Brain endothelial cells promote breast cancer cell extravasation to the brain via EGFR-DOCK4-RAC1 signalling

Galloni, Chiara; Egnuni, Teklu; Zahed Mohajerani, Safoura; Ye, Jiaqi; Mittnacht, Sibylle; Speirs, Valerie; Lorger, Mihaela; Mavria, Georgia

doi:10.1038/s42003-024-06200-x

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Published: 18 May 2024

Brain endothelial cells promote breast cancer cell extravasation to the brain via EGFR-DOCK4-RAC1 signalling

Communications Biology volume 7, Article number: 602 (2024) Cite this article

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Abstract

The role of endothelial cells in promoting cancer cell extravasation to the brain during the interaction of cancer cells with the vasculature is not well characterised. We show that brain endothelial cells activate EGFR signalling in triple-negative breast cancer cells with propensity to metastasise to the brain. This activation is dependent on soluble factors secreted by brain endothelial cells, and occurs via the RAC1 GEF DOCK4, which is required for breast cancer cell extravasation to the brain in vivo. Knockdown of DOCK4 inhibits breast cancer cell entrance to the brain without affecting cancer cell survival or growth. Defective extravasation is associated with loss of elongated morphology preceding intercalation into brain endothelium. We also show that brain endothelial cells promote paracrine stimulation of mesenchymal-like morphology of breast cancer cells via DOCK4, DOCK9, RAC1 and CDC42. This stimulation is accompanied by EGFR activation necessary for brain metastatic breast cancer cell elongation which can be reversed by the EGFR inhibitor Afatinib. Our findings suggest that brain endothelial cells promote metastasis through activation of cell signalling that renders breast cancer cells competent for extravasation. This represents a paradigm of brain endothelial cells influencing the signalling and metastatic competency of breast cancer cells.

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Introduction

Cancer metastasis is the leading cause of cancer-related deaths with brain metastases being particularly devastating as they result in extreme morbidity and mortality^1,2. Breast cancer is commonly associated with brain metastases, particularly in the case of triple-negative breast cancer (TNBC) defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2). Brain metastasis occurs in approximately one-third of TNBC patients³. Following haematogenous dissemination during the metastatic process, circulating breast cancer cells must arrest within the luminal space of brain capillaries, and then extravasate across the endothelium into the brain parenchyma^4,5. This process is enabled by the interaction of cancer cells with the brain endothelium, which is partially characterised. Some molecules that promote breast cancer cell adhesion to the endothelium, and mediate the passage to the brain parenchyma have been identified including ST6GALNAC5, COX2, MMP1, HBEGF and VLA-4 (α4β1)^6,7,8,9. The interaction results in changes in cancer cell morphology, disassembly of endothelial junctions, and alterations within the brain parenchyma that collectively promote successful extravasation^4,5,10.

Breast cancer often exhibits Epidermal Growth Factor Receptor (EGFR) overexpression, and studies have reported that TNBC patients also exhibit EGFR overexpression with a prevalence rate that varies depending on the patient cohort^11,12. EGFR overexpression has been associated with poor prognosis, and an increased rate of metastasis in TNBC patients and has been proposed as a potential therapeutic _target, with some patients demonstrating a response to EGFR inhibitors in clinical trials¹². EGFR signalling is known to activate multiple downstream signalling pathways involved in cancer cell survival, proliferation and migration¹³. Furthermore, during brain metastasis EGFR plays a crucial role in the crossing of the brain endothelium by breast cancer cells, and in promoting their survival and growth in the brain^6,14. However, it is unclear whether the interaction of breast cancer cells with endothelial cells influences EGFR activation, and which downstream signalling molecules are involved. The Rho family GTPases play a critical role in cancer progression and metastasis, partly due to their ability to regulate the actin cytoskeleton of metastasising cancer cells, thus influencing their morphology, polarity and migratory potential^15,16. Their activation downstream of growth factor signalling is largely driven by guanine nucleotide exchange factors (GEFs) responsible for facilitating the exchange of GDP for GTP^15,16. In addition to regulating cancer cell invasion, Rho proteins are also involved in the disruption of the endothelial barrier function, promoting extravasation of cancer cells into the underlying tissue¹⁷.

In this study, we demonstrate that the Dedicator of Cytokinesis 4 (DOCK4), a GEF implicated in RAC1 activation downstream of EGFR signalling¹⁸, is necessary for induction of a cancer cell mesenchymal-like morphology compatible with intercalation of breast cancer cancer cells into brain endothelium in vitro, and brain metastatic extravasation in vivo. We show that the pathway is activated by factors secreted by brain endothelial cells (BEC) in the absence of direct BEC-cancer cell interaction. BEC-conditioned media promote EGFR activation, breast cancer cell elongated morphology and filopodial protrusions; and knockdown of either DOCK4 or RAC1 reverses cancer cell elongation without affecting filopodia. Elongated morphology also requires the GEF Dedicator of Cytokinesis 9 (DOCK9) and Cell Division Control protein 42 (CDC42). Hence, BEC promote metastatic extravasation through activation of EGFR signalling in breast cancer cells that requires DOCK4, DOCK9, RAC1 and CDC42, rendering them competent for extravasation. Understanding the mechanisms of brain extravasation has the potential to identify strategies to prevent the process of metastasis of breast cancer cells to the brain.

