In Situ Single Cell Proteomics Reveals Circulating Tumor Cell Heterogeneity during Treatment,ACS Nano

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In Situ Single Cell Proteomics Reveals Circulating Tumor Cell Heterogeneity during Treatment
ACS Nano ( IF 15.8 ) Pub Date : 2021-07-06 , DOI: 10.1021/acsnano.0c10008
K Kamil Reza ₁ , Shuvashis Dey ₁ , Alain Wuethrich ₁ , Jing Wang ₁ , Andreas Behren _{2,

3} , Fiach Antaw ₁ , Yuling Wang ₄ , Abu Ali Ibn Sina ₁ , Matt Trau _{1,

5}

Affiliation

Cancer is a dynamic disease with heterogenic molecular signatures and constantly evolves during the course of the disease. Single cell proteomic analysis could offer a suitable pathway to monitor cancer cell heterogeneity and deliver critical information for the diagnosis, recurrence, and drug-resistant mechanisms in cancer. Current standard techniques for proteomic analysis such as ELISA, mass spectrometry, and Western blots are time-consuming, expensive, and often require fluorescence labeling that fails to provide accurate information about the multiple protein expression changes at the single cell level. Herein, we report a surface-enhanced Raman spectroscopy-based simple microfluidic device that enables the screening of single circulating tumor cells (CTC) in a dynamic state to precisely understand the heterogeneous expression of multiple protein biomarkers in response to therapy. It further enables identifying intercellular heterogeneous expression of CTC surface proteins which would be highly informative to identify the cancer cells surviving treatment and potentially responsible for drug resistance. Using a bead and cell line-based model system, we successfully detect single bead and single cell spectra when flowed through the device. Using SK-MEL-28 melanoma cells, we demonstrate that our system is capable of monitoring heterogeneous expressions of multiple surface protein markers (MCSP, MCAM, and LNGFR) before and during drug treatment. Integrating a label-free electrochemical system with the device, we also monitor the expression of an intracellular protein (here, BRAF^V600E) under drug treatment. Finally, we perform a longitudinal study with 15 samples from five different melanoma patients who underwent therapy. We find that the average expression of receptor proteins in a patient fails to determine the therapy response particularly when the disease progresses. However, single CTC analysis with our device shows a high level of intercellular heterogeneity in the receptor expression profiles of patient-derived CTCs and identifies heterogeneity within CTCs. More importantly, we find that a fraction of CTCs still shows a high expression of these receptor proteins during and after therapy, indicating the presence of resistant CTCs which may evolve after a certain time and progress the disease. We believe this automated assay will have high clinical importance in disease diagnosis and monitoring treatment and will significantly advance the understanding of cancer heterogeneity on the single cell level.

中文翻译：

原位单细胞蛋白质组学揭示治疗期间循环肿瘤细胞的异质性

癌症是一种具有异质性分子特征的动态疾病，并在疾病过程中不断演变。单细胞蛋白质组学分析可以提供合适的途径来监测癌细胞的异质性，并为癌症的诊断、复发和耐药机制提供关键信息。目前用于蛋白质组学分析的标准技术，如 ELISA、质谱法和蛋白质印迹法，既费时又昂贵，而且通常需要荧光标记，无法提供有关单细胞水平的多种蛋白质表达变化的准确信息。在此处，我们报告了一种基于表面增强拉曼光谱的简单微流体装置，该装置能够在动态状态下筛选单个循环肿瘤细胞 (CTC)，以精确了解多种蛋白质生物标志物对治疗的异质表达。它进一步能够识别 CTC 表面蛋白的细胞间异质表达，这将为识别治疗后存活的癌细胞和可能导致耐药性的癌细胞提供大量信息。使用基于珠子和细胞系的模型系统，我们在流经设备时成功检测到单珠子和单细胞光谱。使用 SK-MEL-28 黑色素瘤细胞，我们证明我们的系统能够在药物治疗之前和期间监测多种表面蛋白标记物（MCSP、MCAM 和 LNGFR）的异质表达。^V600E) 正在接受药物治疗。最后，我们对来自五名接受过治疗的不同黑色素瘤患者的 15 个样本进行了纵向研究。我们发现患者受体蛋白的平均表达无法确定治疗反应，尤其是当疾病进展时。然而，使用我们的设备进行的单个 CTC 分析显示患者来源的 CTC 的受体表达谱中存在高水平的细胞间异质性，并确定了 CTC 内的异质性。更重要的是，我们发现一小部分 CTC 在治疗期间和治疗后仍显示这些受体蛋白的高表达，表明存在耐药性 CTC，这些耐药性 CTC 可能会在一定时间后进化并使疾病进展。

更新日期：2021-07-27

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