# Differential modulation of endothelial cell functionality by LRP1 expression in fibroblasts and cancer-associated fibroblasts via paracrine signals and matrix remodeling

Julie Martin, Auréana Falaise, Sara Faour, Christine Terryn, Cathy Hachet, Émilie Thiébault, Louise Huber, Pierre Nizet, Damien Rioult, Rodolphe Jaffiol, et al.

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## ► To cite this version:

Julie Martin, Auréana Falaise, Sara Faour, Christine Terryn, Cathy Hachet, et al. Differential modulation of endothelial cell functionality by LRP1 expression in fibroblasts and cancer-associated fibroblasts via paracrine signals and matrix remodeling. Matrix Biology, 2025, 139, pp.61-76. 10.1016/j.matbio.2025.05.004. hal-05108300

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Submitted on 21 Jul 2025

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# Differential modulation of endothelial cell functionality by LRP1 expression in fibroblasts and cancer-associated fibroblasts via paracrine signals and matrix remodeling

Julie Martin<sup>a,b,1</sup>, Auréana Falaise<sup>a,b</sup>, Sara Faour<sup>a,b,c</sup>, Christine Terryn<sup>d ID</sup>, Cathy Hachet<sup>a,b ID</sup>, Émilie Thiébault<sup>a,b ID</sup>, Louise Huber<sup>a,b ID</sup>, Pierre Nizet<sup>a,b ID</sup>, Damien Rioult<sup>e ID</sup>, Rodolphe Jaffiol<sup>c</sup>, Stéphanie Salesse<sup>a,b,1,* ID</sup>, Stéphane Dedieu<sup>a,b,1,* ID</sup>, Benoit Langlois<sup>a,b,1,* ID</sup>

## ARTICLE INFO

**Keywords:**

LRP-1

Extracellular matrix

Angiogenesis

Cancer-associated fibroblast

Proteomics

## ABSTRACT

LRP1 is a multifunctional endocytosis receptor involved in the regulation of cancer cell aggressiveness, fibroblast phenotype and angiogenesis. In breast cancer microenvironment, cancer-associated fibroblasts (CAFs) play a crucial role in matrix remodeling and tumor niche composition. LRP1 expression was described in fibroblasts and CAFs but remains poorly understood regarding its impact on endothelial cell behavior and angiocrine signaling. We analyzed the angio-modulatory effect of LRP1 expression in murine embryonic fibroblasts (MEFs) and breast cancer-educated CAF₂ cells. We employed conditioned media and fibroblast-derived matrices to model fibroblastic cells angiogenic effects on human umbilical vein endothelial cells (HUVEC). Neither the extracellular matrix assembled by MEFs knock-out for LRP1 (PEA-13) nor their secretome modify the migration of HUVEC as compared to wild-type. Conversely, LRP1-deficient CAF₂ secretome and matrices stimulate endothelial cell migration. Using spheroids, we demonstrate that PEA-13 secretome does not affect HUVEC angio-invasion. By contrast, CAF₂ secretome invalidated for LRP1 stimulates endothelial sprouting as compared to controls. In addition, it specifically stabilized peripheral VE-cadherin-mediated endothelial cell junctions. A global proteomic analysis revealed that LRP1 expression in CAFs orchestrates a specific mobilization of secreted matricial components, surface receptors and membrane-associated proteins at the endothelial cell surface, thereby illustrating the deep influence exerted by LRP1 in angiogenic signals emitted by activated fibroblasts.

**Abbreviations:** ANX6, annexin VI; CAF, cancer-associated fibroblast; CCR5, C-C chemokine receptor type 5; CKAP4, cytoskeleton-associated protein 4; CLC14/ CLEC14A, C-type lectin domain family 14 member A; DESP, desmoplakin; DSG, desmoglein 1; ECM, extracellular matrix; EGF, epidermal growth factor; FAP, fibroblast activation protein; GO, gene ontology; HIF-1, hypoxia-inducible factor-1; HRNR, hornerin; HUVEC, human umbilical endothelial cell; IGF-1, insulin-like growth factor-1; ITB1, integrin β1; KEGG, Kyoto encyclopedia of genes and genomes; LRP-1, low-density lipoprotein receptor-related protein-1; LEG1, galectin 1; MEF, murine embryonic fibroblast; MOES, moesin; MUC18, mucin 18; NRP1, neuropilin 1; PDGFRa, platelet-derived growth factor alpha; PLAK, junction plakoglobin; PTPRF, receptor-type tyrosine-protein phosphatase F; RAP, receptor-associated protein; RHOG, rho-related GTP-binding protein rhoG; RRAS, ras-related protein r-ras; SEM, scanning electron microscopy; aSMA, alpha smooth muscle actin; TGFβ, transforming growth factor beta; TGM1, transglutaminase 1; TME, tumor microenvironment; TSP1, thrombospondin-1; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; VIM, vimentin.

* Corresponding authors at: UFR Sciences Exactes et Naturelles, Université de Reims Champagne-Ardenne, Reims, France.

E-mail addresses: stephanie.salesse@univ-reims.fr (S. Salesse), stephane.dedieu@univ-reims.fr (S. Dedieu), benoit.langlois@univ-reims.fr (B. Langlois).

¹ These authors contributed equally to this work.

<footnote><sup>a</sup> UFR Sciences Exactes et Naturelles, Université de Reims Champagne-Ardenne, Reims, France</footnote>

<footnote><sup>b</sup> Matrice Extracellulaire et Dynamique Cellulaire, MEDI.C, UMR 7369 CNRS, Reims, France</footnote>

<footnote><sup>c</sup> Light, Nanomaterials, Nanotechnologies, ERL CNRS 7004, Université de Technologie de Troyes, Troyes, France</footnote>

<footnote><sup>d</sup> Plate-Forme Imagerie Cellulaire et Tissulaire (PICT), Université de Reims Champagne-Ardenne, UFR Médecine, Reims, France</footnote>

<footnote><sup>e</sup> Plateau Technique Mobile de Cytométrie Environnementale MOBICYTE, Université de Reims Champagne-Ardenne/INERIS, Reims, France</footnote>
## Introduction

Angiogenesis, consisting in new blood vessel formation from an existing vascular network, is critically mobilized to provide oxygen and nutrients during body development and in various pathological contexts [1,2]. To elaborate new functional vessels, existing endothelial vessels perform multiple adaptations to degrade extracellular matrix (ECM), modify their intercellular connections, differentiate, proliferate and migrate in vessel sprouts. Finally, branching structures coopt each other to form lumenized structures, stabilized by peripheral mural cells and extracellular matrix and become fully perfused to generate normoxic conditions [1,3]. In the frame of cancer development, angiogenesis is a limiting step intimately associated with metastatic dissemination [4–7]. At early stages, uncontrolled tumor growth leads to intense remodeling of the tumor microenvironment (TME), decrease blood supply and generate unresolved hypoxic gradients. These profound reorganizations of the interactions between cancer cells, extracellular matrix and stromal cells induce a biological imbalance that favors the formation of new blood vessels during an angiogenic switch [2,6]. The resulting vascular network is poorly functional leaky and fenestrated, thereby facilitating cancer cell entry into lymph or blood circulation. Moreover, the low perfusion of tumor vessels negatively impacts anticancer drugs delivery, which represents a considerable clinical challenge [5,7–10]. Most of the pharmacological strategies rely on targeting VEGFA by bevacizumab, or its receptor VEGFR2, or on the administration of broader spectra tyrosine kinase inhibitors [10]. Although, a substantial proportion of clinical trials targeting VEGF signaling revealed limited efficacy or even cancer relapse in more aggressive format [4,5,8–10]. Escape mechanisms include vascular rebound after treatment withdrawal [9], endothelial-to-mesenchymal-transition [8] and increased metastatic incidence [7]. Due to acquired plasticity, endothelial cells can perform metabolic adaptation under the influence of microenvironmental cues and paracrine signals emitted by different cellular compartments of the tumor bed [1]. Cancer-associated fibroblasts (CAFs) are crucial actors in that context [6,11–14]. They form a major cell type responsible for ECM deposition and remodeling in the TME [15,16]. Moreover, they secrete various cytokines and growth factors that support tumor progression [6, 12], influencing critical functions of endothelial cell in the TME such as branching angioinvasion, secretion of growth factors and ECM components facilitating angiogenic switch completion and angiogenesis-dependent tumor growth [11,13,14]. A more comprehensive understanding of the dialogue between CAFs and endothelial cells could provide valuable insights to develop combinatorial therapies, thereby circumventing alternative TME-induced proangiogenic pathways.

