Landscape of tumor immune microenvironment in CCLM revealed by single-cell transcriptomics

A. Schematic overview of the experimental design and analytical workflow. Colon T: colon tumor tissue; Colon P: colon paratumor normal tissue; Liver T: liver metastasis tumor tissue; Liver P: liver paratumor normal tissue; LN: lymph node metastasis tissue. B. The UMAP plot of all main immune cell types. C. Dot plots showing average expression of highly variable genes of each cell group. The dot size represents percent of cells expressing the genes in each cluster and the color of dot represents the expression intensity. D. The cell numbers of main immune cells across tissues. E. Proportions of all main immune cells. P values were determined by the paired nonparameter test.

Cellular identification in spatial transcriptomic samples

A. UMAP visualization of cell clusters in spatial transcriptomic samples. B. Dot plots showing average expression of markers in indicated cell clusters. C. Overview of the spatial transcriptomic sections. H&E staining of spatial transcriptomic sections (upper). Tumor tissue and paratumor tissue identification of each section (middle). Spatial cluster distribution of each section (lower). PC: primary cancer; LM: liver metastasis. ST-P1: spatial transcriptomic dataset patient 1, ST-P1: spatial transcriptomic dataset patient 1; ST-P2: spatial transcriptomic dataset patient 2; ST-P3: spatial transcriptomic dataset patient 3; ST-P4: spatial transcriptomic dataset patient 4. D. The signature scores of T cells (upper) and NK cells (lower) in colon cancer and liver metastasis in the spatial transcriptomic sections.

Clinical and biological relationship between NK cells and metastasis of colon cancer revealed by Bulk RNA transcriptomics

A-D. The relationship of immune cell percentage determined by xCell, EPIC, quanTIseq and MCPCounter between metastasis and non-metastasis tumor in TCGA COAD cohort. E. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enriched pathway bar chart of Differentially expressed genes (DEGs) in metastasis versus non-metastasis colon cancer. F. Volcano plot showing nearly all the genes enriched in pathway of NK cell-mediated cytotoxicity were downregulated in the metastasis colon cancer. G. Gene set enrichment analysis (GSEA) of KEGG gene set. H. Natural killer cell mediated cytotoxicity was enriched in the non-metastasis colon cancer.

The landscape of NK cells in the disease progression of CCLM

A. The UMAP plot of NK cells from CCLM. B. Unsupervised clustering identifies 8 subsets of NK cells. C. Expression of key markers that distinguish resting and activated subsets of NK cells. D. Expression of key resting and activated NK cell markers across all samples. E. The UMAP plot of distribution of resting and activated NK cells from ColonP, ColonT, LiverT and LN. F. Cellular landscape of each sample from the ColonP, ColonT, LiverT and LN. The proportion of NK cells subsets in total immune cells (upper). The proportion of NK cells subsets in total NK cells (lower). G. Number of cells identified from each group (ColonP, ColonT, LiverT and LN) by cell type proportion. H. Proportions of resting (upper) and activated (lower) subsets of NK cells. P values were determined by the paired nonparameter test.

Clinical relationship between NK cells subsets and metastasis of colon cancer revealed by Bulk RNA and spatial transcriptomics

A-B. The relationship of activated and resting NK cell percentage determined by CIBERSORT and tumor metastasis in TCGA COAD cohort. M0: non-metastasis colon cancer; M1: metastasis colon cancer. C-D. K-M survival plots show that high resting NK cell and low activated NK cell predicted poor prognosis in TCGA COAD and GSE29623 cohort. PFI: Progression Free Interval; OS: Overall Survival. E. The signature scores of resting (upper) and activated NK cells (lower) in colon cancer and liver metastasis in the spatial transcriptomic sections.

Characterization and developmental course of differential subsets of NK cells in CCLM

A. The UMAP plot of KIR2DL4+ GPR171+ Activated NK cells, GZMK+ resting NK cells from CCLM. B. Proportions of KIR2DL4+ GPR171+ ActiVated NK cells, GZMK+ resting NK cells in total immune cells. P values were determined by the paired nonparameter test. C. The signature scores of GZMK+ resting NK cells (upper) and KIR2DL4+ GPR171+ Activated NK cells (lower) in colon cancer and liver metastasis in the spatial transcriptomic sections. D. Monocle analysis showing the developmental trajectory of NK cells. Color as in Pseudotime, cell state, subsets of NK cells and sample group. E. PseudoLJtemporal change curve of marker genes in each subsets of NK cells. F. The heatmap shows the expression patterns of the top 50 significant genes in branched expression analysis modeling, associated GO terms (using DAVID v6.7) are given on the right of the corresponding gene clusters. G-H. The Kaplan–Meier curve shows COAD patients survival with different GZMK+ resting NK cells and KIR2DL4+ GPR171+ Activated NK cells infiltration.

Colon cancer cells (HCT-116) educated NK cells shift toward tumor promoting status depends on cell-to-cell interaction

A-B. Clone formation assay showed the NK cell-mediated inductive effect on cell proliferation of colon cancer cell (HCT-116). Colon cancer cells were cultured in the supernatant from different co-culture system in transwell devices. CNS: colon cancer cells were cultured in the supernatant from co-culture system that NK cells and cancer cells were cultured in direct contact (CNS); CS: cancer cells cultured directly in supernatant of cancer cells. C. Cell Counting Kit-8 (CCK-8) assay showed the NK cell–mediated inductive effect on cell proliferation of colon cancer cell. D-F: The NK cell–mediated inductive effect on migration and invasion of colon cancer cell. G-H: Clone formation assay CCK-8 assay showed the NK cell–mediated inductive effect on cell proliferation of colon cancer cell. SNS: colon cancer cells were cultured in the supernatant from co-culture system that NK cells were cultured in supernatant of cancer cells; MNS: colon cancer cells were cultured in the supernatant from co-culture system that NK cells were cultured in fresh medium. M-Q. Phenotype switch (KIR2DL4, GZMK, CD9, CD49a, PD-1) of NK cells was induced by cell-to-cell interactions with cancer cells. CN: NK cells were co-cultured with colon cancer cells; SN: NK cells were cultured in supernatant of cancer cells; MN: NK cells were cultured in fresh medium.

