The dissemination of primary solid tumor cells to distant organs, termed metastasis, is a major cause of cancer-related deaths. Circulating tumor cells (CTCs), which can exist as individual cells or multicellular clusters, travel through the bloodstream. Their isolation from liquid biopsy samples is increasingly recognized as a valuable tool for diagnosis, prognosis, and treatment guidance for cancer patients. Current isolation methods typically rely on biomarkers like epithelial cell adhesion molecule (EpCAM) and utilize technologies such as magnetic beads or microfluidic chips. However, these methods face limitations due to tumor heterogeneity. Furthermore, tumor cells that transfer into CTCs often undergo epithelial-to-mesenchymal transition, gaining invasive characteristics while losing epithelial markers. As a result, these cells are difficult to detect using EpCAM-based methods. Label-free microscale isolation technologies tackle the limitations of biomarker-based methods by leveraging the distinctive physical properties of CTCs, such as their size, electrical charge, viscoelasticity, and deformability that contrast them from normal blood cells. This review evaluates primary label-free isolation methods, including deterministic lateral displacement, microfiltration, acoustophoresis, and dielectrophoresis, and whether they can offer a deeper insight into tumor heterogeneity and the dynamics of cancer progression and treatment. Additionally, it highlights automated platforms for high-throughput CTC isolation and analysis.

Epithelial-to-mesenchymal transitions (EMT) and unjamming transitions provide two distinct pathways for cancer cells to become invasive, but it is still unclear to what extent these pathways are connected. Here, we addressed this question by performing 3D spheroid invasion assays on epithelial-like (A549) and mesenchymal-like (MV3) cancer cell lines in collagen-based hydrogels, where we varied both the invasive character of the cells and matrix porosity. We found that the onset time of invasion was correlated with the matrix porosity and vimentin levels, while the spheroid expansion rate correlated with MMP1 levels. Spheroids displayed solid-like (non-invasive) states in small-pore hydrogels and fluid-like (strand-based) or gas-like (disseminating cells) states in large-pore hydrogels or for mesenchymal-like cells. Our findings are consistent with different unjamming states as a function of cell motility and matrix confinement predicted in recent models for cancer invasion, but show that cell motility and matrix confinement are coupled via EMT-related matrix degradation.

Within the tumor microenvironment (TME), cancer cells use mechanotransduction pathways to convert biophysical forces to biochemical signals. However, the underlying mechanisms and functional significance of these pathways remain largely unclear. The upregulation of mechanosensitive pathways from biophysical forces such as interstitial flow (IF), leads to the activation of various cytokines, including transforming growth factor-β (TGF-β). TGF-β promotes in part via a Smad-dependent signaling pathway the epithelial–mesenchymal transition (EMT) in cancer cells. The latter process is linked to increased cancer cell motility and invasion. Current research models have limited ability to investigate the combined effects of biophysical forces (such as IF) and cytokines (TGF-β) in a 3D microenvironment. We used a 3D-matrix based microfluidic platform to demonstrate the potentiating effect of IF on exogenous TGF-β induced upregulation of the Smad-signaling activity and the expression of mesenchymal marker vimentin in A549 lung cancer spheroids. To monitor this, we used stably integrated fluorescent based reporters into the A549 cancer cell genome. Our results demonstrate that IF enhances exogenous TGF-β induced Smad-signaling activity in lung cancer spheroids embedded in a matrix microenvironment. In addition, we observed an increased cell motility for A549 spheroids when exposed to IF and TGF-β. Our 3D-microfluidic model integrated with real-time imaging provides a powerful tool for investigating cancer cell signaling and motility associated with invasion characteristics in a physiologically relevant TME.

In the research of cancer cell invasion and metastasis, recreation of physiologically relevant and faithful three-dimensional (3D) tumor models that recapitulate spatial architecture, spatiotemporal control of cell communication and signaling pathways, and integration of extracellular cues remains an open challenge. Here, a programmable multifunctional 3D cancer cell invasion microbuckets-hydrogel (Mb-H) platform is developed by integrating various function-variable microbuckets and extracellular matrix (ECM)-like hydrogels. Based on this Mb-H micro platform, the aggregation of multi-cancer cells is well controlled to form cancer cell spheroids, and the guiding relationship of single-cell migration and collective cell migration during the epithelial-mesenchymal transition (EMT) of cancer cell invasion are demonstrated. By programming and precisely assembling multiple functions in one system, the Mb-H platform with spatial-temporal controlled release of cytokine transforming growth factor beta (TGF-β) and various functionalized Mb-H platforms with intelligent adjustment of cell-matrix interactions are engineered to coordinate the 3D invasive migration of cancer cell spheroids. This programmable and adaptable 3D cancer cell invasion micro platform takes a new step toward mimicking the dynamically changing (localized) tumor microenvironment and exhibits wide potential applications in cancer research, bio-fabrication, cell signaling, and drug screening.

Epithelial to mesenchymal transition (EMT) is crucial during embryonic development, tissue fibrosis, and cancer progression. Epithelial cells that display a cobblestone-like morphology can undergo a switch to mesenchymal-like phenotype, displaying an elongated spindle shape or a fibroblast-like morphology. EMT is characterized by timely and reversible alterations of molecular and cellular processes. The changes include loss of epithelial and gain of mesenchymal marker expression, loss of polarity, increased cell migratory and invasive properties. Epithelial cells can progress unevenly during this transition and attain hybrid E/M states or metastable EMT states, referred to as epithelial cell plasticity. To gain a deeper insight into the mechanism of EMT, understanding the dynamic aspects of this process is essential. One of the most prominent factors to induce EMT is the cytokine transforming growth factor-β (TGF-β). This chapter discusses molecular and cellular techniques to monitor TGF-β-induced signaling and EMT changes in normal and cancer cell lines. These methods include measuring the TGF-β-induced activation of its intracellular SMAD effectors proteins and changes in epithelial/mesenchymal marker expression and localization. Moreover, we describe assays of cell migration and dynamic reorganization of the actin cytoskeleton and stress filaments that are frequently part of the TGF-β-induced EMT cellular response.

An early step of metastasis requires a complex and coordinated migration of invasive tumor cells into the surrounding tumor microenvironment (TME), which contains extracellular matrix (ECM). It is being appreciated that 3D matrix-based microfluidic models have an advantage over conventional in vitro and animal models to study tumor progression events. Recent microfluidic models have enabled recapitulation of key mechanobiological features present within the TME to investigate collective cancer cell migration and invasion. Microfluidics also allows for functional interrogation and therapeutic manipulation of specific steps to study the dynamic aspects of tumor progression. In this review, we focus on recent developments in cancer cell migration and how microfluidic strategies have evolved to address the physiological complexities of the TME to visualize migration modes adapted by various tumor cells.