The low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) is a large multifunctional receptor, member of the LDL-receptor family [17]. It is composed of a 515 kDa extracellular α-chain involved in the specific recognition of extracellular ligands, uncovalently associated to an 85 kDa β-chain containing a transmembrane domain and a short intracytoplasmic tail responsible for the recruitment of intracellular scaffolds. This provides a dual function in fast substrate endocytosis and/or ligand signal transduction [17,18]. LRP1 was involved in high-affinity binding of over 30 extracellular secreted ligands and membrane-integrated receptors. Through its interaction with ECM compounds, proteases and their inhibitors, growth factors, and cell surface receptors, LRP1 is implicated in the adaptation to diverse microenvironmental changes of various cell types [18–22]. This is especially true in the TME where LRP1 was initially proposed as a tumor suppressor due to its ability to reduce the excess of proinvasive extracellular matrix proteases [18,20]. However, this simple view was mitigated by the discovery of its contribution to signals favoring tumor growth and metastatic dissemination [20,23–26]. For instance, LRP1 supports tumor cells invasion by controlling the cell-surface expression and dynamics of adhesion receptors such as CD44 [26] and β1 integrins through both outside-in and inside-out signals [21,27]. Those functions

in cancerology were mainly documented in tumor cell autonomous pathways [21,23,24]. LRP1 contributions to tumor progression also emerged within stromal compartments [25,28,29]. Interestingly, several angio-active molecules were identified among the diverse endocytic substrates and signaling partners of LRP1 [18,19,22,30–32]. Early lethality of its global deletion in mice was explained by defective embryonic vascular network development [33]. LRP1 also controls the internalization and endosomal traffic of VEGFR2-containing complexes in endothelial cells [32]. These studies highlighted the angio-regulatory functions of LRP1, but to date its contribution to tumor angiogenesis remains elusive and poorly studied. Our team recently showed that the depletion of LRP1 in an invasive cellular model of triple negative breast cancer impairs tumor growth and disturbs tumor vessel functionality in a grafted model of murine mammary cancer [28,34]. LRP1 was already shown to support epithelial-to-mesenchymal transition and metastatic dissemination of breast tumor cells through autocrine pathways [23,24]. However, the mechanistic contribution of LRP1 in breast TME landscaping seems to extend beyond tumor cells [35,36]. LRP1 is abundantly expressed in fibroblasts, and it regulates their contractile phenotype and activation through extracellular ligand binding and signal transduction [37–39]. It was also identified, together with ANXA6 and TSP1, as a biomarker of CAF-derived vesicles predictive of poor cancer patient outcome and increased metastasis incidence in pancreatic ductal carcinomas [25].

The angio-modulatory functions of LRP1 expressed in CAFs have not been directly addressed so far. In this study we used LRP1 knock-out fibroblasts and LRP1 knock-down breast-tumor educated-CAFs to analyze the functional consequences of their secretory and ECM remodeling activities on key endothelial cell parameters. Depletion of LRP1 in CAFs led to a complex angiogenic response towards endothelial cells which included increased random cell migration, stimulation of sprouting angioinvasion and stabilization of VE-cadherin-mediated adhesions. Finally, we applied a proteomic approach to identify cell surface proteins differentially mobilized at endothelial cell surface in response to CAF paracrine signals. This newly identified protein signature provides a comprehensive view of LRP1-dependent angio-modulation resulting from endothelial exposure to CAF-derived secretomes.

## Results

### LRP1 induces paracrine signals from fibroblasts and CAFs that sustain endothelial cell functionality

LRP1 is recognized as a ubiquitously expressed receptor identified in various cellular components within the breast TME [28]. However, the relative extent of its expression in different compartments is not consistently incorporated in functional studies. We analyzed LRP1 expression in cellular models representing the three major compartments of the breast TME critically involved in stromal reaction and tumor angiogenesis, namely tumor cells, fibroblasts, and endothelial cells (Fig. 1A). In the two fibroblast models tested, murine embryonic fibroblasts (MEF-1) and breast cancer-associated fibroblasts (CAF₂), LRP1 was strongly detected. Among the tested cellular panel, CAF₂, a model of activated fibroblasts educated by breast tumors [12], exhibited the highest protein level. A weaker expression was evidenced in macrovascular venous HUVEC cells and cerebral microvascular hCMEC/D3 cells. LRP1 protein expression appeared highly variable in the breast tumor cell lines. To investigate the impact of LRP1 on endothelial/fibroblastic crosstalk induced by LRP1, we employed MEF-1 and CAF₂ cellular models (Fig. 1B). Initially, we characterized the two fibroblast models based on their respective activation level (Fig. 1C-E). We compared the expression of four selected markers related to either cellular plasticity and contractile apparatus (vimentin, αSMA) or fibroblasts activation (FAP, PDGFRα). The protein expression ratio of vimentin, αSMA, FAP and PDGFRα measured in CAF₂ versus MEF-1 revealed a significant induction of 5.8, 2.9, 7.4, and 6-fold,
A

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C

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D

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G

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H

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B

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E

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F

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**Fig. 1.** LRP1-dependent fibroblastic paracrine signals sustain endothelial morphogenesis. LRP1 protein expression was assessed in breast cancer cell lines (MCF-7, MDA-MB-468, MDA-MB-231), MEF-1 and CAF₂ fibroblastic models, in HUVEC and hCMEC/D3 endothelial cells by western blot, using β-actin as a normalization control. A representative picture of immunoblots is presented (A). Functional scheme presenting the general purpose of the study: define the role played by LRP1 in the dialogue of fibroblasts and CAF towards endothelial cells (B). Whole extracts from MEF-1 and CAF₂ cells were analyzed by SDS-PAGE and western blot using specific antibodies against aSMA, vimentin, FAP and PDGFRα. GAPDH was used as normalization control. Pictures are representative of at least 4 experiments. Densitometric analysis of specific bands normalized to GAPDH signal was expressed as ratio of MEF-1 signals (C). MEF-1 and CAF₂ cells were cultivated 24 h on fibronectin coated coverslips, fixed and immunostained with phalloidin and an antibody against aSMA. Representative pictures of 3 independent experiments, acquired with confocal microscope, are presented (D). MEF-1 and CAF₂ were embedded in collagen I lattices and cultivated for 24 h to measure their contractile activity, expressed as a % of reduction of collagen lattices against initial area and quantified from 3 independent experiments each performed in quintuplicate (E). LRP1 protein expression was analyzed by western blot in MEF-1 and PEA-13 cells. CAF₂ cells were transfected with siRNA against LRP-1 expression (siLRP1) or non-targeting control sequences (siCTL). Whole extracts were harvested 48 h post-transfection and analyzed by western blot using LRP1 antibody. β-actin was used as a normalization control (F). HUVEC were cultivated 6 h on Matrigel in the presence MEF-1 or PEA-13 conditioned media (G) and in response to LRP1-expressing (siCTL) or -depleted (siLRP1) CAF₂ conditioned media (H). Endothelial morphogenic activity was assessed by quantifying the number of tubes, their perimeter, and their cumulative areas. Pictures are representative of at least 4 quantified experiments each performed at least in quintuplicate. Each value is mean ± sem. ns, not significant; *, *P* < 0.05; **, *P* < 0.01; ***, *P* < 0.001.

respectively. Consistently, we also observed by confocal imaging a higher expression of αSMA in elongated and spindle shaped CAF₂, mostly distributed in transverse and peripheral fibers coaligned with actin filament network (Fig. 1D). We could also observe that CAF₂ presented an increased number of activated β1-integrins positive clusters than MEF-1 when cultivated on fibronectin, which is in line with strong adhesive capacities of activated fibroblasts to ECM (Fig. S1A). We then compared the ability of MEF-1 and CAF₂ to remodel and contract three-dimensional type I collagen lattices, a commonly cited hallmark of *in vitro* fibroblast activation [15,16,40] (Fig. 1E). After 24 h of culture in collagen hydrogels, MEF-1 did not show any contractile activity. In sharp contrast, CAF₂ cultivated in the same conditions induced a 58.7 % reduction of collagen area. To study the influence of LRP1 expression on fibroblasts-mediated angio-modulation, we used the LRP1 knock-out counterparts of MEF-1, namely PEA-13 cells [41] (Fig. 1F). We also adapted a previously validated method to silence LRP1 in CAF₂ [27], reaching a 70 % decrease of its expression as measured by western blot (Fig. 1F). We analyzed the protein expression of fibroblasts activation markers (Fig. S2A) and we showed that LRP1 silencing induced significant increase of αSMA and FAP in CAF₂ of 1.3 and 1.7 fold, respectively (Fig. S2B). Conditioned media from MEF-1 and PEA-13 were prepared and used to treat HUVEC during tubulogenesis assays [42]. We observed a 32.6 % decrease of the number of closed endothelial tubes in response to PEA-13 secretome, as compared to wild-type MEF1-derived conditioned media (Fig. 1G). We also documented a concordant increase of 14.3 % and 34.2 % of tubes perimeter and area, respectively. A similar strategy was applied to study the consequences of LRP1-silenced CAF₂ secretome on HUVEC tubulogenesis activity. The inhibition of LRP1 expression in CAF₂ led to an 11 % decrease in the number of endothelial tubes in response to fibroblast secretome, without any significant change of either tube perimeter or closed tube area (Fig. 1H).

