3.1 Introduction:
The present study aims for analysing the role of tumour-associated carbohydrate antigen as a potential cancer vaccine. To fulfil this aim, the present study has adopted the concept of formulating a Zwitterionic Polysaccharide capsular protein needed for preparing off-shelf vaccines to do in vitro in vivo tests. Therefore, this study has adopted the Zwitterionic Polysaccharide-based TACA Delivery approach for fulfilling the major aim of the study.
Various studies have revealed that the understanding of ideas and theories adopted regarding the research study is necessary for the conduction of any research along with other motivational implementation. Hence, the research methodology has been considered as the most relevant section of a research study. The research methodology consists of the protocols that require the information regarding the research study according to the selected topic. This chapter illustrates the research methods that are being implemented in the current study for analysing the results. The research study is entirely dependent on the research methodology for fulfilling the major aims and objectives of the study. Also, the research methodology has analysed the appropriate interpretation of the study. This chapter explains the associated sections related to the concerned topic.
3.2 Cell culture:
The present research will use commercial cell lines of animal models along with human monocytes and human tumour models. These animal cell lines will be maintained in DMEM media, human monocytes in RPMI-1640, and human tumour cell lines in FBS. The animal macrophage cell line will be maintained in Dulbecco’s modified Eagle medium (DMEM) being supplemented with 10% FBS and 1% Penicillin-Streptomycin. The cells will be maintained in n 25cm2 and 75cm2 tissue culture flasks and will be harvested using a cell scraper. The human monocytes will be cultured using RPMI-1640 being supplemented with 10% FBS and 1% Penicillin-Streptomycin. The cells will be harvested by performing centrifugation and subculturing for ensuring confluency. The human carcinoma cells will be grown in 10%FBS, 1% Penicillin-Streptomycin supplemented DMEM, and will be harvested using 2-3mL of 0.25% Trypsin-EDTA solution. All the cell cultures will be incubated at 37°C containing 5% CO2 and will be cryopreserved in vapor-phase liquid nitrogen or -80°C using 5% DMSO in fresh culture media.
4.2 Zwitterionic Polysaccharide capsular protein Extraction
Zwitterionic Polysaccharide capsular proteinwill be extracted as per the methodology of Ziegler et al., 2014. Carcinoma plasma membrane fractions will be isolated from differential centrifugation using the partial methodology of Ziegler et al. (2014). All membrane extraction steps will be undertaken at 4°C to inhibit protease action. Approximately 1×108 A549 cells (3x 175cm2 tissue culture flasks) will be washed twice in ice-cold Phosphate buffered saline (PBS) before being harvested using a cell scraper to avoid membrane protein disruption. Cells will be pelleted via centrifugation at 800 x g for 5 minutes. Cells will be resuspended in 10 mL of hypotonic lysis buffer supplemented with a 1% protease inhibitor cocktail (Sigma-Aldrich) and left on ice for 30 minutes. Following this, 2 mL aliquots of cells will be subjected to 30 strokes of a Dounce homogenizer until the entire cell suspension will be processed. The efficiency of lysis will be analyzed via trypan blue staining, when cell lysis was greater than 90% the lysate was spun at 800 x g for 10 minutes. The supernatant will be then collected and spun at 100,000 x g for 1 hour. The supernatant containing the cytosolic portion will be discarded and the crude total membrane pellet will be resuspended in 4mL of PBS and stored at -80°C. Protein concentration will be determined using a BCA protein assay kit (Thermo-Scientific). Membrane protein will be harvested to function as an antigen source for the following experiments.
4.3 Experimental Conditions
Macrophages will be harvested via scraping then seeded at 7 x 105 /mL in 24 well microplates. The in vivo and in vitro experiments will be carried out. Cells will be exposed to varying concentrations of harvested membrane protein (ZPS capsular protein) and CpG ODN (human ligand) with a specific sequence. Monocytes will be seeded in 24 well microplates and grown in RPMI-1640 supplemented with 200nM phorbol1,2-myristate 13-acetate (PMA). Plates will be incubated for 48 hours before washing twice with fresh PMA-free media and rested for a further 24 hours. Macrophage phenotype will be identified upon adherence to microtitre plates. Macrophage phenotype will be identified upon adherence to microtitre plates. Macrophages will be exposed to specific combinations of membrane protein (ZPS capsular protein) and the specific CpG ODN (ligand). Following RAW 264.7 experiments, the experimental protocol will be amended to include a control containing only CpG ODN to clarify the effectiveness of membrane protein (ZPS capsular protein) as an antigen source.
| Group | Conditions |
| Experimental 1 | CPG* + 5ng Antigen |
| Experimental 2 | CPG* + 25ng Antigen |
| Experimental 3 | CPG* + 125ng Antigen |
| Experimental 4 | CPG* + 625ng Antigen |
| Experimental 5 | CPG* + 3125ng Antigen |
| Positive Control | LPS 200ng/mL |
| Negative Control | Unstimulated |
| Non-CPG* | Non-CPG DNA 4µg/mL |
| Antigen Control | 125ng Antigen |
| *CpG oligodeoxynucleotide 4µg/mL
*Non-CpG containing DNA |
|
Table: Macrophage experimental and control groups
4.4 Nitrate Assay
A nitrite standard curve will be generated using sodium nitrite dissolved in fresh culture media. A 1:1 ratio of cell culture media and Griess reagent (Sigma-Aldrich) will be aliquoted into 96 well microplates in a total volume of 100µL. A nitrite standard curve will be generated using sodium nitrite dissolved in fresh culture media serially diluted from 100 – 1.56µM. Samples will run in triplicate and nitrite concentration was measured using a plate reader set to an absorbance of 540nm.
