Department of Chemistry, CB 3290, University of North Carolina, Chapel Hill, NC 27599, USA.
Integr Biol (Camb). 2014 Feb;6(2):164-74. doi: 10.1039/c3ib40249e.
Single-cell methodologies are revealing cellular heterogeneity in numerous biological processes and pathologies. For example, cancer cells are characterized by substantial heterogeneity in basal signaling and in response to perturbations, such as drug treatment. In this work, we examined the response of 678 individual U937 (human acute myeloid leukemia) cells to an aminopeptidase-inhibiting chemotherapeutic drug (Tosedostat) over the course of 95 days. Using a fluorescent reporter peptide and a microfluidic device, we quantified the rate of reporter degradation as a function of dose. While the single-cell measurements reflected ensemble results, they added a layer of detail by revealing unique degradation patterns and outliers within the larger population. Regression modeling of the data allowed us to quantitatively explore the relationships between reporter loading, incubation time, and drug dose on peptidase activity in individual cells. Incubation time was negatively correlated with the number of peptide fragment peaks observed, while peak area (which was proportional to reporter loading) was positively correlated with both the number of fragment peaks observed and the degradation rate. Notably, a statistically significant change in the number of peaks observed was identified as dose increased from 2 to 4 μM. Similarly, a significant difference in degradation rate as a function of reporter loading was observed for doses ≥2 μM compared to the 1 μM dose. These results suggest that additional enzymes may become inhibited at doses >1 μM and >2 μM, demonstrating the utility of single-cell data to yield novel biological hypotheses.
单细胞方法正在揭示许多生物学过程和病理学中的细胞异质性。例如，癌细胞在基础信号和对药物治疗等干扰的反应方面存在很大的异质性。在这项工作中，我们在 95 天的时间内研究了 678 个单独的 U937（人急性髓系白血病）细胞对一种氨肽酶抑制化疗药物（Tosedostat）的反应。使用荧光报告肽和微流控设备，我们定量测量了报告物降解的速度作为剂量的函数。虽然单细胞测量反映了总体结果，但它们通过揭示较大群体中的独特降解模式和异常值，增加了一层细节。对数据的回归建模使我们能够定量探索报告物负载、孵育时间和药物剂量与单个细胞中肽酶活性之间的关系。孵育时间与观察到的肽片段峰的数量呈负相关，而峰面积（与报告物负载成正比）与观察到的片段峰的数量和降解速率呈正相关。值得注意的是，当剂量从 2 到 4 μM 增加时，观察到的峰数的统计学显著变化被识别出来。同样，与 1 μM 剂量相比，在 2 μM 以上剂量下观察到的降解速率存在显著差异。这些结果表明，在剂量大于 1 μM 和大于 2 μM 时，可能会有更多的酶被抑制，这表明单细胞数据可以产生新的生物学假设。
