© 2014. Published by The Company of Biologists Ltd.
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Blocking Hedgehog release from pancreatic cancer cells increases paracrine signaling
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potency
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Helene [Damhofer]a, Veronique L. [Veenstra]a, Johanna A.M.G. [Tol]b, Hanneke W.M. [van
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Laarhoven]a,c, Jan Paul [Medema]a, and Maarten F. [Bijlsma]a*
Journal of Cell Science
Accepted manuscript
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a
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Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105AZ,
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Amsterdam, The Netherlands
Laboratory for Experimental Oncology and Radiobiology, Center for Experimental Molecular
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b
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1105AZ, Amsterdam, The Netherlands
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c
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Meibergdreef 9, 1105AZ, Amsterdam, The Netherlands
Department of Surgery, Academic Medical Center, University of Amsterdam, Meibergdreef 9,
Department of Medical Oncology, Academic Medical Center, University of Amsterdam,
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* Author for correspondence (
[email protected])
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Key words: Sonic Hedgehog, shedding, ADAM, pancreatic cancer
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JCS Advance Online Article. Posted on 29 October 2014
Abstract
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Members of the Hedgehog (Hh) family of morphogens play critical roles in development, but are
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also involved in the progression of certain types of cancer. Despite being synthesized as
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hydrophobic dually lipid modified molecules, and thus strongly membrane-associated, Hh ligands
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are able to spread through tissue and act on target cells several cell diameters away. Various
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mechanism that mediate Hh release have been discussed in recent years, however, little is known
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about dispersion of this ligand from cancer cells. Using coculture models in conjunction with a
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newly developed reporter system, we were able to show that different members of the ADAM
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family of metalloproteases strongly contribute to the release of endogenous, bioactive Hh from
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pancreatic cancer cells, but that this solubilization decreases the potency of cancer cells to signal
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to adjacent stromal cells in direct coculture models. These findings imply that under certain
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conditions, cancer cell-tethered Hh molecules are the more potent signaling activators and that
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retaining Hh on the surface of cancer cells can unexpectedly increase the effective signaling
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range of this ligand depending on tissue context.
Journal of Cell Science
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Accepted manuscript Journal of Cell Science
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Introduction
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Morphogens are locally secreted proteins that spread from producing cells and define the cell fate
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of receiving cells at different distances from the source (Ashe and Briscoe, 2006). In both
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vertebrates and invertebrates, many diffusible signals are essential during development, and
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dysfunction in the dispersion of these proteins gives rise to severe developmental defects. One of
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the best-studied group of such molecules is the Hedgehog (Hh) family, consisting of Sonic
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Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh) in mammals (Echelard et
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al., 1993). Hh proteins are essential for processes like patterning of the ventral neural tube,
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specifying digit identities in the limb bud, as well as cell growth and differentiation (Bijlsma et
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al., 2004). Given this wide array of functions, it is not surprising that aberrant activation of the Hh
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pathway has been implicated in many types of cancer. An especially notorious example of this is
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pancreatic cancer, in which SHH produced by tumor cells activates the pathway in adjacent
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stromal cells to aid in the activation of this compartment (Yauch et al., 2008).
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Hh ligand is generated as a 45 kD precursor protein, which undergoes autocatalytic cleavage
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producing a 19 kD N-terminal peptide that has a cholesterol moiety attached to its C-terminus. In
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addition, a palmitoyl residue is attached at the N-terminus by the transferase Skinny
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Hedgehog/Hhat, yielding a dually lipid-modified ligand that is strongly hydrophobic and hence
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membrane-associated. This has long raised questions as to how it can reach cells located at a
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distance from the cell of origin. Notably, both lipid modifications are essential for normal
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biological activity (Chamoun et al., 2001; Chen et al., 2004; Traiffort et al., 2004).
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Several molecular mechanisms of how Shh travels long distances are currently under intense
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debate. Proposed models of Hh solubilization include release via the transmembrane protein
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dispatched 1 (Disp1), oligomerization of proteins and formation of ‘micelle-like’ structures, co-
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transport with lipoprotein particles, and proteolytic release by metalloproteases (Burke et al.,
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1999; Zeng et al., 2001; Ma et al., 2002; Panakova et al., 2005; Dierker et al., 2009; Queiroz et
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al., 2010). The latter mechanism was described only recently and involves A desintegrin and
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metalloproteases (ADAMs).
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The family of ADAM proteins is important for the release of proteins from producing cells, a
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process termed shedding. Substrates include members of the EGF receptor family ligands, Notch 3
receptors, TNFa, and the amyloid precursor protein (APP) (Murphy, 2008). Not surprisingly,
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ADAMs have been implicated to play an important role in diseases such as Alzheimer’s, chronic
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inflammation, and cancer (Murphy, 2008). ADAMs have also been shown to mediate shedding of
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Hh ligands by cleavage of Hh oligomers from the surface of Hh producing cells, leading to the
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formation of biologically active signaling complexes (Dierker et al., 2009; Ohlig et al., 2011).
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These initial studies were based on overexpression systems and it is still unknown if ADAMs can
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be involved in processing and release of endogenously expressed Hh molecules.
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In the present study, we define the role of ADAM metalloproteases in the release of endogenous
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Hh from pancreatic cancer cells. Using chemical stimulation of sheddase activity, profiling of
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cancer tissue and cells, and knockdown studies, we show that Hh release from cancer cells can be
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mediated by ADAM10, ADAM12 and ADAM17. We further show that blocking the release of
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Hh leads to an increase in cell bound Hh protein, which results in both an enhancement of
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juxtacrine signaling as well as an expansion of the signaling range.
Journal of Cell Science
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Accepted manuscript Journal of Cell Science
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Results
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Pancreatic cancer cells activate Hh responsive fibroblast over several cell diameters
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Developmental biology has yielded elegant models to study Hh distribution, like the neural tube
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or the imaginal discs. These tissues can be imaged to reveal the spread and functional
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consequences of Hh ligand as it forms a gradient. For tumor biology, such imaging was not
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previously available. To monitor Hh pathway activity in a spatial and temporal manner, we
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developed a lentiviral reporter construct that expresses eGFP under the control of a promoter
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containing eight concatemerized Gli-binding site motifs and a TATA box (Fig. 1A). Mouse
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embryonic fibroblasts transduced with the reporter construct (GBS-GFP MEFs; GGM), showed a
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wide dynamic range of reporter activity. Almost no GFP expression was observed under
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unstimulated conditions, as measured by fluorescence microscopy (Fig. 1B) and flow cytometry
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(Fig. 1C), indicating very low basal Hh pathway activity in GGMs. Treatment with ShhN or Smo
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agonists SAG and purmophamine led to a strong induction of GFP expression (Fig 1C, D).
