Mannose synergizes with chemoradiotherapy to cure cancer via metabolically targeting HIF‐1 in a novel triple‐negative glioblastoma mouse model

Dear Editor, The standard care of glioblastoma multiforme (GBM), which is surgery followed by temozolomide concurrent radiotherapy (RT/TMZ), confers limited survival benefit for patients.1 Additionally, current animal models do not faithfully recapitulate humanGBM features, impeding animal-to-human translation.2 Here, we developed and characterized a novel orthotopic murine GBM model, in which we tested the efficacy of the RT/TMZ supplemented with mannose (RT/TMZ/Man). Following the optimized protocol illustrated in Figure 1A, we established a stable subcutaneous-intracranial G422-GBM syngeneic mouse model system. Subcutaneous injection of 1 × 106 fresh G422 cells led to 100% tumor formation, and the tumors reaching 1 cm3 on day 7–9 postimplantation (p.i.) were used for further experiments (Figure 1B). Microinjection of 1 × 103–1 × 105 G422 cells in mouse striatum caused 100% lethality within 30 days (Figure 1C) and 5 × 104 cells, which led to mouse death within 14–23 days (Figure S1A), were selected for establishing the orthotopic model. We then determined the pathology and molecular characters of G422-tumors. The brain slices showed rapid G422-tumor development during day 7–15 p.i. (Figure S1B). The G422tumors were GFAPVimentinCD3 and infiltrated into distant brain areas (Figure 1D-G) on day 5 p.i. They were IDH1/2WTchromosome1/19IntactTERT-promoterWT with ATRXMutant and Trp53Mutant (Figure 1H), determined by the whole genome sequencing, and thus belonged to the triple-negative (TN) primary GBM subtype.3 Our model was therefore named the G422TN-GBM. Next, we determined the therapeutic responses of the orthotopic G422TN-GBM to conventional surgery, radiotherapy, temozolomide, and the therapies of combining these two or three. Surgery alone, performed on day 7 p.i., effectively removed the tumor and slightly extended the median survival of the G422-mice (Figure 2A-C and

