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. 2017 Feb 28:248:1-9.
doi: 10.1016/j.jconrel.2016.12.036. Epub 2017 Jan 3.

Surface engineering tumor cells with adjuvant-loaded particles for use as cancer vaccines

Kawther K Ahmed  1 , Sean M Geary  1 , Aliasger K Salem  2
Affiliations

Surface engineering tumor cells with adjuvant-loaded particles for use as cancer vaccines

Kawther K Ahmed et al. J Control Release. .

Abstract

Cell surface engineering is an expanding field and whilst extensive research has been performed decorating cell surfaces with biomolecules, the engineering of cell surfaces with particles has been a largely unexploited area. This study reports on the assembly of cell-particle hybrids where irradiated tumor cells were surface engineered with adjuvant-loaded, biodegradable, biocompatible, polymeric particles, with the aim of generating a construct capable of functioning as a therapeutic cancer vaccine. Successfully assembled cell-particle hybrids presented here comprised either melanoma cells or prostate cancer cells stably adorned with Toll-like receptor-9 ligand-loaded particles using streptavidin-biotin cross-linking. Both cell-particle assemblies were tested in vivo for their potential as therapeutic cancer vaccines yielding promising therapeutic results for the prostate cancer model. The ramifications of results obtained for both tumor models are openly discussed.

Keywords: Cancer vaccines; Cell surface engineering; PLGA particles; Streptavidin-biotin cross-linking.

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Figures

Figure 1
Figure 1. Particle functionalization and characterization
(A): Schematic of preparation of streptavidin-coated particles. PLGA particles were formed using a double emulsion solvent evaporation method with simultaneous activation of terminal carboxyl groups as described in the methods section. Surface activated particles were lyophilized and coated with streptavidin immediately prior to use. (B) Representative SEM image of PLGA particles, scale bar = 2 microns. (C) EDC/NHS-activated and non-activated (control) particles incubated with streptavidin-PE. (D) EDC/NHS-activated particles treated or untreated (control) with streptavidin and then incubated with biotinylated fluorescein. When applicable, error bars = SD. ** p < 0.01, *** p < 0.001, n = 3.
Figure 2
Figure 2. Fabrication and characterization of cell-particle assemblies
(A1) B16.F10 cells and (A2) RM11 cells treated or not treated (control) with biotinylated anti-CD29 antibodies (anti-CD29-biotin) followed by strepavidin-PE. (B–F) Validation of cell-particle hybrid assembly from B16.F10 or RM11 cells subsequent to surface biotinylation using anti-CD29-biotin and mixed with streptavidin-coated particles loaded with rhodamine B and washed to remove unbound particles (hybrid). Control involved the same conditions except cells were not treated with anti-CD29-biotin. Validation was performed using: (B1–2 and E1–2): flow cytometry where (B1 and E1) representive (n = 1) and (B2 and E2) mean (n = 3) results from measuring relative mean rhodamine fluorescence intensity (RMFI(Rh)) of (B1 and B2) B16.F10 and (E1 and E2) RM11 cells; (C1–2 and F1–2): laser scanning confocal microscopy showing (C1 and F1) hybrid and (C2 and F2) control cell-particle mixtures for (C1 and C2) B16.F10 and (F1 and F2) RM11 cells (blue = DAPI stained cell nuclei, red = rhodamine-labeled PLGA particles); (D): scanning electron microscopy showing (D1) B16.F10 hybrid and (D2) control cell-particle mixtures (arrows in C1, D1, and F1 indicate particles bound to cell surface). Scale bar= 20 micron for C1–2 and F1–2, 10 microns for D1–2. When applicable, error bars = SD. ** p < 0.01, *** p < 0.001, n = 3.
Figure 3
Figure 3. Irradiated murine prostate cancer cells, RM11, are effective therapeutic cancer vaccines when conjugated to adjuvant-loaded particles
(A–B): Irradiated RM11 cells surface engineered with CpG ODN-loaded particles were effective as a therapeutic vaccine (see methods for tumor challenge and vaccination details) as shown by: (A) significantly reducing prostate cancer tumor burden compared to naïve mice (Day 13, * p < 0.05) and mice vaccinated with irradiated RM11 plus soluble CpG ODN (Day 17, * p < 0.05), and (B) significantly extending the survival of mice compared to naïve mice as shown in Kaplan-Meier survival curve (* p < 0.05). Median survival = 28 days for cell-particle hybrid group, 23 days for cell-particle mixture group, 20 days for cells + soluble CpG group, and 16 days for naïve mice. See methods for tumor challenge and vaccination details. Samples are presented as mean ± SEM, n= 5 – 10 mice per group.
Figure 4
Figure 4. Cell-particle hybrid uptake by BMDC in vitro
Laser scanning confocal microscopy imaging of cell-particle hybrid uptake by BMDC. Cell-particle hybrids were incubated with BMDC Yellow arrows indicating colocalization of B16.F10 cells (green) and particles (red) inside BMDC (magenta). Magenta: Alexa flour®700 (CD11c) stained BMDC, green: CFSE labeled B16.F10 melanoma cells, red: rhodamine B-labeled PLGA particles, gray: DAPI stained cell nuclei. Scale bar: 20 micron.
Figure 5
Figure 5. In vitro effect of irradiated tumor cells on the immunostimulatory properties of CpG ODN
(A) CD80 and (B) CD86 expression by BMDC after 24 hour incubation in vitro with media alone (control), CpG ODN alone, CpG ODN + indicated irradiated tumor cells, or indicated irradiated tumors cells alone. Samples are presented mean ± SD. *p< 0.05, ****p<0.0001, n = 3. These experiments did not involve any cell-particle hybrids.
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