Extracellular Hsp90 and TGFβ regulate adhesion, migration and anchorage independent growth in a paired colon cancer cell line model

Leibovitz’s L-15 medium with GlutaMAX™, 10 x Trypsin-Ethylenediaminetetraacetic acid (EDTA), Penicillin/Streptomycin/Amphotericin (PSA) solution and Hoescht-33342 dye were from Gibco, Invitrogen. Heat-inactivated fetal bovine serum (FBS) was from Celtic Molecular Diagnostics and Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and Accutase® were purchased from Sigma-Aldrich. Insulin was obtained from NovoRapid (Novo Nordisk Pharmaceuticals). Hybond nitrocellulose membrane and Clarity Western Enhanced Chemiluminescence [ECL] substrate was from Bio-Rad (UK). Protein A/G PLUS-Agarose (sc-2003) was from Santa Cruz Biotechnology (USA). SB431542 (1614) was from Tocris Bioscience, while 3,3’, 5,5’ Tetramethyl-benzidine (TMB) substrate, dimethyl sulfoxide (DMSO), novobiocin (N1838), 5-fluorouracil (5-FU) and oxaliplatin were from Sigma-Aldrich. Recombinant native endotoxin-free human Hsp90β protein (SPR-102C) was from StressMarq Biosciences Inc., while recombinant endotoxin-free human TGF-β1 (carrier-free) (580704) was from BioLegend and bovine serum albumin (BSA) (10735078001) was from Roche. Mouse anti-human Hsp90α/β [F-8] (sc-13119), goat anti-human Hsp90α/β [N17] (sc-1055), HRP-conjugated donkey anti-mouse (sc-2314), mouse anti-human TGF-β1 [500-M66] (sc-65378), goat anti-human p-Smad2/3 (Ser 423/425) (sc-11769) and rabbit anti-human Smad2/3 (sc-8332) antibodies were from Santa Cruz. The Alexa-Fluor-550 conjugated donkey anti-rat (A21208) antibody was from Invitrogen, while the HRP-conjugated mouse anti-alpha tubulin (ab40742) and Alexa-Fluor-550 conjugated donkey anti-mouse (ab96876), donkey anti-rabbit IgG DyLight 550 (DY550) (ab96892), donkey anti-goat IgG DY660 (ab96934) and anti-human αvβ6 integrin (ab77906) antibodies were from Abcam. The fluorescein isothiocyanate (FITC)-conjugated mouse anti-human TGF-βRII (FAB241F) antibody was from R&D systems and the rat anti-human Hsp90α antibody (ADI-SPA-840-F) was from Enzo Life Sciences. The mouse anti-human Hsp90β (SMC-107A) antibody was from StressMarq Biosciences Inc. and the rabbit anti-histone H3 (sc-10809) antibody was from Cell Signaling Technologies. The FITC-conjugated mouse IgG1 isotype control was from BD Biosciences. The MTT Cell Proliferation kit 1 was from Roche.

The paired colon cancer cell lines SW480 (ECACC: 87092801, colon adenocarcinoma) and SW620 (ECACC: 87051203, lymph node metastasis) were purchased from the European Collection of Cell Cultures (ECACC) and maintained in L-15 Medium with GlutaMAX™ supplemented with 10% [v/v] fetal calf serum (Lonza) and 100 U/ml Penicillin, 100 μg/ml Streptomycin and 12.5 μg/ml Amphotericin at 37 °C in a humidified incubator without the introduction of CO₂.

Unless stated otherwise, whole cell lysates were prepared from confluent 6-well plates (Nunc) by scraping into lysis buffer (0.05 M Tris-HCl, 10% [v/v] glycerol, 2% [w/v] SDS). The total protein content of cell lysates was determined using a NanoDrop 2000™ spectrophotometer (Thermo Scientific). The method described by Laemmli (1970) [36] was used to separate proteins by denaturing electrophoresis according to size. Western blotting was performed according to Towbin et al. (1979) [37]. Proteins were visualised using Clarity Western Enhanced Chemiluminescence [ECL] substrate and detected using the ChemiDoc™ XRS+ system (Bio-Rad) or the film exposure method (Agfa HealthCare). Where relevant, lysates were probed for histone H3 (rabbit ant-histone H3) or tubulin (mouse anti-alpha tubulin) as a loading control.

