Journal: bioRxiv

Article Title: PTPN1/2 inhibits alveolar macrophage-mediated control of lung metastasis

doi: 10.64898/2026.02.25.707995

Figure Lengend Snippet: A. C57BL/6 mice were subcutaneously implanted with 1 × 10⁶ CMT167 cells and treated daily with 20 mg/kg AC484 or vehicle. B. BALB/c mice were implanted with 1 × 10⁵ 4T1 cells in the mammary fat pad and treated as indicated. C. Representative H&E-stained and analysis markup images of lungs collected at the endpoint from A. Yellow, lung parenchyma; red, metastatic tumors; light blue, glass background. Scale bar = 2 mm. D. Tumor area quantification from C. Shown are mean ± SEM from 3 independent sections. E. Visible lung surface metastasis counts in CMT167-bearing mice at the endpoint. F. Representative H&E-stained and analysis markup images of lungs collected at the endpoint from B. Yellow, lung parenchyma; red, metastatic tumors; light blue, glass background. Scale bar = 2 mm. Arrows point to metastatic tumor regions. G. Tumor area quantification from F. Mean ± SEM from 3 sections. H. Surface metastases count in 4T1-bearing mice at the endpoint. I. Western blot of PTPN1/2 double knockout (dKO) and scrambled (Scr) control 4T1 cells treated overnight with IFNγ or IL-6 (100 ng/mL), in the presence or absence of 1 μM AC484. J. BALB/c mice implanted with dKO or Scr 4T1 cells and treated as indicated. Lung surface metastases quantified after India ink staining. K-M. NSG mice implanted with 4T1 or CMT167 cells and treated as indicated. Lung metastases were visualized and quantified separately for 4T1 ( L ) and CMT167 ( M ). N. C57BL/6 mice were treated with depleting antibodies (anti-CD8β, anti-NK1.1, or isotype control) starting on Day -1, followed by CMT167 cell implantation and AC484 treatment. Surface metastases were quantified at the endpoint. For all bar graphs, shown are individual animals (dots), with mean ± SEM (bars). Unpaired t-test for two-group comparisons; one-way ANOVA for multiple groups: ns, p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: 4T1 breast cancer cell line (CRL-2539, ATCC) and CMT167 lung cancer cell line (Clone of CMT 64, Sigma Aldrich) were respectively cultured in RPMI-1640 and Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (complete medium).

Techniques: Staining, Western Blot, Double Knockout, Control

Journal: bioRxiv

Article Title: PTPN1/2 inhibits alveolar macrophage-mediated control of lung metastasis

doi: 10.64898/2026.02.25.707995

Figure Lengend Snippet: A. Experimental scheme showing BALB/c mice were implanted with 4T1 tumor cells and treated daily with AC484 or vehicle starting on Day 7 post-implantation. Lungs were harvested on Day 22 for spatial transcriptomics. B . H&E-stained lung sections on Day 22, corresponding to A. C . Spatial mapping of cell types in the serial lung section in B. D . CellChat analysis integrates all signaling pathways to quantify the relative communication strength among cell types. E . Chord diagram illustrating cell-cell communication, with outer nodules communicating to and from immune cell types. String color indicates whether the relative signal strength is increased (Red) or decreased (Blue) in the AC484-treated lung. Edge weight corresponds to the relative increase or decrease in signal strength. Arrow directionality corresponds to whether the differentially represented signaling strength is incoming or outgoing. F . Quantification of distances between alveolar macrophages (AMs) and disseminated tumor cells across the tissue section from C. G. Representative immunofluorescence images show the classification of macrophages around metastatic lesions in the lung sections of 4T1-bearing mice. Metastatic regions are semi-transparently masked in cyan; CD11b⁻ AMs are shown in magenta, and CD11b⁺ macrophages in green. Scale bar = 100 μm (upper panel) and 50 μm (lower panel). H . Quantification of AM density across three independent lung sections within the indicated regions. Shown are mean ± SEM. Unpaired t-test: ns, p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. I . Differential gene expression analysis comparing AMs from AC484- and vehicle-treated lung from C. Genes are color-coded by the Gene Ontology (GO) pathway. The dotted line indicates the significance cutoff (BH adjusted p < 0.05).