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To test whether this reporter system could also be used to study the activity of Hh endogenously
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expressed in pancreatic ductal adenocarcinoma (PDAC) cells, we cocultured GGMs with the
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PANC-1 cell line and measured GFP positive cells by flow cytometry. PANC-1 cells robustly
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activated the reporter construct in GGMs and co-administration of the Smo inhibitor KAAD-
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cyclopamine or the Hh blocking antibody 5E1 strongly diminished this activation, showing that
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GFP activity in the fibroblasts was specifically mediated by Hh derived from PANC-1 cells (Fig.
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1F, G).
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Intriguingly, we did not only observe pathway activation in cells directly adjacent to H2B-
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mCherry labelled cancer cells, but also 3-4 cell diameters away from the source of Hh ligand in a
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gradient like manner, with the GGMs closest to the cancer cells having stronger GFP signal (Fig.
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1E and Fig. S1). What we inferred from this observation is that we can use this system to
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quantitatively assess the diffusion capacity of cancer cell-produced forms of Hh ligand.
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Sheddases can release Hh protein from pancreatic cancer cells
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Several mechanisms for the release of Hh protein have been described in recent years. In
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overexpression systems it was shown that one of these release mechanisms involved the catalytic
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activity of members of the ADAM family of metalloproteases, especially ADAM17 (Dierker et 5
Accepted manuscript Journal of Cell Science
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al., 2009). The process of metalloprotease-mediated cleavage and release of membrane bound
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substrates, is a regulated process that can be rapidly stimulated, for example by activation of
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protein kinase C (PKC) by PMA, and via G-protein coupled receptor activation through the
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addition of LPA (Gschwind et al., 2003; Matthews et al., 2003; Horiuchi et al., 2007). To test
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whether ADAM sheddases can play a role in solubilization of Hh from pancreatic cancer cells,
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we treated PANC-1 cells with known stimulators of sheddase activity and measured the amount
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of surface bound Hh protein by flow cytometry. All tested stimuli significantly reduced the levels
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of Hh on these cells (Fig. 2A). As a positive control, methyl-β-cyclodextrin, which extracts
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cholesterol and as a consequence sterolated Hh from the cell surface, was included (Tukachinsky
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et al., 2012). In addition, this compound has been demonstrated to activate sheddases (Dierker et
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al., 2009). Treatment with cyclodextrin resulted in a near complete loss of Hh on the cell surface.
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Similar results were obtained with Capan-1 cells, and the patient derived primary cell line 53M,
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(Fig. 2B, C).
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To demonstrate that Hh is indeed released into the medium of treated cells after sheddase
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stimulation, we collected the supernatant of PANC-1 cells treated with PMA and quantified the
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amount of Hh protein in the supernatant and cell lysates. Indeed, the supernatant to lysate protein
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ratio increased over time (Fig. 2D), showing that the addition of PMA results in a displacement of
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Hh ligand from the cell membrane to the aqueous extracellular environment. To formally assess
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the involvement and requirement for ADAMs in the observed release of Hh, we decide to
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pinpoint and ablate the exact molecules involved.
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Expression of ADAMs in pancreatic cancer patients, patient derived xenografts, and cell
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lines
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To identify which ADAM proteins could mediate the release of Hh from pancreatic cancer cells,
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we used two publically available microarray datasets from PDAC patients (Badea et al., 2008; Pei
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et al., 2009) to profile the expression of ADAM10, ADAM12 and ADAM17, which were
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previously implicated in shedding of overexpressed Hh from cells (Ohlig et al., 2011). All
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proteins studied were expressed in healthy pancreatic tissue, but were significantly upregulated in
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tumor tissue, consistent with previous observations (Ringel et al., 2006; Gaida et al., 2010)(Fig.
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3A). We also found similar expression of these ADAMs in pancreatic cancer patient derived
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xenografts (PDX) measured by qPCR, with ADAM10 and ADAM17 being considerably higher 6
Accepted manuscript Journal of Cell Science
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expressed than ADAM12 (Fig. 3B). The pancreatic cancer cell lines used for the shedding assay
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(Fig. 2) displayed comparable expression levels to the PDX tumors (Fig. 3C).
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For ADAMs to be able to act on their substrates, they must be present in an activated form on the
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plasma membrane (Seals and Courtneidge, 2003). To measure this active pool of ADAMs,
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protein levels at the cell surface were determined by staining with ectodomain specific antibodies
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in flow cytometry (Fig. 3D). All ADAMs measured were present on the surface of pancreatic
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cancer cells and could thus potentially mediate Hh release.
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ADAM knockdown leads to retention of biologically active Hh protein on the surface of
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cells
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Having identified ADAM10, 12, and 17 as proteins that could potentially be involved in Hh
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shedding from PDAC cells, we proceeded to ablate these proteins in cancer cells by stable
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shRNA transductions, and assess the amount of Hh protein on the surface of these cells.
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Successful knockdown was confirmed by qPCR and flow cytometry (Fig. S2). Cells carrying an
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shRNA directed against Sonic Hedgehog (SHH) were included as a control for the specificity of
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staining as well as subsequent signaling assays. A positive control for the effects of a blocked Hh
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release came from the knockdown of Dispatched 1 (DISP1). Although the exact working
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mechanism of this protein is not fully understood, its involvement in the spread of lipid modified
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Hh from mammalian cells is well established (Ayers et al., 2010; Caspary et al., 2002; Ma et al.,
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2002; Etheridge et al., 2010) and indeed, knockdown of DISP1 led to an increase in surface levels
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of Hh as measured by immunofluorescent staining (Fig. 4A) and flow cytometry (Fig. 4B).
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Knockdown of any of the three ADAM proteins implicated in the shedding of Shh had similar
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(ADAM10), or even stronger effects (ADAM12 and ADAM17), on the retention of Hh on the
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cell surface compared to DISP1 (Fig. 4A,B). This suggests that these proteases function to release
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Hh from the surface of tumor cells. Surface retention of Hh was also observed in the absence of
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serum (data not shown).
