Low Intensity Ultrasound Combined With Microbubbles Promotes The Uptake Of Targeted Radionuclides In HER-2 Overexpression Breast Cancer

Breast cancer has become the most common cancer in the world,which has a serious impact on human health.The incidence of human epidermal growth factor receptor-2（HER-2）overexpress is about 20-25%in breast cancer,and HER-2 overexpression is associated with poor prognosis of breast cancer.The newly NCCN guidelines recommend that for breast cancer with HER-2 overexpression,patients with primary tumor>2cm（T stage≥2）or regional lymph node metastasis positive（N stage≥1）need neoadjuvant therapy（NAT）to improve the anti-tumor effect.At present,the first-line NAT regimens has a high incidence of grade3-4 toxicity and unsatisfactory anti-tumor efficacy.Therefore,new,more effective and better tolerated treatment strategies are urgently needed to improve the efficacy of NAT,so as to improve the prognosis of patients with HER-2 overexpression breast cancer.Targeted radionuclide therapy（TRT）is an emerging treatment strategy that utilizes targeted radionuclide drugs（TRD）to treat malignant tumors.By labeling radioactive nuclides with radiation damage effects on monoclonal antibodies,the TRD retain the ability to specifically bind to tumor cells,and kill tumors through antibody dependent cell mediated cytotoxicity.At the same time,due to the self-decay of radioactive nuclides,TRD can cause ionizing radiation damage,thus enhancing the anti-tumor effect.TRD has a significant anti-tumor effect in hematology tumors,but in solid tumors,due to the limitation of the blood-tumor barrier（BTB）,the drug dose inside the tumor is low,so the anti-tumor effect is poor.The combination of low intensity ultrasound and microbubbles（USMB）has been proven to promote the uptake of chemotherapy drugs,gene molecules,etc.in tumor tissue.Therefore,the combination of USMB and TRD is expected to increase the dose of TRD in tumor,improve the anti-tumor efficacy of TRD,and make TRT a new NAT strategy for breast cancer with HER-2 overexpression.Objective1.To evaluate the efficacy and toxicity of HER-2 overexpression breast cancer standard NAT protocol in the real world.2.To prepare a TRD,radiolabel trastuzumab（H）with ¹⁷⁷Lu(¹⁷⁷Lu-DOAT-H),and use it in combination with USMB to treat HER-2overexpression breast cancer animal model.3.To explore the feasibility of USMB combined with ¹⁷⁷Lu-DOAT-H as NAT of HER-2 overexpression breast cancer,and provide a new combined treatment strategy for HER-2 overexpression breast cancer NAT.Material and Method1.The NAT regimens was restricted to docetaxel and carboplatin and Herceptin and Pertuzumab（TCHP）or docetaxel and Herceptin and Pertuzumab（THP）for HER-2 overexpression breast cancer patients undergoing NAT at Mianyang central Hospital from January 2020 to September 2022.Gender,age,pathological type,degree of pathological differentiation,estrogen receptor（ER）status,progesterone receptor（PR）status,HER-2 status,primary breast tumor size,clinical stage,NAT regiman,NAT treatment duration,NAT dose,number of NAT cycles,type and dose of adjuvant medication,toxicity during NAT,patient’s weight before and after NAT,and the efficacy of NAT treatment were recorded.p CR rate,toxicity,dose adjustment rate,weight loss rate,using of adjunctive medications during NAT,and completion rate of NAT for different NAT regimens were compared.2.（1）Synthesis of ¹⁷⁷Lu-DOAT-H:Ligand modification of H was carried out through three steps:solvent displacement,coupling reaction,and purification.The bifunctional chelating agent p-SCN-Bn-DOTA was coupled with H（DOTA-H）,and the ligand to-antibody ratio（LAR）of DOTA-H was detected by spectrophotometric method;Subsequently,¹⁷⁷Lu Cl₃ was added to DOTA-H solution at a 2:1 ratio of activity（m Ci）and antibody dose（mg）to label H with radioactivity(¹⁷⁷Lu-DOTA-H).After labeling,the label rate and in vitro stability of ¹⁷⁷Lu-DOAT-H were detected using an instantaneous silica thin layer chromatography（i TLC）,and the radiochemical purity of ¹⁷⁷Lu-DOAT-H was detected using a high performance liquid chromatography（HPLC）.（2）Detection of in vitro targeting binding ability of H,DOTA-H,¹⁷⁷Lu-DOTA-H:Flow cytometry was used to detect the targeting binding ability of H,DOTA-H to HER-2antigen in vitro;Radioimmunoassay was used to detect the targeted binding ability of ¹⁷⁷Lu-DOTA-H to HER-2 antigen in vitro,and the concentration for 50%of maximum effect（EC50）,maximum antibody binding amount（Bmax）,and dissociation constant（Kd）were calculated.