4.5 Enzyme-linked immunosorbent assay (ELISA)
Elisa will be performed on cell culture media following the treatment of cells to detect secreted cytokines. Known cytokine standards will be used to generate standard curves and quantify the level of secreted cytokines using biotin-labeled anti-human and anti-mouse monoclonal antibodies. Protocols will be used varied slightly per cytokine; however, the basic methodology will be performed as follows: 50µL of assay buffer will be combined with 50µL of standards, samples, and zero standard controls in a 1:1 ratio per well. Elisa plates will be covered and incubated at room temperature for 2 hours. Each well will be then aspirated and washed 4 times using a multichannel pipette before 100µL of biotin-labeled antibody will be added to each well and then incubated for a further 2 hours at room temperature. Washing steps will be repeated before 100µL of streptavidin horseradish peroxidase (HRP) conjugate will be added to each well and incubated for 45 minutes.
Following this, each well will be washed 5 times to minimize background detection, and 100µL of TMB substrate will be added and incubated for a further 15 minutes protected from light. Stop solution will be added at the 15-minute mark and absorbance values of each well were read using a microplate reader using a 450nm absorbance. Standard curves will be generated by plotting mean OD values on the y-axis as a function of serially diluted standard concentrations on the x-axis. A 4-parameter logistic curve fit will be used for all analyses and r2 values were 0.99 or higher in each case. All statistical analyses will be performed using Graphpad Prism.
4.6 RNA Extraction
RNA extraction will be performed using the RNeasy Mini kit. Suspension cells will be harvested and pelleted before lysis and adherent cells will be lysed directly within microtitre plate wells. 350µL of Lysis buffer will be added directly to cells and mixed thoroughly. Lysates will then be pipetted into microcentrifuge tubes and homogenized by vortexing for 2 minutes. 350µL of 70% ethanol will be added to each homogenate, mixed, and then transferred to an RNeasy spin column before centrifugation at 8000 x g for 15 seconds. The flow-through will be discarded and 700µL of wash buffer was added and recentrifuged at 8,000 x g for 15 seconds. This centrifugation step will be repeated once more using RPE buffer, before a third washing step at 8,000 x g for 2 minutes. The spin-column will be placed into a fresh microcentrifuge tube and eluted into 30µL of RNAse-free water by spinning at 8,000 x g for 1 minute. Samples will be stored at -20°C.
4.7 cDNA Synthesis
All steps for cDNA synthesis will be undertaken following the manufacturer’s supplied protocol. For each sample 1µg of total RNA will be added to 2µL of 50µM d(T), 1µL of 10mM dNTP mix, 2µL of 10x MMuLV buffer, 1µL of M-MuLV reverse transcriptase, 0.2µL of human placental RNase inhibitor and nuclease-free H2O to a total volume of 20µL. Each cDNA synthesis reaction will be incubated at 42°C for one hour before inactivation at 80°C for 5 minutes.
4.8 Primer Design
Sequence specificity of primers will be all verified using PrimerBLAST, ensuring amplicon length was between 75-250bp; Tm will be between 50°C and 65°C and that forward and reverse primer sequences did not differ by more than 3°C. Sequences will be chosen to avoid secondary structure formation and 3’ complementarity whilst possessing 50-60% GC content. Primer sequences will have either a G or C at the 3’ and 5’ end where possible and no more than triple repeats of either G or C will be present throughout. Melt curves for each gene of interest during real-time PCR reactions will be used to validate primer specificity.
4.9 Real-Time PCR
Relative levels of gene expression will be measured using a thermocycler. Relative quantification analysis will be performed using the associated Quant Studio 3 software (Applied Biosystems). Reaction mixtures will be aliquoted into PCR plates using clear plate seals (Bio-Rad) before vortexing for 30 sec and centrifuging to remove bubbles.
| Component | Volume (µL) | Final Concentration |
| SYBR Green Supermix (2x) | 10 | 1x |
| Forward and reverse primers | 1 | 250-500nM |
| cDNA template | 1 | 100ng-100fg |
| Nuclease-free H2O | 8 | – |
| Total reaction volume | 20 | – |
Table: Real-time PCR reaction mixture
Real-Time PCR thermocycling protocol will be running and the results will be then analyzed. GAPDH will be used as the endogenous control and relative gene expression was performed using the ΔΔCt method. The GAPDH threshold cycle (CT) of each experiment will be taken from the CT value of each gene of interest to give ΔCt. The negative control values for each gene of interest will be used as a calibrator. The calibrator ΔCt value was then subtracted from every unknown ΔCt value per gene to give ΔΔCt values. Fold change expression will be calculated as 2ΔΔCt and graphed.
