Transcription factor activating enhancer-binding protein 2ε (AP2ε) modulates phenotypic plasticity and progression of malignant melanoma.

Malignant melanoma, the most aggressive form of skin cancer, is often incurable once metastatic dissemination of cancer cells to distant organs has occurred. We investigated the role of Transcription Factor Activating Enhancer-Binding Protein 2ε (AP2ε) in the progression of metastatic melanoma. Here, we observed that AP2ε is a potent activator of metastasis and newly revealed AP2ε to be an important player in melanoma plasticity. High levels of AP2ε lead to worsened prognosis of melanoma patients. Using a transgenic melanoma mouse model with a specific loss of AP2ε expression, we confirmed the impact of AP2ε to modulate the dynamic switch from a migratory to a proliferative phenotype. AP2ε deficient melanoma cells show a severely reduced migratory potential in vitro and reduced metastatic behavior in vivo. Consistently, we revealed increased activity of AP2ε in quiescent and migratory cells compared to heterogeneously proliferating cells in bioprinted 3D models. In conclusion, these findings disclose a yet-unknown role of AP2ε in maintaining plasticity and migration in malignant melanoma cells.

A vascularized in vivo melanoma model suitable for metastasis research of different tumor stages using fundamentally different bioinks.

Although 2D cancer models have been the standard for drug development, they don't resemble in vivo properties adequately. 3D models can potentially overcome this. Bioprinting is a promising technique for more refined models to investigate central processes in tumor development such as proliferation, dormancy or metastasis. We aimed to analyze bioinks, which could mimic these different tumor stages in a cast vascularized arteriovenous loop melanoma model in vivo. It has the advantage to be a closed system with a defined microenvironment, supplied only with one vessel-ideal for metastasis research. Tested bioinks showed significant differences in composition, printability, stiffness and microscopic pore structure, which led to different tumor stages (Matrigel and Alg/HA/Gel for progression, Cellink Bioink for dormancy) and resulted in different primary tumor growth (Matrigel significantly higher than Cellink Bioink). Light-sheet fluorescence microscopy revealed differences in vascularization and hemorrhages with no additional vessels found in Cellink Bioink. Histologically, typical human melanoma with different stages was demonstrated. HMB-45-positive tumors in progression inks were infiltrated by macrophages (CD163), highly proliferative (Ki67) and metastatic (MITF/BRN2, ATX, MMP3). Stainings of lymph nodes revealed metastases even without significant primary tumor growth in Cellink Bioink. This model can be used to study tumor pathology and metastasis of different tumor stages and therapies.

Molecular Changes Induced in Melanoma by Cell Culturing in 3D Alginate Hydrogels.

Alginate hydrogels have been used as a biomaterial for 3D culturing for several years. Here, gene expression patterns in melanoma cells cultivated in 3D alginate are compared to 2D cultures. It is well-known that 2D cell culture is not resembling the complex in vivo situation well. However, the use of very intricate 3D models does not allow performing high-throughput screening and analysis is highly complex. 3D cell culture strategies in hydrogels will better mimic the in vivo situation while they maintain feasibility for large-scale analysis. As alginate is an easy-to-use material and due to its favorable properties, it is commonly applied as a bioink component in the growing field of cell encapsulation and biofabrication. Yet, only a little information about the transcriptome in 3D cultures in hydrogels like alginate is available. In this study, changes in the transcriptome based on RNA-Seq data by cultivating melanoma cells in 3D alginate are analyzed and reveal marked changes compared to cells cultured on usual 2D tissue culture plastic. Deregulated genes represent valuable cues to signaling pathways and molecules affected by the culture method. Using this as a model system for tumor cell plasticity and heterogeneity, EGR1 is determined to play an important role in melanoma progression.

Comparison of Hydrogels for the Development of Well-Defined 3D Cancer Models of Breast Cancer and Melanoma.

Bioprinting offers the opportunity to fabricate precise 3D tumor models to study tumor pathophysiology and progression. However, the choice of the bioink used is important. In this study, cell behavior was studied in three mechanically and biologically different hydrogels (alginate, alginate dialdehyde crosslinked with gelatin (ADA-GEL), and thiol-modified hyaluronan (HA-SH crosslinked with PEGDA)) with cells from breast cancer (MDA-MB-231 and MCF-7) and melanoma (Mel Im and MV3), by analyzing survival, growth, and the amount of metabolically active, living cells via WST-8 labeling. Material characteristics were analyzed by dynamic mechanical analysis. Cell lines revealed significantly increased cell numbers in low-percentage alginate and HA-SH from day 1 to 14, while only Mel Im also revealed an increase in ADA-GEL. MCF-7 showed a preference for 1% alginate. Melanoma cells tended to proliferate better in ADA-GEL and HA-SH than mammary carcinoma cells. In 1% alginate, breast cancer cells showed equally good proliferation compared to melanoma cell lines. A smaller area was colonized in high-percentage alginate-based hydrogels. Moreover, 3% alginate was the stiffest material, and 2.5% ADA-GEL was the softest material. The other hydrogels were in the same range in between. Therefore, cellular responses were not only stiffness-dependent. With 1% alginate and HA-SH, we identified matrices that enable proliferation of all tested tumor cell lines while maintaining expected tumor heterogeneity. By adapting hydrogels, differences could be accentuated. This opens up the possibility of understanding and analyzing tumor heterogeneity by biofabrication.

Tumor Cells Develop Defined Cellular Phenotypes After 3D-Bioprinting in Different Bioinks.

Malignant melanoma is often used as a model tumor for the establishment of novel therapies. It is known that two-dimensional (2D) culture methods are not sufficient to elucidate the various processes during cancer development and progression. Therefore, it is of major interest to establish defined biofabricated three-dimensional (3D) models, which help to decipher complex cellular interactions. To get an impression of their printability and subsequent behavior, we printed fluorescently labeled melanoma cell lines with Matrigel and two different types of commercially available bioinks, without or with modification (RGD (Arginine-Glycine-Aspartate)-sequence/laminin-mixture) for increased cell-matrix communication. In general, we demonstrated the printability of melanoma cells in all tested biomaterials and survival of the printed cells throughout 14 days of cultivation. Melanoma cell lines revealed specific differential behavior in the respective inks. Whereas in Matrigel, the cells were able to spread, proliferate and form dense networks throughout the construct, the cells showed no proliferation at all in alginate-based bioink. In gelatin methacrylate-based bioink, the cells proliferated in clusters. Surprisingly, the modifications of the bioinks with RGD or the laminin blend did not affect the analyzed cellular behavior. Our results underline the importance of precisely adapting extracellular matrices to individual requirements of specific 3D bioprinting applications.