Visualizing HCT116 Colorectal Cancer Cells in CT Cancer

9041 words (36 pages) Dissertation

13th Dec 2019 Dissertation Reference this

Tags: CancerBiomedical Science

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Introduction

Cancer cell spheroids are one of the most common and versatile methods of culturing cells in 3-D and are used in cancer research as an intermediate model between in vitro cancer cell line cultures and in vivo tumor.  Spheroids generated from 3-D cell culture models are microscale, elliptic cell clumps that are established by self-assembly using modified cell culture techniques. Spheroid cell culture systems mimic many of the properties of in vivo tumours so are more physiologically relevant to in vivo tumours than cells derived from 2-D monolayers (Edmonson et al, 2014).

A characteristic feature of in vivo solid tumours is their distinct physiological and biological tumor microenvironment that is extremely complex, heterogeneous and comprises of gradients of oxygen tension, nutrients and waste products which differ as a function of distance from a supporting blood vessel (Ayuso et al., 2016). Spheroids mimic these gradients since they are essentially tightly packed multicellular structures with a radii of 200 micrometers or greater that have been formed by the coming together of proliferating and quiescent cells (Mehta et al., 2012) In addition, larger spheroids include hypoxic and necrotic cells at their core (Kim, 2005) which resembles tumour architecture. Tumour spheroids formed with cancer cell lines have a similar cellular heterogeneity to in vivo tissues due to their tight structure. These spheroids also exhibit tumor-specific characteristics because moving in from the outer layers of the tumour towards the core, the diffusion of oxygen, nutrients, growth factors and drugs to the cells is reduced, resulting in deprivation and different levels of stress which mimics the situation in vivo (Figure 1) (Nagelkerke et al. 2013).Oxygen cannot diffuse past an approximate 200 mm limit (Wilson and Hay., 2011).  This reduced diffusion of oxygen and nutrients beyond the outer cell layers, results in the formation of a necrotic central region (Blitterswijk and Boer, 2014). These properties of both tumours and spheroids differ markedly from tumour cells grown in a 2-D monolayer which are all equally exposed to nutrients, drugs and growth factors that are supplied by the medium and are mainly composed of proliferating cells that are stretched out in an unnatural state on a flat substrate.

                               Figure 1: Spheroid characteristics.

The translation of successful pre-clinical drugs to successful clinical drugs has been particularly disappointing for cancer treatment due to the lack of predictive models. Drug development processes have been especially inefficient especially in oncology research, where approximately 95% of the entire anti-cancer new molecular entities fail in Phase 1 to Phase 3 clinical testing (Unger et al., 2014). The findings may in part be due to the shortcomings of 2D cell culture which is currently widely used for drug screening. There is becoming the increasing incorporation of multiplexed 3-D in vitro cell culture systems into vitro pre-clinical testing of anti-cancer drugs candidates and effort to establish mainstream approaches by developing standard and rapid protocols for using 3-D cultures in drug screening (Vinci et al, 2012). In vivo tumours have the development of drug gradients as a result of poor drug uptake by tumours or poor distribution of drugs within a tumour. The rate at which drugs diffuse into a cancerous mass can lessen the therapeutic effects of a drug at the target site (Ivascu and Kubbies, 2007) but 3D cancer cell spheroids account for basic features of solid cancers, pharmacokinetic features and heterotypic cell interaction resulting in them being a clinically relevant model that give a better approximation to drug action within tissues. Thurber and Wittrup (2008) identified that spheroids were discovered to closely replicate drug diffusion observed in the in vivo state. In addition,  hypoxia has also been identified to affect drug responses as it has been recognised as one reason of drug resistance. Therefore, it is critical this is modelled for testing anti-cancer therapeutics (Breslin and O’Driscoll, 2013). For example, hypoxia can enhance receptor tyrosine kinase-mediated signalling, it can cause pro-survival alterations in expression of genes that suppress apoptosis, and it can affect DNA repair pathways [52]. It is feasible to propose that the routine employment of 3D cultures will improve the predictive capabilities of preclinical drug safety and efficacy assessment (Amann et al. 2015) to effective bridge the gap.

A number of drug therapy studies have concluded that 3-D cancer cell spheroids are more resistant to drug treatment and apoptosis than 2-D monolayer (Weaver et al., 2002).

Since 1957, the conventional anticancer drug, 5-FU, has played a vital part as a chemotherapeutic agent for the treatment of colorectal cancer (Zhang et al., 2008). 5-FU exerts its anticancer effects through inhibition of thymidylate synthase (TS) conversion of fluorodeoxyuridine monophosphate (FdUMP), to deoxythymidine monophosphate (dTMP). dTMP plays a vital role in DNA replication and repair and its depletion consequently results in cytotoxicity (Parker and Cheng., 2008; Longley et al., 2013).

