1 Investigating combination of photodynamic therapy and AXL inhibition for improved treatment outcomes of glioblastoma Nada Fadul1,2,3, Louise Mitchelmore1,2,3,4, Farah Farrag1,2,3, Anna Shaw1,2,3, Dilan Gangar1,2,5, Jennifer Yeon1,2,3, Sumiao Pang1,5, Rebecca Hays1,5, Huang-Chiao Huang1,2,5* 1University of Maryland, College Park, College Park, MD 20742, USA. 2Gemstone Honors College, University of Maryland, College Park, MD 20742, USA. 3College of Computer, Mathematical, and Natural Sciences 4College of Behavioral and Social Sciences 5Fischell Department of Bioengineering *Corresponding Author: Huang-Chiao Huang (hchuang@umd.edu). 8278 Paint Branch Drive, College Park, MD 20742. Phone: 301-405-6961 Thesis submitted in partial fulfillment of the requirements of the Gemstone Honors Program, University of Maryland, 2025. Advisory Committee: Dr. Huang-Chiao Huang Dr. Carla Arnau Del Valle Dr. Anthony Kim Dr. Allison Lansverk Dr. David Lovell Dr. Sumiao Pang 2 © Copyright by Team MetaBio 2025 3 Abstract Glioblastoma multiforme (GBM) is notorious for its aggressive behavior, brain invasion, and poor prognosis, with a median survival of less than 18 months. Limited treatment options contribute to GBM’s status as one of the deadliest cancers. The intricate tumor composition and invasion of vital brain areas render it resistant to standard therapies, necessitating novel approaches to extend post-diagnosis survival. Intraoperative photodynamic therapy (PDT) has emerged as a promising technique, involving the administration of a photosensitizer before surgery and red light application afterward to target residual tumor cells. Recently, an excipient-free nanoparticle formulation of verteporfin (NanoVP) photosensitizer was developed for PDT of GBM, demonstrating superior efficacy in reducing tumor burden and extending animal survival compared to existing photosensitizers. We explored the combined effects of NanoVP-PDT and clinically promising AXL inhibitors on GBM cells. Phospho-AXL, which is highly expressed in GBM tumors and correlates with shorter overall patient survival, represents a compelling therapeutic target for small-molecule inhibition. In this study, we investigated the anti-GBM effects of combining NanoVP-PDT with AXL inhibitors in vitro as a new treatment approach to combat GBM. Keywords: Fluorescence-guided intervention, nanotechnology, photodynamic therapy, chemotherapy, glioblastoma multiforme, AXL-RTK inhibition, brain cancer. 4 Acknowledgements We would like to thank our incredible mentor, Dr. Huang Chiao Huang, for his unwavering support, expert guidance, and dedication to our research. We also extend our gratitude to the members of Dr. Huang’s Optical Therapeutics and Nanotechnology Laboratory, especially Dr. Sumiao Pang and Ms. Rebecca Hays, for their valuable contributions and support of our work. Thank you to our thesis discussants, Dr. Anthony Kim, Dr. Carla Arnau Del Valle, and Dr. Sumiao Pang, for their insightful critique and expertise. We would also like to thank all of our LaunchUMD donors for their generosity, whose contributions made this research possible, as well as our team librarian, Ms. Nedelina Tchangalova, for her assistance. Finally, we would like to thank Dr. David Lovell, Dr. Allison Lansverk, and the entire Gemstone Honors Program for their leadership and support. 5 Contributions HH and NF conceived the project and oversaw the overall direction and planning. HH, NF, and LM conceived and planned the experiments. NF, LM, FF, AS, DG, and JY performed experiments. NF, LM, FF, and HH performed data analysis. NF, LM, FF, and HH contributed to statistical analysis of the data. LM created PDT and Method diagrams. NF, LM, FF, AS, DG, and JY prepared the manuscript. SP, RH, and HH provided critical feedback and helped shape the research, analysis, and manuscript. NF and LM contributed to editing the final manuscript. 6 Table of Contents Abstract……………………………………………………………………………………………3 Acknowledgements……………………………………………………………………………..…4 Contributions……………………………………………………………………………….……..5 Table of Contents………………………………………………………………………………….6 1. Introduction…………………………………………………………………………………..…9 2. Literature Review…………………………………………………………………………...…13 2.1 Glioblastoma Multiforme (GBM)……………………………………………………13 2.2 GBM Standard of Care & Treatment Challenges……………………………………15 2.2.1 Current Standard of Care……………………………………………..……15 2.2.2 Surgical Resection…………………………………………………………15 2.2.3 TMZ Chemotherapy and Radiotherapy……………..…………..…………18 2.3 Chemotherapy with Bemcentinib……………………………………………………21 2.3.1 Targeting Receptor Tyrosine Kinases (RTKs)………………..……………21 2.3.2 RTK-AXL Function and Mechanism………………………………………22 2.3.3 RTK-AXK Expression and Prevalence in GBM Tumors…………….……25 2.3.4 Bemcentinib………………………………………………………..………27 2.4 Photodynamic Therapy………………………………………………………………30 2.4.1 Overview of Photodynamic Therapy (PDT)…………….…………………30 2.4.2 Mechanism…………………………………………………………………31 2.4.3 Photosensitizers………………………………………………………….…33 2.4.4 Limitations…………………………………………………………………34 2.4.5 Current Uses/Combinatorial Approaches with PDT…………………….…36 7 3. Methods……………………………………………………………………………….………39 3.1 Cell Culture………………………………………………………..…………………39 3.2 NanoVP Synthesis and Characterization……………………………….……………39 3.3 Photodynamic Therapy (PDT) Monotherapy Treatment in Cell Culture……………40 3.4 Bemcentinib Preparation………………………………………………………….…40 3.5 Bemcentinib Monotherapy Treatment in Cell Culture………………………………41 3.6 Preliminary Combination Treatment (PDT + BEM) in Cell Culture……….……..…41 3.7 Changing Light and BEM Combination Treatments (PDT + BEM) in Cell Culture..42 3.8 MTT Assay…………………………………………………………………….….…44 3.9 Statistical Analysis………………………………………………………………...…45 3.10 Evaluation of Synergism Using Combination Index (CI) Analysis……………...…46 4. Results……………………………………………………………………………………...….48 4.1 Synthesis and Physicochemical Characterization of Excipient-Free NanoVP Formulation…………………………………………………………………………...….48 4.1.1 Nanoparticle Size, Polydispersity Index (PdI), and Zeta Potential (ZP) Analysis….………………………………………………………..…………...…48 4.1.2 Optical Properties of NanoVP: Absorbance and Fluorescence in DPBS vs. DMSO……………………………………………………………………………49 4.2 Photodynamic Killing of Glioblastoma Cells In Vitro………………………………50 4.3 AXL Inhibition of Glioblastoma Cells In Vitro………………..……………….……51 4.4 Combination Treatment with PDT and AXL Inhibition Enhances Cytotoxicity…….52 4.4.1 0.78 µM Bemcentinib…………………………………………..……….…52 4.4.2 1.56 µM Bemcentinib…………………………………………………...…53 8 4.4.3 2 µM & 2.5 µM Bemcentinib…………………………………………...…54 4.5 Combination Index (CI) Analysis Confirms Synergistic, Additive, and Antagonistic Effects Within Conditions Screened…………………………………………………..…55 5. Discussion and Conclusions………………………………………………………….…….…56 6. Future Directions…………………………………………………………………….……..…61 7. Equity-Impact Statement……………………………………………………………………...62 8. References…………………………………………………………………………….………65 9. Appendices……………………………………………………………………….……………90 9.1 Appendix A………………………………………………………………………..…90 9.2 Appendix B………………………………………………………………………..…91 9 1. Introduction Glioblastoma multiforme (GBM), a notoriously aggressive and fatal brain cancer, presents formidable treatment challenges due to its invasive and infiltrative growth, incomplete surgical resection in or near eloquent regions of the brain, and resistance to chemoradiation (Yalamarty et al., 2023). Classified by the World Health Organization (WHO) as a grade IV astrocytoma, GBM is associated with a poor prognosis, with a median survival 15–18 months post-treatment (Bulbeck et al., 2024; Louis et al., 2021). An analysis of the adult glioma registry from 2000 to 2014 revealed a 5-year relative survival rate of around 5% after chemoradiation, a statistic that has seen little improvement over the past decade (Ostrom et al., 2018; Stupp et al., 2009; Yalamarty et al., 2023). Despite efforts to improve treatment outcomes for the most commonly occurring malignant brain tumor in the United States, GBM remains virtually incurable (Yalamarty et al., 2023). Today, the standard treatment for GBM includes maximal safe resection of the primary or secondary tumor in low-risk regions of the brain, followed by a targeted combination of radiotherapy and adjuvant temozolomide (TMZ) chemotherapy. Although advanced intraoperative image-guided techniques have increased the rate of maximal safe resection to 78.6% in GBM patients, the challenge of postoperative tumor recurrence persists in about 90% of patients post-treatment (Bonosi et al., 2023; Ho et al., 2024). Many patients will relapse largely due to the growth of residual cells within 2–3 cm of the resection border that are difficult to resect without inflicting neurological damage and often develop resistance to standard treatments (Bonosi et al., 2023; Ho et al., 2024; McConville et al., 2024; Roy et al., 2015; Loeffler et al., 1990; Putavet & Keizer, 2021). Novel approaches that integrate targeted therapies https://doi.org/10.3390/cancers15072116 https://doi.org/10.1093/nop/npae058 https://doi.org/10.1093/neuonc/noab106 https://jamanetwork.com/journals/jamaoncology/fullarticle/2685651 https://www.thelancet.com/journals/lanonc/article/PIIS1470-2045(09)70025-7/abstract https://www.thelancet.com/journals/lanonc/article/PIIS1470-2045(09)70025-7/abstract https://doi.org/10.3390/cancers15072116 https://doi.org/10.3390/cancers15072116 https://doi.org/10.3390/brainsci13020216 https://actaneurocomms.biomedcentral.com/articles/10.1186/s40478-024-01790-3 https://doi.org/10.3390/brainsci13020216 https://actaneurocomms.biomedcentral.com/articles/10.1186/s40478-024-01790-3 https://doi.org/10.3390/cancers16173008 https://doi.org/10.4103/2278-330X.175953 https://www.redjournal.org/article/0360-3016(90)90358-Q/abstract https://doi.org/10.3390/cancers13071560 10 with clinically validated chemotherapeutic agents are needed to address residual disease, effectively extending post-diagnosis survival and improving patient outcomes. Despite its role in the standard treatment regimen for GBM, chemotherapy faces significant challenges that limit its efficacy, primarily due to the restrictive nature of the blood-brain barrier (BBB). The BBB’s selective permeability influenced by factors such as molecular size, polarity, ligand specificity to surface enzymes, and hydrophobicity, inhibits the effective delivery of many chemotherapeutic agents (Chen & Liu, 2012). A commonly used chemotherapeutic drug, TMZ benefits from its small size and lipophilic properties, which allow it to cross the BBB more efficiently than larger, more hydrophilic compounds. However, TMZ’s low stability and short half-life make it prone to degradation before reaching the brain, necessitating frequent high doses to maintain optimal therapeutic concentrations, which in turn exposes patients to severe side effects (Doolittle et al., 2014; Krajcer et al., 2023). To address these limitations, alternative strategies such as targeting AXL-Receptor Tyrosine Kinase (AXL-RTK) have been explored. AXL, a member of the TAM (Tyro-3, Axl, and Mer) RTK family, plays a critical role in cell proliferation, adhesion, and inflammatory cytokine regulation (Zhen et al., 2018). Phospho-AXL (pAXL), prevalent in GBM, correlates with shorter overall patient survival (Zhou et al., 2021). Bemcentinib (BEM), the first-in-class orally bioavailable inhibitor of the pAXL-RTK pathway, has demonstrated promise in its ability to penetrate the BBB more effectively than traditional chemotherapeutics and its targeted inhibition of AXL, offering potential for more precise and effective GBM treatment (Nabors et al., 2021; Meel et al., 2020). New approaches to treating primary and recurrent GBMs are emerging with the U.S. Food and Drug Administration (FDA) approval of photosensitive agents to improve visualization https://www.sciencedirect.com/science/article/pii/S0169409X11002900#bb0055 https://pmc.ncbi.nlm.nih.gov/articles/PMC5505259/ https://www.sciencedirect.com/science/article/pii/S0753332223009654 https://www.sciencedirect.com/science/article/pii/S0896841118302555?via%3Dihub https://biosignaling.biomedcentral.com/articles/10.1186/s12964-020-00694-8 