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.

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.

[摘 要] 目的：探索抗HSP90单克隆抗体28C10通过靶向肿瘤干细胞促进顺铂（cisplatin，DDP）对人胃癌细胞PAMC82恶性生物学行为的抑制效果及其可能的作用机制。方法: 28C10单独或与DDP联合处理人胃癌细胞PAMC82，采用不同实验方法检测该细胞的无血清成球能力、迁移和侵袭能力与克隆形成能力，CCK-8法检测28C10对PAMC82细胞恶性生物学行为和协同DDP抗癌能力的影响。采用细胞免疫荧光及流式细胞术检测PAMC82细胞中HSP90及eHSP90（extracellular HSP90）的表达、定位、eHSP90+亚群比例，以及28C10处理后对ALDH+、CD44+、eHSP90+细胞亚群的影响。采用WB实验检测28C10作用后PAMC82细胞中HSP90、干性相关蛋白以及PI3K/AKT/mTOR信号通路蛋白表达的变化。结果：胃癌细胞PAMC82膜表面表达eHSP90，具有2%~3%的eHSP90+细胞亚群，且eHSP90+细胞多为与ALDH+或CD44+共阳性细胞。28C10处理能显著抑制PAMC82细胞的成球、克隆形成、增殖、耐药、迁移及侵袭能力，而且和DDP联用的效果更明显（P<0.05或P<0.01）。流式细胞术分析发现28C10处理显著抑制PAMC82细胞的eHSP90+、ALDH+和CD44+亚群数量（均P<0.01）。免疫荧光实验发现28C10作用后eHSP90发生内吞，WB实验结果显示eHSP90、CD44、ALDH和干性相关蛋白OCT4、SOX2表达量均降低（P<0.05或P<0.01）。结论：抗HSP90单克隆抗体28C10可靶向胃癌PAMC82细胞的ALDH+、CD44+肿瘤干细胞相关亚群、内化eHSP90且降低细胞总HSP90的水平、抑制PI3K/AKT/mTOR信号通路，从而有效地抑制PAMC82细胞的干性、耐药和其他恶性生物学行为，协同DDP显著提高抗癌效果。

[Abstract] Objective: To explore the effect of anti-ENO1 (enolase 1) antibody and metformin (MET) treatment on the proliferation, migration, invasion and stemness of cetuximab (CTX) -resistant non-small cell lung cancer (NSCLC) cells through targeting cancer stem cells and the possible mechanism. Methods: 10 mmol/L MET combined with 40 μg/ml anti-ENO1 antibody was used to treat CTX(35 µg/ml)-resistant NSCLC A549 cells for 4 d, and the effects of combined treatment on A549 cells were detected with proliferation experiment, colony formation assay, migration and invasion experiments and methylcellulose ball formation experiment. In the meanwhile, FCM was used to detect the effects of CTX, MET and anti-ENO1 antibody single-drug treatment as well as the three-drug combination treatment on ALDH+ and CD44+ lung cancer stem cell subsets. Results: CTX combined with MET and anti-ENO1 antibody treatment significantly inhibited the proliferation, migration, invasion and self-renewal capacity of A549 cells. FCM analysis found that MET could significantly inhibit ALDH+ stem cell subpopulations, while anti-ENO1 antibody could significantly inhibit CD44+ stem cell subpopulations, and the three-drug combination treatment could simultaneously suppress ALDH+ and CD44+ stem cell subpopulations. Conclusion: MET and anti-ENO1 antibody respectively target ALDH+ and CD44+ cancer stem cell subsets, and the combined treatment of MET and anti-ENO1 antibody can effectively reverse the resistance of A549 cells to CTX, and thereby more effectively inhibiting stemness, proliferation, metastasis of A549 cells and tumor recurrence.

[摘 要] 具有独特的分子表达、表面标志物、干性相关信号通路和代谢模式等方面特征的肿瘤干细胞（cancer stem cell, CSC）因其具有高致瘤、高转移、高治疗抵抗能力，可能是多种类型恶性肿瘤生长、转移、治疗抵抗的关键因素，也是肿瘤发生和复发的重要根源。正常干细胞在产生了第一个致癌突变之后将逐步发展成为癌前干细胞和CSC，随后在突变和微环境的共同作用下进一步积累突变增加异质性，并与CSC可塑性转变交织在一起推动肿瘤的发生和进展，促进肿瘤的复发、转移及治疗抵抗。为了更好地治疗肿瘤，现已研发了多种类型的靶向CSC的治疗策略，包括靶向CSC的细胞表面标志物、信号转导途径、微环境、代谢模式等，以及促CSC分化、靶向CSC的免疫治疗等其他策略。多个靶向CSC治疗肿瘤的新药在临床试验中已经展现出良好的治疗效果，然而，也有一些抗肿瘤新药的失败为未来研发提供了值得注意的教训。未来肿瘤治疗中，特异地靶向患者肿瘤中所有异质性的CSC，并同时清除癌前干细胞和子代肿瘤细胞，将会更好地抑制肿瘤生长、转移和复发，从而为治愈肿瘤带来新的希望。

