Abstract
Nutritional factors play crucial roles in immune responses. The tumor-caused nutritional deficiencies are known to affect antitumor immunity. Here, we demonstrate that pancreatic ductal adenocarcinoma (PDAC) cells can suppress NK-cell cytotoxicity by restricting the accessibility of vitamin B6 (VB6). PDAC cells actively consume VB6 to support one-carbon metabolism, and thus tumor cell growth, causing VB6 deprivation in the tumor microenvironment. In comparison, NK cells require VB6 for intracellular glycogen breakdown, which serves as a critical energy source for NK-cell activation. VB6 supplementation in combination with one-carbon metabolism blockage effectively diminishes tumor burden in vivo. Our results expand the understanding of the critical role of micronutrients in regulating cancer progression and antitumor immunity, and open new avenues for developing novel therapeutic strategies against PDAC.
Significance:
The nutrient competition among the different tumor microenvironment components drives tumor growth, immune tolerance, and therapeutic resistance. PDAC cells demand a high amount of VB6, thus competitively causing NK-cell dysfunction. Supplying VB6 with blocking VB6-dependent one-carbon metabolism amplifies the NK-cell antitumor immunity and inhibits tumor growth in PDAC models.
This article is featured in Selected Articles from This Issue, p. 5
INTRODUCTION
Tumor-induced immune suppression plays a central role in cancer cells’ evasion of immune surveillance and resistance to immunotherapies (1). Both experimental and epidemiologic studies have shown that nutritional factors, including macronutrients (proteins, carbohydrates, and fatty acids) and micronutrients (vitamins and minerals), can regulate cancer progression and immune homeostasis (2). Activation of oncogenic pathways in the cancer cells rewires their metabolic profiles and increases the consumption of nutrient resources from the tumor microenvironments (TME), resulting in the deprivation of vital metabolites for surrounding noncancerous cells, including immune cells (3). The nutritional competition in the TME has been shown to induce the exhaustion of antitumor immune cells and promote immunosuppression (4, 5). Thus, understanding the mechanism of metabolic cooperation and competition in the TME may provide additional therapeutic opportunities against cancer.
Despite success in other cancers, immunotherapies showed limited efficacy against pancreatic ductal adenocarcinoma (PDAC; 6). PDAC TMEs are immunologically cold, posing a great challenge specifically for T cell–based immunotherapies (7). As central innate immune effectors, natural killer (NK)-cell–mediated elimination of malignant cells is MHC-unrestricted and independent of tumor-associated antigen presentation (8). Loss of MHC-I molecules, which is prevalent in PDAC (9, 10), theoretically sensitizes cancer cells to NK cells. Notably, NK-cell degranulation is a predictive prognostic factor in PDAC (11). Therefore, NK cell–based immunotherapies present promising approaches to improve the clinical outcomes for patients with PDAC.
RESULTS
Pancreatic Cancer Cells Are Resistant to NK-cell Cytotoxicity
The bioinformatic analysis of The Cancer Genome Atlas (TCGA) patient cohort with QuanTIseq deconvolution method (12) showed that PDAC patients with high NK infiltration in the tumor displayed improved survival, compared with the ones with low NK infiltrations (Supplementary Fig. S1A). Consistent with the in silico analysis results, enrichment of NK cell–specific gene transcripts, NCAM1 and KLRK1, was associated with a longer overall survival in PDAC (Supplementary Fig. S1A). Transcripts for NK-cell receptor activating ligands were detectable in 11 PDAC cell lines (Supplementary Fig. S1B). Moreover, these ligands showed increased mRNA expression in most PDAC cell lines than an immortalized human pancreatic normal epithelial cell line (HPNE) (Supplementary Fig. S1B).
Given the cancer-protective role of tumor-infiltrating NKs, we treated PDAC tumor-bearing mice with recombinant IL15, a cytokine that induces the proliferation and activation of NK-cells. However, increased intratumoral NK-cell population post IL15 treatment did not improve the survival of mice orthotopically implanted with KPC tumor cells (murine pancreatic cancer cells derived from an LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre genetically engineered mouse; Supplementary Fig. S1C–S1E). These results indicate that perhaps NK cells in the PDAC TME are functionally incompetent. Furthermore, peripheral blood–derived human NK cells were incompetent in reducing the growth of PDAC organoids in coculture assays (Supplementary Fig. S1F and S1G). Two-dimensional adherent as well as three-dimensional spheroid culture studies indicated that PDAC cells are more resistant to NK cell–mediated cytotoxicity, as compared with HPNE cells (Supplementary Fig. S1H–S1J). Moreover, NK cells displayed reduced expression of activation markers (IFNγ and CD107a), upon coculturing with PDAC cells compared with HPNE cells (Supplementary Fig. S1I).
