. 2021 Apr 12;39(4):480-493.e6.

doi: 10.1016/j.ccell.2020.12.023. Epub 2021 Jan 28.

Stanniocalcin 1 is a phagocytosis checkpoint driving tumor immune resistance

Heng Lin¹, Ilona Kryczek¹, Shasha Li², Michael D Green³, Alicia Ali⁴, Reema Hamasha⁴, Shuang Wei¹, Linda Vatan¹, Wojciech Szeliga¹, Sara Grove¹, Xiong Li¹, Jing Li¹, Weichao Wang¹, Yijian Yan¹, Jae Eun Choi⁵, Gaopeng Li¹, Yingjie Bian¹, Ying Xu¹, Jiajia Zhou¹, Jiali Yu¹, Houjun Xia¹, Weimin Wang¹, Ajjai Alva⁴, Arul M Chinnaiyan⁶, Marcin Cieslik⁷, Weiping Zou⁸

Affiliations

¹ Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, University of Michigan School of Medicine, Ann Arbor, MI, USA.
² Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, University of Michigan School of Medicine, Ann Arbor, MI, USA; Department of Computational Medicine & Bioinformatics, University of Michigan School of Medicine, Ann Arbor, MI, USA.
³ Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, University of Michigan School of Medicine, Ann Arbor, MI, USA; Department of Radiation Oncology and Veterans Affairs Ann Arbor Healthcare System, University of Michigan School of Medicine, Ann Arbor, MI, USA.
⁴ Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, MI, USA.
⁵ Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, University of Michigan School of Medicine, Ann Arbor, MI, USA; Department of Pathology, University of Michigan School of Medicine, Ann Arbor, MI, USA; Michigan Center for Translational Pathology, University of Michigan School of Medicine, Ann Arbor, MI, USA.
⁶ Department of Pathology, University of Michigan School of Medicine, Ann Arbor, MI, USA; Michigan Center for Translational Pathology, University of Michigan School of Medicine, Ann Arbor, MI, USA; Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI, USA; Department of Urology, University of Michigan School of Medicine, Ann Arbor, MI, USA.
⁷ Department of Computational Medicine & Bioinformatics, University of Michigan School of Medicine, Ann Arbor, MI, USA; Department of Pathology, University of Michigan School of Medicine, Ann Arbor, MI, USA.
⁸ Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, University of Michigan School of Medicine, Ann Arbor, MI, USA; Department of Pathology, University of Michigan School of Medicine, Ann Arbor, MI, USA; Graduate Program in Immunology, University of Michigan School of Medicine, Ann Arbor, MI, USA; Graduate Program in Cancer Biology, University of Michigan School of Medicine, Ann Arbor, MI, USA. Electronic address: wzou@med.umich.edu.

PMID: 33513345
PMCID: PMC8044011
DOI: 10.1016/j.ccell.2020.12.023

Stanniocalcin 1 is a phagocytosis checkpoint driving tumor immune resistance

Heng Lin et al. Cancer Cell. 2021.

. 2021 Apr 12;39(4):480-493.e6.

doi: 10.1016/j.ccell.2020.12.023. Epub 2021 Jan 28.

Authors

Affiliations

¹ Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, University of Michigan School of Medicine, Ann Arbor, MI, USA.
² Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, University of Michigan School of Medicine, Ann Arbor, MI, USA; Department of Computational Medicine & Bioinformatics, University of Michigan School of Medicine, Ann Arbor, MI, USA.
³ Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, University of Michigan School of Medicine, Ann Arbor, MI, USA; Department of Radiation Oncology and Veterans Affairs Ann Arbor Healthcare System, University of Michigan School of Medicine, Ann Arbor, MI, USA.
⁴ Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, MI, USA.
⁵ Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, University of Michigan School of Medicine, Ann Arbor, MI, USA; Department of Pathology, University of Michigan School of Medicine, Ann Arbor, MI, USA; Michigan Center for Translational Pathology, University of Michigan School of Medicine, Ann Arbor, MI, USA.
⁶ Department of Pathology, University of Michigan School of Medicine, Ann Arbor, MI, USA; Michigan Center for Translational Pathology, University of Michigan School of Medicine, Ann Arbor, MI, USA; Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI, USA; Department of Urology, University of Michigan School of Medicine, Ann Arbor, MI, USA.
⁷ Department of Computational Medicine & Bioinformatics, University of Michigan School of Medicine, Ann Arbor, MI, USA; Department of Pathology, University of Michigan School of Medicine, Ann Arbor, MI, USA.
⁸ Department of Surgery, University of Michigan School of Medicine, Ann Arbor, MI, USA; Center of Excellence for Cancer Immunology and Immunotherapy, University of Michigan Rogel Cancer Center, University of Michigan School of Medicine, Ann Arbor, MI, USA; Department of Pathology, University of Michigan School of Medicine, Ann Arbor, MI, USA; Graduate Program in Immunology, University of Michigan School of Medicine, Ann Arbor, MI, USA; Graduate Program in Cancer Biology, University of Michigan School of Medicine, Ann Arbor, MI, USA. Electronic address: wzou@med.umich.edu.

