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Objectives: To observe the mitochondrial morphology of normal and triple-negative breast cancer cells, extract mitochondria from normal cells, and investigate the effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of triple-negative breast cancer cells. Methods: The morphology of mitochondria was observed by transmission electron microscope. Mitochondria were extracted by mitochondrial extraction kit, mitochondrial protein was identified by western blot, and mitochondrial activity was detected by mitochondrial membrane potential detection kit. MitoTracker Green or MitoTracker Deep Red fluorescent probes were used to label the mitochondria of living cells, and the degree of mitochondria entering LTT cells was observed by confocal laser microscopy at 12, 24, and 96 hours. The effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of breast cancer cells were examined by CCK8, colony formation assay, flow cytometry, and sphere formation assay after 24 hours of mitochondrial transplantation. Results: The mitochondria of normal cells were rod-shaped or elongated, while the mitochondria of triple-negative breast cancer cells were swollen and vacuolated. Western blot results showed that cytochrome c oxidase subunit I (MT-CO1) protein encoded by mitochondria was present in the isolated mitochondria. The content of heat shock protein 60 (HSP60) was higher in mitochondria than that in cytoplasm. The result of the multi-mode microplate reader showed that the content of mitochondrial J-aggregates/monomer was 1.67±0.06, which was significantly higher than 0.35±0.04 of the control group ( P ＜0.001). Exogenous mitochondria were observed in LTT cells at 12, 24, and 96 hours after mitochondrial transplantation. The results of the CCK8 experiment showed that OD450 of LTT cells was 0.27±0.13 after 48 hours transplantation, which was lower than 0.62±0.36 of the control group ( P =0.023). The OD450 of MDA-MB-468 cells was 0.30±0.03, which was lower than 0.65±0.10 of the control group ( P =0.004). After 120 hours of mitochondrial transplantation, OD450 in both groups was still significantly lower than that in the control group (P ＜0.01). The number of clones formed by mitochondrial transplantation of LTT cells was 21.33±7.31, which was lower than 35.22±13.59 of the control group ( P =0.016). Flow cytometry showed that the early apoptosis rate of LTT cells was (30.07±2.15)% after 24 hours of mitochondrial transplantation, which was higher than 2.07±1.58 of the control group ( P ＜0.001). The proportion of early apoptosis in MDA-MB-468 cells was 24.47%±5.22%, which was higher than (7.83±2.06)% in the control group ( P =0.007). In addition, the number of mitochondria transplanted LTT cells into the cell sphere was 46.25±5.40, which was significantly lower than 62.58±6.43 of the control group ( P ＜0.001). Conclusion: Normal mitochondria can enter triple-negative breast cancer cells by co-culture, inhibit the proliferation and stemness of triple-negative breast cancer cells, and promote the apoptosis of triple-negative breast cancer cells.

Background: Antibody-based platforms ( i.e. , ADC) have emerged as one of the most encouraging tools for the cancer resistance caused by cancer stem cells (CSCs) enrichment. Our study might provide a promising therapeutic direction against drug resistance and serve as a potential precursor platform for screening ADC., Methods: The cell migration, invasion, drug resistance, and self-renewal were assessed by the cell invasion and migration assay, wound healing assay, CCK-8 assay, colony formation assay, and sphere formation assay, respectively. The expression profiles of CSCs (ALDH + and CD44 + ) subpopulations were screened by flow cytometry. The western blot and cell immunofluorescence assay were used to evaluate pathway-related protein expression in both anti-ENO1 antibody, MET combined with DPP/CTX-treated CSCs., Results: In the present study, western blot and flow cytometry verified that anti-ENO1 antibody target the CD44 + subpopulation by inhibiting the PI3K/AKT pathway, while metformin might target the ALDH + subpopulation through activation of the AMPK pathway and thus reverse drug resistance to varying degrees. Subsequently, in vitro investigation indicated that anti-ENO1 antibody, metformin combined with cisplatin/cetuximab could simultaneously target ALDH + and CD44 + subpopulations. The combination also inhibited the CSCs proliferation, migration, invasion, and sphere formation; which may result in overcoming the drug resistance. Then, molecular mechanism exploration verified that the anti-ENO1 antibody, metformin combined with cisplatin/cetuximab inhibited the Wnt/β-catenin signaling., Conclusions: The study preliminarily revealed anti-ENO1 antibody combined with metformin could overcome drug resistance against CSCs by inhibiting the Wnt//β-catenin pathway and might serve as a potential precursor platform for screening ADC. More importantly, it is reasonably believed that antibody-based drug combination therapy might function as an encouraging tool for oncotherapy., Competing Interests: The authors declare that they have no competing interests., (© 2024 Shu et al.)

