Shaping immune landscape of colorectal cancer by cholesterol metabolites

Cancer immunotherapies have achieved unprecedented success in clinic, but they remain largely ineffective in some major types of cancer, such as colorectal cancer with microsatellite stability (MSS CRC). It is therefore important to study tumor microenvironment of resistant cancers for developing new intervention strategies. In this study, we identify a metabolic cue that determines the unique immune landscape of MSS CRC. Through secretion of distal cholesterol precursors, which directly activate RORγt, MSS CRC cells can polarize T cells toward Th17 cells that have well-characterized pro-tumor functions in colorectal cancer. Analysis of large human cancer cohorts revealed an asynchronous pattern of the cholesterol biosynthesis in MSS CRC, which is responsible for the abnormal accumulation of distal cholesterol precursors. Inhibiting the cholesterol biosynthesis enzyme Cyp51, by pharmacological or genetic interventions, reduced the levels of intratumoral distal cholesterol precursors and suppressed tumor progression through a Th17-modulation mechanism in preclinical MSS CRC models. Our study therefore reveals a novel mechanism of cancer–immune interaction and an intervention strategy for the difficult-to-treat MSS CRC.

(A) Intensity of sterols in the interstitial fluid of human MSS CRC tumors (T-IF) and paired adjacent normal tissues (N-IF).Fold change of the mean level of each sterol in tumor interstitial fluid vs normal tissue interstitial fluid (T/N) is labeled under each graph (n = 36).(B) Th1 induction in the presence of desmosterol at different concentrations.Naive CD4 + T cells were stimulated with plate-bound anti-CD3/CD28 and cytokines to induce Th1 differentiation, in the presence of desmosterol or vehicle control.Th1 (IFNγ + gated on CD4 + ) polarization were analyzed on Day 4 (n = 4).(C) Treg induction in the presence of desmosterol at different concentrations.Naive CD4 + T cells were stimulated with plate-bound anti-CD3/CD28 and cytokines to induce Treg differentiation, in the presence of desmosterol or vehicle control.Treg (Foxp3 + gated on CD4 + ) polarization were analyzed on Day 4 (n = 3).(D) Th17 induction in the presence of desmosterol at different concentrations.Naive CD4 + T cells from Rorc -/-(RORγt-KO), Rorc +/-(RORγt-HT) or Rorc +/+ (WT) mice were stimulated plate-bound anti-CD3/CD28 and cytokines to induce Th17, in the presence of desmosterol or vehicle control.Th17 (IL-17a + gated on CD4 + ) percentage was analyzed on Day 4 (n = 4).(E, F) CD8 + T cell function in the presence of desmosterol at different concentrations.Naive CD8 + T cells were stimulated with plate-bound anti-CD3/CD28, in the presence of desmosterol or vehicle control.CD8 + T cell function (E) IFNγ + gated on CD8 + ; (F) GzmB + gated on CD8 + were analyzed on Day 2 (n = 4).(G) CD8 + T cell activation in the presence of desmosterol at different concentrations.Naive CD8 + T cells were stimulated with plate-bound anti-CD3/CD28, in the presence of desmosterol or vehicle control.CD8 + T cell activation (CD44 + gated on CD8 + ) were analyzed on Day 1 (n = 4).(H) Absolute quantification of zymosterol and 7-dehydrocholesterol in the interstitial fluid of human MSS CRC tumors (T-IF) and paired adjacent normal tissues (N-IF).Fold change of the mean level of indicated sterol in tumors interstitial fluid vs normal tissue interstitial fluid (IF T/N) is labeled under graph (n = 9).(I, J) Intensity of indicated distal cholesterol precursors in the control blank medium (Ctrl) and the conditioned media of mouse MSS CRC cell CT26 (n = 10), mouse MSI CRC cell MC38 (n = 10), human MSS CRC cell Caco2 (n = 6), and human MSI CRC cell HCT116 (n = 6).Two-tailed unpaired t test was used when variances were similar, whereas a two-tailed unpaired t test with Welch's correction was used when variances were different.(K) Intensities of the 13 C-labeled sterols in CT26 and MC38 cells (n = 6).Two-tailed unpaired t test was used when variances were similar, whereas a two-tailed unpaired t test with Welch's correction was used when variances were different.(L) Th17 induction in the presence of the conditioned medium of MC38 cells (n = 3).(M) Intensity of the sterols in the plasma samples of human MSS CRC patients before (Pre) and after (Post) surgical resection of tumors.Fold change of the mean level of each sterol in plasma before vs after surgical resection (Pre/ Post) is labeled under each graph (n = 8).Data information: in (A-M), data are presented as mean ± SEM.In (B-G, L) two-tailed unpaired Student's t test; data are representative of two independent experiments.In (A, H, M), two-tailed Wilcoxon matched-pairs signed-rank test.P levels < 0.05*, < 0.01**, < 0.001***, < 0.0001****.Source data are available online for this figure.

