ID1 expressing macrophages support cancer cell stemness and limit CD8+ T cell infiltration in colorectal cancer

Abstract

Elimination of cancer stem cells (CSCs) and reinvigoration of antitumor immunity remain unmet challenges for cancer therapy. Tumor-associated macrophages (TAMs) constitute the prominant population of immune cells in tumor tissues, contributing to the formation of CSC niches and a suppressive immune microenvironment. Here, we report that high expression of inhibitor of differentiation 1 (ID1) in TAMs correlates with poor outcome in patients with colorectal cancer (CRC). ID1 expressing macrophages maintain cancer stemness and impede CD8⁺ T cell infiltration. Mechanistically, ID1 interacts with STAT1 to induce its cytoplasmic distribution and inhibits STAT1-mediated SerpinB2 and CCL4 transcription, two secretory factors responsible for cancer stemness inhibition and CD8⁺ T cell recruitment. Reducing ID1 expression ameliorates CRC progression and enhances tumor sensitivity to immunotherapy and chemotherapy. Collectively, our study highlights the pivotal role of ID1 in controlling the protumor phenotype of TAMs and paves the way for therapeutic targeting of ID1 in CRC.

Fig. 1. Enhanced expression of ID1 in TAMs correlates with poor clinical outcome for CRC patients.

a Schematic diagram for screening the upregulated oncogenic transcription factors (Log₂FC > 1.5) in peritoneal macrophages (PMs) stimulated with CT26 cell-derived conditional medium (CM) compared to the untreated PMs (left) and the screened 10 upregulated genes (right) by using GEO dataset of GSE80065. b, c Immunoblots of indicated proteins in RAW264.7 cells treated with CT26-derived CM (b) or MC38-derived CM (c), n = 3 biologically independent samples. d–f Representative multiple immunohistochemistry (mIHC) images of Id1 and F4/80 in tumor tissues of AOM/DSS-induced CRC model (d), Apc^Min spontaneous CRC model (e) or in the normal colon tissues of C57BL/6J mouse (f). Scale bar, 25 μm, n = 3 biologically independent samples. g, h The strategy of isolating TAMs from orthotopic MC38 tumor-bearing mice or PMs from normal C57BL/6 J mice (g). The mRNA (h, left) and protein abundance (h, right) of Id1 in TAMs and PMs were detected, n = 3 mice per group, Student’s t-test. i, j Representative mIHC staining of ID1 and CD68 in tumor and adjacent normal tissues from CRC patients (i), and the related statistical data of ID1 expression within CD68⁺ cells (j), Mann–Whitney U test. Scale bar, 25 μm. k Kaplan–Meier plot of overall survival of CRC patients stratified by ID1 expression level within CD68⁺ cells, Log-rank test. Unless specified otherwise, the data are presented as means ± SEM. g is created with BioRender.com. Source data are provided as a Source Data file.

Fig. 2. ID1 expressing TAMs promote CRC growth and metastasis.

a Schematic diagram for establishing the MC38 and TAMs mixture s.c. model. b, c Tumor volumes (b) and tumor weight (c) of groups presented in (a), n = 8 mice per group, Mann–Whitney U test is used in (b), and Welch’s test is used in (c). d Representatives of Ki67 immunohistochemical staining in the groups presented in (a), n = 6 mice per group, Student’s t-test. Scale bar, 25 μm. e Schematic diagram for establishing the CT26 spleen–liver metastasis model. f Representative bioluminescence images and statistical data of bioluminescence signal of groups presented in (e), n = 6 mice per group, Kruskal–Wallis test. W/O: without. g, h Representative gross liver images (g) and H&E staining (h) of the liver lobes for groups as presented in (e). Scale bar, 1 cm. i The ratio of liver weight to body weight of groups presented in (e), n = 5 mice per group, one-way ANOVA test. j The largest diameter of metastatic tumor of groups as presented in (e), n = 5 mice per group, one-way ANOVA test. k The number of hepatic lobes with metastatic tumor nodules of groups presented in (e), n = 4 mice per group, Kruskal–Wallis test. l Schematic diagram for establishing the CT26 orthotopic liver metastasis model. m Representative gross liver images of groups presented in (l). Scale bar, 1 cm. n Total metastatic sites of groups presented in (l), n = 6 mice per group, Student’s t-test. o The largest tumor diameter of groups presented in (l), n = 6 mice per group, Student’s t-test. p Schematic diagram for establishing the CT26 orthotopic model. q Representative bioluminescence images and statistical data of bioluminescence signal of groups presented in (p), n = 6 mice per group, one-way ANOVA test. r Representative immunohistochemical Ki67 staining of groups presented in (p). Scale bar, 25 μm. n = 3 mice per group, one-way ANOVA test. a, e, l, and p are created with BioRender.com. Source data are provided as a Source Data file.