It has also become increasingly evident that 5-FU has higher anti-proliferative effects on 2D cultures in comparison to 3D cultures.  The half maximal inhibitory concentration (IC50) is the drug concentration that results in a reduction of cell survival by 50%, compared with the survival of untreated control cells. The half maximal inhibitory concentration (IC50) for 5-FU in 3D cell culture models has been found to be 133Um compared to 100mm for 2D culture models (Wilson S, Abertay University, personal communication). This indicates that drug sensitivities derived from 2-D studies could overvalue the efficacy of therapeutics and the drug appear to be very effective (Godugu et al., 2013; Ekert et al., 2014). In recent studies, Tung et al (2011) presented data demonstrating that A431.H9 cells grown in 2D and 3D show differences in viability when treated with the same concentrations of 5- FU. There was only 5% viability relative to untreated control for 2D cultures, but still 75% viability relative to control for 3D spheroids. This clearly shows that A431.H9 cells are more resistant to 5-FU in 3D than 2D cultures indicating that these 3D spheroids were more resistant to the anti-proliferative effects of 5-FU. Further research by Karlsson et al, 2012 investigated the sensitivity of colon cancer HCT-116 cells to four different anticancer drugs and two promising investigational cancer drugs using both a 3-D tumour spheroid and 2-D tumour cell monolayer. It was found that monolayer cultured cells were sensitive to standard and investigational drugs, whereas the spheroids gradually turned resistant. This data displays that the way in which cells are cultured by 2D and 3D cellular systems has the ability to significantly adjust the effect of a drug on the cells.

Computed Tomography (CT) scanning is a nondestructive imaging procedure that employs x-rays to visualise interior features within solid objects. The scanner fires an electron beam at a metal target that emits a cone-beam of X-rays at the object to be scanned.  Algorisms then create 3D virtual models that are rendered from a series of two-dimensional X- ray images captured at intervals around a single axis of rotation. The 3D imgaes can be used for gaining digital information on their 3-D geometries and properties. The inherent high-contrast resolution of CT means that differences between tissues that differ in physical density by less than 1% can be discriminated between.

Intensity of the X-ray beam that passes through a scanned object depends on the electron and packing density in itself and also energy of radiation (Asseng et al., 2000). The more electron- dense an object is, the attenuation is higher and the intensity of an X-ray that passes through is reduced. X- rays that passes through the object then are detected by a detector system.

It is important to consider the 3D structure and the effects of anti-cancer drugs on the morphology of spheroids. It has previously been illustrated that tumour morphology changes according to classification and drug treatment.

It would be particularly interesting to examine 3D morphology changes in drug-treated spheroids because tumor cells invade collectively as strands, cords and clusters of cells into the stroma, which is dramatically reorganized during cancer progression, therefore they ultimately required morphological change such as tumour budding. Also unlike OPT which was used in the 2012 paper, CT scanning allows for the possibility of  viewing the internal structure such as the hypoxic core of the spheroids and the effects of drugs on it. This infomoration could then be employed for modelling nutrient and waste flow in a multicellular structure. Only problem is that spheroids are not electron dense so that’s where Rb comes in.

Human pathology studies suggest that tumor cells invade collectively as strands, cords and clusters of cells into the stroma, which is dramatically reorganized during cancer progression

Such clusters represent fragmentation of the tumour 2D slice and, because the section is a slice through tissue, this fragmentation might represent a tumour structure that in 3D has invasive protrusions and/or tumour budding.

In order to visualise the spheroids in the CT scanner the process is dependent on the activity of cellular Na+/K+ATPase. It is known that Na+/K+ATPase actively transports Na+ and K+ across the cell membrane and leads to the accumulation of K+ in cells that are metabolically active (Savage, Biffen and Martin 1989). Like potassium(K), Rubidium(Rb) is a group 1 metal and can be used as an analogue of potassium (K), i.e. Rb+, can be used to replace K+ in an extracellular medium will be accumulated by cells via Na+/K+ ATPases instead of K+ as a result of their similar chemical properties. It was first demonstrated that Rb+is accumulated by Na+/K+ATPase by Anner and Moosmayer (1982) when they showed that ATP-driven Rb accumulation was arrested by the drug ouabain that is an established, specific inhibitor of ATP-dependent Na+ /K+ exchange across the cell membrane. Recent studies have further validated the uptake of Rb+ by Na,K-ATPase transport in cells through the incubation of modified physiological buffer, PBS, in which KCl was replaced with RbCl (Metal ion transport quantified by ICP-MS in intact cells) with additional research determining that Na+,K+-ATPase has the ability to transport Rb+ as efficient as K+. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3605618/). The findings of these studies indicate that the spheroids being incubated in an extracellular medium where there is the replacement of KCl with RbCl will result in the cells taking up the contrast agent, Rb+, resulting in a spheroid that is adequately electron dense to block x-rays and result in CT images of the spheroids.Through the use of Rb+ and CT scanning there is also the ability to analyse and examine the spheroids for areas of low electron density that suggests different metabolic states of the spheroids internal regions. Such areas consist of hypoxic cells so cells have not accumulated Rb+ due to reduced metabolism. This is supported by the fact that Rb accumulation has been subsequently exploited to quantify Na/K ATPase activity and hepatocyte viability (Savage et al, 1989). Viable cells are metabolically active cells allowing for the ability to produce more ATP in comparison to apoptotic or hypoxic cells. As a consequence, viable cells will have greater and more efficient Rb accumulation ultimately leading to the cells having a greater electron density and blocking the X-rays to a larger degree.

