早期结直肠癌：侵袭前沿的胎儿样状态如何出现？

Fig. 1 | Metastasis-associated signatures at the invasive front of early-stage colon cancer. a, Spatial transcriptomics on 19 T1 CRCs with cancer-specific (CTA; 373 segments; 5 probes per gene) and whole-transcriptome (WTA; 281 segments; 1 probe per gene) probe panels. Lymph node (N) and distant metastasis status (M) are indicated. b, Example of ROI placement in T1 CRCs. c, Zoom-in of a representative ROI (invasive front no. 2 shown in b), showing epithelial and stromal segmentation for separate profiling. The dashed line shows the tumour border. d, Variance partitioning of a spatial transcriptomics dataset (WTA; n = 2,000 most variable genes; boxes, interquartile range; black bars, median; whiskers, 1.5× interquartile range). e, Violin plot of pairwise correlations between stromal–stromal (blue) or epithelial–epithelial (red) segment pairs within, or between, patients (n = 104 region comparisons across 9 patients; boxes, interquartile range; black bars, median; whiskers, 1.5× interquartile range; ANOVA P = 2.31 × 10−11, Tukey’s honestly significant difference (HSD)). f, Heatmap showing relative expression (log2 fold change) of the top differentially expressed genes (lowest 10 Padj values with Padj < 0.05; Wilcoxon rank-sum test with Bonferroni correction) in epithelial segments (n = 140) for each histopathological region (CTA). Bold font shows fetal markers. g, Correlation matrix of T1 tumour core (red font) and invasive front (yellow font) signatures from this study versus published CRC signatures within tumour core (n = 42) and invasive front (n = 43) epithelial segments (WTA). h, EpiHR signature in epithelial segments of indicated histopathological regions of all 19 spatially profiled T1 CRCs. Connected points denote patient median. EpiHR signature was subset for genes probed in both the CTA and WTA probe panels (boxes, interquartile range; black bars, median; whiskers, 1.5× interquartile range; t-test). i, Immunofluorescence showing oncofetal marker LAMC2 at the invasive front of T1 CRC (n = 2 tumours). j, Immunofluorescence for LAMC2+ oncofetal tumour cells and FAP+ CAFs on tissue microarray of 232 T1 CRCs with 5-year clinical follow-up. k, Composition analysis of j. l, GSEA of epithelial invasive front segments from metastatic versus non-metastatic tumours. Significant immune-related gene ontology biological processes are highlighted in blue (permutation-based test, Benjamini–Hochberg corrected). Ade, adenoma; core, tumour core; CSC, cancer stem cell; GO, gene ontology; inv, invasive front; NES, normalized enrichment score; nor, normal; NS, not significant; pt, patient; TMA, tissue microarray. Scale bars, 1 mm (b); 200 μm (c,i). Illustration in a reproduced from NIH BioArt (https://bioart.niaid.nih. gov/bioart/212); illustrations in j adapted from NIH BioArt (https://bioart. niaid.nih.gov/bioart/214 and https://bioart.niaid.nih.gov/bioart/232).

Fig. 2 | Invasive front tumour cell phenotypes are not genetically driven. a, Overview of organoid derivation from regional punch biopsies (circles) in early-stage CRC. Biopsies are processed to tissue fragments and aliquoted for organoid culture and cryopreservation. Regional identity of biopsies was histologically confirmed on the sampled CRC (FFPE) by a pathologist (circles denote punch locations on haematoxylin and eosin). b, Overview of multiregional early-stage CRC organoid biobank. c, Driver landscape in early-stage CRC organoids. Driver genes with a prevalence of more than 4% in ref. 49 and a driver likelihood score of more than 0.8 are shown. d, Representative phylogenetic tree (patient 14) showing an evolutionary relationship between histopathological regions based on WGS data. Mutations and stage of acquisition are annotated. e, CNAs in early-stage CRC organoids. Top, mean CNA profile of our samples and PCAWG primary CRC reference dataset (dMMR and WGD excluded, statistical comparison is Pearson’s correlation r with P value and cosine similarity). f, Boxplot showing pairwise cosine similarities of inferred CNA profiles (inferCNV) within spatial transcriptomics dataset of early-stage CRCs. Points represent pairwise comparisons between regions (boxes, interquartile range; black bars, median; whiskers, 1.5× interquartile range). g, Quantification method for organoid outgrowth efficiency in various culture conditions using OrganoSeg64. Total organoid area relative to control medium after 9 days of outgrowth from single cells was assessed. h, Outgrowth efficiency of organoids from the early-stage CRC biobank in medium with indicated modifications. i, Pearson correlations of outgrowth efficiencies between organoid lines derived from the same tumour (n = 29 comparisons across regional organoid lines from 13 patients; boxes, interquartile range; black bars, median; whiskers, 1.5× interquartile range; ANOVA P < 0.0001, Tukey’s HSD). AJCC, American Joint Committee on Cancer staging system; cnLOH, copy neutral loss of heterozygosity; ctrl, control; del, deletion; dMMR, deficient mismatch repair; ND, not determined; pMMR, proficient mismatch repair; WGD, whole-genome duplication.

