1644 Research Article

IntroductionPeritoneal carcinomatosis is frequently observed in patients sufferingfrom colorectal and ovarian cancers (van Grevenstein et al., 2007;Slack-Davis et al., 2009). It is initiated by tumor cells exfoliatinginto the abdominal cavity and subsequently adhering to humanperitoneal mesothelial cells (HMCs) that line the intra-abdominalcavity and organs (Mutsaers, 2004). This can result in the formationof intraperitoneal metastases and underlines the importance ofHMCs in the development of peritoneal carcinomatosis (vanGrevenstein et al., 2007; Slack-Davis et al., 2009; Alkhamesi et al.,2005). The median survival period of patients with peritonealcarcinomatosis rarely exceeds 6 months (Levine et al., 2007). Hence,novel therapeutic strategies are currently being developed that targetthe prolongation of life expectancy. Often combined with surgery,all these strategies have the same goal: induction of tumor celldeath. Cytoreductive surgery followed by hyperthermicintraperitoneal chemotherapy, during which chemotherapeutics areapplied locally into the abdominal cavity, has been reported toincrease the median survival period substantially (to an average of22 months) (Levine et al., 2007). For example cisplatin derivates,commonly applied against ovarian tumors, are known to induce celldeath and enhance the basic rates of apoptosis and necrosis incancer cells (Vakifahmetoglu et al., 2008; Bryant et al., 2010).

The current notion is that dying cells in the abdominal cavity areeliminated by professional phagocytes: the peritoneal macrophages(Park et al., 2009; Frey et al., 2009; Gregory and Devitt, 2004).However, aside from macrophages, as the prototype of professionalphagocytes, numerous examples of semi-professional, so-called‘amateur’ phagocytes exist, including fibroblasts, endothelial,epithelial and mesenchymal cells (Gregory and Devitt, 2004; Diniet al., 1995; Cao et al., 2004; Dogusan et al., 2004). These areneighboring cells and have a rather limited capacity of phagocytosisbut do contribute to the removal of cell corpses from theirsurroundings because they commonly outnumber professionalphagocytes (Gregory and Devitt, 2004; Monks et al., 2005).

The phagocytic clearance of apoptotic cell corpses is known toinvolve a multitude of engulfment receptor systems (reviewed byLauber et al., 2004; Mevorach et al., 1998). On the one hand,apoptotic cells can be recognized directly by surface receptors ofthe phagocyte. This is typically mediated by externalizedphosphatidylserine on the apoptotic cell surface and phagocyticphosphatidylserine receptors, including BAI1, Tim1, Tim4 andStab2, or scavenger receptors, such as SR-A1, SR-B1, LOX1,CD68 and CD36. On the other hand, there are phagocytic receptorsystems that require the participation of soluble bridging proteinsfunctioning as opsonins. Examples are the vitronectin receptor

SummaryPeritoneal carcinomatosis is an advanced form of metastatic disease characterized by cancer cell dissemination onto the peritoneum.It is commonly observed in ovarian and colorectal cancers and is associated with poor patient survival. Novel therapies consist ofcytoreductive surgery in combination with intraperitoneal chemotherapy, aiming at tumor cell death induction. The resulting dyingtumor cells are considered to be eliminated by professional as well as semi-professional phagocytes. In the present study, we haveidentified a hitherto unknown type of ‘amateur’ phagocyte in this environment: human peritoneal mesothelial cells (HMCs). Wedemonstrate that HMCs engulf corpses of dying ovarian and colorectal cancer cells, as well as other types of apoptotic cells. Flowcytometric, confocal and electron microscopical analyses revealed that HMCs ingest dying cell fragments in a dose- and time-dependent manner and the internalized material subsequently traffics into late phagolysosomes. Regarding the mechanisms of prey cellrecognition, our results show that HMCs engulf apoptotic corpses in a serum-dependent and -independent fashion and quantitativereal-time PCR (qRT-PCR) analyses revealed that diverse opsonin receptor systems orchestrating dying cell clearance are expressed inHMCs at high levels. Our data strongly suggest that HMCs contribute to dying cell removal in the peritoneum, and future studies willelucidate in what manner this influences tumor cell dissemination and the antitumor immune response.

Key words: Human peritoneal mesothelial cells, Phagocytosis, Apoptosis, Necrosis, Peritoneal carcinomatosis

Accepted 17 January 2011Journal of Cell Science 124, 1644-1654 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.078907

Phagocytosis of dying tumor cells by humanperitoneal mesothelial cellsBritta Janina Wagner1,2, Dennis Lindau1, Dagmar Ripper3, York-Dieter Stierhof3, Jörg Glatzle2, Maria Witte2,Henning Beck4,5, Hildegard Keppeler6, Kirsten Lauber6,7,*, Hans-Georg Rammensee1

and Alfred Königsrainer2

1Department of Immunology, University of Tübingen, Tübingen 72076, Germany2Department of General, Visceral, and Transplantation Surgery, University Hospital Tübingen, Tübingen 72076, Germany3Center of Plant Molecular Biology, University of Tübingen, Tübingen 72076, Germany4Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen,Tübingen 72076, Germany5Graduate School for Cellular and Molecular Neurosciences, University of Tübingen, Tübingen 72076, Germany 6Department of Internal Medicine I, University of Tübingen, Tübingen 72076, Germany7Department of Radiation Oncology, LMU Munich, Munich 81377, Germany*Author for correspondence (Kirsten.lauber@med.uni-muenchen.de)

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(v3 integrin) with the bridging proteins TSP1 or MFG-E8, theMer tyrosine kinase family (Mer, Tyro3 and Axl) with the bridgingproteins Gas6 or protein S (ProtS), the low density lipoprotein-related proteins LRP2 and LRP8 with the bridging protein 2GPI,and the members of the collectin or complement receptor family[calreticulin (CD91) and M2 or X2 integrin] with thecorresponding complement and collectin proteins (C1q, C3, MBL2,SP-A and SP-D). Bridging proteins often derive from body fluids,including serum and surfactant, but they can also be autonomouslyproduced by the phagocytes themselves.

