Cytotoxic, Immunogenic Cell Death-Associated and Immunomodulatory Effects of Ruthenium(II) Terpyridine Complexes in Mesenchymal Stem Cells and CT26 Tumor-Bearing Mice

Ruthenium-based complexes have emerged as promising alternatives to platinum-containing anticancer drugs because of their favorable pharmacological properties and potential immunomodulatory activity. However, their effects on mesenchymal stem cells (MSCs), an important component of the tumor microenvironment, remain poorly understood. The biological effects of two ruthenium(II) terpyridine complexes (Ru-1 and Ru-2), compared with oxaliplatin as a reference compound, were investigated in mouse mesenchymal stem cells (mMSCs) using MTT, LDH, Annexin V/PI, cell-cycle and calreticulin exposure assays. Extracellular ATP and HMGB1 release were additionally assessed as immunogenic cell death-associated markers. The immunomodulatory effects of Ru-1 and Ru-2 were evaluated in healthy and CT26 tumor-bearing mice by flow cytometric analysis of lymphoid and myeloid cell populations. Both ruthenium complexes induced concentration-dependent cytotoxicity in mMSCs, with Ru-1 consistently exhibiting greater cytotoxic activity than Ru-2 and comparable potency to that of oxaliplatin after 24 h of exposure. In mMSC, cell death occurred predominantly through apoptosis and was accompanied by G2/M cell-cycle arrest. Both complexes also induced the emission of ICD-associated DAMPs, including calreticulin exposure and extracellular ATP and HMGB1 release, in mMSCs and CT26 cells. In vivo, Ru-1 increased the proportions of CD8⁺ T lymphocytes, NK cells and NKT cells in CT26 tumor-bearing mice whereas in healthy mice it increased NK and NKT cells without affecting T lymphocytes. Selected myeloid-cell and monocyte populations were also modulated, predominantly in healthy animals. This is the first study to characterize the biological effects of ruthenium(II) terpyridine complexes in mesenchymal stem cells alongside their in vivo immunomodulatory activity. Ru-1 combines direct cytotoxic effects with immunomodulatory activity and induces multiple features associated with immunogenic cell death in both tumor and stromal cell populations. These findings support further preclinical investigation of ruthenium(II) terpyridine complexes as potential immunomodulatory anticancer agents.