Article Snippet: 4T1 breast cancer cell line (CRL-2539, ATCC) and CMT167 lung cancer cell line (Clone of CMT 64, Sigma Aldrich) were respectively cultured in RPMI-1640 and Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (complete medium).

Techniques: Spatial Transcriptomics, Staining, Protein-Protein interactions, Immunofluorescence, Gene Expression

Journal: bioRxiv

Article Title: PTPN1/2 inhibits alveolar macrophage-mediated control of lung metastasis

doi: 10.64898/2026.02.25.707995

Figure Lengend Snippet: A. Heatmap comparison of serum and BAL cytokine levels in 4T1-bearing BALB/c mice treated with AC484 or vehicle (harvested on Day 23; treatment started Day 7). Grey indicates values below the detection threshold; cytokines undetectable in both compartments were excluded. B-C. Quantification of specific cytokines in BAL fluid ( B ) and serum ( C ) from the dataset in A, mean ± SEM (bars). Dotted lines indicate detection limits. D. Intracellular IFNγ and CXCL9 staining in CD170⁺/CD11c⁺ alveolar macrophages (AMs) from AC484 (484-AM) or vehicle treated (Veh-AM) 4T1-bearing BALB/c mice. E. Experimental scheme showing AMs isolated from AC484 or vehicle-treated tumor-bearing mice were stimulated ex vivo with interferons or LPS. F-I. The level of p-STAT1 (Y701) in AMs following stimulation with IFNα or IFNγ (150 ng/mL, 15 minutes). F, H. Representative histograms of AMs from 4T1-bearing ( F ) and CMT167-bearing ( H ) mice. G, I. Fold change of p-STAT1 geometric MFI normalized to unstimulated controls, corresponding to F and H. J-K. The level of p-STAT1 (Y701) in AMs from 4T1-bearing animals with or without LPS stimulation (2.5 µg/mL, 2.5 h). Shown are representative histograms ( J ) and a normalized geometric MFI fold change ( K ). L. Intracellular CXCL9 levels in AMs collected from AC484 or vehicle treated tumor-bearing mice, with or without ex vivo LPS stimulation. For all bar graphs, data represent individual animals (dots) and mean ± SEM (bars). Statistical significance was determined using unpaired t-test: ns, p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: 4T1 breast cancer cell line (CRL-2539, ATCC) and CMT167 lung cancer cell line (Clone of CMT 64, Sigma Aldrich) were respectively cultured in RPMI-1640 and Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (complete medium).

Techniques: Comparison, Staining, Isolation, Ex Vivo

Journal: bioRxiv

Article Title: PTPN1/2 inhibits alveolar macrophage-mediated control of lung metastasis

doi: 10.64898/2026.02.25.707995

Figure Lengend Snippet: A. Experimental scheme showing alveolar macrophages (AMs) isolated from tumor-bearing mice were cocultured with tumor cells in vitro . B-E. The level of p-STAT1 (Y701) in AMs cocultured with tumor cells ± AC484 (100 nM). Representative histograms of CD170⁺/CD11c⁺ AMs ( B, D ) and geometric MFI fold change normalized to non-cocultured controls ( C, E ) are shown for cells derived from 4T1-bearing (B, C) or CMT167-bearing (D, E) mice. F-G. Viability of 4T1 ( F ) and CMT167 ( G ) cells after 3-day coculture with or without AMs, in the presence or absence of AC484 (300 nM). H-J. Effects of AM depletion on metastasis in the 4T1 model. H. Experimental scheme showing BALB/c mice were administered intranasally with clodronate (50 µL/mouse) or PBS liposomes starting Day -1, followed by 4T1 tumor implantation and the indicated treatment. I. Flow cytometry analysis of lung-resident CD45⁺ immune cells the day after the 3rd clodronate dose. J. Lung surface metastases were quantified at the endpoint in mice from H. K-M. Effects of AM depletion on metastasis in the CMT167 model. K. Experimental scheme for intranasal clodronate or PBS liposomes (50 µL/mouse) administration and treatment schedule in CMT167-bearing C57BL/6 mice. L. Flow cytometry analysis of lung-resident CD45⁺ immune cells. M. Lung surface metastases were quantified at the endpoint in mice from K. For all bar graphs, data represent individual animals (dots) and mean ± SEM (bars). Unpaired t-test for two-group comparisons; one-way ANOVA for more than two groups: ns, p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: 4T1 breast cancer cell line (CRL-2539, ATCC) and CMT167 lung cancer cell line (Clone of CMT 64, Sigma Aldrich) were respectively cultured in RPMI-1640 and Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (complete medium).

