Animals were housed in a non-barrier animal facility under pathogen-free conditions, 12-hour light/dark cycle, and access to standard rodent diet and water ad libitum. Experiments were performed in accordance with AAALAC guidelines and with Vanderbilt University Medical Center Institutional Animal Care and Use Committee approval. All mice used in this study were immunocompetent BALB/c mice. Ephrin-A1 knockout ( Efna1 ^-/-) mice were previously characterized by our lab ⁴⁴ . To generate littermate controls, wild-type BALB/c mice were purchased from Jackson Laboratory and mated with Efna1 ^-/- mice to generate heterozygote mating pairs. Efna1 ^+/+, Efna1 ^+/- and Efna1 ^-/- animals were identified by PCR analysis of genomic DNA using the following primers: Forward primer (5’-TGGTTATATCCCCCCACCTCACAC-3’) and two allele-specific reverse primers (WT 5’-AAGGACTCCCATATCTCAGCGACG-3’) and (KO 5’-AGACTGCCTTGGGAAAAGCG-3’). Mice were co-housed with one to four littermates for at least two weeks prior to and during all experiments and compared with littermate controls whenever possible. All mice used for tumor experiments were six to ten weeks old at the onset on the experiment. Experimental cohorts were limited to litters that were born within two consecutive weeks and that also had at least one Efna1 ^-/- and Efna1 ^+/+ gender-matched littermate pair and, when applicable, at least one Efna1 ^+/- gender-matched littermate. Sample sizes are as shown in the figures and range from three to twelve mice per group. At experimental endpoints, mice were euthanized by cervical dislocation.

4T1 murine mammary adenocarcinoma cells were purchased from ATCC and maintained in DMEM (Corning #MT10013CV) supplemented with penicillin/streptomycin (Gibco #15140163) and 10% FBS (Gibco #A3160502). 4T1-GFP-luciferase clones were generated by serial dilutions of 4T1 cells with lentiviral overexpression of GFP and luciferase genes.

To reflect human breast cancer, only female mice were used for tumor experiments. For orthotopic mammary tumor implantations, 1×10 ⁵ 4T1 cells suspended in a 1:1 mixture of PBS and Growth Factor-Reduced Matrigel (Corning #354230) were injected through the nipple into the fourth mammary fat pads of six to eight-week-old female mice. Tumor dimensions were measured by digital caliper at given time points every other day, and volume was calculated using the following formula: volume = length × width ² × 0.52. To observe spontaneous lung metastases and primary tumor recurrence, mammary tumors were resected at day 14 post-implantation, along with draining inguinal lymph nodes and surrounding fat pads, and mice were ultimately sacrificed at day 32. At the time of surgical resection of primary tumors on day 14, tumors were weighed and cut in half to provide tissue for both flow cytometry analysis and cryosection staining. At the experimental endpoint on day 32, tumors were weighed, and lung metastases were counted in a blinded manner. For lung colonization experiments, 4T1-GFP-luciferase cells suspended in PBS were injected via tail vein, and mice underwent in vivo bioluminescence imaging with a PerkinElmer IVIS Spectrum several hours post-injection to verify successful and equal delivery of 4T1 cells. To observe gradual formation of GFP+ metastases, 1×10 ⁵ 4T1-GFP-luciferase cells were injected via tail vein, and mice were sacrificed at day 17. GFP+ lung metastases were counted in a blinded manner. The left lung lobe of each mouse was fixed in 10% formalin for subsequent formalin-fixed paraffin-embedded (FFPE) processing, sectioning, and H&E staining, while the other lung lobes were processed for flow cytometry analysis. To observe early colonization and proliferation of 4T1 cells, 5×10 ⁵ 4T1-GFP-luciferase cells labeled with CellTrace Violet dye (Invitrogen #C34571) were injected via tail vein. At 24 hours, mice were sacrificed, and lungs were perfused with PBS and processed for flow cytometry analysis.

