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Objective: In 2007, entosis was proposed as a form of programmed cell death, distinct from apoptosis. This process involves a living cell (internalized cell) actively invading a neighboring live cell of the same type (host cell), forming a cell-in-cell structure. Recently, entosis has been increasingly associated with cancer, leading to significant advancements in research. Despite this progress, a comprehensive and unbiased review of the current state of entosis research is lacking. This study aims to evaluate the developments in the field of entosis over the past decade and highlight emerging research trends. Materials and methods: We performed a literature search for studies published since the introduction of the entosis concept, using the Web of Science Core Collection database. The bibliometric analysis was conducted using VOSviewer, CiteSpace, Microsoft Excel, and the Bibliometrix R package. Results: A total of 196 articles from 39 countries and 346 institutions were included. Between 2007 and 2024, research on entosis has seen rapid growth, with most publications originating from China and the United States. The United States also leads in total citations, with Memorial Sloan Kettering Cancer Center emerging as the top research institution. Sun Qiang is the most prolific author in this field, while Overholtzer M has the highest number of citations. Current Molecular Medicine has published the most articles related to entosis. Frequently occurring keywords include "entosis," "cannibalism," "autophagy," and "apoptosis." In recent years, keywords such as "phagocytosis," "drug resistance," and "human cancers" have surged, indicating a growing focus on understanding the role of entosis in tumor progression and exploring its potential as a therapeutic target for cancer treatment. Conclusions: This study provides the first bibliometric analysis of entosis, detailing its evolution over the last decade. It highlights critical areas of interest, including the development of inhibitors targeting entosis and their potential clinical applications. This research aims to guide future investigations and serve as a valuable resource for scholars exploring entosis in cancer biology. [ABSTRACT FROM AUTHOR]

We describe a case of a young patient with a recurrent pleomorphic xanthoastrocytoma (PXA) showing unusual cell-in-cell (CiC) phenomena. We observed mostly viable but also necrotic neutrophils engulfed within tumor cells. The recurrent tumor was immunopositive for BRAFV600E mutant protein and showed CDKN2 homozygous deletions typical of PXA. Both genetic alterations were also reported in the original primary tumor. Unlike the original tumor that was GFAP and Olig-2 immunopositive, the recurrent neoplasm was largely negative for GFAP and Olig-2 suggesting dedifferentiation. The large malignant cells that contained the neutrophils were negative for histiocytic and lymphohematopoietic markers. Whereas CDKN2 homozygous deletion is common in PXA, its presence is rare in histiocytic neoplasms. Both reactive astrocytes and glial neoplasms very rarely may engulf neutrophils in a process resembling emperipolesis or cellular cannibalism. Future work may clarify which type of CiC pathway is involved.

Fenglin Zhang,* Jingliang Ye,* Junle Zhu, Wenbo Qian, Haoheng Wang, Chun Luo Department of Neurosurgery, Tongji Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Chun Luo, Email luoc_sh@sina.comPurpose: This study aimed to explore the roles of cell-in-cell (CIC)-related genes in glioblastoma (GBM) using bioinformatics and experimental strategies.Patients and Methods: The ssGSEA algorithm was used to calculate the CIC score for each patient. Subsequently, differentially expressed genes (DEGs) between the CIClow and CIChigh groups and between the tumor and control samples were screened using the limma R package. Key CIC-related genes (CICRGs) were further filtered using univariate Cox and LASSO analyses, followed by the construction of a CIC-related risk score model. The performance of the risk score model in predicting GBM prognosis was evaluated using ROC curves and an external validation cohort. Moreover, their location and differentiation trajectory in GBM were analyzed at the single-cell level using the Seurat R package. Finally, the expression of key CICRGs in clinical samples was examined by qPCR.Results: In the current study, we found that CIC scorelow group had a significantly better survival in the TCGA-GBM cohort, supporting the important role of CICRGs in GBM. Using univariate Cox and LASSO analyses, PTX3, TIMP1, IGFBP2, SNCAIP, LOXL1, SLC47A2, and LGALS3 were identified as key CICRGs. Based on this data, a CIC-related prognostic risk score model was built using the TCGA-GBM cohort and validated in the CGGA-GBM cohort. Further mechanistic analyses showed that the CIC-related risk score is closely related to immune and inflammatory responses. Interestingly, at the single-cell level, key CICRGs were expressed in the neurons and myeloids of tumor tissues and exhibited unique temporal dynamics of expression changes. Finally, the expression of key CICRGs was validated by qPCR using clinical samples from GBM patients.Conclusion: We identified novel CIC-related genes and built a reliable prognostic prediction model for GBM, which will provide further basic clues for studying the exact molecular mechanisms of GBM pathogenesis from a CIC perspective.Keywords: glioblastoma, bioinformatics, cell-in-cell, prognosis

Cell-in-cell (CIC) is a phenomenon described as the active infiltration of one or more living cells into another living cell, forming intracellular structures and producing biological effects. This phenomenon was first observed and detailed in the early 20th century. However, it has only become an active area of research in cell biology in the past decade. As research advanced, scientists gradually recognized that the phenomenon of CIC exists in many biological systems and diseases, and has important physiological and pathological significance in evolution, development, homeostasis, and diseases, especially in tumors, which has important clinical research value. Currently, five forms of CIC structures have been identified: cell cannibalism, phagoptosis, enclysis, entosis, and emperipolesis. Each form represents a distinct interaction between cells, characterized by unique cell types, biological characteristics, molecular mechanisms, and implications in pathophysiology. This article aims to review the cell types involved in each CIC structure, their biological characteristics and the molecular mechanisms driving these interactions, and to explore the potential clinical significance in the diagnosis, treatment and prognosis of human diseases. [ABSTRACT FROM AUTHOR]

A recent article in Cell reported a new peculiar interaction between mast cells (MCs) and neutrophils. Upon IgE/Ag-mediated degranulation, MCs produce leukotriene B4, which attracts migrating neutrophils. Some neutrophils are able to establish close contact with MCs and end up trapped inside the MC, forming a cell-in-cell structure. While the neutrophil eventually dies inside the vacuole, the MC benefits from the remains of its prisoner, using it as a nutrient reserve and reusing its antimicrobial weapons. [ABSTRACT FROM AUTHOR]

Cell-in-cell (CIC) structures have been suggested to mediate intracellular substance transport between cells and have been found widely in inflammatory lung tissue of asthma. The aim of this study was to investigate the significance of CIC structures in inflammatory progress of asthma. CIC structures and related inflammatory pathways were analyzed in asthmatic lung tissue and normal lung tissue of mouse model. In vitro, the activation of inflammatory pathways by CIC-mediated intercellular communication was analyzed by RNA-Seq and verified by Western blotting and immunofluorescence. Results showed that CIC structures of lymphocytes and alveolar epithelial cells in asthmatic lung tissue mediated intercellular substance (such as mitochondria) transfer and promoted pro-inflammation in two phases. At early phase, internal lymphocytes triggered inflammasome-dependent pro-inflammation and cell death of itself. Then, degraded lymphocytes released cellular contents such as mitochondria inside alveolar epithelial cells, further activated multi-pattern-recognition receptors and NF-kappa B signaling pathways of alveolar epithelial cells, and thereby amplified pro-inflammatory response in asthma. Our work supplements the mechanism of asthma pro-inflammation progression from the perspective of CIC structure of lymphocytes and alveolar epithelial cells, and provides a new idea for anti-inflammatory therapy of asthma. [ABSTRACT FROM AUTHOR]

