Stem cells can be used to affect the biology of tumors and to deliver anti-tumor drugs for the treatment of cancers [1,2]. Breast cancer is one of the most common types of malignancy among women worldwide and has therefore been the subject of research by scientists to diagnose and treat for decades [3]. In addition to the conventional therapies (e. g., surgery, radiotherapy, and chemotherapy by natural and synthetic chemicals), extensive studies have been conducted to develop effective and low-risk targeted therapies, such as immunological-based therapy, nanoparticles-based anti-cancer drug delivery and stem cell-based therapy, for the treatment of different types of cancers [4,5]. Mesenchymal stem cells (MSCs) are important immune-compatible cells for stem cell-based therapies, and drug delivery applications to treat human diseases such as cancer. It has been reported that MSCs exert their therapeutic effects through direct interaction with surrounding cells or paracrine activities by the production and secretion of various factors [6,7]. Interestingly, the therapeutic effect of MSCs is primarily achieved through the production of microvesicles (0.1–1 mm in diameter) and exosomes (50–150 nm in diameter) and thus their paracrine activities. The MSCs-derived microvesicles and exosomes, through delivering RNAs and proteins into the target cells, can affect the biological characteristics of different cells [[8], [9], [10], [11], [12]]. MSCs can also able to produce and secrete various bioactive molecules in the conditioned culture medium (CM) in vitro. CM is a medium kept in a culture dish with the cultured cells overnight. After culturing MSCs, they secrete a broad range of bioactive molecules, including proteins (such as TGF-β, EGF، VEGF-A، FGF، PDGF، SDF-1α, CSF, GM-CSF, MCP-1, CXCL1, IL-6, and IL-8), nucleic acids, proteasomes, exosomes, microRNA, and membrane vesicles, which collectively known as the secretome [[13], [14], [15], [16], [17]]. In recent years, much attention has been paid to the possible use of MSCs-derived CM for the treatment of some human pathological conditions; because it is easier to be used, and there is no need for MSC transplantation. Furthermore, concerns regarding the unwanted differentiation and tumorigenesis of MSCs after transplantation are eliminated.

It has been reported that MSCs are essential cells that fundamentally, through modulation of the tumor microenvironment (TME), regulate and determine the final destination of tumor cells. However, there are conflicting functions regarding the activity of MSCs in the tumor microenvironment. Indeed, MSCs confer a tumorigenic or anti-tumor potential to the tumor cells. Functionally, MSCs are recognized for homing to TME and promoting or inhibiting cancer cell proliferation, angiogenesis, and metastasis [18]. TME is a complex ecosystem that comprises stromal cells (including adipocytes, endothelial cells, cancer-associated fibroblasts, immune cells, and MSCs), extracellular matrix components, and secreted factors (such as cytokines, chemokines, angiogenic, and growth factors) [19]. Correlation between MSCs and tumor cells in the TME is demonstrated for various cancers [20,21]. For example, MSCs are recruited and activated into carcinoma-associated fibroblasts by prostate cancer microenvironment-derived TGF-β1 [21].

MSCs can affect the biology of breast cancer, however, the exact mechanism by which MSCs homing to the tumor site is still unclear and is apparently dependent on the specific microenvironment of the tumor [[22], [23], [24]]. There are contradictory reports regarding the effect of MSCs on breast cancer [25]. For example, it demonstrated that MSCs significantly reduced the proliferation and migration of MDA-MB231 cell lines in 2D and 3D co-culture systems [26]. On the other hand, it found that the secretion of CXCL1 and IL8 chemokines by MSCs induced angiogenic signaling pathways and thus facilitated breast tumor growth and metastasis [27]. Different studies also examined the effect of the MSCs-derived CM on the proliferation, survival, migration, and apoptosis of breast cancer cells. Contradictory results have also been reported in these studies [[28], [29], [30], [31]]. Therefore, more research is still needed to determine the exact effect of human- and other mammals-derived MSCs and their CMs on the biology of breast cancer.

From the past to the present, much attention has been paid to the use of medicinal plants and their effective compounds for treating of human diseases. The black seed (Nigella sativa), which is belonged to the Ranunculaceae family of plants, has a long history in traditional medicine. Black seed is a valuable source of bioactive compounds. Researchers attribute most of the black seed properties to Thymoquinone (TQ), which is one of the volatile oily elements in the seed. TQ is a potent anti-oxidant molecule that has the potential to scavenge free radicals and superoxide anions. Moreover, TQ has many biomedical properties, including anti-inflammatory, anti-diabetic, anti-aging, anti-cancer, etc. Different studies reported the anti-neoplastic properties of TQ against a variety of cancers by affecting various signaling pathways and cellular behaviors, such as cell survival, proliferation, apoptosis, angiogenesis, and metastasis [[32], [33], [34]]. In addition, previous studies indicated that TQ improves the biological properties of MSCs [35,36]. For example, it has been reported that TQ induces mouse bone marrow-derived MSCs (BM-MSC) proliferation and influences their immunomodulation properties [35]. Also, it promotes mouse BM-MSCs migration in vitro and induces their immunogenicity in vivo [36]. Therefore, considering the potential characteristics and functions of MSC and TQ, we propose that treatment of MSCs with TQ may influence their paracrine production and secretion of bioactive molecules and thus their effects on other cells, especially cancerous cells. Although the effects of MSCs and TQ have separately been studied on the biological characteristics of breast cancer cells in vitro and in vivo, the TQ-treated MSCs-derived CM (TQ-MSC-CM) effects have not still been studied on the breast cancer cells. Therefore, this study aimed to evaluate the effects of untreated MSC-CM and TQ-treated MSC-CM on the biological characteristics of the MCF7 breast cancer cell line.