Oncology

Cancer is one of the leading cause of worldwide death in recent years. In 2012, there were 14.1 million new cancer cases and 8.2 million cancer deaths worldwide as estimated by the International Agency for Research on Cancer (IARC). The cancer statistics is expected to grow to 21.7 million new cancer cases and 13 million cancer deaths by 2030. Hanahan and Weinberg proposed the cell physiology hallmarks for the transformation of normal cells to malignant cells (Hanahan and Weinberg, 2011). These 'hallmarks' are shared by most of the cancer cells, and are essential for their growth and metastasis (Figure 1). Therefore, these hallmarks have been targeted regularly to develop therapeutic modalities to treat cancer.


Source: http://www.cancerresearchuk.org/sites/default/files/cs_report_world.pdf
Figure 1: Therapeutic targeting of the Hallmarks of Cancer.(Hanahan and Weinberg, Cell 144, 2011)

Cell-based functional assays

We support pharmaceutical companies and academic researchers in various aspects of oncology research. We have expertise in developing customised cell-based assay using cell lines of your preference in one dimensional, 2-D or 3-D models (spheroids) to investigate the drug of interest. Some of the cell based assays targeting the different hallmarks are listed below:

CATEGORY ASSAY END-POINT
Resisting cell death,Evading growth suppressors, Avoiding immune destruction Anoikis (Anchorage-Dependent Cell Death) Assays Quantitative
  • Colorimetric/ Fluorometric
Cell viability and apoptosis/cytotoxicity Quantitative
  • Colorimetric/ Fluorometric
DNA/RNA damage and repair Quantitative
  • Colorimetric/ Fluorometric
Membrane Integrity Assay Quantitative
  • Colorimetric/ Fluorometric
Oxidase/Peroxidase Activity assays Quantitative
  • Colorimetric/ Fluorometric
ROS activity Quantitative
  • Colorimetric/ Fluorometric
Sustaining proliferative signalling Cell phosphorylation Quantitative
  • Multiplex immunoassays
  • In-Cell ELISA/In-Cell Western Assay
Tumour promoting inflammation Protein Expression Quantitative
  • Multiplex immunoassays
  • Western blotting
  • ELISA
Activating invasion and metastasis Cell adhesion assays Quantitative
  • Colorimetric/ Fluorometric
Cell Invasion Assay Quantitative
  • Colorimetric/ Fluorometric
Cell migration/chemotaxis; Scratch migration assay (Chemokinesis) Quantitative
  • Colorimetric/ Fluorometric
Inducing Angiogenesis In Vitro Vascular Permeability assessment Quantitative
  • Colorimetric/ Fluorometric
Qualitative
  • Time point images.
Tube formation (Angiogenesis) Quantitative
  • Time point images
Genome instability and mutation Gene Expression Quantitative
  • PCR
  • RT-PCR
  • Multiplex Nucleic Acid Testing
Qualitative
  • Images of PCR products by agarose electrophoresis.

3-D Models for Cancer

  • Normal cells undergo transformation to neoplastic cells via a series of genetic and epigenetic alterations. These transformed neoplastic cells have the ability to proliferate and survive independent of both internal and external signals, such as extracellular growth promoting factors. One of the important characteristic of neoplastic cell is their anchorage-independent growth which leads to colony and spheroid formation.
  • The study of cancer cells on 2-D monolayer formats limits the study of these features. Therefore, we propose 3-D models such as, clonogenic (colony formation) and spheroid assays to study these anchorage-independent growth and transformation of neoplastic cells.

