Cell culture and reagents

Murine HNSCC tumor cell lines MOC2 and LY2 were used for all in vivo studies. The MOC2 cell line was obtained from Ravindra Uppaluri (Dana-Farber Cancer Institute) and is derived from a C57BL/6 mouse with squamous cell carcinoma of the oral cavity that was exposed to DMBA for 25 weeks. The LY2 cell line was obtained from Dr. Nadarajah Vigneswaran (University of Texas Health Science Center). The LY2 cell line was isolated from lymph node metastases that developed in BALB/c mice after inoculation of PAM 212 squamous cell carcinoma cells [117]. All cell lines were cultured at 37 °C and 5% CO2. The LY2 cell lines were cultured in DMEM-F12 media supplemented with 10% fetal bovine serum, 2% Primocin, and 1% Fungin (InvivoGen, San Diego, California, USA). The MOC2 cell line was cultured in DMEM-F12:IMDM (2:1) supplemented with 2% Primocin and 1% Fungin (InvivoGen, San Diego, California, USA), 1.75 ng EGF, 20 ng hydrocortisone per 500 mL of media, and 0.1% insulin (Sigma Aldrich, St. Louis, Missouri, USA). HEK293AD human embryonic cells (#240085, Agilent, Santa Clara, California, USA) and PC3 prostate cancer cells (#CRL-1435, ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; #10-013-CV, Corning, Corning, New York, USA) and RPMI 1640 medium(#11875093, ThermoFisher Scientific, Waltham, Massachusetts, USA), supplemented with 10% fetal bovine serum and 1% antimycotics and antibiotics (#A5955, Sigma-Aldrich, St. Louis, Missouri, USA). HEK293AD cells were stably transfected with pLVX-IRES-Neo encoding full-length human EphB4 with an N-terminal FLAG tag using Lipofectamine 2000 reagent according to the manufacturer’s instruction (#11668019, ThermoFisher Scientific). Forty-eight hours after transfection, the cells were selected with 1 mg/ml G418 (#10131035, ThermoFisher Scientific) for 15 days to generate stably transfected cells. T cells were cultured in RPMI media supplemented with recombinant human IL-2 (Biological Resources Branch National Cancer Institute, Frederick, Maryland, USA), 10% fetal bovine serum, 50 nM 2-mercaptoethanol, 1X MEM non-essential amino acids, 1X penicillin-streptomycin, 1 M HEPES, and 100 mM sodium pyruvate (Gibco, Billings, Montana, USA).

Fc fusion proteins

Commercial purified mouse ephrinB2-Fc was purchased from R&D Systems (#496-EB-200, which contains a C-terminal His tag). The ephrinB2-Fc-His plasmid was obtained by cloning the cDNA encoding the mouse ephrinB2 extracellular region (residues 3-227), a GSGDP linker, the Fc portion of human IgG1, a glycine, and a His tag into the PT3-EF1a-C-Myc plasmid. The Fc-TNYL-RAW-GS plasmid was obtained by cloning a cDNA encoding the CD5 signal peptide followed by the Fc portion of human IgG1 and the sequence ARTNYLFSPNGPIARAWGS, with the underlined sequence representing the TNYL-RAW peptide [75]. The Fc control plasmid was obtained by cloning a cDNA encoding the CD5 signal peptide followed by the Fc portion of human IgG1. The PT3-EF1a-C-Myc and pCMV/SB11 plasmids were kindly provided by Dr. Gen-Sheng Feng (University of California, San Diego) [118].

Purified ephrinB2-Fc-His and Fc-TNYL-RAW-GS proteins for cell stimulation were generated by transiently transfecting HEK293 cells with the respective plasmids and the pCMV/SB11 plasmid at a 1:10 ratio. Cells were passaged into a larger plate the next day. Upon reaching 70% confluence, the culture medium was replaced with fresh Opti-MEM (#31985088, ThermoFisher Scientific) after washing the cells twice with warm DPBS (#21-030-CV, Corning). After three days, the culture medium was collected, and HEPES, pH 7.5 was added to a final concentration of 10 mM before centrifugation at 1000 g for 10 min to remove cell debris. The resulting supernatant was incubated with GammaBind Plus Sepharose beads (#17088602, Cytiva, Marlborough, Massachusetts, USA) overnight at 4 °C. Beads were washed with cold PBS once before bound Fc fusion proteins were eluted with 0.1 M glycine HCL, pH 2.5, and the low pH in the eluates was neutralized with 1 M Tris HCl buffer, pH 7.5.

