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.

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.

Epstein-Barr virus (EBV) can infect both susceptible B lymphocytes and non-susceptible epithelial cells (ECs). Viral tropism analyses have revealed two intriguing means of EBV infection, either by a receptor-mediated infection of B cells or by a cell-to-cell contact-mediated infection of non-susceptible ECs. Herein, we report a novel “in-cell infection” mechanism for EBV infection of non-susceptible ECs through the formation of cell-in-cell structures. Epithelial CNE-2 cells were invaded by EBV-infected Akata B cells to form cell-in-cell structures in vitro. Such unique cellular structures could be readily observed in the specimens of nasopharyngeal carcinoma. Importantly, the formation of cell-in-cell structures led to the autonomous activation of EBV within Akata cells and subsequent viral transmission to CNE-2 cells, as evidenced by the expression of viral genes and the presence of virion particles in CNE-2 cells. Significantly, EBV generated from in-cell infected ECs displayed altered tropism with higher infection efficacy to both B cells and ECs. In addition to CNE-2 tumor cells, cell-in-cell structure formation could also mediate EBV infection of NPEC1-Bmi1 cells, an immortalized nasopharyngeal epithelial cell line. Furthermore, efficient infection by this mechanism involved the activation of the PI3K/AKT signaling pathway. Thus, our study identified “in-cell infection” as a novel mechanism for EBV infection. Given the diversity of virus-infected cells and the prevalence of cell-in-cell structures during chronic infection, we speculate that “in-cell infection” is likely a general mechanism for EBV and other viruses to infect non-susceptible ECs.

Highlights d Mechanical ring (MR) is interfacing between the invading and the engulfing cells d MR is a multi-molecular complex enriched in the mechanical sensor vinculin d Vinculin coordinates the polarized AJ and CA to promote entosis d MR, in addition to AJ and CA, is the third core element essential for entosis

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.