Introduction. In recent years tumor microenvironment has been recognized as one of the main actors in cancer relapses and metastasis. Indeed, the crosstalk between malignant cells and the niche, as well as the extracellular matrix (ECM) remodeling, not only favor tumor mass growth, but are also preliminary mechanisms to dissemination. These processes have been mainly investigated in tumors of epithelial origin, for which many 3D models have been produced, while little is still known about sarcoma microenvironment. This is true in particular for rhabdomyosarcoma, the most frequently diagnosed soft tissue sarcoma in pediatric patients. Thus, the development of a suitable 3D model to recapitulate the onset of metastasis, which is mostly driven by an altered ECM deposition, is in need. Macromolecular crowding (MMC) has been shown to enhance the process of ECM deposition. The aim of this work was the application of MMC to rhabdomyosarcoma cell lines. A 2D monolayer culture and a 3D spheroid model were compared with a 3D bioprinted construct.
Methodology. RH30 and RD cell lines were cultured under MMC in 2D in presence of a Ficoll cocktail. MMC was also applied to spheroids produced in ultra-low attachment (ULA) plates. Finally, a 3D bioprinted model was produced using the inkjet printing technology and Matrigel as scaffold, to which also MMC was applied. ECM deposition in these samples was analyzed by immunofluorescence and qRT-PCR, and shape parameters were described for the 3D samples.
Results. MMC induced ECM deposition in both cell lines in 2D, which resulted in increased fibronectin signal in immunofluorescence images. MMC-treated spheroids displayed not only augmented ECM protein presence, but also reduced dimensions and higher solidity compared to untreated spheroids. Interestingly, only RH30 spheroids treated with MMC attached to ULA culture surfaces, depositing collagen and fibronectin on the bottom of the culture well. Also, the 3D printed constructs treated with MMC were smaller and more compact than the untreated ones, probably due to the increased amount of ECM components; furthermore, after day 10 of culture small multicellular aggregates detached from the core of these samples, mimicking a dissemination mechanism. This release was less evident in the MMC-treated condition compared to control, but interestingly in both conditions RH30 samples released a higher number of spheroids compared to RD. Of note, CXCR4 levels increased in RH30 samples under MMC stimulation, a gene involved in metastasis promotion.
Conclusions. These observations indicate that MMC stimulate ECM deposition in rhabdomyosarcoma cells and induce a metastatic behavior. As future perspective, the MMC-treated and 3D printed model developed in this work could find applications in drug testing.