). A single dose (10 Gy) of whole brain irradiation did not enhance the animal survival, while the TMZ monotherapy started on day 7 p.i. significantly increased the survival ( Figure 2D and E and Figure S3). RT/TMZ further improved the survival compared to either RT or TMZ monotherapy (RT/TMZ vs RT, P = .0002; RT/TMZ vs TMZ, P = .0166; Figure 2E). However, surgery did not offer extra survival benefit for the G422-mice treated with the RT/TMZ (surgery/RT/TMZ vs RT/TMZ, P = .5770; Figure 2F). Therefore, the RT/TMZ treatment, without being combined with the surgical resection, was used for the following therapeutic studies.
In clinic, GBM patients diagnosed and treated early exhibit better therapeutic responses. 4 Bioluminescent imaging and H&E staining showed progressive growth and infiltration of the G422 tumors on day 5, 7, and 9 p.i. (Figure 2G and H and Figure S4), which could represent the early, middle or late stages of GBM, respectively. The survivals of the G422-mice that received the RT/TMZ regimen started on either day 5, 7, or 9 p.i. were all significantly improved compared to that of the control group ( Figure 2I and J). Importantly, the RT/TMZ treatment started on day 5 was significantly more effective in extending animal survival compared to the one started on day 9 (P = .0147, Figure 2I). Nevertheless, with this most effective regimen that we had tested thus far, the G422-mice still exhibited 100% lethality within 35 days p.i. Taken together, our G422 TN− GBM model recapitulates the refractory character of human GBM to standard therapies in clinic, which is superior for therapeutic studies to our previous G422model. 5 We next used the G422 TN− GBM model as a preclinical tool to explore treatments for GBM. The monosaccharide mannose, found naturally in high amounts in many fruits, was recently highlighted as an effective anti-cancer drug by impeding glucose metabolism. 6 The PET/CT imaging with [18F] FDG verified that oral administration of F I G U R E 1 Establishment and characterization of the preclinical orthotopic mouse G422 glioma model. A, Schematic diagram of the process of establishing the stable orthotropic G422-glioma murine model. 1 × 10 6 G422 cells freshly isolated from subcutaneous tumors were used in the establishment of stable subcutaneous G422-model. G422 tumors of 1 cm 3 after 7-9 days of subcutaneous growth were used for subsequent experiments. After a short incubation of G422 cells in vitro, 5 × 10 4 living G422 cells were microinjected into the right striatum of syngeneic mice to establish stable orthotopic G422-model. B, The growth curve of subcutaneous G422 tumor (n = 6). C, The Kaplan-Meier survivals of the orthotopic G422-mice (n = 6-11/group, * P < .05; ** P < .01; ns, not statistically significant). H, The Kaplan-Meier survival curves of the G422-mice treated with the indicated therapies started on the fifth day (n = 8-9/group) (*P < .05; **P < .01; ns, not statistically significant) mannose indeed inhibited glucose metabolism of subcutaneous or intracranial G422 gliomas ( Figure 3A and Figure S5). Mannose monotherapy started on day 5 p.i. slightly but significantly prolonged the survival of the G422-mice as compared to the control and glucose-treated groups (P = .0074 and .0282, respectively) ( Figure 3B). In addition, mannose supplement significantly enhanced the therapeutic efficacy of both TMZ monotherapy (TMZ/Man vs TMZ, P = .0260) and RT/TMZ (RT/TMZ/Man vs RT/TMZ, P = .0085), but did not synergize with RT (RT/Man vs RT, P = .1100) ( Figure 3C and Figure S6). Remarkably, 50% or 25% of G422-mice that received the RT/TMZ/Man started on day 5 or 7 p.i. achieved long-term survival (LTS), defined by animal survival of over 100 days, while none of the G422-mice receiving RT/TMZ/Man on day 9 survived over 35 days ( Figure 3C-E and Figure S6). In contrast to mannose, neither metformin 7 nor disulfiram 8 enhanced the RT/TMZ started on day 7 p.i. to achieve LTS for the G422mice ( Figure S7). In summary, mannose can achieve disease cure in combination with RT/TMZ at the early and middle tumor developmental stages.
The RT/TMZ/Man therapy suppresses cell proliferation and invasion and increases DNA damage, as indicated by the decreased Ki-67, vimentin/PD-L1, and increased γ-H2AX in the G422 tumors ( Figure S8). Lastly, we explored the molecular mechanisms through which RT/TMZ/Man cures the G422-mice by metabolomic analysis. The identified metabolites that are significantly differentially expressed between the RT/TMZ/Man and the RT/TMZ, Man, or untreated-subcutaneous G422 gliomas were listed in the heat maps ( Figure 3F and Figure S9). KEGG pathway enrichment analysis of these differential metabolites identified the HIF-1 signaling pathway as the only significantly altered pathway between the RT/TMZ/Man and RT/TMZ groups ( Figure 3G). Western blot assay verified the downregulation of HIF-1α/VEGF and the increase of PHD2 that signals HIF degradation ( Figure S10). Pharmacological restoration of HIF-1α/VEGF by using the PHD2 inhibitor Molidustat (Mol) completely abolished the LTS endowed by RT/TMZ/Man (RT/TMZ/Man/Mol vs RT/TMZ/Man, P = .0037) and there was no statistical difference between the RT/TMZ and RT/TMZ/Man/Mol groups (P = .2045, Figure 3H and Figure S10). These results demonstrated the dominant role of the HIF-1 pathway underlying the RT/TMZ/Man efficacy.
In conclusion, we established a highly reproducible syngeneic G422 TN -GBM mouse model that faithfully recapitulates the aggressiveness and therapeutic responses of human GBM, which still requires further verification. We discovered that the RT/TMZ/Man could offer disease cure for GBM in our model through metabolically abolishing the HIF-1-mediated resistance. Our work highlights a novel and superior preclinical mouse model for drug screening and the potential therapeutic value of the RT/TMZ/Man regimen for GBM.

A C K N O W L E D G M E N T
This work was supported by the National Natural Science Foundation of China (Grant Nos. 81672504 and 81972362 to X.Q.C.).

C O N F L I C T O F I N T E R E S T
The authors declare that they have no conflict of interest.

E T H I C S A P P R O VA L A N D C O N S E N T T O PA R T I C I PAT E
All animal handling and experiments were performed in accordance with the NIH guidelines and approved by the Institutional Ethics Committees of Huazhong University of Science and Technology.