ObjectiveIn 2007, entosis was proposed as a form of programmed cell death, distinct from apoptosis. This process involves a living cell (internalized cell) actively invading a neighboring live cell of the same type (host cell), forming a cell-in-cell structure. Recently, entosis has been increasingly associated with cancer, leading to significant advancements in research. Despite this progress, a comprehensive and unbiased review of the current state of entosis research is lacking. This study aims to evaluate the developments in the field of entosis over the past decade and highlight emerging research trends.Materials and methodsWe performed a literature search for studies published since the introduction of the entosis concept, using the Web of Science Core Collection database. The bibliometric analysis was conducted using VOSviewer, CiteSpace, Microsoft Excel, and the Bibliometrix R package.ResultsA total of 196 articles from 39 countries and 346 institutions were included. Between 2007 and 2024, research on entosis has seen rapid growth, with most publications originating from China and the United States. The United States also leads in total citations, with Memorial Sloan Kettering Cancer Center emerging as the top research institution. Sun Qiang is the most prolific author in this field, while Overholtzer M has the highest number of citations. Current Molecular Medicine has published the most articles related to entosis. Frequently occurring keywords include “entosis,” “cannibalism,” “autophagy,” and “apoptosis.” In recent years, keywords such as “phagocytosis,” “drug resistance,” and “human cancers” have surged, indicating a growing focus on understanding the role of entosis in tumor progression and exploring its potential as a therapeutic target for cancer treatment.ConclusionsThis study provides the first bibliometric analysis of entosis, detailing its evolution over the last decade. It highlights critical areas of interest, including the development of inhibitors targeting entosis and their potential clinical applications. This research aims to guide future investigations and serve as a valuable resource for scholars exploring entosis in cancer biology.

Abstract Background In our previous study, we identified a population of adiponectin expressing regulatory T cells (Tregs) residing within thymic nurse cell complexes, which were capable of inhibiting the development of breast cancer in vitro. Triple‐negative breast cancer (TNBC) with no proper treatment at present is characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor‐2. In this study, we aimed to investigate the potential of a cultured T cell fraction comprising adiponectin‐expressing Tregs, referred to as A‐TregTF (adiponectin‐expressing Treg‐containing T cell fraction), in inhibiting the progression of TNBC in vivo. Methods The efficacy of a spontaneously expanding T cell fraction comprising adiponectin‐expressing Treg in inhibiting tumor growth was analyzed in a murine orthotopic 4 T1‐Luc TNBC model. Results The treatment with T cell fraction containing adiponectin‐expressing Tregs significantly inhibited the growth and metastasis of orthotopically transplanted 4 T1‐Luc tumor cells. Histopathological examination further revealed that the adiponectin‐expressing Tregs infiltrated the tumor tissue via a cell‐in‐cell mechanism and were found to be specifically localized around the necrotic areas. Conclusions Based on our findings, the T cell fraction comprising adiponectin‐expressing Tregs, represents a potential candidate for adoptive cell therapy against TNBC.

Abstract Background A population of regulatory T cells (Treg), which reside within thymic nurse cell complexes, express adiponectin and abrogate breast cancer development in transgenic mice. In this study, we examined whether adiponectin‐expressing Treg could impair triple‐negative breast cancer, which is defined by a lack of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor‐2. Methods CD4‐ and CD25‐positive cells were sorted from cultured T lymphocytes of a previously characterized experimental thymic tumor model composed of thymic nurse cells and abundant lymphoid stroma. These sorted cells were examined for FOXP3 and adiponectin immunoreactivity and subsequently exposed to triple‐negative breast cancer MDA‐MB‐157 and ‐231 cells. Results Adiponectin‐expressing Treg were obtained by CD4‐ and CD25‐positive sorting and cell death was induced in triple‐negative breast cancer cells through the cell‐in‐cell phenomenon. Conclusions Adiponectin‐expressing Treg may be candidates for adoptive cell therapy against triple‐negative breast cancer.

Background: In our previous study, we identified a population of adiponectin expressing regulatory T cells (Tregs) residing within thymic nurse cell complexes, which were capable of inhibiting the development of breast cancer in vitro. Triple‐negative breast cancer (TNBC) with no proper treatment at present is characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor‐2. In this study, we aimed to investigate the potential of a cultured T cell fraction comprising adiponectin‐expressing Tregs, referred to as A‐TregTF (adiponectin‐expressing Treg‐containing T cell fraction), in inhibiting the progression of TNBC in vivo. Methods: The efficacy of a spontaneously expanding T cell fraction comprising adiponectin‐expressing Treg in inhibiting tumor growth was analyzed in a murine orthotopic 4 T1‐Luc TNBC model. Results: The treatment with T cell fraction containing adiponectin‐expressing Tregs significantly inhibited the growth and metastasis of orthotopically transplanted 4 T1‐Luc tumor cells. Histopathological examination further revealed that the adiponectin‐expressing Tregs infiltrated the tumor tissue via a cell‐in‐cell mechanism and were found to be specifically localized around the necrotic areas. Conclusions: Based on our findings, the T cell fraction comprising adiponectin‐expressing Tregs, represents a potential candidate for adoptive cell therapy against TNBC. [ABSTRACT FROM AUTHOR]

The cell-in-cell (CIC) phenomenon has received increasing attention over recent years because of its wide existence in multiple cancer tissues. The mechanism of CIC formation is considerably complex as it involves interactions between two cells. Although the molecular mechanisms of CIC formation have been extensively investigated, the process of CIC formation remains ambiguous. Currently, CIC is classified into four subtypes based on different cell types and inducing factors, and the underlying mechanisms for each subtype are distinct. Here, we investigated the subtypes of CIC and their major mechanisms involved in cancer development. To determine the clinical significance of CIC, we reviewed several clinical studies on CIC and found that CIC could serve as a diagnostic and prognostic biomarker. The implications of CIC on the clinical management of cancers also remain largely unknown. To clarify this aspect, in the present review, we highlight the findings of recent investigations on the causal link between CIC and cancer treatment. We also indicate the existing issues that need to be resolved urgently to provide a potential direction for future research on CIC. [ABSTRACT FROM AUTHOR]

