Sex as a biological variable. This study exclusively examined female human breast tissue microarrays and female mice, because the disease modeled — breast cancer — is mainly relevant in females.

Animals. Six-week-old female BALB/c (stock jlc0003), BALB/c-nu/nu (jlc0005), and NOD-SCID (jlc0008) mice were purchased from Shanghai Lingchang Biotechnology Co. All mice were housed under specific pathogen–free conditions (12-hour light/12-hour dark cycle, 50% relative humidity, and 22°C ± 2°C) with free access to a normal laboratory diet (SZS9126, Xietong Pharmaceutical Bioengineering) and sterile water and were monitored by inspection twice each day.

Cell lines and culture conditions. The human breast cancer cell lines MDA-MB-231 (stock HTB-26), SUM159 (Y-XB-2391), MDA-MB-468 (HTB-132), and MCF7 (CRL-12584), the murine TNBC cell line 4T1 (CRL-2539), and the human cell lines HEK293T (CRL-3216) and HEK293FT (PTA-5077) were all purchased from ATCC. These cells were cultured in DMEM (L110KJ, BasalMedia) supplemented with 10% FBS) (F8318, Sigma-Aldrich) and 1% penicillin/streptomycin (C100C5, NCM Biotech) at 37°C in a humidified incubator with 5% CO2.

Generation of the monoclonal antibody for human PEAR1. Various anti–human PEAR1 monoclonal antibodies were developed by our research group by the hybridoma technique and screened by ELISA. After sequencing, the antibody was expressed in HEK293S cells and purified by protein A beads (17). For the therapeutic antibody, to avoid the possibility of PEAR1 clustering caused by the 2 Fab arms of the normal antibody and to prolong the lifespan of the antibody in vivo, 1 Fab of the antibody was coupled to HSA, and Fab-HSA targeting the PEAR1-EMI domain was obtained.

Generation of the anti–phospho-PEAR1 Ser891 antibody. An anti–phospho-PEAR1 Ser891 antibody (p-PEAR1 Ser891 Ab) was generated by Shanghai GL Biochem Co. Rabbit antiserum against PEAR1 Ser891 phosphorylation was generated using the peptide Cys-RGSSRLDRSY(pS)YSYSNGP coupled to the carrier protein keyhole limpet hemocyanin (KLH), the Ser891 of which is phosphorylated and indicated as pS. The antiserum was precleaned with affinity chromatography using the corresponding nonphosphorylated peptide (Cys-RGSSRLDRSYSYSYSNGP) coupled to Pierce NHS-Activated Agarose and purified by affinity chromatography. The nonphosphorylated PEAR1 antibody was purified using nonphosphorylated peptide and used as a basal control for the p-PEAR1 Ser891 antibody. The affinity and specificity of this anti–phospho-PEAR1 Ser891 antibody were evaluated as shown in Supplemental Figure 9, A–D.

Plasmid construction and transfection. To generate stable PEAR1-overexpressing and PEAR1-knockdown cells and stable CD44-knockdown cells, we used a lentiviral system. Specifically, PEAR1-overexpressing plasmids (WT, mutated-SA, and EMI deletion) and their corresponding negative controls were synthesized by Shanghai Saiheng Biotechnology Co. Lentiviral vectors encoding human PEAR1/murine Pear1–based shRNA (shPEAR1/shPear1) and human CD44–based short hairpin shRNA (shCD44), as well as control hairpins (shnc), were designed and synthesized by Shanghai Genechem Co. Lentivirus was produced by cotransfecting HEK293FT cells with the target plasmids alongside psPAX2 and pMD2.G. After 48 and 72 hours, the lentiviral supernatants were collected, centrifuged at 2,000 g for 15 minutes, and filtered through 0.45 μm sterile filters (SLHVR33RB, Millex). Subsequently, breast cancer cells were infected with lentivirus overnight in the presence of 8 μg/mL polybrene (40804ES76, Yeasen) and selectively cultured with 2 μg/mL puromycin (60210ES60, Yeasen). The PEAR1-ICD–Flag cDNA sequence was synthesized and cloned and inserted into the pcDNA3.4 vector, and the CD44-ICD-HA cDNA sequence was synthesized and cloned and inserted into the pcDNA3.1+ vector. At approximately 70% confluence, cell transfections were performed using Lipofectamine 2000 (11668019, Thermo Fisher Scientific) according to the manufacturer’s instructions. Transfection efficiency was assayed after 48 hours using RT-qPCR and Western blotting. The sequences for the plasmids and shRNA are listed in Supplemental Table 1.

