Four cases of cell cannibalism in highly malignant feline and canine tumors
© Ferreira et al. 2015
Received: 6 April 2015
Accepted: 17 October 2015
Published: 2 November 2015
Four cases of tumors in which cell internalization was frequently visualized are reported: one feline mammary carcinoma, one feline cutaneous squamous cell carcinoma, one canine pulmonary squamous cell carcinoma and one canine pleural mesothelioma. Cell internalization was observed by cytology in two of these cases (the feline mammary tumour and the pleural effusion in the canine mesothelioma) and by histopathology in all but the canine mesothelioma. Immunohistochemical staining for pancytokeratin was positive for both internalized and host cells, while E-cadherin expression was frequently absent, although internalized cells occasionally stained positive. This cell-to-cell interaction seems to be associated with tumors displaying a strong epithelial-mesenchymal transitional phenotype, in which cancer cells become engulfed by other cancer cells. Such event could be regarded as an important hallmark of very high malignancy.
Cell-to-cell interaction is a common phenomenon reported in inflammatory tissues, where macrophages engulf dying neutrophils and foreign material in a cooperative process during inflammatory/infectious diseases . So far, three different mechanisms of cell-to-cell interaction have been described involving tumor cells: cannibalism, emperipolesis and entosis . Briefly, cannibalism is the active internalization and destruction of dead or living tumor cells by other engulfing cells; emperipolesis is the phagocytosis of intact hematopoietic cells, mainly neutrophils, lymphocytes and plasma cells by host cancer cells; and entosis is a mechanism of homogenous live-cell invasion resembling a parasite-cell interaction, such that the invading cell seems to take the initiative in being internalized . Recent studies have proven that these mechanisms have different cell recognition and cell penetration strategies [3–5]. However, there is much speculation around the possible benefits of such cell internalization events to either tumor or host.
In this study, the authors describe and discuss four cases of highly malignant tumors in which cell internalization was a frequent phenomenon. To the best of our knowledge, these internalization mechanisms have not been considered relevant in establishing diagnosis or prognosis in veterinary oncology.
A 9-year-old spayed European shorthair female cat was euthanized at the owner’s request, after a two-month history of a rapidly growing right inguinal mammary carcinoma diagnosed by cytology. At necropsy, a 4 cm nodule was present in the right inguinal mammary gland, along with several metastatic nodules identified in lymph nodes, lungs and in various muscles. Tissue samples were processed as usual for routine microscopical observation.
Antibodies used and technique details
Dilution, incubation time
Clone AE1/AE3 (DAKO)
Microwave 900W 5 min + 600W 15 min (Tris EDTA pH 9.0, Novocastra™ Epitope Retrieval Solution, Leica Biosystems, Wetzlar, Germany
1:100 60min RT
Novolink (Leica Biosystems)
Clone V9 (DAKO)
Microwave 900W 15 min (Tris EDTA pH 9.0, Novocastra™ Epitope Retrieval Solution, Leica Biosystems, Wetzlar, Germany)
1:50 60 min RT
Novolink (Leica Biosystems)
Clone 36 (Ventana, Tucson, USA)
Microwave 600W 15 min (citrate buffer pH 6.0)
Ready to Use 60 min RT
Novolink (Leica Biosystems)
Immunohistochemical staining for pancytokeratin and E-cadherin were again performed as described in case one. Both tumor and internalized cells exhibited a strong cytoplasmic staining for pancytokeratin (Fig. 2c), while only cannibalizing cells consistently stained positively for E-cadherin. Internalized cells only occasionally stained for this marker (Fig. 2d).
The cat was euthanized at the owner’s request one month after surgery, due to severe ulceration of the skin in the area were the tumor was first removed, with no response to therapy.
To confirm the epithelial nature of internalized cells, immunohistochemistry for pancytokeratin was performed according to the protocol described in Table 1. Tumor cells exhibited a strong cytoplasmic staining for pancytokeratin, along with the internalized cells (Fig. 3b).
Immunohistochemical labeling with pancytokeratin and vimentin was employed to confirm the diagnosis of mesothelioma (protocol available in Table 1). The sarcomatoid cells were positive for vimentin (Fig. 4c) and the epithelial cells positive for cytokeratin (Fig. 4d).