### LRP1 governs the CAF-mediated ECM remodeling to stimulate endothelial cell migration

Fibroblasts and CAFs are main contributors of ECM production, assembly, and remodeling in various tissular microenvironments including in breast cancer TME [11,16]. To address whether LRP1 controls the functional properties of fibroblasts towards endothelial cells, we set-up MEF-1 and CAF₂-derived matrices model, in the presence or absence of LRP1 (Fig. 2A). Cell-derived matrices produced by fibroblasts recapitulate key biochemical and architectural features of the *in vivo* breast TME [16,40,43]. Biphotonic confocal imaging of cellularized CDM (Fig. S1B) revealed CAF₂ with elongated nuclei that control the assembly of a dense and oriented network of fibrilar collagen fibers as compared to anisotropic matrices assembled and remodeled by MEF-1. Scanning electron microscope visualization of PEA-13-derived matrices revealed the presence of delineated and individualized superficial fibers as compared to amorphous surface of matrices assembled by wild-type MEF-1 cells (Fig. 2B). CAF₂-derived matrices from LRP1

silenced cells exhibited contrasted intermeshed network of filamentous material. Control CAF₂-derived matricial network revealed almost similar pattern with less surface irregularities. We analyzed the migratory capacities of HUVEC seeded on decellularized fibroblast-derived matrices by videomicroscopy and single cell tracking (Fig. 2C and 2D). The global migratory behavior of HUVEC was assessed through quantitative measurements of migrating cell velocity, Euclidean distance travelled and cell migration directionality. HUVEC migration on PEA-13 compared to MEF-1-derived matrices showed similar directionality but a downward trend in velocity and Euclidean distance measured (Fig. 2B). Conversely, the tracking of HUVEC seeded on LRP1-depleted CAF₂-derived matrices revealed a significant increase of Euclidean distance and migration directionality compared with wild type CAF₂-derived matrices (Fig. 2B). Similar data were obtained using the brain microvascular hCMEC/D3 endothelial cells (Fig. S3).

### LRP1-mediated paracrine signals emitted by CAF₂ affect endothelial cell migration

LRP1 expression in fibroblasts could modify the balance of secreted or internalized diffusible factors at cellular vicinity, thereby influencing other cellular compartments through paracrine signals. Therefore, we tested the impact of conditioned media from LRP1-depleted fibroblasts on endothelial cell migration. PEA-13 secretome weakly decreased HUVEC velocity by 9 % as compared to control MEF-1 (Fig. 3A), but no difference was noticed on Euclidean distance or migration. By contrast, the Euclidean distance covered by HUVEC and migration directionality were increased by 79.8 % and 88.5 % respectively when using conditioned media from LRP1-depleted CAF₂ (Fig. 3B). However, HUVEC velocity was not impacted. Quantification of VEGF-A by ELISA in media conditioned by CAF₂ did not reveal a differential level of its secretion by LRP1-depleted or control cells (Fig. S2C). Although, similar to data acquired from fibroblast-derived matrices, LRP1-mediated CAF₂ secretome appears to impede endothelial cell migration.

### Limitation of *in vitro* endothelial cell angioinvasion by LRP1-dependent paracrine signals from CAFs

Vessel sprouting in a three-dimensional environment relies on a complex balance between proliferation, intercellular connections and spatially resolved specialization of endothelial cells to prospect microenvironment. To address the potential impact of LRP1 fibroblastic expression on angioinvasive process, endothelial cell spheroids were generated and incorporated into collagen I lattice. The peripheral invasion of HUVEC from spheroids in presence of conditioned media from fibroblasts expressing or not LRP1 was followed for 24 h (Fig. 4). Confocal imaging analysis of endothelial cell invasion allowed to record the number of emitted HUVEC sprouts, their cumulative length and the number of cells detached from the spheroid. The angioinvasion parameters do not differ whether PEA-13 or wild-type MEF-1 conditioned
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**Fig. 2. Silencing of LRP1 stimulates endothelial cell migration on CAF-derived matrices.** Schematic representation of the method used to generate decellularized MEF-1 and CAF₂-derived matrices (A). Scanning electron microscopy pictures of metalized surfaces of MEF-1 and CAF₂ derived matrices (B). Random migration of HUVEC seeded on MEF-1 or PEA-13-derived matrices was tracked for 24 h under EVOS videomicroscope. Trajectory plots were mounted, and the analyzed parameters included velocity, Euclidean distance, and directionality, and presented as histograms (C). Similar experiments were conducted with HUVEC seeded on siCTL or siLRP1 CAF₂-derived matrices (D). Results from (C) and (D) were obtained from at least 4 experiments performed in 6 replicates. Each value is mean ± sem. ns, not significant; **, P < 0.01; ***, P < 0.001.
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<figure><img src="image_18.png" /><figcaption>**Fig. 3.** LRP1 limits endothelial cell motility through CAF-diffused factors. Random migration of HUVEC treated by MEF-1 or PEA-13 conditioned media. Media were conditioned for 24 h and migration was tracked for 24 h. Trajectory plots were mounted and the analyzed parameters presented as histograms (A). Similar experiments were conducted with HUVEC treated with conditioned media from CAF siCTL or siLRP1 CAF₂ (B). All the results were obtained from at least 4 experiments performed in 6 replicates. Each value is mean ± sem. ns, not significant; *, P < 0.05; ***, P < 0.0001.</figcaption></figure>

media were used (Fig. 4A). By contrast, the number of invasive structures emitted by HUVEC spheroids in presence of LRP1-depleted CAF₂ conditioned media was 2.1-fold higher than that observed with control cell secretome (Fig. 4B). In accordance, the cumulative length of invaded sprouts and the number of detached cells were also increased by 2.4 and 1.8-fold, respectively, in response to LRP1-silenced CAF₂ secretome. These findings were further confirmed by examining hCMEC/D3 spheroid sprouting (Fig. S4). The results obtained from two distinct endothelial models highlight the specific regulatory role of LRP1 expression in CAFs in restricting the invasion of endothelial cells.

### LRP1-dependent modulation of CAF secretome controls VE-cadherin incorporation into peripheral intercellular adhesions

The dynamics and stability of intercellular connections between endothelial cells constitute key parameters of angioinvasive processes, tightly regulated by environmental cues. We performed immunostainings to analyze the cellular distribution of VE-cadherin-enriched complexes in confluent monolayer of HUVEC, exposed for 24 h to fibroblastic cell conditioned media (Fig. 5). Confocal acquisitions revealed that VE-cadherin-mediated peripheral cell-cell connections were only partially closed after HUVEC exposure to MEF-1 conditioned media (Fig. 5A). Similar patterns were observed in response to PEA-13 secretome. Fluorescent signal quantification did not show any significant difference in VE-cadherin immunostaining intensities between HUVEC treated with either MEF-1 or PEA-13-derived conditioned media. The transcriptional level of VE-cadherin expression was not

significantly different between HUVEC responding to PEA-13 or MEF-1 conditioned media (Fig. 5B). We then analyzed the VE-cadherin expression at protein level, both from HUVEC whole cell extracts and cell-surface enriched fractions (Fig. 5C). Immunoblot analysis did not reveal differential levels of VE-cadherin protein following addition of MEF-1 or PEA-13 supernatants. Thus, exposure of endothelial monolayer to conditioned media from LRP1 expressing or knock-out MEFs did not exert substantial influence on VE-cadherin-mediated intercellular adhesions. A similar scheme of experiments was applied to test whether CAF₂ secretomes may influence VE-cadherin expression and distribution (Fig. 5D–F). Immunofluorescent staining revealed that peripheral adherent junctions were well delineated in HUVEC challenged by LRP1-expressing CAF₂ conditioned media, as we could observe partial closure at several cell-cell connection sites (Fig. 5D). Interestingly, confocal imaging analysis of VE-cadherin distribution in HUVEC treated by secretome from LRP1-silenced CAF₂ revealed a reinforcement of peripheral adhesions and less discontinuities. We could not obtain exploitable staining to analyze β-catenin distribution in our experimental setting (data not shown). Quantification of fluorescent signal intensities showed that media conditioned by LRP1-depleted CAF₂ led to a 1.35-fold increase of VE-cadherin positive complexes, as compared to controls. Of note, VE-cadherin expression remains unchanged at either transcriptional (Fig. 5E) or post-transcriptional level (Fig. 5F) in response to LRP1-expressing or depleted CAF₂ secretome. We conclude that LRP1 expression limits VE-cadherin stabilization in HUVEC peripheral adherent junctions, exclusively in CAFs and without affecting its expression at transcriptional or protein level.
<figure><img src="image_19.png" /><figcaption>**Fig. 4.** Diffusible factors emitted by LRP1-depleted CAF stimulate endothelial cells 3D angioinvasion. HUVEC spheroids formed by the hanging drop method were harvested and incorporated in collagen I lattices for 20 h in the presence of MEF-1 or PEA-13 conditioned media (A) or treated with conditioned media from CAF siCTL or siLRP1 CAF₂ (B). Nuclei of fixed spheroids were stained and confocal acquisition were performed to visualize all endothelial cells invading collagen. 10–17 images were used to quantify the mean number of sprouts per spheroid, the cumulative sprouts length and the number of detached cells. Results were obtained from at least three independent experiments. Each value is mean ± sem. ns, not significant; *, P < 0.05; **, P < 0.01.</figcaption></figure>

