(C-112) EAT: Electro-Antibacterial-Therapy To Target Intracellular Bacteria

Introduction:: The presence of gram-negative anaerobic bacteria, Fusobacterium nucleatum (Fn), in the tumor microenvironment (TME) is associated with worse prognosis in patients with pancreatic ductal adenocarcinoma [1]. We have previously shown that intracellular invasion of Fn into host pancreatic cancer cells (PANC1 and BxPC3) can increase migration and proliferation of host cells through the secretion of IL-8, CXCL1, MIP-3α, GM-CSF [2]. This invasion, however, makes the intracellular bacteria difficult to target using traditional antibacterial treatments such as systemic antibiotic delivery. The protection of the cell membrane from otherwise effective and clinically-relevant antibiotics such as metronidazole (MTZ) suggests the need for alternative therapies that enhance the local delivery of antibiotics into the cell. Electroporation is the increase of cell permeability in response to an applied electric field by increasing the transmembrane potential (TMP). The magnitude of the TMP determines whether the cell membrane experiences a reversible formation of pores in the membrane. The cell membrane may be temporarily disrupted, this is known as reversible electroporation (RE), or a larger disruption leading to cell death, which is known as irreversible electroporation (IRE). Within the TME, characteristic pulsing schemes for both RE and IRE have been used clinically to enhance the delivery of chemotherapeutics and soft tissue ablation, respectively. While these methods target the whole tumor, there have been little advances on its use for treating the entirety of the TME. Here, we present Electro-Antibacterial Therapy (EAT) as a local treatment modality to enhance antibiotic delivery to pathogenic intracellular bacteria in the tumor microenvironment.


Materials and Methods:: Pancreatic cancer cells (Panc1) seeded in T-175 flasks were washed with phosphate buffered solution (PBS) and media was replaced with serum-free DMEM. Overnight cultures of Fusobacterium nucleatum nucleatum (ATCC 23726) were back-diluted and used at an OD₆₀₀ of 0.2-0.4 for cancer host infection experiments. Panc1 cells were infected at a multiplicity of infection of 50:1 (Bacteria:Host) for 4 hours under anaerobic conditions. Infected cells were then treated with complete DMEM containing penicillin-streptomycin (PenStrep) for 30 minutes, washed with PBS, trypsinized, and collected. A stock MTZ solution (30mg/mL in DMSO) was made fresh and varying concentrations were made in serum-free DMEM with a constant DMSO concentration of 0.33%. Similarly, a stock of PenStrep (10,000U/mL Penicillin and 10,000 ug/mL Streptomycin) was used to create antibiotic dilutions and additional DMSO was added to match the 0.33% found in MTZ dilutions. For electroporation experiments, infected host cells were spun down at 150g x 5mins and resuspended in MTZ solution before being transferred to a 4mm gap electroporation cuvette. Using a BTX^TM ECM^TM 830, eight 100µs pulses at 1200 V/cm or eight 1ms pulses at 1200V/cm were applied, samples were then incubated for 1 hour before washing with PBS and plating onto CBHK agar plates. Colony forming units (CFU) were counted 72 hours later.


Results, Conclusions, and Discussions::

As seen in Figure 1, intracellular Fn remains viable when no antibiotic is present in the surrounding medium regardless of pulse width. While the electric field alone is not effective at reducing the viability of intracellular Fn, there is a significant decrease when 1ms pulses are applied for both PenStrep and MTZ but not when 100µs pulses are applied. This suggests that the combination of longer 1ms electric field pulses and antibiotics significantly enhances their delivery inside the cell and proves effective against intracellular Fn. Traditional 100µs pulses, do not enhance delivery and in fact, appear to lower the efficacy of both PenStrep and MTZ compared to their respective antibiotic controls.



Albeit the standard of care, systemic delivery of antibiotics such as metronidazole has limited efficacy against Fusobacterium nucleatum that has invaded pancreatic cancer cells due to transport limitations and potential metabolic changes of intracellular bacteria that are yet to be understood. Here, we show Electro-Antibacterial Therapy (EAT), or the use of pulsed electric fields to enhance the permeability of the cell membrane to ambient antibiotics can significantly reduce the viability of intracellular bacteria when longer 1ms pulses are applied compared to pulsed electric fields alone, antibiotics alone and even the combination of the two with shorter 100µs pulses.



Electro-chemotherapy typically uses 100µs pulses to aid in the delivery of chemotherapies to cancer cells yet in our study this pulsing regime was ineffective at delivering antibiotics to the intracellular niche at levels effective against Fn. Clearance of intracellular Fn was only seen with a 1ms pulsing regime. To test the specificity of the 1ms pulsing regime we looked at both metronidazole and PenStrep. While metronidazole is effective against clinical isolates, PenStrep usually is not. The specific strain of Fusobacterium used in the study is susceptible to PenStrep showing the increased delivery of antibiotics using the 1ms pulsing regime is not metronidazole specific. Interestingly the combination of antibiotic and 100µs pulses led to higher Fn viability compared to antibiotic alone. This is potentially due to sublethal intracellular metronidazole levels while increasing nutrients within the intracellular niche from the surrounding serum-free media.


Acknowledgements (Optional): : This work was supported by a grant for Electro-Antibacterial-Therapy from the Wake Forest Comprehensive Cancer Center and NIH R21 1R21CA238630-01A1


References (Optional): : [1] Mitsuhashi et al. 2015 Oncotarget

[2] Udayasuryan et al. 2022 Science Signaling