In the present study, we have analyzed the role of HMCs assemi-professional phagocytes in an in vitro model of peritonealcarcinomatosis in which confluent HMC monolayers wereincubated with ovarian and colorectal tumor cell suspensions atvarying ratios over different time periods. By employing flowcytometry, and confocal and electron microscopy, we show for thefirst time that HMCs phagocytose corpses of apoptotic and necroticcancer cells. We show that HMCs internalize fragments of dyingcells, by engaging an active actin cytoskeleton, and subsequentlytraffic the internalized prey to late phagolysosomes. HMCs clearlydistinguished between viable, apoptotic and necrotic tumor cells;the highest rate of phagocytosis was observed when HMCs wereco-incubated with apoptotic prey cells. Regarding the underlyingmechanisms of prey cell recognition, our results show that HMCsengulf apoptotic prey cells in a serum-dependent as well as-independent fashion. Finally, quantitative real-time PCR (qRT-PCR) was employed to analyze the expression levels of differentengulfment genes.

In conclusion, our study demonstrates that HMCs can act asamateur phagocytes that internalize dying tumor cell corpses. Giventhat HMCs are far more predominant, in terms of numbers, thanperitoneal macrophages, we propose that HMCs play a pivotal rolein the removal of dead cells from the abdominal cavity.

ResultsHMCs engulf tumor cell particlesIn order to study the role of HMCs in peritoneal carcinomatosis,with special regard to the interaction between HMCs and cancercells, we have established an in vitro model, in which primaryHMCs [CellTrace Far Red DDAO-succinimidyl ester (FR)-labeled;red] were co-cultured with ovarian SKOV-3 or colorectal HT29tumor cells [carboxyfluorescein succinimidyl ester (CFSE)-labeled;green]. The adherent tumor cells were enzymatically detached andadded, in suspension, to confluent HMC monolayers to mimicperitoneal metastasis at the point of tumor cell spread. After 48hours of incubation in the presence of 15% fetal calf serum (FCS)and 5% human serum (HS), cells were fixed and analyzed byconfocal microscopy (Fig. 1). Cancer cells and HMCs could beclearly distinguished in the single-channel measurements (Fig. 1A)and displayed close cell-to-cell contacts (Fig. 1B,C). Surprisingly,we observed scattered green SKOV-3 and HT29 cell fragmentswithin the red HMCs (Fig. 1B,C). The z-stacks unambiguouslydemonstrated that these green fragments (1 to 25 m in diameterand generally smaller than whole tumor cells) indeed were insidethe HMCs, suggesting that they had been engulfed. By contrast,uptake of HMC fragments into tumor cells was not detected.

Preparations of primary HMCs always contain small fractionsof contaminating peritoneal macrophages (in the present studyconsistently <5%) (Stylianou et al., 1990). To exclude the possibilitythat these macrophages were responsible for the observedphagocytic phenomenon, we examined the uptake of SKOV-3 cell

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fragments by HMCs after magnetic macrophage depletion by livecell imaging (supplementary material Movie 1). SKOV-3 cellswere again incubated with HMCs for 24 hours and photographswere taken at 15-minute intervals for a period of 14 hours. Overtime, several SKOV-3 cells rounded up and displayed blebbingfeatures. These structural changes typically appear during apoptosis(Mills et al., 1999). Most importantly, the video sequence clearlyrevealed two red HMCs engulfing green tumor cell fragments,which were constricted from the SKOV-3 cytoplasm, supportingthe notion that in our co-culture system some SKOV-3 cells undergoapoptosis (~10–15%; data not shown) and subsequently arephagocytosed by HMCs.

Electron microscopy analysis of pseudopod formation byHMCsOur confocal microscopy data strongly favor the idea that HMCsphagocytose (dying) tumor cell fragments (Fig. 1). Therefore, wenext analyzed the anatomy of this engulfment process. Duringphagocytosis, scavenger cells form protrusions of the plasmamembrane, so-called pseudopods (Booth et al., 2001). Hence, weexamined the cell-to-cell contacts between HMCs and HT29 cells,after 48 hours of co-incubation, by transmission electronmicroscopy (Fig. 2A,B) and compared these contacts withintermesothelial cell contacts (Fig. 2C,D). As clearly shown, HMCsformed cellular extensions, which reached far into the cytoplasmof the HT29 cells, similar to the formation of pseudopods (Fig. 2B;HMC, red; HT29, green). The intercellular contacts formed betweentwo HMCs were fundamentally different, as the cellular extensionsonly touched the cell surfaces (Fig. 2C,D).

Internalized tumor cell fragments colocalize withphagosome markers in HMCsThe degradation of ingested cellular material involves phagosomematuration through homotypic and heterotypic fusion of earlyendosomes, late endosomes and lysosomes (Zhou and Yu, 2008).The classical markers employed for the discrimination of thesedifferent phases are Ras-related protein 5 (Rab5), a small GTPaseassociated with early phagosomes (Kinchen et al., 2008; Desjardinset al., 1994), and annexin I (Anx1) or lysosomal-associatedmembrane protein 1 (LAMP1), two proteins that are utilized toshow later phagosomal stages and phagosomes after lysosomalfusion, respectively (May and Machesky, 2001; Alvarez-Dominguezet al., 1997; Tjelle et al., 2000). We examined the colocalization ofphagocytosed tumor fragments with the above-mentionedphagosomal marker proteins by confocal microscopy (Fig. 3).Tumor cells were co-incubated with macrophage-depleted HMCs,and intracellular staining for Rab5 (Fig. 3A,B), annexin I (Fig.3C,D) or LAMP1 (Fig. 3E,F) was performed after 24 hours (Rab5)or 48 hours (annexin I, LAMP1). The micrographs clearly showedthat HMCs (stained with CellTracker Orange CMRA; blue)internalized tumor cell fragments (FR; green) and that thesefragments intracellularily colocalized with the Rab5 signal[fluoresceinisothiocyanate (FITC)-labeled; red] (Fig. 3A,B). Thesefindings were illustrated even more clearly by the histogramsdisplaying the distribution of tumor-cell- and Rab5-derivedfluorescence intensities. In the case of annexin I and LAMP1, weobserved an accordingly convincing association of the markerproteins with the internalized tumor cell material (Fig. 3C–F).Here, the signals for annexin I (FITC; red) and LAMP1[phycoerythrin–cyanine 5 (PE–Cy5); red] in fact surrounded theingested tumor cell fragments, as the maximal fluorescence

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intensities for both protein stainings always paralleled the increaseor decrease in the tumor-cell-derived CFSE fluorescence. Theseresults reflect the anatomical structure of phagosomes where themembrane-associated proteins surround the tumor cell fragmentsin the phagosomal lumen. Note, all the antibodies employedspecifically stained their target proteins; in stainings with thecorresponding isotype controls no considerable fluorescence signalwas detected (data not shown).

