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. 2022 Sep;10(9):e004717.
doi: 10.1136/jitc-2022-004717.

Complement downregulation promotes an inflammatory signature that renders colorectal cancer susceptible to immunotherapy

Carsten Krieg  1   2 , Lukas M Weber  3   4 , Bruno Fosso  5 , Marinella Marzano  5 , Gary Hardiman  6 , Monica M Olcina  7 , Enric Domingo  7 , Sahar El Aidy  8 , Khalil Mallah  9 , Mark D Robinson  3   4 , Silvia Guglietta  10   11   12
Affiliations

Complement downregulation promotes an inflammatory signature that renders colorectal cancer susceptible to immunotherapy

Carsten Krieg et al. J Immunother Cancer. 2022 Sep.

Abstract

Background and aims: The role of inflammatory immune responses in colorectal cancer (CRC) development and response to therapy is a matter of intense debate. While inflammation is a known driver of CRC, inflammatory immune infiltrates are a positive prognostic factor in CRC and predispose to response to immune checkpoint blockade (ICB) therapy. Unfortunately, over 85% of CRC cases are primarily unresponsive to ICB due to the absence of an immune infiltrate, and even the cases that show an initial immune infiltration can become refractory to ICB. The identification of therapy supportive immune responses in the field has been partially hindered by the sparsity of suitable mouse models to recapitulate the human disease. In this study, we aimed to understand how the dysregulation of the complement anaphylatoxin C3a receptor (C3aR), observed in subsets of patients with CRC, affects the immune responses, the development of CRC, and response to ICB therapy.

Methods: We use a comprehensive approach encompassing analysis of publicly available human CRC datasets, inflammation-driven and newly generated spontaneous mouse models of CRC, and multiplatform high-dimensional analysis of immune responses using microbiota sequencing, RNA sequencing, and mass cytometry.

Results: We found that patients' regulation of the complement C3aR is associated with epigenetic modifications. Specifically, downregulation of C3ar1 in human CRC promotes a tumor microenvironment characterized by the accumulation of innate and adaptive immune cells that support antitumor immunity. In addition, in vivo studies in our newly generated mouse model revealed that the lack of C3a in the colon activates a microbiota-mediated proinflammatory program which promotes the development of tumors with an immune signature that renders them responsive to the ICB therapy.

Conclusions: Our findings reveal that C3aR may act as a previously unrecognized checkpoint to enhance antitumor immunity in CRC. C3aR can thus be exploited to overcome ICB resistance in a larger group of patients with CRC.

Keywords: Gastrointestinal Neoplasms; Immunity, Innate; Immunotherapy.

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Conflict of interest statement

Competing interests: None declared.

