Spatial Engineering of Mammary Epithelial Cell Cultures with 3D Bioprinting Reveals Growth Control by Branch Point Proximity.

The three-dimensional (3D) structure of the ductal epithelium and the surrounding extracellular matrix (ECM) are integral aspects of the breast tissue, and they have important roles during mammary gland development, function and malignancy. However, the architecture of the branched mammary epithelial network is poorly recapitulated in the current in vitro models. 3D bioprinting is an emerging approach to improve tissue-mimicry in cell culture. Here, we developed and optimized a protocol for 3D bioprinting of normal and cancerous mammary epithelial cells into a branched Y-shape to study the role of cell positioning in the regulation of cell proliferation and invasion. Non-cancerous cells formed continuous 3D cell networks with several organotypic features, whereas the ductal carcinoma in situ (DCIS) -like cancer cells exhibited aberrant basal polarization and defective formation of the basement membrane (BM). Quantitative analysis over time demonstrated that both normal and cancerous cells proliferate more at the branch tips compared to the trunk region of the 3D-bioprinted cultures, and particularly at the tip further away from the branch point. The location-specific rate of proliferation was independent of TGFß signaling but invasion of the DCIS-like breast cancer cells was reduced upon the inhibition of TGFß. Thus, our data demonstrate that the 3D-bioprinted cells can sense their position in the branched network of cells and proliferate at the tips, thus recapitulating this feature of mammary epithelial branching morphogenesis. In all, our results demonstrate the capacity of the developed 3D bioprinting method for quantitative analysis of the relationships between tissue structure and cell behavior in breast morphogenesis and cancer.

IGFBP2 secretion by mammary adipocytes limits breast cancer invasion.

The progression of noninvasive ductal carcinoma in situ to invasive ductal carcinoma for patients with breast cancer results in a significantly poorer prognosis and is the precursor to metastatic disease. In this work, we have identified insulin-like growth factor-binding protein 2 (IGFBP2) as a potent adipocrine factor secreted by healthy breast adipocytes that acts as a barrier against invasive progression. In line with this role, adipocytes differentiated from patient-derived stromal cells were found to secrete IGFBP2, which significantly inhibited breast cancer invasion. This occurred through binding and sequestration of cancer-derived IGF-II. Moreover, depletion of IGF-II in invading cancer cells using small interfering RNAs or an IGF-II-neutralizing antibody ablated breast cancer invasion, highlighting the importance of IGF-II autocrine signaling for breast cancer invasive progression. Given the abundance of adipocytes in the healthy breast, this work exposes the important role they play in suppressing cancer progression and may help expound upon the link between increased mammary density and poorer prognosis.

Laminin matrix adhesion regulates basal mammary epithelial cell identity.

Mammary epithelium is a bilayered ductal network composed of luminal and basal epithelial cells, which together drive the growth and functional differentiation of the gland. Basal mammary epithelial cells (MECs) exhibit remarkable plasticity and progenitor activity that facilitate epithelial expansion. However, their activity must be tightly regulated to restrict excess basal cell activity. Here, we show that adhesion of basal cells to laminin α5-containing basement membrane matrix, which is produced by luminal cells, presents such a control mechanism. Adhesion to laminin α5 directs basal cells towards a luminal cell fate, and thereby results in a marked decrease of basal MEC progenitor activity in vitro and in vivo. Mechanistically, these effects are mediated through ß4-integrin and activation of p21 (encoded by CDKN1A). Thus, we demonstrate that laminin matrix adhesion is a key determinant of basal identity and essential to building and maintaining a functional multicellular epithelium.

MYO10-filopodia support basement membranes at pre-invasive tumor boundaries.

Ductal carcinoma in situ (DCIS) is a pre-invasive stage of breast cancer. During invasion, the encapsulating DCIS basement membrane (BM) is compromised, and tumor cells invade the surrounding stroma. The mechanisms that regulate functional epithelial BMs in vivo are poorly understood. Myosin-X (MYO10) is a filopodia-inducing protein associated with metastasis and poor clinical outcome in invasive breast cancer (IBC). We identify elevated MYO10 expression in human DCIS and IBC, and this suggests links with disease progression. MYO10 promotes filopodia formation and cell invasion in vitro and cancer-cell dissemination from progressively invasive human DCIS xenografts. However, MYO10-depleted xenografts are more invasive. These lesions exhibit compromised BMs, poorly defined borders, and increased cancer-cell dispersal and EMT-marker-positive cells. In addition, cancer spheroids are dependent on MYO10-filopodia to generate a near-continuous extracellular matrix boundary. Thus, MYO10 is protective in early-stage breast cancer, correlating with tumor-limiting BMs, and pro-invasive at later stages, facilitating cancer-cell dissemination.