Phagocytosis of tumor cells by HMCs is inhibited bycytochalasin DAfter having examined the structural characteristics of HMC-mediated phagocytosis, we utilized a flow cytometric assay toquantify the process of phagocytosis. Prior to co-incubation, HMCsand tumor cells were labeled with FR and CFSE, respectively.Both cell populations could be clearly discriminated by FACSanalysis (HMCs, gate 1, red; SKOV-3, gate 3, green; Fig. 4A,upper row). After 48 hours of co-incubation, FR-labeled HMCswere positive for tumor-cell-specific CFSE-fluorescence asreflected by their considerable shift on the x-axis. Assuming thatthis shift displays HMCs that have internalized tumor cell

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fragments (Figs 1 and 3), this population was defined asphagocytosing HMCs (gate 2, highlighted in turquoise; Fig. 4A,lower row). From the FACS data alone, it cannot be ruled out thatthe right-hand half of gate 2 also contained SKOV-3 cells that hadphagocytosed HMCs. However, we never observed uptake ofHMC fragments into tumor cells in our confocal microscopyanalyses (Fig. 1). Note that doublet exclusion was performed onthe basis of FSC-A and FSC-H. Hence, the detection of FR andCFSE double-positive cells could not be due to an artifact of cellpairing. The population of double-positive cells could be furthersubdivided into phagocytosing HMCs (gate 4, turquoise) andperitoneal macrophages positive for CD11c (PE) and/or CD86[Pacific Blue (Pablue)] (gate 5, purple) (Fig. 4A, dot plot on thelower right-hand side). Notably, the percentage of thesecontaminating macrophages was always below 5% and they wereexcluded from further analyses. The gating strategy, and colordisplay of the different cell populations (red, HMCs; green, tumorcells; turquoise, phagocytosing HMCs; and purple, phagocytosingmacrophages), described here, was accordingly applied to theHT29 and HMC co-culture system (Fig. 4C) and was usedthroughout the whole study.

Fig. 1. Confocal microscopical analysis of HMCsengulfing tumor cell particles. (A)Cultures of singlecell types: HMCs (FR, red), HT29 and SKOV-3 (CFSE,green). (B)Co-incubation of HMCs with SKOV-3 cells.(C)Co-culture of HT29 cells and HMCs. HMCs wereincubated with tumor cells in a ratio of 1:1 for 48 hours.The z-stacks demonstrate that HMCs internalize CFSE-labeled tumor cell fragments of ~1–25m diameter.

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As displayed in Fig. 4B, ~30% of HMCs phagocytosed SKOV-3 cell fragments (Fig. 4B). Phagocytosis was significantly anddose-dependently reduced in the presence of cytochalasin D, anactin polymerization inhibitor widely used to substantiate theinvolvement of active cytoskeletal rearrangements in the contextof phagocytosis (P<0.001) (May and Machesky, 2001; Akya et al.,2009). Similar effects were detected for the co-incubation of HMCswith HT29 cells (Fig. 4C), although the total percentage ofphagocytosing HMCs was slightly lower.

The above data confirm that HMCs indeed phagocytose tumorcell fragments. Nevertheless, the ‘quality’ of the ingested tumormaterial still remained elusive, although the video data(supplementary material Movie 1) suggested that it was apoptosing,rather than living, tumor cells that were being engulfed. To clarifythis issue, viable and apoptotic [staurosporine-treated with ~60% ofcells annexin V (AnxV)–FITC-positive and propidium iodide (PI)-negative] tumor cells were co-incubated with HMCs, and thepercentage of phagocytosing HMCs was assessed by flow cytometry.Fig. 4D depicts unambiguously that HMCs preferentially ingestedfragments of apoptotic rather than viable HT29 cells; the same wastrue for SKOV-3 (dot plots not shown). Again, the addition ofcytochalasin D strongly inhibited phagocytosis (Fig. 4D–F).

Note that, irrespective of the nature of prey cells (i.e. apoptoticor viable), HMCs internalized profoundly less prey cell fluorescencethan CD11c- and/or CD86-positive macrophages (Fig. 4A,D;turquoise population compared with the purple population). Thisparallels our microscopical findings that the phagocytic capacity ofHMCs is limited to subcellular fragments, whereas macrophagesare well-known to engulf whole prey cells (Aderem, 2002).However, in the co-incubations with SKOV-3 cells a substantialnumber of HMCs were detected in the right-half of gate 2 suggestingthat more prey cell fluorescence was taken up. Hence, it is reasonableto assume that more HMCs phagocytosed whole SKOV-3 cells thanwas the case with HT29 prey cells. Nevertheless, the percentage ofHMCs that internalized only fragments of dying cells was alwayshigher than the percentage of HMCs that engulfed whole cells.

