Cell lines and cell culture
Mycoplasma-free breast cancer cell lines, MDA-MB-231 (ATCC® HTB-26™) and 4T1 (ATCC® CRL-2539™) cells, were obtained from American Type Culture Collection (ATCC) and maintained in DMEM (MDA-MB-231; Sigma-Aldrich) or RPMI-1640 (4T1; Sigma-Aldrich) with 10% fetal bovine serum (FBS; Corning) and 1% penicillin/streptomycin (P/S) (Invitrogen). SUM159 and 4T1-luciferase-tagged (4T1-Luc) cells were kindly provided by the Sukumar lab and were cultured in Ham’s F12 medium supplemented with 5% FBS, 1% P/S, and 5% insulin/hydrocortisone (SUM159) or in DMEM with 10% FBS and 1% P/S (4T1-Luc). Cells were maintained in a humidified environment at 37 °C and 5% CO2. MMTV-PyMT cells were derived from a tumor excised from a female triple-transgenic mouse as previously described [31] and maintained in 50% DMEM, 50% DMEM/F12, 10% FBS, 5% insulin, and 1% P/S. Fluorescent MDA-MB-231 cells were developed and maintained as previously described. [32]
Proliferation assays
Half-maximal inhibitory concentration (IC50) values were obtained for MMTV-PyMT, MDA-MB-231, 4T1, and SUM159 cells by seeding 1000 cells/well in a 96-well plate for 24 h (h) and then treated with 0.01–500 µM of mebendazole (MBZ) or less than 1% dimethyl sulfoxide (DMSO) as vehicle control. After 48 h of treatment, PrestoBlue (Thermo Fisher) was added to achieve a 10% (v/v) concentration in each well, incubated for 4 h, and fluorescence was measured using a Cytation5 (BioTek Instruments). The IC50 was calculated using a nonlinear fit log vs. response model. To calculate % cell survival, cells were fixed with 70% ethanol, stained with DAPI, and imaged using a Cytation 5 (BioTek Instruments) equipped with an Olympus–UPLFLN 4XPh phase objective and DAPI filter. A 4 × 3 montage was used to capture the entire area of each well, and NIS Elements software (Nikon Instruments Inc.) was used to threshold the DAPI positive area of the image which is presented as % survival.
Colony formation assays
MDA-MB-231, 4T1, and SUM159 cells were plated in a 24-well plate (250 cells/well) and exposed to 0.01–1 µM of MBZ or < 1% DMSO as vehicle control for 10–14 days (refreshed every 3 days) or pre-treated with MBZ for 48 h and then seeded in plates without further drug treatment. The cells were washed with 1 × phosphate-buffered-saline (PBS), fixed with 4% paraformaldehyde (PFA) in PBS for 15 min (min), and washed again with PBS. Crystal violet solution (1% (w/v) crystal violet diluted in water containing 20% methanol) was added to each well and incubated for 10 min at room temperature. Wells were washed with distilled water and dried. Well plates were imaged using a Cytation 5 (BioTek Instruments) equipped with an Olympus–UPLFLN 4XPh phase objective in color bright field. A 4 × 3 montage of the acquired images was used to capture an image of the entire well. Colonies were quantified by manually counting individual colonies in each well.
Cell cycle analysis
MDA-MB-231 cells were treated with 0.25 µM, 0.5 µM, 1 µM, and 5 µM of MBZ or DMSO (vehicle control) for 48 h, washed with PBS, fixed in 70% ethanol, and pelleted and stained with 100 µg/mL RNase and 50 µg/mL of propidium iodide in the dark at 4 °C overnight. The distribution of cells in G1, S, and G2/M phases was determined by flow cytometry using a CytoFLEX flow cytometer (Beckman Coulter), and data analysis was performed using FlowJo software V10.
Western blotting
Cell homogenates were prepared from MDA-MB-231 and SUM159 cell lines or primary tumors formed by MDA-MB-231 cells lysed in IGEPAL CA-630 buffer (150 mM sodium chloride (NaCl), 1% IGEPAL CA-630, 50 mM Tris–HCL, pH 8.0, protease and phosphatase inhibitors) for 10 m on ice. The lysate was centrifuged for 10 min at 13,000 g at 4 °C, and the supernatant was collected. Proteins were fractionated by a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane for 30 min using a Trans-blot Turbo (Bio-Rad). The membranes were blocked in 5% milk (% w/v) in 1 × Tris-buffered saline and 0.1% Tween-20 (TBS-T) and incubated overnight in primary antibodies at a dilution of 1:1000 at 4 °C. The membranes were washed three times in TBS-T and incubated in HRP-conjugated secondary antibodies (Cell Signaling Technology) followed by three additional washes in TBS-T. The chemiluminescence signal was detected using an AZURE C300 (Azure™ Biosystems) after incubating the membrane with ECL (Perkin Elmer). A list of antibodies can be found in Additional file 1: Table S1.
