Bovine Holo-lactoferrin inhibits tumor growth and metastasis induced by BPA in murine triple negative breast cancer models

Bovine Holo-lactoferrin inhibits tumor growth and metastasis induced by BPA in murine triple negative breast cancer models | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bovine Holo-lactoferrin inhibits tumor growth and metastasis induced by BPA in murine triple negative breast cancer models Rina Valenzuela-Echeverria, Rocio Castillo-Sanchez, Pablo Torres-Alamilla, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8904788/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Breast cancer is the leading cause of cancer-related deaths in worldwide women. Triple negative breast cancer (TNBC) subtype does not express progesterone, estrogen and Her2 receptors. Bisphenol A (BPA) is an endocrine-disrupting chemical used in plastics and epoxy resins production that is associated with an increased rate of cancer including breast cancer. Lactoferrin (Lf) is a glycoprotein expressed in a variety of species that is found free of Fe 3+ (Apo-Lf) and associated with Fe 3+ (Holo-Lf). Bovine Lf (BLf) exhibits anti-tumor properties through inhibition of proliferation, migration, invasion, matrix metalloproteinases secretion, and epithelial–mesenchymal transition. In this study we demonstrate that Holo-BLf inhibits migration, invasion and assembly of focal adhesions induced by BPA in 4T1 cells, and migration in MDA-MB-231 cells. In addition, Holo-BLf inhibits the growth of mammary tumors and metastasis induced by BPA in two murine models of breast cancer using TNBC 4T1/MDA-MB-231 cells and Balb/cJ/NSG mice. In conclusion, we demonstrate, for first time, that Holo-BLf inhibits cellular processes involved with the metastasis process and the growth of mammary tumors and metastasis in two murine models of breast cancer. Breast cancer Lactoferrin Mammary tumor Metastasis 4T1 Migration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Breast cancer is the most common neoplasia among women in developed countries and the leading cause of mortality in women worldwide. In 2022, breast cancer was diagnosed in 2.3 million women, and 680,000 deaths were recorded in 2020 (Filho et al., 2025 ; Sung et al., 2021 ). Molecular classification of breast cancer considers the expression of estrogen receptor (ER), progesterone receptor (PR) and Her2, and recently it has been considered the expression of Ki67. Triple negative breast cancer (TNBC) is a subtype that does not express ER and PR, presents low levels of Her2 and high expression of Ki67 (Anders, Abramson, Tan, & Dent, 2016 ). TNBC subtype constitutes about 15–20% of all breast cancer cases and has aggressive behavior, early relapses and the highest incidence of metastasis (Bou Zerdan et al., 2022 ). Bisphenol A (BPA) is a synthetic compound used as monomer or additive in the manufacturing of polycarbonate plastics and epoxy resins, which are employed in metal can linings, thermal paper, dental compounds and containers and packaging for food and drink (Cimmino et al., 2020 ; Lehmler, Liu, Gadogbe, & Bao, 2018). Humans are exposed to BPA mainly through ingestion of food and water (90–99%), because high temperature and basic solutions promote the cleavage of ester bonds that hold BPA monomers together and then BPA is released into food and beverages (Colorado-Yohar et al., 2021 ; Sajiki & Yonekubo, 2004 ). BPA is an endocrine disrupting chemical with estrogenic activity and is associated with variations in hormonal homeostasis, sexual differentiation and increasing rates of cancer including breast cancer (Cimmino et al., 2020 ; Nohynek, Borgert, Dietrich, & Rozman, 2013 ). Lactoferrin (Lf) is a highly conserved cationic glycoprotein in mammals, which has a similar tertiary structure between a variety of species including bovine, porcine, murine and human (Kowalczyk et al., 2022 ). Lf structure consists of two homologous globular domains (N and C lobes) connected by an alpha helix, where each lobe has a metal-binding site composed of four conserved amino acid residues (Gonzalez-Chavez, Arevalo-Gallegos, & Rascon-Cruz, 2009 ; Wang, Timilsena, Blanch, & Adhikari, 2019 ). Lf has the ability to bind Fe 2+ or Fe 3+ using the two lobes of the molecule, and then Lf is found free of Fe 3+ (Apo-Lf) and associated with Fe 3+ (Holo-Lf), which generates a different conformation of Apo-Lf and Holo.Lf, stability of the molecule and functional features (Baker & Baker, 2012 ; Gonzalez-Chavez et al., 2009 ). Bovine Lf (BLf) shares 69% homology with human Lf, and the exogenous treatment with BLf presents anti-tumor activity in a variety of cancers (Cutone, Rosa, et al., 2020 ). Particularly, Holo-BLf and Apo-BLf induce apoptosis through inhibition of survivin in MCF-7 and MDA-MB-231 breast cancer cells, whereas BLf partly inhibits migration and invasion in MDA-MB-231 cells (Gibbons, Kanwar, & Kanwar, 2015 ; Rodriguez-Ochoa, Cortes-Reynosa, Rodriguez-Rojas, de la Garza, & Salazar, 2023 ). In this study, we demonstrate that Holo-BLf inhibits cellular processes that mediate invasion/metastasis process including migration, invasion and assembly of focal adhesions induced by BPA in 4T1 cells, and migration in MDA-MB-231 cells. In addition, Holo-BLf inhibits the growth of mammary tumors and metastasis induced by BPA in two murine models of breast cancer. Materials and methods Materials Apo-BLf (97% of purity) was from NutriScience Innovations, LLC (Connecticut, USA). BPA, anti-vinculin antibody (Ab), Bouin’s solution, Tetramethylrhodamine (TRITC)-conjugated phalloidin and 4',6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich (Merck KGaA). Matrigel matrix was from Corning (Bedford, USA). Preparation of Holo-BLf Holo-BLf was obtained by saturation of Apo-BLf with iron as described previously (Avalos-Gomez et al., 2020 ). Concentration of iron in Holo-BLf was of 93%, which was determined by an enzymatic automated method, (MicroTech Laboratories, Mexico). Cell lines and culture Human TNBC MDA-MB-231 cells and murine TNBC 4T1 cells were obtained from the American Type Culture Collection (ATCC). MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) complemented with 5% fetal bovine serum (FBS), 3.7 g/l sodium bicarbonate and antibiotics. 4T1 cells were cultured in RPMI-1640 medium supplemented with 5% FBS and antibiotics. Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 and 95% air. For experimental purposes, MDA-MB-231 cells were starved in DMEM without FBS for 18 h and 4T1 cells were starved in RPMI-1640 medium with 0.5% FBS for 5 h before treatment with BPA and/or Holo-BLf. Scratch wound assays Cultures of 4T1 cells (1.5x10 6 cells/35-mm culture plate) were scratch-wounded, washed twice with PBS, refed with RPMI-1640 medium/0.5% FBS and then untreated and treated with 1250 nM Holo-BLf and/or stimulated with 1 µM BPA for 48 h at 37°C. At the end of incubation, cells were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% violet crystal solution for 10 min. Images were acquired from at least three fields per experimental condition from three independent experiments, and they were analyzed using the ImageJ software v1.52e (NIH, USA). Invasion assays Inserts with 8 µm pores of 24-well plates were covered with Matrigel (15 µg/insert) and incubated for 12 h at 37 ºC. Next, 4T1 cells (1x10 5 cells/well) were plated on Matrigel in RPMI-1640 medium/0.5% FBS enriched without or with 1250 nM Holo-BLf. Lower chambers were filled with RPMI-1640 medium/0.5% FBS supplemented without or with 1 µM BPA. Plates were incubated for 30 h at 37°C in a humidified atmosphere with 5% CO 2 and 95% air. At the end of incubation, Matrigel on the upper surface of membranes was removed using cotton swabs. Cells on the lower surface of membranes were washed with PBS and fixed with methanol for 25 min and then stained with 0.1% crystal violet in PBS for 15 min. Membranes were photographed and images of at least 3 fields of each experimental condition were obtained. Dye was eluted using 750 µl of 10% acetic acid, and absorbance was measured at a wavelength of 600 nm. Immunofluorescence confocal microscopy 4T1 cells were grown on coverslips, washed with PBS, equilibrated in FBS-free RPMI-1640 for 30 min and then untreated and treated with 1250 nM Holo-BLf and/or stimulated with 1 µM BPA for various times as indicated. Cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 for 20 min and blocked with gelatin solution (0.5% gelatin, 1 mM CaCl 2 , 0.5 mM MgCl 2 ) for 20 min at room temperature. Cells were stained by incubation with anti-vinculin Ab (1:1000) for 12 h at 4°C followed by incubation with FITC-labeled anti-mouse secondary Ab for 2 h at room temperature. Fibrillar actin was stained by incubation of cells with TRITC-conjugated phalloidin for 2 h, whereas nucleus was stained by incubation of cells with DAPI for 5 min at room temperature. Cells were mounted on glass slides with Vectashield and analyzed by confocal microscopy (Model TCS SP8; Leica Microsystems, Inc). Ethics statement for experimental procedures with female Balb/cJ and NSG mice Unit for Production and Experimentation of Laboratory Animals (UPEAL) of the Center of Research and Advanced Studies (Cinvestav, Mexico) provided female Balb/cJ mice of 6–8 weeks old and NOD-SCID-gamma (NSG) mice of 4–6 weeks old. Experimental procedures were carried out ethically following the guidelines established by the Institutional Animal Care and Use Committee of UPEAL, which operates under the Mexican Official Standard NOM-062-ZOO-1999 for technical specifications for the production, care, and use of laboratory animals (NOM-062-ZOO-1999, 2001). Protocol numbers 0294 − 19 and 0370 − 24 Formation of mice groups Female Balb/cJ and NSG mice were randomly divided into four groups of 10 and 4 mice per group respectively: 1) Control group was untreated mice; 2) BPA group was mice treated with 10 µg/kg/mouse BPA; 3) Holo-BLf group was mice treated with 50 mg/kg/mouse Holo-BLf; and 4) Holo-BLf + BPA group was mice treated with 50 mg/kg/mouse Holo-BLf and/or 10 µg/kg/mouse BPA. Induction of mammary tumors in female Balb/cJ and NSG mice MDA-MB-231 and 4T1 cells were harvested from semi-confluent cultures (80%), washed once with PBS, collected by centrifugation and 4T1 cells were resuspended in RPMI-1640 medium at 6x10 4 cells/ml, whereas MDA-MB-231 cells were resuspended in DMEM enriched with human type IV collagen (400 mg/ml) at 6x10 4 cells/ml. Balb/cJ mice were inoculated with 3,000 4T1 cells and NSG mice were inoculated with 50,000 cells into the left fourth inguinal mammary fat pads using 0.3 ml tuberculin syringe equipped with a 31G needle. Mice were housed in Super Mouse 750™ ventilated cages with five mice per cage, under regulated temperature and 12 h light/dark cycle. Treatment of Balb/cJ and NSG mice was performed by oral administration of Holo-BLf (50 mg/kg/mouse) and BPA (10 µg/kg/mouse). Mice treated with Holo-BLf + BPA were treated with BPA (10 µg/kg/mouse) in the morning and 6 h later were treated with Holo-BLf (50 mg/kg/mouse). Treatments started on the third day after inoculation with 4T1 cells, and then treatments were administered daily for 18 days. Mice were monitored daily and weighed every third day. Balb/cJ mice were euthanized after 21 days post-inoculation, whereas NSG mice were euthanized after 23 days post-inoculation using carbon dioxide administered through the Euthanex Smartbox Auto CO 2 System EA-3400 (flow rate of 6 l per minute for 5 min). Mammary tumors, lungs, livers and brains were obtained (Fig. S1 ). Volume of mammary tumors was determined by the equation V = (W2 × L)/2, where W represents the width and L the length of tumors (Faustino-Rocha et al., 2013 ). Mammary tumors were fixed in 4% paraformaldehyde for 24 h, whereas lungs, livers and brains were fixed in Bouin's solution for 24 h. Tumors were stored in 70% ethanol. Metastatic nodules in livers, lungs and brains were identified by the presence of spherical white areas on a background of yellow-stained tissue (X. Xu et al., 2016 ). Histopathology Fixed mammary tumors, livers, lungs and brains were paraffin-embedded. One paraffin