Reducing metabolic stress within the tumor microenvironment (TME) could be essential for improving the efficacy of cancer immunotherapy. Using a mouse model of melanoma, we show here that appropriately timed treatment with the PPARα agonist fenofibrate improves the ability of a T cell–inducing cancer vaccine to delay tumor progression. Fenofibrate reduced the use of glucose by tumor and stromal cells in the TME and promoted the use of fatty acids for their metabolic needs. The glucose within the TME was in turn available for use by vaccine-induced tumor-infiltrating CD8+ T cells, which improved their ability to slow tumor progression. Early fenofibrate treatment 3 days after vaccination improved functions of circulating CD8+ T cells but failed to significantly affect tumor-infiltrating lymphocyte (TIL) metabolism or decrease tumor progression. In contrast, delaying treatment until day 5 after vaccination modified TIL metabolism and augmented the vaccine's ability to slow tumor progression. In summary, our findings reveal that a PPARα agonist can increase the efficacy of a cancer vaccine by reprogramming cells within tumors to increase fatty acid metabolism, providing T cells access to glucose in the TME.

Significance:

These findings suggest that metabolic manipulations using already approved drugs may offer an easy pathway to increase the efficacy of vaccines against solid tumors.

CD8+ T cells are able to rapidly eliminate large numbers of antigen-bearing cells thereby controlling viral infections or rejecting organ transplants. Nevertheless, CD8+ T cells commonly fail to eradicate solid tumors. This may in part reflect the poor immunogenicity of tumor cells, which often express only slightly altered self-antigens and/or lack or have reduced levels of MHC class I molecules on their surface (1, 2). Furthermore, the tumor microenvironment (TME) is highly immunosuppressive due to infiltration with inhibitory T cells, fibroblasts, and monocyte populations (3–7). Loss of T-cell functions is further caused by T-cell exhaustion (8, 9); this is in part triggered by the metabolically stressful TME that is low in glucose, a favorite nutrient for energy and biomass production by tumor cells and activated T cells (10–14); the latter cannot compete with tumor cells for glucose and will therefore starve unless they switch to alternative energy sources (15). The interstitial fluid of melanomas and presumably other solid tumors has increased levels of fatty acids (15, 16) that could be used to fuel tumor-infiltrating T lymphocytes (TIL) as well as other cells within the tumors. Several studies have shown that fatty acid catabolism can play an important role in promoting melanoma progression by providing energy to tumor cells that face nutritional challenges within the TME (16, 17). Upon activation, T cells increase glycolysis (11, 12) but as we showed previously, they can be trained to switch to fatty acid catabolism by in vivo or in vitro treatment with fenofibrate (15). This drug is an agonist of peroxisome proliferator activated receptor alpha (PPARα), a transcription factor that regulates the majority of enzymes, receptors, and transporters involved in fatty acid metabolism. Our previous study (15) used an adoptive transfer model where CD8+ T cells expressing a transgenic receptor to a tumor-associated antigen were either stimulated in vitro in presence of fenofibrate or donor mice were treated after vaccination with a melanoma vaccine with fenofibrate prior to adoptive transfer of their lymphocytes into melanoma-bearing vaccinated recipient mice. Switching the metabolism of the adoptively transferred T cells toward fatty acid metabolism improved their functions, reduced their differentiation toward exhaustion and improved their ability to slow tumor progression (15).

