Myeloid cell-derived creatine in the hypoxic niche promotes glioblastoma growth

Abstract

Glioblastoma (GBM) is a malignancy dominated by the infiltration of tumor-associated myeloid cells (TAMCs). Examination of TAMC metabolic phenotypes in mouse models and patients with GBM identified the de novo creatine metabolic pathway as a hallmark of TAMCs. Multi-omics analyses revealed that TAMCs surround the hypoxic peri-necrotic regions of GBM and express the creatine metabolic enzyme glycine amidinotransferase (GATM). Conversely, GBM cells located within these same regions are uniquely specific in expressing the creatine transporter (SLC6A8). We hypothesized that TAMCs provide creatine to tumors, promoting GBM progression. Isotopic tracing demonstrated that TAMC-secreted creatine is taken up by tumor cells. Creatine supplementation protected tumors from hypoxia-induced stress, which was abrogated with genetic ablation or pharmacologic inhibition of SLC6A8. Lastly, inhibition of creatine transport using the clinically relevant compound, RGX-202-01, blunted tumor growth and enhanced radiation therapy in vivo. This work highlights that myeloid-to-tumor transfer of creatine promotes tumor growth in the hypoxic niche.

Keywords: creatine metabolism; glioblastoma; myeloid cells; pseudopalisading necrosis.

Fig. 1:. Tumor-associated myeloid cells (TAMC) produce creatine de-novo from arginine.

In (A-B), Five matched tumor and PBMC samples were obtained from patients with newly diagnosed GBM, and CD163⁺ magnetic bead-based isolation was performed prior to running untargeted metabolomics via LC-MS. In (A), a heat-map of changes in peripheral versus tumoral myeloid cells organized by paired t-test significance (top 25 shown), and in (B), normalized peak area measurements of key creatine metabolites in the five matched samples. In (C), schema of the de-novo creatine biosynthetic pathway. In (D) dimensional reduction of myeloid cells in human GBM . Colors indicate the cluster affiliation based on an iterative shared nearest neighbor approach and the indicated cell types are based on the classification of Ravi et al. In (E), dimensional reduction with gene expression of GATM and GAMT indicating an enhanced expression in the activated myeloid cluster. In (F), spatially weighted correlation analysis of the integrated spatially resolved transcriptomic-metabolomic dataset , performed for the GATM, GAMT and SCL6A8 genes (left panel), the myeloid cell types (right panel). In (G) the spatially distinct transcriptional phenotypes, and in (H) the Verhaak subgroups. In (I) the comparison and validation of GAMT, GATM and SCL6A8 expression with its spatially weighted correlation to the myeloid cell types. In (J), the spatial localization of the θMa population surrounding the hypoxic and pseudopalisading niche. In (K), normalized spatial gene expression for hallmark hypoxia genes overlaid with paired H&E image. In A-B, n=5 paired PBMC and tumor samples with statistics via calculated paired Student’s t-test. Data in D-K is pooled and analyzed from 16 patients with GBM. *=p<0.05; **=p<0.01; ***=p<0.001.

Fig. 2:. Creatine synthesis and uptake is both spatially and cellularly compartmentalized in the hypoxic niche.

In (A), multiplexed immunofluorescence to examine the location of macrophages (CD163⁺) or microglia (TMEM119) in the context of hypoxic regions surrounding necrosis. In (B), immunostaining of flash frozen tumors and (C), MFI of hydroxyprobe in M-MDSCs, microglia, CD8, Tregs, and Tconv from mice implanted intracranially (i.c.) with CT-2A tumors for 14 days and injected i.v. with pimonidazole. In (D-F), single R annotation of all major cell populations from scRNA-sequencng of CD45⁺ cells isolated from CT-2A tumors 2 weeks post i.c. implantation. In (G), bulk metabolomic analysis of splenic macrophage and TAMC populations isolated 2 weeks post i.c. with GL261 or CT-2A tumor cells. In (H), FKPM values of all genes involved in de-novo creatine biosynthesis from our previously published dataset . In (I), percent M+1 creatine in splenic CD8⁺ T-cell and TAMC populations cultured with ¹³C-arginine overnight. Significance was calculated via a paired Student’s t-test in A, and unpaired Student’s t-test in H In A, n=6 GBM tissues with the entire section analyzed. In D-F, n= 2-3 independent experiments with n=5-10 tumors pooled per n. Data shown in G-I is integrated from two independent experiments. Error bars are expressed as mean ± SEM. p<0.05*, p<0.01**, p<0.001***.

