Angiogenesis 7: 97--103, 2004.
Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands.
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Hanging-drop multicellular spheroids as a model of tumour angiogenesis Nicholas E. Timmins, Stefanie Dietmair and Lars K. Nielsen Laboratory for Biological Engineering, Department of Chemical Engineering, University of Queensland, Brisbane, Australia Received 28 March 2004; accepted in revised form 26 April 2004
Key words: angiogenesis, 3-D culture, endothelial cell, HUVEC, multicellular spheroid, tumour
Abstract The establishment of a vascular network within tumours is a key step in the progression towards an aggressive, metastatic state, with poor prognosis. We have developed a novel in vitro model to specifically capture the interaction between endothelial cells and solid tumours. Micro-vascularised in vitro tumour constructs were produced by introducing endothelial cells to multicellular spheroids formed in hanging drops. Upon introduction, the endothelial cells migrated into the tumour spheroid, establishing tubular networks and luminal structures. This system relies on the natural pro-angiogenic capacity of multicellular spheroids, and does not require the addition of exogenous angiogenic factors, or use of extracellular-matrix substitutes. Abbreviations: EC -- endothelial cell; HUVEC -- human umbilical vein endothelial cell; LSCM -- laser scanning confocal microscopy; MCS -- multicellular spheroid; VEGF -- vascular endothelial growth factor; vWF -- von Willebrand factor
Introduction Due to nutritional and oxygen limitations, avascular tumours in vivo are restricted in size and activity. These benign tumours often remain dormant for years, never progressing beyond a few millimetres in size. In the case of malignant tumours, hypoxia and nutrient limitation stimulate production of pro-angiogenic factors, actively recruiting existing vasculature, and establishing a network of blood vessels throughout the tumour [1--3]. Establishment of a vascular network permits tumours to overcome growth restrictions, and provides a means for dissemination of metastatic cells [4]. As a result of unrestricted growth and metastatic potential, vascularised tumours are aggressive and typically have a poor prognosis [3, 5--10]. Since endothelial cells (EC) are readily accessible and genetically stable, tumour angiogenesis has been pursued for decades as a promising therapeutic target, a pursuit vindicated by the recent approval of Avastin (antiVEGF monoclonal antibody) in combination with intravenous 5-fluorouracil-based chemotherapy for treatment of metastatic colorectal cancer [11]. However, previous results with other anti-angiogenesis drugs have been disappointing Correspondence to: Lars Keld Neilsen, PhD, Laboratory of Bioengineering, Department of Chemical Engineering, The University of Queensland, St. Lucia, Brisbane, QLD 4072, Australia. Tel: +61-7-33654682; Fax: +61-733654199; E-mail:
[email protected]
despite pre-clinical indications [12, 13], highlighting a need for better pre-clinical models [14]. No single model can cover the full range of complexity, and it is common to use several models of increasing complexity from cell-culture in vitro assays to animal models. Cell-culture assays are invaluable in early screening of potential drug candidates and in identifying molecular mechanisms underlying angiogenesis. These assays typically address a single aspect of angiogenesis: cell proliferation, matrix dissolution motility, chemotaxis, or tube formation [15--20]. The standard Matrigel or collagen-gel tube-formation assays have been critiqued for failing to capture the most basic features of in vivo tubule formation [14, 17] and several new assays have been developed including co-cultivation of EC with stromal elements [14, 17, 21--23] and cultivation of EC in spheroid monocultures [24, 25]. The next level of complexity in tumour angiogenesis is the interaction between EC and tumours, i.e., the migration of EC into, and tube formation within tumours. The failure in 2002 of Avastin in a phase-III study of refractory metastatic breast cancer highlights the need for tumourspecific assays. One model proposed is the side-by-side monolayer co-cultivation of MCF-7 cells and EC, leading to tubule formation by EC [26]. While this model demonstrates the release of angiogenic factors by MCF-7, it does not capture the physical interaction between tumours and EC.
98 In this study, we have developed a model of tumour angiogenesis based on the co-culture of isolated EC with preformed multicellular spheroids (MCS). MCS are widely considered to be representative of in vivo avascular tumour regions, and thus present an opportunity to recapture aspects of true tumour angiogenesis in vitro. Where previous attempts at utilising MCS in co-culture with isolated EC have had limited success [27--30], we have succeeded in producing micro-vascularised MCS constructs relying exclusively on the their inherent angiogenic potential, without the need for addition of exogenous angiogenic factors or matrix material.
