Micro-region transcriptomics of fixed human tissue using Pick-Seq

Abstract

Spatial transcriptomics and multiplexed imaging are complementary methods for studying tissue biology and disease. Recently developed spatial transcriptomic methods use fresh-frozen specimens but most diagnostic specimens, clinical trials, and tissue archives rely on formaldehyde-fixed tissue. Here we describe the Pick-Seq method for deep spatial transcriptional profiling of fixed tissue. Pick-Seq is a form of micro-region sequencing in which small regions of tissue, containing 5-20 cells, are mechanically isolated on a microscope and then sequenced. We demonstrate the use of Pick-Seq with several different fixed and frozen human specimens. Application of Pick-Seq to a human melanoma with complex histology reveals significant differences in transcriptional programs associated with tumor invasion, proliferation, and immuno-editing. Parallel imaging confirms changes in immuno-phenotypes and cancer cell states. This work demonstrates the ability of Pick-Seq to generate deep spatial transcriptomic data from fixed and archival tissue with multiplexed imaging in parallel.

The spatial landscape of progression and immunoediting in primary melanoma at single cell resolution

“…x 110 µm, selected to provide details of cellular interactions in regions of tumor regression, MIS, and invasion (Figure 1C and S1D). Micro-region transcriptomics (mrSEQ) was performed on a total of 292 mROIs using PickSeq, which recovers smaller regions of tissue, and GeoMX, which is commercially available and samples larger tissue regions (Maliga et al, 2021;Zollinger et al, 2020); Key findings were concordant between the two methods, increasing confidence in these recently developed approaches.…”

“…It is made 1B and S2A). PickSeq recovered 5-20 cells per 40 µm mROI and transcript profiling was performed using cDNA sequencing (Maliga et al, 2021); GeoMX recovered ~200 cells per ~200 µm mROI and transcript profiling was performed by hybridization to a panel of ~1,800 cancer-related genes (Zollinger et al, 2020). Principal Component Analysis (PCA) of the resulting data revealed three primary clusters corresponding to (i) MIS, (ii) malignant tumor (EM plus IM), and (iii) regions of active immune response (IR -which was adjacent to the MIS or a bTIL region adjacent to the invasive boundary of the nodular melanoma) (Figure 2A).…”

“…PickSeq is a method by which 40 µm mROIs of interest is physically extracted using a robotic arm followed by mRNA extraction and RNA sequencing (Maliga et al, 2021). 222 ROIs representing five morphologically distinct sites (MIS, IM, IB, bTIL, EM; Figure S2A) were selected for collection and library preparation.…”

“…We recommend the introductory MINERVA story HTA MEL Atlas 1: Introduction to the MEL Atlas. One of the "discoveries" in Nirmal et al (2021) is the identification of large scale contiguous graded expression patterns displayed by key melanoma markers and distinct localization of specific tumor-cell states within the tumor, both of which were discernable only with whole-slide imaging (see Figure 3, 4). Whole slide imaging is also an FDA requirement for any diagnostic application.…”

“…The spatial organization of the tumor microenvironment (TME) was analyzed using both conventional and high-resolution (wide-field deconvolution) 20 to 30-plex fluorescence microscopy (CyCIF) (Lin et al, 2018) in combination with transcriptional profiling of selected micro-regions using micro-region transcriptomics (mrSEQ: GeoMx and PickSeq) (Demirkan et al, 2020;Maliga et al, 2021). The resulting molecular and morphological data were then analyzed with respect to histopathology annotations.…”