(F-211) Immunogenicity of a Tumor Nano-Lysate Vaccine for Triple-Negative Breast Cancer

Introduction::
In the United States, 1 in 8 women will be diagnosed with breast cancer in her lifetime, with distant or metastatic cancers associated with the lowest survival rates [1]. Metastasis occurs when circulating tumor cells (CTCs) travel through the bloodstream to form a secondary tumor, and CTCs can be rapidly destroyed into nanoscale fragments by therapeutics such as TRAIL-conjugated liposomes [2,3]. Here, we aimed to synthesize this phenomenon artificially to develop a preventative vaccine and to analyze the immune response elicited by the resulting “tumor nano-lysate” (TNL) vaccine [4].




Materials and Methods::
TNL was fabricated by probe sonication of 4T1 cells with three 10 s pulses at 50% amplitude. TNL were characterized by size via NanoSight, polydispersity index and zeta potential via Zetasizer, and morphology via scanning and transmission electron microscopy. An in vivo triple-negative breast cancer mouse model investigated the efficacy of the vaccine, with BALB/c mice injected with one dose of TNL or PBS control 10 d prior to 4T1 tumor inoculation. Tumors were monitored via caliper measurements and bioluminescence imaging. Primary splenocytes were isolated from healthy mice and analyzed for activation of T cells, B cells, macrophages and dendritic cells (DCs) using flow cytometry. Immortalized DC2.4 cells were analyzed for costimulatory molecules via flow cytometry, phagocytosis markers via confocal microscopy and cytokine release via proteome profiler. Temporal analyses were performed to analyze the immune response in vivo over time, with cardiac punctures at 1, 3, 7 and 10 d following PBS or TNL injections. Immune cell populations were analyzed by flow cytometry and a complete blood count was provided by the Vanderbilt Translational Pathology Shared Resource (TPSR).




Results, Conclusions, and Discussions:: Results and Discussion:

TNL was measured to be an average of 205 nm in diameter and -4.1 mV zeta potential. The in vivo study resulted in delayed metastasis, reduced tumor growth and increased survival for TNL-vaccinated mice (Fig. 1A). Costimulatory molecule and major histocompatibility expression for antigen-presenting cells were enhanced with TNL treatment (Fig. 1B). NF-kB and c-Fos phosphorylation of DCs at varying concentrations was comparable to stimulatory controls (Fig. 1C). After 3 d, peripheral T cell populations were significantly reduced in TNL-treated mice compared to controls (Fig. 1D-E). Splenic natural killer and T cell counts were reduced after 7 d in TNL-treated mice (Fig. 1F-G).


Conclusions:

TNL evoke a preventative immune response as demonstrated in vivo and ex vivo. The activation of antigen-presenting cells such as B cells and DCs indicates the significance of these cells in response to TNL. The temporal immune analysis provides important information to the timeline in which the vaccine is functioning, suggesting the importance of introducing multiple doses to enhance the efficacy.

Acknowledgements (Optional): :
This work was supported by NIH Grant R01 CA203991 and the NSFGRFP.




References (Optional): :
[1] CE DeSantis, CA Cancer J Clin, 2019, 69, 438-451; [2] IJ Fidler, Nat Rev Cancer, 2003, 3, 453–458; [3] MJ Mitchell, New J Phys, 2013, 15, 015008. [4] JA Dombroski, Langmuir, 2020, 36, 6531-6539.