block per organ (liver, lung, brain) or mammary tumor from each mouse across all three experimental groups (n = 10/group) were obtained. Three tissue sections of 4 µm were trimmed from each block and mounted in duplicate on separate glass slides. Sections were deparaffinized, rehydrated, and subjected to hematoxylin and eosin staining (Cardiff, Miller, & Munn, 2014 ). Three random fields per tissue section were analyzed using a Nikon H550S microscope. All slides were examined by a pathologist blinded to experimental groups to objectively assess tumor characteristics and micrometastasis across experimental conditions. Statistical analysis Data were evaluated for normal distribution using the Komolgorov-Smirnov statistical test. Data with normal distribution were analyzed using unpaired t test or the one-way ANOVA statistical test followed by Dunnett's multiple comparison post hoc test. Data without normal distribution were analyzed by the Mann-Whitney test and Kruskal-Wallis statistical test followed by uncorrected Dunn´s test or Dunn's post hoc multiple comparison test. Results are presented as the mean ± S.D. of at least three independent experiments. A statistical probability of P ≤ 0.05 was considered significant. Results Holo-BLf inhibits migration and invasion induced by BPA in TNBC cells BPA induces migration and invasion in MDA-MB-231 and 4T1 breast cancer cells (Castillo-Sanchez et al., 2020 ; Castillo Sanchez, Gomez, & Perez Salazar, 2016 ; Torres-Alamilla, Castillo-Sanchez, Cortes-Reynosa, Gomez, & Perez Salazar, 2023). We determined whether Holo-BLf inhibited migration induced by BPA in MDA-MB-231 and 4T1 cells. Cultures of MDA-MB-231 and 4T1 cells were scratched and then untreated and treated with 1250 nM Holo-BLf and/or stimulated with 1 µM BPA for 24 h. Findings showed that treatment with Holo-BLf inhibited migration induced by BPA in MDA-MB-231 and 4T1 cells (Fig. 1 A and B). Next, we determined whether Holo-BLf inhibited invasion induced by BPA. Invasion assays were performed using 4T1 cells untreated and treated with 1250 nM Holo-BLf and/or stimulated with 1 µM BPA for 30 h. As illustrated in Fig. 1 C, treatment with Holo-BLf inhibited invasion induced by BPA in 4T1 cells. Holo-BLf inhibits formation of focal adhesions induced by BPA in 4T1 cells BPA induces migration in 4T1 cells (Torres-Alamilla et al., 2023 ). We determined whether BPA induced the formation of focal adhesions. Cultures of 4T1 cells were cultured on coverslips and then unstimulated and stimulated with 1 µM BPA for 10, 15, 30 and 45 min. Presence of focal adhesions was determined by immunofluorescence analysis of vinculin, which is a protein localized in focal adhesions (Bays & DeMali, 2017 ). Findings showed that BPA induced the formation of focal adhesions in a time-dependent manner reaching a maximum at 15 min of stimulation in 4T1 cells (Fig. 2 ). Next, we determined whether Holo-BLf inhibited the formation of focal adhesions induced by BPA in 4T1 cells. Cultures of 4T1 cells were cultured on coverslips and then untreated and treated with 1250 nM Holo-BLf and/or stimulated with 1 µM BPA for 15 min. Presence of focal adhesions was determined by immunofluorescence analysis of vinculin. Findings showed that BPA promoted an increase in the number of focal adhesions, however treatment with Holo-BLf inhibited the assembly of focal adhesions induced by BPA in 4T1 cells (Fig. 3 ). Holo-BLf inhibits the growth of mammary tumors in Balb/cJ inoculated with 4T1 cells and NSG mice inoculated with MDA-MB-231 cells and treated with BPA BPA promotes an increase in the growth of mammary tumors in a murine model of breast cancer using Balb/cJ mice inoculated with 4T1 cells (Torres-Alamilla et al., 2023 ). We determined whether treatment with Holo-BLf inhibited the growth of mammary tumors induced by treatment with BPA in Balb/cJ mice. 4T1 cells were inoculated into mammary fat pads of Balb/cJ mice, and then mice were untreated and treated with 10 µg/kg/mouse BPA, and/or 50 mg/kg/mouse Holo-BLf for 21 days. As illustrated in Fig. 4 A-C and Table 1, treatment with Holo-BLf inhibited the increase of weight and volume of mammary tumors induced by treatment with BPA in Balb/cJ mice inoculated with 4T1 cells. Histological sections of mammary tumors stained with hematoxylin and eosin were analyzed at low (10x) and high (40x) magnification. As shown in Fig. 4 D, basal, BPA, Holo-BLf and Holo-BLf + BPA groups corresponded to high-grade malignant epithelial neoplasms, which are characterized by solid growth without tubular formation, marked nuclear pleomorphisms and high mitotic index. To substantiate our findings, we studied whether treatment with 50 mg/kg/mouse Holo-BLf inhibited the growth of mammary tumors induced by treatment with 10 µg/kg/mouse BPA in NSG mice inoculated with human MDA-MB-231 cells. Findings showed that treatment with Holo-BLf inhibited the increase of weight and volume of mammary tumors induced by treatment with BPA in NSG mice inoculated with MDA-MB-231 cells (Fig. 8 A-B and Table 1S). Holo-BLf inhibits the metastatic potential of BPA to lungs and liver in Balb/cJ mice inoculated with 4T1 cells We also analyzed whether treatment with Holo-BLf inhibited the increases in the number of Balb/cJ mice inoculated with 4T1 cells with metastasis to lungs, liver and brain. Metastasis was determined by the presence of metastatic nodules. Findings showed that treatment with BPA significantly increased the number of mice with metastasis in lungs (10/10) and liver (6/10) compared with the number of untreated mice (Basal) with metastasis in lungs (5/10) and liver (1/10) and mice treated with Holo-BLf with metastasis in lungs (4/10) and liver (1/10). However, treatment of mice with Holo-BLf completely inhibited the increase in the number of mice with metastasis to lungs (5/10) and liver (1/10) induced by BPA (Fig. 5 A and B, Table 2). We did not find any significant difference in the number of mice with metastasis to brain in all the groups of mice studied (Table 2). Holo-BLf inhibits the increase in the number of metastatic nodules in lungs and liver induced by BPA in Balb/cJ mice inoculated with 4T1 cells We determined whether treatment with Holo-BLf inhibited the increases in the total and average number of metastatic nodules in lungs, liver and brain. Findings showed that treatment of Balb/cJ mice with BPA induced an increase in the total number and average number/mouse of metastatic nodules in lungs and liver compared with total number and average number/mouse of metastatic nodules in lungs and liver from untreated mice (Basal) and mice treated with Holo-BLf. However, treatment of mice with Holo-BLf completely inhibited the increase in the total number and average number/mouse of metastatic nodules in lungs and liver induced by BPA (Fig. 6 A-D and Table 2) Metastatic nodules from lungs, liver and brain were analyzed by histological analysis using histological sections stained with hematoxylin and eosin at high (40x) magnification. As shown in Fig. 7 , lungs, liver and brain from untreated mice (Basal) did not present metastatic cells. However, lungs, liver and brain from mice treated with BPA presented large clusters of metastatic neoplastic cells. Interestingly, mice treated with Holo-BLf and BPA showed a smaller number of metastatic neoplastic cells in lungs, liver and brain, whereas a few isolated clusters of metastatic neoplastic cells were found in the lungs, liver and brain from mice treated with Holo-BLf. Holo-BLf inhibits the increase in the number of metastatic nodules in lungs induced by BPA in NSG mice inoculated with MDA-MB-231 cells To further substantiate our findings, we analyzed the number of metastatic nodules in lungs, liver and brain in NSG mice inoculated with MDA-MB-231 cells and untreated and treated with 50 mg/kg/mouse Holo-BLf and/or 10 µg/kg/mouse BPA. Findings showed that treatment of NSG mice with BPA induced an increase in the total number and average number/mouse of metastatic nodules in lungs compared with the total number and average number/mouse of metastatic nodules in lungs from untreated mice (Basal) and mice treated with Holo-BLf. However, treatment of mice with Holo-BLf completely inhibited the increase in the total number and average number/mouse of metastatic nodules in lungs Induced by BPA (Fig. 8 C, Table 2S). We did not find a significant difference in the total number and average number/mouse of metastatic nodules in liver and brain between NSG mice treated with BPA and NSG mice treated with BPA and Holo-BLf (Table 2S). Discussion BPA promotes an increase of GPER expression and the activation of signal transduction pathways via GPER that mediate migration and invasion in human TNBC MDA-MB-231 and murine TNBC 4T1 cells (Castillo-Sanchez et al., 2020 ; Castillo Sanchez et al., 2016 ; Torres-Alamilla et al., 2023 ). Moreover, oral administration of BPA increases the volume and weight of mammary tumors and promotes metastasis to lung in a murine model of breast cancer using 4T1 cells and female Balb/cJ mice (Torres-Alamilla et al., 2023 ). BLf exhibits anti-tumor activities because it inhibits migration, invasion and proliferation through arresting the cancer cells in G1 to S phase transition (Tsuda et al., 2010 ; Y. Zhang, Lima, & Rodrigues, 2014 ). We previously demonstrated that Holo-BLf inhibits migration induced by FBS and linoleic acid in MDA-MB-231 cells (Rodriguez-Ochoa et al., 2023 ). However, the capacity of Holo-BLf to inhibit cellular processes involved in the migration/invasion process, as well as its capacity to inhibit the growth of mammary tumors and metastasis in breast cancer remains to be studied. In this study, we employed a concentration of 1250 nM Holo-BLf for the “in vitro” assays with 4T1 breast cancer cells because it has been demonstrated that treatment of MDA-MB-231 breast cancer cells with 1250 nM Holo-BLf inhibits migration, invasion, MMP-9 secretion and phosphorylation of FAK at tyrosine-397 induced by FBS (Rodriguez-Ochoa et al., 2023 ). We demonstrate here treatment of MDA-MB-231 and 4T1 breast cancer cells with 1250 nM Holo-BLf inhibits migration induced by BPA. Moreover, treatment of 4T1 cells with 1250 nM Holo-BLf inhibits invasion induced by BPA. In agreement with our findings BLf partly inhibits migration and the expression of Snail and vimentin in human GL-15 and murine GL-261 glioblastoma cells and the invasion in HSC2 and HOC313 oral squamous cells (Chea et al., 2023 ; Cutone, Colella, et al., 2020 ). We propose that Holo-BLf can inhibit migration and invasion induced by a variety of ligands including endocrine disruptors in TNBC cells. Focal adhesions are structures composed of integrin receptors that mediate the interaction between the actin cytoskeleton and the extracellular matrix proteins. The composition of focal adhesions is complex and includes scaffolding proteins, GTPases, phosphatases, kinases, and adaptor proteins including vinculin (Carisey & Ballestrem, 2011 ; Wozniak, Modzelewska, Kwong, & Keely, 2004 ). Particularly, focal adhesions play a pivotal role in a variety of cellular processes including spreading, migration, invasion, differentiation, angiogenesis and survival (Zhao & Guan, 2009 ). We demonstrate here that BPA induces focal adhesions assembly in a time-dependent manner, and treatment with Holo-BLf inhibits the assembly of these focal adhesions in 4T1 cells. In agreement with our findings, Holo-BLf inhibits FAK phosphorylation at tyrosine-397 and disassembly of focal adhesions induced by FBS in MDA-MB-231 cells (Rodriguez-Ochoa et al., 2023 ). Since focal adhesions assembly/disassembly and FAK phosphorylation at tyrosine 397 are required for migration, invasion and the epithelial to mesenchymal transition process (Avizienyte & Frame, 2005 ; Zhao & Guan, 2009 ), we propose that Holo-BLf inhibits tumor progression through inhibition of focal adhesions assembly/disassembly including FAK phosphorylation at tyrosine 397 in breast cancer cells. BPA induces proliferation through inhibition of miR-381-3p and STAT3 in MCF-7 breast cancer cells (Deng et al., 2021 ; W. Zhang et al., 2012 ). Moreover, oral