Collectin-11 promotes cancer cell proliferation and tumor growth

Research ArticleImmunologyOncology Open Access | 10.1172/jci.insight.159452

Jia-Xing Wang,1 Bo Cao,1 Ning Ma,1 Kun-Yi Wu,1 Wan-Bing Chen,1 Weiju Wu,2 Xia Dong,3 Cheng-Fei Liu,1 Ya-Feng Gao,1 Teng-Yue Diao,1 Xiao-Yun Min,1 Qing Yong,1 Zong-Fang Li,4 Wuding Zhou,2 and Ke Li1,4

1Core Research Laboratory, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China.

2Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom.

3Department of Ophthalmology, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China.

4Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, China.

Address correspondence to: Ke Li, Core Research Laboratory, The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, 710004 China. Phone: 86.0.298.632.0788; Email: ke.li@mail.xjtu.edu.cn. Or to: Wuding Zhou, Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom. Phone: 44.0.207.188.1528; Email: wuding.zhou@kcl.ac.uk.

Authorship note: JXW, BC, and NM are co–first authors. KL and WZ are co–senior authors.

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1Core Research Laboratory, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China.

2Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom.

3Department of Ophthalmology, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China.

4Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, China.

Address correspondence to: Ke Li, Core Research Laboratory, The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, 710004 China. Phone: 86.0.298.632.0788; Email: ke.li@mail.xjtu.edu.cn. Or to: Wuding Zhou, Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom. Phone: 44.0.207.188.1528; Email: wuding.zhou@kcl.ac.uk.

Authorship note: JXW, BC, and NM are co–first authors. KL and WZ are co–senior authors.

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1Core Research Laboratory, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China.

2Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom.

3Department of Ophthalmology, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China.

4Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, China.

Address correspondence to: Ke Li, Core Research Laboratory, The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, 710004 China. Phone: 86.0.298.632.0788; Email: ke.li@mail.xjtu.edu.cn. Or to: Wuding Zhou, Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom. Phone: 44.0.207.188.1528; Email: wuding.zhou@kcl.ac.uk.

Authorship note: JXW, BC, and NM are co–first authors. KL and WZ are co–senior authors.

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1Core Research Laboratory, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China.

2Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom.

3Department of Ophthalmology, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China.

4Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, China.

Address correspondence to: Ke Li, Core Research Laboratory, The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, 710004 China. Phone: 86.0.298.632.0788; Email: ke.li@mail.xjtu.edu.cn. Or to: Wuding Zhou, Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom. Phone: 44.0.207.188.1528; Email: wuding.zhou@kcl.ac.uk.

Authorship note: JXW, BC, and NM are co–first authors. KL and WZ are co–senior authors.

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1Core Research Laboratory, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China.

2Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom.

3Department of Ophthalmology, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China.

4Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, China.

Address correspondence to: Ke Li, Core Research Laboratory, The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, 710004 China. Phone: 86.0.298.632.0788; Email: ke.li@mail.xjtu.edu.cn. Or to: Wuding Zhou, Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom. Phone: 44.0.207.188.1528; Email: wuding.zhou@kcl.ac.uk.

Authorship note: JXW, BC, and NM are co–first authors. KL and WZ are co–senior authors.

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1Core Research Laboratory, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China.

2Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom.

3Department of Ophthalmology, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China.

4Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, China.

Address correspondence to: Ke Li, Core Research Laboratory, The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, 710004 China. Phone: 86.0.298.632.0788; Email: ke.li@mail.xjtu.edu.cn. Or to: Wuding Zhou, Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom. Phone: 44.0.207.188.1528; Email: wuding.zhou@kcl.ac.uk.

Authorship note: JXW, BC, and NM are co–first authors. KL and WZ are co–senior authors.

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1Core Research Laboratory, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China.

2Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom.

3Department of Ophthalmology, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China.

4Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, China.

Address correspondence to: Ke Li, Core Research Laboratory, The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, 710004 China. Phone: 86.0.298.632.0788; Email: ke.li@mail.xjtu.edu.cn. Or to: Wuding Zhou, Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom. Phone: 44.0.207.188.1528; Email: wuding.zhou@kcl.ac.uk.

Authorship note: JXW, BC, and NM are co–first authors. KL and WZ are co–senior authors.

