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Acetylcholine promotes the self-renewal and immune escape of CD133+ thyroid cancer cells through activation of CD133-Akt pathway
Journal Pre-proof Acetylcholine promotes the self-renewal and immune escape of CD133+ thyroid cancer cells through activation of CD133-Akt pathway Zhenglin Wang, Wei Liu, Cong Wang, Yinan Li, Zhilong Ai PII:
S0304-3835(19)30615-9
DOI:
https://doi.org/10.1016/j.canlet.2019.12.009
Reference:
CAN 114605
To appear in:
Cancer Letters
Received Date: 26 August 2019 Revised Date:
3 December 2019
Accepted Date: 4 December 2019
Please cite this article as: Z. Wang, W. Liu, C. Wang, Y. Li, Z. Ai, Acetylcholine promotes the selfrenewal and immune escape of CD133+ thyroid cancer cells through activation of CD133-Akt pathway, Cancer Letters, https://doi.org/10.1016/j.canlet.2019.12.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier B.V. All rights reserved.
Nerves infiltrate the tumor microenvironment and stimulate the growth of cancer cells through the secretion of neurotransmitters. However, the contributions of nerves to the self-renewal capacity of cancer stem cells (CSCs) remain largely unknown. In this study, we found that CD133+ cancer cells were responsible for the initiation of thyroid cancer. Neurons were juxtaposed with CD133+ cells in thyroid cancer tissues. Acetylcholine, one of the most abundant neurotransmitters, promoted CD133 Y828 phosphorylation, and subsequently increased the interaction between CD133 and PI3K regulatory subunit p85, resulting in the activation of the PI3K-Akt pathway. Acetylcholine increased the self-renewal ability of CD133+ thyroid cancer cells through activation of CD133-Akt pathway. Furthermore, acetylcholine promoted the expression of the immune regulator PD-L1 through the activation of the CD133-Akt pathway, resulting in the resistance of CD133+ thyroid cancer cells to CD8+ T cells. However, acetylcholine receptor antagonist 4-DAMP blocked the positive effects of acetylcholine on the self-renewal and immune escape of CD133+ thyroid cancer cells. Taken together, these data suggest that acetylcholine increases the self-renewal and immune escape abilities of CD133+ thyroid cancer cells through the activation of the CD133-Akt pathway.
Acetylcholine promotes the self-renewal and immune escape of CD133+ thyroid cancer cells through activation of CD133-Akt pathway Zhenglin Wang1#, Wei Liu1#, Cong Wang1, Yinan Li2, Zhilong Ai1* 1
Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai
200032, China. 2
NHC Key Laboratory of Glycoconjugates Research，Department of Biochemistry
and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China *Corresponding author. Zhilong Ai, Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032, China. E-mail address: [email protected] (Z. Ai) #
These authors contributed equally to this work.
Running Title: Acetylcholine promotes the self-renewal and immune escape of CD133+ thyroid cancer cells
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Abstract Nerves infiltrate the tumor microenvironment and stimulate the growth of cancer cells through the secretion of neurotransmitters. However, the contributions of nerves to the self-renewal capacity of cancer stem cells (CSCs) remain largely unknown. In this study, we found that CD133+ cancer cells were responsible for the initiation of thyroid cancer. Neurons were juxtaposed with CD133+ cells in thyroid cancer tissues. Acetylcholine, one of the most abundant neurotransmitters, promoted CD133 Y828 phosphorylation, and subsequently increased the interaction between CD133 and PI3K regulatory subunit p85, resulting in the activation of the PI3K-Akt pathway. Acetylcholine increased the self-renewal ability of CD133+ thyroid cancer cells through activation of CD133-Akt pathway. Furthermore, acetylcholine promoted the expression of the immune regulator PD-L1 through the activation of the CD133-Akt pathway, resulting in the resistance of CD133+ thyroid cancer cells to CD8+ T cells. However, acetylcholine receptor antagonist 4-DAMP blocked the positive effects of acetylcholine on the self-renewal and immune escape of CD133+ thyroid cancer cells. Taken together, these data suggest that acetylcholine increases the self-renewal and immune escape abilities of CD133+ thyroid cancer cells through the activation of the CD133-Akt pathway. Keywords Neurotransmitter; Neuron; p85; CD8+ T cells; PD-L1 Abbreviations siRNA, small interfering RNA; shRNA, short hairpin RNA; LV, lentiviral; CSC, cancer stem cell; DTC, differentiated thyroid carcinoma; PDTC, poorly differentiated thyroid carcinoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ACh, acetylcholine; M3R， M3 muscarinic acetylcholine receptor; IP, immunoprecipitation; PI3K, Phosphoinositide 3-kinase; PBS, phosphate buffered saline. Funding This work was supported by National Natural Scientific Foundation of China (81272722).
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1. Introduction Thyroid cancer is the most common type of endocrine cancer [1, 2]. Understanding the mechanisms of development of thyroid cancer will aid in improving the treatment of thyroid cancer [3]. Cancer stem cells (CSCs) are responsible for the initiation and recurrence of tumors in various malignancies [4]. CD133+ cells are responsible for the initiation and therapy-resistance of thyroid cancer [5, 6]. Thus, elimination of CD133+ thyroid cancer cells is a promising strategy to improve the outcomes of thyroid cancer treatment. The tumor microenvironment maintains the CSCs in a stem-like state and promotes their self-renewal [7-9]. Understanding the mechanisms through which the microenvironment regulates CSCs fates could help to find clues to eliminate CSCs in vivo. Increasing numbers of studies have shown that nerve fibers regulate the proliferation and migration of cancer cells [10, 11]. Cancer cells attract nerve fibers and stimulate nerve outgrowth by secreting neurotrophic factors [12]. Conversely, nerve fibers infiltrate the tumor microenvironment and stimulate the growth and dissemination of cancer cells [13, 14]. For example, acetylcholine promotes the proliferation and migration of cancer cells through the M3R receptor [15-18]. However, the contribution of neurons to the self-renewal capacity of CSCs remains largely unknown. Here, we found that neurons were juxtaposed with CD133+ thyroid cancer cell. Acetylcholine promoted the self-renewal of CD133+ thyroid cancer cells partly through the activation of the CD133-Akt pathway. Furthermore, acetylcholine promoted PD-L1 expression through the activation of the CD133-Akt pathway and thus promotes the resistance of CD133+ thyroid cancer cells to CD8+ T cells. Together, acetylcholine increases the self-renewal and immune escape abilities of CD133+ thyroid cancer cells through the activation of the CD133-Akt pathway.
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2. Materials and methods 2.1. Cell cultures Thyroid cancer cell lines C643 and 8305C were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 50 µg/ml streptomycin at 37 °C in a CO2 incubator (5% CO2, 95% air). The sorted CD133+ cells were isolated from xenograft formed by thyroid cancer cell lines (C643 or 8305C) and human thyroid cancer tissues as previously described [19]. Human thyroid cancer tissues were obtained in accordance with protocols approved by the Ethics Committees of Zhongshan Hospital of Fudan University (NO. 2018-151). The information of the tissues was shown in Supplementary Table. 1. Briefly, tumor specimens were washed and enzymatically dissociated into single cells. CD133+ cells were separated through magnetic cell sorting with a CD133 cell isolation kit (Miltenyi Biotech). CD133+ cells were cultured in the DMEM/F12 media supplemented with B27 lacking vitamin A (Invitrogen), 2 µg/ml heparin (Sigma), 20 ng/ml EGF (Chemicon) and 10 ng/ml bFGF (Chemicon) as previously described [20]. Tissues were digested with collagenase for 1-1.5 hr at 37 °C. After lysis of RBC, CD8+ T cells were purified using the magnetic cell sorting system (Miltenyi Biotech). 2.2. Sphere formation assay CD133+ thyroid cancer cells were cultured in serum-free DMEM/F12 media (Invitrogen), supplemented with 20 ng/ml human recombinant EGF (Chemicon), 10 ng/ml human recombinant bFGF (Chemicon), and B27 (Invitrogen). Cells were cultured in suspension in 96-well plates at different density. After 10 days, the percent of wells with spheres with diameters larger than 50 µM or the number of spheres with diameters larger than 50 µM were counted under an inverted microscope. 2.3. Dual luciferase assay Dural luciferase assay was performed as previously described [21]. Cells were transiently transfected with pGL3-PD-L1 promoter and pRL-SV40 plasmids. 48-72 h after transfection, cells were rinsed in PBS for three times and lysed in a Passive Lysis Buffer (Promega). Luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega) with Turner luminometer and normalized to the Renilla Luciferase activity for transfection efficiency. Data were represented as the 4
mean from at least three independent experiments. 2.4. Western blot Western blot assay was performed as previously described [20]. Primary antibodies included rabbit monoclonal anti-GAPDH antibody (Cell signaling, cat# 5174), goat polyclonal anti-PD-L1 (R&D, cat# AF156), rabbit anti-Src (Cell signaling, cat# 2108), rabbit anti-phospho-Src (Tyr416) antibody (Cell signaling, cat# 2101), rabbit monoclonal anti-phospho-Akt (Thr308) antibody (Cell signaling, cat# 13038), rabbit anti-p85 antibody (Millipore, cat# 06-195) and rabbit anti-Akt antibody (Cell signaling, cat# 9272, 1:2000). For quantification, the western blot films were scanned and were analyzed using ImageJ Version 1.33u software. 2.5. Immunoprecipitation. Immunoprecipitation was performed as previously described [19]. Briefly, cells were lysated in RIPA buffer (50 mM Tris (pH 7.4), 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM NaF and protease inhibitor mixture). Lysates were centrifuged and cleared by incubation with 20 µL of Protein G-Agarose (Roche) for 1.5-2 h at 4 °C. The pre-cleared supernatant was subjected to immunoprecipitation using anti-FLAG or anti-CD133 (Miltenyi Biotec, W6B3C1 clone) antibodies at 4 °C overnight. Then, the protein complexes were collected by incubation with 30 µL of Protein G-Agarose (Roche) for 4-6 h at 4 °C. The collected protein complexes were washed five times with IP buffer and analyzed by western blot. . 2.6. Tumor formation assay To examine the tumor-initiating capacity of CD133+ thyroid cancer cells, tumor cells was transplanted into 6 to 8-week old immunodeficient mice in accordance with Zhongshan hospital of Fudan University Institutional Animal Care and Use Committee approved protocol concurrent with national regulatory standards. Mice were sacrificed when they were moribund or 180 days after implantation. Tumor formation was determined by histology. To examine the effect of 4-DAMP on the in vivo growth of CD133+ cells, CD133+ cells were injected into the right flank region of 6-week old immunodeficient mice. Five days after injection, 4-DAMP was injected i.p. at doses of 1 mg/kg/day 5
respectively. Tumor volumes of each group were measured every three days. 2.7. Statistical Analysis Results are expressed as the mean ± standard error of the mean (mean ± SEM). In general, significance was tested by unpaired two-tailed Student’s t test or ANOVA test using GraphPad InStat 5.0 software. P value < 0.05 was considered statistically significant.