HMC-mediated phagocytosis of viable, apoptotic andnecrotic tumor cells: target ratio analysisThe results presented in Fig. 4 have shown that HMCs engulffragments of apoptotic tumor cells far more potently than fragments

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of viable ones, suggesting that HMCs contribute to efferocytosis,the process of removing dying cells (Thorp, 2010). Underphysiological and pathophysiological conditions, different formsof cell death occur (Vakifahmetoglu et al., 2008; Byrant et al.,2010). In this scenario, apoptosis and necrosis represent theextremes of programmed and accidental cell death, respectively,on a scale with a multitude of possibilities in between (Galluzzi etal., 2007; Kinchen and Ravichandran, 2007). Therefore, we nextanalyzed, by flow cytometry, the engulfment of viable, dying anddead cells by HMCs at different target ratios. AnxV–FITC andpropidium iodide (PI) staining was employed to confirm the viable,apoptotic and necrotic status of untreated, staurosporine-stimulatedor heat-treated tumor cells, respectively (Fig. 5A). FACS analysisof prey cell engulfment convincingly revealed a positive correlationbetween the percentage of phagocytosing HMCs and the amountof target cells applied (Fig. 5B,C). Most importantly, fragments ofapoptotic prey cells were much more efficiently engulfed thanfragments of necrotic or viable cells. It is reasonable to assumethat, in the case of living HT29 cells, the contaminating 12% ofspontaneously dying cells, rather than the living cells, were theones that were being phagocytosed (Fig. 5A). Notably, the ‘quality’of viable, apoptotic, and necrotic cells did not significantly changeover the time of co-incubation (48 hours) as measured by FACSanalysis of the FSC/SSC profiles and PI exclusion (data not shown).Hence, HMCs apparently discriminate between living, dying anddead tumor cells and display a strong predilection for apoptoticcells in terms of engulfment.

We next examined further the phagocytosis of other differentapoptotic cell types by HMCs. In these studies, we included ovarian(OvCar-29), colorectal (CaCo-2) and leukemic tumor cell lines(Jurkat and THP-1), as well as primary human neutrophils. Asdisplayed in supplementary material Fig. S1, all types of apoptoticcells were internalized. However, there seemed to be a preferenceof HMCs for ovarian cancer cells and neutrophils, as they weremore efficiently engulfed than the other cell types tested(supplementary material Fig. S1).

Phagocytosis of apoptotic tumor cells: time dependencySo far, the data shown have demonstrated that HMCs can contributeto efferocytosis. However, our finding that their phagocytic capacityis limited to subcellular fragments rather than whole cells (Figs 1

Fig. 2. Electron micrographs of HT29 cell engulfment by HMCs. (A,B)Interaction of HT29 (�) and HMCs (*) after incubation for 48 hours at a 1:1 ratio. Bshows a magnification of a representative cellular extension of an HMC that reaches far into the cytoplasm of the HT29 cell (false-colored EM-picture; HMC, red;HT29, green). (C,D)By contrast, the extensions of two HMCs only touch the cell surfaces.

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and 4) underlines their ‘semi-professional’ nature (Dini et al., 1995).Therefore, we next analyzed the kinetics of apoptotic corpseinternalization by HMCs in comparison with that of peritonealmacrophages over a period of up to 48 hours. The percentage ofphagocytosing HMCs increased continuously over time (Fig. 6A,B).However, in contrast to peritoneal macrophages, which phagocytosedapoptotic cells very quickly (Fig. 6C,D), HMCs engulfed apoptotictumor cells with a rather slow kinetics – an observation that furthersubstantiates their amateur phagocyte character (Parnaik et al., 2000).The number of phagocytosing HMCs in the co-incubation withapoptotic SKOV-3 cells was consistently higher than in the co-culture system with apoptotic HT29 cells (see also Figs 4, 5), as wasthe case for phagocytosing macrophages. Because the percentage ofapoptotic cells in both prey cell populations did not differ substantially(70–90%, as determined by AnxV–FITC and PI staining; Fig. 5Aand data not shown), these data indicate that the phagocytic potentialof HMCs also depends on the target cell type.

Serum dependency of HMC-mediated efferocytosisThe phagocytic clearance of apoptotic cells is known to involve amultitude of engulfment receptor systems. Among them, there are

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receptors recognizing apoptotic cells directly (typically mediatedby externalized phosphatidylserine on the apoptotic cell surface),whereas others require the participation of soluble bridging proteins(often serum-derived), functioning as opsonins (Lauber et al., 2004;Mevorach et al., 1998). The intriguing question that needed to beaddressed at this stage was which receptor systems would beengaged in HMC-mediated efferocytosis. To this end, we examinedthe internalization of apoptotic tumor cell fragments in the absenceor presence of serum. HMCs engulfed apoptotic cells under serum-free conditions, suggesting that either opsonin-independent receptorsystems play a role or that HMCs autonomously produce therequired bridging proteins (Fig. 7A,B). However, because additionof serum significantly enhanced the uptake of apoptotic corpses(P<0.001), HMCs apparently also utilize receptor systems involvingserum-derived opsonins. Decomplementation, by heat-inactivationof serum, did not profoundly interfere with its phagocytosis-enhancing effect. Hence, serum-derived complement proteins areobviously dispensable for HMC-mediated efferocytosis. In orderto exclude that the serum-free culture conditions negatively affectedHMC viability, we monitored HMC viability by AnxV–FITC andPI staining. Importantly, cultivation in the absence of serum did

Fig. 3. Colocalization of phagosome-membrane-associated proteins with tumor cell particles internalized by HMCs. Colocalization of Rab5 (FITC signal,red) and tumor cell fragments from (A) SKOV-3 and (B) HT29 cells (FR signal, green) in HMCs (CMRA, always depicted in blue) after 24 hours co-incubation.Co-localization of annexin I (FITC signal, depicted in red) and incorporated tumor cell particles from (C) SKOV-3 and (D) HT29 (FR signal, green) in HMCs after48 hours co-incubation. Colocalization of LAMP1 (PE–Cy5 signal, red) and tumor cell fragments from (E) SKOV-3 and (F) HT29 cells (CFSE signal, green)internalized by HMCs after 48 hours co-incubation. The incubation ratio was always 1:1. The z-stacks show that Rab5, annexin I, LAMP1 and the tumor cellfragments are located intracellularly. The graphs on the right of each subframe (A–F) display the colocalization of the fluorescence signals, given in arbitraryfluorescence units (AFU), for the tumor cell particles and the phagosome membrane proteins (antibody AFU, red; tumor cell fragment AFU, green). The white barsin the pictures on the upper right of each graph show the location of the cross-section corresponding to the fluorescence diagram within the HMCs (note thatdifferent scales were used in these diagrams).

