Abstract
Background
Tumor cells continue to evolve the metastatic potential in response to signals provided by the external microenvironment during metastasis. Platelets closely interact with tumor cells during hematogenous metastasis and facilitate tumor development. However, the molecular mechanisms underlying this process are not fully understood.
Methods
RNA-sequencing was performed to screen differentially expressed genes mediated by platelets. The effects of platelet and CD39 on tumor metastasis were determined by experimental metastasis models with WT, NCG and CD39−/− mice.
Results
RNA-sequencing results showed that platelets significantly up-regulated CD39 expression in tumor cells. CD39 is a novel immune checkpoint molecule and a key driver of immunosuppression. Our data provided evidence that the expression of CD39 was enhanced by platelets in a platelet-tumor cell contact dependent manner. Although the role of CD39 expressed by immune cells is well established, the effect of CD39 expressed by tumor cells on tumor cell behavior, anti-tumor immunity and tumor metastasis is unclear. We found that CD39 promoted tumor cell invasion, but had no effect on proliferation and migration. Notably, we showed that the ability of platelets to prime tumor cells for metastasis depends on CD39 in the experimental tumor metastasis model. CD39 silencing resulted in fewer experimental metastasis formation, and this anti-metastasis effect was significantly reduced in platelet-depleted mice. Furthermore, overexpression of CD39 in tumor cells promoted metastasis. In order to eliminate the effect of CD39 expressed in cells other than tumor cells, we detected tumor metastasis in CD39−/− mice and obtained similar results. Moreover, overexpression of CD39 in tumor cells inhibited antitumor immunity. Finally, the data from human samples also supported our findings.
Conclusions
Our study shows that direct contact with platelets induces CD39 expression in tumor cells, leading to immune suppression and promotion of metastasis.
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Introduction
Accumulating evidence have clearly shown strong interactions between tumor cells and microenvironment, confirming the influence of the surrounding cells on tumor progression [1]. Platelets can adhere to the surface of tumor cells, and have been identified as a vital external stimuli to tumor cells in the process of hematogenous metastasis. As a matter of fact, platelets have been found to promote tumor growth and metastasis. They can form a physical shield surrounding tumor cells to protect against the lysis by natural killer (NK) cells [2,3,4,5], reduce the damage caused by shear stress [6, 7], and promote the adhesion to endothelium [7, 8]. In addition, we speculate that platelets may also provide additional signals for tumor cells to affect their metastatic potential. To explore the mechanisms involved, we treated murine CT-26 colon cancer cells with platelets and then performed RNA-sequencing (RNA-seq) analysis. Among differentially expressed genes, CD39 is one of the most significantly up-regulated genes.
CD39 has attracted much attention as a newly discovered immune checkpoint molecule in recent years. It can hydrolyze extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to adenosine monophosphate (AMP) [9, 10], and AMP is further hydrolyzed by CD73 to adenosine [11]. Adenosine is a potent immune regulatory molecule that exerts immunosuppressive effects by binding to adenosine A2A receptor (A2AR) expressed by lymphocytes [12]. CD39 is identified to be the immunological switch shifting the ATP-induced pro-inflammatory status to the adenosine-driven anti-inflammatory status [13]. CD39 shows a high expression level in tumor-infiltrating immunocytes—especially the effector T cells and regulatory T cells (Tregs). The number of tumor-infiltrating CD39highCD8+ T cells increases as tumor grows, which display exhaustion feature [14]. The presence of high levels of CD39 on regulatory immune subsets is directly correlated with immune dysfunction [15, 16]. In CD39-deficient mice, the growth of several syngeneic tumors is inhibited [17, 18]. In addition, the CD39-deficient mice also show metastasis resistance in spontaneous metastasis or disseminated disease models [19, 20]. The promotion of tumor metastasis is observed in transgenic mice with CD39 over-expression [21]. Besides, blocking CD39 activity pharmacologically using the inhibitor or antagonistic antibody remarkably restricts tumor development and improves the anticancer immunity [17, 19, 22, 23].
