/min.  According to 10x genomics proprietary protocol of the kit According to 10x genomics proprietary protocol of the kit: https://support.10xgenomics.com/single-cell-gene-expression/library-prep/doc/user-guide-chromium-single-cell-3-reagent-kits-user-guide-v2-chemistry) page31ff