Results

DOCK4 is required for breast cancer cell extravasation to the brain in vivo

Previous studies have shown an association of DOCK4 and the metastatic phenotype of cancer cells^19,20. In this study, we investigated the role of DOCK4 in the process of breast cancer cell extravasation and the growth of brain metastases. We first showed that stable knockdown of DOCK4 did not affect the in vitro proliferation of brain homing breast cancer cells²¹ (MDA-MB-231/Brain) (Fig. 1a–c and Supplementary Movie 1). We then implanted these cells intracranially in mice and assessed tumour growth using bioluminescence imaging (IVIS) over a 20-day period (Fig. 1d). Analysis revealed that DOCK4 does not play a role in the growth of breast cancer cells in the brain, as cells lacking DOCK4 are as efficient as control cells in tumour growth in the brain parenchyma (Fig. 1e). Next, we investigated whether DOCK4 is important for extravasation by injecting DOCK4 depleted cells into the internal carotid artery of mice and determining the percentage of breast cancer cells that localised inside or outside the brain blood vessels 5-days post intracarotid injection (Fig. 1f). We found that 5 days post-injection, the majority of DOCK4 depleted cells (>60%) were still trapped inside the mouse brain capillaries, whereas in comparison, approximately 70% of control cells had already extravasated (Fig. 1g, h). These data suggest that while DOCK4 does not play a role in the growth of breast tumours colonising the brain, it is required for effective extravasation of cancer cells cells from the bloodstream into the brain parenchyma.

**Fig. 1: DOCK4 knockdown inhibits breast cancer cell extravasation to the brain.**

EGF and brain endothelial cell-secreted factors promote cancer cell elongation and endothelial intercalation through DOCK4 and RAC1

One key step of the extravasation process is represented by the crossing of the endothelial layer termed transendothelial migration (TEM)²². Prior to this, cancer cells adhere and insert into the endothelial monolayer via a process termed intercalation^22,23. We investigated the ability of breast cancer cells to both adhere to or intercalate through confluent brain endothelial cells (BEC) (hCMEC/D3)²⁴ (Fig. 2a, b). In vitro intercalation assays revealed that DOCK4 depleted breast cancer cells display a significant delay in intercalation compared to control cells (Fig. 2c, d and Supplementary Movie 2); whereas adhesion assays performed in parallel showed that cancer cells lacking DOCK4 are as efficient as control cells in adhering to BEC (Fig. 2e). Hence, we conclude that DOCK4 regulates the extravasation of breast cancer cells into the brain via controlling the stage of intercalation. As DOCK4 is a RAC1 GEF²⁵, and RAC1 has been implicated in TEM²⁶, we investigated the role of RAC1 in the adhesion and intercalation processes of breast cancer cells. RAC1-depleted cells (Fig. 2f) were seeded onto confluent BEC, and adhesion and intercalation assays were performed. The experiments showed that RAC1 is required for intercalation but not for adhesion of breast cancer cells onto BEC (Fig. 2g, h).

**Fig. 2: Knockdown of DOCK4 or RAC1 blocks breast cancer cell intercalation into brain endothelial cells.**

We investigated the cellular mechanism by which DOCK4 mediates breast cancer cell intercalation. Fröse and co-workers previously demonstrated that a mesenchymal-like elongated morphology facilitates cancer cell extravasation during breast cancer metastasis to the lungs²⁷. Thus, we explored the potential role of a mesenchymal-like elongated morphology in brain extravasation. To measure the elongation of breast cancer cells seeded onto confluent BEC, we calculated the aspect ratio, which is the ratio between the cell length and width. Our results showed that control cells adopt an elongated morphology prior to intercalation (Fig. 2b), while DOCK4-depleted cells were significantly less elongated compared to controls 240 min post-seeding onto the BEC monolayer (Fig. 3a). Next, we investigated whether BEC promote elongation via activation of a signalling cascade that involves DOCK4. Breast cancer cells stimulated with conditioned media harvested from BEC (hCMEC/D3) showed an increased aspect ratio compared to cells stimulated with endothelial cell media (EC M) and this phenotype was blocked by DOCK4 depletion (Fig. 3b, c). Furthermore, breast cancer cells did not elongate in response to conditioned media harvested from non-brain endothelial cells (HUVEC) (Fig. 3b, c). These data demonstrate that BEC promote elongation of breast cancer cells via DOCK4, and that activation does not require cell-cell contact but is mediated via secreted factors.

**Fig. 3: DOCK4 and RAC1 control breast cancer cell elongation driven by brain endothelial cells and EGF.**

We explored whether endothelial-derived factors such as EGF and TGFβ (Transforming growth factor β) promote the elongation of triple-negative breast cancer cells (Fig. 3d–f). Previous studies have shown that both EGF and TGFβ can be secreted by endothelial cells and induce the acquisition of a mesenchymal-like phenotype in breast cancer cells^28,29. Our results showed that upon EGF stimulation, DOCK4-depleted cells do not elongate to the same extent as control cells (Fig. 3d, e). Additionally, breast cancer cells do not elongate in response to TGFβ stimulation (Fig. 3f), suggesting that EGF, but not TGFβ, stimulates the acquisition of an elongated phenotype via DOCK4. As previous studies have shown that increased filopodia number and length upon EGF and TGFβ stimulation promote invasiveness of cancer cells^30,31 and DOCK4/RAC1 control lateral filopodia formation²⁵, we quantified the number of filopodia on breast cancer cells cells upon EGF and TGFβ stimulation and their potential dependence on DOCK4/RAC1 (Fig. 3d, g, h). Our results showed that control and DOCK4-depleted cancer cells almost double their filopodia in response to EGF treatment (Fig. 3d, g), whereas upon TGFβ stimulation, the number of filopodia remained unchanged in both control and DOCK4-depleted cells (Fig. 3h). These data indicate that EGF stimulates filopodia formation in via a signalling cascade that does not require DOCK4. We next investigated if RAC1 is essential for breast cancer cell elongation. Our results showed that cells lacking RAC1 do not elongate as control cells in response to EGF (Fig. 3i, j). Quantification of filopodia showed that upon EGF stimulation, both control and RAC1 depleted cells almost double their filopodia (Fig. 3k). These experiments suggest that DOCK4 and RAC1 promote the intercalation of breast cancer cells into the brain endothelium via regulating cell elongation but not filopodial protrusions in response to endothelial-derived growth factors, such as EGF. Taken together, our data, along with previous findings showing the activation of RAC1 downstream of EGFR via DOCK4¹⁸ suggest that BEC-secreted factors, including EGF, activate EGFR and RAC1 via DOCK4 to promote an elongated phenotype and extravasation competency of tripe-negative breast cancer cells.

