A Novel Ex Vivo Method for Visualizing Live-Cell Calcium Response Behavior in Intact Human Tumors

The functional impact of intratumoral heterogeneity has been difficult to assess in the absence of a means to interrogate dynamic, live-cell biochemical events in the native tissue context of a human tumor. Conventional histological methods can reveal morphology and static biomarker expression patterns but do not provide a means to probe and evaluate tumor functional behavior and live-cell responsiveness to experimentally controlled stimuli. Here, we describe an approach that couples vibratome-mediated viable tissue sectioning with live-cell confocal microscopy imaging to visualize human parathyroid adenoma tumor cell responsiveness to extracellular calcium challenge. Tumor sections prepared as 300 micron-thick tissue slices retain viability throughout a >24 hour observation period and retain the native architecture of the parental tumor. Live-cell observation of biochemical signaling in response to extracellular calcium challenge in the intact tissue slices reveals discrete, heterogeneous kinetic waveform categories of calcium agonist reactivity within each tumor. Plotting the proportion of maximally responsive tumor cells as a function of calcium concentration yields a sigmoid dose-response curve with a calculated calcium EC50 value significantly elevated above published reference values for wild-type calcium-sensing receptor (CASR) sensitivity. Subsequent fixation and immunofluorescence analysis of the functionally evaluated tissue specimens allows alignment and mapping of the physical characteristics of individual cells within the tumor to specific calcium response behaviors. Evaluation of the relative abundance of intracellular PTH in tissue slices challenged with variable calcium concentrations demonstrates that production of the hormone can be dynamically manipulated ex vivo. The capability of visualizing live human tumor tissue behavior in response to experimentally controlled conditions opens a wide range of possibilities for personalized ex vivo therapeutic testing. This highly adaptable system provides a unique platform for live-cell ex vivo provocative testing of human tumor responsiveness to a range of physiological agonists or candidate therapeutic compounds.

在缺乏可在人类肿瘤原生组织微环境中探究动态活细胞生化事件的手段时，肿瘤内异质性（intratumoral heterogeneity）的功能影响始终难以评估。常规组织学方法可揭示组织形态与静态生物标志物表达谱，但无法提供探究、评估肿瘤功能行为以及活细胞对实验可控刺激物响应性的途径。本文报道一种将振动切片机（vibratome）介导的活性组织切片技术与活细胞共聚焦显微镜成像相结合的方法，用于可视化人甲状旁腺腺瘤肿瘤细胞对细胞外钙刺激的响应性。制备为300微米厚的组织切片可在超过24小时的观测周期内保持活性，并保留亲本肿瘤的原生组织结构。在完整组织切片中对细胞外钙刺激引发的生化信号通路进行活细胞观测，可揭示每个肿瘤内钙激动剂反应性的离散且异质性的动力学波形类别。以最大响应肿瘤细胞的比例随钙浓度的变化作图，可得到S型剂量反应曲线，其计算得到的钙半数有效浓度（EC50）显著高于已发表的野生型钙敏感受体（calcium-sensing receptor, CASR）敏感性参考值。随后对经功能评估的组织标本进行固定与免疫荧光分析，可将肿瘤内单个细胞的物理特征与特定钙响应行为进行对齐和映射。对不同钙浓度刺激下的组织切片中细胞内甲状旁腺激素（PTH）相对丰度进行评估，可证明该激素的产生可在离体（ex vivo）条件下被动态调控。在实验可控条件下可视化活体人类肿瘤组织行为的能力，为个性化离体治疗测试开辟了广阔的可能性。这套高度可适配的系统为人类肿瘤对一系列生理激动剂或候选治疗化合物的离体激发测试提供了独特的研究平台。