Extended Data Figure 10. In vivo histidine supplementation sensitizes tumors to methotrexate without enhancement of treatment toxicity (part 1).

a. We evaluated whether methotrexate treatment combined with histidine supplementation might be more toxic than methotrexate alone by setting up a longer treatment regime of 15 days with a recovery period of two weeks. During the experiment we monitored weight loss and observed no difference between mice treated with methotrexate alone and those treated with methotrexate and histidine. NOD-SCID mice were injected with HEL cells subcutaneously on day 1, followed by weight measurement every other day, in vivo imaging of HEL cell-derived tumors (on days 7, 15, 20 and 28), treatment on days 12 to 23 (vehicle, histidine and methotrexate injections every other day), and final termination of the experiment after 40 days, unless an early euthanization was required in accordance with the guidelines for humane experimental end-point of the animal care committee at MIT. The experiment included four experimental groups: vehicle-treated (saline) (n=7), histidine supplementation (n=8), methotrexate-treated (n=8), and histidine supplementation combined with methotrexate treatment (n=8). Serum was collected at days zero and day 23 for metabolite profiling and liver diagnostics. Methotrexate dose used was 25 mg/kg based on the weight measured at day 0. Histidine dose was 18 mg per injection in 400 μl saline. b. Significant reduction in tumor size over time in mice treated with the combination of methotrexate and histidine supplementation. Tumors were imaged in vivo by luciferase expression at the indicated days. Fold changes in tumor sizes over measurements done on day 7 are presented. p-values were calculated by non parametric one-way ANOVA. Group size changed over time due to mice euthanization for humane reasons: on day 15: vehicle-treated (n=5), histidine supplementation (n=7), methotrexate-treated (n=7), and histidine supplementation combined with methotrexate treatment (n=8). on day 20: vehicle-treated (n=6), histidine supplementation (n=6), methotrexate-treated (n=7), and histidine supplementation combined with methotrexate treatment (n=8). on day 28: vehicle-treated (n=6), histidine supplementation (n=5), methotrexate-treated (n=7), and histidine supplementation combined with methotrexate treatment (n=8). c. in vivo imaging of luciferase-expressing HEL cell-derived tumors at days 7 (top) and 28 (bottom). Mice are numbered in color by their experimental group. All mice that participated in the experiment are shown, group size is the same as in panel a. d, e. No elevation in the abundance of serum markers indicative of kidney damage in methotrexate plus histidine supplementation-treated mice compared to methotrexate-treated mice. Markers of kidney toxicity (urea and creatinine) were measured by LC/MS in serum samples of the tested mice. Urea and creatinine relative abundance was normalized to isotopically-labeled valine and tryptophan as an internal standard. p-values were calculated using non parametric one-way ANOVA. Group size: vehicle-treated (n=7), histidine supplementation (n=7), methotrexate-treated (n=8), and histidine supplementation combined with methotrexate treatment (n=6). f, g. Some elevation in liver-toxicity markers in mice treated with the combined therapy compared to methotrexate alone. Markers of liver toxicity (ALT and AST) were measured by an external serum diagnostics lab (IDEXX) in serum samples of the tested mice. Measurement units are indicated. p-values were calculated using non parametric one-way ANOVA. Group size: vehicle-treated (n=6), histidine supplementation (n=6), methotrexate-treated (n=6), and histidine supplementation combined with methotrexate treatment (n=5).

Source data for Extended Data Fig. 9 can be found in the file Source Data_5.