Mungai Rozanne W, Hartman Roger J, Jolin Grace E, Piskorowski Kevin W, Billiar Kristen L
Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, MA, 01605, USA.
, Worcester, MA, 01605, USA.
Sci Rep. 2024 Dec 28;14(1):31007. doi: 10.1038/s41598-024-82191-3.
Multicellular spheroids embedded in 3D hydrogels are prominent in vitro models for 3D cell invasion. Yet, quantification methods for spheroid cell invasion that are high-throughput, objective and accessible are still lacking. Variations in spheroid sizes and the shapes of the cells within render it difficult to objectively assess invasion extent. The goal of this work is to develop a high-throughput quantification method of cell invasion into 3D matrices that minimizes sensitivity to initial spheroid size and cell spreading and provides precise integrative directionally-dependent metrics of invasion. By analyzing images of fluorescent cell nuclei, invasion metrics are automatically calculated at the pixel level. The initial spheroid boundary is segmented and automated calculations of the nuclear pixel distances from the initial boundary are used to compute common invasion metrics (i.e., the change in invasion area, mean distance) for the same spheroid at a later timepoint. We also introduce the area moment of inertia as an integrative metric of cell invasion that considers the invasion area as well as the pixel distances from the initial spheroid boundary. Further, we show that principal component analysis can be used to quantify the directional influence of a stimuli to invasion (e.g., due to a chemotactic gradient or contact guidance). To demonstrate the power of the analysis for cell types with different invasive potentials and the utility of this method for a variety of biological applications, the method is used to analyze the invasiveness of five different cell types. In all, implementation of this high-throughput quantification method results in consistent and objective analysis of 3D multicellular spheroid invasion. We provide the analysis code in both MATLAB and Python languages as well as a GUI for ease of use for researchers with a range of computer programming skills and for applications in a variety of biological research areas such as wound healing and cancer metastasis.
嵌入三维水凝胶中的多细胞球体是用于三维细胞侵袭的重要体外模型。然而，仍缺乏高通量、客观且易于使用的球体细胞侵袭定量方法。球体大小和内部细胞形状的变化使得难以客观评估侵袭程度。这项工作的目标是开发一种高通量的细胞向三维基质侵袭的定量方法，该方法可将对初始球体大小和细胞铺展的敏感性降至最低，并提供精确的、综合的、方向依赖性的侵袭指标。通过分析荧光细胞核图像，在像素水平自动计算侵袭指标。分割初始球体边界，并使用从初始边界自动计算的核像素距离来计算同一球体在稍后时间点的常见侵袭指标（即侵袭面积变化、平均距离）。我们还引入了面积惯性矩作为细胞侵袭的综合指标，该指标同时考虑了侵袭面积以及距初始球体边界的像素距离。此外，我们表明主成分分析可用于量化刺激对侵袭的方向影响（例如，由于趋化梯度或接触导向）。为了证明该分析方法对具有不同侵袭潜能的细胞类型的有效性以及该方法在各种生物学应用中的实用性，该方法用于分析五种不同细胞类型的侵袭性。总之，这种高通量定量方法的实施可对三维多细胞球体侵袭进行一致且客观的分析。我们以MATLAB和Python语言提供分析代码以及一个图形用户界面，以便具有不同计算机编程技能水平的研究人员易于使用，并应用于各种生物学研究领域，如伤口愈合和癌症转移。
