# Impact of senescent fibroblasts secretome in ovarian cancer dissemination

Luce Perié, Alexandra Landras, Mickaëla Bignard, Marion Theuret, Mélanie Briand, Franck Carreiras, Cédric R Picot

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Luce Perié, Alexandra Landras, Mickaëla Bignard, Marion Theuret, Mélanie Briand, et al. Impact of senescent fibroblasts secretome in ovarian cancer dissemination. Experimental Gerontology, 2025, 211, pp.112909. 10.1016/j.exger.2025.112909. hal-05469980

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# Impact of senescent fibroblasts secretome in ovarian cancer dissemination

Luce Perié<sup>a</sup>, Alexandra Landras<sup>a</sup>, Mickaëla Bignard<sup>a</sup>, Marion Theuret<sup>a</sup>, Mélanie Briand<sup>b</sup>, Franck Carreiras<sup>a</sup>, Cédric R. Picot<sup>a,∗</sup>

<figure><img src="image_3.png" /></figure>

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## ARTICLE INFO

Section Editor: Anna Picca

Keywords:

Age-related diseases

Ovarian cancer cell dissemination

Senescent fibroblasts

Fibronectin

SASP

Tumor microenvironment

ECM remodeling

## ABSTRACT

Aging is a significant risk factor in ovarian cancers, which remains one of the most lethal gynecological malignancies. Over 50% of ovarian cancer cases occur in women aged 65 and older. During aging, senescent cells progressively accumulate in various tissues, contributing to structural and inflammatory changes in the tumor microenvironment. While recent studies have underscored the role of the senescent mesothelial cells in facilitating cancer cell dissemination, the contribution of senescent fibroblasts that are a major component of the connective tissue, remains elusive. In this study, we first characterized the senescent phenotype of human ovarian fibroblasts, with a focus on extracellular matrix (ECM) remodeling and the senescence-associated secretory phenotype (SASP). Senescent ovarian fibroblasts exhibited an accumulation of intracellular Fibronectin (Fn), impaired fibrillar Fn into ECM assembly, and marked alterations in ECM organization. SASP profiling revealed a pro-inflammatory secretome enriched in immune-modulatory cytokines and factors potentially involved in cancer cell dissemination and metastasis. We then evaluated the influence of the senescent fibroblast secretome on ovarian cancer cell behavior. While it did not alter cancer cell proliferation, it conferred resistance to apoptosis and significantly enhanced cancer cell migration and invasion. Altogether, our findings highlight the role of senescent ovarian fibroblasts in promoting cancer cell dissemination and suggest that age-related stromal changes contribute to disease progression.

## 1. Introduction

Ovarian cancer remains one of the most lethal gynecological malignancies, with a five-year survival rate below 40% (Momenimovahed et al., 2019; Koshiyama et al., 2014). Its poor prognosis is largely attributed to late-stage diagnosis, limited screening methods, and early metastatic spread within the peritoneal cavity (Lopez et al., 2023; Yeung et al., 2015). The disease predominantly affects older individuals, with more than half of the women patients being over 65 years old, classifying it among age-related diseases (Duska et al., 2015; Tew, 2016). Despite the increasing recognition of aging as a major risk factor, the mechanistic links between age-associated tissue changes and ovarian cancer progression have only recently begun to receive significant attention. For instance, senescent peritoneal mesothelial cells have been shown to promote ovarian cancer cell proliferation, migration, and

invasion (Thapa et al., 2024; Mikula-Pietrasik et al., 2016; Ksiązek et al., 2009), highlighting the potential role of senescent stromal cells in creating a pro-tumoral microenvironment.

Ovarian cancer is distinct from other cancers as cells disseminate from the primary tumor site into the peritoneal cavity (Tan et al., 2006; Weidle et al., 2016). During this peritoneal dissemination, cancer cells can undergo an epithelial-to-mesenchymal transition (EMT), detach from the primary tumor and overcome anoikis (Tan et al., 2006). These cells spread either as single cells or multicellular spheroids (Ahmed and Stenvers, 2013; Kellouche et al., 2010), a process strongly supported by the tumor microenvironment (TME). This TME contains a rich mix of inflammatory cytokines, chemokines, matrix metalloproteinases (MMPs), and extracellular matrix (ECM) components, creating a highly permissive niche for tumor progression (Worzfeld et al., 2017; Kim et al., 2016; Zhao et al., 2021; Laurent-Issartel et al., 2024). The ovarian

