| BackgroundThere is a high incidence of esophageal cancer in China,and there is a phenomenon of regional aggregation.The most common sites are the upper and middle segments of the esophagus.The main pathological type is squamous cell carcinoma,which is significantly different from that in Europe and the United States.Because of its high incidence,strong invasiveness and poor prognosis,it has a serious threat and impact on people’s health and life.The onset of esophageal cancer is hidden,often at the time of diagnosis has entered the middle and late stage,the local focus spread obviously,and there is distant lymph node metastasis,the effect of surgical treatment is not good.At present,simultaneous chemoradiotherapy is the important mode for advanced esophageal carcinoma’ treatment,which can effectively improve the local control rate and survival time of the tumor.However,due to the existence of tumor cell clones resistant to radiotherapy and chemotherapy,such as hypoxic cells,the local control rate of tumor is reduced,so that the effect of radiotherapy is far from satisfactory.The choice of drugs for advanced esophageal squamous cell carcinoma is limited.Although the antiangiogenic drugs on the market have achieved certain efficacy in inhibiting angiogenesis,there are also many problems,including:1.Easy to develop drug resistance;2.The specificity is not strong,the side effect is big;3.The antivascular effect is not good;therefore,so far,there are still no effective molecular targeting drugs,molecular targeting is becoming the focus of research to improve the efficacy of treatment.Anlotinib Hydrochloride(AL3818,Anlotinib Hydrochloride)is a small molecular multi-target tyrosine kinase inhibitor independently developed in China.It can inhibit tumor angiogenesis by targeting growth factor receptors(VEGFR1,VEGFR2,VEGFR3,PDGFRα,FGFR1,FGFR2,FGFR3).Moreover,it can inhibit some tumor cell proliferation related receptors(c-Kit)and pathways(Erk and Akt pathways)and directly inhibit tumor cell proliferation,which has dual anti-tumor effects.Clinical studies have found that anlotinib has significant efficacy in the treatment of a variety of tumors,and adverse reaction data show that it is safe and tolerable.However,in the study of esophageal cancer,there are only rare reports of single-drug maintenance therapy for advanced esophageal cancer,which is less available for reference and the curative effect is limited.This study focuses on the role and mechanism of anlotinib combined with radiotherapy in the treatment of esophageal squamous cell carcinoma.Objective:1.Clinical research to observe the safety and effectiveness of Anlotinib 8mg once a day(QD)combined with radiotherapy and chemotherapy for patients with postoperative lymph node recurrence of esophageal squamous cell carcinoma.2.Construct a model of esophageal cancer subcutaneous tumor suppression in nude mice,and explore the anti-tumor effect of Anlotinib on esophageal squamous cell carcinoma and the sensitization effect of radiotherapy.3.Through the determination of tumor vascular morphology,to clarify the effect of anlotinib on tumor vascular normalization,and to find the window period of vascular normalization.4.Using hypoxia probe to explore the improvement of hypoxia in tumor tissue in the time window of tumor vascular normalization.5.To construct 3D co-culture system and explore the mechanism of anlotinib promoting vascular normalization through angiogenesis experiment.6.Once again,through the in vivo experiment,to find the mechanism of anlotinib sensitizing radiotherapy,which may be the normalization of blood vessels.7.Finally,it provides more basis for the clinical transformation of radiotherapy effect of esophageal squamous cell carcinoma sensitized by anlotinib.Methods:1.The clinical data are expressed by numerical value and percentage(%),and the continuous variables are expressed by median.2.Tumor formation experiment in nude mice:esophageal cancer cells Ecal09,in logarithmic growth phase were collected to determine the cell viability above 90%,and the cell density was adjusted to 5 × 107 ml.The single cell suspension was injected subcutaneously into BALB/C nude mice.Under aseptic condition,0.2 ml/was inoculated subcutaneously into the medial scapula of nude mice.When the tumor volume was about 100-300mm3,it was reserved.3.Using the transplanted tumor model of esophageal carcinoma in nude mice,the inhibitory effect of Anlotinib on esophageal squamous cell carcinoma and the effect of radiosensitization on esophageal squamous cell carcinoma were