Homotypic entosis is a phenomenon in which one cancer cell invades a neighboring cancer cell and is closed entirely within its entotic vacuole. The fate of entosis can lead to inner cell death or survival. Recent evidence draws attention to entosis as a novel prognostic marker in breast cancer. Nevertheless, little is known about the quantity and quality of the process of entosis in human cancer specimens. Here, for the first time, we analyze the frequency of entotic figures in a case of NOS (Non-Other Specified) breast cancer with regard to location: the primary tumor, regional lymph node, and distant metastasis. For the identification of entotic figures, cells were stained using hematoxylin/eosin and assessed using criteria proposed by Mackay. The majority of entotic figures (65%) were found in the lymph node, 27% were found in the primary tumor, and 8% were found in the far metastasis. In the far metastases, entotic figures demonstrated an altered, atypic morphology. Interestingly, in all locations, entosis did not show any signs of cell death. Moreover, the slides were stained for E-cadherin or Ki67, and we identified proliferating (Ki67-positive) inner and outer entotic cells. Therefore, we propose additional criteria for the identification of pro-survival entotic structures in diagnostic histopathology. [ABSTRACT FROM AUTHOR]

Background: A population of regulatory T cells (Treg), which reside within thymic nurse cell complexes, express adiponectin and abrogate breast cancer development in transgenic mice. In this study, we examined whether adiponectin‐expressing Treg could impair triple‐negative breast cancer, which is defined by a lack of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor‐2. Methods: CD4‐ and CD25‐positive cells were sorted from cultured T lymphocytes of a previously characterized experimental thymic tumor model composed of thymic nurse cells and abundant lymphoid stroma. These sorted cells were examined for FOXP3 and adiponectin immunoreactivity and subsequently exposed to triple‐negative breast cancer MDA‐MB‐157 and ‐231 cells. Results: Adiponectin‐expressing Treg were obtained by CD4‐ and CD25‐positive sorting and cell death was induced in triple‐negative breast cancer cells through the cell‐in‐cell phenomenon. Conclusions: Adiponectin‐expressing Treg may be candidates for adoptive cell therapy against triple‐negative breast cancer. [ABSTRACT FROM AUTHOR]

This review summarizes information about cell-in-cell (CIC) structures with a focus on gastrointestinal tumors. The phenomenon when one cell lives in another one has attracted an attention of researchers over the past decades. We briefly discuss types of CIC structures and mechanisms of its formation, as well as the biological basis and consequences of the cell-engulfing process. Numerous clinico-histopathological studies demonstrate the significance of these structures as prognostic factors, mainly correlated with negative prognosis. The presence of CIC structures has been identified in all gastrointestinal tumors. However, the majority of studies concern pancreatic cancer. In this field, in addition to the assessment of the prognostic markers, the attempts to manipulate the ability of cells to form CISs have been done in order to stimulate the death of the inner cell. Number of CIC structures also correlates with genetic features for some gastrointestinal tu-mors. The role of CIC structures in the responses of tumors to therapies, both chemotherapy and immunotherapy, seems to be the most poorly studied. However, there is some evidence of involvement of CIC structures in treatment failure. Here, we summarized the current literature on CIC structures in cancer with a focus on gastrointestinal tumors and specified future perspectives for investigation. [ABSTRACT FROM AUTHOR]

Entosis is a non‐apoptotic cell death process that forms characteristic cell‐in‐cell structures in cancers, killing invading cells. Intracellular Ca2+ dynamics are essential for cellular processes, including actomyosin contractility, migration, and autophagy. However, the significance of Ca2+ and Ca2+ channels participating in entosis is unclear. Here, it is shown that intracellular Ca2+ signaling regulates entosis via SEPTIN‐Orai1‐Ca2+/CaM‐MLCK‐actomyosin axis. Intracellular Ca2+ oscillations in entotic cells show spatiotemporal variations during engulfment, mediated by Orai1 Ca2+ channels in plasma membranes. SEPTIN controlled polarized distribution of Orai1 for local MLCK activation, resulting in MLC phosphorylation and actomyosin contraction, leads to internalization of invasive cells. Ca2+ chelators and SEPTIN, Orai1, and MLCK inhibitors suppress entosis. This study identifies potential targets for treating entosis‐associated tumors, showing that Orai1 is an entotic Ca2+ channel that provides essential Ca2+ signaling and sheds light on the molecular mechanism underlying entosis that involves SEPTIN filaments, Orai1, and MLCK. [ABSTRACT FROM AUTHOR]

Abstract Background Pancreatic cancer (PC) is a highly malignant tumor featured with high intra-tumoral heterogeneity and poor prognosis. Cell-in-cell (CIC) structures have been reported in multiple cancers, and their presence is associated with disease progression. Nonetheless, the prognostic values and biological functions of CIC-related genes in PC remain poorly understood. Methods The sequencing data, as well as corresponding clinicopathological information of PC were collected from public databases. Random forest screening, least absolute shrinkage, and selection operator (LASSO) regression and multivariate Cox regression analysis were performed to construct a prognostic model. The effectiveness and robustness of the model were evaluated using receiver operating characteristic (ROC) curves, survival analysis and establishing the nomogram model. Functional enrichment analyses were conducted to annotate the biological functions. The immune infiltration levels were evaluated by ESTIMATE and CIBERSORT algorithms. The expression of KRT7 (Keratin 7) was validated by quantitative real-time PCR (qRT-PCR), western blotting and immunohistochemistry (IHC) staining. The CIC formation, cell clusters, cell proliferation, migration and invasion assays were applied to investigate the effects of silencing the expression of KRT7. Results A prognostic model based on four CIC-related genes was constructed to stratify the patients into the low- and high-risk subgroups. The high-risk group had a poorer prognosis, higher tumor mutation burden and lower immune cell infiltration than the low-risk group. Functional enrichment analyses showed that numerous terms and pathways associated with invasion and metastasis were enriched in the high-risk group. KRT7, as the most paramount risk gene in the prognostic model, was significantly associated with a worse prognosis of PC in TCGA dataset and our own cohort. High expression of KRT7 might be responsible for the immunosuppression in the PC microenvironment. KRT7 knockdown was significantly suppressed the abilities of CIC formation, cell cluster, cell proliferation, migration, and invasion in PC cell lines. Conclusions Our prognostic model based on four CIC-related genes has a significant potential in predicting the prognosis and immune microenvironment of PC, which indicates that targeting CIC processes could be a therapeutic option with great interests. Further studies are needed to reveal the underlying molecular mechanisms and biological implications of CIC phenomenon and related genes in PC progression.

Abstract Entosis is a non‐apoptotic cell death process that forms characteristic cell‐in‐cell structures in cancers, killing invading cells. Intracellular Ca2+ dynamics are essential for cellular processes, including actomyosin contractility, migration, and autophagy. However, the significance of Ca2+ and Ca2+ channels participating in entosis is unclear. Here, it is shown that intracellular Ca2+ signaling regulates entosis via SEPTIN‐Orai1‐Ca2+/CaM‐MLCK‐actomyosin axis. Intracellular Ca2+ oscillations in entotic cells show spatiotemporal variations during engulfment, mediated by Orai1 Ca2+ channels in plasma membranes. SEPTIN controlled polarized distribution of Orai1 for local MLCK activation, resulting in MLC phosphorylation and actomyosin contraction, leads to internalization of invasive cells. Ca2+ chelators and SEPTIN, Orai1, and MLCK inhibitors suppress entosis. This study identifies potential targets for treating entosis‐associated tumors, showing that Orai1 is an entotic Ca2+ channel that provides essential Ca2+ signaling and sheds light on the molecular mechanism underlying entosis that involves SEPTIN filaments, Orai1, and MLCK.

