Characterisation of the Morphological, Functional and Molecular Changes in Sunitinib-Resistant Renal Cell Carcinoma Cells

Sunitinib resistance is a major clinical problem hampering the treatment of renal cell carcinoma (RCC). Studies on the comprehensive characterisation of morphological, functional and molecular changes in sunitinib-resistant RCC cells are lacking. The aim of the current study was to develop sunitinib resistance in four human RCC cell lines (786-0, Caki-1, Caki-2 and SN12K1), and to characterise the changed cell biology with sunitinib resistance. RCC cells were made resistant by continuous, chronic exposure to 10 μM of sunitinib over a period of 12 months. Cell proliferation, morphology, transmigration, and gene expression for interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), Bcl-2 and Bax were studied. There was no significant difference in growth rate or transmigration between the parental and resistant cells. Sunitinib-resistant cells were significantly hypertrophic compared with parental cells as evidenced by increases in the surface areas of the whole cells and the nuclei. IL-6 was significantly increased in all resistant cells. IL-8 was increased in sunitinib-resistant Caki-2 and SN12K1 cells and decreased in 786-0 without any significant changes in Caki-1. VEGF was increased in resistant Caki-2 and SN12K1 cells but not in 786-0 and Caki-1. The Bcl2/Bax ratio was increased in Caki-1, Caki-2 and SN12K1 cells but decreased in 786-0 cells. The increased IL-6 may contribute to sunitinib resistance either via VEGF-mediated angiogenesis or through shifting of the Bcl2/Bax balance in favour of anti-apoptosis.


Cell culture
The RCC cell lines 786-0, Caki-1 and Caki-2 were obtained from American Type Culture Collection (Rockville, MD). Another human RCC cell line, SN12K1, was obtained from Professor D Nicol, Princess Alexandra Hospital, Brisbane, Australia, through his collaborations with Dr IJ Fidler, Cancer Research Institute, MD Anderson Cancer Center, Houston, TX, USA. The RCC cell lines were cultured in DMEM/ F12 (Gibco, Invitrogen, CA, USA) supplemented with foetal bovine serum (10%), penicillin (50 U/ml), streptomycin (50 µg/ml) and amphotericin B (0.125 µg/ml) in a humidified atmosphere of 5% CO 2 in air at 37°C. All cell lines were recurrently tested and determined to be mycoplasma-free.

Development of sunitinib-resistant RCC cell lines
Cells resistant to 10 µM sunitinib were established by exposure to increasing concentrations of sunitinib. In brief, the RCC cell lines were treated with varying concentrations of sunitinib (0, 1, 5, 10, 20, 50 and 100 µM). While all concentrations above 1µM inhibited the growth rate of the RCC cell lines, at 10 µM, more than 98% of cells were dead by 72 h, as measured by MTT assay. It was assumed that the remaining cells were a mix of transient (or tolerant) and stable resistant cells. If this assumption is true, with the passage of time, the transient cells are eliminated, and only stably resistant cells would eventually grow to confluence. With this assumption, these cells were continually maintained in 10 µM sunitinib and passaged every 4 days and the cells that eventually grew to confluence were developed into stable sunitinib-resistant cells over a period of 12 months. At no point during the development process were the cells in sunitinib-free medium. Further experiments showed that these cells were also resistant to 20 and 40 µM sunitinib. However, experiments were conducted in 10 µM. The results presented are from sunitinib-resistant cells that had been in culture for more than 12 months.

Measurement of cell and nuclear size as a marker of hypertrophy
The surface area of whole cell and nucleus, as a marker of hypertrophy, was analysed as per previous reports (35,36). In brief, parental and resistant cells were seeded and cultured on glass cover slips in 24-well plates at a density of 4 × 10 4 cells/ml. After 24 h, the cells were washed in phosphate-buffered saline (PBS), fixed for 20 min at room temperature in 4% formaldehyde, stained with haematoxylin and eosin as per routine methodology and mounted with DePex mounting medium. The cells were viewed under a Nikon Eclipse 50i microscope (Nikon Instruments Inc., NY, USA) at 200× magnification. Images were captured from four random areas of each coverslip using DS-Fi1 colour camera (Nikon Australia, Sydney, Australia). Analyses of cell and nuclear sizes were performed using NIS Elements Software version 2.0 (Nikon Instruments, Melville, NY, USA).

