Abstract: Recent data suggest that vascular endothelial growth factor receptor inhibitor (VEGFRi) can enhance the anti-tumor activity of the anti-programmed cell death-1 (anti-PD-1) antibody in colorectal cancer (CRC) with microsatellite stability (MSS). However, the comparison between this combination and standard third-line VEGFRi treatment is not performed, and reliable biomarkers are still lacking. We retrospectively enrolled MSS CRC patients receiving anti-PD-1 antibody plus VEGFRi (combination group, n=54) or VEGFRi alone (VEGFRi group, n=32), and their efficacy and safety were evaluated. We additionally examined the immune characteristics of the MSS CRC tumor microenvironment (TME) through single-cell and spatial transcriptomic data, and an MSS CRC immune cell-related signature (MCICRS) that can be used to predict the clinical outcomes of MSS CRC patients receiving immunotherapy was developed and validated in our in-house cohort. Compared with VEGFRi alone, the combination of anti-PD-1 antibody and VEGFRi exhibited a prolonged survival benefit (median progression-free survival: 4.4 vs. 2.0 months, P=0.0024; median overall survival: 10.2 vs. 5.2 months, P=0.0038) and a similar adverse event incidence. Through single-cell and spatial transcriptomic analysis, we determined ten MSS CRC-enriched immune cell types and their spatial distribution, including naive CD4⁺ T, regulatory CD4⁺ T, CD4⁺ Th17, exhausted CD8⁺ T, cytotoxic CD8⁺ T, proliferated CD8⁺ T, natural killer (NK) cells, plasma, and classical and intermediate monocytes. Based on a systemic meta-analysis and ten machine learning algorithms, we obtained MCICRS, an independent risk factor for the prognosis of MSS CRC patients. Further analyses demonstrated that the low-MCICRS group presented a higher immune cell infiltration and immune-related pathway activation, and hence a significant relation with the superior efficacy of pan-cancer immunotherapy. More importantly, the predictive value of MCICRS in MSS CRC patients receiving immunotherapy was also validated with an in-house cohort. Anti-PD-1 antibody combined with VEGFRi presented an improved clinical benefit in MSS CRC with manageable toxicity. MCICRS could serve as a robust and promising tool to predict clinical outcomes for individual MSS CRC patients receiving immunotherapy.

单细胞联合空间转录组分析揭示免疫细胞相关特征可以预测微卫星稳定结直肠癌患者接受免疫治疗的临床结局

[1]AndréT, ShiuKK, KimTW, et al., 2020. Pembrolizumab in microsatellite-instability-high advanced colorectal cancer. N Engl J Med, 383(23):2207-2218.

[2]ButlerA, HoffmanP, SmibertP, et al., 2018. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol, 36(5):411-420.

[3]ColapricoA, SilvaTC, OlsenC, et al., 2016. TCGAbiolinks: an R/Bioconductor package for integrative analysis of TCGA data. Nucleic Acids Res, 44(8):e71.

[4]CousinS, CantarelC, GueganJP, et al., 2021. Regorafenib-avelumab combination in patients with microsatellite stable colorectal cancer (REGOMUNE): a single-arm, open-label, phase II trial. Clin Cancer Res, 27(8):2139-2147.

[5]DoroshowDB, BhallaS, BeasleyMB, et al., 2021. PD-L1 as a biomarker of response to immune-checkpoint inhibitors. Nat Rev Clin Oncol, 18(6):345-362.

[6]EngstrandJ, NilssonH, StrömbergC, et al., 2018. Colorectal cancer liver metastases - a population-based study on incidence, management and survival. BMC Cancer, 18:78.

[7]FinnRS, QinSK, IkedaM, et al., 2020. Atezolizumab plus bevacizumab in unresectable hepatocellular carcinoma. N Engl J Med, 382(20):1894-1905.

[8]FukuokaS, HaraH, TakahashiN, et al., 2020. Regorafenib plus nivolumab in patients with advanced gastric or colorectal cancer: an open-label, dose-escalation, and dose-expansion phase Ib trial (REGONIVO, EPOC1603). J Clin Oncol, 38(18):2053-2061.

[9]GrotheyA, van CutsemE, SobreroA, et al., 2013. Regorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): an international, multicentre, randomised, placebo-controlled, phase 3 trial. Lancet, 381(9863):303-312.

[10]HoP, MelmsJC, RogavaM, et al., 2023. The CD58-CD2 axis is co-regulated with PD-L1 via CMTM6 and shapes anti-tumor immunity. Cancer Cell, 41(7):1207-1221.e12.

[11]HodiFS, LawrenceD, LezcanoC, et al., 2014. Bevacizumab plus ipilimumab in patients with metastatic melanoma. Cancer Immunol Res, 2(7):632-642.

[12]HouseIG, SavasP, LaiJY, et al., 2020. Macrophage-derived CXCL9 and CXCL10 are required for antitumor immune responses following immune checkpoint blockade. Clin Cancer Res, 26(2):487-504.

[13]JinSQ, Guerrero-JuarezCF, ZhangLH, et al., 2021. Inference and analysis of cell-cell communication using CellChat. Nat Commun, 12:1088.

[14]KarapetisCS, Khambata-FordS, JonkerDJ, et al., 2008. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med, 359(17):1757-1765.