The aim of this project was to visualize HCT116 colorectal cancer cells in the CT cancer using Rb+ as a contract agent to increase the electron density of the cells. Of particular interest was the presence of the hypoxic core. For comparison, the diameter of the spheroid with and without 5FU will be measured using light microscopy.

Method

Colorectal Carcinoma Cell Culture

The complete growth medium used for the colorectal carcinoma cell line, HCT116 p53+/+, was the Dulbecco’s Modified Eagle Medium, high glucose, GlutaMAX Supplement, pyruvate (DMEM GlutaMAX™) culture medium (Gibco®, USA) supplemented with 10% Foetal Bovine Serum (FBS) (Gibco®, USA) and 1% Penicillin-streptomycin-glutamine antibiotic (1%PSG) (Gibco, USA). The cells were cultured in 75 cm2 (T75) cell culture flasks (Sigma-Aldrich®) containing 12mls of the complete growth media and incubated in a humidified atmosphere at 37°C, 5% CO2, 95% O2, optimal conditions for cell culture growth. The cells were grown to 70–90% confluency before being passaged and disaggregated from the T75 flask using an 5ml aliquot of Trypsin-EDTA (Gibco®). The harvested cells were collected into a 15ml Corning centrifuge tube containing 6ml of DMEMGlutaMAX in order to inhibit the further action of the Trypsin-EDTA and centrifuged at 44g for 10 minutes. The supernatant was removed and the pelleted cells were re-suspended in an appropriate volume of growth media. The subsequent cell suspension was either seeded into a new T75 flask for use in future experiments or excess cells were sub-cultured by centrifuging at 44g for 7 minutes, removing the supernatant and adding 1ml of freeze mix (80% FBS, 12% DMEMGlutaMAX and 8% Dimethylsulfoxide (DMSO) (Sigma-Aldrich®). This suspension was transferred to cryogenic vials and incubated at -80C.

Establishing HC116 +/+ Spheroids in ULA 96-well Plates

The HCT116 +/+ human colorectal cancer cells were trypsinised following the routine protocol. A hemocytometer was employed to count the cells with the required amount of cell suspension being re-suspended in the respective amount of DMEM GlutaMAX. To form spheroids, 200μl of the cell suspension was seeded into the selected wells of a round-bottomed, ultra-low attachment (ULA), 96-well plate (Corning) at a starting density of either 1 x 104, 1.5 x 104, 2 x 104, 2.5 x 104 or 2.8 x 104 cells/well.  Phosphate-buffered saline (PBS) (Gibco®). The plate containing the newly seeded cells were spun at 200g for 5minutes using a plate centrifuge (Thermo Scientific) to facilitate their collection at the bottom of the wells. The 96-well ULA plate was incubated at 37°C, 5% CO2, 95% O2 with the development of stable, tight spheroids with necrotic regions that simulate in vivo tumors generated following 48 hours. Following a 7 day growth period, the spheroids were utilized in experiments. This growth period was reduced in later experiments to 5 days to reduce the time possible for contamination.

Drug Treatment of the HC116 +/+ Spheroids.

For drug treated spheroids, the 96-well ULA plate was seeded at 3 x 103 cells per well with each well having a total volume of 200µl.Following 72 hour’s incubation of the plate at 37°C in a humidified 95% O2/5% CO2/pH = 7.2-7.4 environment, a drug exposure experiment was undertaken with the HCT116+/+ cancer cells spheroids by treating them with medium containing different concentrations of 5-FU (Adrucil®) (dissolved in 100% DMSO (Sigma-Aldrich®)). The drug concentrations were prepared in medium at twice the desired concentration to give a final drug concentration range of 18 to 300µM. The levels of DMSO were adjusted so that each incubation contained the same concentration of DMSO.The concentration of DMSO was < 1% of drug treatment. To make the treatment additions, 100µl of medium was removed from each well and replaced with 100µl of the appropriate treatment. The negative control, just growth medium was used, growth medium with the concentration of DMSO that was applied to all drug concentrations served as the positive control. Assays were carried out in quadruplicate.

