RPPA profiling of ES8 Ewing sarcoma xenografts after Endoglin-targeting antibody-drug conjugate treatments. RPPA profiling of ES8 Ewing sarcoma xenografts after Endoglin-targeting antibody-drug conjugate treatments

Endoglin (EDG) is a cell surface protein with an important role in the establishment of neo-angiogenesis and vasculogenic mimicry. EDG is part of the transforming growth factor-β (TGF-β) family, acting as an important co-receptor. EDG is shed from the cell surface into the extracellular compartment by matrix metalloproteinase 14 (MMP14), in its soluble form (sEDG). Both transmembrane and soluble forms of EDG exert important signaling functions in the development of new blood vessels and tumour progression. To better understand the role of EDG in Ewing sarcoma (ES), a deadly neoplasm of late childhood and adolescence, we test the efficacy of OMTX703, an endoglin-targeting antibody-drug conjugate in ES8 xenograft. Having determined an optimal dose for OMTX703, an additional experiment was conducted to assess the mechanism(s) of OMTX703 action and its potential mechanism(s) of resistance following a 2-week exposure to OMTX703 at 0, 10, 30, and 60 mg/kg; 246 proteins were assessed by reverse-phase protein array (RPPA). Analysis of variance (ANOVA), Pearson’s correlation as distance metric and Ward’s linkage as the clustering method using a false discovery rate (FDR) of 0.01, identified 60 proteins that discriminated between treatment groups (Matrix#1-Normalized Values). To investigate the proteomic changes associated with the heightened clinical activity of the 60 mg/kg dose, a secondary analysis was performed, which grouped the 10 mg/kg OMTX703 samples and the 10 mg/kg OMTX003 ones with the placebo-treated samples (Matrix#2-Normalized Values). Using a FDR of 0.0001, an absolute log2 fold change of 1.5, Pearson’s correlation as distance metric and Ward’s linkage as the clustering method, 22 proteins were discriminately identified between the 3 treatment groups (Matrix#2-Normalized Values). Notably, a protein regulator of altered metabolism (RPS6) was exclusively upregulated following OMTX703 (60mg/kg), and a second metabolism biomarker (LDHA) was down-expressed in the 30 and 60 mg/kg-treated groups. Conversely, BRD4 was one of about a dozen proteins that were preferentially down-regulated in samples treated only by 60 mg/kg. Overall design: An RPPA analysis of ES8 xenografts treated with OMTX003 (n=8, a nacked mAb anti endoglin at 10 mg/kg), OMTX703 (an endoglin-targeting antibody-drug conjugate with cytolisin at 10, 30 and 60 mg/kg (n=8 each)) or control vehicle (n=7) were performed simultaneously using the same array. Lysates were processed, spotted onto nitrocellulose-coated FAST slides, probed with 300 validated primary antibodies, and detected using earlier methods. MicroVigene software program (VigeneTech) was used for automated spot identification, background correction, and individual spot-intensity determination. Expression data was normalized for possible unequal protein loading, taking into account the signal intensity for each sample for all antibodies tested. Raw log2 intensity values were normalized for global protein expression by median centering across 246 antibodies tested. Principal component analysis was used to check for a batch effect and feature-by-feature two-sample t-tests were used to assess differences between treatment and control groups. We also used feature-by-feature one-way analysis of variance (ANOVA) followed by the Tukey test to perform pair comparisons for all groups. Beta-uniform mixture models were used to fit the resulting p value distributions to adjust for multiple comparisons. The cutoff p values and number of significant proteins were computed for several different false discovery rates (FDRs).

内皮糖蛋白（Endoglin, EDG）是一种细胞表面蛋白，在新生血管生成与血管生成拟态的建立过程中发挥重要作用。EDG属于转化生长因子-β（TGF-β）家族成员，作为关键共受体参与相关生物学过程。基质金属蛋白酶14（MMP14）可将EDG从细胞表面剪切释放至细胞外间隙，形成可溶性形式（sEDG）。EDG的跨膜形式与可溶性形式均在新生血管发育及肿瘤进展中发挥重要信号调控功能。
为深入解析EDG在尤因肉瘤（ES，一种好发于儿童及青少年的致死性恶性肿瘤）中的作用，本研究针对ES8异种移植模型，检测靶向内皮糖蛋白的抗体药物偶联物OMTX703的抗肿瘤功效。在确定OMTX703的最优给药剂量后，本研究额外开展实验，以评估OMTX703的作用机制，以及在以0、10、30、60 mg/kg剂量连续给药2周后产生的潜在耐药机制。研究采用反相蛋白微阵列（RPPA）对246种蛋白进行检测。以错误发现率（FDR）=0.01为阈值，通过方差分析（ANOVA）、以皮尔逊相关系数作为距离度量、沃德聚类法进行聚类分析，最终筛选出60种可区分不同给药组的蛋白（矩阵#1-标准化值）。
为探究60 mg/kg剂量组展现出更强临床活性背后的蛋白质组学变化，本研究开展二次分析：将10 mg/kg OMTX703样本、10 mg/kg OMTX003样本与安慰剂处理组样本合并分组（矩阵#2-标准化值）。以FDR=0.0001、绝对log2倍数变化≥1.5、皮尔逊相关系数作为距离度量、沃德聚类法进行聚类分析，最终在3个给药组间筛选出22种差异显著蛋白（矩阵#2-标准化值）。值得注意的是，代谢调控蛋白RPS6仅在OMTX703（60 mg/kg）处理组中显著上调，而另一代谢生物标志物LDHA在30 mg/kg与60 mg/kg给药组中表达下调。与之相反，BRD4是仅在60 mg/kg给药组中优先下调的十余种蛋白之一。
实验整体设计：本研究同步采用相同的蛋白微阵列，对分别接受OMTX003（n=8，10 mg/kg剂量的裸抗内皮糖蛋白单克隆抗体）、OMTX703（靶向内皮糖蛋白的细胞溶素型抗体药物偶联物，10、30、60 mg/kg剂量，每组n=8）以及对照溶剂（n=7）处理的ES8异种移植瘤开展RPPA分析。样本裂解液经处理后点样至硝酸纤维素包被的FAST玻片，采用300种经过验证的一抗进行孵育，并通过此前报道的方法完成信号检测。采用MicroVigene软件（VigeneTech公司）实现自动化斑点识别、背景校正以及单斑点强度定量。为校正蛋白上样量不均一带来的偏差，本研究基于所有待测一抗的各样本信号强度，对表达数据进行标准化处理；原始log2强度值通过对246种待测抗体的信号进行中位数中心化处理，以实现全局蛋白表达水平标准化。采用主成分分析检测批次效应，通过逐特征两样本t检验评估给药组与对照组的表达差异。此外，本研究还采用逐特征单因素方差分析（ANOVA）结合图基检验完成所有组间的两两比较，并通过β均匀混合模型拟合所得P值分布以校正多重比较偏差。针对多种不同的错误发现率阈值，计算对应的临界P值以及显著差异蛋白的数量。