Selective glutamine metabolism inhibition in tumor cells improves antitumor T lymphocyte activity in triple-negative breast cancer

Abstract

Rapidly proliferating tumor and immune cells need metabolic programs that support energy and biomass production. The amino acid glutamine is consumed by effector T cells and glutamine-addicted triple-negative breast cancer (TNBC) cells, suggesting that a metabolic competition for glutamine may exist within the tumor microenvironment, potentially serving as a therapeutic intervention strategy. Here, we report that there is an inverse correlation between glutamine metabolic genes and markers of T cell-mediated cytotoxicity in human basal-like breast cancer (BLBC) patient data sets, with increased glutamine metabolism and decreased T cell cytotoxicity associated with poor survival. We found that tumor cell-specific loss of glutaminase (GLS), a key enzyme for glutamine metabolism, improved antitumor T cell activation in both a spontaneous mouse TNBC model and orthotopic grafts. The glutamine transporter inhibitor V-9302 selectively blocked glutamine uptake by TNBC cells but not CD8+ T cells, driving synthesis of glutathione, a major cellular antioxidant, to improve CD8+ T cell effector function. We propose a "glutamine steal" scenario, in which cancer cells deprive tumor-infiltrating lymphocytes of needed glutamine, thus impairing antitumor immune responses. Therefore, tumor-selective targeting of glutamine metabolism may be a promising therapeutic strategy in TNBC.

Keywords: Amino acid metabolism; Breast cancer; Cancer immunotherapy; Oncology.

Figure 1. Glutamine metabolism inversely correlates with expression of T cell activation markers in glutamine metabolism high human basal-like breast cancer samples.

(A–C) Analysis of mRNA expression (log₂) z-scores in basal-like tumor samples from TCGA BRCA data set. The samples with the greatest glutamine metabolism gene signature (GMGS) combined with the lowest glycolysis gene signature are considered “glutamine metabolism high.” All others are considered to have “mixed metabolism.” (A) Expression levels of individual genes are displayed as a heatmap, with low expression in blue and high expression in red. (B and C) The CTL gene signature ([GZMA + GZMB + PRF1 + INFG]/4) was plotted as a function of the GMGS or GLS for basal-like breast cancer samples that are “glutamine metabolism high” (black) and “mixed metabolism” (pink). Linear regression lines of best fit are shown for both groups. Pearson’s correlation analyses are shown. (D) Kaplan-Meier analysis of overall survival of basal breast tumors stratified based on the GMGS and the CTL gene signature: GMGS^LOW/CTL^HIGH (green, n = 64), GMGS^LOW/CTL^LOW (blue, n = 14), GMGS^HIGH/CTL^HIGH (orange, n = 121), and GMGS^HIGH/CTL^LOW (red, n = 42). Log-rank (Mantel-Cox) test: P values are shown. Red box indicates statistical significance after Bonferroni’s correction for multiple comparisons. Hazard ratio (HR) (log-rank) and 95% confidence intervals are shown.

Figure 2. Loss of tumor cell–specific GLS reduces tumor growth and metastasis in a lymphocyte-dependent manner in an orthotopic model of TNBC.

(A–D) E0771 sgLacZ (E0771-WT, red) or sgGls_3C15 (E0771-GLS^KO, blue) (5 × 10⁵) cells were implanted into the number 4 mammary fat pad of female C57BL/6 mice (Jackson Laboratories), and tumors were harvested after 14 days. (A) Tumor volume (P = 1.19 × 10^–10 by 2-way ANOVA) and (B) tumor weight at harvest (P = 7.94 × 10^–4 by unpaired Student’s t test) are shown. n = 5 mice per group. (C and D) Tumor sections from A and B were stained by (C) immunohistochemistry for GLS (brown) or immunofluorescence for (D) Ki67 (red). Nuclei were stained with (C) hematoxylin (blue) or (D) DAPI (blue). Total nuclei and Ki67⁺ cells were counted from 3 fields of view using ImageJ software. Scale bars: 10 μm (GLS) and 20 μm (Ki67). P = 0.84 by unpaired Student’s t test. n = 3 mice per group. (E–G) E0771-WT or E0771-GLS^KO cells were implanted as described above in wild-type C57BL/6 (Rag1^+/+) or Rag1-deficient (Rag1^–/–) mice. (E) Plot of tumor volumes is shown. P = 2.66 × 10^–6 by 2-way ANOVA. (F) Image of tumors harvested from E (n = 4 mice per group). (G) Immunofluorescence of cleaved caspase-3 (red). Nuclei were stained with DAPI (blue). P = 0.011 by 1-way ANOVA with Tukey’s post hoc analysis for multiple comparisons. n = 3 mice per group. (H and I) C57BL/6 (Rag1^+/+) (H) or Rag1^–/– (I) mice were implanted with E0771-WT or E0771-GLS^KO cells (2.5 × 10⁵). (H) Lungs were harvested when tumors reached 400–600 mm³ (21–27 days). Micrometastases (black arrows) were scored from 3 H&E-stained sections isolated 100 μm apart (n = 4 mice per group). Scale bar: 200 μm. (I) Tumors were resected at 1400–1500 mm³, and lungs were harvested between 13 and 20 days after resection (n = 5 mice per group). Surface metastases (yellow arrows) were scored from whole lung specimens. Unpaired Student’s t test: (H) P = 0.031, (I) P = 0.062. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.