PMID: 33513345
PMCID: PMC8044011
DOI: 10.1016/j.ccell.2020.12.023

Abstract

Immunotherapy induces durable clinical responses in a fraction of patients with cancer. However, therapeutic resistance poses a major challenge to current immunotherapies. Here, we identify that expression of tumor stanniocalcin 1 (STC1) correlates with immunotherapy efficacy and is negatively associated with patient survival across diverse cancer types. Gain- and loss-of-function experiments demonstrate that tumor STC1 supports tumor progression and enables tumor resistance to checkpoint blockade in murine tumor models. Mechanistically, tumor STC1 interacts with calreticulin (CRT), an "eat-me" signal, and minimizes CRT membrane exposure, thereby abrogating membrane CRT-directed phagocytosis by antigen-presenting cells (APCs), including macrophages and dendritic cells. Consequently, this impairs APC capacity of antigen presentation and T cell activation. Thus, tumor STC1 inhibits APC phagocytosis and contributes to tumor immune evasion and immunotherapy resistance. We suggest that STC1 is a previously unappreciated phagocytosis checkpoint and targeting STC1 and its interaction with CRT may sensitize to cancer immunotherapy.

Keywords: PD-1; T cell immunity; calreticulin; checkpoint; dendritic cell; eat-me signal; macrophages; phagocytosis; stanniocalcin 1; tumor.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests The authors declare no conflict of interest.

Figures

**Figure 1.. *STC1* correlates to cancer resistance to immunotherapy**
(A) Transcriptome analysis in patients treated with checkpoint blockade. Upregulated genes in non-responders treated with checkpoint blockade were determined in cohorts 1 (Hugo et al., 2016) and 2 (Riaz et al., 2017). The overlapping upregulated genes in 2 cohorts are shown. Cohort 1, n = 15 (responders), 13 (non-responders); Cohort 2, n = 26 (responders), 25 (non-responders). (B-C) Expression of *STC1* transcripts in Responders (R) and Non-Responders (NR) in cohort 1 (B) n = 14 (R), 13 (NR), p = 0.0287; and cohort 2 (C) n = 26 (R), 25 (NR), p = 0.0301. The dash line represents the median value, the bottom and top of the boxes are the 25^th and 75^th percentiles (interquartile range). Whiskers encompass 1.5 times the inter-quartile range. (D) Association of *STC1* expression levels with cancer patient survival analyzed on combined cohorts 1 and 2, *STC1* high (n = 22) and low (n = 27) expression, p = 0.0385. (E-G) Relationship of *STC1* expression with cancer patient survival in 18 cancer types in TCGA data set. Results are shown as individual cancer survival curves with top 15% high and low *STC1* expression (E); living status (F) Forest plot represents the adjust value of Cox proportional hazard ratio (HR) and 95% confidential interval (CI) of overall survival; and p-values (G) (STAD n = 56, p = 0.00475; HNSC n = 74, p = 0.00272; KIRP n = 42, p= 0.00698; LUAD n = 73, p = 0.0145; CESC n = 39, p = 0.00762; LGG n = 76, p = 0.00521; GBM n = 22, p = 0.00421; BLCA n = 60, p= 0.000765) See also Figure S1.