Homotypic entotic figures, which are a form of "cell-in-cell" structures, are considered a potential novel independent prognostic marker in various cancers. Nevertheless, the knowledge concerning the biological role of this phenomenon is still unclear. Since breast cancer cells are remarkably entosis-competent, we aimed to investigate and compare the frequency of entoses in a primary breast tumor and in its lymph node metastasis. Moreover, as there are limited data on defined molecular markers of entosis, we investigated entosis in correlation with classical breast cancer biomarkers used in routine pathomorphological diagnostics (HER2, ER, PR, and Ki67). In the study, a cohort of entosis-positive breast cancer samples paired into primary lesions and lymph node metastases was used. The inclusion criteria were a diagnosis of NOS cancer, lymph node metastases, the presence of entotic figures in the primary lesion, and/or lymph node metastases. In a selected, double-negative, HER2-positive NOS breast cancer case, entoses were characterized by a correlation between an epithelial–mesenchymal transition and proliferation markers. We observed that in the investigated cohort entotic figures were positively correlated with Ki67 and HER2, but not with ER or PR markers. Moreover, for the first time, we identified Ki67-positive mitotic inner entotic cells in clinical carcinoma samples. Our study performed on primary and secondary breast cancer specimens indicated that entotic figures, when examined by routine HE histological staining, present potential diagnostic value, since they correlate with two classical prognostic factors of breast cancer. [ABSTRACT FROM AUTHOR]

Cell-in-cell (CIC) structures in breast cancer have so far been studied in a small inhomogeneous patient population, suggesting the prognostic importance of CIC. In the present study, we focused on CIC in early hormone-sensitive breast cancer. With in vitro co-culture experiments, we compared the homotypic phagocytic capacity of two breast cancer cell lines to that of primary human fibroblasts. Afterward, we studied 601 tissue specimens from 147 patients participating in an institutional accelerated partial breast irradiation (APBI) phase II trial. Both breast cancer cell lines performed non-professional phagocytosis at a higher rate than primary human fibroblasts. In this study cohort, 93.2% of the patients had T1 tumours, and 6.8% had T2 tumours. CIC was found in 61.2% of the patients, with a CIC rate ranging from <1/mm2 to 556.5/mm2 with a mean of 30.9/mm2 ± 68.4/mm2. CIC structures were prognostically favourable for local recurrence-free survival and disease-free survival. Regarding metastasis-free survival, CIC-positive patients had an unfavourable prognosis. Subgroup analysis indicated a correlation between a high proliferation index and high CIC rates. CIC had the highest prognostic value in young breast cancer patients (p = 0.004). With this study, we provide further evidence of CIC as a prognostic marker in breast cancer. [ABSTRACT FROM AUTHOR]

Background: Pancreatic cancer (PC) is a highly malignant tumor featured with high intra-tumoral heterogeneity and poor prognosis. Cell-in-cell (CIC) structures have been reported in multiple cancers, and their presence is associated with disease progression. Nonetheless, the prognostic values and biological functions of CIC-related genes in PC remain poorly understood.Methods: The sequencing data, as well as corresponding clinicopathological information of PC were collected from public databases. Random forest screening, least absolute shrinkage, and selection operator (LASSO) regression and multivariate Cox regression analysis were performed to construct a prognostic model. The effectiveness and robustness of the model were evaluated using receiver operating characteristic (ROC) curves, survival analysis and establishing the nomogram model. Functional enrichment analyses were conducted to annotate the biological functions. The immune infiltration levels were evaluated by ESTIMATE and CIBERSORT algorithms. The expression of KRT7 (Keratin 7) was validated by quantitative real-time PCR (qRT-PCR), western blotting and immunohistochemistry (IHC) staining. The CIC formation, cell clusters, cell proliferation, migration and invasion assays were applied to investigate the effects of silencing the expression of KRT7.Results: A prognostic model based on four CIC-related genes was constructed to stratify the patients into the low- and high-risk subgroups. The high-risk group had a poorer prognosis, higher tumor mutation burden and lower immune cell infiltration than the low-risk group. Functional enrichment analyses showed that numerous terms and pathways associated with invasion and metastasis were enriched in the high-risk group. KRT7, as the most paramount risk gene in the prognostic model, was significantly associated with a worse prognosis of PC in TCGA dataset and our own cohort. High expression of KRT7 might be responsible for the immunosuppression in the PC microenvironment. KRT7 knockdown was significantly suppressed the abilities of CIC formation, cell cluster, cell proliferation, migration, and invasion in PC cell lines.Conclusions: Our prognostic model based on four CIC-related genes has a significant potential in predicting the prognosis and immune microenvironment of PC, which indicates that targeting CIC processes could be a therapeutic option with great interests. Further studies are needed to reveal the underlying molecular mechanisms and biological implications of CIC phenomenon and related genes in PC progression. [ABSTRACT FROM AUTHOR]

Abstract Melatonin has been proposed as a potent anticarcinogen presents a short half-life for osteosarcoma (OS). Cell-in-cell (CIC) structures play a role in the development of malignant tumors by changing the tumor cell energy metabolism. This study developed a melatonin-loaded 3D printed magnesium–polycaprolactone (Mg–PCL) scaffold and investigated its effect and molecular mechanism on CIC in OS. Mg–PCL scaffold was prepared by 3D-printing and its characteristic was determined. The effect and molecular mechanism of Mg–PCL scaffold as well as melatonin-loaded Mg–PCL on OS growth and progression were investigated in vivo and in vitro. We found that melatonin receptor 1 (MT1) and CIC expressions were increased in OS tissues and cells. Melatonin treatment inhibit the key CIC pathway, Rho/ROCK, through the cAMP/PKA signaling pathway, interfering with the mitochondrial physiology of OS cells, and thus playing an anti-invasion and anti-metastasis role in OS. The Mg–PCL–MT could significantly inhibit distant organ metastasis of OS in the in vivo model. Our results showed that melatonin-loaded Mg–PCL scaffolds inhibited the proliferation, invasion and metastasis of OS cells through the CIC pathway. The Mg–PCL–MT could be a potential therapeutics for OS.