Brain endothelial cell-secreted factors activate EGFR to promote breast cancer cell elongation

We examined whether BEC-secreted factors promote EGFR activation and whether there were differences between parental and brain homing MB-MDA-231 cancer cells that may account for the ability of MB-MDA-231/Brain cells to respond to BEC-secreted factors via cellular elongation (Fig. 4a, b). Previous studies have shown that EGFR is necessary for the crossing of an endothelial barrier by brain-homing breast cancer cells in vitro and for brain metastasis in vivo⁶, and elevated EGFR levels have been reported in triple-negative breast cancer cells with propensity to metastasise^11,13. Our results showed that stimulation of breast cancer cells with BEC (hCMEC/D3)-conditioned media but not control media (MEM or EC M), stimulated EGFR phosphorylation to levels similar to those of EGF stimulation (Fig. 4a). Furthermore, there was increased EGFR prosphorylation in MDA-MB-231/Brain compared to parental MDA-MB-231 cells, which correlated with increased levels of total EGFR (Fig. 4a). To further investigate the role of EGFR in the response of breast cancer cells to BEC-secreted factors, we treated the cells with Afatinib, an inhibitor of the ErbB family kinases, including EGFR³². First we confirmed that Afatinib treatment blocks EGFR activation in MDA-MB-231/Brain cells (Fig. 4c). Our results showed that treatment with Afatinib, but not with the unrelated inhibitors SD208 (an inhibitor of the TGFβ receptor ALK5) or Y27632 (a Rho-kinase inhibitor), reversed the stimulation of elongated morphology by BEC-secreted factors and EGF (Fig. 4d–f). These data support the notion that BEC-secreted factors activate EGFR, which then promotes a DOCK4-dependent elongated morphology compatible with intercalation and metastatic brain extravasation.

**Fig. 4: EGFR is activated by brain endothelial cell factors and is necessary for brain metastatic breast cancer cell elongation.**

DOCK9 and CDC42 control breast cancer cell elongation and endothelial intercalation

Small interfering RNA screens _targeting Rho GTPases in human prostate cancer cells identified CDC42 as a critical regulator of cancer cell-endothelial cell interaction and intercalation³³. In addition, we have previously shown that DOCK4 can interact with DOCK9, a CDC42 GEF, and this signalling cascade controls lateral filopodia and tubule formation in ECs²⁵. Hence, we investigated the role of DOCK9 and CDC42 in elongation and intercalation of breast cancer cells (Fig. 5). Elongation assays showed that, like DOCK4-depleted cells (Fig. 3d, e), cells lacking either DOCK9 or CDC42 did not elongate upon EGF stimulation as control cells (Fig. 5a–d), suggesting that DOCK9 and CDC42, together with DOCK4 and RAC1, are essential for the elongation and subsequent intercalation of breast cancer cells. In addition, as previously observed for DOCK4 and RAC1 (Fig. 3g, k), DOCK9 and CDC42 are not required for filopodia formation in response to EGF stimulation, as both control and DOCK9 or CDC42 depleted cells doubled the number of filopodia upon EGF treatment (Fig. 5e). Intercalation and adhesion assays showed that breast cancer cells depleted of DOCK9 adhere to BEC as efficiently as control cells; however, they show a significant delay in intercalation compared to control cells at the time point where ≥ 50% control cells have intercalated (T₅₀) (Fig. 5f–h). Similar adhesion and intercalation phenotypes were observed upon CDC42 knockdown (Fig. 5i–k). Although cells with CDC42 knockdown adhere to the brain endothelium as efficiently as control cells, they intercalate much slower (Fig. 5i–k). These data suggest that DOCK9 and CDC42 play an essential role in promoting the intercalation of breast cancer cells into brain endothelial cells. Altogether, our data suggest that the brain endothelium and EGF contribute to extravasation by promoting DOCK4/RAC1- and DOCK9/CDC42-mediated elongation and intercalation of breast cancer cells.

**Fig. 5: DOCK9 and CDC42 control breast cancer elongation and intercalation into brain endothelial cell monolayers.**

Discussion

In this study we demonstrate how factors secreted by brain endothelial cells (BEC) activate EGFR signalling via DOCK4 to promote an elongated phenotype in breast cancer cells, thereby facilitating their successful extravasation to the brain. The activation of RAC1 downstream of EGFR also supports this conclusion¹⁸. In line with this, using xenograft mouse models, we show that DOCK4 plays a key role in promoting the extravasation of cancer cells through the brain capillaries in vivo, while it does not affect tumour growth and progression in the brain. Consistent with these findings, previous studies have shown that EGFR ligands, such as EREG and HBEGF, stimulate breast cancer cell transendothelial migration, while EGFR inhibition promotes survival following introduction of brain homing breast cancer cells into the circulation in vivo⁶. The results of our study offer insight into how endothelial cells can influence the metastatic capability of cancer cells. By stimulating breast cancer cells with growth factors released by endothelial cells, such as EGF or TGFβ, we found that EGF, but not TGFβ, can lead to breast cancer cell elongation compatible with extravasation. These results were surprising because both growth factors have previously been shown to promote elongation and invasion of breast cancer cells in vivo and in vitro in spheroid and matrigel invasion assays^34,35. However, in those studies, the elongated morphology was additionally driven by Ras³⁵, or EGF drove downstream TGFβ signalling in MCF-7 cells³⁴. Thus, while TGFβ is a mediator of epithelial-mesenchymal transition (EMT) conferring cancer cell plasticity, it does not drive the elongated, invasive morphology in triple-negative breast cancer cells.