Here we tested whether fenofibrate can improve the efficacy of a cancer vaccine if the drug is given directly to tumor-bearing mice. T-cell activation in vitro fundamentally differs from their stimulation in vivo. In vitro cultures have higher levels of key nutrients and oxygen than plasma or lymphatic tissues. Activation of T cells in vitro occurs rapidly, while in vivo T-cell activation takes place over a period of days. Furthermore, giving a PPARα agonist directly to tumor-bearing mice not only affects TILs but also other cells within the TME. Under the assumption that timing will influence the effectiveness of metabolic manipulations, here we explore different times for onset of fenofibrate treatment relative to vaccination. Our data show that starting fenofibrate treatment early, by 3 days after vaccination, improves functions of circulating CD8+ T cells, but fails to significantly affect TIL metabolism or decrease tumor progression. In contrast, delaying treatment until day 5 after vaccination modifies TIL metabolism and augments the vaccine's ability to slow tumor progression. Unexpectedly, fenofibrate not only increased the TILs' levels of transcripts encoding factors of fatty acid metabolism, but also those of glucose metabolism, and the latter unlike the former, directly correlated with the delay in tumor growth. This was linked to the effects of fenofibrate on tumor cells and other cells present in the tumor stroma, which switched away from glycolysis toward fatty acid metabolism, resulting in increased levels of glucose within tumors, thus allowing TILs enhanced access to this nutrient.

Mice and mouse procedures

Female C57Bl/6 mice (6–8 weeks) were purchased from the Jackson Laboratories and housed at the Wistar Institute Animal Facility. Mice were vaccinated intramuscularly with 1010 virus particles (vp) of AdC68-gDMelapoly. For tumor challenge experiments, B16BrafV600E cells (3–5 × 104 cells/mouse) in PBS were injected subcutaneously into the right flank. Fenofibrate (100–200 mg/kg/day) in DMSO/PBS was given by oral gavage daily for up to 3 weeks starting 3, 5, and 7 days after vaccination or 6–10 days after tumor challenge. Control mice received the DMSO/PBS diluent. Tumor size was monitored by measuring the perpendicular diameter of tumors every other day. Tumors were harvested once they were approximately 1.5 cm in diameter. All mouse experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee of The Wistar Institute (Philadelphia, PA).

Cell lines

The B16BrafV600E cell line was derived from B16.F10 (obtained from ATCC) cells by transduction with the lentivector pLU-EF1a-mCherry expressing mouse BrafV600E (provided by M Herlyn laboratory, The Wistar Institute, Philadelphia, PA). The mutant cell line showed the same in vivo growth characteristics as unmodified B16.F10 cells. HEK293 cells were used to propagate the melanoma vaccine. Cells were grown in DMEM supplemented with 10% FBS. HEK293 cells were purchased from the ATCC and thawed at the 43rd passage and used between the 43rd and the 60th passages. Both cell lines have been tested for Mycoplasma. HEK293 cells have been authenticated using human cell authentication assay, which identifies short tandem repeat markers by multiplex PCR.

AdC68-gDMelapoly vaccine

The vaccine vector has been described previously (18). The Melapoly sequence is composed of eight CD8+ T-cell epitopes from human (h) or mouse (m)Trp-2, mTrp-1, hgp100, and mBrafV600E fused into herpes simplex virus glycoprotein (g)D. AdC68-gDMelapoly, grown on HEK293 cells, was purified by CsCl-gradient centrifugation. The concentration of the virus was determined by measuring vps by spectrophotometry at 260 nm.

Isolation of lymphocytes from mice

Peripheral blood mononuclear cells (PBMC) and lymphocytes from spleens were harvested as described previously (18). Blood samples were collected by submandibular puncture and PBMCs were isolated by Histopaque gradient centrifugation. Single-cell suspension was generated by mincing spleens with mesh screens in Leibovitz's L15 medium followed by passing cells through a 70 μm filter. Red blood cells were lysed by 1× RBC lysis buffer (eBioscience). To obtain TILs, tumors were harvested, cut into small fragments and treated with 2 mg/mL collagenase P, 1 mg/mL DNase I in Hank's Balanced Salt Solution (1×, Thermo Fisher Scientific) with 2% FBS under agitation for 1 hour. Tumor fragments were homogenized, filtered through 70 μm strainers and lymphocytes were purified by Percoll-gradient centrifugation and washed with DMEM supplemented with 10% FBS. Cell viability assay was determined microscopically upon trypan blue staining.