<footnote><sup>a</sup> Equipe de Recherche sur les Relations Matrice Extracellulaire Cellules, ERRMECe (EA 1391), Groupe Matrice Extracellulaire et Physiopathologie (MECuP), Maison Internationale de la Recherche, CY Cergy Paris Université, 1 rue Descartes, 95000, Neuville-sur-Oise, France</footnote>

<footnote><sup>b</sup> INSERM U1086 ANTICIPE (Interdisciplinary Research Unit for Cancers Prevention and Treatment), Precision Medicine for Ovarian Cancers axis, UNICANCER, Comprehensive Cancer Center François Baclesse, PLATON Services Unit, Biological Resources Center 'OvaRessources', Université de Caen Normandie 14000, Caen, France</footnote>

<footnote>* Corresponding author: Cédric R. Picot, Equipe de Recherche sur les Relations Matrice Extracellulaire Cellules, ERRMECe (EA 1391), Groupe Matrice Extracellulaire et Physiopathologie (MECuP), Maison International de la Recherche, CY Cergy Paris Université, 1 rue Descartes, 95000, Neuville-sur-Oise, France.

E-mail address: cedric.picot@cyu.fr (C.R. Picot).</footnote>
cancer TME is particularly complex, comprising immune cells, mesothelial cells, and fibroblasts which play a pivotal role in ovarian cancer dissemination (Lopez et al., 2023; Zhao et al., 2021; Wang et al., 2024a). Recent studies across tumor types have revealed that cancer-associated fibroblasts (CAFs) are heterogeneous and can exert varied effects on tumorigenesis (Licaj et al., 2024). Within this heterogeneity, emerging evidence indicates that senescent fibroblasts can promote tumor progression. For example, in pancreatic and breast cancers, they facilitate dissemination by shaping an immunosuppressive microenvironment (Ye et al., 2024; Pelon et al., 2020; Assouline et al., 2024). In breast cancer, senescent skin fibroblasts were also shown to enhance cancer cell motility and induce EMT (Wang et al., 2024b). In ovarian cancer, early work revealed the presence of senescent fibroblasts within tumor sites, where they promoted the malignant transformation of ovarian epithelial cells (Yang et al., 2006). Lawrenson et al. further demonstrated that senescent fibroblasts enhanced proliferation, migration, and invasion of ovarian epithelial cells (Lawrenson et al., 2010).

Fibroblasts are present both in the ovarian stroma and in deeper layers of the peritoneal mesothelium, placing them at critical sites of tumor initiation and dissemination. A defining feature of fibroblasts is their capacity to produce and remodel ECM components. These cells are essential in wound healing and fibrosis, where they also modulate immune responses (Asif et al., 2021; Plikus et al., 2021; DeLeon-Pennell et al., 2020). In tumor, altered ECM becomes progressively altered not only reflecting tissue disorganization but also promoting cancer cell survival, migration, EMT and resistance to therapy (Carreiras et al., 1999a; Carreiras et al., 1999b; Maubant et al., 2002; Leroy-Dudal et al., 2005; Carreiras et al., 1996; Carduner et al., 2014). In parallel, it facilitates the recruitment of pro-tumoral immune cells, including tumor-associated macrophages (TAMs) (Ye et al., 2024). Several ECM components act as ligands for integrins and other immune receptors, shaping macrophage phenotype and contributing to immune evasion (Puttock et al., 2023; Dalpati et al., 2025; Yuan et al., 2023). Among ECM components, Fibronectin (Fn) has emerged as a key player in the tumour-stroma dialogue. Fn is upregulated in several cancers, including ovarian cancer (Gendrau-Sanclemente et al., 2024; Masi et al., 2025). Recent studies have shown that fragmented, misfolded, or conformationally altered forms of Fn, often produced by stromal cells such as mesothelial cells, are associated with increased cancer cell adhesion, invasion, and immune cell recruitment (Puttock et al., 2023; Gopal et al., 2017; Bascetin et al., 2018; Kenny et al., 2014; Kenny et al., 2008). Altered forms of Fn result from proteolytic cleavage by MMPs or from dysregulated secretion by cancer or surrounding stromal cells (Gopal et al., 2017; Perié et al., 2024), and act via integrin-mediated signaling and ECM remodeling (Bascetin et al., 2018; Stinson et al., 2024).