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3. Results 3.1. Neurons are juxtaposed to CD133+ thyroid cancer cells The expression of the representative neuron marker MAP2 was examined in thyroid cancer tissues. MAP2 was sparsely expressed in thyroid cancer tissues (Fig. 1A-B). Thyroid cancers mainly comprise differentiated thyroid carcinoma (DTC) and poorly differentiated carcinoma (PDTC) [22]. To examine the relationship between neuron numbers and pathological types in DTC and PDTC samples, MAP2 expression was analyzed using immunohistochemical staining. Compared to the DTC samples, MAP2 was expressed at higher levels in the PDTCs (Fig. 1C, p < 0.01). To examine the association between thyroid CSCs and neurons, thyroid cancer tissues were analyzed by immunofluorescence using anti-CD133 (marker of thyroid CSC) and anti-MAP2 (marker of neuron) antibodies. Juxtaposition of neuron with CD133+ thyroid cancer cells was detected in the thyroid cancer samples (patient #1 and #2) (Fig. 1D-E). The percentage of MAP2-positive cells or CD133-positive cells in thyroid cancer samples (patient #1 and #2) ranged from 2 to 3% (Fig. 1F-G). Taken together, these data suggest that neurons are juxtaposed with CD133+ thyroid cancer cells. 3.2. Acetylcholine promotes the self-renewal capacity of CD133+ thyroid cancer cells Neurotransmitters bind to neurotransmitter receptors in cancer cells and promote the growth and invasion of cancer cells [10, 13]. CD133 is a marker for thyroid CSC [5, 6]. The effects of neurotransmitters on the self-renewal capacity of CD133+ thyroid cancer cells were examined. CD133+ and CD133- cells were isolated from C643 and 8305C xenografts and thyroid cancer samples (patient #1 and #2). The CD133+ tumor cells gradually formed spheres of different sizes and shapes (Fig. 2A). Sphere formation from single cells showed that CD133+ tumor cells formed the spheres with different diameters (Supplementary Fig. 1), indicating the phenotypic heterogeneity of the spheres. CD133+ cells displayed characteristics consistent with that of CSCs including expression of stem cell markers (Fig. 2B-E) and high tumorigenicity in immunocompromised mice (Fig. 2F-G). Thus, CD133+ subpopulations are enriched for thyroid CSCs. CD133+ cells were treated with various neurotransmitters including acetylcholine, neuropeptides, Dynorphin B or GABA. Acetylcholine evidently increased the number 7
of spheres formed by CD133+ cells isolated from the C643 xenograft (Fig. 2H-I). Consistent with this, acetylcholine also increased the number of spheres formed by CD133+ cells isolated from 8305C xenograft or thyroid cancer samples (patient #1 and #2) (Fig. 2J). The sphere formation efficiency is one method to determine the self-renewal capacity of CSCs [19]. Acetylcholine increased the sphere formation ability of CD133+ cells isolated from C643 and 8305C xenografts (Fig. 2K). Furthermore, acetylcholine promoted the in vivo growth of CD133+ cells isolated from C643 xenograft (Fig. 2L). These data suggest that acetylcholine increases the self-renewal capacity of CD133+ thyroid cancer cells. 3.3. Acetylcholine activates CD133-Akt pathway Next, the mechanism of acetylcholine-mediated increase the self-renewal capacity of CD133+ thyroid cancer cells was examined. CD133 promotes the self-renewal of CSCs depending on its Y828 phosphorylation [19]. Acetylcholine clearly increased the Y828 phosphorylation of CD133 without affecting the expression of total CD133 (Fig. 3A-B). The phosphorylation of CD133 at the Y828 residue is mediated by c-Src kinase [23]. The kinase activity of c-Src is positively regulated by phosphorylation of Y416 [24, 25]. Acetylcholine promotes cancer cells growth through the acetylcholine receptor M3R [26]. Downregulation of M3R expression using siRNA reduced the acetylcholine-induced Src Y416 phosphorylation and consequently the CD133 Y828 phosphorylation (Fig. 3C). Thus, acetylcholine increased c-Src Y416 phosphorylation and CD133 Y828 phosphorylation. CD133 interacts with p85 depending on its Y828 phosphorylation, consequently increasing PI3K activity and activating Akt phosphorylation in glioma [19]. To examine the significance of Y828 phosphorylation of CD133 on its interaction with p85 in thyroid cancer cells, CD133+ cells expressing CD133 short-hairpin RNA (shRNA) were infected with lentivirus expressing shRNA resistant wild type CD133 or its Y828F or Y852F mutant. Y828F mutant, but not Y852F mutant, visibly reduced the interaction between CD133 and p85 (Fig. 3D). Consistent with this, the mutation of Y828 to F significantly blocked the induction of PI3K kinase activity by CD133 (Fig. 3E). The effect of acetylcholine on CD133-Akt signaling pathway was examined next. Acetylcholine promoted the interaction between CD133 and p85 (Fig. 3F), and increased the activity of PI3K in CD133+ cells isolated from C643 and 8305C xenografts and thyroid cancer samples (patient #1 and #2) (Fig. 3G). Furthermore, 8
acetylcholine increased Akt T308 phosphorylation in CD133+ cells (Fig. 3H). Taken together, these data suggest that acetylcholine increases CD133 phosphorylation and enhances the PI3K-Akt pathway. 3.4. Acetylcholine increases the self-renewal ability of CD133+ thyroid cancer cells through activation of CD133-Akt pathway The Akt pathway promotes the self-renewal of CSCs [27, 28]. This finding motivated
us
to
examine
the
contribution
of
CD133-Akt
pathway
in
acetylcholine-promoted self-renewal of CD133+ cells. First, the effect of CD133 down-regulation on acetylcholine-promoted self-renewal of CD133+ thyroid cancer cells was examined. CD133 shRNA effectively reduced the endogenous CD133 expression (Fig. 4A). ShRNA-mediated down-regulation of CD133 reduced the number of spheres formed by CD133+ cells isolated from the C643 xenograft (Fig. 4B-C), and blocked the positive effects of acetylcholine on the self-renewal ability of CD133+ cells (Fig. 4D). The effect of acetylcholine on the self-renewal ability of CD133+ cells expressing CD133 shRNA could be rescued by overexpression of wild type CD133, but not by overexpression of CD133 Y828F mutant (Fig. 4E). Next, the effect of an Akt pathway inhibitor on acetylcholine-promoted self-renewal of CD133+ cells was examined. Wortmannin is a widely used inhibitor of the PI3K/Akt pathway [29]. CD133 overexpression increased the number of spheres formed by CD133+ cells expressing CD133 shRNA, which was markedly reduced by wortmannin (Fig. 4F). Wortmannin reduced the number of spheres formed by CD133+ cells (Fig. 4G), and reduced the positive effects of acetylcholine on the self-renewal ability of CD133+ cells isolated from C643 xenograft and thyroid cancer sample (patient #1) (Fig. 4H-I). These data suggest that down-regulation of CD133 or inhibition of the Akt pathway reduces the positive effect of acetylcholine on CD133+ thyroid cancer cells self-renewal. 3.5. Acetylcholine up-regulates PD-L1 through activation of CD133-Akt pathway and promotes the immune escape of CD133+ thyroid cancer cells The Akt pathway promotes immune escape by up-regulation of PD-L1 [30, 31]. The interaction between PD-L1 expressed on the tumor cells and PD-1 on CD8+ T cells inhibits anti-tumor immunity [32, 33]. We next examined the effect of acetylcholine on the expression of PD-L1. Wortmannin reduced the expression of 9
PD-L1 in CD133+ cells isolated from C643 xenograft (Fig. 5A), indicating that the Akt pathway promoted the expression of PD-L1 in thyroid CSCs. Acetylcholine induced mRNA expression and the level of PD-L1 protein in CD133+ cells (Fig. 5B-C). Further, acetylcholine induced the activity of PD-L1 promoter in CD133+ cells isolated from C643 and 8305C xenografts and thyroid cancer samples (patient #1 and
#2)
(Fig.
5D).
