Journal: bioRxiv

Article Title: Endothelial ACKR1 expression regulates neutrophil infiltration and breast cancer metastatic engraftment in the lung metastatic niche

doi: 10.64898/2026.03.15.711832

Figure Lengend Snippet: (A) Expression of ACKR1 mRNA in mouse lung endothelial cells (L-ECs), E0771 and E0771.LMB (LMB) cells. Significance determined by one-way ANOVA. (B) Representative image of Day 28 LMB tumor in a wildtype mouse, showing adjacent mammary stroma. Dotted line indicates border between tumor (right side of image) and stroma (left side of image). Rectangle indicates inset area adjacent. CD31 = magenta, ACKR1 = green. ACKR1 is expressed in some stromal vessels (red arrowheads). (C-E) Tumor growth curve and final weights of LMB and PY8119 tumors implanted into the 4 th mammary fat pad of WT and Ackr1 Null mice. Significance determined by multiple unpaired t-test (C, E) or unpaired t-test (D). (F) Representative low magnification overviews of lung sections of wildtype and Ackr1 Null mice implanted with LMB tumors, Day 28 endpoint. Red = RFP pos tumor cells. Same mice as details shown in .

Article Snippet: Primary antibodies used included anti-mouse ACKR1 (1 μg/mL, a kind gift of Dr. Ulrich von Andrian, Harvard Medical School), anti-mouse CD31 (1:100, Cell Signaling, 77699), anti-mouse EphB4 (2.5 μg/mL, R&D Systems, AF446), anti-mouse Pan Cytokeratin (1:200, ThermoFisher/Bioss, BS-10403R), and anti-mouse Ly-6G (E6Z1T) (1:200, Cell Signaling, 87048).

Techniques: Expressing

Journal: bioRxiv

Article Title: Endothelial ACKR1 expression regulates neutrophil infiltration and breast cancer metastatic engraftment in the lung metastatic niche

doi: 10.64898/2026.03.15.711832

Figure Lengend Snippet: (A) Representative images of spontaneous lung metastases from orthotopic E0771.LMB (LMB) mammary tumors implanted into WT and Ackr1 Null mice harvested at humane endpoint (2 cm tumor diameter), Day 28 after tumor injection. Tumor cells are detected with mCherry (red) with individual tumor cells (white arrowheads) indicated in higher magnification inset. (B) Quantification of lung metastases in WT (n = 6) and Ackr1 Null (n = 7) mice. Error bars indicate s.e.m. in all figures. Significance determined by unpaired t-test. Each data point represents a single mouse, in which a minimum of three sections at different depths within the lung have been analyzed and averaged. (C) Representative lung sections from tumor-free (TF) mice and mice bearing LMB and AT3 orthotopic mammary tumors harvested at humane endpoints (Day 28 and Day 40, respectively). Immunofluorescent (IF) staining of endothelial cells with CD31 (magenta), ACKR1 (green) and DAPI (blue). ACKR1-expressing vessels indicated with white arrowheads. (D) Quantification of ACKR1-positive vessels per mm 2 in TF, LMB-bearing, and AT3-bearing mouse lungs. Significance determined by one-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: Primary antibodies used included anti-mouse ACKR1 (1 μg/mL, a kind gift of Dr. Ulrich von Andrian, Harvard Medical School), anti-mouse CD31 (1:100, Cell Signaling, 77699), anti-mouse EphB4 (2.5 μg/mL, R&D Systems, AF446), anti-mouse Pan Cytokeratin (1:200, ThermoFisher/Bioss, BS-10403R), and anti-mouse Ly-6G (E6Z1T) (1:200, Cell Signaling, 87048).

