Penetration of immune cells into tumor cells was believed to be immune-suppressive via cell-in-cell (CIC) mediated death of the internalized immune cells. We unexpectedly found that CIC formation largely led to the death of the host tumor cells, but not the internalized immune cells, manifesting typical features of death executed by NK cells; we named this “in-cell killing” which displays the efficacy superior to the canonical way of “kiss-killing” from outside. By profiling isogenic cells, CD44 on tumor cells was identified as a negative regulator of “in-cell killing” via inhibiting CIC formation. CD44 functions to antagonize NK cell internalization by reducing N-cadherin-mediated intercellular adhesion and by enhancing Rho GTPase-regulated cellular stiffness as well. Remarkably, antibody-mediated blockade of CD44 signaling potentiated the suppressive effects of NK cells on tumor growth associated with increased heterotypic CIC formation. Together, we identified CIC-mediated “in-cell killing” as a promising strategy for cancer immunotherapy.

The nonautonomous cell death by entosis was mediated by the so-called cell-in-cell structures, which were believed to kill the internalized cells by a mechanism dependent on acidified lysosomes. However, the precise values and roles of pH critical for the death of the internalized cells remained undetermined yet. We creatively employed keima, a fluorescent protein that displays different excitation spectra in responding to pH changes, to monitor the pH dynamics of the entotic vacuoles during cell-in-cell mediated death. We found that different cells varied in their basal intracellular pH, and the pH was relatively stable for entotic vacuoles containing live cells, but sharply dropped to a narrow range along with the inner cell death. In contrast, the lipidation of entotic vacuoles by LC3 displayed previously underappreciated complex patterns associated with entotic and apoptotic death, respectively. The pH decline seemed to play distinct roles in the two types of inner cell deaths, where apoptosis is preceded with moderate pH decline while a profound pH decline is likely to be determinate for entotic death. Whereas the cancer cells seemed to be lesser tolerant to acidified environments than noncancerous cells, manipulating vacuolar pH could effectively control inner cell fates and switch the ways whereby inner cell die. Together, this study demonstrated for the first time the pH dynamics of entotic vacuoles that dictate the fates of internalized cells, providing a rationale for tuning cellular pH as a potential way to treat cell-in-cell associated diseases such as cancer.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is highly contagious and causes lymphocytopenia, but the underlying mechanisms are poorly understood. We demonstrate here that heterotypic cell-in-cell structures with lymphocytes inside multinucleate syncytia are prevalent in the lung tissues of coronavirus disease 2019 (COVID-19) patients. These unique cellular structures are a direct result of SARS-CoV-2 infection, as the expression of the SARS-CoV-2 spike glycoprotein is sufficient to induce a rapid (~45.1 nm/s) membrane fusion to produce syncytium, which could readily internalize multiple lines of lymphocytes to form typical cell-in-cell structures, remarkably leading to the death of internalized cells. This membrane fusion is dictated by a bi-arginine motif within the polybasic S1/S2 cleavage site, which is frequently present in the surface glycoprotein of most highly contagious viruses. Moreover, candidate anti-viral drugs could efficiently inhibit spike glycoprotein processing, membrane fusion, and cell-in-cell formation. Together, we delineate a molecular and cellular rationale for SARS-CoV-2 pathogenesis and identify novel targets for COVID-19 therapy.

Though homotypic cell-in-cell (hoCIC) structures are implicated in the development and progression of multiple human tumors, the molecular mechanisms underlying their formation remain poorly understood. We found that the expression of Protocadherin-7 (PCDH7), an integral membrane protein, was negatively associated with the formation of hoCIC structures. Overexpression of PCDH7 efficiently inhibits, while its depletion significantly enhances, hoCIC formation, which was attributed to its regulation on intercellular adhesion and contractile actomyosin as well. Via directly interacting with and inactivating PP1α, a protein phosphatase that dephosphorylates pMLC2, PCDH7 increases the level of pMLC2 leading to enhanced actomyosin at the intercellular region and compromised hoCIC formation. Remarkably, PCDH7 enhanced anchorage-independent cell growth in a hoCIC-dependent manner. Together, we identified PCDH7 as the first trans-membrane protein that inhibits hoCIC formation to promote tumor growth.