During metastatic extravasation cancer cells undergo alterations of their actin cytoskeleton, allowing them to squeeze through openings of the endothelial barrier¹⁰. Our study shows that adoption of elongated morphology, driven by endothelial factors, is tightly associated with the process of extravasation. This is supported by previous studies investigating morphological changes in cancer cells during the metastatic process. Kienast and co-workers used intracranial windows and two-photon microscopy to follow metastasis and observed cancer cells adopting an elongated shape along blood vessel lumens⁴, a step followed by dynamic extensions and retractions of extravascular protrusions during the active process of extravasation⁴. In breast cancer lung metastasis, upregulation of the surface protein podocalyxin promotes transition of cancer cells from a non-polarised, rounded shape to an elongated, invasive morphology necessary for crossing microvascular networks in vitro and tissue colonisation in vivo²⁷. In brain metastasis, breast cancer cell elongation along the vessel lumens precedes the development of endothelial blebs that isolate invading cells from the circulation, facilitating extravasation³⁶.

Because of their function in regulating the cytoskeleton and actomyosin contractility several Rho proteins and their regulators have been implicated in the process of cell invasion and metastasis in vitro and in vivo, but less is known about their role during the process of cancer cell extravasation. Our study shows that factors secreted by brain endothelial cells, including EGF, promote both cell elongation and upregulation of filopodial protrusions associated with the metastatic phenotype³⁰. Blocking the elongated morphology but not filopodial protrusions by knockdown of the RAC GEF DOCK4 inhibits cancer cell intercalation in vitro, and brain extravasation in vivo. Knockdown of the DOCK4 interaction partner DOCK9, or CDC42, had similar effects in blocking elongated morphology and intercalation, while filopodial protrusions remained unaffected. This suggests that in breast cancer cells responding to factors secreted by brain endothelial cells, EGF controls filopodia via a pathway that does not involve CDC42, and filopodia may be dispensible for elongation and invasiveness during metastatic extravasation^37,38; moreover it has been demonstrated previously that filopodia formation may be independent of CDC42³⁸. Interestingly, mechanical signals conveyed by physical constraints within small capillaries can also change the morphology of cancer cells to an elongated phenotype, leading to changes in gene expression compatible with invasiveness and extravasation^39,40. Therefore, both paracrine signalling from the endothelium and mechanical deformation in response to physical constraints can alter the shape and behaviour of cancer cells during extravasation.

Survival of metastasising cancer cells in the microcirculation is influenced by their ability to withstand fluid shear stress and biomechanical constriction forces, a capability attributed to upregulation of actomyosin contractility^41,42. Interestingly, fluid forces remodel the vasculature to generate regions permissive for the arrest of cancer cells surviving in the circulation⁴³. Our study suggests that EGFR and DOCK4-mediated RAC1 activation in response to secreted brain endothelial factors oppose this contractile phenotype, allowing cancer cells to cross the endothelium and propel themselves into the brain parenchyma (Fig. 5l). In support of this notion, patient-derived cancer cells alter their phenotype while in the circulation, exhibiting a predominantly round morphology after release from the primary tumour, and mesenchymal shape after crossing the capillary bed⁴⁴. We postulate that following extravasation, cessation of EGFR-DOCK4-RAC signalling by yet unidentified factors must suppress the invasive phenotype, for initiation of micrometastases at sites of activation of the endothelium⁴⁵. Previous research has identified DOCK4 as a potential biomarker for breast cancer bone metastasis²⁰. Hence, common molecular mechanisms involving DOCK4/RAC1 may promote extravasation of breast cancer cells to the other metastatic sites. This is supported by the study of Klotz and co-workers, who utilised cell lines derived from patient circulating tumour cells and found partial sharing of mediators of metastasis to the brain and bone⁴⁶. In the case of lung metastasis in breast cancer, ezrin plays a crucial role by linking podocalyxin at the plasma membrane to the underlying actin cytoskeleton, which establishes dorsal cortical polarity of breast cancer cells enabling their transition to the mesenchymal-like, extravasation-competent shape. Ezrin is a susbtrate for EGFR and is involved in RAC1 activation through interaction with RhoGDIs (guanine nucleotide dissociation inhibitors) and RAC1 localisation to specific regions of the plasma membrane⁴⁷. Therefore, it may participate in EGFR signalling via DOCK4 during the process of brain extravasation. In future studies, a more comprehensive understading of the signalling pathway can be achieved by conducting knockdown and overexpression experiments in conjunction with GTPase activation assays²⁵. This approach will help elucidate the potential reciprocal regulation of RAC1, CDC42 and their activating GEFs in brain homing breast cancer cells. Moreover, it will be imperative to elucidate the in vivo roles of RAC1, DOCK9 and CDC42 in the process of brain metastatic extravasation.

In conclusion, inhibiting brain extravasation could potentially extend the survival of patients with metastatic disease. Our study highlights the role of endothelial cells in influencing breast cancer cells’ ability to extravasate to the brain and identifies molecules involved in this process. Studies on the pathways of metastatic extravasation have potential to lead to the development of strategies to prevent brain metastasis. It is worth noting that brain metastasis may have not occurred in many patients with metastastic disease to other organs; and cancer cells in the circulation may repopulate the brain following standard of care treatment of metastatic disease.

Methods

Cell culture and antibodies

MDA-MB-231/Parental and MDA-MB-231/Brain cells²¹ were cultured in Eagle’s minimum essential medium supplemented with L- Glutamine, vitamin mix, non essential amino acids, sodium pyruvate, penicillin-streptomycin, and 10% foetal calf serum (FCS). MDA-MB-231/Brain cells were stably transduced with lentiviral vector expressing LifeAct-GFP to allow their detection by immunofluorescence. For in vivo imaging MDA-MB-231/Brain was stably transduced with Firefly luciferase (F-luc)⁵. hCMEC/D3 cells²⁴ (a kind gift from Prof Graham Cook) and HUVEC (TCS CellWorks) were cultured in Human Large Vessel Endothelial Cell Growth Medium (TCS CellWorks). All cells were cultured at 37 °C with 5% CO₂. Antibodies were sourced as follows: Abcam, anti-GFP (ab2090, 1:1000); BD Biosciences, anti-CD31 (clone MEC 13.3, 1:50); Bethyl Laboratories, anti-DOCK4 (A302-263A, 1:1,000); Cell signalling, anti-EGFR (D38B1, 1:500), anti-pEGFR (D7A5, 1:500); Millipore, anti-RAC1 (clone 23A8, 1:1,000); Proteintech, anti-GAPDH (60004, 1:1,000), anti-DOCK9 (18987; 1:1,000); Santa Cruz, anti-CDC42 (sc-8401, 1:50); Thermo Fisher Scientific, Alexa Fluor secondary antibodies 488 and 594 (1:200).

