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Loss of contact inhibition of locomotion in the absence of JAM-A promotes entotic cell engulfment

Summary Entosis is a cell competition process during which tumor cells engulf other tumor cells. It is initiated by metabolic stress or by loss of matrix adhesion, and it provides the winning cell with resources derived from the internalized cell. Using micropatterns as substrates for single cell migration, we find that the depletion of the cell adhesion receptor JAM-A strongly increases the rate of entosis in matrix-adherent cells. The activity of JAM-A in suppressing entosis depends on phosphorylation at Tyr280, which is a binding site for C-terminal Src kinase, and which we have previously found to regulate tumor cell motility and contact inhibition of locomotion (CIL). Loss of JAM-A triggers entosis in matrix-adherent cells but not matrix-deprived cells. Our findings strongly suggest that the increased motility and the perturbed CIL response after the depletion of JAM-A promote entotic cell engulfment, and they link a dysregulation of CIL to entosis in breast cancer cells.


INTRODUCTION
Cancer cells have developed a plethora of mechanisms that allow cells to resist the tumor-suppressive activities of the host organism and to survive under conditions of limited nutrient and oxygen supply. For example, anoikis, a cell death program activated in response to the loss of cell-matrix adhesion to prevent cancer cell dissemination (Frisch and Francis, 1994), is antagonized by multiple signaling pathways upregulated in cancer cells (Buchheit et al., 2014). Also, cancer cells undergo profound metabolic reprogramming enabling the cells to scavenge nutrients from nutrient-poor environments (Finicle et al., 2018), and have developed various mechanisms to outcompete other cancer cells as well as non-cancerous cells in the microenvironment (Merino et al., 2016).
Among the various mechanisms involved in cancer cell competition, engulfment of other cells, which results in cell-in-cell structures (''cellular cannibalism'') has been proposed to confer metabolic advantages to the ''winner'' cells (Fais and Overholtzer, 2018). Cell-in-cell structures can occur through an active uptake of a different living cell by a tumor cell through a process that resembles phagocytosis and that has been named phagoptosis (Brown and Neher, 2012;Lugini et al., 2003). Alternatively, cell-in-cell structures can result from an invasion process during which one tumor cell invades another tumor cell of the same type, a process called entosis (Overholtzer et al., 2007). In both cases, the inner cells are, eventually, degraded within a phagolysosomal compartment (Florey et al., 2011;Lugini et al., 2003), leaving the outer cell as the winner cell which benefits from the energy resources provided by the inner cell. Importantly, cellin-cell structures have been observed in numerous cancers (Fais and Overholtzer, 2018), and the presence of cell-in-cell structures has been associated with advanced cancer stages (Krajcovic et al., 2011;Lugini et al., 2003;Mackay et al., 2018), strongly suggesting that the ability to engulf other cells contributes to the malignant phenotype.
Entosis is characterized by the internalization of a living cell. Internalization is driven by Rho kinase (ROCK)dependent actomyosin contractility at the rear end of the invading cell, which is required to generate the mechanical tension necessary for invasion (Overholtzer et al., 2007). The internalized cell persists in an entotic vacuole in which it can divide and from which it can escape (Fais and Overholtzer, 2018). The default pathway, however, involves the maturation of the entotic vacuolar membrane through lipidation, followed by lysosome fusion, death of the cell, and its degradation (Florey et al., 2011). Although apoptosis of the internalized cell has also been observed, specifically when lysosome function or autophagy proteins required for vacuole lipidation are absent (Florey et al., 2011), entotic events observed in clinical cancer specimens were found to occur in the absence of caspase 3 activity defining entosis as non-apoptotic cell death (Overholtzer et al., 2007).
Entosis has been observed in a number of tumor isolates including melanoma and carcinomas of cervix, colon, stomach, and liver (Bozkurt et al., 2021;Overholtzer et al., 2007). On the other hand, entosis is rarely observed in cells grown under regular tissue culture conditions unless cells are detached from the matrix (Durgan and Florey, 2018;Overholtzer et al., 2007), or cells are deprived of nutrients (Hamann et al., 2017). This is most likely owing to the optimized culture conditions under which cells are normally maintained. For example, in two matrix adherent tumor cell lines which readily undergo entosis when cultured in suspension or under conditions of glucose starvation (Hamann et al., 2017;Overholtzer et al., 2007;Sun et al., 2014a;Wang et al., 2016), the rates of entosis are below 2% when grown under regular culture conditions (Garanina et al., 2017). As tumor cells migrating in tissue are exposed to extracellular matrix (ECM) components (Winkler et al., 2020), it will be important to understand in more detail the mechanisms regulating entotic cell engulfment in matrix-adherent cells.
We have recently found that the depletion of the cell adhesion receptor JAM-A in MCF7 tumor cells strongly increases cell motility and abrogates the contact inhibition of locomotion (CIL) (Kummer et al., 2022), a process that contributes to cancer cell dissemination when inappropriately regulated (Stramer and Mayor, 2016). In this study, we describe that JAM-A depletion strongly enhances entotic cell engulfment in matrix-adherent cells. Our results link deregulated motility and perturbed CIL to entotic cell engulfment, and they identify JAM-A as a central regulator of these processes.