https://academic.oup.com/neuro-oncology/article/23/Supplement_6/vi60/6426814 https://aacrjournals.org/clincancerres/article/26/13/3319/82769/Combined-Therapy-of-AXL-and-HDAC-Inhibition https://aacrjournals.org/clincancerres/article/26/13/3319/82769/Combined-Therapy-of-AXL-and-HDAC-Inhibition 11 of occult brain tumor margins and maximize the extent of tumor resection during surgery. Intraoperative photodynamic therapy (PDT) has emerged as a promising technique, involving the administration of a photosensitizer before surgery and red light application afterward to target residual tumor cells. A minimally invasive procedure, PDT is typically performed during surgical resection within a 30-60 minute time frame. Notably, the recent approval of 5-aminolevulinic acid (5-ALA) for fluorescence-guided surgery (FGS) has significantly improved the extent of tumor resection and extended progression-free survival for GBM patients, minimizing the risk of local recurrence (Miretti et al., 2023; He et al., 2020). In addition to addressing the residual disease, the application of PDT in GBM treatment offers many advantages, particularly in minimizing damage to the healthy brain parenchyma surrounding the invasive tumor. PDT produces reactive oxygen species (ROS) that, due to their limited distance of diffusion and short lifespan, selectively damage tumor cells while minimizing harm to surrounding healthy tissue (Mahmoudi et al., 2019). PDT has also been shown to increase the permeability of the blood-brain barrier (BBB), allowing for the delivery of both small and high-molecular-weight chemotherapeutic agents to previously inaccessible regions, enhancing drug delivery to brain tumors (Semyachkina-Glushkovskaya et al., 2017). The integration of PDT with chemotherapy presents a promising and synergistic approach to GBM treatment development, sensitizing tumors to chemotherapeutic drugs and targeting residual tumor cells that exhibit resistance to standard therapies. Recent advances have led to the development of an excipient-free nanoparticle formulation of verteporfin (NanoVP) for PDT of GBM, which has demonstrated superior efficacy in BBB opening compared to 5-ALA, reducing tumor burden, and extending survival in animal models (Quinlan et al., 2024). In this study, we investigate the combined effects of https://www.sciencedirect.com/science/article/pii/S2666469023000027#bib0085 https://pmc.ncbi.nlm.nih.gov/articles/PMC7823121/ https://link.springer.com/article/10.1007/s11060-019-03103-4 https://opg.optica.org/boe/fulltext.cfm?uri=boe-8-11-5040&id=375358 https://doi.org/10.1002/advs.202302872 12 NanoVP-PDT and clinically tested pAXL inhibitors on GBM cells. First, cytotoxicity was assessed using an ML8500 illumination system and a 4W 690 nm laser for fluorescence-guided intervention in vitro. Further in vitro cytotoxicity studies were conducted to evaluate the inhibitory effects of Bemcentinib and determine its IC50, which was found to be between 2–2.5 μM. To examine the potential synergy of combination therapy, GBM cells were concurrently treated with NanoVP and free Bemcentinib, followed by PDT illumination. Cytotoxic effects were quantified using cell viability assays, and a combination index (CI) was calculated to assess the efficacy of the combined treatment. Preliminary findings indicate that optimization of treatment parameters, including PDT light dose and Bemcentinib concentration, is crucial for improving therapeutic efficacy. Ultimately, the combination of NanoVP-PDT and pAXL inhibitors demonstrates promising synergistic effects in vitro. This investigation of the synergistic anti-GBM effects of NanoVP-PDT and pAXL inhibitors in vitro lays the groundwork for optimizing combination treatment strategies prior to advancing to in vivo studies. We anticipate that this novel combinatorial approach will advance the clinical application of PDT in the treatment of GBM and other malignancies shielded by the BBB, paving the way for more effective management of these challenging tumors. 13 2. Literature Review 2.1 Glioblastoma Multiforme (GBM) Glioblastoma Multiforme (GBM) is the most aggressive and most common malignant brain tumor, making up almost half of all brain and central nervous system (CNS) tumors diagnosed in the United States (Thakkar et al., 2024). GBM’s highly aggressive nature is attributed to several of its key characteristics including its heterogeneity, location, ability of its cells to invade and migrate within the brain, and rapid proliferation, all of which allow it to evade conventional therapies such as chemoradiation and immunotherapy (Yalamarty et al., 2023). Intratumorally, GBM is highly heterogeneous, consisting of a diverse set of cell populations each contributing to the tumor’s complex composition. A project done by The Cancer Genome Atlas (TCGA) has detailed the genomic profile of GBM, allowing for the classification of GBM into four distinct subtypes–classical, mesenchymal, neural, and pro-neural–each defined by unique gene expression patterns (Sottoriva et al., 2013). For example, in the classical subtype, high expression of epidermal growth factor receptor (EGFR), a type of receptor tyrosine kinase (RTK), defines its molecular signature and plays a significant role in tumor growth and invasion. Similarly, the pro-neural subtype is defined by high expression of platelet derived growth factor receptor alpha (PDGFRA), another RTK contributing to similar clinical outcomes. Moreover, high expression of neuronal genes and neurofibromatosis 1 (NF1) are characteristic of the neural and highly aggressive mesenchymal subtypes, respectively (Verhaak et al., 2010; Qazi et al., 2017; Behnan et al., 2019). While these markers are used to define the aforementioned subtypes due to their high prevalence, there are https://www.aans.org/patients/conditions-treatments/glioblastoma-multiforme/ https://pmc.ncbi.nlm.nih.gov/articles/PMC10093719/ https://pmc.ncbi.nlm.nih.gov/articles/PMC10093719/ https://www.pnas.org/doi/10.1073/pnas.1219747110 https://www.cell.com/cancer-cell/fulltext/S1535-6108(09)00432-2?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1535610809004322%3Fshowall%3Dtrue https://www.sciencedirect.com/science/article/pii/S0923753419322665 https://academic.oup.com/brain/article/142/4/847/5420896 14 several other expression patterns that exist within and across these categories, providing insight into future therapeutic targets for the treatment of GBM. Furthermore, the tumor microenvironment (TME) surrounding GBM is also characterized by its heterogeneity in that it is made up of various cell types such as macrophages, T-cells, endothelial cells, microglia, glioma stem cells, myeloid-derived suppressor cells, and fibroblasts (Eisenbarth & Wang, 2023). Each of these cells foster an ideal environment for tumor progression. Most prevalently within the TME, tumor associated macrophages and microglia (TAMs) contribute to increased tumor formation, proliferation, survival, migration and immunosuppression by producing growth factors and cytokines in response to signaling factors sent by the tumor cells (Quail & Joyce, 2017; Liu et al., 2022; Hambardzumyan et al., 2016). These effects are especially evident in the mesenchymal subtype, where TAM genes are notably overexpressed (Chen & Hambardzumyan, 2018). Moreover, increased expression of myeloid-derived suppressor cells (MDSCs) in this subtype further contributes to tumor progression, allowing for immunosuppression, angiogenesis, invasion, and metastasis to occur (Lin et al., 2024). In addition to GBM’s intra- and intertumoral heterogeneity contributing to poor prognosis, the tumor’s location within the brain introduces another layer of complexity, as the surrounding neural environment and blood-brain barrier (BBB) influence tumor behavior and therapeutic response. For example, tumors in contact with the subventricular zone (SVZ) are associated with increased invasion due to their proximity to progenitor and neural stem cell niches, facilitating differentiation into various cell types necessary for the tumor to infiltrate further into the brain (Armocida et al., 2021). Moreover, the BBB, a biophysical and biochemical barrier between the brain and bloodstream, is a key characteristic of GBM’s location and https://www.nature.com/articles/s41388-023-02738-y https://www.cell.com/cancer-cell/fulltext/S1535-6108(17)30052-1?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1535610817300521%3Fshowall%3Dtrue https://www.frontiersin.org/journals/oncology/articles/10.3389/fonc.2022.822085/full https://www.nature.com/articles/nn.4185 https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2018.01004/full https://jhoonline.biomedcentral.com/articles/10.1186/s13045-024-01544-7 https://www.sciencedirect.com/science/article/pii/S2214751921000979 15 response to therapy, selectively constraining the entry of molecules into the brain based on properties such as size, charge, protein interactions, and liposolubility (Sarkaria et al., 2017). Despite GBM tumorigenesis and proliferation disrupting the BBB to form the blood-tumor barrier (BTB), heterogeneity among different regions of the tumor leads to some areas being more permeable than others, offering perspective on drug delivery challenges and therapeutic resistance (Arvanitis et al., 2020). 2.2 GBM Standard of Care & Treatment Challenges 2.2.1 Current Standard of Care Until today, there remains no comprehensive treatment for GBM. Currently, the standard of care for GBM consists of surgical resection of the primary or secondary tumor in low-risk brain regions, followed by adjuvant radiotherapy and chemotherapy for 6 cycles (Nabian et al., 2024). However, the highly invasive and heterogeneous nature of GBM presents significant challenges, limiting the efficacy of each of these monotherapies (Rong et al., 2022). Consequently, multimodal treatment approaches are employed to target distinct aspects of disease progression and improve patient outcomes. 2.2.2 Surgical Resection Surgical resection remains the first-line intervention for GBM, with the primary objective of maximizing tumor debulking to prolong survival (Rong et al., 2022). Gross total resection (GTR), or when feasible, supratotal resection, is considered the optimal surgical approach due to its association with improved patient outcomes (Nabian et al., 2024). Complete resection of the tumor at the cellular level is rarely achievable due to the highly invasive and infiltrative nature of GBM, which enables malignant, residual cells to disseminate into adjacent brain tissue (McConville et al., 2024). The extent of resection and the volume of residual tumor are https://academic.oup.com/neuro-oncology/article/20/2/184/4107399 https://www.nature.com/articles/s41568-019-0205-x https://academic.oup.com/noa/article/6/1/vdae028/7619493 https://academic.oup.com/noa/article/6/1/vdae028/7619493 https://jeccr.biomedcentral.com/articles/10.1186/s13046-022-02349-7 https://jeccr.biomedcentral.com/articles/10.1186/s13046-022-02349-7 https://academic.oup.com/noa/article/6/1/vdae028/7619493 https://doi.org/10.3390/cancers16173008 16 significantly correlated with prognosis (Wen et al. 2021). Greater extent of resection and smaller residual tumor volume have been linked to improved survival and delayed recurrence in patients who undergo adjuvant therapies, such as TMZ chemotherapy and radiotherapy (Chaichana et al., 2014). However, more than 50% of GBM tumors are located within or near eloquent brain regions that govern essential neurological functions, limiting the extent of resection and increasing the risk of postoperative deficits (Gerritsen et al., 2022). A retrospective analysis of 16,530 patients who underwent surgical resection for malignant GBM between 2002 and 2011 revealed that approximately 31% developed new postoperative ischemic lesions, leading to neurological decline (Garza-Ramos et al., 2016). The most prevalent surgical complication was iatrogenic stroke, which increased in-hospital mortality risk ninefold, followed by postoperative hemorrhage or hematoma, both of which