Fig. 3. ID1-expressing TAMs promote CRC progression partially through inhibiting CD8⁺ T cell recruitment.

a Tumor inhibition rate of MC38 and TAMs mixture s.c. model in the indicated groups, n = 8 mice per group, Welch’s test. b, c Percentage of CD8⁺ T cells (b), representative mIHC images, and statistical data of CD8⁺ T cells (c) infiltrated in the tumor tissues as presented in Fig. 2a, n = 4 (b) or 3 (c) mice per group, Student’s t-test, Scale bar, 25 μm. d Representative density dot plots and the statistical data for IFN-γ⁺ and Granzyme B⁺ CD8⁺ T cells in the tumor tissues are presented in Fig. 2a. n = 3 mice per group, Student’s t-test. e Tumor inhibition rate of CT26 spleen-liver metastasis model with i.p. injection of CM from BMDM-derived TAMs in the indicated groups, n = 5 mice per group, Student’s t-test. f Representative mIHC images and statistical data of CD8⁺ T cells in liver metastatic tumor tissues presented in Fig. 2e, n = 3 mice per group, one-way ANOVA test. Scale bar, 20 μm. g, h Percentage of CD8⁺ T cells infiltrated in the tumor tissues from recipient mice presented in Fig. 2l (g) and Supplementary Fig. 1q (h), n = 6 mice per group, Student’s t-test. i Representative mIHC images and statistical data of CD8⁺ T cells in tumors presented in Fig. 2p, n = 3 mice per group, one-way ANOVA test. Scale bar, 20 μm. j Schematic diagram for the deletion of CD8⁺ T cells in tumor models. k–m Tumor volumes (k), tumor weight (l), and representative tumor images (m) of groups presented in (j), n = 8 mice per group, Kruskal–Wallis test. Scale bar, 1 cm. n Tumor inhibition rate of indicated groups, n = 8 mice per group, Student’s t-test. o, p Relative migration of CD8⁺ T cells cocultured with different groups of RAW264.7 cells (o) or TAMs (p), n = 3 biologically independent samples, Welch’s test. q Relative migration of human CD8⁺ T cells cocultured with different groups of TAMs, n = 4 patients, paired t-test. j is created with BioRender.com. Source data are provided as a Source Data file.

Fig. 4. ID1-expressing TAMs are essential for the maintenance of CRC stemness traits through activating FAK-YAP signaling.

