Experimental Study Of Detecting Tumor Repopulation During Fractionated Radiotherapy In FaDu Human Pharyngeal Squamous Cell Carcinoma Bearing Nude Mice

Part One: Histopathologic validation of3’-Deoxy-3’-18F-Fluorothymidine PET fordetecting tumor repopulation during fractionated radiotherapy in FaDu humansquamous cell carcinoma bearing nude miceBackground&Objective:Accelerated tumor repopulation describes the phenomenon by which clonogenic tumorcells acquire increased capacity to regenerate tumor during fractionated radiotherapy and isbelieved to be an important cause for treatment failure. In vivo imaging, tracking andtargeting the repopulation of these cells has been of clinical interests so as to give higherdose to tumor repopulation regions. Functional imaging methods such as3’-Deoxy-3’-18F-fluorothymidine positron emission tomography (18F-FLT PET) areshowing promise in assessing the proliferation activity of tumors in vivo in molecular level.Utilizing previously established xenograft model experiments, coupled with the functionalimaging described in our clinical study, we propose a real-time, non-invasive method formeasuring tumor repopulation. After experimental verification of tumor repopulation asdescribed by Petersen et al., we validate18F-FLT PET as a novel assay for the detection oftumor repopulation in FaDu tumor by comparison to histopathological markers at selectedtime points before and after the switch in Tclon. Methods:Experiments were performed using6-to8-week-old,18-20g, female BALB/c nudemice. FaDu cells were injected in the right hind limbs of the nude mice. After tumors hadgrown to a mean diameter of about10mm, mice were randomized to irradiated andnon-irradiated groups. Local irradiations were administered using6MeV electron. Differentnumbers of3.0Gy fractions (12to18) were applied to mice either daily or every second day.Tumor diameters were measured per three days.18F-FLT micro-PET scans were performedat different time points, FLT parameters(SUVmax, SUVmean, and T/NT) were measured.Tumor sections were stained for Ki-67and BrdUrd, a labeling index (LI) was calculated.Imaging-pathology correlation was determined by comparing FLT parameters andimmunohistochemical results.Results:Measured SUVmax, SUVmean and T/NT decreased significantly after daily irradiationwith12fractions in12days (P<0.05) and18fractions in18days (P<0.05). In contrast,these parameters increased in mice treated with12fractions in24days (P>0.05) and18fractions in36days (P>0.05), suggesting accelerated repopulation. Similarly, Ki-67andBrdUrd LIs demonstrated significant decreases with daily irradiation (P<0.05), andincreases with every-second-day irradiation(P>0.05).18F-FLT parameters correlatedstrongly with proliferation markers(with Ki-67，r:0.728-0.744，P<0.05；with BrdUrd，r:0.770-0.842, P<0.05).Conclusions:1、Tumor repopulation of FaDu squamous cell carcinoma during fractionatedradiotherapy was validated by histopathology.2、18F-FLT PET could detect tumor repopulation during fractionated radiotherapy withpathological validation.3、18F-FLT uptake parameters correlated strongly with proliferation markers. Part Two: Effect of celecoxib on inhibiting tumor repopulation during radiotherapy inhuman FaDu squamous cell carcinomaBackground&Objective:Rapid proliferation of tumor cells is the basis for the development of tumors, andcorelated significantly with the biological behavior of tumor growth, invasion, recurrence,metastasis and prognosis. With the development of radiobiology, more and more animalexperiments and clinical data indicate that accelerated repopulation of tumor cells is one ofthe main reasons for treatment failure in various tumors during conventional fractionatedradiotherapy. The selective cyclooxygenase (COX)-2inhibitor celecoxib can inhibit theexpression of COX-2, reduce endogenous prostaglandin E2(PGE2) produce and therebyinhibit the proliferation of tumor cells. It was reported that the selective cyclooxygenase(COX)-2inhibitor can induce tumor apoptosis and inhibit proliferation, thereby enhancetumor response to radiotherapy. The aim was to investigate whether celecoxib can promotetumor apoptosis and inhibit tumor repopulation during fractionated radiotherapy based onthe verification of tumor repopulation as described in part one.Methods:Experiments were performed using6-to8-week-old,18-20g, female BALB/c nudemice. FaDu cells were injected in the right hind limbs of the nude mice. After tumors hadgrown to a mean diameter of about10mm, mice were randomized to control groups,celecoxib or irradiation alone groups and celecoxib+irradiation groups. Celecoxib(100mg/kg/day) was administrated by daily gavage. Irradiation was delivered with12to18fractions of3.0Gy daily or every second day based on Petersen’s repopulation model. Atdifferent time points, tumors were excised for immunohistochemistry staining.Results:With irradiation prolonged, Ki-67and BrdUrd labeling index(LIs) significantlydecreased (P<0.05), and gradually increased again later(P>0.05). Tumor growth curvesshow that compared with the control groups, radiotherapy or celecoxib alone and thecombined groups all inhibited tumor growth, but the combined groups have the slowesttumor growth. Ki-67LI decreased in celecoxib concurrent with radiotherapy for12 fractions in24days and18fractions in36days compared with irradiated alone (P=0.004and0.042, respectively). BrdUrd LIs were lower in the concurrent groups than irradiatedalone (P=0.001and0.006, respectively). EGFR expression score decreased in theconcurrent groups than irradiated alone (P=0.037and0.031, respectively). Caspase-3expression score were higher in the concurrent groups than irradiated alone (P=0.05and0.006, respectively).Conclusions:1、 Tumor repopulation of FaDu squamous cell carcinoma during fractionatedradiotherapy was validated by histopathology.2、Celecoxib combined with radiotherapy can significantly inhibit tumor growth.3、 Celecoxib combined with radiotherapy can significantly inhibit tumorproliferation(Ki-67and BrdUrd) and EGFR expression, and increase Caspase-3expression.