were associated with significantly increased mortality rates (Garza-Ramos et al., 2016). The primary risk factor for postoperative ischemic damage was identified as tumor proximity to central arteries (Garza-Ramos et al., 2016). Consequently, to mitigate surgical morbidity, most patients undergo subtotal or partial resection rather than GTR, prioritizing the preservation of functional brain tissue and reducing the risk of postoperative neurological damage. To enhance the extent of tumor resection while minimizing damage to eloquent brain regions, a multitude of preoperative and intraoperative adjuncts have been developed. Among these, fluorescence-guided surgery (FGS) has emerged as a valuable tool for enhancing intraoperative visualization of neoplastic tissue (Schupper et al., 2021). FGS utilizes 5-aminolevulinic acid (5-ALA), a Gliolan photosensitizer and hemoglobin precursor that preferentially accumulates in GBM cells, where it is metabolized into fluorescent protoporphyrin IX (PP9), enabling real-time tumor delineation during surgery (Hadjipanayis et al., 2015). A https://bmccancer.biomedcentral.com/articles/10.1186/s12885-021-07800-0 https://doi.org/10.1093/neuonc/not137 https://doi.org/10.1093/neuonc/not137 https://academic.oup.com/nop/article/9/5/364/6541330 https://www.sciencedirect.com/science/article/pii/S0303846715300718?via%3Dihub https://www.sciencedirect.com/science/article/pii/S0303846715300718?via%3Dihub https://www.sciencedirect.com/science/article/pii/S0303846715300718?via%3Dihub https://www.frontiersin.org/journals/neurology/articles/10.3389/fneur.2021.682151/full https://doi.org/10.1227/neu.0000000000000929 17 randomized controlled trial involving 322 GBM patients demonstrated that GTR was achieved in 65% of patients who underwent 5-ALA FGS-guided surgery, compared to only 36% of those who underwent standard white-light surgery (McCracken et al., 2022). Additionally, the 5-ALA FGS cohort exhibited a 20% increase in progression-free survival at six months postoperatively (McCracken et al., 2022). Supporting these findings, a recent systematic review on intraoperative fluorescent imaging in high-grade gliomas concluded that 5-ALA FGS was associated with a greater extent of tumor resection, as well as longer overall and progression-free survival (Eatz et al., 2022). Collectively, these studies highlight the critical role of intraoperative fluorescence imaging in facilitating maximal safe resection and improving patient outcomes. Despite advancements in surgical techniques, resection alone remains insufficient for effectively treating recurrent or progressive GBM, presenting significant challenges for long-term disease control. One of the greatest challenges is the presence of residual disease following surgical resection. GBM cells invade the healthy brain tissue surrounding the primary tumor, making it challenging to establish clear boundaries between the cancerous and normal brain tissue (Patel & Chavda, 2024). This invasion extends beyond the visible tumor margins detected by imaging, resulting in the persistence of undetectable microscopic tumor cells, known as residual cells, even after GTR (Patel & Chavda, 2024). The regrowth of these residual GBM cells ultimately leads to recurrence. Even with aggressive resection strategies, residual tumor cells persist within 2–3 cm of the resection margin, contributing to tumor recurrence in more than 90% of patients with high-grade glioma (Bonosi et al., 2023, Ho et al., 2024, McConville et al., 2024, Roy et al., 2015; Loeffler et al., 1990; Putavet and Keizer, 2021). As a result, the current standard of care includes adjuvant radiotherapy and chemotherapy to target residual disease and minimize the likelihood of recurrence. https://academic.oup.com/neuro-oncology/article/24/Supplement_6/S52/6793936 https://academic.oup.com/neuro-oncology/article/24/Supplement_6/S52/6793936 https://link.springer.com/article/10.1007/s11060-021-03901-9 https://link.springer.com/article/10.1007/s11060-021-03901-9 https://doi.org/10.1016/j.cpt.2023.11.006 https://doi.org/10.1016/j.cpt.2023.11.006 https://doi.org/10.3390/brainsci13020216 https://actaneurocomms.biomedcentral.com/articles/10.1186/s40478-024-01790-3 https://doi.org/10.3390/cancers16173008 https://doi.org/10.3390/cancers16173008 https://doi.org/10.4103/2278-330X.175953 https://www.redjournal.org/article/0360-3016(90)90358-Q/abstract https://doi.org/10.3390/cancers13071560 18 2.2.3 TMZ Chemotherapy and Radiotherapy Chemotherapy with concomitant temozolomide (TMZ) represents a key component of the standard treatment regimen for GBM, typically administered after surgical resection and in combination with radiotherapy (Rong et al., 2022). TMZ chemotherapy is generally well tolerated by GBM patients, with the most frequently reported side effects being fatigue, nausea, vomiting, and myelosuppression (Jezierzański et al., 2024). However, approximately 15% of cases undergo treatment discontinuation due to intolerable adverse side effects (Jezierzański et al., 2024). Among the more severe complications, hematologic toxicities such as neutropenia, thrombocytopenia, lymphopenia, and leukopenia represent the primary concerns during TMZ chemotherapy (Jezierzański et al., 2024). Chemotherapeutic alkylating agents are critical in the therapeutic management of GBM due to their ability to disrupt DNA synthesis and repair mechanisms in tumor cells (Ortiz et al., 2020). TMZ, an orally administered imidazotetrazine derivative of dacarbazine, is a DNA-alkylating agent that is efficiently absorbed into the bloodstream (Jezierzański et al., 2024). Approved by the FDA for GBM treatment in 2005, TMZ is also indicated for the management of other solid tumors, including advanced neuroendocrine malignancies (Jezierzański et al., 2024). While the BBB poses a significant challenge in brain cancer therapy by restricting chemotherapeutic drug penetration and reducing treatment efficacy, TMZ's small molecular size and lipophilic properties allow it to effectively traverse the BBB, achieving a bioavailability of 98% following oral administration (Oraiopoulou et al., 2024; Ahmed et al., 2023). TMZ exerts its cytotoxic effects by methylating the purine bases of DNA, leading to the formation of O6-methylguanine lesions. These lesions, in turn, trigger various forms of cell death, including apoptosis, autophagy, and cellular senescence in GBM cells (Ortiz et al., 2020). https://jeccr.biomedcentral.com/articles/10.1186/s13046-022-02349-7 https://doi.org/10.3390/curroncol31070296 https://doi.org/10.3390/curroncol31070296 https://doi.org/10.3390/curroncol31070296 https://doi.org/10.3390/curroncol31070296 https://pmc.ncbi.nlm.nih.gov/articles/PMC8206461/ https://pmc.ncbi.nlm.nih.gov/articles/PMC8206461/ https://doi.org/10.3390/curroncol31070296 https://doi.org/10.3390/curroncol31070296 https://www.nature.com/articles/s41598-024-53684-y https://doi.org/10.1016/j.neurol.2023.03.013 https://pmc.ncbi.nlm.nih.gov/articles/PMC8206461/ 19 Despite being the primary chemotherapeutic agent for GBM, TMZ shows limited efficacy, with approximately 50% of patients developing treatment resistance (Jezierzański et al., 2024). The clinical efficacy of TMZ is significantly compromised by the expression of the O6-methylguanine DNA methyltransferase (MGMT) enzyme, which confers resistance by repairing the O6-methylguanine lesions that TMZ induces (Ortiz et al., 2020). Following its passage through the BBB, TMZ spontaneously hydrolyzes to release a methyl diazonium ion that alkylates DNA (Ortiz et al., 2020). In the presence of MGMT, this repair mechanism effectively neutralizes the cytotoxic effects of TMZ (Ortiz et al., 2020). A study of 16 GBM cell lines showed that MGMT expression was present in all TMZ-resistant cell lines, while it was absent in TMZ-sensitive lines (Nifterik et al., 2010). This finding confirms that MGMT expression is a major determinant of TMZ resistance, thus making TMZ therapy effective primarily in patients whose tumors exhibit low or absent MGMT levels. In the current standard of care for GBM, radiation is commonly administered after surgical resection, preceding or in combination with TMZ chemotherapy. Radiotherapy utilizes high-energy beams of radiation to damage the DNA of cancerous cells and eventually lead to inhibited cell proliferation and apoptosis (Liu et al., 2021). Although radiotherapy is commonly used for GBM, there has been little to no improvements of survival rates after treatment in the last 20 years (Aiyappa-Maudsley et al., 2022). Tumor hypoxia of GBM is a major barrier to radiotherapy being an effective treatment method. Hypoxia refers to the decrease of oxygen levels found in tumors, which promotes metastasis of the tumor to the surrounding area (Aiyappa-Maudsley et al., 2022). Oxygen is needed for radiotherapy as oxygen generates free radicals that destroy cancer cells (NCI, 2018). The lack of oxygen found in GBM makes radiotherapy an ineffective treatment method. The non-selectivity of radiotherapy also causes https://doi.org/10.3390/curroncol31070296 https://doi.org/10.3390/curroncol31070296 https://pmc.ncbi.nlm.nih.gov/articles/PMC8206461/ https://pmc.ncbi.nlm.nih.gov/articles/PMC8206461/ https://pmc.ncbi.nlm.nih.gov/articles/PMC8206461/ https://www.nature.com/articles/6605712 https://pmc.ncbi.nlm.nih.gov/articles/PMC8554658/ https://pmc.ncbi.nlm.nih.gov/articles/PMC9617255/ https://pmc.ncbi.nlm.nih.gov/articles/PMC9617255/ https://www.cancer.gov/news-events/cancer-currents-blog/2018/microbubbles-radiation-breast-cancer#:~:text=Supplying%20Oxygen%20to%20Boost%20Radiation%20Therapy&text=Much%20of%20this%20resistance%20is,radiation%20therapy%20is%20less%20effective. 20 toxicity as normal tissue is exposed to high levels of radiation, resulting in low patient quality of life and adverse side effects (Majeed & Gupta, 2023). Side effects of toxicity can include memory and concentration issues, blurry vision, fatigue, and nausea (NCI, 2022). Radiotherapy used as monotherapy for GBM was found to have low efficacy as patients became resistant to radiotherapy (Xu et al., 2019). GBM cells that underwent radiotherapy were found to have an upregulated LGMNP1 causing a reduction in DNA damage and decreased apoptosis of cancerous cells (Xu et al., 2019). The clinical efficacy of TMZ chemotherapy is further enhanced when combined with radiation therapy. A large, randomized Phase 3 trial demonstrated improved progression-free and overall survival in GBM patients treated with concurrent and adjuvant TMZ alongside radiotherapy, following maximal safe resection (Mann et al., 2018). In this trial, the median progression-free survival was 6.9 months for patients receiving both TMZ and radiotherapy, compared to 5.0 months for those undergoing radiotherapy alone. Additionally, the median overall survival was 14.6 months in the combination therapy group, whereas it was 12.1 months for the radiotherapy-only group (Mann et al., 2018). These findings highlight the superior efficacy of combining TMZ chemotherapy with radiotherapy, reinforcing this approach as the current standard of care for newly diagnosed GBM. Researchers have attributed this survival benefit, in part, to the radiation-sensitizing effects of TMZ. According to a review of TMZ chemotherapy with radiation therapy in high-grade gliomas, the superior efficacy of chemoradiation is attributed to the interaction between their mechanisms (Koukourakis et al., 2009). In preclinical studies, researchers have explored the radiation-sensitizing effects of TMZ, particularly in glioblastomas with low or absent MGMT expression (Chakravarti et al., 2006). In MGMT-negative GBMs, concurrent https://www.ncbi.nlm.nih.gov/books/NBK563259/#:~:text=Side%20effects%20of%20radiotherapy%20are,of%20common%20complications%20of%20radiotherapy. https://www.cancer.gov/about-cancer/treatment/types/radiation-therapy https://www.spandidos-publications.com/10.3892/or.2019.7128 https://www.spandidos-publications.com/10.3892/or.2019.7128 https://doi.org/10.3389/fneur.2017.00748 https://doi.org/10.3389/fneur.2017.00748 https://www.mdpi.com/1420-3049/14/4/1561 https://doi.org/10.1158/1078-0432.CCR-06-0596 21 TMZ and radiation treatment led to greater DNA damage and reduced DNA repair capacity compared to either radiation alone or TMZ administered sequentially with radiation (Chakravarti et al., 2006). In MGMT-positive GBMs, the study also found that the compound O6-benzylguanine, when used with concurrent TMZ and radiation, improved the antitumor effects by increasing DNA damage and promoting cell death (Chakravarti et al., 2006). This effect was less pronounced when O6-benzylguanine was used alone or without radiation (Chakravarti et al., 2006). In vivo studies using GBM xenografts confirmed these findings, further supporting the superior efficacy of combined TMZ and radiation therapy (Chakravarti et al., 2006). 