N.E. Timmins et al. Spheroid culture
Materials and methods
Multicellular spheroids of non-endothelial cells were generated by the hanging-drop method [31]. Twenty microlitres of cell suspension (DMEM with 10% FBS and antibiotic/ antimycotic mix) was dispensed into each well of a 60-well mini tray (Nunc). Trays were then inverted and incubated under standard conditions. At day 5 trays were up-righted and the desired number of HUVECs (typically between 5000 and 10000 cells/well) were introduced in 6 ll of the same media using a multistep pipette. These cultures were then re-inverted and either incubated further, or after 24 h transferred by pipette to 48-well trays coated with 200 ll of 1% agarose, and further cultivated in fresh growth medium. Co-cultures were performed on a minimum of 10 independent occasions.
Non-endothelial cell culture
Oxygen profiles
The cell lines 293, HepG2, and HCT116 were maintained as monolayers in DMEM medium (GIBCO) supplemented with 10% foetal bovine serum (JRH) and antibiotic/antimycotic mix (GIBCO). Cultures were incubated at 37 C in a 5% CO2, 95% humidity atmosphere. Cell enumeration was achieved by trypan blue exclusion with an improved Neubauer haemocytometer.
A Clark-type oxygen microsensor with tip size of 20 lm (Unisense) was used to measure oxygen profiles in MCS. MCS were harvested at day 10 and transferred to tissue culture dishes, where they were left to attach for at least 4 h. Oxygen profiles were measured at 37 C by moving the sensor stepwise into the MCS using a computer-controlled motorised micromanipulator set-up. The data were transferred to a computer and logged using Profix software (Unisense).
Endothelial cell isolation and culture MCS culture kinetics HUVECs were harvested from freshly collected umbilical cords by collagenase digestion. Cords were rinsed thoroughly and flushed with PBS. The vein was then filled with 0.1% collagenase (GIBCO) solution, clamped, and incubated at 37 C for 30 min. One end of the cord was cut and the vein drained, using gentle massage to expel the cell slurry. The cell slurry was centrifuged for 5 min at 120 RCF, and the pellet resuspended in expansion medium consisting of DMEM/F12 (GIBCO) supplemented with 10% FBS, 100 lg/ml endothelial cell growth supplement (Sigma), 1 ng/ml rhEGF (Sigma), 1 ng/ml rhbFGF (Promega), 1 ng/ml rhVEGF (Chemicon), 100 lg/ml heparin (Sigma), 10 lg/ml hydrocortisone, and antibiotic/antimycotic mix (GIBCO). Cultures were maintained at 37 C, 95% humidity, and 5% CO2. For coculture experiments, banked HUVEC were recovered from storage in liquid nitrogen (passage 5 or earlier) and cultivated in DMEM/F12 supplemented with 10% FBS, 1 ng/ml rhEGF, 2 ng/ml rhbFGF, 5 ng/ml rhVEGF, 100 lg/ml heparin (Sigma), 10 lg/ml hydrocortisone, and antibiotic/antimycotic mix.
Images of MCS were captured in-well using an Olympus BX61 microscope fitted with a CoolSnapcf cCCD camera (MediaCybernetics). These images were analysed using ImagePro v4.5 (MediaCybernetics) to determine MCS diameter, a minimum of 30 images were analysed for each time point. Replicate glucose/lactate measurements were taken using a YSI2300 STAT PLUS glucose/lactate analyser (Yellow Spring Inc.). Immunofluorescence/immunohistochemistry HUVEC monolayers HUVEC monolayers cultivated on SuperFrost Plus glass slides (Menzel-Glaser) were fixed in 3.7% formaldehyde solution for 10 min, blocked in 6% BSA (Sigma) with 0.1% Triton-X100 for 30 min, and incubated with antibodies directed against vWF (Sigma, rabbit IgG 1 : 1000) and CD31 (Chemicon, mouse IgG 1 : 50) for 1 h. Secondary detection was achieved using AlexaFluor 488 and 647 conjugated antibodies (Molecular Probes), incubating for 1 h. Nuclear counterstaining was achieved using DAPI.
Matrigel assay 1 105 HUVECs were plated on Matrigel-coated (BD Biosciences) 35-mm dishes in 2 ml endothelial cell growth medium, and incubated overnight under standard conditions.
