Project description:Neoadjuvant Immune Checkpoint Blockade Before Chemoradiation for Patients with FIGO Stage IB3–IVA Cervical Squamous Carcinoma: GINECO Window-Of-Opportunity COLIBRI Study
Project description:Combining immune checkpoint blockade (ICB) with chemoradiation is effective in locally advanced cervical cancer. However, the impact of dual ICB before any therapy on immune modulation and treatment response is poorly understood.
Project description:In our study, we examined tissue samples from 20 cattle that had been experimentally infected with the FMDV strain O/FRA/1/2001. In 18 of these animals, the infection persisted for more than 28 days. At necropsy, epithelial tissue was collected from the dorsal nasopharynx and dorsal soft palate, the primary sites of persistent infection. Five biological replicates were taken from each location in each animal and analyzed using FMDV-specific RT-qPCR. A subset of these samples was then selected for transcriptome sequencing. Samples were disintegrated using a CP02 cryoPREP (Covaris) and mixed with AL buffer (Qiagen) and TRIzol LS (Invitrogen). After biocontainment removal, trichloromethane was added, and the mixture was centrifuged. About 400 µl of the RNA-containing upper aqueous phase was extracted using the Agencourt RNAdvance Tissue Kit (Beckman Coulter) with a KingFisher Flex processor (Thermo Fisher Scientific). RNA quantity and quality were measured with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). For transcriptomic analysis, 52 samples were selected, including 25 DNP and 23 DSP samples from 14 persistently infected animals and two that cleared the infection before 21 dpc. mRNA was isolated using the Dynabeads mRNA DIRECT Micro Purification Kit (Invitrogen), and libraries were prepared with the Colibri Stranded RNA Library Prep Kit (Invitrogen). ERCC internal control was added before mRNA extraction. The isolated mRNA was fragmented to ~150 nucleotides, adapters were ligated, and the RNA was transcribed into cDNA, which was amplified and indexed for Illumina libraries. The libraries' length and quality were assessed with an Agilent 4150 TapeStation and quantified using Qubit 2.0. Sequencing was performed on a NovaSeq machine in 100 bp single-end mode.
