Novel human lymph node-derived matrix supports the adhesion of metastatic oral carcinoma cells.

BACKGROUND: 3D culture is increasingly used in cancer research, as it allows the growth of cells in an environment that mimics in vivo conditions. Metastases are the primary cause of morbidity and mortality in cancer patients, and solid tumour metastases are mostly located in lymph nodes. Currently, there are no techniques that model the pre-metastatic lymph node microenvironment in vitro. In this study, we prepared a novel extracellular matrix, Lymphogel, which is derived from lymph nodes, mimicking the tumour microenvironment (TME) of metastatic carcinoma cells. We tested the suitability of the new matrix in various functional experiments and compared the results with those obtained using existing matrices. METHODS: We used both commercial and patient-derived primary and metastatic oral tongue squamous cell carcinoma (OTSCC) cell lines. We characterized the functional differences of these cells using three different matrices (human uterine leiomyoma-derived Myogel, human pre-metastatic neck lymph node-derived Lymphogel (h-LG), porcine normal neck lymph node-derived Lymphogel (p-LG) in proliferation, adhesion, migration and invasion assays. We also performed proteomic analyses to compare the different matrices in relation to their functional properties. RESULTS: OTSCC cells exhibited different adhesion and invasion patterns depending on the matrix. Metastatic cell lines showed improved ability to adhere to h-LG, but the effects of the matrices on cell invasion fluctuated non-significantly between the cell lines. Proteomic analyses showed that the protein composition between matrices was highly variable; Myogel contained 618, p-LG 1823 and h-LG 1520 different proteins. The comparison of all three matrices revealed only 120 common proteins. Analysis of cellular pathways and processes associated with proteomes of each matrix revealed similarities of Myogel with h-LG but less with p-LG. Similarly, p-LG contained the least adhesion-related proteins compared with Myogel and h-LG. The highest number of unique adhesion-related proteins was present in h-LG. CONCLUSIONS: We demonstrated that human pre-metastatic neck lymph node-derived matrix is suitable for studying metastatic OTSCC cells. As a whole-protein extract, h-LG provides new opportunities for in vitro carcinoma cell culture experiments.

Human myoma tissue-based extracellular matrix models for testing the effects of irradiation on the HPV positive cells.

BACKGROUND: This study was designed to investigate the invasion of human papillomavirus (HPV) positive human cervical carcinoma cell lines in human leiomyoma-based extracellular matrices in vitro, and to test the suitability of the model for studying the irradiation effects on the cancer cell invasion. METHODS: HPV positive cervical carcinoma cell lines SiHa and CaSki, and HPV negative squamous cell carcinoma cell line HSC-3 were used. CaSki cells contain around 600 copies of HPV 16 virus in the genome, whereas SiHa have only 1-2 copies per cell. Cells were analyzed using two different human tumor derived extracellular matrix methods (3D myoma disc model, and Myogel Transwell invasion assay). Cultures were irradiated with 4 Gy. Myoma invasion area and the depth of invasion were measured with ImageJ 1.51j8 software. Statistical analyses were performed with SPSS Statistics (IBM SPSS® Statistics 25). RESULTS: All cells invaded through Myogel coated Transwell membranes and within myoma discs. In myoma discs, a difference in the invasion depth (p = 0.0001) but not in invasion area (p = 0.310) between the HPV positive cell lines was seen, since SiHa (less HPV) invaded slightly better than CaSki (more HPV). HSC-3 cells (HPV negative) invaded deepest (p = 0.048) than either of the HPV positive cell line cells. No difference was detected in the invasion area (p = 0.892) between HPV positive and HPV negative cells. The ionized radiation significantly reduced the invasion depth of HSC-3 (p = 0.008), SiHa (p = 0.0001) and CaSki (p = 0.005). No significant effect on the invasion area was detected in any of the cell lines. However, a significant difference was observed between SiHa and CaSki in the reduction of the invasion depth after radiation (p = 0.013) as the reduction was greater with SiHa than CaSki. CONCLUSIONS: Both solid and gelatinous human leiomyoma-based extracellular matrix models were suitable platforms to study the invasion of HPV positive cervical carcinoma cells in vitro. SiHa cells with less HPV copy number cells invaded slightly better and were slightly more sensitive to irradiation than CaSki cells with high HPV copy number. However, there was no drastic differences between the invasion properties of these carcinoma cells.

Effects of ionizing radiation and HPSE1 inhibition on the invasion of oral tongue carcinoma cells on human extracellular matrices in vitro.

Chemoradiation is an established approach in the treatment of advanced oral tongue squamous cell carcinoma (OTSCC), but therapy may cause severe side-effects due to signal interchanges between carcinoma and the tumour microenvironment (TME). In this study, we examined the potential use of our human 3D myoma disc and Myogel models in in vitro chemoradiation studies by analysing the effects of ionizing radiation (IR) and the combined effect of heparanase I (HPSE1) inhibitors and IR on OTSCC cell proliferation, invasion and MMP-2 and - 9 production. Finally, we analysed the long-term effects of IR by studying clones of previously irradiated and invaded HSC-3 cells. We found that in both human uterine leiomyoma-based extracellular matrix models IR inhibited the invasion of HSC-3 cells, but blocking HPSE1 activity combined with IR induced their invasion. Low doses of IR increased MMP expression and initiated epithelial-mesenchymal transition in cells cultured on myoma discs. We conclude that myoma models offer consistent methods for testing human carcinoma cell invasion and phenotypic changes during chemoradiation treatment. In addition, we showed that IR had long-term effects on MMP-2 and - 9, which might elicit different HSC-3 invasion responses when cells were under the challenge of HPSE1 inhibitors and IR.