Abstract Background Spindle cell squamous cell carcinoma is an uncommon variant of squamous cell carcinoma; its diagnosis is sometimes challenging because it histopathologically resembles neoplastic or reactive spindle cell lesions of mesenchymal origins. Here, we report a rare case of spindle cell squamous cell carcinoma exhibiting prominent neutrophil phagocytosis. Case presentation A 69-year-old Japanese man presented with pain and a polypoid mass on the lower left gingiva. He had received chemoradiotherapy for squamous cell carcinoma of the buccal mucosa 15 years prior to this consultation. In addition, he was treated for mandibular osteonecrosis 6 years after chemoradiotherapy without evidence of cancer recurrence. A biopsy revealed atypical spindle or pleomorphic cells scattered in the edematous and fibrin-rich stroma; however, no malignant squamous components were apparent. These atypical cells frequently contained neutrophils within their cytoplasm that formed cell-in-cell figures. Immunohistochemically, the atypical cells were negative for cytokeratins, epithelial membrane antigen, and E-cadherin, but positive for p63, vimentin, and p53. Although these findings suggested spindle cell squamous cell carcinoma, it was difficult to reach a definitive diagnosis. Based on a clinical diagnosis of a malignant tumor, the patient underwent a hemimandibulectomy. The surgically resected specimen had a typical spindle cell squamous cell carcinoma histology consisting of biphasic spindle cells and conventional squamous cell carcinoma components. Moreover, the surgical specimen also exhibited spindle tumor cells that frequently included neutrophils, around which intense staining for lysosomal-associated membrane protein 1 and cathepsin B was observed. This suggested that the cell-in-cell figures represent active neutrophil phagocytosis by tumor cells, and not emperipolesis. Conclusion The presence of neutrophil phagocytosis may be a potent indicator of malignancy.

Entosis is a mechanism of cell death that involves neighbor cell ingestion. This process occurs in cancers and promotes a form of cell competition, where winner cells engulf and kill losers. Entosis is driven by a mechanical differential that allows softer cells to eliminate stiffer cells. While this process can be induced by matrix detachment, whether other stressors can activate entosis is unknown. Here, we find that entosis is induced in adherent cells by glucose withdrawal. Glucose withdrawal leads to a bimodal distribution of cells based on their deformability, where stiffer cells appear in a manner requiring the energy-sensing AMP-activated protein kinase (AMPK). We show that loser cells with high levels of AMPK activity are eliminated by winners through entosis, which supports winner cell proliferation under nutrient-deprived conditions. Our findings demonstrate that entosis serves as a cellular response to metabolic stress that enables nutrient recovery through neighbor cell ingestion.

Abstract Background Research on cell-in-cell (CIC) phenomena, including entosis, emperipolesis and cannibalism, and their biological implications has increased in recent years. Homotypic and heterotypic engulfment of various target cells by numerous types of host cells has been studied in vitro and in tissue sections. This work has identified proteins involved in the mechanism and uncovered evidence for CIC as a potential histopathologic predictive and prognostic marker in cancer. Our experimental study focused on non-professional phagocytosis of leukocytes. Results We studied the engulfment of peripheral blood mononuclear cells isolated from healthy donors by counting CIC structures. Two non-tumorigenic cell lines (BEAS-2B, SBLF-9) and two tumour cell lines (BxPC3, ICNI) served as host cells. Immune cells were live-stained and either directly co-incubated or treated with irradiation or with conventional or microwave hyperthermia. Prior to co-incubation, we determined leukocyte viability for each batch via Annexin V-FITC/propidium iodide staining. All host cells engulfed their targets, with uptake rates ranging from 1.0% ± 0.5% in BxPC3 to 8.1% ± 5.0% in BEAS-2B. Engulfment rates of the cancer cell lines BxPC3 and ICNI (1.6% ± 0.2%) were similar to those of the primary fibroblasts SBLF-9 (1.4% ± 0.2%). We found a significant negative correlation between leukocyte viability and cell-in-cell formation rates. The engulfment rate rose when we increased the dose of radiotherapy and prolonged the impact time. Further, microwave hyperthermia induced higher leukocyte uptake than conventional hyperthermia. Using fluorescent immunocytochemistry to descriptively study the proteins involved, we detected ring-like formations of diverse proteins around the leukocytes, consisting, among others, of α-tubulin, integrin, myosin, F-actin, and vinculin. These results suggest the involvement of actomyosin contraction, cell-cell adhesion, and the α-tubulin cytoskeleton in the engulfment process. Conclusions Both non-tumorigenic and cancer cells can form heterotypic CIC structures by engulfing leukocytes. Decreased viability and changes caused by microwave and X-ray irradiation trigger non-professional phagocytosis.

The internalization of apoptotic cells by non-phagocytic cells has been observed in different tissues and could be an important mechanism for the elimination of dying cells. Here, we describe a probable event of phagocytosis of apoptotic cells mediated by urothelial cells in urinary sediment. A 90-years-old male patient was admitted unconscious to the hospital, visible signs included: pale skin and dry mucous membranes, presumptively diagnosed as dehydration. Blood test revealed anaemia (haemoglobin 130 g/L) and hyperglycaemia (glucose 7.8 mmol/L), urinalysis showed a picture of urinary tract infection (leukocyturia and bacteriuria). The microscopic analysis of urinary sediment revealed the presence of urothelial cells and leukocytes internalized in urothelial cells. Anti-CD68 (membrane marker of macrophages) was tested by immunocytochemistry and a negative result was observed. Based on the findings phagocytosis of apoptotic cells mediated by urothelial cells was identified. This phenomenon can be observed in urinary sediment and should not be confused with a neoplastic process since it is a physiological event of cell elimination. [ABSTRACT FROM AUTHOR]

This work describes curious structures formed by the mainly phytopathogenic mycoplasma Acholeplasma laidlawii, as well as the human pathogen Ureaplasma parvum cells which resemble cell-in-cell structures of higher eukaryotes and protists. The probable significance of such structures for the mycoplasma cell is discussed. The possibility of their formation in nature and their potential role in the transformation of genetic material, for example, by maintaining (on the one hand) the stability of the genome in the line of generations during asexual reproduction or (on the other hand) the genome plasticity, are substantiated. It should be especially noted that all the arguments presented are based only on morphological data. However, closer attention to unusual structures, the existence of which was shown by electron microscopy images in this case, may prompt researchers to analyze their data more carefully and find something rare and non-trivial among seemingly trivial things. If it is proven by additional methods that cell-in-cell structures can indeed be formed by prokaryotes without a cell wall, this phenomenon may acquire general biological significance. [ABSTRACT FROM AUTHOR]

Abstract Background Non-professional phagocytosis is usually triggered by stimuli such as necrotic cell death. In tumor therapy, the tumors often disappear slowly and only long time after the end of therapy. Here, tumor therapy inactivates the cells by inducing senescence. Therefore, study focused whether senescence is a stimulus for non-professional phagocytosis or whether senescent cells themselves phagocytize non-professionally. Results Senescence was induced in cell lines by camptothecin and a phagocytosis assay was performed. In tissue of a cohort of 192 rectal cancer patients senescence and non-professional phagocytosis was studied by anti-histone H3K9me3 and anti-E-cadherin staining. Senescent fibroblasts and pancreas carcinoma cells phagocytize necrotic cells but are not phagocytized. In the tissue of rectal carcinoma, senescent cells can phagocytize and can be phagocytized. A high number of senescent cells and, at the same time, high numbers of non-professional phagocytizing cells in the rectal carcinoma tissue lead to an extremely unfavorable prognosis regarding overall survival. Conclusion Senescent cells can be non-professionally phagocytized and at the same time they can non-professionally phagocytize in vivo. In vitro experiments indicate that it is unlikely that senescence is a strong trigger for non-professional phagocytosis. Combined high rates of non-professional phagocytosis and high rates of senescence are an extremely poor prognostic factor for overall survival.