Following treatment, spheroids were incubated for 96 hours and imaged every 24 hours for up to 96 hours at 10x magnification using a Leica DMIRE2 microscope teamed with an ixon camera with an Andor Solis software platform that is specifically designed for image capture and analysis. The 2D images of the spheroids were saved as “TIFF” files and were edited into 8-bit images in ImageJ (Ver.1.49, National Institutes of Health). Images were that thresholded and converted to binary images. From the threshold images, the individual particles in the image were identified and the Feret’s diameter and the circularity of each replicate for each day was measured using ImageJ. The Feret’s diameter is defined as the longest distance between the two parallel tangents touching the outline of the spheroid in all directions.The circularity was used to access the roundness of the spheroids. Image J generated a circularity value for each spheroid with a value of 1.0 indicating a perfect circle. As the value advances towards 0.0, it signifies an increasingly elongated polygon. Statistical analysis was performed on this data by carrying out a linear mixed model (for repeated measures) using IBM SPSS Statistics 22. Differences were considered to be statistically significant for p < 0.05.

Computed Tomography Scanning of Cancer Spheroids

In order to prepare the spheroids to be scanned they were transferred to a 1.5ml Eppendorf tube. DMEMGlutaMAX media was removed and the spheroids were washed twice with PBS, centrifuging at 166g for 3 mins between each washing step. This was ensured sufficient washing and that the spheroids were at the bottom of the tube. The PBS was replaced by modified PBS containing 2.7mM RbCl in place of KCl and supplemented with 5mM glucose The spheroids were incubated at 120 minutes with the PBS+ Rb (Savage et al, 1989) at 37°C, 5% CO2, 95% O2. To reduce the electron density of the extracellular medium and improve the quality of the images produced by CT scanning, the spheroids were washed in standard PBS prior to scanning. This reduced the electron-density of the extracellular medium which would attenuate the x-rays before reaching the spheroid. Five spheroids of each cell density were transferred to a 1.5ml Eppendorf tube that was placed in a clear plastic tube in order to raise the spheroids to the level of the x-ray beam.  The spheroids were transferred as quickly as possible to the CT scanner.  Control spheroids (without Rb+) were also imaged by the CT scanner to assess electron density without the Rb+ contrast agent.

To obtain 3-D volumetric images of the colorectal cancer cell spheroids a Metris X-Tek HMX CT scanner with a tungsten target was used that consisted a Varian Paxscan 2520 V detector and a 225 kV X-ray source (Nikon Metrology X-Tek Systems Ltd) resulting of a resolution of up to 5μm. The apparatus containing the spheroids was placed in the center of a rotary table to aid in enabling obtainment of high resolution tomographic image. The spheroids were scanned at 60 kV and 180 mA with a tungsten filter to obtain 1500 angular projections (based on a 360˚ rotation) that resulted in a 3D image of the spheroid. The set-up of the CT scanner involved the undesired brightness variation in the background being limited through the use of shading correction that utilized that employed 2 reference image and creating a volume of interest. At the respective conditions, the scan took place over a 51 minute period. For reconstruction and analyse of the CT images, VGStudio MAX 2.2 (Volume Graphics, Germany) was employed.

Results

During the course of four constitutive days, untreated spheroids were compared with spheroids that had been treated with a serial dilution of 5-FU. The effect of 5-FU on the morphology of the spheroids was measured using the Feret’s diameter (UNITS).  The 96-well ULA plates helped to create spheroids that were relatively uniform in size and shape (ref Vinci 2013).

Figure: Micrographs Showing the Effects of Different 5-FU Concentrations (300μM-18μM) on the growth of HCT116+/+ Spheroids

Figure ? displays representative micrographs of 5-FU treated (300μM – 18μM) and untreated spheroids (C0 – C2) from Day 1 through to Day 4 taken at 10x magnification using a light microscope. The spheroids were composed of 3 x 103 cells with 4 replicates and were imaged for five consecutive days. Levels of DMS O were held constant (see Methods). The Feret’s diameter was measured using ImageJ.

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                                                                                             Drug Concentration (μM)

  C0                        C2                    18                     26                       40                      59                     89                      133                 200                      300                                                     300  

The final size of the spheroids treated with the higher 5-FU drug concentrations (300 – 59μM) evidently causes inhibition of spheroid growth upon drug exposure and there is ultimately spheroid shrinkage when they are viewed to the size of control spheroids (C0 and C2) and the drug concentrations 49 μM -18μM. In fact, the size of the spheroids within this range increase in size. In order to confirm this, the images were analysed in ImageJ to achieve the Feret’s diameter. The mean diameters are plotted in Figure 2. It is evident that there is an effect of time and drug concentration over time on the growth of the spheroids.