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1Core Research Laboratory, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China.

2Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom.

3Department of Ophthalmology, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China.

4Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, China.

Address correspondence to: Ke Li, Core Research Laboratory, The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, 710004 China. Phone: 86.0.298.632.0788; Email: ke.li@mail.xjtu.edu.cn. Or to: Wuding Zhou, Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom. Phone: 44.0.207.188.1528; Email: wuding.zhou@kcl.ac.uk.

Authorship note: JXW, BC, and NM are co–first authors. KL and WZ are co–senior authors.

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1Core Research Laboratory, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China.

2Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom.

3Department of Ophthalmology, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China.

4Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, China.

Address correspondence to: Ke Li, Core Research Laboratory, The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, 710004 China. Phone: 86.0.298.632.0788; Email: ke.li@mail.xjtu.edu.cn. Or to: Wuding Zhou, Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom. Phone: 44.0.207.188.1528; Email: wuding.zhou@kcl.ac.uk.

Authorship note: JXW, BC, and NM are co–first authors. KL and WZ are co–senior authors.

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1Core Research Laboratory, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China.

2Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom.

3Department of Ophthalmology, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China.

4Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, China.

Address correspondence to: Ke Li, Core Research Laboratory, The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, 710004 China. Phone: 86.0.298.632.0788; Email: ke.li@mail.xjtu.edu.cn. Or to: Wuding Zhou, Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom. Phone: 44.0.207.188.1528; Email: wuding.zhou@kcl.ac.uk.

Authorship note: JXW, BC, and NM are co–first authors. KL and WZ are co–senior authors.

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1Core Research Laboratory, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China.

2Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom.

3Department of Ophthalmology, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China.

4Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, China.

Address correspondence to: Ke Li, Core Research Laboratory, The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, 710004 China. Phone: 86.0.298.632.0788; Email: ke.li@mail.xjtu.edu.cn. Or to: Wuding Zhou, Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom. Phone: 44.0.207.188.1528; Email: wuding.zhou@kcl.ac.uk.

Authorship note: JXW, BC, and NM are co–first authors. KL and WZ are co–senior authors.

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1Core Research Laboratory, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China.

2Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom.

3Department of Ophthalmology, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China.

4Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, China.

Address correspondence to: Ke Li, Core Research Laboratory, The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, 710004 China. Phone: 86.0.298.632.0788; Email: ke.li@mail.xjtu.edu.cn. Or to: Wuding Zhou, Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom. Phone: 44.0.207.188.1528; Email: wuding.zhou@kcl.ac.uk.

Authorship note: JXW, BC, and NM are co–first authors. KL and WZ are co–senior authors.

Find articles by Yong, Q. in: JCI | PubMed | Google Scholar

1Core Research Laboratory, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China.

2Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom.

3Department of Ophthalmology, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China.

4Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, China.

Address correspondence to: Ke Li, Core Research Laboratory, The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, 710004 China. Phone: 86.0.298.632.0788; Email: ke.li@mail.xjtu.edu.cn. Or to: Wuding Zhou, Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom. Phone: 44.0.207.188.1528; Email: wuding.zhou@kcl.ac.uk.

Authorship note: JXW, BC, and NM are co–first authors. KL and WZ are co–senior authors.

Find articles by Li, Z. in: JCI | PubMed | Google Scholar

1Core Research Laboratory, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China.

2Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom.

3Department of Ophthalmology, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China.

4Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, China.

Address correspondence to: Ke Li, Core Research Laboratory, The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, 710004 China. Phone: 86.0.298.632.0788; Email: ke.li@mail.xjtu.edu.cn. Or to: Wuding Zhou, Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom. Phone: 44.0.207.188.1528; Email: wuding.zhou@kcl.ac.uk.

Authorship note: JXW, BC, and NM are co–first authors. KL and WZ are co–senior authors.

Find articles by Zhou, W. in: JCI | PubMed | Google Scholar |

1Core Research Laboratory, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China.

2Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom.

3Department of Ophthalmology, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China.

4Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, China.

Address correspondence to: Ke Li, Core Research Laboratory, The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, 710004 China. Phone: 86.0.298.632.0788; Email: ke.li@mail.xjtu.edu.cn. Or to: Wuding Zhou, Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom. Phone: 44.0.207.188.1528; Email: wuding.zhou@kcl.ac.uk.