Transwell migration assays
Transwell invasion assays were performed using Costar Transwell cell culture inserts (Corning) with an 8-μm pore size PET membrane in a 24-well plate. The lower chambers of the transwell plates were filled with 500 μL of cell-appropriate culture media. MDA-MB-231 or SUM159 cells were pre-treated with 0.5 or 1 μM of MBZ for 48 h. The cells (20,000/well) were resuspended in 150 μL of medium containing only 1% FBS and placed into the upper well. After 24 h, cells in the upper chamber were removed with a cotton swab, and those that migrated through the pores on the lower surface were fixed in 100% ethanol (EtOH) and stained in 0.2% crystal violet dye. Five random fields of each pore were imaged using a Cytation 5 (BioTek Instruments) equipped with an Olympus–UPLFLN 10XPh phase objective. For MDA-MB-231 cells, the percent area and total area were quantified using ImageJ software using a custom macro with the following steps: Each image was converted to 16-bit, the scale was converted from pixel to μm, the background was subtracted using a rolling ball radius of 10 pixels and light background, and a threshold was set to highlight crystal violet positive areas of the wells. The macro was altered to include the color threshold feature, the “hue” slider was used to highlight the cell area, and images were processed at 8-bit to quantify the % area for SUM159 cells.
Wound-healing assays
MDA-MB-231 and SUM159 cells were cultured in a 12-well plate (100,000 cells/well) and allowed to reach near confluence within 48 h. Linear scratches of equal width were introduced using a 200 µL pipette tip. Fresh media containing 0.25 µM, 0.35 µM, 0.5 µM MBZ, or DMSO (vehicle control) was added. Five images of the wounded area were taken at 12 and 24 h using a Cytation 5 (BioTek Instruments) equipped with an Olympus–UPLFLN 4XPh phase objective. A montage of the entire well was constructed by stitching individual images. The average healing speed and area covered were quantified using ImageJ software. The following macro was created: Images were converted to an 8-bit format, scale was set with the appropriate pixel to μm conversion, a Gaussian blur of radium 5 pixels was applied, and a threshold was applied using the measure tool to highlight only the gap created by the wound. The macro code calculates the wound area covered in μm2; this value was then divided by the total hours of the experiment (12 h or 24 h).
DNA extraction from mouse tissues
Mouse liver or lung tissues were placed in Genomic DNA lysis buffer (1 M Tris, pH 8.0, 5 M NaCl, 0.5 M EDTA, 10% Tween-20, 10% NP-40, and 40 μg of proteinase K) at 55 °C for 3 h with intermittent vortex. Proteinase K was inactivated with a 2 min incubation at 95 °C. One volume of phenol/chloroform/isoamyl alcohol (25:24:1) (Sigma) was added to each sample, followed by 20 s vortex. Samples were centrifuged for 10 min at 13,000 × g, and the upper aqueous phase was collected. Glycogen (20 μg), 0.1 volume of 2.5 M NaCl, and 2 × volume of 100% EtOH were added, and samples were incubated for 20 min at − 20 °C. The samples were spun at 13,000 × g for 15 min to pellet the DNA followed by one wash with 70% EtOH. The sample was spun at 13,000 × g for 5 min, the remaining liquid was decanted, and the DNA pellets were air-dried. DNA pellets were resuspended in DNase/RNase-free water. To assess the amount of human DNA content in the mouse lung and liver, the cycle threshold value of human hexokinase 2 (HK2) measured by RT-PCR was normalized to the DNA concentration of the sample (ranged from 180 to 220 ng/µL). The result for each sample was than normalized to the average value of the control group.
Reverse transcription and quantitative polymerase chain reaction (qPCR)
Total RNA was extracted using TRIzol (Invitrogen) followed by cDNA synthesis using Promega’s GoScript™ Reverse Transcriptase. qPCR analysis was conducted on CFX96 Real-Time PCR detection system (Bio-Rad) using SYBR Green qPCR master mix (Bio-Rad). The expression of each target mRNA relative to 18S rRNA control was calculated based on the cycle threshold (Ct) as 2 − Δ(ΔCt), in which ΔCt = Ct (target mRNA) − Ct (18S rRNA), and Δ(ΔCt) = ΔCt (treatment) − ΔCt (control). Primer sequences are listed in Additional file 1: Table S2. (Figs. 1, 2, 3, 4, and 5).