Figure EV2 .
Figure EV2.Accumulation of distal cholesterol precursors in the tumor microenvironment of MSS CRC.

Figure
Figure EV3.MSS CRC and MSI CRC had different programs of cholesterol metabolism.(A) Left: GSEA of cholesterol biosynthesis pathway in MSI-H CRC tumors (n = 89) and MSS CRC tumors (n = 294) in COAD.Right: GSEA rank metric score and transcriptional levels of enzymes in the cholesterol biosynthesis pathway.Blue and red represents downregulation and upregulation in tumor.P value of gene expression between tumor and normal tissue (two tailed Mann-Whitney test) is labeled by the side of each gene.(ns P > 0.05, *P ≤ 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).(B) Transcriptional levels of LDLR in MSI tumors (MSI-H-T, n = 89) and MSS tumors (MSS-T, n = 294) from the COAD database.Whiskers denote minimum to maximum.Box indicates the interquartile range (25-75%); center line indicates the median."+" indicates mean.Two tailed Mann-Whitney test.Source data are available online for this figure.

Figure EV4 .
Figure EV4.Recapitulation of the asynchronous upregulation of the cholesterol biosynthesis pathway in MSI CRC MC38 cells.(A) Schematic illustration of experimental procedure.(B) Transcriptional levels of Sqle, Cyp51 and in wildtype (WT) and the genetic manipulated MC38 as described in panel A (nSrebp2), n = 3. (C) Intensity of indicated distal cholesterol precursors in the blank medium (Ctrl), and the conditioned media of MC38 cells, n = 8.Two-tailed unpaired Student's t test if data fitted a normal distribution, Mann-Whitney test if data did not fit a normal distribution.(D) Th17 induction with CM of above-mentioned cells or CT26-CM as positive control, n = 3, two-tailed unpaired Student's t test.Data information: in (B-D), data are presented as mean ± SEM.P levels < 0.01**, < 0.001***, < 0.0001****.Source data are available online for this figure.

Figure EV5 .
Figure EV5.Single-cell analysis of CT26 tumors (WT or Cyp51KO) in BALB/c mice as described in Fig. 6. (A) Heatmap showing the gene expression levels of chemokines/growth factors and related receptors across all cell clusters.Z-score was calculated by mean expression level and indicated by color.(B) Violin plot showing the MDSC score in all cell clusters.The neutrophils and macrophages are major contributor of the suppressive function of myeloid derived suppressive cells (MDSC) (Dysthe and Parihar, 2020) in tumor.(C) Heatmap showing the expression levels of tumor growth/metastasis related genes across myeloid cell populations.Z-score was calculated by mean expression level and indicated by color.(D) Heatmap showing the expression levels of immunosuppression related genes across myeloid cell populations.Z-score was calculated by mean expression level and indicated by color.(E) Heatmap showing the expression levels of immune checkpoint genes across all cell populations.Z-score was calculated by mean expression level and indicated by color.