Targeting and killing cencer cells in vitro with near-infrared light

Boer, E. de (Esther) (2013) Targeting and killing cencer cells in vitro with near-infrared light. thesis, Medicine.

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Abstract

Introduction: Peritoneal carcinomatosis (PC) of colorectal origin is associated with a poor prognosis. Surgical cytoreduction is pivotal and has shown to positively influence both the effect of chemotherapy and overall prognosis. A novel technique that may be beneficial in the treatment of PC is photo-immuntherapy (PIT). PIT allows for selective destruction of cancer cells. A tumor-selective monoclonal antibody (mAb) is conjugated to a fluorescent photosensitizer that can be activated by light in the near infrared (NIR) range. After activation, reactive oxygen species (ROS) are produced by the target-bound photosensitizer, leading to selective cell death. In this research project, the potential of PIT for the detection and subsequent killing of cancer cells was evaluated in vitro. PIT is expected to improve the prognosis of colorectal cancer (CRC) patients in the future through; I) more sensitive and reliable intraoperative tumor detection using fluorescence optical imaging, thus leading to a more complete surgical tumor elimination; and II) elimination of microscopic residual disease using the phototoxic properties of the fluorescent photosensitizer IRDye700DX. Methods: A target-specific photosensitizer based on the phthalocyanine dye IRDye700DX conjugated to the mAb rituximab (RTX) was constructed. Labeling efficiency, binding specificity and immunoreactivity of the RTX-IRDye700DX conjugate were determined. Cytotoxicity studies of PIT targeting the CD20-positive Ramos lymphoma cells with the RTX-IRDye700DX conjugate were performed. In order to test whether PIT was target-specific, the binding characteristics of the RTX-IRDye700DX conjugate were analyzed in a predefined cell mixture of CD20-positive Ramos and CD20-negative Jurkat lymphoma cells. The cell mixtures were incubated with the RTX-IRDye700DX conjugate, irradiated with NIR light, and subsequently the amount and specificity of cell death were analyzed with a DEAD/LIVE cell viability assay. In an additional experiment, the immunoreactivity and phototoxicity of the cetuximab (CTX) -IRDye700DX conjugate was tested in a head and neck cancer cell line and in a colorectal carcinoma cell line. Results: Labeling efficiencies, binding specificity and immunoreactivity were optimal for the RTX-IRDye700DX conjugate in Ramos cells. Fluorescent microscopy confirmed the presence of the RTX-IRDye700DX on the cellular membrane. Subsequent light from the fluorescent microscope induced immediate cellular swelling and blebbing, which are both indicators of necrotic cell death. Phototoxicity was induced only when the RTX-IRDye700DX conjugate was bound to the target antigen; no additional cytotoxicity was seen in unbound conjugate surrounding the tumor cells. In a predefined cell mixture, the RTX-IRDye700DX conjugate was distributed in a CD20-specific manner and showed immediate cytotoxicity in the CD20-positive cells, whereas CD20-negative cells showed no phototoxicity or cell death after irradiation with NIR-light. Also, the CTX-IRDye700DX conjugate proved to be capable of inducing tumor-specific cell death in the two additional cell lines. Conclusion: We conclude that PIT can be used to target and kill cancer cells in vitro. The phototoxic activity of the RTX-IRDye700DX conjugate may allow for an enhanced elimination of refractory tumor cells, and thus may lead to a better prognosis in patients with PC of CRC due to a more complete cytoreduction. In the near future, the fluorescence properties of tumor-targeted PIT may allow for real-time feedback regarding tumor localization and extent during surgery. Additional experiments in mouse CRC models will provide the in vivo efficacy towards clinical translation of PIT.

Item Type:	Thesis (Thesis)
Supervisor name:	Dam, Dr. G.M. van and Crane, Dr. L.M.A. and Surgical Oncological Research Laboratory, UMC Groningen and
Faculty:	Medical Sciences
Date Deposited:	25 Jun 2020 10:59
Last Modified:	25 Jun 2020 10:59
URI:	https://umcg.studenttheses.ub.rug.nl/id/eprint/1977

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