    Drug screening in 3-D models can reduce the false positives.
    (Figure adapted from Horman, 2016. http://www.intechopen.com/books/special-topics-in-drugdiscovery)
  • Cells in a 2-D culture environment differ physiologically from cells in 3-D cultures. These physiological changes could affect the drug discovery
  • The additional dimension in a 3-D culture influences the spatial and physical aspects of cell interactions which further influences the cell behaviour.
  • 3-D cultures for drug-discovery have gained popularity as it provides a more complete and physiological model to the disease.
  • Clonogenic assays
    • - Ability of a single cell to grow into a colony (at least 50 cells) is utilised in the clonogenic assay
    • - The assay examines every cell in the population for its ability to undergo uncontrolled division
    • - Clonogenic assays are used particularly to determine cell reproductive death after treatment with ionizing radiation and treatment with cytotoxic drugs
    • - Cells are seeded onto semisolid agar to form colonies, before or after treatment with drug.
    • - The colonies are either manually counted or lysed, stained with fluorescent dye to provide quantitative data.
    • - Assay can be used to detect cancer cell proliferation, cytotoxicity and cell viability.
  • Spheroid Forming Assays
    • The similarity in physiology of the tumour and 3-D spheroid.
      (Figure compiled from Horman, 2016. http://www.intechopen.com/books/special-topics-in-drugdiscovery and Jain, R. K.; Forbes, N., PNAS 2001)
    • - Apart from colonies, neoplastic cells also have the ability to form spheroids
    • - Spheroids are more representative of in vivo conditions than cell monolayers.
    • - Cells in these microenvironments exhibit physiological traits similar to that in cancer microenvironment.
    • - Therefore, 3-D spheroids provide a more complete and physiological model to investigate cancer growth, viability, invasion, immune cell response, drug screening and angiogenesis.
    • - Cells are suspended in spheroid forming extracellular matrix (ECM) or any other matrix of choice, plated onto spheroid forming plates and allowed to aggregate.
    • - Assay can be used to detect cancer cell proliferation, cytotoxicity, cell viability and cell invasion, both qualitatively and quantitatively
    • - Histology of the spheroids can also be analysed for expression and distribution of biomarkers

Cell lines relevant for Oncology assays

CELL LINES CELL LINES
KIDNEY BLOOD & CIRCULATION
RPTEC- Renal proximal tubular epithelial cells U937 - human leukemic monocyte lymphoma cell line
Human Mesangial Cells E0L1 - human eosinophil-like cell line
HEK 293 - human embryonic kidney cells HL60 - human neutrophil-like cell line
AIRWAYS PBMC - peripheral blood mononuclear cells
HASM (human airway smooth mucle cells) HPBE - humal peripheral blood eosinophils
Human airway epithelial cells Jurkat - human T cells leukemic cells
MRC5 - human foetal lung fibroblasts peripheral blood CD14+ Monocytes
NHDF - Human airway fibroblasts OVARY
A549 - adenocarcinomic human alveolar basal epithelial cells CHO - Chinese hamster ovary cells (OVARY)
  OVCAR-3 human ovarian adenocarcinoma
SKIN LIVER
Dermal fibroblasts Primary hepatocytes ( LIVER)
GR-M human melanoma cell line VASCULAR CELLS
Human keratinocytes HUVEC - human umbilical vein endothelial cells
A431: Human Epidemoid Carcinoma HMVEC - human microvascular endothelial cells
BREAST COLON
MCF-7 human breast adenocarcinoma cell line Caco2 - human epithelial colorectal adenocarcinoma cells
BT-474 - human breast ductal carcinoma HCT-116 - human epithelial colorectal carcinoma
CERVIX CARDIOVASCULAR
HeLa - human cervical epithelial cells adenocarcinoma HPAEC - human pulmonary artery endothelial cells
STOMACH HCF - human cardiac fibroblasts
AGS - Human gastric adenocarcinoma cell line AC16 Human Cardiomyocyte Cell Line
PROSTATE Primary coronary artery smooth muscle cells
PC3 - human Prostatic small cell carcinoma BRAIN
DU-145 - human prostate carcinoma U87MG - human glioblastoma astrocytoma

Oncology-based assays

1. Cytotoxicity/Apoptosis/Viability Assays

  • Green Cyanine dye assay : assay is based on the principles of measuring the membrane integrity that occur as a result of cell death.
  • Alamar Blue assays : Specifically, the system incorporates an oxidation-reduction (REDOX) indicator that both fluoresces and changes color in response to chemical reduction of growth medium resulting from cell growth.
  • LDH Cytotoxicity Assay : The level of formazan formation is directly proportional to the amount of LDH released into the medium, which is indicative of cytotoxicity.
  • Caspase3/7 : a fluorescence based assay to detect caspase 3/7 activity as an indicator of apoptosis

2. Target validation, qualification

  • Real time PCR
  • Gene silencing/knockdown assays
  • Western Blotting
  • Immunocytochemistry

