Correlation Of Some Glucose Metabolism-related Genes In The Efficacy Of Neoadjuvant Therapy For Triple-positive Breast Cancer

Background:Breast cancer is a highly heterogeneous malignancy,which can be classified into LuminalA,LuminalB,HER-2 overexpressing and basal-like types according to its molecular typing,among which there is a special subtype-Triple positive type in LuminalB.For hormone receptor-positive or HER-2-positive breast cancer alone,endocrine therapy and targeted therapy can be chosen,respectively,but the outcome and prognosis of triple-positive breast cancer with both hormone receptor-positive and HER-2 overexpression are much less than other types after receiving endocrine therapy combined with anti-HER-2 targeted therapy.Cancer metabolism as an emerging perspective has set off a boom in oncology research,and a growing number of studies have demonstrated a clear correlation between metabolism and neoadjuvant treatment access to PCR and survival prognosis in breast cancer patients.However,most previous studies were based on whole sample genetic testing to assess tumor metabolism.With the advent of single-cell sequencing technology,which can clarify the gene expression status of individual cells and intercellular heterogeneity,even the same type of cells can also be divided into different subpopulations,and different subpopulations may also differ in metabolism,and the distribution and proportion of different cell subpopulations respond to treatment or drug resistance.We attempted to analyze at the cell subpopulation level with the help of single-cell sequencing to investigate the impact of metabolic differences in different cell subpopulations on neoadjuvant therapy resistance in triple-positive breast cancer.Objective:Single-cell sequencing was used to identify different cell subpopulations in the epithelial cells of triple-positive breast cancer and further investigate the relationship between the metabolism of these subpopulations and the efficacy and prognosis of neoadjuvant therapy for triple-positive breast cancer.In this study,8 tissue specimens of triple positive breast cancer treated with standard neoadjuvant therapy(4 neoadjuvant sensitive and 4 neoadjuvant insensitive)were screened and analyzed by single cell sequencing.cellranger 3.0 was processed and the data were quality controlled(quality control criteria were nFeature>300,nCount>400,MT<25,batch removal by seurat cca effect),the corresponding single-cell expression profiles were obtained.The assay results were normalized,t-SNE downscaled and visualized.Cell populations were annotated with cell types according to CellMarker,PanglaoDB and other databases.The epithelial cell populations were then extracted and subdivided into their subgroups after re-performing the same clustering analysis.GO functional enrichment analysis and KEGG pathway enrichment analysis of differentially expressed genes was performed using the clusterProfiler package,and finally cellular metabolism of single cell sequencing results was analyzed using the scMetabolism package.Survival analysis of differential genes on metabolic pathways was performed using KM-Plotter in the public database(Logrank test was used for survival analysis and P<0.05 was considered a statistically significant difference)to screen for target genes with significant effects on OS and RFS in breast cancer patients.A retrospective analysis of 66 triple-positive breast cancer patients who received standard neoadjuvant therapy at our breast surgery department from January 2018 to January 2023 was performed to record their underlying clinical case information.The efficacy of neoadjuvant therapy was assessed based on the patients’ comprehensive assessment from the first visit,the last preoperative imaging findings(ultrasound and breast MRI),puncture pathology,and postoperative pathology,and the efficacy of PCR and PR was defined as the treatment-sensitive group,and the efficacy of SD and PD was defined as the treatment-insensitive group.Breast cancer tissue specimens before and after chemotherapy were selected from patients in the sensitive and insensitive groups,immunohistochemical staining of target genes was performed to detect their expression in both groups,and the correlation between target protein expression levels and treatment efficacy was analyzed by chi-square test and Fisher exact test.Results:1.78,750 cells were obtained by single cell sequencing.Based on Marker gene analysis,eight cell types were identified,including fibroblasts,T cells,myeloid cells,epithelial cells,endothelial cells,B cells,macrophages and plasma cells.The epithelial cell population could be divided into 8 subgroups,namely cluster0-7,and different subgroups had their own metabolic characteristics,among which cluster0 was more predominant in the drug-resistant group.The GO and KEGG enrichment analysis of all differential genes revealed that the differential genes were mainly enriched in the metabolic pathways of mitochondrial oxidative phosphorylation,glycolysis/gluconeogenesis,and tricarboxylic acid cycle;similar results were also obtained by scMetabolism package for the metabolic analysis of cells in sensitive and non-sensitive groups.2.In this study,36 differential genes related to metabolism were screened from all differential genes.Using the KM-Plotter database,the OS of PGK1(HR=1.47(1.22-1.78),logrankP=5.5e-05<0.05).the RFS of PGK1(HR=1.42(1.28-1.57),logrankP=1.4e-11<0.05).the OS of ENO1(HR=1.2(0.99-1.45),logrankP=0.059>0.05).the RFS of ENO1(HR=1.19(1.08-1.32),logrankP=0.00061<0.05).the OS of NDUFAB1(HR=1.16(0.96-1.4),logrankP=0.13>0.05),the RFS of NDUFAB1(HR=1.26(1.14-1.4),logrankP=6.8e-0.6<0.05).the OS for MDH2(HR=1.38(1.15-1.67),logrankP=0.00071<0.05).the RFS of MDH2(HR=0.79(0.71-0.87),logrankP=3.7e-06<0.05).3.By immunohistochemical staining,it was found that the immunohistochemical staining of PGK1,ENO1,NDUFAB1,MDH2,the proportion of low expression was significantly increased after neoadjuvant therapy,but had no significant difference in the proportion of distribution of both before neoadjuvant therapy.The protein expression levels of PGK1,ENO1,NDUFAB1,MDH2,were significantly different between sensitive and non-sensitive groups after treatment(P<0.05).By analysis of the basic clinical data,it was found that there were statistically significant differences between the sensitive and non-sensitive groups at different clinical stages(P=0.001<0.05),but there were no significant differences with age,menstrual status,body mass index,HER-2,ER,PR,Ki-67(P>0.05).Conclusions:Tri-positive breast cancer showed metabolic changes before and after neoadjuvant therapy,mainly in the form of increased activity of metabolic pathways such as glycolysis/glycogenesis,mitochondrial oxidative phosphorylation,and tricarboxylic acid cycle.After neoadjuvant therapy,high expression of key proteins of metabolic pathways PGK1,ENO1,NDUFAB1 and MDH2was negatively correlated with drug sensitivity.