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, 7 (13), 17060-73

Combination of Metronomic Cyclophosphamide and Dietary Intervention Inhibits Neuroblastoma Growth in a CD1-nu Mouse Model

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Combination of Metronomic Cyclophosphamide and Dietary Intervention Inhibits Neuroblastoma Growth in a CD1-nu Mouse Model

Raphael Johannes Morscher et al. Oncotarget.

Abstract

Background: MYCN-amplification in high-grade Neuroblastoma (NB) tumors correlates with increased vascularization and therapy resistance. This study combines an anti-angiogenic approach with targeting NB metabolism for treatment.

Methods and results: Metronomic cyclophosphamide (MCP) monotherapy significantly inhibited NB growth and prolonged host survival. Growth inhibition was more pronounced in MYCN-amplified xenografts. Immunohistochemical evaluation of this subtype showed significant decrease in blood vessel density and intratumoral hemorrhage accompanied by blood vessel maturation and perivascular fibrosis. Up-regulation of VEGFA was not sufficient to compensate for the effects of the MCP regimen. Reduced Bcl-2 expression and increased caspase-3 cleavage were evident. In contrast non MYCN-amplified tumors developed resistance, which was accompanied by Bcl-2-up-regulation. Combining MCP with a ketogenic diet and/or calorie-restriction significantly enhanced the anti-tumor effect. Calorie-restricted ketogenic diet in combination with MCP resulted in tumor regression in all cases.

Conclusions: Our data show efficacy of combining an anti-angiogenic cyclophosphamide dosing regimen with dietary intervention in a preclinical NB model. These findings might open a new front in NB treatment.

Keywords: anti-angiogenic; glucose; ketogenic diet; metronomic cyclophosphamide; neuroblastoma.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that no competing interests exist.