In the present study we demonstrated that direct contact with platelets induces CD39 expression in tumor cells and the ability of platelets to prime tumor cells for metastasis depends on CD39. Furthermore, different from previous studies, we explored the effect of CD39 expressed by tumor cells on tumor metastasis. The results show that platelet-mediated CD39 induction inhibits anti-tumor immunity and promotes tumor metastasis, indicating that platelets not only serve as the physical shield to tumor cells, but also offer pro-metastatic signals through inducing CD39 expression.
Material and Methods
Human tissue samples
Tumor tissues were collected from colon cancer patients or patients with colon cancer and lung metastasis. All the samples were obtained from the Affiliated Hospital of Jining Medical University (Shandong, China). The present study was approved by the Ethics Committee of the Affiliated Hospital of Jining Medical University. Informed consent was obtained from all individual participants included in the study.
Mice, cell lines and reagents
BALB/c, NCG and CD39 gene-knockout (CD39−/−) mice provided by Nanjing University Model Animal Institute were raised in Jining Medical University at specific pathogen-free (SPF) conditions. NCG mice lacking mature NK cells and B/T lymphocytes were created through sequential clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) editing of the protein kinase, DNA-activated, Catalytic subunit (PRKDC) and interleukin 2 receptor gamma (IL2RG) genes in the NOD/ShiLtJ Gpt mice. The CD39−/− mice were generated through genome engineering mediated by CRISPR/Cas9. Mice were used at 6–8 weeks of age and randomly divided into different treatment groups. The sample size was determined based on published data or preliminary experiments [24]. Investigators were blinded to the group allocation during the procedure and data analysis. All experiments involving animals were approved by the Medical Animal Care & Welfare Committee of Jining Medical University. Human colon cancer HCT116 cells, murine prostate cancer RM-1 cells and murine colon cancer CT-26 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cultured cells were all mycoplasma negative. SB203580, SCH58261, crystal violet and Calcein-AM were purchased from Beyotime (Shanghai, China). PSB-603 was purchased from MedChemExpress (New Jersey, USA). The antibodies used are detailed in Supplementary Table 1.
Preparation of platelets and platelet fractions
Mouse platelets and platelet fractions were prepared as described previously [25]. Platelets at a concentration of 1.5 × 108 platelets/ml were added to the cell medium when used to treat tumor cells in vitro. Human platelets were obtained from the Affiliated Hospital of Jining Medical University (Shandong, China).
Platelet depletion
To deplete platelets, mice received 2 µg/g rat monoclonal antibodies against mouse glycoprotein (GP) Ibα intravenously. CT-26 cells were injected through tail veins 12 h after antibody administration.
RNA extraction and real-time quantitative PCR (RT-qPCR)
The EasyPure1 RNA Kit (Invitrogen, Carlsbad, CA, USA) was applied in isolating the total RNA following instructions. Reverse transcription was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany). Target gene expression levels were analyzed by SYBR Green I-based RT-qPCR. The sequences of each primer set are shown in Supplementary Table 2.
Western blot analysis
Western blot was performed according to the established protocols described previously [26]. The RIPA buffer was utilized for extracting cellular proteins and BCA protein assay kit (Solarbio, Beijing, China) was utilized for quantification. Proteins were separated through SDS-PAGE, transferred into the PVDF membrane, and incubated with the primary antibody. Afterwards, the membrane was subjected to further incubation using specific horseradish peroxidase (HRP)-conjugated secondary antibodies (Beyotime, Shanghai, China). Quantity One v4.62 was applied in analyzing protein expression after exposure to enhanced chemiluminescence (ECL, Pierce, Rockford, IL).
RNA-Seq
RNA was extracted using the EasyPure1 RNA Kit (Invitrogen, Carlsbad, CA) following specific instructions and RNA quality was assessed by BGI Tech (Shenzhen, China). RNA-Seq was conducted using the BGISEQ-500 sequencing platform in BGI Tech.
