Abstract
Microenvironment stiffening plays a crucial role in tumorigenesis. While filopodia are generally thought to be one of the cellular mechanosensors for probing environmental stiffness, the effects of environmental stiffness on filopodial activities of cancer cells remain unclear. In this work, we investigated the filopodial activities of human lung adenocarcinoma cells CL1-5 cultured on substrates of tunable stiffness using a novel platform. The platform consists of an optical system called structured illumination nano-profilometry, which allows time-lapsed visualization of filopodial activities without fluorescence labeling. The culturing substrates were composed of polyvinyl chloride mixed with an environmentally friendly plasticizer to yield Young's modulus ranging from 20 to 60 kPa. Cell viability studies showed that the viability of cells cultured on the substrates was similar to those cultured on commonly used elastomers such as polydimethylsiloxane. Time-lapsed live cell images were acquired and the filopodial activities in response to substrates with varying degrees of stiffness were analyzed. Statistical analyses revealed that lung cancer cells cultured on softer substrates appeared to have longer filopodia, higher filopodial densities with respect to the cellular perimeter, and slower filopodial retraction rates. Nonetheless, the temporal analysis of filopodial activities revealed that whether a filopodium decides to extend or retract is purely a stochastic process without dependency on substrate stiffness. The discrepancy of the filopodial activities between lung cancer cells cultured on substrates with different degrees of stiffness vanished when the myosin II activities were inhibited by treating the cells with blebbistatin, which suggests that the filopodial activities are closely modulated by the adhesion strength of the cells. Our data quantitatively relate filopodial activities of lung cancer cells with environmental stiffness and should shed light on the understanding and treatment of cancer progression and metastasis.
Highlights
Microenvironment stiffness plays a crucial role in cancer development and progression
We quantified the relationship between the filopodial activities of lung cancer cells and substrate stiffness using a label-free imaging technique
We found that substrate stiffness regulated the filopodial length and density in cancer cells probably via affecting the filopodial retraction rate, which was primarily modulated by varied myosin activities
Summary
Microenvironment stiffness plays a crucial role in cancer development and progression. Stiffening of extracellular matrix resulting from increased collagen crosslinking occurs during tumorigenesis [1,2]. The matrix stiffening affects cell motility, directs the migration of cancer cells, and may further be related to organ-specific metastasis [3]. Stiff matrix promotes the stability of cell focal adhesion, which enhances intracellular growth factor signaling and in turn increases tumor cell transformation and growth [2,4]. It was shown recently that several lung cancer cell lines grew better on stiffer substrates [5], and that reduction of matrix stifferening by inhibiting the lysyl oxidasemediated collagen crosslinking impeded tumor progression [6]. Understanding how cancer cells sense and respond to environmental stiffness should provide valuable insights into the intricacies of cancer progression and assist in the improvement of treatment strategies
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