Although tumor cells contribute to ECM alterations, increasing evidence indicates that a substantial portion of the ECM remodeling is driven by the surrounding stromal cells (Puttock et al., 2023; Gopal et al., 2017; Ye et al., 2023; Ruhland et al., 2016). Among stromal cells, fibroblasts are especially prone to senescence in response to aging or chronic stress. These senescent cells have attracted growing interest due to their altered secretory profile and matrix production capacity (Ye et al., 2024; Ruscetti et al., 2020). Senescent cells accumulate macro-molecular damage that leads to a permanent cell cycle arrest. However, they remain metabolically active and adopt a senescence-associated secretory phenotype (SASP), characterized by the release of inflammatory cytokines, chemokines, and ECM components (Hayflick and Moorhead, 1961; Coppé et al., 2010). This secretory profile collectively alters the tissue environment in ways that can promote tumor progression.

Previous studies have highlighted the importance of senescent fibroblasts in ovarian cancer (Yang et al., 2006; Lawrenson et al., 2010), and additional insights are needed to fully understand their specific contribution to cancer progression. In this study, we provide new evidence that senescent fibroblasts secretome promotes ovarian cancer cell migration and invasion. In addition, we show, to our knowledge for the

first time, that it can confer apoptosis resistance and promote EMT. We further found that senescence in human ovarian fibroblasts is associated with disrupted Fn fibril organization and increased expression of ECM regulators. SASP profiling revealed a pro-inflammatory and immunomodulatory secretome, containing several factors linked to cancer dissemination. Functional assays showed that the senescent fibroblast secretome promotes cancer cell migration, invasion, and resistance to apoptosis, without affecting proliferation. Altogether, our results suggest that senescent ovarian fibroblasts secretome contribute to cancer cell dissemination. These findings provide new insight into the protumoral impact of senescent fibroblasts and may help guide future strategies aimed at targeting senescence-associated pathways in ovarian cancer.

## 2. Material and methods

### 2.1. Cell culture

Human ovarian fibroblasts (HOF) were isolated from human ovary tissue and obtained from CliniSciences (#7330-SC). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen), supplemented with 10 % fetal bovine serum (FBS; Invitrogen), and maintained at 37 °C in a humidified incubator with 5 % CO₂. Senescence was considered established when >80 % of HOF cells were positive for senescence associated-β-Galactosidase (SA-β-Gal) activity. For the preparation of conditioned medium, young and senescent HOF cells were seeded 500 000 cells per 75 cm² flask and incubated for 12 h in serum-free DMEM. The conditioned media were then collected and stored at -20 °C; cells were harvested for subsequent experiments. SKOV3, obtained from ATCC (#HTB-79), were isolated from ascites of a serous cystadenocarcinoma. Human ovarian adenocarcinoma cell lines IGROV1, derived from a stage III ovarian adenocarcinoma, were kindly provided by Dr. J. Benard and Dr. L. Poulain from the BioTICLA unit (INSERM U1086, Biologie et Thérapie Innovantes des Cancers Locale Agressifs, Caen, France) (Wallace and Balkwill, 1987; Bénard et al., 1985). IGROV1 and SKOV3 cells were cultured in RPMI-1640 GlutaMAX medium (Invitrogen) supplemented with 0.07 % (v/v) sodium bicarbonate and 10 % FBS. The human mesothelial cell line Met-5A (#CRL-9444, ATCC) was maintained in M199 medium (Invitrogen) supplemented with 3.3 nM epidermal growth factor, 870 nM insulin, 400 nM hydrocortisone, 1.25 g/L sodium bicarbonate (Invitrogen), and 10 % FBS.

### 2.2. SA-β-galactosidase assays

SA-β-gal activity was assessed in HOF cell cultures using the SA-β-gal staining kit (#CS0030-1KT, Sigma Aldrich) as previously described (Dimri et al., 1995). Briefly, cells were first fixed for 5 min using a solution containing 2 % formaldehyde and 0.2 % glutaraldehyde. Following fixation, cells were incubated in a staining solution composed of 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal, Sigma Aldrich) dissolved in a buffer (40 mM citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl₂). A blue precipitate indicative of SA-β-gal activity became visible in some cells within 2 h, with optimal staining typically achieved after 16 h of incubation. Cells were then rinsed with PBS and examined under bright-field or phase-contrast microscopy. Quantification was performed by counting the proportion of SA-β-gal-positive cells in five randomly selected microscopic fields.