Loss of JAM-A promotes homotypic cell engulfment
Using one-dimensional (1D) kinematic assays, in which cells are cultured on functionalized stripes of 5 mm width to promote cell collisions ( Figure 1A), we have recently found that the absence of JAM-A in MCF7 cells results in increased motility as well as in a severely impaired contact inhibition of locomotion (CIL) response when cells collide with other cells (Kummer et al., 2022). The analysis of the underlying molecular mechanism revealed that JAM-A recruits c-Src regulatory kinase (Csk) to limit the activity of Src which is a key regulator of cell motility (Yeatman, 2004). During these experiments, we observed that when JAM-Adepleted cells migrated across collided cells, these were frequently engulfed resulting in cell-in-cell structures ( Figure 1B). Confocal microscopic analyses confirmed that internalized cells were completely surrounded by the outer cells ( Figure 1C). Quantification of cell engulfment indicated that the fraction of cell-in-cell structures strongly increased when JAM-A KD MCF7 cells collided with wildtype (WT) MCF7 cells (KD -WT collisions) as compared to collisions between control KD MCF7 cells and WT MCF7 cells (Ctrl-WT collisions) ( Figure 1D). These observations suggested that the disturbed CIL response after the depletion of JAM-A facilitates homotypic cell engulfment.

Loss of JAM-A facilitates entosis
We next aimed to characterize the cell-in-cell structures observed after JAM-A depletion in detail. We focused on entosis, a cell-in-cell phenomenon that is characterized as an invasive process and that has been found to occur at low frequencies (1.5-2%) in matrix-adherent MCF7 cells (Garanina et al., 2017) but at relatively high frequencies (20-30%) in matrix-deprived MCF7 cells (Overholtzer et al., 2007). During entosis, the invading cell ends up in a membrane-bounded compartment that is positive for Lamp1 and b-catenin (Overholtzer et al., 2007). In most cases, the internalized cells are degraded by lysosomal enzymes within the Lamp1-positive compartment; however, escape of the inner cell, as well as inner cell division, have also been observed (Krishna and Overholtzer, 2016;Overholtzer et al., 2007). We found that in the vast majority of cell-in-cell structures the internalized cell was present in a compartment positive for both Lamp1 and b-catenin ( Figure 2A). The internalized cells displayed various types of behavior which were previously observed during entosis, including cell fragmentation and cell death, escape from the engulfing cell, and division of the inner cell ( Figure 2B and Videos S1, S2, S3, and S4). In time-lapse movies, we also observed lobe-like structures emanating from the inner cells during the process of internalization prior to complete closure (Video S1), similar to what has recently been observed during developmental entosis in C.elegans (Lee et al., 2019). Ultrastructural analyses indicated a firm association between the outer and the ll OPEN ACCESS iScience 25, 105144, October 21, 2022 iScience Article inner cell by intercellular junctions (Figures 2C and S1), which is typically observed during entosis (Garanina et al., 2017;Overholtzer et al., 2007). The formation of cell-in-cell structures after JAM-A depletion required Rho-associated kinase (ROCK) activity ( Figure 2D), which has been described to be necessary for entotic cell engulfment (Overholtzer et al., 2007). JAM-A KD -WT collisions resulted predominantly in cell-incell structures in which the WT cells were engulfed by the JAM-A KD cells ( Figure 2E), suggesting that the increased motility of single cells combined with a loss of contact inhibition observed after JAM-A depletion (Kummer et al., 2022;Tholmann et al., 2022)

prones cells to be invaded by other cells. Similar
to what has been observed in the regulation of CIL (Kummer et al., 2022), we observed no difference in the rate of entosis between heterotypic JAM-A KD vs WT collisions and homotypic JAM-A KD vs JAM-A KD collisions ( Figure S2), further supporting that trans-homphilic JAM-A interaction is required during entosis. Together, these observations suggested that the depletion of JAM-A facilitates cell engulfment of matrix-adherent cells through an entotic process, most likely as a result of loss of CIL combined with increased cell motility after JAM-A depletion.