Transfection and lentiviral transduction

MDA-MB-231/Brain cells (1.5 ×10⁵ per well) were seeded onto 6-well plates (Corning) and transfected with 10 nM siRNA using Lipofectamine 2000 RNAiMax (Invitrogen) and Opti-MEM I Reduced Serum Medium (Gibco) according to the manufacturer’s recommendations. Assays were performed 48 h after transfection. For stable knockdown MDA-MB-231/Brain cells expressing shRNAs and EGFP, or empty vector EGFP control were generated by lentiviral infection in the presence of polybrene (8 μg ml⁻¹), and EGFP expressing cells were FACS sorted and expanded for use in assays. Lentiviral vectors (pGIPZ) were obtained from Open Biosystems. The sequences of DOCK9 and CDC42 siRNAs, and DOCK4 shRNAs were as previously listed²⁵.

Western blotting

Cells were washed once in PBS before being lysed with lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1% NP-40, 10% Glycerol, 1 mM DTT and EDTA-free complete protease inhibitor). The protein samples were span down at 13,000 rpm at 4 °C prior to determine protein concentration using BSA protein assay kit (Thermo Fisher Scientific) and following the manufacturer instructions. NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) with the addition of 50 mM DTT was then added to the cell lysates and the samples were incubated at 98 °C for 1 min. For EGFR and pEGFR western blots 500 mM TCEPT was added to the lysate sample. Protein samples were run into NuPAGE Midi Protein gels (Thermo Fisher Scientific); 10% Bis-Tris gels were used for the detection of proteins up to 100 kDa and 3–8% Tris-Acetate gels were used for higher molecular weight proteins. Few µl of Precision Plus Protein Dual Colour marker (Bio-rad) were used to track electrophoresis. Samples were electrophoresed at 150 V in either 1× MES or 1× Tris Acetate (Thermo Fisher Scientific). Following transfer, membranes were blocked and probed with primary antibodies in 5% BSA TBS, 0.1% Tween 20 overnight at 4 °C before washing and incubating with horseradish peroxidase-conjugated secondary antibodies. Loading controls were run on the same gel as the proteins under investigation. The blot was either stripped and re-probed for GAPDH (DOCK4, DOCK9, RAC1, CDC42); or the membrane was cut at the expected molecular weight, and the segments were probed separately for the proteins of interest and GAPDH (pEGFR, EGFR). Protein signals were subsequently visualised using ECL Western Blotting Substrate (Thermo Fisher Scientific). The uncropped western blots are provided in Supplementary Fig. 1.

Adhesion Assay

hCMEC/D3 cells (1.5 ×10⁴ per well) were seeded onto 96-well plates (Corning) coated with 1% gelatin and incubated for 72 h until they reached confluency. MDA-MB-231/Brain cells (1.5 × 10⁴ per well) were then seeded onto the hCMEC/D3 monolayer and incubated at 37 °C for 15 min before each plate was washed with PBS to remove non adherent cells. The cultures were then fixed with 4% (w/v) paraformaldehyde (PFA; Sigma-Aldrich) for 10 min at room temperature, and washed with PBS before being imaged with the Incucyte® ZOOM Live-Cell Analysis System (Essen Bioscience) using a 20× objective. The cells adhering onto the monolayer were counted from the images. Each condition was performed in 3 independent experiments and the percentage of adherent cells was calculated.

Intercalation assay

hCMEC/D3 cells (4.5 × 10⁴ per well) were seeded onto 24 well plates (Corning) coated with 1% gelatin and incubated 72 h until they reached confluency. MDA-MB-231/Brain cells (5 × 10⁴ per well) stably expressing LifeAct-GFP were then seeded onto the hCMEC/D3 monolayer and monitored by time-lapse microscopy using the Incucyte® ZOOM Live-Cell Analysis System (Essen Bioscience) using a 20× objective. Images were taken every 30 min over a period of 5 h. Intercalated cells were quantified from the timelapse movies. A cell was considered intercalated if it was no longer round and phase bright, and it was part of the monolayer. Each condition was performed in three independent experiments and the percentage of intercalated cells was calculated.

Elongation assay and immunofluorescence

MDA-MB-231/Parental or MDA-MB-231/Brain cells (1.5 × 10⁵ per well) were seeded onto 6-well plates (Corning) coated with Poly-L-Lysine (1:40, Sigma) and serum-starved overnight. Cells were then trypsinised and resuspended in 30 ng/ml Epidermal Growth Factor (EGF) (Sigma), or 50 ng/ml Transforming Growth Factor (TGFβ) β (Peprotech), or conditioned media harvested from 50% confluent hCMEC/D3, HUVEC, or normal culture media. The cells were then seeded onto glass coverslips coated with 1% gelatin and incubated at 37 °C for 4 h. Cells were then fixed with 4% PFA for 10 min at room temperature and washed with PBS. The cells were permeabilised with 0.1% Triton-X for 5 min, and Phalloidin Alexa Fluor 594 (Invitrogen 1:500) was added for 1 hr at room temperature. After washing with PBS the coverslips were incubated with 4,6-diamidino-2-phenylindole (DAPI) (2 µg/ml) for 1 min, washed with dH₂O, and mounted onto microscope slides using Antifade Mountant (Thermo Fisher Scientific). Coverslips were visualised on a Zeiss Apotome microscope using 10×, 20× or 40× objectives. The aspect ratio (length/width) of cells was determined using Fiji.