Entosis is a cell-in-cell (CIC)-mediated death program. Contractile actomyosin (CA) and the adherens Junction (AJ) are two core elements essential for entotic CIC formation, but the molecular structures interfacing them remain poorly understood. Here, we report the characterization of a ring-like structure interfacing between the peripheries of invading and engulfing cells. The ring-like structure is a multi-molecular complex consisting of adhesive and cytoskeletal proteins, in which the mechanical sensor vinculin is highly enriched. The vinculin-enriched structure senses mechanical force imposed on cells, as indicated by fluorescence resonance energy transfer (FRET) analysis, and is thus termed the mechanical ring (MR). The MR actively interacts with CA and the AJ to help establish and maintain polarized actomyosin that drives cell internalization. Vinculin depletion leads to compromised MR formation, CA depolarization, and subsequent CIC failure. In summary, we suggest that the vinculin-enriched MR, in addition to CA and AJ, is another core element essential for entosis.

Entosis was proposed to promote aneuploidy and genome instability by cell-in-cell mediated engulfment in tumor cells. We reported here, in epithelial cells, that entosis coupled with mitotic arrest functions to counteract genome instability by targeting aneuploid mitotic progenies for engulfment and elimination. We found that the formation of cell-in-cell structures associated with prolonged mitosis, which was sufficient to induce entosis. This process was controlled by the tumor suppressor p53 (wild-type) that upregulates Rnd3 expression in response to DNA damages associated with prolonged metaphase. Rnd3-compartmentalized RhoA activities accumulated during prolonged metaphase to drive cell-in-cell formation. Remarkably, this prolonged mitosis-induced entosis selectively targets non-diploid progenies for internalization, blockade of which increased aneuploidy. Thus, our work uncovered a heretofore unrecognized mechanism of mitotic surveillance for entosis, which eliminates newly born abnormal daughter cells in a p53-dependent way, implicating in the maintenance of genome integrity.

Entosis was proposed to promote aneuploidy and genome instability by cell-in-cell mediated engulfment in tumor cells. We reported here, in non-transformed epithelial cells, that entosis coupled with mitotic arrest functions to counteract genome instability by targeting aneuploid mitotic progenies for engulfment and elimination. We found that the formation of cell-in-cell structures associated with prolonged mitosis, which was sufficient to induce entosis. This process was controlled by the tumor suppressor p53 (wild type) that upregulates Rnd3 expression in response to DNA damages associated with prolonged metaphase. Rnd3 compartmentalized RhoA activities accumulated during prolonged metaphase to drive cell-in-cell formation. Remarkably, this prolonged itosis-duced en (mintosis) selectively targets non-diploid progenies for internalization, blockade of which increased aneuploidy. Thus, our work uncovered a heretofore unrecognized mechanism of mitotic surveillance for entosis, which eliminates newly-born abnormal daughter cells in a p53-depedent way to maintain genome integrity.

目的:研究核内RNA与真核细胞基因组三维结构的关系,并探讨核内RNA对染色体高级结构如拓扑相关结构域(TAD)和A/B区室的影响.方法:利用全基因组染色质构象捕获(Hi-C)技术对正常GM12878细胞和经过RNase处理的GM12878细胞进行染色质相互作用的检测.结果:经RNase处理后,染色质的三维高级结构在整体上变得松散,染色体之间的相互作用增强(秩和检验P<0.05),A/B区室发生改变,并且相比于所有染色体,X染色体在A/B区室上的变化更大.同时,核内RNA的去除也让TAD边界的强度有所减弱(Vilcox校验P<0.05).结论:核内RNA参与了真核细胞基因组三维高级结构的形成,并使TAD和A/B区室更加致密.