Objective: To investigate the diagnostic efficiency of conventional serum tumor markers and their combination with chest CT for stage ⅠA lung cancer. Methods: A total of 1 155 patients with stage ⅠA lung cancer and 200 patients with benign lung lesions (confirmed by surgery) treated at the Cancer Hospital, Chinese Academy of Medical Sciences from January 2016 to October 2020 were retrospectively enrolled in this study. Six conventional serum tumor markers [carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), squamous cell carcinoma associated antigen (SCCA), cytokeratin 19 fragment (CYFRA21-1), neuron-specific enolase (NSE), and gastrin-releasing peptide precursor (ProGRP)] and chest thin-slice CT were performed on all patients one month before surgery. Pathology was taken as the gold standard to analyze the difference of positivity rates of tumor markers between the lung cancer group and the benign group, the moderate/poor differentiation group and the well differentiation group, the adenocarcinoma group and the squamous cell carcinoma group, the lepidic and non-lepidic predominant adenocarcinoma groups, the solid nodule group and the subsolid nodule group based on thin-slice CT, and subgroups of ⅠA1 to ⅠA3 lung cancers. The diagnostic performance of tumor markers and tumor markers combined with chest CT was analyzed using the receiver operating characteristic curve. Results: The positivity rates of six serum tumor markers in the lung cancer group and the benign group were 2.32%-20.08% and 0-13.64%, respectively; only the SCCA positivity rate in the lung cancer group was higher than that in the benign group (10.81% and 0, P =0.022). There were no significant differences in the positivity rates of other serum tumor markers between the two groups (all P >0.05). The combined detection of six tumor markers showed that the positivity rate of the lung cancer group was higher than that of the benign group (40.93% and 18.18%, P =0.004), and the positivity rate of the adenocarcinoma group was lower than that of the squamous cell carcinoma group (35.66% and 47.41%, P =0.045). The positivity rates in the poorly differentiated group and moderately differentiated group were higher than that in the well differentiated group (46.48%, 43.75% and 22.73%, P =0.025). The positivity rate in the non-lepidic adenocarcinoma group was higher than that in lepidic adenocarcinoma group (39.51% and 21.74%, P =0.001). The positivity rate of subsolid nodules was lower than that of solid nodules (30.01% vs 58.71%, P =0.038), and the positivity rates of stageⅠA1, ⅠA2 and ⅠA3 lung cancers were 33.33%, 48.96% and 69.23%, respectively, showing an increasing trend ( P =0.005). The sensitivity and specificity of the combined detection of six tumor markers in the diagnosis of stage ⅠA lung cancer were 74.00% and 56.30%, respectively, and the area under the curve (AUC) was 0.541. The sensitivity and specificity of the combined detection of six serum tumor markers with CT in the diagnosis of stage ⅠA lung cancer were 83.0% and 78.3%, respectively, and the AUC was 0.721. Conclusions: For stage ⅠA lung cancer, the positivity rates of commonly used clinical tumor markers are generally low. The combined detection of six markers can increase the positivity rate. The positivity rate of markers tends to be higher in poorly differentiated lung cancer, squamous cell carcinoma, or solid nodules. Tumor markers combined with thin-slice CT showed limited improvement in diagnostic efficiency for early lung cancer.

Background: Identification of promising targeted antigens that exhibited cancer-specific expression is a crucial step in the development of novel antibody-targeted therapies. We here aimed to investigate the anti-tumor activity of a novel monoclonal antibody (mAb) 11C9 and identify the antibody tractable target in the hepatocellular cancer stem cells (HCSCs)., Methods: The identification of the targeted antigen was conducted using SDS-PAGE, western blot, mass spectrometry, and co-immunoprecipitation. Silence of HSP90 was induced by siRNA interference. Positive cells were sorted by fluorescence-activated cell sorting. Double-immunofluorescent (IF) staining and two-color flow cytometry detected the co-expression. Self-renewal, invasion, and drug resistance were assessed by sphere formation, matrigel-coated Transwell assay, and CCK-8 assay, respectively. Tumorigenicity was evaluated in mouse xenograft models. RNA-seq and bioinformatics analysis were performed to explore the mechanism of mAb 11C9 and potential targets., Results: MAb 11C9 inhibited invasion and self-renewal abilities of HCC cell lines and reversed the cisplatin resistance. HSP90 (~ 95 kDa) was identified as a targeted antigen of mAb 11C9. Tissue microarrays and online databases revealed that HSP90 was overexpressed in HCC and associated with a poor prognosis. FACS and double-IF staining showed the co-expression of HSP90 and CSCs markers (CD90 and ESA). In vitro and in vivo demonstrated the tumorigenic potentials of HSP90. The inhibition of HSP90 by siRNA interference or 17-AAG inhibitor both decreased the number of invasion, sphere cells, and CD90 + or ESA + cells, as well as reversed the resistance. Bioinformatics analysis and western blot verified that HSP90 activated Wnt/β-catenin signaling., Conclusions: The study preliminarily revealed the anti-tumor activity of mAb 11C9. More importantly, we identified HSP90 as a targeted antigen of mAb 11C9, which functions as an oncogene in phenotype shaping, stemness maintenance, and therapeutic resistance by activating Wnt/β-catenin signaling., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Cancer stem cells (CSCs) are responsible for drug resistance, cancer relapse, and metastasis. Here, we report the first analysis of Palladin expression and its impacts on stem cell-like properties in lung cancer., Methods: Tissue microarrays were used to investigate Palladin expression and its association with prognosis. Immunofluorescence (IF), flow fluorescence assay, and Western blot were performed to detect Palladin expression in 6 NSCLC cell lines. Cell phenotypes and drug resistance were evaluated. Xenograft models were constructed to confirm the role of Palladin in vivo., Results: By using the tissue microarrays, Palladin was identified to be highly expressed in the cytoplasm, specifically in the cytomembrane of NSCLC, and its high expression is associated with poor prognosis. Palladin is widely expressed and enriched in the sphere cells. The in vitro and in vivo studies showed that Palladin promoted stem cell-like properties, including cell viability, invasion, migration, self-renewal abilities, taxol resistance, and tumorigenicity. Western blot revealed that Palladin promoted the accumulation of β-catenin and activated Wnt/β-catenin signaling. Tissue microarrays analysis further confirmed the positive correlation between Palladin and β-catenin. Wnt/β-catenin pathway inhibitor blocked the Palladin-induced enhancement of sphere-forming., Conclusions: Palladin might act as an oncogene by promoting CSCs-like properties and tumorigenicity of NSCLC cells via the Wnt/β-catenin signaling pathway. Besides, Palladin was identified to have the potential as a cell surface marker for LCSCs identification. These findings provide a possible target for developing putative agents targeted to LCSCs., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)