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To assess if the pool of Hh that accumulates on the cell surface following silencing of ADAM17
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(or DISP1) is still amenable to dispersal by Disp1, and to confirm that the effects we observe are
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not due to subcellular sorting artefacts that affect Hh distribution prior to its presentation at the
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surface, we overexpressed Disp1 in these cells and assessed if this could reduce the accumulated 7
Accepted manuscript Journal of Cell Science
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surface Hh. Indeed, overexpression of Disp1 was found to rescue the effect of ADAM17 and
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DISP1 silencing (Fig. 4C), and this effect was most obvious in cells with the highest levels of
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surface Hh. A form of Disp1 that carries a mutated residue in the 4th transmembrane region
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critical to its antiporter function was included as a control. This protein (D99Y) was unable to
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rescue the effect of ADAM silencing, and in fact was found to result in increased Hh levels in
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control silenced cells. This is probably a consequence of the dominant negative action such a
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mutant molecule could have in the previously described trimers Disp1 functions in (see
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(Etheridge et al., 2010)). These results show that the fraction of Hh that accumulates on the cell
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surface in the absence of ADAM17 can still be transferred by Disp1 to some other machinery that
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disperses Hh from the cell surface. This in turn implies that in the absence of ADAM17, DISP1 is
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a rate-limiting step in the redirection of Hh and that the two proteins act on the same pool or
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fraction of Hh.
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The strong surface retention of Hh observed in DISP1 or ADAM knockdown cells suggests that
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less Hh is released into the medium of these cells. To confirm this to be the case, we compared
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the amount of Hh in these supernatants and the corresponding lysates. Indeed, ablation of DISP1
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or ADAM resulted in a strongly reduced sup/lysate ratio of Hh (Fig. 4D) and again, ADAM17
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knockdown had the strongest effect. What was also obvious from these experiments was that a
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large fraction of the total Hh present in the cell culture well resided in the supernatant as
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compared to the cell. This seems to suggest that these cells are very actively transporting Hh from
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the cell surface into the medium, for instance by the action of ADAMs. This notion is further
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supported by the finding that although the amount of Hh detected by Western blot or FACS in the
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SHH knockdown cells was very low, some residual ligand could still be detected in the
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supernatant. At the very least, these data strongly suggest that in PDAC cells, ADAMs function
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to shed or otherwise move Hh from the surface into the medium.
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ADAM mediated release of Hh was reported to involve proteolytic processing of Hh proteins,
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resulting in the removal of the cholesterol moiety (Ohlig et al., 2011). A consequence of this
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model holds that any Hh that is medium-borne in the absence of ADAM function should still be
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sterolated. To formally confirm this to be the case in our experimental setup, we loaded PDAC
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cells with 3H-cholesterol and assessed the amount of sterolated Hh in the supernatant. Although
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the total amount of Hh protein in the supernatant from ADAM17 knockdown cells was found to 8
Accepted manuscript Journal of Cell Science
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be strongly reduced (Fig. 4D), the amount of sterolated Hh in the supernatant was found to be
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equal or even higher compared to control, suggesting that indeed ADAM17-mediated processing
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involves removal of cholesterol from the mature Hh protein (Fig. 4E). Addition of cyclodextrin to
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the medium mobilized sterolated Hh from the cell surface, resulting in a strong increase in
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sterolated Hh in the supernatant (Fig. 4E, dark grey bars). This effect was especially apparent in
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the ADAM17 silenced cells. In addition, this confirms that the effect of cyclodextrin on Hh
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release from the surface is at least in part due to extraction of the protein from the membrane due
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to its cholesterol modification (Tukachinsky et al., 2012), rather than acting solely through the
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activation of sheddase activity (Dierker et al., 2009). To assess if palmitoylated Hh is also
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extracted from the cell surface by cyclodextrin, we loaded PANC-1 cells with 3H-palmitate and
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repeated the experiment (Fig. 4F). In control silenced cells, palmitoylated Hh was not extracted
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by cyclodextrin, suggesting that the fractions of sterolated and palmitoylated Hh do not
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necessarily overlap. In ADAM17 silenced cells, however, palmitoylated Hh was extracted by
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cyclodextrin. This suggests that in the absence of ADAM function, the SHH accumulating on the
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cell surface is more likely to have both lipid modifications.
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To assess the functional consequences of Hh solubilization by ADAMs, we treated Shh-LIGHT II
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reporter cells (Taipale et al., 2000) with conditioned medium from the different knockdown cell
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lines. As expected from the reduced Hh protein levels in the supernatant following ADAM
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ablation, we also observed a significant decrease in signaling activity of the conditioned medium
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of all the tested knockdown cells (Fig. 4G). These findings were confirmed in the complete
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absence of serum (Fig. 4H). However, when we performed direct co-culture experiments with the
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knockdown PANC-1 cells and luciferase reporter cells to allow juxtacrine signaling we observed
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a clear increase in Hh pathway activity with cells displaying high Hh levels on the surface (Fig.
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4I). Interestingly, despite the apparent abundance of Hh ligand in the medium, the supernatants
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were less effective inducers of pathway activity than the cocultured cells. The experiments
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mentioned above show that although cells can release biologically active Hh into the medium,
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and that stopping this release strongly reduces activity in medium transfer experiments,
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enhancing the presence of cell-tethered forms of Hh increases the direct signaling capacity of
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cells in coculture. This effect is apparently strong enough to overcome the effects of reduced
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signaling by solubilized Hh in the system. 9
Accepted manuscript Journal of Cell Science
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Blocking release of Hh leads to increase in signaling range
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To confirm our finding that the surface bound form of Hh is a more potent pathway activator than
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shed Hh, we performed cocultures of different knockdown cells with the GGMs, which in
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contrast to the luciferase reporter system allow us to determine the position of responding cells
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relative to the source of ligand. Consistent with the previous observation in the luciferase assays
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(Fig. 4I), we found that coculture with cancer cells silenced for DISP1 or ADAMs lead to a
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strong increase in the percentage of GFP positive reporter cells measured by flow cytometry (Fig.
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5A, B), showing that this effect is independent of the readout used for Hh pathway activation.
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Again, these data seem to suggest that cell-tethered Hh is a strong signaling form and that its
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release from the cell surface is detrimental to its signaling capacity. These findings are
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corroborated by the lack of responsiveness of GGMs to conditioned supernatant from PANC-1
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cells (data not shown) but their high sensitivity to cell-tethered ligand (Fig. 5A, B).
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To test whether this increase in signaling potential of cells with high Hh surface expression is
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related to the range of signaling, we performed the same cocultures and imaged these. DISP1 and
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ADAM silenced cells showed clearly more GFP positive fibroblasts around the cancer cell
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colonies with the reporter cells close to the source displaying higher GFP intensity compared to
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control silenced cells (Fig. 5C). To measure the actual signaling range in distance, we quantified
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the spread of GFP signal from at least 20 colonies of each condition (Fig. 5D). We found that
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especially interference with ADAM17 and ADAM12 increased the intensity and range of Hh
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pathway activity in the cells adjacent to the cancer cells. While coculture with control silenced
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cells yielded a signal reaching around 4 cell diameters, the range in shADAM17 cells was
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doubled to 8 cell diameters, showing that increase in direct transsignaling ability of cells with
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high Hh surface levels can be contributed to an increased signaling range away from the ligand
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source. The effects of ADAM silencing on the signaling capacity of cancer cell-derived Hh were
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confirmed in a primary line (Fig. S3).