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not substantially induce HMC apoptosis within 48 hours (Fig. 7C–E). These results indicate that HMCs employ serum-dependent, aswell as serum-independent, engulfment receptor systems forefferocytosis, with serum-derived complement proteins apparentlyplaying a minor role.

Expression profiling of engulfment genes in HMCsFinally, we examined the expression levels of different engulfmentgenes in HMCs (obtained from four different donors) by qRT-

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PCR. Intriguingly, mRNA transcripts for various known receptorscould be detected. The most abundantly expressed transcripts, withaverage threshold cycle (Ct) values <25, belonged to the scavengerreceptors (SR-B1, LOX1 and CD68), the MFG-E8 and TSP1receptors (v3 or v5 integrin), the Gas6 and ProtS receptors(Axl and Tyro3), the 2-GPI receptors (LRP8), and the collectinreceptor (CD91, also known as calreticulin). High transcript levels(Ct values <25) for the corresponding bridging proteins MFG-E8,TSP1, ProtS and Gas6 were also found in HMCs (supplementary

material Fig. S2). For relative quantification, target genemRNA levels were normalized to that of 18S rRNA andcalibrated to the normalized expression levels of the respectivegene in macrophage colony-stimulating factor (M-CSF)-differentiated peripheral blood monocyte-derivedmacrophages, which served as a positive control. This analysisyielded three different groups of engulfment genes (Fig. 8A):genes expressed in HMCs in comparable amounts tomacrophages, such as the collectins (MBL2, SP-A and SP-D)and the collectin receptor (CD91); genes expressed in HMCsless frequently (factor 2–5 to less than 2–10) than inmacrophages, like the complement receptors or the Fc gammareceptors; and genes expressed in HMCs at much higher levels(factor 22 to more than 210) than in macrophages, includingAxl, Tyro3, v3 integrin and the respective bridgingmolecules Gas6, ProtS, TSP1 and MFG-E8. We conclude,from these results, that it is particularly the members of thelatter group that are the most probable candidates to orchestrateHMC-mediated efferocytosis.

To analyze whether HMCs, in principle, might be able todegrade and/or export the internalized cell fragments, we alsoexamined DNaseII, the cathepsins B and D and ATP-bindingcassette, sub-family A, member 1 (ABCA1) in our qRT-PCRanalyses. As demonstrated in supplementary material Fig. S2,and Fig. 8B, all these genes were expressed at high levels(average Ct values <25), which is approximately comparableto the levels found in macrophages.

Fig. 4. Quantification of phagocytosis by flow cytometry. HMCs andprey cells were incubated separately (upper row in A) or in a 1:1 ratio for48 hours (lower row in A, and D). Subsequently, flow cytometricanalysis was performed as described in the Materials and Methods.(A)After appropriate forward and sideward scatter gating, and doubletexclusion, HMCs and SKOV-3 cells were discriminated by FR (HMCs)and CFSE (SKOV-3) signals (A, upper row; gate 1, HMCs, red; gate 3,tumor cells, green). After 48 hours of co-incubation HMCs becamepositive for CFSE (A, lower row; gate 2, phagocytosing HMCs,highlighted in turquoise). CD11c- and/or CD86-positive contaminatingperitoneal macrophages (gate 5, purple) were excluded from furtheranalyses (gate 4 was defined with the help of the CD11c and CD86isotype control). Co-culture of HMCs and HT29 cells with subsequentflow cytometric analysis of phagocytosis was performed accordingly.HMCs were incubated with living SKOV-3 cells (B) or HT29 cells (C)in the presence or absence of different concentrations of cytochalasin Dto determine the percentage that were phagocytosing (means±s.d.).(D)Representative dot plots of the comparison of viable (alive, left-handpanel) compared with apoptotic (middle panel) tumor cell phagocytosisare shown for HT29 cells in the absence or presence of 10Mcytochalasin D (right-hand panel). Analysis followed the same gatingstrategy as described in A. (E,F)Quantitative analysis (means±s.d.) ofapoptotic SKOV-3 cell or HT29 cell phagocytosis by HMCs after 48hours co-incubation at a 1:1 ratio (note that different scales are used inthe column charts). *P≤0.001.

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DiscussionThe timely and efficient removal of dying cells, efferocytosis, iscrucial for tissue development, homeostasis and protection againstchronic inflammation and autoimmunity. Macrophages, as theprototypical professional phagocytes, play a central role in thisprocess (Gregory and Devitt, 2004; Lauber et al., 2004).Nevertheless, the contribution of amateur phagocytes to dying cellclearance can be demonstrated by two examples. First, in lower-order macrophage-deficient organisms, such as the nematode wormCaenorhabditis elegans, dying cells are engulfed by neighboringcells (Gregory and Devitt, 2004). Second, mice lackingmacrophages develop ostensibly normally (Wood et al., 2000),strongly supporting the notion that non-professional phagocytes

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assist in the clearance of apoptotic cells when macrophages areabsent or when their phagocytic capacity is overwhelmed (Parnaiket al., 2000).

Here, we have identified a hitherto unknown type of amateurphagocyte in the context of dying cell removal: human peritonealmesothelial cells (HMCs). By utilizing an in vitro model ofperitoneal carcinomatosis, we show that HMCs phagocytosecorpses of apoptotic and necrotic ovarian SKOV-3 and OvCar-29cancer cells, and colorectal HT29 and Caco-2 cancer cells, as wellas other types of apoptotic cells. Flow cytometry, confocal andelectron microscopy have revealed that this internalization processis dependent on an active actin cytoskeleton and that it is followedby the typical sequence of phagosome maturation (Figs 1–4). These

Fig. 5. HMC-mediated phagocytosis of viable,apoptotic and necrotic tumor cells: target ratioanalysis. (A)AnxV–FITC and PI staining of HT29 cellsto determine living, necrotic (20 minutes at 56°C) orapoptotic cell populations (5M staurosporine for 12hours). Similar results were obtained for SKOV-3 cells(data not shown). For the target ratio assay, living,necrotic or apoptotic SKOV-3 (B) or HT29 cells (C)were incubated with confluent HMC monolayers for 48hours (means±s.d.). Phagocytosis was quantified by flowcytometry as depicted in Fig. 4.