Animal tumor models

For studies using only wild-type hosts, 5- to 7-week-old female C57BL/6 mice were purchased from the Jax Labs, Bar Harbor, Maine, USA. For studies using ephrinB2fl/flTie2Cre mice (C57BL/6 background), breeding pairs were obtained from Dr. Mohit Kapoor’s lab (University Health Network, University of Toronto, Canada) and maintained at the Anschutz Medical Campus, Aurora mouse facility. Deletion of ephrinB2 from the vasculature of ephrinB2fl/flTie2Cre mice was done as previously described [18]. Experiments were performed once enough ephrinB2fl/flTie2Cre were bred, and 5- to 10-week-old female mice were used and age matched with controls. In vivo orthotopic HNSCC tumor models were established as previously described [96, 119]. For buccal tumor implantations, 1 × 105 MOC2 cells per 50ul of serum-free cell media were prepared. For floor of mouth implantations, 1 × 105 LY2 cells per 50 uL of serum-free cell media were prepared. A 1:1 mixture of cells and Matrigel (10 mg/mL, BD Biosciences, San Jose, California, USA) at a volume of 100ul was injected into the right buccal mucosa of the mice (MOC2) or floor of mouth (LY2). Mice were appropriately age matched and randomized into groups, with treatment beginning when tumors reached ~100–200 mm3 (MOC2) or ~150–300 mm3 (LY2). Tumor size was measured twice weekly using digital calipers, and tumor volume was estimated using the equation V = AxB2/2, where A is the longest diameter of the tumor and B is the shortest. Computed tomography scans were performed once a week as described below. Mice were euthanized when the mice reached study end qualifications approved by the Institutional Animal Care and Use Committee (IACUC). Mice were euthanized under CO2, and intracardiac blood collection was performed. Blood was placed in serum collection tubes (BD, Franklin Lakes, New Jersey, USA) and centrifuged at 6000 rpm for 2 min to collect the serum supernatant. Tumors were harvested and flash frozen in liquid nitrogen, while lung tissues were harvested and placed in 10% formalin for further processing. Both sera and tumors samples were stored in -80°C for subsequent analysis.

For studies using ephrinB2-Fc and Fc-TNYL-RAW-GS plasmids, C57BL/6 J mice were initially implanted with 100k MOC2 cancer cells. 7 days later, mice were randomized into different groups including a control group treated with radiation therapy alone, a control group transfected with 1 µg of Sleeping Beauty transposase plasmid (SB), and experimental groups transfected with 1 µg of SB in addition to 20 µg of EFNB2-Fc or Fc-TNYL-RAW-GS plasmid. Sleeping Beauty transposase plasmid was used to promote cDNA integration into mouse liver genomic DNA. Liver transfection was performed by hydrodynamic tail vein injection as described previously [19]. A repeat liver transfection was performed 3 weeks post-tumor implantation.

All in vivo experiments were performed using a sample size of n = 3-20 mice per group unless noted otherwise. Power analysis was used to decide on the number of mice per group. For effect studies, no mice were excluded from the data shown. Investigators were blinded during group allocation and analysis for the experiments included in the study. All animal protocols used in this study were approved by IACUC of the University of Colorado, Denver.

Irradiation and computed tomography scans

Mice were anesthetized using isoflurane before and during the procedure. The mice were then placed in an X-RAD image guided small animal irradiator (Precision X-Ray, Bradford, Connecticut, USA). For tumor irradiation, the irradiation field was determined using fluoroscopy for each mouse. After the field was established, the mice were irradiated with 225kVp/20 mA with a copper filter, which corresponds to 5.6 Gy/min for 98 s (8 Gy) or 123 s (10 Gy). For computed tomography scans, mice were scanned using an aluminum filter. For MOC2 metastasis studies, 3D lung contouring was generated using ITK-Snap. For flow studies, mice received one fraction of 10 Gy. For MOC2 metastasis studies, three fractions of 8 Gy were administered to the buccal tumor. For LY2 floor of mouth studies, three fractions of 8 Gy were administered to the tumor. In some studies utilizing the MOC2 cell line, tumors were given an additional dose of 8 Gy RT as they approached 500 mm3 in volume to reduce local tumor burden and prolong the study. For transendothelial migration studies, mice received one dose of 10 Gy 12 to 14 days after implantation.