2.3 Chemotherapy with Bemcentinib 2.3.1 Targeting Receptor Tyrosine Kinases (RTKs) Receptor Tyrosine Kinases (RTKs) are transmembrane proteins known for their extensive signaling cascades. Expressed throughout the entire body, RTKs are important for regulating cell survival, proliferation, differentiation, and migration (Wintheiser & Silberstein, 2022). Each RTK contains an extracellular ligand-binding region, as well as a transmembrane alpha helix. Inside the cell, RTKs contain a tyrosine kinase domain (TKD) along with a C-terminal tail. When a ligand binds to the extracellular domain, it initiates signal transduction throughout the cell. The specific ligand-binding domain, the receptor-specific ligand that binds to it, and the subsequent downstream signaling pathway depend on the class of RTK that is expressed. In total, 58 human RTKs have been identified, and mutations in many of these classes have been connected to cancer development and proliferation (Wintheiser & Silberstein, 2022). Under typical physiological conditions, RTKs are activated when growth factor ligands bind to their receptors, inducing receptor dimerization. This conformational change brings the https://doi.org/10.1158/1078-0432.CCR-06-0596 https://doi.org/10.1158/1078-0432.CCR-06-0596 https://doi.org/10.1158/1078-0432.CCR-06-0596 https://doi.org/10.1158/1078-0432.CCR-06-0596 https://doi.org/10.1158/1078-0432.CCR-06-0596 https://doi.org/10.1158/1078-0432.CCR-06-0596 https://www.ncbi.nlm.nih.gov/books/NBK538532/ https://www.ncbi.nlm.nih.gov/books/NBK538532/ 22 two intracellular TKDs into close proximity, resulting in autophosphorylation of tyrosine residues. This phosphorylation event destabilizes and releases autoinhibitory interactions that typically keep the TKD inactive, which subsequently activates the kinase. Once activated, downstream signaling molecules are recruited to the receptor, which serves as a docking site for the initial signal. Due to their large quantity of phosphotyrosine residues and docking proteins, RTKs efficiently sequester signaling molecules to these sites, enabling the production of an amplified cascade for a vast range of signaling pathways (Du & Lovly, 2018) Because RTKs are involved in many signaling pathways controlling cell proliferation, differentiation, and migration, dysfunctional RTKs can function as oncogenes, with their overactivation providing a scaffold for cancer development. RTK overactivation can occur through multiple mechanisms, including gain-of-function mutations, DNA amplifications, and chromosomal rearrangements. Most commonly, they result from gain-of-function mutations in the genome. When this occurs, the concentration of localized receptors increases (Du & Lovly, 2018). This results in dramatic levels of signal amplification, allowing for uncontrolled cell growth and proliferation. In DNA amplification, the RTK receptor becomes overexpressed, multiplying the amount of downstream effects that occur. If chromosomal rearrangement occurs, RTK receptors can become constitutively expressed and constantly send out signals that enhance growth and proliferation. Finally, even the subsequent signaling molecules within RTK pathways can become overactivated, which result in a more narrow scope of effects (Wintheiser & Silberstein, 2022). Overall, the effects of RTK overexpression are multifactorial, including changes in cell differentiation, survival, metabolism, and migration, leading to their designation as oncogenes. 2.3.2 RTK-AXL Function and Mechanism https://molecular-cancer.biomedcentral.com/articles/10.1186/s12943-018-0782-4#:~:text=Mechanisms%20of%20RTK%20activation%20under,1a). https://molecular-cancer.biomedcentral.com/articles/10.1186/s12943-018-0782-4#:~:text=Mechanisms%20of%20RTK%20activation%20under,1a). https://molecular-cancer.biomedcentral.com/articles/10.1186/s12943-018-0782-4#:~:text=Mechanisms%20of%20RTK%20activation%20under,1a). https://www.ncbi.nlm.nih.gov/books/NBK538532/ https://www.ncbi.nlm.nih.gov/books/NBK538532/ 23 Axl, named after the Greek word “anexelekto” (uncontrolled), is a RTK in the TAM subfamily (Zhu et L., 2019). Located on chromosome 19 at position q13.2, the Axl gene (axl) was first detected in patients with chronic myelogenous leukemia (Liu et al., 1988; O’Bryan et al., 1991). Along with the having a similar predicted amino acid sequence to previously identified kinases, the idea that axl encodes a tyrosine-kinase was supported after a 140-kDa tyrosine-phosphorylated protein was detected in transformed NIH 3T3 cells (O’Bryan et al., 1991). Additionally, 104 kDa (unglycosylated) and 120 kDa (partially glycosylated) tyrosine-phosphorylated proteins were detected in axl-baculovirus-infected Sf9 cells. Like all RTKs, Axl contains extracellular (N-terminal), transmembrane, and intracellular (C-terminal) domains. Axl’s extracellular domain contains two immunoglobulin-like (IgL) repeats and two fibronectin type III (FNIII repeats) and the intracellular kinase domain contains a distinct KWIAIE sequence (O’Bryan et al., 1991). While novel at the time, the conserved KW(I/L)A(I/L)ES sequence and adhesion molecule like domains (IgL and FNIII) later became defining features of the TAM family which now consists of Tyro-3, AXL, and Mer (Linger et al., 2008). Axl and its downstream pathways can be activated by several mechanisms such as ligand-dependent dimerization. Ligands for the TAM family include growth arrest-specific gene 6 (Gas6), protein S (Pros1), Tubby, Tulp-1, and Galectin-3 (Yadav et al., 2025). Gas6 and Protein S, the first identified and most well-studied TAM ligands, have high structural homology and are both members of the vitamin K-dependent protein family (Manfioletti et al., 1993). Gas6, can bind to all members of the TAM sub-family, but it has the highest binding affinity to Axl and is considered Axl’s main ligand (Nagata et al., 1996; Stitt et al., 1995; Varnum et al., 1995). Axl/Gas6 dimerization occurs in two steps. First, a 1:1 Gas6/Axl complex forms when Axl’s two https://molecular-cancer.biomedcentral.com/articles/10.1186/s12943-019-1090-3#:~:text=The%20word%20AXL%2C%20coming%20from,and%20intracellular%20domain%20%5B8%5D. https://www.pnas.org/doi/epdf/10.1073/pnas.85.6.1952 https://pmc-ncbi-nlm-nih-gov.proxy-um.researchport.umd.edu/articles/instance/361494/pdf/molcellb00034-0222.pdf https://pmc-ncbi-nlm-nih-gov.proxy-um.researchport.umd.edu/articles/instance/361494/pdf/molcellb00034-0222.pdf https://pmc-ncbi-nlm-nih-gov.proxy-um.researchport.umd.edu/articles/instance/361494/pdf/molcellb00034-0222.pdf https://pmc-ncbi-nlm-nih-gov.proxy-um.researchport.umd.edu/articles/instance/361494/pdf/molcellb00034-0222.pdf https://pmc-ncbi-nlm-nih-gov.proxy-um.researchport.umd.edu/articles/instance/361494/pdf/molcellb00034-0222.pdf https://pmc.ncbi.nlm.nih.gov/articles/PMC3133732/ https://pmc.ncbi.nlm.nih.gov/articles/PMC3133732/ https://www.nature.com/articles/s41392-024-02121-7 https://pubmed.ncbi.nlm.nih.gov/8336730/ https://www.jbc.org/article/S0021-9258(19)79242-4/fulltext https://www.cell.com/cell/pdf/0092-8674(95)90520-0.pdf?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2F0092867495905200%3Fshowall%3Dtrue https://www.nature.com/articles/373623a0 24 IgL domains are linked with Gas6’s LG1 domain. Second, this 1:1 Gas6/Axl complex will dimerize with another 1:1 Gas6/Axl complex forming a 2:2 complex (Sasaki et al., 2002; Sasaki et al., 2006). Following dimerization, trans-autophosphorylation can occur in Axl’s intracellular kinase domain. Phosphorylated sites on Axl include Tyr698, Tyr702, and Tyr703, which are important for autophosphorylation along with Tyr779, Tyr821, and Tyr866 which are important for adaptor protein docking and signal transduction (Tang et al., 2023). Once phosphorylated, Axl can activated several downstream pathways such as PI3K/Akt and MEK/ERK which can increase cancer growth and migration (Yadav et al., 2025). Pros1 was only originally thought to activate Tyro-3 and Mer, but more recent studies have indicated that Pros1 can also bind to and activate Axl (Lew et al., 2014; Sadahiro et al., 2018; Wei et al., 2022). Although the mechanism is not completely clear, the Pros1/Axl pathway plays an important role in NFκB signalling and GBM growth in particular. Axl can also be activated by ligand-independent mechanisms such as self-dimerization or crossphosphorylation by EGFR (Yadav et al., 2025; Vouri et al., 2016;). Oxidative stress (e.g., ROS) may also activate Axl phosphorylation and ROS levels in CL1-0 cells were reduced by BEM, an Axl inhibitor (Huang et al., 2013). The impact of ROS in cancer is concentration-dependent and, while moderate levels of ROS can promote tumor progression, a high level of ROS can decrease tumor growth and result in cell death (Glorieux et al., 2024). Axl is regulated at several levels from transcription to post-translation. For example, several transcription factors (e.g. activation protein-1 and hypoxia-inducible transcription factor-1ɑ) and methylation have been found to regulate Axl transcription and expression (Rankin et al., 2014; Mudduluru et al., 2011; Mudduluru & Allgayer, 2008). Additionally, MicroRNAs (e.g. miR-34a) can inhibit the expression of Axl mRNA and their downregulation can lead to cancer (Li et al., 2015). Alternative splicing by PTBP1 can lead to different isoforms of Axl such https://www.sciencedirect.com/science/article/pii/S0021925819717669 https://pubmed.ncbi.nlm.nih.gov/16362042/ https://pubmed.ncbi.nlm.nih.gov/16362042/ https://link.springer.com/article/10.1186/s13046-023-02726-w#ref-CR19 https://www.nature.com/articles/s41392-024-02121-7 https://elifesciences.org/articles/03385 https://aacrjournals.org/cancerres/article/78/11/3002/625035/Activation-of-the-Receptor-Tyrosine-Kinase-AXL https://wjso.biomedcentral.com/articles/10.1186/s12957-022-02801-0 https://www.nature.com/articles/s41392-024-02121-7 https://pubmed.ncbi.nlm.nih.gov/27775700/ https://www.sciencedirect.com/science/article/pii/S089158491300614X?via%3Dihub https://www.nature.com/articles/s41573-024-00979-4#:~:text=The%20biological%20effect%20of%20ROS,cancer%20cells%20to%20therapeutic%20agents. https://www.pnas.org/doi/full/10.1073/pnas.1404848111 https://www.pnas.org/doi/full/10.1073/pnas.1404848111 https://onlinelibrary.wiley.com/doi/abs/10.1042/BC20100094 https://portlandpress.com/bioscirep/article-abstract/28/3/161/55622/The-human-receptor-tyrosine-kinase-Axl-gene?redirectedFrom=fulltext https://link.springer.com/article/10.1007/s13277-015-3445-8 25 as the Axl-S isoform, which demonstrated stronger interactions with Gas6 and increased Axl phosphorylation (Shen et al., 2020). The stronger interaction therefore led to greater activation of PI3K-AKT and MAPK-ERK pathways (Shen et al., 2020). Axl stability and activity can be altered by post-translational regulation (Tang et al., 2023). The overexpression of Axl has been associated with cancer cell growth, survival, migration, epithelial-to-mesenchymal transition, angiogenesis, treatment resistance, and immune invasion (Wu et al., 2014; Yadav et al., 2025). Axl is involved in activating several signalling pathways (e.g. PI3K/Akt, JAK/STAT, NFκB , RAS/RAF/MEK/ERK, PKC) leading to it tumor promoting effects (Scaltriti et al., 2016; Yadav et al., 2025; Zhu et L., 2019). Given its role in cancers, Axl has become a promising therapeutic target for several cancers including lung cancer, breast cancer, colon cancer, and GBM (Wu et al., 2014; Kim et al., 2025; Zhang et al., 2018; Martinelli et al., 2015). 