Platelets control liver tumor growth through P2Y12-dependent CD40L release in NAFLD

Abstract

Platelets, the often-overlooked component of the immune system, have been shown to promote tumor growth. Non-alcoholic fatty liver disease (NAFLD) is a common disease in the Western world and rising risk for hepatocellular carcinoma (HCC). Unexpectedly, we observed that platelets can inhibit the growth of established HCC in NAFLD mice. Through pharmacological inhibition and genetic depletion of P2Y12 as well as in vivo transfusion of wild-type (WT) or CD40L^-/- platelets, we demonstrate that the anti-tumor function of platelets is mediated through P2Y12-dependent CD40L release, which leads to CD8⁺ T cell activation by the CD40 receptor. Unlike P2Y12 inhibition, blocking platelets with aspirin does not prevent platelet CD40L release nor accelerate HCC in NAFLD mice. Similar findings were observed in liver metastasis models. All together, our study reveals a complex role of platelets in tumor regulation. Anti-platelet treatment without inhibiting CD40L release could be considered for liver cancer patients with NAFLD.

Keywords: CD40L; HCC; NAFLD; P2Y12; liver metastasis; platelets.

Figure 1.. Platelet depletion or P2Y12 inhibiton accleates HCC in NAFLD.

(A, B) Intrahepatic Hep55.1c tumor cells were implanted into MCD diet-fed mice. After tumor implantation, mice were given i.p. injection of 50 μg αGPIb antibody or IgG control every 3 days. Tumor growth was monitored by microCT scan at day 7 after tumor injection (A) or by weight at the experimental end point (B). Data are presented as mean ± s.e.m. from three independent experiments. n=26, *P <0.05, Student’s t-test. (C) Hep55.1c tumor cells were implanted into livers of MCD diet-treated mice with or without clopidogrel (Clo) treatment for 3 weeks. Mice were euthanized, and liver tumor weight was measured. Representative images are shown. Data are presented as mean ± s.e.m. from two independent experiments. n=15, *P <0.05, Student’s t-test. (D) DEN injected mice kept on control diet (CTR) were were treated with clopidogrel or vehicle for 6 months. Mice were euthanized and liver surface tumor nodules were counted. Representative liver images were shown. Cumulative data are presented as mean ± s.e.m, n=14. (E) DEN mice fed with CDAA diet and clopidogrel or vehicle for 6 month. Liver surface tumor nodules were counted. Representative liver images are shown. Cumulative data are presented as mean ± s.e.m, n=10. *p<0.05, Student’s t-test. (F) DEN mice fed with hight fat (HF) diet were treated with clopidogrel or vehicle for 5 months. Liver surface tumor nodules were counted. Cumulative data are presented as mean± s.e.m, n=15 for vehicle, 18 for Clopidogrel. *P <0.05, Student’s t-test. (G) DEN mice were fed with hight fat (HF) diet for 5 months, then mice were given clopidogrel or vehicle. Survival of HCC bearing mice was monitored. n=20 for Clo and 15 for vehicle. *p<0.05, Log-rank Mantel-Cox test. (H) MYC-ON mice fed on control diet were treated with clopidogrel or vehicle for 8 weeks. Liver surface tumor nodules were counted. Cumulative data are presented as mean ± s.e.m. n=9. (I) MYC-ON mice fed with MCD diet were treated with clopidogrel or vehicle for 4 weeks. Representitave images of H&E staining of liver sections are shown. The scale bars represent 2 mm. Microscopic tumor leisions were counted, and cumulative data are presented as mean ± s.e.m. n=13 for vehicle, 10 for Clopidogrel. *P <0.05, Student’s t-test. (J, K) Representative liver images and liver surface tumor count of western diet-CCL4 treated male C57BL/6 mice given clopidogrel or vehicle (J). Largest tumor diameter of each mouse was measured at 26-week time point (K). Data are presented as mean ± s.e.m. n=8 for vehicle 24W,10 for clopidogrel 24W, 15 for vehicle 16W or clopidogrel 26W. *P <0.05, Student’s t-test. See also Figure S1.

Figure 2.. Platelets release more CD40L in NAFLD.