3. Proliferation

  • MTT: indicator of mitochondrial metabolic activity
  • BrdU: detects 5-bromo-2'-deoxyuridine (BrdU) incorporated into cellular DNA during cell proliferation
  • Alamar Blue: Specifically, the system incorporates an oxidation-reduction (REDOX) indicator that both fluoresces and changes color in response to chemical reduction of growth medium resulting from cell growth.
  • Ki-67: Immunoassay to determine percentages of proliferating and non-proliferating cells based on Ki67 expression. Ki67 is a nuclear protein expressed during G1, S, G2 and M phase of the cell cycle but absent in the G0 phase. Ki67 antigen positive cells provide a more specific and accurate indicator of proliferating cell.
  • Caspase3/7: a fluorescence based assay to detect caspase 3/7 activity as an indicator of apoptosis
  • TUNEL assay:
    TUNEL Assay Principle (Courtesy: https://www.rndsystems.com/resources/technical/tunel-assay-principle)

    DNA fragmentation represents a characteristic hallmark of apoptosis. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is an established method for detecting DNA fragments.
    The TACS® TdT kits contain a highly purified form of the TdT enzyme for the enzymatic incorporation of biotinylated nucleotides. Biotin labeling is achieved using Streptavidin-horseradish peroxidase, and colorimetric substrates diaminobenzidine (DAB) or TACS Blue LabelTM.
    TACS XL® kits embody a novel approach for the detection of apoptosis. This assay is based on incorporation of biotinylated nucleotides conjugated to bromodeoxyuridine (BrdU) at the 3' OH ends of the DNA fragments that form during apoptosis. This detection system utilizes a biotin conjugated anti-BrdU antibody and streptavidin-horseradish peroxidase. TACS XL kits are available with colorimetric substrates diaminobenzidine (DAB) or TACS Blue Label.

4. Cell Invasion assays

  • Boyden chamber
    Cells seeded at the top chamber and chemoattractant or different cell line in the bottom chamber - outcome: number of cells migrated to the bottom chamber, compared to controls
  • Matrigel invasion

    Similarly to the Boyden chamber, cells are seeded on top of Matrigel and their activity to degrade the Matrigel is measured on the migrated cells

5. Vascular Permeability assays (airway endothelial cells)/Membrane Integrity Assays (airway epithelial cells)

  • Trans-endothelial epithelial resistance (TEER) Measurement: measurement of electrical resistance across a cellular monolayer and is a very sensitive and reliable method to confirm the integrity and permeability of the monolayer.
    TEER measurement with chopstick electrodes
  • Membrane integrity/Permeability determination using FITC-labelled Dextran: The extent of permeability can be determined by measuring the fluorescence of the receiver plate well solution. This Assay is ideal for measuring compounds that may disrupt or protect an endothelial monolayer.
    Representation of the vascular permeability assay using the FITC Dextran.
  • Tight junction protein expression (V-Cadherin, occludins and claudins) by Immunofluorescence

6. Angiogenesis

  • Scratch assay
    - Monolayer of cells is scratched to form a gap and treated with an anti-proliferative agent and test compound
    - outcome: the gap closure compared to controls.
  • Matrigel network formation
    - to determine the ability of cells to form connections, follow chemoattractive signals, secrete signals.
    - Cells are seeded in a layer of Matrigel (with growth factors or reduced)
    - Incubated at 37oC in presence of activators/inhibitors - blocking antibodies, siRNA, growth factors, etc.
    - Remove media and fix with PFA (optional)
    - Image analysis


7. Immunogenic Cell death Assays

  • CAL (calreticulin) receptor expression by Immunofluorescence/histology, immunoassays
  • Secretion ATP -luminescence assays
  • Release of intracellular HMBG1 measured by immunoassays, localisation of intracellular HMGB1 by flow cytometry
  • HSP70 release measured by immunoassays, western blot
  • HSP 90 release measured by immunoassays, western blot
  • induction of a type-1 interferon response - detection of IFNa1, a2 and β1 by RT-PCR
    - type 1 interferon response includes STAT3 activation by Tyr phosphorylation and tyrosine kinase activation (mainly, but there are more targets) - measure pSTAT3 and pTYK as one of the pathway activation biomarkers suggesting induction of type-1 interferon response.


8. Pathway activation biomarkers

  • Measure phosphorylation of downstream signalling events
    - Multiplexing immunoassays
    - Western Blot
    - In-Cell ELISA


9. Disease Biomarkers

  • Measure release of cytokines, chemokines, growth factors and inflammatory mediators (mRNA and protein)
    - Multiplexing immunoassays
    - ELISA


For a complete list of relevant biomarkers, please click on the pdf link below:
Oncology Assays-CB