Platelet SHARPIN regulates platelet adhesion and inflammatory responses through associations with αIIbß3 and LUBAC.

Platelets form hemostatic plugs to prevent blood loss, and they modulate immunity and inflammation in several ways. A key event during hemostasis is activation of integrin αIIbß3 through direct interactions of the ß3 cytoplasmic tail with talin and kindlin-3. Recently, we showed that human platelets express the adapter molecule Shank-associated RH domain interacting protein (SHARPIN), which can associate directly with the αIIb cytoplasmic tail and separately promote NF-κB pathway activation as a member of the Met-1 linear ubiquitination activation complex (LUBAC). Here we investigated the role of SHARPIN in platelets after crossing Sharpin flox/flox (fl/fl) mice with PF4-Cre or GPIbα-Cre mice to selectively delete SHARPIN in platelets. SHARPIN-null platelets adhered to immobilized fibrinogen through αIIbß3, and they spread more extensively than littermate control platelets in a manner dependent on feedback stimulation by platelet adenosine diphosphate (ADP) (P < .01). SHARPIN-null platelets showed increased colocalization of αIIbß3 with talin as assessed by super-resolution microscopy and increased binding of soluble fibrinogen in response to submaximal concentrations of ADP (P < .05). However, mice with SHARPIN-null platelets showed compromised thrombus growth on collagen and slightly prolonged tail bleeding times. Platelets lacking SHARPIN also showed reduced NF-κB activation and linear ubiquitination of protein substrates upon challenge with classic platelet agonists. Furthermore, the loss of platelet SHARPIN resulted in significant reduction in inflammation in murine models of colitis and peritonitis (P < .01). Thus, SHARPIN plays differential and context-dependent roles in platelets to regulate important inflammatory and integrin adhesive functions of these anucleate cells.

Sortilin-related receptor is a druggable therapeutic target in breast cancer.

In breast cancer, the currently approved anti-receptor tyrosine-protein kinase erbB-2 (HER2) therapies do not fully meet the expected clinical goals due to therapy resistance. Identifying alternative HER2-related therapeutic targets could offer a means to overcome these resistance mechanisms. We have previously demonstrated that an endosomal sorting protein, sortilin-related receptor (SorLA), regulates the traffic and signaling of HER2 and HER3, thus promoting resistance to HER2-targeted therapy in breast cancer. This study aims to assess the feasibility of targeting SorLA using a monoclonal antibody. Our results demonstrate that anti-SorLA antibody (SorLA ab) alters the resistance of breast cancer cells to HER2 monoclonal antibody trastuzumab in vitro and in ovo. We found that SorLA ab and trastuzumab combination therapy also inhibits tumor cell proliferation and tumor cell density in a mouse xenograft model of HER2-positive breast cancer. In addition, SorLA ab inhibits the proliferation of breast cancer patient-derived explant three-dimensional cultures. These results provide, for the first time, proof of principle that SorLA is a druggable target in breast cancer.

Low TGF-ß1 in Wound Exudate Predicts Surgical Site Infection After Axillary Lymph Node Dissection.

