Live slow-frozen human tumor tissues viable for 2D, 3D, ex vivo cultures and single-cell RNAseq

Abstract

Biobanking of surplus human healthy and disease-derived tissues is essential for diagnostics and translational research. An enormous amount of formalin-fixed and paraffin-embedded (FFPE), Tissue-Tek OCT embedded or snap-frozen tissues are preserved in many biobanks worldwide and have been the basis of translational studies. However, their usage is limited to assays that do not require viable cells. The access to intact and viable human material is a prerequisite for translational validation of basic research, for novel therapeutic target discovery, and functional testing. Here we show that surplus tissues from multiple solid human cancers directly slow-frozen after resection can subsequently be used for different types of methods including the establishment of 2D, 3D, and ex vivo cultures as well as single-cell RNA sequencing with similar results when compared to freshly analyzed material.

Fig. 1. Schematic representation of the project workflow.

Cancer tissue was either cut into small tumors (BCC, CRC) or collected as shave biopsies (BCC). The small tumor pieces were either immideately digested to single-cell suspension (BCC, CRC) (F) or slow-frozen (BCC, CRC) (S) and subsequently used for 2D and 3D culture establishment and scRNAseq. Shave biopsies were either used fresh (F) or slow-frozen (S) and used to establish ex vivo cultures. Moreover, melanoma FNAs were either slow-frozen or directly processed for scRNAseq. (Created with BioRender.com).

Fig. 2. 2D, 3D, and ex vivo culture from fresh and slow-frozen matching tumor samples.

a Percentage of successfully established melanoma cell lines from fresh or slow-frozen biopsies. b Representative bright-field micrographs of a successful melanoma cell line and a cell line contaminated with non-melanoma cells. Scale Bar 100 μm and 30 μm (insert). c Microscopic photographs of matched colon carcinoma primary parental tumor tissue and organoids derived from fresh and slow-frozen tissues. Scale bar lengths are 100 μm, for insert microscopic photographs 50 μm. CK20: Keratin 20, PAS-AB: Periodic acid-Schiff Alcian Blue, Ki67. d Microscopic photographs of shave biopsies from matched fresh or slow-frozen BCC stained with Hematoxylin-eosin (H&E) BErEp4 and Ki67 antibodies. Scale bar lengths are 2 mm and 200 μm.

Fig. 3. scRNAseq analysis of matching fresh and slow-frozen tumor samples.

a Stacked barplot showing the cell-type composition of matching fresh and slow-frozen tumor samples. b Dotplot showing the gene expression of cell lineage markers in major cell types in fresh and slow-frozen tumor samples. c UMAP plot of the matching fresh and slow-frozen tumor samples.

Fig. 4. Correlation and differential gene expression analysis of fresh and slow-frozen scRNAseq tumor samples.

a UMAP visualization of fresh and slow-frozen samples according to the preservation method. b Boxplot showing the correlations between the gene expression of cells in fresh samples of different patients (purple) and the correlations between the gene expression of cells of fresh vs frozen samples (orange). Dots depict the correlation value for each sample pairing (e.g., Sample1_fresh vs Sample2_fresh or Sample1_fresh vs Sample1_frozen). c Heatmap showing differentially expressed genes between fresh and frozen samples (genes differentially expressed in at least 9 out of the 18 cell types). d Negative log10 p values of the enrichment analysis for the Reactome pathway “Cellular response to heat stress” for each cell type.

Fig. 5. scRNA sequencing of slow-frozen breast cancer samples.

a UMAP plot showing major cell populations in five breast cancer samples. b UMAP visualization of five breast cancer samples by clinical subtypes. c Heatmap showing the five most differentially expressed genes in the breast cancer samples from different clinical subtypes: UHB129/UHB173/UHB182 are hormone receptor-positive and UHB150/UHB194 are triple-negative subtypes.

Fig. 6. Correlation analysis of genes (from scRNAseq analysis of slow-frozen samples) and proteins (from immunohistochemistry stainings of FFPE sections) expression in breast cancer.

a UMAP visualization of major cell types in UHB129 (hormone receptor positive) breast cancer tissue sample and marker expression on transcriptome level. b IHC images showing corresponding protein expression; scale bar 5 mm, 250 μm, 50 μm. c Correlation of mRNA and protein expression in tumor cells. d Top: Representative IHC staining for neutrophil granulocytes marker, Myeloperoxidase (MPO) on FFPE section of UHB129 (hormone receptor positive) breast cancer tissue, scale bar: 5 mm, 250 μm, 50 μm. Bottom: Tissue classification (red: tumor cells, green: stroma) and cell, MPO staining quantification using HALO imaging analysis software, scale bar: 100 μm.