Fig. 3.. De-novo Creatine synthesis is regulated by HIF1a in TAMCs.

In (A-B) GATM and GAMT mRNA and (C) protein expression of in vitro generated TAMCs cultured under the indicated conditions for 6h (A-B) or 24h (C-D, G-H). In (E-F), densitometry for (C). In (G-H), protein expression of GATM (G) or GAMT (H) normalized to GAPDH under indicated conditions and measured using Protein Simple Wes quantitative western blotting. In (I) Hif1a and Hif2a mRNA expression in TAMCs treated with lipid-nanoparticle (LNP) containing siRNA against murine Hif1a or Hif2a for 24h. In (J-K), GATM mRNA (J) and protein (K) expression in TAMCs treated with siRNA-containing LNP and subjected to indicated conditions for 6h (J) or 24h (K). In A-F, n=4-6, In G-H, n=3-4, in I, n=2, and in J-K, n=3-6 independent experiments. One Way ANOVA with Tukey’s post-hoc was used for all statistical analysis and individual comparisons. Error bars are expressed as mean ± SEM. p<0.05*, p<0.01**, p<0.001***.

Figure 4.. Creatine uptake by GBM is regulated by hypoxia.

In (A), two representative images of the spatially weighted gene expression of GATM, GAMT and SLC6A8 in human GBM tissue. In (B) spatial trajectory analysis of SLC6A8, GATM and GAMT in relationship to the necrotic niche. In (C), Slc6a8 expression in CT-2A, GSC-005 and U251 tumor cells after 24hrs of 1% O₂. In (D) SLC6A8 upregulation in CT-2A, GSC-005, and U251 GBM cell lines under the indicated conditions for 24 hrs. All experiments in (D), are the results of 3-4 independent experiments. In (E), HIF mRNA expression in U251 GBM cells treated with LNP-siRNA against HIF1α and HIF2α for 24 hours. In (F), SLC6A8 mRNA expression in U251 cells treated with siRNA-containing LNPs for 24h prior to indicated treatment for 24h. In (G), the same siRNAs were used on U251 for 24 hours before being placed under normoxia or hypoxia + acidity (1% O₂ + pHe 6.7) for 24 hours for qPCR analysis. In (G-I), 14 days after CT-2A tumor implantation, TAMC and splenic myeloid cells were harvested via magnetic bead isolation and cultured for 24 hours in ¹³C-arginine media. In (G), flux media versus in-vitro generated TAMC supernatant after 24 hours culture in flux media. In (H-I), Cell-free supernatant was harvested from splenic or tumor myeloid cells after 24 hours of culture of flux media, and abundance of M+1 Guanidinoacetate (H), and M+1 Creatine (I) was analyzed via LC-MS. n=2 mice in each group for validation in E, N=3-6 mice for qPCR in C,D,F. In G-I, n=3 replicates representative of two individual experiments. One Way ANOVA with Tukey’s post-hoc was used for all statistical analysis in D-F and unpaired Student’s t-test was used in C, G-I to test for significance. Error bars are expressed as mean ± SEM. p<0.05*, p<0.01**, p<0.001***.

Figure 5.. Creatine uptake via SLC6A8 by tumor cells promotes cell growth, viability, and tumor progression.

