Comparison between samples was performed by a nonparametric Mann-Whitney test or paired section. viability of tumor cells. Therefore, this platform could be applied for a personalized immune-based drug screening, with results after a maximum of 10 days of culture, in order to develop more tailored breast cancer treatments and ameliorate patients’ survival rate. cell culture offer more efficient cell-cell and cell-matrix interactions, which influences cell structure, cell signaling, cell adhesion and mechano-sensing. Moreover, the diffusion of oxygen and nutrients is hampered to the hypoxic core of the structure and produced metabolites are also poorly diffused from the core to the surface of the 3D structure (13, 14). Finally, 3D systems allow for the addition of different cell types, creating a multicellular structure, necessary for the study of the TIME and alternative therapeutic options (15, 16). GDF1 Nevertheless, there are multiple ways to develop a 3D culture system. Here, we disclose a protocol for a liquid overlay technique, which consists in coating the plate with a nonadhesive component to enhance cell-cell interaction, allowing a spontaneous 3D spheroid formation (17). This approach is scaffold-independent, easy to work and handle, reproducible and less expensive, when compared to the use of commercial low adherence plates or even scaffold-based approaches that use Matrigel or collagen, for instance (17, 18). Another advantage is the low number of cells needed to form the 3D structure, which is a beneficial characteristic when working with patient-derived cells. Although other protocols have been established for the formation of breast cancer spheroids with immune cell invasion, here we reveal a protocol specifically for MDA-MB-231 cell line, which is often seen as a difficult cell line to spontaneous form spheroids without scaffolds (19, 20). The employment of this cell line in drug-screening platforms is particularly relevant because it is Alimemazine hemitartrate a highly Alimemazine hemitartrate aggressive, invasive and poorly differentiated TNBC cell line. TNBC lacks the estrogen and progesterone receptor (ER and PR) expression, as well as the human epidermal growth factor receptor 2 (HER2), currently having limited treatment options (21). Interestingly, this cell line, differently from other common breast cancer cell lines, such as MCF-7, express high levels of the immune checkpoint PD-L1 (22), which can bind to its receptor PD-1 at the surface of effector T lymphocytes, therefore acting as a brake and impairing the activation and the assemble Alimemazine hemitartrate of a proper antitumor response. PD-L1, a key component of the tumor immune evasion mechanisms, is indeed highly expressed in many breast cancer patients’ tumors, especially in cases with poorly activated tumor-infiltrating effector T lymphocytes (3). Additionally, with this protocol, it is possible to perform a co-culture in a 1:1 ratio of tumor cells to immune cells and observe the effect of the later in the tumor. This is an advantage when comparing to other protocols with a 10:1 ratio of immune cells to tumor cells (23), since it is more representative of immune cells infiltration into the tumor microenvironment, as we observed previously (3). Thus, besides describing in detail the protocol employed for the establishment of this 3D system, we also demonstrate the utility of this allogeneic system?3D co-culture of MDA-MB-231 cell line with breast cancer patient-derived immune cellsin the development of novel therapies, as treating the immune cells with an external canonical stimulus could improve their cytotoxic activity against the tumor cells. We believe that this system can become extremely useful to test, in a simple and economic fashion, several clinical grade immune-modulators that alone or in combination with chemotherapeutic compounds could improve the anti-tumor response of Alimemazine hemitartrate individual breast cancer patients. Likewise, this protocol also has the advantage of possible modifications to include different cell types such as fibroblasts or even tumor cells, matching the immune cells, to build an autologous system. Materials and Equipment Materials/Reagents MDA-MB-231 (ATCC? HTB-26?) Dulbecco’s Modified Eagle Medium (DMEM, Biowest, catalog number: L0102-500), store at 4C Fetal Bovine Serum (FBS, Sigma Aldrich, catalog number: F9665-0500), store at?20C Penicillin/Streptomycin (Pen/Strep, GE Healthcare, catalog number: SV30010), store at ?20C TrypLE (Gibco, catalog number: 12605028), store at 4C RPMI-1640 (Gibco, catalog number:.