PURPOSE: Surgical site infection (SSI) after axillary lymph node dissection (ALND) for breast cancer increases morbidity and delays the onset of adjuvant treatment. Only a few studies have investigated the feasibility of wound exudate analysis in SSI prediction. This study assessed changes in cytokine levels in postsurgical wound exudate after ALND and examined their predictive value for the early diagnosis of SSI. METHODS: An observational prospective pilot study was conducted in 47 patients with breast cancer undergoing ALND. Wound exudate samples were collected on the first and sixth postoperative days (POD). Interleukin (IL)-1α, IL-1ß, IL-4, IL-10, IL-13, tumor necrosis factor alpha (TNF-α), transforming growth factor beta1 (TGF-ß1) and vascular endothelial growth factor (VEGF) C and D levels were measured by immunoassay. Patients were followed to detect SSI. RESULTS: SSI was diagnosed in 8/47 (17.0%) patients. Four SSI patients were hospitalized and treated with intravenous antibiotics. The concentration of TGF-ß1 in wound exudate was significantly lower on POD#1 in the SSI group compared to the no SSI group (p=0.008). The receiving operator characteristics (ROC) curve for TGF-ß1 showed an area under curve of 0.773 (p=0.0149) indicating good diagnostic potential. On POD#6, the concentration of TGF-ß1 remained significantly lower (p=0.043) and the concentrations of IL-10 (p=0.000) and IL-1ß (0.004) significantly higher in the SSI group compared to the no SSI group. CONCLUSION: To our knowledge, this is the first study suggesting a predictive role of wound exudate TGF-ß1 levels for SSI. Our results suggest that the risk for SSI can be detected already on POD#1 and that the assessment of TGF-ß1 levels in the wound exudate after ALND can provide a usefull method for the early detection of SSI. The key findings of this pilot study warrant verification in a larger patient population.

CIP2A Interacts with TopBP1 and Drives Basal-Like Breast Cancer Tumorigenesis.

Basal-like breast cancers (BLBC) are characterized by defects in homologous recombination (HR), deficient mitotic checkpoint, and high-proliferation activity. Here, we discover CIP2A as a candidate driver of BLBC. CIP2A was essential for DNA damage-induced initiation of mouse BLBC-like mammary tumors and for survival of HR-defective BLBC cells. CIP2A was dispensable for normal mammary gland development and for unperturbed mitosis, but selectively essential for mitotic progression of DNA damaged cells. A direct interaction between CIP2A and a DNA repair scaffold protein TopBP1 was identified, and CIP2A inhibition resulted in enhanced DNA damage-induced TopBP1 and RAD51 recruitment to chromatin in mammary epithelial cells. In addition to its role in tumor initiation, and survival of BRCA-deficient cells, CIP2A also drove proliferative MYC and E2F1 signaling in basal-like triple-negative breast cancer (BL-TNBC) cells. Clinically, high CIP2A expression was associated with poor patient prognosis in BL-TNBCs but not in other breast cancer subtypes. Small-molecule reactivators of PP2A (SMAP) inhibited CIP2A transcription, phenocopied the CIP2A-deficient DNA damage response (DDR), and inhibited growth of patient-derived BLBC xenograft. In summary, these results demonstrate that CIP2A directly interacts with TopBP1 and coordinates DNA damage-induced mitotic checkpoint and proliferation, thereby driving BLBC initiation and progression. SMAPs could serve as a surrogate therapeutic strategy to inhibit the oncogenic activity of CIP2A in BLBCs. SIGNIFICANCE: These results identify CIP2A as a nongenetic driver and therapeutic target in basal-like breast cancer that regulates DNA damage-induced G2-M checkpoint and proliferative signaling.

CC chemokine ligand 2 (CCL2) stimulates aromatase gene expression in mammary adipose tissue.

Obesity is a risk factor for postmenopausal breast cancer. Obesity-related inflammation upregulates aromatase expression, the rate-limiting enzyme for estrogen synthesis, in breast adipose tissue (BAT), increasing estrogen production and cancer risk. The regulation of aromatase gene (CYP19A1) in BAT is complex, and the mechanisms linking obesity and aromatase dysregulation are not fully understood. An obesity-associated factor that could regulate aromatase is the CC chemokine ligand (CCL) 2, a pro-inflammatory factor that also activates signaling pathways implicated in CYP19A1 transcription. By using human primary breast adipose stromal cells (ASCs) and aromatase reporter (hARO-Luc) mouse mammary adipose explants, we demonstrated that CCL2 enhances the glucocorticoid-mediated CYP19A1 transcription. The potential mechanism involves the activation of PI.4 via ERK1/2 pathway. We also showed that CCL2 contributes to the pro-inflammatory milieu and aromatase expression in obesity, evidenced by increased expression of CCL2 and CYP19A1 in mammary tissues from obese hARO-Luc mice, and subcutaneous adipose tissue from obese women. In summary, our results indicate that postmenopausal obesity may promote CCL2 production in BAT, leading to exacerbation of the menopause-related inflammatory state and further stimulation of local aromatase and estrogens. These results provide new insights into the regulation of aromatase and may aid in finding approaches to prevent breast cancer.

Integrin-mediated adhesion and mechanosensing in the mammary gland.