Fig. 6. Phagocytosis of apoptotic tumor cells: time dependency. Forkinetic analysis, HMCs were co-incubated with apoptotic SKOV-3 orHT29 cells for 6–48 hours at a 1:1 ratio. Phagocytosis (means±s.d.) wasassessed by flow cytometry. (A,B)Results obtained from CD11c andCD86 double-negative HMCs. (C,D)Results obtained from CD11c-and/or CD86-positive contaminating peritoneal macrophages (M�).Please note that different scales are used in the curve charts.

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observations add a new facet to HMC biology. Being derived fromthe mesoderm and lining the serous cavities of the body, mesothelialcells are known to exert a variety of functions: the production oflubricating fluid, thus providing a non-adhesive and protectivesurface to facilitate intracoelomic movement; the transport of fluidand cells across the serosal cavities; and acting as a physical barrierand first-line of defense against invading pathogens and harmfulsubstances (Mutsaers, 2004; Mutsaers, 2002). In the context of thelatter process, it has been shown that HMCs are able to phagocytosepathogens, such as Staphylococcus aureus, as well as asbestosfibers (Brinkmann and Muller, 1989; Haslinger-Löffler et al., 2006).Hence, our observation that HMCs internalize fragments of dying

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tumor cells complements these studies and extends the spectrumof HMC function to the field of efferocytosis.

Although the present study has clearly revealed that HMCs candistinguish between viable, apoptotic and necrotic tumor cells (Fig.5), the large proportion of these cells ingested only subcellularfragments and not whole cells, in contrast with the action ofperitoneal macrophages (Figs 1, 4). This is in line with theobservation that other non-professional phagocytes, including liverendothelial cells, also preferentially engulf apoptotic bodies (Diniet al., 1995). Moreover, the internalization process followed a farslower kinetics than macrophage-mediated efferocytosis (Fig. 6), afinding that has accordingly been reported for other types of

Fig. 7. Serum dependency of HMC-mediated efferocytosis.HMCs were co-incubated with apoptotic SKOV-3 (A) or HT29cells (B) with different media at a 1:2 ratio for 48 hours. Themedium was supplemented with active human serum (HS),heat-inactivated human serum or no human serum D todetermine the percentage that were phagocytosing(means±s.d.). (C)HMC viability in the different media wasmonitored by AnxV–FITC and PI staining. *P≤0.001.

Fig. 8. Expression profiling of engulfment genes in HMCs by qRT-PCR. (A,B) Relative engulfment geneexpression levels in HMCs obtained from for different donors (donors A, B, C and D) were determined asdescribed in Materials and Methods. Quantification was performed by employing the Ct algorithm with 18SrRNA serving as housekeeping control and cDNA from M-CSF-differentiated monocyte-derived macrophagesas calibrator. Results are presented as log2 expression ratios.

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amateur phagocytes (Parnaik et al., 2000). The molecularmechanisms underlying this delay remain elusive. Parnaik et al.suggested that non-professional phagocytes, upon encounteringdying cells, first have to acquire the competency to phagocytose,which might be accomplished during an extended period of bindingto their targets (Parnaik et al., 2000). Nevertheless, our qRT-PCRdata have clearly revealed that (at least on the mRNA level)different engulfment genes are readily expressed before HMCsencounter their prey. Hence, it can be hypothesized that either thetranslation of different engulfment systems or their trafficking tothe cell surface are the crucial steps that are responsible for thedelayed engulfment kinetics. Concerning the limited phagocyticcapacity of HMCs in terms of engulfable prey size, one couldspeculate that it was related to a restricted availability of degradativeenzymes, such as DNase II and members of the cathepsin family(Yoshida et al., 2005), or ABCA1, a transporter that is vital for theexport of internalized prey-cell-derived cholesterol (Kiss et al.,2006). However, our qRT-PCR analyses have demonstrated thatHMCs express these enzymes at mRNA levels that are higher thanthe levels found in macrophages (Fig. 8B). Hence, insufficientexpression of degradative enzymes is unlikely to be the cause forthe amateur phagocyte character of HMCs.

The question that arises at this point is: what is the contributionof HMCs to dying cell removal in vivo when, compared withperitoneal macrophages, their capability to engulf dying cells ismuch more limited and follows a rather slow kinetics? Obviously,in the peritoneal cavity HMCs are far more predominant in termsof numbers. Furthermore, Gregory et al. have suggested thatmacrophages serve exclusively as a cellular ‘backup’ compartmentfor professional efferocytes, which will only be recruited shouldthe local semi-professional phagocytic system be overwhelmed(Gregory and Devitt, 2004). In line with this hypothesis, our datasupport the assumption that HMCs do contribute to the clearanceof dying cell corpses in the peritoneal cavity and future in vivostudies will clarify this issue in detail.