FFPE lung sections were prepared and stained for PCNA (1:100, BD Biosciences #555567 raised in mouse, RRID: AB_395947) as described previously ⁴⁵ . Slides were blinded, and the number of metastatic foci per section of lobe was quantified. Nuclear PCNA staining was analyzed using ImageJ v1.52o with the IHC Profiler plugin ⁴⁶ and percentage of PCNA+ tumor cell nuclei were quantified. Each data point is an average of two sections of the left lung from an individual mouse. To prepare cryosections, mammary tumors were frozen in OCT Compound (Thermo Fisher Scientific #23-730-571) on dry ice and stored at -80°C. Sections (8 µm) were cut on a Leica Cryostat CM1950, fixed in 4% PFA, washed with PBS, permeabilized with 0.3% Triton X-100 (Sigma-Aldrich #X100), and blocked using M.O.M. Mouse Ig Blocking Reagent and Protein Concentrate (Vector Laboratories #PK-2200) per manufacturer recommendations and with 2.5% goat serum (Sigma-Aldrich #G9023) in PBS. Slides were then incubated over two nights at 4°C with primary antibodies against CD31 (1:150, Biolegend #102501 raised in rat, RRID: AB_312908) and αSMA (1:150, Dako #M085129-2 raised in mouse, RRID: AB_2811108) in blocking buffer. After washing with PBS, slides were incubated for one hour at room temperature in secondary antibodies goat anti-rat Ax594 (1:500, Invitrogen #A11007, RRID: AB_10561522) and anti-mouse Ax488 (1:500, Invitrogen #A11001, RRID: AB_2534069), washed with PBS, and mounted with ProLong Gold Antifade Mountant with DAPI (Invitrogen #P36931). Slides were blinded, and images were taken by an Olympus DP72 camera through a BX60 inverted fluorescence microscope and processed using CellSens Dimension software. A total of 12-40 20x fields of view were analyzed from each section using ImageJ. For αSMA analysis, images were evaluated for colocalization with CD31 staining, and data was displayed as a percentage of αSMA+ out of CD31+ area or integrated intensity. Each data point is an average of all fields of view of two to three tumor sections from an individual mouse.

Tumors and lungs were minced and dissociated in RPMI-1640 media (Corning #MT10040CV) containing 2.5% FBS, 1 mg/ml collagenase IA (Sigma-Aldrich #C9891), and 0.25 mg/ml DNase I (Sigma-Aldrich #DN25) for 45 minutes at 37°C. Digested tissue was then filtered through a 70-µm strainer, and red blood cells were lysed using ACK Lysis Buffer (KD Medical #RGF-3015). Samples were washed with PBS and stained with Ghost Dye Violet V510 (Tonbo Biosciences #13-0870) to exclude dead cells. After washing with buffer (0.5% BSA, 2mM EDTA in PBS), samples were blocked in αCD16/32 mouse Fc block (Tonbo Biosciences #70-0161) and stained for extracellular proteins using an antibody master mix made in buffer. After washing with buffer, cells were fixed with 2% PFA. For FoxP3 intracellular staining, cells were permeabilized using the FoxP3 Transcription Factor Staining Kit (Tonbo Biosciences #TNB-0607-KIT) per manufacturer protocol. Flow cytometry data was obtained on a BD 4-laser Fortessa using BD FACS Diva software v8.0.1 and analyzed using FlowJo software v10.6.1. Fluorescence minus one (FMO) samples were used as gating controls when needed. Antibodies used in flow panels are detailed in Table 1, and gating strategies used in analysis are detailed in Table 2. Each data point is generated after analyzing at least 5×10 ⁵ viable cells from a specimen from an individual mouse.

All graphs and statistical analysis were completed using GraphPad Prism software v6.07. For comparisons between two groups, an unpaired Mann-Whitney U-test was performed. For comparisons between three groups, a Kruskal-Wallis H-test was performed, followed by post-hoc Mann-Whitney U-tests evaluating differences between the knockout and either the wild-type or heterozygote animals. A P-value less than 0.05 was considered statistically significant.

We initially investigated the impact of ephrin-A1 host deficiency on primary tumor growth by implanting 4T1 cells orthotopically into the mammary fat pads of syngeneic BALB/c female Efna1 ^+/+ and Efna1 ^-/- mice. No difference in primary tumor growth or weight at 21 days post-implantation was observed ( Figure 1A). To test the impact of ephrin-A1 host deficiency on spontaneous metastasis, 4T1 cells were implanted orthotopically as described above and surgically resected on day 14 post-implantation to allow for gradual development of endogenous metastases by day 32 ( Figure 1B). As expected, primary tumors resected from Efna1 ^+/+ and Efna1 ^-/- mice were not different in size ( Figure 1C), and this was additionally verified with Efna1 ^+/+, Efna1 ^+/-, and Efna1 ^-/- littermates ( Figure 1D). However, at the experimental endpoint, the number of visible lung metastases and lung weights were significantly decreased in knockout mice ( Figure 1E). Many of these mice not only harbored lung metastases but also tumors that had regrown at the original site of the resected primary tumor. Similar to our findings in lung metastases, the size of recurrent primary tumors was significantly reduced in knockout mice ( Figure 1F). Together, these results demonstrate that while host deficiency in ephrin-A1 may not affect initial tumor growth, it can impact metastatic spread and recurrence. Underlying data are available ^{47, 48} .