Plasmids and transfection
pCMV3-CD39 plasmid was obtained from Sino Biological Inc (Beijing, China). pGPU6/Hygro shRNA expression vector that contained CD39-targeting DNA oligos was provided by Genepharma Inc (Shanghai, China), and oligonucleotides sequences were shown below: 5’-GCATGCGCTTGCTTAGAATGG-3’ (shRNA1), 5’-GGGCAGATTCACTCAGGAACA-3’ (shRNA2), 5’-GCCTGTTCATCTATAGCAAGC-3’ (shRNA3) and 5’-TTCTCCGAACGTGTCACGT-3’ (negative control shRNA, NC-shRNA). Plasmids were transfected with the Effectene Transfection Reagent (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The cells with stable transfection were obtained by hygromycin B selection.
Flow cytometry
Blood was collected into the sodium heparin-coated tubes. Red blood cells (RBCs) lysis buffer was used to lyse RBCs. The cells were then incubated with mouse antibodies against CD3, CD4, CD8, programmed cell death 1 (PD1), T cell immunoglobulin mucin-3 (TIM3) and lymphocyte-activation gene 3 (LAG3) according to the standard procedure. After washing by PBS, cells were analyzed on a flow cytometer (BD BioSciences, Bedford, MA, USA).
ATP hydrolysis assay
After 30 min of cell incubation with 10 μM ATP, concentration of unhydrolyzed extracellular ATP was determined using the ATP detection kit (Beyotime, Beijing, China) according to the instructions.
Cell viability assay
Cell viability was determined using CCK-8 solution (Beyotime, Beijing, China) according to the manufacturer’s instructions.
Cytotoxic T lymphocyte (CTL) cytotoxicity
CTLs were prepared as described previously [27]. Splenocytes from CT-26 tumor-bearing mice (after inoculation for 10 days) were cocultured with 100 U/ml interleukin 2 (IL-2) and irradiated CT-26 tumor cells at a ratio of 10:1. After 10 days, viable lymphocytes were collected as CTLs.
Cell migration and invasion assay
Cell migration capacity was assessed by wound-healing assay. The cell monolayer was scraped using a micropipette tip and cells were then incubated in serum-free medium for 24 h. All assays were carried out in triplicate. For the invasion assays, 24-well Matrigel invasion chambers (Corning, NY, USA) were used. Cells (5 × 104) were resuspended in 100 µl of serum-free medium in the upper chamber, and 600 μl of medium with 10% fetal bovine serum (FBS) was added to the bottom chamber. The cells that migrated through the pores were fixed with methanol and stained with crystal violet. The cell number was calculated under the phase contrast microscope (Nikon, Tokyo, Japan).
Immunohistochemistry (IHC)
IHC assay was conducted using Histostain Plus Kits (Bioss, China) according to the manufacturer’s instructions. The immunohistochemical staining was semi-quantified using the H-score method [28]. The H-score (range, 0–300) is given as the sum of the percent staining multiplied by an ordinal value corresponding to the intensity degree (where 0, 1, 2 and 3 indicate none, weak, moderate and strong, respectively).
Statistical analysis
Results were presented in the manner of mean ± standard deviation. Two-sided Student’s t test was used to compare the two groups. One-way analysis of variance (ANOVA) was used to analyze differences among three or more groups. The survival curves were determined using the Kaplan-Meier method. A difference of P < 0.05 indicated statistical significance.
Results
Platelets induce CD39 expression in tumor cells
To explore the mechanisms involved in the promotion of tumor metastasis induced by platelets, we treated murine CT-26 colon cancer cells with platelets and performed RNA-seq analysis. Platelets have a significant impact on tumor cells and numerous differentially expressed genes were identified. Among the most significantly up-regulated genes, CD39 has aroused our interest. Considering CD39 is a driver of immunosuppression, we speculate that platelet-induced CD39 expression may contribute to the immune escape of tumor cells during hematogenous metastasis. The expression of CD39 in CT-26 cells was verified by RT-qPCR and western blot, and showed consistent with the results of RNA-seq (Fig. 1a). Furthermore, this finding was reproduced in murine RM-1 prostate cancer cells (Fig. 1b). To avoid possible interference with CD39 detection by residual platelets, we removed platelets prior to RNA or protein extraction of tumor cells. The results of immunofluorescence staining showed that there were very few platelets remain attached to tumor cells treated with platelets (Fig. 1c). We also detected the expression of platelet marker CD41 and obtained similar results (Fig. 1d). In addition, we found that white and red blood cells had no effect on the expression of CD39 in tumor cells (Fig. S1). CD39 collaborates with CD73 to produce adenosine, so we examined CD73 expression levels in CT-26 and RM-1 cells. As shown in Figure S2, CD73 was expressed in both CT-26 and RM-1 cells. We next sought to define the molecular mechanisms underlying platelet-mediated induction of CD39. First, we analyzed whether platelets promoted CD39 expression in tumor cells via direct contact. The results showed that separation of platelets from tumor cells using a semipermeable membrane abolished the up-regulation of CD39 in CT-26 (Fig. 1e) and RM-1 cells (Fig. 1f).
