(H-283) Real-time Near-infrared Fluorescence Guided Protoporphyrin IX Imaging and Photodynamic Therapy for the Treatment of Glioblastoma

Introduction:: Glioblastoma (GBM) is a highly aggressive cancer that accounts for more than 49% of primary malignant brain tumors. Despite advances in brain cancer treatments, the 5-year relative survival rate for GBM patients has remained less than 6% for decades. Tumor removal surgery with the maximal outcome is considered the most effective treatment to reduce the cancer recurrence rate and extend patients’ survival. However, incomplete tumor resection is unavoidable due to the migration and infiltration of glioma cells that extend into surrounding healthy tissue. Fluorescence-guided surgery (FGS) is a prevalent technique employed in various clinical applications that hold a promise to enhance the intraoperative visualization of tumors and enable more resections with high accuracy. The United States Food and Drug Administration (FDA) approved 5-aminolevulinic acid (5-ALA) serving as a precursor of protoporphyrin IX (PpIX) for high-grade GBM treatment. PpIX is a natural fluorophore that emits visible red fluorescence for FGS using violet excitation light (405 nm) and also functions as a photosensitizer that generates reactive oxygen species (ROS) to kill cancer cells through red light (625 nm) photodynamic therapy (PDT). While the combination of 5-ALA-induced PpIX FGS and PDT is expected to eradicate all tumors, its efficacy is limited to superficial lesions due to the shallow light penetration, invisible small satellite tumors, and high autofluorescence contamination in fluorescence visualization. We introduce LS301, a novel FDA-approved near-infrared (NIR) contrast agent targeting to solid tumors, to guide PpIX treatments to overcome those shortcomings and to treat deep tumors.

Materials and Methods:: We first confirmed the co-localization of LS301 and PpIX in delayed brain tumors (DBT) and human brain glioblastoma (U87-MG) in vitro and in vivo studies. We then evaluated the LS301 effect on ALA-PpIX PDT treatment in BalB/C mice bearing subcutaneous DBT tumors. These mice's PpIX and LS301 fluorescence images were acquired on an IVIS® Spectrum CT In Vivo Imaging System using their respective preferred excitation light and emission filter pairs. To further validate the efficacy of the LS301-guided ALA-PpIX treatment approach, we conducted a series of experiments that simulated intraoperative procedures on BalB/C mice with subcutaneous DBT tumors. For this experiment, we built a portable real-time imaging device on the Raspberry Pi platform with an Arducam 12.3MP IMX 477 HQ camera, which is capable of capturing PpIX and LS301 fluorescence images under various combinations of excitation lights and emission filters. Thirty-six Balb/C mice with DBT tumors received LS301 (I.V. injection) and 5-ALA (I.P. injection) 24 hours and 4 hours before surgery, respectively. After surgically removing 80% of the tumors from all 36 mice individually, these mice were segregated into three groups based on their respective treatment for residual tumors that were not detectable through PpIX imaging: a control group in which residual tumors were left untreated, PpIX-PDT treatment for LS301-riched residual tumors, and further resection of LS301-detected residual tumors. Tumor growth was identified by using Bioluminescence imaging on the IVIS system.

Results, Conclusions, and Discussions:: Our preliminary results indicate that LS301 co-localizes with PpIX in DBT and U87-MG in vitro and in vivo studies. We subsequently confirmed that the LS301 has no adverse events on ALA-PpIX FGS and ALA-PpIX PDT treatment in DBT tumor-bearing BALB/c mice. Notably, our results suggest that LS301 may be a promising photosensitizer that can improve the treatment outcomes in ALA-PpIX PDT treatment groups. Regarding the simulated intraoperative experiments, we observed a rapid tumor relapse within one week in the control group that underwent incomplete tumor removal surgery. In contrast, the two treatment groups guided by LS301 imaging exhibited a gradual and slow rate of tumor regrowth. Furthermore, the cell-free analysis of LS301 and PpIX demonstrated varying types of ROS production, which has motivated us to investigate the mechanism of LS301-PDT further.

Incorporating LS301 into the ALA-PpIX treatment is a promising strategy for detecting residual rumors and administering precise PDT treatment. Our portable real-time imaging system efficiently improves the LS301-guided ALA-PpIX treatment, which can be an alternative option for other types of cancer surgery.


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