[Abstract] Objective: To investigate the effect of 18H12, a functional monoclonal antibody that can target gastric cancer stem cells, on the self-renewal and invasion ability of gastric cancer cells. Methods: The gastric cancer cell line PAMC-82 was used as cell model, the expression of ENO1 (enolase-1) on the membrane surface of its parental cells and enriched stem cells by sphere culture was detected by Flow cytometry. Flow cytometry was used to separate ENO1+ cells and ENO1- cells to detect their self-renewal ability and invasion ability. With the commercial ENO1 antigen and antibody as the samples, CoIP (co-immunoprecipitation) was used to verify whether 18H12 antibody targeting ENO1 could able to accurately recognize ENO1. After being treated with 18H12 for 12 h, 24 h and 48 h, the selfrenewal and invasion ability of PAMC-82 cells were detected by methylcellulose pelletization experiment and Transwell chamber assay, respectively. Results: Flow cytometry showed that the expression of ENO1 on the membrane surface of PAMC-82 sphere cells was significantly higher than that of its parental cells (P<0.01), so ENO1 could be a potential target for targeting gastric cancer stem cells. The self-renewal ability and invasion ability of the sorted ENO1+ cells were significantly stronger than those of the ENO1- cells and the parental cells (P<0.05 or P<0.01). 18H12 antibody could accurately recognize ENO1, which was consistent with the commercial antibody recognition band. 18H12 could significantly inhibit self-renewal ability and invasion ability of PAMC-82 cells (P<0.01). Conclusion: Monoclonal antibody 18H12 can significantly inhibit the self-renewal and invasion of gastric cancer stem cells and is expected to be a candidate antibody drug targeting gastric cancer stem cells.

Objective: To compare the preoperative and postoperative dosimetric parameters in the treatment of spinal metastasis, and to verify the accuracy of 3D-printing non-coplanar template (3D-PNCT) combined with CT-guided ¹²⁵I seed implantation for the treatment of spinal metastasis. Methods: The treatment plans of 7 patients with spinal metastasis (9 lesions) from 2016 to 2018 receiving 3D-PNCT in combination with CT-guided ¹²⁵I seed implantation were retrospectively analyzed. The dosimetric parameters including homogeneity index (HI), conformal index (CI), external index (EI), dose of 90% target volume(D₉₀), mPD, volume percent of 100%, 150%, and 200% prescribed dose V₁₀₀、V₁₅₀、V₂₀₀ and D_2cm³ of spinal cord were compared before and after operation. The british columbia cancer ageny particle implantation quality evaluation standard was applied to evaluate the quality of implantation. Results: The HI, EI and CI, D₉₀, mPD, V₁₀₀, V₁₅₀, V₂₀₀ and D_2cm³ of spinal cord did not significantly differ before and after the plan (all P>0.05). Five were evaluated as excellent and 4 were assessed as good. Conclusion The postoperative dosimetric parameters of 3D-PNCT combined with CT guided ¹²⁵I seed implantation of spinal metastasis are basically consistent with preoperative dosimetric parameters. The postoperative plans are evaluated as excellent or good, suggesting that the technology has a good therapeutic accuracy in the treatment of spinal metastasis.