Flow cytometry

Tumors and draining lymph nodes (DLNs) were harvested from mice and immediately put on ice in Hank’s Balanced Salt Solution (HBSS). To facilitate single cell suspension of lymphocytes from tumors, the tumors were minced and then incubated for 30 min at 37 °C with 200U of Collagenase III (Worthington, Lakewood, New Jersey, USA). Both tumors and draining lymph nodes were then filtered through 70 µm nylon filters. The tumors were then spun down at 400 g for 5 min and resuspended in 3 mL of Red Blood Cell Lysis Buffer for 3 min. After 3 min, 6 mL of HBSS was added and all the samples were spun down again at 400 g for 5 min. Blood was taken using an intracardiac puncture. The blood was spun down for 10 min at 400 g before resuspending in 3 mL of Red Blood Cell Lysis Buffer for 3 min. After 3 min, 6 mL of HNSCC was added to the sample and the samples were spun down at 400 g for 5 min. The samples were aspirated and then were plated in 24 well plates in 1 mL of RPMI with 0.1% monensin to prevent cytokine release by the golgi apparatus and 0.2% brefeldin A with cell stim cocktail of PMA and ionomycin to simulate cytokine production and transport in the cells. The cells were incubated in the stim media for 4 hours at 37 °C. The plates were then spun down and the samples were resuspended in 200 µl of Fc Block (anti-CD16/CD32 antibody, Tonbo Biosciences, San Diego, California, USA) for 20 min at room temperature. The samples were then plated into a 96 well plate and spun down at 400 g for 5 min. The samples were then resuspended in 100 µl of PBS containing 5 µl of live/dead aqua viability stain (Invitrogen, Carlsbad, California, USA). The samples were incubated away from light for 15 min at room temperature. The samples were then spun down and resuspended in a mixture of the extracellular antibodies in brilliant stain buffer (BD Biosciences, Franklin Lakes, New Jersey, USA). The cells were incubated for 30 min at room temperature. After incubated the cells were washed twice with FA3 buffer. The cells were then incubated overnight with the Foxp3 transcription factor staining kit (eBioscience, San Diego, California, USA). The next day the cells were spun down and washed twice as per the kit instructions. The cells were then stained with the intracellular antibodies in brilliant stain buffer for 30 min at room temperature. The cells were then washed twice with PBS. Cells were re-suspended and then run on an Aurora Spectral Flow Cytometer (Cytek Biosciences, Fremont, California, USA) at the Barbara Davis Center Diabetes Research Center Cell and Tissue Analysis Core. For all flow cytometry data, if too few events were available for analysis ( < 10 cells), the sample was either not included in the analysis or cell counts were shown for each sample.