Cell-in-cell is a unique phenomenon mostly documented in human cancer tissues. A recent study demonstrated that cell-in-cell might promote lymphopenia by internalizing and killing immune cells in COVID-19, which implicates cell-in-cell as an emerging player in a broader spectrum of pathological processes, such as immune dysregulation.

We describe a case of a young patient with a recurrent pleomorphic xanthoastrocytoma (PXA) showing unusual cell-in-cell (CiC) phenomena. We observed mostly viable but also necrotic neutrophils engulfed within tumor cells. The recurrent tumor was immunopositive for BRAFV600E mutant protein and showed CDKN2 homozygous deletions typical of PXA. Both genetic alterations were also reported in the original primary tumor. Unlike the original tumor that was GFAP and Olig-2 immunopositive, the recurrent neoplasm was largely negative for GFAP and Olig-2 suggesting dedifferentiation. The large malignant cells that contained the neutrophils were negative for histiocytic and lymphohematopoietic markers. Whereas CDKN2 homozygous deletion is common in PXA, its presence is rare in histiocytic neoplasms. Both reactive astrocytes and glial neoplasms very rarely may engulf neutrophils in a process resembling emperipolesis or cellular cannibalism. Future work may clarify which type of CiC pathway is involved., Competing Interests: The authors have no conflicts of interest to report.

Cell-in-cell (CIC) structures in breast cancer have so far been studied in a small inhomogeneous patient population, suggesting the prognostic importance of CIC. In the present study, we focused on CIC in early hormone-sensitive breast cancer. With in vitro co-culture experiments, we compared the homotypic phagocytic capacity of two breast cancer cell lines to that of primary human fibroblasts. Afterward, we studied 601 tissue specimens from 147 patients participating in an institutional accelerated partial breast irradiation (APBI) phase II trial. Both breast cancer cell lines performed non-professional phagocytosis at a higher rate than primary human fibroblasts. In this study cohort, 93.2% of the patients had T1 tumours, and 6.8% had T2 tumours. CIC was found in 61.2% of the patients, with a CIC rate ranging from 2 to 556.5/mm2 with a mean of 30.9/mm2 ± 68.4/mm2. CIC structures were prognostically favourable for local recurrence-free survival and disease-free survival. Regarding metastasis-free survival, CIC-positive patients had an unfavourable prognosis. Subgroup analysis indicated a correlation between a high proliferation index and high CIC rates. CIC had the highest prognostic value in young breast cancer patients (p = 0.004). With this study, we provide further evidence of CIC as a prognostic marker in breast cancer.

Melatonin has been proposed as a potent anticarcinogen presents a short half-life for osteosarcoma (OS). Cell-in-cell (CIC) structures play a role in the development of malignant tumors by changing the tumor cell energy metabolism. This study developed a melatonin-loaded 3D printed magnesium–polycaprolactone (Mg–PCL) scaffold and investigated its effect and molecular mechanism on CIC in OS. Mg–PCL scaffold was prepared by 3D-printing and its characteristic was determined. The effect and molecular mechanism of Mg–PCL scaffold as well as melatonin-loaded Mg–PCL on OS growth and progression were investigated in vivo and in vitro. We found that melatonin receptor 1 (MT1) and CIC expressions were increased in OS tissues and cells. Melatonin treatment inhibit the key CIC pathway, Rho/ROCK, through the cAMP/PKA signaling pathway, interfering with the mitochondrial physiology of OS cells, and thus playing an anti-invasion and anti-metastasis role in OS. The Mg–PCL–MT could significantly inhibit distant organ metastasis of OS in the in vivo model. Our results showed that melatonin-loaded Mg–PCL scaffolds inhibited the proliferation, invasion and metastasis of OS cells through the CIC pathway. The Mg–PCL–MT could be a potential therapeutics for OS. [ABSTRACT FROM AUTHOR]

Background: Spindle cell squamous cell carcinoma is an uncommon variant of squamous cell carcinoma; its diagnosis is sometimes challenging because it histopathologically resembles neoplastic or reactive spindle cell lesions of mesenchymal origins. Here, we report a rare case of spindle cell squamous cell carcinoma exhibiting prominent neutrophil phagocytosis.Case Presentation: A 69-year-old Japanese man presented with pain and a polypoid mass on the lower left gingiva. He had received chemoradiotherapy for squamous cell carcinoma of the buccal mucosa 15 years prior to this consultation. In addition, he was treated for mandibular osteonecrosis 6 years after chemoradiotherapy without evidence of cancer recurrence. A biopsy revealed atypical spindle or pleomorphic cells scattered in the edematous and fibrin-rich stroma; however, no malignant squamous components were apparent. These atypical cells frequently contained neutrophils within their cytoplasm that formed cell-in-cell figures. Immunohistochemically, the atypical cells were negative for cytokeratins, epithelial membrane antigen, and E-cadherin, but positive for p63, vimentin, and p53. Although these findings suggested spindle cell squamous cell carcinoma, it was difficult to reach a definitive diagnosis. Based on a clinical diagnosis of a malignant tumor, the patient underwent a hemimandibulectomy. The surgically resected specimen had a typical spindle cell squamous cell carcinoma histology consisting of biphasic spindle cells and conventional squamous cell carcinoma components. Moreover, the surgical specimen also exhibited spindle tumor cells that frequently included neutrophils, around which intense staining for lysosomal-associated membrane protein 1 and cathepsin B was observed. This suggested that the cell-in-cell figures represent active neutrophil phagocytosis by tumor cells, and not emperipolesis.Conclusion: The presence of neutrophil phagocytosis may be a potent indicator of malignancy. [ABSTRACT FROM AUTHOR]

Background: Research on cell-in-cell (CIC) phenomena, including entosis, emperipolesis and cannibalism, and their biological implications has increased in recent years. Homotypic and heterotypic engulfment of various target cells by numerous types of host cells has been studied in vitro and in tissue sections. This work has identified proteins involved in the mechanism and uncovered evidence for CIC as a potential histopathologic predictive and prognostic marker in cancer. Our experimental study focused on non-professional phagocytosis of leukocytes. Results: We studied the engulfment of peripheral blood mononuclear cells isolated from healthy donors by counting CIC structures. Two non-tumorigenic cell lines (BEAS-2B, SBLF-9) and two tumour cell lines (BxPC3, ICNI) served as host cells. Immune cells were live-stained and either directly co-incubated or treated with irradiation or with conventional or microwave hyperthermia. Prior to co-incubation, we determined leukocyte viability for each batch via Annexin V-FITC/propidium iodide staining. All host cells engulfed their targets, with uptake rates ranging from 1.0% ± 0.5% in BxPC3 to 8.1% ± 5.0% in BEAS-2B. Engulfment rates of the cancer cell lines BxPC3 and ICNI (1.6% ± 0.2%) were similar to those of the primary fibroblasts SBLF-9 (1.4% ± 0.2%). We found a significant negative correlation between leukocyte viability and cell-in-cell formation rates. The engulfment rate rose when we increased the dose of radiotherapy and prolonged the impact time. Further, microwave hyperthermia induced higher leukocyte uptake than conventional hyperthermia. Using fluorescent immunocytochemistry to descriptively study the proteins involved, we detected ring-like formations of diverse proteins around the leukocytes, consisting, among others, of α-tubulin, integrin, myosin, F-actin, and vinculin. These results suggest the involvement of actomyosin contraction, cell-cell adhesion, and the α-tubulin cytoskeleton in the engulfment process. Conclusions: Both non-tumorigenic and cancer cells can form heterotypic CIC structures by engulfing leukocytes. Decreased viability and changes caused by microwave and X-ray irradiation trigger non-professional phagocytosis. [ABSTRACT FROM AUTHOR]

