Introduction:

Glioblastomas are a type of human brain cancer that are usually aggressive and deadly. Even with early detection and advanced treatments, patients only live for about 14 months on average. The glioblastoma cells spread into nearby brain tissue, making it impossible to remove the whole tumor by surgery, which leads to almost certain tumor recurrence for glioblastoma patients. Many techniques have been tried to improve the identification of tumor edges. Preoperative MRI is often used to guide stereotactic surgery. But these methods have problems with low spatial resolution and differences between the preoperative MRI-defined borders and the real tumor edges during surgery due to brain shift. Surgeons who want to remove more glioblastoma face several difficulties, such as irregular and unclear tumor boundaries and tumor growth close to or within vital neurological structures. Prostate-specific membrane antigen (PSMA), a type 2 transmembrane glycoprotein receptor, has become an important target of interest. It is expressed on neovasculature in various solid tumors. Immunohistochemistry studies in clinical settings have shown that in glioblastomas, PSMA is expressed on neovasculature in 31% to 100% of cases, while it is missing in vasculature within normal brain regions. High levels of PSMA expression on neovasculature in glioblastomas have been linked with increased angiogenesis and worse prognosis. Also, PSMA expression has been found, though to a smaller degree, on tumor cells themselves; however, these levels did not relate to patient survival outcomes. It has been shown that the blood-brain barrier (BBB) blocks up to 95% of targeting agents from entering the brain, mainly allowing only hydrophobic and small molecules to pass through. Despite partial disruption during glioblastoma multiforme (GBM) development, the blood-tumor barrier remains a major obstacle, limiting optimal drug accumulation within brain tumors. In this study, we targeted glioblastomas expressing human prostate-specific membrane antigen (hPSMA) using synthesized small molecules PSMA1-Pc413, photodynamic therapy (PDT) only, and PSMA1-MMAE-Pc413, PDT and MMAE chemotherapy, using an in vivo orthotopic xenograft model. Our results confirm the effectiveness of these molecules in targeting hPSMA in vivo, successfully crossing the BTB.

Methodology:

We tested the targeting of small molecules in vitro and in vivo. For in vitro studies cells were administered either agent, kept in the dark or irradiated with light. For in vivo, we implanted orthotopic glioblastoma tumors in mice models. We used Gli36Δ5 EGFR cells, which do not express PSMA, as a control and Gli36Δ5 EGFR (hPSMA), which were transformed with human PSMA, as the test tumor. We injected PSMA1-Pc413 intravenously and imaged the fluorescence of the tumors with a Maestro imaging system. We also used the PSMA1-MMAE-Pc413 probe for intraoperative fluorescence imaging with a Curadel fluorescence image guided surgery camera.

Results:

Both probes showed PSMA specific uptake and inhibition in vitro. In vivo, we saw strong fluorescence of PSMA1-Pc413 in ex vivo Gli36Δ5 EGFR (hPSMA) tumor, which crossed BBB and targeted hPSMA tumor cells. The tumor margin was clearly visible. The curadel images showed the feasibility of PSMA1-MMAE-Pc413 probe for surgical guidance.

Conclusion:

Two imaging probes, one that provides PDT only and one that provides PDT and chemotherapy, were tested for in vivo use in mouse models of glioblastoma. Both PSMA-Pc413 and PSMA-Pc413-MMAE appear to cross the BBB/BTB and effectively target brain tumors. The fluorescence imaging feature of these probes also makes them candidates to improve intraoperative navigation during surgery.

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