Regulation of entosis by JAM-A requires Tyr280 phosphorylation of JAM-A and the activity of Csk
The regulation of CIL by JAM-A requires the phosphorylation of JAM-A at Tyr280, which serves as a binding site for Csk to inhibit the activity of Src both in platelets (Naik et al., 2014) and in tumor cells (Kummer et al., 2022). To test the role of JAM-A Tyr280 phosphorylation during entosis of matrix-adherent cells, we analyzed the rate of entosis in JAM-A-depleted MCF7 cells after their reconstitution with shRNA-insensitive mouse JAM-A constructs, either mJAM-A/WT or phosphorylation-deficient mJAM-A/Y281F. Ectopic expression of mJAM-A/WT almost completely suppressed entotic cell-in-cell structures in JAM-A-depleted cells (Figure 3A). In contrast, mJAM-A/Y281F did not suppress entotic engulfment in JAM-A-depleted cells iScience Article ( Figure 3A). As JAM-A Tyr280 phosphorylation mediates recruitment of Csk to regulate CIL in MCF7 cells (Kummer et al., 2022) and to regulate the premature activation of platelets (Naik et al., 2014), we analyzed the frequency of cell-in-cell structures after the depletion of Csk. Loss of Csk expression strongly increased the number of cell-in-cell structures ( Figure 3B). In the vast majority of cell-in-cell structures, the internalized cell was present in a Lamp1-and b-catenin-positive compartment ( Figures 3C and 3D), indicating that internalization occurred through entosis. In addition, similar to the observations after the depletion of JAM-A, depletion of Csk resulted in the predominant internalization of WT MCF7 cells ( Figure 3E). Finally, in support of the role of Csk in entosis regulated by JAM-A, the inhibition of Src activity in JAM-A-depleted cells prevented the increase in entosis ( Figure S3). Similarly, inhibiting Erk1/2, which is a downstream effector of Src and a key regulator of cell motility (Mendoza, 2013;Mendoza et al., 2011) prevented the increase in entosis in JAM-A-depleted cells ( Figure S3). Together, these observations indicated that the suppression of entosis in matrix-adherent cells requires Tyr280 phosphorylation of JAM-A and the activity of Csk to suppress Src activity. As JAM-A Tyr280 phosphorylation as well as Csk activity are required to regulate CIL (Kummer et al., 2022) and to limit cell motility ( Figures 3F and 3G), and as JAM-A interacts with Csk in a Tyr280-dependent manner (Kummer et al., 2022), we conclude that the loss of CIL in combination with increased cell motility facilitates entotic cell engulfment.

JAM-A is not involved in entosis triggered by matrix deprivation
Loss of cell-matrix adhesion has been identified as a potent inducer of entosis in various tumor cell types (Buchheit et al., 2014;Ishikawa et al., 2015;Sun et al., 2014aSun et al., , 2014b. As our studies in matrix-adherent cells did not exclude the possibility of a direct role of JAM-A during entosis, for example by mediating cell-cell recognition as described for E-cadherin (Sun et al., 2014a), we performed experiments in which entosis was induced by suspension culture, thereby excluding the influence of cell motility. Co-cultures of LA-GFPlabelled WT MCF7 cells and LA-mCherry-labelled JAM-A KD MCF7 cells were kept in suspension for 6 h or 12 h and analyzed for cell-in-cell structures, as described (Sun and Overholtzer, 2013) ( Figure 4A). In control samples (Ctrl KD -WT co-cultures), approximately 20% of cell structures contained entotic cell-in-cell structures, both after 6 and 12 h of suspension culture ( Figure 4B). The same frequency of cell-in-cell structures was observed in JAM-A KD samples (JAM-A KD -WT co-cultures) ( Figure 4B). Also, we did not observe a preference for the internalization of WT cells vs JAM-A KD cells in entotic events resulting from suspension culture ( Figure S4). These observations indicated that JAM-A is not involved in entosis induced by the absence of cell -matrix adhesion. They further support the notion that the increased rate of entosis after the depletion of JAM-A results from enhanced cell motility and disturbed CIL, rather than from altered cell-cell recognition.