2.3.3 RTK-AXL Expression and Prevalence in GBM Tumors Axl is overexpressed in GBM cell lines and tissue samples (Onken et al., 2016; Cheng et al., 2015; Vajkoczy et al., 2006). In fact, phosphorylated Axl (pAxl) was detected in 74% of patients with newly diagnosed GBM (Onken et al., 2017). High pAxl expression was detected specifically in GBM tumor vasculature, pseudo-palisades, herringbone-like regions, and hypercellular regions (Onken et al., 2017; Hutterer et al., 2008; Sadahiro et al., 2018; Onken et al., 2016). Axl was found to be expressed and activated more in mesenchymal GBM stem-like cells compared to proneural GBM stem-like cells (Cheng et al., 2015). Additionally, high Axl expression and phosphorylation was detected in both primary and recurrent GBM samples, but levels were higher in the recurrent samples (Onken et al., 2016). Overexpression of Axl expression was not found to be as high in human astrocytes, normal brain tissue, and low grade https://www.thno.org/v10p5719.htm https://www.thno.org/v10p5719.htm https://link.springer.com/article/10.1186/s13046-023-02726-w#ref-CR41 https://www.oncotarget.com/article/2542/text/ https://www.nature.com/articles/s41392-024-02121-7 https://pmc.ncbi.nlm.nih.gov/articles/PMC4957976/ https://www.nature.com/articles/s41392-024-02121-7 https://molecular-cancer.biomedcentral.com/articles/10.1186/s12943-019-1090-3#:~:text=The%20word%20AXL%2C%20coming%20from,and%20intracellular%20domain%20%5B8%5D. https://www.oncotarget.com/article/2542/text/ https://www.sciencedirect.com/science/article/pii/S1357272525000172?via%3Dihub https://pmc.ncbi.nlm.nih.gov/articles/PMC5778882/#:~:text=Axl%20is%20overexpressed%20in%20the,survival%20advantage%20to%20tumor%20cells. https://pmc.ncbi.nlm.nih.gov/articles/PMC5778882/#:~:text=Axl%20is%20overexpressed%20in%20the,survival%20advantage%20to%20tumor%20cells. https://pmc.ncbi.nlm.nih.gov/articles/PMC4695118/ https://pmc.ncbi.nlm.nih.gov/articles/PMC4891090/ https://www.cell.com/stem-cell-reports/fulltext/S2213-6711(15)00099-5?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2213671115000995%3Fshowall%3Dtrue https://www.cell.com/stem-cell-reports/fulltext/S2213-6711(15)00099-5?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2213671115000995%3Fshowall%3Dtrue https://www.pnas.org/doi/abs/10.1073/pnas.0510923103 https://pmc.ncbi.nlm.nih.gov/articles/PMC5584143/ https://pmc.ncbi.nlm.nih.gov/articles/PMC5584143/ https://aacrjournals.org/clincancerres/article/14/1/130/196285/Axl-and-Growth-Arrest-Specific-Gene-6-Are https://aacrjournals.org/cancerres/article/78/11/3002/625035/Activation-of-the-Receptor-Tyrosine-Kinase-AXL https://pmc.ncbi.nlm.nih.gov/articles/PMC4891090/ https://pmc.ncbi.nlm.nih.gov/articles/PMC4891090/ https://www.cell.com/stem-cell-reports/fulltext/S2213-6711(15)00099-5?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2213671115000995%3Fshowall%3Dtrue https://pmc.ncbi.nlm.nih.gov/articles/PMC4891090/ 26 gliomas (Cheng et al., 2015; Vouri et al., 2015). Together, this could indicate that Axl contributes to the aggressive nature and growth of the mesenchymal GBM subtype and recurrent tumors. High Axl expression in GBM tumors is associated with poor prognosis. GBM patients with high Axl expression had a lower median time to tumor progression (TTP) and lower median overall survival (OS) than those with low Axl expression (Hutterer et al., 2008). Increased Axl expression in patients with GBM has also been associated with therapeutic resistance (Scherschinski et al., 2022). Moreover, pAxl expression in both the tumor vasculature and hypercellular areas was associated with a decrease in OS (Onken et al., 2017). Axl signaling has been associated with cell proliferation, growth, migration, and invasion of GBM (Cheng et al., 2015 ; Vajkoczy et al., 2006; Hutterer et al., 2008). Moderate to high Axl and Gas6 expression along with Axl/Gas6 coexpression has been detected in GBM tissue samples (Hutterer et al., 2008). Furthermore, patients with high coexpression of Axl/Gas6 also had a lower median TTP and OS than those with low coexpression, indicating that the Axl/Gas6 pathway is an important regulator in GBM (Hutterer et al., 2008). In some GBM cell lines (i.e. SNB-19), Gas6 appeared to phosphorylate Axl (Vouri et al., 2015). However, in other cell lines (i.e. UP007) Axl activity appeared to be Gas-6 independent and the expression of Gas6 and Axl in vascular proliferation areas displayed different patterns (Vouri et al., 2015; Sadahiro et al., 2018). This indicates that many mechanisms may contribute to Axl activity in GBM. One alternative mechanism is ligand dependent activation with PROS1. PROS1, produced by tumor-associated microglia/macrophages, has been found to activate Axl in glioma sphere cultures leading to increased growth and aggressive mesenchymal GBM (Sadahiro et al., 2018). Poor prognosis for GBM patients has been associated with high expression of Axl and PROS1 (Sadahiro et al., 2018). Ligand-independent mechanisms are also thought to be involved in Axl https://www.cell.com/stem-cell-reports/fulltext/S2213-6711(15)00099-5?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2213671115000995%3Fshowall%3Dtrue https://pubmed.ncbi.nlm.nih.gov/25980499/ https://aacrjournals.org/clincancerres/article/14/1/130/196285/Axl-and-Growth-Arrest-Specific-Gene-6-Are https://www.mdpi.com/1422-0067/23/2/982 https://pmc.ncbi.nlm.nih.gov/articles/PMC5584143/ https://www.cell.com/stem-cell-reports/fulltext/S2213-6711(15)00099-5?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2213671115000995%3Fshowall%3Dtrue https://www.pnas.org/doi/abs/10.1073/pnas.0510923103 https://aacrjournals.org/clincancerres/article/14/1/130/196285/Axl-and-Growth-Arrest-Specific-Gene-6-Are https://aacrjournals.org/clincancerres/article/14/1/130/196285/Axl-and-Growth-Arrest-Specific-Gene-6-Are https://aacrjournals.org/clincancerres/article/14/1/130/196285/Axl-and-Growth-Arrest-Specific-Gene-6-Are https://pubmed.ncbi.nlm.nih.gov/25980499/ https://pubmed.ncbi.nlm.nih.gov/25980499/ https://aacrjournals.org/cancerres/article/78/11/3002/625035/Activation-of-the-Receptor-Tyrosine-Kinase-AXL https://aacrjournals.org/cancerres/article/78/11/3002/625035/Activation-of-the-Receptor-Tyrosine-Kinase-AXL https://aacrjournals.org/cancerres/article/78/11/3002/625035/Activation-of-the-Receptor-Tyrosine-Kinase-AXL https://aacrjournals.org/cancerres/article/78/11/3002/625035/Activation-of-the-Receptor-Tyrosine-Kinase-AXL 27 activation in GBM including Axl dimerization and autophosphorylation, Axl/Tyro3 heterodimerization, or crossphosphorylation of Axl by EGFR (Vouri et al., 2016 ; Onken et al., 2017; Vouri et al., 2015). Regardless of the mechanisms however, knockdown of Axl or treatment with Axl inhibitors increased survival of mice with GBM tumors indicating that Axl is a promising therapeutic target for GBM specifically. (Vajkoczy et al., 2006; Sadahiro et al., 2018; Cheng et al., 2015). 2.3.4 Bemcentinib Bemcentinib (BEM), also known as BGB324 and R428, is an orally bioavailable selective AXL inhibitor (Holland et al., 2010). Demonstrating anti-tumor activity in several types of cancers, BEM was the first Axl-specific inhibitor to enter clinical trials (Sheridan, 2013). Furthermore, in 2021, BEM was granted Fast Track designation with an anti-PD-(l)1 agent by the U.S. Food & Drug Administration (Asa, 2021). In vivo and in vitro studies have shown that BEM can inhibit metastasis, suppress angiogenesis, prolong survival, induce apoptosis, decrease proliferation, reduce tumor growth, and reduce resistance to chemotherapy and radiation (Holland et al., 2010; Chen et al., 2018; Beitzen-Heineke et al., 2021; Vouri et al., 2015; Scherschinski et al., 2022). However, the full mechanism of BEM is still relatively unknown. After antibody-mediated cross-linking or Gas9 activation, BEM blocked Axl autophosphorylation at the Tyr821 docking site (Holland et al., 2010). Blocking the phosphorylation of Axl could then inhibit downstream signalling pathways. Negative myeloproliferative neoplasm cell lines with Axl expression (SET-2 and BaF3-EpoR-JAK2V617F) demonstrated a dose-dependent reduction in cell viability, decreased proliferation, and increased apoptosis after treatment of BEM (Beitzen-Heineke et al., 2021). A reduction in cell viability was not seen in the cell lines without Axl expression (Beitzen-Heineke https://pubmed.ncbi.nlm.nih.gov/27775700/ https://pmc.ncbi.nlm.nih.gov/articles/PMC5584143/ https://pmc.ncbi.nlm.nih.gov/articles/PMC5584143/ https://pubmed.ncbi.nlm.nih.gov/25980499/ https://www.pnas.org/doi/abs/10.1073/pnas.0510923103 https://aacrjournals.org/cancerres/article/78/11/3002/625035/Activation-of-the-Receptor-Tyrosine-Kinase-AXL https://www.cell.com/stem-cell-reports/fulltext/S2213-6711(15)00099-5?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2213671115000995%3Fshowall%3Dtrue https://aacrjournals.org/cancerres/article/70/4/1544/562308/R428-a-Selective-Small-Molecule-Inhibitor-of-Axl https://www.nature.com/articles/nbt0913-775a https://www.prnewswire.com/news-releases/bergenbio-receives-fda-fast-track-designation-for-bemcentinib-in-stk11-mutated-advancedmetastatic-non-small-lung-cancer-nsclc-301419799.html https://aacrjournals.org/cancerres/article/70/4/1544/562308/R428-a-Selective-Small-Molecule-Inhibitor-of-Axl https://pmc.ncbi.nlm.nih.gov/articles/PMC6129480/#:~:text=Abstract,regulation%20of%20autophagy%20and%20apoptosis. https://pmc.ncbi.nlm.nih.gov/articles/PMC8357258/ https://pubmed.ncbi.nlm.nih.gov/25980499/ https://www.mdpi.com/1422-0067/23/2/982 https://aacrjournals.org/cancerres/article/70/4/1544/562308/R428-a-Selective-Small-Molecule-Inhibitor-of-Axl https://aacrjournals.org/cancerres/article/70/4/1544/562308/R428-a-Selective-Small-Molecule-Inhibitor-of-Axl https://pmc.ncbi.nlm.nih.gov/articles/PMC8357258/ https://pmc.ncbi.nlm.nih.gov/articles/PMC8357258/ 28 et al., 2021). Treatment with BEM decreased phosphorylated Axl levels and specifically reduced STAT5 phosphorylation in Axl+ human SET-2 cells and AKT phosphorylation in Axl+ murine BaF3-EpoR-JAK2V617F cells (Beitzen-Heineke et al., 2021). Both pathways are related to Axl and have been known to promote proliferation and survival of tumor cells (Yadav et al., 2025; Halim et al., 2020; He et al., 2021). Furthermore, BEM has been found to reduce expression of Snail, an epithelial-mesenchymal transition regulator regulated by Axl (Holland et al., 2010). BEM may also operate using additional mechanisms beyond the Axl/Gas6 pathway. After human non-small cell lung carcinoma cells (H1299) were treated with BEM, growth was inhibited and PARP cleavage was induced (indicating apoptosis). Interestingly however, even though Axl phosphorylation was initially inhibited by the treatment, the levels bounced back after six hours and continued to rise without an increase in Gas6 expression (Chen et al., 2018). Since BEM is lysosomotropic this decrease in inhibition could have been due BEM becoming trapped in lysosomes preventing its interaction with Axl (Chen et al., 2018). In cervical cancer cells (Hela), BEM induced PARP cleavage with and without Axl expression indicating that BEM may induce apoptosis using a mechanism independent of Axl (Chen et al., 2018). One alternative mechanism suggests that BEM may lead to lysosomal deformation and apoptosis by altering acidification, inhibiting protein degradation, and blocking lysosome recycling (Chen et al., 2018). In vitro, BEM has reduced GBM cell survival, proliferation, migration, and invasion. However, different cell lines appear to exhibit varying sensitivities to BEM treatment. UP007 cells (IC50 = 1 µM) were more sensitive to BEM when compared to SNB-19 cells (IC50 = 2.5 µM) and mesenchymal GBM stem-like cells (IC50 = 1.027 µM) were more sensitive to BEM than PN_528 GBM stem-like cells (IC50 = 2.035 µM; Vouri et al., 2015; Cheng et al., 2015). The https://pmc.ncbi.nlm.nih.gov/articles/PMC8357258/ https://pmc.ncbi.nlm.nih.gov/articles/PMC8357258/ https://www.nature.com/articles/s41392-024-02121-7 https://pmc.ncbi.nlm.nih.gov/articles/PMC7555335/#:~:text=STAT5%20plays%20a%20crucial%20role,cell%20proliferation%2C%20survival%20and%20metastasis. https://www.nature.com/articles/s41392-021-00828-5 https://aacrjournals.org/cancerres/article/70/4/1544/562308/R428-a-Selective-Small-Molecule-Inhibitor-of-Axl https://pmc.ncbi.nlm.nih.gov/articles/PMC6129480/#:~:text=Abstract,regulation%20of%20autophagy%20and%20apoptosis. https://pmc.ncbi.nlm.nih.gov/articles/PMC6129480/#:~:text=Abstract,regulation%20of%20autophagy%20and%20apoptosis. https://pmc.ncbi.nlm.nih.gov/articles/PMC6129480/#:~:text=Abstract,regulation%20of%20autophagy%20and%20apoptosis. https://pmc.ncbi.nlm.nih.gov/articles/PMC6129480/#:~:text=Abstract,regulation%20of%20autophagy%20and%20apoptosis. https://pmc.ncbi.nlm.nih.gov/articles/PMC6129480/#:~:text=Abstract,regulation%20of%20autophagy%20and%20apoptosis. https://pubmed.ncbi.nlm.nih.gov/25980499/ https://pubmed.ncbi.nlm.nih.gov/25921812/ 29 heterogeneity of GBM could explain the varying sensitivities as both in vitro and in vivo experiments demonstrated that higher Axl expression enhances the effectiveness of BEM in reducing growth and increasing survival (Sadahiro et al., 2018). In vivo, treatment with BEM decreased glioma sphere culture derived mesenchymal GBM-like tumor growth and increased the survival time of mice (Sadahiro et al., 2018). While the mechanism that BEM uses in GBM is unknown, the treatment appears to inhibit Gas6-independent Axl phosphorylation and downstream Akt phosphorylation (Vouri et al., 2015). The PROS1/Axl pathway could be involved here as BEM inhibited PROS1 induced Axl phosphorylation and downstream activation of NFκB in mesenchymal glioma sphere cultures (Sadahiro et al., 2018). BEM did not stop Axl phosphorylation by EGFR however, indicating that BEM’s anti-tumor effects in GBM may be limited by the range of possible pathways involved (Vouri et al., 2016). This is why combination treatments may be more effective. To enhance treatment outcomes BEM is often used in combination with other types of inhibitors or treatments. Treating MCF7 breast cancer cells with BEM, for example, resulted in increased sensitivity to 4-OHT, an estrogen receptor inhibitor (Kim et al., 2025). Treating CMS4 colorectal cancer cells with BEM and galunisertib (a TGFβ inhibitor), reduced colony formation and tumor migration (Ciardiello et al., 2021). Treating diffuse intrinsic pontine glioma cells with BEM and a HDAC inhibitor (panobinostat) decreased expression of DNA damage repair and mesenchymal genes resulting in increased sensitivity to radiation (Meel et al., 2020). Mice with syngeneic GBM tumors displayed prolonged survival after treatment with BEM and a PD-1 antibody (Sadahiro et al., 2018). For GBM, combining BEM with TMZ and radiation has shown enhanced effects (Vouri et al., 2015; Scherschinski et al., 2022). While a single dose of TMZ had minor effects on Axl mRNA and protein expression, repeated doses lead to post-translational Axl https://aacrjournals.org/cancerres/article/78/11/3002/625035/Activation-of-the-Receptor-Tyrosine-Kinase-AXL https://aacrjournals.org/cancerres/article/78/11/3002/625035/Activation-of-the-Receptor-Tyrosine-Kinase-AXL https://pubmed.ncbi.nlm.nih.gov/25980499/ https://aacrjournals.org/cancerres/article/78/11/3002/625035/Activation-of-the-Receptor-Tyrosine-Kinase-AXL https://pubmed.ncbi.nlm.nih.gov/27775700/ https://www.sciencedirect.com/science/article/pii/S1357272525000172?via%3Dihub https://link.springer.com/article/10.1007/s12032-021-01464-3 https://aacrjournals.org/clincancerres/article-abstract/26/13/3319/82769/Combined-Therapy-of-AXL-and-HDAC-Inhibition https://aacrjournals.org/cancerres/article/78/11/3002/625035/Activation-of-the-Receptor-Tyrosine-Kinase-AXL https://pubmed.ncbi.nlm.nih.gov/25980499/ https://www.mdpi.com/1422-0067/23/2/982 30 modification in GBM cell lines indicating that Axl may play a role in therapeutic resistance (Scherschinski et al., 2022). Additionally, elevated Axl expression was associated with increased resistance to radiotherapy (Scherschinski et al., 2022). Treatment with BEM increased the efficacy of TMZ and radiation even in TMZ resistant GBM cell lines (Scherschinski et al., 2022). To date, BEM has been involved in several phase I and II clinical trials (ClinicalTrials.gov ID: NCT06516887, NCT06469138, NCT05469178, NCT03965494, NCT03824080, NCT03654833, NCT03649321, NCT03184571, NCT03184558, NCT02922777, NCT02872259, NCT02488408, NCT02424617). The completed clinical trials indicated that BEM treatment alone or in combination with other drugs (elotinib, cytarabine, decitabine, dabrafenib/trametinib, pembrolizumab, docetaxel) was generally well tolerated and effective for patients with non-small cell lung cancer (NSCLC), acute myeloid leukemia and myelodysplastic syndrome, and metastatic melanoma (Byers et al., 2021; Loges et al., 2025; Straume et al., 2018; Rashdan et al., 2018; Felip et al., 2023; Kubasch et al., 2023). One phase 1 trial (ClinicalTrials.gov ID: NCT03965494) used BEM to treat patients with recurrent Glioblastoma. The plan was to treat some patients with BEM before surgery and the other patients with BEM after surgery. However, this trial was terminated in October 2023 due to redirection. Ongoing clinical trials are currently investigating BEM in combination with chemo-immunotherapy for patients with non-small cell lung cancer (ClinicalTrials.gov ID: NCT05469178) and BEM in combination with pacritinib in patients with advanced lung adenocarcinoma (ClinicalTrials.gov ID: NCT06516887). 2.4 Photodynamic Therapy 2.4.1 Overview of Photodynamic Therapy (PDT) https://www.mdpi.com/1422-0067/23/2/982 https://www.mdpi.com/1422-0067/23/2/982 https://www.mdpi.com/1422-0067/23/2/982 https://www.mdpi.com/1422-0067/23/2/982 https://clinicaltrials.gov/study/NCT06516887?id=NCT06516887,NCT06469138,NCT05469178,NCT04890509,NCT03965494,NCT03824080,NCT03654833,NCT03649321,NCT03184571,NCT03184558,NCT02922777,NCT02872259,NCT02488408,NCT02424617&rank=1 https://clinicaltrials.gov/study/NCT06469138?id=NCT06516887,NCT06469138,NCT05469178,NCT04890509,NCT03965494,NCT03824080,NCT03654833,NCT03649321,NCT03184571,NCT03184558,NCT02922777,NCT02872259,NCT02488408,NCT02424617&rank=2 https://clinicaltrials.gov/study/NCT05469178?id=NCT06516887,NCT06469138,NCT05469178,NCT04890509,NCT03965494,NCT03824080,NCT03654833,NCT03649321,NCT03184571,NCT03184558,NCT02922777,NCT02872259,NCT02488408,NCT02424617&rank=3 https://clinicaltrials.gov/study/NCT03965494?id=NCT06516887,NCT06469138,NCT05469178,NCT04890509,NCT03965494,NCT03824080,NCT03654833,NCT03649321,NCT03184571,NCT03184558,NCT02922777,NCT02872259,NCT02488408,NCT02424617&rank=5 https://clinicaltrials.gov/study/NCT03824080?id=NCT06516887,NCT06469138,NCT05469178,NCT04890509,NCT03965494,NCT03824080,NCT03654833,NCT03649321,NCT03184571,NCT03184558,NCT02922777,NCT02872259,NCT02488408,NCT02424617&rank=6 https://clinicaltrials.gov/study/NCT03654833?id=NCT06516887,NCT06469138,NCT05469178,NCT04890509,NCT03965494,NCT03824080,NCT03654833,NCT03649321,NCT03184571,NCT03184558,NCT02922777,NCT02872259,NCT02488408,NCT02424617&rank=7 https://clinicaltrials.gov/study/NCT03649321?id=NCT06516887,NCT06469138,NCT05469178,NCT04890509,NCT03965494,NCT03824080,NCT03654833,NCT03649321,NCT03184571,NCT03184558,NCT02922777,NCT02872259,NCT02488408,NCT02424617&rank=8 https://clinicaltrials.gov/study/NCT03184571?id=NCT06516887,NCT06469138,NCT05469178,NCT04890509,NCT03965494,NCT03824080,NCT03654833,NCT03649321,NCT03184571,NCT03184558,NCT02922777,NCT02872259,NCT02488408,NCT02424617&rank=9 https://clinicaltrials.gov/study/NCT03184558?id=NCT06516887,NCT06469138,NCT05469178,NCT04890509,NCT03965494,NCT03824080,NCT03654833,NCT03649321,NCT03184571,NCT03184558,NCT02922777,NCT02872259,NCT02488408,NCT02424617&rank=10 https://clinicaltrials.gov/study/NCT02922777?id=NCT06516887,NCT06469138,NCT05469178,NCT04890509,NCT03965494,NCT03824080,NCT03654833,NCT03649321,NCT03184571,NCT03184558,NCT02922777,NCT02872259,NCT02488408,NCT02424617&rank=11 https://clinicaltrials.gov/study/NCT02872259?id=NCT06516887,NCT06469138,NCT05469178,NCT04890509,NCT03965494,NCT03824080,NCT03654833,NCT03649321,NCT03184571,NCT03184558,NCT02922777,NCT02872259,NCT02488408,NCT02424617&rank=12 https://clinicaltrials.gov/study/NCT02488408?id=NCT06516887,NCT06469138,NCT05469178,NCT04890509,NCT03965494,NCT03824080,NCT03654833,NCT03649321,NCT03184571,NCT03184558,NCT02922777,NCT02872259,NCT02488408,NCT02424617&rank=13 https://clinicaltrials.gov/study/NCT02424617?id=NCT06516887,NCT06469138,NCT05469178,NCT04890509,NCT03965494,NCT03824080,NCT03654833,NCT03649321,NCT03184571,NCT03184558,NCT02922777,NCT02872259,NCT02488408,NCT02424617&rank=14 https://ascopubs.org/doi/10.1200/JCO.2021.39.15_suppl.9110 https://pubmed.ncbi.nlm.nih.gov/40122885/ https://ascopubs.org/doi/10.1200/JCO.2018.36.15_suppl.9548 https://ascopubs.org/doi/10.1200/JCO.2018.36.15_suppl.e21043 https://www.annalsofoncology.org/article/S0923-7534(23)03308-2/fulltext https://www.nature.com/articles/s41375-023-02029-1 https://clinicaltrials.gov/study/NCT03965494?id=NCT06516887,NCT06469138,NCT05469178,NCT04890509,NCT03965494,NCT03824080,NCT03654833,NCT03649321,NCT03184571,NCT03184558,NCT02922777,NCT02872259,NCT02488408,NCT02424617&rank=5 https://clinicaltrials.gov/study/NCT05469178?id=NCT06516887,NCT06469138,NCT05469178,NCT04890509,NCT03965494,NCT03824080,NCT03654833,NCT03649321,NCT03184571,NCT03184558,NCT02922777,NCT02872259,NCT02488408,NCT02424617&rank=3 https://clinicaltrials.gov/study/NCT06516887?id=NCT06516887,NCT06469138,NCT05469178,NCT04890509,NCT03965494,NCT03824080,NCT03654833,NCT03649321,NCT03184571,NCT03184558,NCT02922777,NCT02872259,NCT02488408,NCT02424617&rank=1 31 Phototherapy, a method of using light to treat disease, has been used since ancient civilizations where sunlight exposure was used as treatment for various types of skin diseases (Correia et al., 2021). This concept led to the development of photodynamic therapy (PDT) in 1900 in Munich Germany where Oscar Raab discovered the basis of PDT when he found acridine red dye and light combined resulted in a greater cytotoxic effect than the dye or light alone in paramecium (Hamblin, 2020). Further research and advancements in PDT led to its first approval in 1993 in Canada for prophylactic treatment of bladder cancer using porfimer sodium (Photofrin), then leading to approvals of PDT and Photofrin in European countries, Japan, and the United States later on (Dougherty et al., 1998). PDT is a method that utilizes photosensitizers (PS) and light to selectively kill cancer cells, making the treatment minimally invasive. PDT can be used in combination with other treatment methods such as surgical resection. In a study with 77 patients with GBM, the median survival for patients that received PDT after resection was 11 months and the patients that received only resection had a median survival of 8 months (Aebisher et al., 2024). Another clinical trial utilized a combination treatment with PDT and tumor-treating fields therapy (TTF) on 14 eligible patients, where median survival for treated patients was 13.4 months as compared to 11 months for those untreated, and 6 patients surpassing the 2 year mark (Fukami et al. 2025). Along with the improved patient survival rate, patient quality of life was increased with PDT with reduced toxicity to healthy tissue and side effects post-treatment (Kim & Chang, 2023). Future advancements to PDT will improve the efficacy of the treatment so that it may be applied to complex cancer types and improve outcomes for patients (Allamyradov et al., 2024). 