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. 2023 Dec 19:14:1278000.
doi: 10.3389/fimmu.2023.1278000. eCollection 2023.

Light-enhanced VEGF121/rGel induce immunogenic cell death and increase the antitumor activity of αCTLA4 treatment

Ane Sager Longva  1 , Kristian Berg  1 , Anette Weyergang  1
Affiliations

Light-enhanced VEGF121/rGel induce immunogenic cell death and increase the antitumor activity of αCTLA4 treatment

Ane Sager Longva et al. Front Immunol. .

Erratum in

Abstract

Background: Immune-checkpoint inhibitors (ICIs) represent a revolution in cancer therapy and are currently implemented as standard therapy within several cancer indications. Nevertheless, the treatment is only effective in a subset of patients, and immune-related adverse effects complicate the improved survival. Adjuvant treatments that can improve the efficacy of ICIs are highly warranted, not only to increase the response rate, but also to reduce the therapeutic ICI dosage. Several treatment modalities have been suggested as ICI adjuvants including vascular targeted treatments and photodynamic therapy (PDT). Photochemical internalization (PCI) is a drug delivery system, based on PDT. PCI is long known to generate an immune response in murine models and was recently shown to enhance the cellular immune response of a vaccine in a clinical study. In the present work we evaluated PCI in combination with the vascular targeting toxin VEGF121/rGel with respect to induction of immune-mediated cell death as well as in vitro ICI enhancement.

Methods: DAMP signaling post VEGF121/rGel-PCI was assessed in CT26 and MC38 murine colon cancer cell lines. Hypericin-PDT, previously indicated as an highly efficient DAMP inducer (but difficult to utilize clinically), was used as a control. ATP release was detected by a bioluminescent kit while HMGB1 and HSP90 relocalization and secretion was detected by fluorescence microscopy and western blotting. VEGF121/rGel-PCI was further investigated as an αCTLA enhancer in CT26 and MC38 tumors by measurement of tumor growth delay. CD8+ Dependent efficacy was evaluated in vivo using a CD8+ antibody.

Results: VEGF121/rGel-PCI was shown to induce increased DAMP signaling as compared to PDT and VEGF121/rGel alone and the magnitude was found similar to that induced by Hypericin-PDT. Furthermore, a significant CD8+ dependent enhanced αCTLA-4 treatment effect was observed when VEGF121/rGel-PCI was used as an adjuvant in both tumor models.

Conclusions: VEGF121/rGel-PCI describes a novel concept for ICI enhancement which induces a rapid CD8+ dependent tumor eradication in both CT26 and MC38 tumors. The concept is based on the combination of intracellular ROS generation and vascular targeting using a plant derived toxin and will be developed towards clinical utilization.

