Remember me
Research ArticleCell biologyImmunology
Open Access | 
10.1172/JCI186648
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Wei, W. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Li, H. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Tian, S. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Zhang, C. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Liu, J. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Tao, W. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Cai, T. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Dong, Y. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Wang, C. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Lu, D. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Ai, Y. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Zhang, W. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Wang, H. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Liu, K. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Fan, Y. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Gao, Y. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Huang, Q. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Ma, X. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Wang, B. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Zhang, X. in: JCI | PubMed | Google Scholar
1Department of Urology, The Third Medical Center and
2Department of Urology Laboratory, Chinese PLA General Hospital, Beijing, China.
3Medical School of PLA, Beijing, China.
4State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.
5Department of Pathology, The Third Medical Center, Chinese PLA General Hospital, Beijing, China.
Address correspondence to: Yan Huang, Xu Zhang, or Baojun Wang, Department of Urology, The Third Medical Centre, Chinese PLA General Hospital, Beijing, 100039, China. Phone: 86.10.66938008; Email: dr.huangyan301@foxmail.com (YH); xzhang301@163.com (XZ); baojun40009@126.com (BW).
Authorship note: WW and HL contributed equally as co-authors of this article.
Find articles by Huang, Y. in: JCI | PubMed | Google Scholar
Authorship note: WW and HL contributed equally as co-authors of this article.
Published February 18, 2025 - More info
Published in Volume 135, Issue 8 on April 15, 2025
AbstractTumor cells often employ many ways to restrain type I IFN signaling to evade immune surveillance. However, whether cellular amino acid metabolism regulates this process remains unclear, and its effects on antitumor immunity are relatively unexplored. Here, we found that asparagine inhibited IFN-I signaling and promoted immune escape in bladder cancer. Depletion of asparagine synthetase (ASNS) strongly limited in vivo tumor growth in a CD8+ T cell–dependent manner and boosted immunotherapy efficacy. Moreover, clinically approved L-asparaginase (ASNase),synergized with anti–PD-1 therapy in suppressing tumor growth. Mechanistically, asparagine can directly bind to RIG-I and facilitate CBL-mediated RIG-I degradation, thereby suppressing IFN signaling and antitumor immune responses. Clinically, tumors with higher ASNS expression show decreased responsiveness to immune checkpoint inhibitor therapy. Together, our findings uncover asparagine as a natural metabolite to modulate RIG-I–mediated IFN-I signaling, providing the basis for developing the combinatorial use of ASNase and anti–PD-1 for bladder cancer.
Graphical Abstract
Introduction
Bladder cancer is one of the most prevalent tumors in the urinary system and is responsible for nearly 165,000 deaths every year worldwide (1). Approximately 25% of patients present with muscle invasive disease and the relative 5-year overall survival rate of advanced-stage bladder cancer is low, with limited therapeutic advances (2, 3). Radical cystectomy and cisplatin-based chemotherapy are recommended for the standard treatment option for muscle-invasive bladder cancer (MIBC) (4). In recent years, immune checkpoint inhibitors (ICIs) have achieved tremendous clinical breakthroughs in cancer immunotherapy (5, 6). Though ICIs have shown durable responses in a subset of patients with bladder cancer, the overall response rate is only approximately 15%–25% (7, 8), which increases the demand for biomarkers of response and effective targeted therapies to enhance ICI therapy.
Emerging evidence indicates that the tumor microenvironment (TME) is a complex system that determines the occurrence of tumor immune responses (9, 10). CD8+ T cells play a central role in cancer immunotherapy and elicit antitumor activity by directly recognizing and killing tumor cells (11, 12). Patients with high CD8+ T cell infiltration in TME are associated with a survival benefit in several tumor types and better response to immunotherapy (13). Previous studies have reported that IFN-I is essential for activating adaptive immune response and plays a crucial role in CD8+ T cell infiltration and immunogenic tumor rejection (14, 15). Given that retinoic acid-inducible gene-I–like (RIG-I–like) receptors (RLRs) are critical for activating the IFN pathway and triggering immunogenic cell death, stimulation of RIG-I or melanoma differentiation–associated gene 5 (MDA5) signaling has emerged as a strategy for antitumor immunity (16, 17). However, tumor cells often employ multiple strategies to evade immunosurveillance by inhibiting RLR-mediated signaling pathway (18, 19). Therefore, a better comprehension of the regulatory mechanism of RLR-mediated IFNs in tumor immunity is of great clinical importance for patients with bladder cancer.
