PD-L1 positive platelets mediate resistance to immune checkpoint inhibitors in patients with colorectal cancer

Jiacheng Li; Jia Liu; Shifeng Yang; Yu Xia; Qingzhe Meng; Biying Sun; Yansong Liu; Bin Zhao; Jiaqi Jin; Hui Xu; Lihong Wang; Pengxia Zhang; Zhuoxin Cheng

doi:10.1186/s12964-025-02034-0

. 2025 Jan 15;23:29. doi: 10.1186/s12964-025-02034-0

PD-L1 positive platelets mediate resistance to immune checkpoint inhibitors in patients with colorectal cancer

Jiacheng Li ^1,^2,^3,^#, Jia Liu ^3,^#, Shifeng Yang ^4,^#, Yu Xia ¹, Qingzhe Meng ¹, Biying Sun ¹, Yansong Liu ¹, Bin Zhao ¹, Jiaqi Jin ⁵, Hui Xu ^3,^✉, Lihong Wang ^2,^✉, Pengxia Zhang ^3,^✉, Zhuoxin Cheng ^1,^3,^✉

PMCID: PMC11737274 PMID: 39815258

Abstract

Background

Programmed cell death ligand 1 (PD-L1) expression on immune cells is correlated with the efficacy of immune checkpoint inhibitor (ICI) therapy in various types of cancer. Platelets are important components of the tumour microenvironment (TME) and are widely involved in the development of many types of cancer including colorectal cancer (CRC). However, the role of PD-L1 positive platelets in ICI therapy for CRC remains unknown. We hypothesized that PD-L1 positive platelets trigger and sustain CRC immunosuppression.

Methods

The functional depletion effects of PD-L1 positive platelets on TME and immune cells were measured via western blotting, immunofluorescence staining, qRT-PCR, ELISpot and flow cytometry. In vivo, CD274 knockout (KO), CD8a KO, platelet-specific KO (PF4-Cre-Hsp90b1^flox/flox) mouse models and a subcutaneous tumour model treated with aspirin and PD-L1 mAb were established in C57BL/6 N mice.

Results

We found that PD-L1 positive platelets are correlated with a poor prognosis, CD8 + T cell exhaustion and serve as a novel noninvasive biomarker for predicting immunotherapy efficacy in patients with CRC. The transfer of PD-L1 from tumour cells to platelets in the TME depends on direct cell contact via the fibronectin-1/GPIbα/integrin α5β1 pathway. In turn, platelets can also induce PD-L1 expression on cancer cells. Animal experiments revealed that antiplatelet pharmacological agents and genetic knockout of platelets potentiated the antitumour effect of the PD-L1 mAb treatment in a CD8 + T cell dependent manner.

Conclusions

Our data suggest that PD-L1 positive platelets suppress CD8 + T cell immunity. Clinical combination treatment with ICIs and antiplatelet agents may be an effective therapeutic strategy for treating CRC.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12964-025-02034-0.

Keywords: PD-L1, Platelet, Colorectal cancer, Immune checkpoint inhibitors

Introduction

Colorectal cancer (CRC) is a prevalent form of cancer and ranks second in terms of mortality among cancer patients worldwide [1]. Presently, the widely accepted treatment approach for CRC involves a combination of radical surgical resection, adjuvant radiotherapy, and chemotherapy [2]. However, in recent years, immune checkpoint inhibitors (ICIs), such as programmed cell death protein-1 (PD-1)/programmed cell death ligand 1 (PD-L1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) inhibitors, have emerged as novel therapeutic agents for various solid tumours, including CRC [3]. In 2017, the Food and Drug Administration (FDA) approved pembrolizumab as a second-line therapeutic option for patients with metastatic CRC and a microsatellite instability-high (MSI-H) status. However, it is worth noting that only a minority, approximately 15%, of CRC patients exhibit deficient DNA mismatch repair (dMMR) which results in MSI, and a subset of these individuals may eventually develop resistance to immunotherapy [4]. This finding was investigated further in a cohort study comprising 73 CRC patients with MSI-H status to assess the correlation between the efficacy of navolumab and the expression of PD-L1 on tumour cells or immune cells [5]. A previous revealed that the presence of PD-L1 on tumour cells was not significantly correlated with the immune response [5]. However, a substantial increase in the objective response rate (ORR) was observed when PD-L1 was highly expressed on immune cells. Further investigations are needed to identify novel biomarkers that can effectively predict the therapeutic response of CRC patients to ICIs.

The tumour microenvironment (TME) supports tumour proliferation and distant metastasis [6]. The composition and heterogeneity of the TME are intricately linked to the development of resistance to ICIs [7]. Within the TME, tumour cells interact with platelets, which leads to their activation and aggregation [8]. Through extensive investigations, scholars have discovered that platelets not only serve as vital contributors to the coagulation system but also exert regulatory effects on both innate and adaptive immunity [8]. Furthermore, platelets actively participate in tumour immune evasion and the process of tumour angiogenesis [9]. Complex and reciprocal interactions occur between tumour cells in the TME and circulating platelets. Previous research has shown that platelets can shield tumour cells from immune responses mediated by natural killer (NK) cells [10]. However, the precise impact of platelets on tumour-infiltrating lymphocytes (TILs) within the TME remains incompletely understood.

Platelets, the smallest cellular constituent of the haematopoietic system, rank second in quantity only to erythrocytes [11]. Platelets exhibit numerous cell surface receptors responsible for adhesion and aggregation, including the glycoprotein (GP) Ib-IX-V complex, which functions as a receptor for von Willebrand factor (vWF) and GPIIb-IIIa integrin, which binds to fibrinogen and fibronectin [12]. The primary unresolved questions pertain to the mechanism by which CRC cells acquire the platelet phenotype within the TME, the role of activated platelets in facilitating tumour immune evasion, and the potential for reversing immunosuppression through blockade of the interaction between tumour cells and platelets. Aspirin, which was first synthesized in 1897, has been extensively employed as a pharmaceutical agent to mitigate platelet activation and aggregation. Increasing amounts of evidence have shown that aspirin can reduce cancer metastasis and mortality rates, providing support for the application of aspirin in cancer treatment [13, 14]. However, it is important to consider the potential adverse effects, such as bleeding tendencies and gastrointestinal diseases, associated with prolonged aspirin use must be considered [15]. Consequently, the role of aspirin in primary cancer treatment and its effectiveness in blocking immunotherapy resistance have not been fully substantiated.