Proteomic profiling of endothelial membrane rearrangement induced by LRP-1-mediated CAF secretomes

Most of the LRP1-dependent functional effects documented on endothelial cells in this study were observed in CAF₂ rather than in non-activated MEFs. We therefore focused our further study on CAF₂ to identify the protein changes involved in HUVEC response to LRP1. We established a mass spectrometry-based procedure for the analysis of cell-surface proteins from HUVEC treated with CAF secretomes during a tubulogenic assay (Fig. 6A). This allowed to assemble two groups of 25 and 18 proteins specifically enriched at HUVEC surface in response to conditioned media from LRP1-silenced CAF₂ and wild-type CAF₂, respectively (Fig. S5). We also extracted proteins unique to each experimental condition, allowing us to identify 20 proteins present only in controls and one specific to conditioned media from LRP1-deficient CAF₂, the LRP1 family member LRP2. Through this method we managed to assemble a pool of 64 proteins discriminately expressed at HUVEC surface after incubation with secretomes from LRP1-depleted or -expressing CAFs. We then employed DAVID (Database for Annotation, Visualization and Integrated Discovery) to explore Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms specifically enriched in HUVEC membrane proteome mobilized in response to our experimental conditions, considering both differentially regulated and mutually exclusive hits (Fig. 6B). Among the GO category “Biological processes”, we could identify a specific enrichment of proteins involved in regulation of cell adhesion, migration and sprouting angiogenesis. Analysis of the GO category “Cellular components”

confirmed the specific and significant enrichment of membrane tethered components in both experimental conditions (p-value in CAF₂ siLRP1 = 2.8 × 10⁻⁰⁶; 9.4 × 10⁻³³ in controls), thereby supporting the validity of our approach and its biological relevance. Interestingly, membrane fractions of HUVEC treated with media conditioned by LRP1-silenced CAF₂ were enriched with proteins involved in cell-cell adhesion. Several GO terms (processes, components, and functions) concern adherent junctions and cadherin binding proteins what is consistent with data reported on VE-cadherin-mediated intercellular connections in Fig. 5D. Interestingly, HIF-1 and glucose metabolism were enriched in KEGG pathways when using LRP1-silenced CAF₂, which could be an indicator of specific endothelial cell metabolic adaptations to LRP1-depleted CAF-derived signals. As illustrated by volcano plot of differentially expressed proteins (Fig. 6C) and the corresponding colored heatmap of the protein hits presenting the strongest amplitude of expression at HUVEC surface (Fig. 6D), some of the identified proteins were membrane integral, such as CKAP4, ITB1 and MUC18, enriched in the control condition. Other proteins can be stabilized through lipid anchor such as TGM1, enriched at HUVEC surface when cultured with LRP1-depleted CAF₂ conditioned media, and a substantial number of proteins can be associated to peripheral membranes and cytoskeleton. Of note, several proteins identified and significantly modulated can contextually be stabilized at juxtamembrane position, on both inner and external membrane side to modulate signaling platforms critical for endothelial cell behavior. A substantial proportion are bona fide effectors of intercellular cohesion (RHOG, MOES, PLAK, DSC3, DESP, DSG1), highlighting the importance of this regulation in LRP1-dependent
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Conditioned media:
MEF-1

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PEA-13

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Conditioned media:
CAF₂ siCTL

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CAF₂ siLRP1

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<figure><img src="image_31.png" /></figure>

(caption on next page)
**Fig. 5.** LRP-1-dependent CAF signals disturb VE-cadherin-based endothelial cell junctionality. HUVEC cells were cultivated in quiescent monolayer and treated for 24 h with media conditioned by MEF-1 or PEA-13 for 24 h. Cells were fixed and immunostained by using antibody specific of VE-cadherin. Representative confocal pictures are shown. The mean intensity of VE-cadherin fluorescent signal acquired in the presence of PEA-13 conditioned media was quantified and expressed as a ratio of fluorescent signal measured in the presence of MEF-1 conditioned media (A). The transcriptional level of VE-cadherin was assessed by RT-qPCR (B). HUVEC cells were biotinylated at 4 °C and membrane fraction was purified by precipitation of surface proteins using avidin-sepharose beads. Surface expression of VE-cadherin was analyzed by SDS-PAGE and immunoblotting. Whole extracts were loaded to control efficient enrichment of membrane fraction. Densitometric analysis of specific bands was expressed as percentage of MEF-1 signals, using β-actin as a normalization control (C). Similar experiments were conducted to study the consequences of HUVEC treatment with conditioned media from CAF siCTL or siLRP1 CAF₂ on VE-cadherin immunostaining distribution and fluorescent intensity (D), VE-cadherin transcriptional level (E). Protein expression of VE-cadherin in HUVEC surface or whole extracts was assessed by western blot (F). All results were obtained from at least 3 independent experiments. Each value is mean ± sem. ns, not significant; **, P < 0.01.

endothelial cell adaptation to CAF-emitted factors. The results of this proteomic analysis bring a detailed portrait on specific surface proteins and biological functions engaged under the dependence of LRP1 in the frame of this dialogue.

Galectin-1 and hornerin are two endothelial cell effectors that contribute to LRP1-dependent in vitro angiogenic response to CAF secretomes

Among the top proteins differentially present in endothelial cell
membranome we were able to identify that LEG1 (galectin-1) was 2.32
fold more abundant at HUVEC surface in response to LRP1-expressing
CAF₂ supernatants as compared to HUVEC treated with secretomes
from LRP1-depleted CAF₂. Conversely, we were able to evidence a 3.18
fold accumulation of HRNR (hornerin) in response to LRP1-depleted
CAF₂ as compared to controls (Fig. 6C). In order to investigate the
functional contribution of these two potential effectors of LRP1-
dependent angiomodulation by CAF₂ we applied a siRNA specific
strategy to invalidate their expression in HUVEC. We observed a 76 %
decrease of LEG1 and an inhibition of 58 % of HRNR protein expressions
in HUVEC as measured by western blot (Fig. 7A). We then tested the
potential of LEG1 and HRNR depletion to reverse the specific morpho-
genic response of endothelial cells to CAF₂ secretomes. We performed
tubulogenesis assays with LEG1-depleted or control cells in the presence
of LRP1-expressing CAF₂ conditioned media. In parallel, we studied the
same activity by using HRNR-silenced or control HUVEC treated with
LRP1-silenced CAF secretomes (Fig. 7B). The analysis of LEG1-depleted
HUVEC revealed a 129 % increase of the number of endothelial tubes in
response to control CAF conditioned media, without any significant
change of either tube perimeter or area. In HUVEC treated with LRP1-
silenced CAF₂ secretome, HRNR silencing resulted in a 45 % reduction
of tubes number associated to a 41 % decrease of endothelial loops area.
Considering that the depletion of a potential LRP1 effectors could be
balanced by other angioactive molecules in the context of complex CAF₂
secretomes, we also tested the autonomous influence of LEG1 and HRNR
on tubulogenesis activities of endothelial cells. Since galectin-1 is mainly
secreted and could be present in serum we tested its impact in basal
medium (Fig. 7C). Our analysis revealed a 7,5 fold increase of
the number of endothelial tubes formed by LEG1-depleted HUVEC cells as
compared to controls. We also observed a 3,5 fold and a 5,5 fold increase
of tubes perimeter and area, respectively. By contrast, the depletion of
HRNR resulted in a 44,7 % reduction of the number of HUVEC tubes,
without any significant changes of perimeter nor area, as compared to
control cells (Fig. 7D). Together, these data strongly support that LEG1
and HRNR constitute two molecular effectors mobilized at endothelial
surface involved in the LRP1-dependent angiogenic response to CAF
secretomes.

Discussion

LRP1 had recently emerged as a regulator of fibroblast phenotype and pathophysiological angiogenesis, but the link between LRP1 expression in fibroblasts and angiogenic signaling is poorly characterized. In this study we provide a comprehensive description of *in vitro* angio-modulatory response of endothelial cells to LRP1-dependent fibroblast and CAF-derived signals.

