Purpose

Emergent cancer cells likely secrete factors that inhibit anti-tumor immunity. To identify such factors, we applied a functional assay with proteomics to an immunotherapy resistant syngeneic mouse melanoma model. Four secreted factors were identified that potentially mediate immunosuppression and could become targets for novel immunotherapies. We tested for consistent clinical correlates in existing human data and verified in vivo whether knocking out tumor cell production of these factors improved immune-mediated control of tumor growth.

Methods

Existing human data was analyzed for clinical correlates. A CRISPR/Cas9 approach to generate knockout cell lines and a kinetic analysis leveraging a Markov Chain Monte Carlo (MCMC) approach quantified the various knockouts’ effect on cells’ intrinsic growth rate. Flow cytometry was used to characterize differences in immune infiltration.

Results

While all four gene products were produced by malignant melanocytes, only increased CCN4 expression was associated with reduced survival in primary melanoma patients. In immunocompetent C57BL/6 mice the CCN4 knockout increased survival while the other knockouts had no effect. This survival advantage was lost when the CCN4 knockout cells were injected into immunocompromised hosts, indicating that the effect of CCN4 may be immune mediated. Parameter estimation from the MCMC analysis shows that CCN4 was the only knockout tested that decreased the net tumor growth rate in immunocompetent mice. Flow cytometry showed an increase in NK cell infiltration in CCN4 knockout tumors.

Conclusions

The results suggest that CCN4 is a mediator of immunosuppression in the melanoma tumor microenvironment and a potential collateral immunotherapy target.

中文翻译：

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CCN4、DNMT3A、PTPN11 和 SPARC 作为抗肿瘤免疫抑制剂的头对头比较

目的

新出现的癌细胞可能会分泌抑制抗肿瘤免疫的因子。为了识别这些因素，我们将蛋白质组学的功能测定应用于免疫治疗耐药的同基因小鼠黑色素瘤模型。确定了四种可能介导免疫抑制的分泌因子，并可能成为新型免疫疗法的靶标。我们测试了现有人类数据中一致的临床相关性，并在体内验证了敲除肿瘤细胞产生这些因子是否可以改善免疫介导的肿瘤生长控制。

方法

对现有的人类数据进行了临床相关性分析。采用 CRISPR/Cas9 方法生成敲除细胞系，并利用马尔可夫链蒙特卡罗 (MCMC) 方法进行动力学分析，量化了各种敲除对细胞内在生长速率的影响。流式细胞术用于表征免疫浸润的差异。

结果

虽然所有四种基因产物都是由恶性黑色素细胞产生的，但只有 CCN4 表达增加与原发性黑色素瘤患者的生存率降低相关。在免疫功能正常的 C57BL/6 小鼠中，CCN4 敲除可提高存活率，而其他敲除则没有效果。当CCN4敲除细胞被注射到免疫功能低下的宿主中时，这种生存优势就消失了，这表明CCN4的作用可能是免疫介导的。MCMC 分析的参数估计表明，CCN4 是唯一经过测试的可降低免疫活性小鼠中肿瘤净生长率的敲除试验。流式细胞术显示 CCN4 敲除肿瘤中 NK 细胞浸润增加。

结论

结果表明，CCN4 是黑色素瘤肿瘤微环境中免疫抑制的介质，也是潜在的附带免疫治疗靶点。