Authorship note: JXW, BC, and NM are co–first authors. KL and WZ are co–senior authors.

Find articles by Li, K. in: JCI | PubMed | Google Scholar |

Authorship note: JXW, BC, and NM are co–first authors. KL and WZ are co–senior authors.

Published March 8, 2023 - More info

Published in Volume 8, Issue 5 on March 8, 2023
JCI Insight. 2023;8(5):e159452. https://doi.org/10.1172/jci.insight.159452.
© 2023 Wang et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published March 8, 2023 - Version history
Received: February 16, 2022; Accepted: January 25, 2023 View PDF Abstract

Collectin-11 (CL-11) is a recently described soluble C-type lectin that has distinct roles in embryonic development, host defence, autoimmunity, and fibrosis. Here we report that CL-11 also plays an important role in cancer cell proliferation and tumor growth. Melanoma growth was found to be suppressed in Colec11–/– mice in a s.c. B16 melanoma model. Cellular and molecular analyses revealed that CL-11 is essential for melanoma cell proliferation, angiogenesis, establishment of more immunosuppressive tumor microenvironment, and the reprogramming of macrophages to M2 phenotype within melanomas. In vitro analysis revealed that CL-11 can activate tyrosine kinase receptors (EGFR, HER3) and ERK, JNK, and AKT signaling pathways and has a direct stimulatory effect on murine melanoma cell proliferation. Furthermore, blockade of CL-11 (treatment with L-fucose) inhibited melanoma growth in mice. Analysis of open data sets revealed that COLEC11 gene expression is upregulated in human melanomas and that high COLEC11 expression has a trend toward poor survival. CL-11 also had direct stimulatory effects on human tumor cell proliferation in melanoma and several other types of cancer cells in vitro. Overall, our findings provide the first evidence to our knowledge that CL-11 is a key tumor growth–promoting protein and a promising therapeutic target in tumor growth.

Graphical Abstractgraphical abstract Introduction

Collectins are group of soluble C-type lectins that represent an important group of pattern-recognition molecules. Collectins are composed of a carbohydrate-recognition domain, a neck region, and a collagen-like region, and they are involved in carbohydrate recognition and innate immunity (1, 2). Collectins can bind to carbohydrate structures on pathogens to enhance phagocytosis of pathogen by phagocytes. Some of collectins can also trigger the lectin pathway of complement activation and generate complement effector functions (3, 4). Mannose-binding lectin (MBL) and lung surfactant proteins (SP-A, SP-D) are well known members among the group.

Collectin-11 (CL-11, also known as CL-K1 and encoded by COLEC11) is a recently described member of the collectin family. CL-11 displays structural similarities with other collectins but has some distinct features such as having a wide tissue distribution, relatively lower serum concentrations (~300 ng/mL), and the ability to bind a wide range of ligands (58). These features lead to suggestions that CL-11 could be involved in a wide range and different types of cellular processes through local action, and CL-11 produced by different tissues/cells can participate in those cellular processes. CL-11 is highly conserved among species; human and mice are 92% homologous at the amino acid level (6). Carbohydrate specificity studies have shown that CL-11 preferentially binds to fucose, mannose, and high-mannose oligosaccharides present on both self- and non–self-structures (6, 8, 9). CL-11 has been shown to play important roles in embryonic development, host defence, debris removal, and regulation of cytokine secretion (1012). CL-11 has also been shown to contribute to tissue injury and fibrosis in sterile inflammation (13, 14). More recently, in a mouse model of collagen-induced rheumatoid arthritis, we have shown that Colec11–/– mice developed more severe arthritis than WT littermates. Disease severity was associated with significantly enhanced antigen-presenting cell (APC) activation, Th1/Th17 responses, and pathogenic IgG2a production, as well as elevated circulating levels of inflammatory cytokines, indicating an important role for CL-11 in regulating APC activation and limiting autoimmunity (15). Taken together, these findings suggest that CL-11 is a multifunctional molecule, not only playing roles in homeostasis and host defence, but also participating in the pathogenesis of inflammatory and immunological diseases. So far, it is unknown whether CL-11 plays an important role in tumor growth.

