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e14660 Background: Combining laser capture microdissection (LCM) with proteogenomics in cancer research offers a targeted way to way to explore the intricate interactions between the tumor microenvironment and tumor cells proper. For immuno-oncology (IO) research, this is especially important, since improvements in the predictability of patient response to IO-based drugs is greatly needed. An improved understanding of the spatial relationships involving tumor cells, stromal cells, blood supply and immune cell interactions may help to inform drug development and improve the indicators of success for IO-based drug regimens. Methods: From fresh frozen tissue, LCM is used to isolate and obtain distinct histological cell types. Nucleic acids and proteins are extracted from captured cells for in-depth multi-modality molecular profiling assays. This includes multiple genomic modalities and a customized solution-based mass spectrometry proteomics approach for samples or specimens with a limited amount of tissue. Results: Optimizing the analysis of minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, DNA sequencing can be performed for the discovery and analysis of actionable mutations, copy number variation, and methylation profiles. RNA-seq may be performed for gene expression. Regarding genomic-based studies, commercial kits are often utilized for these steps due to their general availability, rapid innovation, and the robust performance of these products. However, this kit-based adaptive approach is not true for proteomics. There remains a need for highly sensitive proteomic methods targeting small-sized samples. Our novel proteogenomics approach is focused around a customized enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. This includes an in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Conclusions: A proteogenomic approach is reported that is utilizing amount limited tissue quantities from microdissected fresh frozen tissue. The methodology may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.
Johann et al. (Sat,) studied this question.