Fig 2: 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 3: 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.

Fig 4: Fibrocyte depletion reduces lung tumor progression in LLC1 syngeneic mouse lung tumor models.A Photographs (upper panel) of whole lung (scale bar, 2 mm) and Hematoxylin and eosin (H&E) stainings (down panel) of lung sections (scale bar, 2.5 mm) that represents intravenous (i.v.) lung metastasis model in fibrocyte-depleted (HSV-TK/Col1+Ganciclovir) and control mice (HSV-TK/Col1+BM and control), n = 5. B Quantification of macroscopic and microscopic lung tumor nodules from fibrocyte-depleted and control lung tumors, n = 5. C Quantification of PCNA+ proliferating cells (n = 5, 7 images per lung tumor) and vWF+ vessels (n = 5, 5 images per lung tumor) from fibrocyte-depleted and control lung tumors by immunohistochemistry. D Quantification of fibrocytes (CD45+CD162+F4/80+CD9+CCR2/5+) and macrophages (CD45+CD162+F4/80+CD9–CCR2/5–) from fibrocyte-depleted and control lung tumors by FACS analysis, n = 5. E Quantification of fibroblasts (CD45–CD163–Col1+), fibrocytes (CD45+CD163+CCR2+Col1+), and macrophages (CD45+CD163+CCR2–Col1–), M2-like (CD206+) and M1-like (TNF+) macrophages from fibrocyte-depleted and control lung tumors by multiplex immunofluorescence, n = 3. F Representative photographs (upper panel) of whole lung (scale bar, 2 mm) and H&E stainings (down panel) of lung sections (scale bar, 2.5 mm) to represent tumor relapse (t.r.) lung metastasis model in fibrocyte-depleted and control mice, n = 5. G Quantification of macroscopic and microscopic lung tumor nodules from fibrocyte-depleted and control lung tumors, n = 5. H Quantification of PCNA+ proliferating cells (n = 4, 7 images per lung tumor) and vWF+ vessels (n = 4, 5 images per lung tumor) from fibrocyte-depleted and control lung tumors by immunohistochemistry. I Quantification of fibrocytes (CD45+CD162+F4/80+CD9+CCR2/5+) and macrophages (CD45+CD162+F4/80+CD9–CCR2/5–) with the markers described above from fibrocyte-depleted and control lung tumors by FACS analysis, n = 4. J Quantification of fibroblasts (CD45-CD163–Col1+), fibrocytes (CD45+CD163+CCR2+Col1+), macrophages (CD45+CD163+CCR2–Col1–), M2-like (CD206+) and M1-like (TNF+) macrophages from fibrocyte-depleted and control lung tumors by multiplex immunofluorescence, n = 3. B–E, G–J p-values were determined using One-way ANOVA with Fisher’s LSD test. Source data are provided in the source data file.

Fig 5: Co-injection of fibrocytes and fibroblasts with lung cancer cells augments tumor growth.Human fibrocytes were isolated from PBMCs. A Fibrocytes were depicted by bright field microscopy with different magnifications (n = 10, scale bar, 200 µm, and 50 µm). B Representative flow cytometry plots show the purity of fibrocytes using CD45+, Col1+, CD44+, CD163+ CCR5+, CD33+, S100A8+ CD162+, CXCR3+, CCR2+ as markers compared to fibroblasts. Col1 fluorescence minus one (FMO) control indicates efficacy of the Col1 antibody. C–G Co-injection of human lung cancer A549 cells with fibrocytes and fibroblasts into BALB/c nude mice. C Tumor size from mice injected with A549 (cancer cells) alone or co-injected with fibrocytes and fibroblasts. p values were determined using Two-way ANOVA with Fisher’s LSD test, n = 5. Representative pictures (scale bar, 2 mm) and (D) tumor weight of tumors from mice injected with A549 or co-injected with fibrocytes and fibroblasts after 32 days. n = 5, p-values were determined using One-way ANOVA with Fisher’s LSD test. E Quantification of PCNA+ proliferating cells (n = 5, 5 images per lung tumor) and vWF+ vessels (n = 5, 5 images per lung tumor) in tumor tissues from mice injected with A549 or co-injected with fibrocytes and fibroblasts by immunohistochemistry. p-values were determined using One-way ANOVA with Fisher’s LSD test. F Quantification of fibrocytes (CD45+CD11b+CD162+F4/80+CCR2/5+CD9+CD44+Col1+) and macrophages (CD45+CD11b+CD162+F4/80+CCR2/5–CD9–) in tumors from mice injected with A549 or co-injected with fibrocytes and fibroblasts by FACS analysis, n = 5, p-values were determined using One-way ANOVA with Fisher’s LSD test. G Quantification of fibroblasts (CD45–CD163–Col1+), fibrocytes (CD45+CD163+CCR2+Col1+), and macrophages (CD45+CD163+CCR2–Col1–), M2-like (CD206+) and M1-like (TNF+) in tumors from mice injected with A549 or co-injected with fibrocytes and fibroblasts by multiplex immunofluorescence, n = 3. p-values were determined using One-way ANOVA with Fisher’s LSD test. Source data are provided in the source data file.