Cancer is the one of the most important causes of morbidity and mortality worldwide [2, 17]. The side effects of chemotherapy, radiotherapy and surgical practices, which are used as the first treatment options, off-target effects or factors such as tumor recurrence have directed the researches towards the cell therapy [4, 17]. The recent studies have shown that the mesenchymal stem cells are effective therapeutic agents for various diseases, including cancer [14, 18, 53, 54]. Besides, there are many studies showing the proliferation enhancing effects of MSCs on hematological malignancies  and antitumor functions and tumor growth inhibitor effects of MSCs obtained from different sources for various cancer types [56, 57].
Bone marrow is an important source that is commonly used in MSC isolation. However, the searches for new sources continue due to the difficulty of isolation process, ethical problems and the scarcity of MSC in bone marrow . Therefore, tonsillar tissue obtained after tonsillectomy, which is the most frequent surgical procedure in childhood, is an alternative source of MSC since it can be obtained with a less invasive procedure. In the tonsillar microenvironment, which is also the secondary lymphoid organ, MSCs present here have potential implications for initiating and maintaining effective immunity responses .
In this study, we showed that MSCs obtained from the secondary lymphoid organ, the tonsil, were positive for MSCs specific cell surface antigens and negative for hematopoietic stem cell surface antigens, as well as their multi-lineage differentiation potential. It was also shown that co-cultures of TMSCs with hematological cancer cells show anti-proliferative activity in cancer cells in a time dependent manner. We also showed that febrile temperature status induces the rate of apoptosis in hematological cancer K562 and MOLT 4 cell lines. While no significant change was observed after 5 days of co-culture of K562 cells with T-MSC at normal culture temperature, a 1,4-fold increase was observed in pre-apoptotic cell number, especially at the end of the 7-day culture period. Apoptosis-inducing effect of mild hyperthermia was observed with a 2.5-fold increase in the number of pre-apoptotic cells and a 2-fold increase in the number of post-apoptotic cells under 40 degrees febrile conditions created in vitro. The apoptotic effect induced under febrile temperature was confirmed at gene expression level through the finding of 2-fold increase in pro-apoptotic Bax gene expression. The increased c-myc expression under mild hyperthermia conditions supports the apoptotic effect of co-culture with T-MSCs. However, stable expression of p53 suggests that the role of c-myc on apoptosis may be independent of p53. Pre and post apoptotic cell rates under normal temperature and febrile temperature conditions after 7-day long culture showed approximately 2-fold increase in MOLT-4 cells. Unlike K562 cells, it was observed that co-culture of MOLT 4 cells with T-MSCs under febrile temperature conditions induced apoptosis in a shorter time (5 days). Different studies conducted have shown that MSCs obtained from different sources induce apoptosis in cancer cells at different rates [17, 26]. It has also been reported that tonsil derived MSCs induced apoptosis in Head and Neck Squamous Cell Carcinoma Cells . However, our study is the first study to show that tonsil-derived MSCs induced apoptosis in hematological cancer cell lines. p53 is a tumor suppressor gene that inhibits cell proliferation by stimulating apoptosis . The apoptotic effect of T-MSC co-culture on MOLT4 cells under febril temperature was confirmed with the expression of decreased Bcl-2 and up-regulation of apoptotic Bax gene expression after hyperthermia stimulation. Besides, similar changes in p53 and c-myc expressions suggest that c-myc contributes to apoptosis by a p53-related mechanism in MOLT-4 cells of T-MSC co-culture under mild hyperthermia conditions.
We also performed the cell cycle analysis to determine the effects of TMSCs on cancer cell cycling ability. Under mild hyperthermia conditions, despite up-regulation of cyclin-dependent kinase inhibitor p21 in K562 cells co-cultured with T-MSCs, we did not find any significant changes in cell cycle progression. Unlike our study, in a study conducted after the culture of K562 cells with BM- MSCs, while a significant increase was found in the percentage of G0/G1-phase (subG1), decrease was found in G1, S and G2/M stages. In addition, it was reported in the study that after 7 days of culture, K562 cells were mostly in the post-apoptotic stage . The reason for the difference here is likely to be due to the difference in the source from which the MSCs were obtained. We know that it has been reported MSCs obtained from different sources can show different cellular and functional characteristics although they are cultured under the same conditions [61, 62]. The effects of MSCs on malignant tumor cells are controversial. It is thought that the changes in the data obtained from studies may be due to the heterogeneity of MSCs depending on the sources from which they were obtained, experimental changes (time, amount) of MSC applications or other unknown molecules or mechanisms . However, we demonstrated 2- and 2.6-fold G2-M phase cell cycle arrest in MOLT -4 cells under normal temperature and febrile temperature conditions, respectively. It is thought that the cell cycle arrest in the G2-M phase found in MOLT-4 cells causes the growth inhibition. In addition, it was observed that the rate of cells in the sub-G0-G1 phase increased 1.6-fold at febrile temperature. This results, supports the contribution on apoptosis in MOLT-4 cells of stimulation by hyperthermia.