All tumors described here were diagnosed according to standardized classification criteria and were shown to be particularly aggressive. Indeed, all cases reported correspond to highly malignant categories within the tissues they affected: a solid carcinoma of the mammary gland , a squamous cell carcinoma of the skin [7, 8], a poorly differentiated squamous carcinoma of the lung  and a biphasic mesothelioma of the pleura . Two of them (cases 1 and 2) showed lymphatic invasion and in two cases (1 and 3) strong metastatic potential was proven. In the first three cases, images of cell internalization were identified by histology of the primary tumors and also in the lung metastasis of case 1. Regarding mesothelioma in case 4, images of cell internalization were only found in the pleural effusion fluid. In fact, phagocytosis of dying tumor cells by human peritoneal mesothelial cells has been demonstrated , as well as cannibalism in malignant effusions, but this does not necessarily mean that cannibal cells were tumor cells themselves . The large number of cannibal cells present in the pleural effusion could represent cell cannibalism of tumor cells by other tumor cells, or the phagocytosis of exfoliating tumor cells by mesothelial cells. Morphology and immunohistochemistry are not helpful in distinguishing normal from neoplastic mesothelial cells , Cell cannibalism as the internalization of cells by other cells was first reported in human malignant tumors over a century ago, having been described as a diagnostic marker of malignancy in cytology specimens . The potent phagocytic activity of tumor cells has been referred to as a determinant of aggressive behavior in metastatic melanoma cells, with proteins such as ezrin being expressed on phagocytic vacuoles . The internalized cells may remain alive for some time, but will eventually be digested by specific enzymes within a cannibalistic vacuole; the inhibition or down regulation of these enzymes seems to affect the phagocytic activity of tumor cells [3, 5]. It has been suggested that cannibal cells feed off the internalized cells, becoming particularly resistant to unfavorable conditions caused by low nutrient supply in quickly growing solid tumors .
The other described cell-in-cell event is emperipolesis, which affects hematopoietic cells, mainly lymphocytes that establish close connections with the target cell’s membrane that culminate in their internalization. Although emperipolesis has been described in human mammary tumors  and lymphoma , the fact that the internalized cells in the presented case were not lymphocytes, or any other type of hematopoietic cell, excludes this hypothesis. In fact, cytokeratin staining was detected in both internalized and engulfing cells, demonstrating their identical epithelial nature.
The less referred to cell-in-cell phenomenon is entosis, through which live epithelial tumor cells detach from the extracellular matrix and invade the neighboring cells, also of epithelial origin . Live internalized cells may either be degraded by lysosomal enzymes or be released. The process has been described as a non-apoptotic cell death program driven by compaction force associated with adherens junction formation, representing an intrinsic tumor suppression mechanism for cells that are detached from the extracellular matrix . This event was reported in pleural suspensions of human metastatic mammary tumor cells, showing negative staining for apoptotic markers, leading to the conclusion that the internalization process could be associated with entosis . E-cadherin is the main member of the cadherin superfamily involved in epithelial cellular adhesion, and its altered expression has been related to poor differentiation and poor prognosis, in human and canine mammary tumors [17, 18]. In feline mammary metastatic tumors, the loss of both E-cadherin and β-catenin expression was shown to be more pronounced than in non-metastatic neoplasia . Cells in culture were demonstrated to require adherens junctions to initiate entosis, the mechanism being directly linked to cell-cell compaction force in the absence of integrin . In the cases reported here, the generally negative staining obtained for E-cadherin in internalized cells could be due to its altered expression, which would be in accordance with the strong malignant behavior of the tumor. However, no conclusion can be drawn as to whether such cells had functional adherens junctions prior to engulfment.
It may be easily accepted that, similarly to what is reported in humans, cell-in-cell events also take place in tumors in animals. To the best of the authors’ knowledge, these phenomena have been rarely described and valued in veterinary oncology. The only reference to the phenomenon is very recent report involving three cases of cell cannibalism of which only one is of homogenous internalization of cancer cells .
The authors believe that the cell internalization phenomena in the presently described tumors could be qualified as cannibalism, as there is evidence of degradation of the internalized cells. Conversely, it could also be classified as entosis as this is a case of epithelial-in-epithelial cell internalization, eventually evading apoptosis. Clear distinction between both events is beyond the scope of the present work, and does not affect its prime objective: to draw the attention of pathologists to a characteristic that seems to be associated with tumors in which the epithelial-mesenchymal transition is very marked.
Written informed consent was obtained from the animal owners' for the publication of this report and any accompanying images.
The authors wish to thank Dr. Rute Noiva and Dr. Shabir Najmudin for the English revision. This work was supported by ‘Fundação para a Ciência e Tecnologia’ through the Project PEst-OE/AGR/U10276/2014 and through PhD fellowship SFRH/BD/70720/2010.
