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. Author manuscript; available in PMC: 2019 Jan 11.
Published in final edited form as: Nature. 2018 Jul 11;559(7715):632–636. doi: 10.1038/s41586-018-0316-7

Figure 4. In vivo histidine supplementation increases flux through the histidine degradation pathway and sensitizes tumors to methotrexate.

Figure 4

a. Workflow for the in vivo assessment of the methotrexate-sensitivity of tumor xenografts following histidine supplementation. HEL or SEM cells were injected subcutaneously into NOD-SCID mice. Three weeks later tumor size was assessed by in vivo imaging and mice were randomly divided into four experimental groups: vehicle-treated, histidine supplementation, methotrexate-treated, and histidine supplementation combined with methotrexate treatment. The treatments were followed by a second imaging session. b, c. The combination of methotrexate and histidine supplementation is the only treatment that resulted in a significant reduction in tumor size. Fold changes in tumor size as measured in vivo before and after the different treatments are presented for HEL cell-derived tumors (b), and SEM cell-derived tumors (c) (non-parametric one-way ANOVA, KW test). d. The combination of methotrexate treatment and histidine supplementation caused cancer cell death. Hematoxylin-eosin (H&E) staining of tumors from mice injected with HEL cells and treated with the different regimes is presented at two magnifications. Mitotic cells (blue arrows) and apoptotic or necrotic cells (orange arrows) are marked in the bottom panel. e, f. The combination of methotrexate and histidine supplementation was the only treatment that resulted in significant necrosis in tumors. Necrotic areas as detected by H&E staining (d, top three rows, right column) were measured in the different groups (non parametric one-way ANOVA, KW test). g. THF levels decreased following methotrexate treatment and decreased even further when methotrexate was combined with histidine supplementation. Methotrexate levels were not different in tumors from mice treated with methotrexate alone or in combination with histidine supplementation. Nucleotide abundance was significantly lower in tumors from mice treated with methotrexate and histidine supplementation compared to tumors from vehicle-treated mice (non parametric one-way ANOVA, KW test. Methotrexate –two tailed t test).

b, d, e - vehicle n=5, histidine n=4, methotrexate n=6, methotrexate + histidine n=6. c, f - n=6, n=7, n=7, n=7 for the same groups. g - n=5, n=4, n=5, n=6, for the same groups.

Abbreviations: AMP – adenosine monophosphate. GMP – guanosine monophosphate. TMP – thymidine monophosphate.