Extended Data Fig. 6: HRCs are enriched in invasion fronts and micrometastases.
From: Metastatic recurrence in colorectal cancer arises from residual EMP1+ cells
a, Primary tumour outlined by cyan line and coloured in 4 different regions identified with HALO image analysis classifier (tumour-red, stroma-green, background-yellow, necrosis-blue). Scale bar, 1 mm. b, TOM cell intensity analysis in the tumour area after segmentation into individual cells. B’ and B’’ show magnified regions corresponding to tumour core (B’) and invasion fronts + tumour buds (B’’). Scale bars, 1 mm (B), 100 µm (B’ and B’’). c, Representative immunostaining of TOM and E-CADH in the tumour core and in tumour buds of primary tumours derived from Emp1-iCT MTOs 4 weeks post implantation in the caecum. TOM fluorescence is shown with mpl-inferno LUT. The dashed line delimits the caecum edge. Arrows point to tumour buds. Scale bars, 100 µm (tumour core) 50 µm (tumour buds). d, Quantification of Emp1-TOMhigh (defined as cells in percentile 90 for TOM expression) in the tumour core (submucosal area), invasion fronts (inside muscular layer) and isolated glands (over muscular layer). Boxes represent the first, second (median) and third quartiles. Whiskers indicate maximum and minimum values. Two-sided Wilcoxon test on percentages. n = 8 mice. e, Immunofluorescence of TOM, CD31 and DAPI in primary tumours. Amplified insets show the tumour core and invasive glands intermingled in mucosal layers (ML) next to blood vessels. Dashed lines outline healthy intestinal epithelium. Scale bars, 250 µm, 100 µm (tumour core) and 50 µm (tumour buds). f, Representative flow cytometry plot of TOM expression in wild-type and Emp1-iCT AKP MTOs. g, Relative mRNA expression of indicated genes in Emp1-TOMhigh and Emp1-TOMlow sorted cell populations from Emp1-iCT AKP MTOs. Two-sided t-test after normalizing by Ppia. n = 3 technical replicates. Mean +/− SD. h, Representative immunostaining for TOM and E-CADHERIN in Emp1-iCT AKP tumours implanted in the caecum 4 weeks post-implantation. Emp1-TOM fluorescence is shown with an mpl-inferno LUT. Dashed lines delimit the edge of the caecum. Scale bar: 250 µm. i, Representative images of TOM and E-CADHERIN staining in micro (left) and medium (right) size metastases. Scale bars: 50 µm and 250 µm. j, Percentage of tumour area containing TOM-high and TOM-low fluorescent pixels versus metastases size (in pixels). Each dot represents an individual metastasis. k, TOM, KRT20 and E- CADHERIN staining in primary tumours generated by Emp1-iCasp9-tdTomato AKTP MTOs. Dashed lines encompass invasion fronts and tumour buds. KRT20 staining is observed in normal mucosa (NM) and to a lesser extent in the tumour core. Tumour cell clusters invading the muscular layer (ML) express high levels of TOM and no KRT20. Amplified insets show an example of tumour core (K’) and invasion fronts (K’’) with TOM (left) and KRT20 (right) stainings. Scale bars, 500 µm (k) and 100 µm (K’ and K’’). l, Immunofluorescence of TOM and E-CADHERIN (left) and KRT20 and E-CADHERIN (right) in a cluster of tumour cells that enter the liver through a portal vein (PV, delimited with dashed lines). Scale bar, 50 µm.
