Cell migration through confined spaces is a critical step in cancer metastasis, yet the spatial regulation of endocytosis and adhesion dynamics during this process remains poorly understood. To investigate this, we adapted a microfluidic platform that generates stable, spatially linear biochemical gradients across 5 μm-tall migration channels. COMSOL simulations and optical calibration using FITC-dextran confirmed that gradients form reliably within 5 min. The microdevice also supports long-term live imaging and is compatible with both spinning disk confocal and total internal reflection fluorescence structured illumination microscopy modalities, enabling high-resolution visualization of adhesion and endocytic structures. By leveraging this platform for spatially restricted drug delivery, we locally applied the endocytic inhibitor Dyngo-4a to either the front or rear of migrating cells. This revealed that front-targeted endocytic inhibition preserved or increased leading-edge enrichment of paxillin and the clathrin adaptor AP-2, whereas rear-targeted inhibition eliminated paxillin polarity and reduced AP-2 polarity. These changes were accompanied by a significant increase in cell migration speed under front-targeted inhibition, while rear-targeted inhibition had no significant effect on speed and neither treatment altered persistence. Together, these findings suggest that endocytic polarity regulates adhesion dynamics and cell migration under confinement, offering a mechanistic insight into processes relevant to cancer cell invasion.

The extracellular matrix (ECM) provides structural support to cells, thereby forming a functional tissue. In cancer, the growth of the tumor creates internal mechanical stress, which, together with the remodeling activity of tumor cells and fibroblasts, alters the ECM structure, leading to an increased stiffness of the pathological ECM. The enhanced ECM stiffness, in turn, stimulates tumor growth and activates tumor-promoting fibroblasts and tumor cell migration, leading to metastasis and increased therapy resistance. While the relationship between matrix stiffness and migration has been studied before, their connection to internal tumor stress remains unresolved. Here we used 3D ECM-embedded spheroids and hydrogel particle stress sensors to quantify and correlate internal tumor spheroid pressure, ECM stiffness, ECM remodeling, and tumor cell migration. We note that 4T1 breast cancer spheroids and SV80 fibroblast spheroids showed increased invasion—described by area, complexity, number of branches, and branch area—in a stiffer, cross-linked ECM. On the other hand, changing ECM stiffness only minimally changed the radial alignment of fibers but highly changed the amount of fibers. For both cell types, the pressure measured in spheroids gradually decreased as the distance into the ECM increased. For 4T1 spheroids, increased ECM stiffness resulted in a further reach of mechanical stress into the ECM, which, together with the invasive phenotype, was reduced by inhibition of ROCK-mediated contractility. By contrast, such correlation between ECM stiffness and stress-reach was not observed for SV80 spheroids. Our findings connect ECM stiffness with tumor invasion, ECM remodeling, and the reach of tumor-induced mechanical stress into the ECM. Such mechanical connections between tumor and ECM are expected to drive early steps in cancer metastasis.

Exosomes have gained attention as promising therapeutic agents in cancer treatment due to their ability to influence target cell phenotypes and modulate immune responses. Their role in tumor biology, however, is influenced by several factors, including the source of mesenchymal stem cells (MSCs), culture conditions, and the tumor microenvironment. This study aimed to evaluate the effects of exosomes derived from bone marrow MSCs of Sprague-Dawley rats, incorporated into alginate hydrogels (AH), on the migration and viability of murine melanoma (B16-F10) cells. Scanning electron microscopy revealed that the hydrogels preserved their structural integrity after exosome incorporation. Both AH and exosome-loaded AH (AHE) exhibited no cytotoxic effects, as the viability and colony-forming capacity of B16-F10 cells remained comparable to untreated controls. Notably, AHE significantly suppressed tumor cell migration, a critical step in cancer metastasis, whereas AH alone had no effect. These findings indicate that exosomes retained their functionality within the hydrogel matrix, effectively modulating cell migration. This study underscores the therapeutic potential of exosome-loaded hydrogels in regulating cancer cell behavior. Nonetheless, further research is needed to elucidate the molecular mechanisms involved and optimize the clinical application of exosome-integrated hydrogels.