RNA sample preparation and sequencing
MDA-MB-231 and SUM159 cells were collected from culture, and subsequently, total RNA was extracted. Following RNA purification, samples were confirmed to have a RIN value > 9.0 using an Agilent Bioanalyzer. Total RNA was further qualified upon receipt to Novogene (Sacramento, CA). The samples were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA). One μg of RNA was used as input for mRNA library preparation. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads followed by double-stranded cDNA synthesis. The cDNA fragments were adenylated, and sequencing adaptors were added. Each 150–200 bp length fragment was purified, and PCR was performed per Novogene established protocols. Sequencing was performed on a NovaSeq 6000 system with 150-bp paired-end run by Novogene (Sacramento, CA). The reads were mapped to Homo Sapiens (GRCh38/hg38) using STAR (v2.5) with the parameter mismatch set to 2. Quantification was done using HTSeq (v0.6.1) software with the parameter -m union. Differentially expressed genes were identified using EdgeR (v3.16.5) with padj < 0.005 and [log2(FoldChange)] > 1. FASTQ files and read counts have been uploaded to GEO with the accession number: GSE190845. For the gene ontology enrichment analysis, the p value cutoff was set at p < 0.05. In order for a GO pathway to be included in Additional file 2: Fig. S5, the pathway had to be enriched (p < 0.05) in both the SUM159 and MDA-MB-231 cell lines. Differentially expressed genes (DEGs) consisted of genes that increased or decreased with a fold change of 1.5 or higher. Genes meeting this criteria were included in the VENN diagram in Fig. 6. Additional file 3 (SuppDataRNAseq.xls) includes all GO enrichment scores and differential expression values for each cell line.
Immunofluorescence staining
Immunofluorescence staining to detect ITGβ4 was performed on 10-μm-thick tissue cryo-sections after rehydrating them by immersing them into 1 × phosphate-buffered saline and 0.1% Tween-20 (PBS-T). All slides were then blocked with 2% BSA for 60 min and incubated overnight at 4 °C with a primary antibody against ITGβ4 (50–1049-80, Invitrogen) (dilution at 1:200). The next day, slides were washed 3× with PBS-T and then incubated with DAPI (1 ug/mL) for 15 m at room temperature. Sudan Black (0.1% w/v) was applied to the slides for 25 min to quench autofluorescence followed by washing in PBS-T. ITGβ4 and DAPI were visualized in Cy5 and DAPI channels, respectively, using Cytation 5 (BioTek Instruments) equipped with an Olympus–UPLFLN 10XPh phase objective.
Mammosphere formation assay
Fluorescently tagged MDA-MB-231 cells in culture or freshly resected from tumors that were processed (enzymatic digestion in 2 mg/mL collagenase for 1 h at 37 °C with orbital shaking at 160 rpm) were passed through a cell strainer (70 μm) and plated at 2,000 cells/well in 12-well plates previously coated with polyHema (12 g/L in 95% EtOH) and air-dried for 48 h, with 2 mL of mammosphere formation media per well (MammoCult basal medium (human) containing 4 μg/mL heparin, and 0.48 μg/mL hydrocortisone) (STEMCELL Technologies; Vancouver, BC, Canada). Fluorescent images were captured using a Cytation 5 (BioTek Instruments) equipped with an Olympus–UPLFLN 4XPh phase objective. A montage of the entire well was constructed by stitching individual images. The total number and average size of each mammosphere were then calculated using Gen5 software (BioTek instruments) automated counting algorithm.