ObjectiveTo analyze the role of frequency of heterotypic neutrophil-in-tumor structure (FNiT) in the prognosis of patients with buccal mucosa squamous cell carcinoma (BMSCC).MethodsIn vitro, we cocultured BMSCC cell line-H157 with neutrophils to form heterotypic neutrophil-in-tumor structures, which were then subject to fluorescence staining. Clinically, 145 patients were retrospectively enrolled. Associations between FNiT and clinicopathological variables including age, sex, smoking history, drinking history, betel nut chewing, tumor stage, node stage, metastasis, disease stage, lymphovascular invasion, extranodal extension, perineural invasion, and tumor grade were analyzed by chi-square test, and the main endpoints of interest were recurrence-free survival (RFS) and disease-specific survival (DSS) which were analyzed by the Kaplan-Meier method and Cox model.ResultsFluorescent staining results of typical heterotypic neutrophil-in-tumor structure showed that well-differentiated H157 cells had a stronger ability to internalize more neutrophils than poorly-differentiated H157 cells, with the latter often internalizing only one neutrophil or nothing. The mean FNiT was 4.2‰, with a range from 2.3‰ to 7.8‰. A total of 80 patients relapsed and 84 patients died of the disease. The 5-year RFS and DSS rate was 42% and 42%, respectively. Patients with an FNiT≥4.2‰ had a significantly higher risk for locoregional recurrence and cancer-caused death than those with an FNiT

The liver is our largest internal organ and it plays major roles in drug detoxification and immunity, where the ingestion of extracellular material through phagocytosis is a critical pathway. Phagocytosis is the deliberate endocytosis of large particles, microbes, dead cells or cell debris and can lead to cell-in-cell structures. Various types of cell endocytosis have been recently described for hepatic epithelia (hepatocytes), which are non-professional phagocytes. Given that up to 80% of the liver comprises hepatocytes, the biological impact of cell-in-cell structures in the liver can have profound effects in liver regeneration, inflammation and cancer. This review brings together the latest reports on four types of endocytosis in the liver -efferocytosis, entosis, emperipolesis and enclysis, with a focus on hepatocyte biology.

Background: Non-professional phagocytosis is usually triggered by stimuli such as necrotic cell death. In tumor therapy, the tumors often disappear slowly and only long time after the end of therapy. Here, tumor therapy inactivates the cells by inducing senescence. Therefore, study focused whether senescence is a stimulus for non-professional phagocytosis or whether senescent cells themselves phagocytize non-professionally. Results: Senescence was induced in cell lines by camptothecin and a phagocytosis assay was performed. In tissue of a cohort of 192 rectal cancer patients senescence and non-professional phagocytosis was studied by anti-histone H3K9me3 and anti-E-cadherin staining. Senescent fibroblasts and pancreas carcinoma cells phagocytize necrotic cells but are not phagocytized. In the tissue of rectal carcinoma, senescent cells can phagocytize and can be phagocytized. A high number of senescent cells and, at the same time, high numbers of non-professional phagocytizing cells in the rectal carcinoma tissue lead to an extremely unfavorable prognosis regarding overall survival. Conclusion: Senescent cells can be non-professionally phagocytized and at the same time they can non-professionally phagocytize in vivo. In vitro experiments indicate that it is unlikely that senescence is a strong trigger for non-professional phagocytosis. Combined high rates of non-professional phagocytosis and high rates of senescence are an extremely poor prognostic factor for overall survival. [ABSTRACT FROM AUTHOR]

Objective: To analyze the role of frequency of heterotypic neutrophil-in-tumor structure (FNiT) in the prognosis of patients with buccal mucosa squamous cell carcinoma (BMSCC). Methods: In vitro , we cocultured BMSCC cell line-H157 with neutrophils to form heterotypic neutrophil-in-tumor structures, which were then subject to fluorescence staining. Clinically, 145 patients were retrospectively enrolled. Associations between FNiT and clinicopathological variables including age, sex, smoking history, drinking history, betel nut chewing, tumor stage, node stage, metastasis, disease stage, lymphovascular invasion, extranodal extension, perineural invasion, and tumor grade were analyzed by chi-square test, and the main endpoints of interest were recurrence-free survival (RFS) and disease-specific survival (DSS) which were analyzed by the Kaplan-Meier method and Cox model. Results: Fluorescent staining results of typical heterotypic neutrophil-in-tumor structure showed that well-differentiated H157 cells had a stronger ability to internalize more neutrophils than poorly-differentiated H157 cells, with the latter often internalizing only one neutrophil or nothing. The mean FNiT was 4.2‰, with a range from 2.3‰ to 7.8‰. A total of 80 patients relapsed and 84 patients died of the disease. The 5-year RFS and DSS rate was 42% and 42%, respectively. Patients with an FNiT≥4.2‰ had a significantly higher risk for locoregional recurrence and cancer-caused death than those with an FNiT<4.2‰ (p=0.001 and p<0.001, respectively). The FNiT alone was independently significant in predicting poor RFS, and the FNiT along with tumor grade was an independent predictor for DSS. Conclusion: The FNiT as a novel predictor is significantly negatively associated with both the RFS and DSS of patients with BMSCC. [ABSTRACT FROM AUTHOR]

The liver is our largest internal organ and it plays major roles in drug detoxification and immunity, where the ingestion of extracellular material through phagocytosis is a critical pathway. Phagocytosis is the deliberate endocytosis of large particles, microbes, dead cells or cell debris and can lead to cell-in-cell structures. Various types of cell endocytosis have been recently described for hepatic epithelia (hepatocytes), which are non-professional phagocytes. Given that up to 80% of the liver comprises hepatocytes, the biological impact of cell-in-cell structures in the liver can have profound effects in liver regeneration, inflammation and cancer. This review brings together the latest reports on four types of endocytosis in the liver -efferocytosis, entosis, emperipolesis and enclysis, with a focus on hepatocyte biology. [ABSTRACT FROM AUTHOR]

A phenomenon known for over 100 years named “cell-in-cell” (CIC) is now undergoing its renaissance, mostly due to modern cell visualization techniques. It is no longer an esoteric process studied by a few cell biologists, as there is increasing evidence that CICs may have prognostic and diagnostic value for cancer patients. There are many unresolved questions stemming from the difficulties in studying CICs and the limitations of current molecular techniques. CIC formation involves a dynamic interaction between an outer or engulfing cell and an inner or engulfed cell, which can be of the same (homotypic) or different kind (heterotypic). Either one of those cells appears to be able to initiate this process, which involves signaling through cell–cell adhesion, followed by cytoskeleton activation, leading to the deformation of the cellular membrane and movements of both cells that subsequently result in CICs. This review focuses on the distinction of five known forms of CIC (cell cannibalism, phagoptosis, enclysis, entosis, and emperipolesis), their unique features, characteristics, and underlying molecular mechanisms.