Melanoma is a malignant tumor that arises from uncontrolled proliferation of melanocyte pigment–producing cells (16). Melanoma is a potentially lethal form of cancer that accounts for the majority of skin cancer deaths (17). The worldwide incidence of melanoma has steadily increased over the last several decades (18). Global cases of melanoma skin cancer will reach nearly half a million by 2040, an increase of 62% on 2018 figures (19). In the past decade, new immunotherapies, together with early diagnosis and better prevention, significantly improved cancer treatment. Immune checkpoint blockade (ICB) with monoclonal antibodies directed at the inhibitory immune receptors CTLA-4, PD-1, and PD-L1 has emerged as a successful treatment approach for patients with melanoma and other cancers (20, 21). However, these immunotherapies have only shown significant benefit for a minority of patients; the general toxicity- and immune-related adverse effects seen in the majority of patients who receive the combination therapies significantly limit their clinical use (22). Therefore, better understanding of the immunological mechanisms involved in cancer cell proliferation and tumor growth, and identifying novel tumor growth promoting molecules, are a still unmet need.

Given the role of CL-11 in stimulating fibroblast proliferation, immune regulation, and suppressing autoimmunity (12, 14, 15), we hypothesized that CL-11 may play important roles in tumor cell proliferation and tumor growth. Mouse melanoma models have been widely used for studying tumor growth, regulating tumor microenvironment (TME) and immune responses to tumors, and evaluating the efficacy in cancer therapies (23). In the present study, we employed a murine s.c. B16 melanoma model (24) combining Colec11–/– mice (25) and blockade of CL-11 with its preferential ligand approaches to determine the roles of CL-11 in melanoma cell proliferation and tumor growth. The effect of CL-11 in tumor growth was also assessed in a more clinically relevant model using YUMM1.7 murine melanoma cell line (26). We also investigated the underlying mechanisms by which CL-11 promotes tumor growth. Specifically, we explored the direct effect of CL-11 on melanoma cell proliferation and the involved receptors/signal transduction pathways and investigated the influence of CL-11 on immunosuppressive TME. Furthermore, we evaluated the relevance of CL-11 in human melanomas by analyzing COLEC11 gene expression in human melanomas and its association with patient survival using several open data sets and assessing whether CL-11 has stimulatory effects on human melanoma cells in vitro. Moreover, we assessed the generality of CL-11 in cancer cell proliferation by examining the effects of CL-11 on cell proliferation in several human cancer cell lines in vitro.

Results

Melanoma growth is suppressed in Colec11–/– mice. To determine the role of CL-11 in tumor growth, we assessed tumor growth in Colec11–/– mice and their WT littermates after s.c. inoculation with B16 melanoma cells by performing bioluminescence imaging and measuring tumor volume and weight (Figure 1A). Bioluminescence imaging was performed in living animals 14 days after the inoculation of luciferase-labeled melanoma cells. Colec11–/– mice had significantly reduced tumor burden compared with WT mice (Figure 1, B and C). Tumor volume changes was measured in living animals daily from day 6 (d6) to d14 after the inoculation; tumor weight was measured in excised melanomas at the end time point (d14). The tumor volume and weight were significantly reduced in Colec11–/– mice compared with WT mice (Figure 1, D–F). Similar results were observed when the YUMM1.7 melanoma model was used (Figure 1, G–I). These results demonstrate that melanoma growth is suppressed in Colec11–/– mice, indicating a role for CL-11 in promoting tumor growth.