The resting NK cell promote tumor malignant phenotype via elevating tumor-derived sSCF

A. luminex liquid suspension chip detection of 48 common chemotactic and inflammatory cytokines in CNS, SNS and MNS group. B-C. Concentration of SCF determined by luminex liquid suspension chip and Elisa in CNS, SNS and MNS group. D-E. CCK-8 assay showed the proliferation of HCT-116 cells was inhibited by imatinib mesylate, evaluated by a CCK-8 assay. Cells were incubated in the supernatant from different co-culture system with DMSO or 2 µM imatinib mesylate. F-I. Clone formation assay showed the proliferation of HCT-116 cells was inhibited by imatinib mesylate. J-M. The NK cell–mediated inductive effect on migration and invasion of HCT-116 was inhibited by imatinib mesylate.

Schematic diagram

Colon cancer cells (HCT-116 cells) educate NK cells as resting status depends on cell-to-cell interaction. Tumor-educated NK cells subsequent enhances tumor malignancy in a paracrine manner by elevating tumor-derived sSCF.

Schematic overview of the in vitro experimental design.

A. Schematic overview of coculture experiments design. B. Schematic overview of coculture experiments design in transwells.

Cluster characterization of the global landscape of CCLM revealed by single-cell transcriptomics.

A. The UMAP plot of all 12 cell clusters. B. Dot plots showing average expression of marker genes in indicated cell clusters. C. The pie chart of the proportion of main immune cells across tissues. D. The proportion of NK cells in total immune cells of each sample (including PR, PD/SD and untreated patients) from the Liver P, Liver T, Colon P, Colon T. E. Proportions of NK cells in PR, PD/SD and untreated patients among Liver P, Liver T, Colon P, Colon T groups.

Cellular identification in spatial transcriptomic samples.

A. The UMAP plot of all 11 cell clusters. B. Dot plots showing average expression of marker genes in indicated cell clusters. C. Expression of key markers across all samples.

Gene expression features of each sample. The feature plots showed the expression distributions of ALB, CD24, COL1A1, MYH11, CXCL8, CD14, CD79A AND CD3D in each spatial transcriptomic section.

Clinical relationship between NK cells subsets and colon cancer revealed by TCGA COAD cohort.

A. The relationship of activated and resting NK cell and clinical characteristics of colon cancer in TCGA COAD cohort. B. The relationship of 22 immune cells percentage determined by CIBERSORT and prognosis of colon cancer in TCGA COAD cohort.

The proportions of eight clusters of NK cells in Colon P, Colon T, Liver T and LN.

Figure.S7 NK cell-mediated tumor promoting effect in colon cancer cells (DLD-1).

A. Clone formation assay showed the NK cell–mediated inductive effect on cell proliferation of DLD-1 cell between CNS and CS groups. B-C: CCK-8 assay showed the NK cell–mediated inductive effect on cell proliferation of DLD-1 cell between CNS and CS groups. D-F. The NK cell–mediated inductive effect on migration and invasion of DLD-1 cell between CNS and CS groups. G. CCK-8 assay showed the NK cell-mediated inductive effect on cell proliferation of DLD-1 cell among CNS, SNS, MNS groups. H-I. Clone formation assay showed the NK cell-mediated inductive effect on cell proliferation of DLD-1 cell among CNS, SNS, MNS groups. J-L The NK cell–mediated inductive effect on migration and invasion of DLD-1cell among CNS, SNS, MNS groups.

The positive gate locations of CD56, GZMK, KIR2DL4, CD9, CD49a, PD-1 defined according to the FMO control.

Phenotype switch (CD56+, GZMK+) of NK cells was analyzed by FACS after fixation and permeabilization in different co-cultured groups.

Phenotype switch of NK cells in different co-cultured system and the corresponding NK cell-mediated effect on cell migration of fresh colon cancer cell (HCT-116).

A-B: NK cells underwent phenotype switch (high expression of CD9) when cocultured with HCT and HFF, the phenotype switch was more obvious when co-cultured with HCT. CN: NK cells cocultured with HCT/HFF; SN: NK cells cocultured with supernatant of HCT/HFF; MN: NK cells cocultured in fresh medium. C-E: Transwell assay showed the only tumor co-cultured NK mediated the inductive effect on cell migration of colon cancer cell (HCT-116). CNS: Colon cancer cells were cultured in the supernatant from co-culture system that NK and HCT/HFF were cultured in direct contact; SNS: Colon cancer cells were cultured in the supernatant from co-culture system that NK cocultured with supernatant of HCT/HFF; MNS: Colon cancer cells were cultured in the fresh medium.

Relative KITLG expression in different groups.

A. RT-qPCR analysis of KITLG expression in HCT-116 with and without co-cultured with NK cells. B. RT-qPCR analysis of KITLG expression in HCT-116 and DLD-1 cells.