In the present study, we observed elevated levels of PD-L1 positive platelets in CRC patients who tested positive for PD-L1 expression, which was associated with an unfavourable prognosis and CD8 + T cell exhaustion. Furthermore, our research revealed that CRC cells expressing PD-L1 can transfer PD-L1 to platelets within the TME. Concurrently, PD-L1 positive platelets contribute to the maintenance of immunosuppression by impeding the recruitment and activation of CD8 + T cells in the TME. Notably, our findings demonstrated that compared with drug monotherapy, the combination of PD-L1 monoclonal antibody and aspirin significantly reduced the tumour size in mice with established tumours.

Materials and methods

Patient data and tissue specimens

This study enrolled a total of 211 patients diagnosed with CRC and 30 healthy individuals from the First Affiliated Hospital of Jiamusi University between 2018 and 2021. The participants provided complete clinical and pathological data. Blood samples, as well as tumour and adjacent normal tissue samples, were collected from the CRC patients who agreed to participate in the study. Among these patients, 171 were free of disease after undergoing D2 radical surgery, whereas 40 were diagnosed with liver and/or lung metastasis. All patients were followed up for at least one year. Overall survival (OS) was calculated by determining the time from the pathological diagnosis to death, whereas disease-free survival (DFS) was determined by measuring the time from radical resection to disease recurrence or death. The evaluation of a partial response (PR) to ICI treatment was based on a reduction of at least 30% in the sum of the maximum diameters of the target tumour lesion sustained for a minimum of 4 weeks. This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the First Affiliated Hospital of Jiamusi University.

Animal models

The animal protocol was designed to minimize pain or discomfort to the animals. Wild-type C57BL/6 N mice (6–7 weeks old, weighing 18–22 g) were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). Platelet-specific Hsp90b1 KO mice were generated by crossing PF4-Cre mice with Hsp90b1^flox/flox mice. PF4-Cre, Hsp90b1^flox/flox, CD274 KO and CD8a KO mice were purchased from Cyagen Bioscience, Inc. (Suzhou, China). All the mice were housed in a specific pathogen-free mouse facility. All the animal procedures described in this study were approved by the First Affiliated Hospital of Jiamusi University Animal Care Committee and were performed under SPF conditions.

For the detection of bleeding time, the mice were restrained in a 50 mL tube. A superficial incision was made approximately 1 inch from the base of the tail. The incision was then blotted with Whatman paper every 5 s until bleeding ceased. The time from the incision to the cessation of bleeding was recorded.

For the xenograft models, the mice were subcutaneously injected with MC38 colon cancer cells (2 × 10⁶ cells/mouse) into the right axilla. We administered the PD-L1 mAb (Bio X Cell, USA) by intraperitoneal injection 7 days after the MC38 cell injection and then every 48 h until day 28 to test the effects of antiplatelet agents and a PD-L1 monoclonal antibody (mAb) on tumour growth. Aspirin (MCE, USA) was administered through the drinking water (150 mg/L) starting 2 days before the MC38 cell injection and was replaced every 48 h thereafter. The mRNA translation inhibitor cycloheximide (1 mg/kg, MCE, USA) was intraperitoneally injected daily after the MC38 injection. The mice were sacrificed on day 28 after the MC38 cell injection. Tumour volumes were calculated by measuring the length (L) and width (W) of the tumours: tumour volume = π/6×L×W².

Isolation of human plasma and platelets

Fresh whole venous blood was collected into tubes containing 0.5% EDTA using 21-gauge needles. The patients fasted overnight before blood collection. The blood was centrifuged at 150×g for 20 min at room temperature (RT) to obtain platelet-rich plasma (PRP) within 1 h of collection. The top PRP layer was separated into a new tube, diluted with platelet wash buffer (TBD, Tianjin, China) and centrifuged at 460×g for 20 min at RT to obtain clean platelets and platelet-free plasma (PFP). The platelets were resuspended in prewarmed modified Tyrode’s buffer (137 mmol/L NaCl, 2.8 mmol/L KCl, 1.0 mmol/L MgCl2, 12 mmol/L NaHCO3, 0.4 mmol/L Na2HPO4, and 5.5 mmol/L glucose, pH 7.4; Solarbio, Beijing, China).

Flow cytometry

PD-L1 expression on circulating platelet was measured using flow cytometry. Here, whole blood from healthy donors (HDs) and CRC patients was first diluted with 1× PBS and incubated in the dark at RT for 30 min with antibodies. For the assessment of functional changes in immune cells in tumour tissues, mouse samples derived from different treatment groups were harvested with collagenase I, and then the cells were resuspended in PBS and cocultured with antibodies. For the detection of platelet activation and PD-L1 translation, platelets derived from healthy individuals were cocultured with CRC cells or conditioned media (CM) from CRC cells for 1 h and then incubated with antibodies. The following antibodies were used in this study: PerCP-Cy5.5-conjugated CD41, PE-conjugated CD274, FITC-conjugated CD274, PE-Cy7-conjugated CD274, PE-Cy7-conjugated CD45, FITC-conjugated CD45, FITC-conjugated CD4, PE-conjugated CD3, APC-Cy7-conjugated CD3, APC-conjugated CD8, PerCP-Cy5.5-conjugated CD8, PerCP-conjugated CD8a, PerCP-conjugated NK1.1, PE-conjugated Gzmb, APC-conjugated Gzmb and APC-conjugated CD62P antibodies. All of these antibodies were purchased from eBioscience (USA).

In vitro tumour cell and platelet coculture and platelet adhesion assay

Briefly, tumour cells were incubated in media supplemented with 10% FBS in glass-bottom wells of 24-well plates at 37 °C with 5% CO₂ in a humidified environment. Isolated platelets were added to 24-well plates when the tumour cells were cultured to 90% confluence, and the tumour-platelet mixture was incubated for 1 h in an incubator at 37 °C with 5% CO₂. The tumour-to-platelet ratio was 1:100. For the platelet adhesion assay, 24-well plates were coated with the purified fibronectin protein (Bio-Rad, USA) for 2 h. Then, freshly isolated platelets were pretreated with anti-CD41 (ab134131, UK), anti-CD42b (ab61402, UK), anti-integrin β1 (Sigma, USA), anti-integrin α5 (Sigma, USA) or isotype control IgG antibodies for 1 h at 37 °C with 5% CO₂. For immunofluorescence staining and FACS analysis, cells were fixed with 2% PFA in PBS for 15 min at RT.

Immunofluorescence staining

For tissue samples, 5 μm optimal cutting temperature (OCT) compound-embedded tissue sections were fixed with ice-cold acetone for 15 min, followed by an incubation with Triton X-100 for 30 min. The following primary antibodies were used for IF staining: rabbit anti-CD8 (ab217344, UK), rat anti-Gzmb (ab289888, UK), mouse anti-pan cytokeratin (ab77533, UK), rabbit anti-CD41 (ab134131, UK) and mouse anti-CD274 (ab279292, UK) antibodies. The tissues were then incubated with fluorescent dye-conjugated secondary antibodies.