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These data again suggest that, at least in this model, cell-tethered Hh is a more effective form of
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this ligand as opposed to its soluble counterparts. To confirm this in another imaging setup that
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more strictly distinguishes between cells in close proximity on one hand, and medium-borne
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crosstalk on the other, we devised a coculture model in which there is a fairly strong physical
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separation between the producer and responder cells. By first placing cancer cells, mixed in a 10
Accepted manuscript
Matrigel cushion, in a culture dish and subsequently placing GGMs on top, we found that the
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latter cell type only contacted the cancer cells after migrating into the Matrigel laterally (Fig. 5E).
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Following this, we could observe reporter activity only in those GGMs in or under the Matrigel
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cushion, but not in those that are merely in close proximity to the border of this structure (Fig.
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5E, F). Adding cyclopamine to the cultures abrogated the response, proving the observed GFP
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expression to be mediated through Smo rather than some non-canonical activation of the Gli
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transcription factors as a consequence of the seeding conditions (Fig. 5G). Thus, we conclude that
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Hh that is released from cancer cells will not activate signaling in the GGMs and again argues in
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favor of a model of Hh distribution that does not include solubilization.
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Accepted manuscript Journal of Cell Science
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Discussion
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A wide range of release and transport mechanisms for Hh proteins from source cells have been
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described over the years in different, mainly developmental, model systems. It appears that Hh
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secreting cells can make use of not one, but several of these mechanisms to achieve differential
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biological output over short and long distances. The mode of Hh distribution can be influenced by
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the environmental context (Therond, 2012). However, in the setting of malignancies in which the
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ligand is aberrantly expressed in tumor cells and mostly signals to adjacent stroma, like
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pancreatic cancer (Yauch et al., 2008), no studies have addressed the exact signaling range of Hh.
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By using chemical inducers of sheddase activity together with interference of ADAM activity on
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a transcriptional level, a clear role of ADAM sheddases in the release of endogenous Hedgehog
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from pancreatic cancer cells has now become apparent.
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All three ADAMs previously reported to release overexpressed Hh from Bosc23 cells were found
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to be involved in Hh release from pancreatic cancer cells, but some interesting things were noted
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in these experiments: Silencing of the various paralogs did not result in equally strong surface
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retention of Hh, and the silencing of some ADAMs seemed to affect Hh surface levels to an
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extent that is incompatible with a possible redundancy between the three ADAMs tested. One
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explanation for this could be the difference in expression levels of the various ADAM proteins.
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For instance, the very high expression levels of ADAM10, even after knockdown, could be
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responsible for some residual catalytic activity of this protein resulting in an incomplete surface
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retention of Hh. Conversely, ADAM12 was expressed at much lower levels, and as a
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consequence it was reduced to almost undetectable, and possibly inactive, levels following
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knockdown. A second explanation comes from a potential cross-regulation in our knockdown
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experiments: the activity or surface localization of one particular ADAM might depend on the
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presence of other proteases. Indeed, the surface localization and activation of MMP-14 has
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recently been shown to depend on ADAM12 (Albrechtsen et al., 2013), and conceivably such an
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interaction could also exist between the different ADAM family members in our system. A third
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possibility is that proper processing of Hh relies on the sequential action of several ADAMs, and
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that taking out one of the ADAM proteins will affect this sequential action, resulting in more
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surface retention of Hh than would be expected. Although these considerations make it more
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Accepted manuscript Journal of Cell Science
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difficult to pinpoint the exact roles of the individual ADAMs, they do not detract from the
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conclusion that ADAM sheddases are involved in the release of Hh from pancreatic cancer cells.
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Previous studies regarding the activity of proteins involved in Hh transport or dispersion has
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relied on biochemical methods like size exclusion chromatography. However, the amount of
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endogenously expressed Hh in our studies was rather limited and currently not amenable to in-
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depth biochemical analysis to determine for instance the cleavage sites or aggregate status. As a
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consequence, the use of the conformational epitope antibody 5E1 in conjunction with flow
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cytometry has been crucial to this study and we found it to be more sensitive and less technically
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challenging than conventional biochemical methods. It also allows easy quantification on overall
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cell populations.
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A question from developmental biology that remains unanswered in our study is why the
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phenotypes of Adam mutant mice described in literature do not show any of the classical Hh
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pathway mutant hallmarks such as holoprosencephaly, digit patterning abnormalities, or laterality
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defects (Blobel, 2005). Interpretation of these mouse mutants, however, is difficult and could be
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complicated by the compensatory activities of different sheddases with shared ligand specificity,
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resulting in very weak phenotypes. Furthermore, our data suggest that loss of Adam function
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would result in an enhanced Hh signaling, and Hh pathway gain-of-function phenotypes are
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typically much harder to discern. It is conceivable that more targeted observations of for instance
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neural tube patterning in these animals would demonstrate a role for Adams in modulating Hh
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levels in vivo. In the absence of such data at this moment, our results should be interpreted with
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care.
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Although at first glance counterintuitive, our finding that retention of Hh protein on the surface of
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cancer cells leads to an increase in signaling range is perfectly compatible with a direct transport
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model of membrane-bound ligand. One such transport mechanism comes from the filopodia-like
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cell protrusions named cytonemes, initially described in fruit fly (Sato and Kornberg, 2002).
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These dynamic actin-based extensions span several cell diameters and can transport Hh
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molecules to target tissue (Bischoff et al., 2013). Also in the chick neural tube, Hh was shown to
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be transported via long cytoplasmic extensions that span several cell diameters (Sanders et al.,
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2013). The exact location, inside or outside the cytoneme, as well as the actual form of the ligand
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transported, is still under debate.
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Increasing the amount of Hh on cell protrusions, by knocking down ADAMs for instance, would
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allow this ligand to reach the threshold necessary to activate the pathway in receiving cells,
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therefore extending the range of signaling in our reporter cell system. Although we were able to
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detect actin based extensions from PANC-1 cells carrying a LifeACT-mCherry construct, these
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extensions only bridged over maximally 2 cell diameters towards reporter cells, which is not far
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enough to explain the activation observed up to 8 cell diameters away as observed in the case of
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ADAM17 silenced PANC-1 cells. Reminiscent of a model proposed in Drosophila for Dpp
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signaling (Sato and Kornberg, 2002), it could also be the case that responsive fibroblasts reach
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out for ligand with their own cytonemes, therefore shortening the necessary reach of the cancer
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cells to activate the receiving cells, as they would 'meet in the middle'. Also, we cannot exclude
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the trivial explanation that our current life imaging microscopic capacities are inadequate for
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visualizing finer structures that could potentially reach far enough.
