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.

No abstract

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

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 anchorageindependent 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.

32Entosis was proposed to promote aneuploidy and genome instability by cell-in-cell mediated 33 engulfment in tumor cells. We reported here, in non-transformed epithelial cells, that entosis 34 coupled with mitotic arrest functions to counteract genome instability by targeting aneuploid mitotic 35 progenies for engulfment and elimination. We found that the formation of cell-in-cell structures 36 associated with prolonged mitosis, which was sufficient to induce entosis. This process was 37 controlled by the tumor suppressor p53 (wild type) that upregulates Rnd3 expression in response to 38 DNA damages associated with prolonged metaphase. Rnd3 compartmentalized RhoA activities 39 accumulated during prolonged metaphase to drive cell-in-cell formation. Remarkably, this 40 prolonged mitosis-induced entosis (mintosis) selectively targets non-diploid progenies for 41 internalization, blockade of which increased aneuploidy. Thus, our work uncovered a heretofore 42 unrecognized mechanism of mitotic surveillance for entosis, which eliminates newly-born abnormal 43 daughter cells in a p53-depedent way to maintain genome integrity. 44