In (A), ATP Luminescence assay in CT-2A tumor cells were cultured in 21% or 1% O₂ hypoxia for 4 hours. In (B), MTT assay assessing the viability of CT-2A under hypoxia and reducing concentrations of glucose. In (C) cell-titer glo analysis of CT-2A (left panel), GSC-005 (right panel), or GL261(bottom panel) tumor cells under hypoxia/glucose restriction (1mM) given equimolar (1mM) creatine, Creatine + β -GPA, or Creatine + Cyclocreatine for 24 hrs. In (D) Slc6a8 mRNA expression in CT-2A tumor cells treated with CRISPR against SLC6A8. In (E), the amount of unlabeled and ¹³C-labelled Creatine after 4 hours of culture in vector control (Vc) and SLC6A8 KO tumor cell lines. In (F), the kinetics of M+1 labeled Creatine (Cr) and M+1 labelled phosphocreatine (PCr) over time in control and KO tumor cell lines. In (G-I), 7.5x10⁴ of Vc and SLC6A8KO CT-2A were implanted orthotopically into WT mice and one cohort of mice was euthanized for Ki-67 immunostaining (G,H), and another was monitored for survival and plotted in a Kaplan Meier curve (I). In (J) SLC6A8 expression in U251 tumor cell lines treated with shRNA targeting SLC6A8 (and non-targeting controls). In (K) and Cell titer Glo luminescence assays in U251 tumor cell lines treated as in (J). In (L), Cell titer Glo luminescence assay in U251 tumor cells treated with 1mM Creatine. In A, n=10 per group; In B, n=4-6 per group; in C, n=6 per group; in D-E, n=3 per group. In G-I, n= 9 for Vc tumors, and n=5 for Slc6a8 KO tumors; In J, n=1 per group; In K-L, n=6 per group. A one Way ANOVA with Tukey’s post-hoc was used for all statistical analysis in (C,E,K,L) a unpaired Student’s t-test was used in (A,B,D,G). In F, a two-way ANOVA was performed followed by Sidak’s multiple comparisons test for significance. In (I) Log-Rank analysis was performed. Error bars are expressed as mean ± SEM. p<0.05*, p<0.01**, p<0.001***.

Fig. 6:. Inhibition of creatine transport perturbs multiple aspects of GBM biology.

In (A), a schematic demonstrating the assay performed in (B). In (B), Peak area of M+1 creatine (left) and M+1 phospo-creatine (right) in CT-2A tumor cells plated as shown in (A) and incubated for 8 hours in normoxia, 1% O₂ hypoxia, +/− 5mM β-GPA. In (C) Extreme Limiting Dilution Analysis (ELDA) of GSC-005 neurospheres with a diameter greater than 200mm treated either with 1mM Creatine in the presence or absence of equimolar SLC6A8 inhibitor, 3-Guanidinopropionic acid for 7-10 days. After 7 – 10 days, neurospheres with a diameter greater than 200 μm was counted. In (D-F) Kaplan Meier curves of mice implanted with i.c. with 7.5x10⁴ CT-2A, 1x10⁵ GL261 cells, or 5x10⁴ GSC-005 cells (respectively) and administered RGX-202-01 chow and/or 3Gy/3day (9Gy total radiation) or the combination, 7 days post-implantation. In (G), western blot (top) or qPCR (bottom) of GATM in TAMC generated in-vitro from Ctrl or GATM KO mice. In (H), ¹³C-arginine flux on control and GATM KO in-vitro generated TAMCs over 24 hours. In (I), Kaplan Meier curve Control vs Gatm-cKO mice implanted with 100x10⁵ CT-2A tumor cells. In B, n=3-4 per group, representative of two independent experiments. In C, n=2 independent experiments. In D-F and I, n=10 per group (equal gendered), were analyzed. In G, n=2 independent experiments performed in duplicate. In H, n=3 per group, representative of two independent experiments. In B (left panel), an unpaired Student’s t-test was performed. In H, a two-way ANOVA was performed followed by Sidak’s multiple comparisons test for significance. In B (right panel) a one-way ANOVA followed by Tukey’s post hoc was performed for significance. In D-F and I, Log-Rank analysis was performed. In C, Statistics calculated used Walter & Eliza Hall Institute of Medical Research platform (https://bioinf.wehi.edu.au/software/elda/). Error bars are expressed as mean ± SEM. p<0.05*, p<0.01**, p<0.001***.