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. 2021 Mar 4;13(5):1097. doi: 10.3390/cancers13051097

Figure 4.

Figure 4

Spatial distribution of metabolic activity and distinct cell types in individual colorectal cancer (CRC) spheroids. (a) Histological sections of CRC spheroids co-stained for representative lineage-specific marker genes by RNA fluorescence in situ hybridization (FISH). Left: Overview images. Scale bar, 50 μm. Right: Magnified images representing dashed box regions in overview images (4× digital zoom). Scale bar, 10 μm. DEFA5: Paneth-like, FABP1: terminally differentiated-like, LGR5: stem-like cells. Colored arrowheads mark associated subtypes in magnified images. Images represent z projections from 10 μm slices and DNA is counterstained by 6′-diamidino-2-phenylindole (DAPI). (b) Histological section of a spheroid (P1) stained for cell type-specific marker genes (RNA-FISH) and mitochondria (Mitotracker). Top left: Merged overview image. Scale bar, 50 µm. Bottom left: Magnification of dashed region in top left image (4× digital zoom). Scale bar, 10 µm. Center and right: Single channels. Scale bar, 10 µm. Images represent z projections from 10 μm slices and DNA is counterstained by DAPI. (c,d) Fraction of Mitotracker ‘ON’ cells as determined by automated image analysis pipeline. (c) LGR5+ or DEFA5+ cells (total number of cells analyzed: P1: n = 7379; P4: n = 2670; P5: n = 2213). (d) LGR5+ or FABP1+ cells (total number of cells analyzed: P1: n = 3403; P4: n = 1580; P5: n = 1601).