Oncology

Oncology

Neoplastic growth could arise from different cell types within the body, creating a spectrum of disease types that vary in pathological behaviour and treatment approaches. The development of cancer arises from a dysregulation in cellular proliferation, which usually arises from inappropriate responses to external stimuli. Through the years, discovery of mutations in key oncogenes and tumour suppressor genes has been pivotal in characterising biological aberrancies that favour oncogenesis. For instance, many cancers harbour activating mutations in components of several survival pathways (e.g. PI3K and MAPK pathways) leading to constitutive signalling. Downstream effects include increased production of growth factors for inter and intracellular interactions, signalling cross talks and loss of response to inhibitory stimuli.  The acquisition of cancer hallmarks, as outlined by Hanahan and Weinberg (2011), has been the foundation of studying cancer biology and determining various approaches for treatment purposes. For years, therapeutic regimes have been developed to target the following oncogenic drivers:

  • Sustaining proliferative signalling
  • Resisting cell death
  • Inducing angiogenesis
  • Enabling replicative immortality
  • Activating invasion and metastasis
  • Evading growth suppressors

Cellomatics Biosciences Ltd. provides expertise in generating preclinical data for clients within the oncology area of interest. Alongside the ongoing development of disease models, our team routinely performs high throughput drug screens followed by a wide range of assays to measure certain biological responses that complement the hallmarks of cancer.

Cell proliferation

Most chemotherapeutic agents inhibit oncogenesis by reducing the rate of proliferation, usually as a result of cell cycle arrest. Cell proliferation is commonly measured through an ELISA-based approach called BrdU assay. Its working principle involves the incorporation of BrdU in actively proliferating cells. The amount of incorporated BrdU is detected by incubating with anti-BrdU antibody following fixing, permeabilization and DNA denaturation. Horseradish peroxidase-conjugated antibodies bind to the primary antibody and this catalyses TMB to a blue substrate which could be measured by absorbance.

With a reduction in proliferation, cells could undergo senescence in response to a treatment. Increased activity of β-galactosidase is a marker in senescent cells. The CellEvent Senescence Green Detection Kit employs β-galactosidase’s enzymatic activity with which hydrolytic cleavage of fluorescein-based substrate leads to emission of a fluorescent signal. Positively stained cells specify the proportion of cells that have entered cellular senescence.

Cell viability

Cancers are known to exhibit resistance to conventional treatments, which contribute to relapses and disease progression. Quantification of the proportion of cells surviving a treatment is performed through cell viability assays. One approach is MTT assay which involves the conversion of the water-soluble MTT substrate into formazan salts by actively metabolising cells. Solubilisation of the formazan salts produces a coloured solution that is measured by absorbance.

CellTiter Fluor is an alternative means of determining cell viability. Unlike MTT assay, this approach uses live-cell measurements by employing fluorogenic substrate that is proteolytically cleaved following entry into viable cells. The fluorescent signal emitted is proportional to the number of living cells.

Cell death            

Screening for compound toxicity is a fundamental step in analysing the cytotoxic capability of a compound towards cancer cells. Measures from these assays are indicative of the drug’s efficiency in cell killing, or if it only induces cytostaticity. Many of the in-house assays available to measure cell death work based on compromised cell membrane in response to drug treatment. For instance, LDH assays measure enzymatic activity that is released into the reaction mixture by cells undergoing cell death. Alternatively, CellToxTM Green Cytotoxicity Assay involves binding of reporter molecules to released DNA and the fluorescence serves as a parameter to measure the amount of cell death post-treatment.

Spheroid assays

The shift to utilising 3D cancer models remains an attractive option to investigate more clinically relevant features. Previously, breast and ovarian cancer cells have been grown as spheroids as part of our in-house projects. Following drug treatment, fluorescently labelled dyes enabled a detailed annotation of the spheroids to delineate regions that are either viable or have undergone cell death. Complementing this work is our two imaging platforms, JuLITM Stage and ImageXpress Pico System, which can perform an array of functions including live imaging and time lapse capture.

Invasion and Migration

Disease management is often plagued by the detection of migration of cancer cells to distant locations from the primary growth site and invading into nearby tissues to establish secondary tumours. Disabling cell motility brought about by treatments is one strategy to delay disease progression. In vitro analysis of invasion could be mediated by wound healing assays. A scratch is introduced on a pre-formed monolayer of adherent cells. The duration from treatment to closure of the gap then provides an indication of the drug’s efficiency at inhibiting cellular movement.