Techniques: Isolation, In Vitro, Derivative Assay, Liposomes, Tumor Implantation, Flow Cytometry

Journal: bioRxiv

Article Title: PTPN1/2 inhibits alveolar macrophage-mediated control of lung metastasis

doi: 10.64898/2026.02.25.707995

Figure Lengend Snippet: A, B. Viability of 4T1 ( A ) and CMT167 ( B ) cells after a 3-day coculture with or without AMs, in the presence of AC484 (50 nM) and/or IFNγ at indicated concentrations (ng/mL). C, D. Comparison of cytokine levels in conditioned medium collected from AMs cocultured with 4T1 ( C ) or CMT167 ( D ) tumor cells, supplemented with AC484 or DMSO controls. Cross makes indicate values beyond the detection range. Cytokines in the 13-plex panel that did not reach the detection limit in either condition were not plotted. E . Viability of 4T1 cells after coculture with or without AMs, comparing direct cell-to-cell contact versus separation by a transwell insert. F . Representative histograms of p-STAT1 (Y701) in AMs from wild-type (WT) or IFNγ knockout (KO) mice after 48 h coculture with CMT167 cells. AMs cultured alone serve as controls. G . Analysis of the fold change in p-STAT1 geometric MFI normalized to non-cocultured AMs from F. H . Viability of CMT167 cells after coculture with or without AMs harvested from WT or IFNγ KO mice, in the presence or absence of AC484. I . Representative histograms of p-STAT1 (Y701) in AMs after 24 h coculture with 4T1 tumor cells, with AC484 (100 nM) and/or the JAK inhibitor ruxolitinib (3 μM). AMs cultured alone with DMSO served as controls. J . Analysis of the fold change in p-STAT1 geometric MFI, normalized to DMSO-treated, non-cocultured control AMs, from experiments in I. K . Viability of 4T1 cells after coculture with or without AMs, in the presence or absence of AC484 (100 nM) and/or ruxolitinib (3 μM). For all bar graphs, data represent individual animals (dots), with mean ± SEM (bars). Unpaired t-test for two-group comparisons; one-way ANOVA for more than two groups: ns, p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: 4T1 breast cancer cell line (CRL-2539, ATCC) and CMT167 lung cancer cell line (Clone of CMT 64, Sigma Aldrich) were respectively cultured in RPMI-1640 and Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (complete medium).

Techniques: Comparison, Knock-Out, Cell Culture, Control

Journal: bioRxiv

Article Title: PTPN1/2 inhibits alveolar macrophage-mediated control of lung metastasis

doi: 10.64898/2026.02.25.707995

Figure Lengend Snippet: A-C , Effect of IFNγ neutralization in the 4T1 model. A. Experimental scheme showing BALB/c mice received anti-IFNγ antibody (200 µg/mouse) or isotype control on Day -1, followed by 4T1 implantation and the indicated treatment. B. Primary tumor growth kinetics are shown as mean ± SEM. C. Quantification of lung surface metastases at the endpoint. D-E, Effect of host IFNγ deletion in the CMT167 model. D. Experimental scheme showing IFNγ knockout (KO) or wild-type (WT) mice implanted with CMT167 cells and treated as indicated. E. Primary tumor growth kinetics are shown as mean ± SEM. F. Lung surface metastases were quantified at the endpoint in CMT167-bearing mice. G. Graphical summary showing that AC484 suppresses lung metastasis by increasing local IFNγ in the lung microenvironment and sensitizing AMs to IFNγ signaling, thereby enabling them to control metastatic tumor cells. Figure created with BioRender.com. For metastasis counting, shown are individual animals (dots) with mean ± SEM (bars). One-way ANOVA test: ns, p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: 4T1 breast cancer cell line (CRL-2539, ATCC) and CMT167 lung cancer cell line (Clone of CMT 64, Sigma Aldrich) were respectively cultured in RPMI-1640 and Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (complete medium).

Techniques: Neutralization, Control, Knock-Out