Abstract Breast cancer is the most common malignancy among women worldwide, and its recurrence, metastasis, and drug resistance remain significant challenges in systemic treatment. Metabolic alterations are a hallmark of cancer, with breast cancer cells reprogramming glucose metabolism, lipid metabolism, and amino acid metabolism to adapt their nutrient acquisition pathways. This metabolic reprogramming enables cancer cells to meet the energy demands for rapid proliferation and adaption to the microenvironment of metastatic sites, which is closely associated with resistance to chemotherapy, targeted therapies, and immunotherapies. This review summarizes the features of metabolic remodeling in breast cancer, the mechanisms by which metabolic adaptation contributes to treatment resistance, and emerging individualized strategies targeting metabolic vulnerabilities. Particular focus is placed on energy metabolism, cholesterol homeostasis, and glutamine utilization in relation to tumor invasion, metastasis, and drug resistance. A better understanding of metabolic adaptation and accurate monitoring of metabolic dynamics may offer new insights into metabolic therapies and improve clinical outcomes.

Abstract Alternative splicing(AS) is one of the important ways of post-transcriptional regulation, which can generate multiple mRNA isoforms from a single gene. In recent years, many studies have shown that AS can occur in almost all types of tumors, and the abnormal expression of splicing factors is related to the disease progression of tumor patients, which may become a potential marker for judging tumor progression. Therefore, this review summarizes the role, molecular mechanism, clinical relevance and treatment response of AS in tumors. It is found that AS can participate in the progression of malignant tumors by regulating tumor invasion, metastasis, apoptosis, cell metabolism, and promoting tumor immune escape and treatment resistance, which provides a theoretical basis for the clinical application of AS. The precise identification of tumor-specific AS and the development of small molecule inhibitors targeting specific AS isoforms may provide a new direction for precise and personalized cancer treatment.

Objective : To explore the role of semaphoring 6d(SEMA6D) in the malignant progression of triple-negative breast cancer(TNBC). Methods : Bioinformatics and Immunohistochemistry(IHC) were used to analyze the expression level of SEMA6D in TNBC and paracancer non-tumor tissues and its relationship with patients′ clinicopathological features. MDA-MB-231 cell line stably knocking down the expression of SEMA6D was constructed, and the effects of SEMA6D on migration and invasion of TNBC cells were investigated by Wound-healing assays and Transwell assays. cBioPortal and GEPIA2 databases were used to screen out the gene negatively associated with it, namely aurora kinase A(AURKA). Bioinformatics and IHC were used to analyze the expression level of AURKA in TNBC and paracancer non-tumor tissues and its relationship with patients' clinicopathological features. Western blot assay was used to analyze the expression of AURKA and the effect of epithelial-mesenchymal transition(EMT) makers Claudin-1, N-cadherin and Vimentin after knocking downSEMA6D. Results: Bioinformatics analysis and IHC results showed that the expression of SEMA6D in TNBC tissues was significantly lower than that in paracancer non-tumor tissues(bothP<0.05). The expression of AURKA in TNBC tissues was significantly higher than that in paracancer non-tumor tissues(bothP<0.05), SEMA6D and AURKA were significantly negatively correlated in TNBC(P<0.01). Both low expression of SEMA6D and high expression of AURKA were positively correlated with tumor size, tumor histological grade, clinical stage and lymph node metastasis in TNBC patients(allP<0.05). The knockdown ofSEMA6Dsignificantly promoted the migration and invasion ability of TNBC cells(bothP<0.01). Western blot results showed that the knockdown ofSEMA6Dupregulated AURKA expression, promoted the expression of N-cadherin and Vimentin, and inhibited the expression of Claudin-1 in tumor cells. Conclusion Down-regulation of SEMA6D expression in TNBC may be involved in the malignant progression of TNBC through up-regulation of AURKA expression and promotion of EMT.