Gelatin (5% [w/v]) was used to coat 15-well μ-Slide angiogenesis plates (81506, Ibidi). SW480 and SW620 cells were seeded onto μ-Slide plates and fixed with methanol and incubated with appropriate primary antibodies followed by the respective secondary antibodies as described in figure legends. Nuclei were stained with 1 μg/ml Hoechst-33342 dye, after which Dako mounting medium was added to each well. Immunofluorescence was detected using the Zeiss LSM 780 laser scanning confocal microscope and the images analysed using Zen Lite Software 2012 (Zeiss). Images were captured using the 63x oil objective. Colocalisation between the nucleus and pSMAD2/3 staining was performed using the Image J colocalisation plug-in (National Institutes of Health). The proportion of total pSMAD2/3 that is found in the nucleus was determined by developing profiles of the intensity of pSMAD2/3 in a line through the cells using Zen Lite Software.

Cells were seeded into 6-well plates and allowed to adhere overnight. The spent media was removed and centrifuged to remove cell debris. A human TGF-β1 DuoSet Development ELISA kit (R&D Systems Inc.) was utilised to analyse the expression of TGF-β1 in the cell culture supernatant (spent media) of the SW480 and SW620 cell lines. This was carried out according to manufacturer’s specifications. Samples of spent medium were assayed in triplicate and the concentration of TGF-β1 determined from a standard curve generated by assaying purified recombinant human TGF-β1 diluted to an eight-point range between 0 pg/ml and 2000 pg/ml in duplicate. Hsp90β secretion by cells was determined by ELISA from spent media according to Hunter et al., (2014) [26]. Briefly, spent media or pure protein in buffer A (20 mM Tris-HCl, 150 mM NaCl, pH 7.4, 1 mM ATP, 5 mM CaCl2, 0.05% [v/v] Tween 20) was incubated in a high binding 96-well microplate (655061, Greiner Bio-One, UK) overnight. Wells were blocked with 3% (w/v) bovine serum albumin (BSA) in buffer A for 1 h and washed with 1% (w/v) BSA in buffer A, before incubating with mouse anti-Hsp90β in buffer A (1:1000) for 2 h. After further washing, incubation with rabbit anti-mouse IgG-HRP secondary antibody in buffer A (1:10 000) was carried out for 1 h. Detection was carried out using TMB substrate (in 0.05 M Phosphate-Citrate Buffer [0.2 M Na₂HPO₄, 0.1 M citric acid, pH 5.0] with 2 μl of 30% [v/v] hydrogen peroxide) and quantification was carried out from a standard curve using recombinant Hsp90β at a six point concentration range from 0 to 500 ng/ml.

Cells were harvested using Accutase® solution (Sigma-Aldrich), collected by centrifugation and resuspended to a final concentration of 5 × 10⁶ cells/ml. Thereafter 1 μg fluorescein isothiocyanate (FITC)-conjugated mouse anti-human TGF-βRII antibody was added to 1 × 10⁶ cells and incubated at 4 °C for 1 h in the dark. The stained cell suspension was washed, collected by centrifugation and resuspended in phosphate-buffered saline. The stained cells were analysed using the 488 nm laser of a FACSAria™ III flow cytometer (BD Biosciences) using wavelengths of 488 nm and 519 nm for excitation and emission, respectively. A FITC-conjugated mouse IgG1 isotype control was used to account for any non-specific binding of primary antibodies. A further negative control without primary antibody was used to assess cell autofluorescence. Data was analysed using FlowJo software version 10.0.4 (Tree Star Inc., 2013).

SW480 and SW620 cells were seeded at a density of 1.2 x 10⁵ cells/well into a 96-well plate and treated with combinations of TGF-β1, Hsp90β, SB431542, novobiocin and αvβ6 integrin blocking antibody as indicated in figure legends. The cells were left to adhere for 8 h at 37 °C before removing the media and washing the wells with PBS three times. Adherent cells were fixed with 4% [v/v] paraformaldehyde in PBS before washing with deionised water and staining with 10% [w/v] crystal violet in 5% [v/v] ethanol. Wells were again washed with deionised water, allowed to air-dry and crystal violet dye solubilised in 5% [w/v] SDS and 1% [v/v] Triton-X100. Absorbance was read at 590 nm using a microtitre plate reader (PowerWaveXTM, BioTek).