(A) C57BL/6 mice were fed with CDAA, western diet or control diet for 6 months. Plasma CD40L levels were measured. Data are presented as mean± s.e.m. from two independent experiments. n=7 for CTR and CDAA, 9 for Western. *P < 0.05, one-way ANOVA. (B) Plasma CD40L levels of mice fed with MCD diet for different time periods. Cumulative data are presented as mean± s.e.m. n=5 for control, 4 for 1 week and 3 weeks, 5 for 2 and 4 weeks. *P < 0.05, one-way ANOVA. (C) Plasma CD40L level of liver Hep55.1c tumor-bearing mice fed with MCD or control diet. Data are presented as mean± s.e.m. n=10, *P <0.05, Student’s t-test. (D) Plasma CD40L level of MCD diet-fed mice given i.p. injection of α-GPIbα or IgG control. Data are presented as mean± s.e.m. from two independent experiments. n=6 for control, 8 for MCD IgG, and 6 for MCD α-GPIbα. *P < 0.05, one-way ANOVA. (E) Control diet- or MCD diet-fed mice were treated with or without clopidogrel. Mouse plasma samples were collected and incubated with MS-1 cells. MCP1 mRNA expression of MS-1 cells was measured by real-time PCR. Data are mean± s.e.m of two independent experiments. n=6 for control diet with or without clopidogrel, n=12 for MCD diet with or without clopidogrel. *P < 0.05, one-way ANOVA. (F) Freshly isolated platelets from NAFLD patients or healthy donors were incubated with different concentration of ADP for 15 minutes. Surface CD40L⁺ platelet percentage was measured by flow cytometry analysis. Cumulative data are presented as mean± s.e.m. n=15 for NAFLD, 7 for healthy. *P < 0.05, two-way ANOVA. (G) Isolated platelets were incubated with or without 5 μM of ADP for 2 hours. After centrifugation, the released CD40L was measured. Cumulative data are presented as mean± s.e.m. n=10 for NAFLD, 7 for healthy. *P < 0.05, two-way ANOVA. See also Figure S2.

Figure 3.. Platelet-derived CD40L inhibits HCC in NAFLD.

(A) MYC-ON MCD mice given i.p. injection of CD40L neutralizing antibody or IgG control. Fixed liver tissues were subjected to H&E staining. Microscopic liver tumor lesions were counted. Cumulative data are presented as mean± s.e.m. n=10 for IgG, 11 for αCD40L. *P <0.05, Student’s t-test. (B) MYC-ON CTR mice were injected with CD40L-neutralizing antibody or IgG control. Surface liver tumor nodules were counted. Representative imagines are shown in d. Cumulative data are presented as mean± s.e.m. (e). n=10, *P <0.05, Student’s t-test. (C) MYC-ON MCD mice were given i.p. injection of CD40L neutralizing antibody, control IgG or combination of CD40L neutralization with clopidogrel. Four weeks after treatment, mice were euthanized, and fixed liver tissues were subjected to H&E staining. Microscopic tumor lesions were counted. Cumulative data are presented as mean± s.e.m. n=6 for IgG, 8 for αCD40L, 8 for αCD40L+clopidogrel. *P <0.05, one-way ANOVA. (D) Hep55.1c tumor cells were orthotopically injected into the livers of CD40L^−/− mice fed with MCD diet together with or without clopidogrel. Three weeks later, mice were euthanized. Liver tumor burden was measured. Representative imagines are shown. Data are presented as mean± s.e.m. of two independent experiments. n=9 for vehicle and 8 for Clo. (E) MCD diet-fed CD40L^−/− mice were given intrahepatic injection of hep55.1c tumor cells. Tumor-bearing mice were injected i.p. with αGPIb antibody or IgG control. Liver tumor burden was measured at the end point. Data are presented as mean± s.e.m. of two independent experiments. (F,G) MCD diet-fed PF4-DTR mice were given s.c. injection of diphtheria toxin (DT) to deplete endogenous platelets. Two days after DT treatment, mice were given intrahepatic injection of hep55.1c tumor cells followed by adoptive transfer of WT or CD40L^−/− platelets isolated from MCD diet treated mice. Treatment scheme was depicted in F. At experimental end point, mice were euthanized, and liver tumors were weighed. Data are presented as mean± s.e.m. of two independent experiments. n=9 for wt platelets and 10 for CD40L^−/− platelets. *P <0.05, Student’s t-test. See also Figure S3.

Figure 4.. CD40 and CD8⁺T cells mediate the platelet dependent tumor inhibition.