To complement our findings in our model of spontaneous metastasis, we evaluated the impact of ephrin-A1 host deficiency on experimental metastasis. 4T1 cells engineered to express GFP and luciferase (4T1-GFP-luciferase) were injected into the tail veins of Efna1 ^+/+, Efna1 ^+/-, and Efna1 ^-/- littermates. In vivo bioluminescence imaging several hours after injection illustrated comparable signal across all mice ( Figure 2A, B), indicating ephrin-A1 host deficiency did not impact tumor cell trafficking and lodging within the lung, at least in this short time frame. After harvesting the lungs 17 days later, we observed decreased GFP+ metastases in Efna1 ^-/- mice, compared to both Efna1 ^+/+ and Efna1 ^+/- littermates, which was additionally confirmed by flow cytometry ( Figure 2C, D). Similarly, histological analysis revealed fewer metastatic foci in lungs from Efna1 ^-/- mice ( Figure 2E). These data align with our previous observations on endogenous metastasis and suggest that host deficiency in ephrin-A1 inhibits circulating cancer cells from colonizing the lung. Underlying data are available ^{49, 50} .

Ephrin-A1 is expressed in several types of host cells, including immune cells and endothelial cells ^{39– 41} . Thus, we sought to determine how the ephrin-A1-deficient host immune system and endothelium may mitigate metastasis. Among immune cells, Ephrin-A1 can be expressed in B and T cells, monocytes, and macrophages ³⁹ . The role of ephrin-A1 in B cells is largely unknown ⁵¹ . However, in T cells, monocytes, and macrophages, ephrin-A1 has been shown to regulate cell adhesion and migration ^{11, 12, 15, 27, 52– 55} . These immune cell populations play a critical role in overall anti-tumor immunity and immunosurveillance. Dendritic cells and T cells, particularly CD8 cytotoxic T cells, are the primary drivers of the adaptive anti-tumor response in solid tumors and increased infiltration of these cell types is correlated with better prognosis and enhanced response to immunotherapies ^{56, 57} . Conversely, T regulatory cells (Tregs) suppress effector functions of T cells and typically inhibit the anti-tumor response ^{58, 59} . Between the two ends of this spectrum, myeloid populations, such as monocytes, macrophages, and granulocytes, can either promote or suppress an anti-tumor response, depending on their polarization and functionality ^{60, 61} .

Because of ephrin-A1’s known role in adhesion and chemotaxis of immune cells, we performed flow cytometry analysis on 4T1 primary tumors harvested from Efna1 ^+/+, Efna1 ^+/-, and Efna ^-/- littermates. To our surprise, we found no significant differences in total infiltrating immune cells, CD4 or CD8 T cells, dendritic cells, Tregs, or myeloid populations in Efna1 ^+/+, Efna1 ^+/-, and Efna ^-/- littermates ( Figure 3A, B). While there were no apparent differences in the immune microenvironment of the mammary tumors, the immune microenvironment of the lung is distinct from that of the mammary gland and may impact the metastatic niche. To investigate this, we performed flow cytometry analysis on 4T1 tumor-bearing lungs generated from our model of experimental metastasis. Similar to the results we obtained from the 4T1 primary tumors, we did not see significant differences in immune populations in tumor-bearing lungs harvested from Efna1 ^+/+, Efna1 ^+/-, and Efna1 ^-/- littermates, except for a modest increase in macrophages in knockout mice ( Figure 3C, D). Underlying data are available ^{62, 63} .