SW480 and SW620 cells were seeded at a density of 1.2 x 10⁴ cells into each individual well of a micro-insert 4-well chamber (80409, Ibidi) and left to adhere overnight. Each insert was filled with L-15 medium containing combinations of TGF-β1, Hsp90β, SB431542, novobiocin or an αvβ6 integrin blocking antibody as indicated in figure legends. After 24 h, the inserts were lifted and the cell area was washed four times to remove floating cells. Photos of the intersection between the vertical and horizontal wounds generated by the micro-inserts were taken under a 10x objective using an iPhone 5 in conjunction with the SkyLight microscope adapter at 0, 12, and 24 h. Images were analysed in Image J (National Institutes of Health (NIH)) to determine the number of cells in the wound area (calculated as particles per mm²).

The ability of colon cancer cell lines to grow as tumourspheres in suspension was determined as previously described [38]. Briefly, cells were lifted with trypsin, washed with phosphate-buffered saline (PBS) and passed through a 40 μM cell strainer (BD Biosciences) to produce a single cell suspension. Cells were resuspended in anchorage-independent growth (AIG) medium containing DMEM with Glutamax™ supplemented with 1% (v/v) PSA, 2% (v/v) B-27 supplement, 20 ng/ml EGF and bFGF, 4 ng/ml heparin and 10 μg/ml insulin and seeded into ultralow attachment plates (Corning). Seeding was carried out at a density of 100 cells per well in a 96-well plate for analysis of effects of proteins/compounds on tumoursphere formation and 2000 cells/well in a 96-well plate for collection of spent media. Tumourspheres were fed every 3 days by the addition of 50 μl of fresh AIG medium to each well and processed on Day 7 post seeding.

SW480 and SW620 cells were seeded into anchorage-independent conditions as described above and treated with either 2 ng/ml TGF-β1, 20 ng/ml Hsp90β, 100 nM SB431542 or 100 μM novobiocin. After one week, tumourspheres were dissociated with Accutase® (Sigma-Aldrich) solution and reseeded into adherent condition in a 96 well plate at a density of 1.2 x 10⁵ cells/ml, allowed to adhere overnight and then treated with either 75 μM 5-FU or 550 μM oxaliplatin for 72 h. Cell viability was assessed compared to an untreated control for each pre-treatment using a MTT Cell Proliferation kit (Roche) according to manufacturer’s specifications as previously described [39].

We analysed the status of the TGF-β pathway in the SW480 cell line derived from a primary colon adenocarcinoma and its SW620 lymph node metastasis-derived counterpart (Fig. 1). Analysis of the spent media of the cell lines by ELISA revealed that SW480 primary tumour cells and their SW620 secondary tumour counterparts secrete similar levels of TGF-β1 (2.50 ± 0.12 vs. 2.12 ± 0.05 ng/ml, Fig. 1a). Analysis of the cell surface receptors by flow cytometry showed that SW480 cells display twice the level of TGF-βRII compared to SW620 cells (Fig. 1b). SW620 cells, on the other hand, display greater levels of intracellular TGF-β1, specifically in the form of pre-pro-TGF-β1, compared to SW480 cells (Fig. 1c. Images cropped from the original full length western blots provided in Additional file 1). Confocal analysis revealed that SW620 cells also have a greater proportion of nuclear pSmad2/3 than SW480 cells (Fig. 1d), suggesting greater pathway activation in the metastatic cell line. Due to the fact that extracellular Hsp90 has been linked to the TGF-β pathway and is able to bind both TGF-βRI and TGF-βRII [31], we also examined the extracellular levels of Hsp90 in the spent medium of SW480 and SW620 cells. While we were unable to detect Hsp90α in the spent media (data not shown), we found that SW620 cells secrete significantly more Hsp90β than SW480 cells (19.51 ± 4.35 vs. 10.70 ± 3.60 ng/ml, respectively; Fig. 1e). In addition, we noted that the levels of Hsp90β secreted by the cells were 5-10 fold higher than those of TGF-β1 (Fig. 1a). The intracellular levels of both cytoplasmic isoforms of Hsp90 did not appear to differ between the two lines (Fig. 1f).