observed.4.The morphology of tumor vessels was observed by immunofluorescence histochemical staining and laser confocal microscope to find the time window of vascular normalization.5.Tumor hypoxia in the time window of vascular normalization was observed by hypoxia probe(HypoxyprobeTM Kit)and immunofluorescence staining.6.The tumor size was compared by radiotherapy inside and outside the time window of vascular normalization.7.TUNNEL immunofluorescence staining was used to compare the apoptosis of tumor tissue after internal and external radiotherapy in time window.8.The effects on ESCC’s proliferation,survival and migration in different concentrations of Anlotinib were detected by CellCountingKit-8 method(CCK-8)and scratch test.9.A three-dimensional cell culture model was constructed by 3D co-culture of cells in vitro to simulate the morphology of blood vessels and to observe the angiogenesis after treatment with Anlotinib.Results:1.All the 10 patients had postoperative lymph node recurrence of esophageal squamous cell carcinoma,and the short-term curative effect was higher than that of previously reported ORR and DCR,suggesting that Anlotinib combined with simultaneous radiotherapy and chemotherapy is effective and safe in the treatment of postoperative recurrent esophageal squamous cell carcinoma.2.Compared with the control group,the tumor volume of the Anlotinib group grew slowly,and the tumor volume decreased on the 11th day.Anlotinib could inhibit the growth of tumor.3.Compared with the control group,the distribution of vascular endothelium was more uniform,the proportion of mature pericytes increased,the thickening of basement membrane decreased,and the tight connection with surrounding cells increased after treatment with Anlotinib.The statistical results showed that the time window for vascular normalization of Anlotinib was 5-7 days.4.The hypoxia in the control group improved gradually from 5 to 14 days,and again on the 21st day,and there were periodic changes in hypoxia and oxygen enrichment in tumor tissue during the process of tumor enlargement,while in Anlotinib group,hypoxia improved from 3 to 7 days,and then inhibited tumor by inhibiting angiogenesis.5.The transplanted tumor was treated with radiotherapy inside and outside the vascular normalization time window,and the tumor size of the five groups was observed.it was found that the tumor retreated obviously in the vascular normalization time window.6.Through the apoptotic particles labeled by TUNEL fluorescence,compared with the control group,the apoptosis of tumor cells was most significant after radiotherapy during the window of vascular normalization.7.When the concentration of Anlotinib was higher than 100μM,the survival number of esophageal cancer cells decreased significantly,and the toxicity was too high,and when the concentration was less than 50μM,the viability of esophageal cancer cells decreased in a gradient with the increase of the concentration of Anlotinib.It was found that the cell proliferation decreased when the concentration of Anlotinib was 20 and 40μM.8.Using 3D co-culture system,by calculating the coverage rate of pericytes,it was found that the treatment with Anlotinib could weaken the angiogenesis in tumor microenvironment,and this effect was stronger with the increase of concentration,and it was found that the tumor microenvironment at 20uMAn concentration was significant in vascular normalization group.Conclusions:1.Anlotinib hydrochloride has a good short-term effect in the treatment of postoperative lymph node recurrence of esophageal cancer,and the side effects are tolerable and safe.2.Anlotinib can inhibit the growth of tumor blood vessels,promote the normalization of tumor blood vessels,and has anti-tumor effect.3.Anlotinib can promote the normalization of blood vessels,and the sensitization effect is the most significant in combination with radiotherapy in the time window.4.In the initial stage of the use of Anlotinib,due to the mechanism of "abnormal vascular normalization",a "oxygenation time window" appeared on the 5th-7th day,which could be sensitized and irradiated.At this stage,hypoxia in the tumor tissue was improved,giving full play to the synergistic effect of Anlotinib and radiotherapy.5.In the later stage of the use of Anlotinib,due to the antivascular effect,the intratumor vascular degeneration,internal hypoxia and radiosensitivity decreased.At this stage,it is more likely to exert the combined anti-tumor effect of Anlotinib and radiotherapy. |
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