Introduction

Colorectal cancer is the third most commonly diagnosed cancer in males and the second in females worldwide[1],with an increasing incidence rate.Surgery,radiation,chemotherapy,and targeted therapy are common treatments.However,intra-and intertumoral heterogeneity leads to marked difference in prognosis and response to therapy of patients with the same clinical staging,posing a major challenge for tumor therapy[2,3].Heterogeneity is a concept that distinct sub-populations of cancer cells exist within and between tumors of individual patients;each with different growth rate,response to treatment,and metastatic ability through the process of evolution[4].Liver metastasis is the main cause of cancer-related death.Thus,it is important to ascertain metastatic capacity of colorectal cancer cells.Positron emission tomography(PET),as a noninvasive imaging modality,is recently used to quantify tumor heterogeneity,with the ability to display molecular and functional characteristics of tumors in vivo[5,6].The application of PET as a promising biomarker of tumor heterogeneity is a new technique in the field of molecular imaging in recent years.

18F- fludeoxyglucose(18F-FDG)is widely used to differentiate non-malignant from malignant tumors.It is used for staging and prediction of prognosis because of its high sensitivity for detecting metabolically active tumor,but may result in false positive because of a lower speci ficity.The shortcoming may be overcome by using a more speci fic tracer.The radiolabeled thymidine analog 3′-deoxy-3′-18F- fluorothymidine(18F-FLT)is a tracer used for the visualization of tumor cell proliferation[7,8].The metabolism of 18F-FLT is mainly determined by the DNA salvage pathway.18F-FLT is phosphorylated by thymidine kinase(TK1),which is a critical enzyme in this pathway,and consequently trapped in the tumor cell.Elevated TK1 expression is observed during the S-phase of the cell cycle,and,hence,the tracer retained in tumor cells may represent proliferation.The diagnostic value and evaluation of early response to therapy with 18F-FLT have been widely reported in many tumors[9,10].However,only few studies[11,12]investigated the relationship between the two tracers and the characteristics and the aggressive features of tumor cells.This study was to explore these relationships.

SW480 and SW620 cell lines were derived from a primary colorectal adenocarcinoma and its lymph node metastasis,respectively[13,14].The migration and invasion abilities are different between the two cell lines[15,16].The aim of this study was to evaluate the value of 18F-FDG and 18F-FLT in the identi fication of different colorectal cancer liver metastasis models in nude mice on the basis of tracer uptake by tumor cells in vitro and to verify the relationship between tracer uptake and tumor biology.

Methods

Cell culture and tracer uptake assay in vitro

The medium used is serum-free RPMI 1640 medium with 0.2%BSA.SW480 and SW620 cells were cultured in the medium.After 24 h of incubation,100 μL of 18F-FLT medium was mixed with a cell suspension diluted with the medium.After incubation for 15 min,30 min,1 h,2 h,and 4 h at 37 °C,700 μL medium was added to the mixture.The cell suspension was subsequently centrifuged for 5 min,the supernatant was removed,and radioactivity counts of the collected fractions(precipitated cells)were measured in aγ automated counter(5×105 cells per tube).

Experimental liver metastasis model

Forty nude mice were randomly divided into two groups(n=20).The colon cancer cells in the logarithmic growth phase were collected and made into single cell suspension at a concentration of 2.5×107 cells/mL.Nude mice were anesthetized by intraperitoneal injection of 10%chloral hydrate(0.4 mL/100 g)and placed in a supine position.After disinfection of the skin in the area of surgery,a flank incision was made to expose the spleen.SW480 or SW620 cells(2.5×106 cells)were injected into the spleen.Subsequently,the needle hole was pressed with a sterile cotton swab for 1–2 min to prevent tumor cell leakage.The spleen was returned to the abdominal cavity and the abdominal wall was sutured.The incidence of liver metastasis and the number of metastatic nodules in the liver were examined 35 days later after the implantation of SW480 and SW620 cells to the spleen.The incidence of liver metastasis was de fined as the total number of nude mice with con firmed liver metastasis divided by the total number of nude mice with identi fied primary spleen tumor.