### 2.3. Cell proliferation assay

SKOV3 and IGROV1 cells were harvested using 0.25 % trypsin-EDTA, seeded at a density of 50,000 cells per well and incubated for 24 h in complete RPMI culture medium. Proliferation was then assessed over 72 h in DMEM supplemented with 10 % FBS, serum-free DMEM, or
conditioned medium from young or senescent HOFs. At each time point, cells were detached using 0.25 % trypsin-EDTA and counted using a Malassez counting chamber.

## 2.4. Cell migration assay

SKOV3 and IGROV1 cells were seeded at 20,000 cells per well in Ibiidi® 2-well culture inserts (#81176, Ibiidi®) and grown as a monolayer for 24 h in complete RPMI culture medium. After this incubation, the inserts were removed to create a cell-free gap. Cells were then incubated in DMEM supplemented with 10 % FBS or in conditioned medium from young or senescent HOFs. Cell migration into the gap was monitored using inverted time-lapse microscopy (DMI6000B, Leica, Germany) with a 10× objective over a period of 24 h for SKOV3 cells and 72 h for IGROV1 cells, with images captured at regular intervals (every 6 or 24 h).

## 2.5. Multicellular aggregate (MCA) formation

The generation of multicellular aggregates (MCA) was conducted according to previously published methods (Kellouche et al., 2010). To prevent cell adhesion, 25 cm² culture flasks were coated with 4 mL of 1 % agarose in serum-free medium and allowed to solidify for 30 min at room temperature. Subconfluent SKOV3 cells were harvested using 0.25 % trypsin-EDTA and resuspended at a concentration of 1.5 × 10⁶ cells/ flask in either DMEM supplemented with 10 % FBS or in conditioned medium from young or senescent HOF cells. MCAs were cultured for up to 2 days. Cells were then collected, and aggregates were dissociated by incubation in trypsin-EDTA for 10 min at 37 °C. Cell were harvested for protein analysis.

## 2.6. Spheroid formation, coculture and mesothelial implantation

Ovarian cancer spheroids were generated using the hanging-drop method, following a protocol described in Laurent-Issartel et al. (Worzfeld et al., 2017). Briefly, 500 SKOV3 cells were resuspended in 20 µL of ascites and placed as drops on the underside of a Petri dish lid. Ascites were obtained with informed consent from patients with high-grade serous ovarian cancer at the F. Baclesse Cancer Center (Caen, France) and stored at the OvaRessources BRC (NF-S 96900, AFNOR N°2016:72860.5). Collection was authorized by the MESR (N°DC-2010-1243 and amendments) and approved by the CPP Northwest III Ethics Committee (Sept 17, 2011). The setup was incubated in a humidified chamber at 37 °C for 5 days to allow the formation of calibrated 3D ovarian cancer spheroids. To model cancer cell dissemination onto the mesothelium, a co-culture system was established. Met-5A cells were first seeded onto glass coverslips in 24-well plates at a density of 30'000 cells per well and cultured for 5 days in growth medium until confluence. The spheroids were then deposited onto the mesothelial monolayer and cultured in either complete medium or conditioned media from young or senescent cells. The area of disintegration of SKOV3 spheroids into the Met-5A monolayer was assessed at different time points by outlining them on Image J software. Each image was normalized to the same dimensions so that the areas could be compared.

## 2.7. Immunofluorescence microscopy

Immunofluorescence labelling of Fibronectin was performed in young and senescent HOF cells as previously described (Perié et al., 2024). Cells were seeded on glass coverslips in 24-well plates at a density of 2 × 10⁴ cells per well and incubated for 24 h. After fixation with 4 % paraformaldehyde in PBS, cells were washed with PBS containing 0.5 % BSA and permeabilized using 0.1 % Triton X-100 in PBS. Fibronectin was detected by indirect immunofluorescence using a polyclonal anti-Fibronectin antibody (#F3648, Sigma Aldrich) diluted 1:50 in PBS supplemented with 2 % BSA. After washing, coverslips were incubated

with Alexa Fluor® 568-conjugated anti-mouse secondary antibodies (Invitrogen), and nuclei were counterstained with DAPI (#D9542, Sigma Aldrich). Coverslips were mounted with ProLong® Gold Antifade Reagent (#P36930, Invitrogen) and imaged using a Zeiss LSM710 laser-scanning confocal microscope. Controls, in which primary antibodies were replaced with PBS, were negative. Fluorescence intensity in selected regions of interest was quantified using the Zen software (LSM710 system). Intensity values, linearly correlated with the number of pixels, were normalized to the number of cell nuclei stained with DAPI.