Fig. 3 | Trophocyte-like CAFs at the invasive front. a, scRNA-seq-based cell type identification in early-stage CRC TME. b, UMAP of scRNA-seq data from regional biopsies of biobanked CRCs (pt5, pt11, pt13, pt14 and pt16 (Fig. 2b)). Left, cell type annotation. Right, relative contribution of core and invasive front biopsies per cluster. c, Marker gene expression level across fibroblast subtypes from early-stage CRCs and tumour-adjacent normal tissue. Colours, expression level; dot size, percentage of cells expressing transcript. d, TME-HR signature14 scores across TME cell types in early-stage CRCs (n = 1,612 cells; 5 patients). Right panels, cells (black points) with highest TME-HR signature scores among CAFs and trophocytes originate from invasive front biopsies (boxes, interquartile range; black bars, median; whiskers, 1.5× interquartile range); t-test). e, CD8 T cell signature expression in tumours with indicated metastasis status from WTA spatial transcriptomics dataset of early-stage CRC (Fig. 1) (n = 9 patients; boxes, interquartile range; black bars, median; whiskers, 1.5× interquartile range; ANOVA P = 0.0015, Tukey’s HSD test). f, Disease-free survival of patients with CMS4 (ref. 49) stratified by low or mid (bottom 66%) versus high (top 33%) expression of indicated fibroblast subtype signatures (top 100 region-specific markers, Supplementary Table 5) (log-rank test). g, scRNA-seq cell–cell correlation matrix of fibroblasts from core and invasive front biopsies. Three CAF subtypes (green shades) can be recognized within the FAP+ CAF cluster. Colour scale represents cell–cell transcriptome similarity (Pearson correlation coefficient calculated over the 8,000 most variable genes). Bottom tracks, telocyte and trophocyte signature scores for each cell. h, Expression level of markers among fibroblast subtypes. Colours, expression level; dot size, percentage of cells expressing transcripts. Benjamini–Hochberg. i, GSEA of GeoMx (WTA; n = 9 T1 CRCs; permutation test with FDR) bulk expression profiles between tumour core and invasive front using cell type- specific signatures from scRNA-seq. Lollypop length, normalized enrichment score; dot size, nominal probability (−log[P]). j, Immunofluorescence for trophocyte marker SFRP2 (magenta) at the invasive front of early-stage CRC (n = 4 tumours). Tumour cells in green (PanCK+), nuclei in blue (SYTO13). Top panels show zoom-ins of indicated regions. The dashed line shows the tumour border. k, Immunofluorescence against FAP (purple) and HRC marker LAMC2 (red) at the invasive front of early-stage CRC (n = 3 tumours). Tumour cells in green (PanCK), nuclei in grey (SYTO13). Costaining of PanCK and LAMC2 is shown in yellow. The white dashed line shows the tumour border. l, Single-cell spatial transcriptomics of T1 CRC specimen showing oncofetal cells (high expressors of High Relapse signature, bright green)14 and WNT-driven cancer (stem) cells (dark green)29 The dashed line shows the tumour border. m, As in l, but showing trophocytes (orange) and telocytes (yellow). The dashed line shows the tumour border. n, As in l, but showing FAP + CAF subtypes. B, B cell; IQR, interquartile range; lympho.prolif, proliferative lymphocytes; MΦ, macrophage; myo, myofibroblast; NES, normalized enrichment score; NS, not significant; onf, oncofetal; S1, stromal 1; SMC, smooth muscle cell; TCD8+, CD8+ T cell; TCD4+, CD4+ T cell; transcr., transcriptomics; Treg, regulatory T cell. Scale bars, 200 μm (j,k); 1 mm (l–n).