Immunofluorescent labelling of whole-mount spheroids Samples for whole-mount microscopy were harvested from culture in PBS and fixed in 3.7% formaldehyde solution for 2 h at room-temperature. Samples were blocked in 6% BSA with 0.25% Triton-X100 in PBS overnight at room temper-
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ature. This was followed by overnight incubation with rabbit anti-vWF IgG (Sigma) at a 1 : 500 dilution in PBS, with 1% BSA and 0.25% Triton-X100. Secondary detection was achieved using AlexaFluor 647 conjugated antibodies, again in PBS with 1% BSA and 0.25% Triton-X100, overnight. Samples were rinsed in PBS and mounted in 80% glycerol in the chambers of a gasketed microscope slide, and left to clear for at least 4 h. Imaging was achieved using a BioRad Radiance 2000 confocal microscopy system. Negative controls were processed without the use of primary antibodies, or as monocultures without HUVECs. Histological sectioning Samples for histological sectioning were harvested and fixed as above. They were then suspended in 30% sucrose solution in PBS until the MCS had sunk to the bottom of the tube, at which point OCT (TissueTek) was added and mixed, suspending the MCS in a 1 : 1 OCT : sucrose solution. This was then left overnight at room temperature. Samples were transferred to moulds, embedded in OCT, and stored at )80 C until sectioning. Frozen sections were obtained using a Leitz cryomicrotome, and transferred to SuperFrost Plus glass slides. Immunohistochemistry VEGF labelling was achieved using mouse anti-VEGF IgG (1 : 100, Sigma), and poly-HRP secondary antibodies (Chemicon). EC detection was achieved using anti-vWF (1:1000) primary antibodies and poly-AlkPhos secondary antibodies (Chemicon). Sections were blocked and permeablised with 6% BSA in PBS with 0.1% Triton-X100 for 15 min. Labelling was achieved by incubation with primary antibody for 1 h, followed by 45 min incubation with readyto-use secondary antibody. Colour was developed by incubation with DAB (poly-HRP, Chemicon) or NBT/BCIP (poly-AlkPhos, Roche) for 10 min, and lightly counter stained with Mayers hematoxylin (Sigma) or neutral red (Sigma).
Figure 1. (a) Expression of CD31 (green) and vWF (red) in cells isolated from umbilical veins by collegenase digestion. Nuclei counterstained with DAPI (blue). (b) Tubule network formation by HUVEC on Matrigel. (c--k) Monolayers and MCS labelled for VEGF expression (brown). (c) 293 monolayer, (d and e) 293 MCS, (f) HepG2 monolayer, (g and h) HepG2 MCS, (i) HCT116 monolayer, (j and k) HCT116 MCS. Bar ¼ 100 lm.
been demonstrated in MCS [32, 33]. Profiles of oxygen concentration show that chronic hypoxia also arises in HepG2 and HCT116 MCS, Figure 2. Steep gradients in oxygen concentration were observed, falling to near zero in central regions of the MCS.
Results Endothelial cell identification and Matrigel assay Following collagenase digestion of human umbilical vein, primary cultures were established. These cultures were assessed for expression of the endothelial cell markers CD31 and vWF by immunofluorescent labelling. Cultures showed strong positive staining for both antigens with the expected localisation, Figure 1a. Isolated HUVECs were also cultured overnight on matrigel, illustrating the tubule networks formed by this common assay, Figure 1b. Oxygen profiles and VEGF expression The up-regulation of several pro-angiogenic factors is known to occur in response to the hypoxic environment that arises in avascular tumour regions, and has previously
Figure 2. Profiles of oxygen concentration in HepG2 (closed circle) and HCT116 (open circle) MCS show no significant difference. Steep gradients exist within the MCS, with O2 concentrations approaching zero in central regions. The dashed line at a depth of 0 lm corresponds to the surface of MCS.
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Figure 3. Growth kinetics and glucose/lactate levels in hanging drop culture. MCS show a typical Gompertz-type growth pattern, with an initial exponential increase in volume follow by a decline in growth rate and subsequent plateau phase. Following an initial lag, glucose levels (closed circle) decline rapidly, and are mirrored by increasing concentrations of lactate (open circle). (a) HepG2, (b) HCT116.
The upregulation of VEGF is commonly observed in hypoxic tissues. The presence or absence of VEGF in monolayers and MCS composed of 293, HepG2, and HCT116 cells was detected by immunohistochemistry, Figures 1c--k. While no VEGF was observed for any of the three cell lines when cultivated as monolayers (Figures 1c, f, and i), it was detected in all MCS, with localisation varying between
Figure 4. HCT116/HUVECs hanging-drop co-cultures. (a) Prior to addition of HUVECs. (b) 15 min after addition of HUVECs, which have begun to settle at the medium/air interface. (c) 2 h after addition of HUVECs, which have begun to aggregate. (d) 4 h after addition of HUVECs. Aggregates are starting to consolidate into larger clusters. (e) 8 h after addition of HUVEC. (f) 24 h after addition of HUVEC. Large HUVEC clusters are in contact with the MCS. Upon transfer to 48-well plates after 24 h, it can clearly be seen that these clusters have begun to integrate with the MCS (g). With continued cultivation HUVEC progressively migrate into the primary MCS, becoming less distinct over time, (h) 48 h, (i) 72 h, (j) 96 h, (k) 120 h. At later stages the presence of HUVEC is indicated by a lumpy morphology. (l) Control spheroid without HUVECs at 72 h. Bar ¼ 100 lm.