Mouse experiments. For the xenograft models, 6-week-old female BALB/c-nu/nu mice were randomly divided into 2 groups (n = 5 per group). Either negative control–transfected MDA-MB-231 cells or shPEAR1-transfected MDA-MB-231 cells (5 × 106) suspended in 50 μL sterile PBS were orthotopically injected into the inguinal mammary fat pads of the mice. Similarly, 6-week-old female BALB/c mice were randomly assigned to 2 groups (n = 5 per group). Either negative control–transfected 4T1 cells or shPear1-transfected 4T1 cells (2 × 106), diluted in 50 μL sterile PBS, were orthotopically injected into the inguinal mammary fat pads of the mice. Tumor volumes were assessed using caliper measurements and calculated using the following formula: V = a × b2/2 (a, longer diameter; b, shorter diameter). For the metastasis models, 6-week-old female BALB/c-nu/nu mice were randomly divided into 2 groups (n = 5 per group). Either negative control–transfected MDA-MB-231 cells or shPEAR1-transfected MDA-MB-231 cells (5 × 105) were diluted in 100 μL sterile PBS and injected via the tail vein. The same procedures were employed for oe-PEAR1-WT–transfected MDA-MB-231 cells and oe-PEAR1–mutated–all SA/S891A-transfected MDA-MB-231 cells. For PEAR1 Fab-HSA treatment, 6-week-old female NOD-SCID mice were divided into 3 groups (n = 5 per group). The mice were injected with MDA-MB-231 cells (5 × 105), diluted in 100 μL sterile PBS, and then treated concurrently with i.v. administration of PEAR1 Fab-HSA (3.35 mg/kg), control HSA (3.35 mg/kg), or vehicle (PBS) via the tail vein. This treatment was continued every 4 days for 40 days until the mice were sacrificed. At the end of the experiments, mice were euthanized, and the tumors, lungs, and livers were resected for analysis.

Tissue microarray analysis. Human breast tissue microarrays containing 126 breast tumor samples (stock HBre-Duc090Sur-01), 86 normal adjacent breast samples (HBreD145Su01), and 80 corresponding paracancer tissue samples (TNBC-1602) were acquired from Shanghai Outdo Biotech Co. IHC staining of the tissues for PEAR1 (HPA035217, Sigma-Aldrich), phospho-PEAR1 Ser891 (developed as described above), LOXL2 (67139-1-Ig, Proteintech), and CD44 (3570, Cell Signaling Technology) was performed. Subsequently, the stained tissue microarrays were scanned with an Aperio ScanScope and analyzed with Aperio ImageScope software version 12.3.2. The results were scored according to the intensity and area of the cellular staining. Staining intensity was scored as 0 (negative particles), 1 (faintly yellow particles), 2 (brownish-yellow particles), and 3 (brown particles). The final score was determined by multiplying the staining color scores by the proportion of positively stained area. Survival data were evaluated with Kaplan-Meier curves and subjected to statistical analyses through log-rank tests. Correlations between the parameters were determined using Pearson’s correlation analysis. The prognostic effects of the risk scores were assessed using the AUC in time-dependent ROC curve analysis using 10 years as the judgment period. The risk score was the product of the IHC staining score and the hazard ratio [HR = risk function h1(t) in the exposed group/risk function h2(t) in the nonexposed group, where t refers to the same point in time]. The clinical and pathological characteristics of the patients’ tumors are presented in Supplemental Tables 2 and 3.

