Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

National Health Research Institutes

Frequent metastasis to the cervical lymph nodes leads to poor survival of patients with oral squamous cell carcinoma (OSCC). To understand the underlying mechanisms of lymph node metastasis, two sublines were successfully isolated from cervical lymph nodes of nude mice through in vivo selection, and identified as originating from poorly metastatic parental cells. These two sublines specifically metastasized to cervical lymph nodes in 83% of mice, whereas OEC-M1 cells did not metastasize after injection into the oral cavity. After gene expression analysis, we identified insulin-like growth factor binding protein 3 (IGFBP3) as one of the significantly up-regulated genes in the sublines in comparison with their parental cells. Consistently, meta-analysis of the public microarray datasets and IGFBP3 immunohistochemical analysis revealed increased both levels of IGFBP3 mRNA and protein in human OSCC tissues when compared to normal oral or adjacent nontumorous tissues. Interestingly, the up-regulated IGFBP3 mRNA expression was significantly associated with OSCC patients with lymph node metastasis. IGFBP3 knockdown in the sublines impaired and ectopic IGFBP3 expression in the parental cells promoted migration, transendothelial migration and lymph node metastasis of orthotopic transplantation. Additionally, ectopic expression of IGFBP3 with an IGF-binding defect sustained the IGFBP3-enhanced biological functions. Results indicated that IGFBP3 regulates metastasis-related functions of OSCC cells through an IGF-independent mechanism. Furthermore, exogenous IGFBP3 was sufficient to induce cell motility and extracellular signal-regulated kinase (ERK) activation. The silencing of integrin β1 was able to impair exogenous IGFBP3-mediated migration and ERK phosphorylation, suggesting a critical role of integrin β1 in IGFBP3-enchanced functions. 

Insulin-like growth factor-independent insulin-like growth factor binding protein 3 promotes cell migration and lymph node metastasis of oral squamous cell carcinoma cells by requirement of integrin β1.

Mobilisation of endothelial progenitor cells (EPCs) from the bone marrow is a crucial step in the formation of de novo blood vessels, and levels of peripheral blood EPCs have been shown to be elevated in certain malignant states. Using flow cytometry and a Hill-based colony forming unit (CFU) assay, the present study indicated that higher levels of CD34 and vascular endothelial growth factor receptor 2 (VEGFR2) double-positive EPCs, as well as increased formation of endothelial cell colony-forming units (EC-CFUs) are associated with benign and malignant breast diseases, providing possible indicators for breast disease detection. Gene expression profiles revealed a genetic difference between CD34 VEGFR2 EPCs and EC-CFUs. Decreased expression of tumour necrosis factor receptor 2 (TNFR2) signalling-related genes and inhibition of tumour necrosis factor (TNF)-induced signalling were demonstrated in EC-CFUs derived from patients with malignant breast disease in comparison with those from healthy controls. Interestingly, our data provided the first evidence that EC-CFUs derived from patients with malignant breast disease were resistant to TNF-α-induced apoptosis, indicating a plausible target for future therapeutic interventions.

Endothelial cell colony forming units derived from malignant breast diseases are resistant to tumor necrosis factor-α-induced apoptosis.

Oral squamous cell carcinoma (OSCC) LN1-1 cells previously showed greater capacities for lymphangiogenesis and lymph node metastasis compared to their parental OEC-M1 cells, in addition to an ability to enhance the migration and tube formation of lymphatic endothelial cells (LECs). Purified by a series of differential centrifugations and characterized using electron microscopy, dynamic light scattering and western blot, LN1-1 cell-derived extracellular vesicles (LN1-1 EVs) were shown to promote LEC migration, tube formation and uptake by LECs more effectively than did OEC-M1 cell-derived EVs (OEC-M1 EVs). Using stable isotope labeling with amino acids in cell culture/liquid chromatography-tandem mass spectrometry-based proteomic platform, the laminin-332 proteins, including laminin α3, β3 and γ2, were validated as highly expressed proteins in LN1-1 EVs. Clinically, a higher level of laminin-332 was detected in plasma EVs from OSCC patients with lymph node metastasis than in both healthy controls and OSCC patients without lymphatic metastasis, suggesting EV-borne laminin-332 as a novel and noninvasive biomarker for the detection of lymph node metastasis in OSCC. The knockdown of laminin γ2 and inhibition by anti-laminin-332 neutralizing antibodies impaired LN1-1 EV-mediated LEC migration, tube formation and uptake by LECs. Importantly, laminin γ2-deficient EVs showed a reduced ability to drain into lymph nodes in comparison with the control EVs. In addition, the laminin 332/γ2-mediated EV uptake was dependent on integrin α3 but not β1, β4 or α6. Collectively, the uptake of laminin γ2-enriched EVs by LECs enhanced in vitro lymphangiogenesis and EV-borne laminin-332 is thus a viable biomarker for OSCC.

Laminin γ2-enriched extracellular vesicles of oral squamous cell carcinoma cells enhance in vitro lymphangiogenesis via integrin α3-dependent uptake by lymphatic endothelial cells.

In an effort to understand the underlying mechanisms of lymph node metastasis in oral squamous cell carcinoma (OSCC), through in vivo selection, LN1-1 cells were previously established from OEC-M1 cells and showed enhanced lymphangiogenesis and lymphatic metastasis capabilities. In the current study, we use a stable isotope labeling with amino acids in cell culture (SILAC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic platform to compare LN1-1 to OEC-M1 cells. Interferon-stimulated gene 15 (ISG15) was found highly expressed in LN1-1 cells. Immunohistochemical analysis and meta-analysis of publicly available microarray datasets revealed that the ISG15 level was increased in human OSCC tissues and associated with poor disease outcome. Knockdown of ISG15 had minimal effects on tumor growth but did decrease tumor lymphangiogenesis and lymphatic metastasis of LN1-1 cells. Consistent with the in vivo assay, ISG15 knockdown did not impair cell growth but diminished cell migration, invasion, and transendothelial migration in vitro. ISG15-induced cell migration was independent of ISGylation and associated with membrane protrusion. Ectopic expression of ISG15 increased Rac1 activity and knockdown of Rac1 impaired ISG15-enhanced migration. Furthermore, Rac1 colocalized with ISG15 to a region of membrane protrusion and ISG15 coimmunoprecipitated with Rac1, especially with the Rac1-GDP form. Importantly, as shown by proximity ligation assays, ISG15 and Rac1 physically interacted with each other. Our results indicated that ISG15 affects cell migration by interacting with Rac1 and regulating Rac1 activity and contributes to lymphatic metastasis in OSCC.

Interferon-stimulated gene 15 modulates cell migration by interacting with Rac1 and contributes to lymph node metastasis of oral squamous cell carcinoma cells.

Ssu-Han Wang

National Institute of Cancer Research

National Health Research Institutes

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Ssu-Han Wang

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National Health Research Institutes

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

National Institute of Cancer Research