Investigating Spleen-cell-conditioned Medium Treated with Different Concentrations of Adenosine on 4T1 Breast Cancer Cells

 The present study aimed to assess the effects of spleen-cell-conditioned medium treated with different concentrations of adenosine on the 4T1 breast cancer cell line.

For this purpose, mouse spleens were isolated in completely sterile conditions, and splenocytes were then isolated for culture. Different concentrations of adenosine (25, 50, and 100 μM doses), phytohemagglutinin solution, and culture medium containing Fetal Bovine Serum (FBS) were added to splenocytes and incubated for 72 h. Thereafter, splenocytes were incubated by adding fresh culture medium without serum for 24 h. This fluid was collected and stored in a freezer at -80 for further assessment. The conditioned medium was mixed with an equal volume of the culture medium containing 4T1 cancer. The studied groups included control containing untreated 4T1 cancer cells, spleen-cell-conditioned medium untreated with adenosine, spleen-cell-conditioned medium treated with adenosine ∼25 μM, spleen-cell-conditioned medium treated with adenosine ∼5 μM, and the fifth group: spleen-cell-conditioned medium treated with adenosine ∼10 μM. After 48 h, the viability of 4T1 cells was measured by MTT and NR tests, and the amount of apoptosis in the cells was evaluated by vital dye staining.

The spleen-cell-conditioned medium is able to damage cancer cells and causes a significant reduction in neutral red uptake or damage to the cancer cell membrane. In the MTT test, a decrease was observed in the mitochondrial power; that is to say, a decrease in cell viability of cancer cells compared to the control group. In the apoptosis test, a percentage of cancer cells changed color from green to red. This function of the conditioned medium was strengthened by adding adenosine ∼25 μM to the spleen cell conditioned medium so that the amount of neutral red uptake was markedly reduced and caused a further decrease in the mitochondrial power of cancer cells. It also increased apoptosis in cancer cells so that most cancer cells changed color to red. The beneficial effect of the spleen-cell-conditioned medium decreased by increasing the concentration of adenosine to 5 µM so that there was no statistically significant difference with the untreated spleen-cell-conditioned medium. When we increased the concentration of adenosine to 10 µM, the killing efficacy disappeared, and there was no significant difference with the control group.

Based on the results, the spleen-cell-conditioned medium was able to kill a percentage of cancer cells. When we treat the spleen-cell-conditioned medium with low concentrations of adenosine, we observe a marked improvement in the killing efficacy of cancer cells, as well as an interaction between immune cells and cancer cells. Nonetheless, when the concentration of adenosine increases, the killing efficacy of cancer cells is lost and completely reversed, resulting in the growth of cancer cells.