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Immune checkpoint blockade (ICB) targeting the programmed cell death protein 1 (PD-1) and its ligand 1 (PD-L1) fails to provide clinical benefit for most cancer patients due to primary or acquired resistance. Drivers of ICB resistance include tumor antigen processing/presentation machinery (APM) and IFNγ signaling mutations. Thus, there is an unmet clinical need to develop alternative therapies for these patients. To this end, we have developed a CRISPR/Cas9 approach to generate murine tumor models refractory to PD-1/-L1 inhibition due to APM/IFNγ signaling mutations. Guide RNAs were employed to delete B2m, Jak1, or Psmb9 genes in ICB-responsive EMT6 murine tumor cells. B2m was deleted in ICB-responsive MC38 murine colon cancer cells. We report a detailed development and validation workflow including whole exome and Sanger sequencing, western blotting, and flow cytometry to assess target gene deletion. Tumor response to ICB and immune effects of gene deletion were assessed in syngeneic mice. This workflow can help accelerate the discovery and development of alternative therapies and a deeper understanding of the immune consequences of tumor mutations, with potential clinical implications.

There is an increasing interest in using microRNAs (miRNA) as biomarkers in autoimmune diseases. They are easily accessible in many body fluids but it is controversial if they are circulating freely or are encapsulated in microvesicles, particularly exosomes. We investigated if the majority of miRNas in serum and saliva are free-circulating or concentrated in exosomes. Exosomes were isolated by ultracentrifugation from fresh and frozen human serum and saliva. The amount of selected miRNAs extracted from the exosomal pellet and the exosome-depleted serum and saliva was compared by quantitative RT-PCR. Some miRNAs tested are ubiquitously expressed, others were previously reported as biomarkers. We included miRNAs previously reported to be free circulating and some thought to be exosome specific. The purity of exosome fraction was confirmed by electronmicroscopy and western blot. The concentration of miRNAs was consistently higher in the exosome pellet compared to the exosome-depleted supernatant. We obtained the same results using an equal volume or equal amount of total RNA as input of the RT-qPCR. The concentration of miRNA in whole, unfractionated serum, was between the exosomal pellet and the exosome-depleted supernatant. Selected miRNAs, which were detectable in exosomes, were undetectable in whole serum and the exosome-depleted supernantant. Exosome isolation improves the sensitivity of miRNA amplification from human biologic fluids. Exosomal miRNA should be the starting point for early biomarker studies to reduce the probability of false negative results involving low abundance miRNAs that may be missed by using unfractionated serum or saliva.