（3）Ultrasound toxicity testing:MTS reagent kit was used to detect cell toxicity at different ultrasound intensities,and non-toxic ultrasound intensities were selected for subsequent research.（4）The effect of USMB on the stability of H,DOAT-H,¹⁷⁷Lu-DOTA-H:HPLC was used to detect the effect of USMB on the structure of H;Spectrophotometric method was used to detected the effect of USMB on DOAT-H LAR;i TLC was used to detecte the effect of USMB on the stability of ¹⁷⁷Lu-DOTA-H.（5）The effect of USMB on the in vitro targeting binding ability of H and ¹⁷⁷Lu-DOTA-H:Flow cytometry was used to detect the effect of USMB on the in vitro binding of HER-2 to H,and gamma counter was used to detect the effect of USMB on the in vitro binding of HER-2 to ¹⁷⁷Lu-DOTA-H.（6）The effect of ultrasound on the internalization function of antibodies in SKBR-3 cells after binding to H:Flow cytometry and gamma counter were used to detect the internalization amount of antibodies entering the cells after H and ¹⁷⁷Lu-DOTA-H binding to SKBR-3.（7）The effect of ultrasound on the cytotoxicity of ¹⁷⁷Lu-DOTA-H:The MTS kit was used to detect the cytotoxicity of ultrasound on ¹⁷⁷Lu-DOTA-H acting on SKBR-3.3.（1）Biological distribution of ¹⁷⁷Lu-DOTA-H:A USMB group and a control group were set up to evaluate the effect of USMB on the biological distribution of ¹⁷⁷Lu-DOTA-H in tumor bearing mice,with 15 mice in each group.Tumor bearing mice in the USMB group and control group were euthanized at 4 hours,24 hours,72 hours,120 hours,and 168 hours after the end of USMB sonication.Three tumor bearing mice were sacrificed at each time point in each group.Blood,heart,liver,spleen,pancreas,kidneys,muscles（non ultrasound sonication area,ultrasound sonication area）,bones（non ultrasound sonication area,ultrasound sonication area）,skin（non ultrasound sonication area,ultrasound sonication area）,tumors were obtained,the weight（g）and radiation dose（CPM value）of each tissue were measured by high-precision electronic scales and gamma counter,the percentage activity of injection dose per gram of tissue（%ID/g）of those samples were calculated.The%ID/g in those samples between the USMB group and the control group were compared.（2）Evaluation of anti-tumor efficacy and safety of USMB combined with ¹⁷⁷Lu-DOTA-H(USMB+¹⁷⁷Lu-DOTA-H):A control group,USMB group,¹⁷⁷Lu-DOTA-H group,and USMB+¹⁷⁷Lu-DOTA-H group were set up to evaluate the anti-tumor efficacy and safety of USMB+¹⁷⁷Lu-DOTA-H,with 5 tumor bearing mice in each group.The weight and tumor size of tumor bearing mice（every Monday and Thursday）were monitored.When the tumor bearing mice reach the predetermined endpoint of the experiment,all tumor bearing mice were euthanized,tumors were retained for therapeutic evaluation,and normal tissues and blood were retained for safety evaluation.（3）Possible mechanism of USMB opening BTB:A USMB group and a control group were set up to explore the possible mechanism of USMB opening BTB respectively,with 3 tumor bearing mice in each group.Western Blot was used to detect ZO-1,Occludin,AKT/p-AKT,PKCδ/p-PKC-δin the USMB group and control group.The interaction between ZO-1 and Occludin in the USMB group and control group were detected by co-immunoprecipitation.The distribution of ZO-1 and Occludin along vascular wall in the USMB group and control group were detected by immunofluorescence.Result1.81 patients were included in this study（38 TCHP patients and 43THP patients）.The p CR rates of the TCHP and THP groups were 44.7%（95%CI:30.2%?60.3%）and 51.2%（95%CI:36.8%?65.4%）,respectively.The incidence rates of any level of toxic reactions in the TCHP group and THP group were 100%and 95.3%,respectively.The incidence of≥grade 3 toxicity in the TCHP group was significantly higher than that in the THP group（68.4%vs 39.5%,P=0.009）.The incidence of NAT dose adjustment in the TCHP group was significantly higher than that in the THP group（26.3%vs 7.0%,P=0.039）.The incidence of weight loss in the TCHP group was numerically higher than that in the THP group（31.6%vs 18.6%,P=0.117）.The proportion of patients with NAT<6cycles in the TCHP group was significantly higher than that in the THP group（31.6%vs 4.7%,P=0.004）.The results of multivariate analysis showed that HER-2 3+（OR:4.14,95%CI:11.12?15.35）and patients with primary tumors>5cm（OR:3.035,95%CI:1.12?8.26）were significantly correlated with higher p CR