Tremendous efforts have recently been dedicated to the investigation of metabolic reprogramming in the regulation of tumor immunity (20–22). Among them, amino acid metabolism has attracted widespread attention. Asparagine synthetase (ASNS) catalyzes the conversion of aspartate to asparagine in an ATP-dependent reaction. L-asparaginase (ASNase), a drug that deprives asparagine in patients’ plasma, is considered to be a first-line therapy for childhood acute lymphoblastic leukemia (ALL) (23). Accumulating studies have revealed that asparagine is tightly linked to the activation and differentiation of CD8+ T cells, thereby affecting antitumoral functionality (24–26). In addition, ASNS is elevated in many cancer types, and increased asparagine contributes to cancer cell survival and metastasis (27–29). Nonetheless, the specific roles of asparagine metabolism on the cancer cell–intrinsic functions and on the regulation of RLR-mediated antitumor immunity have not been explored.
Here, we report that asparagine restriction enhances RIG-I–mediated IFN signaling and potentiates antitumor immunity in bladder cancer. Clinically, ASNS is upregulated and is associated with a poor response to immunotherapy in bladder cancer. Limiting asparagine by knockdown of ASNS or treatment with ASNase increases intratumoral CD8+ T cell infiltration and effector function, thus boosting the efficacy of PD-1 blockade. Mechanistically, asparagine can directly facilitate the interaction between E3 ligase CBL and RIG-I, consequently inducing RIG-I degradation to suppress IFN signaling, thereby limiting antitumor immune responses. Our study highlights a role of asparagine in regulating RIG-I stability and connects asparagine metabolism to the IFN-I signaling that modulates antitumor immunity, suggesting that targeting ASNS is a promising approach to enhance immunotherapy for bladder cancer.
ResultsAsparagine restriction attenuates tumor growth in an immunity-dependent manner. Previous studies report that ASNS functions as a critical enzyme that catalyzes the biosynthesis of asparagine from aspartate in an ATP-dependent reaction (23). To interrogate the biological functions of ASNS on cancer cells, we knocked down the Asns gene in 2 malignant mouse bladder cancer cell lines (MB49 and MBT2) and a human bladder cancer cell line, UMUC3 (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/JCI186648DS1). Loss of ASNS did not alter the in vitro growth rates and migration abilities of tumor cells (Supplemental Figure 1, C–F). Consistent with this, we found that asparagine also had no obvious difference on the proliferation abilities of bladder cancer cells compared with the control cells (Supplemental Figure 1G).
To investigate the effect of ASNS on tumor growth in vivo, ASNS-deficient mouse bladder cancer cells were injected subcutaneously into the flanks of immunocompromised mice. Our results demonstrated that loss of ASNS had no effect on tumor growth in BALB/c nude mice (Figure 1, A and B and Supplemental Figure 1, H and I). To determine the involvement of the immune system, we inoculated ASNS-deficient and control bladder cancer cells into syngeneic mouse hosts. Of note, silencing of ASNS inhibited tumor growth in both immunocompetent mice (Figure 1, C and D). To assess the effect of asparagine on the tumor growth in vivo, we subcutaneously injected murine bladder cancer cells into mice and followed with oral administration of PBS or asparagine. We observed no significant difference in immunocompromised mice administrated with asparagine (Figure 1, E and F). However, asparagine treatment promoted tumor growth in both immunocompetent murine bladder cancer models (Figure 1, G and H). Collectively, these data suggest that asparagine restriction could suppress tumor growth, which requires the presence of an intact immune system.