Cell growth assay using MTT
Cells were seeded in 96-well plates (5 × 10 3 cells/well/100 µl) and MTT assay was performed as per previous reports (35)(36)(37). After pre-determined time periods (24 h-120 h), 5 µl of MTT (Sigma, MO, USA), from a 5 mg/ml stock in PBS, were added to each well of the culture plates and incubated for 90 min at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . The culture medium was removed, and the purple crystals formed were dissolved in 100 µl of dimethyl sulfoxide (DMSO). The absorbance was read at 570 nm with a background correction of 690 nm in a Multiscan Go Microplate Reader (Thermo Scientific, Waltham, MA, USA). The percentage of cell viability was calculated relative to the control wells, which were designated as 100%.

Transmigration
Transmigration assay was performed using transwell migration chambers following the protocol of the supplier (Thermo Fisher Scientific Australia Pty Ltd; Cat # NUN140629; 8 µm pore size). Briefly, the cells were suspended in serum-free DMEM/F12 medium and seeded on the upper compartment of the transwell. Five hundred microlitres of culture medium containing 10% FBS were added as chemoattractant to the bottom chamber. After 24 h, non-migrated cells from the upper surface of the membrane were removed using a wet cotton swab. The migrated cells on the lower surface of the membrane were fixed in 4% formaldehyde, stained with toluidine blue (1% in a 1% aqueous solution of borax) and mounted with DePex mounting medium. The cells were viewed under a 40 × objective and counted from five random fields.

Statistical analyses
The results were expressed as mean ± standard deviation of mean. Comparisons between groups were analysed using analysis of variance (ANOVA) with Tukey's post hoc test or Student's t-test, where appropriate. Analyses were performed using Graphpad Instat software (San Diego, CA, USA). A p-value of <0.05 was considered significant. The results presented are representatives of three independent experiments.

Morphological changes
Morphometric studies showed significant hypertrophy in sunitinib-resistant cell lines, as evidenced by increases in the surface areas of the whole cells and the nuclei (Figure 1). Representative H&E stained cells from each group are shown in Figure 2. While all cell types were hypertrophic, considerable morphologic heterogeneity could be observed in the resistant cells. In particular, 786-0 showed spindle morphology, whereas the SN12K1 cells displayed multi-lobulated nuclei. Observation of live cells under phase contrast microscope further confirmed hypertrophic and heterogenic features of resistant cells (Figure 3).

Growth and transmigration
Although the resistant cells showed a slightly decreased growth rate, at no point in time during the course of the study was this difference statistically significant (Figure 4). Similarly, there was no statistically significant difference in transmigration between the parental and sunitinib-resistant cells ( Figure 5). Whether this lack of difference is the reflection of increased size of the sunitinib-resistant cells preventing them from migrating or actual decrease in transmigration is not clear.

Gene expression
To investigate the possible mechanisms behind resistance, we studied the expression patterns of the proangiogenic factors IL-6, IL-8 and VEGF, and apoptosis-regulatory molecules Bcl-2 and Bax. In all resistant cells, IL-6 was the only common molecule that was overexpressed ( Figure 6). IL-8 was increased in Caki-2 and SN12K1 cells and decreased in 786-0. No significant change was observed in Caki-1. Interestingly, VEGF was increased in resistant Caki-2 and SN12K1 cells, without