Antibody staining and flow cytometry

Cells were stained with a PE-labeled Trp-1-specific MHC class I (H-2Db) tetramer carrying the TAPDNLGYM peptide (NIAID Tetramer Facility). Lymphocytes were stained with a Brilliant violet (BV) 510-labeled live cell stain, and for surface markers with anti-CD8-APC-Cy7, anti-LAG-3-BV786, anti-PD-1-BV605 (clone 29F.1A12), CD127-PerCP-Cy5.5, CD62L-FTIC, KLRG1-PE-Cy7, (all from BioLegend or eBioscience). For staining of intracellular markers, cells were fixed and permeabilized with cytofix/cytoperm solution (BD Biosciences) and stained with anti-T-bet-AF450 (clone 4B1, Invitrogen), anti-HIF1α-AF750, (clone 4B10, RD Systems), and anti-PPARα AF647 (clone 3B6/PPAR, Novus Biologicals). For intracellular cytokine staining of ex vivo stimulated lymphocytes, approximately 106 cells per samples were cultured in DMEM containing 2% FBS and Golgiplug (Thermo Fisher Scientific, 1.5 μL/mL) for 5 hours with the peptide carrying the immunodominant mTrp-1 epitope (5 μg/mL). Cells were washed once with PBS, stained with a live cell stain, anti-CD44-AF700 and anti-CD8-PerCP-Cy5.5 for 30 minutes. They were washed once and then fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) for 20 minutes. Cells were then incubated for 30 minutes with anti-IFNγ-FITC, anti-TNFα-BV605m, anti-GrmB-AF647, anti-perforin-PE-A, and anti-IL2-Pe-Cy7.

After staining, cells were washed and analyzed by an LSRII (BD Biosciences). Data were analyzed with FlowJo (TreeStar).

Gene expression analysis

Lymphocytes were isolated from spleens and tumors and stained with dyes and antibodies to live cells, CD8+ and CD44+. Cells were sorted (Mono Astrios, Beckman Coulter) on ice for CD44+CD8+ T cells (spleens) or CD44+CD8+ T cells (tumors) into RLT lysis buffer (QIAGEN). RNA was isolated from purified cells using RNeasy Mini kits (Qiagen) and RNA concentrations were determined using Nanodrop (Thermo Fisher Scientific). cDNAs were obtained by reverse transcription using the high-capacity cDNA reverse transcription kit (Applied Biosystems). Relative qRT-PCR analyses were performed using 7500 Fast Real-Time PCR system (Life Technologies). 18S rRNA or GAPDH were used as internal controls. Vector NTI was used for primers design (Supplementary Table S1). Differences in transcript expression levels were evaluated based on their cycle thresholds (Cτ) and are visualized in heatmaps.

Glucose measurement in sera and tumor interstitial fluids

Tumors were harvested from fenofibrate- and DMSO-treated mice once tumors reached approximately 1.5 cm in diameter. Tumor interstitial fluids were collected by centrifugation as described previously (15). Sera were collected from the same mice. Glucose concentrations were measured by Advanced Glucose Meter.

Testing for glucose uptake

We used the 2-NBDG glucose uptake assay kit, to measure glucose uptake in cell cultures. Briefly, 106 lymphocytes per sample (obtained from tumors as described above), were incubated with a fluorescent-labeled deoxyglucose analog 2-NBDG (ab235976) at a final concentration of 200 μg/mL for 5 hours at 37°C and 5% of CO2. Cells were washed once with PBS, stained with a live cell stain, anti-CD44-AF700, anti-CD8-APCY7, anti-CD4-PerCP-cy5.5, anti-CD3-PE, anti-CD14-BV421, and anti-CD11c-AF674 (all from BioLegend or eBioscience). They were washed once and resuspended in stabilizing fixative solution (BD Biosciences). Cells were analyzed by an LSRII (BD Biosciences), and glucose uptake was measured by the fluorescence at Ex/Em = 485/535 nm, which is equivalent to FITC. Data were analyzed with FlowJo (TreeStar).