Melanoma growth is suppressed in Colec11–/– mice.Figure 1

Melanoma growth is suppressed in Colec11–/– mice. (A) Schematic diagram of experimental design. Colec11–/– (WT) or Colec11–/– (KO) mice received melanoma cells (luciferase-labelled B16 [B16-luc] or unlabeled B16 or YUMM1.7) by s.c. injection. (B) Bioluminescence images of s.c. tumors in the 2 groups of mice 14 days after injection of B16-luc cells. (C) Quantification of total flux (photons per second [p/s]) (the bioluminescent signal is expressed in p/s/cm2/sr in the mice shown in B; data are analyzed by unpaired t test with Welch’s correction (n = 6 mice per group). (DF) B16 tumor growth. (D) Tumor volume (d6–d14). Data were analyzed by 2-way ANOVA with multiple-comparison test. Each symbol represents the mean of a group of mice (n = 9 or 10 mice/group, pooled from 2 experiments). (E) Tumor weight (d14). Data were analyzed by unpaired t test (n = 18 mice/group, pooled from 4 experiments). Each dot represents an individual mouse. (F) Images of excised tumors from the 2 groups of mice. Scale bar: 5 mm. Representative images from 2 independent experiments are shown. (GI) YUMM1.7 tumor growth. (G) Tumor volume (d6–d14). Data were analyzed by 2-way ANOVA with multiple-comparison test. Each symbol represents the mean of a group of mice (n = 6 or 7 mice/group). (H) Tumor weight (d14). Data were analyzed by unpaired t test (n = 6 or 7 mice/group). Each dot represents an individual mouse. (I) Images of excised tumors from the 2 groups of mice. Scale bar: 5 mm. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Colec11–/– mice have reduced melanoma cell proliferation and angiogenesis. Tumor cell proliferation and angiogenesis are among the hallmarks of tumor growth; we therefore examined these 2 parameters in excised melanomas (d14). Tumor cell proliferation was assessed by immunochemical staining of Ki67 and CD45 (used for identifying/excluding non-B16 cells). Compared with WT mice, Colec11–/– mice displayed a significant reduction of Ki67+ cells in the tumor core, most of the Ki67+ staining was not associated with CD45+ staining, confirming that Ki67+ cells are mainly tumor cells (Figure 2, A and B). Angiogenesis was assessed by immunochemical staining of endothelial markers (CD31, von Willebrand factor [VWF]). CD31 and VWF were markedly reduced in melanomas of Colec11–/– mice compared with WT mice. CD31 is an adhesion molecule and is expressed on endothelial cells, while VWF is stored in Weibel-Palade bodies of endothelial cells, which are released upon endothelial cell activation. Accordingly, different patterns of endothelial staining were observed, while CD31 was mainly detected on larger vessels, and VWF seemed mainly on smaller vessels (Figure 2, C and D). These results demonstrate that Colec11–/– mice have reduced melanoma cell proliferation and angiogenesis, indicating that CL-11 is required for tumor cell proliferation and angiogenesis.

Colec11–/– mice have reduced melanoma cell proliferation and angiogenesis.Figure 2

Colec11–/– mice have reduced melanoma cell proliferation and angiogenesis. Tumors excised from Colec11+/+ (WT) or Colec11–/– (KO) mice (d14) were used for analysis of tumor cell proliferation, angiogenesis, and CL-11 expression. (A) Representative microscopy images of immunochemical staining for Ki67 (green)/CD45 (red)/DAPI (blue) in tumor core area. The bottom panel show higher-magnification images of boxed regions in the panel above. Scale bars: 50 μm (top panel), 25 μm (bottom panel). (B) Quantification of Ki67+/CD45– cells corresponding to the 2 groups of mice in A. Data were analyzed by unpaired t test (n = 12; 3 mice, 4 image regions from each tumor section each mouse). (C) Representative microscopy images of immunochemical staining for CD31 (green)/DAPI (blue) or VWF (green)/DAPI (blue) in tumor core area. Scale bar: 50 μm. (D) Quantification of CD31- or VWF-stained areas corresponding to the WT and KO mice in C. Data were analyzed by unpaired t test (n = 18; 3 mice, 6 image regions from each tumor section each mouse). **P < 0.01; ****P < 0.0001.