## 2.8. Gelatin zymography

To characterize MMP enzymatic activity in the secretome of senescent ovarian fibroblasts, gelatin zymography was performed. Conditioned media from young and senescent HOF cells were mixed with loading buffer containing 25 % Tris-HCl (0.5 M), 1 % SDS, 0.01 % bromophenol blue, and 20 % glycerol, and loaded onto a 10 % acrylamide gel containing 0.4 % gelatin. After electrophoresis, the gel was washed with 2.5 % Triton X-100. To activate gelatinase activity, the gel was incubated for 18 h in a developing buffer containing Tris-HCl (1 M), ZnCl₂, and CaCl₂. Gels were then stained with Coomassie Brilliant Blue R-250 for 15 min and destained using a methanol-glacial acetic acid solution. Zymograms were scanned with a GS-800 densitometer (BioRad, USA), and densitometric analysis was performed using ImageJ software.

## 2.9. Western blotting

Whole cell extracts were obtained from cultured cells with RIPA buffer (20 mM Tris, 150 mM NaCl, and 1 % NP-40), supplemented with a cocktail of protease inhibitors (Roche). The lysates were centrifuged at 15,000 g for 15 min at 4 °C to pellet the cellular debris and the protein concentration of the supernatant was determined by the BCA method (Sigma Aldrich). Total protein (25 µg) was separated on sodium dodecyl sulfate-polyacrylamide gel. The gels were electrotransferred onto a Hybond nitrocellulose membrane (GE Healthcare) at room temperature. Western blotting was done with monoclonal anti-cellular Fibronectin (#F6140), polyclonal anti-Fibronectin (#F3648), anti-p21<sup>waf1/cip1</sup> (#05-655, Millipore), anti-PARP (#9542, Cell Signaling Technology) anti-E-cadherin (#ab1416, Abcam), anti-N-cadherin (#ab79057, Abcam), anti-NGAL (neutrophil gelatinase-associated lipocalin) (#AF1757, Biotechne), anti-MMP-9 (#AF911, Biotechne), and anti-actin (#A5441, Sigma Aldrich) antibodies in a 0.1 % TBS-Tween (TBST) 0.1 % (v/v) containing 1 % BSA at 1:1000 dilution. After primary antibody incubation, membranes were washed in TBST 0.1 % and incubated for 1 h at RT with an anti-rabbit (#P0217, Dako) or anti-mouse (#P0161, Dako) IgG horseradish peroxidase-conjugated antibody in TBST 0.1 % containing 2 % non-fat dry milk (w/v) at a 1:5000 dilution. After four additional washes in TBST 0.1 %, blots were developed using enhanced chemiluminescence (#WBKLS0500, Millipore). Images were captured with the ImageQuant LAS 500 system (GE Healthcare). Densitometric analysis was performed using ImageJ Software.

## 2.10. Cytokines arrays

To characterize the senescent human ovarian fibroblast secretome, we performed a Cytokine Proteome Profiler Array targeting 106 proteins (#ARY022B, R&D Biotechne, Supplementary Table S1), according to the manufacturer's instructions. The arrays were incubated overnight with conditioned medium from young and senescent HOF cells at 4 °C. The blots were detected using an enhanced chemiluminescence. Images were captured with the ImageQuant LAS 500 system (GE Healthcare). Densitometric analysis of the arrays were performed using ImageJ Software.
## 2.11. Statistical analysis

Results, expressed as mean ± S.E.M were compared by analysis of variance followed by Student's *t*-test for unpaired data. A value of *p* < 0.05 was considered statistically significant.