Discussion
There is no universal consensus or guidelines on how drugresistant cells should be developed in vitro. The underlying principle is to develop cells that are resistant to concentrations that would otherwise kill the parental cells. There are many methods, each with its own advantages and disadvantages, and the choice is often at the discretion of the investigators. Furthermore, there is no agreement on the terminology on whether it is drug tolerance or resistance, which also varies from investigator to investigator. In our procedure, we assumed that the cells that do not die in response to a particular concentration are indeed resistant to that concentration and decided to develop these cells as the resistant cells. We were seeking for the highest concentration of sunitinib that would kill most of the cells, but not all. In this regard, 10 µM sunitinib killed most of the cells (approximately 98%) within 72 h and the remaining 2% of cells were developed to sunitinib resistance. At this stage, the most common practice is to provide a drug holiday period in which the drug is removed from the culture, and the cells are allowed to recover and are rechallenged again (38,39). Thus, cells undergo multiple cycles of drug rechallenge and drug holiday period before being developed into resistant cells. In our method, the cells were never without drugs and it took approximately 12 months to develop stable sunitinib-resistant cells. Although they were developed with 10 µM sunitinib, they were eventually resistant to up to 40 µM sunitinib. The experiments were performed with 10 µM sunitinib because, translationally, dose escalation is not a common practice and could cause much toxicity.
Morphologically, all resistant cell lines showed hypertrophy. Hatakeyama et al. (40) reported similar findings for  786-0 cells in which the nuclei of sunitinib-resistant cells were increased threefold when compared to parental cell lines; however, the size of the whole cells was not reported in this study. The relevance of hypertrophy in sunitinib-resistant cells is not clear. It could be an adaptation of the cells to redistribute the drug so that the overall intracellular drug concentration is less thus somehow "diluting" the effect of the drug. However, the flaw in this argument is that it could also work the other way around, for example, more surface area is present to absorb more drug. The intracellular concentration of sunitinib in these cells is worth pursuing. Nuclear atypia, including larger size and multilobulation (or nuclear bleb formation) as seen in SN12K1 cells, is not uncommon in laminopathies and cancers, and it is associated with highgrade cancers (41).
Despite larger nucleus or nuclear atypia, the surprising finding was the lack of functional aspects in resistant cells that are often considered as "aggressive" in cancer biology, as they did not show any significant difference when compared with the parental cells. For example, Burtz et al. (42), in an in vivo experimental model, reported that sunitinib-resistant renal tumours exhibited aggressive behaviour such as sarcomatoid differentiation, extensive vascular and local invasion, and liver and lung metastases. In our study, resistant cells did not show any significant changes in growth or transmigration when compared with parental cells. Our results on growth are in agreement with the report of Sakai et al. (43) who developed ACHN cells that are resistant to 10 µM sunitinib. Taken together, the functional data appear to suggest that resistance is not necessarily a concern in terms of aggressiveness. Perhaps, these are simply nonresponsive cells without any added aggression. This proposal warrants further exploration. RCC is a highly heterogeneous disease. While no significant heterogeneity could be observed on the functional aspects, it was obvious in the molecular signature. IL-6 was the only molecule that was increased in all cells. As IL-6 is a regulator of VEGF, with the consensus on a mechanism of angiogenesis restoration or neovascularisation (22,39,44), the expression of VEGF was assessed. Interestingly, only two cell lines (Caki-2 and SN12K1) had a significant increase in VEGF mRNA. Thus, VEGF, or angiogenesis, is not necessarily the sole mediator of resistance. To find alternative mechanisms, we explored molecules that regulate apoptosis because apoptosis has been reported to be a mechanism of cell death in RCC in response to sunitinib treatment (45, 46). The results showed that the equilibrium was shifted in favour of the anti-apoptotic molecule Bcl2. Thus, it is not only angiogenesis but also resistance to apoptosis that appear to play a role. To the best of our knowledge, this is the first experimental evidence for the role of anti-apoptosis mechanisms in sunitinib resistance of RCC.

Conclusion
The question arises regarding the best way to combat resistance. One way is to combine other antiangiogenic agents with sunitinib. Although clinical trials combining bevacizumab and sunitinib showed anti-tumour activity, toxicity precludes further clinical development (47)(48)(49). Bcl2 inhibition has long been tried but has not progressed beyond experimental stage (50). Inhibition of IL-6 could be a promising way to overcome drug resistance. In this regard, a recent study showed the beneficial effect of IL-6 receptor inhibition to overcome sunitinib resistance (28). Combination of sunitinib with the ILR-6 inhibitor tocilizumab to overcome sunitinib warrants further research.