Fig 2: Stable expression of human PSGL-1 in the murine EL4.2 lymphoma cell line. A) Surface detection of human and mouse PSGL-1 (left and middle panels) and CD44 and CD90.2 (right panels) in parental EL4.2 and cell lines carrying stably hPSGL-1 or empty vector (EV). MFI, mean fluorescence index. B) Immunoblot detection of human PSGL-1 in parental EL4.2, EL4.2/EV (clone 6), EL4.2/hPSGL-1 (clone 3) and HUT-78 cell lines. C) In vitro growth of cultured parental and stably transduced EL4.2 cells after 48 h. Pooled data of five independent experiments are plotted as mean ± standard error of the mean (SEM). Indicated P values were obtained from two-tailed unpaired t-tests. D) Migration Transwell assays were performed for 24 h. Data from two pooled experiments performed each with three technical replicates are plotted as mean. Indicated P values were obtained from two-tailed unpaired t-tests. E) Flow cytometry detection of recombinant mouse P-selectin bound in vitro to parental EL4.2, EL4.2/EV and EL4.2/hPSGL1 cells under static conditions. Negative control (grey histograms) corresponds to cell incubation with secondary reagents without recombinant selectin. F) Mean fluorescence index (MFI) levels of mouse P-selectin bound to each cell line. Data are expressed as means of two pooled independent experiments. Indicated P values were obtained from two-tailed unpaired t-tests.

Fig 3: PSGL-1 expression in malignant human T cells. A) PSGL-1 surface detection (using Accuri C6 cell analyzer) in T-ALL (CEM, PF-382, DND41, P12, HPB-ALL, SupT1, and Jurkat), cutaneous T cell lymphoma (HUT-78) and B-ALL (Nalm6) cell lines. B) Immunoblot detection of PSGL-1 protein (arrowheads mark homodimeric (~240 kDa) and monomeric (~120 kDa) forms) in the indicated cell lines. Total protein loading was assessed by Ponceau and α-tubulin staining. C) PSGL-1 surface detection (using FACSCanto II cell analyzer) in primary T-ALL patient cells, Jurkat cell line, and normal T cells (CD3+CD19− gating) from healthy donor PBLs. MFI, mean fluorescence index.

Fig 4: High fibrocyte infiltrates negatively correlate with overall and disease-free survival time and positively correlate with pT.A, B Representative multispectral images of tissue samples from healthy lung and lung tumor tissue (left panel). Tissues were stained with CD45 (cyan) and Col1 (green), CD44 (yellow) and CD163 (red), CCR2 (blue) and CD162 (magenta) antibodies. Nuclei were counterstained with DAPI (white). Right side panel represents zoomed-in images from the squared area. Arrows indicate fibrocytes (CD45+Col1+CD44+CD163+CCR2+CD162+) scale bar, 200 µm (healthy), 100 µm (tumor). Quantification of fibrocytes in lung tissue from healthy donors (n = 59) relative to individuals with adenocarcinoma (ADC, n = 86), adenosquamous carcinoma (ADSCC, n = 7), mucous adenocarcinoma (MADC, n = 3), Papillary adenocarcinoma (PADC, n = 6), small cell carcinoma (SCLC, n = 8) and squamous cell carcinoma (SCC, n = 94) using a TMA. P-values were determined using One-way ANOVA with Fisher’s LSD test. C Kaplan-Meier plots reveal patient survival according to fibrocyte infiltrates in the lung tumor tissue (n = 80). D Heatmap showing the correlation of tissue categories or cell subsets with clinical parameters. Non-parametric correlation analysis (Spearman) was performed, and p-values for survival analyses were calculated using log-rank test (n = 80). Fields with significant correlations (p < 0.05) are indicated in bold script. E Representative contour plots for identification of circulating CD45+Col1+ fibrocytes from PBMCs of healthy donors and lung cancer patients. F Relative quantification of CD45+Col1+ fibrocytes from PBMCs of healthy donors (n = 20) and lung cancer patients (n = 30). p-values were determined using two-tailed unpaired t-test with Welsh’s correction. G Representative contour plots for identification of circulating (CD45+CD33+CD162+CD44+CCR2/5+CXCR3+Col1+) fibrocytes from PBMCs of healthy donors and lung cancer patients. H Relative quantification of fibrocytes from PBMCs of healthy donors (n = 4) and lung cancer patients (n = 4). p-values were determined using two-tailed unpaired t-test. Source data are provided in the source data file.

Fig 5: Single-cell RNA-seq identifies a combination of fibrocyte specific markers.A Schematic diagram of CD45+ cell isolation from the bone marrow of a C57BL/6 mouse. B Uniform manifold approximation and projection (UMAP) representation of the data clustered by the Leiden algorithm revealing 17 clusters (left). Expression of Prprc (CD45) is shown in the panel on the right. C UMAP representation of all cells belonging to cluster 13, 14, 16, and 17 by the Leiden algorithm. Expression of Serpinh1, Ccr5, Cxcr3, Selplg, Cd44, Ccr2, Cd163, S100a8 and Cd9 are shown. D Representative FACS contour plots of fibrocytes (CD45+SiglecH–, CD162+CD9+CD11b+CD44+CCR2/5+) in bone marrow of C57BL/6 mice. (E) Representative FACS contour plot and histogram showing Ly6C, F4/80, and Col1 expression in monocytes (CD11b+/Ly6G-; orange), lymphocytes (CD11b–; blue) and fibrocytes (CD45+/SiglecH–, CD162+CD9+CD11b+CD44+CCR2/5+; red). F Quantification of fibrocytes in fibrocyte-depleted mice (HSV-TK/Col1+Ganciclovir) and control mice (HSV-TK/Col1), n = 4. p-values were determined by two-tailed unpaired t–test with Welch’s correction. G Representative FACS contour plots of fibrocytes in fibrocyte-depleted (HSV-TK/Col1+Ganciclovir) and control (HSV-TK/Col1) mice. Source data are provided in the source data file.