In this work, we developed collagen hydrogels blended with variable amounts of agarose (from 0.5% to 0.125%) and we investigated the possibility to grow within them tumor spheroids of three different breast cancer cell lines, i.e., the luminal estrogen receptor positive cells, MCF-7 and MDA-MB-361, and the triple negative model, MDA-MB-231. The gene discussed is ESR1; the disease is breast carcinoma.