Next,
the
contribution
of
CD133-Akt
pathway in
acetylcholine-promoted PD-L1 transcription was examined. Down-regulation of CD133 blocked the positive effects of acetylcholine on the activity of PD-L1 promoter in CD133+ cells isolated from 8305C xenograft (Fig. 5E). To examine the significance of Y828 phosphorylation in acetylcholine-induced PD-L1 promoter activity, CD133+ cells expressing CD133 shRNA and shRNA resistant wild type CD133 or its mutant were transiently transfected with the PD-L1 promoter, followed by treatment of acetylcholine. The Y828F mutant significantly blocked the positive effect of CD133 or acetylcholine on the activity of PD-L1 promoter (Fig. 5F). CD133 overexpression increased the activity of PD-L1 promoter in CD133+ cells expression CD133 shRNA, which was clearly blocked by wortmannin (Fig. 5G). Furthermore, wortmannin inhibited acetylcholine-induced PD-L1 promoter activity in CD133+ cells (Fig. 5H). These data suggest that acetylcholine promotes PD-L1 expression through the activation of the CD133-Akt pathway. To examine the effects of acetylcholine on the immune escape of CD133+ thyroid cancer cells, CD133+ cells isolated from C643 xenografts were pre-treated with acetylcholine and then co-cultured with activated CD8+ T cells. Acetylcholine reduced the percentage of apoptotic CD133+ cells isolated from C643 and 8305C xenografts and thyroid cancer sample (patient #1) induced by CD8+ T cells (Fig. 5I-K). Anti-PD-L1 antibody clearly reduced the positive effect of acetylcholine on the resistance of CD133+ cells to CD8+ T cells (Fig. 5L). Thus, acetylcholine promotes the resistance of CD133+ thyroid cancer cells to CD8+ T cells through up-regulation of PD-L1. 3.6. Acetylcholine receptor (M3R) antagonist, 4-DAMP, inhibits the self-renewal and immune escape abilities of CD133+ thyroid cancer cells induced by acetylcholine The contribution of acetylcholine to self-renewal of CD133+ thyroid cells motivated us to explore the effects of the acetylcholine receptor (M3R) inhibitor 10
4-DAMP on self-renewal of CD133+ thyroid cancer cells. 4-DAMP inhibited the positive effect of acetylcholine on the self-renewal of CD133+ thyroid cancer cells. However, a selective M1 antagonist VU0255035 and a M2/M4 selective antagonist AFDX-116 did not reduce the effect of acetylcholine on the self-renewal capacity of CD133+ cells (Fig. 6A-B). Next, the effect of 4-DAMP on the in vivo growth of CD133+ cells were examined. The in vivo growth of CD133+ cells isolated from 8305C xenograft was visibly reduced by 4-DAMP (Fig. 6C). Further, 4-DAMP reduced the positive effect of acetylcholine on the resistance of CD133+ cells isolated from C643 xenograft to CD8+ T cells (Fig. 6D). Together, these data suggest that acetylcholine receptor M3R antagonist 4-DAMP inhibits the self-renewal and immune escape of CD133+ thyroid cancer cells induced by acetylcholine.
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4. Discussion In this study, we found that acetylcholine promoted the self-renewal capacity of CD133+ thyroid cancer cells through the activation of the CD133-Akt pathway. Furthermore, acetylcholine promoted PD-L1 expression through the activation of the CD133-Akt pathway and enhanced the resistance of CD133+ cells to CD8+ T cells (Fig. 6E). Together, acetylcholine promotes the self-renewal and immune escape of CD133+ thyroid cancer cells through activation of the CD133-Akt pathway. Acetylcholine promotes the self-renewal and immune escape of CD133+ thyroid cancer cells through enhancing CD133 phosphorylation. CD133 is phosphorylated at Y828 and Y852 residues by Src kinase [23]. CD133 Y828F mutation clearly reduced the interaction between CD133 and p85. CD133 Y828F mutation blocked acetylcholine-induced self-renewal of CD133+ cells and the activity of PD-L1 promoter. Thus, we speculate that CD133 Y828 phosphorylation contributes to acetylcholine-induced CD133+ cells self-renewal and immune escape. Our data showed that M3R down-regulation inhibited the activation of Src phosphorylation and the CD133 phosphorylation by acetylcholine. We speculate that acetylcholine promotes CD133 Y828 phosphorylation through increasing c-Src activity. However, the mechanism of acetylcholine promoting Src Y416 phosphorylation in CD133+ thyroid cancer cells needs further explored. Nerves have recently been recognized as key regulators of tumor growth [11, 34]. Neurotrophic factors released from cancer cells promote the outgrowth of neuron in tumors [35]. It is widely known that neurons promote the growth and migration of cancer cells [10]. However, the contributions of neurons to the niche of CSCs remain largely unknown. Here, we found that neurons were juxtaposed with CD133+ thyroid cancer cells. Acetylcholine promoted the self-renewal and immune escape of CD133+ thyroid cancer cells. Thus, neuron might be an important component in the niche of CSCs. The tumor microenvironment is crucial to the self-renewal of CSCs [36, 37]. Therefore, microenvironment-targeting strategies might eliminate the CSCs. Our findings have showed that acetylcholine promoted the self-renewal of CD133+ thyroid cancer cells. Acetylcholine receptor M3R antagonist, 4-DAMP, reduced acetylcholine-induced self-renewal and immune escape of CD133+ thyroid cancer cells. Furthermore, 4-DAMP inhibited the in vivo growth of CD133+ thyroid cancer cells. Thus, inhibition of acetylcholine signaling pathway may be a promising 12
approach for the treatment of thyroid cancer. In summary, we found that neurons were juxtaposed with CD133+ thyroid cancer cells. Acetylcholine promoted the self-renewal and immune escape of CD133+ thyroid cancer cells at least partly through the activation of the CD133-Akt pathway. These findings identify neuron as an important component in the niche of thyroid CSCs. Competing interest Authors have no conflicts of interests. Author contributions ZLW and WL performed most experiments; WC performed cell sorting; YNL performed signaling pathway analysis and plasmids construction; AZL discussed the data and wrote the manuscript Acknowledgements This work was supported by National Natural Scientific Foundation of China (81272722). We are grateful to Dr. Jing Li (Shanghai Medical College of Fudan University) for providing flow cytometry technology. We are grateful to Dr. Jianhai Jiang (Shanghai Medical College of Fudan University) for providing CD133 Y828 phosphorylation antibody and CD133-FLAG plasmids and its mutants.
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phosphorylated in the closed, repressed conformation of c-Src, PLoS One 8 (2013) e71035. [25] L. Kong, Z. Deng, H. Shen, Y. Zhang, Src family kinase inhibitor PP2 efficiently inhibits cervical cancer cell proliferation through down-regulating phospho-Src-Y416 and phospho-EGFR-Y1173, Mol Cell Biochem 348 (2011) 11-19. [26] P. Song, H.S. Sekhon, A. Lu, J. Arredondo, D. Sauer, C. Gravett, G.P. Mark, S.A. Grando, E.R. Spindel, M3 muscarinic receptor antagonists inhibit small cell lung carcinoma growth and mitogen-activated protein kinase phosphorylation induced by acetylcholine secretion, Cancer Res 67 (2007) 3936-3944. [27] J.W. Jang, Y. Song, S.H. Kim, J.S. Kim, K.M. Kim, E.K. Choi, J. Kim, H.R. Seo, CD133 confers cancer stem-like cell properties by stabilizing EGFR-AKT signaling in hepatocellular carcinoma, Cancer Lett 389 (2017) 1-10. [28] S. Almozyan, D. Colak, F. Mansour, A. Alaiya, O. Al-Harazi, A. Qattan, F. Al-Mohanna, M. Al-Alwan, H. Ghebeh, PD-L1 promotes OCT4 and Nanog expression in breast cancer stem cells by sustaining PI3K/AKT pathway activation, Int J Cancer 141 (2017) 1402-1412. [29] L. Madi, B. Rosenberg-Haggen, A. Nyska, R. Korenstein, Enhancing pigmentation via activation of A3 adenosine receptors in B16 melanoma cells and in human skin explants, Exp Dermatol 22 (2013) 74-77. [30] C. Kasikara, S. Kumar, S. Kimani, W.I. Tsou, K. Geng, V. Davra, G. Sriram, C. Devoe, K.N. Nguyen, A. Antes, A. Krantz, G. Rymarczyk, A. Wilczynski, C. Empig, B. Freimark, M. Gray, K. Schlunegger, J. Hutchins, S.V. Kotenko, R.B. Birge, Phosphatidylserine
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[34] J. Bauman, K. McVary, Autonomic nerve development contributes to prostate cancer progression, Asian J Androl 15 (2013) 713-714. [35] B. Boilly, S. Faulkner, P. Jobling, H. Hondermarck, Nerve Dependence: From Regeneration to Cancer, Cancer Cell 31 (2017) 342-354. [36] Y. Luo, Z. Yang, L. Su, J. Shan, H. Xu, Y. Xu, L. Liu, W. Zhu, X. Chen, C. Liu, J. Chen, C. Yao, F. Cheng, C. Zhang, Q. Ma, J. Shen, C. Qian, Non-CSCs nourish CSCs through interleukin-17E-mediated activation of NF-kappaB and JAK/STAT3 signaling in human hepatocellular carcinoma, Cancer Lett 375 (2016) 390-399. [37] K.E. Ritchie, J.E. Nor, Perivascular stem cell niche in head and neck cancer, Cancer Lett 338 (2013) 41-46.