Micro-PET protocol

本项目所选用的PE管电熔焊机主要有2种：第1种型号为THE-9C、输入电压为400V、输出电压为40V、频率为50Hz，第2种型号为HTA-3B，输入电压为240V、输出电压为40V、频率为50Hz，2种型号焊机均采用发电机供电。第1种可以进行多个时段不同参数的熔接；第2种为单时段单参数熔接，具有较小的温差补偿功能。它们的熔接原理一致，都是为电熔连接件提供加热的电流，该电流通过电熔连接件内部的电热丝并产生热量，热量累积温度上升，使管件和管材的连接界面熔融。

Immunohistochemistry assay

Liver metastases of nude mice were fixed overnight in 10%formalin and embedded in paraffin blocks,from which 4-μm thick sections were cut for immunohistochemical staining.After deparaffinization,antigen retrieval was performed by boiling tissue section in 10 mmol/L citrate buffer,pH 6.0 for 10 min followed by cooling at room temperature for 20 min.Endogenous peroxidase was blocked with 3%hydrogen peroxide.Sections were then incubated overnight at 4°C with primary antibodies.The primary antibodies were as follows:anti-CD44 mouse monoclonal antibody(1:100 dilution,3570S,Cell Signaling Technology,MA,USA),anti-VEGF rabbit polyclonal antibody(1:200 dilution,ab46154,Abcam,London,UK),and anti-Ki67 mouse monoclonal antibody(1:100 dilution,ab16667,Abcam).

The next day,tumor sections were incubated with horseradish peroxidase-conjugated secondary antibodies(dextran coupled with peroxidase molecules and goat secondary antibody molecules against rabbit and mouse immunoglobulins).Immunostaining was completed with 3,3′-diaminobenzidine(DAB).The density of the staining was described as negative(–),weak(+),moderate(++),and strong(+++).

Western blotting

Mouse tissue sample of liver metastases were lysed in RIPA buffer(Beyotime,Nanjing,China)and an equal amount of protein(20μg)was loaded and separated by 10%SDS-PAGE and subsequently transferred onto the polyvinylidene fluoride membranes(Merck Millipore,Darmstadt,Germany).The membranes were incubated overnight at 4°C with the primary antibodies which were the same as those described for immunohistochemistry assay.Next,the membrane was probed with the appropriate horseradish peroxidase-coupled secondary antibodies(Zsbio)for 1 h at room temperature.Protein bands were visualized by using a chemiluminescent substrate ECL kit(Merck Millipore).

Statistical analysis

All statistical analyses were performed with IBM SPSS statistic version 19.0.Data were expressed as mean±SD.The Mann-Whitney U and unpaired Student’s t test were used to estimate the differences of PET parameters between SW480 and SW620 metastatic tumors.The diagnostic performance of FDG-PET and FLT-PET in differentiating SW480 and SW620 metastatic tumors was examined using receiver operating characteristic(ROC)curve.The Pearson correlation coefficient was employed to evaluate the correlation of both FDG uptake and FLT uptake with tumor marker levels and metastatic potential of SW480 and SW620 tumors.A P value less than 0.05 was considered statistically signi ficant.

Results

Comparative study of the metastatic capability of SW480 and SW620 cells

Two(13.3%)in 15 of surviving mice with spleen injection of SW620 cells had liver metastases.This number was 9(47.4%)in 19 with the injection of SW480 cells(P=0.034).

Fig.1.Cellular uptake of 18F-FDG and 18F-FLT in SW480 and SW620 cells.A:Differences in 18F-FDG uptake were found between the two cell lines at different intervals.B:Relative to 18F-FDG uptake,differences in 18F-FLT uptake were more apparent at 60 min.

Radiotracer uptake assay in vitro

Radiotracer cell uptake in SW480 and SW620 cells was analyzed in 4 h.The uptake of 18F-FDG and 18F-FLT continuously increased during the experiment(Fig.1).The uptake of 18F-FDG and 18F-FLT was lower in SW620 cells than that in SW480 cells at all time points.Moreover,the cellular uptake ratio of 18F-FLT was obviously higher than that of 18 F-FDG in both SW480 and SW620 cells.After a 60-min incubation at 37°C,the uptakes of 18F-FDG in SW480 and SW620 cells reached 6.07%±1.19%and 2.82%±0.15%,respectively(t=4.69,P=0.04);the uptakes of 18F-FLT in SW480 and SW620 cells were 24.81%±0.45%and 15.57%±0.66%,respectively(t=19.99,P＜0.001).

