Fig 1: CXCL4 induce myeloid-derived suppressor cells, which inhibit CD8+ T cells and promote metastasis.CXCL4 expressed in metastatic tumours makes non-metastatic 67NR tumours to become metastatic in wild-type BALB/c mice (a–c). a Heatmap of mouse cytokine expression array shows CXCL4 (highlighted in yellow) being expressed selectively in metastatic tumours. 67NR cell line (i) and 4T1 cell line (ii); 67NR tumours (iii) and 4T1 tumours (iv) harvested from wild-type BALB/c mice; 67NR tumours (v) and 4T1 tumours (vi) tumours harvested from Nude mice lacking T cells. b Flow cytometry analysis for MDSCs in bone marrow cell samples treated with vehicle, CXCL4, platelets or GM-CSF. c Quantification of MDSCs in bone marrow cell samples treated with vehicle, CXCL4, platelets or GM-CSF (n = 4). In wild-type BALB/c mice, CXCL4 induces non-metastatic 67NR cells to become metastatic from the mammary fat pad to the lung (d–k). d A cartoon showing the mammary fat pad injection (top). Representative images of whole-body bioluminescence of BALB/c wild-type mice harbouring 67NR tumour in the mammary fat pad and treated with vehicle or CXCL4 (middle) and of tumours excised from these mice (bottom) (n = 10). e The mean tumour volume of the tumours excised from these mice (n = 10). f Representative H&E staining of tumours excised from these mice (n = 5). Scale bar: 200 µm. g Representative bioluminescence images of lungs excised from BALB/c wild-type mice harbouring 67NR tumour in the mammary fat pad and treated with vehicle or CXCL4 (top). h The mean total flux from these lungs (bottom) (n = 10). i The number of visible lung nodules in vehicle- or CXCL4-treated tumour-bearing BALB/c wild-type mice (n = 7). j H&E staining of representative lungs from BALB/c mice harbouring 67NR tumour in the mammary fat pad and treated with vehicle or CXCL4. Scale bar: 200 µm. k The number of CTCs isolated from BALB/c wild-type mice harbouring 67NR tumour in the mammary fat pad and treated with vehicle or CXCL4 (n = 5). Quantitative data are mean ± SEM. Statistical analyses were performed by unpaired two-tailed Student’s t test for panels c, e, i and k or by F test for panel h; ns indicates not significant, ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗∗P ≤ 0.0001.
Fig 2: CD8+ T cells inversely correlate with platelets and predict metastasis-free survival.a CXCL4 is selectively expressed in platelets (CD61+) and not by cultured 67NR and 4T1 cells, or the 67NR or 4T1 tumour cells isolated from BALB/c mouse harbouring these tumours, or by CD45+ fractions from these mice (n = 3). b Representative images of 67NR and 4T1 tumours grown in wild-type BALB/c mice stained with CD8a and CD61 (platelets). Scale bars: 200 µm. c Quantification of immunohistochemistry of 67NR and 4T1 tumours grown in wild-type BALB/c mice stained with CD8a and CD61(n = 20, analysed five areas per tumour, a total of four tumours). d Representative images of human triple-negative breast cancer tissues stained with CD8a and CD61 (platelets), scale bars, 200 µm. e Quantification of immunohistochemistry of human triple-negative breast cancer tissues stained with CD8a and CD61 (n = 68). f Negative correlation between platelets and CD8+ T cells in breast cancer using TCGA data. g Metastasis-free survival probability of patients with a low frequency of CD8+ T cells and high frequency of platelets (CD61+ cells) relative to breast cancer patients with a high frequency of CD8+ T cells and low frequency of CD61+ cells (n = 68) in breast tissue TMA. h Overall survival probability of breast cancer patients with high levels of CD8+ T cells and low levels of platelets compared to patients with low levels of CD8+ T cells and high levels of platelets (data obtained from TCGA, n = 1093). Quantitative data are mean ± SEM. Statistical analyses were performed by unpaired two-tailed Student’s t test for panels a and c or by log-rank test for panel e; ns indicates not significant, ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001, ∗∗∗∗P ≤ 0.0001.
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