Next, we investigated tumor cell–induced transcriptomic changes in NK cells by performing RNA sequencing (RNA-seq). Principal component analysis (PCA) of RNA-seq data from NK cells pre-cocultured with K562, HPNE, or pancreatic tumor cells (T3M4, CFPAC1, or Capan2) showed distinct transcriptional profiles (Fig. 1A). Gene set enrichment analysis (GSEA) suggested that pancreatic tumor cells reduce the inflammatory response signals in NKs (Fig. 1B). In coculture assays, PDAC cells downregulated the mRNA expression of genes involved in NK cell–mediated cytotoxicity (GZMA, PRF1, and IFNG) with a parallel increase of key inhibitory receptors (TIGIT, LAG3, and KLRC1) on NK cells (Fig. 1C). Correspondingly, mouse KPC tumor–infiltrating NK cells displayed significantly higher levels of NKG2A and TIGIT compared with peripheral NK cells from healthy control mice (Fig. 1D and E). These results suggest that PDAC cells significantly impair NK-cell functions that translate into a limited cytotoxic effect of NK cells during tumor growth progression.
PDAC Cells Regulate NK-Cell Activity via Metabolic Reprogramming
PDAC cell lines showed differential susceptibility to NK cell–mediated killing (Supplementary Fig. S1K). However, the susceptibility to NK cells did not correlate with the expression levels of NK cell–activating or inhibitory ligands in PDAC cells (Supplementary Fig. S1L and S1M), indicating that PDAC cell–mediated inhibition of NK-cell activity is likely through indirect interactions. Accordingly, coculturing of NK cells with PDAC cells or PDAC organoids, prior to the subsequent cytotoxicity assay, significantly reduced NK cell–mediated killing of other sensitive target cells (K562) and attenuated the expression of IFNγ in NK cells upon coculturing with K562 (Supplementary Fig. S1N–S1P). Moreover, NK-cell activating ligands (UBLP-Fc, MICA-Fc) and anti-NKp30 failed to induce IFNγ expression in the NK cells that were previously cocultured with PDAC cells (Supplementary Fig. S1Q). These results suggest that the immunosuppressive effect is potentially caused by the conditioned media (CM) from PDAC cells. Indeed, replacing the PDAC cell CM with fresh media (FM) drastically increased the NK cell–mediated killing of PDAC cells (Fig. 1F and H; Supplementary Fig. S1R).
To identify PDAC CM–mediated immunosuppressive mechanisms that control NK-cell function, PDAC CM was fractionated by a 3 kDa cutoff centrifugal filter into >3 kDa (enriched mainly for proteins) and <3 kDa (enriched for metabolites) parts. Metabolites (<3 kDa enriched fraction), rather than proteins, contributed to PDAC CM–mediated suppression of NK-cell function as evidenced by attenuated killing, degranulation (CD107a expression), and IFNγ production (Fig. 1I–K; Supplementary Fig. S1S).
To identify tumor-derived metabolites that contribute to NK-cell dysfunction, we performed LC-MS/MS-based metabolomics of NKs post-coculture with PDAC cells or control HPNE cells. Of note, partial least squares discriminant analysis (PLS-DA) showed distinct intracellular metabolite profiles among NK cells cultured alone or cocultured with PDAC or HPNE cells (Fig. 1L). NK cells cocultured with PDAC cells displayed a significant alteration in Tryptophan (Trp), arginine, alanine, and Vitamin B6 (VB6) metabolic pathways (Fig. 1M; Supplementary Fig. S1T and S1U). Variable importance projection (VIP) analysis, which ranks the metabolites based on their overall importance for separating different samples, showed that the intermediates of Trp–Kyn metabolic pathway [Tryptophan (Trp), Kynurenine (Kyn), and 3-hydroxyanthralinic acid (3-HAA)] were differentially altered in NK cells precocultured with or without PDAC or HPNE cells (Supplementary Fig. S1T). NK cells demonstrated significantly higher levels of Kyn upon coculture with PDAC cells as compared with HPNE or K562 (Supplementary Fig. S1V). Consistent with the in vitro results, the KPC mouse model as well as patients with PDAC displayed significantly higher serum Kyn levels as compared with controls (Supplementary Fig. S1W and S1X). In line with previous reports (13–15), NK cells pretreated with Kyn significantly reduced NK cell–mediated cytotoxicity to K562 cells (Supplementary Fig. S1Y). Besides the Trp/Kyn pathway, we noted that vitamin B6 (pyridoxine) was also important for distinguishing NK cells cocultured with