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FIGURE LEGENDS Fig. 1. Neurons are juxtaposed with CD133+ thyroid cancer cells. (A-B) IHC staining for MAP2 in thyroid cancer tissues of patient #1 (A) and patient #2 (B). Representative images were shown. Scale bar, 20 µM. (C) The scores for quantitative staining of MAP2 in DTC samples (n = 12) and PDTC samples (n = 10) were determined. Results are expressed as mean ± SEM; **p < 0.01. (D-E) Confocal images of thyroid cancer sections of patient #1 (D) and #2 (E) co-stained for CSC marker CD133 (red) and neuron marker MAP2 (green) showed the juxtaposition of thyroid CSCs with neurons. Scale bar, 10 µM. (F-G) The percent of MAP2-positive cell (F) or CD133-positive cell (G) in D-E was was calculated from five different fields. Results are expressed as mean ± SEM. Fig. 2. Acetylcholine increases the self-renewal capacity of CD133+ thyroid cancer cells. (A) Representative image of spheres formed by CD133+ cells isolated from C643 and 8305C xenografts and thyroid cancer samples (patient #1 and #2). Scale bar represents 50 µM. (B-E) QPCR analysis was performed for stem cell-associated genes in CD133+ cells versus CD133- cells isolated from C643 (B) and 8305C xenografts (C) and thyroid cancer samples (patient #1 and #2) (D-E). CD133- cells were defined as 1, and the relative expression of each gene in CD133+ cells was expressed as the fold difference over CD133- cells. (F-G) An in vivo limiting dilution tumor formation assay (employing 10,000, 5,000, 1,000 or 500 cells per mouse) was performed to compare the tumor-initiating capacity of CD133+ cells or CD133- cells isolated from C643 and 8305C xenografts and thyroid cancer sample (patient #1). Mice were sacrificed when they were moribund or 180 days after implantation. Tumor formation was determined by histology. (F) The table displays the number of mice developing tumors. (G) H&E staining of mouse thyroids shows tumors formation by 1,000 CD133+ cells but not by 1,000 CD133cells. Scale bar represents 20 µM. (H-I) Sphere formation assay of CD133+ cells. 10 CD133+ cells isolated from C643 xenograft
were
cultured
in
96-well
plates,
followed
by treatment
with
neurotransmitters (10 µM). After 10 days, the number of sphere was counted. (H) Representative images of sphere are shown. (I) Results are expressed as mean ± SEM from four separate experiments; ***p < 0.001, #p > 0.05. Scale bar represents 50 µM. 18
Ach, Acetylcholine. (J) Sphere formation assay of CD133+ cells. 10 CD133+ cells isolated from 8305C xenograft or thyroid cancer samples (patient #1 and #2) were cultured in 96-well plates, followed by treatment with Ach (10 µM). After 10 days, the number of sphere was counted. Results are expressed as mean ± SEM from four separate experiments; ***p < 0.001. (K) Sphere formation assay of single CD133+ cell/well isolated from C643 xenograft or 8305C xenograft treated with or without Ach. Data means the percentage of sphere containing wells in each group. (L) CD133+ cells isolated from C643 xenograft were subcutaneously co-implanted with or without Ach into immunocompromised mice. Tumor volumes were measured every 3 days. Results are expressed as mean ± SEM (n = 6 mouse); **p < 0.01. Fig. 3. Acetylcholine activates the CD133-Akt pathway. (A-B) Western blot analysis of the level of Y828 phosphorylation of CD133 in CD133+ cells treated with control or Ach. GAPDH was blotted as a loading control. (A) Representative image from three separate experiments was shown. (B) The relative densities of pY828-CD133 to total CD133 in A were quantified using densitometry. Values are normalized to that of cells treated with control. Results are expressed as mean ± SEM from three separate experiments; ***p < 0.001. (C) Western blot analysis of the levels of CD133 phosphorylation and Src phosphorylation in CD133+ cells expressing control siRNA or M3R siRNA treated with acetylcholine. GAPDH was blotted as a loading control. (D) The effect of Y828 mutation on the interaction between p85 and CD133. The lysates of CD133+ cells isolated from C643 xenograft expressing CD133 shRNA + FLAG, CD133 shRNA + shRNA-resistant wild type CD133-FLAG, or CD133 shRNA + shRNA-resistant Y828F-FLAG or CD133 shRNA + shRNA-resistant Y852F-FLAG were subjected to immunoprecipitation using anti-FLAG antibody, followed by immunoblotting with anti-FLAG or anti-p85 antibodies. shR, shRNA-resistant. (E) The PI3K activity of CD133+ cells isolated from C643 xenograft expressing CD133 shRNA + FLAG, CD133 shRNA + shRNA-resistant wild type CD133-FLAG, or CD133 shRNA + shRNA-resistant Y828F-FLAG or CD133 shRNA + shRNA-resistant Y852F-FLAG were assessed using a PI3 kinase ELISA kit. Values 19
are normalized to that of control cells. Results are expressed as mean ± SEM from three separate experiments; ***p <0.001. (F) Co-IP analysis to determine the effect of Ach on the interaction between CD133 and p85. Lysates of CD133+ cells pretreated with or without Ach were subjected to IP using anti-CD133 antibody, followed by immunoblotting (IB) with anti-CD133 or anti-p85 antibodies. Whole cell lysates were analyzed by IB with anti-CD133, anti-p85 or anti-GAPDH antibodies as input. (G) The PI3K activity of CD133+ cells isolated from xenograft or thyroid cancer samples treated with control or Ach were assessed using a PI3 kinase ELISA kit. Values are normalized to that of control cells. Results are expressed as mean ± SEM from three separate experiments; ***p <0.001. (H) Western blot analysis of Akt T308 phosphorylation level in CD133+ cells treated with or without ACh. GAPDH was blotted as a loading control. Fig. 4. Acetylcholine increases the self-renewal ability of CD133+ thyroid cancer cells through the activation of the CD133-Akt pathway. (A)Western blot analysis of CD133 expression in CD133+ cells expressing LacZ shRNA or CD133 shRNA. GAPDH was blotted as a loading control. (B-C) 10 CD133+ cells isolated from C643 xenograft expressing LacZ shRNA or CD133 shRNA were cultured in 96-well plates. After 10 days, the number of sphere was counted. (B) Representative images of sphere are shown. (C) Results are expressed as mean ± SEM from three separate experiments; ***p < 0.001. Scale bar represents 50 µM. (D) 10 CD133+ cells isolated from C643 xenograft expressing LacZ shRNA or CD133 shRNA were cultured in 96-well plates, followed by treatment with control or Ach. Results are expressed as mean ± SEM from four separate experiments; ***p < 0.001, *p < 0.05. (E) 10 CD133+ cells isolated expressing CD133 shRNA + FLAG, CD133 shRNA + shRNA-resistant wild type CD133-FLAG, or CD133 shRNA + shRNA-resistant Y828F-FLAG or CD133 shRNA + shRNA-resistant Y852F-FLAG were cultured in 96-well plates, followed by treatment with control or Ach. After 10 days, the number of spheres was counted. Results are expressed as mean ± SEM from four separate experiments; *p < 0.05, ***p < 0.001. (F) 10 CD133+ cells expressing CD133 shRNA + FLAG, CD133 shRNA + 20
shRNA-resistant wild type CD133-FLAG treated with control or wortmannin (1 µM) were cultured in 96-well plates. After 10 days, the number of sphere was counted. Results are expressed as mean ± SEM from four separate experiments; **p < 0.01, ***p < 0.001. (G) 10 CD133+ cells isolated from C643 xenograft treated with control or wortmannin (1 µM) were cultured in 96-well plates. After 10 days, representative images of sphere were shown. Scale bar represents 50 µM. (H-I) 10 CD133+ cells isolated from C643 xenograft (H) or thyroid cancer sample (patient #1) (I) treated with wortmannin (1 µM) and/or Ach (10 µM) were cultured in 96-well plates. After 10 days, the number of sphere was counted. Results are expressed as mean ± SEM from three separate experiments; *p < 0.05, ***p < 0.001. Fig. 5. Acetylcholine induces the expression of PD-L1 through the activation of the CD133-Akt pathway and promotes the immune escape of CD133+ thyroid cancer cells. (A)Western blot analysis of the level of PD-L1 protein in CD133+ cells treated with DMSO or wortmannin. GAPDH was blotted as a loading control. (B) Western blot analysis of the level of PD-L1 protein in CD133+ cells treated with control or Ach. GAPDH was blotted as a loading control. (C) RT-PCR analysis of the level of PD-L1 mRNA in CD133+ cells treated with control or Ach. (D) The PD-L1 promoter construct pGL3-PD-L1 was transiently co-transfected with pRL-SV40 into CD133+ cells isolated from xenografts and thyroid cancer samples. Then, cells were treated with ACh. The luciferase activity was determined. The values were presented as fold activation over CD133+ cells treated with control. Values are mean ± SEM; ***p < 0.001. (E) CD133+ cells expressing control shRNA or CD133 shRNA were transiently co-transfected with pGL3-PD-L1 and pRL-SV40. Then, cells were treated with ACh. The luciferase activity was determined. The values were presented as fold activation over CD133+ cells expressing LacZ shRNA treated with control. Values are mean ± SEM; ***p < 0.001. (F) CD133+ cells expressing CD133 shRNA + FLAG, CD133 shRNA + shRNA-resistant wild type CD133-FLAG, or CD133 shRNA + shRNA-resistant Y828F-FLAG or CD133 shRNA + shRNA-resistant Y852F-FLAG were transiently 21
co-transfected with pGL3-PD-L1 and pRL-SV40. Then, cells were treated with ACh (10 µM). The luciferase activity was determined. The values were presented as fold activation over CD133+ cells expressing CD133 shRNA + FLAG treated with control. Values are mean ± SEM from three separate experiments; *p < 0.05, ***p < 0.001. (G) CD133+ cells isolated from C643 xenograft expressing CD133 shRNA+FLAG, CD133
shRNA+shRNA-resistant
wild
type
CD133-FLAG were
transiently
co-transfected with pGL3-PD-L1 and pRL-SV40. Then, cells were treated with wortmannin. The luciferase activity was determined. The values were presented as fold activation over CD133+ cells expressing CD133 shRNA+FLAG treated with control. Values are mean ± SEM from three separate experiments; **p < 0.01, ***p < 0.001. (H) CD133+ cells were transiently transfected with pGL3-PD-L1 and pRL-SV40. Then, cells were treated with wortmannin and/or ACh. The luciferase activity was determined. The values were presented as fold activation over CD133+ cells treated with control. Values are mean ± SEM from three separate experiments; ***p < 0.001. (I-K) CD133+ cells isolated from C643 (I) and 8305C xenografts (J) and thyroid cancer sample (patient #1) (K) pretreated with or without ACh (10 µM) were co-cultured with activated CTLs (cytotoxic T lymphocytes) for 4 hours. The percentage of apoptotic CD133+ cells was determined by flow cytometry. Results are expressed as mean ± SEM (n = 4); **p < 0.01, ***p < 0.001. (L) CD133+ cells isolated from 8305C xenograft pretreated with control or ACh were co-cultured with CTL cells in the presence of anti-PD-L1 Ab. The percentage of apoptotic CD133+ cells was determined by flow cytometry. Results are expressed as mean ± SEM (n = 4); **p < 0.01, ***p < 0.001. Fig. 6. Acetylcholine receptor (M3R) antagonist, 4-DAMP, reduces the self-renewal and immune escape abilities of CD133+ thyroid cancer cells induced by acetylcholine. (A-B) CD133+ cells were treated with Ach and VU0255035 or 4-DAMP or AFDX-110 (10 µM) for 10 days. The number of sphere was counted. (A) Representative images of sphere are shown. (B) Results are expressed as mean ± SEM from three separate experiments; ***p < 0.001. Scale bar represents 50 µM. (C) CD133+ cells isolated from 8305C xenograft were subcutaneously co-implanted into immunocompromised mice. Five day after injection, mice were injected with or 22
without 4-DAMP. Tumor volumes were measured every 3 days. Results are expressed as mean ± SEM (n = 6 mouse); **p < 0.01. (D) CD133+ cells isolated from C643 xenograft pretreated with ACh and/or 4-DAMP were co-cultured with CTLs for 4 hours. The percentage of apoptotic CD133+ cells was determined by flow cytometry. Results are expressed as mean ± SEM (n = 4); ***p < 0.001. (E) Schematic model of acetylcholine promoting the self-renewal and immune escape of CD133+ thyroid cancer cells.