The following antibodies were used in these studies: PerCP-CD45 (Clone: 30-F11 Biolegend, San Diego, California, USA, 557235), PerCP-Cy5.5- CD3 (Clone: 17A2 Biolegend, 100217), BUV805-CD3 (Clone: 17A2 BD Biosciences, 741982), SB436-CD4 (Clone: GK1.5 eBioscience, 17-0041-82), eF450-CD4 (Clone: GK1.5 eBioscience, 48-0041-82), BUV496-CD4 (Clone: GK1.5 BD Biosciences, 612952), BB515-CD8 (Clone: 53-6.7 BD Biosciences, 564422), BV570-CD44 (Clone: IM7 Biolegend, 103037), AF532-Foxp3 (Clone: FJK-16s eBioscience, 58-5773-82), eF660-CD11b (Clone: M1/70 Invitrogen, 50-112-4253), BUV661-CD11b (Clone: M1/70 BD Biosciences, 612977), BV711-CD25 (Clone: PC61, Biolegend 102049), NovaFluor Yellow 590-CD11b (Clone: ICRF44, eBioscience, H036T03Y02-A), eF450-LY6C (Clone; HK1.4, eBioscience, 48-5932-82), BV650-LY6C (Clone: HK1.4 Biolegend, 128049), BV570-LY6C (Clone: HK1.4 Biolegend, 128029), BV605-LY6C (Clone: HK1.4 Biolegend, 128035), PerCP Cy5.5-LY6G (Clone: 1A8 Biolegend, 127615), BV421-LY6G (Clone: 1A8 Biolegend, 127627), BV650-LY6G (Clone: 1A8 BD Biosciences, 740554), BV605-LY6G (Clone: 1A8 Biolegend, 127639), AF594-ephrinB2 (1° Catalogue number AF496 R&D; 2° Ref A21468), BV480-F4/80 (Clone: T45-2342 BD Horizon, 565635), AF-647-IL2 (Clone: JES6-5H4, Biolegend 503814), BV605-IL-2 (Clone: JES6-5H4 Biolegend, 503829), PE-IL-2 (Clone: JES6-5H4 Biolegend, 503807), BV421-IL-10 (Clone: JES5-16E3 Biolegend 505021), BUV395-PD-1 (Clone: J43 BD Biosciences, 744549), APC-R700-CTLA-4 (Clone: UC10-4F10-11 BD Biosciences, 565778), APC-Fire 750-Granzyme B (Clone: QA16A02 Biolegend, 372209), APC-iNOS (Clone: CXNFT eBioscience, 17-5920-82), PerCP-eFlour 710-Arginase 1 (Clone: A1exF5 eBioscience, 46-3697-82), APC-eFluor 780-Ki-67 (Clone: SoIA15, Invitrogen 47-5698-82), BV480-Ki-67 (Clone: B56 BD Biosciences, 566109), BV786-Ki-67 (Clone: B56 BD Biosciences, 563756), BUV805-MHCII (Clone: M5/114.15.2 BD Biosciences, 748844), PE-Dazzle 594-MHCII (Clone: M5/114.15.2 Biolegend, 107648), PE-Cy7-CD31 (Clone: 390 Biolegend, 102418), SB436-PDPN (Clone: eBio.1.1(8.1.1) Invitrogen, 62-5381-82), Live Dead Aqua (Invitrogen, L34965).

The following antibodies were used in intravital flow studies: PerCP-CD45 (Clone: 30-F11 Biolegend, 557235), PerCP-Cy5.5- CD3 (Clone: 17A2 Biolegend, 100217), eF450-CD4 (Clone: GK1.5 Invitrogen, 48-0041-82), AF532-Foxp3 (Clone: FJK-16s Invitrogen, 58-5773-82), Live Dead Aqua (Invitrogen, L34965).

Human HNSCC Single-Cell RNA sequencing analysis

Single cell RNA-Seq data [66] was downloaded from the UCSC Cell Browser [120] as an expression matrix, metadata, and UMAP coordinates. R software (v4.2.3) was used with package Seurat75 (v4.3.0) to visualize cell populations and import UMAP coordinates and metadata. EphrinB2, SELE, and SELP expression were assessed on cells phenotyped as endothelial cells in the UCSC-provided metadata, and TPM values followed a bimodal distribution where cells could be assumed as ephrinB2 low and high expressors. Log2-transformed fold changes and student’s t-test were calculated to create a volcano plot comparing endothelial cells with high ephrinB2 expression versus low ephrinB2 expression.

T cell isolation and ex vivo activation

CD4+ and regulatory T cells were isolated from dissociated spleens using a CD4 + CD25+ regulatory T cell isolation kit according to manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany). CD4 + T cells were activated in a 24-well plate (Corning, New York, USA) coated with 2 μg/mL anti-CD3 (clone 2C11, Invitrogen, Waltham, Massachusetts) and 2 μg/mL anti-CD28 (clone PV-1, BioXCell, Lebanon, New Hampshire, USA). Two days after activation, CD4 + T cells were removed from the coated plate and split to a concentration of 1 × 106 cells/mL. Subsequent splits were performed as needed. 30 U/mL recombinant human IL-2 (Biological Resources Branch National Cancer Institute) was added on day 0 and every other day of activation.

T cell fluorescent dye-labeling

7 to 10 days following ex vivo activation, T cells were stained with carboxy-fluorescein diacetate succinimidyl ester (CFSE), CellTrace Yellow, or CellTrace Violet proliferation dyes (Invitrogen, Waltham, Massachusetts, USA) at a 1:1000 ratio in serum-free RPMI for 15–20 min at 37 °C. Excess stain was quenched by incubation with 1X volume FBS at room temperature for 2 min, then cells were washed twice with PBS prior to use.