Figure 3. Mammary-specific loss of GLS in a spontaneous TNBC tumor model delays tumor initiation and improves activation of T cells.

C3(1)-TAg/GLS^fl/fl (GLS^fl/fl, red) or C3(1)-TAg; MMTV-Cre; GLS^fl/fl (GLS^Δ/Δ, blue) mice were palpated weekly for tumor formation and progression. (A) Tumor latency for GLS^fl/fl or GLS^Δ/Δ mice was recorded as the age (weeks) of initial tumor detection. P = 0.031 by unpaired Student’s t test. n = 11–12 mice per group. (B) Survival (weeks) was determined by the humane endpoint for the GLS^fl/fl or GLS^Δ/Δ from A, plotted as the percentage of surviving mice as a function of age (weeks). P = 0.0082 by Gehan-Breslow-Wilcoxon test. Hazard ratio was calculated using log-rank analysis, with the 95% confidence interval shown. (C) Plot of tumor volume after tumor initiation in mice described in A. (D) H&E images of GLS^fl/fl or GLS^Δ/Δ tumors. Scale bars: 200 μm (top) and 100 μm (bottom). (E) Immunohistochemistry for GLS (brown) of GLS^fl/fl or GLS^Δ/Δ tumors. Nuclei were stained with hematoxylin (blue). Scale bar: 20 μm. (F) Tumors were harvested from GLS^fl/fl (red) or GLS^Δ/Δ (blue) mice at 1 to 2 weeks after initial tumor detection. Tumor volume (mm³) (left) and tumor mass (grams) (right) were recorded at harvest. Unpaired Student’s t test: P = 0.581 (volume), P = 0.581 (mass). n = 7–9 mice per group. (G–N) Flow cytometric analyses of whole tumor preparations. n = 5–6 mice per group. (G) CD4⁺ (left) or CD8⁺ (right) T cells, plotted as percentage of CD45⁺ immune cells. Unpaired Student’s t test: P = 0.226 (CD4⁺), P = 0.043 (CD8⁺). (H–N) Flow cytometric analyses of (H) CD8⁺GZMB⁺, (I) CD8⁺CD107a⁺, (J) CD8⁺IFN-γ⁺, (K) CD4⁺IFN-γ⁺, (L) CD4⁺IL-4⁺, (M) CD4⁺IL-17A⁺, and (N) CD4⁺FoxP3⁺ T cells in GLS^fl/fl (red) or GLS^Δ/Δ (blue) tumors, plotted as a percentage of CD45⁺ cells. Unpaired Student’s t test: P = 0.0085 (CD8⁺GZMB⁺), P = 0.0056 (CD8⁺CD107a⁺), P = 4.94 × 10^–5 (CD8⁺IFN-γ⁺), P = 0.011 (CD4⁺IFN-γ⁺), P = 0.212 (CD4⁺IL-4⁺), P = 0.322 (CD4⁺IL-17A⁺), P = 0.093 (CD4⁺FoxP3⁺). *P < 0.05, **P < 0.01, ****P < 0.001.

Figure 4. Loss of tumor cell–specific GLS increases T cell activation in an orthotopic TNBC tumor model.