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Aug 26, 2019 Conflict of Interest Disclosure Form Article Title: Acetylcholine promotes the self-renewal and immune escape of CD133+ thyroid cancer cells through activation of CD133-Akt pathway Authors: Zhenglin Wang, Wei Liu, Cong Wang, Yinan Li, Zhilong Ai
We submit our article to Cancer Letters for consideration and make the statement below: On behalf of all the authors above, I declare that all authors have no financial and personal relationships with other people or organizations that could inappropriately influence (bias) our work in this study.
Zhilong Ai, PhD. Zhongshan Hosptial, Fudan University Shanghai, China
1
Neurons are juxtaposed with CD133+ thyroid cancer cells ACh promotes CD133+ thyroid cancer cells self-renewal and immune escape ACh activates Akt phosphorylation through enhancing CD133 phosphorylation ACh promotes CD133+ thyroid cancer cells self-renewal via CD133-Akt pathway
S0304-3835(19)30615-9
DOI:
https://doi.org/10.1016/j.canlet.2019.12.009
Reference:
CAN 114605
To appear in:
Cancer Letters
Received Date: 26 August 2019 Revised Date:
3 December 2019
Accepted Date: 4 December 2019
Please cite this article as: Z. Wang, W. Liu, C. Wang, Y. Li, Z. Ai, Acetylcholine promotes the selfrenewal and immune escape of CD133+ thyroid cancer cells through activation of CD133-Akt pathway, Cancer Letters, https://doi.org/10.1016/j.canlet.2019.12.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier B.V. All rights reserved.
Nerves infiltrate the tumor microenvironment and stimulate the growth of cancer cells through the secretion of neurotransmitters. However, the contributions of nerves to the self-renewal capacity of cancer stem cells (CSCs) remain largely unknown. In this study, we found that CD133+ cancer cells were responsible for the initiation of thyroid cancer. Neurons were juxtaposed with CD133+ cells in thyroid cancer tissues. Acetylcholine, one of the most abundant neurotransmitters, promoted CD133 Y828 phosphorylation, and subsequently increased the interaction between CD133 and PI3K regulatory subunit p85, resulting in the activation of the PI3K-Akt pathway. Acetylcholine increased the self-renewal ability of CD133+ thyroid cancer cells through activation of CD133-Akt pathway. Furthermore, acetylcholine promoted the expression of the immune regulator PD-L1 through the activation of the CD133-Akt pathway, resulting in the resistance of CD133+ thyroid cancer cells to CD8+ T cells. However, acetylcholine receptor antagonist 4-DAMP blocked the positive effects of acetylcholine on the self-renewal and immune escape of CD133+ thyroid cancer cells. Taken together, these data suggest that acetylcholine increases the self-renewal and immune escape abilities of CD133+ thyroid cancer cells through the activation of the CD133-Akt pathway.
Acetylcholine promotes the self-renewal and immune escape of CD133+ thyroid cancer cells through activation of CD133-Akt pathway Zhenglin Wang1#, Wei Liu1#, Cong Wang1, Yinan Li2, Zhilong Ai1* 1
Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai
200032, China. 2
NHC Key Laboratory of Glycoconjugates Research，Department of Biochemistry
and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China *Corresponding author. Zhilong Ai, Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032, China. E-mail address: [email protected] (Z. Ai) #
These authors contributed equally to this work.
Running Title: Acetylcholine promotes the self-renewal and immune escape of CD133+ thyroid cancer cells
1
Abstract Nerves infiltrate the tumor microenvironment and stimulate the growth of cancer cells through the secretion of neurotransmitters. However, the contributions of nerves to the self-renewal capacity of cancer stem cells (CSCs) remain largely unknown. In this study, we found that CD133+ cancer cells were responsible for the initiation of thyroid cancer. Neurons were juxtaposed with CD133+ cells in thyroid cancer tissues. Acetylcholine, one of the most abundant neurotransmitters, promoted CD133 Y828 phosphorylation, and subsequently increased the interaction between CD133 and PI3K regulatory subunit p85, resulting in the activation of the PI3K-Akt pathway. Acetylcholine increased the self-renewal ability of CD133+ thyroid cancer cells through activation of CD133-Akt pathway. Furthermore, acetylcholine promoted the expression of the immune regulator PD-L1 through the activation of the CD133-Akt pathway, resulting in the resistance of CD133+ thyroid cancer cells to CD8+ T cells. However, acetylcholine receptor antagonist 4-DAMP blocked the positive effects of acetylcholine on the self-renewal and immune escape of CD133+ thyroid cancer cells. Taken together, these data suggest that acetylcholine increases the self-renewal and immune escape abilities of CD133+ thyroid cancer cells through the activation of the CD133-Akt pathway. Keywords Neurotransmitter; Neuron; p85; CD8+ T cells; PD-L1 Abbreviations siRNA, small interfering RNA; shRNA, short hairpin RNA; LV, lentiviral; CSC, cancer stem cell; DTC, differentiated thyroid carcinoma; PDTC, poorly differentiated thyroid carcinoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ACh, acetylcholine; M3R， M3 muscarinic acetylcholine receptor; IP, immunoprecipitation; PI3K, Phosphoinositide 3-kinase; PBS, phosphate buffered saline. Funding This work was supported by National Natural Scientific Foundation of China (81272722).
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1. Introduction Thyroid cancer is the most common type of endocrine cancer [1, 2]. Understanding the mechanisms of development of thyroid cancer will aid in improving the treatment of thyroid cancer [3]. Cancer stem cells (CSCs) are responsible for the initiation and recurrence of tumors in various malignancies [4]. CD133+ cells are responsible for the initiation and therapy-resistance of thyroid cancer [5, 6]. Thus, elimination of CD133+ thyroid cancer cells is a promising strategy to improve the outcomes of thyroid cancer treatment. The tumor microenvironment maintains the CSCs in a stem-like state and promotes their self-renewal [7-9]. Understanding the mechanisms through which the microenvironment regulates CSCs fates could help to find clues to eliminate CSCs in vivo. Increasing numbers of studies have shown that nerve fibers regulate the proliferation and migration of cancer cells [10, 11]. Cancer cells attract nerve fibers and stimulate nerve outgrowth by secreting neurotrophic factors [12]. Conversely, nerve fibers infiltrate the tumor microenvironment and stimulate the growth and dissemination of cancer cells [13, 14]. For example, acetylcholine promotes the proliferation and migration of cancer cells through the M3R receptor [15-18]. However, the contribution of neurons to the self-renewal capacity of CSCs remains largely unknown. Here, we found that neurons were juxtaposed with CD133+ thyroid cancer cell. Acetylcholine promoted the self-renewal of CD133+ thyroid cancer cells partly through the activation of the CD133-Akt pathway. Furthermore, acetylcholine promoted PD-L1 expression through the activation of the CD133-Akt pathway and thus promotes the resistance of CD133+ thyroid cancer cells to CD8+ T cells. Together, acetylcholine increases the self-renewal and immune escape abilities of CD133+ thyroid cancer cells through the activation of the CD133-Akt pathway.