Objective : To explore the expression of prohibitin2(PHB2) in breast cancer and its effect on the biological behaviors of tumor cells. Methods : Immunohistochemistry was used to detect the expression of PHB2 protein in breast cancer tissues and its relationship with clinicopathologic features. Breast cancer stable transient cell lines were constructed with knockdown and overexpression ofPHB2, respectively. The effects of PHB2 on cell proliferation, migration and invasion ability were detected by clone formation assay, scratch assay and Transwell assay. Western blot(WB) was used to detect the effects of PHB2 on the expression of epithelial-mesenchymal transition(EMT)-related markers, including E-cadherin, N-cadherin, Snail family transcriptional repressor 1(Snail) protein, Vimentin, and Claudin-1. The effect of PHB2 on tumorigenicityin vivowas detected by subcutaneous tumor formation assay in nude mice. Results: The result of immunohistochemical showed that PHB2 was highly expressed in breast cancer and the expression of PHB2 was significantly positive correlated with tumor size, human epidermal growth factor receptor-2(HER-2) status and proliferation index Ki-67 levels(P<0.05). Clone formation assay, scratch assay and Transwell assay revealed that knockdown ofPBH2significantly inhibited the proliferation, migration and invasion ability of breast cancer cells(P<0.01), while the overexpression ofPHB2significantly promoted cell proliferation, migration and invasion(P<0.01). The result of subcutaneous tumor formation experiment in nude mice revealed a significant decrease in tumor volume and weight in knockdownPHB2mice(P<0.000 1), whilePHB2overexpression tumors significantly increased in volume and weight(P<0.001).WB assay showed that the protein expression of epithelial marker E-cadherin increased, while the expressions of mesenchymal markers N-cadherin, Snail and Vimentin decreased significantly afterPHB2knockdown with them in control cells(P<0.01). The expression of Claudin-1 decreased, while the expressions of N-cadherin, Snail and Vimentin increased significantly inPHB2overexpression cells(P<0.05). Conclusion PHB2 is highly expressed in breast cancer and promotes multiple malignant biological behaviors in tumor cells, suggesting PHB2 may be a potential target for breast cancer diagnosis and treatment.

Objective : To investigate the expression of Keratin 14(KRT14) in Basal-like Breast Cancer(BLBC) and its biological functions and mechanisms. Methods : The expression levels of KRT14 mRNA in BLBC and para-cancer breast tissues were analyzed using The Cancer Genome Atlas(TCGA) database. qPCR, Western blot(WB), and immunohistochemistry were employed to detect KRT14 expression in BLBC and adjacent normal tissues, and its correlation with clinicopathological features was analyzed. KRT14 overexpression and knockdown were performed in breast cancer cells, and cell scratch and transwell assays were performed to evaluate changes in migration and invasion abilities. To investigate the expression of proteins related to the Wnt/β-catenin signaling pathway, including catenin Beta 1(β-catenin), wingless-type MMTV integration site family, member 1(Wnt1), matrix metallopeptidase 7(MMP7), and cellular myelocytomatosis viral oncogene homolog(c-Myc), as well as the cellular localization of β-catenin, WB and immunofluorescence(IF) techniques were employed. Additionally, a Wnt/β-catenin signaling pathway inhibitor was used to verify the mechanism of action of KRT14. Results : The expression of KRT14 was significantly higher in BLBC tissues compared to normal tissues(P<0.05), and was associated with higher T stage and histological grade(P<0.05). The overexpression of KRT14 significantly enhanced the migration and invasion abilities of breast cancer cells, while the knockdown of KRT14 significantly reduced those abilities(P<0.01). The overexpression of KRT14 can increase the expression levels of Wnt/β-catenin pathway-related proteins β-catenin, Wnt1, MMP7, and c-Myc, thereby activating the Wnt/β-catenin pathway. Moreover, the inhibition of this pathway can eliminate the effects of KRT14 on cell migration and invasion. Conclusion The high expression of KRT14 in BLBC may promote the migration and invasion of breast cancer cells through the Wnt/β-catenin signaling pathway.