(A–I) E0771 sgLacZ (WT, red) or sgGls_3C15 (GLS^KO, blue) (5 × 10⁵) cells were implanted into the number 4 mammary fat pad of female C57BL/6 mice (Jackson Laboratories). Tumors were harvested after 14 days. n = 4–5 mice per group. (A and B) Flow cytometric analyses of CD4⁺ (left) or CD8⁺ (right) T cells, plotted as percentage of (A) CD45⁺ immune cells or (B) total cells normalized to tumor mass (gram). Unpaired Student’s t test: CD4⁺, (A) P = 0.091, (B) P = 0.0063; CD8⁺, (A) P = 0.040, (B) P = 8.23 × 10^–4. (C–I) Flow cytometric analyses of tumors for (C) CD8⁺GZMB⁺, (D) CD8⁺CD107a⁺, (E) CD8⁺IFN-γ⁺, (F) CD4⁺IFN-γ⁺, (G) CD4⁺IL-4⁺, (H) CD4⁺IL-17A⁺, and (I) CD4⁺FoxP3⁺ CD45⁺CD3⁺ T cells. Total counts were normalized per tumor mass (grams). Unpaired Student’s t test: P = 0.0064 (CD8⁺GZMB⁺), P = 0.027 (CD8⁺CD107a⁺), P = 0.0091 (CD8⁺IFN-γ⁺), P = 0.0065 (CD4⁺IFN-γ⁺), P = 0.065 (CD4⁺IL-4⁺), P = 0.017 (CD4⁺IL-17A⁺), P = 0.198 (CD4⁺FoxP3⁺). (J) Tumor interstitial fluid was collected from E0771-WT (red) or E0771-GLS^KO (blue) tumors via centrifugation of harvested tumors. Glutamine (gln) was measured and concentration (μM) was calculated against a glutamine standard. P = 0.021 by paired Student’s t test. n = 5 mice per group. (K) IFN-γ ELISA of supernatants collected from Tc1 CD8⁺ T cells activated (anti-CD3/anti-CD28) in media supplemented with 0.1, 0.5, or 2 mM glutamine (n = 3). Two-way ANOVA; unpaired Student’s t test. (L–N) E0771(OVA) sgLacZ (WT, red) or sgGls3 (GLS^KO, blue) (5 × 10⁵) cells were bilaterally implanted into the number 4 mammary fat pads of female C57BL/6 mice. (L) Experimental timeline is shown. (M) Immunofluorescence of GFP⁺CD8⁺ OT-I (GFP⁺CTLs) cells in tumors. Nuclei were stained with DAPI (blue). Arrows indicate GFP⁺CD8⁺ OT-I T cells. Scale bar: 20 μm. (N) Flow cytometric analysis of GFP⁺CD8⁺ T cells from whole cell tumor preparations, plotted as percentage of all live cells. P = 0.043 by unpaired Student’s t test. n = 3–4 mice per group. *P < 0.05, **P < 0.01, ***P < 0.005.

Figure 5. The glutamine transporter inhibitor V-9302 suppresses tumor growth and increases T lymphocyte activation in a model of TNBC.

E0771 cells (2.5 × 10⁵) were bilaterally injected into the number 4 mammary fat pads of female C57BL/6 mice (Taconic). Beginning on day 11, mice were treated with vehicle (DMSO, red) or 50 mg/kg V-9302 (blue) daily for 5 days. (A) Tumor volume was measured over time. Arrow indicates beginning of treatment. P < 1 × 10^–15 by 2-way ANOVA. n = 9–10 mice per group. (B) Average tumor mass per mouse at harvest. P = 0.0060 by unpaired Student’s t test. n = 9–10 mice per group. (C) Immunofluorescence of tumor sections for Ki67 (top) or cleaved caspase-3 (bottom), both red. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. Ki67⁺, cleaved caspase-3⁺, and nuclei were averaged from 3 fields of view. Unpaired Student’s t test: P = 0.283 (Ki67), P = 0.023 (cleaved caspase-3). (D) Flow cytometric analyses of CD4⁺ (left) or CD8⁺ (right) T cells, plotted as a percentage of CD45⁺ immune cells, in vehicle- (red) or V-9302–treated (blue) tumors. Unpaired Student’s t test: P = 0.023 (CD4⁺), P = 0.340 (CD8⁺). n = 5 mice per group. (E) Immunohistochemistry of CD8a (brown) from vehicle- or V-9302–treated (50 mg/kg) tumors. Nuclei were stained with hematoxylin (blue). Edge (black) is considered <500 μm (denoted by solid line) from tumor margin, core (red) is >500 μm. Scale bars: 500 μm and 50 μm for expanded and enlarged images, respectively. CD8⁺ cells (denoted by arrows) were averaged from 3 fields of view and normalized to field-of-view area. P = 0.019 by unpaired Student’s t test. n = 3 mice per group. (F–K) Flow cytometric analyses of (F) CD8⁺GZMB⁺, (G) CD8⁺CD107a⁺, (H) CD8⁺IFN-γ⁺, (I) CD4⁺IFN-γ⁺, (J) CD4⁺IL-4⁺, and (K) CD4⁺FoxP3⁺CD25⁺CD127^loCD45⁺CD3⁺ T cells in vehicle- (red) or V-9302–treated (blue) tumors. Unpaired Student’s t test: P = 0.014 (CD8⁺GZMB⁺), P = 0.047 (CD8⁺CD107a⁺), P = 0.007 (CD8⁺IFN-γ⁺), P = 0.0021 (CD4⁺IFN-γ⁺), P = 0.057 (CD4⁺IL-4⁺), P = 0.034 (CD4⁺FoxP3⁺CD25⁺CD127^lo). n = 3–8 mice per group. *P < 0.05, **P < 0.01, ****P < 0.001.

