Supplementary Figure from Spatiotemporal Immune Landscape of Colorectal Cancer Liver Metastasis at Single-Cell Level
Authors: Wu Yingcheng , Yang Shuaixi , Ma Jiaqiang , Chen Zechuan , Song Guohe , Rao Dongning , Cheng Yifei , Huang Siyuan , Liu Yifei , Jiang Shan , Liu Jinxia , Huang Xiaowu , Wang Xiaoying , Qiu Shuangjian , Xu Jianmin , Xi Ruibin , Bai Fan , Zhou Jian , Fan Jia , Zhang Xiaoming , Gao Qiang 
Published: April 2023

<p>Supplementary Figure S1. The Cellular and Spatial Features of Infiltrated Immune Cells in CRLM, Related to Figure 1. (A) UMAP plot reveals the immune cell difference among patients, treatment types, and tissues. (B) Dot plot reveals the selected marker gene expression of main cell types. (C) UMAP plot reveals the selected marker expression distribution of main cell types. (D) Histogram showing the immune cell infiltration level of all patients. (E) Histogram showing the immune cell type difference between untreated samples and NAC treated samples. (F) The UMAP plot showing the sub populations of B cells, CD4 T cells, and NK cells. (G) The CD4 T cell score of spatial transcriptomics in CRC & LM. (H) The Plasma cell score of spatial transcriptomics in CRC & LM. - Supplementary Figure S2. Discovery and Validation of Tissue-Specific Immune Cells, Related to Figure 2. (A) UMAP plot reveals the tissue-specific or tissue-shared immune cells in adjacent colon, CRC, adjacent liver, LM, LN, and PBMC, respectively. (B) Kupffer cell signature of all LM-infiltrated myeloid cells reveals that MRC1+ CCL18+ M2-like macrophages potentially derived from hepatic Kupffer cells. (C) Validation of immune cells in another independent bulk microarray dataset of CRLM (Methods). *, P < 0.05; ***, P < 0.005; N.S.., not significant. P values were determined by Wilcox test. (D) The survival plot of SPP1+ Macrophages in TCGA CRC patients. P values were determined by log-rank test. - Supplementary Figure S3. Suppressive Immune Cells in Primary and Metastatic Tumors Showed Unique States, Related to Figure 3. (A) Cancer cell pathway enrichment analysis in CRC and LM. Cancer hallmark gene sets were used to perform the pathway enrichment analysis (Methods). (B) The volcano plot represents the differentially expressed genes of SPP1+ macrophages between CRC and LM. (C) The selected gene expression of SPP1+ macrophages in CRC and LM. ***, P < 0.005. P values were determined by Wilcox test. - Supplementary Figure S4. Metabolic Activity of Suppressive Immune Cells, Related to Figure 4. (A) The correlation between VISION, AUCell, and ssGSEA generated metabolism pathway score in scMetabolism pipeline. Peripheral Blood Mononuclear Cells (PBMC), melanoma, Head and Neck Squamous Cell Carcinoma (HNSCC), and testis scRNA-seq data were tested. (B-E) The correlation between metabolism pathway score of simulated dropout, imputed, and raw HNSCC (B), PBMC (C), melanoma (D), and testis (E) scRNA-seq data. We tested the simulated dropout of 5%, 10%, 20%, and 30% of expressed genes. (F) Multiplex immunohistochemistry shows the proportions of SPP1+ macrophages which harbor high metabolic enzyme expression (GSTO1, IL4l1, and MIF). The y axis represents the proportion of triple positive cells of all CD68+ cells. P values were determined by Wilcox test. (G) Multiplex immunohistochemistry shows the selected imaging results of spatial distribution of metabolic enzyme expression (GSTO1, IL4l1, and MIF) in SPP1+ macrophages and CCL18+ macrophages. (H) The UMAP plot of cell type distribution and tissue distribution of all CD45+ cells in mouse CRLM samples. (I) The expression of selected markers of all CD45+ cells in mouse CRLM samples. (J) The cell type proportion among all CD45+ cells in mouse CRLM samples. (K) The score of phenylalanine metabolism of Mrc1+ macrophages and expression of Mif of Mrc1+ macrophages between CRC and LM. P values were determined by Wilcox test. (L) The phenotypic score of MRC1+ CCL18+ macrophages in LM and CRC. n.s., not significant. P values were determined by Wilcox test. (M) The phenotypic score of SPP1+ macrophages in LM and CRC. n.s., not significant. P values were determined by Wilcox test. (N) The selected gene expression associated with antigen processing & presentation and complement in CRC and LM -infiltrated MRC1+ CCL18+ macrophages. ***, P < 0.005. P values were determined by Wilcox test. (O) Correlation between phenotypic score and metabolic activity score in CRC MRC1+ CCL18+ macrophages. Only tumor high metabolic pathways were used for analysis. *, P < 0.05 and Rho > 0.3. P values were determined by Spearman's rank test. - Supplementary Figure S5. The Immune Cell Changes After Neoadjuvant Chemotherapy, Related to Figure 5. (A) The cell clustering of untreated samples.. (B) Pathway enrichment analysis of differentially expressed genes of untreated and NAC-treated cancer cells in CRC and LM. Cancer hallmark gene sets are used to perform the pathway enrichment analysis (Methods). (C) The dot plot shows the alterations of immune cells in adjacent colon, LN, adjacent liver and PBMC bwtween untreated and NAC-PR samples. (D) The dot plot shows the alterations of immune cells in adjacent colon and adjacent liver bwtween untreated and NAC-PD/SD samples. - Supplementary Figure S6. Neoadjuvant Chemotherapy Reprogrammed the Suppressive Immune Cells, Related to Figure 6. (A) Cellular proportional changes of SPP1+ macrophages in untreated and NAC-treated samples revealed by scRNA-seq. The y axis represents the proportion of SPP1+ M2 macrophages among all CD45+ cells. ***, P < 0.005; N.S., not siginicant. P values were determined by Wilcox test. (B) Cellular proportional changes of SPP1+ CD68+ cells in untreated and NAC-treated samples revealed by multiplex immunohistochemistry. The y axis represents the proportion of CD68+ SPP1+ cells among all CD68+ cells. The boxplot represents the mean value and the error bar represents the standard error value. P values were determined by Wilcox test. (C) Trajectory plot shows the treatment status, pseudotime, cell type, tissue, MRC1 expression and S100A9 expression of all myeloid cells in untreated and treated-PD/SD group. (D) Pathway enrichment analysis of highly expressed genes of myeloid cells in CRC (Untreated v.s. PD/SD and untreated v.s. PR respectively). KEGG gene sets were used to perform the pathway enrichment analysis (Methods). - Supplementary Figure S7. Immune Cells in Multifocal Metastasis Harbored Distinct Metabolic and Phenotypic Activity, Related to Figure 7. (A) The significantly differential metabolic activity of MRC1+ CCL18+ macrophages in P10. ***, P < 0.005. P values were determined by Wilcox test. (B) The phenotypic activity of MRC1+ CCL18+ macrophages in P10. ***, P < 0.005. P values were determined by Wilcox test.</p>