As platelets can be activated by tumor cells and release a variety of cytokines [29], we next investigated whether exposure to the releasates or pellets from activated platelets would be sufficient to promote the expression of CD39 in tumor cells. Platelets were therefore activated with thrombin, and platelet fractions, including the releasates and pellets, were separated by centrifugation. The results showed that cell pellets induced the expression of CD39 in CT-26 (Fig. 2a) and RM-1 cells (Fig. 2b), whereas releasates had no significant effect. We also treated CT-26 and RM-1 cells with cytokines that could be released by platelets, including epidermal growth factor (EGF), fibroblast growth factor (FGF)-2, insulin-like growth factor (IGF)-1, transforming growth factor β (TGFβ)-1 and platelet-derived growth factor (PDGF), and no significant changes in CD39 expression were observed (data not shown). Meanwhile, thrombin also did not affect CD39 expression (Fig. S3). Next, we explored the signaling pathway through which platelets regulated the expression of CD39. As shown in Fig. 2c, d, platelets activated the p38 signaling pathway in CT-26 and RM-1 cells. Moreover, p38 signaling pathway inhibitor SB203580 suppressed platelet-mediated up-regulation of CD39 in CT-26 (Fig. 2e) and RM-1 cells (Fig. 2f), demonstrating that platelets enhanced the expression of CD39 through p38 signaling pathway.
The Effects of CD39 on tumor cells
We next evaluated the effects of CD39 on tumor cells. First, we constructed CD39- overexpression and -silencing CT-26 cell lines (Fig. S4). To assess broad effects mediated by CD39, we performed RNA-seq analysis. Differential gene expression analysis revealed significant changes induced by CD39 overexpression (Fig. 3a). We further examined the effects of CD39 on the behaviors of tumor cells. Neither overexpression nor silencing of CD39 affected the proliferation and migration of tumor cells (Fig. 3b–d). However, we found that overexpression of CD39 promoted cell invasion (Fig. 3e), possibly because it up-regulated the expression of matrix metalloproteinase (MMP)-2 and MMP-3, as demonstrated by RNA-seq analysis. Instead, silencing of CD39 had no significant effect on cell invasion (Fig. 3f), which may be due to the low background expression of CD39 in CT-26 cells.
Platelets prime tumor cells for metastasis depends on CD39
We first examined whether the pro-metastatic effects of platelets was related to the induction of CD39 in tumor cells. In the experimental tumor metastasis model by injecting tumor cells intravenously, pretreatment of CT-26 with platelets increased lung metastasis, while silencing CD39 in CT-26 cells reduced this effect (Fig. 4a). This suggests that the ability of platelets to prime tumor cells for metastasis depends on CD39 expressed by tumor cells. Moreover, we examined tumor metastasis in platelet-depleted mice which received platelet depletion antibody against mouse GPIbα. CD39 silencing resulted in reduced tumor metastasis in mice that received isotype control antibody, and this difference was significantly reduced in platelet-depleted mice (Fig. 4b). This result further confirmed the important role of platelets in promoting tumor metastasis by regulating CD39 expression.