A stock solution of 5-FU was made by dissolving the 5-FU in the organic solvent, DMSO. However, DMSO is considered toxic to cells. DMSO towards cells is frequently tolerated with slight or no observable effects observed up to 0.1% final concentration (v/v) with the cell viability rate still remaining greater than 90% (Jamalzadeh et al., 2016). Whereas, at DMSO concentrations of >1% the toxicity of DMSO is known to dramatically increase and have an adverse effect on cell behaviour and proliferation (Galvao, J., Davis, B., Tilley, M., Normando, E., Duchen, M. R., Cordeiro, M. F. Unexpected low-dose toxicity of the universal solvent DMSO). In order to access that the 5-FU was having an adverse impact on the morphology of the spheroids, the final concentration of DMSO was kept constant through the different drug concentrations and an untreated control group was included as a negative control in order to test for solvent toxicity.

Figure: The Effect of 5-FU (M) on the Mean Diameter of Spheroids Over 4 Days

Figure ? shows the results of different concentration of 5-FU(M) on the diameter (mm) of the cancer cell spheroids over a period of 4 days. The means of the data with +/-  SE error bars are included.  It can be interpreted from the graph that the lower concentrations of 5-FU (26M and 18M) had no impact on reducing the diameter of the spheroids and led to the diameter increasing in roughly the same trend to the control spheroids. The higher concentration of the drug resulted in the spheroid shrinking over time. The data was analysed using a linear mixed model (for repeated measures). Time had an effect on the diameter of the spheroids but the extent of this effect is dependent on the drug concentration. The mean diameters of the spheroids treated with higher concentrations of 5FU decreased over time but the diameter of the control spheroids or the spheroids treated with lower drug concentrations did not change perceptively over the 4 days. This was confirmed using a linear mixed model (for repeated measures) at the 5% significance level using Feret’s diameter as a dependent variable, spheroid ID as a random factor and drug concentration and day as covariates.

Drug alone had no significant effect (F(1,191) = 6.804  p = 0.010) which was expected since drugs require time to produce their effects. Similarly day alone (without the presence of drugs) had no significant effect on the diameter (F(1,191) = 0.069 p = 0.794). However, there was a significant interaction between drug and day (F(1,191) = 97.895 p<0.001). This is an expected result as it would not have expected that the drug would have significant effect on its own because 5-FU only has an effect if it is given time to develop that effect. Model assumptions were tested for linearity, normality of residuals (Shapiro Wilks = P=0.066>0.05) and independence of residuals and all assumptions were met (see supplementary materials for plots).

The IC50 of 5-FU in HCT 116+/+ was determined from the drug exposure experiment by calculating  the mean Feret’s diameter for each drug concentration for Day 4.

 

Visualising Spheroids in a CT Scanner

One of the obstacle in relation to scanning the spheroids in the CT scanner was finding a suitable arrangement that allowed the spheroids to be imaged at 360. In order to scan the spheroids, they need to be place in the centre of a rotary table designed for a CT scanner. In previous years, the spheroids were placed in an Eppendorf tube and were located at the base of the tube. For the CT scanner, it was required that the lower proportion of the base was rooted in ‘blu-tak®’ to hold the tube in place within the scanner resulting in the spheroids being obscured on occasions (Savage A., Personal Communication). This lead to the creation of a collagen plug to raise the spheroid to the height of the X-ray beam and allow full 360 view of the spheroids.

Figure – Original set-up for visualising the spheroids in the CT scanner.

Figure ? is a diagram highlighting the design for the CT scanner. It involves an Eppendorf tube filled with liquid with a collagen plug at the base (red) with blu-tak® (blue) securing the tube to the rotary table (black). The spheroids (represented by grey circles_ sit on top of the plug

 

Figure – Electron Dense HCT116 p53+/+ Spheroid visualized using VG Studio Max Software.

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Top 2D view

Figure  (a-b) displays the 3D images of four HCT116 p53+/+ spheroids generated using ultra-low attachment 96-well plates at 1 x 104 cells per well. Cells were allowed to aggregate and form 3D structures for 6 days prior to imaging.  They were scanned at 60kV and 180μA. Images shown are not to scale and the images display two different planes with the 3D image of the tube not being acquired. From the images the spheroids (light grey with very bright voxels) can be distinguished from the tube and the medium that appear darker in colour.  The spheroids were incubated in an Eppendorf tube for 120 minutes at an RbCl concentration of 2.7mM. Following the incubation time, the PBS/Rb was removed from the tube and the spheroids were washed twice with PBS in order to reduce the electron density of the extracellular medium. On analyse of the CT images this technique appears to be sufficient with the spheroids being clearly visible. Reconstruction and analyses of the scan was performed using VG Studio Max. The decision was made to minimise ring artifact when concluding the set-up of the CT scan. This increased the time of the scan from 60 minutes to 120 minutes. It was expected that this would result in better quality images of the spheroids with no ring artifacts obstructing the view of the spheroids.