目的:构建绿色荧光蛋白(EGFP)与有丝分裂阻滞缺陷蛋白2(MAD2)融合基因表达载体,并初步验证融合基因的功能.方法:采用PCR技术扩增EGFP-MAD2融合基因,连接入T载体进行序列测定,随后克隆入逆转录表达载体pQCXIP-EGFP-N1获得重组质粒,采用脂质体转染293FT细胞,荧光显微镜观察融合蛋白的表达,流式细胞仪分析细胞周期.结果:获得序列正确的EGFP-MAD2融合基因及其表达载体pQCXIP-EGFP-MAD2;EGFP-MAD2基因可在293FT细胞中表达;流式分析显示EGFP-MAD2表达的细胞G2/M期比例增加.结论:构建了EGFP-MAD2融合基因表达载体,EGFP-MAD2融合蛋白的表达可以诱导细胞发生G2/M期阻滞.

Cell-in-cell (CIC) structures, characterized by enclosure of one or more cells within another cell, were extensively documented in human cancers. Although elevated CIC formation was found in cancers with CDKN2A inactivation, a causal link between them remains to be established. We reported here that inhibiting CDKN2A expression effectively promoted homotypic CIC formation, whereas ectopic overexpression of p16INK4a or p14ARF, two proteins encoded by CDKN2A gene, significantly suppressed CIC formation in MCF7 cells. The regulation of CIC formation by CDKN2A was tightly correlated with subcellular redistribution of E-cadherin, F-actin rearrangement and reduced phosphorylation of myosin light chain 2 (p-MLC2), consistent with which, CDKN2A expression imparted cells winner/outer identity in competition assay. Moreover, CIC formation negatively correlates with p16INK4a expression in human breast cancers. Thus, our work identifies CDKN2A as the first tumor suppressor whose inactivation promotes homotypic CIC formation in human cancer cells.

BACKGROUND:Islet beta cell replacement therapy is one of the most promising approaches for treating type 1 diabetes mellitus. However, its large scale application is hampered by a shortage of islet beta cells for transplantation. Pluripotent stem cells are one of ideal seed cells for islet beta cell replacement therapy, but pancreatic beta-cell differentiation is time-consuming and labor-intensive. OBJECTIVE:To construct a high efficient pancreatic and duodenal homeobox 1 (Pdx1)/insulin dual-reporter vector and to monitor the key genes expression during pancreatic beta-cell differentiation from pluripotent stem cells. METHODS:In order to construct a high efficient Pdx1/insulin dual-reporter vector, puromycin resistance gene was firstly introduced into pTiger vector, and then the original 410 bp mouse Ins1 promoter of the vector was replaced by 646 bp mouse Ins1 promoter. Finally, the dual-reporter vector was transduced into INS-1 and human induced pluripotent stem cells to testify its function. RESULTS AND CONCLUSION:The high efficient Pdx1/insulin dual-reporter vector was constructed successfully. The vector successfully acquired puromycin resistance gene and high gene expression efficacy of insulin in INS-1 cells. The specific gene expression pattern of Pdx1/insulin was first found in INS-1 cells. To conclude, the real-time monitoring function of Pdx1/insulin expression is preliminarily confirmed during pancreatic beta-cell differentiation.

Objective:To construct the lentiviral expression vector pLVX-EF1α-CD19t-IRES-Puro for truncated CD19(CD19t) in cytoplasmic region,and examine its expression in K562 cells.Methods:CD19t gene was synthesized and cloned into pLVX-EF1α-IRES-Puro vector followed by virus packaging in 293FT cells.Human erythroleukemia K562 cells were infected with recombinant lentivirus containing CD19t to construct K562/CD19t cells.The expression of CD19t was examined by Western blot and immunofluorescence staining.ELISA was used to analyze the secreted IL-2 in co-culture supernatant of K562/CD19t with CAR-Jurkat.Results:Lentiviral expression vector pLVX-EF1α-CD19t-IRES-Puro was confirmed by double enzymatic digestion and sequencing.The expression of CD19t in K562 cells was detected by Western blot and immunofluorescence staining.ELISA showed that CAR-Jurkat secreted IL-2,which was stimulated by CD19t expression.Conclusion:The lentiviral vector for CD19t expression has been constructed and CD19t expression can be detected in K562/CD19t cells,which would be helpful to make animal model for B cell leukemia and test CAR-T performance.