IHC

A number of 4-μm sections of paraffin-embedded tumors were mounted on charged slides and baked in a 60°C oven for 1 hour. Tissues were deparaffinized and rehydrated in two changes of Xylene substitute, two changes of 100% EtOH, two changes of 95% EtOH, and two changes of deionized water. Then hydrophobic barriers were drawn around the tissue sections with Vector ImmEdge Pen. Antigen retrieval was achieved with DAKO EDT (pH 9) under pressure at 110°C for 10 minutes. Slides were cooled on the counter for 20 minutes, then rinsed in deionized water. After that, they were immersed in a 3% hydrogen peroxide solution for 10 minutes, then rinsed in deionized water. First primary antibody (anti-CD8 and anti-CD4 from Cell Signaling Technology) was applied to the bottom tissue section. Antibody is diluted with Cell Signaling Technology Signal Stain Ab diluent. Top section serving as negative control is covered with TBST. Overnight incubation at 4°C. After rinsing, secondary antibody (Vector HRP anti-rabbit polymer) was applied to all sections and incubated for 30 minutes at room temperature. AEC (red) Vector peroxidase substrate (CD4) and second chromagen, Abcam Stay Blue (CD8), were applied and incubated for 30 and 10 minutes, respectively.

Sections were analyzed with a Nikon Eclipse Ti Inverted Microscope, Light Engine SOLA SE II 365, PSF (Perfect Focus System), Motorized FL Filter Turret, Prior Stage, Piezo Stage, using a Nikon 20X Plan Apo, N.A. .95 objective and the Nikon NIS Elements AR Version 5.30.02 (Build 1545) software. Nis Elements Ar (Scan Large Image) defined two regions of interest, size 8 × 8 fields, total 88–128 frames (2088 × 2048 pixel) in X & Y per section, with no overlap between images. Images were taken with a Nikon Fi3 color camera (2048 × 2048 pixel resolution).

Statistical analyses

All statistical analyses were conducted using GraphPad Prism 6 (GraphPad). Differences between two populations were calculated by Student t test. Multiple comparisons between two groups were performed by multiple t tests with type I error correction. Differences among multiple populations were calculated by one- or two-way ANOVA. Differences in survival were calculated by log-rank Mantel–Cox test. Type I errors were corrected by Holm-Šídák method. Significance was set at P values of or below 0.05. For all figures, *, P ≤ 0.05 to 0.01; **, P ≤ 0.01 to 0.001; ***, P ≤ 0.001 to 0.0001; ****, P ≤ 0.0001. Unless noted in the figure legend, all data are shown as mean ± SEM.

Initially we tested whether fenofibrate affects progression of a transplantable melanoma in vivo if given without a vaccine (Supplementary Fig S1). We injected mice subcutaneously with 5 × 104 (Fig. 1A) or in a follow-up experiment 3 × 104 B16BrafV600E cells (Fig. 1B). Starting 6 or 8 days later, mice were treated orally for up to 3 weeks with fenofibrate given once daily at 100 or 200 mg/kg, respectively. Control mice received diluent instead. Tumor progression was monitored over time (Fig. 1A and B) and found to be indistinguishable between fenofibrate- and diluent-treated mice regarding onset of palpable tumors and speed of tumor progression. These results indicate that under the chosen experimental conditions fenofibrate by itself does not inhibit tumor progression, confirming previous results that showed fenofibrate reduced metastasis formation without changing the growth parameters of the primary tumors (19).