2.4.2 Mechanism https://pmc.ncbi.nlm.nih.gov/articles/PMC8470722/ https://onlinelibrary.wiley.com/doi/10.1111/php.13190#:~:text=The%20phenomenon%20underlying%20the%20scientific,then%20added%20to%20the%20paramecium. https://pmc.ncbi.nlm.nih.gov/articles/PMC4592754/#:~:text=Regulatory%20Status%E2%80%94Photofrin,11%20additional%20countries%20in%20Europe. https://pmc.ncbi.nlm.nih.gov/articles/PMC10886821/#:~:text=Median%20survival%20was%2011%20months,31%2C196%2C198%5D. https://pmc.ncbi.nlm.nih.gov/articles/PMC11906418/ https://pmc.ncbi.nlm.nih.gov/articles/PMC10535460/ https://www.mdpi.com/2673-7256/4/4/27 32 PDT is a therapeutic modality dependent on three factors: a photosensitizer (PS), light with a specific wavelength, and the presence of a molecular oxygen (Jiang et al., 2023). The mechanism of action of PDT is based on a series of biochemical and photophysical events that occur upon the administration of a photosensitizer and its subsequent activation by light of a specific wavelength. The first step is the administration of a PS, a compound that absorbs the photon of light at the specified wavelength. The administration can be done topically or intravenously, enabling the PS to aggregate around the treatment site (Correia et al., 2021). Specificity of the PS to its target typically varies with respect to its molecular properties, or conjugation to site-specific antibodies, peptides, proteins and other ligands (Abrahamse & Hamblin, 2016). Once the PS has accumulated in the target area, light activation raises the energy state of the PS to its excited state. As the energy state stabilizes, intersystem crossing and stabilization to a rovibrational triplet excited state occurs. This highly reactive triplet excited state reacts with endogenous substances to form reactive oxygen species (ROS) in two ways: Type I and Type II. During a Type I reaction, the triplet excited state reacts directly with the surrounding molecules, yielding free radicals and radical ions such as superoxides (Jiang et al., 2023). Alternatively, during Type II reactions, the PS reacts with surrounding molecular oxygen, and this energy transfer yields a highly reactive singlet state oxygen (Correia et al., 2021). Ultimately, the products from both Type I and Type II reactions induce biological lesions through oxidative damage, resulting in a degradation of the target. The ROS induces oxidative damage by disrupting the structure of DNA, protein and lipids. As a result, ROS disrupts cellular membranes, ultimately reducing the integrity of the cell and causes the release of intracellular contents and inducing inflammation. In addition, ROS serves as signaling molecules which https://www.mdpi.com/2072-6694/15/3/585 https://www.mdpi.com/1999-4923/13/9/1332 https://pmc.ncbi.nlm.nih.gov/articles/PMC4811612/ https://www.mdpi.com/2072-6694/15/3/585 https://www.mdpi.com/1999-4923/13/9/1332 33 regulate many vital biological pathways, suggesting that ROS plays a key role in apoptotic and necrotic regulation (Schieber & Chandel, 2014). Figure 1: Diagram displaying the mechanism of PDT with NanoVP (photosensitizer) for GBM. (a) GBM tumor is detected and highlighted. (b) NanoVP is administered intravenously. (c) NanoVP becomes distributed via the circulatory system. (d) NanoVP localizes to the target site in the GBM tissue. (e) NanoVP is activated to its triplet excited state via light irradiation. (f) Generation of ROS through Type I or II reactions leads to GBM cell death. 2.4.3 Photosensitizers One of the most important characteristics of an effective PDT treatment is the selection of an adequate photosensitizer to ensure site-specific targeting. It is well known that porphyrins and expanded porphyrins are one of the most well studied types of photosensitizers due to their ability to strongly absorb waves of light leading to high tissue penetration coupled with high singlet oxygen production (Pushpan et al., 2002). In the context of GBM, one such porphyrin called 5-aminolevulinic acid (5-ALA), an intermediate metabolite in the hemoglobin metabolic https://pmc.ncbi.nlm.nih.gov/articles/PMC4055301/ https://www.eurekaselect.com/article/37008 34 pathway, has been shown to accumulate in malignant brain tumors. In 2017, 5-ALA was given FDA approval for intraoperative optical imaging agent in patients with suspected high-grade gliomas (HGGs), due to the fluorescent capabilities of its metabolized form in protoporphyrin IX (PpIX) (Hadjipanayis & Stummer, 2019) . However, under its metabolized state, PpIX has also demonstrated photosensitizing capabilities, which has since led to its use as a PDT photosensitizer in subsequent clinical studies. The preliminary results from the INDYGO clinical trial, composed of ten patients with newly diagnosed GBM who underwent PDT adjuvant therapy beginning between May 2017 and June 2018, indicate an improved median survival rate of 23.1 months, as compared to 15 months under the current standard of care of surgical resection, followed by chemo- and radiotherapy (RT). Additionally, 60% of patients demonstrated no statistically significant progression in the first 12 months of the treatment (Vermandel et al., 2021). Another applied porphyrin called Verteporfin (VP) is primarily utilized as a photosensitizer for the FDA approved PDT treatment of age related macular degeneration (Wei & Li, 2020). However, VP has demonstrated up to 99% uptake by glioma cells, leading to further investigation of VP as a photosensitizer for GBM. In an in vitro study where glioma cell lines were treated with varied concentrations of VP, there was a statistically significant reduction in VP-PDT treated cells vs VP alone and related controls (Jeising et al., 2022). Current studies are being done to investigate the opportunity to utilize VP as an intraoperative dye during GBM surgical resection, similar to how 5-ALA is used in the same context. 2.4.4 Limitations The depth of light penetration remains a large issue in PDT. The phototherapeutic window of 600-800 nm is the optimal wavelength range where there is enough energy to excite https://pubmed.ncbi.nlm.nih.gov/30644008/ https://link.springer.com/article/10.1007/s11060-021-03718-6 https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2020.557429/full https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2020.557429/full https://www.sciencedirect.com/science/article/pii/S1572100022003350?via%3Dihub 35 the PS and sufficient penetration into the tissue. Wavelengths exceeding 800 nm have deeper penetration but not enough energy to generate a strong photodynamic effect (Huis et al., 2023). Due to the short wavelengths of the phototherapeutic window, the depth of penetration is limited to about 6 mm, making PDT not suitable to treat deeper tumors (Li & Lin, 2022). PDT relies heavily on the oxygen of the surrounding environment as the treatment method generates reactive oxygen species to induce a cytotoxic effect that leads to cell death (Correia et al., 2021). Tumors have low levels of oxygen due to rapid cell proliferation and abnormal structure of blood vessels that reduce the level of oxygen and nutrient transport to the tumors. Due to this, tumor hypoxia is a major factor in poor prognosis in various types of cancer treatments (Muz et al., 2015). The use of PDT itself can worsen tumor hypoxia as the already limited oxygen is consumed during treatment, reducing the photodynamic effect. The specific side effects associated after PDT treatment depends on the area being treated. The common side effects include pain and skin irritation. In a study analyzing the pain level of PDT for actinic keratoses, many patients reported feeling a burning, stinging sensation during treatment. In some cases, the treatment had to be paused before continuing due to the severe pain felt (Halldin et al., 2013). The area where the laser was exposed to can become irritated and blistered, causing further pain post-surgery. Although both 5-ALA and Verteporfrin demonstrate promise as effective photosensitizers for the treatment of GBM, there are limitations to the scope of studies that have been done and inherent properties of the compounds themselves. From a resources standpoint, VP is undergoing global storage, where almost all production is centered in a single American factory (Sirks et al., 2024). Additionally, red-light wavelengths used in PDT have poor penetration through thick https://www.mdpi.com/1999-4923/15/2/330 https://www.nature.com/articles/s41377-022-00780-1 https://www.mdpi.com/1999-4923/13/9/1332 https://pmc.ncbi.nlm.nih.gov/articles/PMC5045092/#:~:text=Tumor%20hypoxia%20develops%20due%20to,transport%20of%20oxygen%20and%20nutrients.&text=Hypoxia%20is%20one%20of%20the,poor%20prognosis%20of%20cancer%20patients. https://www.medicaljournals.se/acta/content/html/10.2340/00015555-1500 https://link.springer.com/article/10.1007/s40123-024-00952-9 https://link.springer.com/article/10.1007/s40123-024-00952-9 36 tissues or deep tumors, limiting the scope of PDT inclusive treatments to surgical candidates and intraoperative procedures (Nordmann & Michael, 2021). 2.4.5 Current Uses/Combinatorial Approaches with PDT After the first approval of PDT with Photofrin in Canada, the application of PDT on various types of cancers expanded to other countries. At the time, the Netherlands and France had approved PDT and Photofrin as treatment for advanced esophageal and lung cancers, Germany for early stage lung cancer, and Japan for early stage lung, esophageal, gastric, and cervical cancers (Dougherty et al., 1998). The Food and Drug Administration (FDA) in the United States has currently approved the use of PDT for actinic keratosis (AK), basal cell carcinoma (BCC), squamous cell carcinoma (SCC), advanced cutaneous T-cell lymphoma, Barrett esophagus, esophageal cancer, and non-small cell lung cancer (NSCLC) (National Cancer Institute, 2021). The application of PDT for various types of cancers are currently being researched, including GBM. PDT as a treatment method for cancers have been involved in various phase I/II clinical trials including BCC, NSCLC, and non-melanoma skin cancer (ClinicalTrials.gov ID: NCT00985829, NCT02916745, NCT02872909). A large number of clinical trials involving PDT and GBM are currently recruiting (NCT05363826, NCT04391062, NCT03897491, NCT05736406) with various methods (post-resection, stereotactic, and intraoperative PDT) and PSs (Photobac®, 5-ALA, GLIOLAN®). A completed phase II clinical trial (NCT03048240) treated 10 patients with GBM 5-ALA FGS followed by intraoperative PDT treatment. The patients did not experience serious adverse side effects and had a 12-months progression-free survival rate of 60% and a 12-months overall survival rate of 80% (Vermandel et al., 2021). PDT is particularly well-suited for treating GBM, as it is capable of targeting tumors that are unable to https://www.sciencedirect.com/science/article/pii/S0303846720307733 https://pmc.ncbi.nlm.nih.gov/articles/PMC4592754/#:~:text=Regulatory%20Status%E2%80%94Photofrin,11%20additional%20countries%20in%20Europe. https://www.cancer.gov/about-cancer/treatment/types/photodynamic-therapy#cancer-and-precancers-treated-with-photodynamic-therapy https://www.cancer.gov/about-cancer/treatment/types/photodynamic-therapy#cancer-and-precancers-treated-with-photodynamic-therapy https://clinicaltrials.gov/study/NCT00985829 https://clinicaltrials.gov/study/NCT02916745?cond=NSCLC&intr=photodynamic%20therapy&rank=3 https://clinicaltrials.gov/study/NCT02872909?cond=Non-Melanoma%20Skin%20Cancer&intr=photodynamic%20therapy&rank=1 https://clinicaltrials.gov/study/NCT05363826?cond=Cancer&intr=photodynamic%20therapy&rank=7 https://clinicaltrials.gov/study/NCT04391062?cond=Glioblastoma%20Multiforme&intr=photodynamic%20therapy&page=1&viewType=Table&rank=4 https://clinicaltrials.gov/study/NCT03897491?cond=Glioblastoma%20Multiforme&intr=photodynamic%20therapy&page=1&viewType=Table&rank=5 https://clinicaltrials.gov/study/NCT05736406?cond=Glioblastoma%20Multiforme&intr=photodynamic%20therapy&page=1&viewType=Table&rank=6 https://clinicaltrials.gov/study/NCT03048240?cond=Glioblastoma%20Multiforme&intr=photodynamic%20therapy&page=1&rank=3&tab=results https://pubmed.ncbi.nlm.nih.gov/33743128/ 37 be resected surgically and it minimizes damage to surrounding cells, which is vital when addressing cancers in the brain. More importantly, it can be used in conjunction with the current treatment modalities for GBM, allowing scientists to formulate an amplified treatment approach (Bhanja et al., 2023). The current standard of care for GBM includes surgical resection, chemotherapy, and radiotherapy combined or as monotherapy. A combinatorial approach with PDT demonstrates a more advantageous outcome compared to conventional monotherapies (Yang et al., 2020). Treatment of GBM using a combination of FGS and PDT was found to increase mean survival to 52.8 weeks and decrease mean tumor progression to 8.6 months compared to surgical resection monotherapy with 24.6 weeks and 4.8 months respectively (Ferres et al., 2022). FGS allows visualization of tumors using fluorescent dyes for a safer and more accurate surgical resection of a tumor (Schupper et al., 2021). 