For tumour cells and platelets, the cells were fixed with 2% PFA for 15 min, followed by incubation with Triton X-100 for 30 min. The samples were incubated first with mouse anti-CD274 (ab279292, UK), rat anti-CD41 (ab33661, UK), and rabbit anti-fibronectin 1 (ab199056, UK) primary antibodies and then with fluorescent dye-conjugated secondary antibodies. For platelet adhesion, the cells were incubated with an Alexa Fluor 488-conjugated phalloidin primary antibody (Thermo Fisher, USA). All images were acquired using a confocal microscope (Zeiss, LSM 800, Germany) and analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cell migration and colony formation assays

The above experiments were performed as previously described [16].

Statistical analysis

Clinical data were available for all CRC patients and healthy individuals. The survival of these patients was analyzed using the Kaplan‒Meier method and compared using the log-rank test. Cox proportional hazards regression analysis was performed to determine the effects of plasma PD-L1 levels on OS and DFS. All the data are presented as the means ± standard deviations (SDs). Statistical significance was analyzed using Student’s t test, one-way analysis of variance (ANOVA), the Mann‒Whitney test and the Kruskal‒Wallis test. All analyses were performed using GraphPad Prism v. 8.0 statistical software. P < 0.05 was considered to indicate statistical significance.

Results

PD-L1 positive platelets are correlated with CRC patient outcomes

Prior research has indicated a correlation between activated platelets within the TME and tumour metastasis in patients with CRC [17]. However, the impact of PD-L1 positive platelets on the efficacy of immunotherapy for CRC remains unexplored in the literature. We conducted a histopathological analysis of tissue sections obtained from patients with PD-L1 negative and PD-L1 positive CRC to investigate the potential influence of PD-L1 positive platelets on the composition of the CRC microenvironment. The findings revealed a significant presence of platelets in both PD-L1 negative tumour tissue and adjacent tumour tissue. However, no discernible expression of PD-L1 was observed on these platelets (Fig. 1A, B). In contrast, a high abundance of PD-L1 positive platelets was observed in tissue sections from CRC patients who tested positive for PD-L1 (Fig. 1A, B). We subsequently employed flow cytometry, ELISA, and western blot assays to determine PD-L1 expression in platelets from peripheral blood samples obtained from HDs and CRC patients. Our findings revealed a significant increase in the expression of PD-L1 on the platelets of CRC patients who tested positive for PD-L1 (Fig. 1C-E and Fig. S1A). Additionally, compared with patients with different TNM stages of CRC, patients with advanced CRC exhibited increased numbers of PD-L1 positive platelets (Fig. 1C-E and Fig. S1A). Furthermore, we found that with increasing of tumour TNM stage, the number of PD-L1 positive platelets gradually increased, which was negatively correlated with the a decreased degree of CD8 + T cell infiltration (Fig. S1B-D).

Fig. 1 — PD-L1 positive platelets are correlated with cancer specific outcomes of CRC patients. A and B Representative images of PD-L1 positive platelets in tumour tissue samples and normal tissue samples from the PD-L1 negative and positive CRC patients detected with PD-L1 and CD41 staining (n = 8 each group). The white arrows indicate bona fide PD-L1 positive platelets. Magnification: 20×. Scale bars: 50 μm. Red: PD-L1, Green: CD41, Blue: DAPI. **C-E** The level of PD-L1 positive platelets were detected by ELISA, western-blot and flow cytometry. F CRC patients with higher plasma PD-L1 positive platelets levels (n = 33) had poorer DFS than those with lower PD-L1 positive platelets levels (n = 57). G CRC patients with higher plasma PD-L1 positive platelets levels (n = 49) had poorer OS than those with lower PD-L1 positive platelets levels (n = 59). Log-rank test and univariate Cox regression analysis, as indicated. All values are the means ± SDs. ns = not significant, ***p < 0.001 and ****P < 0.0001

Consequently, our study aimed to examine the correlation between the presence of PD-L1 positive platelets and the prognosis of CRC patients. Our findings revealed a significant association between increased numbers of PD-L1 positive platelets in plasma and shorter DFS among patients who underwent D2 resection (HR: 2.810, 95% CI: 1.623–4.867, p < 0.0001; Fig. 1F; Table S2-4). Furthermore, the univariate analysis demonstrated a correlation between high PD-L1 positive platelet counts and reduced OS in all CRC patients (HR: 3.413, 95% CI: 2.196–5.305, p < 0.0001; Fig. 1G; Table S2-4). Notably, the results of the multivariate Cox regression analysis indicated that of the number of PD-L1 positive platelets in plasma served as an independent prognostic indicator for both DFS and OS, even after adjusting for other established prognostic factors, such as the TNM stage, vascular cancer thrombus, and perineural invasion (DFS, HR: 2.541, 95% CI: 1.664–5.623, p = 0.003; OS, HR: 2.896, 95% CI: 1.782–5.533, p = 0.002; Table S2-4). Collectively, our findings indicate a correlation between the number of PD-L1 positive platelets in both the tissue and blood samples of CRC patients and an unfavourable prognosis.

PD-L1 positive platelets predict the immunotherapy response in patients with CRC

Traditional genetic testing has the ability to predict the therapeutic efficacy of ICIs for CRC patients with MSI-H/dMMR mutations. In addition, the ability of PD-L1 expression in CRC cells to predict ICI therapeutic efficacy hass not been consistent across studies. Moreover, the scarcity of gene mutations and the exorbitant expenses associated with genetic testing necessitate the development of a noninvasive novel detection index to predict the therapeutic response to ICIs in clinical settings. Consequently, we conducted an in-depth investigation to ascertain whether PD-L1 positive platelets can serve as a predictive indicator for the therapeutic outcome of ICI treatment. In the cohort recruited for this study, 45 out of 211 patients received ICI treatment. Among them, 24 patients had dMMR mutations, and 21 patients had pMMR mutations and were HER-2 negative. Our findings indicated that the number of PD-L1 positive platelets in the dMMR cohort was significantly higher than that in the pMMR cohort (dMMR, n = 24; pMMR, n = 99; Fig. S1E). Notably, we used the level of PD-L1 positive platelets and performed receiver-operating characteristic (ROC) analysis to determine the level of PD-L1 positive platelets that would predict the immunotherapy efficacy in CRC patients, and the results showed that the level of PD-L1 positive platelets can serve as a biomarker to predict immunotherapy efficacy (AUC: 0.7565, 95% CI: 0.6095–0.9034, p = 0.0048; Fig. S1F). Interestingly, we observed that among the patients with vascular thrombotic disease who received a combination of antiplatelet drugs and ICIs, three had a greater partial response (PR) than did those who received ICIs alone (Fig. S1G). These findings indicate that PD-L1 positive platelets may serve as a novel biomarker for predicting outcomes and evaluating the effectiveness of ICI therapy in patients with CRC.