Angiogenesis developed *in vivo* engages cellular processes that are commonly involved in the frame of endothelial morphogenic assays, such as cell-matrix and cell-cell interaction dynamics, pericellular proteolysis, cellular plasticity, and differentiation. We first showed that LRP1 depletion in both MEFs and breast-educated CAF₂ models induced a significant decrease of HUVEC morphogenesis. LRP1 was described for its capacity to control endothelial tubulogenesis, through endothelial cell autonomous regulation [44], or in response to indirect stimuli conditioned by tumor cells [34]. Interestingly, our data are in line with a recent study demonstrating that RAP, a LRP1 family antagonist, reduced the morphogenic activities of HUVEC co-cultivated with mesenchymal stromal cells [30]. This effect was linked to the activity of VEGFR2 and uPAR containing signaling platform, LRP1 having been identified at HUVEC surface in molecular complex with β1 integrin, uPAR and VEGFR2 [32]. However, we could not detect a differential level of VEGFA in CAF secretomes (Fig. S2C) nor a differential abundance of VEGFR2 at the surface of HUVEC responding to CAF signals. Future work will aim at analysing VEGFR2 platform assembly and activation under the dependence of LRP1 angiogenic targets that we newly identified. We studied the paracrine consequences of LRP1 depletion on 2D migration, and 3D angioinvasion of endothelial spheroids in both HUVEC and cerebral microvascular endothelial cells. The strongest effects were obtained in response to the breast-educated CAF₂ model. Our thorough *in vitro* analysis of HUVEC 2D migration demonstrates a stimulating effect of LRP1-depleted CAF’s secretome and matrices specifically. This stimulation of endothelial migration is consistent with that of HUVEC invasion out of spheroids in 3D collagen lattices. These results might contrast with the morphogenesis decrease observed previously under LRP1 silencing. One explanation of this discrepancy could rely on the different matrix environments used in each assay. Matrigel (Fig. 1G, H) and CAF-derived matrices (Fig. 2 and Suppl. Fig. 1) possess their own biochemical and biophysical properties that lead to specific HUVEC responses. In particular, the CAF-synthesized and remodeled matrices stiffness might be significantly higher than the rigidity of fibrillated Matrigel. On the other hand, Matrigel is a more deformable hydrogel, highly susceptible to secreted and membrane focalized ECM proteases [16,42,45,46]. It is tempting to speculate that LRP1 expression modifies the composition of CAF-secreted factors and thus influences the rigidity of ECM networks assembled by cells as well as the deformability of collagen I lattices (Fig. 4 and Suppl. Fig. 2). An ongoing analysis of CAF-derived matrices stiffness by atomic force microscopy will aim at exploring the consequences of LRP1 expression in CAF on the rheological properties of the matrices they assemble. This approach will allow to investigate the functional link between ECM rigidity and endothelial cells behavior. Endothelial morphogenesis relies on a subtle balance between cell matrix interaction dynamics and cell-cell connections. Among the differentially expressed proteins at HUVEC surface we identified ITB1 (β1 integrin subunit) as less abundant in endothelial cells treated with LRP1-depleted CAFs. We had previously described that LRP1 controls the endocytic recycling and inside/out signaling of β1 integrin to sustain the migration of invasive tumor cells [21,27]. Here we show that active forms of β1 integrin are more abundant and mainly localized in membrane clusters in activated fibroblasts than in embryonic fibroblasts. This direct regulation of cell surface integrin β1 modulates adhesion strength, influenced by cellular scaffolds present on
A

Secretome
CAF₂
+/- LRP1

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

HUVEC
morphogenesis

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

Endothelial membrane
proteome

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

64
proteins

43

Differentially
abundant

18 UP via siCTL

Only via CAF₂ siCTL

* NDUS3
* ETHE1
* RRAS
* PTPRF
* ERG
* PCP
* AT1B3
* RS6
* RHOG
* NUCB1
* GOGA2
* AIMP2
* SF01
* SRSF7
* ZN326
* CLC14
* ASCC3
* AP3S1
* EXOC4
* EXOC2

25 UP via siLRP1

* LEG1
* KHDR1
* PRDX1
* RL3
* CKAP4
* ITB1
* RL8
* TTXP
* H2B1L
* PHB1
* MYH9
* VIME
* GRP75
* PML
* NUC18
* RS2
* IMB1
* HSDL2

* IF4A1
* MMS19
* PFKAP
* AT1A1
* SPTC1
* RS25
* PRDX6
* MOES
* LDHA
* PLAK
* SF3B3
* TCPG
* NUI55
* DPYL2
* ANM1
* UBA1
* TGM1
* GJP
* LPPRC
* DSC3
* DESP
* HNRPO
* DSG1
* DCD
* HORN

1
Only via CAF₂ siLRP1
* LRP2

From 619 proteins

B

GO/KEGG enriched upon CAF2 siCTL secretome treatment

<figure><img src="image_38.png" /></figure>

GO/KEGG enriched upon CAF₂ siLRP1 secretome treatment

<figure><img src="image_39.png" /></figure>

C

Differentially abundant proteins

<figure><img src="image_40.png" /></figure>

D

<figure><img src="image_41.png" /></figure>

(caption on next page)
**Fig. 6.** Molecular profiling of CAF-derived angiocrine signals driven by LRP1. Schematic view of the methodology applied: membrane fraction of HUVEC performing morphogenesis under the influence of siCTL or siLRP1 CAF₂ media, conditioned for 24 h, were analyzed by nanoLC-MS/MS using an Orbitrap HF-X (illustration partly created with BioRender http://biorender.com). Among 619 detected proteins, 20 were exclusively detected in controls (yellow circles, left), 1 was specific to HUVEC challenged by siLRP1 CAF₂ supernatants (orange circle, right), and 43 were significantly different between the two experimental conditions (circle intersection, up in response to siCTL or siLRP1 conditioned media). Proteins with a minimal p-value of 0.05 [-Log₁₀(p value) > 1.3], used as a cut-off, and a positive or negative fold change of 1.3 [Log₂(fold change > 0.4) were considered as differentially abundant (A). Gene Ontology terms (Biological processes, Cellular component, and Molecular function) and KEGG pathways that are significantly enriched among the 64 proteins constitutive of the specific proteomic signature of HUVEC membranome influenced by LRP1-dependent CAF₂ paracrine signals are presented. 38 proteins enriched in response to siCTL CAF₂ conditioned media (yellow range) and 26 proteins specifically associated to HUVEC in response to siLRP1 CAF₂ conditioned media (orange range) were loaded (B). Volcano plot of proteins differentially expressed at endothelial surface. Protein that are more abundant in HUVEC treated by siCTL CAF₂ conditioned media are in yellow whereas proteins enriched under siLRP1 CAF₂ conditioned media are in orange (C). Color heatmap of proteins presenting the greatest amplitude of expression in five distinct siCTL CAF₂ treated samples (top, 6 proteins) or in three siLRP1 CAF₂ treated HUVEC membranome (bottom, 9 proteins) (D).

integrin intracellular domain and nearby coreceptors laterally incorporated in adhesive structures [32,47]. In this study we therefore propose that LRP1 also modulates β1 integrin surface expression in endothelial cells through CAF-dependent paracrine signals. Previous reports identified extracellular ligands of LRP1 involved in the control of its association with β1 integrin [37,48]. Moreover, the accumulation of TGM1 (tissue transglutaminase 1) that we identified at HUVEC surface in response to LRP1-silenced CAF signals could participate in the control of β1 integrin turnover, thereby influencing endothelial cell plasticity and migration [48]. Interestingly, the VE-cadherin peripheral stabilization we detected in response to LRP1-depleted CAF signals was associated to the recruitment at HUVEC surface of several proteins that could participate to VE-cadherin engagement in cell-cell adhesion complexes, such as MOES (moesin) and PLAK (junction plakoglobin). These proteins recruitment in adherent junctions might thus be induced by VE-cadherin and limit cooption of multicellular cords during tubulogenesis [49,50]. Studies based on LRP1 loss-of-function in endothelial cells already illustrated its potential to influence complex 3D angiogenic processes [44,51]. The depletion of LRP1 in retinal microvascular cells induced a PARP-dependent proliferative response and supported retinal neovascularization under hypoxic conditions, which stimulate the expression of the receptor [44]. Interestingly, some LRP1 specific ligands can be secreted in breast TME under hypoxic conditions to promote tumor development and metastatic colonization of distant organs [52,53]. CAF-derived diffusible factors can also be specifically secreted under hypoxic culture conditions to stimulate endothelial sprouting [13]. Our data illustrate that, even under normoxia, the LRP1 expression by CAF mediates paracrine signals limiting the angio-invasive response of both macrovascular and microvascular cells in a 3D model of collagen invasion. Our ongoing effort to characterize LRP1 action on CAF phenotypic transition, specially on the nature of their secretomes, will allow to reconstruct key signaling pathways converging to endothelial cells functional regulation.