For comparison, two further scans were performed the at corresponding energy value of 60 kV and a current of 180μA. The first scan involved an Eppendorf tube consisting of media and four spheroids that were incubated for 2 hours with ordinary PBS. The second scan simply consisted of an Eppendorf tube with media. No electron dense areas were visualized following reconstruction of the tomographic images in VGStudioMAX generated from both the scans. These findings confirm that the electron dense areas in Figure ? are certainly spheroids and that in the absence of Rb+ the spheroids are not visible.

Several problems were encountered with this approach. For example, an initial scan of the spheroids was performed with the spheroids being raised into the beam of the x-ray using a collagen plug. On inspection of the tube and images it was noted that the collagen plug failed to raise the spheroid from the bottom of the tube. Other collagen plugs from the same batch of collagen were checked by adding PBS to the tube and it was apparent that the collagen had not set. Further collagen plugs were made up. However, setting problems persisted and in addition the solution appeared cloudy which indicated there had been contamination. It was also noticed that from the “Front 1” view of the spheroid that there was slight movement with the tube during the scan so complete stability of the tube with the use of ‘blu-tak®’ alone could not be guaranteed.

These issues limited the number of scans that could be performed during the available time so as back up images of spheroids were taken using light microscopy and the effects of drugs on morphology quantified using the Feret’s diameter as discussed above.

Image

The region growing tool (RGT) is one of several segmentation tool that VGStudio MAX offers. It is a particularly useful tool for further analysis of the spheroids as it has the ability to subdivide volume data from the CT dataset into different sections which can be employed to define a so-called ‘Regions of Interest’ (ROI). The RGT selects voxels with similar gray values, but only if they are connected.  However, this segmentation tool was unable to distinguish the spheroids from the background suggesting that the spheroids are too similar in electron density to the surroundings. This may have been as a result of scanning the spheroid in within DMEMGlutaMAX which contains phenol red which has an electron dense structure. The OH group in phenol red. The oxygen atom oin the OH group has a lone pair of p orbital electrons. The lone pair becomes part of the declocialation increasing the electron density in the ring. This give the benzene ring in phenol a high electron density  high electron density due to the lone pairs.

It is known that spheroids with a diameter greater than 400–500 μm are concentrically layered structures with a necrotic core REF. From the reconstructed CT scan of the spheroids there is evidence of a more transparent area of the spheroid that is closer in colour to the media within the tube which suggests the hypoxic core can be visualized. To further investigate this area, an image stack was created in VGStudio MAX of the 2D top plane of the spheroids. The image stack was opened with ImageJ where it was possible to generate a plot profile from a line drawn through the middle of the spheroid. In imageJ, a line graph was generated which was analysed to identify possible hypoxic areas through the evaluation of electron density decline. Electron density in the image is related to the grayscale value with lighter areas (more electron dense) being higher grayscale values and darker areas (less electron dense) being lower grayscale values.

Figure : Line graph of a plot profile from one colorectal cancer spheroid seeded at 1 x 104.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure ? (a)is a magnified image of an isolated spheroid from one image of the image stack generated. The yellow line was drawn in ImageJ using the straight line tool to incorporate a section of the exterior, the center of the spheroid and a further section of the exterior. Different areas within the image are also highlighted.  (b) The plot profile values generated by ImageJ were inputted into Excel. An 8-point moving average was calculated to reduce the noise. A line graph displaying the grayscale values of pixels lying along the measured line was produced. The grayscale value varied according to the electron density of the area.

It is evident from the graph that the grayscale value increases when the line comes into contact with the spheroid. The increase in gray scale value denotes an increase in electron density due to rubidium within the cells which has been actively accumulated by Na/K ATPase. Thus, this higher electron density indicates metabolically active cells.  Continuing towards the center of the spheroid, there is a sharp decrease in the grayscale value which corresponds to the center of the spheroid. In this area the cells are believed to be hypoxic (REF) so less metabolically active and as a consequence, the cells have not actively accumulated Rb+. On leaving the center, the grayscale value rises indicating that the electron density is increasing as metabolically active cells are once more encountered towards the outer layers of the spheroid.  On leaving the spheroid and entering the exterior (media within tube), the grayscale value drops due to lower electron density.  The period of decreased grayscale value of the spheroid may be indicative of the hypoxic core. The random fluctuation in the graphs is due to noise produced from the CT scanner. These images suggest areas of low electron density within the spheroid which may correspond to the hypoxic core of spheroids. However, there remains the possibility that the center of the spheroid is less electron dense because Rb+ has not penetrated as far as the cells at the center of the spheroid. This point is worth further investigation.

Generating Large Spheroids for CT Scanning

The resolution of the CT scanner is about 3m but this is with augmentation from image enhancing software. A working size of 10 m would be at the limit of visibility without software augmentation. A considerably larger item would be required to take measurements. Preliminary work with spheroids of about 0.2mm has been carried out but considerable software augmentation was required to visualise the structure of the spheroid. The aim the next part of this study was to achieve a spheroid greater than 1mm. This was achieved at the 28,000 and 25,000 cell densities. The 96-well plate was centrifuged following cell seeding. The purpose of this was to concentrate the cells near the bottom of well, and minimize cell death encouraging the formation of larger spheroids (Ivascu and Kubbies., 2006; Vinci et al., 2012).