a, b Flow cytometry analysis of CD44^high Lgr5⁺ cell ratio in CD45^- Epcam⁺ tumor cells isolated from MC38-derived tumor nodules (a) in Supplementary Fig. 2a or CT26-derived liver metastases (b) presented in Fig. 2l, n = 4 biologically independent samples, Student’s t-test. c Schematic diagram for determining the tumor initiation capacity and the statistical data of CSC frequency. d Flow cytometry analysis of CD44^high LGR5⁺ cell ratio in HCT116 cells cocultured with Ctrl or ID1^KD TAMs, n = 4 patients, paired t-test. e Flow cytometry analysis of CD44^high Aldh⁺ cell ratio in different groups of MC38 cells, n = 3 biologically independent samples, Student’s t-test. f Images and quantification of HCT116 tumor spheres with different treatments, n = 3 biologically independent samples, Welch’s test. g Schematic diagram for the transcriptome sequencing. h, i GSEA analysis on differentially expressed genes between CD45^- Epcam⁺ tumor cells of indicated groups presented in (g) with a predefined gene set of FAK (h) and YAP signaling (i). n = 3 biologically independent samples per group in the RNA-seq data. j Immunoblots of indicated proteins in different groups of CD45^- Epcam⁺ tumor cells presented in Supplementary Fig. 1e, n = 3 biologically independent samples. k Immunoblots of indicated proteins in DLD-1 cells cocultured with different THP-1 cells with or without Y15 treatment, n = 3 biologically independent samples. l Immunoblots of indicated proteins in MC38 cells with different treatments, n = 3 biologically independent samples. m Immunoblots of cytosolic and nuclear Yap in MC38 cells cocultured with different TAMs, n = 3 biologically independent samples. n Relative luciferase activity of TEAD in MC38 cells cocultured with different TAMs, n = 3 biologically independent samples, Student’s t-test. o Relative mRNA expression of Yap downstream genes in different groups of MC38 cells, n = 3 biologically independent samples, Student’s t-test. p Effects of Verteporfin on CD44^high ALDH⁺ ratio of HCT116 cells cocultured with different groups of THP-1 cells, n = 3 biologically independent samples. q Schematic diagram for tumor model (q, up) and tumor volumes (q down) of the indicated groups, one-way ANOVA test, n = 12, 10, 10, and 8 mice in the groups of Ctrl, Id1^OE, Ctrl-Y15, Id1^OE-Y15. r Schematic diagram for tumor weight of the indicated groups, n = 8 mice per group, one-way ANOVA test. s Effects of Y15 on CD44 expression of DLD-1 cells cocultured with different groups of THP-1 cells, n = 3 biologically independent samples. c, g, and q are created with BioRender.com. Source data are provided as a Source Data file.

Fig. 5. ID1 in TAMs mediates tumor immune evasion and CRC stemness maintenance by inhibiting CCL4 and SerpinB2 transcription.

a Schematic diagram of the transcriptome sequencing. b Volcano plot of differentially expressed genes of the indicated groups presented in (a), n = 3 biologically independent samples per group in the RNA-seq data. c Log₂FC heatmap of differentially expressed chemokines encoding genes (left). Schematic diagram for the screening of Ccl3, Ccl4, Cxcl9, and Cxcl16 (right). d Relative mRNA level of Ccl3, Ccl4, Cxcl9 and Cxcl16 in different groups of RAW264.7 cells, n = 3 biologically independent samples, Student’s t-test. e CCL4 abundance in the CM from Ctrl and ID1^KD TAMs, n = 5 patients, paired t-test. f Ccl4 abundance in the CM from different groups of RAW 264.7 cells, n = 3 biologically independent samples, Student’s t-test. g CD8⁺ T cells migration under the attraction of CM from Id1^f/f or Id1^Lyz-KO BMDM-derived TAMs with or without Ccl4 knocking down, n = 3 biologically independent samples, one-way ANOVA test. h Log₂FC heatmap of differentially expressed genes encoding secretory tumor suppressive factors (left). Schematic diagram for the screening of Serpinb2 (right). i SerpinB2 abundance in the CM from Ctrl and ID1^KD TAMs, n = 5 patients, paired t-test. j Serpinb2 abundance in the CM from different groups of RAW 264.7 cells, n = 3 biologically independent samples, Student’s t-test. k Flow cytometry analysis of CD44 in MC38 cells pretreated with CM from Id1^f/f or Id1^Lyz-KO BMDM-derived TAMs, which were infected with or without Serpinb2-specific shRNA lentivirus (Serpinb2^KD), n = 3 biologically independent samples. l, m Images and quantification of MC38 tumor spheres (l), and invasiveness of indicated CT26 cells (m) in the indicated groups as mentioned in (k), n = 3 biologically independent samples, one-way ANOVA test. Scale bar, 100 μm. n, o Tumor volumes (n), representative tumor images, and tumor weight (o) of the indicated groups in CT26 s.c. model, n = 6 mice per group, Student’s t-test. p Representative bioluminescence images and the statistical data of indicated groups in CT26 spleen-liver metastasis model, n = 6 mice per group, Student’s t-test. Source data are provided as a Source Data file.

