ABSTRACT
Owing to spatial segregation of tumor subclones, solid tumor sampling using formalin-fixed, paraffin-embedded blocks is often inadequate to represent the genomic heterogeneity of solid tumors. We present an approach, representative sampling, to dissect and homogenize leftover residual surgical tissue prior to sequencing. We also detail optional tumor cell enrichment and DNA preparation. This method, applicable only to surgically removed tumors with leftover tissue, facilitates robust sampling to avoid missing or over-representing actionable variants. For complete details on the use and execution of this protocol, please refer to Litchfield et al. (2020).
Subject(s)
High-Throughput Nucleotide Sequencing/standards , Neoplasms/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , Neoplasms/pathology , Reproducibility of ResultsABSTRACT
Improving vaccine immunogenicity remains a major challenge in the fight against developing country diseases like malaria and AIDS. We describe a novel strategy to identify new DNA vaccine adjuvants. We have screened components of the Toll-like receptor signalling pathways for their ability to activate pro-inflammatory target genes in transient transfection assays and assessed in vivo adjuvant activity by expressing the activators from the DNA backbone of vaccines. We find that a robust increase in the immune response necessitates co-expression of two activators. Accordingly, the combination of tak1 and tram elicits synergistic reporter activation in transient transfection assays. In a mouse model this combination, but not the individual molecules, induced approximately twofold increases in CD8+ T-cell immune responses. These results indicate that optimal immunogenicity may require activation of distinct innate immune signalling pathways. Thus this strategy offers a novel route to the discovery of a new generation of adjuvants.