3
2. Materials and methods 2.1. Cell cultures Thyroid cancer cell lines C643 and 8305C were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 50 µg/ml streptomycin at 37 °C in a CO2 incubator (5% CO2, 95% air). The sorted CD133+ cells were isolated from xenograft formed by thyroid cancer cell lines (C643 or 8305C) and human thyroid cancer tissues as previously described [19]. Human thyroid cancer tissues were obtained in accordance with protocols approved by the Ethics Committees of Zhongshan Hospital of Fudan University (NO. 2018-151). The information of the tissues was shown in Supplementary Table. 1. Briefly, tumor specimens were washed and enzymatically dissociated into single cells. CD133+ cells were separated through magnetic cell sorting with a CD133 cell isolation kit (Miltenyi Biotech). CD133+ cells were cultured in the DMEM/F12 media supplemented with B27 lacking vitamin A (Invitrogen), 2 µg/ml heparin (Sigma), 20 ng/ml EGF (Chemicon) and 10 ng/ml bFGF (Chemicon) as previously described [20]. Tissues were digested with collagenase for 1-1.5 hr at 37 °C. After lysis of RBC, CD8+ T cells were purified using the magnetic cell sorting system (Miltenyi Biotech). 2.2. Sphere formation assay CD133+ thyroid cancer cells were cultured in serum-free DMEM/F12 media (Invitrogen), supplemented with 20 ng/ml human recombinant EGF (Chemicon), 10 ng/ml human recombinant bFGF (Chemicon), and B27 (Invitrogen). Cells were cultured in suspension in 96-well plates at different density. After 10 days, the percent of wells with spheres with diameters larger than 50 µM or the number of spheres with diameters larger than 50 µM were counted under an inverted microscope. 2.3. Dual luciferase assay Dural luciferase assay was performed as previously described [21]. Cells were transiently transfected with pGL3-PD-L1 promoter and pRL-SV40 plasmids. 48-72 h after transfection, cells were rinsed in PBS for three times and lysed in a Passive Lysis Buffer (Promega). Luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega) with Turner luminometer and normalized to the Renilla Luciferase activity for transfection efficiency. Data were represented as the 4
mean from at least three independent experiments. 2.4. Western blot Western blot assay was performed as previously described [20]. Primary antibodies included rabbit monoclonal anti-GAPDH antibody (Cell signaling, cat# 5174), goat polyclonal anti-PD-L1 (R&D, cat# AF156), rabbit anti-Src (Cell signaling, cat# 2108), rabbit anti-phospho-Src (Tyr416) antibody (Cell signaling, cat# 2101), rabbit monoclonal anti-phospho-Akt (Thr308) antibody (Cell signaling, cat# 13038), rabbit anti-p85 antibody (Millipore, cat# 06-195) and rabbit anti-Akt antibody (Cell signaling, cat# 9272, 1:2000). For quantification, the western blot films were scanned and were analyzed using ImageJ Version 1.33u software. 2.5. Immunoprecipitation. Immunoprecipitation was performed as previously described [19]. Briefly, cells were lysated in RIPA buffer (50 mM Tris (pH 7.4), 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM NaF and protease inhibitor mixture). Lysates were centrifuged and cleared by incubation with 20 µL of Protein G-Agarose (Roche) for 1.5-2 h at 4 °C. The pre-cleared supernatant was subjected to immunoprecipitation using anti-FLAG or anti-CD133 (Miltenyi Biotec, W6B3C1 clone) antibodies at 4 °C overnight. Then, the protein complexes were collected by incubation with 30 µL of Protein G-Agarose (Roche) for 4-6 h at 4 °C. The collected protein complexes were washed five times with IP buffer and analyzed by western blot. . 2.6. Tumor formation assay To examine the tumor-initiating capacity of CD133+ thyroid cancer cells, tumor cells was transplanted into 6 to 8-week old immunodeficient mice in accordance with Zhongshan hospital of Fudan University Institutional Animal Care and Use Committee approved protocol concurrent with national regulatory standards. Mice were sacrificed when they were moribund or 180 days after implantation. Tumor formation was determined by histology. To examine the effect of 4-DAMP on the in vivo growth of CD133+ cells, CD133+ cells were injected into the right flank region of 6-week old immunodeficient mice. Five days after injection, 4-DAMP was injected i.p. at doses of 1 mg/kg/day 5
respectively. Tumor volumes of each group were measured every three days. 2.7. Statistical Analysis Results are expressed as the mean ± standard error of the mean (mean ± SEM). In general, significance was tested by unpaired two-tailed Student’s t test or ANOVA test using GraphPad InStat 5.0 software. P value < 0.05 was considered statistically significant.
6
3. Results 3.1. Neurons are juxtaposed to CD133+ thyroid cancer cells The expression of the representative neuron marker MAP2 was examined in thyroid cancer tissues. MAP2 was sparsely expressed in thyroid cancer tissues (Fig. 1A-B). Thyroid cancers mainly comprise differentiated thyroid carcinoma (DTC) and poorly differentiated carcinoma (PDTC) [22]. To examine the relationship between neuron numbers and pathological types in DTC and PDTC samples, MAP2 expression was analyzed using immunohistochemical staining. Compared to the DTC samples, MAP2 was expressed at higher levels in the PDTCs (Fig. 1C, p < 0.01). To examine the association between thyroid CSCs and neurons, thyroid cancer tissues were analyzed by immunofluorescence using anti-CD133 (marker of thyroid CSC) and anti-MAP2 (marker of neuron) antibodies. Juxtaposition of neuron with CD133+ thyroid cancer cells was detected in the thyroid cancer samples (patient #1 and #2) (Fig. 1D-E). The percentage of MAP2-positive cells or CD133-positive cells in thyroid cancer samples (patient #1 and #2) ranged from 2 to 3% (Fig. 1F-G). Taken together, these data suggest that neurons are juxtaposed with CD133+ thyroid cancer cells. 3.2. Acetylcholine promotes the self-renewal capacity of CD133+ thyroid cancer cells Neurotransmitters bind to neurotransmitter receptors in cancer cells and promote the growth and invasion of cancer cells [10, 13]. CD133 is a marker for thyroid CSC [5, 6]. The effects of neurotransmitters on the self-renewal capacity of CD133+ thyroid cancer cells were examined. CD133+ and CD133- cells were isolated from C643 and 8305C xenografts and thyroid cancer samples (patient #1 and #2). The CD133+ tumor cells gradually formed spheres of different sizes and shapes (Fig. 2A). Sphere formation from single cells showed that CD133+ tumor cells formed the spheres with different diameters (Supplementary Fig. 1), indicating the phenotypic heterogeneity of the spheres. CD133+ cells displayed characteristics consistent with that of CSCs including expression of stem cell markers (Fig. 2B-E) and high tumorigenicity in immunocompromised mice (Fig. 2F-G). Thus, CD133+ subpopulations are enriched for thyroid CSCs. CD133+ cells were treated with various neurotransmitters including acetylcholine, neuropeptides, Dynorphin B or GABA. Acetylcholine evidently increased the number 7
of spheres formed by CD133+ cells isolated from the C643 xenograft (Fig. 2H-I). Consistent with this, acetylcholine also increased the number of spheres formed by CD133+ cells isolated from 8305C xenograft or thyroid cancer samples (patient #1 and #2) (Fig. 2J). The sphere formation efficiency is one method to determine the self-renewal capacity of CSCs [19]. Acetylcholine increased the sphere formation ability of CD133+ cells isolated from C643 and 8305C xenografts (Fig. 2K). Furthermore, acetylcholine promoted the in vivo growth of CD133+ cells isolated from C643 xenograft (Fig. 2L). These data suggest that acetylcholine increases the self-renewal capacity of CD133+ thyroid cancer cells. 3.3. Acetylcholine activates CD133-Akt pathway Next, the mechanism of acetylcholine-mediated increase the self-renewal capacity of CD133+ thyroid cancer cells was examined. CD133 promotes the self-renewal of CSCs depending on its Y828 phosphorylation [19]. Acetylcholine clearly increased the Y828 phosphorylation of CD133 without affecting the expression of total CD133 (Fig. 3A-B). The phosphorylation of CD133 at the Y828 residue is mediated by c-Src kinase [23]. The kinase activity of c-Src is positively regulated by phosphorylation of Y416 [24, 25]. Acetylcholine promotes cancer cells growth through the acetylcholine receptor M3R [26]. Downregulation of M3R expression using siRNA reduced the acetylcholine-induced Src Y416 phosphorylation and consequently the CD133 Y828 phosphorylation (Fig. 3C). Thus, acetylcholine increased c-Src Y416 phosphorylation and CD133 Y828 phosphorylation. CD133 interacts with p85 depending on its Y828 phosphorylation, consequently increasing PI3K activity and activating Akt phosphorylation in glioma [19]. To examine the significance of Y828 phosphorylation of CD133 on its interaction with p85 in thyroid cancer cells, CD133+ cells expressing CD133 short-hairpin RNA (shRNA) were infected with lentivirus expressing shRNA resistant wild type CD133 or its Y828F or Y852F mutant. Y828F mutant, but not Y852F mutant, visibly reduced the interaction between CD133 and p85 (Fig. 3D). Consistent with this, the mutation of Y828 to F significantly blocked the induction of PI3K kinase activity by CD133 (Fig. 3E). The effect of acetylcholine on CD133-Akt signaling pathway was examined next. Acetylcholine promoted the interaction between CD133 and p85 (Fig. 3F), and increased the activity of PI3K in CD133+ cells isolated from C643 and 8305C xenografts and thyroid cancer samples (patient #1 and #2) (Fig. 3G). Furthermore, 8
acetylcholine increased Akt T308 phosphorylation in CD133+ cells (Fig. 3H). Taken together, these data suggest that acetylcholine increases CD133 phosphorylation and enhances the PI3K-Akt pathway. 3.4. Acetylcholine increases the self-renewal ability of CD133+ thyroid cancer cells through activation of CD133-Akt pathway The Akt pathway promotes the self-renewal of CSCs [27, 28]. This finding motivated
us
to
examine
the
contribution
of
CD133-Akt
pathway
in
acetylcholine-promoted self-renewal of CD133+ cells. First, the effect of CD133 down-regulation on acetylcholine-promoted self-renewal of CD133+ thyroid cancer cells was examined. CD133 shRNA effectively reduced the endogenous CD133 expression (Fig. 4A). ShRNA-mediated down-regulation of CD133 reduced the number of spheres formed by CD133+ cells isolated from the C643 xenograft (Fig. 4B-C), and blocked the positive effects of acetylcholine on the self-renewal ability of CD133+ cells (Fig. 4D). The effect of acetylcholine on the self-renewal ability of CD133+ cells expressing CD133 shRNA could be rescued by overexpression of wild type CD133, but not by overexpression of CD133 Y828F mutant (Fig. 4E). Next, the effect of an Akt pathway inhibitor on acetylcholine-promoted self-renewal of CD133+ cells was examined. Wortmannin is a widely used inhibitor of the PI3K/Akt pathway [29]. CD133 overexpression increased the number of spheres formed by CD133+ cells expressing CD133 shRNA, which was markedly reduced by wortmannin (Fig. 4F). Wortmannin reduced the number of spheres formed by CD133+ cells (Fig. 4G), and reduced the positive effects of acetylcholine on the self-renewal ability of CD133+ cells isolated from C643 xenograft and thyroid cancer sample (patient #1) (Fig. 4H-I). These data suggest that down-regulation of CD133 or inhibition of the Akt pathway reduces the positive effect of acetylcholine on CD133+ thyroid cancer cells self-renewal. 3.5. Acetylcholine up-regulates PD-L1 through activation of CD133-Akt pathway and promotes the immune escape of CD133+ thyroid cancer cells The Akt pathway promotes immune escape by up-regulation of PD-L1 [30, 31]. The interaction between PD-L1 expressed on the tumor cells and PD-1 on CD8+ T cells inhibits anti-tumor immunity [32, 33]. We next examined the effect of acetylcholine on the expression of PD-L1. Wortmannin reduced the expression of 9
PD-L1 in CD133+ cells isolated from C643 xenograft (Fig. 5A), indicating that the Akt pathway promoted the expression of PD-L1 in thyroid CSCs. Acetylcholine induced mRNA expression and the level of PD-L1 protein in CD133+ cells (Fig. 5B-C). Further, acetylcholine induced the activity of PD-L1 promoter in CD133+ cells isolated from C643 and 8305C xenografts and thyroid cancer samples (patient #1 and
#2)
(Fig.