Assuming that timing of PPARα activation is crucial for the performance of vaccine-induced tumor antigen-specific CD8+ T cells, we explored different schedules for the fenofibrate treatment starting on days 3, 5, or 7 following vaccination of tumor-free mice with 1010 vp of the AdC68-gDMelapoly vaccine. The drug was given at 200 mg/kg dose for 15 days orally; control mice received DMSO. Mice were euthanized 18 days after vaccination and splenocytes were stained and sorted onto CD44+CD8+ T cells; their transcripts were isolated and upon reverse transcription probed by a comparative qPCR for those encoding factors and enzymes involved in glucose and fatty acid metabolism (Supplementary Fig. S2). Figure 2, which illustrates the cycle thresholds (Cτ) of the PCR reaction, with higher thresholds reflecting lower levels of transcripts, shows significant increases in some of the transcripts involved in fatty acid metabolism. Transcript levels for PPARα and acyl-CoA oxidase 1 (ACOX-1), the first enzyme of the fatty acid beta-oxidation pathway did not change when fenofibrate was given 3 days after vaccination but showed increases if drug treatment was delayed till day 5. Transcripts for carnitine palmitoyltransferase 1a (CPT1a), a key factor that is essential to transport long chain fatty acids into mitochondria where they are converted to acetyl-CoA, which can feed the citric acid cycle, increased after fenofibrate treatment regardless of the time frame of drug treatment. We observed some other trends that failed to reach significance such as decreased transcript levels for 3-hydroxybuttyrate dehydrogenase 1 (BDH1) an enzyme that is involved in the conversion of acetyl-CoA into ketone bodies and phosphoglycerate kinase 1 (PGK1), an enzyme of the glycolysis pathway, upon early drug treatment, confirming that timing of drug-induced PPARα activation affects the metabolism of vaccine-induced CD8+ T cells.

Mice were then injected with B16BrafV600E cells 3 days prior to AdC68-gDMelapoly vaccination. Mice were fed DMSO or fenofibrate daily for up to 21 days starting 3, 5, or 7 days after vaccination and tumor progression was tracked (Supplementary Fig. S3). As shown in Fig. 3A, fenofibrate treatment delayed onset of tumor development and this reached significance if the drug treatment was started on days 5 or 7 after vaccination. Fenofibrate treatment also slowed tumor progression and by day 23 after challenge tumor diameters of all fenofibrate-treated groups were significantly lower than those of control mice. The delay was most pronounced if fenofibrate was given 5 days after vaccination. There were no significant differences between the fenofibrate groups; the day 5 protocol was selected for the additional experiments as it tended to perform best. We next determine whether fenofibrate given on day 5 to vaccinated mice affected numbers of T cells that were present in the tumors. As shown in Fig. 3B and C, numbers of CD4+ and CD8+ T cells were higher within tumors of fenofibrate than DMSO-treated mice, which likely reflects increased recruitment, retention, or survival.

Once we determined the optimal time for onset of fenofibrate treatment, that is, day 5 after vaccination, which equates to day 8 after tumor cell challenge, we tested whether fenofibrate could induce melanoma-specific CD8+ T cells in unvaccinated tumor-bearing mice or changed the phenotypes of activated splenocytes or TILs or if these changes were specific for vaccine-induced T cells. Mice were either given AdC68-gDMelapoly or the diluent and then 5 days later they were treated daily for 3 weeks with fenofibrate (Supplementary Fig S4A). Splenocytes and TILs were isolated 1 day later and tested by tetramer staining for frequencies of CD8+ T cells to the immunodominant epitope of Trp-1. Fenofibrate given without vaccination did not induce Trp-1-specific CD8+ T cells (Supplementary Fig. S4B). To assess phenotypes we compared naïve and recently activated, that is, PD1hiCD62LlowCD8+ T cells from the unvaccinated mice to tetramer+CD8+ or tetramer+ PD1hiCD62LlowCD8+ T cells from the vaccinated group from spleens and tumors. Activated CD8+ T cells from unvaccinated mice had after fenofibrate treatment similar percentages of cells high for levels for Tbet, CD127, LAG-3, and HIF1α and only slightly lower frequencies for PPARα, indicating that the drug independent of the vaccine affected these changes in all activated CD8+ T cells but not in naïve T cells. KLRG1 was only high on tet+CD8+ T cells from the vaccine group (Supplementary Fig. S4C).