administration of BPA (10 µg/kg/mouse) increases the volume and weight of mammary tumors in a murine model of breast cancer using 4T1 cells and female Balb/cJ mice (Torres-Alamilla et al., 2023 ), whereas subcutaneous administration of Apo-BLf (50 mg/kg/mouse) in C57BL/6 and CDF mice inoculated with B16-B16 melanoma cells and L5178Y-ML25 lymphoma cells inhibits liver and/or lungs metastasis (Yoo et al., 1997 ). In this study, we demonstrate that oral administration of Holo-BLf (50 mg/kg/mouse) inhibits the increase of weight and volume of mammary tumors induced by BPA (10 µg/kg/mouse) in murine models of breast cancer using human MDA-MB-231 cells and murine 4T1 cells inoculated in NSG and Balb/cJ mice respectively. We propose that Holo-BLf inhibits the proliferation of MDA-MB-231 and 4T1 cells in the mammary tumor, but it does not modify the mammary tumor microenvironment. Supporting our proposal, Holo-BLf inhibits proliferation through cell cycle arrest, increases of phospho-AMPKα, decreases of phospho-mTOR, but it does not induce apoptosis in T-47D, MDA-MB-231, Hs578T and MCF-7 breast cancer cells (Y. Zhang, Nicolau, Lima, & Rodrigues, 2014 ). In addition, intratumoral administration of lactoferricin B, a 25-amino acid peptide released from bovine lactoferrin by acid-pepsin hydrolysis, promotes a reduction in the volume and weight of mammary tumors in a murine model of breast cancer using MDA-MB-231 cells and immunodeficient NSG mice (Rahman et al., 2021 ). Moreover, our findings demonstrate that stroma of mammary tumors is similar in Balb/cJ mice inoculated with 4T1 cells and treated with Holo-BLf and BPA, and the mice treated with BPA. Metastasis involves the dissemination of cancer cells from primary lesions to distant organs, which is mediated by a variety of cellular processes including migration, invasion into adjacent connective tissue and then intravasation into blood and lymphatic vessels, extravasation at distant organs, and the formation of secondary tumors in premetastatic niches (Chambers, Groom, & MacDonald, 2002 ; Friedl & Wolf, 2003 ). Our findings demonstrate that BPA significantly increases the number of Balb/cJ mice inoculated with 4T1 cells with metastasis in lungs and liver, as well as the total number and average number/mouse of metastatic nodules in lungs and liver. Histological analysis of metastatic nodules from lungs and liver shows a similar morphology of neoplastic cells in mice treated with BPA and BPA plus Holo-BLf. However, Holo-BLf promotes the formation of smaller number of metastatic neoplastic cells. In agreement with these findings, we also demonstrate here that BPA significantly increases the total number and average number/mouse of metastatic nodules in lungs from NSG mice inoculated with MDA-MB-231 cells. We propose that Holo-BLf partly inhibits some specific cellular processes that mediate metastasis, such as migration, invasion, hydrolysis of extracellular matrix, intravasation/extravasation, epithelial to mesenchymal transition process and formation of premetastatic niches. Supporting our proposal, we demonstrate here that Holo-BLf inhibits invasion and/or migration induced by BPA in MDA-MB-231 and 4T1 breast cancer cells, whereas it has been demonstrated that Holo-BLf inhibits migration, invasion, MMP-2/MMP-9 secretion and downregulates vimentin expression in MDA-MB-231 breast cancer cells (Rodriguez-Ochoa et al., 2023 ). Moreover, Apo- and Holo-BLf prevent the epithelial to mesenchymal transition process through inhibition of interleukin-6/Stat3 in human glioblastoma GL-15 cells, whereas expression of Lf inhibits proliferation, migration, invasion and downregulates vimentin and N-cadherin expression in HONE-1 nasopharyngeal carcinoma cells (Cutone, Colella, et al., 2020 ; M. Xu, Fan, Zou, Yang, & Xu, 2024 ). Conclusions Holo-BLf inhibits cellular processes involved with the invasion/metastasis process induced by BPA in TNBC MDA-MB-231 and 4T1 breast cancer cells. Moreover, Holo-BLf prevents the increase in the growth of mammary tumors and metastasis induced by BPA in two murine models of breast cancer. We propose that Holo-BLf can be used for the improvement of chemotherapeutic agents used in the treatment of TNBC. Declarations Author contributions Rina Valenzuela-Echeverria, Rocio Castillo-Sanchez, Pablo Torres-Alamilla and Pedro Cortes-Reynosa: Generation of results, methodology, design, Formal analysis of data, validation, Data curation, original draft. Rina Valenzuela-Echeverria, Fernando Candanedo-Gonzalez and Eduardo Perez Salazar: Conceptualization, analysis of data, methodology, writing-review & editing. Eduardo Perez Salazar; Administration of the project, acquisition of funding, supervision, Data curation. Declaration of Competing Interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments We are grateful for her technical assistance in histology studies to Brenda Medina-Rodriguez from Histology Laboratory of Cell Biology and Tissue Department, School of Medicine. UNAM, Mexico. Data availability All data generated or analyzed during this study are included in this published article and its supplementary information files. Funding R. V-E is supported by a SECIHTI predoctoral training grant (839850). R. C-S is supported by a SECIHTI Post-Doctoral grant. Ethics approval All experimental procedures conducted on animals were approved and followed the ethical guidelines and recommendations of the UPEAL of Cinvestav Zacatenco unit. The protocol number endorsed by the UPEAL for the current study is 0294-19. References Anders, C. K., Abramson, V., Tan, T., & Dent, R. (2016). The Evolution of Triple-Negative Breast Cancer: From Biology to Novel Therapeutics. Am Soc Clin Oncol Educ Book, 35 , 34-42. doi:10.1200/EDBK_159135 Avalos-Gomez, C., Reyes-Lopez, M., Ramirez-Rico, G., Diaz-Aparicio, E., Zenteno, E., Gonzalez-Ruiz, C., & de la Garza, M. (2020). Effect of apo-lactoferrin on leukotoxin and outer membrane vesicles of Mannheimia haemolytica A2. Vet Res, 51 (1), 36. doi:10.1186/s13567-020-00759-z Avizienyte, E., & Frame, M. C. (2005). Src and FAK signalling controls adhesion fate and the epithelial-to-mesenchymal transition. Curr Opin Cell Biol, 17 (5), 542-547. doi:10.1016/j.ceb.2005.08.007 Baker, H. M., & Baker, E. N. (2012). A structural perspective on lactoferrin function. Biochem Cell Biol, 90 (3), 320-328. doi:10.1139/o11-071 Bays, J. L., & DeMali, K. A. (2017). Vinculin in cell-cell and cell-matrix adhesions. Cell Mol Life Sci, 74 (16), 2999-3009. doi:10.1007/s00018-017-2511-3 Bou Zerdan, M., Ghorayeb, T., Saliba, F., Allam, S., Bou Zerdan, M., Yaghi, M., . . . Nahleh, Z. (2022). Triple Negative Breast Cancer: Updates on Classification and Treatment in 2021. Cancers (Basel), 14 (5). doi:10.3390/cancers14051253 Cardiff, R. D., Miller, C. H., & Munn, R. J. (2014). Manual hematoxylin and eosin staining of mouse tissue sections. Cold Spring Harb Protoc, 2014 (6), 655-658. doi:10.1101/pdb.prot073411 Carisey, A., & Ballestrem, C. (2011). Vinculin, an adapter protein in control of cell adhesion signalling. Eur J Cell Biol, 90 (2-3), 157-163. doi:10.1016/j.ejcb.2010.06.007 Castillo-Sanchez, R., Ramirez-Ricardo, J., Martinez-Baeza, E., Cortes-Reynosa, P., Candanedo-Gonzales, F., Gomez, R., & Salazar, E. P. (2020). Bisphenol A induces focal adhesions assembly and activation of FAK, Src and ERK2 via GPER in MDA-MB-231 breast cancer cells. Toxicol In Vitro, 66 , 104871. doi:10.1016/j.tiv.2020.104871 Castillo Sanchez, R., Gomez, R., & Perez Salazar, E. (2016). Bisphenol A Induces Migration through a GPER-, FAK-, Src-, and ERK2-Dependent Pathway in MDA-MB-231 Breast Cancer Cells. Chem Res Toxicol, 29 (3), 285-295. doi:10.1021/acs.chemrestox.5b00457 Chambers, A. F., Groom, A. C., & MacDonald, I. C. (2002). Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer, 2 (8), 563-572. doi:10.1038/nrc865 Chea, C., Miyauchi, M., Inubushi, T., Okamoto, K., Haing, S., & Takata, T. (2023). Molecular Mechanisms of Inhibitory Effects of Bovine Lactoferrin on Invasion of Oral Squamous Cell Carcinoma. Pharmaceutics, 15 (2). doi:10.3390/pharmaceutics15020562 Cimmino, I., Fiory, F., Perruolo, G., Miele, C., Beguinot, F., Formisano, P., & Oriente, F. (2020). Potential Mechanisms of Bisphenol A (BPA) Contributing to Human Disease. Int J Mol Sci, 21 (16). doi:10.3390/ijms21165761 Colorado-Yohar, S. M., Castillo-Gonzalez, A. C., Sanchez-Meca, J., Rubio-Aparicio, M., Sanchez-Rodriguez, D., Salamanca-Fernandez, E., . . . Chirlaque, M. D. (2021). Concentrations of bisphenol-A in adults from the general population: A systematic review and meta-analysis. Sci Total Environ, 775 , 145755. doi:10.1016/j.scitotenv.2021.145755 Cutone, A., Colella, B., Pagliaro, A., Rosa, L., Lepanto, M. S., Bonaccorsi di Patti, M. C., . . . Musci, G. (2020). Native and iron-saturated bovine lactoferrin differently hinder migration in a model of human glioblastoma by reverting epithelial-to-mesenchymal transition-like process and inhibiting interleukin-6/STAT3 axis. Cell Signal, 65 , 109461. doi:10.1016/j.cellsig.2019.109461 Cutone, A., Rosa, L., Ianiro, G., Lepanto, M. S., Bonaccorsi di Patti, M. C., Valenti, P., & Musci, G. (2020). Lactoferrin's Anti-Cancer Properties: Safety, Selectivity, and Wide Range of Action. Biomolecules, 10 (3). doi:10.3390/biom10030456 Deng, P., Tan, M., Zhou, W., Chen, C., Xi, Y., Gao, P., . . . Zhou, Z. (2021). Bisphenol A promotes breast cancer cell proliferation by driving miR-381-3p-PTTG1-dependent cell cycle progression. Chemosphere, 268 , 129221. doi:10.1016/j.chemosphere.2020.129221 Faustino-Rocha, A., Oliveira, P. A., Pinho-Oliveira, J., Teixeira-Guedes, C., Soares-Maia, R., da Costa, R. G., . . . Ginja, M. (2013). Estimation of rat mammary tumor volume using caliper and ultrasonography measurements. Lab Anim (NY), 42 (6), 217-224. doi:10.1038/laban.254 Filho, A. M., Laversanne, M., Ferlay, J., Colombet, M., Pineros, M., Znaor, A., . . . Bray, F. (2025). The GLOBOCAN 2022 cancer estimates: Data sources, methods, and a snapshot of the cancer burden worldwide. Int J Cancer, 156 (7), 1336-1346. doi:10.1002/ijc.35278 Friedl, P., & Wolf, K. (2003). Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer, 3 (5), 362-374. doi:10.1038/nrc1075 Gibbons, J. A., Kanwar, J. R., & Kanwar, R. K. (2015). Iron-free and iron-saturated bovine lactoferrin inhibit survivin expression and differentially modulate apoptosis in breast cancer. BMC Cancer, 15 , 425. doi:10.1186/s12885-015-1441-4 Gonzalez-Chavez, S. A., Arevalo-Gallegos, S., & Rascon-Cruz, Q. (2009). Lactoferrin: structure, function and applications. Int J Antimicrob Agents, 33 (4), 301 e301-308. doi:10.1016/j.ijantimicag.2008.07.020 Kowalczyk, P., Kaczynska, K., Kleczkowska, P., Bukowska-Osko, I., Kramkowski, K., & Sulejczak, D. (2022). The Lactoferrin Phenomenon-A Miracle Molecule. Molecules, 27 (9). doi:10.3390/molecules27092941 Lehmler, H. J., Liu, B., Gadogbe, M., & Bao, W. (2018). Exposure to Bisphenol A, Bisphenol F, and Bisphenol S in U.S. Adults and Children: The National Health and Nutrition Examination Survey 2013-2014. ACS Omega, 3 (6), 6523-6532. doi:10.1021/acsomega.8b00824 Nohynek, G. J., Borgert, C. J., Dietrich, D., & Rozman, K. K. (2013). Endocrine disruption: fact or urban legend? Toxicol Lett, 223 (3), 295-305. doi:10.1016/j.toxlet.2013.10.022 Rahman, R., Fonseka, A. D., Sua, S. C., Ahmad, M., Rajendran, R., Ambu, S., . . . Chitra, E. (2021). Inhibition of breast cancer xenografts in a mouse model and the induction of apoptosis in multiple breast cancer cell lines by lactoferricin B peptide. J Cell Mol Med, 25 (15), 7181-7189. doi:10.1111/jcmm.16748 Rodriguez-Ochoa, N., Cortes-Reynosa, P., Rodriguez-Rojas, K., de la Garza, M., & Salazar, E. P. (2023). Bovine holo-lactoferrin inhibits migration and invasion in MDA-MB-231 breast cancer cells. Mol Biol Rep, 50 (1), 193-201. doi:10.1007/s11033-022-07943-8 Sajiki, J., & Yonekubo, J. (2004). Leaching of bisphenol A (BPA) from polycarbonate plastic to water containing amino acids and its degradation by radical oxygen species. Chemosphere, 55 (6), 861-867. doi:10.1016/j.chemosphere.2003.11.065 Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A., & Bray, F. (2021). Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin, 71 (3), 209-249. doi:10.3322/caac.21660 Torres-Alamilla, P., Castillo-Sanchez, R., Cortes-Reynosa, P., Gomez, R., & Perez Salazar, E. (2023). Bisphenol A increases the size of primary mammary tumors and promotes metastasis in a murine model of breast cancer. Mol Cell Endocrinol, 575 , 111998. doi:10.1016/j.mce.2023.111998 Tsuda, H., Kozu, T., Iinuma, G., Ohashi, Y., Saito, Y., Saito, D., . . . Iigo, M. (2010). Cancer prevention by bovine lactoferrin: from animal studies to human trial. Biometals, 23 (3), 399-409. doi:10.1007/s10534-010-9331-3 Wang, B., Timilsena, Y. P., Blanch, E., & Adhikari, B. (2019). Lactoferrin: Structure, function, denaturation and digestion. Crit Rev Food Sci Nutr, 59 (4), 580-596. doi:10.1080/10408398.2017.1381583 Wozniak, M. A., Modzelewska, K., Kwong, L., & Keely, P. J. (2004). Focal adhesion regulation of cell behavior. Biochim Biophys Acta, 1692 (2-3), 103-119. doi:10.1016/j.bbamcr.2004.04.007 Xu, M., Fan, Y., Zou, G., Yang, Q., & Xu, F. (2024). Lactoferrin mediates epithelial-mesenchymal transformation by regulating the PI3K/AKT/mTOR pathway to inhibit nasopharyngeal carcinoma metastasis. Cell Mol Biol (Noisy-le-grand), 70 (3), 130-135. doi:10.14715/cmb/2024.70.3.19 Xu, X., Zhang, Y., Jasper, J., Lykken, E., Alexander, P. B., Markowitz, G. J., . . . Wang, X. F. (2016). MiR-148a functions to suppress metastasis and serves as a prognostic indicator in triple-negative breast cancer. Oncotarget, 7 (15), 20381-20394. doi:10.18632/oncotarget.7953 Yoo, Y. C., Watanabe, S., Watanabe, R., Hata, K., Shimazaki, K., & Azuma, I. (1997). Bovine lactoferrin and lactoferricin, a peptide derived from bovine lactoferrin, inhibit tumor metastasis in mice. Jpn J Cancer Res, 88 (2), 184-190. doi:10.1111/j.1349-7006.1997.tb00364.x Zhang, W., Fang, Y., Shi, X., Zhang, M., Wang, X., & Tan, Y. (2012). Effect of bisphenol A on the EGFR-STAT3 pathway in MCF-7 breast cancer cells. Mol Med Rep, 5 (1), 41-47. doi:10.3892/mmr.2011.583 Zhang, Y., Lima, C. F., & Rodrigues, L. R. (2014). Anticancer effects of lactoferrin: underlying mechanisms and future trends in cancer therapy. Nutr Rev, 72 (12), 763-773. doi:10.1111/nure.12155 Zhang, Y., Nicolau, A., Lima, C. F., & Rodrigues, L. R. (2014). Bovine lactoferrin induces cell cycle arrest and inhibits mTOR signaling in breast cancer cells. Nutr Cancer, 66 (8), 1371-1385. doi:10.1080/01635581.2014.956260 Zhao, J., & Guan, J. L. (2009). Signal transduction by focal adhesion kinase in cancer. Cancer Metastasis Rev, 28 (1-2), 35-49. doi:10.1007/s10555-008-9165-4 Additional Declarations No competing interests reported. Supplementary Files Figure1S.tiff Supplementary figures Fig. S1A Protocol of inoculation of Balb/cJ and NSG mice with 4T1 and MDA-MB-231 cells and treatment with BPA and/or Holo-BLf Table1S.docx Table2S.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 25 Feb, 2026 Editor assigned by journal 18 Feb, 2026 Submission checks completed at journal 18 Feb, 2026 First submitted to journal 17 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8904788","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":597173136,"identity":"48034bc8-cd9e-401a-8740-bec072297b2c","order_by":0,"name":"Rina Valenzuela-Echeverria","email":"","orcid":"","institution":"Cinvestav-IPN. 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Ciudad de Mexico","correspondingAuthor":true,"prefix":"","firstName":"Eduardo","middleName":"Perez","lastName":"Salazar","suffix":""}],"badges":[],"createdAt":"2026-02-18 00:08:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8904788/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8904788/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104400081,"identity":"89646b56-c336-4b97-a431-c3d63bb3cb80","added_by":"auto","created_at":"2026-03-11 12:08:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1014491,"visible":true,"origin":"","legend":"\u003cp\u003eHolo-BLf inhibits migration and invasion induced by BPA in TNBC cells\u003c/p\u003e\n\u003cp\u003e(A-B) Migration assays of MDA-MB-231 and 4T1 cells unstimulated and stimulated with 1 mM BPA and/or treated with 1250 nM Holo-BLf. (C) Invasion assays of 4T1 cells unstimulated and stimulated with 1 mM BPA and/or treated with 1250 nM Holo-BLf. Graphs represent the mean ± S.D. of at least three independent experiments and indicate the fold of migration or invasion above Ctrl value. Asterisks denote comparisons to BPA value. \u003cem\u003e*P\u0026lt;0.5, ***P\u0026lt;0.001, ****P\u0026lt;0.0001\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8904788/v1/f2802a7ed741f329db04c82e.png"},{"id":103735140,"identity":"c9d84555-c9cb-4c36-baad-18f6874b1eb1","added_by":"auto","created_at":"2026-03-02 09:46:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3782950,"visible":true,"origin":"","legend":"\u003cp\u003eBPA induces focal adhesions assembly in 4T1 cells\u003c/p\u003e\n\u003cp\u003e4T1 cells cultured on coverslips were unstimulated and stimulated with 1 mM BPA for 10, 15, 30 and 45 min. Cells were fixed and focal adhesions were analyzed by staining with anti-vinculin Ab. F-actin structures were stained with TRITC-conjugated phalloidin. Focal adhesions are shown in green and F-actin structures are shown in red. Representative images of at least three independent experiments are shown.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8904788/v1/ad6a9bb05ba674002e10b4b4.png"},{"id":104400171,"identity":"2138d930-8f7f-45b8-af6a-40b8ffa7a1c6","added_by":"auto","created_at":"2026-03-11 12:09:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4001995,"visible":true,"origin":"","legend":"\u003cp\u003eHolo-BLf inhibits focal adhesions assembly induced by BPA in 4T1 cells\u003c/p\u003e\n\u003cp\u003e4T1 cells cultured on coverslips were unstimulated and stimulated with 1 mM BPA and/or treated with 1250 nM Holo-BLf for 15 min. Cells were fixed and focal adhesions were analyzed by staining with anti-vinculin Ab. F-actin structures were stained with TRITC-conjugated phalloidin. Focal adhesions are shown in green and F-actin structures are shown in red. Magnifications are shown in squares. Representative images of at least three independent experiments are shown.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8904788/v1/dfb23e6c4f9ed4a6597ef057.png"},{"id":103735142,"identity":"2cf2fdd9-a0f6-4146-8d50-7d812cf1a34a","added_by":"auto","created_at":"2026-03-02 09:46:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2115504,"visible":true,"origin":"","legend":"\u003cp\u003eHolo-BLf inhibits the volume and weight of mammary tumors in Balb/cJ mice inoculated with 4T1 cells and treated with BPA\u003c/p\u003e\n\u003cp\u003e4T1 cells were inoculated in mammary fat pads of Balb/cJ mice, and then mice were untreated and treated with BPA (10 μg/kg/mouse) and/or Holo-BLf (50 mg/kg/mouse). Mammary tumors were obtained and analyzed. (A and B) Graphs represent the mean ± S.D. of weight and volume of mammary tumors of ten mice per group. (C) Representative images of mammary tumors from mice untreated and treated with BPA and/or Holo-BLf are shown. (D) Representative images of mammary tumor sections stained with hematoxylin and eosin. Magnifications at 10x and 40x are shown. Scale bars: 100 mm. Asterisks denote comparisons to BPA value. \u003cem\u003e****P\u0026lt;0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8904788/v1/85feeccf238c40c6979c1161.png"},{"id":104400069,"identity":"ce243e9f-28cb-4766-bb9e-da46499f34d3","added_by":"auto","created_at":"2026-03-11 12:08:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1204605,"visible":true,"origin":"","legend":"\u003cp\u003eHolo-BLf decreases the number of Balb/cJ mice with metastasis to lungs and liver induced by BPA\u003c/p\u003e\n\u003cp\u003e4T1 cells were inoculated in mammary fat pads of Balb/cJ mice, and then mice were untreated and treated with BPA (10 μg/kg/mouse) and/or Holo-BLf (50 mg/kg/mouse). Metastasis to lungs and liver was analyzed. (A and B) Graphs represent the number of mice with metastasis to lungs and liver. Representative images of metastatic nodules in lungs and liver are shown. Asterisks denote comparisons to BPA value. \u003cem\u003e*P\u0026lt;0.5, **P\u0026lt;0.01.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8904788/v1/9ca40aa40428006ac2eae9c2.png"},{"id":103735146,"identity":"06f096bf-17be-495f-91b8-fc7f27d5fc28","added_by":"auto","created_at":"2026-03-02 09:46:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":302085,"visible":true,"origin":"","legend":"\u003cp\u003eHolo-BLf inhibits the increase in the total number and the average number of metastatic nodules/mouse in lungs and liver induced by BPA\u003c/p\u003e\n\u003cp\u003e4T1 cells were inoculated in mammary fat pads of Balb/cJ mice, and then mice were untreated and treated with BPA (10 μg/kg/mouse) and/or Holo-BLf (50 mg/kg/mouse). Lungs and liver were obtained and the number of metastatic nodules was obtained. (A and C) Graphs represent the total number of metastatic nodules in lungs and liver. (B and D) Graphs represent the average number of metastatic nodules/mouse in lungs and liver. Asterisks denote comparisons to BPA value \u003cem\u003e*P\u0026lt;0.5, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8904788/v1/234728ad2fcd61cde0c47a5b.png"},{"id":103735148,"identity":"a70313b9-2332-4604-a91a-61d7319f8c1b","added_by":"auto","created_at":"2026-03-02 09:46:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":11749472,"visible":true,"origin":"","legend":"\u003cp\u003eHistological assessment of metastatic nodules in lungs and liver\u003c/p\u003e\n\u003cp\u003eRepresentative images of metastatic nodules sections of lungs, liver and brain stained with hematoxylin and eosin from Balb/cJ mice inoculated with 4T1 cells and treated with BPA (10 μg/kg/mouse) and/or Holo-BLf (50 mg/kg/mouse). Magnifications at 40x are shown. Scale bars: 100 mm.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8904788/v1/e3d8a2e9e9d37abb53eb9f78.png"},{"id":104400259,"identity":"e1ce263a-ef39-4e71-b580-5b0072332c79","added_by":"auto","created_at":"2026-03-11 12:09:22","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":851303,"visible":true,"origin":"","legend":"\u003cp\u003eHolo-BLf inhibits the growth of mammary tumors and metastasis induced by BPA in NSG mice inoculated with MDA-MB-231 cells\u003c/p\u003e\n\u003cp\u003eMDA-MB-231 cells were inoculated in mammary fat pads of NSG mice, and then mice were untreated and treated with BPA (10 μg/kg/mouse) and/or Holo-BLf (50 mg/kg/mouse). Mammary tumors were obtained and analyzed. (A) Representative images of mammary tumors from mice untreated and treated with BPA and/or Holo-BLf are shown. (B and C) Graphs represent the mean ± S.D. of weight and volume of mammary tumors of four mice per group. (D) Graphs represent the average number of metastatic nodules/mouse and the total number of metastatic nodules in lungs. Asterisks denote comparisons to BPA value. \u003cem\u003e****P\u0026lt;0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8904788/v1/8a14635201034d3bc0a98f4a.png"},{"id":105032987,"identity":"77632034-8b9e-4e4f-b8de-135826c0ac18","added_by":"auto","created_at":"2026-03-20 07:10:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23367429,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8904788/v1/4049bf4a-2169-4fc1-9f77-d131b0255e92.pdf"},{"id":103735149,"identity":"f506ec20-2255-4946-a6c0-3cadcee00d35","added_by":"auto","created_at":"2026-03-02 09:46:50","extension":"tiff","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":35494416,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S1A\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtocol of inoculation of Balb/cJ and NSG mice with 4T1 and MDA-MB-231 cells and treatment with BPA and/or Holo-BLf\u003c/p\u003e","description":"","filename":"Figure1S.