We next examined the effect of the TGF-β pathway and Hsp90 on adhesion and migration in vitro, both of which are biological processes crucial for metastasis. In particular it has been reported that changes in the cytoskeleton and extracellular matrix proteins as well as weak adhesions may cause an increase in migration and may be required for cell motility, revealing that adhesion and migration are two sides of the same coin in terms of metastasis [40‐42]. Our analyses revealed that SW480 cells displayed a significantly higher level of adhesion compared to SW620 cells after 8 h incubation (A₅₉₀ values of 0.91 ± 0.27 vs. 0.58 ± 0.24, respectively; p = 0.0027; Fig. 2a), while SW620 cells appeared to be nearly three-fold more migratory than SW480 cells after 24 h (30.15 ± 12.43 vs. 10.58 ± 1.65 particles per mm², respectively; p = 0.0016; Fig. 2b). These opposing trends between the cell lines stand to reason and lend weight to the hypothesis that if there is less adhesion of the cells they are more likely to migrate [40‐42]. The migration results also agree with published in vivo studies where SW620 cells were found to be more migratory when injected into mice and analysed for liver metastases [43]. The decreased adhesion and increased migration of SW620 cells observed in this study therefore validates the use of the SW480-SW620 system as an in vitro model of cancer progression.

The effect of addition or inhibition of TGF-β1 and Hsp90β on the adhesion of SW480 and SW620 cells was analysed by normalisation to untreated cells (taken as a 100%) as depicted in Fig. 3. In the assessment of adhesion, migration and AIG of SW480 and SW620, TGF-β1 and Hsp90β were used at 2 and 20 ng/ml, which corresponded to the levels of proteins detected in spent media (Fig. 1). Treatment with the TGF-βRI inhibitor SB431542 and the C-terminal Hsp90 inhibitor novobiocin was carried out at 100 nM and 100 μM, respectively, in all cases, as these concentrations were not toxic to either cell line (data not shown). Adhesion studies in SW480 cells (Fig. 3a), at 8 h post seeding, revealed that treatment with TGF-β1 did not significantly affect cell adhesion in SW480 cells in comparison to the untreated cells (85.90 ± 13.98% vs. 100.00 ± 8.42% adhesion, respectively). Similarly, Hsp90β treatment did not have any effect on the adhesion of SW480 cells (106.51 ± 13.41% adhesion relative to control). Treatment with a combination of TGF-β1 and Hsp90β, known to bind the common receptor TGF-βRI [31], again showed no significant change in the adhesion of SW480 cells (89.94 ± 16.93%) compared to untreated cells. Inhibiting the receptor TGF-βRI with SB431542, either with or without the addition of exogenous TGF-β1, also had no significant effect on the adhesion of SW480 cells (83.44 ± 9.72% and 81.52 ± 8.77%, respectively). On the other hand, when SB431542 treatment was combined with both TGF-β1 and Hsp90β, this caused a decrease in the adhesion of SW480 (71.80 ± 13.55%) in comparison to the untreated cells, although this was not significantly different to the effect seen after treatment with the two proteins TGF-β1 and Hsp90β alone. The inhibition of Hsp90 by the addition of novobiocin caused a significant decrease in the adhesion of SW480 cells relative to the normalised untreated control (53.09 ± 4.54%). The addition of exogenous Hsp90β and TGF-β1 in combination with novobiocin was not able to overcome the inhibition of total Hsp90 by the drug in terms of adhesion (56.08 ± 2.19%), although it was observed that this decrease in adhesion was significantly less than TGF-β1 and Hsp90β treatments alone. Since αvβ6 integrin is known to be linked to the TGF-β pathway as an alternate TGF-β1 receptor [44], we assessed the effect of inhibition of this integrin on adhesion. We found that treatment with a blocking antibody against αvβ6 (10 μg/ml) had no significant effect on the adhesion of SW480 cells on its own (83.29 ± 10.75%), but that the antibody reversed the decrease in adhesion by TGF-β1, Hsp90β and SB431542 when combined with these treatments (100.29 ± 14.04%), in the SW480 cells.