Inhibitor treatment

MDA-MB-231/Brain cells were treated with the following inhibitors: Afatinib (5 μM), SD208 (0.1 μM or 1 μM), and Y27632 (5 μM) for 8 h prior to overnight serum starvation. MDA-MB-231/Brain cells (6 × 10³ cells per well) seeded onto glass coverslips in 12-well plates were stimulated with 30 ng/ml EGF (Sigma), or hCMEC/D3-conditioned media, or control media (MEM or E CM) in the presence or absence of inhibitors, or vehicle (DMSO). The cells were then fixed, stained, and the aspect ratio was determined using Fiji.

Confluency assay

MDA-MB-231/Brain cells were seeded at a density of 20,000 cells per cm² and imaged at 37 °C and 5% CO₂ using the Incucyte® Zoom Live-Cell Analysis System (Essen Bioscience). For confluency curve analysis, images were acquired longitudinally at set time points and processed using the Incucyte® Zoom Confluence processing tool to quantify the increase in cellular density over background in each condition.

In vivo intracranial growth and bioluminescence imaging

Six- to 8-wk-old female C57BL/6J mice were purchased from Charles River Laboratories, UK. MDA-MB-231/Brain cells (1 × 10⁵ in 2 µl MEM without supplements) were stereotactically injected into the striatum (2-mm right from the midline, 2-mm anterior from bregma, 3-mm deep). During surgery, animals were anesthesized with isofluorane. Mice were monitored by noninvasive bioluminescence imaging after luciferin injection using IVIS Spectrum and Living Image software (PerkinElmer). All animal procedures performed in the study were approved by the University of Leeds Animal Welfare and Ethical Review Committee (AWERB) and performed under an approved UK Home Office project license according to Home Office Regulations and the CCCR guidelines. We have complied with all relevant ethical regulations for animal use.

In vivo intracarotid injection and quantification of extravasation

Six- to 7-wk-old female CB17- SCID mice were purchased from Charles River Laboratories, UK. MDA-MB-231/Brain cells (2 × 10⁴ in MEM without supplements) were injected into the left internal carotid artery in MEM without supplements in a total volume of 50 μl. During surgery, mice were anesthesized with isofluorane. On Day 5 post tumour cell injection, brains were harvested after perfusing anesthetised animals with 20 ml 0.9% NaCl followed by 4% Paraformaldehyde. Isolated brain tissue fixed in 4% PFA at 4 °C overnight, then incubated in 25% sucrose and 0.1 M sodium phosphate buffer at 4 °C overnight. Brains were then snap-frozen on dry ice, cut into 30-µm floating cryosections using Thermo Scientific™ Cryotome™ FSE Cryostat (Fisher Scientific), and stored in Walter’s antifreeze (30% (v/v), ethylenglycol, 30% (v/v) glycerol, and 0.5 M phosphate buffer) at −20 °C. Floating sections were washed in PBS and blocked in blocking buffer (10% FCS, 0.3% Triton-X-100 in PBS) for 1 hr at room temperature. Primary antibodies detecting GFP and CD31 diluted in blocking buffer were added and the cryosections were incubated overnight at room temperature gently shaking. After washing in PBS, the cryosections were incubated for 2 h with Alexa Fluor secondary antibodies, DAPI (2 µg/ml) was added, and the slides were mounted using anti-fade gel mount. Images were acquired with a Zeiss ApoTome microscope using ×10 and ×20 objectives. The position of the cancer cells inside or outside the brain blood vessels was determined for individual cells and 100 events were analysed per tumour. The percentage of cells in and out of blood vessels was calculated.

Statistics and reproducibility

The data presented represent the mean of three independent experiments. Statistical significance was evaluated using an unpaired Student’s t test.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data supporting the findings of this study are available in Supplementary Data 1.