慢性荨麻疹通过临床的精心护理，通过辩证措护，确定相应的护理方法，对护理的效果进行临床检验，根据荨麻疹的不同类型，安排不同的护理措施，因人、因时、因地进行护理。实践结果表明，通过穴位埋线方法治疗慢性荨麻疹加上中医护理贯穿于整个治疗过程，可以有效地提高患者的治愈率，减少患者复发。

Micro-PET imaging of nude mice

Based on the pilot study,we performed micro-PET imaging of nude mice 5 weeks after tumor cell inoculation.The uptake of 18F-FDG and 18F-FLT was evaluated in colorectal liver metastasis models(Fig.2).Both 18F-FDG and 18F-FLT PET displayed the contour of liver metastases.Among 55 SW620 liver metastases confirmed by pathology,only 23 were detected by 18F-FLT PET.The features of FLT PET images for SW620 liver metastases showed slight differences in terms of activity intensity between displayed lesions and normal liver tissue,which allowed the visualization of the lesion borders.In contrast,the majority of SW480 liver metastases displayed on 18F-FLT PET images,which was also supported by histology.Another discrimination criterion was that,under the same color scaling conditions,the borders of SW480 liver metastases were clearer than those of SW620 liver metastases.Compared with FLT PET images,we did not find obvious visual differences of PET activity between SW480 and SW620 metastatic tumors on FDG PET images.

Table 1 Comparison of FDG and FLT indices between SW480 and SW620 metastatic tumors.

∗metastatic tumor SUVmax divided by the liver SUVmax.

Index SW480(n=30) SW620(n=30) P value FDG PET/CT FDG SUVmax 1.81±0.47 1.96±0.61 0.329 FDG SUVmax ratio∗ 2.84±0.72 3.04±0.83 0.344 FLT PET/CT FLT SUVmax 1.57±0.40 1.28±0.31 0.0089 FLT SUVmax ratio∗ 1.68±0.23 1.49±0.19 0.0013

Comparison of FDG and FLT parameters between SW480 and SW620 metastatic tumors

18F-FLT was considered a useful tool to measure tumor cell proliferation;therefore we attempted to con firm whether 18F-FLT uptake in SW480 and SW620 tumors can reflect the variation trend of proliferation markers,which was analyzed by Western blotting(Fig.5B).However,the study showed that the differences of Ki67 expression levels between SW480 and SW620 tumors(1.17±0.24 versus 1.75±0.38)could not explain variances in 18F-FLT uptake.In addition,the study revealed that the expression levels of VEGF in SW480 and SW620 tumors were similar(0.39±0.14 versus 0.47±0.15).This may partly explain why no correlation was established between 18F-FLT uptake and expression levels of VEGF.Obvious differences in CD44 expression were observed between SW480 and SW620 tumors(2.77±0.49 versus 0.76±0.07).The higher 18FFLT accumulation in SW480 tumors was consistent with high expression of CD44 in this tumor.A signi ficant positive correlation between CD44 levels and 18F-FLT uptake was observed.

All FLT parameters were signi ficantly higher in SW480 than those in SW620 metastatic tumors(SUVmax,P=0.0089;SUVmax ratio,P=0.0013),whereas FDG parameters did not differ between SW480 and SW620 metastatic tumors(P＞0.05 for both parameters)(Table 1).

Diagnostic performance of FLT parameters to differentiate SW480 from SW620 tumors

我不再说什么，愤恨地离开她的办公室。晚上，有人对公司不景气产生了怀疑，骂付玉是一只鸡，还说自己不见兔子不撒鹰，是把她睡了之后，才加入她的团队。另外几个男人，也都说把付玉给睡了。要是以往，谁这么侮辱付玉，我会毫不犹豫地跟他拼命，这次，我有些无动于衷。一个大黄牙烟鬼，是个农村来的老光棍，他说，我花了一万块钱，日了付玉，这一万块挣不回来，也值了。小伙子，你是个大学生，好好的大学不读，怎么掺合这屌事？你日付玉没，如果连点腥气都没沾上，这亏可吃大了。我实在听不下去他这样侮辱付玉，刚要下床揍他，门被咣当一声踢开了，几个警察闯进来。没人敢反抗，我们一起进了派出所。

Tumor markers of metastasis,proliferation,and angiogenesis:immunohistochemistry and Western blotting

Staining was rather heterogeneous within tumors for all markers,except for VEGF.SW480 liver metastases showed a more intense CD44 staining compared to that of SW620 liver metastases.CD44 staining was predominantly detected at the cell membrane,whereas VEGF was mainly expressed in the cytoplasm of colon cancer cells and occasionally in the nucleus.A nuclear staining pattern was observed for Ki67(Fig.5A).

