Metformin has an undoubtedly anticancer value 4,5, but its mechanism of action is complex and not fully understood. Its immunomodulatory effect should be taken into account to optimize its clinical use 4. This could be more relevant on hematological cancers in which effector immune cells are in close contact to malignant cells because they niche in the same tissues. Several clinical approaches such as CAR T 38 or CAR NK 39 cells have shown a relevant success to treat blood-borne cancers. However, roughly 50% of patients undergo relapse and new clinical protocols are required to improve the clinical activity of these genetically-modified cytotoxic lymphocytes. Expression of the PD-1 immune checkpoint ligand PD-L1 in target cells efficiently blocks CTL killing. Metformin can promote PD-L1 phosphorylation and degradation 14. This could increase cytotoxic lymphocyte-mediated tumor cell death. The PD-1/PD-L1 immune checkpoint is less relevant in NK-mediated killing and, additionally, we have observed that our eNK cells express low levels of PD-1 26. Moreover, in MM samples pembrolizumab does not increase tumor killing by our eNK 26. Hence, we do not believe that this immune checkpoint plays a main role in our observations.
Initially, we believed that metformin sensitizes leukemic cells to NK by induction of stress molecules in target cells. We certainly reproduce previous findings 15 showing that metformin increases expression of stress ligands in multiple cell lines. Our eNK express similar NKG2D levels to naïve cells 25, but blocking NKG2D or MICA/B and ULBP1 does not decrease metformin effect. However, we have used activated NK cells. Hence, it is possible that naïve NK cells can better sense NKG2DL upregulation on tumor targets. If naïve or activated cells are more representative of the situation in the tumor microenvironment is unclear. It is believed that once in the tumor microenvironment NK cells should be activated by the targets and/or the pro-inflammatory cytokines. In contrast, for tumor cells outside of this environment, e.g. metastatic cells, it could be the opposite. Moreover, in patients the effect of metformin can be different than in our mouse models. In summary, we can not exclude a major role of metformin in patients.
Interestingly, the cell lines U266 and U937, which are not sensitized to cytotoxic lymphocytes by metformin, do not upregulate ICAM-1 after metformin treatment. Moreover, Mec-1 cells overexpressing the Bcl-2 family members Bcl-XL or Mcl-1 also failed to upregulate ICAM-1 and are resistant to metformin-induced sensitization. Finally, blocking ICAM-1/LFA-1 interaction strongly decreases metformin effect. Hence, our results support that metformin mediates upregulation of integrin ligands, i.e. ICAM-1, which allows the effective binding and activity of cytotoxic lymphocytes on tumor cells 40. Cytotoxic lymphocytes travel and roll over multiple substrates and sample different cellular environments. They need to be able to stop, process the stimulating signals of transient cellular contacts and move on 40. However, when these stimulatory signals are strong enough they should stop, form an adherent junction, stable and strong with the target cell and eliminate it. Metformin-induced ICAM-1 expression on target cells should favor that NK cells establish close contact with them. If the stimulating signals are strong enough, NK cells will proceed to kill the target 30,31. This has been described in in vitro activated NK cells using a similar expansion protocol as the one we have used here 31. Interestingly, metformin decreases ICAM-1 expression in non-transformed cells 41–43, including polycystic ovary syndrome (PCOS) subjects 44 and patients with T2D 45. Hence, it should not favor cytotoxic lymphocyte binding to healthy tissue that could induce autoimmunity.
We did not find any effect in our system. However, we used activated NK cells which are those that should infiltrate the tumor. Hence, it is possible that naïve NK cells can better sense NKG2DL upregulation. Moreover, in patients the effect can be different than in mouse models. We have included these comments in the discussion section.
Metformin only slightly delays tumor growth and does not improve mice survival either alone or together with eNK. In contrast, it improves survival and decreases tumor growth in the presence of an anti-CD20 mAb and eNK. In our mouse model, eNK are in contact with metformin during their antitumor function, which is not the case of the in vitro experiments. It is well-known that cytotoxic lymphocytes require a glycolytic metabolism for their maximal activity and educated NK cells display a high glycolytic rate 46, which is essential for their antitumor function 47. Hence, metformin could affect NK cell metabolism in vivo impairing their function. The efficient anti-CD20 mAb could overpass this effect by inducing maximal NK cell activation through the legation of CD16, which recognizes the Fc moiety of Abs. In this context, metformin improves outcome of DLBCL patients at least partially by sensitizing cells to the anti-CD20 rituximab 48. However, we do not know if our results apply to naïve NK cells. eNK have a relatively high basal cytotoxicity compared to naïve NK cells and show a mature phenotype 24–26. The more mature NK subsets, which possess higher cytotoxic potential, show the highest activation by LFA-1 30. Therefore, it is possible that metformin-induced NK sensitization is specific of already activated cytotoxic lymphocytes, something that perhaps is uncommon in cancer patients that carry impaired NK activity 22.
The concentration of metformin in plasma in T2D patients is around 30 µM 49. A daily dose of metformin could reach 2,000 mg a day 4, which is basically 15 millimoles and could perhaps locally give higher metformin concentrations. For example, it can reach 140 µM in liver 4. The intratumor concentration of metformin is difficult to evaluate, but it accumulates in ovarian cancer patient biopsies 50. Moreover, metformin concentration reaches on average 0.41 mmol/kg in the colon of colorectal cancer patients daily treated with an oral low dose of 250 mg/d of metformin, with some patients reaching 1.87 mmol/kg 51,52. In addition, and remarkably, metformin effects are higher at low glucose concentration (1 mM), which are found in tumor microenvironment 50. This is probably the case of AML and MM patient bone marrow, which is highly infiltrated by glucose-consuming tumor cells. Hence, metformin concentration in the tumor can be significantly higher than in plasma and/or it can produce specific effects at lower doses in the tumor microenvironment.
The antitumor function of metformin is very heterogenous. As previously described, the large variety on age and type of disease of the patients engaged in the clinical trials could explain this fact. We show here that p53 status and/or overexpression of Bcl-xL or Mcl-1 could make tumor cells resistant to the metformin-induced sensitization to cytotoxic lymphocytes. Moreover, the activity of NK cells is largely impaired in leukemia patients 22 and our results suggest that metformin could give better clinical results in patients with sufficient NK activity. Hence, we believe that metformin together with allogeneic activated NK cells could be a future relevant treatment. The ongoing clinical studies of metformin in nondiabetic, cancer, patients will soon show the effect of metformin in several clinical contexts and perhaps support the use of such co-treatment.