Detection of CL-11 in melanomas. In order to understand how CL-11 influences tumor growth, we examined whether CL-11 is present within melanomas, whether CL-11 can be produced locally, and their cellular sources. We performed IHC staining of CL-11 in melanomas of WT and Colec11–/– mice to evaluate protein expression. CL-11 was abundantly detected in the melanomas of WT mice, while it was detected much less in the melanomas of Colec11–/– mice (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.159452DS1). It is known that CL-11 can be produced by a wide variety of tissues/cell types, including skin, spleen, bone marrow, and lymph node (27); in the circulation, CL-11 is often complexed with another collectin (CL-10) forming high-order oligomers at sizes up to 750 kDa that may not be sufficiently extravasated (28). Therefore, it is conceivable that immune-infiltrating cells and nonimmune cells within melanomas could contribute to the local pool of CL-11. We therefore assessed local production of CL-11 and their cellular sources. Quantitative PCR (qPCR) showed that Colec11 mRNA was detected in the melanomas of both WT and Colec11–/– mice; however, significantly higher expression was observed in WT mice, compared with Colec11–/– mice (Supplemental Figure 1B). Costaining of CL-11 and CD45 showed that, in the melanomas of WT mice, the majority of CD45+ cells were positively stained with CL-11, whereas in the melanomas of Colec11–/– mice, CD45+ cells appeared to be negative for CL-11 staining (Supplemental Figure 1C). Cellular sources of CL-11 in the melanoma were further analyzed by qPCR in CD45+ (immune-infiltrating) cells and CD45– (nonimmune) cells obtained from melanomas of WT and Colec11–/– mice by FACS. In the cell preparations from WT mice, Colec11 mRNA was mainly detected in CD45+ cells, while it was detected at very low levels in CD45– cells. In the cell preparations from Colec11–/– mice, no Colec11 mRNA was detected in CD45+ cells, and — just as in WT mice — only very low levels were detected in CD45– cells, implying there is a small amount of CL-11 being produced by melanoma cells (Supplemental Figure 1D). Collectively, these results demonstrate that CL-11 — which can be produced locally — is present in the melanomas; CD45+ infiltrating leukocytes are the major cellular source of CL-11 in the melanoma; and melanoma cell can produce small amount of CL-11.

Colec11–/– mice exhibit less immunosuppressive TME. The TME is an important regulator of tumor growth. Although, in general, the TME exhibits immunosuppressive features, it could be more or less immunosuppressive, depending on cancer-immunoediting processes that could potentially be regulated by factors in the TME. Therefore, we investigated whether CL-11 has an effect on the TME. In doing this, we performed several cellular and molecule analyses using excised B16 melanomas from WT and Colec11–/– mice to assess: (a) composition of tumor-infiltrating leukocytes, (b) intratumor gene expression of cytokines/chemokines, and (c) phenotype of tumor-associated macrophages (TAMs).

The composition of tumor-infiltrating leukocytes in melanomas was evaluated by flow cytometry and immunochemical staining. Flow cytometry analysis showed that the percentage of tumor-infiltrating CD45+ cells was comparable between WT and Colec11–/– mice (Figure 3A). However, the subsets of CD45+ cells were markedly different between the 2 groups of mice. Compared with WT mice, Colec11–/– mice had higher proportions of lymphocytes (CD4+, CD8+, NK1.1+) and lower proportions of myeloid lineage cells (CD11b+, CD11b+Ly6G+, CD11b+Ly6G–Ly6G+) (Figure 3B). A lower percentage of CD25+ cells within the CD4+ T cell compartment was detected in melanomas of Colec11–/– mice compared with WT mice (Supplemental Figure 2, A and B). There was no significant difference in CD19+ B cells within the CD45+ population between the 2 groups of mice (Supplemental Figure 2C). This led to a relatively higher lymphocytes-to-myeloid cells ratio in Colec11–/– mice than that in WT mice (Figure 3C). The costaining of CD3 and CD11b or CD3 and F4/80 showed that, compared with WT mice, Colec11–/– mice exhibited clearly more CD3+ infiltrates and much less CD11b+ and F4/80+ infiltrates in the tumor core and in the outskirt of the tumors (Figure 3D). Furthermore, the staining of CD8 showed that, compared with WT mice, Colec11–/– mice exhibited markedly increased CD8+ infiltrates in the tumor core and tumor edge (Figure 3E). More CD3+ infiltrates and fewer CD11b+ infiltrates were also observed in Colec11–/– mice when YUMM1.7 melanoma model was used (Supplemental Figure 3). Collectively, these results demonstrate that Colec11–/– mice have a higher lymphocytes/myeloid lineage cells ratio and a better cytotoxic T cell infiltration in the melanoma.