The mammary gland is dynamically remodelled during its postnatal development and the reproductive cycles. This inherent plasticity has been suggested to increase the susceptibility of the organ to carcinogenesis. Morphological changes in the mammary epithelium involve cell proliferation, differentiation, apoptosis, and migration which, in turn, are affected by cell adhesion to the extracellular matrix (ECM). Integrin adhesion receptors function in the sensing of the biochemical composition, patterning and mechanical properties of the ECM surrounding the cells, and strongly influence cell fate. This review aims to summarize the existing literature on how different aspects of integrin-mediated adhesion and mechanosensing, including ECM composition; stiffness and topography; integrin expression patterns; focal adhesion assembly; dynamic regulation of the actin cytoskeleton; and nuclear mechanotransduction affect mammary gland development, function and homeostasis. As the mechanical properties of a complex tissue environment are challenging to replicate in vitro, emphasis has been placed on studies conducted in vivo or using organoid models. Outright, these studies indicate that mechanosensing also contributes to the regulation of mammary gland morphogenesis in multiple ways.

Erratum: Integrin Binding Dynamics Modulate Ligand-Specific Mechanosensing in Mammary Gland Fibroblasts.

[This corrects the article DOI: 10.1016/j.isci.2020.100907.].

Integrin Binding Dynamics Modulate Ligand-Specific Mechanosensing in Mammary Gland Fibroblasts.

The link between integrin activity regulation and cellular mechanosensing of tissue rigidity, especially on different extracellular matrix ligands, remains poorly understood. Here, we find that primary mouse mammary gland stromal fibroblasts (MSFs) are able to spread efficiently, generate high forces, and display nuclear YAP on soft collagen-coated substrates, resembling the soft mammary gland tissue. We describe that loss of the integrin inhibitor, SHARPIN, impedes MSF spreading specifically on soft type I collagen but not on fibronectin. Through quantitative experiments and computational modeling, we find that SHARPIN-deficient MSFs display faster force-induced unbinding of adhesions from collagen-coated beads. Faster unbinding, in turn, impairs force transmission in these cells, particularly, at the stiffness optimum observed for wild-type cells. Mechanistically, we link the impaired mechanotransduction of SHARPIN-deficient cells on collagen to reduced levels of collagen-binding integrin α11ß1. Thus integrin activity regulation and α11ß1 play a role in collagen-specific mechanosensing in MSFs.

Preperitoneal Fat Grafting Inhibits the Formation of Intra-abdominal Adhesions in Mice.

BACKGROUND: Adhesion formation contributes to postoperative complications in abdominal and gynaecological surgery. Thus far, the prevention and treatment strategies have focused on mechanical barriers in solid and liquid form, but these methods are not in routine use. As autologous fat grafting has become popular in treatment of hypertrophic scars because of its immunomodulatory effects, we postulated that fat grafting could also prevent peritoneal adhesion through similar mechanisms. METHODS: This was a control versus intervention study to evaluate the effect of fat grafting in the prevention on peritoneal adhesion formation. An experimental mouse model for moderate and extensive peritoneal adhesions was used (n = 4-6 mice/group). Adhesions were induced mechanically, and a free epididymal fat graft from wild type or CAG-DsRed mice was injected preperitoneally immediately after adhesion induction. PET/CT imaging and scaling of the adhesions were performed, and samples were taken for further analysis at 7 and 30 days postoperation. Macrophage phenotyping was further performed from peritoneal lavage samples, and the expression of inflammatory cytokines and mesothelial layer recovery were analysed from peritoneal tissue samples. RESULTS: Fat grafting significantly inhibited the formation of adhesions. PET/CT results did not show prolonged inflammation in any of the groups. While the expression of anti-inflammatory and anti-fibrotic IL-10 was significantly increased in the peritoneum of the fat graft-treated group at 7 days, tissue-resident and repairing M2 macrophages could no longer be detected in the fat graft at this time point. The percentage of the continuous, healed peritoneum as shown by Keratin 8 staining was greater in the fat graft-treated group after 7 days. CONCLUSIONS: Fat grafting can inhibit the formation of peritoneal adhesions in mice. Our results suggest that fat grafting promotes the peritoneal healing process in a paracrine manner thereby enabling rapid regeneration of the peritoneal mesothelial cell layer.