This mini-review addresses the critical issue of cancer metastasis, a process that significantly contributes to cancer-related mortality. The study delves into the molecular and cellular mechanisms underpinning cancer cell migration and metastasis, focusing on recent advances and key factors involved in each step of metastatic progression. The research highlights the complexity of cell migration, comparing single-cell migration with collective cell migration and emphasizing their roles in developmental processes and disease progression. The study also provides a detailed analysis of the five key steps in cancer metastasis: infiltration, intravasation, survival in the circulatory system, extravasation, and colonization, elucidating the cellular and molecular events driving each step. Overall, the findings underscore the importance of understanding cell migration in cancer biology, offering insights into potential therapeutic targets and strategies to prevent tumor dissemination and enhance cancer treatment outcomes.

Background: Breast cancer (BC) is a kind of cancer that starts in the breast. patients in addition to doctors are disturbed by survival prognosis. Besides, the prognostic factor has been the clinic pathological parameter linked to tumor consequences. Also, this cancer type possesses the biggest uprising rate of frequency as well as number two in rates of mortality in female’ cancers. migration of cancer cells starting the primary tumor and the invasion of cancer cells in the adjacent tissue are the primary steps in cancer metastasis. The present education aims to evaluate Chitinase-3-Like Protein 1 (YKL-40) levels in Babylon women with breast cancer and control groups. Material and methods: The case-control group was made up of 60 subjects, presumably healthy women, whereas the sick group was made up of 60 women with breast cancer. A study was conducted in Marjan Medical City, Imam Al-Sadiq Hospital, and AL-Hilla Teaching Hospital in Hilla City, between 2/11/2023 and April 29/4/2024. An ELISA was used to calculate the plasma concentrations that referred to Chitinase-3-Like Protein 1 (YKL-40). SPSS software was used to conduct the statistical analysis. Results: Despite a considerable increase in Chitinase-3-Like Protein 1 levels, with patients comparing it together with the control group (p < 0.05). Conclusion: women with breast cancer had considerably increased serum levels of Chitinase-3-Like Protein 1 levels. Based on the results of this investigation, this indicates the part of Chitinase-3-similar to Protein 1 as a prognostic marker within females with BC disease.

Cancer metastasis is the deadliest event in tumorigenesis. Despite extensive research, there are still unsolved challenges regarding early metastasis detection and targeting strategies. Extracellular vesicles (EVs) and their impact on tumorigenic-related events are in the eye of current investigations. EVs represent a plethora of biomarkers and information, and they are considered key determinants in tumor progression and for tumor prognosis and monitoring. EVs are one of the key mediators for inter-cellular communications between tumor cells and their nearby stroma. They are involved in different steps of metastasis from invasion toward formation of pre-metastatic niches (PMNs), and final growth and colonization of tumor cells in desired organ/s of the target. Membrane components of EVs and their cargo can be traced for the identification of tumor metastasis, and their targeting is a promising strategy in cancer therapy. In this review, we aimed to discuss the current understanding of EV-based metastatic predilection in cancer, providing updated information about EV involvement in different metastatic steps and suggesting some strategies to hamper this devastating condition.

A novel thin plate spline methodology to model tissue surfaces and quantify tumor cell invasion in organ-on-chip models

A SERS-assisted 3D organotypic microfluidic chip for in-situ visualization and monitoring breast cancer extravasation process

Inhibition of galectin-3-mediated interactions by modified citrus pectin (MCP) could affect several rate-limiting steps in cancer metastasis, but the ability of MCP to antagonize galectin-8 function remains unknown. We hypothesized that MCP could bind to galectin-8 in addition to galectin-3. In this study, a combination of gradual ethanol precipitation and DEAE-Sepharose Fast Flow chromatography was used to isolate several fractions from MCP. The ability of these fractions to antagonize galectin-8 function was studied as well as the primary structure and initial structure-function relationship of the major active component MCP-30-3. The results showed that MCP-30-3 (168 kDa) was composed of Gal (13.8%), GalA (63.1%), GlcA (13.0%), and Glc (10.1%). MCP-30-3 could specifically bind to galectin-8, with an MIC value of 0.04 mg mL-1. After MCP-30-3 was hydrolyzed by β-galactosidase or pectinase, its binding activity was significantly reduced. These results provide new insights into the interaction between MCP structure and galectin function, as well as the potential utility in the development of functional foods.