The effect of addition or inhibition of TGF-β and Hsp90 on adhesion was also assessed in SW620 cells (Fig. 3b), revealing that, in a number of cases, the metastasis-derived cell line did not respond in the same way as the SW480 primary tumour-derived line to these treatments. We found that, unlike in SW480 cells, addition of TGF-β1 caused a subtle increase in the adhesion of SW620 cells compared to the untreated control (119.63 ± 17.16 vs. 100.00 ± 14.30% adhesion). On the other hand, treatment with Hsp90β caused a significant increase in adhesion of SW620 cells over the control (142.99 ± 17.13%, p < 0.01) which was once more not seen for SW480 cells. Unexpectedly, despite the fact that they each increased adhesion, treatment with a combination of TGF-β1 and Hsp90β caused a significant decrease in the adhesion of SW620 cells (79.69 ± 4.09%). This decrease in adhesion of SW620 cells was even greater when comparing Hsp90β treatment alone to the combination treatment with TGF-β1 (p < 0.001). As was the case for SW480 cells, inhibition of TGF-βRI with SB431542, either with or without the addition of exogenous TGF-β1, did not have a significant effect on the adhesion of SW620 cells in comparison to the untreated cells (83.55 ± 7.43% and 82.91 ± 5.01%, respectively). Again, it was only when SB431542 treatment was combined with both TGF-β1 and Hsp90β, that a significant decrease in the adhesion of the cells was observed (70.68 ± 5.56%). Similar to the effect seen for SW480 cells, this effect was not significantly different to that seen after treatment with the two proteins TGF-β1 and Hsp90β alone. As was the case for SW480 cells, the inhibition of Hsp90 by novobiocin caused a marked decrease in the adhesion of SW620 cells relative to the normalised untreated control (56.45 ± 5.35%, p < 0.001). The addition of exogenous Hsp90β and TGF-β1 in combination with novobiocin was once more not able to overcome the inhibition of Hsp90 on adhesion (54.53 ± 5.02%). As for SW480 cells, treatment with a αvβ6 blocking antibody had no significant effect on the adhesion of SW620 cells on its own (74.68 ± 10.12%); however, when combined with TGF-β1, Hsp90β and SB431542, the antibody reversed the decreased adhesion by a combination of the latter three treatments alone (81.25 ± 7.02%).

Migration analysis was performed and the wound area covered by cells calculated using ImageJ [particles per area (mm²)] and normalised to each treatment at 0 h (given as 1) in each of the two cell lines (Fig. 4a and b). In this case, statistical analysis was carried out between treatments at 24 h as this was when the greatest migration was observed. For the SW480 cells (Fig. 4a), treatment with TGF-β1 alone, Hsp90β alone as well as the combination of TGF-β1 with Hsp90β, caused no significant changes in the migration of the cells relative to the untreated control (9.26 ± 3.08, 10.72 ± 1.71 and 13.44 ± 2.28, respectively vs. 10.58 ± 1.65 normalised particles per mm²/NPPM). In contrast to this, the inhibition of the TGF-βRI using SB431542 caused a two-fold increase in migration of SW480 cells in comparison to the untreated cells (23.93 ± 2.07 NPPM). The effect of addition of TGF-β1 in combination with SB431542 on migration (28.67 ± 5.58 NPPM) did not differ significantly to that of treatment with SB431542 alone, suggesting that the addition of TGF-β1 was unable to overcome the inhibition of the canonical TGF-β/TGF-βR pathway in these cells. The addition of both TGF-β1 and Hsp90 with SB431542 treatment, on the other hand, caused a significant three-fold increase compared the untreated cells (36.18 ± 6.24 NPPM). This treatment was also greatly increased in comparison to the combination TGF-β1 and Hsp90β treatment (p < 0.001). The effect of the combination of TGF-β1, Hsp90 and SB431542 on migration, however, was not significantly different to that of SB431542 treatment alone. Novobiocin treatment caused an increase in migration in SW480 cells relative to the untreated control (29.76 ± 25.17 NPPM). The effect on migration of TGF-β1 and Hsp90β in combination with the novobiocin (34.25 ± 29.79 NPPM) did not differ significantly in comparison to novobiocin treatment alone, suggesting that the addition of endogenous Hsp90β and TGF-β1 could not overcome the inhibition of Hsp90 by novobiocin. These treatments did however cause a substantial increase in migration, in comparison to the treatment using a combination of only TGF-β1 and Hsp90β (p < 0.01). This highlights that it is the novobiocin specifically that is causing the increase in migration. The inhibition of the αvβ6 integrin with a blocking antibody (10 μg/ml) alone did not have any effect on the migration of SW480 cells (11.33 ± 5.20 NPPM), including when TGF-β1, Hsp90β and SB431542 were added (11.67 ± 1.25 NPPM), effectively returning migration to baseline compared to treatment with TGF-β1, Hsp90β and SB431542 alone. This suggests that αvβ6 integrin may be an alternate receptor mediating migration in response to a combination of TGF-β1 and Hsp90β in these cells.