Colec11–/– mice exhibit less immunosuppressive TME.Figure 3

Colec11–/– mice exhibit less immunosuppressive TME. Tumors excised from Colec11+/+ (WT) or Colec11–/– (KO) mice (d14) were used for analyzing TME. (AC) Tumor infiltrates analyzed by flow cytometry. (A) CD45+ cells. (B) Subsets of tumor-infiltrating leukocytes analyzed by flow cytometry. Data were analyzed by unpaired t test (n = 18 mice per group, pooled from 4 experiments). Each dot represents an individual mouse. (C) A bar chat representing proportion of subsets in CD45+ cells shown in B. (D) Representative microscopy images of immunochemical staining for CD11b (green)/CD3 (red)/DAPI (blue) and F4/80 (green)/CD3 (red)/DAPI (blue). Scale bar: 50 μm. (E) Representative microscopy images of immunochemical staining for CD8 (red)/DAPI (blue) in tumor edge and core areas. Scale bar: 50 μm. (F). qPCR analysis in tumor tissues. Data were analyzed by unpaired t test (n = 8 mice per group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Intratumor mRNA levels of cytokines/chemokines highly relevant to the TME were analyzed by qPCR. Significantly higher levels of Ifng, Nos2, Il12, Ccl5, Cx3cl1, and Cxcl9 — and, by contrast, significantly lower levels of Arg1 — were detected in the melanomas of Colec11–/– mice, compared with WT mice (Figure 3F). These results demonstrate that the production of cytokines/chemokines having antitumor properties is upregulated in the melanomas of Colec11–/– mice.

TAMs are considered as main components of the TME and are present in high numbers in the microenvironment of solid tumors. TAMs affect most aspects of tumor cell biology and drive pathological phenomena, including tumor cell proliferation, tumor angiogenesis, invasion, and metastasis, as well as suppression of antitumor immune responses (29). We therefore performed RNA-Seq in TAMs isolated from melanomas of WT and Colec11–/– mice by sorting CD45+F4/80+ cells by FACS. RNA-Seq analysis revealed 625 differentially expressed genes (DEGs) (log2FC ≥ 1). These genes are mainly classified into “Immune system,” “Signal transduction,” “Signaling molecules and interaction” and “Cancer” pathways (Supplemental Figure 4). DEGs in Immune system and Cancer pathways were further analyzed focusing on genes involved in tumor progression/invasion/metastasis and MΦ phenotype. Compared with TAMs from WT mice, TAMs from Colec11–/– mice exhibited: (a) a lower expression of genes coding for procancer molecules (Spp1, Igf1, Plgs2, Plau, Ctsk, Areg, Hbegf, Il1a, Il1b); (b) a lower expression of genes coding for chemoattractant of myeloid-derived suppressor cells (MDSC) and neutrophil (Cxcl1, Cxcl2, Cxcl12, Cxcl3, Pbbp) and MO/MF (Ccl3, Ccl4, Ccl6, Ccl12) and, by contrast, a higher expression of genes coding for potent chemoattractant of T cell and NK cell (Cxcl9, Ccl5); and (c) a higher expression of M1 marker genes (H2.Aa, H2.Ab1, H2.Eb1, H2.DMa, Fcgg4, CD64, Stat1, Stat2, Socs1, Ifng) and, by contrast, a lower expression of M2 marker genes (Cd36, Cd163, Cd206, Cd14, Il10) (Figure 4, A and B). These results demonstrate that TAMs from Colec11–/– mice exhibit less immunosuppressive and tumor-suppressing features, while TAMs from WT mice exhibit more immunosuppressive and tumor-promoting features.

TAMs from Colec11–/– mice exhibit less immunosuppressive features.Figure 4

TAMs from Colec11–/– mice exhibit less immunosuppressive features. Tumors excised from Colec11+/+ (WT) or Colec11–/– (KO) mice (d14) were used for analyzing TAMs phenotypes by RNA-Seq. (A) Heatmaps of RNA-Seq data showing genes with significant differential expression (with fold change ≥ 1.5) between TAMs (CD45+F4/80+) from WT mice (TAM-WT) and KO mice (TAM-KO). Left panel: procancer pathway genes. Middl

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