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8904788/v1/f4d79adb8c5655a2f3e0dd9c.tiff"},{"id":103735147,"identity":"2a3ba958-88b4-4b93-b72d-ee64861bd155","added_by":"auto","created_at":"2026-03-02 09:46:49","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":18612,"visible":true,"origin":"","legend":"","description":"","filename":"Table1S.docx","url":"https://assets-eu.researchsquare.com/files/rs-8904788/v1/e7010048ce6f64b1d89d414c.docx"},{"id":103735144,"identity":"27ff1ce6-6625-4bfe-9651-35652b7ebf52","added_by":"auto","created_at":"2026-03-02 09:46:49","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":18963,"visible":true,"origin":"","legend":"","description":"","filename":"Table2S.docx","url":"https://assets-eu.researchsquare.com/files/rs-8904788/v1/2444bcf4745abb29332d46cc.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bovine Holo-lactoferrin inhibits tumor growth and metastasis induced by BPA in murine triple negative breast cancer models","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer is the most common neoplasia among women in developed countries and the leading cause of mortality in women worldwide. In 2022, breast cancer was diagnosed in 2.3\u0026nbsp;million women, and 680,000 deaths were recorded in 2020 (Filho et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Sung et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Molecular classification of breast cancer considers the expression of estrogen receptor (ER), progesterone receptor (PR) and Her2, and recently it has been considered the expression of Ki67. Triple negative breast cancer (TNBC) is a subtype that does not express ER and PR, presents low levels of Her2 and high expression of Ki67 (Anders, Abramson, Tan, \u0026amp; Dent, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). TNBC subtype constitutes about 15\u0026ndash;20% of all breast cancer cases and has aggressive behavior, early relapses and the highest incidence of metastasis (Bou Zerdan et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBisphenol A (BPA) is a synthetic compound used as monomer or additive in the manufacturing of polycarbonate plastics and epoxy resins, which are employed in metal can linings, thermal paper, dental compounds and containers and packaging for food and drink (Cimmino et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lehmler, Liu, Gadogbe, \u0026amp; Bao, 2018). Humans are exposed to BPA mainly through ingestion of food and water (90\u0026ndash;99%), because high temperature and basic solutions promote the cleavage of ester bonds that hold BPA monomers together and then BPA is released into food and beverages (Colorado-Yohar et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sajiki \u0026amp; Yonekubo, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). BPA is an endocrine disrupting chemical with estrogenic activity and is associated with variations in hormonal homeostasis, sexual differentiation and increasing rates of cancer including breast cancer (Cimmino et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nohynek, Borgert, Dietrich, \u0026amp; Rozman, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLactoferrin (Lf) is a highly conserved cationic glycoprotein in mammals, which has a similar tertiary structure between a variety of species including bovine, porcine, murine and human (Kowalczyk et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Lf structure consists of two homologous globular domains (N and C lobes) connected by an alpha helix, where each lobe has a metal-binding site composed of four conserved amino acid residues (Gonzalez-Chavez, Arevalo-Gallegos, \u0026amp; Rascon-Cruz, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Wang, Timilsena, Blanch, \u0026amp; Adhikari, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Lf has the ability to bind Fe\u003csup\u003e2+\u003c/sup\u003e or Fe\u003csup\u003e3+\u003c/sup\u003e using the two lobes of the molecule, and then Lf is found free of Fe\u003csup\u003e3+\u003c/sup\u003e (Apo-Lf) and associated with Fe\u003csup\u003e3+\u003c/sup\u003e (Holo-Lf), which generates a different conformation of Apo-Lf and Holo.Lf, stability of the molecule and functional features (Baker \u0026amp; Baker, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Gonzalez-Chavez et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Bovine Lf (BLf) shares 69% homology with human Lf, and the exogenous treatment with BLf presents anti-tumor activity in a variety of cancers (Cutone, Rosa, et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Particularly, Holo-BLf and Apo-BLf induce apoptosis through inhibition of survivin in MCF-7 and MDA-MB-231 breast cancer cells, whereas BLf partly inhibits migration and invasion in MDA-MB-231 cells (Gibbons, Kanwar, \u0026amp; Kanwar, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Rodriguez-Ochoa, Cortes-Reynosa, Rodriguez-Rojas, de la Garza, \u0026amp; Salazar, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we demonstrate that Holo-BLf inhibits cellular processes that mediate invasion/metastasis process including migration, invasion and assembly of focal adhesions induced by BPA in 4T1 cells, and migration in MDA-MB-231 cells. In addition, Holo-BLf inhibits the growth of mammary tumors and metastasis induced by BPA in two murine models of breast cancer.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eMaterials\u003c/p\u003e \u003cp\u003eApo-BLf (97% of purity) was from NutriScience Innovations, LLC (Connecticut, USA). BPA, anti-vinculin antibody (Ab), Bouin\u0026rsquo;s solution, Tetramethylrhodamine (TRITC)-conjugated phalloidin and 4',6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich (Merck KGaA). Matrigel matrix was from Corning (Bedford, USA).\u003c/p\u003e \u003cp\u003ePreparation of Holo-BLf\u003c/p\u003e \u003cp\u003eHolo-BLf was obtained by saturation of Apo-BLf with iron as described previously (Avalos-Gomez et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Concentration of iron in Holo-BLf was of 93%, which was determined by an enzymatic automated method, (MicroTech Laboratories, Mexico).\u003c/p\u003e \u003cp\u003eCell lines and culture\u003c/p\u003e \u003cp\u003eHuman TNBC MDA-MB-231 cells and murine TNBC 4T1 cells were obtained from the American Type Culture Collection (ATCC). MDA-MB-231 cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) complemented with 5% fetal bovine serum (FBS), 3.7 g/l sodium bicarbonate and antibiotics. 4T1 cells were cultured in RPMI-1640 medium supplemented with 5% FBS and antibiotics. Cultures were incubated at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% air. For experimental purposes, MDA-MB-231 cells were starved in DMEM without FBS for 18 h and 4T1 cells were starved in RPMI-1640 medium with 0.5% FBS for 5 h before treatment with BPA and/or Holo-BLf.\u003c/p\u003e \u003cp\u003eScratch wound assays\u003c/p\u003e \u003cp\u003eCultures of 4T1 cells (1.5x10\u003csup\u003e6\u003c/sup\u003e cells/35-mm culture plate) were scratch-wounded, washed twice with PBS, refed with RPMI-1640 medium/0.5% FBS and then untreated and treated with 1250 nM Holo-BLf and/or stimulated with 1 \u0026micro;M BPA for 48 h at 37\u0026deg;C. At the end of incubation, cells were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% violet crystal solution for 10 min. Images were acquired from at least three fields per experimental condition from three independent experiments, and they were analyzed using the ImageJ software v1.52e (NIH, USA).\u003c/p\u003e \u003cp\u003eInvasion assays\u003c/p\u003e \u003cp\u003eInserts with 8 \u0026micro;m pores of 24-well plates were covered with Matrigel (15 \u0026micro;g/insert) and incubated for 12 h at 37 \u0026ordm;C. Next, 4T1 cells (1x10\u003csup\u003e5\u003c/sup\u003e cells/well) were plated on Matrigel in RPMI-1640 medium/0.5% FBS enriched without or with 1250 nM Holo-BLf. Lower chambers were filled with RPMI-1640 medium/0.5% FBS supplemented without or with 1 \u0026micro;M BPA. Plates were incubated for 30 h at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% air. At the end of incubation, Matrigel on the upper surface of membranes was removed using cotton swabs. Cells on the lower surface of membranes were washed with PBS and fixed with methanol for 25 min and then stained with 0.1% crystal violet in PBS for 15 min. Membranes were photographed and images of at least 3 fields of each experimental condition were obtained. Dye was eluted using 750 \u0026micro;l of 10% acetic acid, and absorbance was measured at a wavelength of 600 nm.\u003c/p\u003e \u003cp\u003eImmunofluorescence confocal microscopy\u003c/p\u003e \u003cp\u003e4T1 cells were grown on coverslips, washed with PBS, equilibrated in FBS-free RPMI-1640 for 30 min and then untreated and treated with 1250 nM Holo-BLf and/or stimulated with 1 \u0026micro;M BPA for various times as indicated. Cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 for 20 min and blocked with gelatin solution (0.5% gelatin, 1 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e) for 20 min at room temperature. Cells were stained by incubation with anti-vinculin Ab (1:1000) for 12 h at 4\u0026deg;C followed by incubation with FITC-labeled anti-mouse secondary Ab for 2 h at room temperature. Fibrillar actin was stained by incubation of cells with TRITC-conjugated phalloidin for 2 h, whereas nucleus was stained by incubation of cells with DAPI for 5 min at room temperature. Cells were mounted on glass slides with Vectashield and analyzed by confocal microscopy (Model TCS SP8; Leica Microsystems, Inc).\u003c/p\u003e \u003cp\u003eEthics statement for experimental procedures with female Balb/cJ and NSG mice\u003c/p\u003e \u003cp\u003eUnit for Production and Experimentation of Laboratory Animals (UPEAL) of the Center of Research and Advanced Studies (Cinvestav, Mexico) provided female Balb/cJ mice of 6\u0026ndash;8 weeks old and NOD-SCID-gamma (NSG) mice of 4\u0026ndash;6 weeks old. Experimental procedures were carried out ethically following the guidelines established by the Institutional Animal Care and Use Committee of UPEAL, which operates under the Mexican Official Standard NOM-062-ZOO-1999 for technical specifications for the production, care, and use of laboratory animals (NOM-062-ZOO-1999, 2001). Protocol numbers 0294\u0026thinsp;\u0026minus;\u0026thinsp;19 and 0370\u0026thinsp;\u0026minus;\u0026thinsp;24\u003c/p\u003e \u003cp\u003eFormation of mice groups\u003c/p\u003e \u003cp\u003eFemale Balb/cJ and NSG mice were randomly divided into four groups of 10 and 4 mice per group respectively: 1) Control group was untreated mice; 2) BPA group was mice treated with 10 \u0026micro;g/kg/mouse BPA; 3) Holo-BLf group was mice treated with 50 mg/kg/mouse Holo-BLf; and 4) Holo-BLf\u0026thinsp;+\u0026thinsp;BPA group was mice treated with 50 mg/kg/mouse Holo-BLf and/or 10 \u0026micro;g/kg/mouse BPA.