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- Silva MT. Macrophage phagocytosis of neutrophils at inflammatory/infectious foci: a cooperative mechanism in the control of infection and infectious inflammation. J Leuk Biol. 2010;89:675–83.View ArticleGoogle Scholar
- Yang YLJ. Progress of research in cell-in-cell phenomena. Anat Rec. 2012;295:372–7.View ArticleGoogle Scholar
- Fais S. Cannibalism: a way to feed on metastatic tumors. Cancer Letter. 2007;258:155–64.View ArticleGoogle Scholar
- Mailleux AA, Krajcovic M, Overholtzer M. Le cannibalisme cellulaire par entose. Médecine Sci. 2011;27:689–91.View ArticleGoogle Scholar
- Overholtzer M, Mailleux AA, Mouneimne G, Normand G, Schnitt SJ, King RW, et al. A nonapoptotic cell death process, entosis, that occurs by cell-in-cell invasion. Cell. 2007;131:966–79.View ArticlePubMedGoogle Scholar
- Misdorp W, Else RW, Hellmén E, Lipscomb TP. Histological Classification of Tumors of Mammary Tumors of Domestic Animals. Washington D.C., USA: Armed Forces Institute of Pathology; 1999.Google Scholar
- Goldschmidt MH, Dunstan RW, Stannard AA, Von Tscharner C, Yager JA. Histological Classification of Epithelial and Melanocytic Tumors of the Skin of Domestic Animals. Washington D.C., USA: Armed Forces Institute of Pathology; 1998.Google Scholar
- Gross TL, Ihrke PJ, Walder EJ, Affolter VK. Skin diseases of the dog and cat: clinical and histopathologic diagnosis. 2nd ed. Ames, Iowa, USA: Blackwell Science; 2005.View ArticleGoogle Scholar
- Dungworth DL, Hauser B, Hahn FF, Wilson DW, Haenichen T, Harkema JR. Histological Classification of Tumors of the Respiratory System of Domestic Animals. Washington D.C., USA: Armed Forces Institute of Pathology; 1999.Google Scholar
- Head KW, Else RW, Dubielzig RR. Tumors of the Alimentary Tract. In: Meuten DJ, editor. Tumors in Domestic Animals. 4th ed. Iowa: Iowa State Press; 2002. p. 401–81.Google Scholar
- Wagner BJ, Lindau D, Ripper D, Stierhof YD, Glatzle J, Witte M, et al. Phagocytosis of dying tumor cells by human peritoneal mesothelial cells. J Cell Sci. 2011;124:1644–54.View ArticlePubMedGoogle Scholar
- Gupta K, Dey P. Cell cannibalism: diagnostic marker of malignancy. Diag Cytopath. 2003;28:86–7.View ArticleGoogle Scholar
- Hammar SP. Lung and Pleural Neoplasms. In: Dabbs DJ, editor. Diagnostic Immunohistochemistry. Philadelphia, Pennsylvania, USA: Churchill Livingstone; 2002. p. 267–312.Google Scholar
- Lugini L, Lozupone F, Matarrese P, Funaro C, Luciani F, Malorni W, et al. Potent phagocytic activity discriminates metastatic and primary human malignant melanomas: a key role of Ezrin. Lab Investig. 2003;83:1555–67.View ArticlePubMedGoogle Scholar
- Saxena S, Beena KR, Bansal A, Bhatnagar A. Emperipolesis in a common breast maligancy. a case report. Acta Cytol. 2002;46:883–6.View ArticlePubMedGoogle Scholar
- Lopes LF, Bacchi MM, Coelho KI, Antico Filho A, Bacchi CE. Emperipolesis in a case of B-cell lymphoma: a rare phenomenon outside of Rosai-Dorfman disease. Annals Diag Pathol. 2003;7:310–3.View ArticleGoogle Scholar
- Asgeirsson KS, Jonasson JG, Tryggvadorttir L, Olafsdottir K, Sigurgeirsdottir JR, Ingvarsson S, et al. Altered expression of E-cadherin in breast cancer: patterns, mechanisms and clinical significance. Eur J Cancer. 2000;36:1098–106.View ArticlePubMedGoogle Scholar
- Matos AJF, Lopes C, Carvalheira J, Santos M, Rutteman GR, Gartner F. E-cadherin expression in canine malignant mammary tumours: relationship to other clinico-pathological variables. J Comp Pathol. 2006;134:182–9.View ArticlePubMedGoogle Scholar
- Penafiel-Verdu C, Buendia AJ, Navarro JA, Ramirez GA, Vilafranca M, Altimira J, et al. Reduced expression of e-cadherin and β-catenin and high expression of basal cytokeratins in feline mammary carcinomas with regional metastasis. J Pathol. 2012;49:979–87.Google Scholar
- Meléndez- Lazo A, Cazzini P, Camus M, Doria-Torra G, Valle AJM, Solano-Gallego L et al. Cell cannibalism by malignant neoplastic cells: three cases in dog and a literature review. Vet Clin Pathol. 2015, doi: 10.1111/vcp.12242.