Invasion and Migration

Disease management is often plagued by the detection of migration of cancer cells to distant locations from the primary growth site and invading into nearby tissues to establish secondary tumours. Disabling cell motility brought about by treatments is one strategy to delay disease progression. In vitro analysis of invasion could be mediated by wound healing assays. A scratch is introduced on a pre-formed monolayer of adherent cells. The duration from treatment to closure of the gap then provides an indication of the drug’s efficiency at inhibiting cellular movement.

Transwell systems also allow quantification of cell migration following drug treatment between the apical and basal chambers across a membrane, mimicking physiological conditions. This method has been employed for our suspension cell lines wherein movement between the two compartments is mediated by chemoattractants but inhibited by drug agents.

Angiogenesis

Neoplastic growths maintain their survival through creation of their own blood supply system. Tumours secrete stimulatory factors that allow the recruitment of cells necessary for the vascularisation of the tumour mass. HUVEC cells are used to model angiogenesis by stimulating with VEGF in a gel-based extracellular matrix. Viable stains allow visualisation using our imaging platforms to facilitate measurements of parameters such as tubule formation, lengths and nodes.

Immune-related oncology assays

Cancer cell survival could be mediated through evasion of immune surveillance. Immune cells could be ‘trained’ to detect and eliminate cancer cells, making it an alternative treatment to conventional cancer therapies. Priming of the host’s immune system to mediate cancer cell killing is considered more favourable towards minimising patient mortality. Activated natural killer (NK) cells and T-cells have been employed to initiate killing of suspension and adherent cancer cells, respectively. NK cell cytotoxicity assays use luminescent signals to indicate caspase-3 activity as a function of cell death. T-cell cytotoxicity assays use intensities of red-green viable fluorescent signals to determine the proportion of dead from live cells.

1. Angiogenesis

2. Cell Adhesion

3. Cell Apoptosis

HeLa cells were seeded at a density of 10,000 cells/well and treated with staurosporine for 4 hours at concentrations of: 10µM, 5µM, 2µM, 1µM, 500nM, 250nM, and 125nM. The Caspase-Glo 3/7 Reagent was added directly to cells in 96-well plates; the total volume was 80µl per well. The assay was incubated at room temperature for 30 minutes before recording luminescence. Each point represents the average of 3 replicates (error bars represent ±SEM). The blank control value has been subtracted from each point.

4. Dose Response for Talazoparib and Minocycline of PARP activity and Proliferation in UWB1.289

The ovarian cancer cell line UWB1.289 was treated for 24 hours with increasing concentrations of Talazoparib and Minocycline. PARP activity was measured by in vitro cell free enzymatic assay (A-B). Proliferation was assessed by BrdU incorporation (C-D). DMSO was used as vehicle control and represented 100% PARP activity and proliferation respectively. All experiments were conducted as n=3. Bars represent the Standard Mean Error (SEM).

5. Oncology Biomarkers of relevance

6. PARP assays

Dose Response for Talazoparib and Minocycline of PARP activity and Proliferation in UWB1.289

The ovarian cancer cell line UWB1.289 was treated for 24 hours with increasing concentrations of Talazoparib and Minoccline. PARP activity was measured by in vitro cell free enzymatic assay (A-B). Proliferation was assessed by BrdU incorporation (C-D). DMSO was used as vehicle control and represented 100% PARP activity and proliferation respectively. All experiments were conducted as n=3. Bars represent the Standard Mean Error (SEM).

7. Proliferation Assay

8. Protein analysis from Tumour Tissue lysates

The mouse tumour tissues were weighed and homogenised in appropriate volumes of the assay buffer. The tumour lysates were collected and the cytokine levels analysed using Luminex platform.

Note: similar method can also be applied for protein analysis by ELISA platform.

9. Senescence

Primary human chondrocytes were treated with H2O2 for 24 hours followed by 72 hours recovery period. Cells were fixed and immunofluorescence staining for DAPI and β-galactosidase was performed according to the manufacturer instructions.

  • Untreated human primary chondrocytes; B- H2O2 treated human primary chondrocytes

10. Spheroid Assays

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Cellomatics Biosciences Ltd. is a specialised Contract Research Organisation (CRO) providing bespoke preclinical laboratory based services.

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