5-ALA is administered orally a few hours before surgery and produces a fluorescent molecule called protoporphyrin IX (PplX) (Nagaya et al., 2017). The ability of 5-ALA to penetrate the BBB and selectively accumulate in GBM cells makes the compound favorable for FGS for GBM (Hadjipanayis et al., 2015). PplX then absorbs light between 375 to 440 nm and fluoresces violet-red at 635 nm (Schupper et al., 2021). The different fluorescence emitted allows differentiation between normal tissue and the tumor for an increased overall tumor resection. In a study with 322 patients with GBM, 65% of patients had complete resection of the tumor using 5-ALA and FGS while only 36% of patients had complete resection using only white light (Stummer et al., 2006). Chemotherapy and radiotherapy are not highly selective treatment methods, often causing chemotoxicity from the damaged DNA and inflammation of healthy cells from the chemotherapy drugs. Chemotoxicity has adverse side effects and reduces patient quality of life (Juthani et al., 2024). In addition, the conventional https://pmc.ncbi.nlm.nih.gov/articles/PMC10341187/ https://pmc.ncbi.nlm.nih.gov/articles/PMC7053190/#:~:text=The%20combined%20therapies%20demonstrate%20superior,greatly%20affect%20the%20overall%20outcomes. https://pmc.ncbi.nlm.nih.gov/articles/PMC9755173/ https://www.frontiersin.org/journals/neurology/articles/10.3389/fneur.2021.682151/full https://www.frontiersin.org/journals/oncology/articles/10.3389/fonc.2017.00314/full https://pmc.ncbi.nlm.nih.gov/articles/PMC4615466/#S13 https://www.frontiersin.org/journals/neurology/articles/10.3389/fneur.2021.682151/full https://www.thelancet.com/journals/lanonc/article/PIIS1470-2045(06)70665-9/abstract https://www.nature.com/articles/s44276-024-00064-8 38 treatment methods as monotherapy for GBM are often resistant and are associated with low survival rate (Yalamarty et al., 2023). Combinatorial therapies using PDT are less toxic yet provide a synergistic, effective treatment approach. The future of PDT includes advances in the development of PSs. Development of new PSs with enhanced selectivity and delivery can improve treatment outcomes as well as be applied to other cancer types. With an improved and advanced PS and delivery system, limitations such as light penetration and PS distribution can be addressed (Allamyradov et al., 2024). Additionally, the use of nanoparticles as a mode of delivery for photosensitizers is being examined, particularly for the treatment of GBM. These aim to improve drug delivery, and thus enhance PDT effectiveness (Aebisher et al., 2024). Overall, PDT is a highly promising and constantly developing new field in GBM treatment, offering solutions that mitigate the typical consequences of the current standard of care while potentially improving survival outcomes for patients with this challenging cancer. https://pmc.ncbi.nlm.nih.gov/articles/PMC10093719/#sec3-cancers-15-02116 https://www.mdpi.com/2673-7256/4/4/27 https://www.mdpi.com/2227-9059/12/2/375 39 3. Methods 3.1 Cell Culture The human glioblastoma cell line, U87, was obtained from the American Type Culture Collection. This particular cell line has high expression of the AXL target of interest (Guo, 2017). The U87 cells were maintained in 5% CO2 at 37℃ and were cultured in Eagle’s Minimal Essential Medium (EMEM; Quality Biological) which contained 10% v/v fetal bovine serum (Gibco), 1% penicillin/streptomycin, and L-glutamine. On the first day of all experiments (Day 1), cells were seeded in black-walled, clear bottom 96-well plates. Media was first aspirated out of the 75 cm2 flask and the cells (70-90% confluent) were washed one time with Dulbecco’s Phosphate-Buffered Saline without calcium and magnesium (DPBS; Corning). To detach the cells, 0.05% Trypsin/0.53 mM EDTA (Corning) was added and the cells. The cells were incubated for five minutes and then centrifuged for five minutes at 1000 RPM/4℃. Once a pellet was achieved, the Trypsin was aspirated out and the cells were resuspended in EMEM and counted using Cellometer® K2 image cytometer. Using the concentration acquired, the cell mixture was diluted with EMEM to achieve a seeding density of 4 x 104 cells/mL (8000 cells/well). The final cell solution was transferred to the 96-well plate using a pipette. To allow the cells to adhere to the plate, they were then placed back in the incubator for 24 hours in 5% CO2 at 37℃. 3.2 NanoVP Synthesis and Characterization NanoVP was synthesized using solvent-antisolvent precipitation with Verteporfin (VP; US Pharmacopeia). The solid VP was dissolved in DMSO. This solution was then dropped into deionized water and stirred resulting in nano-sized particles termed NanoVP (Quinlan et al., 2024). The final concentration of NanoVP (459.11 µM) was determined using using the molar https://www.nature.com/articles/nn.4584/figures/1 https://www.nature.com/articles/nn.4584/figures/1 https://advanced.onlinelibrary.wiley.com/doi/10.1002/advs.202302872 https://advanced.onlinelibrary.wiley.com/doi/10.1002/advs.202302872 40 extinction coefficient and UV-Vis absorbance in DMSO at 435 nm, 687 nm, and 800 nm (BioTek Synergy Neo2 Hybrid Multi-Mode Microplate Reader). Dynamic light scattering (DLS) was employed to measure nanoparticle size, polydispersity index (PdI), and zeta potential (ZP). 3.3 Photodynamic Therapy (PDT) Monotherapy Treatment in Cell Culture To begin the PDT treatment (Day 2), NanoVP (see “NanoVP Synthesis and Characterization”) was removed from the 3℃ refrigerator. An aliquot was taken from this stock and diluted with EMEM to achieve a final concentration of 0.25 µM. The U87 cell plate seeded on Day 1 (see “Cell Culture”) was removed from the incubator (5% CO2 at 37℃) and viewed under the microscope to check for even seeding. Since NanoVP is light sensitive, this was the last time that the cell could be viewed with the microscope. The cells were then washed with 0.2 mL of DPBS prior to treatment. Depending on their assigned group (see Appendix A), the cells were then treated with 0.2 mL of the 0.25 µM NanoVP+EMEM solution, 0.2 mL of a 1% DMSO solution, or 0.2 mL of EMEM. The cells were then placed back into the incubator for the 90-minute photosensitizer light interval. After the interval, NanoVP was activated using the ML8500 laser system (irradiance 50 mW/cm2 , 689 nm). Light dosage used to treat each well ranged from 0 J/cm2 - 10 J/cm2, depending on the assigned group. Following this treatment, cells were placed back into the incubator for about 48 hours. 3.4 Bemcentinib Preparation BEM (507 g/mol) was obtained from MedChemExpress and stored at -20 ℃. Prior to the cytotoxicity study, a 19.7 mM BEM solution was created by dissolving solid BEM in 100% DMSO. For the later combination experiments (Preliminary Combination, CLO, and CLB), a 12.5 mM BEM stock solution was created. Aliquots were made from this 12.5 mM BEM stock before being stored at -20 ℃ for up to one month as recommended by MedChemExpress. For all 41 experiments, the BEM + DMSO sample was only thawed once before the experiment and the thawed samples were disposed of after each experiment. 3.5 Bemcentinib Monotherapy Treatment in Cell Culture On treatment day (Day 2) for the BEM monotherapy, the BEM + DMSO solution (see “Bemcentinib Preparation”) was removed from the freezer and thawed at room temperature. A serial dilution was then performed mixing the BEM + DMSO solution with EMEM to achieve the desired final concentrations (12.5 µM BEM, 6.25 µM BEM, 3.125 µM BEM, 1.56 µM BEM, 0.781 µM BEM, 0.391 µM BEM, 0.195 µM BEM, 0.098 µM BEM) all of which had <0.1% DMSO. In addition to the eight treatment groups (see Appendix A), the cytotoxicity studies also included two control groups: No Treatment (EMEM only) and 0.1% DMSO (DMSO + EMEM). After creating the treatment solutions, the cells seeded on Day 1 (see “Cell Culture”) were removed from the incubator (5% CO2 at 37℃). To check for even seeding, the plate was viewed under the microscope. Following this, the cells were washed one time with 0.2 mL of DPBS. The cells were then treated with 0.2 mL of the appropriate treatment or control solutions. All cells were incubated for 48 hours in 5% CO2 at 37 ℃ following treatment. 3.6 Preliminary Combination Treatment (PDT + BEM) in Cell Culture Preliminary combination experiments were first performed to compare the combination treatment to the PDT and BEM monotherapies. BEM was prepared by thawing a 12.5 mM aliquot (see “Bemcentinib Preparation”) and performing a serial dilution with EMEM to achieve a final concentration of 5 µM and 4 µM . The NanoVP solution was created by diluting a sample of NanoVP stock (see “NanoVP Synthesis and Characterization”) with EMEM to achieve a final concentration of 0.5 µM. A 0.1% DMSO control solution was also created as a control. Cells seeded on Day 1 (see “Cell Culture”) were taken out of the incubator, checked under the 42 microscope for even seeding, and then washed with 0.2 mL DPBS. Each well was then treated with the appropriate EMEM, BEM, NanoVP, and DMSO solutions (see Appendix A for all treatment groups). For the combination treatment 2.5 µM BEM + 0.25µm NanoVP + 0.25 J/cm^2 hv, 0.1 mL of both the 5 µM BEM and the and 0.5 µM NanoVP solutions were added to the wells to achieve the desired final concentrations of 2.5 µM Bem + 0.25µm NanoVP. For the combination treatment 2µM BEM + 0.25µM NanoVP + 0.25 J/cm^2 hv, 0.1 mL of the 4 µM BEM and then 0.5 µM NanoVP solutions were added. The additional dilution performed in the wells was also done for the control wells. For example, the 2µM BEM monotherapy wells received 0.1 mL of the 4 µM BEM solution and 0.1 mL of EMEM to achieve the final concentration of 2µM BEM. After treatment, the cells were incubated (5% CO2 at 37℃) for the 90-minute photosensitizer light interval. The ML8500 laser system (irradiance 50 mW/cm2 , 689 nm) was then used to activate the NanoVP (light dosages ranging from 0 J/cm2 - 10 J/cm2). The plate was then placed back into the incubator for about 48 hours. 3.7 Changing Light and BEM Combination Treatments (PDT + BEM) in Cell Culture After the preliminary combination experiments, additional experiments were performed to investigate the impact of changing light dosage at a constant BEM concentration. The first set of experiments to test this (titled “Changing Light Only” or “CLO”) involved two 96-well plates, both seeded with U87 cells on Day 1 (see “Cell Culture”). The first plate, “Plate A,” was a PDT monotherapy experiment (see “Photodynamic Therapy (PDT) Monotherapy Treatment in Cell Culture”). The second plate, “Plate B,” was a combination experiment where the final concentration of BEM was 2µM. For these plates, an aliquot from the 459.11 µM NanoVP stock (see “NanoVP Synthesis and Characterization”) was diluted with EMEM to achieve a final concentration of 0.5 µM. Additionally, a 12.5 mM BEM aliquot previously prepared (see 43 “Bemcentinib Preparation”), was thawed and diluted with EMEM to achieve a final concentration of 4 µM. A 0.1% DMSO control solution was also created by diluting DMSO in EMEM. The U87 cell plate was removed from the 5% CO2 at 37 ℃ incubator and assessed for even seeding under the microscope. The cells were washed one time with 0.2 mL of DPBS. Following this, the cells were treated (see Appendix A for all treatment groups). As done in the Preliminary Combination experiments, the solutions were diluted a second time in the wells of the plate. For example, cells receiving the combination tr