Techniques: Injection, Staining, Expressing

Journal: bioRxiv

Article Title: Endothelial ACKR1 expression regulates neutrophil infiltration and breast cancer metastatic engraftment in the lung metastatic niche

doi: 10.64898/2026.03.15.711832

Figure Lengend Snippet: (A) Representative IF images of lungs from C57BL6/J wildtype mice at 3, 7, 13, and 28 days after orthotopic injection with LMB cells or tumor-free (TF) PBS mock injection. CD31 = magenta, ACKR1 = green, DAPI = blue in all panels. ACKR1-positive endothelial cells indicated by white arrowheads. Fainter ACKR1 staining can also be seen in red blood cells. (B) Representative IF images of lungs from non-transgenic or MMTV-PyMT transgenic C57BL6/J mice at 8-10 weeks (hyperplasia stage) and 18-22 weeks (late stage carcinoma). (C) Number of disseminated LMB tumor cells per mm 2 in lungs at indicated timepoints after tumor implantation. One-way ANOVA with Sidak’s multiple comparisons test. (D) Average number of ACKR1-positive vessels per lung section at indicated timepoints after tumor implantation. Unpaired t-test. (E) Quantification of ACKR1-positive vessels per mm 2 in lungs of non-transgenic (18 week old), MMTV-PyMT transgenic at hyperplasia stage (8-10 week old, HYP) and MMTV-PyMT transgenic at late stage carcinoma (18-22 week old, LSC) mice. One-way ANOVA with Sidak’s multiple comparisons test. For all panels, each data point represents a single mouse, in which a minimum of three sections at different depths within the lung have been analyzed and averaged.

Article Snippet: Primary antibodies used included anti-mouse ACKR1 (1 μg/mL, a kind gift of Dr. Ulrich von Andrian, Harvard Medical School), anti-mouse CD31 (1:100, Cell Signaling, 77699), anti-mouse EphB4 (2.5 μg/mL, R&D Systems, AF446), anti-mouse Pan Cytokeratin (1:200, ThermoFisher/Bioss, BS-10403R), and anti-mouse Ly-6G (E6Z1T) (1:200, Cell Signaling, 87048).

Techniques: Injection, Staining, Transgenic Assay, Tumor Implantation

Journal: bioRxiv

Article Title: Endothelial ACKR1 expression regulates neutrophil infiltration and breast cancer metastatic engraftment in the lung metastatic niche

doi: 10.64898/2026.03.15.711832

Figure Lengend Snippet: (A) Schematic of tumor-conditioned media production and administration. (B) Representative IF images of lungs from C57BLJ/6 wildtype mice treated daily for 7 days with 300µl tumor-free media (TFM) or media conditioned by incubation with LMB tumor cells (TCM-LMB) (CD31 = magenta, ACKR1 = green, DAPI = blue); ACKR1-expressing venule is indicated by white arrowhead. (C) Quantification of ACKR1-positive vessels per mm 2 in lungs of mice treated with TFM or TCM for 7 days. Unpaired t-test. (D) qPCR of ACKR1 mRNA expression in HUVEC monolayers treated with TFM, MDA-MB-231-conditioned media (TCM-MDA), or TCM-LMB. One-way ANOVA with Sidak’s multiple comparisons test. (E-F) Flow cytometry analysis of cell surface ACKR1 expression in HUVEC treated with TFM or TCM-MDA for 24 hours. ACKR1 pos or ACKR1 high numbers expressed as a percentage of CD31 pos cells. One-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: Primary antibodies used included anti-mouse ACKR1 (1 μg/mL, a kind gift of Dr. Ulrich von Andrian, Harvard Medical School), anti-mouse CD31 (1:100, Cell Signaling, 77699), anti-mouse EphB4 (2.5 μg/mL, R&D Systems, AF446), anti-mouse Pan Cytokeratin (1:200, ThermoFisher/Bioss, BS-10403R), and anti-mouse Ly-6G (E6Z1T) (1:200, Cell Signaling, 87048).

Techniques: Incubation, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Endothelial ACKR1 expression regulates neutrophil infiltration and breast cancer metastatic engraftment in the lung metastatic niche