p21 from CDKIs causes cell cycle arrest in the G1, S and G2 by inhibiting cyclin-dependent kinase 2 or cyclin-dependent kinase 4 . Expression of the cell cycle inhibitor Cdkn1a was significantly increased (15-fold) at 37°C in MOLT 4 cells co-cultured with T-MSC compared to control cells, but remained unchanged at 40°C. Accordingly, it is thought that up-regulation of p21 on co-cultured MOLT-4 cells causes cell cycle arrest at S and G2-M phases under 37°C culture conditions. Cell cycle arrest found in MOLT-4 cells co-cultured with T-MSCs under febrile temperature conditions may be related with other cell cycle control points. Gene expression level refers to transcriptional regulation; however, cross talks between proteins have many roles in cellular processes. For this reason, the cellular profile often may not be explained at transcriptional level. Ultimately, T-MSC co-culture resulted in differential expression of apoptotic and cell cycle-related genes in K562 and MOLT-4 cells. Many highlight the mechanism of changes in cellular processes and they should be examined in more detail. The mechanisms by which MSCs cause anti-proliferative effect on leukemia cells have not been fully explained. In the studies of Song et al., it was shown that the expression level of the cell cycle negative regulator p21 was up-regulated similar to our study, the pre-apoptotic cell fraction was increased and this led to cell cycle arrest .
Hyperthermia is a therapeutic application that raises the temperature of tumor tissue to 40°C -44°C. The key role of fever in the immune system is realized through activating the innate immune system such as the release of neutrophils in the periphery, the production of cytokines and nitric oxides from macrophages or dendritic cells and the stimulation of leukocyte transport . However, an increase in body temperature of 1°C in endothermic animals causes a 10-12.5% increase in metabolic rate and results in a high metabolic activity. At the same time, uncontrolled fever has been reported to cause worse outcomes in patients with sepsis or neurological injuries .
P53, which is a tumor suppressive gene, controls different processes in cells against different cellular stress factors. Apart from its known cell cycle functions, P53 is also known to play a role in cell migration and cancer cell metastasis. P53 has been reported to occur against epithelial-mesenchymal transition (EMT) and cell migration. In a study by Goyal and Ta, an increase in p53 protein expression of WJ-MSC was reported under 40°C febrile temperature conditions. The effect of heat stress on MSC properties has not been extensively studied. In the study conducted in 2020 by the same group, they obtained data that several adhesion-related Extracellular Matrix Protein (ECM) genes such as Vitronectin (VTN) regulated p53 expression in MSCs under heat stress at 40°C and they noted that p53 was up-regulated at 40°C . In our study,, p53 expression was up-regulated at 40°C defined as mild hyperthermia condition compared to normal temperature conditions and this gene may be realated to induction of the apoptosis and suppression of the tumor proliferation in mild hyperthermia conditions in K562 and MOLT4 cells.
There are studies with positive results conducted to determine the effects of hyperthermia induced mesenchymal stem cells on cancer cell growth. In the present study, it was shown that MSC conditioned media stimulated with heat (45 minutes at 43°C) modulated the homeostatic balance of cancer cells towards cell death. It has also been found that soluble factors secreted by hyperthermia-stimulated MSCs can provide an anti-tumorigenic microenvironment and this makes the tumor cells more sensitive to chemotherapeutic therapeutic by effectively transmitting the exogenous cell death signals to the cells. CMs obtained from different tissue-derived MSCs under hyperthermic conditions (at 43 ° C) were applied to human cancer cell lines and they were reported to have a negative effect on cancer cell proliferation [37, 65]. In their study, Park et al. showed that CM obtained under 45 minutes of temperature stimulation at 43°C inhibited cell growth by inducing cell cycle arrest in G2/M phase of breast cancer cells . In this study, where the authors wanted to determine the effects of T-MSCs on hematological cancers at 40°C as mild febrile temperature, they showed that cell cycle arrest was induced in the G2/M phase. The apoptosis rate of K562 cells increased significantly compared to 37°C, especially during the 7-day long culture. The apoptosis was induced in MOLT 4 cells in shorter culture time compared to 37°C. A similar effect to 37°C was found on day7.
IL-6 has roles in immune regulation, regulation of cell growth, hematopoiesis and performing a wide variety of cellular functions such as oncogenesis. It is known that high expression of IL-6 in cancer cells causes cancer to progress by inhibition of the apoptosis and stimulation of the angiogenesis in cancer cells [66, 67]. The data obtained from clinical studies have been associated with the increase of IL-6 level in serum, advanced tumour stages in various solid cancers and low survival rates. Thus, it has been suggested that IL-6 inhibitors may be a potential therapeutic agent [67–69]. The role of IL6 in the pathogenesis of Chronic Myelogenous Leukemia (CML) and Chronic Lymphocytic Leukemia (CLL) and its contribution to CML progression are also known [68, 70]. It has also been shown that IL-6 blocking strategy can significantly delay CML initiation and increase survival rates . Therapeutic hyperthermia used in cancer treatment causes increase in the levels of some cytokines and IL-6. Therefore, it has been reported that anticancer efficacy increased in vitro and in vivo when hyperthermia is not applied as an independent treatment, but with conventional methods such as immunotherapy, radiotherapy, chemotherapy and surgery .
In our study, IL-6 gene expression levels was increased 2- and 2.5-fold, respectively at 37°C in hematological cancer cells (both K562 and MOLT4) co-cultured with T-MSCSs under normal temperature. Besides, the mild hyperthermia significantly downregulated of its expression at 40°C. This downregulation has been associated with the decreased proliferation and antitumor function in cancer cells co-cultured with TMSC under mild hyperthermia conditions.
On the light of these findings, it is reported that tonsil-derived MSCs, as a new source of MSC, have antitumor function on hematological cancer cells and also, this function was correlated with hyperthermia stimulation. These findings reveal a promising therapeutical cellular application for the cancer therapy.