A Quantitative Comparison of Human HT-1080 Fibrosarcoma Cells and Primary Human Dermal Fibroblasts Identifies a 3D Migration Mechanism with Properties Unique to the Transformed Phenotype

Abstract

Here, we describe an engineering approach to quantitatively compare migration, morphologies, and adhesion for tumorigenic human fibrosarcoma cells (HT-1080s) and primary human dermal fibroblasts (hDFs) with the aim of identifying distinguishing properties of the transformed phenotype. Relative adhesiveness was quantified using self-assembled monolayer (SAM) arrays and proteolytic 3-dimensional (3D) migration was investigated using matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) (PEG) hydrogels (“synthetic extracellular matrix” or “synthetic ECM”). In synthetic ECM, hDFs were characterized by vinculin-containing features on the tips of protrusions, multipolar morphologies, and organized actomyosin filaments. In contrast, HT-1080s were characterized by diffuse vinculin expression, pronounced β1-integrin on the tips of protrusions, a cortically-organized F-actin cytoskeleton, and quantitatively more rounded morphologies, decreased adhesiveness, and increased directional motility compared to hDFs. Further, HT-1080s were characterized by contractility-dependent motility, pronounced blebbing, and cortical contraction waves or constriction rings, while quantified 3D motility was similar in matrices with a wide range of biochemical and biophysical properties (including collagen) despite substantial morphological changes. While HT-1080s were distinct from hDFs for each of the 2D and 3D properties investigated, several features were similar to WM239a melanoma cells, including rounded, proteolytic migration modes, cortical F-actin organization, and prominent uropod-like structures enriched with β1-integrin, F-actin, and melanoma cell adhesion molecule (MCAM/CD146/MUC18). Importantly, many of the features observed for HT-1080s were analogous to cellular changes induced by transformation, including cell rounding, a disorganized F-actin cytoskeleton, altered organization of focal adhesion proteins, and a weakly adherent phenotype. Based on our results, we propose that HT-1080s migrate in synthetic ECM with functional properties that are a direct consequence of their transformed phenotype.

Biomaterial arrays with defined adhesion ligand densities and matrix stiffness identify distinct phenotypes for tumorigenic and non-tumorigenic human mesenchymal cell types

“…Previous studies on fibronectin-coated polyacrylamide substrates have identified a role for substrate stiffness in migration for glioma cells 23 , neutrophils 24 and vascular smooth muscle cells 25 , while HT-1080 and DU-145 prostate carcinoma cell motility is dependent on a balance of biochemical and biophysical properties in 3D culture 26 . Importantly, transformation by Rous sarcoma virus reduces adhesiveness on fibronectin-coated surfaces due to altered integrin function 4 , while HT-1080s are quantitatively less adherent than primary fibroblasts 12 , which demonstrates that transformed cell types are characterized by altered adhesion properties compared to non-tumorigenic cells. While adhesion properties have been proposed as defining features of tumor cell migration modes 1-3 , most quantitative models of cell adhesion-dependent motility describe non-tumorigenic adherent cell types 27-29 on tissue culture polystyrene (TCP) or glass substrates with moduli orders of magnitude higher (~10 9 Pa) 30, 31 than most tissues (~10 2 -10 4 Pa) 30, 32, 33 .…”

“…However, transformation to an aggressive tumorigenic phenotype alters many of the distinguishing features for cell motility, including cytoskeletal structure and cell adhesion properties 4-11 . Direct comparisons between tumorigenic and non-tumorigenic cell types - thought to utilize analogous migration modes - have revealed important differences in function 12-16 . For example, while HT-1080 fibrosarcoma cells (HT-1080s) were previously characterized by a mesenchymal mode that was qualitatively similar to motile fibroblasts in collagen 1, 17 , pronounced differences in cytoskeletal structure and cell adhesion properties have been identified through comparisons with human dermal fibroblasts (hDFs) cultured using identical 2D and 3D cell culture platforms 12-14 .…”

“…Direct comparisons between tumorigenic and non-tumorigenic cell types - thought to utilize analogous migration modes - have revealed important differences in function 12-16 . For example, while HT-1080 fibrosarcoma cells (HT-1080s) were previously characterized by a mesenchymal mode that was qualitatively similar to motile fibroblasts in collagen 1, 17 , pronounced differences in cytoskeletal structure and cell adhesion properties have been identified through comparisons with human dermal fibroblasts (hDFs) cultured using identical 2D and 3D cell culture platforms 12-14 . These studies highlight the importance of providing quantitative measures of cell function to classify tumor cell migration mechanisms, since features for identifying distinct modes of motility describe qualitative characteristics common to most motile cell types 18 , and are not well-defined 19 .…”

Development and Applications of Microfluidic Devices for Cell Culture in Cell Biology

“…The recent development of 3D microfluidic culture in tissue engineering has resulted in the evolution of 3D in vitro models for cell biology studies [134]; meanwhile, it calls for the development of tissue engineering and biomaterials to maximize the utility and functionality of the models. For example, smartly designed biomaterials can be degraded by growth factors at desired rates that relevant to physiological condition or be cleaved only by specific proteases like in vivo ECM [135][136][137]. Several pharmaceutical companies are moving toward adapting 3D in vitro cancer models as anti-cancer drug testing tools.…”

Heralding a new paradigm in 3D tumor modeling

“…The use of PEG-based hydrogels to precisely simulate the tumor microenvironment in vitro has since then been demonstrated for glioblastoma [98] and pancreatic cancer [99]. Of mention, the Anseth group has also employed these PEG-based hydrogels to study tumor cell migration in 3D under controlled biochemical and physical in vitro conditions [100, 101]. In seeking to understand the differences in migration mechanisms between normal and malignant cells, the migration, morphologies, adhesiveness, expression of adhesion proteins and, cytoskeletal structure of malignant fibrosarcoma cells were compared against that of primary human dermal fibroblasts.…”