Fluorescence-activated cell sorting (FACS)
MDA-MB-231 and SUM159 cells were treated with fresh media containing 0.05 µM, 0.125 µM, 0.5 µM, 1 µM, or 5 µM MBZ or DMSO (vehicle control) for 72 h. Cells were then trypsinized, resuspended in culture media, washed with 1xPBS, and collected in FACS buffer (1xPBS, 1%BSA, 0.5 mM EDTA, and 25 μg/ml DNase). Live cells were stained with APC-conjugated antibody against ITGβ4 (50-1049-80, Invitrogen) diluted at 1:300 for 30 min on ice. Sytox™ Blue (Invitrogen, dilution 1:300) was added immediately before analyzing each sample using flow cytometry. APC (ITGβ4) was detected in the FL-4 channel, and FITC (Sytox™ Blue) was detected in FL-1 channel. Or cells were stained with conjugated antibodies against ITGβ4, CD44, and CD24 diluted at 1:300 for 30 min on ice. APC (ITGβ4) was detected in the FL-4, FITC (CD44) was detected in FL-1 channel, and PE (CD24) was detected in FL-2 channel. Single stained cells were used for compensation controls. Data were analyzed using FlowJo V10 software (Tree Star, Inc.). For primary tumors that were sorted, tumors were subjected to enzymatic digestion prior to staining and ultimately resuspended in sorting buffer (1xPBS, 1%BSA, 0.5 mM EDTA, and 25 μg/ml DNase). Samples were sorted on an SH800 cytometer (Sony) into ITGβ4+ or ITGβ4− expressing populations directly into media. APC (ITGβ4) was detected in the APC channel, and Alexa Fluor 405 (Sytox™ Blue) was detected in Pacific Blue channel. Single stained cells were utilized as compensation controls. A list of antibodies can be found in Additional file 1: Table S1.
Breast cancer patient-derived xenograft (PDX) maintenance
The HCI-001 PDX was kindly provided by the Zahnow Lab and developed by the Welm lab (https://uofuhealth.utah.edu/huntsman/labs/welm-labs/research.php). The individual demographical data can be found in the Baylor College of Medicine PDX Portal (https://pdxportal.research.bcm.edu/). The tumor fragments from the HCI-001 PDX were maintained in NOD-SCID Gamma (NSG) mice. When tumors reached the maximum diameter approved by Johns Hopkins University Animal Care and Use Committee (ACUC), they were collected and re-implanted into new mice as ~ 1 mm size fragments or cryopreserved. The initial tumor was termed “p.x+1,” and passages were tracked. For experiments, primary tumors were manually dissociated and incubated for 1 h at 37 °C in 2 mg/mL collagenase on an orbital shaker and then digested with DNase (0.4 U/ml) (Sigma-Aldrich) for 5 min at room temperature. Next, the tumor-dissociated cell suspension containing organoids was centrifuged and resuspended in fresh media followed by differential centrifugation (×4) at 520 g for 2 s. Finally, the cell suspension was strained through a 100 μm Nylon filter, counted, and then, 5000 organoids were re-implanted into each recipient NSG mouse. This process has been described in-depth previously [33].
In vivo orthotopic breast cancer models
All animal research complied with relevant ethical regulations within protocols approved by the Johns Hopkins University ACUC. Female 5- to 7-week-old NOD-SCID Gamma (NSG) mice or BALB/c mice were anesthetized by the intraperitoneal injection (i.p.) of 100 mg/kg ketamine and 16 mg/kg xylazine. MDA-MB-231 cells (2 × 106; NSG), 4T1-luciferase-tagged cells (500; BALB/c), or HCI-001 tumor organoids (5000; NSG) were injected into the mammary fat pad closest to the second nipple. Tumor removal surgery was performed 5.5 weeks (MDA-MB-231 model only) post-tumor implantation as described previously [34]. At the end of the experiment, tumors, livers, and lungs were excised. A portion of each organ was first formalin-fixed (Sigma-Aldrich) overnight and then saturated in 30% sucrose (Sigma-Aldrich) at 4 °C overnight. Each organ was then placed into a cryomold and covered completely with OCT media (Fisher Scientific). After flash freezing the OCT embedded organ in liquid nitrogen, they were sectioned onto Superfrost Plus Microscope Slides (Fisher Scientific) using a cryotome CM11000 (Leica). A second portion of the same tumor, liver, and lung was flash-frozen and processed for DNA or RNA extraction. Primer sequences are listed in Additional file 1: Table S2. For mice injected with the HCI-001 PDX, rather than embedding in OCT, the lungs from NSG mice were inflated with agarose, formalin-fixed, embedded in paraffin and H&E stained.
Bioluminescence assay to measure metastatic burden
Intratumoral bioluminescence was measured weekly throughout the study by injecting the BALB/c mice bearing 4T1-Luciferase expressing tumors with 2 µg/mouse (150 µL) of D-luciferin potassium salt (Gold Bio) diluted in 1 × PBS. Mice were then briefly anesthetized with Fluorido (Isoflurane, USP) and imaged using a Xenogen imaging system (IVIS 200, equipped with Living Image Software) within 10 min of luciferin injection, as described previously [20]. All tumor bioluminescence values were expressed as radiance (photons.s-1.cm2.103). At the end of the study, lungs and livers were excised and immediately imaged to detect bioluminescence.