**Figure 2.. Tumor STC1 is critical for intrinsic resistance to tumor immunity**
(A-B) Tumor growth curves of control MC38 and *Stc1*^OE MC38 in (A) C57BL/6J mice and (B) NSG mice (n = 8). (C-F) Tumor growth curves of *Stc1*^+/+ and *Stc1*^−/− LLC, and *Stc1*^+/+ and *Stc1*^−/− B16-F10 in (C, D) C57BL/6J mice and (E, F) NSG mice (n = 5–8). (G-H) Tumor growth curves of control LLC and shStc1 LLC in (G) C57BL/6J mice and (H) NSG mice (n = 4–5). (I) Tumor growth curves of *Stc1*^+/+ and *Stc1*^−/− LLC tumors in (I) C57BL/6J mice with anti-PD-L1 or isotype control antibodies treatments every 3 days starting day 3 (n = 4–7). Data are shown as mean ± SEM, 2 tail t-test was used for two-way comparisons (A-I) (*p < 0.05, **p < 0.01). See also Figure S2.

**Figure 3.. Tumor STC1 impairs anti-tumor CD8⁺ T cell responses**
(A-B) Percentages of Ki67⁺CD8⁺ T cells in tumor drained lymph nodes (TdLNs) and tumor tissues in mice bearing *Stc1*^+/+ and *Stc1*^−/− LLC tumors determined by FACS. (C-F) Percentages of IFNγ⁺, granzyme B⁺, and TNFα⁺CD8⁺ T cells in *Stc1*^+/+ and *Stc1*^−/− LLC tumor tissues (C, D) and B16-F10 tumor tissues (E, F) determined by FACS. (G-H) Percentages of IFNγ⁺ and TNFα⁺ CD8 T cells in control and *Stc1*^OE B16-F10 tumor tissues determined by FACS. Data are shown as mean ± SEM, 2 tail T-test was used for two-way comparisons (n = 4–5, *p < 0.05; **p<0.01) (A-H). See also Figure S3.

**Figure 4.. STC1 abrogates tumor immunogenicity via targeting APCs**
(A-L) Effect of *Stc1* on OT-I cell activation *in vitro*. *Stc1*^+/+ or *Stc1*^−/− LLC cells were loaded with OVA and killed with UV-irradiation. CFSE-labeled OT-I cells were cultured with different numbers of dead LLC cells in the presence of macrophages (A-F) or DCs (G-L) for 3 days. CSFE dilution (A-B; G-H), IFNγ⁺ (C-D; I-J), and granzyme B⁺ (E-F; K-L) OT-I cells determined by FACS. Data are presented as mean ± SEM, 2 tail T-test was used for two-way comparisons (n = 3–5, *p < 0.05, **p < 0.01). (M-O) Effect of *Stc1* on OT-I cell activation *in vivo*. B16-F10 tumor bearing mice initially received intratumoral injection of dead tumor cells from OVA loaded-B16-F10 cells and OVA loaded-*Stc1*^OE-B16-F10 cells, and were subsequently intravenously transfused with CSFE labeled OT-I cells (M). CFSE dilution and OT-I cell divisions in TdLNs (N, O) were determined by FACS. Data are presented as mean ± SEM, 2 tail T-test was used for two-way comparisons (n = 3, *p < 0.05). (P-S) Effect of APCs on tumor growth curves. *Stc1*^+/+ and *Stc1*^−/− LLC cells were inoculated into *Batf3*^+/+ or *Batf3*^−/− mice. Starting 3 days before tumor inoculation, anti-CSF1R or isotype control antibodies were given every 3 days (n = 8). Data are shown as mean ± SEM, 2 tail t-test was used for two-way comparisons (P-R) (*p < 0.05, **p < 0.01). (T) Percentages of Ki67⁺ and granzyme B⁺ CD8⁺ T cells in *Stc1*^+/+ and *Stc1*^−/− LLC tumor tissues from wild type or *Batf3*^−/− mice under anti-CSF1R or isotype control antibody treatment (***p < 0.001, *Stc1*^+/+ vs. *Stc1*^−/− LLC tumor; p < 0.05, wild type vs *Batf3*^−/− mice; p < 0.001, anti-CSF1R vs. isotype control antibodies). See also Figure S4.