Objective : To investigate the expression of(heat shock protein a member 8,HSPA8) in breast cancer and its effect on tumor biological behaviors. Methods: Bioinformatics analysis and immunohistochemistry assays were used to detect the expression of HSPA8 in breast cancer and adjacent non-tumor breast tissues,and the relationship between its expression and clinicopathological characteristics of breast cancer patients was analyzed.Correlation between HSPA8 expression and prognosis of breast cancer patients was analyzed by Kaplan-Meier Plotter database.HSPA8 knockdown and over expression breast cancer stabilized cells were constructed,respectively.CCK-8,clone formation,Transwell,cell scratch,Western blot and immunofluorescence assay were used to detect the effects of HSPA8 on the proliferation,invasion and migration of breast cancer cells,and its effect on epithelial-mesenchymal transition(EMT). Results : Bioinformatics analysis and immunohistochemistry assay revealed that the expression of HSPA8 in breast cancer tissues was higher than that in adjacent non-tumour tissues(P<0.05),and its expression level of the protein was significantly and positively correlated with the tumor size,histological grade,lymph node metastasis and Ki-67 proliferation index(P<0.05).The Kaplan-Meier survival curve showed that high expression of HSPA8 was significantly associated with poor prognosis in breast cancer patients(P<0.05).CCK-8,clone formation,transwell,cell scratch,Western blot and immunofluorescence assay showed that knockdown of HSPA8 expression could significantly inhibit the proliferation,invasion,migration function and EMT of breast cancer cells(P<0.05),while overexpression of HSPA8 could significantly promote the proliferation,invasion,migration function and EMT of breast cancer cells(P<0.05). Conclusion HSPA8 is highly expressed in breast cancer tissues,which is closely related to disease progression and the malignant phenotype of breast cancer,suggesting that HSPA8 may be a potential biological target for breast cancer treatment.

Objective: To simulate the tumor microenvironment though the 3D microtumor model which was constructed using droplet microfluidics.To explore its feasibility as a model for in vitro breast cancer research through3D microtumour fabrication,characterisation and sensitivity testing to chemotherapeutic drugs. Methods: Breast cancer cells were encapsulated with hydrogel shells in collagen-rich microencapsulated cores to obtain breast cancer microtumours in vitro;breast cancer microtumours were co-cultured with 3D microencapsulated endothelial cells by Transwell system.The structure and growth characteristics of the microtumours were directly observed by microscopy;the CCK-8 assay was used to detect the proliferation of the cells under different culture models and the drug sensitivity to doxorubicin;flow cytometry was used to compare the differences in apoptosis during the proliferation process;and the differences in the migratory and invasive abilities of the cells were assessed by scratch assay and Transwell assay;the expression of epithelial-mesenchymal transition-related proteins was detected by Western blot. Results: Breast cancer cells grew well in hydrogel nucleus-shell microcapsules;cell proliferation assays showed that 3D culture and 3D co-culture cells proliferated at a significantly lower rate than 2D culture;3D culture and 3D co-culture cells had enhanced migration and invasion ability and showed higher expression of EMT-related proteins compared to 2D culture;3D culture and 3D co-culture cells were significantly less sensitive to chemotherapeutic drugs compared to 2D culture.The sensitivity of 3D and 3D co-cultured cells to chemotherapeutic drugs was significantly reduced compared to 2D culture. Conclusion 3D cultures show similar morphology and biology to in vivo tumours and are more resistant to chemotherapeutic agents.

