In the present study, the MACS magnetic cell separation device system used in breast cancer stem cell isolation worked successfully in accordance with the literature [12, 13, 21]. In characterization studies, the obtained results were found to be compatible with the studies reported in previous years[14].

In antiproliferative activity studies carried out under monolayer (2D) culture conditions, significant cell death in cancer cells was determined for all drug applications. In this context, in line with the literature, it has been determined that Dox is more effective than 5-Fu[3]. It inhibited cell viability and proliferation even at low doses. Experimental data obtained in this study showed that doxorubicin was more effective than 5-fluorouracil on both the MCF-7 cell line and breast cancer stem cells. When the literature was scanned to explain this result, it was seen that ER-positive/HER2-negative tumors had high chemosensitivity to DOX [22, 23]. The relevant data are consistent with the result obtained in this study using MCF-7, an ER-positive/HER2-negative breast cancer cell line. Therefore, in this study, it is thought that one of the reasons why the antiproliferative activity of DOX is higher than 5-Fu may be the characteristic feature of the MCF-7 cell line (ER-positive/HER2-negative). Additionally, the combined drug preparation has a higher antiproliferative effect than the single drug[2]. Similar results were obtained in 3D culture conditions; similar results were obtained with 2D culture by longer exposure time (120 h) and higher doses of drugs. This result is consistent with the scientific article published by Gong et al. in 2015[9]. In addition, when 2D and 3D culture conditions were evaluated together, it was determined that breast cancer stem cells were more resistant than the MCF-7 cancer cell line and required higher drug doses and longer incubation periods to observe a significant antiproliferative effect [24, 25]. This result also shows high compatibility with the relevant literature [4, 6, 23, 24]. In this study designed with MCF-7, a HER2-negative breast cancer cell line, the observation of a synergistic effect for 5-Fu and Dox is of extra importance, together with the data presented in another study published by Mody et al. in 2023. According to this publication, when DOX was administered with another drug that has a synergistic effect with it, side effect markers decreased in the HER2-positive breast cancer cell line, while such an effect was not observed in the MCF-7 cell line [27]. This shows how important breast cancer types are for treatment, and therefore the in vitro results of synergistic or antagonistic effects may produce misleading results when transferred to the clinic. Therefore, the data in this manuscript will make a significant contribution to the studies on the transfer of chemotherapeutics, which have synergistic effects, specifically Dox and 5-Fu, to the clinic [28].

The possible effects of Dox and 5-Fu compounds (as monotherapy and in combination) on the relative expression of genes related to apoptosis were studied. In this context, the expression levels of Bax, Bcl-2 and some caspases were analyzed. As a result, data showed that these drugs (Dox and 5-Fu), alone or together, increased the expression of Bax and Bax/Bcl-2, revealing that the apoptosis mechanism was induced. The data obtained are very valuable, especially since clinically increasing Bax levels are associated with a good response to chemotherapy [29]. Therefore, the experimental process carried out in this study ended with results consistent with the literature [26, 27]. The expressional activities of caspases, another enzyme family involved in apoptosis, known as one of the important mechanisms used in cancer treatment, against the applied chemotherapeutic drug doses were also examined in this study. Changes in the expression levels of caspase 7, 8 and 9, which have an important place in this process, were specifically examined and it was determined that all of them were upregulated, consistent with the literature [28,29,30]. Caspases-8 and 9 are known as initiators of the apoptosis process. However, activation of caspase-8 constitutes a crucial step in the initiation of the death receptor-mediated apoptosis pathway (extrinsic pathway) [31,32,33]. Caspase-9 affects the apoptotic process by playing a very important role in the intrinsic pathway mediated by mitochondria[34]. Considering this basic molecular information, in this study, it has been clearly seen that apoptosis was activated through both extrinsic and intrinsic pathways.

In addition, it has been seen that apoptosis-resistant phenotype associated with 3D tumor spheroids is caused primarily by growth, drug penetrance, or lower oxygen tension and induction of hypoxia in cells embedded within multicellular tumors. For example, hypoxia and reduced cell growth could lead to loss of functional p53 and impede stress-induced apoptosis induction. Because cells incorporated into 3D tissue structures are able to effectively resist apoptosis, and life and death decisions depend upon a delicate balance between proapoptotic and anti-apoptotic signals, cell adhesion in 3D must titrate cell fate in favor of cell survival. Mitochondrial integrity is central to apoptosis regulation, and therefore, it is highly likely that tissue architecture enhances cell survival by modulating mitochondria homeostasis [35,36,37].

In conclusion, the polychemotherapeutic approach was much more effective than the monotherapy. The fact that this effect was seen not only in breast cancer cells, but also in breast cancer stem cells. In addition, it was very promising that the results obtained were similar in both two-dimensional and three-dimensional tumoroids.