rates.The proportion of patients in the TCHP group who used>30mg granisetron to control vomiting was significantly higher than that in the THP group（76.3%vs 9.3%,P<0.001）.The proportion of patients in the TCHP group who used>1500ug of recombinant human granulocyte colony-stimulating factor（GM-CSF）to elevate white blood cells was significantly higher than that in the THP group（52.6%vs 30.2%,P=0.041）.2.Ligand modification was performed on H.The LAR of DOTA-H was 4.32±0.59.Subsequently,this study successfully labeled ¹⁷⁷Lu on H(¹⁷⁷Lu-DOTA-H),and the radionuclide labeling rate and radiochemical purity of ¹⁷⁷Lu-DOTA-H reached 100%.The in vitro cytotoxicity experiment of ultrasound showed that compared with the group without ultrasound sonication,the viability of SKBR-3 was similar when the ultrasound intensity was≤4W/cm²（P>0.05）.Based on published literatures,the selection of ultrasound parameters for subsequent experiments in this study were:intensity:3 W/cm²,sonication time:5minutes,frequency:1MHz and duty cycle:50%.Under the selected ultrasound parameters,USMB did not affect the structure of H,LAR of DOAT-H,and radioactive nuclide labeling rate of ¹⁷⁷Lu-DOTA-H;USMB did not affect the ability of H and ¹⁷⁷Lu-DOTA-H targeting HER-2;USMB did not affect the antibody internalization function of SKBR-3,¹⁷⁷Lu-DOTA-H binding to SKBR-3 cells;USMB did not affect the cytotoxicity of¹⁷⁷Lu-DOTA-H on SKBR-3 cells.3.USMB significantly increased the dose of ¹⁷⁷Lu-DOTA-H in tumor at 24 hours（%ID/g:20.60%±6.19%vs 9.33%±2.00%,P<0.001）and72 hours（%ID/g:19.58%±2.28%vs 10.49%±1.57%,P<0.001）after USMB sonication;At 4 hours,24 hours,72 hours,120 hours,and 168hours after USMB sonication,the dose of ¹⁷⁷Lu-DOTA-H in the blood,heart,liver,spleen,pancreas,kidney,muscle（non ultrasound sonication area）,bone（non ultrasound sonication area）,and skin（non ultrasound sonication area）of the USMB group and the control group were similar（P>0.05）.Considering the presence of skin,muscle,and bone in the USMB sonication area,this study used the skin,muscle,and bone in the USMB ultrasound sonication area as self-control to evaluate the effect of USMB on the uptake of ¹⁷⁷Lu-DOTA-H in normal tissues.The study found that the dose of ¹⁷⁷Lu-DOTA-H in the muscle of the ultrasound sonication area was significantly higher than that in the non sonication area（5.12%±1.55%vs 2.09%±0.55%,P<0.001）at 24 hours after USMB sonication,and the dose of ¹⁷⁷Lu-DOTA-H in the muscle of the ultrasound sonication area was similar to that in the muscle of the non ultrasound sonication area at 4h,72h,120h,168h after USMB sonication（P>0.05）.USMB did not affect the uptake of ¹⁷⁷Lu-DOTA-H in skin and bones（P>0.05）.The evaluation results of anti-tumor efficacy showed that the average tumor growth curve of the USMB group was similar to that of the control group,and USMB had no tumor inhibitory effect;Compared with the control group and USMB group,¹⁷⁷Lu-DOTA-H significantly inhibited tumor growth（P<0.001）,while the USMB+¹⁷⁷Lu-DOTA-H group had a more significant tumor growth inhibition effect（P=0.011）.Meanwhile,safety evaluation experiments showed that USMB+¹⁷⁷Lu-DOTA-H did not increase the blood toxicity of tumor bearing mice（blood routine,P>0.05）,and USMB+¹⁷⁷Lu-DOTA-H did not cause damage to the heart,liver,lung,kidney,and muscle of tumor bearing mice.Compared with the control group,the total expression of ZO-1 and Occludin in the USMB group was similar（P>0.05）,but the interaction between ZO-1 and Occludin was weakened.The expression level of p-AKT protein in the USMB group was significantly higher than that in the control group（3.74±0.72 vs 1.07±0.25,P=0.004）.AKT phosphorylation inhibitor MK2206 can reverse the effect of USMB on the interaction between ZO-1 and Occludin.Conclusion1.In the Chinese population,the overall p CR rate of HER-2overexpression breast cancer patients receiving NAT standard protocol（TCHP and THP）is close to 50%,and the incidence of grade 3 to 4 serious toxicity was high.Therefore,it is urgent to find new NAT strategies to further improve the efficacy of HER-2 overexpressed breast cancer.2.USMB could open BTB,promoted the uptake of ¹⁷⁷Lu-DOAT-H in SKBR-3 breast cancer,and improved the anti-tumor efficacy of ¹⁷⁷Lu-DOAT-H.3.USMB combined with ¹⁷⁷Lu-DOAT-H was a safe and effective combined anti-tumor strategy.This study provided a new idea for exploring the combined treatment strategy of NAT for breast cancer with HER-2overexpression.