References

Berghoff, A. S. et al. Descriptive statistical analysis of a real-life cohort of 2419 patients with brain metastases of solid cancers. ESMO Open 1, e000024 (2016).
Article PubMed PubMed Central Google Scholar
Sacks, P. & Rahman, M. Epidemiology of Brain Metastases. Neurosurg. Clin. N. Am. 31, 481–488 (2020).
Article PubMed Google Scholar
Kadamkulam Syriac, A., Nandu, N. S. & Leone, J. P. Central Nervous System Metastases from Triple-Negative Breast Cancer: Current Treatments and Future Prospective. Breast Cancer 14, 1–13 (2022).
CAS PubMed PubMed Central Google Scholar
Kienast, Y. et al. Real-time imaging reveals the single steps of brain metastasis formation. Nat. Med. 16, 116–122 (2010).
Article CAS PubMed Google Scholar
Lorger, M. & Felding-Habermann, B. Capturing changes in the brain microenvironment during the initial steps of breast cancer brain metastasis. Am. J. Pathol. 176, 2958–2971 (2010).
Article PubMed PubMed Central Google Scholar
Bos, P. D. et al. Genes that mediate breast cancer metastasis to the brain. Nature 459, 1005–1009 (2009).
Article CAS PubMed PubMed Central Google Scholar
Wu, K. et al. Roles of the cyclooxygenase 2 matrix metalloproteinase 1 pathway in brain metastasis of breast cancer. J. Biol. Chem. 290, 9842–9854 (2015).
Article CAS PubMed PubMed Central Google Scholar
Soto, M. S., Serres, S., Anthony, D. C. & Sibson, N. R. Functional role of endothelial adhesion molecules in the early stages of brain metastasis. Neuro Oncol. 16, 540–551 (2014).
Article CAS PubMed Google Scholar
Fares, J., Kanojia, D., Rashidi, A., Ulasov, I. & Lesniak, M. S. Genes that Mediate Metastasis across the Blood-Brain Barrier. Trends Cancer 6, 660–676 (2020).
Article CAS PubMed PubMed Central Google Scholar
Reymond, N., d’Agua, B. B. & Ridley, A. J. Crossing the endothelial barrier during metastasis. Nat. Rev. Cancer 13, 858–870 (2013).
Article CAS PubMed Google Scholar
Reis-Filho, J. S. & Tutt, A. N. Triple negative tumours: a critical review. Histopathology 52, 108–118 (2008).
Article CAS PubMed Google Scholar
Nakai, K., Hung, M. C. & Yamaguchi, H. A perspective on anti-EGFR therapies _targeting triple-negative breast cancer. Am. J. Cancer Res. 6, 1609–1623 (2016).
CAS PubMed PubMed Central Google Scholar
Martin, V. et al. Molecular characterization of EGFR and EGFR-downstream pathways in triple-negative breast carcinomas with basal-like features. Histol. Histopathol. 27, 785–792 (2012).
CAS PubMed Google Scholar
Nie, F. et al. Involvement of epidermal growth factor receptor overexpression in the promotion of breast cancer brain metastasis. Cancer 118, 5198–5209 (2012).
Article CAS PubMed Google Scholar
Maldonado, M. D. M., Medina, J. I., Velazquez, L. & Dharmawardhane, S. _targeting Rac and Cdc42 GEFs in Metastatic Cancer. Front. Cell Dev. Biol. 8, 201 (2020).
Article PubMed PubMed Central Google Scholar
Kazanietz, M. G. & Caloca, M. J. The Rac GTPase in Cancer: From Old Concepts to New Paradigms. Cancer Res. 77, 5445–5451 (2017).
Article CAS PubMed PubMed Central Google Scholar
Rodenburg, W. S. & van Buul, J. D. Rho GTPase signaling networks in cancer cell transendothelial migration. Vasc. Biol. 3, R77–R95 (2021).
Article CAS PubMed PubMed Central Google Scholar
Hiramoto-Yamaki, N. et al. Ephexin4 and EphA2 mediate cell migration through a RhoG-dependent mechanism. J. Cell Biol. 190, 461–477 (2010).
Article CAS PubMed PubMed Central Google Scholar
Yu, J. R. et al. TGF-beta/Smad signaling through DOCK4 facilitates lung adenocarcinoma metastasis. Genes Dev. 29, 250–261 (2015).
Westbrook, J. A. et al. Identification and validation of DOCK4 as a potential biomarker for the risk of bone metastasis development in patients with early breast cancer. J. Pathol. 247, 381–391 (2019).
Article CAS PubMed PubMed Central Google Scholar
Yoneda, T., Williams, P. J., Hiraga, T., Niewolna, M. & Nishimura, R. A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in vitro. J. Bone Min. Res. 16, 1486–1495 (2001).
Article CAS Google Scholar
Reymond, N., Riou, P. & Ridley, A. J. Rho GTPases and cancer cell transendothelial migration. Methods Mol. Biol. 827, 123–142 (2012).
Article CAS PubMed Google Scholar
Wrobel, J. K. & Toborek, M. Blood-brain Barrier Remodeling during Brain Metastasis Formation. Mol. Med. 22, 32–40 (2016).
Article CAS PubMed PubMed Central Google Scholar
Weksler, B., Romero, I. A. & Couraud, P. O. The hCMEC/D3 cell line as a model of the human blood-brain barrier. Fluids Barriers CNS 10, 16 (2013).
Article PubMed PubMed Central Google Scholar
Abraham, S. et al. A Rac/Cdc42 exchange factor complex promotes the formation of lateral filopodia and blood vessel lumen morphogenesis. Nat. Commun. 6, 7286 (2015).
Article PubMed Google Scholar
van Golen, K. L. et al. CCL2 induces prostate cancer transendothelial cell migration via activation of the small GTPase Rac. J. Cell Biochem. 104, 1587–1597 (2008).
Article PubMed Google Scholar
Frose, J. et al. Epithelial-Mesenchymal Transition Induces Podocalyxin to Promote Extravasation via Ezrin Signaling. Cell Rep. 24, 962–972 (2018).
Article CAS PubMed PubMed Central Google Scholar
Wendt, M. K., Smith, J. A. & Schiemann, W. P. Transforming growth factor-beta-induced epithelial-mesenchymal transition facilitates epidermal growth factor-dependent breast cancer progression. Oncogene 29, 6485–6498 (2010).
Article CAS PubMed PubMed Central Google Scholar
Hao, Y., Baker, D. & Ten Dijke, P. TGF-beta-Mediated Epithelial-Mesenchymal Transition and Cancer Metastasis. Int J. Mol. Sci. 20, 2767 (2019).
Article CAS PubMed PubMed Central Google Scholar
Jacquemet, G., Hamidi, H. & Ivaska, J. Filopodia in cell adhesion, 3D migration and cancer cell invasion. Curr. Opin. Cell Biol. 36, 23–31 (2015).
Article CAS PubMed Google Scholar
Yasui, H., Ohnishi, Y., Nakajima, M. & Nozaki, M. Migration of oral squamous cell carcinoma cells are induced by HGF/c-Met signalling via lamellipodia and filopodia formation. Oncol. Rep. 37, 3674–3680 (2017).
Article CAS PubMed Google Scholar
Roskoski, R. Jr. Small molecule inhibitors _targeting the EGFR/ErbB family of protein-tyrosine kinases in human cancers. Pharm. Res. 139, 395–411 (2019).
Article CAS Google Scholar
Reymond, N. et al. Cdc42 promotes transendothelial migration of cancer cells through beta1 integrin. J. Cell Biol. 199, 653–668 (2012).
Article CAS PubMed PubMed Central Google Scholar
Kim, J., Kong, J., Chang, H., Kim, H. & Kim, A. EGF induces epithelial-mesenchymal transition through phospho-Smad2/3-Snail signaling pathway in breast cancer cells. Onco_target 7, 85021–85032 (2016).
Article PubMed PubMed Central Google Scholar
Pang, M. F. et al. TGF-beta1-induced EMT promotes _targeted migration of breast cancer cells through the lymphatic system by the activation of CCR7/CCL21-mediated chemotaxis. Oncogene 35, 748–760 (2016).
Article CAS PubMed Google Scholar
Hasko, J. et al. Response of the neurovascular unit to brain metastatic breast cancer cells. Acta Neuropathol. Commun. 7, 133 (2019).
Article PubMed PubMed Central Google Scholar
Zhang, Y. et al. Focus on Cdc42 in Breast Cancer: New Insights, _target Therapy Development and Non-Coding RNAs. Cells 8, 146 (2019).
Article CAS PubMed PubMed Central Google Scholar
Sigal, Y. J., Quintero, O. A., Cheney, R. E. & Morris, A. J. Cdc42 and ARP2/3-independent regulation of filopodia by an integral membrane lipid-phosphatase-related protein. J. Cell Sci. 120, 340–352 (2007).
Article CAS PubMed Google Scholar
Varotsos Vrynas, A., Perea Paizal, J., Bakal, C. & Au, S. H. Arresting metastasis within the microcirculation. Clin. Exp. Metastasis 38, 337–342 (2021).
Article PubMed PubMed Central Google Scholar
Perea Paizal, J., Au, S. H. & Bakal, C. Squeezing through the microcirculation: survival adaptations of circulating tumour cells to seed metastasis. Br. J. Cancer 124, 58–65 (2021).
Article PubMed Google Scholar
Huang, X. et al. RhoA-stimulated intra-capillary morphology switch facilitates the arrest of individual circulating tumor cells. Int J. Cancer 142, 2094–2105 (2018).
Article CAS PubMed Google Scholar
Moose, D. L. et al. Cancer Cells Resist Mechanical Destruction in Circulation via RhoA/Actomyosin-Dependent Mechano-Adaptation. Cell Rep. 30, 3864–3874.e3866 (2020).
Article CAS PubMed PubMed Central Google Scholar
Follain, G. et al. Impairing flow-mediated endothelial remodeling reduces extravasation of tumor cells. Sci. Rep. 11, 13144 (2021).
Article CAS PubMed PubMed Central Google Scholar
Sun, Y. F. et al. Circulating Tumor Cells from Different Vascular Sites Exhibit Spatial Heterogeneity in Epithelial and Mesenchymal Composition and Distinct Clinical Significance in Hepatocellular Carcinoma. Clin. Cancer Res. 24, 547–559 (2018).
Article CAS PubMed Google Scholar
Ghajar, C. M. et al. The perivascular niche regulates breast tumour dormancy. Nat. Cell Biol. 15, 807–817 (2013).
Article CAS PubMed PubMed Central Google Scholar
Klotz, R. et al. Circulating Tumor Cells Exhibit Metastatic Tropism and Reveal Brain Metastasis Drivers. Cancer Discov. 10, 86–103 (2020).
Article CAS PubMed Google Scholar
Bretscher, A. Regulation of cortical structure by the ezrin-radixin-moesin protein family. Curr. Opin. Cell Biol. 11, 109–116 (1999).
Article CAS PubMed Google Scholar