Entosis is a mechanism of cell death that involves neighbor cell ingestion. This process occurs in cancers and promotes a form of cell competition, where winner cells engulf and kill losers. Entosis is driven by a mechanical differential that allows softer cells to eliminate stiffer cells. While this process can be induced by matrix detachment, whether other stressors can activate entosis is unknown. Here, we find that entosis is induced in adherent cells by glucose withdrawal. Glucose withdrawal leads to a bimodal distribution of cells based on their deformability, where stiffer cells appear in a manner requiring the energy-sensing AMP-activated protein kinase (AMPK). We show that loser cells with high levels of AMPK activity are eliminated by winners through entosis, which supports winner cell proliferation under nutrient-deprived conditions. Our findings demonstrate that entosis serves as a cellular response to metabolic stress that enables nutrient recovery through neighbor cell ingestion.

The term cell-in-cell, morphologically, refers to the presence of one cell within another. This phenomenon can occur in tumors but also among non-tumor cells. The cell-in-cell phenomenon was first observed 100 years ago, and it has since been found in a variety of tumor types. Recently, increasing attention has been paid to this phenomenon and the underlying mechanism has gradually been elucidated. There are three main related process: cannibalism, emperipolesis, and entosis. These processes are affected by many factors, including the tumor microenvironment, mitosis, and genetic factors. There is considerable evidence to suggest that the cell-in-cell phenomenon is associated with the prognosis of cancers, and it promotes tumor progression in most situations. Notably, in pancreatic cancer, the cell-in-cell phenomenon is associated with reduced metastasis, which is the opposite of what happens in other tumor types. Thus, it can also inhibit tumor progression. Studies show that cell-in-cell structure formation is affected by the tumor microenvironment, and that it may lead to changes in cellular characteristics. In this review, we summarize the different cell-in-cell processes and discuss their role in tumor progression and how they are regulated by different mechanisms.

Heterotypic cell-in-cell structure (CICs) is the definition of the entry of one type of living cells into another type of cell. CICs between immune cells and tumor cells have been found to correlate with malignancy in many cancers. Since tumor immune microenvironment promotes non-small cell lung cancer (NSCLC) progression and drug resistance, we wondered the potential significance of heterotypic CICs in NSCLC. Heterotypic CICs was analyzed by histochemistry in an expanded spectrum of clinical lung cancer tissue specimens. In vitro study was performed using the mouse lung cancer cell line LLC and splenocytes. Our results revealed that CICs formed by lung cancer cells and infiltrated lymphocytes were correlated with malignancy of NSCLC. In addition, we found CICs mediated the transfer of lymphocyte mitochondria to tumor cells, and promoted cancer cell proliferation and anti-cytotoxicity by activating MAPK pathway and up-regulating PD-L1 expression. Furthermore, CICs induces glucose metabolism reprogramming of lung cancer cells by upregulating glucose intake and glycolytic enzyme. Our findings suggest that CICs formed by lung cancer cell and lymphocyte contribute to NSCLC progression and reprogramming of glucose metabolism, and might represent a previously undescribed pathway for drug resistance of NSCLC. [ABSTRACT FROM AUTHOR]

Cell-in-cell structure is prevalent in human cancer, and associated with several specific pathophysiological phenomena. Although cell membrane adhesion molecules were found critical for cell-in-cell formation, the roles of other membrane components, such as lipids, remain to be explored. In this study, we attempted to investigate the effects of cholesterol and phospholipids on the formation of cell-in-cell structures by utilizing liposome as a vector. We found that Lipofectamine-2000, the reagent commonly used for routine transfection, could significantly reduce entotic cell-in-cell formation in a cell-specific manner, which is correlated with suppressed actomyosin contraction as indicated by reduced β-actin expression and myosin light chain phosphorylation. The influence on cell-in-cell formation was likely dictated by specific liposome components as some liposomes affected cell-in-cell formation while some others didn't. Screening on a limited number of lipids, the major components of liposome, identified phosphatidylethanolamine (PE), stearamide (SA), lysophosphatidic acid (LPA) and cholesterol (CHOL) as the inhibitors of cell-in-cell formation. Importantly, cholesterol treatment significantly inhibited myosin light chain phosphorylation, which resembles the effect of Lipofectamine-2000, suggesting cholesterol might be partially responsible for liposomes' effects on cell-in-cell formation. Together, our findings supporting a role of membrane lipids and cholesterol in cell-in-cell formation probably via regulating actomyosin contraction. [ABSTRACT FROM AUTHOR]

The cell-in-cell (CIC) phenomenon has received increasing attention over recent years because of its wide existence in multiple cancer tissues. The mechanism of CIC formation is considerably complex as it involves interactions between two cells. Although the molecular mechanisms of CIC formation have been extensively investigated, the process of CIC formation remains ambiguous. Currently, CIC is classified into four subtypes based on different cell types and inducing factors, and the underlying mechanisms for each subtype are distinct. Here, we investigated the subtypes of CIC and their major mechanisms involved in cancer development. To determine the clinical significance of CIC, we reviewed several clinical studies on CIC and found that CIC could serve as a diagnostic and prognostic biomarker. The implications of CIC on the clinical management of cancers also remain largely unknown. To clarify this aspect, in the present review, we highlight the findings of recent investigations on the causal link between CIC and cancer treatment. We also indicate the existing issues that need to be resolved urgently to provide a potential direction for future research on CIC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Liu and Yang.)

Entosis is a non-apoptotic cell death process that forms characteristic cell-in-cell structures in cancers, killing invading cells. Intracellular Ca 2+ dynamics are essential for cellular processes, including actomyosin contractility, migration, and autophagy. However, the significance of Ca 2+ and Ca 2+ channels participating in entosis is unclear. Here, it is shown that intracellular Ca 2+ signaling regulates entosis via SEPTIN-Orai1-Ca 2+ /CaM-MLCK-actomyosin axis. Intracellular Ca 2+ oscillations in entotic cells show spatiotemporal variations during engulfment, mediated by Orai1 Ca 2+ channels in plasma membranes. SEPTIN controlled polarized distribution of Orai1 for local MLCK activation, resulting in MLC phosphorylation and actomyosin contraction, leads to internalization of invasive cells. Ca 2+ chelators and SEPTIN, Orai1, and MLCK inhibitors suppress entosis. This study identifies potential targets for treating entosis-associated tumors, showing that Orai1 is an entotic Ca 2+ channel that provides essential Ca 2+ signaling and sheds light on the molecular mechanism underlying entosis that involves SEPTIN filaments, Orai1, and MLCK., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)