or without PDAC or HPNE cells (Supplementary Fig. S1T and S1U). Vitamins actively participate in the maintenance of immune response, where they serve as cofactors and play vital roles in immune regulation (16). VB6 metabolism showed significant alterations in PDAC cell–cocultured NKs (Fig. 1M; Supplementary Fig. S1T and S1U). PDAC cell–cocultured NKs demonstrated a significant decrease in the intracellular levels of pyridoxine (a common form of VB6 in food) and pyridoxal phosphate (PLP, the active form of VB6; Fig. 1N and O). The level of pyridoxine, the form of VB6 in cell culture media, was significantly lower in PDAC-NK coculture-conditioned media (Supplementary Fig. S1Z). Additionally, attenuated levels of pyridoxine and PLP were recorded in the interstitial fluid from primary pancreatic tumors and the plasma samples from orthotopic PDAC tumor-bearing mice compared with healthy controls (Fig. 1P and Q). Also, patients with PDAC displayed significantly lower PLP levels in serum compared with control gallbladder patients (Fig. 1R). Of note, removal or reduction of pyridoxine levels in cell culture medium impaired NK cell–mediated cytotoxicity against K562 and HPNE cells (Fig. 1S). Together, these results indicate that PDAC cells regulate NK-cell function by altering PLP and Kyn levels in the coculture milieu and TME.
Targeting VB6 and Trp/Kyn Metabolism Impedes PDAC-Induced NK-cell Dysfunction
Our study showed that alterations in VB6 and Kyn levels regulate NK-cell activity. Trp/Kyn have an inhibitory impact on NK-cell function (14, 15). Importantly, VB6 is a coenzyme for multiple enzymatic steps in the Trp/Kyn pathway and other metabolic pathways such as one-carbon (1C) metabolism and glycogenolysis (16, 17). Thus, we next examined if targeting Trp/Kyn and supplementing VB6 alone or in combination could lower the deleterious effects of PDAC cells on NK cells. VB6 (PLP, active form of VB6, 100 nmol/L) supplementation and treatment with IDO1 inhibitor (IDO1i, 100 nmol/L, Epacadostat) elevated the intracellular PLP and reduced Kyn to control levels in NK cells cocultured with PDAC cells (CFPAC1 or T3M4; Supplementary Fig. S2A andS2B). Treatment of VB6 and IDO1i enhanced the NK-cell cytotoxicity against CFPAC1 and T3M4 cells (Fig. 2A and B). The treatment also increased the percentage of CD107a and IFNγ-positive NK cells (Fig. 2C). Similarly, adding VB6 and IDO1i to the CM of PDAC cells successfully restored the NK-cell cytotoxicity against K562 cells (Supplementary Fig. S2C). In the presence of VB6 and IDO1i, NK cells significantly restricted the growth of PDAC cell 3D spheroids and patient-derived organoids (Supplementary Fig. S2D–S2F). To further examine the effects of VB6 and IDO1i on NK cell–mediated immunity against PDAC in vivo, we utilized a syngeneic orthotopic mouse model of PDAC (Supplementary Fig. S2G). IDO1i, 100 mg/kg/day, and VB6 (PLP,100 mg/kg) treatment led to a significant peripheral PLP enrichment and Kyn reduction in tumor-bearing mice compared with saline-treated counterparts (Supplementary Fig. S2H). Comparable tumor volume and tumor weight were observed in vehicle (CTL), VB6, IDO1i, and IL15 treatment alone groups. In contrast, a significant tumor growth delay was observed in mice treated with a therapeutic combination of IDO1i and VB6 and a triple combination of IL15, IDO1i, and VB6 (Fig. 2D and E).
We then assessed the impact of the triple combination on the immune microenvironment in treated tumors. Combined treatment with IDO1i and VB6 triggered a significant accumulation of tumor-infiltrating NK cells (Fig. 2F). IL15 treatment further increased the NK-cell population in IDO1i and VB6-treated pancreatic tumors. Furthermore, NK cells derived from double or triple combination-treated tumors displayed an activated phenotype (Fig. 2G). However, all the treatment cohorts displayed a comparable number of total tumor-infiltrating T cells (Supplementary Fig. S2I and S2J). VB6-supplemented tumor-bearing mice showed a modestly increased level of Th17 (IL17+,CD4+ T cell) ratio, but other subtypes of CD4+ T cells were not altered (Supplementary Fig. S2K). Although the proportion of IFNγ+ CD8+ T cells was increased in IDO1i and VB6-treated tumors, the proportions of CD69+CD8+ T cells, PD-1+CD8+ T cells, and TIGIT+CD8+ T cells were similar in all cohorts (Supplementary Fig. S2L).