H&E staining. Tissues were fixed for 36 hours in 4% PFA (E672002-0500, Sangon Biotech), embedded in paraffin, and sectioned at a thickness of 5 μm. The paraffin-embedded tissue sections were deparaffinized and rehydrated, incubated with hematoxylin (BA4027, BASO) for 5 minutes, incubated with eosin (BA4027, BASO) for 1 minute, and then thoroughly washed with ddH2O between each step. After dehydration, the slides were sealed with neutral balsam (36313ES60, Yeasen). Histologic characteristics were described and metastatic nodules were enumerated under a light microscope.

RNA isolation and RT-qPCR. Total RNA extraction from cells was performed using RNA Isolater Total RNA Extraction Reagent (R401-01, Vazyme), following the manufacturer’s instructions. cDNA was synthesized from 1 μg total RNA using PrimeScript RT Master Mix (Perfect Real Time, RR036A, Takara) and amplified with TB Green Premix Ex Taq II (Tli RNaseH Plus, RR820A, Takara) using the LightCycler 96 Real-Time PCR System. Relative expression of mRNA was determined after normalization to 18S rRNA and calculated using the 2−ΔΔCT method. For a detailed list of primer sequences, see Supplemental Table 4.

Western blotting. Cultured cells were collected with a scraper and then lysed with RIPA buffer (P0013B, Beyotime; composed of 50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS with phosphatase and protease inhibitors) on ice for 15 minutes, and the samples were sonicated, followed by centrifugation at 12,000 g for 20 minutes. Nuclear and cytoplasmic protein extraction (P0028, Beyotime) and membrane and cytosol protein extraction (P0033, Beyotime) were performed following the corresponding kit protocols. The resulting cell lysates were collected and quantified with a BCA Protein Assay Kit (20201ES90, Yeasen). Following denaturation at 100°C for 10 minutes, equivalent amounts of protein samples were loaded and separated using 8%–12% SDS-PAGE and then transferred onto 0.45 μm PVDF membranes (IPVH00010, Millipore). The immunoblots were incubated in blocking buffer (consisting of 5% [wt/vol] skim milk in TBST, 10 mM Tris-HCl [pH 7.5], 500 mM NaCl, and 0.1% Tween 20) for 2 hours at room temperature, followed by incubation with specific antibodies overnight at 4°C. Then the immunoblots were washed 3 times for 10 minutes in TBST, incubated with HRP-conjugated secondary antibodies diluted in blocking buffer for 1 hour at room temperature, and washed 3 times in TBST again. The detected signals were visualized with enhanced chemiluminescence (P10300, NCM Biotech) using the Tanon 2500 Luminescence imaging system.

co-IP. The transfected cells were lysed with cell lysis buffer (P0013, Beyotime; composed of 20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 1% sodium with phosphatase and protease inhibitors) on ice for 15 minutes, followed by centrifugation at 12,000 g for 20 minutes. For endogenous co-IP, the lysates were incubated with the anti-PEAR1 antibody (developed as described above) or anti-CD44 antibody (15675-1-AP, Proteintech) under gentle rotation overnight at 4°C, while corresponding isotype IgG (5415&2729, Cell Signaling Technology) was used as a negative control. The next day, protein A/G plus-agarose beads (sc-2003, Santa Cruz Biotechnology) were added to the lysates and incubated for 4 hours. For exogenous co-IP, the lysates were incubated with anti-Flag agarose beads (A2220, Sigma-Aldrich) or anti-HA agarose beads (26182, Thermo Fisher Scientific ) overnight under gentle rotation at 4°C. Afterward, the beads coupled to the immunocomplexes were collected and washed 3 times with TBS (composed of 10 mM Tris-HCl [pH 7.5] and 500 mM NaCl) by centrifugation at 12,000 g for 1 minute. Thereafter, the eluted proteins were subjected to denaturation at 100°C for 10 minutes and analyzed by MS (performed by the Public Platform of Basic Medicine, Shanghai Jiao Tong University School of Medicine) and Western blotting to detect the interacting proteins.