PD-L1 positive platelets mediate immune cell exhaustion in patients with CRC

Previous studies have reported that platelets in the TME can promote tumour progression. Therefore, we determined the protumoural effect of PD-L1 positive platelets on CRC cells by establishing a PD-L1 positive platelet and CRC cell coincubation model and performing colony formation and Transwell assays. The present study revealed that the proliferation and migration capacities of CRC cells did not increase (Fig. S2A-D).

After the relevance of PD-L1 positive platelets in CRC was investigated, experiments were subsequently conducted to determine the potential impact of these PD-L1 positive platelets on immune cell functionality within the CRC microenvironment. Notably, our study revealed a substantial decrease in the number of activated CD8 + T cells expressing Gzmb in the presence of PD-L1 positive platelets (Fig. 2A, B). Based on these findings, our subsequent study aimed to elucidate the immune inhibitory effects of PD-L1 positive platelets. Platelets were isolated from CRC patients who exhibited PD-L1 positive or PD-L1 negative phenotypes to achieve this objective. These platelets were then cocultured with T cells derived from healthy individuals. The activation of T cells was evaluated using ELISpot assays, which enabled the quantification of the effector cytokine IFN-γ. The findings from this study undeniably indicate that, compared with PD-L1 negative platelets, PD-L1 positive platelets significantly impeded the infiltration of CD8 + T cells and the secretion of IFN-γ, and treatment with an anti-PD-L1 mAb reversed these effects (Fig. 2C-E). Moreover, the flow cytometry analysis confirmed that intratumoural T cells presented decreased CD8 + expression and reduced Gzmb secretion with high levels of PD-L1 positive platelets (Fig. 2F-H). Furthermore, flow cytometry revealed that CD8 + T cells exhibited signs of exhaustion, such as PD-1, Lag3, and Tim3 expression (Fig. S2E-G). These results provide compelling evidence that platelets expressing PD-L1 have a substantial effect on the induction of immune cell exhaustion rather than directly affecting CRC cells.

Fig. 2 — PD-L1 positive platelets mediate immune cell exhaustion in patients with CRC cancer. **A-D** Representative images of CD8 + T cell infiltration and Gzmb secreting cells in tumour tissue samples of the PD-L1 negative and positive CRC patients detected with pan-CK, CD8 and Gzmb staining (n = 5 each group). Magnification: 20×. Scale bars: 50 μm. Red: CK, Yellow: CD8, Green: Gzmb, Blue: DAPI. E PBMCs co-incubated with PD-L1 positive platelets (red) or PD-L1 negative platelets (green) with or without PD-L1 mAb pre-treatment and detected by IFN-γ ELISPOT assay (n = 3). The results were expressed as the number Spot-Forming Units (SFU) per 250,000 seeded cells after subtracting the background of unstimulated cells. **F-H** Flow cytometry analysis of CD8 + T cells infiltration and Gzmb secreting cells in tumour tissues from PD-L1 positive and PD-L1 negative CRC patients (n = 5 each group). All values are the means ± SDs. ns = not significant, **p < 0.01, ***p < 0.001 and ****P < 0.0001

Antiplatelet pharmacological agents and genetic blockade of platelets overcome resistance to ICIs

We pharmacologically inhibited platelets and subsequently developed a PF4-Cre-Hsp90b1^flox/flox mouse model, which is defined as exhibiting platelet dysfunction, for this investigation to ascertain the clinical significance of the inhibitory impact of PD-L1 positive platelets on antitumoural immunity [18]. As anticipated, compared with WT mice, PF4-Cre-Hsp90b1^flox/flox mice presented markedly reduced platelet levels in the bloodstream and an extended duration of bleeding (Fig. 3A, B). Colorectal carcinoma was induced in both WT C57BL/6 mice and PF4-Cre-Hsp90b1^flox/flox mice through the subcutaneous injection of MC38 cells. On day 0, aspirin was administered either alone or in combination with the PD-L1 mAb on day 7, with subsequent administration every two days. Our findings indicate that treatment with aspirin alone did not result in a reduction in tumour size in WT mice (Fig. 3C, D). Furthermore, compared with the PD-L1 mAb or aspirin treatment alone, the combination of aspirin and PD-L1 mAb resulted in a markedly greater reduction in tumour size (Fig. 3C, D). In addition, the tumour sizes of PF4-Cre-Hsp90b1^flox/flox mice that received the PD-L1 mAb treatment were comparable to those of WT mice that received the combination of the two drugs (Fig. 3C, D). All the mice responded to the combined treatment of aspirin and the PD-L1 mAb, whereas only 1 of 9 mice responded to aspirin treatment alone, and 2 of 9 mice responded to PD-L1 mAb treatment alone (Fig. 3C, D). Moreover, the histopathological analysis revealed a noteworthy reduction in the proliferation index of the mice subjected to combination treatment with aspirin and an anti-PD-L1 mAb compared with that of the mice treated with either drug alone (Fig. 3E, F). Taken together, these findings provide compelling evidence that antiplatelet agents exert synergistic effects on the antitumour efficacy of PD-L1 mAb treatment.

Fig. 3 — Targeting platelets genetically potently enhances the efficacy of ICI treatment in CRC. A Platelet counts from the peripheral blood of WT and PF4-Cre-Hsp90b1^flox/flox (PLT KO) mice (n = 10 each group). B Bleeding time was measured in WT and PLT KO mice by pricking the lateral tail vein (n = 10 each group). C and D Subcutaneous MC38 tumour growth was established in WT and PF4-Cre-Hsp90b1^flox/flox mice in the presence of aspirin and PD-L1 mAb treatment alone or combination in vivo (n = 5 each group). Average tumour growth curves are shown. E and F Decreased Ki67 expression in tumours of aspirin and PD-L1 mAb treated mice and PF4-Cre-Hsp90b1^flox/flox mice treated with PD-L1 mAb compared to control mice. Representative images of CD31 staining are shown. Magnification: 20×. Scale bars: 50 μm. All values are the means ± SDs. ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001 and ****P < 0.0001

The antitumour effect of combination treatment with antiplatelet therapy and PD-L1 blockade is depends on CD8 + T cells

The presence of immune cell effectors in tumour tissues from mice was assessed using IF staining and flow cytometry to obtain a deeper understanding of the underlying mechanism responsible for the synergistic antitumoural effects resulting from the combination of antiplatelet therapy and PD-L1 inhibition. Our findings indicate that the concurrent administration of aspirin and the PD-L1 mAb led to a significant increase in the recruitment of CD8 + T cells, particularly those that secrete the cytotoxic protein Gzmb, compared with that in the control groups (Fig. 4A-F). Furthermore, a correlation analysis was conducted to examine the relationship between the tumour volume and the infiltration of CD8 + T cells across the various treatment groups. A statistically significant correlation was not observed between the two variables in the control and single drug treatment groups (Fig. S3A-C). However, an inverse correlation was identified between the tumour volume and the infiltration of CD8 + T cells in WT mice treated with a combination of aspirin and the anti-PD-L1 mAb (Fig. S3D). Next, we analyzed the numbers of CD4 + T cells and NK cells in tumour tissues derived from mice which were treated with aspirin or the PD-L1 mAb alone or in combination via flow cytometry. We did not observe significant changes in the numbers of CD4 + T and NK cells within the tumour tissues from the different groups (Fig. S3E, F).