The combination of IDO1i and VB6 significantly increased the survival of KPC tumor-bearing mice. IL15 further improved survival in mice treated with IDO1i and VB6 (Fig. 2H). These results suggest that VB6 supplementation and inhibition of Trp/Kyn metabolism decreases PDAC tumor burden in vivo. Increased accumulation and activity of intratumoral NKs in IDO1i and VB6-treated tumors indicate that perhaps NK cells contribute to the treatment efficacy. To interrogate the contribution of NKs in antitumor efficacy of IDO1i and VB6-treated mice, we eliminated NKs by using anti-NK1.1 antibody (clone PK136; Supplementary Fig. S2M). NK-cell depletion significantly attenuated the increased survival in the IDO1i and VB6 combination cohort (Fig. 2I). To determine if T cells play a role in controlling the tumor growth in the combination treatment group, CD4+ and CD8+ T cells were depleted in mice administered with IL15+IDO1i+VB6 combination (Supplementary Fig. S2N). Depletion of CD4+ and CD8+ T cells in the mice with combination treatment slightly increased tumor growth compared with the isotype control; however, IL15+IDO1i+VB6 treatment significantly reduced tumor weight even in the absence of CD4 and CD8 T cells (Supplementary Fig. S2O), suggesting the limited or inexistent role of CD8/CD4 T in our model.
VB6 Regulates NK-cell Activity via Catalyzing Glycogenolysis
Given the failure of IDO inhibitors against PDAC in the clinic, we interrogated the other VB6-dependent pathways that might underlie NK-cell activity in our model. To investigate this, we performed LC-MS/MS analysis of human primary NKs after being stimulated with HPNE target cells under different concentrations of VB6 (pyridoxine; Supplementary Fig. S3A). Reduction of pyridoxine to 10% of the standard alpha-MEM medium (1.0 mg/mL) significantly reduced the PLP level in NK cells and weakened the killing activity of NK cells against HPNE and K562 cells (Supplementary Fig. S3B and S3C). PLS-DA models showed distinct intracellular metabolic profiles for NKs cultured at different pyridoxine levels with or without HPNE stimulation (Supplementary Fig. S3D). Notably, VB6 reduction significantly affected glycolysis, gluconeogenesis, and VB6 metabolic pathways in HPNE-stimulated NK cells (Supplementary Fig. S3E). Metabolic intermediates of glycolysis pathways (such as F/G-6P, Lactate, and 3-PG/2-PG) were important for segregating the metabolic profiles of nonstimulated and HPNE-stimulated NK cells with or without VB6 supplementation (Supplementary Fig. S3F).
Subsequently, we assessed the metabolic profile of NKs precultured in PDAC cell CM (CFPAC1 CM and T3M4 CM) for 48 hours at different VB6 doses and then cultured with or without HPNE stimulation. (Supplementary Fig. S3G). Supplementation with extra pyridoxine restored the PLP level in NK cells cultured in PDAC CM (Supplementary Fig. S3H and S3I). The PLS-DA analysis demonstrated that NK cells derived from HPNE CM, PDAC CM, and PDAC CM with or without pyridoxine supplementation displayed different metabolic profiles (Supplementary Fig. S3J). Overall, VB6 supplementation significantly affected glycolysis, gluconeogenesis, and VB6 metabolic pathways in HPNE-stimulated NK cells irrespective of prior culture conditions (Supplementary Fig. S3K).
Although PLP is not a cofactor for glycolysis enzymes, it actively participates in glycogen catabolism as a coenzyme for glycogen phosphorylase (PYG; ref. 18), which releases glucose from glycogen (Supplementary Fig. S3L). In our data, two key intermediates in glycogen catabolism, glucose-1-phosphate (Glc-1-P) and UDP-glucose, were among the top metabolites in PLS-DA-VIP analysis of NK cells with VB6 reduction (Supplementary Fig. S3F). Of note, Glc-1-P and UDP-glucose (suggesting less glycogen breakdown) were drastically downregulated in NKs upon coculturing with PDAC cells but not under conditions of monoculture or coculture with HPNE cells (Fig. 3A and 3B)….