(A) Control diet fed-MYC-ON mice were treated with agonistic CD40 antibody or control IgG. Representative liver images are shown. Liver surface tumor numbers were measured. Cumulative data are shown as mean± s.e.m. n=10, *P <0.05, Student’s t-test. (B) Hep55.1c tumor cells were orthotopically injected into the livers of CD40^−/− mice fed with MCD diet together with or without clopidogrel. Three weeks later, mice were euthanized, and liver tumor weight was measured. Data are presented as mean± s.e.m. of two independent experiments. n=13 for vehicle and 11 for Clo. (C) MCD diet-fed mice were injected i.p. with α-GPIbα or IgG control. CD69 expression on intrahepatic CD8⁺ T cells was measured by flow cytometry. Data are presented as mean± s.e.m. from two independent experiments. n=9 for control, 8 for MCD IgG, and 6 for MCD α-GPIbα. *P < 0.05, one-way ANOVA. (D) IFNγ expression of intrahepatic CD8⁺ T cells from MCD diet-fed mice treated with or without clopidogrel. Data are presented as mean± s.e.m. n=3 for control, 5 for MCD vehicle, and 5 for MCD Clo. *P < 0.05, one-way ANOVA. (E) IFNγ expression of intrahepatic CD8⁺ T cells was measured from the mice that received adoptively transferred platelets described in Fig. 3F. Data are presented as mean± s.e.m. of two independent experiments. n=9 for wt platelets and 10 for CD40L^−/− platelets. *P <0.05, Student’s t-test. (F,G) Hep55.1c tumor cells were implanted into livers of MCD diet-fed mice treated with or without clopidogrel, then tumor-bearing mice were injected i.p. with αCD8 (F) or αCD4 (G). Liver tumor burden was measured at end point. Data are presented as mean± s.e.m. from two independent experiments. n=20, *P <0.05, Student’s t-test. (H) Hep55.1c tumor cells were orthotopically injected into the livers of Batf3^−/− mice fed with the MCD diet together with or without clopidogrel. Liver tumor burden was measured at experimental end point. Data are presented as mean± s.e.m. n=5. See also Figure S4.

Figure 5.. P2Y12 controls platelet CD40L release and HCC growth.

(A, B) Plasma CD40L levels of MCD-fed mice treated with or without clopidogrel (A) or aspirin (Asp) (B). Cumulative data are presented as mean± s.e.m. *P < 0.05, one-way ANOVA. (C) P2Y12^−/− mice were fed with MCD diet or control diet for 4 weeks. Plasma CD40L levels were measured. Cumulative data are presented as mean± s.e.m. (D) Cysteinyl leukotrienes (LTs) levels in liver tissue lysates prepared from mice fed on MCD, CDAA or Western diet were measured by ELSIA. Data are presented as mean± s.e.m. n=10 for CTR, 5 for CDAA, 6 for Western, 7 for MCD. *P < 0.05, one-way ANOVA. (E) Plasma CD40L levels of native mice injected i.v. with a mix of cysteinyl leukotrienes. Data are presented as mean± s.e.m. from two independent experiments. n=8 for vehicle, 9 for LTs. *P <0.05, Student’s t-test. (F, G) Plasma CD40L levels (F) and intrahepatic CD8 T cell CD69 expression(G) of MCD diet-fed mice treated with or without zileuton. Data are presented as mean± s.e.m. *P < 0.05, one-way ANOVA. (H-J) Hepatic hep55.1c tumor-bearing MCD diet-fed mice were treated with or without ticagrelor (Tic, H), aspirin (Asp, I) or cilostazol (Cil, J) for three weeks. Liver tumor burden was measured. Data are presented as mean± s.e.m. from two independent experiments. n=20 for ticagrelor study, n=9 for Aspirin or cilostazol study. *P <0.05, Student’s t-test. (K) MCD diet-fed wildtype or P2Y12−/− mice with or without clopidogrel were given intrahepatic injection of hep55.1c tumor cells. Liver tumor burden was measured. Data are presented as mean± s.e.m. *P < 0.05, one-way ANOVA. (L) CD40L expression levels of bone marrow megakaryocytes from mice fed with MCD or control diet were measured by flow cytometry. Data are presented as mean± s.e.m. from two independent experiments. *P <0.05, Student’s t-test. (M) Bone marrow smears prepared from MCD or control diet-fed mice were subjected to immunohistochemistry staining of CD40L (red) and CD41 (green). Nuclei were stained by DAPI. Organ scale bars represent 200μM. Representative imagines are shown. (N) MCD diet-fed mice were injected with IL-12-neutralizing antibody or IgG control. CD40L expression levels of bone marrow megakaryocytes were measured. Data are presented as mean± s.e.m. from two independent experiments. n=6, *P <0.05, one-way ANOVA. (O) Clodronate liposome was used to deplete macrophage in MCD diet-fed mice. CD40L expression levels of bone marrow megakaryocytes were measured. Data are presented as mean± s.e.m. n=9 for CTR, 10 for MCD-PBS, 8 for MCD-Clodronate, *P <0.05, one-way ANOVA. (P) Mice were fed with MCD diet or control diet. CD40L of lung and bone marrow megakaryocytes were measured. Data are presented as mean± s.e.m. from two independent experiments. n=5, *P <0.05, one-way ANOVA. See also Figure S5.