Fig. 4 — The antitumoural effect of the combination of aspirin and an anti-PD-L1 mAb depends on CD8 + T cells. **A-C** Representative images of CD8 + T cells infiltration and Gzmb secreting cells in tumour tissue samples from mice with aspirin and PD-L1 mAb treatment alone or combination by cytokaretin, CD8 and Gzmb staining (n = 5 each group). Magnification: 20×. Scale bars: 50 μm. Red: CK, Yellow: CD8, Green: Gzmb, Blue: DAPI. **D-F** Flow cytometry analysis of CD8 + T cells infiltration and Gzmb secreting cells in tumour tissues from mice with aspirin and PD-L1 mAb treatment alone or combination (n = 5 each group). The flow cytometry strategy of experiment in tumour tissues are shown. All values are the means ± SDs. ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001 and ****P < 0.0001

Based on the aforementioned observations, we hypothesized that the antitumoural effect achieved through combined treatment with aspirin and an anti-PD-L1 mAb is mediated primarily by CD8 + T cells. To validate this hypothesis, we conducted an experiment in which WT mice were treated with a neutralizing antibody targeting CD8 + T cells prior to the injection of MC38 cells. The mice were subsequently administered a combination of aspirin and the PD-L1 mAb. The results obtained indicated that the combined treatment with aspirin and the PD-L1 mAb was ineffective at reducing the tumour volume in the absence of CD8 + T cells (Fig. S3G). To further validate these observations, we subsequently generated CD8a KO mice injected with MC38 cells, and the efficacy of the combination therapy with aspirin and the anti-PD-L1 mAb in reducing the tumour volume was significantly decreased compared with that in the control group (Fig. S3H). Taken together, these findings provide evidence that the antitumoural efficacy of aspirin and PD-L1 mAb combination therapy is contingent upon the presence of CD8 + T cells.

Tumour cells transfer PD-L1 to platelets in the TME of CRC

To investigate the potential mechanism underlying PD-L1 expression on platelets in CRC patients, we conducted coculture experiments using platelets obtained from healthy donors and various CRC cell lines. Our findings revealed a significant increase in PD-L1 expression on platelets cocultured with PD-L1 positive CRC cells (HCT-116, HT29, and SW480), whereas platelets cocultured with PD-L1-negative CRC cells (Lovo) did not express PD-L1 (Fig. 5A, B). Furthermore, we observed the transfer of PD-L1 when platelets were cocultured with Lovo cells that had undergone PD-L1 knock-in (Fig. 5A, B). Flow cytometry was used to identify PD-L1 positive CRC cells, which were defined as those exhibiting PD-L1 expression in more than 5% of all tumour cells. Consistent with our expectations, the flow cytometry analysis revealed significant platelet activation when cocultured with CRC cells, as evidenced by the expression of CD62P. Notably, coculture with PD-L1 positive CRC cells alone led to the upregulation of PD-L1 expression on platelets (Fig. 5C-E). Subsequently, platelets obtained from HDs were cocultured with conditioned media derived from PD-L1 positive or PD-L1 negative CRC cells to determine the mechanism by which PD-L1 is transferred from tumour cells to platelets, specifically through intercellular contact. Notably, the conditioned media of CRC cells induced substantial platelet activation but did not increase PD-L1 expression on platelets (Fig. 5F-H), suggesting that the transfer of PD-L1 from tumour cells to platelets is contingent upon a direct cell‒cell contact.

Fig. 5 — Tumour cells transfer PD-L1 to platelets in the TME of CRC. A and B Platelets derived from healthy individuals co-cultured with PD-L1 positive CRC cells (HCT-116), PD-L1 negative CRC cells (Lovo) or Lovo with PD-L1 knocked in and then detected with PD-L1 and CD41 staining (n = 5 each group). Magnification: 20×. Scale bars: 50 μm. Red: PD-L1, Green: CD41, Blue: DAPI. **C-E** CD62P and PD-L1 expression on platelets when platelets were co-cultured with PD-L1 positive CRC cells by flow cytometry (n = 5 each group). **F-H** CD62P and PD-L1 expression on platelets when platelets were co-cultured with CM of PD-L1 positive CRC cells by flow cytometry (n = 5 each group). I Experimental protocol for subcutaneous implantation of MC38 cells into WT, PD-L1 KO and PD-L1 KO treated with cycloheximide mice. J Flow cytometry analysis of PD-L1 positive platelets in whole blood of mice (n = 5 each group). All values are the means ± SDs. ns = not significant, *p < 0.05 and ****P < 0.0001

Although platelets do not have a nucleus, protein translation from RNA can still occur [19, 20]. We investigated the effects of cycloheximide, an RNA translation inhibitor, on the transfer of PD-L1 from CRC cells to platelets by coculturing platelets with PD-L1 positive CRC cells in the presence of cycloheximide. Our results indicated that cycloheximide did not affect the transfer of PD-L1 from CRC cells to platelets (Fig. S4A-D). Furthermore, we established an animal subcutaneous CRC model using CD274 KO mice with mouse PD-L1 positive CRC cells (MC38) to further elucidate the mechanism of PD-L1 transfer. We found that mice showed significantly higher expression of PD-L1 on circulating platelets after the injection of MC38 cells, as measured by flow cytometry (Fig. 5I, J). In addition, the treatment of CD274 KO mice or CD274 KO mice with cycloheximide did not affect PD-L1 expression on platelets (Fig. 5I, J). These findings collectively indicate that the transfer of PD-L1 from CRC cells to platelets is primarily mediated by intercellular contact rather than mRNA translation within platelets.