5D).
Next,
the
contribution
of
CD133-Akt
pathway in
acetylcholine-promoted PD-L1 transcription was examined. Down-regulation of CD133 blocked the positive effects of acetylcholine on the activity of PD-L1 promoter in CD133+ cells isolated from 8305C xenograft (Fig. 5E). To examine the significance of Y828 phosphorylation in acetylcholine-induced PD-L1 promoter activity, CD133+ cells expressing CD133 shRNA and shRNA resistant wild type CD133 or its mutant were transiently transfected with the PD-L1 promoter, followed by treatment of acetylcholine. The Y828F mutant significantly blocked the positive effect of CD133 or acetylcholine on the activity of PD-L1 promoter (Fig. 5F). CD133 overexpression increased the activity of PD-L1 promoter in CD133+ cells expression CD133 shRNA, which was clearly blocked by wortmannin (Fig. 5G). Furthermore, wortmannin inhibited acetylcholine-induced PD-L1 promoter activity in CD133+ cells (Fig. 5H). These data suggest that acetylcholine promotes PD-L1 expression through the activation of the CD133-Akt pathway. To examine the effects of acetylcholine on the immune escape of CD133+ thyroid cancer cells, CD133+ cells isolated from C643 xenografts were pre-treated with acetylcholine and then co-cultured with activated CD8+ T cells. Acetylcholine reduced the percentage of apoptotic CD133+ cells isolated from C643 and 8305C xenografts and thyroid cancer sample (patient #1) induced by CD8+ T cells (Fig. 5I-K). Anti-PD-L1 antibody clearly reduced the positive effect of acetylcholine on the resistance of CD133+ cells to CD8+ T cells (Fig. 5L). Thus, acetylcholine promotes the resistance of CD133+ thyroid cancer cells to CD8+ T cells through up-regulation of PD-L1. 3.6. Acetylcholine receptor (M3R) antagonist, 4-DAMP, inhibits the self-renewal and immune escape abilities of CD133+ thyroid cancer cells induced by acetylcholine The contribution of acetylcholine to self-renewal of CD133+ thyroid cells motivated us to explore the effects of the acetylcholine receptor (M3R) inhibitor 10
4-DAMP on self-renewal of CD133+ thyroid cancer cells. 4-DAMP inhibited the positive effect of acetylcholine on the self-renewal of CD133+ thyroid cancer cells. However, a selective M1 antagonist VU0255035 and a M2/M4 selective antagonist AFDX-116 did not reduce the effect of acetylcholine on the self-renewal capacity of CD133+ cells (Fig. 6A-B). Next, the effect of 4-DAMP on the in vivo growth of CD133+ cells were examined. The in vivo growth of CD133+ cells isolated from 8305C xenograft was visibly reduced by 4-DAMP (Fig. 6C). Further, 4-DAMP reduced the positive effect of acetylcholine on the resistance of CD133+ cells isolated from C643 xenograft to CD8+ T cells (Fig. 6D). Together, these data suggest that acetylcholine receptor M3R antagonist 4-DAMP inhibits the self-renewal and immune escape of CD133+ thyroid cancer cells induced by acetylcholine.
11
4. Discussion In this study, we found that acetylcholine promoted the self-renewal capacity of CD133+ thyroid cancer cells through the activation of the CD133-Akt pathway. Furthermore, acetylcholine promoted PD-L1 expression through the activation of the CD133-Akt pathway and enhanced the resistance of CD133+ cells to CD8+ T cells (Fig. 6E). Together, acetylcholine promotes the self-renewal and immune escape of CD133+ thyroid cancer cells through activation of the CD133-Akt pathway. Acetylcholine promotes the self-renewal and immune escape of CD133+ thyroid cancer cells through enhancing CD133 phosphorylation. CD133 is phosphorylated at Y828 and Y852 residues by Src kinase [23]. CD133 Y828F mutation clearly reduced the interaction between CD133 and p85. CD133 Y828F mutation blocked acetylcholine-induced self-renewal of CD133+ cells and the activity of PD-L1 promoter. Thus, we speculate that CD133 Y828 phosphorylation contributes to acetylcholine-induced CD133+ cells self-renewal and immune escape. Our data showed that M3R down-regulation inhibited the activation of Src phosphorylation and the CD133 phosphorylation by acetylcholine. We speculate that acetylcholine promotes CD133 Y828 phosphorylation through increasing c-Src activity. However, the mechanism of acetylcholine promoting Src Y416 phosphorylation in CD133+ thyroid cancer cells needs further explored. Nerves have recently been recognized as key regulators of tumor growth [11, 34]. Neurotrophic factors released from cancer cells promote the outgrowth of neuron in tumors [35]. It is widely known that neurons promote the growth and migration of cancer cells [10]. However, the contributions of neurons to the niche of CSCs remain largely unknown. Here, we found that neurons were juxtaposed with CD133+ thyroid cancer cells. Acetylcholine promoted the self-renewal and immune escape of CD133+ thyroid cancer cells. Thus, neuron might be an important component in the niche of CSCs. The tumor microenvironment is crucial to the self-renewal of CSCs [36, 37]. Therefore, microenvironment-targeting strategies might eliminate the CSCs. Our findings have showed that acetylcholine promoted the self-renewal of CD133+ thyroid cancer cells. Acetylcholine receptor M3R antagonist, 4-DAMP, reduced acetylcholine-induced self-renewal and immune escape of CD133+ thyroid cancer cells. Furthermore, 4-DAMP inhibited the in vivo growth of CD133+ thyroid cancer cells. Thus, inhibition of acetylcholine signaling pathway may be a promising 12
approach for the treatment of thyroid cancer. In summary, we found that neurons were juxtaposed with CD133+ thyroid cancer cells. Acetylcholine promoted the self-renewal and immune escape of CD133+ thyroid cancer cells at least partly through the activation of the CD133-Akt pathway. These findings identify neuron as an important component in the niche of thyroid CSCs. Competing interest Authors have no conflicts of interests. Author contributions ZLW and WL performed most experiments; WC performed cell sorting; YNL performed signaling pathway analysis and plasmids construction; AZL discussed the data and wrote the manuscript Acknowledgements This work was supported by National Natural Scientific Foundation of China (81272722). We are grateful to Dr. Jing Li (Shanghai Medical College of Fudan University) for providing flow cytometry technology. We are grateful to Dr. Jianhai Jiang (Shanghai Medical College of Fudan University) for providing CD133 Y828 phosphorylation antibody and CD133-FLAG plasmids and its mutants.
13
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17
FIGURE LEGENDS Fig. 1. Neurons are juxtaposed with CD133+ thyroid cancer cells. (A-B) IHC staining for MAP2 in thyroid cancer tissues of patient #1 (A) and patient #2 (B). Representative images were shown. Scale bar, 20 µM. (C) The scores for quantitative staining of MAP2 in DTC samples (n = 12) and PDTC samples (n = 10) were determined. Results are expressed as mean ± SEM; **p < 0.01. (D-E) Confocal images of thyroid cancer sections of patient #1 (D) and #2 (E) co-stained for CSC marker CD133 (red) and neuron marker MAP2 (green) showed the juxtaposition of thyroid CSCs with neurons. Scale bar, 10 µM. (F-G) The percent of MAP2-positive cell (F) or CD133-positive cell (G) in D-E was was calculated from five different fields. Results are expressed as mean ± SEM. Fig. 2. Acetylcholine increases the self-renewal capacity of CD133+ thyroid cancer cells. (A) Representative image of spheres formed by CD133+ cells isolated from C643 and 8305C xenografts and thyroid cancer samples (patient #1 and #2). Scale bar represents 50 µM. (B-E) QPCR analysis was performed for stem cell-associated genes in CD133+ cells versus CD133- cells isolated from C643 (B) and 8305C xenografts (C) and thyroid cancer samples (patient #1 and #2) (D-E). CD133- cells were defined as 1, and the relative expression of each gene in CD133+ cells was expressed as the fold difference over CD133- cells. (F-G) An in vivo limiting dilution tumor formation assay (employing 10,000, 5,000, 1,000 or 500 cells per mouse) was performed to compare the tumor-initiating capacity of CD133+ cells or CD133- cells isolated from C643 and 8305C xenografts and thyroid cancer sample (patient #1). Mice were sacrificed when they were moribund or 180 days after implantation. Tumor formation was determined by histology. (F) The table displays the number of mice developing tumors. (G) H&E staining of mouse thyroids shows tumors formation by 1,000 CD133+ cells but not by 1,000 CD133cells. Scale bar represents 20 µM. (H-I) Sphere formation assay of CD133+ cells. 10 CD133+ cells isolated from C643 xenograft
were
cultured
in
96-well
plates,
followed
by treatment
with
neurotransmitters (10 µM). After 10 days, the number of sphere was counted. (H) Representative images of sphere are shown. (I) Results are expressed as mean ± SEM from four separate experiments; ***p < 0.001, #p > 0.05. Scale bar represents 50 µM. 18