 

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Figure ? displays representative images of 2.5 x 104 and 2.8 x 104 cells per well. Following 48 hours, the spheroids had formed tight, clearly defined spheroid tissue cultures. The spheroids were imaged following 96 hours incubation using a light microscope at 4x magnification. The average Feret’s diameter was calculated for each cell density using ImageJ. At 2.8 x 104 and 2.5 x 104 cells per well the average Feret’s diameter was 1.897mm and 1.438mm respectively.

Development for New Set-up for CT-Scanner

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Figure ? demonstrates the new arrangement for the CT scanner consisted of a clear plastic tube that raised the Eppendorf tube. Although the spheroids are situated at the bottom of the tube, the set-up meant they were in-line with the x-ray beam and were not obscured by any object/substance.

Three-dimension images of Cancer Cell Spheroids

 

Figure ? demonstrates 3D images achieved at an energy value of 60kV and a current of 180?o9 and a cell density of 2.8 x 104. The Eppendorf tube consisted of 5 spheroids loaded with the contrast agent, Rb+. The hypoxic core can again by identified from the 3D images obtained (indicated by the red arrows).

Discussion

In this study, it has been possible to view spheroids in a CT scanner using Rb as an electron dense contrast agent.  Since Rb is actively accumulated via the Na+/K+ ATPase (Di Carli et al. 2007; Tian et al. 2009) by metabolically active cells, the visible cells were viable but there was some evidence of a hypoxic core —expand. This project demonstrates that rubidium not only has the potential as contrast agent but could also be used as an indicator of metabolic activity within the spheroid since rubidium blocks x-rays in a concentration-dependent manner (fig.3.1). It is envisaged that areas with lower metabolic activity such as the hypoxic core will accumulate less rubidium due to lower ATP production and Na+/K+ ATPase activity and hence be more transparent to x-rays. This is an area for future development.

The fact that only viable cells were visible offers a distinct advantage over imaging spheroids via light microscopy or Optical Projection Tomography (OPT) since these methods cannot distinguish living and dead cells. The CT images were examined for areas of low electron density. The images were reconstructed and analysed through VGStudio MAX at each cell density revealed a possible hypoxic core that was identified through the presence of a light-grey area that was similar to that of the surrounding media. This result gained from this project makes evident that it achievable to view different regions within the spheroid where there has been hypoxia-induced changes in cancer cell metabolism. Subsequently, in cancer cells of in vivo tumours are found to adjust their metabolism to assist in constant growth and proliferation in challenging hypoxic environments. This suggests that the Rb+ does not have the sole ability of acting as a contrast agent but also can be employed as an indicator of metabolic activity within the spheroid in view of the fact that Rb obstructs X-rays in a concentration- dependent manner. Metabolic changes in response to hypoxia are elicited through direct mechanisms, such as the reduction in ATP generation by oxidative phosphorylation. It is known that cells with an oxygen shortage have enhanced glucose usage in a bid to produce ATP via the less efficient anaerobic glycolysis to lactate (Pasteur effect) (Solaini et al., 2010). Taking this into account, areas with lower metabolic activity such as the hypoxic core will accumulate a reduced amount Rb as a result of reduced ATP production and Na+/K+ ATPase activity. The distinct areas identified could reflect the different metabolic states of the internal regions of the spheroids.

However, there is the question that is there a lack of Rb intake in the centre of the spheroid due to lack of oxygen or is it in fact due to the Rb+ no penetrating that far into the spheroid. This is an area for future development. There would need to be studies performed looking at diffusion gradients to confirm the hypoxic core. This could be explored through seeding cells and spinning them down in order to form a ball of cells. The hypoxic core of the spheroids scanned in the experiment took 4 days to develop. Therefore, if the cells were incubated in oxygenated media and loaded with Rb+ straight after formation, it may be possible to image the balls of cells to see if the Rb+ had diffused to the center of the spheroid and was taken up by the cells. This would help us determine whether the Rb+ could penetrate to the core of the spheroids and may indicate the  4-day old spheroids with Rb+-free centers contained a hypoxic core. Another approach could be the use of a highly electron dense ion or dye that is not taken up by the cells of the spheroid. Instead the spheroid could be incubated with this ion or dye and could be analysed for electron dense areas between the cells in the center of the spheroid, indicating that it is possible for a substance to penetrate the full spheroid. Trypan blue could potentially be used as if if it was found at the centre, this suggests that diffusion to the core is possible.