Intravascular staining

Staining of lymphocytes in the vasculature was done by intravascular staining as previously described [71]. APC-conjugated anti-CD4 antibody (clone GK1.5, Biolegend, San Diego, California, USA, 100412) was administered by tail vein injection 3 min prior to CO2 euthanasia. After euthanizing, perfusions were performed by injection of PBS through the heart to wash out excess stain.

Transendothelial migration flow

C57BL/6 and ephrinB2fl/flTie2Cre mice were implanted with 100k MOC2 control or EphB4 shRNA tumors and irradiated 12 to 14 days post-implantation as described above. Two days later, ex vivo activated CD4 + T cells were fluorescently stained with CFSE, and 1 × 107 cells were adoptively transferred by tail vein injection. 30 min, 60 min, 90 min, 2 h, 6 h, 12 h, and 24 h following adoptive transfer, intravascular staining and perfusions were conducted as described above. Tumors were then harvested and processed for flow cytometry as described above. Optimization studies demonstrated that 1 h was the appropriate time for capturing T cell accumulation in this tumor model. Extravasation flow was therefore performed 1 h after adoptive transfers for subsequent experiments.

RNA-sequencing analysis

Sample preparation and RNA-sequencing analysis were conducted as described previously [18, 97].

Boyden chamber invasion assay

MOC2 control or EphB4 shRNA cells were plated to 70% confluency, then the media was replaced with a 1:1 ratio of complete media and Fc-containing conditioned media and the cells were incubated overnight. Cells were then serum starved and 24-well plate inserts with 8 µM pores were coated with a 1:10 ratio of Matrigel (Corning) and serum-free media for 4 h in a 37 °C incubator. Following incubation, 750 µL complete media containing 10% FBS was added to the bottom chamber, then 500k serum-starved cells were seeded in the Matrigel-coated top chambers. For controls, 750 µL of media containing 0% FBS was added to the bottom chamber. Cells were incubated in a 37°C incubator and allowed to migrate for 24 h. Next, cells were fixed in 3.7% formaldehyde diluted in PBS for 2 min at room temperature followed by 2 washes in PBS. The cells were permeabilized in 100% methanol for 20 min, washed twice with PBS, then stained with 0.1% crystal violet for 15 min at room temperature. Non-invasive cells on the top of the insert were scraped off with cotton swabs, then the inserts were allowed to dry. Inserts were imaged at 10x magnification, and the number of migrated cells was counted using ImageJ (National Institutes of Health, Bethesda, Maryland, USA). The number of invaded cells in each experimental condition was normalized to the number of invaded cells in the control groups where serum-free media was added to the bottom chamber. Each condition was performed in five replicates.

Cancer cell and CD4 + T cell coculture assay

LY2 control or EphB4 shRNA cancer cells were initially cultured in complete DMEM/F12 medium described above. Subsequently, 100,000 cells were plated overnight in 6-well plates. Cells were then treated with 10 ng/ml of IFN-gamma for 48 h, after which 10 µg/ml of OVA peptide was added overnight. CD4 + T cells were then isolated from the spleen and lymph nodes of BALB/c mice (DO11.10) using a CD4 + T cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany). These isolated CD4 + T cells were activated as described above before being co-cultured with LY2 cells at a ratio of 1:1 for 72 h. Finally, the cells were harvested and stained for flow cytometric analysis.

Whole-cell lysate preparation

Tumor tissues from the indicated group were homogenized in RIPA buffer (Millipore) with a protease inhibitor cocktail (Thermo Fisher Scientific) and phosphatase inhibitors (Sigma-Aldrich) on ice for 30 min. Lysates were then collected, and protein concentration was measured using a standard BCA assay as previously described [121].