cell lines. In 293 MCS, strong staining was observed throughout, with more densely stained central regions, Figures 1d and e. In HepG2 MCS, detection of VEGF was more patchy with apparent accumulation in acellular regions, Figures 1g and h. VEGF was also detected in the outer-most cell layers. For HCT116 MCS, VEGF was observed most strongly in the outer 2 or 3 cell layers, with patches detected in deeper regions, and a weak diffuse signal throughout, Figures 1j and k. It has previously been reported that localisation of VEGF expression to the outer cell layers of MCS occurs in response to glucose deficiency [33]. To establish if this was the reason for the observed patterns of VEGF localisation in our MCS, the glucose/lactate profiles and growth kinetics were determined for hanging drop cultures for HepG2 and HCT116, Figure 3. Cultures displayed a Gompertz-type growth pattern typical of MCS and avascular tumour regions [34--36], with an initial exponential increase in volume followed by a gradual decline in growth rate and subsequent plateau phase. At no time during the culture period did glucose levels fall below 1.7 mg/ml, while lactate
Figure 5. Optical section of HCT116 co-culture MCS labelled for vWF. Three-dimensional branching EC networks are clearly observed in localised regions of the MCS. Bar ¼ 50 lm.
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Figure 6. Histological sections of HCT116 co-cultures reveal that HUVEC are predominantly localised to outer regions of the MCS (a, bar ¼ 100 lm). Branching EC networks (b), and large luminal structures (c) are also observed (bar ¼ 25 lm).
steadily accumulated over the duration of the culture. Although not shown here, 293 MCS display similar kinetics. HCT116/HUVEC co-culture MCS cultures were generated by the hanging-drop method, and at day 5, HUVECs were introduced in 6 ll of fresh medium. Within 15 min of introduction to HCT116 hanging-drop cultures, HUVECs were observed to settle at the medium/air interface. In the subsequent 24 h, HUVECs aggregate and collect at the edge of the primary MCS, Figures 4a--f. At 24 h, MCS were transferred to individual wells of a 48-well plate, permitting clearer imaging, Figures 4g--k. Immediately after transfer, aggregated HUVECs are clearly observed to be merging with the outer regions of MCS. These aggregates gradually move deeper within the MCS, becoming less distinct in the process. By 120 h, the main sign of the HUVECs is a lumpy morphology not observed in control spheroids without HUVEC, Figure 4l.
observed (Figures 7e, f), however, these are typically narrower in diameter. O2 measurements of HepG2 and HCT116 co-culture MCS show no statistically significant dierence in the rates of oxygen consumption compared to monoculture MCS (data not shown).
Discussion We have developed a novel in vitro model of EC-tumour interactions in tumour angiogenesis. By employing the hanging-drop method for co-cultivation of HUVECs with MCS, it is possible to generate microvascularised constructs without the addition of angiogenic factors, or basement membrane substitutes such as matrigel or collagen gels.
HUVEC establish 3-D networks in HCT116 spheroids When harvested at day 10--12, optical sectioning of vWFlabelled HCT116 whole-mount MCS by LSCM revealed the presence of branching endothelial networks, Figure 5. Histological sections show that HUVECs are predominantly localised to outer regions of the MCS, Figure 6a. The network structures visualised by LSCM are again observed, as are large luminal structures, Figures 6b, c. 293 and HepG2/HUVECs co-culture In contrast to HCT116 co-cultures, when added to HepG2 hanging drops HUVECs did not assemble into secondary aggregates; instead HUVECs collect around the MCS and migrate into it (data not shown). When added to 293 cultures, HUVEC initially aggregated in a similar fashion to HCT116 co-cultures. However, over the first 24 h these aggregates coalesced, surrounding the MCS before migrating into it. Histological sections show that in contrast to HCT116 cocultures, large numbers of HUVEC are present throughout the depth of both HepG2 and 293 MCS (Figures 7a, b). Closer observation reveals network structures similar to, although less defined than those in HCT116 co-cultures, Figures 7c, d. Likewise, luminal structures were also
Figure 7. In 293 (a) and HepG2 (b) co-cultures, HUVECs are present throughout the MCS (bar ¼ 100 lm). Closer examination reveals EC networks (c--293, and d--HepG2), and small luminal structures (e--293, and f--HepG2), bar ¼ 10 lm.