Subsequently, we explored the effects fenofibrate on the metabolism of cells within tumors of mice that had not been vaccinated. Tumors were harvested once they reached approximately 1.5 cm in diameter from mice that been treated with DMSO or fenofibrate. Sera were collected from the same mice. The tumor interstitial fluids were isolated and, together with sera, tested for levels of glucose (Supplementary Fig. S5). As shown in Fig. 4A, glucose levels were significantly higher within tumors of mice that received fenofibrate compared with those that were treated with DMSO. T cells isolated from tumors of fenofibrate treated mice in turn unlike tumor-infiltrating myeloid cells or neutrophils showed increased glucose uptake compared with those from tumors of DMSO-treated mice (Fig. 4B; Supplementary Fig. S6). To test this further, we isolated RNA from the tumors of mice that had been treated with fenofibrate or diluent but that had not been vaccinated (Supplementary Fig. S6). Melanomas of mice that are not vaccinated have modest numbers of T-cell infiltrates and we therefore assume that the cellular composition reflects mainly tumor cells, tumor stroma cells, and subsets of myeloid cells. Upon reverse transcription, we tested the cDNA for transcripts of fatty acid metabolism, that is, PPARα, ACOX1, PNPLA2, and BDH, and transcripts encoding factors of glucose metabolism, that is, GLUT1, HK2, and PGK1. In most mice, transcripts for markers of fatty acid increased while transcripts encoding markers of glucose metabolism decreased upon fenofibrate treatment (Fig. 4C). We observed strong correlations between different transcripts of fatty acid metabolism, that is, PPARα with ACOX1 and BDH and transcripts of glucose metabolism such as GLUT1 with HK2 and HK2 with PGK1 (Fig. 4D). This in turn indicates that fenofibrate reduced glucose consumption by tumor and tumor stroma cells thus increasing the overall content of this nutrient within the TME.

To assess the effect of fenofibrate given at different times after vaccination on vaccine-induced T cells, we measured frequencies and functions of circulating vaccine-induced melanoma antigen-specific CD8+ T cells in melanoma bearing mice (Fig. 5; Supplementary Fig. S3). Frequencies of melanoma antigen-specific IFNγ-producing CD44+CD8+ T cells circulating in blood were significantly higher in the fenofibrate than the DMSO groups; this was more pronounced if fenofibrate treatment was started on day 3 than on day 5 (Fig. 5A). Frequencies of CD44+CD8+ T cells producing TNFα for all timepoints, or IL2 for day 5, were also increased in the fenofibrate-treated groups, while those of T cells producing perforin decreased. The latter differences failed to reach significance. Boolean gating showed that in all four groups the majority of responding CD8+ T cells exhibited 1 or 2 functions (Fig. 5B) and produced IFNγ and about 25%–30% also produced TNFα (Fig. 5C). Fenofibrate did not change this profile.

To test whether fenofibrate given as of day 5 after vaccination affected T-cell differentiation pathways and metabolism, TILs and splenocytes from tumor-bearing AdC68-gDMelapoly–vaccinated mice that had been treated with the drug or the diluent were isolated and stained with a live cells stain, an antibody to CD8, a tetramer specific for an immunodominant epitope within the vaccine insert, and antibodies to the metabolic markers PPARα and HIF1α, which regulate fatty acid and glucose metabolism, respectively, the exhaustion markers PD-1 and LAG-3, T-bet, which controls T-cell functions, KLRG1, CD127, and CD62L, three markers that are indicators of terminal T-cell differentiation, memory formation, or activation (Supplementary Fig. S5). Tetramer+CD8+ T cells were compared with naïve CD62LhiCD8+ T cells (Fig. 6). Vaccine-induced CD8+ splenocytes showed decreases in HIF1α and CD62 L combined with increases in the expression of PD-1, LAG3, T-bet, and KLRG1, compared with naïve CD8+ splenocytes. Comparing splenocytes with TILs showed tetramer+ CD8+ TILs cells had higher expression of PPARα, HIF1α and the two exhaustion makers, and lower expression of KLRG1. CD127 tended to be lower on TILs than splenocytes, although this failed to reach significance. Comparing tetramer+CD8+TILs from fenofibrate-treated mice with those from diluent-treated mice showed that the drug increased expression of PPARα, KLRG1, and unexpectedly HIF1α a transcription factor that is induced upon hypoxia and promotes glycolytic energy production. PD-1 tended to be higher upon fenofibrate treatment.

