| BackgroundBreast cancer has surpassed lung cancer as the most common cancer among women,accounting for 30% of all female cancer cases.Of these cases,20-25% are positive for human epidermal growth factor receptor 2(HER2),which often indicates a faster disease progression and poorer prognosis.Trastuzumab is a first-line treatment for this type of breast cancer and plays an irreplaceable role in both postoperative targeted therapy and neoadjuvant therapy.The anti-tumor mechanism of trastuzumab mainly includes three aspects: inhibiting the heterodimerization or homodimerization of HER proteins on the cell surface,thus inhibiting downstream HER2 signaling at the source;inhibiting the activity of proliferation-related signaling pathways in tumor cells;and mediating the antibody-dependent cell-mediated cytotoxicity(ADCC)of immune cells in the tumor microenvironment,with natural killer(NK)cells being the primary immune cells involved in this process in HER2-positive breast cancer.However,many patients develop resistance within one year of treatment,and the efficacy for advanced patients with metastases is minimal.Therefore,exploring the mechanism of trastuzumab resistance and finding new ways to improve efficacy are of great significance.Currently,research on trastuzumab resistance mainly focuses on the inability of the drug to effectively bind due to HER2 loss on the surface of tumor cells,which leads to ineffective downstream pathway inhibition,as well as estrogen receptor signal interference or PTEN gene mutations leading to abnormal activation of downstream HER2 signaling pathways.In fact,in addition to these factors at the tumor cell level,changes in the tumor immune microenvironment are also an important factor leading to trastuzumab resistance.Literature has suggested that HER2-positive breast cancer is a typical "cold tumor," with fewer and weaker immune cells in the tumor microenvironment,which may reduce the efficacy of trastuzumab through ADCC.However,there is little research on whether trastuzumab resistance is related to ADCC and whether intervening in the number and activity of NK cells in the tumor microenvironment to enhance ADCC efficacy can improve the efficacy of trastuzumab.AimsThis study aims to clarify the relationship between the intensity of ADCC in the tumor microenvironment of HER2-positive breast cancer and the efficacy of trastuzumab treatment.We will validate whether increasing the number and function of NK cells in the tumor microenvironment can enhance ADCC and improve the efficacy of trastuzumab treatment.Furthermore,we will design in vivo interventions using nanomaterials to provide new directions and insights for improving the therapeutic efficacy of trastuzumab in HER2-positive breast cancer,as well as to lay a theoretical foundation for improving the prognosis of HER2-positive breast cancer patients.Methods1.Collect clinical case data of HER2-positive breast cancer patients who resistant to trastuzumab in,and analyze HER2 immunohistochemical results before and after treatment.Then use single-cell sequencing to generate a tumor microenvironment map for trastuzumab-resistant and-sensitive breast cancer patients and verify the relationship between NK cell activity and their ADCC effect with trastuzumab resistance.2.Screen and synthesize peptides that activate NK cells without affecting their ADCC process,assess peptide purity using high-performance liquid chromatography,and determine the peptide’s molecular weight using mass spectrometry.Then conduct Transwell migration experiments to determine if the IP-10 p peptide effectively attracts NK cells,and use flow cytometry to determine if the TKD peptide effectively activates NK cell cytotoxicity.Next,perform Calcein release assays to determine if the TKD peptide effectively enhances NK cell ADCC effect.Finally,real-time monitor through Ju LI Stage living cell imaging system if the TKD peptide can effectively enhance the anti-tumor effects of trastuzumab by increasing NK cell functional activity and ADCC effect in vitro.3.Design nanomaterials to effectively deliver two peptides in vivo,with characteristics including specific targeting of HER2,spontaneous release at the tumor site,and effective protection of the peptides.At last,conduct in vivo experiments to verify if the nano delivery system can attract NK cells to the tumor site,enhance NK cell activity,improve ADCC effect,and ultimately improve the therapeutic effect of trastuzumab in mice with a complex TME.Results1.By analyzing the tumor characteristics of HER2-positive breast cancer patients who developed resistance to neoadjuvant trastuzumab treatment,it was found that more than half of the patients’ resistance was not due to downregulation of HER2 expression in the tumor itself.Obtain samples from such patients for single-cell sequencing,draw the TME map of sensitive patients and resistant patients respectively and found that the functional activity of NK cells in the resistant breast cancer tumor microenvironment was decreased.Further,through bioinformatics analysis,we found that the activity,proliferation ability and biological process mediated by NK cells in TME of trastuzumab resistant breast cancer are inhibited.2.Peptides IP-10 p and TKD were selected for their chemotactic and activating effects on NK cells,respectively.Their purities were greater than 94% as determined by high-performance liquid chromatography,and their molecular weights as determined by mass spectrometry were consistent with their theoretical values,so peptide synthesis is successful.Transwell migration assays demonstrated that the peptide IP-10 p had a clear chemotactic effect on NK cells,and flow cytometry assays showed that the peptide TKD enhanced NK cell cytotoxicity so IP-10 p and TKD are suitable NK cell immune activators.Then,Calcein AM assays confirmed that the peptide TKD had a clear enhancing effect on ADCC.Finally,real-time monitor through Ju LI Stage living cell imaging system demonstrated that the peptide TKD enhanced ADCC efficacy,thereby improving the anti-tumor effect of trastuzumab in vitro.3.Acid-sensitive liposome nanomaterials targeting HER2-positive breast cancer were successfully constructed for the delivery of peptides TKD and IP-10 p to the tumor site.In addition,hydrodynamic size and zeta potential measurements indicated appropriate material size,and transmission electron microscopy showed normal morphology and size of 100-200 nm.At last,animal experiments showed that using nano-materials to recruit NK cells and improve NK cell killing activity can enhance the ADCC effect in the TME effectively improved the efficacy of trastuzumab.Conclusion1.Through single-cell RNA sequencing,it was found that NK cell function and ADCC effect activity in the TME of patients whose HER2 expression remained unchanged before and after treatment were closely related to trastuzumab resistance.2.In vitro cellular experiments have demonstrated that the peptide IP-10 p has a chemotactic effect on NK cells,while the peptide TKD has an activating effect on NK cells and can further activate NK cell ADCC effect.Enhancing ADCC effect in vitro can improve the anti-tumor effect of trastuzumab.3.In vivo experiments with trastuzumab-resistant HER2-positive tumors in animal models have demonstrated that the peptides TKD and IP-10 p,which were targeted to the tumor site by nanomaterial delivery,effectively exert their respective NK cell activation and chemotactic effects.The number of NK cells in the tumor microenvironment increased,NK cell activation improved,and ADCC effect was enhanced,significantly improving the efficacy of trastuzumab treatment in mice. |