There is now accumulating evidence that LRP1 expression in the TME is not restricted to tumor cells compartments [25,29,35,36,54,55]. Although, to date the consequences of its expression in stromal cells and specifically in activated fibroblasts has been underestimated. In addition to the angiocrine influence of LRP1 through CAF paracrine signals, we demonstrated that its expression during the assembly and remodeling of CAF-derived matrices limits endothelial cell migration. Surprisingly, we could only observe a non-significant trend in the ability of MEF-1-derived matrices to stimulate HUVEC migration, compared to matrices from LRP1-KO-fibroblasts. Other studies have indeed demonstrated that LRP1 expression in MEFs exerts strong influence on the secretion and activity of pro-fibrotic factors as well as on the production of matricellular glycoproteins and fibrillar collagens, including angiogenic proteins such as fibronectin, periostin or TGFβ [38,41,56,57]. Despite previous demonstrations of clinically relevant pathways affected by LRP1 expression in murine fibroblasts, the differences of ECM composition between LRP1 expressing versus KO MEFs could be limited, particularly concerning the balance of pro- and anti-angiogenic molecules, in contrast to CAF matrices. We are currently investigating additional fibroblasts and CAF models to enlarge the translational potential

of the new mechanistic contribution of LRP1 to the stromal control of cancer angiogenesis we identified. Additional biochemical and biophysical parameters such as topography, stiffness, angiogenic factors retention as well as fibers alignment could undoubtedly explain the angiogenic consequences of fibroblast-derived matrices [15,16,40,43, 58]. It is therefore tempting to speculate that two fibroblastic models - MEFs and CAF₂ - clearly discriminated for their contractile properties will differentially impact the specific endothelial response obtained from their self-assembled matrices. Accordingly, our preliminary analysis of fibrillar collagen I and III content of MEFs and CAF₂-derived matrices by biphotonic confocal analysis of second harmonic generation (SHG) signal suggests that LRP1 induces substantial modifications of collagen incorporation and organization (Fig. S1B). LRP1 has been involved in several pro-fibrotic pathways accumulated in pathological situations [37,38] but its direct role on CAF activation and CAF specific control of breast TME matrix niche parameters has not been investigated so far. Of note, the anisotropic nature of collagen and fibronectin fibers can exert consequences on both tumor and endothelial cells [16,43]. In breast TME, densification and condensation of peritumoral collagen fibers, under physical constrains applied by tumor and stromal cells, were linked to poor prognosis due to increased metastatic colonization [15, 16]. Increased alignment of MEF-derived matrices collagen content was correlated to proangiogenic response of human endothelial cells *in vitro* [43]. Consistently, the increased alignment of collagen fibers we observed could explain the longer distance travelled by endothelial cells onto CAF-derived *versus* MEF-derived matrices. The angiomodulation by CAF₂ secretome and CAF₂-derived matrices we have described provides a strong rationale to study the impact of LRP1 on ECM niche constitution and its consequences on breast tumor vasculature. Our current research is now focused on the functional and preclinical validation of the two newly identified angioactive targets of LRP1-dependent CAF signals: LEG1 and HRNR, especially regarding their perivascular distribution in breast cancer microenvironment and their impact on endothelial cells junctionality and its potential consequences on tumor cell dissemination.

The global proteomic approach we applied to identify endothelial membrane events induced by LRP1-dependent CAF₂ paracrine signals enabled us to detect a significant enrichment of membrane spanning molecules. β1-integrin accumulation in HUVEC in response to LRP1 expressing CAF₂ could contribute to an endothelial migration slow down. Indeed, if initial steps of adhesions are critically involved in tip cell prospection activities, a reinforcement of endothelial cell attachment could constitute a negative signal limiting angioinvasion in 3D environment [21,27,47]. Consistently, proteins that can be contextually associated to both intracellular and extracellular surfaces of reactive endothelium were also identified and are expected to influence angiogenic signaling platforms. CLC14 (also known as CLEC14A) is an endothelial cell marker involved in the control of sprouting angiogenesis and was described in negative regulation of tumor growth through complex modulation of VEGFR2 and VEGFR3 receptors [46,59]. It was exclusively present in HUVEC conditioned by LRP1-expressing CAF supernatants. Similarly, RHOG, RRAS and PTPRF are also involved in the control of endothelial cell directed signals. We identified LEG1
<figure><img src="image_42.png" /><figcaption>**Fig. 7.** Galectin-1 and Hornerin contribute to LRP1-mediated endothelial cell response to CAF secretome. HUVEC cells were transfected with siRNA against Galectin-1 (siLEG1), Hornerin (siHRNR) or non-targeting control sequences (siCTL). Whole extracts were harvested 48 h post-transfection and analyzed by western blot using LEG1 or HRNR specific antibodies. GAPDH was used as a normalization control (A). HUVEC cells transfected with siRNA were harvested 42 h post-transfection and cultivated 6 h on Matrigel in the presence of LRP1-expressing (siCTL) or -depleted (siLRP1) CAF₂ conditioned media (B), in basal (EBM-2) (C) or growth (EGM2) medium (D). Endothelial morphogenic activity was assessed by quantifying the number of tubes, their perimeter, and their cumulative areas. Pictures are representative of at least 3 quantified experiments each performed at least in quadruplicate. Each value is mean ± sem. ns, not significant; *, P < 0.05; **, P < 0.001; ***, P < 0.0001.</figcaption></figure>
(galectin-1) as the top protein specifically mobilized at HUVEC surface in response to LRP1-expressing CAF supernatants. Galectin-1 is a known biomarker of breast cancer microenvironment involved in the regulation of multiple angiogenic pathways. Interestingly, this protein was enriched in breast TME after radiotherapy and in response to sorafenib treatment [60]. It was also involved in vascular permeabilization through direct control of NRP1/VEGFR1 signaling in endothelial cells [61]. Hornerin, the most abundant protein accumulated in response to LRP1-depleted CAF₂ secretome, was described as an angioactive molecule highly expressed in pancreatic cancer vasculature where its depletion, associated to VEGFR2 inhibition, efficiently countered tumor progression [62]. In accordance with our data, a recent study demonstrated that hornerin depletion impairs *in vitro* angiogenesis [63]. Among the endothelial cell specific markers identified, some are already used in preclinical studies aiming at developing combinatorial anti-angiogenic therapies [59,60,62]. Here we describe for the first time that the control of endothelial cell surface expression of LEG1 and HRNR through paracrine signals dependent of LRP1 expression in CAF₂ is functionally linked to their *in vitro* angiogenic functions. Additional biochemical analysis will aim to specify the positive or negative angiocrine events triggered by those newly identified CAF₂-specific targets controlled by LRP1. Gene ontology analysis of the CAF₂ induced membrane proteome revealed a significant enrichment of proteins involved in the control of endothelial cell intercellular connections (e.g., cadherin binding, cell-cell adhesion and junction, adherent junction), via both LRP1 expressing (CKAP4, MUC18) or LRP1-depleted CAF₂ (MOES, PLAK, DESP, DSG). Those endothelial surface adaptations could participate in the modulation of VE-cadherin distribution identified in our study. LRP1 was previously reported in the regulation of endothelial layer permeability, in brain and pulmonary microvascular endothelial cells [64,65]. In the frame of cancer progression, LRP1 expression in melanoma contributes to their endothelial recruitment and metastatic dissemination [66]. In a pancreatic cancer model, myeloid depletion of LRP1 induced an imbalance of intratumoral inflammatory cytokines which stimulated the recruitment of CCR5 pro-angiogenic population of monocytes [29]. The original LRP1-dependent dialogue between CAF and HUVEC monolayer junctionality that we identified could be relevant to several key tumor vessel parameters. It could play instrumental role in vascular plasticity and endothelial cell fitness to CAF-derived signals that are critically engaged in mammary cancers ecosystems and can culminate into the dissemination of tumor cells [5,13,14]. It would therefore be interesting to test the potential of newly identified endothelial targets to influence the opening of vascular barrier in the frame of transendothelial migration assays and the consequence of their expression on metastasis occurrence. The endothelial membrane protein signature that we identified in response to CAF paracrine signals will constitute a valuable biological reservoir of potential angiogenic targets to study the multiple angiogenic functions regulated by LRP1 in breast cancer ecosystem.

