Cancer, as a cause of death, is only surpassed by cardiovascular diseases. Thus, it is critical to achieve progress in its treatment and prevention. Given the complexity and heterogeneity of cancer, various therapeutic targets are being investigated, including components of the tumor milieu. The tumor microenvironment (TME) consists of several types of cells (vascular cells, tumor-associated fibroblasts, immune cells, mesenchymal stem cells, and adipocytes) embedded in extracellular matrix soaked by interstitial fluid rich in soluble factors secreted by cells . Increasing evidence indicates that tumor progression depends on the interaction between the tumor and its microenvironment and that the effectiveness of anti-cancer therapies is modulated by changes in the TME [1-3]. Therefore, extensive research efforts are devoted to investigating the spatial organization of the native TME and to build in vitro models of the TME using three-dimensional (3D) bioprinting  and tissue-on-a-chip techniques .
In this work, we report the 3D bioprinting of avascular structures that recapitulate several features of the TME . In our model, the tumor is represented by a hydrogel droplet uniformly loaded with breast cancer cells, whereas the microenvironment is modelled by rings of hydrogel loaded with peritumoral cells: tumor associated fibroblasts and peripheral blood mononuclear cells. The tumor cells used in our experiments came from a commercial cell line (SK-BR-3), while the peritumoral cells were obtained from breast cancer female patients in different carcinoma stages. The cells were embedded in CELLINK Universal Bioink at concentrations of 1 million cells per milliliter, and the tumor models were fabricated using extrusion bioprinters (INKREDIBLE and BIO X, CELLINK, Sweeden). For the optimization and precise control of the printing process, we developed in-house Python scripts able to generate the G-code instructions for the two bioprinters based on the geometries of the digital models. Our workflow was designed to permit the subsequent bioprinting of desired constructs on multiwell plates of different dimensions. After two weeks of in vitro culture, histological cryosections of the tumor models showed that the hydrogel used in this study was appropriate for sustaining cell growth and proliferation. When tumor models were implanted subcutaneously, in the dorsal region of CD1 Nu/Nu immunosuppressed mice, within 28 weeks in vivo they became vascularized and grew about 5 times in diameter. In conclusion, our work presents a reliable methodology for building models of the TME using extrusion bioprinting. Such models can be used for fundamental research or, if built from patient-derived cells, for testing the effectiveness of anti-cancer therapies, thereby contributing to personalized treatment plans.
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