Although the percentage of tumor infiltrating T cells in Efna1 ^+/+ and Efna1 ^-/- mice is comparable, their activation status and effector function may still be different. Tumor-infiltrating T cells with upregulated expression of activation markers, such as CD44, CD69, and CD25, and downregulated expression of antigen-naïve markers like CD62L and exhaustion markers like PD-1 and CTLA-4 indicate a higher T cell functional status that mediates a stronger and more enduring anti-tumor response ^{58, 59} . We assessed these markers on T cells in 4T1 primary tumors and tumor-bearing lungs from Efna1 ^+/+ and Efna1 ^-/- littermates using flow cytometry. However, we did not observe consistent increases in activation or decreases in naïve or exhaustion markers in knockout-derived T cells (data not shown, included in Underlying data) ⁶² . In summary, host deficiency in ephrin-A1 does not significantly affect tumor-infiltrating immune cells in both primary tumors and tumor-bearing lungs. Thus, the reduction of lung metastases in Efna1 ^-/- hosts in vivo is unlikely due to host immunity.

In addition to the anti-tumor immune response, another host factor that can impact metastasis is the tumor vasculature. Angiogenesis is the formation of new blood vessels from a pre-existing network and is required for solid tumor growth and progression. Blood vessels can supply nutrients that support tumor growth and provide an entry for hematological dissemination and invasion ^{1, 64} . These new blood vessels are typically hastily constructed in response to the high release of growth factors, such as vascular endothelial growth factor (VEGF), from tumor cells ^{65, 66} . Thus, tumor vessels tend to be disorganized, leaky, and poorly covered by pericytes, which normally support the integrity of the endothelium. Ephrin-A1 is expressed in the vascular endothelium and has been shown to promote angiogenesis in vitro and in several in vivo models ^{67– 72} . Therefore, we hypothesized that tumors in Efna1 ^-/- mice may have reduced tumor vasculature and increased endothelial pericyte coverage compared to Efna1 ^+/+ controls.

To evaluate tumor vascularity and vessel function, we co-stained cryosections of 4T1 primary tumors from Efna1 ^+/+ and Efna1 ^-/- littermates with CD31 and αSMA, markers for endothelial cells and pericytes, respectively. Colocalization of αSMA with CD31 acts as an indicator for functional endothelium within tumors. Surprisingly, we did not observe a change in CD31+ area or intensity in 4T1 tumors from Efna1 ^+/+ and Efna1 ^-/- littermates ( Figure 4A, B). Furthermore, pericyte coverage on tumor vessels remained the same in tumors from Efna1 ^+/+ and Efna1 ^-/- littermates ( Figure 4A, C). Together, these data suggest that loss of ephrin-A1 in the host does not affect tumor vessel formation and function in the primary tumor. Underlying data are available ⁷³ .

Our results from analysis of the immune infiltrate and vasculature of primary tumors, coupled with the significant difference in experimental lung metastasis between Efna1 ^+/+ and Efna1 ^-/- mice, suggest that host factors critical to this metastatic phenotype are more likely to lie downstream of the primary tumor site. These steps include tumor cell trafficking to the lung vasculature, extravasation, and adaptation to new selective pressures of the lung microenvironment. 4T1 cell trafficking to the lung was not significantly different between Efna1 ^+/+ and Efna1 ^-/- littermates after tail vein injections ( Figure 2A, B). Thus, we aimed to evaluate extravasation and adaptation to the lung metastatic niche in Efna1 ^+/+ and Efna1 ^-/- hosts.

To test extravasation of 4T1 cells in vivo, we injected 4T1-GFP-luciferase cells labeled with CellTrace Violet dye into the tail veins of Efna1 ^+/+ and Efna1 ^-/- littermates. This dye is retained only in the labeled tumor cells and diminished after subsequent cell divisions, enabling quantification of short-term cell proliferation. At 24 hours after injection, we perfused the lungs with PBS to flush out remaining cells in the pulmonary vasculature and processed the lungs for flow cytometry. Decreased GFP+ 4T1 cells were found in ephrin-A1-deficient lungs compared to wild-type controls ( Figure 5A), suggesting that fewer cancer cells had extravasated into the lung parenchyma at this timepoint. This result was not due to decreased vascularity of ephrin-A1-deficient lungs at baseline, as CD31+ endothelial cells were not significantly different in knockout and wild-type lungs ( Figure 5B). Moreover, this was not due to decreased proliferation of the 4T1 cells within the 24-hour timeframe, as the amount of retained CellTrace Violet dye was not increased in 4T1 cells that had extravasated in knockout lungs compared to wild-type lungs ( Figure 5C). Together, these data suggest that extravasation of 4T1 cells is inhibited in knockout mice, compared to wild-type controls, and ephrin-A1 deficiency in the host lung may play a role in this process.

