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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. assays are excellent approaches to extrapolate to situations and study live cells behavior. Here we present four protocols that describe step-by-step cell migration, invasion and adhesion strategies and their corresponding image data quantification. These current protocols are based on wound healing assays (comparing traditional pipette tip-scratch assay vs. culture insert assay), 2D individual cell-tracking experiments by live cell imaging and 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.

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. Cell metabolism must adapt to the energy needs of migrating cells. This study finds that fast amoeboid migrating cells harbor high AMPK activity, which controls both mitochondrial dynamics and cytoskeletal remodeling, enabling reduced energy needs.

Melanoma is a highly aggressive tumour that can metastasize very early in disease progression. Notably, melanoma can disseminate using amoeboid invasive strategies. We show here that high Myosin II activity, high levels of ki-67 and high tumour-initiating abilities are characteristic of invasive amoeboid melanoma cells. Mechanistically, we find that WNT11-FZD7-DAAM1 activates Rho-ROCK1/2-Myosin II and plays a crucial role in regulating tumour-initiating potential, local invasion and distant metastasis formation. Importantly, amoeboid melanoma cells express both proliferative and invasive gene signatures. As such, invasive fronts of human and mouse melanomas are enriched in amoeboid cells that are also ki-67 positive. This pattern is further enhanced in metastatic lesions. We propose eradication of amoeboid melanoma cells after surgical removal as a therapeutic strategy. Amoeboid cells are associated with melanoma invasive capacity. Here, the authors show that the WNT11-FZD7-DAAM1 pathway regulates tumour-initiating potential, invasion and metastasis lead by amoeboid cells in the invasive front of melanoma tumours.

The extracellular matrix (ECM) controls tumour dissemination. We characterise ECM organization in human and mouse tumours, identifying three regions: tumour body, proximal invasive front and distal invasive front. Invasive areas show increased matrix density, fibre thickness, length, and alignment, with unique radial fibre orientation at the distal invasive front correlating with amoeboid invasive features. Using patient samples and murine models, we find that metastases recapitulate ECM features of the primary tumour. Ex vivo culture of murine cancer cells isolated from the different tumour regions reveals a spatial cytoskeletal and transcriptional memory. Several in vitro models recapitulate the in vivo ECM organisation showing that increased matrix induces 3D confinement supporting Rho-ROCK-Myosin II activity, while radial orientation enhances directional invasion. Spatial transcriptomics identifies a mechano-inflammatory program associated with worse prognosis across multiple tumour types. These findings provide mechanistic insights into how ECM organization shapes local invasion and distant metastasis. Interactions with the extracellular matrix (ECM) control tumor proliferation, invasion and metastasis. Here, authors provide spatial information on ECM organization and how it influences tumor cell invasive and metastasis properties through induction of cytoskeletal and transcriptional memory.

Acral lentiginous melanoma (ALM) is a rare melanoma subtype primarily located in acral regions. However, ALMs exhibit a distinctive genetic profile characterized by a high number of copy number variations (CNVs) and limited point mutations. Late diagnosis and restricted therapeutic efficacy contribute to its poor prognosis. The secretome within the tumor microenvironment (TME) influences immune modulation and plays a vital role in melanoma progression. We aim to analyze the role of ALM secretome and CNVs profile with prognosis in primary ALM patients. Here, we demonstrated that high CNV burden (CNVsHigh) was associated with worse clinicopathological characteristics and poor prognosis. Furthermore, our study also revealed that conditioned media (CM) of CNVsHigh genetic profile ALM cell line was associated with pro-tumoral, pro-angiogenic, and immunosuppressive secretome profiles. In addition, CM of CNVsHigh cell lines in vitro promotes macrophage polarization to immunosuppressive phenotype. Moreover, we observed an increased presence of immunosuppressive tumor-associated macrophages (TAMs) at the invasive front (IF) of CNVsHigh ALM biopsies. This research reveals the adverse prognostic impact of CNVsHigh in ALM patients, establishing a novel link with a pro-tumor secretome, offering potential biomarkers for prognosis and personalized treatment to enhanced disease monitoring in ALM patients.

Cutaneous melanoma is the most aggressive of skin tumors. In order to discover new biomarkers that could help us improve prognostic prediction in melanoma patients, we have searched for germline DNA variants associated with melanoma progression. Thus, after exome sequencing of a set of melanoma patients and healthy control individuals, we identified rs1042602, an SNP within TYR, as a good candidate. After genotyping rs1042602 in 1025 patients and 773 healthy donors, we found that the rs1042602-A allele was significantly associated with susceptibility to melanoma (CATT test: p = 0.0035). Interestingly, we also observed significant differences between patients with good and bad prognosis (5 years of follow-up) (n = 664) (CATT test for all samples p = 0.0384 and for men alone p = 0.0054). Disease-free-survival (DFS) analyses also showed that patients with the A allele had shorter DFS periods. In men, the association remained significant even in a multivariate Cox Proportional-hazards model, which was adjusted for age at diagnosis, Breslow thickness, ulceration and melanoma subtype (HR 0.4; 95% confidence interval (CI) 0.20–0.83; p = 0.0139). Based on our results, we propose that rs1042602-A is a risk allele for melanoma, which also seems to be responsible for a poorer prognosis of the disease, particularly in men.