Ach, Acetylcholine. (J) Sphere formation assay of CD133+ cells. 10 CD133+ cells isolated from 8305C xenograft or thyroid cancer samples (patient #1 and #2) were cultured in 96-well plates, followed by treatment with Ach (10 µM). After 10 days, the number of sphere was counted. Results are expressed as mean ± SEM from four separate experiments; ***p < 0.001. (K) Sphere formation assay of single CD133+ cell/well isolated from C643 xenograft or 8305C xenograft treated with or without Ach. Data means the percentage of sphere containing wells in each group. (L) CD133+ cells isolated from C643 xenograft were subcutaneously co-implanted with or without Ach into immunocompromised mice. Tumor volumes were measured every 3 days. Results are expressed as mean ± SEM (n = 6 mouse); **p < 0.01. Fig. 3. Acetylcholine activates the CD133-Akt pathway. (A-B) Western blot analysis of the level of Y828 phosphorylation of CD133 in CD133+ cells treated with control or Ach. GAPDH was blotted as a loading control. (A) Representative image from three separate experiments was shown. (B) The relative densities of pY828-CD133 to total CD133 in A were quantified using densitometry. Values are normalized to that of cells treated with control. Results are expressed as mean ± SEM from three separate experiments; ***p < 0.001. (C) Western blot analysis of the levels of CD133 phosphorylation and Src phosphorylation in CD133+ cells expressing control siRNA or M3R siRNA treated with acetylcholine. GAPDH was blotted as a loading control. (D) The effect of Y828 mutation on the interaction between p85 and CD133. The lysates of CD133+ cells isolated from C643 xenograft expressing CD133 shRNA + FLAG, CD133 shRNA + shRNA-resistant wild type CD133-FLAG, or CD133 shRNA + shRNA-resistant Y828F-FLAG or CD133 shRNA + shRNA-resistant Y852F-FLAG were subjected to immunoprecipitation using anti-FLAG antibody, followed by immunoblotting with anti-FLAG or anti-p85 antibodies. shR, shRNA-resistant. (E) The PI3K activity of CD133+ cells isolated from C643 xenograft expressing CD133 shRNA + FLAG, CD133 shRNA + shRNA-resistant wild type CD133-FLAG, or CD133 shRNA + shRNA-resistant Y828F-FLAG or CD133 shRNA + shRNA-resistant Y852F-FLAG were assessed using a PI3 kinase ELISA kit. Values 19
are normalized to that of control cells. Results are expressed as mean ± SEM from three separate experiments; ***p <0.001. (F) Co-IP analysis to determine the effect of Ach on the interaction between CD133 and p85. Lysates of CD133+ cells pretreated with or without Ach were subjected to IP using anti-CD133 antibody, followed by immunoblotting (IB) with anti-CD133 or anti-p85 antibodies. Whole cell lysates were analyzed by IB with anti-CD133, anti-p85 or anti-GAPDH antibodies as input. (G) The PI3K activity of CD133+ cells isolated from xenograft or thyroid cancer samples treated with control or Ach were assessed using a PI3 kinase ELISA kit. Values are normalized to that of control cells. Results are expressed as mean ± SEM from three separate experiments; ***p <0.001. (H) Western blot analysis of Akt T308 phosphorylation level in CD133+ cells treated with or without ACh. GAPDH was blotted as a loading control. Fig. 4. Acetylcholine increases the self-renewal ability of CD133+ thyroid cancer cells through the activation of the CD133-Akt pathway. (A)Western blot analysis of CD133 expression in CD133+ cells expressing LacZ shRNA or CD133 shRNA. GAPDH was blotted as a loading control. (B-C) 10 CD133+ cells isolated from C643 xenograft expressing LacZ shRNA or CD133 shRNA were cultured in 96-well plates. After 10 days, the number of sphere was counted. (B) Representative images of sphere are shown. (C) Results are expressed as mean ± SEM from three separate experiments; ***p < 0.001. Scale bar represents 50 µM. (D) 10 CD133+ cells isolated from C643 xenograft expressing LacZ shRNA or CD133 shRNA were cultured in 96-well plates, followed by treatment with control or Ach. Results are expressed as mean ± SEM from four separate experiments; ***p < 0.001, *p < 0.05. (E) 10 CD133+ cells isolated expressing CD133 shRNA + FLAG, CD133 shRNA + shRNA-resistant wild type CD133-FLAG, or CD133 shRNA + shRNA-resistant Y828F-FLAG or CD133 shRNA + shRNA-resistant Y852F-FLAG were cultured in 96-well plates, followed by treatment with control or Ach. After 10 days, the number of spheres was counted. Results are expressed as mean ± SEM from four separate experiments; *p < 0.05, ***p < 0.001. (F) 10 CD133+ cells expressing CD133 shRNA + FLAG, CD133 shRNA + 20
shRNA-resistant wild type CD133-FLAG treated with control or wortmannin (1 µM) were cultured in 96-well plates. After 10 days, the number of sphere was counted. Results are expressed as mean ± SEM from four separate experiments; **p < 0.01, ***p < 0.001. (G) 10 CD133+ cells isolated from C643 xenograft treated with control or wortmannin (1 µM) were cultured in 96-well plates. After 10 days, representative images of sphere were shown. Scale bar represents 50 µM. (H-I) 10 CD133+ cells isolated from C643 xenograft (H) or thyroid cancer sample (patient #1) (I) treated with wortmannin (1 µM) and/or Ach (10 µM) were cultured in 96-well plates. After 10 days, the number of sphere was counted. Results are expressed as mean ± SEM from three separate experiments; *p < 0.05, ***p < 0.001. Fig. 5. Acetylcholine induces the expression of PD-L1 through the activation of the CD133-Akt pathway and promotes the immune escape of CD133+ thyroid cancer cells. (A)Western blot analysis of the level of PD-L1 protein in CD133+ cells treated with DMSO or wortmannin. GAPDH was blotted as a loading control. (B) Western blot analysis of the level of PD-L1 protein in CD133+ cells treated with control or Ach. GAPDH was blotted as a loading control. (C) RT-PCR analysis of the level of PD-L1 mRNA in CD133+ cells treated with control or Ach. (D) The PD-L1 promoter construct pGL3-PD-L1 was transiently co-transfected with pRL-SV40 into CD133+ cells isolated from xenografts and thyroid cancer samples. Then, cells were treated with ACh. The luciferase activity was determined. The values were presented as fold activation over CD133+ cells treated with control. Values are mean ± SEM; ***p < 0.001. (E) CD133+ cells expressing control shRNA or CD133 shRNA were transiently co-transfected with pGL3-PD-L1 and pRL-SV40. Then, cells were treated with ACh. The luciferase activity was determined. The values were presented as fold activation over CD133+ cells expressing LacZ shRNA treated with control. Values are mean ± SEM; ***p < 0.001. (F) CD133+ cells expressing CD133 shRNA + FLAG, CD133 shRNA + shRNA-resistant wild type CD133-FLAG, or CD133 shRNA + shRNA-resistant Y828F-FLAG or CD133 shRNA + shRNA-resistant Y852F-FLAG were transiently 21
co-transfected with pGL3-PD-L1 and pRL-SV40. Then, cells were treated with ACh (10 µM). The luciferase activity was determined. The values were presented as fold activation over CD133+ cells expressing CD133 shRNA + FLAG treated with control. Values are mean ± SEM from three separate experiments; *p < 0.05, ***p < 0.001. (G) CD133+ cells isolated from C643 xenograft expressing CD133 shRNA+FLAG, CD133
shRNA+shRNA-resistant
wild
type
CD133-FLAG were
transiently
co-transfected with pGL3-PD-L1 and pRL-SV40. Then, cells were treated with wortmannin. The luciferase activity was determined. The values were presented as fold activation over CD133+ cells expressing CD133 shRNA+FLAG treated with control. Values are mean ± SEM from three separate experiments; **p < 0.01, ***p < 0.001. (H) CD133+ cells were transiently transfected with pGL3-PD-L1 and pRL-SV40. Then, cells were treated with wortmannin and/or ACh. The luciferase activity was determined. The values were presented as fold activation over CD133+ cells treated with control. Values are mean ± SEM from three separate experiments; ***p < 0.001. (I-K) CD133+ cells isolated from C643 (I) and 8305C xenografts (J) and thyroid cancer sample (patient #1) (K) pretreated with or without ACh (10 µM) were co-cultured with activated CTLs (cytotoxic T lymphocytes) for 4 hours. The percentage of apoptotic CD133+ cells was determined by flow cytometry. Results are expressed as mean ± SEM (n = 4); **p < 0.01, ***p < 0.001. (L) CD133+ cells isolated from 8305C xenograft pretreated with control or ACh were co-cultured with CTL cells in the presence of anti-PD-L1 Ab. The percentage of apoptotic CD133+ cells was determined by flow cytometry. Results are expressed as mean ± SEM (n = 4); **p < 0.01, ***p < 0.001. Fig. 6. Acetylcholine receptor (M3R) antagonist, 4-DAMP, reduces the self-renewal and immune escape abilities of CD133+ thyroid cancer cells induced by acetylcholine. (A-B) CD133+ cells were treated with Ach and VU0255035 or 4-DAMP or AFDX-110 (10 µM) for 10 days. The number of sphere was counted. (A) Representative images of sphere are shown. (B) Results are expressed as mean ± SEM from three separate experiments; ***p < 0.001. Scale bar represents 50 µM. (C) CD133+ cells isolated from 8305C xenograft were subcutaneously co-implanted into immunocompromised mice. Five day after injection, mice were injected with or 22
without 4-DAMP. Tumor volumes were measured every 3 days. Results are expressed as mean ± SEM (n = 6 mouse); **p < 0.01. (D) CD133+ cells isolated from C643 xenograft pretreated with ACh and/or 4-DAMP were co-cultured with CTLs for 4 hours. The percentage of apoptotic CD133+ cells was determined by flow cytometry. Results are expressed as mean ± SEM (n = 4); ***p < 0.001. (E) Schematic model of acetylcholine promoting the self-renewal and immune escape of CD133+ thyroid cancer cells.
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Aug 26, 2019 Conflict of Interest Disclosure Form Article Title: Acetylcholine promotes the self-renewal and immune escape of CD133+ thyroid cancer cells through activation of CD133-Akt pathway Authors: Zhenglin Wang, Wei Liu, Cong Wang, Yinan Li, Zhilong Ai
We submit our article to Cancer Letters for consideration and make the statement below: On behalf of all the authors above, I declare that all authors have no financial and personal relationships with other people or organizations that could inappropriately influence (bias) our work in this study.
Zhilong Ai, PhD. Zhongshan Hosptial, Fudan University Shanghai, China
1
Neurons are juxtaposed with CD133+ thyroid cancer cells ACh promotes CD133+ thyroid cancer cells self-renewal and immune escape ACh activates Akt phosphorylation through enhancing CD133 phosphorylation ACh promotes CD133+ thyroid cancer cells self-renewal via CD133-Akt pathway