这是一个怎样的家啊？就是黑黢黢的两间房，满是灰尘和杂物，唯一的电器就是一台黑白电视机，还是搜不到信号做摆设的。

All PET parameters are summarized in Table 1 and Fig.3.The SUVmax ratio was signi ficantly higher on FDG PET than that on FLT PET for SW620 metastatic tumors(3.04±0.83 versus 1.49±0.19,P＜0.05)and SW480 metastatic tumors(2.84±0.72 versus 1.68±0.23,P＜0.05).

The areas under the ROC curves(AUC)and optimal cut-off values of FLT SUVmax and FLT SUVmax ratio to differentiate SW480 from SW620 tumors are summarized in Table 2 and Fig.4.Each optimal cut-off value to differentiate SW480 from SW620 tumors on FLT PET was＞1.55 for FLT SUVmax and ＞1.65 for FLT SUVmax ratio.On FLT PET,AUC for differentiating between SW480 and SW620 tumors was 0.70 for FLT SUVmax and 0.74 for FLT SUVmax ratio.

Fig.2.Micro-PET images of 18 F-FDG and 18F-FLT in SW480 and SW620 liver metastasis models.A:Micro-PET images of 18F-FLT in SW480 and SW620 liver metastasis models at 2 h after tracer injection and the image showing pathologically con firmed metastasis(arrows).B:Micro-PET images of 18F-FDG in SW480 and SW620 liver metastasis models.

Table 2 ROC curve analysis for FLT SUVmax and FLT SUVmax ratio in metastatic tumors.

∗metastatic tumor SUVmax divided by the liver SUVmax.

Parameters AUC 95%CI P value Optimal cut-off Sensitivity Speci ficity FLT SUVmax SW480 versus SW620 0.70 0.56–0.83 0.01 1.55 0.63 0.80 FLT SUVmax ratio∗SW480 versus SW620 0.74 0.61–0.86 0.002 1.65 0.70 0.77

Linear regression analysis

18F-FDG PET is routinely used for the detection and staging of tumors.In the present study,all liver metastases of colorectal cancer xenografts were visible on 18F-FDG PET images without distinct differences in tracer uptake between SW480 and SW620 tumors.These findings suggest that the glucose consumption of these metastatic tumors was similar and 18F-FDG PET alone cannot discriminate differences between the various tumor types.

Discussion

Colorectal cancer is one of the most common causes of cancer-related death worldwide.The death of many patients with colorectal cancer is associated with liver metastases,which can be attributed to a more aggressive biological behavior of colon cancer cells[17].Inter-individual differences in the same type of tumors and tumor response to clinical therapy are well-known [18].The visualization of such molecular biology-speci fic changes can be ful filled by molecular imaging,which can also be used for tumor staging and to monitor the treatment response.18F-FDG PET provides measurement of cellular glucose metabolism.A large number of studies[19–21]suggested the application of this tracer to differentiate tumor,tumor staging,and prediction of prognosis,but the higher rate of false positive and lower speci ficity of 18F-FDG PET limited its application.However,several studies[22,23]revealed that 18F-FLT,regarded as being more proliferation-speci fic,seems more appropriate for tumor detection.The present study took advantage of both 18F-FDG and 18F-FLT to discriminate colorectal cancer cell lines with different biologic behaviors and to analyze the association between the two types of tracers and tumor aggressiveness.

The most difficult problem encountered during the process of establishing liver metastasis xenografts was to determine a proper“window period”for PET imaging.The main reason for the selection included not only the shorter survival time of the mouse model with liver metastasis of colorectal cancer,but also PET imaging for liver metastasis models as early as possible,because it took a long time to complete multi-nuclide imaging for all mouse models with liver metastasis of colorectal cancer.Once the imaging time of the colorectal cancer liver metastasis model was delayed,a large number of nude mice would die in the shortterm.Therefore,we determined that the optimal imaging time was 5 weeks after the tumor cells were implanted in preliminary experiments.

