Fig. 6. The combination treatment affects the serum levels of glycine and the downstream metabolite homocysteine and reduces LLC tumor growth in vivo.

a Inverse correlations between baseline absolute lymphocyte counts before RT in patients with stage I/III NSCLC and ser/gly pathway metabolites in the blood plasma. Pearson’s correlations between metabolites (arbitrary units) and baseline absolute lymphocyte counts (10³/µl), n = 16. b Schematic representation of in vivo experiment and treatment schedule. LLC cells were cultured for 2 days in media depleted of serine and glycine to induce ser/gly synthesis. C57BL/6 mice were injected in the right flank with LLC cells. Half of the tumor bearing mice were locally irradiated with 15 Gy. All groups received intraperitoneal treatment injections, either DMSO (10%) or sertraline (15 mg/kg). Sertraline treatment was initiated 3 days prior to RT, i.e., on day 7, and repeated every 3 days. End-point tumors on day 17 were processed and used for further analyses. c Growth curves of LLC cells cultured in control media and media depleted of serine and glycine amino acids. Data are presented as mean ± SD of 6 replicate wells. d Box plot showing tumor growth as tumor weight in the different treatment groups: DMSO, RT 15 Gy, sertraline 15 mg/kg, and combination. Kruskal–Wallis test with a Dunn’s multiple comparisons test was performed. e SHMT2 protein expression on day 17 measured by immunoblot in n = 3 LLC tumors and n = 2 technical replicates. An unpaired two-tailed t test has been used to compare DMSO treated mice and irradiated tumors. f Box plots showing the serum levels of serine, glycine, threonine, and homocysteine in mice treated with DMSO, RT, sertraline as monotherapies and the combination of sertraline and RT. A Jonckheere-Terpstra test was performed. g Pearson correlations of glycine, threonine and GSH with tumor weight. Statistical analysis *p-value  <  0.05.