Background

Photodynamic therapy (PDT) is an FDA-approved light-based treatment modality that utilizes a photoactivatable molecule, termed a photosensitizer (PS). Upon irradiation with an appropriate wavelength of light, the PS generates reactive molecular species (RMS) within the cytosol. These RMS, often reactive oxygen species, induce photodamage and are toxic to the cell (Celli et al., 2010; Obaid et al., 2016). Certain formulations of PS preferentially accumulate in the malignant tissue and thus PDT often achieves dual selectivity as a result of confined PS accumulation in the tumor and its spatiotemporal control over the light irradiation. PDT is clinically approved for several indications, including esophageal cancer, lung cancer, age-related macular degeneration, and is being investigated for many more cancerous and noncancerous indications (Celli et al., 2010).

Ovarian cancer is a deadly gynecologic malignancy often associated with treatment-resistant metastases and poor outcomes in patients. While surgery and chemotherapy are the current standard of care for ovarian cancer, the efficacy of PDT to manage ovarian cancer is currently being investigated in pre-clinical studies by many research groups (Goff et al., 1996; Rizvi et al., 2010).

Like many other malignancies, ovarian cancer metastases display complex morphological and biochemical characteristics that are lost in traditional monolayer (2D) cell culture. 3D tumor model is an emerging technique for evaluating tumor properties and treatment response in vitro. 3D cultures of tumor cells create a more physiologically relevant tumor microenvironment than traditional 2D cell culture because they retain the biological and structural features of tumors that are found in vivo (Rizvi et al., 2016). Cells in monolayer experience contact inhibition and a homogeneous oxygen and nutrient environment, while 3D cultures display heterogeneity within and across spheroids and restore extracellular matrix-related cues that are important in treatment response. Like in vivo tumors, 3D cultured tumors proliferate into heterogeneous clusters with varying levels of oxygen and nutrients, making them useful tools for studying PS uptake and PDT efficacy within oxygen gradients. Increased PDT resistance has been found in 3D tumor models compared with 2D, providing a platform for testing alternative treatment regimens and combination therapies to overcome this resistance. The complex factors that contribute to treatment failure in patients necessitate an in vitro model that mimics the structural, biological, and extracellular cues that play a major role in treatment outcomes (Rizvi et al., 2016).

Here, we describe an in vitro PDT protocol using a 3D model of ovarian cancer. 3D nodules of human ovarian cancer cells (NIH: Ovcar5) that stably express a fluorescent reporter were grown on beds of Growth Factor-Reduced Matrigel^®. PDT was performed using FDA-approved PS verteporfin (Visudyne^®). The treatment response was evaluated by imaging of the fluorescent 3D nodules. The protocol described here has been adapted from a previously published article (Rizvi et al., 2019).