Purpose To explore the expression of autoph-agy-related 7(ATG7)in breast cancer and its effect on the breast cancer development.Methods Immunohistochemistry(IHC)was used to detect ATG7 protein expression in breast cancer tissues and the relationship between ATG7 and clinico-pathological features was analyzed.ShRNA was used to interfere with the expression of ATG7 in breast cancer cell line MCF-7.Puromycin was used to screen for stably transfected cells and Western blot was used to detect transfection efficiency.The effect of ATG7 knockdown cells on proliferation ability was de-tected by CCK8 and clone formation experiments.The effect of ATG7 knockdown cells on tumorigenicity in vivo was detected by subcutaneous tumor formation experiment in nude mice.Results IHC showed that ATG7 expression in breast cancer tissues was mainly localized in cytoplasm,and its expression was significant-ly correlated with tumor size and Ki67 expression(P＜0.05).ATG7-shRNA significantly interfered with ATG7 expression in breast cancer cells MCF-7.CCK8 and clone formation experi-ments showed that ATG7 knockdown promoted the cell prolifera-tion compared with the control group.The experiment of subcu-taneous tumor formation in nude mice showed that the tumor for-mation ability of mice was significantly increased after ATG7 knockdown compared with the control group.Conclusion ATG7 may inhibit the proliferation capacity of breast cancer and could be a potential target for breast cancer therapy.

Objective To analyze the medication law of Professor Yuan Jinsheng,a renowned TCM practitioner in China,for treating palpitations through data mining methods.Methods Clinical prescriptions for treating palpitations by Professor Yuan Jinsheng from January 2016 to May 2023 were collected.The prescriptions were screened based on inclusion and exclusion criteria,a prescription compatibility network was constructed based on R Studio 4.3.1,and the medication law of prescriptions was analyzed.Results Totally 331 prescriptions were screened,involving 180 types of Chinese materia medica,with a total frequency of 3 625.The most frequently used drugs(≥30 times)were mainly tonics.The main properties were warm and neutral,the main tastes were sweet,bitter,and pungent,which belonged to heart,spleen and lung meridians.The top 5 drugs with high correlation were tonifying,blood circulation-activating and stasis-resolving,qi-regulating,and heat-clearing.Correlation analysis reveals high-frequency drugs,which were mainly Glycyrrhizae Radix et Rhizoma Praeparata cum Melle,Rehmannine Radix,Ophiopogonis Radix,Astragali Radix and Codonopsis Radix.The clustering analysis results showed that the efficacy was mainly tonifying deficiency,regulating qi,activating blood circulation,and resolving stasis.Conclusion Professor Yuan Jinsheng's prescription compatibility for treating palpitations primarily focuses on tonics,qi-regulating,and blood circulation-activating and stasis-resolving herbs,embodying the principles of treating palpitations from the perspective of the heart and spleen and the combined use of multiple organs.

Purpose To investigate the expression of PD-1,PD-L1,CD3 and CD8 in the immune microenvironment of non-small cell lung cancer(NSCLC)and their clinical signifi-cance.Methods The clinical data of 39 patients with NSCLC brain metastasis(BM)were collected.The expression of PD-1,PD-L1,CD3 and CD8 in the tumor and stroma of BM was detec-ted using an immunofluorescence-based tissue microenvironment analysis panel.Targeted sequencing was carried out to catalog cancer-related genes.The clinical pathological features were an-alyzed with review of relevant literature.Results Thity-nine patients with NSCLC presented with tumor-infiltrating lympho-cytes(TIL)in different degree.CD3+TIL(P=0.000 7)and CD8+TIL(P=0.0006)were more prominent in the tumor stroma,and the positive PD-L1 expression was significantly higher in the interstitial tissues of tumor(P=0.025 8).Com-pared with the whole wild-type driver gene cohort,the expres-sion of PD-L1 in the stroma of BM was significantly increased in the EGFR mutation cohort(P=0.039).Patients with high in-filtration of stroma CD8+TIL had longer median overall survival than those with low infiltration(16 months vs 6 months,P=0.032);PD-L1-positive patients(36 months)were longer sur-vival time than PD-L1-negative patients(5.5 months,P=0.056).Compared with lung primary lesions,the variant allele frequencies(VAFs)in BM generally increased,and samples with higher VAFs corresponded to higher expression of PD-1,PD-L1,CD3 and CD8.Conclusion The TIL infiltration is most prominent in the stroma of NSCLC BM.The EGFR muta-tion of a tumor might affect the immune microenvironment of me-tastases;PD-L1 expression and TIL infiltration were correlated with overall survival.