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An alternative explanation for the increased signaling range would be a model in which the block
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of release of Hh from the surface of producer cells leads to more Hh protein accessible to
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transport from one reporter cell to the next via planar diffusion on heparan sulfate proteoglycan
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(HSPG) molecules. Especially studies in Drosophila have shown a crucial requirement of the
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glypicans dally and dally-like, the fruitfly equivalent of mammalian HSPG molecules, as well as
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the glycosaminoglycan transferase tout-velu, in activating target cells at large distances from the
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source of Hh ligand (Bellaiche et al., 1998; Han et al., 2004). But also in vertebrate systems
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HSPGs have been shown to affect the spread of Hh ligands (Burke et al., 1999; Ma et al., 2002).
23
In agreement with this model is the previous observation that ADAM-mediated processing
24
decreases the binding capacity of Hh to HSPGs by inactivating the heparin sulfate binding motif
25
(Ohlig et al., 2012). Decreasing ADAM-mediated processing would therefore enhance Hh
26
binding to HSPG molecules on producer cells, fitting the results in our study from ADAM
27
knockdown cells. As a consequence, more HSPG sequestered ligand can be handed to reporter
28
cells and the threshold needed for activation is reached in reporter cells further away from the
29
ligand source. The data shown in Figure S4 at least suggest that release of Hh by competing with
30
HSPG binding is detrimental to transsignaling, but whether this is due to solubilization or 14
Accepted manuscript Journal of Cell Science
1
masking of Ptch1-binding residues cannot be concluded. Both models of cytoneme transport and
2
HSPG bound lateral diffusion are not mutually exclusive, as Hh transported on cytonemes could
3
be HSPG associated, allowing for enrichment of Hh on these structures.
4
Concluding, our data show that ADAMs can act on Hh expressed in cancer cells, and that they
5
generate soluble, biologically active forms of this protein. More importantly however, is the
6
functional consequence of this solubilization: In our experimental setups, diffusion of Hh limits
7
its concentration in the relevant compartment where it exerts its biological effect and conversely,
8
retaining Hh on the surface of cells increases the effective signaling range in trans. To exclude
9
the possibility that our conclusions are based on models that possibly overestimate the
10
contribution of cell-tethered Hh or otherwise skew the results in a non-physiological way, future
11
studies will be needed to address the relevance of different ligand distribution mechanisms as
12
well as effect of ADAM manipulation on Hh target genes in the context of pancreatic cancer. In
13
addition, a better understanding of how the stroma in this disease is activated by tumor cell-
14
derived Hh is especially relevant in light of recently published work that shows that outright
15
ablation of Hh signaling in the stroma can have unexpected detrimental outcomes (Rhim et al.,
16
2014; Lee et al., 2014).
17
Figure Legends
18
Fig. 1. Pancreatic cancer cells active the HH pathway several cell diameters away from the
19
source in an eGFP reporter cell line.
20
(A) Schematic representation of the lentiviral 8x-GLI binding site (of which the consensus logo is
21
shown) reporter construct driving the expression of eGFP. Construct was used to create the stable
22
mouse embryonic fibroblast reporter cell line (GBS-GFP MEF, GGM). (B) Representative
23
fluorescence images of GGMs treated for 72h with 500nM SAG. Scale bar: 100 µm. (C) FACS
24
plots of reporter MEFs treated 72h with conditioned ShhN supernatant, 500nM SAG or 1 µM
25
purmorphamine. (D) Mean percentage of GFP positive reporter cells from treatment experiments
26
as shown in panel C. Shown is mean ± s.d., n≥8. (E) 2.000 H2B-mCherry labeled PANC-1 were
27
seeded on top of confluent GGMs and imaged after 72h. Scale bar: 50 µm. (F) GGMs and
28
PANC-1 cells were cocultured, and 100nM KAAD-cyclopamine or 2 µg/ml Hh-blocking
29
antibody 5E1 was added for the duration of the experiment to show specificity of the pathway 15
Accepted manuscript Journal of Cell Science
1
activation. 9E10 is an anti-Myc antibody clone. (G) Mean percentage of GFP positive reporter
2
cells from treatment experiments as shown in panel F. Shown is mean ± s.d., n≥7. All
3
experiments were performed in 0.5% FCS.
4
Fig. 2. Hedgehog protein can be released from the surface of pancreatic cancer cells in
5
response to stimulation of sheddases.
6
Pancreatic cancer cell lines PANC-1 (A) and Capan-1 (B), or primary pancreatic cancer cells
7
53M (C) were treated with 100 ng/ml phorbol myristate acetate (PMA), 10 µM lysophosphatidic
8
acid (LPA), or 1 mg/ml methyl-β-cyclodextrin (MbCD) for 1 h in serum free medium. Surface
9
expression of HH ligand was detected by flow cytometry using 5E1 a-SHH antibody and results
10
are displayed as geometric mean fluorescence intensity (gMFI) after subtraction of isotype gMFI
11
relative to control treatment. Data show the mean ± s.d., n≥3, *P<0.05, **P<0.01, ***P<0.001.
12
(D) PMA mediated release of endogenous HH into serum-free supernatant was measured over
13
time. Plot shows quantification of HH protein levels in supernatant relative to lysates of PANC-1
14
cells treated with 100 ng/ml PMA for 30, 60, and 120 min. Supernatant was subjected to 5E1
15
immunoprecipitation before detection of Hh protein by Western blot. Cells were lysed directly
16
prior to blotting. Data show the mean ± s.d., n≥3.
17
Fig. 3. ADAM sheddases are expressed in pancreatic cancer patients, patient derived
18
xenografts and pancreatic cancer cell lines.
19
(A) Box plots showing log2 transformed gene expression values from two microarray datasets of
20
pancreatic cancer patients comparing normal and tumor tissue. Badea set (GSE15471); n=36
21
paired biopsies, Pei set (GSE16515); n=15 (normal), n=36 (tumor). *P<0.05, **P<0.01,
22
***P<0.001. (B) Indicated transcript levels were measured on 7 different patient derived
23
xenografts by qPCR using species-specific primers. Box plots show min to max distribution of
24
ADAM transcripts relative to human GAPDH. (C) qPCR analysis of ADAM gene expression
25
relative to human GAPDH in PANC-1, Capan-1 and 53M cells. Data show the mean ± s.d., n=3.