\u003c/p\u003e \u003cp\u003eInduction of mammary tumors in female Balb/cJ and NSG mice\u003c/p\u003e \u003cp\u003eMDA-MB-231 and 4T1 cells were harvested from semi-confluent cultures (80%), washed once with PBS, collected by centrifugation and 4T1 cells were resuspended in RPMI-1640 medium at 6x10\u003csup\u003e4\u003c/sup\u003e cells/ml, whereas MDA-MB-231 cells were resuspended in DMEM enriched with human type IV collagen (400 mg/ml) at 6x10\u003csup\u003e4\u003c/sup\u003e cells/ml. Balb/cJ mice were inoculated with 3,000 4T1 cells and NSG mice were inoculated with 50,000 cells into the left fourth inguinal mammary fat pads using 0.3 ml tuberculin syringe equipped with a 31G needle. Mice were housed in Super Mouse 750\u0026trade; ventilated cages with five mice per cage, under regulated temperature and 12 h light/dark cycle. Treatment of Balb/cJ and NSG mice was performed by oral administration of Holo-BLf (50 mg/kg/mouse) and BPA (10 \u0026micro;g/kg/mouse). Mice treated with Holo-BLf\u0026thinsp;+\u0026thinsp;BPA were treated with BPA (10 \u0026micro;g/kg/mouse) in the morning and 6 h later were treated with Holo-BLf (50 mg/kg/mouse). Treatments started on the third day after inoculation with 4T1 cells, and then treatments were administered daily for 18 days. Mice were monitored daily and weighed every third day. Balb/cJ mice were euthanized after 21 days post-inoculation, whereas NSG mice were euthanized after 23 days post-inoculation using carbon dioxide administered through the Euthanex Smartbox Auto CO\u003csub\u003e2\u003c/sub\u003e System EA-3400 (flow rate of 6 l per minute for 5 min). Mammary tumors, lungs, livers and brains were obtained (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eVolume of mammary tumors was determined by the equation V = (W2 \u0026times; L)/2, where W represents the width and L the length of tumors (Faustino-Rocha et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Mammary tumors were fixed in 4% paraformaldehyde for 24 h, whereas lungs, livers and brains were fixed in Bouin's solution for 24 h. Tumors were stored in 70% ethanol. Metastatic nodules in livers, lungs and brains were identified by the presence of spherical white areas on a background of yellow-stained tissue (X. Xu et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHistopathology\u003c/p\u003e \u003cp\u003eFixed mammary tumors, livers, lungs and brains were paraffin-embedded. One paraffin block per organ (liver, lung, brain) or mammary tumor from each mouse across all three experimental groups (n\u0026thinsp;=\u0026thinsp;10/group) were obtained. Three tissue sections of 4 \u0026micro;m were trimmed from each block and mounted in duplicate on separate glass slides. Sections were deparaffinized, rehydrated, and subjected to hematoxylin and eosin staining (Cardiff, Miller, \u0026amp; Munn, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Three random fields per tissue section were analyzed using a Nikon H550S microscope. All slides were examined by a pathologist blinded to experimental groups to objectively assess tumor characteristics and micrometastasis across experimental conditions.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were evaluated for normal distribution using the Komolgorov-Smirnov statistical test. Data with normal distribution were analyzed using unpaired t test or the one-way ANOVA statistical test followed by Dunnett's multiple comparison post hoc test. Data without normal distribution were analyzed by the Mann-Whitney test and Kruskal-Wallis statistical test followed by uncorrected Dunn\u0026acute;s test or Dunn's post hoc multiple comparison test. Results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. of at least three independent experiments. A statistical probability of \u003cem\u003eP\u0026thinsp;\u0026le;\u0026thinsp;0.05\u003c/em\u003e was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eHolo-BLf inhibits migration and invasion induced by BPA in TNBC cells\u003c/p\u003e \u003cp\u003eBPA induces migration and invasion in MDA-MB-231 and 4T1 breast cancer cells (Castillo-Sanchez et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Castillo Sanchez, Gomez, \u0026amp; Perez Salazar, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Torres-Alamilla, Castillo-Sanchez, Cortes-Reynosa, Gomez, \u0026amp; Perez Salazar, 2023). We determined whether Holo-BLf inhibited migration induced by BPA in MDA-MB-231 and 4T1 cells. Cultures of MDA-MB-231 and 4T1 cells were scratched and then untreated and treated with 1250 nM Holo-BLf and/or stimulated with 1 \u0026micro;M BPA for 24 h. Findings showed that treatment with Holo-BLf inhibited migration induced by BPA in MDA-MB-231 and 4T1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we determined whether Holo-BLf inhibited invasion induced by BPA. Invasion assays were performed using 4T1 cells untreated and treated with 1250 nM Holo-BLf and/or stimulated with 1 \u0026micro;M BPA for 30 h. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, treatment with Holo-BLf inhibited invasion induced by BPA in 4T1 cells.\u003c/p\u003e \u003cp\u003eHolo-BLf inhibits formation of focal adhesions induced by BPA in 4T1 cells\u003c/p\u003e \u003cp\u003eBPA induces migration in 4T1 cells (Torres-Alamilla et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). We determined whether BPA induced the formation of focal adhesions. Cultures of 4T1 cells were cultured on coverslips and then unstimulated and stimulated with 1 \u0026micro;M BPA for 10, 15, 30 and 45 min. Presence of focal adhesions was determined by immunofluorescence analysis of vinculin, which is a protein localized in focal adhesions (Bays \u0026amp; DeMali, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Findings showed that BPA induced the formation of focal adhesions in a time-dependent manner reaching a maximum at 15 min of stimulation in 4T1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we determined whether Holo-BLf inhibited the formation of focal adhesions induced by BPA in 4T1 cells. Cultures of 4T1 cells were cultured on coverslips and then untreated and treated with 1250 nM Holo-BLf and/or stimulated with 1 \u0026micro;M BPA for 15 min. Presence of focal adhesions was determined by immunofluorescence analysis of vinculin. Findings showed that BPA promoted an increase in the number of focal adhesions, however treatment with Holo-BLf inhibited the assembly of focal adhesions induced by BPA in 4T1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHolo-BLf inhibits the growth of mammary tumors in Balb/cJ inoculated with 4T1 cells and NSG mice inoculated with MDA-MB-231 cells and treated with BPA\u003c/p\u003e \u003cp\u003eBPA promotes an increase in the growth of mammary tumors in a murine model of breast cancer using Balb/cJ mice inoculated with 4T1 cells (Torres-Alamilla et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). We determined whether treatment with Holo-BLf inhibited the growth of mammary tumors induced by treatment with BPA in Balb/cJ mice. 4T1 cells were inoculated into mammary fat pads of Balb/cJ mice, and then mice were untreated and treated with 10 \u0026micro;g/kg/mouse BPA, and/or 50 mg/kg/mouse Holo-BLf for 21 days. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C and Table\u0026nbsp;1, treatment with Holo-BLf inhibited the increase of weight and volume of mammary tumors induced by treatment with BPA in Balb/cJ mice inoculated with 4T1 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHistological sections of mammary tumors stained with hematoxylin and eosin were analyzed at low (10x) and high (40x) magnification. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, basal, BPA, Holo-BLf and Holo-BLf\u0026thinsp;+\u0026thinsp;BPA groups corresponded to high-grade malignant epithelial neoplasms, which are characterized by solid growth without tubular formation, marked nuclear pleomorphisms and high mitotic index.\u003c/p\u003e \u003cp\u003eTo substantiate our findings, we studied whether treatment with 50 mg/kg/mouse Holo-BLf inhibited the growth of mammary tumors induced by treatment with 10 \u0026micro;g/kg/mouse BPA in NSG mice inoculated with human MDA-MB-231 cells. Findings showed that treatment with Holo-BLf inhibited the increase of weight and volume of mammary tumors induced by treatment with BPA in NSG mice inoculated with MDA-MB-231 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B and Table\u0026nbsp;1S).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHolo-BLf inhibits the metastatic potential of BPA to lungs and liver in Balb/cJ mice inoculated with 4T1 cells\u003c/p\u003e \u003cp\u003eWe also analyzed whether treatment with Holo-BLf inhibited the increases in the number of Balb/cJ mice inoculated with 4T1 cells with metastasis to lungs, liver and brain. Metastasis was determined by the presence of metastatic nodules. Findings showed that treatment with BPA significantly increased the number of mice with metastasis in lungs (10/10) and liver (6/10) compared with the number of untreated mice (Basal) with metastasis in lungs (5/10) and liver (1/10) and mice treated with Holo-BLf with metastasis in lungs (4/10) and liver (1/10). However, treatment of mice with Holo-BLf completely inhibited the increase in the number of mice with metastasis to lungs (5/10) and liver (1/10) induced by BPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B, Table\u0026nbsp;2). We did not find any significant difference in the number of mice with metastasis to brain in all the groups of mice studied (Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHolo-BLf inhibits the increase in the number of metastatic nodules in lungs and liver induced by BPA in Balb/cJ mice inoculated with 4T1 cells\u003c/p\u003e \u003cp\u003eWe determined whether treatment with Holo-BLf inhibited the increases in the total and average number of metastatic nodules in lungs, liver and brain. Findings showed that treatment of Balb/cJ mice with BPA induced an increase in the total number and average number/mouse of metastatic nodules in lungs and liver compared with total number and average number/mouse of metastatic nodules in lungs and liver from untreated mice (Basal) and mice treated with Holo-BLf. However, treatment of mice with Holo-BLf completely inhibited the increase in the total number and average number/mouse of metastatic nodules in lungs and liver induced by BPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D and Table\u0026nbsp;2)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMetastatic nodules from lungs, liver and brain were analyzed by histological analysis using histological sections stained with hematoxylin and eosin at high (40x) magnification. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003e, lungs, liver and brain from untreated mice (Basal) did not present metastatic cells. However, lungs, liver and brain from mice treated with BPA presented large clusters of metastatic neoplastic cells. Interestingly, mice treated with Holo-BLf and BPA showed a smaller number of metastatic neoplastic cells in lungs, liver and brain, whereas a few isolated clusters of metastatic neoplastic cells were found in the lungs, liver and brain from mice treated with Holo-BLf.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHolo-BLf inhibits the increase in the number of metastatic nodules in lungs induced by BPA in NSG mice inoculated with MDA-MB-231 cells\u003c/p\u003e \u003cp\u003eTo further substantiate our findings, we analyzed the number of metastatic nodules in lungs, liver and brain in NSG mice inoculated with MDA-MB-231 cells and untreated and treated with 50 mg/kg/mouse Holo-BLf and/or 10 \u0026micro;g/kg/mouse BPA. Findings showed that treatment of NSG mice with BPA induced an increase in the total number and average number/mouse of metastatic nodules in lungs compared with the total number and average number/mouse of metastatic nodules in lungs from untreated mice (Basal) and mice treated with Holo-BLf. However, treatment of mice with Holo-BLf completely inhibited the increase in the total number and average number/mouse of metastatic nodules in lungs Induced by BPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, Table\u0026nbsp;2S). We did not find a significant difference in the total number and average number/mouse of metastatic nodules in liver and brain between NSG mice treated with BPA and NSG mice treated with BPA and Holo-BLf (Table\u0026nbsp;2S).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBPA promotes an increase of GPER expression and the activation of signal transduction pathways via GPER that mediate migration and invasion in human TNBC MDA-MB-231 and murine TNBC 4T1 cells (Castillo-Sanchez et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Castillo Sanchez et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Torres-Alamilla et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, oral administration of BPA increases the volume and weight of mammary tumors and promotes metastasis to lung in a murine model of breast cancer using 4T1 cells and female Balb/cJ mice (Torres-Alamilla et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). BLf exhibits anti-tumor activities because it inhibits migration, invasion and proliferation through arresting the cancer cells in G1 to S phase transition (Tsuda et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Y. Zhang, Lima, \u0026amp; Rodrigues, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). We previously demonstrated that Holo-BLf inhibits migration induced by FBS and linoleic acid in MDA-MB-231 cells (Rodriguez-Ochoa et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the capacity of Holo-BLf to inhibit cellular processes involved in the migration/invasion process, as well as its capacity to inhibit the growth of mammary tumors and metastasis in breast cancer remains to be studied.