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Acknowledgements

We gratefully acknowledge the support of project grant PR748 from Breast Cancer Now, awarded to G.M., V.S. and M.L. We are also thankful to the staff at St James’ Biological Services for their assistance with animal husbandry and in vivo work, as well as Adam Davison and Elizabeth Straszynski for FACS sorting. We thank Professor Francesco Del Galdo for generously providing the inhibitor SB208. We are grateful to Professor Ingunn Holen for her valuable comments on the manuscript. Finally, we acknowledge the support of the British Heart Foundation, which provided funding for S.Z.M.

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Chiara Galloni
Present address: Sheffield Institute for Nucleic Acids (SInFoNiA) and School of Biosciences, University of Sheffield, Sheffield, UK
Safoura Zahed Mohajerani
Present address: Leeds Centre for Disease Models, University of Leeds, Leeds, UK

Authors and Affiliations

Leeds Institute of Medical Research, University of Leeds, Leeds, UK
Chiara Galloni, Teklu Egnuni, Safoura Zahed Mohajerani, Jiaqi Ye, Mihaela Lorger & Georgia Mavria
UCL Cancer Institute, University College London, London, UK
Sibylle Mittnacht
Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK
Valerie Speirs

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Chiara Galloni
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Teklu Egnuni
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Safoura Zahed Mohajerani
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Jiaqi Ye
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Sibylle Mittnacht
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Valerie Speirs
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Mihaela Lorger
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Georgia Mavria
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Contributions

The experimental design was conceived by C.G., G.M., V.S. and M.L., S.M. contributed to the proposal and provided insights on the EGFR inhibition. T.E. carried out and analysed the in vivo intracranial growth experiment, and provided support for the in vivo carotid artery injection experiments conducted by M.L., S.Z.M. and J.Y. conducted and analysed the experiments with Afatinib, while C.G. executed and analysed all other experiments. C.G. and G.M. wrote the manuscript with input from all authors. G.M. provided project supervision and administration.

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Correspondence to Georgia Mavria.

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Communications Biology thanks Hironori Katoh and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Joao Valente.

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Galloni, C., Egnuni, T., Zahed Mohajerani, S. et al. Brain endothelial cells promote breast cancer cell extravasation to the brain via EGFR-DOCK4-RAC1 signalling. Commun Biol 7, 602 (2024). https://doi.org/10.1038/s42003-024-06200-x

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Received: 26 October 2023
Accepted: 15 April 2024
Published: 18 May 2024
DOI: https://doi.org/10.1038/s42003-024-06200-x

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Abstract

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Introduction

Results

DOCK4 is required for breast cancer cell extravasation to the brain in vivo

EGF and brain endothelial cell-secreted factors promote cancer cell elongation and endothelial intercalation through DOCK4 and RAC1

Brain endothelial cell-secreted factors activate EGFR to promote breast cancer cell elongation

DOCK9 and CDC42 control breast cancer cell elongation and endothelial intercalation

Discussion

Methods

Cell culture and antibodies

Transfection and lentiviral transduction

Western blotting

Adhesion Assay

Intercalation assay

Elongation assay and immunofluorescence

Inhibitor treatment

Confluency assay

In vivo intracranial growth and bioluminescence imaging

In vivo intracarotid injection and quantification of extravasation

Statistics and reproducibility

Reporting summary

Data availability

References

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Supplementary information

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