Organ-on-chip (OOC) models can be useful tools for cancer drug discovery. Advances in OOC technology have led to the development of more complex assays, yet analysis of these systems does not always account for these advancements, resulting in technical challenges. A challenging task in the analysis of these two-channel microfluidic models is to define the boundary between the channels so objects moving within and between channels can be quantified. We propose a novel imaging-based application of a thin plate spline method – a generalized cubic spline that can be used to model coordinate transformations – to model a tissue boundary and define compartments for quantification of invaded objects, representing the early steps in cancer metastasis. To evaluate its performance, we applied our analytical approach to an adapted OOC developed by Emulate, Inc., utilizing a two-channel system with endothelial cells in the bottom channel and colorectal cancer (CRC) patient-derived organoids (PDOs) in the top channel. Initial application and visualization of this method revealed boundary variations due to microscope stage tilt and ridge and valley-like contours in the endothelial tissue surface. The method was functionalized into a reproducible analytical process and web tool – the Chip Invasion and Contour Analysis (ChICA) – to model the endothelial surface and quantify invading tumor cells across multiple chips. To illustrate applicability of the analytical method, we applied the tool to CRC organoid-chips seeded with two different endothelial cell types and measured distinct variations in endothelial surfaces and tumor cell invasion dynamics. Since ChICA utilizes only positional data output from imaging software, the method is applicable to and agnostic of the imaging tool and image analysis system used. The novel thin plate spline method developed in ChICA can account for variation introduced in OOC manufacturing or during the experimental workflow, can quickly and accurately measure tumor cell invasion, and can be used to explore biological mechanisms in drug discovery.

A basic process in cancer is the breaching of basement-membrane barriers to permit tissue invasion. Cancer cells can use proteases and physical mechanisms to produce initial holes in basement membranes, but how cells squeeze through this barrier into matrix environments is not well understood. We used a 3D invasion model consisting of cancer-cell spheroids encapsulated by a basement membrane and embedded in collagen to characterize the dynamic early steps in cancer-cell invasion across this barrier. We demonstrate that certain cancer cells extend exceptionally long (~30-100 μm) protrusions through basement membranes via actin and microtubule cytoskeletal function. These long protrusions use integrin adhesion and myosin II-based contractility to pull cells through the basement membrane for initial invasion. Concurrently, these long, organelle-rich protrusions pull surrounding collagen inward while propelling cancer cells outward through perforations in the basement-membrane barrier. These exceptionally long, contractile cellular protrusions can facilitate the breaching of the basement-membrane barrier as a first step in cancer metastasis.

The Presence of Extensive Lymphovascular Invasion (LVI) is Associated with Higher Risk of Recurrence in Curatively Treated Breast Cancer Patients

Cancer cell extravasation is a crucial step in cancer metastasis. However, many of the mechanisms involved in this process are only now being elucidated. Thus, in the present study we analysed the trans-endothelial invasion of melanoma cells by a high throughput label-free cell impedance assay applied to transwell chamber invasion assay. This technique monitors and quantifies in real-time the invasion of endothelial cells by malignant tumour cells, for a long time, avoiding artefacts due to preparation of the end point measurements. Results obtained by impedance analysis were compared with endpoint measurements. In this study, we used human melanoma M14 wild type (WT) cells and their drug resistant counterparts, M14 multidrug resistant (ADR) melanoma cells, selected by prolonged exposure to doxorubicin (DOX). Tumour cells were co-cultured with monolayers of human umbilical vein endothelial cells (HUVEC). Results herein reported demonstrated that: (i) the trans-endothelial migration of resistant melanoma cells was faster than sensitive ones; (ii) the endothelial cells appeared to be strongly affected by the transmigration of melanoma cells which showed the ability to degrade their cytoplasm; (iii) resistant cells preferentially adopted the transcellular invasion vs. the paracellular one; (iv) the endothelial damage mediated by tumour metalloproteinases seemed to be reversible.

