Rescue of dendritic cells from glycolysis inhibition improves cancer immunotherapy in mice

Abstract

Inhibition of glycolysis in immune cells and cancer cells diminishes their activity, and thus combining immunotherapies with glycolytic inhibitors is challenging. Herein, a strategy is presented where glycolysis is inhibited in cancer cells using PFK15 (inhibitor of PFKFB3, rate-limiting step in glycolysis), while simultaneously glycolysis and function is rescued in DCs by delivery of fructose-1,6-biphosphate (F16BP, one-step downstream of PFKFB3). To demonstrate the feasibility of this strategy, vaccine formulations are generated using calcium-phosphate chemistry, that incorporate F16BP, poly(IC) as adjuvant, and phosphorylated-TRP2 peptide antigen and tested in challenging and established YUMM1.1 tumours in immunocompetent female mice. Furthermore, to test the versatility of this strategy, adoptive DC therapy is developed with formulations that incorporate F16BP, poly(IC) as adjuvant and mRNA derived from B16F10 cells as antigens in established B16F10 tumours in immunocompetent female mice. F16BP vaccine formulations rescue DCs in vitro and in vivo, significantly improve the survival of mice, and generate cytotoxic T cell (Tc) responses by elevating Tc1 and Tc17 cells within the tumour. Overall, these results demonstrate that rescuing glycolysis of DCs using metabolite-based formulations can be utilized to generate immunotherapy even in the presence of glycolytic inhibitor.

Fig. 1. Fructose-1,6-biphosphate (F16BP), a glycolytic metabolite, can be formulated into microparticles (MPs).

a Schematic representation of F16BP MPs rescuing dendritic cells (DCs) from glycolytic inhibition. b Scanning electron microscopy-energy dispersive X-ray analysis (SEM-EDX) mapping demonstrating the presence of Ca and P in the microparticles. c Scanning electron microscopy image indicating spherical morphology. d Dynamic light scattering (DLS) suggests a size of 2200 ± 300 nm of F16BP MPs.

Fig. 2. F16BP MPs rescue DC glycolysis and function.

a–d DCs treated with F16BP MPs rescued glycolysis and glycolytic capacity from glycolytic inhibition (PFK15), in vitro (n = 6; One-way ANOVA Tukey’s test), e–h DCs treated with F16BP MPs accelerate basal and maximal respiration even under glycolytic inhibition (PFK15), in vitro (n = 6; One-way ANOVA Tukey’s test). i Vaccine particles induced significantly higher frequency of MHCII + CD86+ in CD11c+ DCs as compared to the individual component controls of the MPs (n = 6; One-way ANOVA Tukey’s test). j F16BP MPs were able to rescue the activation of DCs even in the presence of PFK15 (PFK15 conc. =  25 µM) (n = 6; One-way ANOVA Tukey’s test). Data represented as mean ± std error.

Fig. 3. F16BP(pTRP2+PolyIC) MPs promote a pro-inflammatory response against melanoma, in vivo.

a Schematic representation of subcutaneous injection of Vacc MPs, in vivo, b Kaplan–Meir curve demonstrating significantly higher survival of mice treated with Vacc MPs, c, d Mice treated with Vacc MPs had a significantly higher percentage of the total as well as activated DCs in the draining lymph (n = 5; One-way ANOVA Tukey’s test), e, f Mice treated with Vacc MPs had significantly higher number of Tc and activated and proliferating Tc as compared to other treatment groups (n = 4 or 5; One-way ANOVA Tukey’s test), g No significant differences in the number of Tregs was observed across treatment groups (n = 5; One-way ANOVA Tukey’s test), h–k Mice treated with Vacc MPs had significantly higher number of Tc, Tc1, activated and proliferating Tc1 and Tc17 cells (n = 4; One-way ANOVA Tukey’s test), l Significantly higher Tc1/Treg ratio was observed in mice treated with Vacc MPs as compared to the control groups (n = 4; One-way ANOVA Tukey’s test). Data represented as mean ± std error.

Fig. 4. F16BP MPs rescue DCs in adoptive cellular transfer (ACT) therapy model, in vivo.

a Schematic representation of the adoptive cellular therapy model employed. b, c Mice injected with adoptively transferred DCs along with F16BP MPs were able to rescue glycolysis and glycolytic capacity from glycolytic inhibition (PFK15), in vivo (n = 6; One-way ANOVA Tukey’s test), d, e Mice injected with adoptively transferred DCs along with F16BP MPs accelerated basal and maximal respiration even under glycolytic inhibition (PFK15), in vivo (n = 6; One-way ANOVA Tukey’s test). Data represented as mean ± std error.

Fig. 5. F16BP MPs-based vaccines are compatible with adoptively transferred DCs and improve survival in melanoma.

a Kaplan–Meir curve demonstrating significantly higher survival of mice treated with adoptively transferred Vacc MPs (n = 10, p < 0.001), b Representative tumour images of different treatment groups on day 16. c Mice treated with adoptively transferred Vacc DCs had significantly lower tumour weights as compared to other treatment groups, in vivo (n = 3; One-way ANOVA Tukey’s test). d, e Significantly higher total, as well as activated DCs, were observed in mice treated with adoptively transferred Vacc DCs as compared to other treatment groups, in vivo (n = 4; One-way ANOVA Tukey’s test). f Significantly higher MHCI+ activated DCs were observed in mice treated with adoptively transferred Vacc DCs as compared to other treatment groups, in vivo (n = 4; One-way ANOVA Tukey’s test). g Significantly higher MHCII+ activated DCs were observed in mice treated with adoptively transferred Vacc DCs as compared to F16BP DCs, in vivo (n = 4; One-way ANOVA Tukey’s test). Data represented as mean ± std error.

Fig. 6. Adoptively transferred F16BP MPs-based vaccines DCs generated robust anti-tumour adaptive immune responses.

a, b Significantly modulation of total (CD8+) as well as activated and proliferating (Ki67+CD44+ in CD8+) cytotoxic T cells were observed in mice treated with adoptively transferred Vacc DCs as compared to other treatment groups, in vivo (n = 4; One-way ANOVA Tukey’s test). c–f Significantly higher total Tc1 (Tbet+ in CD8+), activated and proliferating Tc1 (Tbet+Ki67+CD44+ in CD8+), total Tc17 (RORɣT+ in CD8+), activated and proliferating Tc17 (RORɣT+Ki67+CD44+ in CD8+) were observed in mice treated with adoptively transferred Vacc DCs as compared to other treatment groups, in vivo (n = 4; One-way ANOVA Tukey’s test). g Significantly higher ratio of cytotoxic to regulatory T cells (Tc1/Treg) was observed in mice treated with adoptively transferred Vacc DCs as compared to other treatment groups, in vivo (n = 4; One-way ANOVA Tukey’s test). Data represented as mean ± std error.