## Experimental procedures

**Cell culture.** Human umbilical vein endothelial cells (HUVEC) and HUVEC-GFP, purchased from Promocell and Cellworks respectively, were cultured in Endothelial cell Growth Medium 2 (Lonza). Blood-Brain Barrier hCMEC/D3 cells, purchased from CELLution Biosystems were cultured in EBM-2 medium supplemented with VEGF, IGF-1, EGF, basic FGF, hydrocortisone, ascorbate, gentamycin and 2.5 % fetal bovine serum as recommended by the manufacturer. Normal mouse embryonic fibroblasts, designated MEF-1, and its deficient counterpart for LRP-1, designated PEA-13, were obtained from ATCC and cultured in Dulbecco's Modified Eagles Medium (Invitrogen, Cergy Pontoise, France) with 4.5 g/L glucose and L-glutamine, supplemented with 1 % (v/v) penicillin-streptomycin and 10 % FBS. CAF₂ used as cancer-associated fibroblast were obtained from Professor Akira Orimo of Juntento University in Japan [12] and cultured in DMEM with 1 g/L glucose and

L-glutamine, supplemented with 1 % (v/v) penicillin-streptomycin and 10 % FBS. These cells were generated *in vivo* in immunodeficient nude mice after co-injection of immortalized primary normal human mammary fibroblasts and MCF-7-ras human mammary adenocarcinoma cells expressing an activated ras oncogene. MDA-MB-231 (DSMZ no. ACC 732), MDA-MB-468 (DSMZ no. ACC 738) and MCF-7 (ATCC no. HTB-22) cell lines were purchased from DSMZ, and ATCC and cultured in DMEM Medium with 1 g/L glucose and L-glutamine, supplemented with 1 % (v/v) penicillin-streptomycin and 10 % FBS.

**siRNA transfection.** Small interfering RNA (siRNA)-mediated silencing of LRP1 was described elsewhere [27]. Specific LRP-1 targeting sequences, designed as follows: GACUUGCAGCCCCAAGCAGUU (sense), CUGCUU GGGGUGCAAGUCUU (antisense), validated ON-TARGETplus siRNA sequences specifically targeting LEG1 (J011718050020) or HRNR (J027450170020) were purchased from Dharmacon (distributed by Horizon Discovery, Cambridge, UK). Control transfection experiments were achieved by using SiGENOME Non-targeting siRNA (D00121001–20) from Dharmacon. SiRNA were transfected using Lipofectamine RNAiMAX Reagent (Invitrogen) according to the manufacturer's protocol.

**3D collagen I gel contraction assay.** $2.5 \times 10^4$ MEF-1 and CAF₂ were embedded in 100 µL of rat tail collagen I at 1,5mg/mL (Corning) and seeded in quintuplicate in 96 well plate. Once polymerized, the fibroblasts-collagen gels were allowed to contract in 100 µL of complete media. After 24 h, images of the collagen lattices were acquired using EVOS microscope (ThermoFisher). The relative diameter of the well and the collagen lattices were measured using ImageJ (ImageJ software, National Institutes of Health, Bethesda, MD, USA), and the percentage of gel contraction was calculated using the formula $100 \times (\text{well area} - \text{gel area}) / \text{well area}$.

**Fibroblasts conditioned media.** Fibroblasts were seeded on petri dish ($5 \times 10^6$ for MEF-1 and PEA-13 and $2.5 \times 10^6$ for CAF₂) in complete media during 24 h. The next day, after two washes with PBS, the media was replaced by EBM2. Conditioned media from CAF₂ needed an extra day before changing media and conducting siRNA transfection. After 24 h, the media were collected, centrifuged, filtered with 0.45 µm filters and stored at -80 °C. Secreted VEGF-A was quantified on paired secretome samples using RayBio® Human VEGF-A ELISA Kit (Catalogue no. ELH-VEGF, RayBiotech, GA, USA), according to the manufacturer's protocol.

**Western blot.** Cells were lysed in ice-cold lysis buffer (25 mM Tris-HCl, pH 7.7, 150 mM NaCl, 0.5 % glycerol, 1 % Triton X-114, 5 mM EDTA, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM orthovanadate supplemented with proteinase inhibitor cocktail). Protein concentration in supernatants was quantified by Bradford method (BioRad Laboratories). Proteins were separated by electrophoresis in a 10 % sodium dodecyl sulfate-polyacrylamide gel, transferred onto a nitrocellulose membrane (Whatman GmbH, Dassel, Germany), and incubated overnight at 4 °C with primary antibodies against LRP1, LEG1 or HRNR (abcam). Immunoreactive bands were revealed using Odyssey® Imaging System (LI-COR). Immunoblots presented are representative of at least three separate experiments. The specific signals of β-actin or GAPDH were used to ensure equal loading.

**Tube formation assay.** HUVECs stained with CMFDA cell tracker (Invitrogen) were seeded on µ-Slide Angiogenesis plate from Ibidi ($10^4$ cells/well) coated with 10 µL growth factor-reduced Matrigel (Corning) in 50 µL of pure EBM-2, EGM-2 or fibroblast conditioned media. After 6 h incubation, images were acquired with EVOS microscope (Thermo-Fisher). Tube formation was quantified using ImageJ (ImageJ software, National Institutes of Health, Bethesda, MD, USA).

**Motility assay.** HUVEC stained with CFMDA cell tracker were seeded in pure fibroblast conditioned media ($10^4$ cells in 500 µL) on 24-well plate or on fibroblasts-derived matrices in complete media. After 6 h incubation at 37 °C and 5 % CO₂, images were taken during 24 h with EVOS videomicroscope. Velocity, distances, and directionality were evaluated with Chemotaxis Tool on ImageJ software.
**Cell-derived matrice assay.** The procedure was adapted from previous studies with minor modifications [45,67]. 24 plates with coverslips were coated with 1 % gelatin for 1 h at 37 °C, followed by crosslinking with 1 % (v/v) glutaraldehyde (Sigma-Aldrich) for 30 min at room temperature. Crosslinker was then quenched with 1 M glycine (Sigma-Aldrich) for 20 min at room temperature. Wells were washed with PBS and complete media was added during 1 h at 37 °C. Based on their respective proliferation kinetics, MEF-1 or PEA-13 (4.10⁵ cells), CAF₂ siCTL (2.10⁵ cells) or CAF₂ siLRP1 (3.10⁵ cells) were then seeded and ascorbic acid (50µg/mL; Sigma-Aldrich) treatment started 48 h after on confluent cell layer. Ascorbic acid containing medium was changed every day for 5 (MEF-1 and PEA-13) or 7 days (CAF₂). Fibroblasts were then removed by extraction solution treatment (50 mM (MEFs) and 10 mM (CAF₂) NH₄OH, Sigma-Aldrich), 0.5 % (v/v) Triton X-100 (Fisher Scientific) in PBS). One hour DNase (10µg/mL; Sigma-Aldrich) treatment at 37 °C allowed remaining DNA removal. The resulting cell-derived matrices were stored at 4 °C in PBS supplemented with 1 % (v/v) penicillin-streptomycin. Alternatively, MEF-1 and CAF₂-derived matrices were fixed 10 min with 4 % paraformaldehyde and washed with PBS before fluorescent stainings and confocal microscopy imaging.

**Scanning electron microscopy.** Decellularized cell-derived matrices were fixed with 2.5 % (v/v) glutaraldehyde for 1 h at room temperature. After 2 washes of 10 min in PBS, samples were dehydrated in ascending ethanol baths, dried through evaporation of hexamethydisilazane solution (Sigma-Aldrich) and coated with gold using SPT 20 sample metallizer (Scientec). SEM images were acquired using an EVO10 scanning electron microscope equipped with a LaB6 source (Zeiss).

**3D spheroid angiogenesis assay.** 3D spheroid-based angiogenesis assay was adapted from a previously described procedure with modifications [68,69]. Endothelial cells stained with CFMDA cell tracker were suspended in EGM2 medium with 10 % FBS and 20 % methocell (1.2 % methylcellulose in EBM2) and aggregated overnight in hanging drops to form cellular spheroids (500 cells/spheroid). Spheroids were embedded in 3 mg/mL rat acid-treated collagen (Corning) and 0.5 % methylcellulose. Pure fibroblasts conditioned media were added 30 min after embedding. After 20 h incubation at 37 °C, spheroids were fixed with 4 % paraformaldehyde for 10 min at room temperature and nuclei were stained with 2,5 µM DRAQ5 in PBS (Invitrogen). 10 to 17 images of spheroids, from three different experiments, were acquired with Zen software on a Zeiss LSM 880 Airyscan confocal microscope (Oberkochen, Germany) using a x20 Plan Apochromat M27 objective (0.8 NA) and *in vitro* angiogenesis was quantified using ImageJ software.

**Immunofluorescence analysis.** Cells were rinsed out with PBS and then fixed with 4 % paraformaldehyde for 10 min at room temperature. Subsequently, samples were permeabilized with 0.3 % Triton X-100 during 20 min and blocked with 2 % bovine serum albumin (BSA; diluted with PBS) for 30 min at room temperature. Samples were incubated with anti αSMA (abcam), anti-active β1 integrin (BD Transduction Laboratories) or anti VE-cadherin (Cell Signaling Technology) primary antibodies diluted in 2 % BSA in PBS during 1 h at 4 °C. After 3 PBS washes, samples were incubated with goat anti-rabbit A647, goat anti-rat or anti-rabbit A568 (Invitrogen) secondary antibodies for 1 h. Nuclei were stained using DAPI (Sigma Aldrich). Images were acquired with Zen software on a Zeiss LSM 880 Airyscan confocal microscope (Oberkochen, Germany) using a x63 Plan Apochromat objective (oil immersion, 1.40 NA). Fixed cell-derived matrices were permeabilized with 0.1 % Triton X-100 during 10 min, washed with PBS and incubated 1 h at room temperature with A568-conjugated phalloidin. Nuclei were stained with DRAQ5 (Invitrogen). Images were acquired with Zen software on a Zeiss LSM 710 NLO using a x20 Plan Apochromat objective (0.8 NA). Second harmonic generation signal was acquired at 430 nm by using linear unmixing mode after excitation at 860 nm.

