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. 2024 Feb 17;27(3):109248.
doi: 10.1016/j.isci.2024.109248. eCollection 2024 Mar 15.

A1-reprogrammed mesenchymal stromal cells prime potent antitumoral responses

Marina Pereira Gonçalves  1 , Roudy Farah  2 , Jean-Pierre Bikorimana  2 , Jamilah Abusarah  3 , Nehme El-Hachem  4 , Wael Saad  3 , Sebastien Talbot  5 , Daniela Stanga  6 , Simon Beaudoin  6 , Sebastien Plouffe  6 , Moutih Rafei  1   2   3
Affiliations

A1-reprogrammed mesenchymal stromal cells prime potent antitumoral responses

Marina Pereira Gonçalves et al. iScience. .

Abstract

Mesenchymal stromal cells (MSCs) have been modified via genetic or pharmacological engineering into potent antigen-presenting cells-like capable of priming responding CD8 T cells. In this study, our screening of a variant library of Accum molecule revealed a molecule (A1) capable of eliciting antigen cross-presentation properties in MSCs. A1-reprogrammed MSCs (ARM) exhibited improved soluble antigen uptake and processing. Our comprehensive analysis, encompassing cross-presentation assays and molecular profiling, among other cellular investigations, elucidated A1's impact on endosomal escape, reactive oxygen species production, and cytokine secretion. By evaluating ARM-based cellular vaccine in mouse models of lymphoma and melanoma, we observe significant therapeutic potency, particularly in allogeneic setting and in combination with anti-PD-1 immune checkpoint inhibitor. Overall, this study introduces a strong target for developing an antigen-adaptable vaccination platform, capable of synergizing with immune checkpoint blockers to trigger tumor regression, supporting further investigation of ARMs as an effective and versatile anti-cancer vaccine.

Keywords: Cancer; Classification Description; Immunology; Pharmaceutical engineering.

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Conflict of interest statement

Daniela Stanga and Sebastien Plouffe are employees of Defense Therapeutics and declare competing financial interests. Moutih Rafei is part of Defense Therapeutics Board.