Desmoglein 3 - Influence on oral carcinoma cell migration and invasion.

Desmoglein 3 (Dsg3) is an adhesion receptor in desmosomes, but its role in carcinoma cell migration and invasion is mostly unknown. Our aim was to quantitatively analyse the motion of Dsg3-modified carcinoma cells in 2D settings and in 3D within tumour microenvironment mimicking (TMEM) matrices. We tested mutant constructs of C-terminally truncated Dsg3 (∆238 and ∆560), overexpressed full-length (FL) Dsg3, and empty vector control (Ct) of buccal mucosa squamous cell carcinoma (SqCC/Y1) cells. We captured live cell images and analysed migration velocities and accumulated and Euclidean distances. We compared rodent collagen and Matrigel® with human Myogel TMEM matrices for these parameters in 3D sandwich, in which we also tested the effects of monoclonal antibody AK23, which targets the EC1 domain of Dsg3. In monolayer culture, FL and both truncated constructs migrated faster and had higher accumulated distances than Ct cells. However, in the 3D assays, only the mutants invaded faster relative to Ct cells. Of the mutants, the shorter form (Δ238) exhibited faster migration and invasion than Δ560 cells. In the Transwell, all of the cells invaded faster through Myogel than Matrigel® coated wells. In 3D sandwich, AK23 antibody inhibited only the invasion of FL cells. We conclude that different experimental 2D and 3D settings can markedly influence the movement of oral carcinoma cells with various Dsg3 modifications.

Evaluation of in vitro and in vivo personalized cancer treatment assays for oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a common cancer with a high heterogeneity and few approved treatments. OSCC is one of the least explored areas for precision oncology. In this study, we aimed to test the reliability of our three established rapid cancer systemic treatment-testing assays: human tumour-derived matrix (Myogel)-coated well-plates, zebrafish xenografts, and 3D microfluidic chips. METHODS: Chemo-, radio- and targeted-therapy testing in Myogel-coated wells and zebrafish xenografts was conducted nine times using five samples; two primary and three metastatic lymph node samples from three OSCC patients. Peripheral blood mononuclear cells (PBMNCs) were isolated from the patients' blood. The response of the tumour cells to radio-, chemo-, and targeted therapy was tested using Myogel-coated wells and zebrafish larvae xenografts. The tumour cells' response to immunotherapy was tested using 3D microfluidic chips. The cells' sensitivity to the treatments was compared with the patients' clinical response. Primary and metastatic lymph node tissue-derived DNA samples from two patients underwent whole exome sequencing to compare the mutational profiles of the samples. RESULTS: Test results were in line with patients' responses in 7/9 (77%) zebrafish xenograft assays and 5/9 (55%) Myogel-coated wells assays. Immunotherapy testing was done using one metastatic patient sample which matched the patients' response. Differences in responses to treatments between primary and metastatic samples of the same patient were detected in 50% of the zebrafish larvae assays. CONCLUSIONS: Our results show the potential of using personalized cancer treatment testing assays - specifically zebrafish xenografts that revealed promising results - in OSCC patient samples.

Transcriptomic Profiling of JEG-3 cells using human leiomyoma derived matrix.

Oxygen tension varies during placental and fetal development. Although hypoxia drives early trophoblast invasion, low placental oxygen levels during pregnancy show association with pregnancy complications including fetal growth restriction and preeclampsia. JEG-3 cells are often used as a trophoblast model. We studied transcriptional changes of JEG-3 cells on a uterine leiomyoma derived matrix Myogel. This might be the closest condition to the real uterine environment that we can get for an in vitro model. We observed that culturing JEG-3 cells on the leiomyoma matrix leads to strong stimulation of ribosomal pathways, energy metabolism, and ATP production. Furthermore, Myogel improved JEG-3 cell adherence in comparison to tissue culture treated plastic. We also included PDMS microchip hypoxia creation, and observed changes in oxidative phosphorylation, oxygen related genes and several hypoxia genes. Our study highlights the effects of Myogel matrix on growing JEG-3 cells, especially on mitochondria, energy metabolism, and protein synthesis.

Comparative Analysis of Vascular Mimicry in Head and Neck Squamous Cell Carcinoma: In Vitro and In Vivo Approaches.

Tissue vasculature provides the main conduit for metastasis in solid tumours including head and neck squamous cell carcinoma (HNSCC). Vascular mimicry (VM) is an endothelial cell (EC)-independent neovascularization pattern, whereby tumour cells generate a perfusable vessel-like meshwork. Yet, despite its promising clinical utility, there are limited approaches to better identify VM in HNSCC and what factors may influence such a phenomenon in vitro. Therefore, we employed different staining procedures to assess their utility in identifying VM in tumour sections, wherein mosaic vessels may also be adopted to further assess the VM-competent cell phenotype. Using 13 primary and metastatic HNSCC cell lines in addition to murine- and human-derived matrices, we elucidated the impact of the extracellular matrix, tumour cell type, and density on the formation and morphology of cell-derived tubulogenesis in HNSCC. We then delineated the optimal cell numbers needed to obtain a VM meshwork in vitro, which revealed cell-specific variations and yet consistent expression of the EC marker CD31. Finally, we proposed the zebrafish larvae as a simple and cost-effective model to evaluate VM development in vivo. Taken together, our findings offer a valuable resource for designing future studies that may facilitate the therapeutic exploitation of VM in HNSCC and other tumours.