研究背景及目的：在我国以及全球，结直肠癌一直是公共卫生健康严重的负担，其发病率和死亡率均居于前列。尽管随着肠镜筛查的应用以及癌症早诊早治项目的推广，仍有约25%的患者在首次就诊时被诊断为转移性结直肠癌；同时，约有50%的患者在术后会出现疾病进展。传统的手术、放化疗、介入以及靶向治疗等带来的生存获益较为有限，转移性结直肠癌的5年生存率仍不足15%。因此，寻求新的药物治疗是提高晚期结直肠癌总生存的关键。大量研究证实以PD-1/PD-L1抑制剂为主的免疫治疗，可显著改善晚期结直肠癌的总生存期，但只有微卫星不稳定型结直肠癌能从中获益，而该部分患者仅占晚期结直肠癌的5%。因此，如何寻找其他潜在的免疫治疗靶点是破解当前结直肠癌免疫治疗瓶颈的关键。前期，我们利用TCGA数据库分析了B7-CD28家族成员在结直肠癌的表达情况，发现B7-H3的表达量最高，且相比于正常肠黏膜组织，B7-H3在肠癌组织的表达量明显升高，提示B7-H3可作为潜在的免疫治疗靶点。在这里，我们拟在大样本量的组织芯片队列中进一步探究B7-H3在结直肠癌的表达及预后意义；并通过体外实验，初步探究其促进血管形成的作用，为后期的临床转化应用奠定基础。

方法：回顾性收集2010-2014年在我院接受根治性手术的结直肠癌患者，调取相应的病理组织蜡块制作组织芯片队列。进行B7-H3、B7-H4、PD-L1以及CD3、CD8、CD45RO等分子的免疫组化染色，并分析上述分子在结直肠癌的表达、临床病理特征以及预后意义。体外实验构建B7-H3的过表达和敲降的稳转结直肠癌细胞系，分析其对人脐静脉内皮细胞成管作用的影响。

结果：本研究队列共纳入805例患者，平均年龄58.3岁，男性446例（55.4%）；其中I期101例（12.5%），II期241例（29.9%），III期319例（39.6%）以及IV期144例（17.9%）。免疫组化结果显示，50.9%的结直肠癌原发灶呈现B7-H3的阳性表达，明显高于B7-H4（29.1%）和PD-L1（29.2%）；且B7-H3在III-IV期患者中的表达明显高于I-II期。B7-H3与B7-H4以及PD-L1的共表达率分别仅为6.3%和5.7%。尽管超过16%的患者出现了不一致的表达，B7-H3、B7-H4和PD-L1在原发灶以及配对转移灶的表达仍呈正相关性（ρ＞0.6; p＜0.001）。生存分析显示，B7-H3与无病生存期降低显著相关，而B7-H4、PD-L1与预后无相关性。B7-H3与高密度浸润的CD45RO+ T细胞相关，而B7-H4、PD-L1与高密度浸润的CD3+ T细胞相关。体外实验证实，过表达B7-H3会促进人脐静脉内皮的成管能力，而敲降B7-H3会抑制该作用。

结论：与B7-H4、PD-L1相比，B7-H3在结直肠癌中的阳性表达率更高，而B7-H3与B7-H4、PD-L1共表达率较低。同时，B7-H3在原发灶中的表达与其在配对转移灶的表达呈正相关。B7-H3与肿瘤的恶性程度、不良预后、CD45RO+ T细胞浸润以及血管形成等密切相关，提示B7-H3可作为结直肠癌潜在治疗的靶点。

Background and Aims: Colorectal cancer (CRC) has been a serious public health burden in China and globally, with a high rate of morbidity and mortality. Despite the promotion of cancer screening and the improvement of people's health awareness, about a quarter of patients are diagnosed with synchronous metastases and ~50% of cases suffer from metastases after initial radical surgery during long-term follow-up. For metastatic CRC, the use of conventional surgery, chemotherapy, ablative methods and targeted therapy has improved survival. However, the prognosis of metastatic CRC is still poor, with 5-year survival of less than 15%. Thus, there is an urgent need for new therapies to treat metastatic CRC patients. A large number of studies have verified that immunotherapy, including PD-1/PD-L1 inhibitors, has significantly improved the prognosis of metastatic CRC. However, only patients with deficient mismatch repair can benefit from immunotherapy, and this group only accounts for 5% of metastatic CRC. Thus, targeting other immune checkpoints may offer more immunotherapeutic choices for patients, who are excluded from current PD-1/PD-L1 inhibitor therapy. During the early stage of this study, we found B7-H3 to have the highest expression among B7-CD28 family members in CRC and B7-H3 expression was elevated in CRC tissues compared to normal mucosal tissues from TCGA database, indicating that B7-H3 may be a potential immunotherapy target. Here, the expression, prognostic significance and angiogenesis of B7-H3 was further investigated in our large CRC cohort and in vitro, laying the foundation for the later clinical transformation application.

Methods: Patients diagnosed with CRC that were treated from 2010 to 2014 at the Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College were retrospectively obtained, and the corresponding HE-stained paraffin blocks were collected to construct the tissue microarrays. B7-H3, B7-H4, and PD-L1 protein levels and CD3+, CD8+, and CD45RO+ T cells were evaluated in tissue microarrays, and the relationships between immune markers, patient characteristics, and survival outcomes were determined. In vitro, stable B7-H3 overexpression and knockdown CRC cells were constructed to explore the effect on angiogenesis of human umbilical vein endothelial cells (HUVECs).

Results: A total of 805 patients were enrolled in this study, with the mean age of 58.3 years and 446 (55.4%) males. The stage I, II, III, IV patients were 101 (12.5%), 241 (29.9%), 319 (39.6%) and 144 (17.9%) respectively. B7-H3 (50.9%) was detected in more primary tumors than B7-H4 (29.1%) or PD-L1 (29.2%), and elevated B7-H3 expression was associated with advanced overall stage. Co-expression of B7-H3 only with B7-H4 or PD-L1 was infrequent in primary tumors (6.3%, 5.7%, respectively). Although discordant expression in primary tumors versus metastases was identified in over 16% of patients, B7-H3, B7-H4 and PD-L1 in primary tumors were positively correlated with their respective expression at metastatic sites (ρ＞0.6; p<0.001). No significant relationships between B7-H4 and PD-L1 and survival were observed; however, B7-H3 overexpression in primary tumors was significantly related to decreased disease-free survival. A positive relationship between B7-H3 expression and high density of CD45RO T-cell was observed in primary tumors, whereas B7-H4 and PD-L1 overexpression were related to CD3 T-cell infiltration. In vitro, overexpression of B7-H3 promotes the angiogenesis of HUVECs, however, knockdown of B7-H3 can abolish this effect.

Conclusions: Compared with B7-H4 and PD-L1, B7-H3 expression exhibited a higher prevalence and co-expression of B7-H3 with B7-H4 and PD-L1 was infrequent in primary tumors. In addition, B7-H3 expression in primary tumors correlated with their corresponding expression in metastatic sites. Moreover, B7-H3 expression in primary tumors was significantly related to advanced overall stage, worse prognosis, CD45RO T-cell infiltration and angiogenesis, supporting B7- H3 as a potential immunotherapeutic target in CRC patients.