Fig.3.Box and Whisker plots of PET parameters in SW480 and SW620 metastatic tumors.

Fig.4.ROC curves analysis of FLT SUVmax and SUVmax ratio were used to differentiate SW480 and SW620 tumors.FLT SUVmax ratio yielded a greater area under the curve and better performance relative to FLT SUVmax.

First,in vitro cellular uptake experiments of 18F-FDG and 18FFLT were carried out to establish a basis for the detection of differ-ent biological behaviors in vivo by PET.The results con firmed that the uptake of 18F-FDG and 18F-FLT in SW480 cells was higher than that in SW620 cells,and the uptake of 18F-FLT was always higher than that of 18F-FDG,irrespective of the cell type.Results of small animal PET imaging showed that FLT SUVmax and FLT SUVmax ratio were signi ficantly higher in SW480 cells than those in SW620 cells,which was in agreement with the results of in vitro 18F-FLT cellular uptake experiments.Compared with 18F-FLT PET imaging,no statistical difference was observed in FDG SUVmax and FDG SUVmax ratio for all metastatic tumors,which contrasted with the results from in vitro 18F-FDG uptake experiments.We speculated that this discrepancy may be associated with the internal environment and a smaller difference of 18F-FDG uptake between SW480 and SW620 cells compared with18F-FLT uptake[24].

18F-FDG and 18F-FLT were from Sumitomo Heavy Industries,Ltd.(Tokyo,Japan).PET images were obtained from Inveon PET(Siemens Preclinical Solutions).Nude mice were injected with 18FFLT or 18F-FDG via the tail vein,a median dose was 5.0 MBq(range 3.23–6.67).Anesthesia was maintained with 1.5%iso flurane in 100%oxygen with a flow rate of 2 L/min.Mice were positioned prone on the scanner bed.Images were acquired from 60 to 75 min after injection of 18 F-FDG and from 120 to 135 min after injection of 18F-FLT.Images were analyzed with the software ASIProVM(Siemens Preclinical Solutions).Regions of interest(n=30)were drawn on the liver metastases of every group and the background was de fined as a circular region manually contoured in the liver.The maximum standardized uptake value(SUVmax)ratio was calculated as the tumor SUVmax divided by the liver SUVmax.

Cellular uptake of 18F-FLT in vitro correlated with liver metastatic tumor uptake in vivo(r=0.64,P=0.0083).A significant correlation was observed between 18F-FLT SUV ratio and CD44 expression in liver metastases(r=0.81,P=0.0049,Fig.6).A good correlation was detected between 18F-FLT uptake in the spleen primary tumor and the number of liver metastases(r=0.73,P=0.0019)(Fig.7A),but no correlation was observed with 18FFDG(r=0.16,P＞0.05)(Fig.7B).A signi ficant correlation was observed between CD44 expression and distant metastatic potential(r=0.86,P=0.0034),but Ki67 and VEGF expression did not correlate with metastases(r=0.27,P＞0.05;r=0.19,P＞0.05;respectively).

Fig.5.Examination of tumor markers in liver metastases.A:Immunohistochemistry from 2 representative nude mouse models of human colorectal cancer liver metastasis(left column).The CD44 expression in SW480 liver metastasis is positively correlated with 18F-FLT uptake(right column).Compared to SW480 liver metastases,higher FDG uptake values and lower FLT uptake values can be observed in SW620 liver metastases.Scale bars=50μm.B:Nuclear and cytoplasmic proteins were collected from SW480 and SW620 liver metastases in nude mice.Western blotting was performed to detect the expression of CD44,Ki67,and VEGF.GAPDH was used as a loading control.