Figure 1Asparagine restriction attenuates tumor growth in syngeneic mice. (A) Tumor image and tumor weight of immunodeficient nude mice (n = 6) injected subcutaneously with scramble or shAsns MB49 cells. (B) Tumor image and tumor weight of immunodeficient nude mice (n = 6) injected subcutaneously with scramble or shAsns MBT2 cells. (C) Tumor growth curves and tumor weight of immunocompetent C57BL/6 mice (n = 6) injected subcutaneously with scramble or shAsns MB49 cells. (D) Tumor growth curves and tumor weight of immunocompetent C3H mice (n = 6) injected subcutaneously with scramble or shAsns MBT2 cells. (E) Tumor growth curves and tumor weight of immunodeficient nude mice (n = 6) injected subcutaneously with MB49 cells administrated with PBS or Asn. (F) Tumor growth curves and tumor weight of immunodeficient nude mice (n = 6) injected subcutaneously with MBT2 cells administrated with PBS or Asn. (G) Tumor growth curves and tumor weight of immunocompetent C57BL/6 mice (n = 6) injected subcutaneously MB49 cells administrated with PBS or Asn. (H) Tumor growth curves and tumor weight of immunocompetent C3H mice (n = 6) injected subcutaneously with MBT2 cells administrated with PBS or Asn. Data were mean ± SD. Statistical significance was calculated by 2-tailed unpaired Student’s t tests for E–H; 1-way ANOVA for A–D. **P < 0.01, ***P < 0.001.
Knockdown of ASNS potentiates recruitment and activation of CD8+ T cells. We next attempted to quantify immune effector cells in control and ASNS-deficient bladder tumors by flow cytometry. The gating strategy to analyze the population of immune cells in mouse-transplanted tumors is shown in Supplemental Figure 2A. We found that knockdown of ASNS could significantly increase CD8+ T cell infiltration in both tumors established by MB49 and MBT2 cells (Figure 2A). However, there were no consistent differences in CD4+ T cells and NK cells in both models (Supplemental Figure 3, A–D). Previous studies have shown that T cell exclusion from the tumor parenchyma is one of the mechanisms underlying immunosuppression in the TME and is associated with poor response to current immunotherapies (30, 31). Notably, depletion of ASNS also increased the infiltration of CD8+ T cells in tumor parenchyma (Figure 2B) and enhanced their cytokine production including GZMB, TNF-α, and IFN-γ (Figure 2, C and D, and Supplemental Figure 2B). On the contrary, asparagine treatment decreased the infiltration of CD8+ T cells and impaired function of CD8+ T cells to secrete GZMB (Figure 2, E and F and Supplemental Figure 3E).
Figure 2Knockdown of ASNS potentiates antitumor function of CD8+ T cells. (A) Tumor infiltrating CD8+ T cells from transplanted shAsns-MB49 tumors (n = 6) in C57BL/6 mice and shAsns-MBT2 tumors (n = 6) in C3H mice were analyzed by flow cytometry. (B and C) Representative images and quantification of immunofluorescence for CD8 (B) and GZMB (C) in scramble and shAsns-MB49 tumors (n = 6). Scale bars: 50 μm. (D) Flow staining and frequency of CD8+TNFα+ and CD8+IFNγ+ cells in shAsns-MB49 and control tumors (n = 5). (E) Tumor-infiltrating CD8+ T cells were analyzed by flow cytometry from transplanted MB49 and MBT2 tumors (n = 6) in syngeneic mice administrated with PBS or Asn. (F) Representative images and quantification of immunofluorescence for CD8 and GZMB in MB49 tumors administrated with PBS or Asn (n = 6). Scale bars: 50 μm. (G) C57BL/6 mice were subcutaneously inoculated with MB49 tumor cells and treated with anti-CD8 antibody. Flow cytometry analysis of CD8+ T cell content in peripheral blood of mice (n = 5) at the end of experiment. (H) Tumor growth curves and tumor weight from scramble and shAsns-MB49 tumor cells in C57BL/6 mice (n = 5) followed by intraperitoneal injection with anti-CD8 antibody. (I) Tumor growth curves and tumor weight from scramble and shAsns-MBT2 tumor cells in C3H mice (n = 5) followed by intraperitoneal injection with anti-CD8 antibody. Data were mean ± SD. Statistical significance was calculated by 2 tailed unpaired Student’s t tests for E and F. 1-way ANOVA for A–D; 2-way ANOVA for G–I. *P < 0.05, **P < 0.01, ***P < 0.001.