Figure 6. V-9302 induces ATB^0,+ expression to sustain glutamine uptake and glutathione synthesis in activated CD8⁺ T cells.

(A) LDH cytotoxicity assay of E0771(OVA) and CD8⁺ OT-I CTLs cocultured in vehicle (red) or V-9302 (blue) (n = 3). P = 0.0018 by 1-way ANOVA with Tukey’s post hoc test. (B) Tumor interstitial fluid glutamine in vehicle- or V-9302–treated tumors. P = 0.0027 by unpaired Student’s t test. n = 6 mice per group. (C and D) ³H-glutamine uptake assay in (C) E0771 and C567BL/6 CD8⁺ CTLs or (D) HCC1806 and human CD8⁺ CTLs isolated from PBMCs in vehicle or V-9302 (n = 3–4). Averaged triplicate radioactivity (CPM) was normalized to vehicle. Two-way ANOVA (E0771: P = 2.18 × 10^–4; HCC1806: P = 1.43 × 10^–4) with Tukey’s post hoc test. (E) Relative expression of ATB^0,+/Slc6a14 (n = 3). P = 0.036 by 2-way ANOVA with Tukey’s post hoc test. (F) ATB^0,+ Western blot of cells from E. Relative ATB^0,+ protein was normalized as indicated (n = 3). Two-way ANOVA (bottom, P = 0.0014; right, P = 0.0026) with Tukey’s (bottom) or Sidak’s (right) post hoc test. (G and H) ³H-glutamine uptake of CD8⁺ CTLs (G) after ATB^0,+ knockdown or (H) in the presence of glutamine transporter inhibitors, as indicated (n = 4). One-way ANOVA: P = 4.98 × 10^–5 (G), P = 3.41 × 10^–4 (H); Holm-Sidak post hoc test. (I and J) CD8⁺ CTLs overexpressing ATB^0,+ were evaluated for (I) ³H-glutamine uptake or (J) LDH cytotoxicity (n = 3–4). Empty vector (EV) was used as a control. Unpaired Student’s t test (P = 7.39 × 10^–4) (I) or 1-way ANOVA (P = 4.13 × 10^–5) with Tukey’s post hoc test (J). (K and L) Intracellular (K) glutathione (GSH) and (L) cysteine in CD8⁺ CTLs (n = 4–5). Paired (P = 0.020) (K) or unpaired (P = 0.027) (L) Student’s t test. (M) xCT/Slc7a11 expression in activated CD8⁺ CTLs (n = 3). P = 0.025 by unpaired Student’s t test. (N) GCLC protein expression in CD8⁺ TILs in tumors of vehicle- or V-9302–treated mice. Dotted line denotes vehicle peak in representative data shown. P = 0.0034 by unpaired Student’s t test. n = 7 mice per group. (O) ROS in activated CD8⁺CD44⁺CD62L^– effector T cells determined by H₂-DCFDA (n = 4). P = 0.016 by unpaired Student’s t test. (P) Proposed model of V-9302–mediated increases (bold) and decreases (gray) in CD8⁺ TILs. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.