In the above experiments, tumour cells transfer PD-L1 from their surface to platelets and activate them. Next, we wanted to investigate whether platelets affect the expression of PD-L1 on the surface of tumour cells. We cocultured Lovo cells with platelets derived from CRC patients and detected the PD-L1 expression on the cancer cell surface. The results indicated that PD-L1 expression on Lovo cells was significantly increased when these cells were cocultured with platelets (Fig. S4E). These results strongly suggest that complex interactions and synergistic relationships between tumour cells and platelets to promote tumour immunosuppression.

PD-L1 protein is transferred from tumours to platelets via the fibronectin-1/GPIbα/integrin α5β1 pathway

According to the aforementioned experiments, intercellular contact significantly influences the translation of PD-L1. Consequently, we propose that the expression of adhesion molecules by CRC cells might be involved in the transfer of PD-L1 from tumours to platelets. To test this possibility, we assessed the expression of FN-1, ICAM-1, and TF through qRT‒PCR analysis. Our findings revealed a positive correlation between the rate of PD-L1 transfer and the level of FN-1 expression, whereas no significant association was observed between the rate of PD-L1 transfer and the expression of ICAM-1 or TF (Fig. 6A, B). Moreover, IF staining revealed a close association between the platelet count and FN-1 expression in CRC cells (Fig. 6C-E). Notably, the transfer of PD-L1 from CRC cells to platelets was significantly hindered when platelets were cocultured with CRC cells treated with an FN-1 siRNA (Fig. 6C-F), indicating the crucial involvement of FN-1 in the transfer of PD-L1 from CRC cells to platelets. Additionally, the presence of adhesion molecules on the surface of platelets also plays a role in the interaction between tumours and platelets. Therefore, our study aimed to investigate the potential role of adhesion molecules on platelets in mediating the transfer of PD-L1 from CRC cells to platelets. The findings revealed a significant reduction in platelet adhesion when GPIbα and integrin α5β1 were blocked, as opposed to GPIIbIIIa blockade (Fig. 6G). Furthermore, pretreatment of platelets with FN-1, GPIbα and integrin α5β1 antagonist effectively abrogated the transfer of PD-L1 from CRC cells to platelets (Fig. 6G, Fig. S5A, B).

Fig. 6 — The PD-L1 protein is transferred from tumour cells to platelets via the fibronectin-1/GPIbα/integrin α5β1 pathway. A Heatmap of the relative FN-1, TF and ICAM1 mRNA levels in all tested CRC cell lines (n = 3). B Relative mRNA level of FN-1 in all tested CRC cell lines (n = 3). C Representative images of platelets adhension to HCT-116, HT-29 and HT-29 with FN-1 KD cells by FN-1 and CD41 staining (n = 5 each group). The white arrows indicate bona fide PD-L1 positive platelets. Magnification: 20×. Scale bars: 50 μm. Red: FN-1, Green: CD41, Blue: DAPI. D Quantification of adhesive platelets after co-cultured with CRC cells. Quantified as CD41 + area in %/FoV (n = 5). E Quantification of FN-1 converage area in %/FoV in CRC cells (n = 5). F CD41 + area was positively correlated with FN-1 converage area in CRC cells. G Representative images of platelet adhesion to fibronectin-coated surface in the presence or absence of different pathways (n = 3). Magnification: 20×. Scale bars: 50 μm. Green: phalloidin. All values are the means ± SDs. ns = not significant, **p < 0.01, ***p < 0.001 and ****P < 0.0001

Discussion

The TME and pathological subtypes of CRC are highly complex and heterogeneous, with multiple mechanisms contributing to the initiation and perpetuation of immunosuppression [21, 22]. The advent of ICIs has substantially increased the number of treatment options available for patients with advanced CRC. However, the benefits of ICIs are limited to a small subset of patients with a dMMR or MSI-H status. In non-small cell lung cancer patients, a histopathological assessment of intratumoural PD-L1 expression is routinely conducted as a biomarker to predict the response to ICI therapy [23]. However, a significant amount of data from clinical trials fail to provide substantial evidence for the utilization of intratumoural PD-L1 as a biomarker for predicting the effectiveness of ICIs in patients with CRC [24, 25]. Furthermore, the correlation between the levels of MSI-H/dMMR and PD-L1 expression remains uncertain. Consequently, a pressing need exists to identify alternative noninvasive biomarkers that can accurately predict the efficacy of ICI treatment and establish a logical basis for implementing immunotherapy strategies in the context of this prevalent gastrointestinal malignancy.

Platelets play a protumourigenic role in the initiation and progression of CRC in patients [26]. Notably, approximately 10–15%, of individuals diagnosed with cancer experience cancer-associated thrombosis (CAT), particularly venous thromboembolism (VTE), which is the second most prevalent cause of mortality among cancer patients [27, 28]. The aberrant activation of platelets within the circulatory system of cancer patients frequently results in CAT. The notion of tumour cell-induced platelet aggregation (TCIPA) can be traced back to the late 19th century. However, its precise mechanism remains to be fully elucidated. The activation of platelets in the context of malignant tumours may be attributed primarily to platelet agonists, including thrombin and ADP, produced by tumour cells and the TME [27]. Additionally, interactions between different platelet receptors and ligands may also play an important role in this activation process. Moreover, the release of chemokines and cytokines from platelet organelles not only aids in the initiation of inflammation and immune reactions but also plays a role in the progression of TCIPA, which is linked to malignant tumours [12]. Previous research has shown that platelets can protect tumour cells from immune responses mediated by NK cells [29]. Furthermore, recent research has demonstrated that the genetic manipulation of platelets or the use of antiplatelet pharmacological agents can augment the efficacy of adoptive T-cell therapy for cancer by targeting the GARP-TGFβ axis [18]. However, no previous investigations have explored the potential involvement of PD-L1 positive platelets in conferring resistance to ICIs in CRC patients. Histopathological findings revealed that PD-L1 positive platelets significantly impair the antitumoural capabilities of CD8 + T cells within the TME of CRC patients. Moreover, an analysis of the clinical data revealed a significant correlation between increased numbers of PD-L1 positive platelets and a poor prognosis, as evidenced by shorter OS and DFS. Additionally, three patients who received a combination of antiplatelet drugs and ICIs exhibited a better PR than did patients who received ICIs alone. These findings suggest that the presence of PD-L1 positive platelets may contribute to resistance to ICIs in CRC patients. Although the number of patients in this particular subset of CRC patients was limited within this cohort, the clinical data of these patients still provide promising insights for the future treatment of CRC.