[15]KimRD, KovariBP, MartinezM, et al., 2022. A phase I/Ib study of regorafenib and nivolumab in mismatch repair proficient advanced refractory colorectal cancer. Eur J Cancer, 169:93-102.

[16]LangfelderP, HorvathS, 2008. WGCNA: an R package for weighted correlation network analysis. BMC Bioinf, 9:559.

[17]LeDT, UramJN, WangH, et al., 2015. PD-1 blockade in tumors with mismatch-repair deficiency. N Engl J Med, 372(26):2509-2520.

[18]LeDT, DurhamJN, SmithKN, et al., 2017. Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science, 357(6349):409-413.

[19]LeDT, KimTW, van CutsemE, et al., 2020. Phase II open-label study of pembrolizumab in treatment-refractory, microsatellite instability-high/mismatch repair-deficient metastatic colorectal cancer: KEYNOTE-164. J Clin Oncol, 38(1):11-19.

[20]LeeHO, HongY, EtliogluHE, et al., 2020. Lineage-dependent gene expression programs influence the immune landscape of colorectal cancer. Nat Genet, 52(6):594-603.

[21]LiJ, QinSK, XuRH, et al., 2018. Effect of fruquintinib vs placebo on overall survival in patients with previously treated metastatic colorectal cancer: the FRESCO randomized clinical trial. JAMA, 319(24):2486-2496.

[22]LiYS, ShenZY, ChaiZ, et al., 2023. Targeting MS4A4A on tumour-associated macrophages restores CD8⁺ T-cell-mediated antitumour immunity. Gut, 72(12):2307-2320.

[23]LiebermeisterW, NoorE, FlamholzA, et al., 2014. Visual account of protein investment in cellular functions. Proc Natl Acad Sci USA, 111(23):8488-8493.

[24]MariathasanS, TurleySJ, NicklesD, et al., 2018. TGFβ attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells. Nature, 554(7693):544-548.

[25]McDermottDF, HuseniMA, AtkinsMB, et al., 2018. Clinical activity and molecular correlates of response to atezolizumab alone or in combination with bevacizumab versus sunitinib in renal cell carcinoma. Nat Med, 24(6):749-757.

[26]MintsM, LandinD, NäsmanA, et al., 2021. Tumour inflammation signature and expression of S100A12 and HLA class I improve survival in HPV-negative hypopharyngeal cancer. Sci Rep, 11:1782.

[27]ModestDP, PantS, Sartore-BianchiA, 2019. Treatment sequencing in metastatic colorectal cancer. Eur J Cancer, 109:70-83.

[28]OvermanMJ, McDermottR, LeachJL, et al., 2017. Nivolumab in patients with metastatic DNA mismatch repair-deficient or microsatellite instability-high colorectal cancer (CheckMate 142): an open-label, multicentre, phase 2 study. Lancet Oncol, 18(9):1182-1191.

[29]OvermanMJ, LonardiS, WongKYM, et al., 2018. Durable clinical benefit with nivolumab plus ipilimumab in DNA mismatch repair-deficient/microsatellite instability-high metastatic colorectal cancer. J Clin Oncol, 36(8):773-779.

[30]ParikhAR, SzabolcsA, AllenJN, et al., 2021. Radiation therapy enhances immunotherapy response in microsatellite stable colorectal and pancreatic adenocarcinoma in a phase II trial. Nat Cancer, 2(11):1124-1135.

[31]QiuXJ, MaoQ, TangY, et al., 2017. Reversed graph embedding resolves complex single-cell trajectories. Nat Methods, 14(10):979-982.

[32]RahmaOE, HodiFS, 2019. The intersection between tumor angiogenesis and immune suppression. Clin Cancer Res, 25(18):5449-5457.

[33]ShiR, WangX, WuY, et al., 2022. APOBEC-mediated mutagenesis is a favorable predictor of prognosis and immunotherapy for bladder cancer patients: evidence from pan-cancer analysis and multiple databases. Theranostics, 12(9):4181-4199.

[34]SocinskiMA, JotteRM, CappuzzoF, et al., 2018. Atezolizumab for first-line treatment of metastatic nonsquamous NSCLC. N Engl J Med, 378(24):2288-2301.

[35]SungH, FerlayJ, SiegelRL, et al., 2021. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 71(3):209-249.

[36]WuYC, YangSX, MaJQ, et al., 2022. Spatiotemporal immune landscape of colorectal cancer liver metastasis at single-cell level. Cancer Discov, 12(1):134-153.

[37]ZhangL, YuX, ZhengLT, et al., 2018. Lineage tracking reveals dynamic relationships of T cells in colorectal cancer. Nature, 564(7735):268-272.

[38]ZhangL, LiZY, SkrzypczynskaKM, et al., 2020. Single-cell analyses inform mechanisms of myeloid-targeted therapies in colon cancer. Cell, 181(2):442-459.e29.

[39]ZhengLT, QinSS, SiW, et al., 2021. Pan-cancer single-cell landscape of tumor-infiltrating T cells. Science, 374(6574):abe6474.

[40]ZhengRS, ZhangSW, SunKX, et al., 2023. Cancer statistics in China, 2016. Chin J Oncol, 45(3):212-220 (in Chinese).

[41]ZhouH, ZhuLY, SongJ, et al., 2022. Liquid biopsy at the frontier of detection, prognosis and progression monitoring in colorectal cancer. Mol Cancer, 21:86.