Poster S16 - Microfluidic Phenotypic Sorting Enables Discovery That Metastatic Breast Cancer Cells with High Strain Energy are Less Proliferative and Less Invasive

Introduction: For metastasis to occur, cancer cells must possess a few advantageous mechanical properties that allow them to survive the multiple steps of the metastatic cascade. It has been shown by multiple groups that metastatic cancer cells are often softer than their benign counterparts [1]. However, a causal link between metastasis and a cells’ mechanical properties has yet to be established. If such a link were to be identified, it could provide a mechanism through which metastasis could be slowed or stopped. As metastasis is responsible for the vast majority of cancer-related deaths, such a finding would be of great relevance [2].

In an effort to explore this link, a multiplexed microfluidic device was used to sort breast cancer cells by their stiffness and size [3]. Stiffer and larger cells cannot easily squeeze under the device’s ridges and are translated along the diagonal ridges resulting in an altered trajectory. Meanwhile, smaller and softer cells proceed through the device relatively undeflected (Figure 1a and 1b). The sorting process was performed repeatedly on the heterogenous MDA-MB-231 breast cancer cell line until stable subpopulations of different mechanical properties were established. Upon conclusion of this serial sorting process, the resulting subpopulations were compared for their nuclear size, in vitro proliferation rate, and invasive abilities after spheroid formation. This comparison brings us closer to understanding the role of cell size and stiffness in metastasis.

Materials and

Methods: MDA-MB-231 cells were fractionated into subsets of cells that differed in mechanical properties. Cells that demonstrated a low strain energy (a product of volume, stiffness, and experienced strain under compression) exited the second microfluidic outlet (LSE subpopulation: softer, smaller), while cells with high strain energy were sorted to the fifth outlet (HSE subpopulation: stiffer, larger). These sorted cells were expanded and re-sorted, establishing stable subpopulations. Atomic force microscopy, micropipette aspiration, and brightfield imaging confirmed these subpopulations differed in strain energy (Figure 1c).
A Click-iT EdU proliferation assay was used to compare the subpopulation proliferation rates. Cells were exposed to EdU for 2 hours, and those which entered the S phase of the cell cycle incorporated the nucleoside into their DNA. Cells were fixed and permeabilized and those with incorporated EdU were fluorescently labeled. After counterstaining nuclei with DAPI, cells were imaged using an LSM 800 confocal fluorescent microscope. Nuclear area and proliferation rate (ratio of EdU-positive to total cells) was determined through QuPath software analysis (Figure 2a).

Additionally, each of the three populations were organized into spheroids (5,000 cells each) using a hanging drop method and encapsulated in 3 or 4.5 mg/mL collagen. These spheroids provided an in vitro model for potential invasive and metastatic ability. Spheroids were imaged over two days post-encapsulation with a BioTek Cytation 5 Cell Imaging Multi-mode Reader (Figure 3a). ImageJ software was used to quantify spheroid outgrowth, radial distance (measured at regular intervals), and area (Figure 3b).

Results, Conclusions, and Discussions: As the nucleus is a large physical barrier to confined-space cell migration [4], nuclear size can impact migration, navigation, and invasion in the tumor microenvironment. When comparing the nuclear sizes of the different cell subtypes, the HSE cells’ nuclear size was approximately two times that of the LSE cells and the parental MDA-MB-231 cells (respectively: 151.2±57.9 μm2, 75.9±21.4 μm2, 75.6±21.6 μm2) (Figure 2b).

Because alterations to nuclear size have been associated with cancer malignancy and proliferation [5], the subpopulations’ proliferation rates were compared. The HSE cells proliferated most slowly (46.4±5.0%), while the LSE cells proliferated most quickly (52.6±7.4%) (Figure 2c). The unsorted MDA-MB-231 line demonstrated an intermediate proliferation rate (49.8±5.7%). The LSE cell proliferation rate is significantly faster than that of HSE cells (p=0.02, Tukey post-hoc test).

Beyond differences in cell proliferation, we hypothesized cell mechanics would also affect cell migration and invasion. The cell subpopulations exhibited vast differences in spheroid outgrowth: LSE cells demonstrated the most outgrowth (average radial projection length of 162.3±101.8 μm in 3 mg/mL collagen, 136.0±86.4 μm in 4.5 mg/mL), HSE cells demonstrated least outgrowth (average radial projection length of 74.7±56.3 μm in 3 mg/mL and 74.2±59.4 μm in 4.5 mg/mL), and the parental cells demonstrated moderate outgrowth (average radial length of 98.0±68.0 μm in 3 mg/mL and 121.2±83.4 μm in 4.5 mg/mL) (Figures 3c). Upon normalizing final spheroid outgrowth areas to their initial areas, the HSE spheroids’ final outer area increased by an average of 161±15%, the parental spheroids by 218±11%, and the LSE spheroids by 356±28% (Figure 3d).

These subpopulations and the parental cell line thus demonstrated phenotypic differences in cell proliferation and spheroid outgrowth. We are continuing to explore in vitro and in vivo differences in cell behavior (including investigating a link between nuclear size and genomic differences), with the goal of establishing a causal link between cell mechanical properties and metastatic ability to improve future cancer treatments.

Acknowledgements (Optional): This work was supported by NIH Award 1F32CA281162-01.

References
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