26
(D) Surface expression of ADAM10, ADAM12 and ADAM17 on cell lines was confirmed by
27
flow cytometry using antibodies directed against the ectodomain of these proteins. Representative
28
histograms are shown. Legends shown in panel C indicate the measured proteins.
16
Accepted manuscript Journal of Cell Science
1
Fig. 4. Knockdown of ADAMs in pancreatic cancer cells leads to retention of HH on the cell
2
surface and increased juxtacrine signaling.
3
(A) Indicated genes were silenced in PANC-1 cells and following confirmation of knockdown,
4
cells were seeded on coverslips, grown in 8% FCS, and immunofluorescence using 5E1 on live
5
cells was performed. Detection, mounting, and imaging were performed after fixing. shRNA
6
against SHH was included as a control for the specificity of the staining. Scale bar: 30 µm. (B)
7
Hh surface levels of the cells used in panel A were measured by flow cytometry analysis using
8
5E1. All shRNA clones targeting the ADAM proteases as well as DISP1 showed an accumulation
9
of HH protein on the surface. The isotype corrected gMFI of several experiments is plotted
10
relative to control silenced cells. Data show the mean ± s.d., n≥3. Experiments were performed in
11
8% FCS. (C) Cells silenced for DISP1, ADAM17, or control were cotransfected with eGFP and
12
Renilla luciferase (control), mDisp1, or mDisp1D99Y as indicated. After 3 days, cells were
13
harvested and stained for surface Hh. Data show the average isotype corrected geometric mean of
14
the GFP + population, ± s.d., n=6. Significance was calculated compared to respective Renilla
15
control in every cell line.*P<0.05, ***P<0.001. Experiments were performed in 8% FCS. (D)
16
Serum free medium was conditioned on cells silenced for the indicated genes for 48 h and
17
supernatant was subjected to immunoprecipitation before Western blotting. Cells were lysed
18
directly in Laemmli buffer. Note that the membranes containing lysates or immunoprecipitated
19
samples were ran simultaneously, but imaged separately, and that the loaded fraction of total
20
protein was approximately 3 times higher in the immunoprecipitation membrane (10% of lysate
21
ran vs. 33% of IP eluate). (E) Control or ADAM17 silenced PANC-1 cells were seeded, loaded
22
with 3H-cholesterol or 3H-palmitate (F) and serum-free supernatant was conditioned for 4 days
23
with the addition of 300 µg/ml MbCD during the last 24 h. Supernatant was subjected to
24
immunoprecipitation with 5E1 and radioactivity was measured in the precipitated protein. Graph
25
shows disintegration events per minute relative to untreated control cells. Shown is mean ± s.d.,
26
n=3. (G) Supernatant containing 0.5% FCS or 0% FCS (H) was conditioned on indicated cells
27
and Shh-LIGHT II cells were treated with 1:2 diluted supernatants for 24 h after which pathway
28
activation was measured by firefly luciferase activity normalized to constitutive Renilla
29
luciferase, and expressed relative to supernatant from control silenced PANC-1 cells. Data are
30
shown as mean ± s.d., n≥3, *P<0.05, **P<0.01, ***P<0.001. (I) Shh-LIGHT II cells were
31
cocultured with indicated PANC-1 cell in 0.5% FCS and pathway activity was measured after 24 17
Accepted manuscript Journal of Cell Science
1
h. Data are displayed relative to control silenced cells and show the means ± s.d., n≥6, *P<0.05,
2
**P<0.01, ***P<0.001.
3
Fig. 5. Cell membrane tethered HH protein increases the range of signaling to adjacent
4
reporter cells.
5
(A) Cells silenced for the indicated genes were seeded on top of GGM reporter cells as for Figure
6
4, and after 3 days cells were harvested and GFP positive cells were quantified by flow
7
cytometry. Data are displayed as percentage relative to control silenced PANC-1 cells, and shown
8
is the mean ± s.d., n≥14. Experiments were performed in 0.5% FCS. (B) As for panel A, using
9
0% FCS. (C) Representative images of cocultures 72 h after seeding. Upper panels show GFP
10
positive cells activated by Hh ligand from the tumor cells. Lower panel shows corresponding
11
bright field image with cancer cell colonies demarcated by dashed line. Scale bar: 100 µm. (D)
12
Images of cocultures with indicated PANC-1 cells were taken on a fluorescence microscope and
13
GFP intensity reaching from the cancer cell colonies was quantified using ImageJ. One cell
14
diameter equals 15 µm on x-axis. Graph represents measurements of over 20 cells. (E) 4x105
15
H2B-mCherry labeled PANC-1 cells were seeded in 50 µl growth factor reduced Matrigel and
16
after 48 h, GGMs were seeded on top. Representative image of cocultures 3 d after seeding is
17
shown. Green and red bars indicate the border of the Matrigel cushion. Scale bar: 50 µm (F) As
18
for panel D, 6 d after seeding. (G) As for panel E, including 100nM KAAD-cyclopamine. Scale
19
bar in E and F: 200 µm. Experiments were performed in 0.5% FCS.
20
Material and Methods:
21
Cell Culture and Chemicals
22
PANC-1, Capan1, HEK293T (ATCC, Manassas, VA), and mouse embryonic fibroblasts
23
(Goodrich, science 1997), were cultured in high-glucose DMEM containing 8% fetal bovine
24
serum (FBS), L-glutamine, penicillin and streptomycin (all from Lonza, Basel, Switzerland)
25
according to routine cell culture procedures. Shh-LIGHT II cells ((Taipale et al., 2000), ATCC)
26
were grown in the abovementioned medium supplemented with 400 µg/mL neomycin (Sigma, St
27
Louis, MO) and 150 µg/mL zeocin (Invitrogen, Carlsbad, CA). 9E10 and 5E1 hybridoma cells
28
(Developmental Studies Hybridoma Bank, Iowa City, Iowa) were maintained in RPMI containing
29
10% FBS, L-glutamine, penicillin and streptomycin (all from Lonza). Hybridoma cells were 18
Accepted manuscript Journal of Cell Science
1
switched to serum free medium for antibody production. Treatment of pancreactic cancer cell
2
lines with 100 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma), 1 mg/ml methyl-β-
3
cyclodectrin (MbCD, sigma), or 10 µM lysophosphaditic acid (LPA, Sigma) was performed in
4
serum free DMEM for 1 h at 37°C. Subsequently, cells were washed twice with phosphate-
5
buffered saline (PBS) and processed for flow cytometry analysis.