Objective:To explore effect of supplementing melatonin into medium of in vitro culture on development potential of aging oocytes.Methods:The MII oocytes retrieved from ovaries were incubated with M2 culture medium without (as control) or with H2O2 at different concentrations (10,50,100,150 μmol/L) for 3 hours to induce oocyte aging.The 2-cell rate and blastocyst rate were counted after in vitro fertilization (IVF).The mitochondrial mass and activity (Mitotracker Red and JC-1),reactive oxygen species (ROS) levels,as well as mitochondrial DNA (mtDNA) copy number were also detected in control and aging oocytes.After treated with 100 μmol/L H2O2,the aging oocytes were in vitro fertilized and cultured in medium without or with melatonin at different concentrations (10-5,10-7,10-9 mol/L).Then,the 2-cell rate and blastocyst rate were counted,and the cell number of blastocysts and apoptotic rate were detected in the each groups.Results:Different concentrations of H2O2 induced aging of oocyte showed that the blastocyst development rate was decreased along with the increase of H2O2 concentration.The blastocyst rate of aging oocytes induced by 50 μmol/L and 100 μmol/L H2O2 was (26.27±0.06)% and (28.46±3.45)%,respectively,which were significantly lower than that in the control group [(34.90±1.77)%] (P<0.05).H2O2 at higher concentration (100 or 150 μmol/L) induced significantly higher intracellular reactive oxygen species (ROS) in aging oocytes (P<0.05),as well as a tendency of reduced mitochondrial mass and increased mitochondrial membrane potential (MMP),but the difference was not significant (P>0.05).The blastocyst rate [(29.42±2.39)% vs.(20.87±4.12)%,P<0.05) and the cell number of blastocysts from aging oocytes treated with 100 μmol/L H2O2 were significantly increased [(39.36±9.78) vs.(37.91±4.25),P<0.05) and the apoptotic rate was significantly decreased (2.57% vs.3.18%,P<0.05)by supplementing 10-9 mol/L melatonin into culture medium.And these indicators reached a similar level of development without aging treatment group.Conclusions:The lower development rate and development quality in aging oocytes in vitro fertilization can be reversed and improved by supplementing melatonin into culture medium.

Objective:To construct the retroviral expression vector for RRAGD-EGFP fusion protein,and examine its subcellular localization in MDA-MB-436 cells.Methods:Total RNA was extracted from HEK293 cells and reverse transcribed into cDNA.RRAGD(Ras-related GTP binding protein D) gene was amplified by PCR and cloned into the retroviral expression vector pQCXIP-EGFP-N1 to construct pQCXIP-RRAGD-EGFP,followed by retrovirus was made and infected MDA-MB-436,a cell line of breast cancer.Afterwards,time lapse imaging was performed.Results:The retroviral expression vector for RRAGD-EGFP fusion protein was successfully constructed.RRAGD-EGFP fusion protein was found in cytoplasmic vesicles and nucleus.Conclusion:Localization of RRAGD-EGFP fusion protein in cytoplasmic vesicles is consistent with its role in lysosomal biogenesis,which has laid foundation for the further study on the role of RRAGD in cell-in-cell.

Objective:To construct retroviral vector pQCXIP-EGFP-PEST-N1 for the expression of EGFP-PEST protein,and examine the effects of PEST motif on EGFP stability.Methods:DNA fragment containing EGFP-PEST was amplified by PCR from NFATx6-mPro-EGFP-PEST-YES as template,and then cloned into the retroviral expression vector pQCXIP-EGFP-N1 to generate pQCXIP-EGFP-PEST-N1,which was packaged into retrovirus to infect 293FT cells.Western blot was employed to examine changes at protein level upon treatment of proteasome inhibitor (MG-132).Results:Compared with pQCXIP-EGFP-N1 virus,retroviral vector of pQCXIP-EGFP-PEST-N1 produced significantly lower EGFP in 293FT cells,and MG-132 treatment reverted the expression of EGFP-PEST but not EGFP,suggesting effective PEST-mediated degradation via proteasome.Conclusion:A vector has been constructed to express EGFP-PEST,and PEST motif has been proved to effectively mediate EGFP degradation in a proteasome-dependent manner.This work set a basis for visualing target screen in gene edition.

