Boyden chamber-based method for characterizing the distribution of adhesions and cytoskeletal structure in HT1080 fibrosarcoma cells

Abstract

A 2D model was previously presented that describes the gliding motility of human fibrosarcoma cells. The model was based on the observation that adhesions are present only on the outer rim of the leading lamella of the semicircular cell. The present model describes the organization of adhesions and the cytoskeleton of migrating HT1080 fibrosarcoma and LX2 hepatic stellate cells in three dimensions. The migration assays were performed in a modified Boyden chamber using fibronectin, Matrigel, or collagen I as chemoattractants. The distribution of the adhesions was analyzed by confocal laser scanning microscope, and following decoration with heavy meromyosin, the organization of actin filaments was analyzed by electron microscopy. Double labeling was performed to study the relationship of the actin and vimentin filament network in the moving cells. Vinculin containing adhesions were observed only at the front of the cell in the form of a ring while passing through a filter pore of the Boyden chamber. Actin filaments were present only below the plasma membrane, except the very tip of the leading lamella. Vimentin intermediate filaments were localized around the cell nucleus behind the actin filament-rich lamella. This paper describes a model of the organization of adhesions and the cytoskeleton of migrating cells in the Boyden chamber. The model is based on the observation that adhesions are present only at the leading edge of the cell. The results extend the earlier 2D model of cell locomotion into 3D.

Rear-polarized Wnt5a-receptor-actin-myosin-polarity (WRAMP) structures promote the speed and persistence of directional cell migration

“…We next asked whether WRAMP structures could be observed in nonmelanoma cells, which also express MCAM and in which Wnt5a signaling has been shown to promote cell migration. One such model is HT1080 fibrosarcoma cells, which are often used in studies of migration and invasion ( Michl et al , 2005 ; Ripka et al , 2007 ; Schwartz et al , 2013 ; Wei et al , 2013 ; Tovari et al , 2014 ). HT1080 cells were treated with Wnt5a for 30 min and then fixed and immunostained for endogenous MCAM and myosin IIB, with phalloidin staining of F-actin.…”

A Role for the Cavin-3/Matrix Metalloproteinase-9 Signaling Axis in the Regulation of PMA-Activated Human HT1080 Fibrosarcoma Cell Neoplastic Phenotype

“…[53][54][55][56] Conceptual limitations of our current study must be forthrightly acknowldged as the results presented and conclusions raised may not necessarily be generalized to other cellular models. However, HT-1080 fibrosarcoma cells are considered as a relatively well-established cellular model and validated for studies related to in vivo mice models, 57 methodology development, 58 PMA impact assessment, 59 and in models assessing metastasis and cancer cell motility. 60 In conclusion, our study still demonstrates that cavin-3 expression potentiated a decrease in PMA-mediated AKT dephosphorylation and that cavin-3 loss of function restored increased MMP-9 expression upon PMA treatment.…”

Cell Culture Based in vitro Test Systems for Anticancer Drug Screening

“…Wessely et al (2019) also tested the use of the Boyden chamber to evaluate and compare the invasive activity of spheroids containing only tumor cells and spheroids containing a mixture of tumor and stem cells. Another study examined the adhesion and cytoskeletal migration of HT1080 fibrosarcoma cells and LX2 line stellate cells in a three-dimensional system using fibronectin, Matrigel and type I collagen as chemoattractants (Tovari et al, 2014). However, despite the ease of use of the Boyden chamber, researchers are increasingly turning to more advanced systems that take into account a greater number of TME conditions, in particular, microfluidic systems.…”