Fig. 6. ID1 interacts with STAT1 to inhibit the transcription of CCL4 and SerpinB2.

a Screening for ID1-interacting transcription factors responsible for CCL4 and SerpinB2 transcription. b Co-immunoprecipitation (Co-IP) of ID1 with STAT1 in HEK 293T cells transfected with STAT1-DDK and ID1-GFP expressing plasmids. n = 1 biologically independent sample. c Immunofluorescent staining of Id1 and Stat1 in TAM-like RAW264.7 cells. Scale bar, 10 μm, n = 3 biologically independent samples. d Proximity ligation assay (PLA) for defining the interaction between Id1 and Stat1 in TAM-like RAW264.7 cells. Scale bar, 10 μm, n = 10 biologically independent samples. e Immunofluorescence staining of ID1, STAT1 and CD68 in tumor tissue from CRC patients. Scale bar, 20 μm, n = 3 biologically independent samples. f Analysis of the luciferase activity of truncated promoter sequences of Ccl4 and Serpinb2 in Ctrl or Id1^OE RAW264.7 cells, n = 3 biologically independent samples, Student’s t-test. g Effects of Id1 on Stat1 and p-Stat1 subcellular localization under IFN-γ stimulation in RAW264.7 cells, n = 3 biologically independent samples. h The effects of ID1 on the interaction between STAT1 and CRM1, n = 3 biologically independent samples. i Representative images of PLA and the statistical data showing the protein interaction between STAT1 and CRM1 with ID1 ectopic expression or not, n = 20 biologically independent cells. Scale bar, 5 μm. j Representative images of PLA showing the effects of ID1 on the dimerization and subcellular localization of STAT1 under the treatment of leptomycin B (100 nM), Scale bar, 5 μm, n = 3 biologically independent samples. Source data are provided as a Source Data file.

Fig. 7. Targeting ID1 inhibits CRC progression and sensitizes tumor cells to chemotherapy and immunotherapy.

a Schematic diagram for evaluating the therapeutic effects of ML323. b, c Tumor volumes (b), representative images (left) and quantification (right) of CD8⁺ T cell infiltration (c) of the indicated groups presented in (a), n = 8 (b), or 5 (c) mice per group, Welch’s test. Scale bar, 20 μm. d CD44 expression in the indicated groups. e The therapeutic effects of ML323 in the liver metastasis model. f–i Representative gross images of metastatic sites (f), number of hepatic lobes with metastatic tumor (g), the percentage of CD8⁺ T cells (h), and flow cytometry of CD44 in CD45^- Epcam⁺ cells (i) of the indicated groups presented in (e). n = 5 mice per group in f–h, Welch’s test. n = 3 biologically independent samples in (i), Student’s t-test. Scale bar, 1 cm. j, k Therapeutic effects of ML323 in Id1^f/f or Id1^Lyz-KO tumor-bearing mice. Tumor weight (j) and tumor inhibition rate (k) were shown, n = 6 mice per group, one-way ANOVA test (j), Student’s t-test (k). l Schematic diagram for isolating TAMs from indicated mice. m The migration rate of CD8⁺ T cells cocultured with TAMs isolated as presented in (l), n = 5 mice per group. n CD44^high Aldh⁺ cell ratio of CT26 cells cocultured with TAMs isolated as presented in (l), n = 3 mice per group. o Migration of CD8⁺ T cells when cocultured with Id1^f/f or Id1^Lyz-KO TAMs under ML323 (10 μM) treatment, n = 3 biologically independent samples, one-way ANOVA test. p CD44 expression in CT26 cells cocultured with Id1^f/f or Id1^Lyz-KO TAMs under ML323 (10 μM) treatment, n = 3 biologically independent samples. q, r Effects of ML323 on expression of Serpinb2 (q) and Ccl4 (r) in the indicated groups, n = 4 biologically independent samples. s Therapeutic effects of ML323 with 5-FU. Comb: ML323 + 5-FU. t–v Tumor volumes (t), tumor weight (u), and representative tumor images (v) of indicated groups in (s), n = 6 mice per group, one-way ANOVA test. Scale bar, 1 cm. w Therapeutic effects of ML323 with CTLA4-Ab. Comb: ML323 + CTLA4-Ab. x Tumor volumes of indicated groups in (w), n = 6 mice per group, one-way ANOVA test. y Tumor weight of indicated groups in (w), n = 8, 7, 8 and 7 in the groups of Vehicle, ML323, CTLA4-Ab, and Comb, one-way ANOVA test. z Representative tumor images of indicated groups in (w). Scale bar, 1 cm. Elements of a, e, j, l, s, and w are created with BioRender.com. Source data are provided as a Source Data file.