| Polymerase Activation and DNA Denaturation | Denaturation at 95°C | Annealing/Extension + 60°C Plate read | Cycles | Melt Curve Analysis |
| 98°C for 2 minutes | 10 seconds | 15 seconds | 40 | 65°C -95°C (0.5°C/2sec) |
Table: Real-time PCR thermocycling protocol
4.10 Immunofluorescence
4.10.1 Nuclear Factor Kappa B (NF-kB)
Monocytes will be seeded at 5 x 104 cells per well in optically clear bottomed 96-well microtitre plates (Perkin Elmer) and differentiated with 100ng/mL PMA (Berges et al., 2005) Following a 24-hour incubation with CpG ODN 4µg/mL and 125ng/mL of membrane protein (ZPS capsular protein), RAW 264.7 and macrophages will be fixed with 4% paraformaldehyde (PFA) at room temperature for 10 minutes. Cells will be then washed 3 times with ice-cold PBS before a 10-minute incubation in 0.25% Triton X-100 in PBS. Cells will be washed three times with PBS for 5 minutes. Following this, macrophages will be incubated in 1% Bovine serum albumin (BSA) PBS Tween-20 (PBST: 0.1% Tween-20 PBS) for 30 minutes to block any nonspecific antibody binding. This solution will be aspirated, and cells will be again incubated in 1% BSA PBST, this time containing 1µg/mL of the primary rabbit Anti-NF-κB p65 human antibody. Cells will be incubated in a humidified chamber for 1 hour at room temperature. The solution will be decanted, and cells will be washed with PBS three times for 5 minutes. The cells will be incubated in 2µg/ml Anti-Rabbit IgG H&L (Alexa Fluor® 488) secondary antibody in 1% BSA at room temperature and away from light for 1 hour. Three subsequent 5-minute PBS washing steps will be also performed in the dark. Macrophages will be then incubated with HOECHST 33342 (1µg/mL) away from light for 10 minutes, followed by 2 final washes with PBS to remove the excess nuclear stain.
4.10.2 MHCII expression
The protocol for the same will be followed and used. Confocal microscopy will be used for analyzing the images. Monocytes will be seeded in optically clear bottomed 96-well microtitre plates (Perkin Elmer) and differentiated with 100ng/mL PMA (Berges et al., 2005). Following differentiation macrophages will be incubated with CPG ODN 4µg/mL and 125ng/mL of membrane protein (ZPS capsular protein) for 24 hours. Cells will be then fixed with 4% PFA at 37°C for 10 minutes and washed thrice with PBS. Blocking will be performed using 2% BSA in PBST for 30 minutes before a 1-hour incubation with 500ng/mL Anti-HLA-DR antibody for 1 hour in a humidified chamber. The solution will be decanted, and cells will be washed three times for five minutes in PBS. Images will be taken using a confocal microscope.
4.11 Phase Contrast Microscopy
Phase-contrast microscopy will be performed using an inverted microscope to observe morphological changes within macrophages upon exposure to immunostimulatory molecules. Images will be taken at 100x and 200x total magnification.
4.12 Confocal Microscopy
The protocol for confocal microscopy will be used. Macrophages will be treated with 4µg/mL and 8µg/mL 6- 6-Carboxyfluorescein (6-FAM) tagged with sequence. Following a 24-hour incubation at 37°C 5% CO2, cells will be incubated for another 2 hours with 50nM LysoTracker deep red. Following endosomal staining, macrophages will be fixed with 4% PFA for 10 minutes before washing twice with PBS. The cells will then counterstained with 1µg/mL HOECHST-3342 (Thermo Scientific) (excitation 350nm, emission 451nm) and washed twice more. Imaging will be performed on the confocal microscope to observe cellular uptake of 6FAM conjugated CpG ODN about late endosomes and lysosomes, using LysoTracker.
4.13 Statistical Analyses
All samples will run in triplicate and presented as the mean ± SE of the mean (SEM) unless otherwise specified. One-way ANOVA and Tukey’s posthoc statistical tests will be used to analyze all observed results for statistical significance. All data will be subjected to Kolmogorov-Smirnov (KS), Shapiro-Wilk, and Anderson-Darling tests to ensure a normal distribution. Linear and nonlinear ANOVA trend analyses will be used to evaluate dose relationships. All statistical analyses will be performed using the SPSS.
References
- Ziegler, Y.S., et al., Plasma membrane proteomics of human breast cancer cell lines identifies potential targets for breast cancer diagnosis and treatment. PloS one, 2014. 9(7).
- Berges, C., et al., A cell line model for the differentiation of human dendritic cells. Biochemical and biophysical research communications, 2005. 333(3): p. 896-907.