Background: Cutaneous melanoma shows high variability regarding clinicopathological presentation, evolution and prognosis. Methods: Next generation sequencing was performed to analyze hotspot mutations in different areas of primary melanomas (MMp) and their paired metastases. Clinicopathological features were evaluated depending on the degree of variation of the BRAFV600E mutant allele frequency (MAF) in MMp. Results: In our cohort of 14 superficial spreading, 10 nodular melanomas and 52 metastases, 17/24 (71%) melanomas had a BRAFV600E mutation and 5/24 (21%) had a NRASQ61 mutation. We observed a high variation of BRAFV600E MAF (H-BRAFV600E) in 7/17 (41%) MMp. The H-BRAFV600E MMp were all located on the trunk, had lower Breslow and mitotic indexes and predominantly, a first nodal metastasis. Regions with spindled tumor cells (Spin) and high lymphocytic infiltrate (HInf) were more frequent in the H-BRAFV600E patients (4/7; 57%), whereas regions with epithelial tumor cells (Epit) and low lymphocytic infiltrate (LInf) were predominant (6/10; 60%) and exclusive in the low BRAFV600E MAF variation tumors (L-BRAFV600E). The H-BRAFV600E/Spin/HInf MMp patients had better prognostic features and nodal first metastasis. Conclusions: The H-BRAFV600E MMp were located on the trunk, had better prognostic characteristics, such as lower Breslow and mitotic indexes as well as high lymphocytic infiltrate.

Metastasis involves dissemination of cancer cells away from a primary tumour and colonization at distal sites. During this process, the mechanical properties of the nucleus must be tuned since they pose a challenge to the negotiation of physical constraints imposed by the microenvironment and tissue structure. We discovered increased expression of the inner nuclear membrane protein LAP1 in metastatic melanoma cells, at the invasive front of human primary melanoma tumours and in metastases. Human cells express two LAP1 isoforms (LAP1B and LAP1C), which differ in their amino terminus. Here, using in vitro and in vivo models that recapitulate human melanoma progression, we found that expression of the shorter isoform, LAP1C, supports nuclear envelope blebbing, constrained migration and invasion by allowing a weaker coupling between the nuclear envelope and the nuclear lamina. We propose that LAP1 renders the nucleus highly adaptable and contributes to melanoma aggressiveness. Jung-Garcia et al. dissect the contributions of isoforms of the inner nuclear membrane protein LAP1 to nuclear envelope blebbing, constrained migration and invasion in melanoma cells.

T-type calcium channels (TTCCs) are overexpressed in several cancers. In this review, we summarize the recent advances and new insights into TTCC biology, tumor progression, and prognosis biomarker and therapeutic potential in the melanoma field. We describe a novel correlation between the Cav3.1 isoform and the increased basal autophagy in BRAFV600E-mutant melanomas and after acquired resistance to BRAF inhibitors. Indeed, TTCC blockers reduce melanoma cell viability and migration/invasion in vitro and tumor growth in mice xenografts in both BRAF-inhibitor-sensitive and -resistant scenarios. These studies open a new, promising therapeutic approach for disseminated melanoma and improved treatment in BRAFi relapsed melanomas, but further validation and clinical trials are needed for it to become a real therapeutic option.

Cowden syndrome is an autosomal dominant genodermatosis, characterized by the presence of multiple hamartomas in the skin, breast, thyroid, gastrointestinal tract, central nervous system, and an increased risk in developing breast and thyroid carcinomas. Over 80 germline mutations of the tumor suppressor gene PTEN, on chromosome 10q23, have been reported in more than 100 unrelated patients and families; however, questions regarding distribution of the mutations in populations from different geographic areas, and phenotypic expression are still unclear. In this study the results are reported of mutation analysis of PTEN in 13 families from Spain and one family of Brazilian origin with Cowden syndrome. PTEN germline mutations were detected in nine of them (64%). Five mutations were located in exon 5, one in exon 6, two in exon 7, and one in exon 8. Four of the mutations were novel. In another case, an identical change had been previously reported as a somatic mutation in an endometrial carcinoma. In one family, the patient presented a de novo mutation, which was not detected in his parents. In five patients, the detection of the PTEN germline mutation confirmed their condition, even in the absence of sufficient criteria to make the clinical diagnosis of Cowden syndrome.