\u003c/p\u003e \u003cp\u003eIn this study, we employed a concentration of 1250 nM Holo-BLf for the \u0026ldquo;in vitro\u0026rdquo; assays with 4T1 breast cancer cells because it has been demonstrated that treatment of MDA-MB-231 breast cancer cells with 1250 nM Holo-BLf inhibits migration, invasion, MMP-9 secretion and phosphorylation of FAK at tyrosine-397 induced by FBS (Rodriguez-Ochoa et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). We demonstrate here treatment of MDA-MB-231 and 4T1 breast cancer cells with 1250 nM Holo-BLf inhibits migration induced by BPA. Moreover, treatment of 4T1 cells with 1250 nM Holo-BLf inhibits invasion induced by BPA. In agreement with our findings BLf partly inhibits migration and the expression of Snail and vimentin in human GL-15 and murine GL-261 glioblastoma cells and the invasion in HSC2 and HOC313 oral squamous cells (Chea et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Cutone, Colella, et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). We propose that Holo-BLf can inhibit migration and invasion induced by a variety of ligands including endocrine disruptors in TNBC cells.\u003c/p\u003e \u003cp\u003eFocal adhesions are structures composed of integrin receptors that mediate the interaction between the actin cytoskeleton and the extracellular matrix proteins. The composition of focal adhesions is complex and includes scaffolding proteins, GTPases, phosphatases, kinases, and adaptor proteins including vinculin (Carisey \u0026amp; Ballestrem, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Wozniak, Modzelewska, Kwong, \u0026amp; Keely, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Particularly, focal adhesions play a pivotal role in a variety of cellular processes including spreading, migration, invasion, differentiation, angiogenesis and survival (Zhao \u0026amp; Guan, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). We demonstrate here that BPA induces focal adhesions assembly in a time-dependent manner, and treatment with Holo-BLf inhibits the assembly of these focal adhesions in 4T1 cells. In agreement with our findings, Holo-BLf inhibits FAK phosphorylation at tyrosine-397 and disassembly of focal adhesions induced by FBS in MDA-MB-231 cells (Rodriguez-Ochoa et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Since focal adhesions assembly/disassembly and FAK phosphorylation at tyrosine 397 are required for migration, invasion and the epithelial to mesenchymal transition process (Avizienyte \u0026amp; Frame, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Zhao \u0026amp; Guan, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), we propose that Holo-BLf inhibits tumor progression through inhibition of focal adhesions assembly/disassembly including FAK phosphorylation at tyrosine 397 in breast cancer cells.\u003c/p\u003e \u003cp\u003eBPA induces proliferation through inhibition of miR-381-3p and STAT3 in MCF-7 breast cancer cells (Deng et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; W. Zhang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Moreover, oral administration of BPA (10 \u0026micro;g/kg/mouse) increases the volume and weight of mammary tumors in a murine model of breast cancer using 4T1 cells and female Balb/cJ mice (Torres-Alamilla et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), whereas subcutaneous administration of Apo-BLf (50 mg/kg/mouse) in C57BL/6 and CDF mice inoculated with B16-B16 melanoma cells and L5178Y-ML25 lymphoma cells inhibits liver and/or lungs metastasis (Yoo et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). In this study, we demonstrate that oral administration of Holo-BLf (50 mg/kg/mouse) inhibits the increase of weight and volume of mammary tumors induced by BPA (10 \u0026micro;g/kg/mouse) in murine models of breast cancer using human MDA-MB-231 cells and murine 4T1 cells inoculated in NSG and Balb/cJ mice respectively. We propose that Holo-BLf inhibits the proliferation of MDA-MB-231 and 4T1 cells in the mammary tumor, but it does not modify the mammary tumor microenvironment. Supporting our proposal, Holo-BLf inhibits proliferation through cell cycle arrest, increases of phospho-AMPKα, decreases of phospho-mTOR, but it does not induce apoptosis in T-47D, MDA-MB-231, Hs578T and MCF-7 breast cancer cells (Y. Zhang, Nicolau, Lima, \u0026amp; Rodrigues, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In addition, intratumoral administration of lactoferricin B, a 25-amino acid peptide released from bovine lactoferrin by acid-pepsin hydrolysis, promotes a reduction in the volume and weight of mammary tumors in a murine model of breast cancer using MDA-MB-231 cells and immunodeficient NSG mice (Rahman et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, our findings demonstrate that stroma of mammary tumors is similar in Balb/cJ mice inoculated with 4T1 cells and treated with Holo-BLf and BPA, and the mice treated with BPA.\u003c/p\u003e \u003cp\u003eMetastasis involves the dissemination of cancer cells from primary lesions to distant organs, which is mediated by a variety of cellular processes including migration, invasion into adjacent connective tissue and then intravasation into blood and lymphatic vessels, extravasation at distant organs, and the formation of secondary tumors in premetastatic niches (Chambers, Groom, \u0026amp; MacDonald, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Friedl \u0026amp; Wolf, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Our findings demonstrate that BPA significantly increases the number of Balb/cJ mice inoculated with 4T1 cells with metastasis in lungs and liver, as well as the total number and average number/mouse of metastatic nodules in lungs and liver. Histological analysis of metastatic nodules from lungs and liver shows a similar morphology of neoplastic cells in mice treated with BPA and BPA plus Holo-BLf. However, Holo-BLf promotes the formation of smaller number of metastatic neoplastic cells. In agreement with these findings, we also demonstrate here that BPA significantly increases the total number and average number/mouse of metastatic nodules in lungs from NSG mice inoculated with MDA-MB-231 cells. We propose that Holo-BLf partly inhibits some specific cellular processes that mediate metastasis, such as migration, invasion, hydrolysis of extracellular matrix, intravasation/extravasation, epithelial to mesenchymal transition process and formation of premetastatic niches. Supporting our proposal, we demonstrate here that Holo-BLf inhibits invasion and/or migration induced by BPA in MDA-MB-231 and 4T1 breast cancer cells, whereas it has been demonstrated that Holo-BLf inhibits migration, invasion, MMP-2/MMP-9 secretion and downregulates vimentin expression in MDA-MB-231 breast cancer cells (Rodriguez-Ochoa et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, Apo- and Holo-BLf prevent the epithelial to mesenchymal transition process through inhibition of interleukin-6/Stat3 in human glioblastoma GL-15 cells, whereas expression of Lf inhibits proliferation, migration, invasion and downregulates vimentin and N-cadherin expression in HONE-1 nasopharyngeal carcinoma cells (Cutone, Colella, et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; M. Xu, Fan, Zou, Yang, \u0026amp; Xu, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eHolo-BLf inhibits cellular processes involved with the invasion/metastasis process induced by BPA in TNBC MDA-MB-231 and 4T1 breast cancer cells. Moreover, Holo-BLf prevents the increase in the growth of mammary tumors and metastasis induced by BPA in two murine models of breast cancer. We propose that Holo-BLf can be used for the improvement of chemotherapeutic agents used in the treatment of TNBC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRina Valenzuela-Echeverria, Rocio Castillo-Sanchez, Pablo Torres-Alamilla and Pedro Cortes-Reynosa: Generation of results, methodology, design, Formal analysis of data, validation, Data curation, original draft. Rina Valenzuela-Echeverria, Fernando Candanedo-Gonzalez and Eduardo Perez Salazar: Conceptualization, analysis of data, methodology, writing-review \u0026amp; editing. Eduardo Perez Salazar; Administration of the project, acquisition of funding, supervision, Data curation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful for her technical assistance in histology studies to Brenda Medina-Rodriguez from Histology Laboratory of Cell Biology and Tissue Department, School of Medicine. UNAM, Mexico.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR. V-E is supported by a SECIHTI predoctoral training grant (839850). R. C-S is supported by a SECIHTI Post-Doctoral grant.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures conducted on animals were approved and followed the ethical guidelines and recommendations of the UPEAL of Cinvestav Zacatenco unit. The protocol number endorsed by the UPEAL for the current study is 0294-19.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAnders, C. K., Abramson, V., Tan, T., \u0026amp; Dent, R. (2016). The Evolution of Triple-Negative Breast Cancer: From Biology to Novel Therapeutics. \u003cem\u003eAm Soc Clin Oncol Educ Book, 35\u003c/em\u003e, 34-42. doi:10.1200/EDBK_159135\u003c/li\u003e\n\u003cli\u003eAvalos-Gomez, C., Reyes-Lopez, M., Ramirez-Rico, G., Diaz-Aparicio, E., Zenteno, E., Gonzalez-Ruiz, C., \u0026amp; de la Garza, M. (2020). Effect of apo-lactoferrin on leukotoxin and outer membrane vesicles of Mannheimia haemolytica A2. \u003cem\u003eVet Res, 51\u003c/em\u003e(1), 36. doi:10.1186/s13567-020-00759-z\u003c/li\u003e\n\u003cli\u003eAvizienyte, E., \u0026amp; Frame, M. C. (2005). Src and FAK signalling controls adhesion fate and the epithelial-to-mesenchymal transition. \u003cem\u003eCurr Opin Cell Biol, 17\u003c/em\u003e(5), 542-547. doi:10.1016/j.ceb.2005.08.007\u003c/li\u003e\n\u003cli\u003eBaker, H. M., \u0026amp; Baker, E. N. (2012). A structural perspective on lactoferrin function. \u003cem\u003eBiochem Cell Biol, 90\u003c/em\u003e(3), 320-328. doi:10.1139/o11-071\u003c/li\u003e\n\u003cli\u003eBays, J. L., \u0026amp; DeMali, K. A. (2017). Vinculin in cell-cell and cell-matrix adhesions. \u003cem\u003eCell Mol Life Sci, 74\u003c/em\u003e(16), 2999-3009. doi:10.1007/s00018-017-2511-3\u003c/li\u003e\n\u003cli\u003eBou Zerdan, M., Ghorayeb, T., Saliba, F., Allam, S., Bou Zerdan, M., Yaghi, M., . . . Nahleh, Z. (2022). Triple Negative Breast Cancer: Updates on Classification and Treatment in 2021. \u003cem\u003eCancers (Basel), 14\u003c/em\u003e(5). doi:10.3390/cancers14051253\u003c/li\u003e\n\u003cli\u003eCardiff, R. D., Miller, C. H., \u0026amp; Munn, R. J. (2014). Manual hematoxylin and eosin staining of mouse tissue sections. \u003cem\u003eCold Spring Harb Protoc, 2014\u003c/em\u003e(6), 655-658. doi:10.1101/pdb.prot073411\u003c/li\u003e\n\u003cli\u003eCarisey, A., \u0026amp; Ballestrem, C. (2011). Vinculin, an adapter protein in control of cell adhesion signalling. \u003cem\u003eEur J Cell Biol, 90\u003c/em\u003e(2-3), 157-163. doi:10.1016/j.ejcb.2010.06.007\u003c/li\u003e\n\u003cli\u003eCastillo-Sanchez, R., Ramirez-Ricardo, J., Martinez-Baeza, E., Cortes-Reynosa, P., Candanedo-Gonzales, F., Gomez, R., \u0026amp; Salazar, E. P. (2020). Bisphenol A induces focal adhesions assembly and activation of FAK, Src and ERK2 via GPER in MDA-MB-231 breast cancer cells. \u003cem\u003eToxicol In Vitro, 66\u003c/em\u003e, 104871. doi:10.1016/j.tiv.2020.104871\u003c/li\u003e\n\u003cli\u003eCastillo Sanchez, R., Gomez, R., \u0026amp; Perez Salazar, E. (2016). Bisphenol A Induces Migration through a GPER-, FAK-, Src-, and ERK2-Dependent Pathway in MDA-MB-231 Breast Cancer Cells. \u003cem\u003eChem Res Toxicol, 29\u003c/em\u003e(3), 285-295. doi:10.1021/acs.chemrestox.5b00457\u003c/li\u003e\n\u003cli\u003eChambers, A. F., Groom, A. C., \u0026amp; MacDonald, I. C. (2002). Dissemination and growth of cancer cells