PD-L1 positive platelets have been shown to contribute to the reconstruction of the immune microenvironment in CRC, suggesting that PD-L1 positive platelets may play a distinct role in systemic immunomodulation. In this study, we provide evidence that the infiltration of CD8 + T cells and the secretion of Gzmb were significantly lower in tumour tissues with a microenvironment characterized by PD-L1 positive platelets than in those with a microenvironment characterized by PD-L1-negative platelets. The immunosuppressive effect of PD-L1 positive platelets has been identified through the use of an ELISpot assay and flow cytometry in vitro. However, the precise mechanism by which this effect occurs, whether through direct cell‒cell contact or the release of indirect factors, remains to be elucidated. Previous research has indicated that soluble factors derived from platelets can impair the functions of immune cells [18]. A previous study showed that platelet-derived RGS18 mediated the immunotherapy efficacy by depressing NK cells in circulating tumour cells of pancreatic ductal adenocarcinoma [30]. A recent study reported that platelets mediate tumour cell evasion from NK cell killing via the immune checkpoint CD155 [31]. In this study, we established subcutaneous tumour models by injecting MC38 cells into WT, CD8a KO, and PF4-Cre-Hsp90b1^flox/flox mice. These model mice were then treated with either an antiplatelet pharmacological agent, a PD-L1 mAb, or the combination of both agents. Interestingly, we observed that the tumour size did not change significantly in the WT mice treated with aspirin or the PD-L1 mAb alone. However, when aspirin and the PD-L1 mAb were administered together, the tumour size decreased significantly, and CD8 + T cells were more closely localized to cancer cells. Of note, we did not observe significant changes in the numbers of CD4 + T and NK cells within the tumour tissues derived from the mice treated with aspirin or the PD-L1 mAb. The observed outcome aligns with the findings from PF4-Cre-Hsp90b1^flox/flox tumour-bearing mice that were administered a PD-L1 mAb. Despite the significant immunomodulatory effects of the combination of aspirin and ICI, their antitumoural efficacy as individual agents was not altered. These results suggest that aspirin can be exploited to enhance cancer immunotherapy, indicating that the effects of antiplatelet agents are immune-mediated. Furthermore, our use of a tumour-bearing CD8a KO mouse model revealed that the synergistic antitumoural effect of the combination of aspirin and the PD-L1 mAb is contingent upon CD8 + T cells activation. A previous study reported that antiplatelet agents conferred no benefit when the transferred T cells lacked IFNγ or when anti-IFNγ neutralizing antibodies were administered to tumour-bearing mice with melanoma [18], which was consistent with our results in this study. Our work focused on the overexpression of inhibitory receptors, diminished production of effector cytokines and a reduction in antitumour immunity of T cells and NK cells, but the alterations in the metabolic functions of T cells and NK cells are still unknown.

We subsequently observed that when cocultured with platelets obtained from healthy individuals, PD-L1 positive CRC cells, as opposed to PD-L1-negative CRC cells, ransfer the PD-L1 protein exclusively to platelets. Importantly, we conducted further analyses employing both direct and indirect coculture methods to investigate the association between platelet activation and the transfer of PD-L1 from CRC cells to platelets. Our findings revealed that platelets were activated in all experimental groups, whereas the transfer of PD-L1 was solely mediated through direct cell‒cell contact. Notably, previous reports have indicated that cancer patients who exhibit negative PD-L1 expression, as detected by immunohistochemistry, may still exhibit PD-L1 expression in their platelets. In this study, we unequivocally showed that the high expression of PD-L1 on platelets is a result of PD-L1 transfer from CRC cells in CD274 KO tumour-bearing mice, indicating that PD-L1 is transferred to platelets from CRC cells. PD-L1 is expressed by various cells, including macrophages, DCs, T cells, B cells, mast cells, and endothelial cells. Notably, PD-L1 can be upregulated in a wider range of cell types in response to inflammatory cytokines and other stimuli, and is typically overexpressed in various types of cancer cells and tumour stromal cells, forming an immunosuppressive TME and promoting tumour development [32, 33]. However, due to the heterogeneity of the TME, it does not exclude that some expression of PD-L1 on platelets might be derived from other nonmalignant cell types. Previous study has reported that PD-L1 was highly expressed in vascular endothelial cells of metastatic lung cancer, and treatment with osimertinib can improve the tumor microenvironment and enhance the antitumor effect of bevacizumab by reducing the expression of PD-L1 in tumor blood vessels [34]. In addition, PD-L1 was contained within neutrophils extracellular traps (NETs) derived from human and murine neutrophils of metastatic colorectal cancer [35]. Our previous study also demonstrated that NETs was accumulated in the TME of cancer patients, and promote cancer-associated thrombosis through platelets activation and endothelial cells damage [36], suggesting that the complex interactions between platelets and various cells in the TME. In our present data, the vast majority of PD-L1 does not colocalize with CD41 in the TME of CRC patients, indicating that PD-L1 expression on the platelets might not only derived from tumor cells, but also derived from endothelial cells or neutrophils. In this study, we only explore the transfer of PD-L1 from tumor cells to platelets in TME is limited.

Through the aforementioned experiments, qRT‒PCR was employed to elucidate the differences among the CRC cell lines, revealing a positive correlation between the expression of adhesion molecules on CRC cells and the efficiency of PD-L1 transfer. Moreover, when platelets were cocultured with FN-1 gene knockout CRC cells, the transfer of PD-L1 from CRC cells to platelets was significantly diminished compared with that in control cells, indicating that FN-1 was necessary for the process of PD-L1 transfer between CRC cells and platelets, it was evident that CRC cells can transfer PD-L1 to platelets. However, the precise mechanism underlying the transfer of PD-L1 to platelets remains unidentified. Platelets exhibit a diverse array of cell surface adhesion and aggregation receptors, including the GPIb-IX-V complex, which serves as the primary receptor for vWF, as well as the integrin GPIIb-IIIa, which binds to fibrinogen and fibronectin [37, 38]. Other activated receptors, such as thromboxane A2 receptors, adenosine diphosphate (ADP) receptors P2Y1 and P2Y12, and protease-activated receptors PAR1 and PAR4, are also expressed on the surface of the cell [39]. These receptors collectively contribute to various pathological processes, including thrombotic disease, chronic inflammation, and tumour cell metastasis [40, 41]. In our current study, we observed that the transfer of the PD-L1 from CRC cells to platelets can be significantly inhibited by blocking GPIbα and integrin α5β1. This finding suggested that the adhesion molecules expressed on platelets are also involved in this process (Fig. 7). This evidence is derived from the observation that the transfer of PD-L1 protein from CRC cells to platelets is contingent upon the presence of adhesion molecules expressed on both CRC cells and platelets. A previous study revealed robust PD-L1 expression on platelet α-granules in NSCLC patients [23]. Furthermore, changes in energy production through glycolysis and mitochondrial respiration in platelets are important for platelet aggregation, phosphatidylserine exposure and Ca²⁺ mobilization in CAT and cancer metastasis [42–44]. Our data revealed that most PD-L1-positive platelets were activated and exhibited CD62P expression. These findings indicate that other platelet anatomical structures and processes, such as platelet phospholipids, dense granules, mitochondria, ADP production and Ca²⁺, may also be involved in the transfer of PD-L1 between tumour cells and platelets. In addition, we found that PD-L1 expression on the tumour cells was significantly increased when cocultured with platelets from CRC patients. Interestingly, cancer cells are reprogrammed to a metastatic state through the acquisition of platelet mitochondria via the PINK1/Parkin-Mfn2 pathway [43]. Activated platelets donate major histocompatibility class I molecules to cancer cells to facilitate their evasion from immune cells [45]. These findings suggest that complex interactions and synergistic relationships between tumour cells and platelets to promote tumour progression and immunosuppression. Certainly, further research is needed to predict the efficacy of immunotherapy for CRC patients by using the levels of PD-L1 positive platelets, and the intracellular signaling pathways that mediate immunosuppressive of PD-L1 positive platelets still need to be addressed.

