Cell migration is a key procedure involved in many biological processes including embryological development, tissue formation, immune defense or inflammation, and cancer progression. How physical, chemical, and molecular aspects can affect cell motility is a challenge to understand migratory cells behavior. In vitro assays are excellent approaches to extrapolate to in vivo situations and study live cells behavior. Here we present four in vitro protocols that describe step-by-step cell migration, invasion and adhesion strategies and their corresponding image data quantification. These current protocols are based on two-dimensional wound healing assays (comparing traditional pipette tip-scratch assay vs. culture insert assay), 2D individual cell-tracking experiments by live cell imaging and three-dimensional spreading and transwell assays. All together, they cover different phenotypes and hallmarks of cell motility and adhesion, providing orthogonal information that can be used either individually or collectively in many different experimental setups. These optimized protocols will facilitate physiological and cellular characterization of these processes, which may be used for fast screening of specific therapeutic cancer drugs for migratory function, novel strategies in cancer diagnosis, and for assaying new molecules involved in adhesion and invasion metastatic properties of cancer cells.

ROCK-Myosin II drives fast rounded-amoeboid migration in cancer cells during metastatic dissemination. Analysis of human melanoma biopsies revealed that amoeboid melanoma cells with high Myosin II activity are predominant in the invasive fronts of primary tumors in proximity to CD206+CD163+ tumor-associated macrophages and vessels. Proteomic analysis shows that ROCK-Myosin II activity in amoeboid cancer cells controls an immunomodulatory secretome, enabling the recruitment of monocytes and their differentiation into tumor-promoting macrophages. Both amoeboid cancer cells and their associated macrophages support an abnormal vasculature, which ultimately facilitates tumor progression. Mechanistically, amoeboid cancer cells perpetuate their behavior via ROCK-Myosin II-driven IL-1α secretion and NF-κB activation. Using an array of tumor models, we show that high Myosin II activity in tumor cells reprograms the innate immune microenvironment to support tumor growth. We describe an unexpected role for Myosin II dynamics in cancer cells controlling myeloid function via secreted factors.

Cell migration underlies metastatic dissemination of cancer cells, and fast "amoeboid" migration in the invasive fronts of tumors is controlled by high levels of actomyosin contractility. How amoeboid migration is regulated by extracellular signals and sustained over time by transcriptional changes is not fully understood. Transforming growth factor β (TGF-β) is well known to promote epithelial-to-mesenchymal transition (EMT) and contribute to metastasis, but melanocytes are neural crest derivatives that have undergone EMT during embryonic development. Surprisingly, we find that in melanoma, TGF-β promotes amoeboid features such as cell rounding, membrane blebbing, high levels of contractility, and increased invasion. Using genome-wide transcriptomics, we find that amoeboid melanoma cells are enriched in a TGF-β-driven signature. We observe that downstream of TGF-β, SMAD2 and its adaptor CITED1 control amoeboid behavior by regulating the expression of key genes that activate contractile forces. Moreover, CITED1 is highly upregulated during melanoma progression, and its high expression is associated with poor prognosis. CITED1 is coupled to a contractile-rounded, amoeboid phenotype in a panel of 16 melanoma cell lines, in mouse melanoma xenografts, and in 47 human melanoma patients. Its expression is also enriched in the invasive fronts of lesions. Functionally, we show how the TGF-β-SMAD2-CITED1 axis promotes different steps associated with progression: melanoma detachment from keratinocytes, 2D and 3D migration, attachment to endothelial cells, and in vivo lung metastatic initial colonization and outgrowth. We propose a novel mechanism by which TGF-β-induced transcription sustains actomyosin force in melanoma cells and thereby promotes melanoma progression independently of EMT.

Purpose: We have studied a consecutive case series of patients with multiple primary melanoma (MPM) for the involvement of the melanoma susceptibility loci CDKN2A and CDK4. Patients and Methods: One hundred four MPM patients (81 patients with two primary melanomas, 14 with three, five with four, one with five, two with six, and one with seven) were included. Results: Seven different CDKN2A germline mutations were identified in 17 patients (16.3%). In total, we identified 15 CDKN2A exon 2, one exon 1α missense mutation, and one exon 1β frameshift mutation. The age of onset was significantly lower and the number of primary melanomas higher in patients with mutations. CDKN2A mutations were more frequent in patients with familial history of melanoma (35.5%) compared with patients without (8.2%), with a relative risk (RR) of 4.32 (95% CI, 1.76 to 10.64; P = .001), and in patients with more than two melanomas (39.1%) compared with patients with only two melanomas (10%) with an RR of 3.29 (95% CI, 1.7 to 6.3; P = .002). The A148T polymorphism was more frequent in patients with MPMs than in the control population (P = .05). A variant of uncertain significance, A127S, was also detected in one patient. No CDK4 mutations were identified, suggesting that it has a low impact in susceptibility to MPM. Conclusion: MPM patients are good candidates for CDKN2A mutational screening. These patients and some of their siblings should be included in a program of specific follow-up with total body photography and digital dermoscopy, which will result in the early detection of melanoma in this subset of high-risk patients and improve phenotypic characterization.

Cell migration is crucial for cancer dissemination. We find that AMP-activated protein kinase (AMPK) controls cell migration by acting as an adhesion sensing molecular hub. In 3-dimensional matrices, fast-migrating amoeboid cancer cells exert low adhesion/low traction linked to low ATP/AMP, leading to AMPK activation. In turn, AMPK plays a dual role controlling mitochondrial dynamics and cytoskeletal remodelling. High AMPK activity in low adhering migratory cells, induces mitochondrial fission, resulting in lower oxidative phosphorylation and lower mitochondrial ATP. Concurrently, AMPK inactivates Myosin Phosphatase, increasing Myosin II-dependent amoeboid migration. Reducing adhesion or mitochondrial fusion or activating AMPK induces efficient rounded-amoeboid migration. AMPK inhibition suppresses metastatic potential of amoeboid cancer cells in vivo, while a mitochondrial/AMPK-driven switch is observed in regions of human tumours where amoeboid cells are disseminating. We unveil how mitochondrial dynamics control cell migration and suggest that AMPK is a mechano-metabolic sensor linking energetics and the cytoskeleton.