Loss of CIL after the depletion of JAM-A does not results in entosis in polarized epithelial cells
To test if the increased occurrence of entosis after disturbed CIL is specific to tumor cells, we performed cell collision experiments with MDCKII cells. MDCKII cells are derived from kidney distal tubules (Herzlinger et al., 1982) and are characterized by a pronounced apico-basal polarity (Dukes et al., 2011). MDCKII cells with doxycycline-regulated JAM-A shRNA expression (Tuncay et al., 2015) stably transfected with LifeAct-GFP were mixed with MDCKII WT cells stably transfected with LifeAct-mCherry and cultured on functionalized micropatterns. Collision events were classified as ''opposite migration'', ''anergy'', ''contact formation'', and ''continuous migration'' ( Figure 5A), as described (Kummer et al., 2022). Depletion of JAM-A increased the number of events in which cells continued migration after collision ( Figure 5B), which was very similar to what was observed in MCF7 cells (Kummer et al., 2022), indicating that JAM-A is required iScience Article for CIL in polarized epithelial cells. In contrast to MCF7 cells, however, the loss of CIL was not accompanied by increased entosis in these cells ( Figure 5C). These observations indicated that entosis is not a general response to the loss of CIL.

DISCUSSION
Entosis has been observed in a large variety of cancer including breast cancer (Krajcovic et al., 2011;Overholtzer et al., 2007), but is not frequently observed in matrix-adherent breast cancer cells under standard culture conditions in vitro (Garanina et al., 2017). Two explanations might account for this. First, we have used micropatterned substrates to perform 1D kinematic assays , in which cells migrate on stripes of 5 mm widths. Under these spatially confined culture conditions, cells cannot simply bypass other cells in response to collisions. Second, we have used MCF7 cells in which the cell adhesion receptor JAM-A has been downregulated, which results in increased cell motility and in a dysregulated CIL response in colliding cells (Kummer et al., 2022). Our observations thus provide an experimental framework to study entosis of matrix-adherent cells in vitro, which is expected to contribute to a better understanding of the molecular mechanisms that underly the regulation of entosis.
From our observations that JAM-A downregulation increases the rate of entosis in matrix-adherent cells but not suspension-cultured cells, we infer that JAM-A's role in the regulation of entosis is indirect and based on its regulatory function in cell motility and CIL, rather than on a function in cell-cell recognition during the entotic process. However, our recent observations on a cell-autonomous function of JAM-A suggest that JAM-A could be involved in the entotic process through additional functions independent of cell-cell adhesion. After the depletion of JAM-A, MCF7 cells display increased cell motilities associated with lower traction forces, have increased stiffness, and show increased Rac1 activities (Kummer et al., 2022). Interestingly, an increased Rac1 activity has been associated with the outer cell in entotic cell-in-cell structures, which probably serves to downregulate actomyosin contractility to facilitate the internalization of neighboring cells (Sun et al., 2014b). Through these cell-autonomous functions JAM-A could thus contribute to the entotic process. As to the mechanism of cell-cell recognition during entosis, published works indicate that entosis strongly depends on the expression of classical cadherins, such as E-cadherin or P-cadherin, and their link to the actin cytoskeleton (Sun et al., 2014a;Wang et al., 2015). Although additional cell-cell adhesion systems cannot be truled out, it seems that the cadherin-catenin complex is the primary regulator of cell-cell recognition during entosis.
Our findings thus suggest that entosis is caused by dysregulated CIL. A loss of CIL has been proposed to contribute to cancer development (Abercrombie, 1979). A loss of CIL between tumor cells and stromal cells (heterotypic CIL) facilitates cancer cell invasion into the stromal environment and thereby promotes iScience Article metastases formation (Astin et al., 2010;Paddock and Dunn, 1986). As suggested by the observations of this study, a loss of CIL between two breast cancer cells of the same type (homotypic CIL) facilitates entotic cell engulfment and thus might promote selective advantages under competitive growth conditions, which is supported by the high incidence of entosis in breast cancer (Krajcovic et al., 2011). Our findings link a dysregulation of CIL to the onset of entosis in matrix-adherent breast cancer cells.