Figures

Figure 1
Figure 1
C3aR methylation and downregulation in patients with CRC. (A) C3aR expression in patients with RC and colon cancer from four independent datasets (TGCA: 22 healthy, 55 RC; Skrzypczak cohort: 24 healthy, 45 CoAdK; Sebates-Bellver cohort: 32 healthy, 25 CoAd; Ki cohort: 41 healthy, 76 CoAdK). (B) N shelf, (C) S shore and (D) N shore methylation of c3ar1 in patients with CRC. Significance was calculated using t-test. *=P>0.05, ****=P>0.0001. C3aR, C3a receptor; CoAd, colon adenoma; CoAdK, colon adenocarcinoma; CRC, colorectal cancer; RC, rectal cancer.
Figure 2
Figure 2
Loss of C3aR switches tumorigenesis from small intestine to colon in APCMin/+ mice and promotes increased immune cell infiltration. (A) Tumor numbers in the colon of APCMin/+ and APCMin/+/C3aR−/− from weeks 5 to 28 weeks of age. (B) Representative picture of tumor number and distribution in the colon of APCMin/+ and APCMin/+/C3aR−/− mice. Single-cell suspensions from cLP of APCMin/+ and APCMin/+/C3aR−/− mice were analyzed by flow cytometry. Shown are the total number of (C) CD4+ T cells, (D) Th17 cells, (E) Th1 cells, (F) Th1/Th17 cells, (G) CD8+ T cells, and (H) Tc cells (CD3+CD8+IFN-γ+). A significance was calculated using two-way analysis of variance with Bonferroni post-test and a minimum of 10 mice/group. In panels, (C–H) Results are pooled from two independent experiments with a minimum of nine mice/group. Significance was calculated using unpaired t-test. *P<0.05, **P<0.01, ****P<0.0001. C3aR, C3a receptor; cLP, colon lamina propria; IFN-γ, interferon gamma; IL, interleukin; ns, not significant.
Figure 3
Figure 3
Transplantation of the APCMin/+/C3aR−/− microbiota transfers inflammation and colon tumorigenesis to APCMin/+ mice. (A) Treatment scheme for 5-week-old APCMin/+ mice (recipients) transplanted with the microbiota of 12-week-old APCMin/+ or APCMin/+/C3aR−/− mice (donors). (B) Tumor counts in the colon of recipient mice. (C–H) Flow cytometry analysis of cLP infiltrating lymphocytes showing total cell number of (C) CD4+ T cells, (D) Th17 cells, (E) Th1 cells, (F) Th1/17 cells, (G) CD8+ T cells, and (H) cytotoxic T (Tc) cells. Shown are the results of two independent experiments with 11 mice/group. Significance was calculated using unpaired t-test. **P<0.01, ***P<0.001, ****P<0.0001. C3aR, C3a receptor; cLP, colon lamina propria; FMT, fecal microbiota transplantation; IFN-γ, interferon gamma; IL, interleukin
Figure 4
Figure 4
Loss of C3aR affects the composition of the fecal microbiota in APCMin/+mice. Bacterial DNA was extracted from the feces of 8-and 12-week-old APCMin/+, APCMin/+/C3aR−/−, C3aR−/− and WT mice, and bacterial profiling was performed by sequencing the V5-V6 hypervariable of 16S rDNA using Illumina MiSeq platform. (A) Plots showing alpha diversity evaluated by observed ASV, Faith, and Shannon Index in 8-and 12-week-old mice. (B) Principal Coordinates Analysis (PCoA) showing the beta diversity assessed by the three inferred Beta Diversity metrics (weighted UniFrac, unweighted UniFrac, and Bray-Curtis) in mice 8 and 12 weeks old. (C) Doughnut charts showing phylum abundance at 8 and 12 weeks. Four to eight mice/group were used. Significance was calculated using (A) the Kruskal-Wallis test, followed by a pairwise Wilcoxon as post-test; (B) PERmutational Multivariate ANOVA (PERMANOVA) test; and (C) DESeq R-package. C3aR, C3a receptor.
Figure 5
Figure 5
Tumors in APCMin/+/C3aR−/− show enrichment in innate and adaptive inflammatory pathways. (A) Heatmap from log counts per million values using data from colon polyps from APCMin/+ and APCMin/+/C3aR−/− mice. (B) Summary plot showing the number of significant differentially expressed genes in polyps of APCMin/+ and APCMin/+/C3aR−/− mice. The plot displays log fold change against log counts per million for each gene. The red points represent significant differentially expressed genes, and the horizontal blue lines indicate a twofold increase or decrease in expression. (C) Visualization of the gene-associated GO biological processes in the polyps. (D) Volcano plot showing the fold change magnitude for all genes differentially expressed in the polyps of APCMin/+C3aR−/− versus APCMin/+ mice (n=4). Significantly upregulated and downregulated genes belonging to the GO biological processes (B) are highlighted in yellow. Vertical and horizontal red dotted lines indicate the threshold. Significance was calculated using the edgeR function decideTestsDGE with Benjamini-Hochberg correction for FDR; a default FDR threshold of 0.05 and a log2 fold change threshold of 0.6 were applied. C3aR, C3a receptor; FDR, false discovery rate; GO, Gene Ontology.
Figure 6
Figure 6
C3aR expression in patients with cancer affects the infiltration of innate and adaptive immune cells. Plots showing negative (A–C) and positive (B–D) correlation between C3ar1 expression and innate and adaptive immune cell populations assessed by CIBERSORT in a retrospective cohort of 231 patients with rectal cancer in the S:CORT WS3 Grampian Set and 97 patients with colon cancer in the FOxTROT cohort. Data were visualized and downloaded through the private S:CORT cBioPortal and analyzed using the Spearman and Pearson correlation tests. C3aR, C3a receptor; DC, dendritic cell; NK, natural killer.
Figure 7
Figure 7
Lack of C3aR confers susceptibility to treatment with a-PD-1 treatment. (A) Tumor number and load in individual APCMin/+/C3aR−/− mice treated for 5 weeks with a-PD1 expressed as % reduction over the mice treated with the control antibody. (B) Visualization of 70 000 pooled events from a-PD1 and control mice using the uMAP algorithm. (C) The functional profile of cell clusters that were significantly upregulated or downregulated in anti-PD1-treated mice compared with control was visualized by using the bimatrix approach. The results of two independent experiments with a minimum of 11 mice/group are shown. DC, dendritic cell; IFN, interferon; IL, interleukin; ILC, innate lymphoid cells; NK, natural killer.
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