A Portable Controllable Compressive Stress Device to Monitor Human Breast Cancer Cell Protrusions at Single-Cell Resolution

Abstract

In vitro devices offer more numerous methods than in vivo models to investigate how cells respond to pressure stress and quantify those responses. Several in vitro devices have been developed to study the cell response to compression force. However, they are unable to observe morphological changes of cells in real-time. There is also a concern about cell damage during the process of harvesting cells from 3D gels. Here we report a device employing transparent, thin gel layers to clamp cells between the interfaces and applied a controllable compression force by stacking multiple layers on the top. In this approach, cells can be monitored for alteration of cellular protrusions, whose diversity has been proven to promote cancer cell dissemination, with single-cell resolution under compression force. Furthermore, p-Rac-1 and rhodamine staining on the device directly to confirm the actin filaments of lamellipodia. The method was able to fulfill real-time live-cell observation at single-cell resolution and can be readily used for versatile cell analysis. MDA-MB-231 and MCF7 breast cancer cells were utilized to demonstrate the utility of the device, and the results showed that the stimuli of compression force induce MDA-MB-231 and MCF7 to form lamellipodia and bleb protrusions, respectively. We envision the device may be used as a tool to explore mechanisms of membrane protrusion transitions and to screen drug candidates for inhibiting cancer cell protrusion plasticity for cancer therapy.

Rac1 promotes kidney collecting duct repair by mechanically coupling cell morphology to mitotic entry

“…S29). To assess the mechanistic relevance of this finding, we used a well-established in vitro cell compression model where confluent cells are covered with a 1:1 mixture of low gelling agarose and full medium creating a thin gel layer with a defined height and weight that provides low but constant pressure deforming the cell layer (fig, S30, A and B) (76)(77)(78)(79). Long (4 hours)-but not short (1 hour)-cell compression reduced Rac1 and increased Hace1 levels (fig.…”

“…Control plates were not subjected to stretch. Cell compression was performed as previously described (76)(77)(78)(79). Briefly, low-gelling temperature agarose (Sigma-Aldrich, #A9045) was dissolved in PBS at 3% concentration by heating in a microwave oven, followed by mixing the solution with DMEM cell culture medium with 20% FBS in a 1:1 ratio to form a 1.5% agarose gel solution containing full medium (DMEM and 10% FBS), and poured into a round petri dish.…”