Quantitative FRET Imaging to Visualize the Invasiveness of Live Breast Cancer Cells

Matrix metalloproteinases (MMPs) remodel tumor microenvironment and promote cancer metastasis. Among the MMP family proteases, the proteolytic activity of the pro-tumorigenic and pro-metastatic membrane-type 1 (MT1)-MMP constitutes a promising and targetable biomarker of aggressive cancer tumors. In this study, we systematically developed and characterized several highly sensitive and specific biosensors based on fluorescence resonant energy transfer (FRET), for visualizing MT1-MMP activity in live cells. The sensitivity of the AHLR-MT1-MMP biosensor was the highest and five times that of a reported version. Hence, the AHLR biosensor was employed to quantitatively profile the MT1-MMP activity in multiple breast cancer cell lines, and to visualize the spatiotemporal MT1-MMP activity simultaneously with the underlying collagen matrix at the single cell level. We detected a significantly higher level of MT1-MMP activity in invasive cancer cells than those in benign or non-invasive cells. Our results further show that the high MT1-MMP activity was stimulated by the adhesion of invasive cancer cells onto the extracellular matrix, which is precisely correlated with the cell’s ability to degrade the collagen matrix. Thus, we systematically optimized a FRET-based biosensor, which provides a powerful tool to detect the pro-invasive MT1-MMP activity at single cell levels. This readout can be applied to profile the invasiveness of single cells from clinical samples, and to serve as an indicator for screening anti-cancer inhibitors.基质金属蛋白酶（Matrix metalloproteinases, MMPs）可重塑肿瘤微环境并促进癌症转移。在MMP家族蛋白酶中，兼具促瘤性与促转移性的膜型1（membrane-type 1, MT1）-MMP的蛋白水解活性，是侵袭性肿瘤极具潜力且可靶向的生物标志物。本研究系统性开发并表征了多款基于荧光共振能量转移（FRET）的高灵敏特异性生物传感器，用于可视化活细胞内的MT1-MMP活性。其中AHLR-MT1-MMP生物传感器的灵敏度最高，为已报道同类传感器的五倍。据此，研究团队利用该AHLR生物传感器对多种乳腺癌细胞系中的MT1-MMP活性进行了定量分析，并在单细胞水平上同时可视化了MT1-MMP的时空活性及其对应的下层胶原基质。研究发现，侵袭性癌细胞中的MT1-MMP活性显著高于良性或非侵袭性癌细胞。本研究结果进一步显示，侵袭性癌细胞黏附于细胞外基质可刺激MT1-MMP的高活性，这与细胞降解胶原基质的能力精准相关。综上，本研究系统性优化了一款基于FRET的生物传感器，该工具可在单细胞水平上有效检测促侵袭性MT1-MMP活性。该检测手段可用于分析临床样本中单细胞的侵袭能力，亦可作为抗癌抑制剂筛选的检测指标。