Although Cell-in-cell structures (CICs) had been documented in human tumors for decades, it is unclear what types of CICs were formed largely due to low resolution of traditional way such as H&E staining. In this work, we employed immunofluorescent method to stain a panel of human tumor samples simultaneously with antibodies against E-cadherin for Epithelium, CD68 for Macrophage and CD45 for Leukocytes, which we termed as “EML method” based on the cells detected. Detail analysis revealed four types of CICs, with tumor cells or macrophage engulfing tumor cells or leukocytes respectively. Interestingly, tumor cells seem to be dominant over macrophage (93% vs 7%) as the engulfer cells in all CICs detected, whereas the overall amount of internalized tumor cells is comparable to that of internalized CD45+ leukocytes (57% vs 43%). The CICs profiles vary from tumor to tumor, which may indicate different malignant stages and/or inflammatory conditions. Given the potential impacts different types of CICs might have on tumor growth, we therefore recommend EML analysis of tumor samples to clarify the correlation of CICs subtypes with clinical prognosis in future researches.

Cell-in-cell structures (CICs), characterized by the presence of one or more viable cells inside another one, were recently found important player in development, immune homeostasis and tumorigenesis etc. Incompatible with ever-increasing interests on this unique phenomenon, reliable methods available for high throughput quantification and systemic investigation are lacking. Here, we report a flow cytometry-based method for rapid analysis and sorting of heterotypic CICs formed between lymphocytes and tumor cells. In this method, cells were labeled with fluorescent dyes for fluorescence-activated cell sorting (FACS) by flow cytometry, conditions for reducing cell doublets were optimized such that high purity (>95%) of CICs could be achieved. By taking advantage of this method, we analyzed CICs formation between different cell pairs and found that factors from both internalized effector cells and engulfing target cells affect heterotypic CICs formation. Thus, flow cytometry-based FACS analysis would serve as a high throughput method to promote systemic researches on CICs.

Although cell-in-cell structures (CICs) could be detected in a wide range of human tumors, homotypic CICs formed between tumor cells occur at low rate for most of them. We recently reported that tumor cells lacking expression of E- and P-cadherin were incapable of forming homotypic CICs by entosis, and re-expression of E- or P-cadherin was sufficient to induce CICs formation in these tumor cells. In this work, we found that homotypic CICs formation was impaired in some tumor cells expressing high level of E-cadherin due to loss expression of alpha-catenin (α-catenin), a molecular linker between cadherin-mediated adherens junctions and F-actin. Expression of α-catenin in these tumor cells restored cell-cell adhesion and promoted CICs formation in a ROCK kinase-dependent way. Thus, our work identified α-catenin as another molecule in addition to E- and P-cadherin that were targeted to inactivate homotypic CICs formation in human tumor cells.

To establish a gene regulation system compatible with biopharmaceutical industry and gene therapy, we constructed a fusion protein of biotin ligase from Bacillus subtilis (BS-BirA) and the trans-activation domain, and used its expression vector as the regulatory vector. Meanwhile, BS-BirA-specific operators were ligated upstream of attenuated CMV promoter to obtain the response vector. In this way, a novel eukaryotic gene regulation system responsive to biotin was established and named BS-Biotin-On system. BS-Biotin-On system was further investigated with the enhancing green fluorescent protein (EGFP) as the reporter gene. The results showed that our system was superior to the current similar regulation system in its higher induction ratio, and that the expression of interest gene could be tuned in a rapid and efficient manner by changing the biotin concentrations in the cultures, Our results show that the established system may provide a new alternative for the exogenous gene modulation.