Immunoblot

For western blotting of tumor tissue, proteins were denatured at 95 °C for 7 min and then stored at −20 °C. Equal amounts of protein (15 μg) were separated using 10% sodium dodecyl sulfate–polyacrylamide (SDS) gels and transferred onto PVDF membranes (#10600023, Amersham Biosciences, Piscataway, New Jersey, USA). To block nonspecific binding sites, the membranes were incubated with 5% Bovine Serum Albumin in Tris-buffered saline containing 1% Tween for 1 h at room temperature. The membranes were then probed with the specified primary antibodies. Blots were probed overnight at 4 °C with respective antibodies. Primary antibodies anti-EFNB2 (#131536, Abcam, Cambridge, United Kingdom), anti-pEFNB2- pTyr316 (SAB 4300631, Sigma), anti-EphB4 (#37-1800, Invitrogen), anti-pEphB4-Tyr987 (PA5-64792, Invitrogen) and anti-β-actin -HRP conjugate (#5125, Cell Signaling Technology, Danvers, Massachusetts, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Sigma. Membranes were washed thrice and the antibodies visualized with enhanced chemiluminescent HRP substrate (#R-03031-D25 and R-03025-D25, Advansta, San Jose, California, USA). For detection of signals, X-ray films or versa doc were used. To confirm loading control after detection of phosphorylated protein, membranes were stripped using ReBlot Plus strong antibody stripping solution (#2504, Merck, Germany), blocked and re-probed with different antibodies against whole protein. Protein bands were quantified using Image lab & ImageJ software. Results are shown as the ratio of total protein or phospho-protein to B actin normalized to the control group. To assign the right protein size, Precision Plus Protein Dual Color Standards Protein Marker was used as a marker (6 μl, #1610374, BIO-RAD).

For immunoblotting of Fc fusion proteins in vivo, mouse serum and purified human IgG Fc (#0855911, MP Biomedicals, Irvine, California, USA) were diluted in LDS sample buffer (#B0007, Life Technologies, Carlsbad, California, USA) with 2.5% β-mercaptoethanol, heated at 95 °C for 2 min, and run on Bolt Bis-Tris Plus gels (#NW04125Box, ThermoFisher Scientific). After semi-dry transfer, the Immobilon membranes were blocked with 5% BSA in 0.1% Tween 20 in Tris buffered saline (TBS) for 1 h and then incubated in the cold overnight with an anti-human IgG antibody at a 1:1000 dilution (#109-005-098, Jackson ImmunoResearch Labs, West Grove, Pennsylvania, USA). Blots were washed 3 times with 0.1% Tween 20 in TBS before incubating with a horseradish peroxidase-conjugated anti-goat secondary antibody at a 1:4000 dilution (#A16005, Life Technologies) for 1 h. After 3 washes, the chemiluminescence signal was captured using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, California, USA). Chemidoc images were quantified using Image Lab (Bio-Rad) to determine the approximate concentration of Fc proteins in the mouse serum using known concentrations of human Fc as standards.

ELISA

HEK293AD cells stably expressing EphB4 plated on poly-D-Lysine coated plates, or PC3 cells plated without coating, were cultured to 70–80% confluency. Cells were serum starved for 1 h before treating with ephrinB2-Fc, Fc-TNYL-RAW, or Fc for 30 min. Cells were rinsed once with cold DPBS (#21-030-CV, Corning), and collected with lysis buffer (0.5% TX-100 in PBS with Halt Protease and Phosphatase Inhibitor Cocktail (#78443, ThermoFisher Scientific)). After 15 min of incubation on ice, cell lysates were centrifuged at 16,000 g at 4 °C for 10 min, and supernatants were collected. The supernatants were used with the Human Phospho-EphB4 DuoSet IC ELISA kit (#DYC4057-2, R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer’s instructions.

Hematoxylin and eosin staining and imaging

Lung samples were submitted to the Dermatology Histology Core at CU Anschutz for cutting and hematoxylin and eosin staining.

Statistical analysis

Statistical tests were chosen appropriate to sample sizes and the number of group comparisons. A student’s t-test or one or two-way ANOVA was used when comparing groups for tumor growth curves. A log-rank Mantel-Cox test was used to determine significance in survival studies. The Dunnett post hoc test was used after one-way ANOVA where multiple experimental groups were involved. For flow analysis, a student’s t-test or Mann Whitney test was used to compare control and experimental groups. For western blot analysis, a Mann Whitney test was performed to compare relative protein expression between control and experimental groups. A Chi-square test was used to compare incidence of metastasis between groups. All statistical analysis was done in Prism Software (v9.1.0). Significance was determined by p-values: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.