Figures

Figure 1
Figure 1. Dietary intervention enhances the growth inhibitory effect of MCP on NB xenografts
After establishing tumors, mice were randomized to therapy and control groups as indicated. For xenografts of both cell lines the MCP regimen significantly inhibited tumor growth compared to the SD group w/o CTx (p < 0.001). (A1) SH-SY5Y and (B1) SK-N-BE(2) tumor growth curves. Data points represent mean values ± SEM of the corresponding therapy group (n = 8–12). (A2) and (B2) show Kaplan–Meier survival curves for mice with SH-SY5Y and SK-N-BE(2) xenografts respectively. Survival was significantly prolonged in all therapy groups when compared to the SD group w/o CTx (p < 0.001). The effect of dietary intervention on tumor growth was evaluated by comparing diet groups to the corresponding SD on MCP. Significance levels are given for each dietary intervention group compared to SD on MCP and are stacked from the group with lowest to highest tumor volume. Statistics: ANOVA (p < 0.05) followed by two-tailed Dunnett's test correcting for multiple comparisons; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Differences in survival were determined in a univariate analysis with the log-rank test. Death is coded: tumor volume above 3000 mm3, tumor ulceration or impaired health condition. Abbrev.: SD, standard diet; CR, calorie restriction; KD, ketogenic diet; w/o CTx, without chemotherapy; MCP, metronomic cyclophosphamide.
Figure 2
Figure 2. Blood glucose reduction and induction of ketosis goes with reduced tumor weight in mice under MCP
(2A) SH-SY5Y groups and (2B) SK-N-BE(2) groups. (A1) and (B1) CR significantly reduced blood glucose levels in mice with both xenograft types. (A2) and (B2) Ketone body levels (beta-hydroxybutyrate) were consistently elevated in the CR-KD groups and the KD group of SK-N-BE(2). The trend in the KD group of SH-SY5Y did not reach statistical significance. (A3) and (B3) Mean Glucose Ketone Index over the treatment period was significantly reduced in all dietary intervention groups (p < 0.001). (A4) and (B4) Tumor weight was significantly reduced in all groups on MCP when compared to the SD group w/o CTx (p < 0.001; data not shown) and dietary intervention groups as given. The results are consistent with the tumor volumes calculated in Figure 1A and 1B. Data are shown for day 36 or the last day of therapy. Mean values ± SEM of the corresponding therapy group are given (n = 8–12). Statistics: ANOVA (p < 0.05) followed by two-tailed Dunnett's test correcting for multiple comparisons; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Abbrev.: SD, standard diet; CR, calorie restriction; KD, ketogenic diet; w/o CTx, without chemotherapy; MCP, metronomic cyclophosphamide.
Figure 3
Figure 3. Reduced proliferation contributes to the growth inhibitory effect of CR but not MCP
(3A) SH-SY5Y groups and (3B) SK-N-BE(2) groups. IHC evaluation of proliferation markers on day 36 or the last day of therapy. (A1) and (B1) The fraction of Ki67 positive stained cells was not significantly altered by MCP or diet when compared to SD w/o CTx. (A2) and (B2) In xenografts of both cell lines, PHH3 positive cells per high power field were significantly lower in the CR groups. (A3) and (B3) Exemplary tumor sections stained for PHH3 from mice on SD w/o CTx (left) and CR-KD (right) are shown. Scale bar = 50 μm. Mean values ± SEM of the therapy group are given (n ≥ 8). Statistics: ANOVA (p < 0.05) followed by two-tailed Dunnett's test correcting for multiple comparisons; **p ≤ 0.01; ***p ≤ 0.001. Abbrev.: Ad libitum: SD/KD; restricted: CR-SD/CR-KD. SD, standard diet; CR, calorie restriction; KD, ketogenic diet; w/o CTx, without chemotherapy. Proliferation indices for individual dietary subgroups are given in Supplementary Figure S3.
Figure 4
Figure 4. Predominant intratumoral hemorrhage in MYCN-amplified tumors is reduced upon MCP treatment
(4A) SH-SY5Y and (4B) SK-N-BE(2) tumors. Images of xenograft sites, as well as tumors after explantation are shown. Pronounced macroscopic hemorrhage in the SK-N-BE(2) SD group w/o CTx compared to all other therapy groups of both cell lines was evident. (A1) and (B1) Specimens from SD w/o CTx compared to SD on MCP are shown. (A2) and (B2) Relative quantification of macroscopically visible hemorrhagic tumors w/o CTx (SH-SY5Y n = 9, SK-N-BE(2) n = 8) and groups on MCP (SH-SY5Y n = 35, SK-N-BE(2) n = 25). Intratumoral hemorrhage did not vary between dietary subgroups and is given in Supplementary Figure S4. Abbrev.: SD, standard diet; w/o CTx, without chemotherapy; MCP, metronomic cyclophosphamide.
Figure 5
Figure 5. Microscopic evaluation supports an anti-angiogenic effect of MCP and vessel maturation in NB xenografts
(5A1) SH-SY5Y and (5B1) SK-N-BE(2). Hematoxylin/Eosin staining of NB sections on day 36 or the last day of therapy was scored for angiogenesis and hemorrhage on a scale from 0-3. In the SK-N-BE(2) xenografts, blood vessel morphology changed to a more mature pattern as described in detail in Figure 6B1 and 6B2. (C) IHC staining for HIF1A of a SK-N-BE(2) xenograft section from the SD group w/o CTx (upper) and SD on MCP (lower). Blood vessel (arrow) rarefication and maturation caused a hypoxic pattern in SK-N-BE(2) xenografts with increased nuclear HIF1A accumulation and areas of marked cell death in the center of hypoxic areas (asterisk). (D) This correlated to VEGFA up regulation in SK-N-BE(2) xenografts exposed to MCP, as depicted by immunoblotting. Scale bars = 100 μm (overview) and 50 μm (detail). Mean values ± SEM of the therapy group are given (n ≥ 8). Statistics: unpaired t test (p < 0.05); *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Angiogenesis scoring across all subgroups is given in Supplementary Figure S5. Densitometry for VEGFA levels is given in Supplementary Figure S8A. Abbrev.: SD, standard diet; w/o CTx, without chemotherapy; MCP, metronomic cyclophosphamide.
Figure 6
Figure 6. Evaluation of blood vessels by IHC for msCD-31 endothelial cell marker (A1, B1) and Van Gieson staining of collagen fibers (A2, B2)
Images show exemplary sections of (6A) SH-SY5Y and (6B) SK-N-BE(2) xenografts at day 36 or the last day of therapy. The left upper (overview) and lower (detail) images are taken from tumors w/o CTx, the right upper and lower images are from tumors exposed to MCP. (A1) and (B1) show IHC staining for the endothelial marker msCD-31. Under both conditions SH-SY5Y xenografts show well-formed blood vessels, as can be appreciated best in the detail section (A1, lower). In comparison SK-N-BE(2) xenografts show marked difference in blood vessel morphology between the w/o CTx group and the MCP group (B1). (A2) and (B2) Van Gieson staining identified the strong perivascular connective tissue in the SK-N-BE(2) group on MCP as collagen deposition (red). Blood vessels are marked by arrows, Scale bars = 100 μm (overview) and 50 μm (detail). Abbrev.: SD, standard diet; w/o CTx, without chemotherapy; MCP, metronomic cyclophosphamide.
Figure 7
Figure 7. SH-SY5Y (A) and SK-N-BE(2) (B) cell lines show divergent regulation of Bcl-2 levels under MCP
(A1) and (B1) Bcl-2 levels on IHC staining were scored on day 36 or the last day of therapy. In the untreated state, SH-SY5Y xenografts showed significantly lower Bcl-2 levels when compared to SK-N-BE(2) xenografts (p < 0.001). On MCP treatment, the two cell lines showed an opposing response. (A2) and (B2) Exemplary Bcl-2 IHC-stained sections w/o CTx (left) and on MCP (right). (C) On western blot analysis, along with the reduction in Bcl-2 levels, increased cleaved caspase 3 protein was detected in the SK-N-BE(2) xenografts exposed to MCP. Densitometry for western blots is given in Supplementary Figure S8B. Scale bar = 100 μm; Mean values ± SEM of the therapy group are given (n ≥ 8). Statistics: Stundent's t test (p < 0.05); **p ≤ 0.01, ***p ≤ 0.001. Abbrev.: SD, standard diet; w/o CTx, without chemotherapy; MCP, metronomic cyclophosphamide.

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