Pulldown assay. MDA-MB-231 cells were cultivated with the PEAR1 ligands until they reached approximately 85% confluence in a 10 cm culture dish. Subsequently, the cells were gently washed 3 times with serum-free medium and then cultured in serum-free medium at 37°C with 5% CO2 for 24 hours. Afterward, the supernatants were collected, centrifuged at 2,000 g for 15 minutes, and then filtered through 0.22 μm filters (SLGVR33RB, Millex). The resulting filtrate was then concentrated using Amicon Ultra-4 (3 kDa MWCO; UFC8003, Millipore). The secreted proteins in supernatant were incubated with PEAR1-ECD–His protein (17), and Dynabeads His-tag (10103D, Thermo Fisher Scientific) were used to pull down the His-tagged protein and its interacting proteins per the manufacturer’s instructions. The eluate was subjected to silver staining. Then the differential bands were cut for further MS analysis and Western blotting. Analogously, the combination between PEAR1-ECD-His (17) and CD44-ECD–Fc (221334, Abcam) was examined using His-tag pulldown with Dynabeads.

Confocal IF. Cells were seeded onto microscopic glass coverslips (VWRI631-0149, VWR) in 24-well culture plates and cultured overnight. Following drug administration, the cells were gently rinsed with PBS and fixed with 4% PFA for 1 hour. Then the cells were rinsed twice with PBS, followed by permeabilization with 0.2% Triton X-100 (93443, Sigma-Aldrich) in PBS for 10 minutes at room temperature. Thereafter, the cells were blocked in 2% BSA (B2064, Sigma-Aldrich) in PBS for 1 hour at room temperature. Then the cells were incubated with primary antibodies in PBS containing 1% BSA overnight at 4°C. The next day, the cells were rinsed twice with PBS and then incubated with fluorochrome-conjugated secondary antibodies for 2 hours at room temperature in the dark. Next, the cells were rinsed and sealed with DAPI Fluoromount-G (36308ES20, Yeasen). Finally, imaging was performed under a Leica TCS Sp8 STED confocal microscope with a 100× magnification objective.

Mammosphere formation. Cells were trypsinized (C100C1, NCM Biotech) and cultured in 24-well Nunclon Sphera plates (174930, Thermo Fisher Scientific), with a precise seeding of 1,000 cells per well in serum-free conditional medium (consisting of DMEM/nutrient mixture F-12 [DMEM/F12; L310KJ, BasalMedia] supplemented with 20 ng/mL EGF [CYT-217, Prospec], 20 ng/mL basic FGF [bFGF; CYT-288, Prospec], 4 μg/mL insulin [40107ES25, Yeasen], 0.4 ng/mL hydrocortisone [40109ES08, Yeasen], 0.4% BSA, 1× B27 [12587010, Gibco], and 1% penicillin/streptomycin) at 37°C in a humidified incubator with 5% CO2. The mammospheres were formed for the first time after approximately 10 days. Then the mammospheres were trypsinized again, prepared into a single-cell suspension with serum-free medium, and cultured under ultra-low adhesion condition. After 20–25 days, mammospheres were imaged and counted under a microscope.

Transwell assay. Matrigel (BD 356234; Matrigel/DMEM 1:3, serum-free) was added to the upper chamber of an 8-μm-pore-size insert (CLS3422, Corning) and allowed to gel at 37°C for 3 hours. Then the cells were suspended and seeded with serum-free medium containing diverse treatments into the upper chamber (1 × 105 cells per well), whereas the lower 24-well plates were filled with 750 μL DMEM containing 20% FBS to support cell health. Following 48-hour incubation at 37°C, the noninvasive cells were removed by wiping with cotton swabs, whereas the cells that adhered to the underside of the chamber were fixed with methanol and stained with 0.1% crystal violet (C8470, Solarbio) for 30 minutes. The number of invading cells was calculated using microscope images taken from 5 random fields.

Wound healing assay. Cells were seeded into 6-well plates (2 × 106 cells per well) and cultured to full confluence in complete DMEM. Subsequently, media were replaced by serum-free media for 24 hours, and a sterile 200 μL pipette tip was used to create “wounds’” by gently scratching the confluent cell monolayers. Samples were gently washed with PBS to remove cell debris. The cultured cells were then cultured in serum-free media supplemented with various drugs. Images of the wounds were captured using a microscope at 0, 12, 24, 36, and 48 hours, and the healing rates were analyzed with ImageJ software (NIH).