To further analyze how fenofibrate and timing of onset of drug treatment affect TIL metabolism, TILs were isolated from vaccinated, tumor-bearing mice that had been treated with drugs or diluent as of days 3, 5, or 7 after vaccination (Supplementary Fig. S3). TILs were sorted into CD44+CD8+ cells. RNA was isolated and, upon reverse transcription, levels of transcripts for factors involved in fatty acid and glucose metabolism were determined by qPCR. As shown in Fig. 7A fenofibrate given early on day 3 after vaccination did not have a significant effect on T-cell transcripts, while treatment as of day 5 after vaccination increased levels of transcripts for PPARα and CPT-1a. A further delay of drug treatment until day 7 after vaccination resulted in increased expression of transcripts for BDH, an enzyme involved in ketone body formation, GLUT1, the receptor involved in glucose uptake, PGK1, an enzyme of glycolysis, and IDH3, an enzyme that converts isocitrate to 2-oxoglutarate in the tricarboxylic acid cycle. Fenofibrate-induced changes in transcripts within CD8+ TILs were similar but not identical to those in the corresponding cells from spleens of mice that did not carry tumors (Fig. 2A and B). This most likely reflects that levels of transcripts encoding proteins that affect metabolism are driven by the availability of nutrients, which differs between spleens and the more hypoxic and hypoglycemic tumors. Fenofibrate-induced increases of GLUT1 and PGK1 in TILs suggest that the drug paradoxically augmented the use of glucose by TILs, which was presumably caused by the increased presence of glucose within the tumors of fenofibrate-treated mice. To test whether increased use of glucose contributed to the improvement of the vaccine-induced delay in tumor progression, we correlated the AUC of the tumor diameter measured over time to the Cτ values we obtained for the different metabolic markers. There were no significant correlations for mice that had been treated with DMSO. For fenofibrate-treated mice upon combining the data for all three treatment regimens tumor progression directly correlated with Cτ values for several markers of fatty acid metabolism including PPARα but negatively correlated with transcripts for enzymes of glycolysis (Fig. 7B). This was largely driven by the results obtained from the day 5 fenofibrate treatment. As Cτ values are inversely proportional to the amount of cDNA in a sample, this indicates that the improved performance of vaccine-induced TILs was not driven by their increased fatty acid catabolism as in the previously published adoptive transfer models (15) but rather by the increased availability of glucose, which allowed TILs to fuel their bioenergetic needs through this nutrient.

Tumor cells use glycolysis to fuel their high demand for energy (20, 21). This pathway is bioenergetically wasteful (22) but allows tumor cells to gain energy very rapidly and to reserve their mitochondria for anabolic processes needed for proliferation (21). As a result, the TME becomes depleted of glucose while levels of lactic acid increase causing acidosis (23). Low vascularization of rapidly growing tumors prevents replenishing nutrients from the periphery and also reduces the availability of oxygen. Hypoxia in turn by inducing HIF1α expression further promotes anaerobic glycolysis (24, 25). Metabolic stress within the TME impairs CD8+ TIL functions (26) and leads to active immunotherapy failures (15, 27–29). This could potentially be circumvented by metabolic manipulations as is being explored (30–32).

Here we show in a mouse melanoma model that combining a T cell–inducing cancer vaccine with a PPARα agonist that promotes the use of fatty acids for energy and biomass production improves the ability of vaccine-induced CD8+ TILs to delay tumor progression. This confirms previous studies with adoptively transferred T cells, which suggested that therapies with in vitro expanded TILs or chimeric antigen receptor T cells could be improved by adding fenofibrate, a PPARα agonist, to the cultures (15). Studies presented here extend these results by demonstrating that the same approach is effective if applied to tumor-bearing mice, which opens the way to use fenofibrate or a similar drug to augment the efficacy of cancer vaccines. Unexpectedly, the mechanism that increased the efficacy of the tumor vaccine upon fenofibrate treatment was not linked to increased fatty acid metabolism by vaccine-induced TILs but rather to the reduced glucose consumption by tumor and tumor stroma cells increasing the availability of this nutrient.