Keywords: immune check point inhibitor (ICI); immunogenic cell death (ICD); photochemical internalization (PCI); photodynamic therapy; targeted toxin; vascular endothelial growth factor; vascular targeting.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Schematic illustration of mechanisms induced by VEGF121/rGel-PCI in combination with αCTLA4 treatment Created with BioRender.com.
Figure 2
Figure 2
Schematic in vivo treatment schedule for VEGF121/rGel-PCI enhanced αCTLA4, including experiments with αCD8+ depletion. Created with BioRender.com.
Figure 3
Figure 3
VEGF121/rGel-PCI induces secretion of DAMP signals from CT26 cells. (A) Relative cell viability (MTT) 48 hrs post VEGF121/rGel-PCI with indicated controls. (B) Normalized ATP secretion (bioluminescence assay) 24 and 48 hrs post VEGF/Gel-PCI with indicated controls. (C, D) HSP90 and HMGB1 secretion (quantification of western blots) 24 and 48 hrs post VEGF121/rGel-PCI with indicated controls. The graphs show averages of 3 independent experiments with error bars indicating SD. Bar labeled with * indicate p < 0.05 as compared to non-treated control (NT) (t-test). Significance between two treatments is indicated with * and dotted line (t-test). ** indicate significance with paired t-test. (E) Representative western blots of 3 independent experiments of HSP90 and MHGB1 in cell media harvested 24 and 48 hrs post treatment. VEGF121/rGel-PCI was performed with the same light dose as used with PDT while PDThigh was executed at a higher light dose.
Figure 4
Figure 4
Intracellular localization of HMGB1 post VEGF121/rGel-PCI. Fluorescence images of CT26 cells showing intracellular localization of HMGB1 (green) 3 and 24 hrs post VEGF121/rGel-PCI with indicated controls. Fluorescence from DAPI (blue), staining the nucleus, and Phalloidin (red), staining the actin filaments, is included for orientation. Yellow color indicates co-localization between green and red. Bar: 10μm. VEGF121/rGel-PCI was performed with the low light dose. All images are representative for 3 independent experiments.
Figure 5
Figure 5
VEGF121/rGel-PCI induces secretion of DAMP signals from MC38 cells. (A) Relative cell viability (MTT) 48 hrs post VEGF121/rGel-PCI with indicated controls. (B) Normalized ATP secretion (bioluminescence assay) 24 and 48 hrs post VEGF/Gel-PCI with indicated controls. (C, D) HSP90 and HMGB1 secretion (quantification of western blots) 24 and 48 hrs post VEGF121/rGel-PCI with indicated controls. The graphs show an average of 3 independent experiments with error bars indicating SD. Bar labeled with * indicate p < 0.05 as compared to non-treated control (NT) (t-test). Significance between two treatments is indicated with * and dotted line (t-test). ** indicate significance with paired t-test. (E) representative western blots of 3 independent experiments of HSP90 and MHGB1 in cell media harvested 24 and 48 hrs post treatment. VEGF121/rGel-PCI was performed with the same light dose as used with PDT while PDThigh was executed at a higher light dose.
Figure 6
Figure 6
Photoactivation of hypericin induces secretion of DAMP signals from CT26 cells. (A) Relative cell viability (MTT) 48 hrs post photoactivation of hypericin at two light doses (high and low). (B) Normalized ATP secretion (bioluminescence assay) 24 and 48 hrs post photoactivation of hypericin at high and low light dose. (C, D) HSP90 and HMGB1 secretion (quantification of western blots) 24 and 48 hrs post photoactivation of hypericin at two light doses. The graphs show an average of 3 independent experiments with error bars indicating SD. Bar labeled with * indicate p < 0.05 as compared to non-treated control (NT) (t-test). Significance between two treatments is indicated with * and dotted line (t-test). (E) representative western blots of 3 independent experiments of HSP90 and MHGB1 in cell media harvested 24 and 48 hrs post treatment.
Figure 7
Figure 7
VEGF121/rGel-PCI enhances αCTLA-4 treatment in CT26 tumors dependent on CD8+ T-cells. (A) Median relative tumor volume at day 4 as compared to day 0 in each treatment group. (B) Relative change in tumor volume from day 0 to day 4 for each individual animal within the treatment groups (The tumor volume distribution is shown in Figure S4 ). (C) Average AUC integrated from day 0 to day 16 for each treatment group. The panels represent increasing doses of αCTLA-4 antibody. Error bars: SE. (D) Kaplan-Meier plots illustrating overall treatment response of VEGF121/rGel-PCI +αCTLA4 (5µgx3) as measured by tumors with volume < 1000mm3 with indicated controls. (E) Relative change in tumor volume from day 0 to day 9 for each individual animal within the indicated treatment groups. αCTLA-4 antibody was used at a total dose of 150µg (50µgx3). Within the figure panels, significant difference between the treatment groups is evaluated by ANOVA and the p value is indicated when significant. * indicate significant difference (p<0.05) from the other groups.
Figure 8
Figure 8
VEGF121/rGel-PCI enhances αCTLA-4 treatment in MC38 tumors. (A) Median relative tumor volume at day 7 as compared to day 0 in each treatment group. (B) Relative number of animals with decreased tumor size at day 7 as compared to day 0 in each treatment group. The numbers above the bars indicate number of animals with decreased size per total number of animals within the group. (C) Relative change in tumor volume from day 0 to day 7 for each individual animal within the treatment groups (The tumor volume distribution is shown in Figures S9A, B ). The 2 panels represent 0 and 50µgx2 αCTLA-4 antibody. (D) Average AUC integrated form day 0 to day 16 for each treatment group. The panels represent 0 and 50µgx3 αCTLA-4 antibody. Error bars: SE. (E) Kaplan-Meier plots illustrating overall treatment response of VEGF121/rGel-PCI +αCTLA4 (50µgx3) as measured by tumors with volume < 1000mm3 with indicated controls. (F) Relative change in tumor volume from day 0 to day 9 for each individual animal within the indicated treatment groups. αCTLA-4 antibody was used at 50µgx3.
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