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Description
The aim of the project is to develop a universal molecular probe for the PD-L1 protein (programmed death receptor -1 ligand), which will facilitate the diagnosis of various types of cancer. PD-L1 is a protein present on the surface of many cancer cells, allowing them to bypass the natural defense system of the immune system. One of the components of the immune system are T lymphocytes, which recognize and attack cancer cells. These cells have structures called receptors on their outer surfaces, which act as keys to lock onto the molecules of attacking organisms. This molecular recognition is a major component of the immune response. One of the elements of this mechanism are so-called "checkpoints", which prevent T cells from attacking normal cells. A key part of this mechanism is the PD-L1 / PD-1 system (programmed death protein -1). PD-L1 on normal cells recognizes and attaches to PD-1 on T cells, preventing them from attacking healthy cells. Unfortunately, some cancers have learned to produce large amounts of PD-L1 in order to trick the immune system into avoiding detection. Hence, the designed probes that target PD-L1 binding will be able to detect and locate neoplastic cells at a very early stage of the disease. These probes can be used in early diagnosis, increasing the chances of detecting the disease and starting treatment.
Summary of project results
Nanotechnology is the design and assembly of submicroscopic devices called nanoparticles. Its application in medicine can bring significant improvements in the diagnosis of diseases. Cancer diseases are one of the main causes of the high mortality rate, which is related to the late detection of neoplastic changes and the low effectiveness of standard treatments. Biomedical imaging, one of the key pillars of modern cancer diagnosis and treatment, is tightly integrated into clinical decision making. Early diagnosis is known to be a major factor in reducing mortality, treatment costs and hospital stays. Imaging probes are a key component of molecular imaging and must offer high sensitivity, low background noise, low toxicity and relative stability. Application of nanoparticles in molecular imaging techniques enables earlier detection and treatment of tumors by increasing sensitivity of signals and targeting of contrast agents directly into cancer cells. A number of studies have shown that nanoparticles known as aptamers are among the best imaging tools due to their high stability, target specificity and affinity. The aim of the current project is to the development of an aptamer-based molecular probe with a single-stranded DNA structure capable of specifically binding human PD-L1 (Programmed death-ligand 1). The development of a universal probe would create the possibility of imaging various types of tumors depending on their ability to overexpress PD-L1, and would provide a tool / finished product as a probe for future diagnostic and even therapeutic clinical trials.
In these studies, it has been demonstrated the development of a single-stranded aptamer-based molecular probe specifically recognizing human PD-L1. Target-engaging aptamers were selected by iterative enrichment from a random ssDNA pool, and binding was biochemically characterized. We demonstrated specificity and dose dependence in vitro in cell culture using human renal cell carcinoma cells (786-0), human melanoma cells (WM115 and WM266.4), and human LN18 glioblastoma carcinoma cells. We demonstrated the in vivo utility of the probe using two murine tumor models, where we showed that the probe showed excellent imaging potential. We postulate that further development of the probe may allow for universal imaging of various types of tumors depending on their PD-L1 status, which may be used in cancer diagnostics.
As part of the work at WP1, a single-stranded DNA (ssDNA) sequence was selected as an aptamer-based probe that specifically recognizes human PD-L1, which is present in many types of cancer cells. The development of the probe included several stages: selection using the SELEX method, bioinformatics analysis, and aptamer binding tests to PD-L1. In the next stage of WP2 research, the specificity of the selected fluorescently labeled aptamer as well as the dose dependence were examined using in vitro tests. First, the usefulness of the selected aptamer for the detection of PD-L1 in the context of a living cell was assessed using cell lines overexpressing the target protein (kidney cancer, melanoma, glioma) and the kinetics of binding of the aptamer to PD-L1 on the cell surface was assessed. The obtained results showed that the selected aptamer specifically recognizes PD-L1 expressed on the cell surface. The resulting partial epitope masking was only observed after ectopic overexpression of the human antigen in a murine cell line and was unrelated to physiological expression in the cancer cell lines examined. In the next stage of WP3 research, the functionality of the aptamer was checked in in vivo models. First, the use of the aptamer as a diagnostic probe in ex vivo tumor imaging was tested. For this purpose, cells without or with overexpression of PD-L1 were implanted subcutaneously in mice, and after tumor development, histochemical diagnosis of the collected tumor was performed, which showed that the aptamer allows the selective detection of PD-L1 in tumor tissue. Additionally, before tumor removal, mice were intravenously administered a selected fluorescently labeled aptamer and a spot signal measurement was performed within the tumor, which also confirmed the specificity of the aptamer. To determine whether physiological expression of PD-L1 in tumor tissue is sufficient for aptamer monitoring, animals were implanted with luciferase-labeled renal tumor cells directly into the renal capsule (orthotopic model), inducing tumor growth. Luciferase made it possible to track the growth and localize the tumor by measuring luminescence. Intraperitoneal administration of the Cy5.5-labeled aptamer showed a strong correlation between tumor location (luminescence) and the signal from the PD-L1-specific aptamer (fluorescence), indicating that the aptamer is capable of detecting PD-L1-expressing tumors in vivo. The research results showed that the selected aptamer can be considered as a new diagnostic probe in cancer detection and therapy.