102 The upregulation of VEGF within avascular tumour regions is known to occur in response to hypoxia, and a positive association between increased micro-vessel density and the expression of VEGF in biopsy samples has been well documented [37, 38]. Similarly, hypoxia and elevated expression of VEGF by MCS has been demonstrated [27, 32, 33, 37, 39], indicative of their angiogenic potential. HepG2 and HCT116 MCS exhibited steep gradients in oxygen concentration, resulting in near-total depletion at the centre, Figure 2. In all three of the cell lines used in this study, upregulation of VEGF expression in MCS was observed, Figures 1c--k. The pattern of VEGF localisation varied widely between 293, HepG2, and HCT116 spheroids. Unexpectedly, for HCT116 VEGF was observed most strongly in the outer cell layers that are directly exposed to the growth medium. This cannot be explained by a difference in oxygen profiles between cell lines, as none was observed. Shweike et al. [33] report that for C6 glioma MCS peripheral VEGF expression arises under glucose deficiency. Analysis of the glucose levels present in hangingdrop cultures shows that at no point did glucose levels fall below 1.7 mg/ml. Burns and Wilson [40] have previously demonstrated that elevated levels of lactate induce an angiogenic response from cultured HUVEC, probably via up-regulation of VEGF expression. In hanging-drop cultures, lactate steadily accumulated during the course of cultivation. At day 10, lactate concentrations were approximately 1.5 times higher in HCT116 cultures than HepG2 cultures. This accumulation of lactate within hanging-drop cultures may contribute to the overall pro-angiogenic phenotype observed, however it seems unlikely that this could account for the observed differences in localisation of VEGF. While the possibility of localised expression of VEGF cannot be discounted, the observed differences in our system may simply reflect differences in the binding of VEGF to cells and the extra-cellular matrix. When introduced to HCT116 cultures, HUVEC formed secondary aggregates and collected against the MCS. Subsequently, these secondary aggregates integrated with the MCS, and vWF-positive capillary-like networks formed, Figures 5 and 6. Compared to HUVECs cultivated on Matrigel, the networks observed within MCS were more highly branched and irregular, both characteristics of tumour vasculature in vivo [12]. Large luminal structures were also observed, Figure 6c. The response of HUVECs to HepG2 and 293 MCS was markedly different to that for HCT116. Many HUVECs were located throughout the MCS, dominating some regions, Figures 7a and b. Observation of regions less densely populated by HUVEC reveals that network structures are formed, as are individual luminal structures, Figures 7c--f. This behaviour may be linked to the localisation of VEGF within central regions of HepG2 and 293 MCS compared to HCT116. However, VEGF is only one of a number of angiogenic factors released by tumour cells, and several factors may be involved in a single malignancy as demonstrated by Pavlakovic et al. [41]. The observed differences in response do, however, highlight the need for tumour/tissue-specific assays of angiogenesis.
N.E. Timmins et al. In the generation of EC/MCS co-cultures, care must be taken in the selection of functional EC that are clearly identifiable by the expression of endothelial markers. In our hands, HUVECs obtained from the American Type Cell Culture Collection (ATCC) and Cascade Biologics failed to perform (data not shown), while those isolated in-house (Figures 1a, b) performed well. Some variability in the performance of different endothelial cell media was also observed. Both the ATCC- and Cascade-recommended media resulted in poor performance of HUVECs in coculture, while exclusion of ECGS during HUVECs expansion led to reduced HUVECs integration in HCT116 co-cultures. Migration, network assembly and architecture, and lumen formation, are all key aspects of tumour angiogenesis. Co-cultivation of HUVECs with MCS in hanging drops recaptures these features within a single system, without stimulation from exogenous factors. While at this time we have only studied this model of tumour angiogenesis at a relatively superficial level, we present this cultivation method as a platform from which to undertake further in-depth studies in an environment reminiscent of the in vivo situation. Additional optimisation of culture parameters to enhance reproducibility and quantitation, will lend this approach to screening type applications for the identification and testing of angiogenesis modulating agents.
Acknowledgement We gratefully acknowledge the assistance of Dr Rikke L. Meyer (AWMC, University of Queensland) in the collection of oxygen concentration profiles.
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