Macrophages identify apoptotic prey cells by employing a varietyof sensor systems. Within the engulfment synapse, ‘eat-me’ signalsdisplayed on the apoptotic cell surface are recognized bycorresponding phagocyte receptors, either directly or indirectlyunder participation of soluble bridging proteins (Gregory andDevitt, 2004; Lauber et al., 2004). Regarding the mechanismsunderlying HMC-mediated prey cell recognition, our results showthat HMCs potently engulf apoptotic cell corpses in the absence ofserum, suggesting that they either utilize opsonin-independentengulfment receptors or that they autonomously produce solublebridging factors themselves. qRT-PCR analyses in fact revealedthat HMCs express high levels of several scavenger receptors (SR-B1, LOX1 and CD68), as well as the receptors for MFG-E8 andTSP1 (v3 integrin), and Gas6 and ProtS (Axl and Tyro3), withthe corresponding opsonins MFG-E8, TSP1, Gas6 and ProtS (Fig.8A). Our observation that the addition of serum significantlyenhanced the uptake of apoptotic cells by HMCs (Fig. 7) is in linewith the qRT-PCR finding that HMCs express high levels of theProtS receptors (Axl and Tyro3), the collectin receptor CD91-calreticulin and one of the 2-GPI receptors (LRP8).Decomplementation of serum did not considerably reduce theserum-mediated phagocytosis-promoting effect, which can beexplained by the qRT-PCR observation that complement receptorsare only weakly expressed by HMCs. Taken together, our datasupport the notion that serum-independent HMC-mediatedefferocytosis is coordinated by the MFG-E8-TSP1-v3 or v5

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integrin system, the Gas6-ProtS-Axl-Tyro3 system and severalscavenger receptors (SR-B1, LOX1 and CD68), whereas serum-dependent HMC-mediated efferocytosis is governed by Axl, Tyro3,the collectin receptor CD91 and the 2-GPI receptor LRP8. Ofnote, previous reports have identified v3 and v5 integrin,CD36 and LOX1 as crucial contributors in amateur phagocyte-mediated efferocytosis (Hughes et al., 1997; Oka et al., 1998;Walsh et al., 1999; Sexton et al., 2001).

In summary, our findings provide proof-of-principle evidencethat HMCs indeed phagocytose dying ovarian and colorectal tumorcells and thereby might contribute to the clearance of dead cancercells in the peritoneal cavity. This is of special importance asHMCs are known to secrete various chemokines and cytokinesinvolved in leukocyte recruitment and/or activation, and there isaccumulating support for a role of HMCs in antigen presentation(Mutsaers and Wilkosz, 2007). Thus, future studies are required inorder to clarify how far HMC-mediated efferocytosis influencestumor cell dissemination into the peritoneal cavity and how itmodulates the anti-tumor-immune response, especially duringchemotherapeutic intervention.

Materials and MethodsCell isolation and cultureHMCs were isolated from greater omentum tissue obtained from consenting patientsundergoing elective surgery. This method was approved by the ethics committee ofthe Faculty of Medicine and University Hospital at the University of Tübingen,Germany. The greater omentum samples were stored in isotonic saline solution for30 minutes transportation time, followed by directly continued processing. Theisolation procedure was based on the method described by Stylianou et al. (Stylianouet al., 1990). After enzymatic disaggregation with type II collagenase (Invitrogen),HMCs were grown on fibronectin-coated dishes (Sigma–Aldrich) in mediumcontaining M199 supplemented with 20 mmol/l HEPES pH 7.4, 2 mmol/l glutamine(Invitrogen), 5% (v/v) human serum (HS), endothelial cell growth supplement (150g/ml) (both PromoCell, Heidelberg, Germany), 15% (v/v) fetal calf serum (FCS),100 IU/ml penicillin, 100 g/ml streptomycin (all PAA, Pasching, Austria) and 5IU/ml heparin (Ratiopharm, Ulm, Germany). HMCs displayed a uniform cobblestone-like appearance at confluence and were tested for expression of intracellularcytokeratin 18 (mouse anti-human-cytokeratin-18–FITC; clone DC10, Abcam),surface staining of ICAM-1 (mouse anti-human-ICAM-1–FITC; clone 1H4, Abcam)and VCAM-1 (mouse anti-human-VCAM-1–PE; clone STA, BioLegend, San Diego,California, CA) using flow cytometry (data not shown). Mouse IgG2b and mouseIgG1 (clones MPC-11 and MPC-21, respectively; BioLegend) served as isotypecontrols.

The following tumor cell lines were chosen for this study: SKOV-3 and OvCar-29 (subclone of OvCar-3) ovarian cancer cells, and HT29 and Caco-2 colorectalcancer cells, as well as the leukemic tumor cell lines Jurkat and THP-1. The cell lineswere purchased from the American Type Culture Collection (ATCC; HTB-77, HTB-161, HTB-38, HTB 37, TIB-152 and TIB-202, respectively). They were cultured inIscove’s modified Dulbecco’s medium (Lonza) supplemented with 10% FCS (v/v),penicillin (100 IU/ml) and streptomycin (100 g/ml). If not stated otherwise, allexperiments were performed in M199 medium supplemented with 15% FCS and 5%HS but lacking endothelial cell growth supplement, which did not affect cell viabilityas monitored by Trypan Blue staining.

Macrophage depletionMagnetic depletion of CD11b-positive macrophages was performed with mouseanti-human-CD11b magnetic micro beads (Miltenyi Biotec, Bergisch Gladbach,Germany) according to the manufacturer’s instructions. Successful depletion wasensured by anti-human-CD11b–PE staining (clone ICRF44, BD Biosciences) andflow cytometry. HMCs were only used if the CD11b-positive fraction was <0.5%.Prior to further processing HMCs were allowed to recover for 24 hours.

Fluorescence microscopyHMCs of the first passage were grown to confluence on Lab-Tek chambered 1.0borosilicate coverglass systems (Thermo Scientific) after labeling with CellTrace FarRed DDAO-SE (FR) or CellTracker Orange CMRA (CMRA, both from Invitrogen)as recommended by the manufacturer. A total of 1�106 HMCs were stained in 1 mlPBS containing 8 M or 25 M dye, respectively.