The impact of CD39 on tumor growth has mostly been delineated using global CD39 targeted mice; previous studies have shown that tumor growth and metastasis were significantly inhibited in CD39 knockout mice [17, 18, 23, 30]. However, the effect of CD39 expressed by tumor cells on tumor metastasis is unclear. Next, we evaluated the effect of overexpression or silencing of CD39 on tumor metastasis in the experimental tumor metastasis model. The results showed that overexpression of CD39 promoted lung metastasis (Fig. 5a), whereas silencing of CD39 resulted in fewer lung metastases (Fig. 5b) and improved overall survival in mice (Fig. 5c). In addition to tumor cells, CD39 is also expressed in immune cells and vascular endothelial cells. In order to eliminate the effect of CD39 expressed by these cells, we detected the effect of CD39 expressed by tumor cells on tumor metastasis in CD39−/− mice and obtained similar results as in wild type (WT) mice (Fig. 5d–f). These results indicate that platelets prime tumor cells for metastasis depends on CD39 and CD39 expressed by tumor cells has pro-metastatic effects.
CD39 suppresses antitumor immunity
We first evaluated whether the pro-metastatic effect of CD39 is achieved primarily through the modulation of the immune response. We intravenously injected CD39-overexpressing or -silencing CT-26 cells into immunodeficient NCG mice. There was no significant difference in the number of pulmonary nodules between different groups, although NCG mice showed a significantly higher number of pulmonary nodules than WT mice (Fig. 6a, b). These data suggest that CD39 affects tumor metastasis depending on an active immune system. In addition, we found that A2AR antagonist SCH58261 could significantly inhibit CD39-mediated promotion of tumor metastasis (Fig. 6c), whereas adenosine A2B receptor (A2BR) antagonist PSB-603 showed no significant effect (Fig. S5). These results indicate that the ability of CD39 to prime tumor cells for metastasis depended on adenosine and A2AR. CD39 expressed by immune cells can exert immunosuppressive effects [14, 31]. However, the effect of CD39 expressed by tumor cells on anti-tumor immune response is not clear. First, we examined whether CD39 affects cancer cell killing by CTLs. CT-26-specific CTLs were generated as previously described [27] and added to CT-26 cell cultures. As expected, CD39-overexpressing CT-26 cells resisted killing by CTLs (Fig. 6d). Next, we further examined the effect of CD39 on CD8+ T cell exhaustion. Results showed that overexpression of CD39 in tumor cells enhanced the expression of inhibitory receptors such as PD1, LAG3 and TIM3 in circulating CD8+ T cells (Fig. 6e). In contrast, CD39 overexpression had no effect on CD4+ T cells (Fig. S6). Collectively, the above data shows that CD39 expressed by tumor cells can suppress CD8+ T cell-mediated antitumor immunity, which may be contributing to tumor cell immune escape.
Expression of CD39 in human tumor cells and tissues
Based on the animal experiments, we then assessed CD39 expression in human tumor cells and tissues. Results showed that human platelets also could up-regulate the expression of CD39 in human HCT116 colon cancer cells (Fig. 7a). IHC analysis of tumor samples from patients showed that CD39 expression was significantly higher in lung metastases from colon cancer than in colon cancer in situ (Fig. 7b, c). Considering that colon cancer cells metastasize to lung through the blood, these results indicate that the enhanced expression of CD39 in lung metastases may be related to the interaction between tumor cells and platelets during hematogenous metastasis.
Discussion
Tumor microenvironment (TME) plays a key role in tumorigenesis and development [32]. The primary TME provides signals for the tumor cells to invade and access the vasculature [33]. Nonetheless, the metastatic potential of tumor cells can be further defined responding to signals provided during the intravascular transit [25]. The present study suggests that the interaction between platelets and tumor cells increases tumor cells metastatic ability through inhibiting antitumor immune response. Immune surveillance poses a major threat to tumor cells during hematogenous metastasis, so platelet-mediated immunosuppression may play a critical role in tumor metastasis. Our findings give evidence that platelets not only serve as the physical shield to tumor cells, and reveal a new mechanism of tumor metastasis mediated by platelets.
