B7-H3 (CD276) has two isoforms (2Ig and 4Ig), no confirmed cognate receptor, and physiological functions that remain elusive. While differentially expressed on many solid tumors correlating with poor survival, mechanisms of how B7-H3 signals in cis (tumor cell) versus in trans (immune cell co-regulator) to elicit pro-tumorigenic phenotypes remain poorly defined. Herein, we characterized a tumorigenic and signaling role for tumor cell-expressed 4Ig-B7-H3, the dominant human isoform, in gynecological cancers that could be abrogated upon CRISPR/Cas9 knockout of B7-H3; tumorigenesis was rescued upon re-expression of 4Ig-B7-H3. Size exclusion chromatography revealed dimerization states for the extracellular domains of both human 4Ig- and murine 2Ig-B7-H3. mEGFP lifetimes of expressed 4Ig-B7-H3-mEGFP fusions determined by FRET-FLIM assays confirmed close-proximity interactions of 4Ig-B7-H3 and identified two distinct homo-FRET lifetime populations, consistent with monomeric and homo-dimer interactions. In live cells, bioluminescence imaging of 4Ig-B7-H3-mediated split luciferase complementation showed dimerization of 4Ig-B7-H3. To separate basal from dimer state activities in the absence of a known receptor, C-terminus (cytosolic) chemically-induced dimerization of 4Ig-B7-H3 increased tumor cell proliferation and cell activation signaling pathways (AKT, Jak/STAT, HIF1α, NF-κβ) significantly above basal expression of 4Ig-B7-H3 alone. These results revealed a new, dimerization-dependent intrinsic tumorigenic signaling role for 4Ig-B7-H3, likely acting in cis, and provide a therapeutically-actionable target for intervention of B7-H3-dependent tumorigenesis.

B7-H3 ( CD276 ) 有两种同种型（2Ig 和 4Ig），没有已证实的同源受体，其生理功能仍然难以捉摸。虽然在许多实体瘤上差异表达与较差的生存率相关，但 B7-H3顺式（肿瘤细胞）与反式（免疫细胞共调节因子）信号如何引发促肿瘤表型的机制仍不清楚。在此，我们描述了肿瘤细胞表达的 4Ig-B7-H3（主要的人类亚型）在妇科癌症中的致瘤和信号传导作用，该作用可通过 CRISPR/Cas9 敲除 B7-H3 消除。 4Ig-B7-H3 重新表达后肿瘤发生得以挽救。尺寸排阻色谱揭示了人 4Ig-和鼠 2Ig-B7-H3 细胞外结构域的二聚化状态。通过 FRET-FLIM 测定确定的表达的 4Ig-B7-H3-mEGFP 融合体的 mEGFP 寿命证实了 4Ig-B7-H3 的紧密相互作用，并鉴定了两个不同的同源 FRET 寿命群体，与单体和同源二聚体相互作用一致。在活细胞中，4Ig-B7-H3 介导的分裂荧光素酶互补的生物发光成像显示 4Ig-B7-H3 的二聚化。为了在缺乏已知受体的情况下分离基础活性和二聚体状态活性，C 端（胞质）化学诱导的 4Ig-B7-H3 二聚化增加了肿瘤细胞增殖和细胞激活信号通路（AKT、Jak/STAT、HIF1α、NF） -κβ) 显着高于单独的 4Ig-B7-H3 的基础表达。这些结果揭示了 4Ig-B7-H3 的新的、二聚化依赖性内在致瘤信号传导作用，可能以顺式作用，并为干预 B7-H3 依赖性肿瘤发生提供了治疗上可行的靶点。