6
Flow Cytometry
7
Cell were harvested with trypsin-EDTA solution (Lonza) and washed in FACS buffer (PBS
8
containing 1% FBS). Antibodies were diluted in FACS buffer and incubated 30 min at 4°C.
9
Concentrations used were: a-ADAM10; 1:500 (MAB1427 from R&D, Minneapolis, MN), a-
10
ADAM17; 1:100 (R&D, MAB9301), a-ADAM12; 1:500 (Sigma, SAB2100046), conditioned
11
hybridoma supernatant containing either a-SHH antibody 5E1 (0.08 µg/ml) or a-Myc antibody
12
9E10 (1 µg/ml); diluted 1:5 in FACS buffer before used for staining. Secondary APC labeled a-
13
mouse (BD, 550826) or a-rabbit antibodies (southern biotechnology, 4050-11S) were diluted
14
1:500. After washing cells were resuspended in FACS buffer containing 1ug/ml propidium Iodide
15
(PI) (Sigma) and acquired on a FACSCanto II (BD, Franklin Lakes, NJ). Data were analyzed
16
with FlowJo 7 (Tree Star, Ashland, OR). From the PI negative fraction the geometric mean
17
fluorescence intensity (gMFI) in the APC channel was calculated and the gMFI from the isotype
18
control was subtracted from the respective staining yielding the delta gMFI.
19
Immunoprecipitation and detection of Hedgehog by Western blot
20
Equal amount of cells were seeded in 6-well plates and the next day, 1 mL serum free medium
21
was added and incubated for 48h. Supernatants were harvested and cleared of cells by
22
centrifugation at 500 g for 5 minutes. Shh was precipitated from the supernatants by addition of
23
5E1 hybridoma supernatant 1:5 overnight, and 30 µL Protein A/G beads (Santa Cruz
24
Biotechnology, Santa Cruz, CA) for 2h. Elution was performed using Laemmli sample buffer
25
(Biorad) with DTT. For detection of cell-bound Shh, cells were directly lysed. Samples were
26
loaded on 4-15% gradient SDS PAGE gels. Following transfer and blocking with 5% milk/Tris-
27
buffered saline with 0.1% Tween-20 (TBS-T), membranes were incubated in a-Shh (H160, Santa
28
Cruz) at 1:2,000 overnight). All appropriate secondary antibodies were used at 1:5,000. Proteins
29
were visualized using a FujiFilm LAS 4000 imager (GE Life Sciences, Pittsburgh, PA). Band
30
intensity was quantified in ImageJ using the Gel Analyzer plugin, and sup/lysate ratio was 19
Accepted manuscript Journal of Cell Science
1
calculates. For determining the input corrected ratio between cell- and supernatant Shh, sup and
2
lysate signals of control shRNA transduced PANC-1 cells were quantified from the same blot and
3
exposure, and corrected for fraction of sample loaded on gel. This ratio was used to normalize the
4
ratios in control transduced PANC-1 cells in all other experiments.
5
Immunofluorescence staining
6
Transduced PANC-1 cells were grown on coverslips, incubated with 5E1 supernatant (0.016
7
µg/ml) for 1h at 37 °C, and fixed using 4% formaldehyde. Following blocking and
8
permeabilization in 5% goat serum/phosphate-buffered saline with 0.1% Triton X100 (PBS-T),
9
Alexa 488 conjugated a-mouse secondary antibody (Invitrogen) was added at 1:2,000 for 1h at
10
room temperature and slides were mounted using ProLong Gold (Invitrogen) and images were
11
taken on a Zeiss AxioVert microscope.
12
Constructs
13
The 8x3’GLI binding site sequence (GBS) was isolated from the GBS-luciferase reporter
14
construct (Sasaki et al., 1997) by PCR using the following primers (forward 5’-
15
TATAACCGGTACAGATTCGCGATCGACC-3’; reverse: 5’-
16
ACTTGCTAGCTGCAGGTCGACTCTAGAGGAT-3’) introducing AgeI and NheI restriction
17
sites. The digested PCR fragment was cloned into the lentiviral reporter vector pRRL TOP-
18
d2GFP after excision of the TCF/LEF biding sites using XmaI and XbaI. The d2GFP was then
19
replaced by eGFP from the pRRL TOP-GFP vector (Reya nature 2003) using XhoI and EcoRI.
20
All enzymes were purchased from New England Biolabs (Ipswich, MA). The H2B-mCherry
21
expression construct was obtained by removing the TOP site from the Wnt reporter vector TOP-
22
GFP-PGK-H2BmCherry.The expression construct for mDisp was a kind gift from Dr P. Beachy.
23
The D99Y proton channel mutant was made using the QuikChange II XL Site-Directed
24
Mutagenesis Kit (Agilent, Santa Clara, CA), and the following primers (forward 5'-
25
TCGGGATTGGCGCGTACGACGCTTTTGTC-3'; reverse 5'-
26
GACAAAAGCGTCGTACGCGCCAATCCCGA-3').
27
Lentiviral transduction
28
Lentiviral particles were produced by transiently transfecting HEK293T cells with pLKO
29
constructs from the Sigma Mission library. TurboGFP-targeting control shRNA (shc004) or the 20
Accepted manuscript Journal of Cell Science
1
GBS-GFP reporter and the packaging plasmids pMD2.G and psPAX2 with calcium phosphate.
2
48 h and 72 h after transfection the supernatant was harvested, filtered through a 0.45 µm filter
3
(Millipore, Germany) and subsequently used to transduce 60% confluent PANC-1 cells or MEFs
4
in the presence of 7 µg/ml polybrene (Sigma) overnight. Two days after transduction, cells were
5
selected for stable expression of shRNA with 2 µµg/ml puromycin (Sigma) and knockdown
6
efficiency was analyzed by qRT-PCR and flow cytometry.
7
GGM reporter cell line
8
MEFs transduced with GBS-GFP reporter construct were sorted on a BD FACSAria for GFP
9
expression after stimulation with ShhN conditioned supernatant from HEK293T cells for 4d.
10
Sorted GFP positive cells were grown under 8% FCS conditions, which resulted in loss of GFP
11
activity. For reporter assays, GGM cells were seeded in 96 well plates (Greiner) and upon
12
growing confluent, treated with 500 nM Smoothened agonist SAG (EMD Millipore, Billerica,
13
MA), 1 µM purmorphamine (EMD Millipore), ShhN conditioned supernatant or 2x103 cancer
14
cells per well in DMEM containing 0.5% FCS. For blocking experiments 100nM KAAD-
15
cyclopamine (Merck Millipore), or hybridoma supernatant containing 5E1 or 9E10 was added to
16
the cocultures. After 3-5 days, cells were imaged on a Zeiss fluorescence microscope or on a
17
Operetta High Content Imaging System (PerkinElmer, Waltham, MA) and percentage of GFP
18
positive cells was determined by flow cytometry on a FACSCanto II.
