Effects Of Rapamycin On The Peritumor And Intratumor Lymphangiogenesis Of Melanoma Xenograft Tumor In The Immunodeficient Mice

BACKGROUND AND PURPOSE:Cutaneous melanoma is one of the common malignant skin tumors.Its incidence is still rising in recent years.Melanoma tends to metastasize distantly in early stage and lead to poor prognosis.Although great achievements have been made in the immunotherapy and targeted therapy of melanoma,there still have many problems to be solved,such as ineffective treatment,drug resistance,and so on.Therefore,novel treatment is still necessary.Lymphatic metastasis is the major route by which the primary tumor disseminates to distant organs.Lymphangiogenesis in and around the tumor is the premise for lymphatic metastasis.Theoretically speaking,inhibiting peri tumor and intratumor lymphangiogenesis and reducing the number of lymph vessels can suppress tumor's lymphatic metastasis and prolong the patients' survival time.Rapamycin,one of mTOR inhibitors,was originally used in the immunosuppression after kidney transplant.Due to the extensive role of mTOR signaling pathway,other effects of rapamycin are constantly discovered,including anti-angiogenic effects.In vitro and in vivo studies demonstrated its ability to inhibit the vasculogenic potential of hemangioma-derived stem cells and the ability to inhibit tube formation of lymphatic vessels.Therefore,we hypothesized that rapamycin may could be used in the treatment of melanoma by above mechanism.In this study,we established a melanoma xenograft tumor model in immunodeficient mice,treated them with rapamycin,and observed the changes of peritumor and intratumor lymphangiogenesis.The purpose of this study is to provide experimental evidences for the application of rapamycin in the treatment of melanoma.METHODS:(1)A375 human melanoma cells were cultured and injected subcutaneously into both sides of the back in nude mice.Two weeks later,the mice were devided into two groups(NS-controlled group and rapamycin-treated group),given i.p.injections of 0.1 ml NS or rapamycin solution at a dose of 1.5 mg/kg every other day.Two weeks after intervention,the tumors were harvested and bisected.Half of the samples were fixed in 4%formalin,embedded in paraffin for hematoxylin and eosin(H&E)staining,immunohistochemical(IHC)staining,and immunofluorescence(IF)staining.Other samples were stored in liquid nitrogen for quantitative PCR and Western blot analysis.(2)IHC and IFC staining were used to detect the changes of peritumor and intratumor lymph vessels.(3)IHC staining was used to detect the expression of mTORCl/p70S6K/4EBP1.Western blot was used to detect the expression of mTORCl/p70S6K/p-p70S6K/4EBP1/p-4EBPl and VEGF-C/VEGFR-3.(4)Quantitative-PCR was used to detect the expression of VEGF-C/VEGFR-3 genes.RESULTS:(1)IFC staining showed the LYVE-1 expression in peritumor and intratumor regions,which suggested the lymphangiogenesis in and around tumor.(2)IHC staining and quantitative analysis showed the peritumor and intratumor ALVD(average lymph vessel density)in rapamycin-treated group were significantly lower than that in NS-controlled group.(3)IHC staining and Western blot showed the expression of mTOR/p70S6K/4EBPl in rapamycin-treated group was similar to that in NS-controlled group.But the expression of p-p70S6K/p-4EBPl in rapamycin-treated group was significantly lower than that in NS-controlled group.(4)Western blot and quantitative PCR showed the expression of VEGF-C/VEGFR-3 was down-regulated in rapamycin-treated group.CONCLUSIONS:Rapamycin inhibits the peritumor and intratumor lymphangiogenesis in the melanoma xenograft model.The possible mechanism is to block the mTOR signal passway and then down-regulate the expression of VEGF-C/VEGFR-3.These findings provide the preliminary experimental evidences for the treatment of melanoma with rapamycin.