Pick-Seq®: a novel technology to retrieve tissue micro-regions for RNA sequencing


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Rebecca Podyminogin1,Nolan Ericson1,Jennifer Chow1,Jia-Ren Lin2,Yu-An Chen2,Zoltan Maliga2,Kyla Teplitz1,Tessa Pei-Ching Tsai1,Adrian Quintanilla1,Peter Sorger2,Eric Kaldjian1,Tad George1
1RareCyte, Seattle, US,2Harvard Medical School, Cambridge, US

Abstract

Background

Tumour tissue imaging allows for contextual understanding of tumour cells in the immune microenvironment. Pick-Seq combines high-resolution multi-parameter imaging with micro-region retrieval for RNA sequencing.

Method

Frozen breast carcinoma and formalin-fixed, paraffin-embedded tonsil sections were stained with immunofluorescence (IF) for cytokeratin and B and T cell markers. CyteFinder® Imaging System performed whole-slide scanning. 40μm diameter micro-regions were retrieved with CytePicker® Retrieval Module. RNA was amplified, sequenced, and differential gene expression analysis was performed. Identified genes were selected for Pick-Seq-informed IF panels. Cell compositions of each micro-region were deconvolved with CIBERSORT.

Results

Breast cancer micro-regions containing T cells, tumour cells, and tumour-infiltrating lymphocytes were retrieved for RNA sequencing. CIBERSORT showed an association between T-cell quantity observed by IF imaging and relative abundance of T-cell RNA. Tonsil micro-regions from one T-cell zone and two adjacent follicles were retrieved for RNA sequencing. Hierarchical clustering of differentially expressed genes confirmed B-cell markers in follicles and T-cell markers in the T-cell zone. CIBERSORT revealed distinct cellular compositions between T-cell zones and the B-cell follicles. Principle component analysis of gene expression found that micro-regions picked from the two follicles clustered independently from each other, and from the T-cell zone micro-regions. Fragments per kilobase million analysis revealed differing expression of genes between the adjacent follicles. Staining confirmed differential protein expression, indicating that only one follicle contained a germinal center in the section.

Conclusion

We demonstrate high-resolution multi-parameter imaging of both frozen and FFPE sections for retrieval of micro-regions for transcriptomic analysis using Pick-Seq. RNA expression analysis differentiated the tumour and T cell micro-regions in breast carcinoma, and between follicle and T-cell areas in tonsil and uncovered differences between adjacent follicles that were confirmed by IF imaging. These data demonstrate the power of Pick-Seq as a tool for spatially-focused RNA sequencing and biomarker discovery.