When analysing the migration of SW620 cells (Fig. 4b), it was found that treatment with either TGF-β1 or Hsp90β caused minor decreases in the migration of these cells in comparison to the untreated cells (11.46 ± 2.53 and 17.30 ± 4.66 vs. 30.15 ± 12.43 NPPM, respectively). On the other hand, treatment with a combination of TGF-β1 and Hsp90β caused a statistically significant two-fold increase in migration of SW620 cells in comparison to untreated cells (61.21 ± 16.24 NPPM). This increase in migration of SW620 cells caused by the combination treatment was significantly higher than either TGF-β1 or Hsp90β treatment alone (p < 0.001 and p < 0.01, respectively). Unlike in SW480 cells, SB431542 treatment alone did not cause a significant change in the migration of SW620 cells relative to the untreated control (43.73 ± 17.25 NPPM). However, similarly to SW480 cells, treatment of SW620 cells with a combination of SB431542 and TGF-β1 as well as a combination of SB431542, TGF-β1 and Hsp90β caused a substantial increase in migration (90.50 ± 17.03 and 77.28 ± 16.89 NPPM, respectively). This increase in migration was however not significantly different to the combination treatment of TGF-β1 and Hsp90β alone. These data suggest that the pro-migratory effect of the combination of TGF-β1 and Hsp90β is not disrupted by inhibition of the receptor, suggesting that the canonical pathway is not being used. Treatment with novobiocin alone and in combination with TGF-β1 and Hsp90β had no significant effect on the migration of SW620 cells relative to the untreated control (37.27 ± 25.75 and 42.15 ± 1.82 NPPM, respectively), unlike in SW480 where the drug caused a substantial increase in migration. While disruption of αvβ6 integrin using a blocking antibody did not cause any change in the migration of SW620 cells (28.98 ± 16.34 NPPM), this was able to overcome the stimulation of migration caused by a combination of TGF-β1, Hsp90β and SB431542, (31.71 ± 12.16 NPPM for treatment with αvβ6 integrin + TGF-β1 + Hsp90β + SB- 431542), thereby returning migration to a level comparable with untreated cells. This trend was the same as that observed in SW480 cells.

Since the ability of malignant cells to survive and grow anchorage-independently is a requirement for metastasis, we investigated the secretion of and response to TGF-β1 and Hsp90β of the paired colon cancer cell lines under such conditions. The cell lines were grown in suspension as well defined three-dimensional structures known as tumourspheres, with both cell lines forming spheres of similar morphologies and size (Fig. 5a). When comparing the secretion of TGF-β1 and Hsp90β by these tumourspheres (Fig. 5b) we found that SW480 tumourspheres produced much higher levels (p < 0.001) of both proteins in the spent media compared to SW620 tumourspheres (TGF-β1: 11.68 ± 1.03 vs. 5.85 ± 2.35 ng/ml, respectively; Hsp90β: 81.28 ± 0.43 vs. 0.10 ± 0.23, respectively) as well as to either cell line grown adherently (TGF-β1: 2.97 ± 0.05 ng/ml in SW480, 2.52 ± 0.04 ng/ml in SW620; Hsp90β: 0.44 ± 0.18 ng/ml in SW480, 8.96 ± 0.66, in SW620 cells).

