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. 2012 Nov;138(11):1879-90.
doi: 10.1007/s00432-012-1270-7. Epub 2012 Jun 27.

Metronomic chemotherapy in combination with antiangiogenic treatment induces mosaic vascular reduction and tumor growth inhibition in hepatocellular carcinoma xenografts

Affiliations

Metronomic chemotherapy in combination with antiangiogenic treatment induces mosaic vascular reduction and tumor growth inhibition in hepatocellular carcinoma xenografts

Fan Zhou et al. J Cancer Res Clin Oncol. 2012 Nov.

Abstract

Background: In addition to sprouting angiogenesis, other mechanisms, such as mosaic tumor vessel formation, have been recognized to contribute to tumor vascularization. We sought to examine vascular alteration as well as tumor growth inhibition after treatment with antiangiogenic therapy, chemotherapy alone or in combination.

Methods: Hepatocellular carcinoma cells (Hep3B) expressed green fluorescent protein were utilized to establish orthotopic xenograft model in nude mice. The formation and distribution of mosaic vessels was analyzed quantitatively by immunolabeling. Next, changes in tumor microcirculation and therapeutic effects on tumor growth were evaluated in several different treatment groups: control, conventional doxorubicin, metronomic doxorubicin, bevacizumab, bevacizumab plus conventional doxorubicin, and bevacizumab plus metronomic doxorubicin. In addition, we examined the effects of combined regimens on lung metastasis using a highly metastatic human hepatocellular carcinoma (HCCLM3) mouse model.

Results: Approximately 62 % of the vessels were present in the central part or near the midsection of the tumor and were mosaic. Only the combined antiangiogenic treatment and chemotherapy (metronomic schedule, P = 0.00; conventional schedule, P = 0.02) had a significant effect on the degree of mosaic vasculature. Metronomic doxorubicin in combination with bevacizumab had an even more profound effect than bevacizumab plus conventional doxorubicin (P < 0.05) on tumor growth inhibition and survival. However, bevacizumab plus metronomic doxorubicin failed to inhibit lung metastasis compared with antiangiogenic monotherapy.

Conclusions: Metronomic chemotherapy in combination with antiangiogenic treatment results in the reduction of mosaic tumor vasculature, inhibition of tumor growth, and enhanced survival of mice. Further investigation of drug scheduling is required to optimize antitumor activity.

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Conflict of interest statement

We declare that we have no conflict of interest.

Figures

Fig. 1
Fig. 1
Formation and distribution of mosaic tumor vessels in hepatocellular carcinoma xenografts. a, b Merged two-color images of mosaic tumor vessels (red CD31/CD105 immunoreactivity of endothelium, green GFP-expressing tumor cells) showed that tumor cells were apparent and involved in the formation of vessel wall (a, b scale bar 15 μM). c, d Fluorescence micrographs showing the vasculature of HCC xenografts stained by intravenous injection of Ricinus communis agglutinin I lectin (blue d) and the same vessels stained for CD31/CD105 immunoreactivity (red c). Mosaic tumor vessels were perfused with fluorescent labeled lectin (arrow), indicating that tumor cells in HCC mosaic vessels could be in contact with flowing blood (c, d scale bar 50 μM). e Density and frequency of mosaic tumor vessels in the periphery, sub-central and central parts of the tumors were recorded. Analysis revealed low mosaic vascular density and mosaicism frequency in the tumor periphery with a progressive increase toward the tumor center. f The mosaic vessel was formed with tumor cells (blue) and endothelial cells (brown), and red blood cells appeared inside the vessel (scale bar 15 μM). Data are the mean of 10 images per section and 15 sections per tumor
Fig. 2
Fig. 2
Intratumoral penetration of doxorubicin (10 mg/kg) when delivered at different days after bevacizumab (5 mg/kg) in mice bearing established HCC xenografts (P = 0.25, day 0; P = 0.20, day 1; *P = 0.00, day 3; *P = 0.04, day 5; P = 0.32, day 7) for up to 7 days were shown. DOX doxorubicin, BV bevacizumab, PBS phosphate-buffered saline
Fig. 3
Fig. 3
Confocal images show alteration in tumor microcirculation after different treatment regimens. Group 1 served as control (a); group 2 received conventional doxorubicin (5 mg/kg, every 2 weeks for two times, b); group 3 received metronomic doxorubicin (0.5 mg/kg, three times a week, c); group 4 received bevacizumab monotherapy (5 mg/kg, twice a week, d); group 5 received conventional doxorubicin (5 mg/kg, every 2 weeks for two times) plus bevacizumab (5 mg/kg, twice a week, e); and group 6 received metronomic doxorubicin (0.5 mg/kg, three times a week) in combination with bevacizumab (5 mg/kg, twice a week, f). Blood vessel structures (red) were immunostained with CD31/105 and detected with a Cy5-conjugated antibody; tumor cells (green) were identified by FITC-labeled antibody (n = 7 mice per group; scale bar 50 μM). Area density of vessel was counted in orthotopic xenografts of various treatment groups (g). There was no significant difference in area density of total vessel between the control and the conventional doxorubicin groups. Tumor vessel numbers were reduced by metronomic doxorubicin or bevacizumab monotherapy. The metronomic doxorubicin plus bevacizumab group showed the most inhibitory effect of tumor vessel count among all the groups (*P < 0.01 compared with the control arm and conventional doxorubicin group). However, the metronomic doxorubicin group did not show any significant difference in the number of mosaic tumor vessel. Only combined antiangiogenic treatment and chemotherapy (metronomic schedule, P = 0.00; conventional schedule, P = 0.02) had a significant effect on mosaic vasculature. Con DOX conventional doxorubicin, met DOX metronomic doxorubicin, BV bevacizumab
Fig. 4
Fig. 4
Mice bearing tumors were treated with PBS, bevacizumab, bevacizumab plus the conventional doxorubicin, and with bevacizumab in combination with the metronomic doxorubicin for up to 6 weeks. Afterward, mice were euthanized and lung tissues were collected to investigate distant metastasis. Analysis of serial lung sections revealed that bevacizumab or combination treatments significantly decrease the number of metastatic foci compared with the control arm (*P < 0.001). Combination treatments failed to improve inhibitory effect on lung metastasis compared with antiangiogenic monotherapy (conventional doxorubicin plus bevacizumab, P = 0.56; metronomic doxorubicin plus bevacizumab, P = 0.38). Con DOX conventional doxorubicin, met DOX metronomic doxorubicin, BV bevacizumab
Fig. 5
Fig. 5
Survival of mice treated with conventional doxorubicin (5 mg/kg, every 2 weeks for two times) or metronomic doxorubicin (0.5 mg/kg, three times weekly) and in combination with bevacizumab (5 mg/kg, twice a week, n = 10 per group). Treatment was continued until mice were moribund, and days of life were recorded. Survival data were compared for significance with the log-rank statistic. Treatment was initiated 7 days (a) or 21 days (b) after tumor cell injection. Therapies consisted of six groups: (1) control group (blue line); (2) conventional doxorubicin (green line); (3) metronomic doxorubicin (brown line); (4) bevacizumab monotherapy (purple line); (5) conventional doxorubicin plus bevacizumab (yellow line); or (6) metronomic doxorubicin plus bevacizumab (red line). The metronomic doxorubicin in combination with bevacizumab group provided the most effective therapy with longest survival times among all the groups (P = 0.000)

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