in metastatic sites. \u003cem\u003eNat Rev Cancer, 2\u003c/em\u003e(8), 563-572. doi:10.1038/nrc865\u003c/li\u003e\n\u003cli\u003eChea, C., Miyauchi, M., Inubushi, T., Okamoto, K., Haing, S., \u0026amp; Takata, T. (2023). Molecular Mechanisms of Inhibitory Effects of Bovine Lactoferrin on Invasion of Oral Squamous Cell Carcinoma. \u003cem\u003ePharmaceutics, 15\u003c/em\u003e(2). doi:10.3390/pharmaceutics15020562\u003c/li\u003e\n\u003cli\u003eCimmino, I., Fiory, F., Perruolo, G., Miele, C., Beguinot, F., Formisano, P., \u0026amp; Oriente, F. (2020). Potential Mechanisms of Bisphenol A (BPA) Contributing to Human Disease. \u003cem\u003eInt J Mol Sci, 21\u003c/em\u003e(16). doi:10.3390/ijms21165761\u003c/li\u003e\n\u003cli\u003eColorado-Yohar, S. M., Castillo-Gonzalez, A. C., Sanchez-Meca, J., Rubio-Aparicio, M., Sanchez-Rodriguez, D., Salamanca-Fernandez, E., . . . Chirlaque, M. D. (2021). Concentrations of bisphenol-A in adults from the general population: A systematic review and meta-analysis. \u003cem\u003eSci Total Environ, 775\u003c/em\u003e, 145755. doi:10.1016/j.scitotenv.2021.145755\u003c/li\u003e\n\u003cli\u003eCutone, A., Colella, B., Pagliaro, A., Rosa, L., Lepanto, M. S., Bonaccorsi di Patti, M. C., . . . Musci, G. (2020). Native and iron-saturated bovine lactoferrin differently hinder migration in a model of human glioblastoma by reverting epithelial-to-mesenchymal transition-like process and inhibiting interleukin-6/STAT3 axis. \u003cem\u003eCell Signal, 65\u003c/em\u003e, 109461. doi:10.1016/j.cellsig.2019.109461\u003c/li\u003e\n\u003cli\u003eCutone, A., Rosa, L., Ianiro, G., Lepanto, M. S., Bonaccorsi di Patti, M. C., Valenti, P., \u0026amp; Musci, G. (2020). Lactoferrin\u0026apos;s Anti-Cancer Properties: Safety, Selectivity, and Wide Range of Action. \u003cem\u003eBiomolecules, 10\u003c/em\u003e(3). doi:10.3390/biom10030456\u003c/li\u003e\n\u003cli\u003eDeng, P., Tan, M., Zhou, W., Chen, C., Xi, Y., Gao, P., . . . Zhou, Z. (2021). Bisphenol A promotes breast cancer cell proliferation by driving miR-381-3p-PTTG1-dependent cell cycle progression. \u003cem\u003eChemosphere, 268\u003c/em\u003e, 129221. doi:10.1016/j.chemosphere.2020.129221\u003c/li\u003e\n\u003cli\u003eFaustino-Rocha, A., Oliveira, P. A., Pinho-Oliveira, J., Teixeira-Guedes, C., Soares-Maia, R., da Costa, R. G., . . . Ginja, M. (2013). Estimation of rat mammary tumor volume using caliper and ultrasonography measurements. \u003cem\u003eLab Anim (NY), 42\u003c/em\u003e(6), 217-224. doi:10.1038/laban.254\u003c/li\u003e\n\u003cli\u003eFilho, A. M., Laversanne, M., Ferlay, J., Colombet, M., Pineros, M., Znaor, A., . . . Bray, F. (2025). The GLOBOCAN 2022 cancer estimates: Data sources, methods, and a snapshot of the cancer burden worldwide. \u003cem\u003eInt J Cancer, 156\u003c/em\u003e(7), 1336-1346. doi:10.1002/ijc.35278\u003c/li\u003e\n\u003cli\u003eFriedl, P., \u0026amp; Wolf, K. (2003). Tumour-cell invasion and migration: diversity and escape mechanisms. \u003cem\u003eNat Rev Cancer, 3\u003c/em\u003e(5), 362-374. doi:10.1038/nrc1075\u003c/li\u003e\n\u003cli\u003eGibbons, J. A., Kanwar, J. R., \u0026amp; Kanwar, R. K. (2015). Iron-free and iron-saturated bovine lactoferrin inhibit survivin expression and differentially modulate apoptosis in breast cancer. \u003cem\u003eBMC Cancer, 15\u003c/em\u003e, 425. doi:10.1186/s12885-015-1441-4\u003c/li\u003e\n\u003cli\u003eGonzalez-Chavez, S. A., Arevalo-Gallegos, S., \u0026amp; Rascon-Cruz, Q. (2009). Lactoferrin: structure, function and applications. \u003cem\u003eInt J Antimicrob Agents, 33\u003c/em\u003e(4), 301 e301-308. doi:10.1016/j.ijantimicag.2008.07.020\u003c/li\u003e\n\u003cli\u003eKowalczyk, P., Kaczynska, K., Kleczkowska, P., Bukowska-Osko, I., Kramkowski, K., \u0026amp; Sulejczak, D. (2022). The Lactoferrin Phenomenon-A Miracle Molecule. \u003cem\u003eMolecules, 27\u003c/em\u003e(9). doi:10.3390/molecules27092941\u003c/li\u003e\n\u003cli\u003eLehmler, H. J., Liu, B., Gadogbe, M., \u0026amp; Bao, W. (2018). Exposure to Bisphenol A, Bisphenol F, and Bisphenol S in U.S. Adults and Children: The National Health and Nutrition Examination Survey 2013-2014. \u003cem\u003eACS Omega, 3\u003c/em\u003e(6), 6523-6532. doi:10.1021/acsomega.8b00824\u003c/li\u003e\n\u003cli\u003eNohynek, G. J., Borgert, C. J., Dietrich, D., \u0026amp; Rozman, K. K. (2013). Endocrine disruption: fact or urban legend? \u003cem\u003eToxicol Lett, 223\u003c/em\u003e(3), 295-305. doi:10.1016/j.toxlet.2013.10.022\u003c/li\u003e\n\u003cli\u003eRahman, R., Fonseka, A. D., Sua, S. C., Ahmad, M., Rajendran, R., Ambu, S., . . . Chitra, E. (2021). Inhibition of breast cancer xenografts in a mouse model and the induction of apoptosis in multiple breast cancer cell lines by lactoferricin B peptide. \u003cem\u003eJ Cell Mol Med, 25\u003c/em\u003e(15), 7181-7189. doi:10.1111/jcmm.16748\u003c/li\u003e\n\u003cli\u003eRodriguez-Ochoa, N., Cortes-Reynosa, P., Rodriguez-Rojas, K., de la Garza, M., \u0026amp; Salazar, E. P. (2023). Bovine holo-lactoferrin inhibits migration and invasion in MDA-MB-231 breast cancer cells. \u003cem\u003eMol Biol Rep, 50\u003c/em\u003e(1), 193-201. doi:10.1007/s11033-022-07943-8\u003c/li\u003e\n\u003cli\u003eSajiki, J., \u0026amp; Yonekubo, J. (2004). Leaching of bisphenol A (BPA) from polycarbonate plastic to water containing amino acids and its degradation by radical oxygen species. \u003cem\u003eChemosphere, 55\u003c/em\u003e(6), 861-867. doi:10.1016/j.chemosphere.2003.11.065\u003c/li\u003e\n\u003cli\u003eSung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A., \u0026amp; Bray, F. (2021). Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. \u003cem\u003eCA Cancer J Clin, 71\u003c/em\u003e(3), 209-249. doi:10.3322/caac.21660\u003c/li\u003e\n\u003cli\u003eTorres-Alamilla, P., Castillo-Sanchez, R., Cortes-Reynosa, P., Gomez, R., \u0026amp; Perez Salazar, E. (2023). Bisphenol A increases the size of primary mammary tumors and promotes metastasis in a murine model of breast cancer. \u003cem\u003eMol Cell Endocrinol, 575\u003c/em\u003e, 111998. doi:10.1016/j.mce.2023.111998\u003c/li\u003e\n\u003cli\u003eTsuda, H., Kozu, T., Iinuma, G., Ohashi, Y., Saito, Y., Saito, D., . . . Iigo, M. (2010). Cancer prevention by bovine lactoferrin: from animal studies to human trial. \u003cem\u003eBiometals, 23\u003c/em\u003e(3), 399-409. doi:10.1007/s10534-010-9331-3\u003c/li\u003e\n\u003cli\u003eWang, B., Timilsena, Y. P., Blanch, E., \u0026amp; Adhikari, B. (2019). Lactoferrin: Structure, function, denaturation and digestion. \u003cem\u003eCrit Rev Food Sci Nutr, 59\u003c/em\u003e(4), 580-596. doi:10.1080/10408398.2017.1381583\u003c/li\u003e\n\u003cli\u003eWozniak, M. A., Modzelewska, K., Kwong, L., \u0026amp; Keely, P. J. (2004). Focal adhesion regulation of cell behavior. \u003cem\u003eBiochim Biophys Acta, 1692\u003c/em\u003e(2-3), 103-119. doi:10.1016/j.bbamcr.2004.04.007\u003c/li\u003e\n\u003cli\u003eXu, M., Fan, Y., Zou, G., Yang, Q., \u0026amp; Xu, F. (2024). Lactoferrin mediates epithelial-mesenchymal transformation by regulating the PI3K/AKT/mTOR pathway to inhibit nasopharyngeal carcinoma metastasis. \u003cem\u003eCell Mol Biol (Noisy-le-grand), 70\u003c/em\u003e(3), 130-135. doi:10.14715/cmb/2024.70.3.19\u003c/li\u003e\n\u003cli\u003eXu, X., Zhang, Y., Jasper, J., Lykken, E., Alexander, P. B., Markowitz, G. J., . . . Wang, X. F. (2016). MiR-148a functions to suppress metastasis and serves as a prognostic indicator in triple-negative breast cancer. \u003cem\u003eOncotarget, 7\u003c/em\u003e(15), 20381-20394. doi:10.18632/oncotarget.7953\u003c/li\u003e\n\u003cli\u003eYoo, Y. C., Watanabe, S., Watanabe, R., Hata, K., Shimazaki, K., \u0026amp; Azuma, I. (1997). Bovine lactoferrin and lactoferricin, a peptide derived from bovine lactoferrin, inhibit tumor metastasis in mice. \u003cem\u003eJpn J Cancer Res, 88\u003c/em\u003e(2), 184-190. doi:10.1111/j.1349-7006.1997.tb00364.x\u003c/li\u003e\n\u003cli\u003eZhang, W., Fang, Y., Shi, X., Zhang, M., Wang, X., \u0026amp; Tan, Y. (2012). Effect of bisphenol A on the EGFR-STAT3 pathway in MCF-7 breast cancer cells. \u003cem\u003eMol Med Rep, 5\u003c/em\u003e(1), 41-47. doi:10.3892/mmr.2011.583\u003c/li\u003e\n\u003cli\u003eZhang, Y., Lima, C. F., \u0026amp; Rodrigues, L. R. (2014). Anticancer effects of lactoferrin: underlying mechanisms and future trends in cancer therapy. \u003cem\u003eNutr Rev, 72\u003c/em\u003e(12), 763-773. doi:10.1111/nure.12155\u003c/li\u003e\n\u003cli\u003eZhang, Y., Nicolau, A., Lima, C. F., \u0026amp; Rodrigues, L. R. (2014). Bovine lactoferrin induces cell cycle arrest and inhibits mTOR signaling in breast cancer cells. \u003cem\u003eNutr Cancer, 66\u003c/em\u003e(8), 1371-1385. doi:10.1080/01635581.2014.956260\u003c/li\u003e\n\u003cli\u003eZhao, J., \u0026amp; Guan, J. L. (2009). Signal transduction by focal adhesion kinase in cancer. \u003cem\u003eCancer Metastasis Rev, 28\u003c/em\u003e(1-2), 35-49. doi:10.1007/s10555-008-9165-4\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biometals","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biom","sideBox":"Learn more about [BioMetals](http://link.springer.com/journal/10534)","snPcode":"10534","submissionUrl":"https://submission.nature.com/new-submission/10534/3","title":"BioMetals","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Breast cancer, Lactoferrin, Mammary tumor, Metastasis, 4T1, Migration","lastPublishedDoi":"10.21203/rs.3.rs-8904788/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8904788/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBreast cancer is the leading cause of cancer-related deaths in worldwide women. Triple negative breast cancer (TNBC) subtype does not express progesterone, estrogen and Her2 receptors. Bisphenol A (BPA) is an endocrine-disrupting chemical used in plastics and epoxy resins production that is associated with an increased rate of cancer including breast cancer. Lactoferrin (Lf) is a glycoprotein expressed in a variety of species that is found free of Fe\u003csup\u003e3+\u003c/sup\u003e (Apo-Lf) and associated with Fe\u003csup\u003e3+\u003c/sup\u003e (Holo-Lf). Bovine Lf (BLf) exhibits anti-tumor properties through inhibition of proliferation, migration, invasion, matrix metalloproteinases secretion, and epithelial\u0026ndash;mesenchymal transition. In this study we demonstrate that Holo-BLf inhibits migration, invasion and assembly of focal adhesions induced by BPA in 4T1 cells, and migration in MDA-MB-231 cells. In addition, Holo-BLf inhibits the growth of mammary tumors and metastasis induced by BPA in two murine models of breast cancer using TNBC 4T1/MDA-MB-231 cells and Balb/cJ/NSG mice. In conclusion, we demonstrate, for first time, that Holo-BLf inhibits cellular processes involved with the metastasis process and the growth of mammary tumors and metastasis in two murine models of breast cancer.\u003c/p\u003e","manuscriptTitle":"Bovine Holo-lactoferrin inhibits tumor growth and metastasis induced by BPA in murine triple negative breast cancer models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-02 09:46:43","doi":"10.21203/rs.3.rs-8904788/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-02-25T22:01:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-19T02:31:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-19T02:30:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioMetals","date":"2026-02-17T23:54:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biometals","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biom","sideBox":"Learn more about [BioMetals](http://link.springer.com/journal/10534)","snPcode":"10534","submissionUrl":"https://submission.nature.com/new-submission/10534/3","title":"BioMetals","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0048196a-575d-44cc-aa09-1e8334268629","owner":[],"postedDate":"March 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T09:46:44+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-02 09:46:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8904788","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8904788","identity":"rs-8904788","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}