We next assessed the effect of addition or inhibition of TGF-β1 and Hsp90β on the ability of both cell lines to form tumourspheres as a measure of anchorage-independent growth (Fig. 5c). Sphere forming efficiency (SFE) was defined as the percentage of the total number of cells seeded that are able to form tumourspheres after 7 days and this was normalized to that of untreated cells for each cell line (taken as 100%). In SW480 cells (Fig. 5c), we found that addition of either TGF-β1 or Hsp90β alone caused a statistically significant decrease in SFE compared to untreated cells (72.00 ± 9.60 and 74.13 ± 2.44%, respectively vs. 100.00 ± 6.40%; p < 0.001 in each case). On the other hand, a combination of Hsp90β and TGF-β1 led to a significant increase in SFE (108.27 ± 6.65%) relative to SW480 cells treated with either protein alone (p < 0.05 for TGF-β1, p < 0.01 for Hsp90β), returning SFE to the level observed in untreated cells. While the addition of SB431542 alone had no significant effect on SFE in these cells (94.93 ± 1.85%), the inhibitor was able to reverse the effect of TGF-β1 on SFE (107.73 ± 6.66%). Interestingly, the combination of TGF-β1, Hsp90β and SB431542 caused a marked increase in SFE relative to the untreated control (111.47 ± 5.11%, p < 0.01). An even greater increase in SFE was observed for cells treated with the novobiocin (137.07 ± 6.06%, p < 0.001), and this was not affected by the addition of both TGF-β1 and Hsp90β (138.67 ± 22.19%). Since αVβ6 integrin is an alternate receptor in the TGF-β machinery, we assessed the effect of a blocking antibody against this integrin on SFE. While the antibody alone had no effect on SFE of SW480 cells (99.20 ± 9.99%), it was able to reverse the increase in SFE caused by the combination of TGF-β1, Hsp90β and SB431542 (82.66 ± 19.29% for cell treated with the αVβ6 integrin antibody, TGF-β1, Hsp90β and SB431542).

In terms of anchorage-independent growth in the SW620 cell line (Fig. 5d), it was clear that these cells responded very differently not only to addition but also inhibition of TGF-β and Hsp90 compared to the SW480 line and that, in general, these treatments had less of an impact on SFE in SW620 cells. In particular we found that, unlike in SW480 cells, anchorage-independent growth in these cells was not affected by the addition of TGF-β1 (SFE of 97.11 ± 6.00% in treated cells vs. 100.00 ± 11.69% in untreated cells). Hsp90, on the other hand, caused a significant increase in SFE in SW620 cells (129.87 ± 5.21%), a trend which is opposite to that observed in SW480 cells. Interestingly, this increase in SFE upon Hsp90 treatment was reversed when combined with TGF-β1 (84.39 ± 2.00% SFE). Treatment with SB431542 had no real effect on tumoursphere formation either alone (90.56 ± 8.98% SFE) or when combined with TGF-β1 alone (108.67 ± 2.00%) or TGF-β1 with Hsp90β (105.20 ± 4.00%). Unexpectedly, inhibition with novobiocin also increased AIG in SW620 cells (120.23 ± 8.73%) in the same manner as Hsp90β addition but this effect was no longer significant when the drug was combined with TGF-β1 and Hsp90β (119.85 ± 10.74%). It is important to note however that this inhibitor is not specific to the Hsp90β isoform or extracellular Hsp90. An αVβ6 integrin blocking antibody had no effect on SFE of SW620 cells either alone (96.34 ± 5.82%), or when combined with TGF-β1, Hsp90β and SB 431542 (91.71 ± 5.95%).

