Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.

High School Student with a Traditionally Underrepresented Background

HS Poster

Poster 62 - Engineered Endosymbionts for Targeted Delivery of Tumor Suppressor Proteins to Histiocytic Sarcoma

Saturday, October 26, 2024
10:00 AM - 12:00 PM EST
Location: Exhibit Hall E, F & G

Introduction: Histiocytic sarcoma (HS) is a rare and aggressive tumor with < 1% incidence in humans, but a relatively high incidence in some dog breeds, e.g. the Bernese Mountain Dog. The prognosis for human and veterinary patients with HS is quite poor and there are no effective therapies for this disease, and so we propose to develop a targeted therapy for this rare cancer enabled by the relatively high incidence of the canine disease. HS is a cancer of histiocytes that are capable of engulfing substances by phagocytosis, and we will exploit its phagocytic nature to deliver tumor suppressor proteins intracellularly using engineered endosymbionts (EES). EES are otherwise innocuous free-living bacteria engineered to persist inside the cytoplasm of mammalian cells acting as synthetic organelles that can direct cell fate and function. Previously, we have utilized EES based on a Bacillus subtilis chassis expressing mammalian transcription factors to direct mouse macrophages towards a pro-inflammatory state in vitro1 and in vivo2. Using this EES chassis, we hypothesized that we could deliver tumor suppressor proteins such as p53 and azurin inside canine histiocytic sarcoma cells, leading to tumor cell death. Specifically, we evaluated two research questions: can codon-optimized versions of canine p53 and azurin be efficiently secreted by EES; and will delivery of tumor suppressor proteins intracellularly by EES in the DH82 HS cell line lead to cell death.

Materials and

Methods: To investigate the first question, codon optimized DNA fragments for canine p53 and azurin were assembled into a vector library of 6 secretion signal peptides (SP) by Gibson assembly, and transformed into NEB 5 alpha E. coli. After verifying successful assembly by colony PCR, 12 plasmids were transformed into B. subtilis LLO, and selected by antibiotic resistance conferred by the plasmid, transformation was re-verified by colony PCR targeting either the p53 or azurin sequences. After overnight growth of positive colonies in LB broth supplemented with antibiotic, bacteria were re-inoculated into fresh media and allowed to grow for 5 hours. At this time point, the cultures were centrifuged to separate cells and supernatant and the proteins in the supernatant, and cell pellets were analyzed. Proteins in the supernatant and cell lysate samples were denatured by SDS in 4x loading buffer, separated by SDS-PAGE, and transferred onto a nitrocellulose membranes and antibodies to the proteins were used in a Western blot to assess the levels of proteins.

Results, Conclusions, and Discussions: The Western blots for (A, B) dog p53 and (C) azurin are shown in Figure 1. We observed that dog p53 is cleaved at the C terminus in the supernatant, likely due to extracellular or membrane-bound proteases produced by B. subtilis (Fig. 1A). However, the smaller fragment of dog p53 is still present in the extracellular fraction (Fig. 1B). Cleavage does not occur for intracellular dog p53, and the SPs phoD and amyQ are optimal signal peptides for dog p53. Interestingly, extracellular cleavage of p53 occurs in both the protease positive (SCK6) and deficient (KO8-S) B. subtilis strains. Azurin is effectively secreted using all SPs except phoD (Fig. 1C), possibly due to its small size. In conclusion, we have shown that dog p53 and azurin can be secreted from EES. We determined the optimal signal peptides for these proteins and will use phoD as the SP for dog p53 and LLO-rigid for azurin. We will further evaluate these strains in co-culture with DH82 cells at various endosymbiont to mammalian cell ratios and measure cytostatic and/or cytotoxic effects by live cell imaging to answer the second research question proposed in this study.

Acknowledgements (Optional): This research was financially supported by the James and Kathleen Cornelius endowment.

References (Optional): 1. Madsen, C. S., Makela, A. V., Greeson, E. M., Hardy, J. W., & Contag, C. H. (2022). Engineered endosymbionts that alter mammalian cell surface marker, cytokine and chemokine expression. Communications Biology, 5(1), 888.
2. Madsen, C. S., Makela, A. V., Maduka, C. V., Greeson, E. M., Tundo, A., Ural, E., ... & Contag, C. H. (2024). Engineered endosymbionts that modulate primary macrophage function and attenuate tumor growth by shifting the tumor microenvironment. bioRxiv, 2024-07.