Objective The objectives of this study were to screen and identify monoclonal antibodies against hepatoma stem cells by screening for hepatoma spheroid cells,and to provide candidate therapeutic monoclonal antibodies for targeting cancer stem cells to treat hepatic cancer. Methods Hepatic cancer stem cells were enriched by serum-free suspension culture. Immunofluores-cence,cisplatin resistance assay, Real -time qPCR, subcutaneous tumor formation in nude mice, and other methods were used to screen and identify anti-hepatocarcinoma stem cell monoclonal antibodies. Immunohistochemistry was used to identify the expression of antigen recognized by monoclonal antibody in liver cancer tissues. The antigen was identified by mass spectrometry. Results MH-CC97L cells were able to form cell spheres in serum -free suspension culture and were labeled with PKH26 dye. Flow cytometry showed that the expression of CD90 + in MHCC97L spheroid cells was 3. 4 times higher than that in the parental cells. In the inhibition experiment of serum-free spheroid,6 monoclonal antibodies significantly inhibited MHCC97L cells in serum-free medium,and in-hibitory rates were 54. 67% ,50. 33% ,45. 73% ,42. 26% ,39. 11% ,and 37. 63% ,respectively. The results of immunofluorescence showed that monoclonal antibodies 28C10 and CD90 were colocalized in MHCC97L cells. The results of real-time qPCR showed that the expression of Sox-2 and Oct-4 in MHCC97L 28C10 + cells was significantly higher than those of MHCC97L 28C10 - cells. Flow cytometry showed that the ratio of 28C10 + in MHCC97L cells and its sphere cells were 7. 98% and 10. 7% ,respectively. The ratio of 28C10 + cells was increased by 1. 34 times. The in vitro globing ability and invasive ability of 28C10 + cells obtained by flow cytometry were significantly higher than those of 28C10 - cells. The results of CCK-8 assay showed that 28C10 + cells were resistance to cispla-tin in 28C10 - cells,which are 1. 96 g/ml and 1. 16 g/ml,respectively. Tumorigenic assay showed that 28C10 + cells were inoculated subcutaneously with 2×104 cells into the nude mice,and tumors were formed in 2 months,with 40% of tumor formation rate. Another nude mouse that did not form a tumor had formed a lung metastasis(1/5). Immunohistochemistry showed that the target antigen posi-tive rate of monoclonal antibody 28C10 in hepatic cancer tissues was about 72. 0% (77/107),while it was lowly expressed in adjacent tissues,and the difference was significant. Mass spectrometry showed that the antigen recognized by 28C10 was HSP90α. Conclusion The MHCC97L spheroid cell model is successfully used to identify a monoclonal antibody that specifically recognizes hepatoma stem cells,which provides a foundation for antibody therapy targeting hepatic cancer stem cells.

Objective: To investigate the expression of programmed death ligand 1 (PD-L1) in liver cancer tissues and its clinical significance. Methods: The expression levels of PD-L1 in 110 liver cancer tissues, including 95 cases of hepatocellular carcinoma and 15 cases of cholangiocarcinoma were detected by using immunohistochemical staining method, and the relationship between PD-L1 expression and the clinicopathological characteristics of patients with hepatocellular carcinoma was analyzed. Results: Immunohistochemistry results showed that the positive rate of PD-L1 in liver cancer tissues was 69.1% (76/110), and the positive rate of membrane and cytoplasm was 46.4% (51/110) and 22.7% (25/110), respectively. The positive rate of PD-L1 expression in hepatocellular carcinoma was higher than that in cholangiocarcinoma [78.9% (75/95) vs. 6.7% (1/15)], and the difference was statistically significant (χ ² = 31.693, P < 0.01). The positive expression of PD-L1 was closely associated with the depth of invasion and TNM stage of hepatocellular carcinoma (both χ² = 4.629, both P = 0.031), but there was no relationship with the patients'age, gender and pathological grade (all P > 0.05). Further analysis showed that protein expression localization of PD-L1 was closely related to the pathological grade in hepatocellular carcinoma (P = 0.013). Conclusion The positive expression of PD-L1 in hepatocellular carcinoma is closely associated with depth of invasion and TNM stage, which provides a theoretical basis for the immunotherapy of liver cancer targeting PD-L1.

Objective To verify the accuracy of three-dimensional printing coplanar coordinate template (3D-PCCT) technology for 125I seeds implantations in chest tumor at dosimetry level.Methods From January 2016 to June 2017,a total of 22 patients (15 males,7 females;median age 62 years) with chest tumors who received 3D-PCCT assisted 125I seeds implantation in Peking University Third Hospital were enrolled in this retrospective study.There were 8 patients with primary lung cancer and 14 with metastatic carcinoma.The sites for implantation included lung (12 cases),mediastinum (2 cases) and chest wall (8 cases).The prescribed dose was 110-180 Gy.The preoperative plan design,puncture and seeds implantation guided by template were carried out and the dose distribution of postoperative plan was compared with that of preoperative plan.Dose parameters included dose delivered to 90％ gross tumor volume (D90),minimum peripheral dose,the percentage of GTV receiving 100％ prescription dose (V100),the percentage of GTV receiving 150％ prescription dose (V150),conformity index,external index of target volume,uniformity index,D2 cm3 of spinal cord and aorta,and V20 of affected side lung.Wilcoxon signed rank test was used to analyze the data.Results The median D90 was 150.4(125.6,187.0) Gy.Postoperative D90 was higher than the prescribed dose in 68％ (15/22) cases.For median value of most parameters,there were no significant differences between the postoperative plans and preoperative plans (all P＞0.05) except for the actual V100,which was lower than the preplanned (95.5％ vs 97.2％;P=O.040).Conclusion 3D-PCCT could provide good accuracy in 125I seeds implantation for chest tumor.