doi: 10.64898/2026.03.15.711832

Figure Lengend Snippet: (A) Representative image of ACKR1-positive lung vessel from LMB-bearing mouse 28 days after implantation, showing colocalization of ACKR1 with venous endothelial marker EphB4. CD31 = magenta, EphB4 = yellow, ACKR1 = green. All ACKR1 pos vessels also expressed EphB4. 22.7±1.4% of EphB4 pos venules expressed ACKR1. Quantitation shown in . (B) Representative annotated IF image for QuPath-based proximity analysis of tumor cell distance to nearest venule (EphB4 = yellow, Pan-CK = tumor cell marker pancytokeratin in red, ACKR1 = green, and DAPI = blue). For QuPath annotations, yellow line = vessel border, red circle = disseminated tumor cell (DTC), red line = shortest distance between DTC and nearest venule border. (C) Quantification of the number of DTCs within indicated distance from the border of ACKR1-positive or ACKR1-negative venules in Day 28 LMB-bearing mouse lungs (n = 116 ACKR1 pos venules, n = 100 ACKR1 neg venules, across 4 individual mice). Unpaired t-tests. Summed data shown here for clarity, individual data points shown in . (D) Percentage of ACKR1-positive and ACKR1-negative venules with at least one DTC within 50 µm of the venule border. Chi-squared test. (E) Representative IF images for QuPath proximity analysis of neutrophil distance to nearest venule, as described in panel B except Ly6G = neutrophil marker (cyan) and red lines indicate shortest distance between neutrophil and nearest venule border. (F) Quantification of the number of neutrophils within indicated distance from the border of ACKR1-positive or ACKR1-negative venules in Day 28 LMB-bearing mouse lungs (n = 16 ACKR1 pos venules, n = 16 ACKR1 neg venules, across 3 individual mice). Unpaired t-tests. Summed data shown here for clarity, individual data points shown in .

Article Snippet: Primary antibodies used included anti-mouse ACKR1 (1 μg/mL, a kind gift of Dr. Ulrich von Andrian, Harvard Medical School), anti-mouse CD31 (1:100, Cell Signaling, 77699), anti-mouse EphB4 (2.5 μg/mL, R&D Systems, AF446), anti-mouse Pan Cytokeratin (1:200, ThermoFisher/Bioss, BS-10403R), and anti-mouse Ly-6G (E6Z1T) (1:200, Cell Signaling, 87048).

Techniques: Marker, Quantitation Assay

Journal: bioRxiv

Article Title: Endothelial ACKR1 expression regulates neutrophil infiltration and breast cancer metastatic engraftment in the lung metastatic niche

doi: 10.64898/2026.03.15.711832

Figure Lengend Snippet: (A) Schematic of the Ackr1 floxed allele and its excision in endothelial cells following tamoxifen-induced Cdh5CreERT2 recombination to generate the Ackr1 ECKO mouse model. Consensus GATA/TAL binding sequences (black triangles) were not disrupted when inserting the loxP sites (red triangles). (B) Representative IF images (n=2 mice per genotype) of peripheral lymph nodes, which constitutively express ACKR1 in endothelial cells, confirming the loss of ACKR1 protein in Ackr1 ECKO mice (CD31 = magenta, ACKR1 = green). (C) Flow cytometry analysis of peripheral blood confirms retention of ACKR1 expression in red blood cells of Ackr1 ECKO mice. TER-119 = red blood cell marker, anti-ACKR1 = blue peaks, isotype control = red peaks. Control = Cdh5CreERT2;Ackr1 WT , Null = Ackr1 Null (negative control), Flox = Ackr1 flox/flox (no cre control), ECKO = Cdh5CreERT2;Ackr1 flox/flox (experimental). (D) Representative images of Day 28 LMB mammary tumor-bearing Control and Ackr1 ECKO mouse lungs confirming loss of endothelial ACKR1 in Ackr1 ECKO mouse lungs after induction by primary mammary tumors (CD31 = magenta, ACKR1 = green). (E) Quantification of loss of ACKR1-positive vessels in Day 28 LMB tumor-bearing Control (n=3) and Ackr1 ECKO (n=4) mouse lungs. Unpaired t-test.

Article Snippet: Primary antibodies used included anti-mouse ACKR1 (1 μg/mL, a kind gift of Dr. Ulrich von Andrian, Harvard Medical School), anti-mouse CD31 (1:100, Cell Signaling, 77699), anti-mouse EphB4 (2.5 μg/mL, R&D Systems, AF446), anti-mouse Pan Cytokeratin (1:200, ThermoFisher/Bioss, BS-10403R), and anti-mouse Ly-6G (E6Z1T) (1:200, Cell Signaling, 87048).

Techniques: Binding Assay, Flow Cytometry, Expressing, Marker, Control, Negative Control