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 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.

Adhesion

Cell adhesion is involved in stimulating signals that regulate cell differentiation, cell cycle, cell migration, and cell survival. This has application in cancer metastasis study, and in the determination of the adhesion properties of normal and cancerous cells.

Leukocyte adhesion using HUVEC co-culture

A statistically significant increase in leukocyte adhesion was observed on TNFα stimulated HUVEC monolayers when compared to HUVEC (untreated), HUVEC+ leukocyte (untreated) and IC87114 treatment (PI3K Inhibitor) (*p<0.05; **p<0.01; ***p<0.001±SEM).

Invasion

Cell invasion are finely regulated processes that are critical in many normal physiological processes including during embryonic development, wound repair and immune surveillance (Lauffenburger and Horwitz, 1996; Pollard and Borisy, 2003; Ridley et al., 2003). However, these dynamic cell movements are also crucial in cancer progression and metastasis (Hamidi and Ivaska, 2018).

Migration & Invasion

Migration: HT-1080 and 3T3-LI were seeded at 100,000 cells/well into Corning Transwell™ inserts (Pore size: 0.8 μm) inserum-free media and allowed to migrate towards FBS for 48hrs in the presence or absence of 2µM Cytochalsain D. Migration assay showed that treatment with 2µM Cytochalsain D resulted in a significant decrease in migration with highly invasive HT-1080 cells, as indicated in the relative fluorescent values.

Invasion: HT-1080 and 3T3-LI were seeded at 300,000 cells/well (HT-1080) and 100,000 cells/well (3T3-LI) into Corning Transwell™ inserts (Pore size: 0.8 μm) and were coated with Cultrex® Basement Membrane Extract. Cells seeded in serum-free media invaded towards FBS for 48hrs in the presence or absence of 2µM Cytochalsain D. Invasion assay showed that treatment with 2µM Cytochalsain D resulted in a significant decrease in invasion with highly invasive HT-1080 cells, as indicated in the relative fluorescent values.

 

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.

Chemotaxis

Neutrophil migration through the HUVEC monolayer

A statistically significant increase in neutrophil migration towards the fMLP chemoattractant was observed in HUVEC monolayers treated with TNFα when compared to untreated HUVEC monolayers (ns=not significant; **p<0.01; ***p<0.001±SEM).

Chemokinesis

Neutrophil migration through the HUVEC monolayer

A statistically significant increase in neutrophil migration towards the fMLP chemoattractant was observed in HUVEC monolayers treated with TNFα when compared to untreated HUVEC monolayers (ns=not significant; **p<0.01; ***p<0.001±SEM).

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.

BrdU Assay

HLF (human lung fibroblasts) cells were seeded at a density of 4000 cells/well and incubated overnight at 37oC in a humidified incubator. The cells were treated with PDGF-BB (3ng/mL; 10ng/mL) and Mitomycin C (10µM; 100µM). The BrdU assay showed that treatment with PDGF-BB and 10% FBS resulted in a significant increase in the cell proliferation, as indicated by the increase in the absorbance values. Treatment with Mitomycin C significantly reduced the absorbance, suggesting an inhibition in cell proliferation (***p<0.001 ± SEM).

MTT

HMC1.2 cells were treated with a (or the) test compound at 6 different concentrations. The % cell viability post-compound-treatment was determined by means of an MTT assay. A significant reduction in cell viability was observed with δ-Tocopherol when compared to the positive and vehicle controls (***p<0.001; n=5; ±SEM)

Alamar Blue

HUVEC cells were seeded at different densities and the signal for Alamar Blue dye was monitored over 24 hours.

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.

Apoptosis/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.

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.

Caspase 3/7 Assay

HeLa cells were treated with Staurosporine for 4 hours. The Caspase-Glo 3/7 Reagent was added directly to cells in 96-well plates and incubated at room temperature for 30 minutes before recording luminescence (RLU). Each point represents the average of 3 replicates (error bars represent ±SEM).

CellTitre-Glo (Cell Viability)

LDH (Cytotoxicity)

A549 cells were treated with drug compounds at 8 different concentrations. Cell Viability and cytotoxic responses were assessed using the CellTitre-Glo and LDH assays. Staurosporine (positive control compound) induced a significant reduction in cell viability when compared to untreated or vehicle control (***p<0.001; ±SEM).

Senescence

It is a potential mechanism for a cell to avoid malignant transformation. However, senescence can also promote cancer development by altering the cellular microenvironment through a senescence-associated secretory phenotype (SASP). At least, three types of cellular stress such as activation of oncogenes, loss of tumor suppressor genes, and chemo/radiotherapy can induce cell senescence (Zeng et al., 2018).

Senescence Assay

Primary human chondrocytes were treated with H2O2 for 24 hours followed by a 72 hours recovery period and then treated with ABT 263 (a known senolytic). Cells were fixed followed by immunofluorescence staining for DAPI and β-galactosidase.

PARP Assays

Poly (ADP-ribose) polymerases (PARPs) are a family of enzymes present in all somatic cells that help damaged cells repair themselves. PARPs are also involved in the transcriptional regulation of several signalling pathways, including genes involved in inflammation. PARPs, and particularly the tankyrases have become important cancer drug targets. Scientific evidence suggests that PARP is overexpressed in lung, ovarian, and breast cancer, and PARP inhibitors are showing dramatic therapeutic potential for breast and ovarian cancer treatments.

PARP Assay

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 an in vitro cell free enzymatic assay. Proliferation was assessed by BrdU incorporation (n=3±SEM).

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.

3D spheroid models

MDA-MB436 cells cultured in 3D cell culture media were allowed to form spheroids 5 days prior to Talazoparib and Bleomycin drug treatment. Following the drug treatment, spheroids were stained with DEAD Red and SYTO Green fluorescent dye every 72 hours. Spheroid growth and morphology were assessed using JuLITM Stage Automated cell imaging system to determine the effect of drug treatments. Combination of Talazoparib and Bleomycin resulted in an increase in cell death and an observable loss of spheroids.

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