The Eleventh ENBDC Workshop: Advances in Technology Help to Unveil Mechanisms of Mammary Gland Development and Cancerogenesis.

The eleventh annual workshop of the European Network for Breast Development and Cancer, Methods in mammary gland biology and breast cancer, took place on the 16th to 18th of May 2019 in Weggis, Switzerland. The main topics of the meeting were high resolution genomics and proteomics for the study of mammary gland development and cancer, breast cancer signaling, tumor microenvironment, preclinical models of breast cancer, and tissue morphogenesis. Exciting novel findings in, or highly relevant to, mammary gland biology and breast cancer field were presented, with insights into the methods used to obtain them. Among others, the discussed methods included single-cell RNA sequencing, genetic barcoding, lineage tracing, spatial transcriptomics, optogenetics, genetic mouse models and organoids.

SORLA regulates endosomal trafficking and oncogenic fitness of HER2.

The human epidermal growth factor receptor 2 (HER2) is an oncogene targeted by several kinase inhibitors and therapeutic antibodies. While the endosomal trafficking of many other receptor tyrosine kinases is known to regulate their oncogenic signalling, the prevailing view on HER2 is that this receptor is predominantly retained on the cell surface. Here, we find that sortilin-related receptor 1 (SORLA; SORL1) co-precipitates with HER2 in cancer cells and regulates HER2 subcellular distribution by promoting recycling of the endosomal receptor back to the plasma membrane. SORLA protein levels in cancer cell lines and bladder cancers correlates with HER2 levels. Depletion of SORLA triggers HER2 targeting to late endosomal/lysosomal compartments and impairs HER2-driven signalling and in vivo tumour growth. SORLA silencing also disrupts normal lysosome function and sensitizes anti-HER2 therapy sensitive and resistant cancer cells to lysosome-targeting cationic amphiphilic drugs. These findings reveal potentially important SORLA-dependent endosomal trafficking-linked vulnerabilities in HER2-driven cancers.

Integrin signaling and mechanotransduction in regulation of somatic stem cells.

Somatic stem cells are characterized by their capacity for self-renewal and differentiation, making them integral for normal tissue homeostasis. Different stem cell functions are strongly affected by the specialized microenvironment surrounding the cells. Consisting of soluble signaling factors, extracellular matrix (ECM) ligands and other cells, but also biomechanical cues such as the viscoelasticity and topography of the ECM, these factors are collectively known as the niche. Cell-ECM interactions are mediated largely by integrins, a class of heterodimeric cell adhesion molecules. Integrins bind their ligands in the extracellular space and associate with the cytoskeleton inside the cell, forming a direct mechanical link between the cells and their surroundings. Indeed, recent findings have highlighted the importance of integrins in translating biophysical cues into changes in cell signaling and function, a multistep process known as mechanotransduction. The mechanical properties of the stem cell niche are important, yet the underlying molecular details of integrin-mediated mechanotransduction in stem cells, especially the roles of the different integrin heterodimers, remain elusive. Here, we introduce the reader to the concept of integrin-mediated mechanotransduction, summarize current knowledge on the role of integrin signaling and mechanotransduction in regulation of somatic stem cell functions, and discuss open questions in the field.

68Ga-DOTA-E[c(RGDfK)]2 PET Imaging of SHARPIN-Regulated Integrin Activity in Mice.