Fig. 7 — Diagram of the mechanism by which PD-L1-positive platelets mediate ICI resistance in CRC

Conclusions

In conclusion, this study provides novel insights into the role of platelets in the resistance of CRC to ICI therapy. This study elucidated the mechanism of PD-L1 transfer from CRC cells to platelets via FN-1/GPIbα/integrin α5β1 and demonstrated that PD-L1 positive platelets can induce T-cell exhaustion in the TME of CRC patients. In turn, platelets can also induce PD-L1 expression on the surface of CRC cells. Considering the compelling evidence from clinical data, animal models, and the potential for acceptable adverse reactions, combination therapy with pharmacological agents targeting both platelets and PD-L1 mAbs may be a viable treatment strategy for CRC patients.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1^{(130.2MB, doc)}

Supplementary Material 2^{(6.5MB, doc)}

Acknowledgements

Not applicable.

Abbreviations

CM: Conditioned media
CTLA-4: Cytotoxic T lymphocyte associated antigen-4
Dmmr: Deficient DNA mismatch repair
DFS: Disease-free survival
FN: Fibronectin
ICIs: Immune checkpoint inhibitors
IF: Immunofluorescence
IFN-γ: Interferon-γ
ICAM-1: Intercellular cell adhesion molecule-1
GP: Glycoprotein
Gzmb: Granzymeb
mCRC: Metastatic colorectal cancer
MSI-H: Microsatellite instability-high
OS: Overall survival
PD-1: Programmed cell death protein-1
PD-L1: Programmed cell death-ligand 1
PR: Partial response
PRP: Platelet rich plasma
PFP: Platelet free plasma
PBMCs: Peripheral blood monocellular cells
TILs: Tumour infiltrating lymphocytes
TF: Tissue factor
TCIPA: Tumour cell induced platelet aggregation
TME: Tumour microenvironment
NETs: Neutrophils extracellular traps

Author contributions

ZXC, JCL, LHW and PXZ designed the study, completed the experiments and drafted the manuscript. HX, JL, YX, QZM and YSL collected the patient clinical data and part of experiments. BZ and YSL participated in the animal experiments. JQJ, BYS and SFY performed the statistical analysis. All authors read and approved the final manuscript.

Funding

This study was funded by the Research Projects of Basic Scientific Research Business Expenses of Provincial Colleges and Universities in Heilongjiang Province (grant no. 2023-KYYWF-0605), the Basic Research Support Program for Excellent Young Teachers in Provincial Undergraduate Universities in Heilongjiang Province (grant no. YQJH2023222), the Doctoral Special Research Fund Project of Jiamusi University (grant no. JMSUBZ2022-09), the National Fund Cultivation Project of Jiamusi University (grant no. JMSUGPZR2023-005), the Excellent Scientific Research Team Project of the First Affiliated Hospital of Jiamusi University (grant no. 202301), the Longjiang Technology Talent Spring Goose Support Program (grant no. 2022CYQN0140), the Innovation Team Project of Heilongjiang Provincial Department of Education (grant no. 2024-KYYWF-0613) and the Dongji Academic Team of Jiamusi University (grant no. DJXSTD202410).

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Ethical approval and consent to participate

This study involving human tissue samples was approved by the Ethics Committee of the First Affiliated Hospital of Jiamusi University (approval number: 2022-600-01) and was conducted in accordance with the Declaration of Helsinki. The animal experiments were approved by the Animal Ethical and Welfare Committee of Jiamusi University and were performed in accordance with the Basel Declaration.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jiacheng Li, Jia Liu and Shifeng Yang contributed equally to this work.

Contributor Information

Hui Xu, Email: xuhui19782003@163.com.

Lihong Wang, Email: wlh_6663@163.com.

Pengxia Zhang, Email: pengxiaz@jmsu.edu.cn.

Zhuoxin Cheng, Email: czx6892551@yeah.net.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1^{(130.2MB, doc)}

Supplementary Material 2^{(6.5MB, doc)}

Data Availability Statement

Data is provided within the manuscript or supplementary information files.

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PERMALINK

PD-L1 positive platelets mediate resistance to immune checkpoint inhibitors in patients with colorectal cancer

Jiacheng Li

Jia Liu

Shifeng Yang

Yu Xia

Qingzhe Meng

Biying Sun

Yansong Liu

Bin Zhao

Jiaqi Jin

Hui Xu

Lihong Wang

Pengxia Zhang

Zhuoxin Cheng

Abstract

Background

Methods

Results

Conclusions

Supplementary Information

Introduction

Materials and methods

Patient data and tissue specimens

Animal models

Isolation of human plasma and platelets

Flow cytometry

In vitro tumour cell and platelet coculture and platelet adhesion assay

Immunofluorescence staining

Cell migration and colony formation assays

Statistical analysis

Results

PD-L1 positive platelets are correlated with CRC patient outcomes

Fig. 1.

PD-L1 positive platelets predict the immunotherapy response in patients with CRC

PD-L1 positive platelets mediate immune cell exhaustion in patients with CRC

Fig. 2.

Antiplatelet pharmacological agents and genetic blockade of platelets overcome resistance to ICIs

Fig. 3.

The antitumour effect of combination treatment with antiplatelet therapy and PD-L1 blockade is depends on CD8 + T cells

Fig. 4.

Tumour cells transfer PD-L1 to platelets in the TME of CRC

Fig. 5.

PD-L1 protein is transferred from tumours to platelets via the fibronectin-1/GPIbα/integrin α5β1 pathway

Fig. 6.

Discussion

Fig. 7.

Conclusions

Electronic supplementary material

Acknowledgements

Abbreviations

Author contributions

Funding

Data availability

Declarations

Ethical approval and consent to participate

Consent for publication

Competing interests

Footnotes

Contributor Information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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