Fig. 4 | Trophocyte-like CAFs induce oncofetal plasticity to EMP1+ tumour cell states. a, Schematic of coculture experiment. b, GSEA of coreHRC signature in organoids across culture conditions (permutation test with FDR). Left, 2D (n = 6) versus monoculture (n = 7). Right, 3D (n = 6) versus 2D (n = 6). c, The log2 fold change of differentially expressed genes in coculture. Oncofetal markers annotated. d, 3D fibroblasts signature (top 100 differentially expressed genes ranked by log2 fold change, Padj < 0.01) expression in GeoMx regions (WTA; n = 9 patients). Boxes, interquartile range; black bars, median; whiskers, 1.5× interquartile range; t-test). e, Differentially expressed genes in 3D (n = 6) versus 2D (n = 6) cocultured fibroblasts. Ligand-mediated signalling genes are annotated (Wald test with Benjamini–Hochberg correction). f, Top, EMP1 reporter knock-in schematic. Bottom, fluorescence image of EMP1 reporter organoid. g, Organoid-based screen for inducers of oncofetal state. h, Top, fold change in EMP1-mNeon+ cells (%) versus control. Data points, independent experiments (exact n shown by number of data points; error bars, s.e.m.). Bottom, scRNA-seq dot plot of screened ligands in invasive front biopsies (Fig. 3) (PGE2, PTGES; PGD2, PTGDS). i, Percentage of EMP1-mNeon+ cells across conditions. Prostaglandin, PGD2 + PGE2. TGFβ, TGFβ1 + TGFβ3 (n = 5; mean + s.d.; ANOVA, Bonferroni correction). j, GSEA of oncofetal and CSC signatures in organoids treated with TGFβ (TGFβ1 + TGFβ3) and prostaglandins (PGD2 + PGE2) (n = 2 patients; 3 replicates; permutation-based test, Benjamini– Hochberg corrected). k, Like j, but relative expression of coreHRC signature genes. l, qPCR of oncofetal markers and LGR5, following 24 h of treatment with PGD2, PGE2, TGFβ1 and TGFβ3 in 7 independent organoid lines from 4 early- stage CRCs. Values normalized to DMSO, mean + s.d. Points, independent measurements (n = 3; *P < 0.05, ratio paired t-test). Illustration in g adapted from NIH BioArt (https://bioart.niaid.nih.gov/bioart/160); illustration in l reproduced from NIH BioArt (https://bioart.niaid.nih.gov/bioart/661). DMSO, dimethylsulfoxide; IRES, internal ribosomal entry site, mono, monoculture; NLS, nuclear localization signal; pA, polyA; PuroR, Puromycin Resistance cassette. Scale bar, 20 μm (f).

Fig. 5 | Trophocyte-like CAFs and oncofetal plasticity co-emerge at the birth of malignancy. a, Pseudo-longitudinal single-cell spatial transcriptomics of 11 early-stage CRCs (CosMx; 6,000 probe panel). b, UMAP of epithelial (left) and microenvironmental (right) compartments. c, Relative abundance (%) of tumour cell types and fibroblasts in single-cell spatial transcriptomics of intramucosal (n = 3), T1 sm1 (n = 5) and T1 sm3 (n = 3) specimens. d, Single-cell spatial plots showing localization of WNT-driven cancer (stem) cells (CSC, dark green), oncofetal cells (bright green, HRC signature), trophocytes (orange) and trophocyte-like CAFs (purple) in representative intramucosal, T1 sm1 and T1 sm3 specimens. e, Percentage of LAMC2+ oncofetal tumour cells (within PanCK+ epithelium) and FAP+ CAFs (within PanCK− stroma) just before (intramucosal carcinoma, n = 3) and shortly after (T1 sm1 n = 3 and T1 sm3 n = 3) malignant transformation. f, Immunofluorescence of tumour specimens just before and after malignant transformation. Nuclei (grey, SYTO13), epithelial cells (green, PanCK), oncofetal tumour cells (red, LAMC2) and CAFs (purple, FAP) are stained. Orange, PanCK and LAMC2 co-expression. White dashed line, muscularis mucosae. g, Differential cell type composition of oncofetal and CSC neighbourhoods (50 µm radius; n = 11 patients). Whiskers, 95% credible interval, coloured by significance (FDR < 0.05). h, ssGSEA scores for TGFβ and prostaglandin signalling in stromal neighbourhoods of oncofetal and CSC tumour cells per tumour stage (n = 11 patients; boxes, interquartile range; grey bars, median; whiskers, 1.5× interquartile range). i, UMAP of integrated CRC TME scRNA-seq datasets45,54,55 (n = 110 patients). Left, unsupervised clustering and cell type annotations. Right, zoom-in of fibroblasts, split over normal and tumour tissue. j, UMAP of fibroblasts from integrated scRNA-seq with streamlines showing inferred trajectories of Monocle, Slingshot and CytoTRACE pseudotime. k, Relative contribution of fibroblast subtypes to total fibroblast compartment in normal tissue, and early (stage I–II) and advanced (stage III–IV) CRCs. Im, intramucosal; MM, muscularis mucosae. Scale bars, 1 mm (d);  200 μm (f).