Cancer Biology

Photodynamic therapy (PDT), is a clinically-approved light-based anti-cancer treatment modality in which a photoactivatable photosensitizer is irradiated with an appropriate wavelength of light to generate cytotoxic molecules to kill cancer cells. In this article, we describe an in vitro PDT protocol using a 3-dimensional (3D) model of ovarian cancer that was established on beds of Matrigel. PDT was performed using a liposomal formulation of verteporfin photosensitizer (Visudyne^®). The cancer cells were genetically-labeled with the fluorescent protein mCherry to facilitate the evaluation of the treatment response. This protocol is advantageous because the mCherry fluorescence is an indicator of cell viability, eliminating the need for external dyes, which often exhibit limited penetration and diffusion into 3D organoids. Additionally, Visudyne PDT achieves significant tumor-killing efficacy in a 3D model for ovarian cancer.

The expression of genes is frequently manipulated in cell lines to study their cellular functions. The use of exogenous small Interfering RNAs (siRNAs) is a very efficient technique to temporarily downregulate the expression of genes of interest [reviewed by Hannon and Rossi (2004)]. A genome-wide siRNA library allows the user to study both the effect of each individual gene on a particular cell phenotype in a high throughput manner and also assess its phenotypic effect relative to all other genes targeted. Several factors that potentially influence the outcome of a screen need to be considered when performing a large siRNA screen (Jiang et al., 2011). Here we present a detailed protocol for a genome-wide screen to identify genes involved in anti-cancer drug resistance using the human siGENOME library from Dharmacon. In this protocol, we focus on resistance to treatment with the Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor (EGFR-TKI) erlotinib in the lung cancer cell line PC9, which is exquisitely sensitive to EGFR-TKIs (de Bruin et al., 2014). This protocol can be used for other cell lines and other drug treatments, as we expand in the Notes below.

Thymidine Kinase from human Herpes simplex virus type 1 (HSV1-TK) in combination with specific substrate prodrug nucleotide analogue ganciclovir (GCV) has been widely used as suicidal therapeutic gene for cancer gene therapy. HSV1, and its mutant (HSV1-sr39TK) with improved substrate specificity, were used as reporter genes for PET-imaging of various biological functions in small animals, by combining with radiolabeled substrates such as ¹⁸F-FHBG and ¹²⁴I-FIAU. ³H-Penciclovir (PCV) uptake assay is a method of choice used to determine the expression level of HSV1-TK in mammalian cells and tissues. HSV1-TK phosphorylate PCV and result in the formation of penciclovir monophosphate, and its subsequent phopsphorylation by cellular TK lead to the formation of penciclovir triphosphate, which is trapped selectively in cells express HSV-TK. ³H-Penciclovir enables the detection of penciclovir uptake of mammalian cells and tissues by radioactive procedures such as scintillation counting. Here we describe the protocol to carry out ³H-Penciclovir uptakes in mammalian cells.