Cell counting kit-8 assay. Cell proliferation was determined by employing the Cell Counting Kit-8 (CCK-8) assay. Following diverse treatments, cells were seeded into 96-well plates (5,000 cells per well) and incubated with 10% CCK-8 reagent (CK04, Dojindo) for 2 hours. Subsequently, cell viability was measured at 6 distinct time points: 0, 24, 48, 72, 96, and 120 hours. OD was detected at 450 nm by a microplate reader.

EdU cell proliferation assay. The method was based on the incorporation of the thymidine analog EdU (5-ethynyl-2′-deoxyuridine) during DNA synthesis and the subsequent click reaction to label EdU with biotin. Then HRP-labeled streptavidin was added to biotin. To assess TMB (3, 3’, 5, 5’-Tetramethylbenzidine) color, we directly measured absorbance at 370 nm or 620–650 nm. Alternatively 2 M H2SO4 was added to terminate the reaction, and absorbance was subsequently measured at 450 nm. The kit instructions were followed to achieve a simple, rapid, and highly sensitive method for quantitative detection of cell proliferation in porous plates (C0088S, Beyotime Biotechnology).

ELISA. Briefly, 96-well EIA/RIA (enzyme immunoassay/radio immunoassay) plates (CLS3361, Corning) were coated with recombinant streptavidin (10 μg/mL, 100 μL; P5084, Beyotime) in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) for 1.5 hours at 37°C. Then the wells were washed 3 times with washing buffer (PBST, 0.05% Tween 20 in PBS), blocked with 2% BSA for 1 hour at 37°C, and coated with capture biotin-conjugated PEAR1-ECD domain peptides (EMI and EGF-like 1 through EGF-like 15; 10 μg/mL, 100 μL) in PBS overnight at 4°C. In contrast, the capture proteins were diluted in coating buffer directly and coated overnight at 4°C. The next day, the wells were washed 3 times with PBST, blocked with 2% BSA for 1 hour at 37°C, and washed 3 times with PBST again. Then 100 μL samples were added to the wells and incubated for 1 hour at 37°C, after which the plates were washed 4 times with PBST. Next, HRP-conjugated secondary antibodies (1:10,000) in 1% BSA in PBS were added to the wells for 30 minutes at 37°C, and the wells were washed 6 times with PBST before the addition of TMB (34029, Thermo Fisher Scientific). Ten minutes later, 2 M H2SO4 was added to the wells to stop the reaction, and OD was immediately measured at 450 nm using a microplate reader. The sequences of the recombinant proteins and peptides are listed in Supplemental Table 5.

Quantification and statistical analyses. All experiments were repeated 3 times independently, with similar results obtained, and representative data are shown in the figures. Statistical analyses were performed using GraphPad Prism 7.0 software or IBM SPSS Statistics 20.0 software. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the MS data (Figure 1B) were performed using DAVID and KOBAS online. Differences between 2 groups were assessed by employing an unpaired 2-tailed Student’s t test. For comparisons, 1-way ANOVA followed by Dunnett’s post hoc test was used. To compare overall survival curves, Kaplan-Meier plots and the log-rank test were used. Correlations between parameters were assessed using Pearson’s correlation analysis. Prognostic effects of the risk scores were assessed by the AUC in a time-dependent ROC curve analysis. Details regarding data presentation and statistical analyses are provided within the figure legends. A 2-sided P value less than 0.05 was considered statistically significant.

Study approval. This study was approved by the Shanghai Jiao Tong University School of Medicine Science and Technology Ethics Committee. All experimental procedures were conducted in accordance with protocols approved by the Shanghai Jiao Tong University School of Medicine IACUC. All human tissue specimens were collected by Shanghai Outdo Biotech Co. in compliance with their informed consent policy. All animal studies were conducted in full accordance with the guidelines for the care and use of laboratory animals and were approved by the Shanghai Jiao Tong University School of Medicine IACUC.

Data availability. Values for all data points in graphs are reported in the Supporting Data Values file. The MS proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD052128. Data can also be obtained from the corresponding author upon reasonable request.