19
Patient derived xenografts and establishment of primary cell line 53M
20
Collection of material from patients undergoing surgical resection for pancreatic adenocacinoma
21
in our institute was approved by the institute’s ethical committee. Informed consent was obtained.
22
Tumor pieces originating from primary tumor or liver metastasis were washed several times in
23
PBS and grafted with Matrigel (BD) subcutaneously into the flank of immunocompromised NSG
24
mice (JAX 005557). Animals were bred and maintained at the local animal facility according to
25
the legislation and ethical approval was gained for the establishment of patient derived
26
xenografts. Upon reaching a size of 500 mm3, PDX tumors were harvested and transplanted in a
27
new animal. After the second passage of the liver metastasis derived 53M PDX, xenograft tissue
28
was dissociated with collagenase IV (0,5 mg/ml, Sigma) and hyaluronidase (20 µg/ml, Sigma)
29
and isolated cells where grown in 8% FBS containing DMEM. Purity of the epithelial culture was
30
assessed by human specific EpCAM antibody staining (DAKO, F0860 at 1:100). 21
Accepted manuscript Journal of Cell Science
1
Quantitative real-time PCR
2
PDX tissue pieces or cells grown in culture were homogenized and lysed in Trizol (Invitrogen)
3
and RNA was isolated according to the manufacturer’s protocol. cDNA was synthesized using
4
Superscript III (Invitrogen) and random primers (Invitrogen). Real-time quantitative RT-PCR was
5
performed with SYBR green (Roche, Basel, Switzerland) on a Lightcycler LC480 II (Roche).
6
Relative expression of genes was calculated using the comparative threshold cycle (Cp) method
7
and values were normalized to reference gene GAPDH.
8
Luciferase reporter assay
9
Shh-LIGHT II cells were grown confluent in a 96 well plate (Greiner), starved overnight in 0,5%
10
FBS containing DMEM and treated with conditioned supernatant or 50.000 cancer cells per well.
11
After 24h of treatment, cells were lysed in 20 µL of lysis buffer, scraped, freeze-thaw cycled, and
12
luciferase activity was assayed according to the Dual-Glo Luciferase Assay System (Promega,
13
Madison, WI) protocol on a Biotek Synergy HT plate reader. Each firefly luciferase value was
14
corrected for its CMV-driven Renilla luciferase standard to correct for nonspecific effects. For
15
production of conditioned supernatant, equal amount of PANC-1 cells with different knockdown
16
constructs were seeded in a 6 well plate, adhered overnight and supernatant containing 0,5% FCS
17
was harvested after 24 h. Conditioned supernatant was spun at 500 g for 10 min in a conventional
18
table top centrifuge and diluted 1:10 in 0.5% FCS medium for treatment of Shh-LIGHT II cells.
19
Transfection and ShhN production
20
HEK293T cells were transfected with expression plasmid for full length Shh (ShhWT), ShhN or
21
ShhC (in pRK5, kind gift from Henk Roelink) using the calcium phosphate method. For
22
production of ShhN conditioned medium used in signaling assays, DMEM containing 0.5% FCS
23
was incubated on cells starting 24h after transfection for 2-3 days before harvest. PANC-1 cells
24
were transfected in 12 well plates (Greiner) with 1 µg plasmid DNA per well (0.25 µg spectrin-
25
GFP and 0.75 µg CMV-Renilla, mDisp1 or mDisp1D99Y) using 3 µg polyethylenimine (Sigma).
26
Tritium labeling
27
Cells were plated in a 10 cm dishes (Greiner), and upon reaching 70% confluency, medium was
28
changed to serum free DMEM containing 55.5 KBq of [1,2(n)-3H] cholesterol or [3H] palmitate
29
(GE Healthcare). After 72 hours, 300 µg/ml MbCD (Sigma) was added to the dishes and medium 22
Accepted manuscript Journal of Cell Science
1
was collected 24h later. Supernatant was cleared by centrifugation for 5 min at 500 g, and
2
immunoprecipitation was performed as described above. Washed agarose beads were
3
resuspended in 4 ml Scintillation liquid (PerkinElmer). Samples were vigorously shaken and
4
disintegrations per minute (DPM) were measured by liquid scintillation analyser TRI-carb
5
2000CA (PerkinElmer).
6
Proliferation assay
7
PANC-1 cells were seeded in 96 well plates at a density of 1000 cells per well. After adhesion
8
overnight, cells were washed with PBS, medium containing 8%, 0.5% or 0% FCS was added and
9
cell viability on the same day (= day 1) was measured by addition of 20 µl CellTiter-Blue reagent
10
(Promega) to the respective wells. After 4 hours incubation at 37°C, fluorescence signal was
11
measured on a Biotek Synergy HT plate reader. Measurements were repeated on day 2 and day 3
12
after seeding.
13
Statistical analysis
14
Data were analyzed by Students t-test using Graph Pad Prism 5 (GraphPad Software, La Jolla,
15
CA). The mean ± s.d. was determined with a significance level of p>0.05.
16
Acknowledgments
17
We thank Dr. M.P. Scott (Stanford University, School of Medicine) for the wild type (Ptch1+/+)
18
mouse embryonic fibroblasts and T.van Leusden for technical assistance and mouse work, and H.
19
van Andel (Academic Medical Center, Amsterdam, The Netherlands) for providing us with the
20
TOP-GFP-PGK-H2BmCherry construct.
21
Footnotes
22
Competing interests
23
The authors declare no competing interests.
23
Author contributions
2
H.D., V.V., and M.F.B. designed experiments, analyzed data and wrote the manuscript. J.P.M.
3
and H.v.L. helped with manuscript preparation and experimental design. J.A.M.G.T. performed
4
patient inclusion and helped obtaining patient derived xenografts.
5
Funding
6
This work was supported by a KWF Dutch Cancer Society Research Grant (UVA 2012-5607) for
7
MFB.
Journal of Cell Science
Accepted manuscript
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Accepted manuscript Journal of Cell Science
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Journal of Cell Science
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Journal of Cell Science
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Journal of Cell Science
Accepted manuscript
Journal of Cell Science
Accepted manuscript
Journal of Cell Science
Accepted manuscript
