Characteristics of Cancer-Cell Filopodia on Substrates with Different Stiffness Observed by Structured Illumination Nano-Profilometry
Date Issued
2012
Date
2012
Author(s)
Liou, Yu-Ren
Abstract
In this thesis, we observed the filopodia formation and dynamics of lung cancer cells (CL1-5) on substrates with different stiffness by structured illumination nano-profilometry (SINAP).
Cell migration plays a key role in various physiological and pathological processes, such as tissue development, wound healing, angiogenesis, inflammation, and cancer metastasis. Most of the previous studies focused on the effects of environmental chemical cues on the cell migration. Recently, some studies have reported that the cell migration could also respond to the environmental mechanical stimuli; for example, the substrate stiffness is one of the most important mechanical stimuli.
The traditional method to measure the cell motility is to trace the moving distance of a cell for an accumulated time. This method however should take a long time thus hard to be exerted in vivo and in real-time. Therefore, a way to improve is to observe the cell morphology and relate it to the cell motility. Evidences have shown that the formation of filopodia is related to the cell migration. Furthermore, the CL1-5, which has been shown to be the high invasive lung cancer cell line, has more filopodia than CL1-0, which is low invasive lung cancer cell line. In this study, we made the correlation between the formation of filopodia and the substrate stiffness. We speculated that the substrate stiffness could modulate the filopodia formation and dynamics through the effects on cell adhesions.
The structured illumination nano-profilometry (SINAP), which is developed by Dr. Lee’s group, has lateral resolution of 140 nm and depth resolution of 6 nm. This super-resolution microscopy is advanced in the observation of filopodia, whose diameters are only between 100 and 300 nm. Without fluorescence labeling and two-dimensional scanning, the imaging of SINAP is high speed and very suitable for live-cells observation. However, these techniques require culturing cells on materials of refractive index close to that of glass, while most studies regarding the effects of mechanical cues on cellular dynamics were conducted on hydrogel-based substrates.
Here we report the development of culturing substrates of tunable rigidity and refractive index suitable for SINAP studies. Polyvinyl chloride (PVC)-based substrates were mixed with a softener called Di(isononyl) Cyclohexane-1,2-Dicarboxylate (DINCH). The volume ratios of PVC to DINCH were varied from 1:1 to 3:1. The Young’s moduli of the resulting substrates ranged from 20 kPa to 60 kPa. Human lung adenocarcinoma cells CL1-5 were cultured on the composite substrates and cell viability was examined using the MTT assay. The results showed that the PVCs were successfully applied to the SINAP system and had high biocompatibility. Thus in this thesis, the observation of filopodia formation and dynamics were conducted on the PVC substrates.
The results of cells on different stiffness showed that the cells on soft substrates had more filopodial density and length than those on the stiff substrates. Inhibiting the contractility of the stress fibers with blebbistatin treatment increased the density and length of filopodia on stiff substrates, and mimicked the situation of the cells on the soft substrates. Therefore, a possible but indirect mechanism of the effects of the substrate stiffness on filopodia formation might be the formation of the stress fibers, which antagonize to the formation of the filopodia. On the other respects, we also measured the stretching rate of filopodia on different stiffness. The results showed no difference in the protrusion rate and retraction rate between the soft and stiff substrates. This might resulted from the interval times we took between images. Thus in the future, we should shorten the interval time to observe the filopodia with higher stretching speed.
From this study, we can understand the effects of the substrate stiffness on the filopodial density and length. Further studies are needed to determine the underlying mechanism of these effects. With their medical importance, the results would shed new light on the therapy and diagnosis of the cancer disease.
Subjects
Filopodia
SINAP
Lung Cancer Cell
Substrate Stiffness
SDGs
Type
thesis
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