Comparative Cytotoxicity Of ¹⁷⁷Lu On Different Lung Cancer Cells And Targeting Of ¹⁷⁷Lu-DOTA-Cetuximab In Nude Mice With Lung Cancer

Objective:As a kind of malignant tumor,lung cancer is a major cause of cancer deaths in the world,fine non-small cell lung cancer（NSCLC）accounts for about 85%of lung cancer^[1].Part of the lung cancer radiotherapy curative effect and radiation sensitivity,irradiation dose,irradiation time,the ray energy and other factors can also affect radiation effect^[2,3].¹⁷⁷Lu is a kind of excellent nuclide,with long half-life time,short radiation radius,appropriate radiation energy,can also launch gamma ray for imaging.At home and abroad on the cellular radiosensitivity research mainly focus on irradiation,cell radiation sensitivity study of ¹⁷⁷Lu is still a blank field.Cetuximab is a kind of commonly used in clinical EGFR（Epidermal Growth Factor Receptor）monoclonal antibody,in patients with NSCLC,cetuximab is usually combined with radiotherapy in order to improve the efficacy of radiotherapy^[4-6].The purpose of this study is to select sensitive to ¹⁷⁷Lu lung cancer cell lines,and cetuximab as ¹⁷⁷Lu effective carrier,the ¹⁷⁷Lu targeted antibody in ¹⁷⁷Lu tags sensitive non-small cell lung cancer properties in vivo and in vitro,and in the biological distribution and the small animal SPECT（Single-Photon Emission Computed Tomography）as experiment ¹⁷⁷Lu-DOTA-cetuximab in targeting a tumor-burdened mice.Methods:1.Evaluation of radiation sensitivity:We studied the radiosensitivity of lung cancer cells to radionuclide ¹⁷⁷Lu,and measured the survival rate of different lung cancer cells under different radioactivity of ¹⁷⁷Lu by MTS method.2.γ-H2AX:To further investigate the relationship between¹⁷⁷Lu and DNA double-strand breaks in lung cancer cells,we quantified the breaks in lung cancer cells in the presence of ¹⁷⁷Lu usingγ-H2AX.3.We evaluated the binding affinity of cetuximab,an anti-EGFR non-small cell lung cancer monoclonal antibody,to NSCLC cells and the effect of chelating agents on cell binding.4.Saturation binding test and radioactive antibody toxicity test:In order to enhance the toxicity of antibody or only ¹⁷⁷Lu,we selected HCC827as the target cell line,labeled ¹⁷⁷Lu with cetuximab,and further performed saturation binding experiments.5.Biodistribution:According to the results of cytotoxicity test,γ-H2AX and flow cytometry,the mice model of HCC827 was established.The mice were divided into two groups according to the tumor size.¹⁷⁷Lu-DOTA-cetuximab was injected into the tail vein of the mice/the mice were killed at 6h,24h,48h and 72h,and the tissues and organs were weighed and the radioactivity was counted.6.In vitro SPECT imaging:¹⁷⁷Lu-DOTA-cetuximab was injected into the tail vein of the successfully constructed tumor model mice at 6h,24h,48h,72h respectively to explore the distribution of ¹⁷⁷Lu-DOTA-cetuximab in vivo.Results:1.The radiosensitivity of different lung cancer cells to ¹⁷⁷ Lu varied greatly.A549 was not sensitive to ¹⁷⁷Lu,and had a strong radioresistance.The survival rate was higher than 95%after incubation for 48h in the high-radioactive culture medium,compared with 66±8.9%for H292,60.3%±1.5%for HCC827 and 57%±11.2%for H446.Our results suggest that radiosensitivity is related to cell type.2.Apoptosis induced by ¹⁷⁷Lu-labeled lung cancer targeting drugs is related to DNA double-strand breaks and cell types.There was no significant DNA damage was observed in all cells incubated without ¹⁷⁷Lu Cl₃ cell culture medium.NCIH292 showed the highest radiosensitivity among the cells incubated with ¹⁷⁷Lu Cl₃,and H446 and HCC827 showed similar radiosensitivity.Consistent with cytotoxicity results at equivalent concentrations of radioactivity.3.Cetuximab and DOTA-cetuximab had similar binding affinities for HCC827and NCI-H292 cells,with DOTA-cetuximab binding affinity slightly decreased with NCI-H446 and A549.However,the EC50 of all cells was 0.1-0.4 n M,and the Kd was 0.1-0.5 n M.We considered that the effect of DOTA on the binding ability of m Ab was negligible.4.We selected HCC827 cells to represent EGFR-positive non-small cell lung cancer.In the radio-antibody saturation binding assay,the radio-antibody binding of HCC827 lung cancer cells increased with increasing concentration of radio-antibody,and the EC50 value obtained by calculation was 2.6±1.19 n M.5.According to SPECT/CT imaging results,¹⁷⁷Lu-DOTA-cetuximab accumulates in lung cancer cell xenograft tumors,some types such as HCC827 or NCI-H292 are more sensitive to ¹⁷⁷Lu radiation,¹⁷⁷Lu-DOTA-cetuximab has high cytotoxicity and can effectively target HCC827-bearing xenografts.6.The biodistribution results were similar to those of SPECT/CT imaging,with high radioactivity appearing in the tumor and accumulating in the tumor over time,and radioactivity gradually decreasing in various normal tissues over time.Conclusion:The results showed that there was a certain correlation between cell types and radiosensitivity,which was mainly reflected in DNA breakage.When the cells were damaged by radiation,the DNA in the cells would be destroyed,which would lead to cell apoptosis,thus affecting the survival rate of the whole cells.Flow cytometry and antibody saturation experiments showed that EGFR was highly expressed in many lung cancer cells,suggesting that the chelating agent DOTA did not affect the binding of antibody to NSCLC.Cetuximab could be an effective carrier for radionuclide ¹⁷⁷Lu.¹⁷⁷Lu-DOTA-cetuximab can be retained and accumulated in lung cancer tumor for a long time,and the radioactive antibody gradually disappears in non-tumor tissues with time,which can effectively reduce side effects.Therefore,we believe that ¹⁷⁷Lu-DOTA-cetuximab has high potential in imaging and radiotherapy of lung cancer.