Platelets can adhere to the surface of tumor cells, and serve as an important external stimulating factor for tumor cells during hematogenous metastasis. As discovered in this work, platelets increased the CD39 level within tumor cells in the manner of direct contact. Notably, tumor cells can activate platelets to release various cytokines, such as PDGF, FGF2, EGF, TGFβ1, and IGF1 [29]. To elucidate whether platelets regulated the expression of CD39 by releasing cytokines, this study adopted thrombin to activate platelets in vitro, and then collected the supernatant and pellet fractions to treat tumor cells. As a result, the activated platelet pellets, rather than supernatants, induced CD39 expression. In addition, EGF, FGF2, IGF1, TGFβ1, and PDGF were unable to modulate CD39 expression (data not shown). Interestingly, platelets can be activated by tumor cells and release endogenous ADP [34, 35]. Therefore, it is reasonable to assume that platelet-released ADP can be subsequently hydrolyzed on the surface of tumor cells by CD39, which is up-regulated by platelets (Fig. 7d). This indicates that platelets and tumor cells are interrelated and interact with each other, leading to the generation of adenosine.
Our data showed that SCH58261 inhibited CD39-mediated pro-metastatic effects. However, SCH58261 could not completely abolish the effect of CD39 on tumor metastasis, potentially because CD39 could also facilitate metastasis through other ways, such as promoting cell invasion. The effects of CD39 expressed by immune cells on antitumor immunity have been evaluated in prior studies [14, 15]. This work demonstrated that, CD39 expression within tumor cells had certain effect on the antitumor immune responses. Notably, overexpression of CD39 led to the exhaustion of circulating CD8+ T cells. The underlying mechanism may be similar to that of Tregs, which can mediate immune suppression via the CD39-CD73-adenosine axis. CD39 and CD73 are coexpressed in Tregs, and Treg-produced adenosine can induce CD8+ T cell exhaustion through the A2AR pathway [36, 37]. These results indicate that CD39 may facilitate tumor metastasis through suppressing antitumor immunity. Interestingly, both CT-26 and RM-1 cells are resistant to anti-PD1 therapy [23], suggesting that CD39 may be a potential target for “cold” anti-PD1 refractory tumors.
Finally, IHC analysis of tumor samples from patients showed that CD39 expression was higher in lung metastases from colon cancer than in colon cancer in situ. Although this may be related to the interactions between colon cancer cells and platelets during hematogenous metastasis, it may also be caused by other factors. Therefore, more investigation is needed to explore the underlying mechanisms.
Evidence that the platelets contribute to cancer progression is substantial and continues to mount. Here, we show that platelets provide pro-metastatic signals to tumor cells by enhancing CD39 expression. The findings identify a new mechanism of tumor immune evasion mediated by platelet-tumor interaction, and highlight a novel cancer immunotherapy strategy focused on targeting the enzymatic activity of tumor CD39.
Data availability
All data generated or analyzed during this study are available from the corresponding author on reasonable request. The RNA sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) database under the accession number PRJNA1056323 and PRJNA1066901.
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Acknowledgements
We thank the members of Institute of Immunology and Molecular Medicine for discussions and technical help; Wei Wang and Yuzhong Wang for their assistance in tumor tissues collection.
Funding
The present study was funded by the National Natural Science Foundation of China (82003027, 81874169, 82171810), Doctoral Startup Fund of Jining Medical University (2017JYQD24). Grants from Tai Shan Young Scholar Foundation of Shandong Province (tsqn202211234). Project of Shandong Province Higher Educational Youth Innovation Science and Technology Program (2021KJ074).
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Zhaochen Ning led the main research work. Keyan Liu, Hui Zhang and Xiaotong Wang participated in discussions on study design, analysis, and interpretation. Guanjun Dong contributed to revise the manuscript. Huabao Xiong contributed to the overall design and guidance of the study.
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All experiments involving animals were approved by the Medical Animal Care & Welfare Committee of Jining Medical University. Human tissue samples were acquired following the relevant guidelines and regulations approved by the Ethics Committee of the Affiliated Hospital of Jining Medical University. Informed consent was obtained from all individual participants included in the study.
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Ning, Z., Liu, K., Zhang, H. et al. Platelets induce CD39 expression in tumor cells to facilitate tumor metastasis. Br J Cancer 130, 1542–1551 (2024). https://doi.org/10.1038/s41416-024-02640-8
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DOI: https://doi.org/10.1038/s41416-024-02640-8