Shank-associated RH domain-interacting protein (SHARPIN) is a cytosolic protein that plays a key role in activation of nuclear factor κ-light-chain enhancer of activated B cells and regulation of inflammation. Furthermore, SHARPIN controls integrin-dependent cell adhesion and migration in several normal and malignant cell types, and loss of SHARPIN correlates with increased integrin activity in mice. Arginyl-glycyl-aspartic acid (RGD), a cell adhesion tripeptide motif, is an integrin recognition sequence that facilitates PET imaging of integrin upregulation during tumor angiogenesis. We hypothesized that increased integrin activity due to loss of SHARPIN protein would affect the uptake of αvß3-selective cyclic, dimeric peptide 68Ga-DOTA-E[c(RGDfK)]2, where E[c(RGDfk)]2 = glutamic acid-[cyclo(arginyl-glycyl-aspartic acid-D-phenylalanine-lysine)], both in several tissue types and in the tumor microenvironment. To test this hypothesis, we used RGD-based in vivo PET imaging to evaluate wild-type (wt) and SHARPIN-deficient mice (Sharpincpdm , where cpdm = chronic proliferative dermatitis in mice) with and without melanoma tumor allografts. Methods:Sharpincpdm mice with spontaneous null mutation in the Sharpin gene and their wt littermates with or without B16-F10-luc melanoma tumors were studied by in vivo imaging and ex vivo measurements with cyclic-RGD peptide 68Ga-DOTA-E[c(RGDfK)]2 After the last 68Ga-DOTA-E[c(RGDfK)]2 peptide PET/CT, tumors were cut into cryosections for autoradiography, histology, and immunohistochemistry. Results: The ex vivo uptake of 68Ga-DOTA-E[c(RGDfK)]2 in the mouse skin and tumor was significantly higher in Sharpincpdm mice than in wt mice. B16-F10-luc tumors were detected 4 d after inoculation, without differences in volume or blood flow between the mouse strains. PET imaging with 68Ga-DOTA-E[c(RGDfK)]2 peptide at day 10 after inoculation revealed significantly higher uptake in the tumors transplanted into Sharpincpdm mice than in wt mice. Furthermore, tumor vascularization was increased in the Sharpincpdm mice. Conclusion:Sharpincpdm mice demonstrated increased integrin activity and vascularization in B16-F10-luc melanoma tumors, as demonstrated by RGD-based in vivo PET imaging. These data indicate that SHARPIN, a protein previously associated with increased cancer growth and metastasis, may also have important regulatory roles in controlling the tumor microenvironment.

Integrin beta 1 inhibition alleviates the chronic hyperproliferative dermatitis phenotype of SHARPIN-deficient mice.

SHARPIN (Shank-Associated RH Domain-Interacting Protein) is a component of the linear ubiquitin chain assembly complex (LUBAC), which enhances TNF-induced NF-κB activity. SHARPIN-deficient (Sharpincpdm/cpdm) mice display multi-organ inflammation and chronic proliferative dermatitis (cpdm) due to TNF-induced keratinocyte apoptosis. In cells, SHARPIN also inhibits integrins independently of LUBAC, but it has remained enigmatic whether elevated integrin activity levels in the dermis of Sharpincpdm/cpdm mice is due to increased integrin activity or is secondary to inflammation. In addition, the functional contribution of increased integrin activation to the Sharpincpdm/cpdm phenotype has not been investigated. Here, we find increased integrin activity in keratinocytes from Tnfr1-/- Sharpincpdm/cpdm double knockout mice, which do not display chronic inflammation or proliferative dermatitis, thus suggesting that SHARPIN indeed acts as an integrin inhibitor in vivo. In addition, we present evidence for a functional contribution of integrin activity to the Sharpincpdm/cpdm skin phenotype. Treatment with an integrin beta 1 function blocking antibody reduced epidermal hyperproliferation and epidermal thickness in Sharpincpdm/cpdm mice. Our data indicate that, while TNF-induced cell death triggers the chronic inflammation and proliferative dermatitis, absence of SHARPIN-dependent integrin inhibition exacerbates the epidermal hyperproliferation in Sharpincpdm/cpdm mice.

Epithelial vimentin plays a functional role in mammary gland development.

In the mammary gland, vimentin intermediate filaments are expressed in stromal cells and in basal epithelial cell populations, including gland-reconstituting mammary stem cells, with largely undefined functions. Here, we have studied how vimentin deficiency affects mouse mammary gland development. We find that, in adult vimentin knockout mice (Vim-/- ), mammary ductal outgrowth is delayed. The adult Vim-/- glands display dilated ducts and a reduced basal-to-luminal mouse mammary epithelial cell (MMEC) ratio indicative of altered progenitor cell activity. Accordingly, isolated Vim-/- MMECs form fewer mammospheres and basal-like organoids in vitro than their wild-type counterparts. Importantly, reduced basal MMEC number translates into defects in Vim-/- mammary gland regeneration in vivo Global gene expression profiling of basal MMECs reveals that lack of vimentin alters multiple pathways, including adhesion, cancer and Wnt signalling. Furthermore, vimentin contributes to stem-like cell properties in MDA-MB-231 breast cancer cells, wherein vimentin depletion reduces tumoursphere formation and attenuates expression of breast cancer stem cell-associated surface markers. Together, our findings identify vimentin as a positive regulator of stemness in the developing mouse mammary gland and in breast cancer cells.