X14 - Axon-specific Neuroprotection using FA in CIPN model

Introduction: : Chemotherapy-induced peripheral neuropathies (CIPNs) are common, severe sequelae of anti-cancer therapies, and there are no available treatments currently. The side-effects and complications of chemotherapy drugs have significant consequences on a patient's quality of life and can cause discomfort, nausea, cyclothymia, and withdrawals alongside other common side effects. This axonal degeneration, known as "dying-back neuropathy," commonly starts at the distal ends of peripheral nerves and can lead to reduced chemotherapy dosage or stopping the treatment. In this study, we investigated the site-specific neurotoxicity of four chemotherapeutic agents—Paclitaxel (PTX), Monomethyl auristatin E (MMAE), Vincristine (VCR), and Cisplatin (CDDP)—using compartmentalized microfluidic culture platform that isolates cell bodies from axons. PTX, MMAE, and VCR, target microtubule structures, were more toxic when focally applied to axons. In contrast, CDDP, a DNA replication inhibitor, exhibited greater toxicity when applied to cell bodies. In this research, we have shown that FDA-approved drug, fluocinolone acetonide (FA), is neuroprotective against all these chemotherapy drugs in vitro.
We studied the FA-mediated neuroprotection using innovative mitochondrial trafficking analysis methods along axons in the microchannels. FA co-treatment with these drugs restored anterograde mitochondrial trafficking, suggesting that enhanced organelle transport may underlie its neuroprotective mechanism. Mitochondrial trafficking analysis showed that all chemotherapy drugs significantly reduced mitochondrial movement, an important function for axonal health. These findings emphasize the importance of identifying drug-specific sites of action and targeting mitochondrial health as a therapeutic approach. This study provides a foundation for developing effective, focal treatments to CIPN and preserving neuronal integrity during chemotherapy.

Materials and

Methods: : We use a compartmentalized microfluidic system to isolate the cell bodies and axons for focal administration of cancer drugs. The compartmentalized system was developed using a photolithographic method, with the dimensions of microchannels; width of 10 μm, length of 500 μm, and depth of 3 μm. These dimensions will not allow cell bodies to enter into the other side of the microchannel, thus separating the cell body from the axonal side. Dorsal Root Ganglion (DRG) neurons were isolated from an E-15 Sprague Dawley Rat, and cultured in the microfluidic device, and left to grow for 5-6 days till the axons reach to the other side of the microchannel. The cells were stained using calcein AM (ThermoFisher) dye for 30 min–1 h and imaged using a Leica DMi8 Thunder Fluorescence Microscope immediately after the drug was administered as well as 24 h after treatment. Mitoview Green was obtained from Biotium, USA, and was used to stain the somal chamber containing cell bodies prior to drug treatment for 30 min–1 h. Mitochondria were imaged immediately after dilution of the dye using the Leica DMi8 Thunder Fluorescence and then again, 24 h after initial imaging. Data was collected using Fiji-ImageJ to measure axon lengths in triplicate samples. Microsoft Office Excel 2022 was used to compile all measurement data, where average and standard deviation was calculated. Axon integrity was found by calculating percent difference compared to the average control (0 h) for each sample.

Results, Conclusions, and Discussions:: Global (cell body and axonal) and focal (cell body or axonal) treatments showed significant susceptibility to PTX, VCR, and MMAE while CDDP primarily affected the cell body because of its DNA-targeting mechanism. CDDP, also caused axonal damage suggesting it may disrupt mitochondrial DNA highlighting the importance of axonal mitochondrial trafficking in the study of CIPN. The cotreatment of the chemotherapy drugs with FA significantly improved axon integrity, with the neuroprotection ranging from 32-35% across all drugs. Additionally, FA did not interfere with the efficacy of these anticancer drugs in SKOV-3 ovarian cancer cells.

Mitochondrial trafficking was studied along the microchannels through fluorescent imaging and time-lapse microscopy. The results showed a significant decrease in the mitochondrial trafficking in treatment with the chemotherapy drugs. However, FA treatment restored mitochondrial numbers and motility, especially in CDDP-treated axons, where 83.45 ± 9.57 % increase is seen. This suggests FA enhances anterograde mitochondria transport and possibly suppress membrane permeability, reducing drug-induced mitochondrial damage.

The results showed important findings regarding the mechanism of CIPN and neuroprotection by FA. The study of sites of action for each chemotherapy drug helped us gain a better understanding of CIPN targeting mechanisms. FA treated alongside each chemotherapy drug led to an enhancement of the anterograde movement of mitochondria. Additional studies need to be performed to understand the signaling pathways related to enhanced mitochondrial trafficking.

Acknowledgements and/or References (Optional):: This Research was also supported by the National Science Foundation (NSF) Career Award (2238723).