**Figure 5.. Tumor STC1 traps CRT to inhibit macrophage function**
(A) Effect of *Stc1* on macrophage-mediated phagocytosis. Macrophages were incubated with dead cells from fluor-647 labeled B16-F10 cells and fluor-647 labeled *Stc1*^OE B16-F10 cells. Mean fluorescence intensity (MFI) of fluorescence 647 in macrophages, gated on CD11b⁺ cells, determined by FACS. Data are presented as mean ± SEM, 2 tail T-test was used for two-way comparisons (n = 6, *p < 0.05, **p < 0.01). (B-C) Effect of *Stc1* on macrophage-mediated bead up-taking. Macrophages were incubated with dead cells from B16-F10 cells and *Stc1*^OE B16-F10 cells (B) or *Stc1*^+/+ and *Stc1*^+/+ LLC cells (C) for 20 hours. pHrodo^™-SE labeled 3 μm latex beads were added for 1 hour. Red pHrodo signals in macrophages were determined by FACS. Data are shown as mean ± SEM (n = 4, *p < 0.05, **p < 0.01). (D) Effect of *Stc1* on antigen presentation. Macrophages were incubated with dead cells from OVA-loaded B16-F10 cells (control) and OVA-loaded *Stc1*^OE B16-F10 cells for 48 hours. Surface OVA-binding-H2b complex expression (MFI) in macrophages were determined by FACS (n = 3, **p < 0.01). (E) Mass spectrum showing STC1 interactive proteins. FLAG-IP was conducted in dead cells from STC1-FLAG expressing B16-F10 cells. Mass spectrum was subsequently performed in the FLAG-IP proteins. Control, cell lysates from B16-F10 cells without STC1-FLAG. Top 5 hits are shown. (F) Interaction between endogenous CRT and STC1. Co-IP of STC1-FLAG was conducted with endogenous CRT, ERp57, and IRE1α in B16-F10 cells. One of 2 representative experiments is shown. (G) Interaction between exogenous CRT and STC1. Co-IPs of CRT-FLAG with STC1-GFP and ERp57 were performed in cell lysates from UV-treated or non-treated B16-F10 cells. One of 2 representative experiments is shown. (H) Duo-link showing the interactions (Red) of CRT and STC1-GFP, co-localizing with mito-tracker (white) in B16-F10 cells transfected with GFP or STC1-GFP with or without UV-treatment. Scale bars: 10 μm. Duo-link dots per cell merged or unmerged with mito-tracker were counted from over 20 images, mean ± SEM (n = 20, ***p < 0.001, STC1-GFP vs. GFP; # p <0.05, UV vs. No UV treatment). (I) Membrane CRT in B16-F10 cells. UV-treated B16-F10 and *Stc1*^OE B16-F10 cells were labeled with biotin. Western blot showed cell membrane CRT and Na⁺, K⁺-ATPase α1 in biotin-labeled proteins. One of 2 representative experiments is shown. (J) Membrane CRT in UV-treated *Stc1*^+/+ and *Stc1*^−/− LLC cells. Confocal images showed membrane CRT expression. Scale bars: 10 μm. The intensity of CRT expression was analyzed through ImageJ software. Data are shown as mean ± SEM (n = 12, **p < 0.01). (K) Western blots showing CRT distribution in mitochondria (TOM20) and cytosol (GAPDH) from UV-treated B16-F10 cells and *Stc1*^OE B16-F10 cells. Mito, mitochondria; Cyto, cytosol. One of 2 experiments is shown. (L-M) Effect of *Stc1* on T cell activation in the context of *Calr*. *Calr*^+/+ or *Calr*^−/− vehicle control and *Stc1*^OE B16-F10 cells were loaded with OVA and killed by UV-irradiation. CFSE-labeled OT-I cells were cultured with different numbers of dead B16-F10 cells with macrophages for 3–4 days. CSFE dilution (L), and granzyme B⁺ and IFNγ⁺ (M) OT-I cells were determined by FACS. Data are presented as mean ± SEM, 2 tail T-test was used for two-way comparisons (n = 3–5, **p < 0.01). See also Figure S5.

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Comment in

Subversion of calreticulin exposure as a strategy of immune escape.
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Stanniocalcin 1 is a phagocytosis checkpoint driving tumor immune resistance

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Stanniocalcin 1 is a phagocytosis checkpoint driving tumor immune resistance

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