Limiting dilution assays (LDA)
Animal experiments complied with Johns Hopkins ACUC protocols. In vitro pre-treatment (Fig. 7E): MDA-MB-231 cells were grown in fresh media containing 1 µM MBZ or DMSO (vehicle control) for 72 h. Cells were trypsinized, counted, and resuspended at 106, 105, 104, or 103 cells per 100 µL in a 1:1 ratio of Matrigel to PBS. Nude mice (female, 6–8 weeks of age) were implanted in the 2nd, 3rd, 4th, and 5th mammary fat pad on either side of the mice. The right mammary fat pads were injected with cells treated with vehicle control, and the left mammary fat pads were injected with MBZ-treated cells as follows: 2nd—106 cells, 3rd—105 cells, 4th—104, and 5th—103. In vivo pre-treatment (Fig. 7G): MDA-MB-231 cells were injected into the right and left mammary fat pads of three NSG mice. Five days post-injection, mice were treated with 30 mg/kg MBZ in a sesame oil suspension (N = 2) or sesame oil alone (N = 1) 4 × per week. After 30 days, tumors and lungs were excised for further processing. Primary tumors underwent enzymatic digestion in 2 mg/mL collagenase for 1 h at 37 °C at 160 g and strained through a 70 µm filter. Cells were counted and resuspended at 0.8 × 106, 0.8 × 105, 0.8 × 104, or 0.8 × 103 cells per 100 µL in a 1:1 ratio of Matrigel to PBS. Secondary recipient nude mice (female, 6–8 weeks of age) were implanted in the 2nd, 3rd, 4th, and 5th mammary fat pad on either side of the mice. The right mammary fat pads were injected with tumor-derived cells from NSG mice treated with sesame oil alone. In contrast, the left mammary pads were injected with the following dilutions of tumor-derived cells from NSG mice treated with 30 mg/kg MBZ in a sesame oil suspension as follows: 2nd—0.8 × 106 cells, 3rd—0.8 × 105 cells, 4th—0.8 × 104, and 5th—0.8 × 103. In both the in vitro and in vivo pre-treated LDA experiments, mouse weights and tumor growth were regularly measured. At the end of the experiment, tumors were excised and weighed. Stem cell frequency was calculated based on tumor incidence in all treatment groups using ELDA: Extreme Limiting Dilution Analysis. [35]
Mebendazole in vivo dosages
MBZ pure polymorph C powder (Batch No. 1180916-WC, K A Malle Pharmaceuticals LTD) was utilized in all in vitro experiments and utilized for oral gavage dosing of mice. The mice were weighed on a weekly basis with their body masses averaged in treatment groups to calculate the 30 mg/kg MBZ dosage in a sesame oil (Sigma-Aldrich) suspension. The mice were treated 4× a week for the experiment and exposed to a weekly dosage of 120 mg/kg of MBZ. A second route of MBZ treatment utilized was MBZ incorporated into mouse feed. Research Diets, Inc. (New Brunswick, NJ) formulated rodent diets with 45% Kcal fat and modified version with MBZ at 215 ppm (0.18% MBZ of total mixture) and 250 ppm (0.22% MBZ of total mixture). The ingredients are as follows: protein, carbohydrate, fat, casein, l-cystine, corn starch, maltodextrin 10, sucrose, cellulose, soybean oil, lard, mineral mix S10026, dicalcium phosphate, calcium carbonate, potassium citrate, vitamin mix V10001, choline bitartrate, FD&C yellow dye #5, and red dye #40. The mice were weighed, and food consumed was monitored to calculate the dosages administered of MBZ. The mice were able to eat the feed incorporated with MBZ seven days a week and on average consumed the following dosages: 215 ppm–152 mg/kg MBZ weekly intake (25 mg/kg/day) and 250 ppm–197 mg/kg MBZ weekly intake (32 mg/kg/day). These routes of MBZ dosages differ in dose rates since the mice exposed to the feed have consistent exposure unlike the oral gavage dosed mice that are given treatments 4 × per week.
Statistical analysis
All data are presented as mean ± standard error of the mean (SEM), and statistical analysis was performed using GraphPad Prism 9 with statistical tests appropriate for each experimental setup. Each comparison of two variables was performed using an unmatched two-way ANOVA with Bonferroni multi-comparison tests. A comparison of a single variable measured in a sample at two different locations was performed via paired two-tailed Student’s t test. A comparison of “yes” or “no” metastases categorical data was performed via a chi-squared test. Significance levels are reported as ****P < 0.0001, ***P < 0.001, **P < 0.01 *P < 0.5.