Immuno-Target Selection Of Infiltrating Immune Cells And Laser Capture Microdissection Mediated Transcriptional Profiling

Immune checkpoints define an evolving class of inhibitory mediators that are expressed by tumour cells and infiltrating leucocytes to down-modulate the effector immune response against tumour cells. Therefore, the most recent efforts in cancer therapy centre on targeting immune-mediated mechanisms of tumour evasion. The ability to assess gene expression profiles from isolated populations of tumour-infiltrating lymphocytes (TILs) can assist in monitoring checkpoint inhibitor therapy efficacy and provide a better understanding of clinical outcomes. Ideally, transcriptional profiles should be obtained from isolated cell populations of interest, free from contaminating cell populations that may create adverse background noise. Micro sampling by laser capture microdissection is a powerful tool that allows for specific analysis from whole tissues, particularly in a heterogeneous microenvironment such as in a tumour, where less well represented target cells are dwarfed in abundance by tissue stroma and other cell types. However, whilst the application is potentially powerful for target discovery and mechanistic understanding, the process is notoriously difficult, particularly in clinical specimens. We developed a methodology to successfully overcome these challenges in analysing target cells, organelles or other tissue subsets by transcriptional profiling following immunostain-mediated laser capture microscopy (LCM). Thorough optimisation of traditional immunohistochemistry techniques enabled us to select target cell populations of interest from tissue sections whilst minimising degradation of mRNA and miRNA allowing us to perform genomic and pathway interrogation using microarray and/or RT-qPCR analysis. Our technique allows multiple capture types per slide, sample pooling when required, high capture throughput and capture image documentation. Multiple target selection, to differentiate lymphocytes, can be performed routinely using comparative serial sectioning. Here we exhibit simple staining and immunostaining histology images using methods sympathetic to RNA integrity which allow for target selection by specific staining or target morphology, and demonstrate comparative analysis of matched pairs of disease and healthy sections of colon tumours. We used a Palm MicroBeam 4 LCM platform to identify, cut and specifically capture CD8+ tumour-infiltrating lymphocytes from frozen embedded tissue. This approach enabled us to isolate discrete targeted cell populations free from contaminating cell populations. Epistem’s Single Cell RNA-Amp™ was then used to provide robust and linear amplification of RNA and enable comparative analysis in applications such as target discovery and pharmacodynamics. The results indicated that transcriptional profiling was technically robust with a good replicate correlation despite the limited input obtained by micro-sampling. Citation Format: Ross Haggart, Chaxiraxi Arzola-Donate, Elliott Harrison, Benjamin J. Reed, Saba Alzabin, Gino Miele. Immuno-target selection of infiltrating immune cells and laser capture microdissection mediated transcriptional profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1677. doi:10.1158/1538-7445.AM2017-1677