Our study indicates that there was a little difference in the data expressed as mean±SD between SW480 and SW620 tumors;however,statistical analysis suggested a signi ficant difference in FLT SUVmax ratio between the two types of metastatic tumors.The data with a skewed distribution led to the results.In brief,the FLT SUVmax ratio was below 1.45 in SW620 tumors compared with SW480 tumors.A reasonable explanation for this is a higher background activity in the liver,caused by hepatic glucuronidation of FLT[25,26].Our results for 18F-FLT PET imaging con firmed this hypothesis,indicating a higher liver FLT SUVmax(mean 0.94,range 0.53–1.29)in 18F-FLT scans than that in 18F-FDG scans(mean 0.65,range 0.38–0.82).Accordingly,there should be a minimum value that must be surpassed to allow visualization of metastatic tumors based on FLT SUVmax in SW620 tumors(mean 1.28,range 0.74–1.86).Owing to above reasons,a sizeable portion of SW620 metastatic tumors may not be detected on FLT PET.Tumor aggressiveness is a key factor that affects the prognosis of colorectal cancer,including differentiation,growth rate,metastatic potential,and response to chemotherapeutic agents.It is important to analyze the molecular markers closely related to tumor invasiveness to predict the prognosis,monitor metastasis and recurrence,and to select the clinical treatment strategies.Three tumor markers(VEGF,Ki67 and CD44)were chosen to represent several facets of tumor aggressiveness,metastasis,proliferation,and angiogenesis,respectively.

异化与归化两种翻译策略不可能是对立的，翻译实践中二者之间是相互交融的。 同一句话可以在不同的语境中采取不同的的意译方式而有所侧重，也可以采用保留原文化痕迹的直译手法，如：

Our study demonstrated that there was no signi ficant difference in VEGF expression between SW480 and SW620 metastatic tumors,which was not represented by 18F-FLT uptake or by 18F-FDG uptake.This finding may con firm the theory that angiogenesis may not play a critical role during the process of invasion and metastasis of colon cancer cells as most of the liver metastases are poorly vascularized.18F-FDG uptake in SW620 tumors was slightly higher than that in SW480 tumors,but it was not statistically signi ficant.Although 18F-FDG cannot be expected to reflect the metastatic capacity of SW480 and SW620 tumors to the liver,18F-FDG still provide a higher diagnostic sensitivity compensating the lower detection of liver metastases with 18F-FLT.

CD44,a well-recognized multifunctional cell surface adhesion molecule that is highly expressed in many cancers,including colorectal cancer,plays important roles in regulating biologic activities of tumor cells,including adhesion,proliferation,survival,metastases,and differentiation.Its interaction with the appropriate extracellular matrix ligands promotes tumor progression,leading to tumor metastases by evoking different signal transduction pathways.Many studies[34–36]revealed that CD44 is a more reliable marker for predicting liver metastases and survival.Indeed,we showed a signi ficant positive correlation between tumor CD44 levels and the metastatic ability of the tumor.In other words,SW480 cells highly expressing CD44 have a stronger metastatic ability.Furthermore,we also found that 18F-FLT uptake can be directly linked to tumor CD44 levels,at least in our model of liver metastasis xenografts.One can speculate that a correlation exists between 18F-FLT uptake and metastatic ability of the tumor;namely,higher 18F-FLT uptake can predict greater invasion to the liver.Despite the differences detected in 18F-FDG uptake in vitro between SW480 and SW620 cells,18F-FDG uptake was not associated with metastatic potential.

许多时候常爱兰都觉得很恍惚，似乎一切都是陌生的。几年下来，这个叫寿包子的男人是让人陌生的，酗酒打架赌博样样都来，把自己与小羽也打得好不了伤疤忘不了痛；而这个小羽呢，却似乎是从生下来就没有过好脾气，从小哭到大，一直到寿包子死掉，然后突然就不哭了。整个人一天到晚也不说什么话；来到岭北镇，这个被人叫麻糍的男人却是个可以随手被人捏被人捣的软麻糍，这个真名叫周财富的男人，不仅没什么财富，却被叫成了麻糍。这一切的一切似乎都陌生得很。

Uptake of 18F-FLT varied signi ficantly between SW480 and SW620 metastatic tumors,with SW480 tumors showing a higher accumulation of the radial activity.However,immunohistochemistry and Western blotting analysis showed that Ki67 expression did not differ between SW480 and SW620 metastatic tumors.As a proliferation marker,Ki67 is expressed preferentially during late G1,S,G2,and M phases of the cell cycle,while the antigen cannot be detected in cells in G0 phase.A positive correlation between Ki67 expression and 18F-FLT uptake has been reported in a number of reports in which breast,brain,and lung cancer were studied[27–29].However,the correlation has also been questioned because Ki67 expression occurs in all phases of the cell cycle,except for G0.18F-FLT uptake is considered to be under the control of TK1 activity,which is closely linked with proliferation during the S phase of the cell cycle[8,30].Consequently,no positive correlation existed between Ki67 expression and 18F-FLT uptake,consistent with reports that claim that 18F-FLT uptake does not necessarily correlate with Ki67 expression and cannot be considered a non-invasive Ki67 index[31–33].