Cancer metastasis is still a major challenge in clinical cancer treatment. The migration and invasion of cancer cells into surrounding tissues and blood vessels is the primary step in cancer metastasis. However, the underlying mechanism of regulating cell migration and invasion are not fully understood. Here, we show the role of malic enzyme 2 (ME2) in promoting human liver cancer cell lines SK-Hep1 and Huh7 cells migration and invasion. Depletion of ME2 reduces cell migration and invasion, whereas overexpression of ME2 increases cell migration and invasion. Mechanistically, ME2 promotes the production of pyruvate, which directly binds to β-catenin and increases β-catenin protein levels. Notably, pyruvate treatment restores cell migration and invasion of ME2-depleted cells. Our findings provide a mechanistic understanding of the link between ME2 and cell migration and invasion.

Extravasation of circulating tumor cells (CTCs) from the vasculature is a key step in cancer metastasis. CTCs bind to cell adhesion molecules (CAMs) expressed by endothelial cells (ECs) for flow arrest prior to extravasation. While a number of EC-expressed CAMs have been implicated in facilitating CTC binding, this work investigated the efficacy of inhibiting cancer cell binding to human lung microvascular ECs via antibody blocking of E-selectin using antibody-functionalized gold nanoshells (NS). The antibody-functionalized gold NS were synthesized using both directional and non-directional antibody conjugation techniques with variations in synthesis parameters (linker length, amount of passivating agents, and ratio of antibodies to NS) to gain a better understanding of these properties on the resultant hydrodynamic diameter, zeta potential, and antibody loading density. We quantified the ability of E-selectin antibody-functionalized NS to bind human lung microvascular endothelial cells (HMVEC-Ls) under non-inflamed and inflamed (TNF-α) conditions to inhibit binding of triple-negative MDA-MB-231s. E-selectin-targeted NS prepared using non-directional conjugation had higher antibody loading than those prepared via directional conjugation, resulting in the conjugates having similar overall binding to HMVEC-Ls at a given antibody concentration. E-selectin-targeted NS reduced MDA-MB-231 binding to HMVEC-Ls by up to 41% as determined using an in vitro binding assay. These results provide useful insights into the characteristics of antibody-functionalized NS prepared under different conditions while also demonstrating proof of concept that these conjugates hold potential to inhibit CTC binding to ECs, a critical step in extravasation during metastasis.

Cell-substratum and cell-cell adhesion forces and single-cell mechanical properties in mono- and multilayer assemblies from robotic fluidic force microscopy

Abstract Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, is activated by growth factors and integrin clustering, promoting cell migration, a critical step for cancer metastasis. The activity of FAK is closely associated with the phosphorylation of diverse residues, which are mediated by various protein tyrosine kinases and phosphatases. Previous studies have demonstrated that serine/threonine protein phosphatase 5 (PP5) promotes cell migration, while protein phosphatase 2A (PP2A) inhibits cell migration. However, whether PP5 and PP2A regulate cell migration by mediating FAK phosphorylation is poorly understood. In this study, we found that overexpression of PP5 decreased FAK phosphorylation at S722, and knockdown of PP5 increased p-FAK (S722) in a spectrum of tumor cell lines. Our co-immunoprecipitation (co-IP) results showed an interaction between PP5 and FAK. In addition, we observed that overexpression of PP5 induced S178 phosphorylation of paxillin, an FAK interacting protein. However, there was no physical interaction between PP5 and paxillin, indicating an indirect regulation. In addition, knockdown of PP2A enhanced the phosphorylation of FAK and conferred resistance to rapamycin inhibition of IGF-I-stimulated phosphorylation of FAK. To our surprise, by co-IP, we failed to detect any direct interaction between FAK and PP2A. Further research will determine how PP5 and PP2A regulate cell migration through FAK and paxillin. Acknowledgements This work was supported by the Feist-Weiller Cancer Center (FWCC) Bridge Award (S.H.), Shreveport, LA, USA Citation Format: Lin Li, Lie Liu, Shile Huang. Regulation of FAK phosphorylation by serine/threonine protein phosphatases in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2403.