**Sample preparation and mass spectrometry (MS) analysis.** For label-free quantitative proteomics, five independent biological replicates of HUVEC membrane treated with siCTL CAF₂ conditioned

media and three corresponding samples treated with siLRP1 CAF₂ secretome were prepared. HUVEC at passage 5 were plated on growth factor reduced Matrigel® (Corning) in presence of fibroblasts conditioned media for 24 h at 37 °C, 5 % CO₂. Cells were then biotinylated with 0.5 mg/mL of sulfo-NHS-biotin during 30 min at 4 °C and 100 mM Glycine was added during 30 min at 4 °C. To remove Matrigel®, cell recovery solution (Fischer scientific) was added to samples and left on ice until complete dissolution. Cell pellet was obtained after centrifugation and 250 µL of lysis buffer was added. After 2 h solubilization samples were clarified by centrifugation and 50 µL was kept as whole extract part. Remaining volume was mixed with avidin beads during 45 min at 4 °C on rotator disk. Avidin and protein complexes mixes were centrifuged and supernatant was kept as no surface extracts fraction. Laemli 1.5X + β-mercaptoethanol were added on beads, heated and spun for 5 min at 10 000 g and supernatant was collected as surfaces extracts fraction. Samples were separated on SDS-PAGE gel and strips were cut and processed (washing and reduction/alkylation) before enzymatic digestion with trypsin. Samples were analysed on a nanoUPLC-system (nanoAcquity, Waters) coupled to a quadrupole-Orbitrap hybrid mass spectrometer (Q-Exactive HF-X, Thermo Scientific, San Jose, CA). The UPLC system was equipped with a Symmetry C18 precolumn (20 x 0.18 mm, 5 µm particle size, Waters, Milford, USA) and an ACQUITY UPLC® BEH130 C18 separation column (75 µm × 250 mm, 1.7 µm particle size, Waters). The solvent system consisted of 0.1 % formic acid in water (solvent A) and 0.1 % formic acid in acetonitrile (solvent B). Peptides were trapped during 3 min at 5 µL/min with 99 % A and 1 % B. Elution was performed at 60 °C at a flow rate of 350 nL/min, using a 79 min linear gradient from 1–35 % B. To minimize carry-over, a column wash (50 % ACN during 20 min.) was included in between each sample in addition to a solvent blank injection, which was performed after each sample. The mass spectrometer was operated in positive mode, with the following settings: spray voltage 1800 V and capillary temperature 250 °C. The MS scan had a resolution of 70,000, the AGC target was 3 × 10⁶ and the maximum IT was 50 ms on *m/z* [300–1800] range. The MS/MS scans had a resolution of 17,500, the AGC target was 1 × 10⁵ and the maximum IT was 100 ms with fixed first mass of 100 *m/z* and Isolation window of 2 *m/z*. Top 10 HCD was selected with MS2 Identity threshold of 2 × 10⁵ and dynamic exclusion of 60 s. The normalized collision energy (NCE) was fixed at 27 V. The complete system was fully controlled by Thermo Scientific™ Xcalibur™ software (4.0.27.19). Raw data collected were processed and converted with MSConvert into.mgf peak list format. The peak list has been searched against a Human SwissProt combined target-decoy database (created 2022–09–26, containing 40,992 target sequences with reversed decoy sequences) using Mascot (version 2.6.2, Matrix science, London, England). During database search, up to one missed cleavage by trypsin, one fixed modification (carbamidomethylation of Cysteine (+57 Da)) and one variable modification (oxidation of Methionine (+16 Da), were considered. The search window was set to 10 ppm for precursor ions and 0.07 Da for fragment ions. Protein lists from Mascot were validated by Proline software with an FDR of 1 % on peptides and proteins [70]. A quantitative analysis was performed with MaxQuant. MaxQuant parameters were set as follows: MS tolerance set to 20 ppm for the first search and 10 ppm for the main search, MS/MS tolerance set to 20 ppm, maximum number of missed cleavages set to 1, Carbamidomethyl (C) set as a fixed modification, Oxidation (M) set as variable modifications [71]. Identifications were validated at a false discovery rate (FDR) below 1 % at both peptide spectrum match and protein levels. Data normalization and protein quantification were performed using label-free quantification (LFQ) with a “minimal ratio count” of two. The “Match between runs” option was enabled using a 1-minute time window after retention time alignment. All other MaxQuant parameters were set as default. Differential data analysis was performed using the open-source ProStaR software version 1.30.2 [72]. To be considered for differential analysis, proteins must be identified in all replicates of each experimental group. For residual missing values, the slsa algorithm was used for the POV
(Partially Observed Values) imputation, and MEC (Missing on the Entire Condition) values were replaced by the 2.5 percentile value. A Limma moderated *t*-test was applied to the data set to perform differential analysis. Benjamini-Hochberg method was used to adjust *p*-values for multiple testing, and differentially expressed proteins were sorted out using a *p*-value threshold of 0,05. Contaminant proteins were excluded from analysis. Only proteins with at least 2 peptides uniquely assigned to the respective sequence were considered. Complete data set has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository [73] with the data set identifier PXD047525 and 10.6019/PXD047525. Annotations were obtained from the Gene Ontology (GO) Project and Kyoto Encyclopedia of Genes and Genomes (KEGG) using the DAVID Gene-enrichment and functional annotation platform (version DAVID 2021: https://david.ncifcrf.gov), loading official gene symbols of protein hits against Homo sapiens database (9606). Binomial statistics were used to ensure that the biological processes presented were significantly enriched with a *p*-value < 0.05.

**Real-time qPCR.** Cell lysates were prepared 24 h after conditioned media treatment. Total RNAs isolated using the RNeasy Mini kit (Qiagen, Courtaboeuf, France) were reverse transcribed into cDNA with the ABsolute Blue verso 2-step kit and subjected to quantitative real-time PCR by using the Absolute SYBR Green mix. Following this, quantitative real-time PCR were performed using the qPCRBIO SyGreen Blue Mix (PCR biosystems, USA). The relative levels of expression were quantified by using Opticon Monitor software (Bio-Rad). For each specific gene, the amount of target RNA (2-ΔΔCt) was normalized to the internal actin reference (ΔCt) and related to the amount of target RNA in control sample, set to 1. Statistical analysis of each target gene relative expression was done after three separate conditioned media treatments and duplicate PCR reactions.

**Statistical analysis.** Statistical analyses were performed using GraphPad Prism 9.5.1 software (GraphPad, Bethesda, MD, USA). Student's *t*-test was used to evaluate the difference between two groups. One-way ANOVA with Tukey's multiple comparisons test was used to analyze data from three or more groups. For the analysis of MS data, protein lists from Mascot were validated by Proline software with an FDR of 1 % on peptides and proteins. For DAVID analysis (GO and KEGG) binomial statistics were used to ensure that the biological processes presented were significantly enriched. *p* < 0.05 was considered significant and used as cut-off. Lists of proteins enriched in presented terms are produced in supplemental material. For some terms presenting FDR *q*-value > 0,05 due to low protein count, all proteins were individually verified for their specific involvement in the described categories.

## Funding

This work was supported by fundings from Université de Reims Champagne-Ardenne (URCA), CNRS, Ligue Contre Le Cancer (CCIR-Est) and the Région Grand-Est. J. Martin was PhD fellow supported by URCA and Région Grand-Est.

## Declaration of competing interest

SD is co-founder of Apmonia-Therapeutics and acts as the chair of the Scientific and Clinical Advisory Board. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

## Acknowledgements

The proteomic analysis was conducted at the Plateforme Protéomique Strasbourg Grand Est (PSGE), Strasbourg, France. We address our acknowledgements to Dr Jean-Marc Strub and Dr Christine Schaeffer-Reiss (CNRS, Université de Strasbourg, France) for the proteomic data analysis. We acknowledge Dr Nicolas Richet for technical

assistance. We thank Professor Akira Orimo (Jutento University, Japan) for having kindly shared the CAF₂ cell line.

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.matbio.2025.05.004.

## Data availability

Characterization of HUVEC angiogenic surface proteome responding to breast cancer-associated fibroblasts secretome, with or without LRP1 expression; contact: Jean-Marc Strub; data repository: PRIDE; accession: PXD047525, doi:10.6019/PXD047525; username: reviewer_pxd047525@ebi.ac.uk, password: uTseC8L9; 2023.

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