Pérez-Treviño Perla, la Cerda Héctor Hernández-De, Pérez-Treviño Jorge, Fajardo-Ramírez Oscar Raúl, García Noemí, Altamirano Julio
Tecnologico de Monterrey, Escuela de Medicina, Av. Morones Prieto No. 3000 Pte., Monterrey, NL, Mexico, 64710.
Tecnologico de Monterrey, Escuela de Medicina, Av. Morones Prieto No. 3000 Pte., Monterrey, NL, Mexico, 64710.
Transl Oncol. 2018 Jun;11(3):672-685. doi: 10.1016/j.tranon.2018.03.004. Epub 2018 Apr 5.
Patients with breast cancer (BC) overexpressing HER2 (HER2+) are selected for Trastuzumab treatment, which blocks HER2 and improves cancer prognosis. However, HER2+ diagnosis, by the gold standard, immunohistochemistry, could lead to errors, associated to: a) variability in sample manipulation (thin 2D sections), b) use of subjective algorithms, and c) heterogeneity of HER2 expression within the tissue. Therefore, we explored HER2 3D detection by multiplexed imaging of Affibody-Quantum Dots conjugates (Aff-QD), ratiometric analysis (RMA) and thresholding, using BC multicellular tumor spheroids (BC-MTS) (~120 μm of diameter) as 3D model of BC. HER2+, HER2- and hybrid HER2+/- BC-MTS (mimicking heterogeneous tissue) were incubated simultaneously with two Aff-QD probes (anti-HER2 and negative control (NC), respectively, (1:1)). Confocal XY sections were recorded along the Z distance, and processed by automatized RMA (anti-HER2 Aff-QD/ NC). Quantifying the NC fluorescence allowed to predict the fraction of non-specific accumulation of the anti-HER2 probe within the thick sample, and resolve the specific HER2 level. HER2 was detected up to 30μm within intact BC-MTS, however, permeabilization improved detection up to 70μm. Specific HER2 signal was objectively quantified, and HER2 3D-density of 9.2, 48.3 and 30.8% were obtained in HER2-, HER2+ and hybrid HER2+/- permeabilized BC-MTS, respectively. Therefore, by combining the multiplexing capacity of Aff-QD probes and RMA, we overcame the challenge of non-specific probe accumulation in 3D samples with minimal processing, yielding a fast, specific spatial HER2 detection and objective quantification.
过表达HER2（HER2+）的乳腺癌（BC）患者会被选作曲妥珠单抗治疗对象，该药物可阻断HER2并改善癌症预后。然而，按照金标准免疫组织化学进行的HER2+诊断可能会出现误差，这些误差与以下因素有关：a）样本处理的变异性（薄二维切片），b）主观算法的使用，以及c）组织内HER2表达的异质性。因此，我们利用BC多细胞肿瘤球体（BC-MTS，直径约120μm）作为BC的三维模型，通过Affibody-量子点缀合物（Aff-QD）的多重成像、比率分析（RMA）和阈值处理来探索HER2的三维检测。将HER2+、HER2-和HER2+/-混合型BC-MTS（模拟异质性组织）同时与两种Aff-QD探针（分别为抗HER2和阴性对照（NC），比例为1:1）孵育。沿Z轴距离记录共聚焦XY切片，并通过自动化RMA（抗HER2 Aff-QD/NC）进行处理。对NC荧光进行定量可预测抗HER2探针在厚样本中的非特异性积累比例，并解析特定的HER2水平。在完整的BC-MTS中，HER2的检测深度可达30μm，然而，通透处理可将检测深度提高至70μm。对特定的HER2信号进行了客观定量，在通透处理后的HER2-、HER2+和HER2+/-混合型BC-MTS中，HER2的三维密度分别为9.2%、48.3%和30.8%。因此，通过结合Aff-QD探针的多重检测能力和RMA，我们以最少的处理克服了三维样本中非特异性探针积累的挑战，实现了快速、特异性的HER2空间检测和客观定量。