作为一种新型经济模式和商业模式，物流业发展共享经济中难免会遇到质疑和困难，有些问题会引起政府或者有关部门的干涉和禁止。在共享经济的快速发展与应用中，其更新速度远远超出了目前政府所监管的范畴和能力，政府还没有一个针对性地健全监管制度，一定程度上阻碍了物流业共享经济的发展。基于这种背景，为了更好地利用共享经济促进我国绿色物流的建设、实现对社会物流费用的有效降低，政府要充分发挥出其监管作用，建立起针对物流共享平台搭建以及合理运行的相关监管制度，形成一个允许试错、宽容、多元化、开放的经济环境，打破一味禁止和限制的监管弊端，完善经济环境，为物流共享经济的发展提供必要保障。

Fig.6.Tumor CD44 expression signi ficantly correlates with 18F-FLT SUVmax ratio in liver metastases of colorectal cancer xenografts(r=0.81,P=0.0049).CD44 levels were determined by Western blotting.

Fig.7.Linear regression analysis for tracer uptake and metastatic potential of colorectal cancer.A:Correlation between 18F-FLT uptake and metastatic activity in the liver metastatic model(r=0.73).B:The lack of correlation between 18F-FDG uptake and metastatic activity in the liver metastatic model demonstrated that 18F-FDG was inappropriate to predict the metastatic ability of colorectal cancer.

In the present study,we not only validated the hypothesis that 18F-FLT can discriminate SW480 and SW620 cells and metastatic tumors,but also determined that 18F-FLT uptake is a biomarker for biological characteristics of tumor cells or,more precisely,the metastatic ability of two types of tumor cells.In addition,18F-FDG may complement the inherent disadvantages of 18F-FLT as a liver imaging agent,although 18F-FDG PET cannot differentiate SW480 and SW620 metastatic tumors and cannot predict liver metastasis.More explicitly,combined with FDG PET may be superior to FLT PET alone for detecting and distinguishing liver metastases because of additional information regarding not only metabolism,but also higher sensitivity for detection of liver metastases[25,26].Interindividual differences between tumor phenotypes will complicate choices of treatment protocols for tumors[37,38].FLT PET is not only capable of visualizing heterogeneities between tumors,but also has the ability to differentiate individual tumor biology,as reported in this study.Consequently,18F-FLT should be considered a valuable tracer to monitor changes in biological activity following tumor therapy.

In conclusion,18F-FLT PET may be superior to 18F-FDG PET in differentiating between SW480 and SW620 metastatic tumors;however,combining 18F-FLT with 18F-FDG PET can possibly improve diagnostic performance based on some defects of 18F-FLT when used for liver imaging.Moreover,18F-FLT allows the distinction of individual tumor biology.18F-FLT uptake is positively correlated with metastatic potential of colorectal cancer and should be considered a valuable biomarker.

《麦克白》是莎士比亚四部悲剧作品中最短的一部作品，也是最震撼人心的一部作品。《麦克白》这部作品给人们带来很多思考，中外读者对作品中麦克白的悲剧分析也众口不一，有的人说是因为命运，有的人认为是性格所致。笔者试图从人性这一角度揭示麦克白悲剧的主要归因。

Contributors

XHL and JHJ contributed to the conception and design of the study.XHL and ZRJ performed the experiment.LM performed the statistical analysis.JHJ revised the manuscript.XHL accomplished the paper.All authors read and approved the final manuscript.JHJ is the guarantor.

Funding

This study was supported by grants from the National Natural Science Foundation of China(81471736 and 81671760),the National Science and Technology Pillar Program during the Twelfth Five-Year Plan Period(2015BAI01B09),and Project of Research Foundation of the Talent of Scienti fic and Technical Innovation of Harbin City(2016RAXYJ063).

Ethical approval

This study was approved by the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University(KY2016-028).

③大多数居民的防灾意识较弱，对内涝灾害的重视程度远远不够，减灾措施欠缺，已有的防灾减灾措施很传统，一旦大灾到来，防灾减灾效率低下。

Competing interest

No bene fits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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