Tumor cells were labeled with CFSE or FR (both from Invitrogen) according tothe manufacturer’s recommendations. Briefly, CFSE staining was conducted in 0.5ml PBS containing 12 M or 16 M dye for 1�106 HT29 or SKOV-3 cells,respectively. FR staining was performed in 1 ml PBS containing 5 M dye (HT29)or 10 M dye (SKOV-3) for 1�106 cells in each case. Subsequently, labeled tumor

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cells were added in suspension to the confluent HMC monolayers (ratio 1:1). After24–48 hours co-incubation, cells were gently washed, fixed in PBS containing 1%formaldehyde (Fluka) and, in some experiments, subjected to further antibodystaining. After permeabilization with Cytofix/Cytoperm solution (BD Biosciences)for 20 minutes antibody staining was conducted at 4°C for 30 minutes in Perm/Washbuffer (BD Biosciences) with anti-human-LAMP-1–PE–Cy5 antibody (cloneeBioH4A3; eBioscience, San Diego, CA), anti-human annexin I antibody (clone29), anti-human-Rab5 mouse IgG2a antibody (clone 1/Rab5) (both from BDBiosciences) and the corresponding secondary antibody (goat anti-mouse-IgG–FITC antibody, Jackson ImmunoResearch). Isotype controls were stained in parallel.Confocal microscopy was performed using an inverted LSM 510 confocal laserscanning microscope (Carl Zeiss) endowed with a Plan-Apochromat 63� 1.4 NAoil objectives. CFSE and FITC were exited at 488 nm with an argon-ion laser andemission was detected from 505 to 530 nm using bipolar filter sets. CMRA and PE–Cy5 were exited at 543 nm and FR at 633 nm with helium-neon lasers. Emissionwas detected with appropriate filtersets: PE–Cy5 and FR at wavelengths longerthan 650 nm and CMRA at 585 nm. The photographs were taken at 630�magnification (ocular �10, objective �63). z-stacks were taken in slices of 1–2m. Two-color images were acquired by single-track measurement. Three-colorimages were taken in the multi-track mode. All images were analyzed using LSM5 Image Examiner (Carl Zeiss).

Flow cytometryHMCs from the first to third passage were used for the experiments. HMCs werestained with the FR dye (4 M), and SKOV-3 and HT29 cells with the CFSE dye(6 M or 5 M, respectively) as described above. HMCs and tumor cells were eitherincubated separately or in co-culture at different ratios over the indicated times. Insome incubations, cells were pretreated with 1, 10 or 100 M cytochalasin D(Calbiochem) for 1 hour. Dimethyl suloxide (DMSO) served as a vehicle control.

After co-incubation all cells were detached with accutase (PAA) and subjected toantibody staining at 4°C for 20 minutes with anti-human-CD86–Pacific-Blue (cloneIT2.2) and anti-human CD11c-PE (clone 3.9, both from BioLegend). After staining,cells were fixed in 1% formaldehyde and examined using the FACSCanto II flowcytometer. The data were analyzed with FACSDiva software 6.0 (both BDBiosciences). Contaminating macrophages were identified by CD11c and/or CD86positivity (see Fig. 4).

Induction of tumor cell deathTumor cells were stimulated to undergo apoptosis by incubation with 5 Mstaurosporine (Sigma–Aldrich) for 12 hours. Necrosis was induced by heat-treatmentat 56°C for 20 minutes. Phosphatidylserine (PtdSer) exposure (with AnxV–FITC,BD Biosciences) and loss of membrane integrity (with PI, Sigma-Aldrich) werestained in accordance with the manufactures’ guidelines and examined using aFACSCalibur flow cytometer (BD Biosciences). The data were analyzed with Summitversion 4.1 software (Dako Cytomation, Glostrup, Denmark). Apoptotic cellpopulations comprised 70–90% of the AnxV–FITC-positive, PI-negative cells,whereas necrotic cell populations comprised 70–90% of the PI-positive cells (Fig.5).

Electron microscopyHT29 cells were incubated for 48 hours on confluent HMC monolayers. Owing tothe clear structural differences between HMCs and HT29, only the colorectal cancercell line was used for this experiment. Cells grown on coverslips were fixed in 2.5%glutaraldehyde for 2 hours and treated with 1% osmium tetroxide (both Sigma–Aldrich) in PBS for 90 minutes on ice. Thereafter, samples were stained with 1%aqueous uranyl acetate (Sigma–Aldrich) and dehydrated in a graded series of ethanolbefore infiltration and embedding in epoxy resin (Sigma–Aldrich). Before ultrathinsectioning, coverslips were removed from the cell layer by dipping the polymerizedsample into liquid nitrogen. The sections were viewed in a LEO 906 transmissionelectron microscope.

Quantitative real-time PCR analysisQuantification of engulfment gene mRNA levels was performed by qRT-PCR analysiswith an ABI Prism 7000 Sequence Detection System (Applied Biosystems) andMaxima SYBR Green qPCR Mastermix (Fermentas St. Leon-Rot, Germany) asdescribed previously (Peter et al., 2008). The primer pairs employed (Sigma–Aldrich)are listed in supplementary material Table S1.

Total RNA from approximately 2�106 HMCs from four different donors wasextracted with the NucleoSpin RNA II-kit (Macherey & Nagel, Dueren, Germany).1 g of total RNA was reversely transcribed with 200 units RevertAid reversetranscriptase (Fermentas) in the presence of 50 M random hexamers (GEHealthcare), 400 M dNTPs (Promega, Heidelberg, Germany), and 1.6 units/lRibolockRNase inhibitor (Fermentas). 20 ng of the resulting cDNA was applied forthe following qRT-PCR analyses (20 l final volume) with 300 nM primers. Relativequantification was performed according to the Ct algorithm with 18S rRNAserving as housekeeping control and cDNA from M-CSF-differentiated monocyte-derived macrophages as calibrator. Results are presented as log2 expression ratios.

Statistical analysisFlow cytometry experiments were conducted at least three times in triplicate. HMCswere always obtained from three different individuals. The experimental group wasanalyzed using Student’s t-test. Findings were considered statistically significant atP≤0.001 (*).

We thank Bernd Knöll (Department of Molecular Biology, Universityof Tübingen, Germany) and Karin Blume (Department of InternalMedicine I, University of Tübingen, Germany) for excellent technicaladvice. This work was supported by the fortune funding (1793-0-1) ofthe Medical Faculty, University of Tübingen, Germany and the SFB685, Department of Immunology, University of Tübingen, Germany.The authors declare no conflict of interests.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/124/10/1644/DC1

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