Past research has recognized that cells rapidly uptake rubidium (Roelcke et al 1996). The short incubation period of two hours and a concentration of 2.7mM did not only allow efficient uptake of Rb+ it also maintained the quality of the cancer cell spheroid. . Also from previous work it is known that cells rapidly uptake rubidium (Roelcke et al 1996) so it is hoped that shorter incubation periods will be possible which will be more efficient and will be better for the quality of the cell. The RbCl was kept as 2.7mM as previous research identified higher concentrations effected the the Na+ gradient and lower concentration aid in preserving the membrane potential The addition of glucose to the modified PBS aided in the process of loading the cells with as the cells would of ended up being depleted of ATP. It was observed that the highest affinity was reached as after 30min in potassium-limiting condition, and the highest Vmax for potassium-sufficient condition was observed after 2 hours incubation (Navarrete et al. 2010). However as tumour cells are correlated with increased Na/K- ATPase activity (Kaplan et al. 1978; Shen et al. 1978) it is expected that malignant cells will reach a Vmax of Rb+ uptake after much shorter incubation times.

One aim of the project was to visualise the spheroids in 3D dimensions which was achieved. However, through the use of VGStudio MAX it was not possible to single out the spheroids and segment them from their background to get a close look at the hypoxic core. An alternative technique for 3D imaging of spheroids is optical projection tomograph (OPT). There is the possibility of 2D images generated by Optical projection tomography to be reconstructed to produce 3D images. From this the images could to be filtered and then segmented to produce clear boundaries of the spheroids

HCT116+/+ cancer cells spheroids by treating with 5-FU showed that there was a visible and calculated decrease in the Feret’s diameter of the 300 – 59μM drug treated spheroids. If 3D images were achievable of the drug treated and non-drug treated spheroids it would be possible to in quantify tumour topology in the absence of and in response to therapeutic intervention by using Minkowski functionals that can obtain point estimates of volume, surface area, integral mean curvature and integral total curvature (Savage et al, 2012). However, one downfall with OPT is that it only achieves an image of the surface of an objectas it uses light as oppose to x-rays. Therefore, it would not be possible to view the internal structure of the spheroid including the hypoxic core.

The IC50 of 5FU, the concentration of 5-FU where the Feret’s diameter is reduced by half, was calculated to be considerably higher, ———-, than the IC50 of 5FU at the chosen spheroid sizes than the state value of 133mM (Wilson S, personal communication). Potential explanations for this could be that there is no possible way to distinguish between the dead and live cells from the images taken by the light microscope. When obtaining the Feret’s diameter using ImageJ it picks up noise including the dead cells. From the high concentration drug treated spheroids there is a large ring of dead cells present around the boundary of the spheroid. It is not possible to completely eliminate this and this would ultimately lead to a greater Feret’s diameter. The dead cells may also still be attached to the spheroid adding to the diameter. In future development, the drug treated spheroids could be loaded with Rb and scanned in the CT scanner to identify if the cells are actually in fact dead leaving. Due to the 3D integrity of spheroids, it is more difficult for 5-FU to diffuse and penetrate into the center cell mass. Furthermore, 5-FU specifically targets proliferating cells, and thus would not kill the quiescent cells in the spheroids. Whereas in 2D mono- layer cultures, cells proliferate at a faster rate and thus 5-FU inhibits cellular growth more effectively. However, 3D spheroids were susceptible to 10 M TPZ treatment compared to 2D controls, possibly because active oxygen consumption by cells and limits in diffusive oxygen transport creates a hypoxic core similar to in vivo tumors. When treated with these drugs together, there was an additive effect on the cell death in 3D spheroids, since 5-FU targets proliferating cells in the peripheral layers of spheroids and TPZ kills cells in the hypoxic core of spheroids [59]. 5-FU primarily targets proliferating cells. Therefore, it is more effective against the rapidly proliferating cells in 2D monolayer culture and would not kill the quiescent.

Hypoxia is a feature of most tumours, albeit with variable incidence and severity within a given patient population. It is a negative prognostic and predictive factor owing to its multiple contributions to chemoresistance, radioresistance, angiogenesis, vasculogenesis, invasiveness, metastasis, resistance to cell death, altered metabolism and genomic instability. Given its central role in tumour progression and resistance to therapy, tumour hypoxia might well be considered the best validated target that has yet to be exploited in oncology. However, despite an explosion of information on hypoxia, there are still major questions to be addressed if the long-standing goal of exploiting tumour hypoxia is to be realized. Here, we review the two main approaches, namely bioreductive prodrugs and inhibitors of molecular targets upon which hypoxic cell survival depends. We address the particular challenges and opportunities these overlapping strategies present, and discuss the central importance of emerging diagnostic tools for patient stratification in targeting

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Tests of Normality
Kolmogorov-Smirnova Shapiro-Wilk
Statistic df Sig. Statistic df Sig.
Residuals .082 156 .013 .984 156 .066
a. Lilliefors Significance Correction

P=0.066>0.05

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