This study proposes an approach to cancer therapy by utilizing the regenerative properties of the aquatic flatworm Schmidtea Mediterannea and the targeting properties of protein-activatable photosensitizers in photodynamic therapy (PDT). The primary goal was to develop an in-vitro model that simulated the process of cancer metastasis by inducing hyperproliferation in planarian regenerative cells (neoblasts), and as a result, tissue, by silencing the planarian homolog for the stem-cell-regulating gene smed-p53. Then, an activatable photosensitizer would be developed such that reactive oxygen species would be selectively released into the “tumor” environment once exposed to the hyperproliferating cells. The natural rate of planarian regeneration after fragmentation was measured as a means to collect preliminary data, which would be used as a negative control in comparison with the later collected data on hyperproliferated regeneration. Results from the preliminary data collection yielded consistent patterns of regeneration for all planarian fragments (head and tail) across all planarian populations. Hyperproliferation of planarian stem cells was achieved through the overexpression of the Epidermal Growth Factor Receptor (EGFR) gene, which was a direct result of the silencing of smed-p53. The photosensitizer was developed as two separate components conjugated by a chemical linker. Chlorin e6 was chosen as the photosensitizer for the photosensitizing component, and GE11 peptide was chosen as the targeting component that would activate the photosensitizer once exposed to the overexpressed EGFR. Staining markers were added to the photosensitizer conjugate as part of a proliferation assay that determined the efficacy of the conjugate’s targeting capacity as well as the photodynamic therapy in general.
How will an activatable photosensitizer affect planaria neoblast hyperproliferation during photodynamic therapy?
The use of a protein-activated photosensitizer will result in reduced neoblast proliferation and reduced tissue regeneration as a result.
Cancer is a highly pressing public health hazard and one of the leading causes of death in the United States, accounting for over 600,000 deaths each year, according to the American Cancer Society. Cancer treatment, which encompasses a variety of processes including surgery, chemotherapy, radiation therapy, and thermotherapy, pose significant limitations despite their benefits. Breast, Ovarian, Uterine, Vaginal, and Vulvar Cancer Care in Low- and Middle-Income Countries: Prevalence, Screening, Treatment, Palliative Care, and Human Resources Training estimates that over 80% of cancer patients will require surgery, some several times, but less than 20% of them actually receive affordable and timely surgery due to a lack of medical professionals as well as knowledge of the cancer itself. Since very few early-stage symptoms of cancer are effectively detected, surgery treatment very rarely becomes completely successful. Metastasis, in the context of cancer, is defined as the process by which cancer cells spread from a primary tumor to form new tumors in other parts of the body and remains a significant challenge to current oncological therapies. Chemotherapy, radiation therapy, and thermotherapy, despite being popular modes of therapy, amongst their benefits, pose the primary limitation of being generally non-targeting, meaning that they act broadly and have a great potential to harm healthy, non-diving cells along with tumor cells. It is of utmost importance then, that more tumor-targeting forms of therapy are developed in order to minimize the side-effects that come with harming healthy cells. Oftentimes, due to the highly resistive genetic characteristics of metastases, patients become unresponsive to existing therapies. It is also often the case that once a tumor is metastasized, each additionally grown tumor will have a unique tumor microenvironment that responds differently to treatment. It then becomes imperative that models with a further understanding of the mechanisms of metastases are developed in order to minimize side-effects while maximizing efficacy and tolerability. While traditional mammalian models for metastasis research exist, they are often resource-intensive and constrained by ethical considerations, which further necessitates the need for alternative systems. Photodynamic therapy (PDT) is a form of cancer treatment employing photosensitizers and near-infrared light (NIR) exposure to cause apoptotic cell death in tumor cells. Once administered, photosensitizers, which are light-absorbing chemical agents, must collect around the target tissue before being exposed to activation-specific wavelengths of NIR. The exposure to NIR allows for the release of cytotoxic reactive oxygen species (ROS) from the photosensitizer, which then, as a result, induces selective apoptotic cell death in the tumor tissue. Although a variety of different animal models and in vitro systems have been utilized to understand metastasis and regeneration, planaria have emerged to the forefront as a species with incredible limb-regenerating abilities and unique capacities for chemically induced carcinogenesis. Planaria, characterized by their three-branched intestines, are a group of freshwater flatworms whose regenerative abilities come primarily from a population of stem cells called neoblasts. Neoblasts share behavioral similarities with cancerous cells, which include rapid proliferation, where they divide rapidly to produce progenitor cells that split off into various cell types, migration to injury or fragmentation sites, and a resistance to various types of stress, which can include but are not limited to environmental stress and apoptosis. Planaria are also physically transparent, which makes them ideal for real-time imaging and visual biological analyses. This, along with the fact that planaria are known elicit measurable responses from light radiation, make them a highly optimal model for understanding the cellular dynamics and mechanisms of metastasis, and the potential efficacy of drug therapy. Previous research on planaria as cancer models have looked into the impact of molecular signaling pathways, some of which have behavioral parallels with those involved in cancer formation in humans, on planarian regeneration. A study focusing on the β-Catenin-dependent Wnt pathway yielded results that showed a direct correlation between the pathway and anterior-posterior polarity in planaria, as well as head-tail regeneration (Sureda-Gomez et. al, 2016). Since the pathway is also implicated in cancer cell proliferation and migration, the study placed emphasis on utilizing planaria pathway models as a means to imitate cancer progression in humans. Another study utilizing planaria regeneration as a model for cancer focused on inducing carcinogenesis through a cadmium chloride agent in order to understand the function of tumor suppressor genes at play. The researchers found that Smed-MmpB in particular, a protein displaying tumor-suppressing properties, inhibited regeneration of cancerous stem cells in the planaria, and also found correlative inhibitive behaviors between similar tumor-suppressing proteins and cancerous cell division in humans. The figure below shows that after exposure to the cadmium chloride, planarian fragments with the added Smed-MmpB did not experience any phenotypic changes inconsistent with natural outgrowths. There has not been much prior research This study aims to use planaria as a novel in-vitro model to simulate metastatic behavior and determine the efficacy of photodynamic therapy in a controlled and measured system. The regenerative aspect of planaria will be incorporated alongside activated PDT by using regenerative fragmentation as a representation of cancer metastasis and observing neoblast proliferation and survival rate while in the regeneration process. An activatable photosensitizer will be synthesized such that it binds strictly to EGFR-overexpression by conjugating the photosensitizer Chlorin e6 to the EGFR-binding peptide GE11. The efficacy of photodynamic therapy will be evaluated by looking at how photodynamic therapy impacts regenerating tissue and employs a targeted approach to inducing cell death by selectively releasing ROS into EGFR-overexpressing, hyperproliferating cells while maintaining the health of non-dividing cells. The targeting ability of the PDT will be controlled by assessing a variety of variables, including photosensitizer concentration, light intensity, and NIR exposure duration. Although the enhancement of photodynamic therapy has been studied previously, this is a novel meta-analytical approach that effectively models metastasis in planaria while incorporating key photodynamic therapy aspects to explore the relationship between regeneration and cancer, as well as ways to optimize and maximize efficacy and tolerability in cancer therapy overall.