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.

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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.

Though current pathological methods are greatly improved, they provide rather limited functional information. Cell-in-cell structures (CICs), arising from active cell–cell interaction, are functional surrogates of complicated cell behaviors within heterogeneous cancers. In light of this, we performed the subtype-based CIC profiling in human breast cancers by the “EML” multiplex staining method, and accessed their values as prognostic factors by Cox univariate, multivariate, and nomogram analysis. CICs were detected in cancer specimens but not in normal breast tissues. A total of five types of CICs were identified with one homotypic subtype (91%) and four heterotypic subtypes (9%). Overall CICs (oCICs) significantly associated with patient overall survival (OS) (P = 0.011) as an independent protective factor (HR = 0.423, 95% CI, 0.227–0.785; P = 0.006). Remarkably, three CICs subtypes (TiT, TiM, and MiT) were also independent prognostic factors. Among them, higher TiT, from homotypic cannibalism between tumor cells, predicted longer patient survival (HR = 0.529, 95% CI, 0.288–0.973; P = 0.04) in a way similar to that of oCICs and that (HR = 0.524, 95% CI, 0.286–0.962; P = 0.037) of heterotypic TiM (tumor cell inside macrophage); conversely, the presence of MiT (macrophage inside tumor cell) predicted a death hazard of 2.608 (95% CI, 1.344–5.063; P = 0.05). Moreover, each CIC subtype tended to preferentially affect different categories of breast cancer, with TiT (P < 0.0001) and oCICs (P = 0.008) targeting luminal B (Her2+), TiM (P = 0.011) targeting HR− (Her2+/HR− and TNBC), and MiT targeting luminal A (P = 0.017) and luminal B (Her−) (P = 0.006). Furthermore, nomogram analysis suggested that CICs impacted patient outcomes in contributions comparable (for oCICs, TiT, and TiM), or even superior (for MiT), to TNM stage and breast cancer subtype, and incorporating CICs improved nomogram performance. Together, we propose CICs profiling as a valuable way for prognostic analysis of breast cancer and that CICs and their subtypes, such as MiT, may serve as a type of novel functional markers assisting clinical practices.

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.