Limitations of the study
Our study reveals a hitherto unexplored link between loss of CIL and the occurrence of entosis. Our experiments were performed on two-dimensional linear micropatterns composed of ECM-functionalized stripes of 5-mm width that are separated by cytophobic spacers. Under these conditions repolarization and migration in other directions in response to cell-cell collision is impeded, thus favoring continuous migration and, eventually, entotic engulfment. In a three-dimensional tissue, individually migrating metastatic tumor cells may have the possibility to bypass other cells after collision, thus preventing entosis. In addition, our study involved only homotopic collisions between MCF7 tumor cells but not heterotypic collisions between MCF7 cells and other cells. In a three-dimensional tissue, individually migrating metastatic tumor cells will interact with cells of different origins. These issues should be kept in mind to avoid an overinterpretation of the findings.  d Original western blot images and microscopy data reported in this paper will be shared by the lead contact upon request. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon reasonable request.

EXPERIMENTAL MODELS AND SUBJECT DETAILS
MCF7 cells (ATCC #HTB-22) are human adenocarcioma cells derived from a female patient. Cells were grown in at 37 C in a humidified atmosphere in RPMI 1640 medium without phenol red (Thermo Fisher #32404014) containing 10% FCS, 2 mM glutamine, 100 U/ml penicillin and 100 U/ml streptomycin, 1 mM Na-pyruvate and 1:100 non-essential amino acids (Capricorn Scientific). The generation of MCF7 cell lines expressing JAM-A shRNAs, Csk shRNAs, and fluorescent proteins (EGFP, LifeAct-mCherry, LifeAct-GFP) have been described in detail before (Kummer et al., 2022). MCF7 WT cells were used as collision partners in 1D micropattern collision assays and in suspension assyas; MCF7 cells with constitutive (pLVTHM) or inducible JAM-A KD (pEmU6proT, + Dox, shRNA expression On, -Dox, shRNA expression off) were used as collision partners in 1D micropattern collision assays and in suspension assays. HEK293T (ATCC #CRL-3216) cells are human epithelial-like cells derived from embryonic kidney. Cells were grown in DMEM (PAN Biotech #P04-03500) containing 10% FCS, 2 mM glutamine, 100 U/ml penicillin and 100 U/ml streptomycin. Cells were used for the generation of pseudovirus particles. MDCKII cells are canine epithelial cells derived from the collecting duct of a female cocker spaniel (Herzlinger et al., 1982). MDCKII wildtype cells (Sigma-Aldrich, Munich, Germany, Sigma cat. #00062107) were grown in DMEM containing 10% FCS, 1% glutamine, 100 U/ml penicillin, 100 mg/mL streptomycin. MDCKII wildtype cells were stably transfected with LifeAct-mCherry and used as collision partners in 1D micropattern collision assays. Cells were grown in DMEM containing 10% FCS, 1% glutamine, 100 U/ml penicillin, 100 mg/mL streptomycin. The generation of MDCKII cell lines expressing canine-specific JAM-A shRNAs under a doxycycline-regulated promoter have been described in detail before (Tuncay et al., 2015). Cells were stably transfected with LifeAct-GFP. Stably LifeAct-GFP-transfected MDCKII cells with inducible JAM-A shRNA expression were used in collision assays as JAM-A KD cells (+ doxycycline) or control KD cells (-doxycycline). All cell lines used in this study were routinely tested and found to be negative for mycoplasm contamination.

Cell culture and transfections
Transient transfections of siRNAs were performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's instructions. Lentiviral particles for the generation of stably transfected cell lines expressing either shRNAs or cDNAs were generated by cotransfection of HEK293T cells with the lentiviral vector and the packaging vectors psPAX2 and pMD2.G (kindly provided by Dr. Didier Trono, Addgene plasmids #12260 and #12259) in a ratio of 3:2:1 into HEK293T cells. Lentiviral particles-containing HEK293T supernatants were harvested one day after transfection. MCF7 and MDCKII target cells were incubated with lentiviral supernatants for 24 h. Transduced cells were maintained in standard culture medium supplemented with 1 mg/mL puromycin (AppliChem). MCF7 cells and MDCKII cells expressing JAM-A shRNAs under a doxycycline-regulated promoter were generated by electroporation of cells with plasmid pEmU6-proT (provided by Dr. Karl Matter). Transfectants were selected with 500 ug/ml G418 (AppliChem) and 7 ug/ml blasticidin (InvivoGen).