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.
Highlights
The uncontrollable growth of malignant cells within the tumor generates mechanical compression forces that enhance the capacity of cell motility and invasiveness (Shieh, 2011; Chaudhuri et al, 2018; Kalli et al, 2018)
The plasticity of cell membrane protrusions as an adaption to the cellular microenvironment plays a critical role during cell migration. These results demonstrated that a typically adhesive cell membrane protrusion phenotype can be induced by mechanical compression force in high-invasive cells (MBAMD-231) (Hooshmand et al, 2013; Aumsuwan et al, 2016; FIGURE 4 | Compression force regulates cell protrusions to transition from no protrusions to lamellipodia or blebs in MDA-MB-231 and MCF7 cells on a lowadhesive surface. (A, B) Representative images of the three cell membrane protrusions, including (1) no protrusions, (2) lamellipodia, and (3) blebs, in (A) MDA-MB-231 and (B) MCF7 cells on a low-adhesive surface with or without compression force
Our results show that in 2D low-adhesion conditions, compression force induces a transition from no protrusions to lamellipodia for MDA-MB-231 and no protrusions to blebs for MCF7
Summary
The uncontrollable growth of malignant cells within the tumor generates mechanical compression forces that enhance the capacity of cell motility and invasiveness (Shieh, 2011; Chaudhuri et al, 2018; Kalli et al, 2018). Tse et al (2012) seeded cells on a conventional Transwell insert membrane and covered them with a thin film agarose cushion, allowing oxygen and nutrition transmission This was followed by placing an opaque piston of specific weight on top of the gel to apply a constant force on the cells. Kim et al (2017) embedded suspension cells into agarose gel to form a thin film on the bottom of the dish and applied force to the cells using a cube filled with iron ball bearings on the top They proved that compression force triggers mechanotransduction to induce vascular endothelial growth factor A (VEGFA) overexpression, promoting tumor progression. Their applications are limited in end-point analysis because the setup of pressure applied by loads is opaque and real-time observation under microscopy cannot be performed
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