To determine the extent to which ASNS inhibition promoted an antitumor reaction dependent on CD8+ T cells in vivo, CD8+ T cells were deleted using anti-CD8 antibody (Figure 2G and Supplemental Figure 3, F and G). We found that ASNS deficiency–mediated antitumor function was largely abolished in the CD8+ T cell–depleted group (Figure 2, H and I). Collectively, these results indicate that the tumor-suppressive effect of asparagine restriction might be mediated by tumor-infiltrating CD8+ T cells.
Silencing of ASNS triggers RIG-I–induced IFN-I signaling. To further decipher the mechanism of immunoactivation mediated by ASNS inhibition, we performed RNA-seq analysis in ASNS-deficient MBT2 cells (Figure 3A and Supplemental Figure 4A). Gene ontology (GO) analysis and gene set enrichment analysis (GSEA) showed that differentially expressed genes were enriched in biological pathways related with the IFN pathway and adaptive immune response process, including response to IFN-I, regulation of T cell–mediated cytotoxicity, and so on (Figure 3, B and C and Supplemental Figure 4B). Then, we confirmed RNA-seq data through qRT-PCR that knockdown of ASNS elevated the expression of IFN-stimulated genes (ISGs) (e.g., OAS2, OAS3, RIG-I, CCL5, and ISG15) and IFN-β (Figure 3D and Supplemental Figure 4C). We also applied Luminex system multiple immunoassays to detect multiple cytokines and chemokines in cell culture supernatants from ASNS-deficient and control MBT2 cells. Supporting the results obtained in RNA-seq, knockdown of ASNS upregulated the expression of CCL5 and promoted its secretion (Figure 3E), which is robustly correlated with CD8+ T cell infiltration in solid tumors (32). Moreover, the secretion levels of IFN-β and CCL5 protein were improved in ASNS-deficient bladder cancer cells by using ELISA (Figure 3F and Supplemental Figure 4D). In contrast, asparagine attenuated the expression of IFN-β and CCL5 in bladder cancer cells (Supplemental Figure 4, E and F).
Figure 3Silencing of ASNS activates RIG-I–induced IFN-I signaling. (A) Heat map depicted the differentially expressed mRNA in the indicated MBT2 cells. (B) Enrichment analysis for representative GO pathways in shAsns-mediated target genes. (C) GSEA plots of individual pathways enriched in shAsns-deficient MBT2 cells. (D) qRT-PCR showed the relative expression levels of ISGs genes in the indicated MBT2 cells. (E) Heatmap of multiple cytokines and chemokines detected by Luminex protein biochip testing system between Asns knockdown and the control groups in MBT2 cell culture supernatants. (F) ELISA experiment revealed the expression levels of Ifn-β and Ccl5 in culture supernatants of the indicated MBT2 cells. (G) qRT-PCR (left) and ELISA (right) assays showed the expression levels of Ifn-β in the indicated MBT2 cells. Western blot analysis of cell lysates from the indicated MBT2 cells. (H and I) Scramble or shAsns MBT2 cells were transfected with poly (I:C) (2 μg/mL) for 8 hours and the protein levels of Ifn-β (H) and Ccl5 (I) were determined by ELISA. (J) ELISA assay showed the expression levels of Ifn-β protein in the indicated MBT2 cells. (K) ELISA assay showed the expression levels of Ifn-β and Ccl5 in MBT2 cells treated with ASNase for 48 hours. (L) Western blot analysis of cell lysates from the MBT2 cells stably transfected with scramble and shAsns. (M) Western blot analysis of cell lysates from the indicated MBT2 cells. (N) Western blot analysis of cell lysates from the indicated MBT2 cells. (O and P) Tumor image and tumor weight of immunocompetent C57BL/6 mice (n = 5) injected subcutaneously with indicated MB49 cells. Data were mean ± SD. Statistical significance was calculated by 2 tailed unpaired Student’s t tests for J; 1-way ANOVA for D, F, H, I, and K; 2-way ANOVA for G and P. *P < 0.05, **P < 0.01, ***P < 0.001.
Previous studies have reported that several innate sensing pathways can stimulate the induction of IFN-I. For example, cGAS-STING of dsDNA sensors, RIG-I/MDA5-MAVS of dsRNA sensors, and Toll-like r
Comments (0)