Background
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 [
1]. So far, three different mechanisms of cell-to-cell interaction have been described involving tumor cells: cannibalism, emperipolesis and entosis [
2]. 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 [
2]. 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.
Discussion
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 [
6], a squamous cell carcinoma of the skin [
7,
8], a poorly differentiated squamous carcinoma of the lung [
9] and a biphasic mesothelioma of the pleura [
10]. 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 [
11], as well as cannibalism in malignant effusions, but this does not necessarily mean that cannibal cells were tumor cells themselves [
12]. 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 [
13], 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 [
11]. 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 [
14]. 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 [
2].
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 [
15] and lymphoma [
16], 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 [
2]. 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 [
5]. 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 [
11]. 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 [
19]. 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 [
5]. 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 [
20].
Conclusions
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.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LB and GV were responsible for the clinical part of two of the cases. MCP, JC and SB carried out the pathology, being responsible for the necropsies and the histological diagnosis. MJS was responsible for the clinical part of one of the cases and, together with SC, carried out the immunohistochemistry. FCF and MCP wrote the manuscript. All authors read and approved the final manuscript.