While decreased extravasation of tumor cells may explain in part the decreased lung metastases in Efna1 ^-/- mice, another possibility is that once tumor cells have extravasated and established in the lung, they have reduced fitness of survival in ephrin-A1-deficient lungs, compared to wild-type lungs. There are many stressors in the lung metastatic niche that could impact the adaptability of the tumor cell. We used tumor proliferation index as a marker to evaluate how well tumor cells have adapted to a metastatic niche. Since no differences were observed in proliferation of 4T1 cells that had newly extravasated into the lung parenchyma of Efna1 ^+/+ and Efna1 ^-/- littermates within the short 24-hour timeframe ( Figure 5C), we next assessed proliferation of tumor cells in lung micrometastases that had established over 17 days after tail vein injection ( Figure 2). There was a significant decrease in cell proliferation in metastatic foci established in Efna1 ^-/- mice, compared to Efna1 ^+/+ controls, as indicated by PCNA staining ( Figure 5D). These findings suggest that reduced tumor cell lung colonization in Efna1 ^-/- hosts is due to both decreased extravasation of cancer cells and decreased proliferation in the metastatic niche. Underlying data are available ^{74, 75} .

Harvard Dataverse: Host deficiency in ephrin-A1 inhibits breast cancer metastasis; https://dataverse.harvard.edu/dataverse/hostEfna1metastasis.

This project contains the following underlying data:

Harvard Dataverse: 4T1 primary tumor dimensions and weights. https://doi.org/10.7910/DVN/AGKDWV ⁴⁷ . (4T1 primary tumor dimensions from digital caliper measurements, volume calculations, and weights (related to Figure 1A, C, D.)

Harvard Dataverse: 4T1 recurrent primary and spontaneous lung metastases. https://doi.org/10.7910/DVN/FU8JEY ⁴⁸ . (Spontaneous 4T1 lung metastases quantification and recurrent primary tumor weights (related to Figure 1E, F.)

Harvard Dataverse: Images and quantification of 4T1-GFP-luciferase spontaneous lung metastases. https://doi.org/10.7910/DVN/2ANDYX ⁴⁹ . (Experimental 4T1-GFP-luciferase lung metastases quantification and images (related to Figure 2C-E.))

Harvard Dataverse: 4T1-GFP-luciferase bioluminescence images and quantification post-tail vein injection. https://doi.org/10.7910/DVN/39D0YR ⁵⁰ . (4T1-GFP-luciferase bioluminescence quantification and images (related to Figure 2A, B).)

Harvard Dataverse: 4T1 primary tumor flow cytometry. https://doi.org/10.7910/DVN/ZRX2RG ⁶² . (Flow cytometry files (fcs), gating and analysis (wsp), and panels (xlsx) containing immune profiling of 4T1 primary mammary tumors from Efna1 ^+/+, Efna1 ^+/-, and Efna1 ^-/- littermate mice (related to Figure 3A, B))

Harvard Dataverse: 4T1-GFP-luciferase tumor-bearing lung flow cytometry. https://doi.org/10.7910/DVN/S06NQ1 ⁶³ . (Flow cytometry files (fcs), gating and analysis (wsp), and panels (xlsx) containing immune profiling of 4T1-GFP-luciferase tumor-bearing lungs from Efna1 ^+/+, Efna1 ^+/-, and Efna1 ^-/- littermate mice (related to Figure 2D, 3C, D).)

Harvard Dataverse: 4T1-GFP-luciferase 24-hr lung colonization flow cytometry. https://doi.org/10.7910/DVN/G7TAAE ⁷⁴ . (Flow cytometry files (fcs), gating and analysis (wsp), and panels (xlsx) containing profiling of 24-hr 4T1-GFP-luciferase tail vein injected lungs from Efna1 ^+/+ and Efna1 ^-/- littermate mice (related to Figure 5A-C).)

Harvard Dataverse: CD31 and aSMA images and quantification of 4T1 primary tumors. https://doi.org/10.7910/DVN/MOYPE7 ⁷³ . (4T1 primary tumor CD31 and αSMA staining quantification and images (related to Figure 4A-C).)

Harvard Dataverse: PCNA images and quantification of 4T1-GFP-luciferase lung metastases. https://doi.org/10.7910/DVN/8AJKFM ⁷⁵ . (Lung metastasis PCNA staining quantification and images (related to Figure 5D).)

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).