We assessed the effect of pre-treatment under AIG conditions by either addition or inhibition of TGF-β1 and Hsp90 on chemosensitivity of SW480 and SW620 cells to oxaliplatin and 5-FU (Fig. 6). Pre-treatment was carried out under AIG conditions since this has been demonstrated to enrich for cancer stem cells (CSC) by ourselves and others [38, 45] and this sub-population is reportedly resistant to traditional chemotherapeutic agents due to the overexpression of several drug transporters [46]. The chemosensitivity assay used high concentrations of the colon cancer drugs as previously described [47], namely 550 μM 5-FU and 75 μM oxaliplatin, in order to see any chemoresistance which may result from the various pre-treatments. In the assay, pre-treatment with TGF-β1, Hsp90, SB 431542 or novobiocin was carried out upon seeding into AIG conditions. The resultant tumourspheres after 7 days of culture were dissociated and seeded into regular culture conditions, allowed to adhere overnight, and then treated with vehicle control (representing “untreated” cells), 5-FU or oxaliplatin. For each pre-treatment, viability was calculated relative to its own vehicle-treated control (taken as 100%). A comparison was also made with untreated cells (i.e. not pre-treated) that had previously either been grown adherently (first bar in each graph of Fig. 6) or been cultured as tumourspheres and then dissociated (second bar in each graph of Fig. 6). In each case, statistical significance was assessed in comparison to cells with had been cultured as tumourspheres but not pre-treated (second bar in each graph). We report that pre-culturing as tumourspheres under AIG conditions appears to infer chemoresistance of SW480 primary tumour cells to 5-FU (47.16 ± 0.54% survival for tumoursphere-derived untreated cells vs. 11.27 ± 2.64% for adherently cultured untreated cells, Fig. 6ai). This was expected since a previous study by [47] reported that colon CSC are 60-fold more resistant to 5-FU. In terms of pre-treatment during AIG, we found that TGF-β1, Hsp90β, SB431542 and novobiocin all significantly increased chemosensitivity of SW480 cells to 5-FU (26.04 ± 1.30%, 15.31 ± 3.15%, 16.79 ± 1.96% and 39.20 ± 0.17% survival, respectively, Fig. 6ai).

Converse to that observed for SW480 cells, pre-culture as tumourspheres rendered SW620 cells significantly more sensitive to 5-FU (22.81 ± 2.17 for tumoursphere-derived cells vs. 31.53 ± 1.94% for adherent cells, Fig. 6aii). On the other hand, similar trends were noted in the SW620 cells compared to the SW480 cells in terms of the effect of pre-treatment with TGF-β1, Hsp90β and SB431542, though the resultant chemosensitivity was less pronounced (19.63 ± 0.45%, 15.69 ± 1.38% and 17.04 ± 0.85%, respectively, Fig. 6aii). The major difference between the cell lines in terms of pre-treatment was the effect of novobiocin which, unlike in SW480 cells, significantly decreased the sensitivity of SW620 cells to 5-FU (38.36 ± 3.48% survival).

We report that, unlike for 5-FU, AIG pre-culture did not significantly influence the chemosensitivity of SW480 cells to oxaliplatin (37.16 ± 2.30% survival in tumoursphere-derived cells vs. 36.35 ± 3.24% in adherent cells, Fig. 6bi). This was unexpected since [47] reported that colon CSC are 15-fold more resistant to oxaliplatin than bulk colon tumour cells. The various pre-treatments had less of an effect on chemosensitivity in general of SW480 cells to oxaliplatin compared to 5-FU. In particular, pre-treatment with TGF-β1 and SB431542 did not influence chemosensitivity to oxaliplatin (37.72 ± 1.82%, 38.74 ± 2.02% survival, respectively) while Hsp90β and novobiocin pre-treatment rendered SW480 cells significantly more resistant to the drug (63.00 ± 1.63% and 82.91 ± 4.90% survival, respectively, Fig. 6bi), the latter effect being opposite to that observed for 5-FU.

In the case of SW620 cells, as for SW480 cells AIG pre-culture did not influence the chemosensitivity of the cells to oxaliplatin (67.22 ± 3.56% in tumoursphere-derived cells vs. 59.73 ± 2.42% in adherent cells, Fig. 6bii). In terms of pre-treatment, only SB431542 treatment had a significant effect on sensitivity to oxaliplatin, causing a slight increase in sensitivity of SW620 cells (55.09 ± 5.03% survival), with no significant effects on chemosensitivity noted for pre-treatment with TGF-β1, Hsp90β or novobiocin (62.93 ± 3.62%, 68.17 ± 2.35% and 82.91 ± 4.90% survival, respectively, Fig. 6bii).