Fenofibrate is a drug that is licensed for treatment of high levels of cholesterol and triglycerides (33). It activates PPARα but also impairs mitochondrial functions by inhibiting complex I of the electron transfer chain (34). Fenofibrate is hydrolyzed by serum enzymes into fenofibric acid, which retains PPARα agonist activity but cannot cross mitochondrial membranes and thereby has no effects on energy production in the citric acid cycle. In our previous adoptive transfer studies (15), in which only the transferred lymphocytes were treated with fenofibrate, we initiated treatment immediately at the onset of CD8+ T cells' activation by a vaccine in vivo or by CD8/CD28 ligation in vitro. T-cell activation by a genetic vaccine such as the AdC68-gDMelapoly vector used here is delayed till antigen is produced, processed, and presented. Switching the metabolism of lymphocytes cells too early before they encounter their antigen and start the activation process may blunt T-cell activation responses. We therefore explored the effect of fenofibrate given at various times after vaccination on tumor progression and vaccine-induced T-cell functions and metabolism. Fenofibrate given without a vaccine did not affect the growth of the melanoma although it affected the metabolism of cells within the tumor stroma, which switched from glucose to fatty acid catabolism. This metabolic switch, which could have been further promoted by the availability of nutrients within the tumors, has been observed in previous studies; one showed that CPT-1 is upregulated in melanomas (35), the other reported that expression of fatty acid binding proteins, which regulate fatty acid uptake and lipid trafficking, promotes melanoma progression (36). Notwithstanding, in our study, increased fatty acid metabolism by cells within the TME did not accelerate tumor progression, as opposed to another study that linked a high-fat diet to accelerated growth of B16V600E melanomas (37).

Regardless of when it was initiated, fenofibrate treatment increased frequencies of vaccine-induced CD8+ T cells and delayed tumor progression. The latter was most pronounced when fenofibrate treatment was started 5 days after vaccination, which also resulted in increases in numbers of T cells, which could be isolated from tumors of fenofibrate as compared with DMSO-treated mice. The 5-day delay in treatment also caused the highest increases in the TILs' transcripts for PPARα and CPT-1 and, in some mice, an increase in factors involved in glucose metabolism. The latter became more pronounced upon delaying drug treatment for 7 days, indicating that fenofibrate paradoxically increased the CD8+ TIL's glucose metabolism. Our data suggest that increases in factors involved in glycolysis reflect that the reduced consumption of glucose by tumor cells and other cells within the tumor stroma enhances the glucose content within tumors thus allowing TILs more access to this nutrient, which could potentially increase their survival. Tumor growth showed indirect correlations with levels of transcripts involved in glucose metabolism while PPARα and molecules downstream of this transcription factor were positively correlated, suggesting that the mechanism of delay in tumor progression in presence of fenofibrate given to tumor-bearing mice fundamentally differs from that which we previously reported for fenofibrate-treated adoptively transferred T cells (15).

In summary, our findings reveal that a PPARα agonist increases the efficacy of a cancer vaccine by instructing cells within tumors to increase fatty acid metabolism, which enhances glucose content within tumors thus allowing TILs access to this nutrient.

A. Chekaoui is supported by a grant from Department of Defense (W81XWH-19-1-0485 CA180191). H.C.J. Ertl reports grants from Department of Defense during the conduct of the study; grants from Virion Therapeutics; personal fees from Freeline, Biogen, Takadea, and Regenxbio; and non-financial support from Gamalaya Institute outside the submitted work; in addition, H.C. Ertl has a patent for Fenofibrate as an adjuvant for cancer therapy pending.

A. Chekaoui: Resources, data curation, formal analysis, investigation, writing–original draft, writing–review and editing. H.C.J. Ertl: Conceptualization, formal analysis, supervision, funding acquisition, investigation, methodology.

This study was supported by a grant from the Department of Defense (W81XWH-19-1-0485 CA180191). The authors acknowledge the Wistar Flow Cytometry Facility help with cell sorting and flow cytometry, and the Wistar Imaging Facility for analyzing tumor sections.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data