Figures

None
Graphical abstract
Figure 1
Figure 1
Screening Accum variants capable of inducing cross-presentation in MSCs (A) Schematic diagram of the antigen cross-presentation assay in which MSC are pulsed using different Accum-linked OVA variants. Treated MSCs are co-cultured with B3Z, a T cell hybridoma expressing a TCR recognizing the OVA-derived SIINFEKL peptide in the context of MHC I. B3Z express beta-galactosidase (lacZ) driven by NF-AT elements activated after recognition and activation. (B) Same as in (A), except that the Accum variants were admixed with OVA protein. (C) Antigen presentation assay screening the Accum-linked OVA constructs. SIINFEKL is used as a positive technical control for B3Z, MSCs (untreated), OVA, and Accum are used as negative controls. (D) Same as in (C), except that the antigen presentation assay is screening the Accum variants admixed with OVA. (E) Cartoon structure of the original Accum molecule, comprising cholic acid (CA) as the bile acid module and SV40 as the NLS moiety. (F) Cartoon structure of the A1 Accum variant, which maintains the CA, but linked to the NLS hnRNPA1.
Figure 2
Figure 2
Characterizing the cross-presentation capacity of the A1 variant (A) Antigen cross-presentation assay conducted using A1 diluted in PBS or water to compare the impact of A1 formulations in the capacity to induce cross-presentation. (B) Antigen cross-presentation assay conducted using different pulsing time points to optimize the treatment period. (C) Antigen cross-presentation assay conducted using A1 admixed with SIINFEKL to assess the impact on antigen presentation, and A1 admixed with OVA to confirm induction of cross-presentation. (D) Similar to(C) but using OT-T-derived CD8 T cells as responding lymphocytes. (E) Evaluating the effect of A1 on antigen uptake by MSCs using OVA-AF647. (F) Assessing the effect of A1 on antigen processing by MSCs using OVA-DQ. (G) Assessing the endosomal damaging properties of A1 on MSCs co-treated with Cyt-C. After endocytosis, Cyt-C can induce apoptosis if reaching the cytosol upon endosomal break. Annexin-V staining was used to analyze changes in cell death. (H) Representative flow cytometry analysis of H2-Kb (top panel) and I-Ab (lower panel). Gray histograms show isotype controls, whereas test-stained samples are in white. For panels A-C, n = 4/group. Asterisks refers to statistical differences between the group labeled by the symbol and the group of untreated MSCs. Results are presented as average mean with standard deviation (S.D.) error bars, and statistical significance is represented with asterisks: ∗p ˂ 0.05, ∗∗p ˂ 0.01, ∗∗∗p ˂ 0.001. See also Figures S1–S3.
Figure 3
Figure 3
The A1-induced antigen cross-presentation capacity increases and requires ROS production (A) Flow cytometry assessment of ROS production by MSCs in response to A1 using the dye DHE. NAC was used as the negative control whereas Dp44mt was used as a positive control. (B) Antigen cross-presentation assay performed to investigate the effect of neutralizing ROS on A1-related activity. (C) Same as (B) except that it was conducted using NOX inhibitors. (D) A graphical abstract summarizing the mechanistic insights of A1-driven endosomal disruption obtained from the data above. Results are presented as average mean with standard deviation (S.D.) error bars, and statistical significance is represented with asterisks: ∗p ˂ 0.05, ∗∗p ˂ 0.01, ∗∗∗p ˂ 0.001.
Figure 4
Figure 4
Molecular characterization of the ARMs List of top Reactome pathways that are enriched for both up-regulated (A) and down-regulated (B) genes in the A1 treated group versus control MSCs. The circle’s color corresponds to adjusted p values; the size of the circles corresponds to the ratio count of genes in the tested set. (C) A representative unfolded-protein response (UPR) heatmap displaying the genes that contribute the most to the pathway enrichment and modulated in response to A1 treatment (FDR <5%); gene expression is scaled between −1 and +1, followed by color code indicated in the figure. (D) A turbidity assay to evaluate the A1 capacity to form protein aggregation when mixed with the OVA protein. The groups and controls are depicted according to the color code. Results are presented as average mean with standard deviation (S.D.) error bars, and statistical significance is represented with asterisks: ∗p ˂ 0.05, ∗∗p ˂ 0.01, ∗∗∗p ˂ 0.001. See also Figures S4–S6.
Figure 5
Figure 5
Therapeutic vaccination using the ARM vaccine can induce regression of established tumors (A) Experimental design represented by the timeline of the ARM therapeutic vaccination as a monotherapy or combination treatment approach with the ICI anti-PD-1. (B) Evaluation of E.G7 tumor growth in response to syngeneic ARM vaccination (MSCs were obtained from and administered to C57BL/6 mice). The group conditions are indicated by the color code. E.G7 is an OVA-expressing T cell lymphoma, and the protein OVA is used as the stimulating antigen. (C) Kaplan-Meier survival curve of the experiment shown in (B). (D) Evaluation of E.G7 tumor growth in response to allogeneic ARM vaccination (MSCs were obtained from BALB/c mice and administered to tumor-bearing C57BL/6 mice), using OVA as the stimulating antigen. (E) Kaplan-Meier survival curve of the experiment shown in panel D. (F) Evaluation of B16 tumor growth in response to allogeneic ARM vaccination using B16 lysate as stimulating antigen. B16 is a melanoma cell line, and the protein lysate of B16 cells was used as the pulsing antigen. (G) Kaplan-Meier survival curve of the experiment shown in (F). For panels (B–E), n = 5/group. PD-1 refers to the antibody anti-PD-1. Results are presented as average mean with standard deviation (S.D.) error bars, and statistical significance is represented with asterisks: ∗p ˂ 0.05, ∗∗p ˂ 0.01, ∗∗∗p ˂ 0.001. See also Figure S7.
Figure 6
Figure 6
Optimizing the therapeutic potency of the ARM vaccine using a dimeric form of A1 (A) Structure of the A1 monomer (left) versus dimer (right). (B) Comparing the antigen cross-presentation capacities of ARM cells generated in response to the monomer versus different concentrations of dimer form of A1. (C) Experimental design used for the therapeutic vaccination used to test the ARM cells generated using the A1 dimer, as a monotherapy or combination treatment approach with the ICI anti-PD-1. (D) EG.7, an OVA-expressing T cell lymphoma, tumor growth in response to allogeneic ARM vaccination. The vaccination consisted of BALB/c MSCs pulsed with A1 dimer and the antigen OVA, administered to E.G7-bearing C57BL/6 mice. (E) Kaplan-Meier survival curve of the experiment shown in (G). For (B), n = 6/group. For (D and E), n = 10/group. Results are presented as average mean with standard deviation (S.D.) error bars, and statistical significance is represented with asterisks: ∗p ˂ 0.05, ∗∗p ˂ 0.01, ∗∗∗p ˂ 0.001.
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