Cinnamomum bejolghota, used in Thai traditional medicine remedies, has several biological activities including antimicrobial, antifungal, and anticancer. In colorectal cancer, epithelial-mesenchymal transition (EMT) is an initial step of cancer metastasis. Thus, this study investigated the effects of C. bejolghota bark extract (CBE) on colorectal cancer cell metastasis and transforming growth factor-β1 (TGF-β1) induced EMT in LoVo cells. The results showed that CBE could reduce cell migration, invasion, and adhesion of LoVo cells in a dose-dependent manner. In addition, our studies also showed that CBE could reverse TGF-β1-induced morphological changes as well as increase an epithelial marker, E-cadherin, while the expression of the mesenchymal marker, N-cadherin, was decreased in TGF-β1-treated LoVo cells. MMP-2 expression was effectively decreased but TIMP-1 and TIMP-2 expression was increased by the CBE treatment in LoVo cells. CBE also inhibited Smad2/3 phosphorylation and nuclear translocation as well as decreased the expression of Snail, Slug, and TCF8/ZEB1 transcription factors in LoVo cells. Moreover, CBE could inhibit TGF-β1-induced Smad-independent signaling pathway by decreased phosphorylation of ERK1/2, p38, and Akt. These findings suggest that CBE inhibited TGF-β1-induced EMT in LoVo cells via both Smad-dependent and Smad-independent pathways. Therefore, CBE may function as an alternative therapeutic treatment for colorectal cancer metastasis.

Non-proteincoding transcripts bearing 200 base pairs known as long non-coding RNAs (lncRNAs) play a role in a variety of molecular mechanisms, including cell differentiation, apoptosis and metastasis. Previous studies have suggested that frequently dysregulated lncRNAs play a crucial role in various aspects of cancer metastasis. Metastasis is the main leading cause of death in cancer. The role of lncRNAs in different stages of metastasis is the subject of this review. Based on in vitro and in vivo investigations on metastasis, we categorized lncRNAs into distinct stages of metastasis including angiogenesis, invasion, intravasation, survival in circulation, and extravasation. The involvement of lncRNAs in angiogenesis and invasion has been extensively studied. Here, we comprehensively discuss the role and functions of these lncRNAs with a particular focus on the molecular mechanisms.

Adhesion of cancer cells to vascular endothelial cells in target organs is an initial step in cancer metastasis. Our previous studies revealed that amphoterin-induced gene and open reading frame 2 (AMIGO2) promotes the adhesion of tumor cells to liver endothelial cells, followed by the formation of liver metastasis in a mouse model. However, the precise mechanism underlying AMIGO2-promoted the adhesion of tumor cells and liver endothelial cells remains unknown. This study was conducted to explore the role of cancer cell-derived AMIGO2-containing extracellular vesicles (EVs) in the adhesion of cancer cells to human hepatic sinusoidal endothelial cells (HHSECs). Western blotting indicated that AMIGO2 was present in EVs from AMIGO2-overexpressing MKN-28 gastric cancer cells. The efficiency of EV incorporation into HHSECs was independent of the AMIGO2 content in EVs. When EV-derived AMIGO2 was internalized in HHSECs, it significantly enhanced the adhesion of HHSECs to gastric (MKN-28 and MKN-74) and colorectal cancer cells (SW480), all of which lacked AMIGO2 expression. Thus, we identified a novel mechanism by which EV-derived AMIGO2 released from AMIGO2-expressing cancer cells stimulates endothelial cell adhesion to different cancer cells for the initiate step of liver metastasis.