# 3. Results

## 3.1. Assessment of replicative senescence in human ovarian fibroblasts

We assessed the senescent state of human ovarian fibroblasts (FBs) cells to characterize their SASP composition and ECM remodeling. To induce replicative senescence, fibroblasts were cultured until they reached a senescent state, as confirmed by SA-β-gal activity. Approximately 70 % of the cells were SA-β-gal-positive, compared to only 15 %

in early-passage young fibroblasts (Fig. 1A and B). In parallel, we measured $p21^{Waf1/Cip1}$ protein levels, which showed pronounced increases in senescent fibroblasts compared to young FBs, further confirming their senescent status (Fig. 1C and D). These results validate that the fibroblasts used in subsequent experiments were indeed in a senescent state. We have previously shown that Fn protein expression is induced during senescence in skin fibroblasts (Perié et al., 2024). Furthermore, our studies revealed that Fn distribution within the extracellular matrix is altered in senescent cells, with distinctly shorter Fn fibrils. To investigate whether similar alterations occur in senescent ovarian fibroblasts, we compared Fn levels in young and senescent cells. Western blot analysis demonstrated a significant 3-fold increase in total intracellular Fn protein in senescent cells compared to young cells (Fig. 1E and F). Additionally, several intracellular Fn isoforms were detected in senescent FBs but not in young FBs. To assess Fn secretion,

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Senescent state of human ovarian fibroblasts.</figcaption></figure>

(A) Representative images of young and senescent ovarian fibroblasts stained for the SA-β-gal. Scale bar is 100 µm. (B) Percentage of SA-β-gal positive cells in each group. (C—D) Relative $p21^{cif/waf}$ protein levels shown by Western blot (C) and densitometric analysis (D) in cell lysates. (E-F) Relative cellular Fn protein levels shown by Western blot (E) and densitometric analysis (F) in cell lysates. (G-H) Relative Fn levels in conditioned medium shown by Western blot (G) and densitometric analysis (H) in conditioned medium of young and senescent ovarian fibroblasts. (I-J) Immunofluorescent staining (I) and relative fluorescence intensity (J) of Fibronectin expressed in young and senescent fibroblasts. Staining was examined with immunofluorescence microscopy. Arrows indicate altered Fn fibril formation. Scale bar is 100 µm. Results are expressed as a mean ± SEM from three to five independent experiments. \* *p* value < 0.05; \*\* *p* value < 0.005; \*\*\* *p* value < 0.001. SA-β-gal, Senescence-associated β-galactosidase; Fn, Fibronectin; FBs, Fibroblasts; CM, conditioned medium.
we measured cellular and total Fn levels in conditioned media collected from fibroblasts incubated for 12 h in serum-free medium. Western blot analysis using an antibody specific to the ED-A domain of Fn (cellular Fn) revealed significantly reduced levels in the conditioned medium (CM) from senescent fibroblasts (Fig. 1G and H). When using a polyclonal antibody recognizing multiple epitopes shared by all Fn isoforms (total Fn), including ED-A negative forms, we detected additional lower molecular weight bands in the conditioned media of senescent fibroblasts, suggesting the presence of smaller Fn isoforms or proteolytic degradation products. Immunofluorescence staining further revealed that Fn fibrils were less abundant, shorter, and more disorganized in the ECM of senescent FBs, compared to those in young cells, indicating impaired matrix assembly (Fig. 1I and J). These observations suggest that replicative senescence in ovarian fibroblasts is associated with reduced Fn secretion and fibril assembly into the ECM, along with intracellular Fn accumulation. This altered Fn profile may serve as a potential marker of cellular senescence.

## 3.2. Characterization of the SASP composition of senescent ovarian fibroblasts

To profile the secretory phenotype of young and senescent human ovarian fibroblasts, we collected CM after incubating the cultures in serum-free medium for 12 h. The CM was analyzed using a cytokine array targeting 100 proteins. Among these, 18 proteins showed significant changes in senescent CM: 4 were downregulated (Serpin E1, IL-8, MCP-1, and uPAR), while 14 were upregulated (CHI3L1, CD26, CD147, FGF-7, GDF-15, IL-6, IL-18Bpa, CXCL9, MMP-9, RAGE, CXCL12,

THBS1, and CD106) (Fig. 2A and B, Supplementary Fig. S1). Several of the oversecreted factors identified are known components of the SASP, including IL-6 and CXCL9, which are involved in macrophage recruitment and polarization recruitment (Xuan et al., 2015; Beider et al., 2014), as well as MMP-9, a key extracellular matrix (ECM) remodeling enzyme (Winkler et al., 2020). Among the proteins detected in the array, MMP-9 was further validated by western blot, confirming its upregulation in senescent cells (Fig. 2C and D). Taken together, our results highlight a complex SASP signature in senescent FBs that encompasses inflammatory and immune-modulatory cytokines, chemokines, and ECM remodeling regulators, including several molecules with potential roles in cancer dissemination and metastasis.

## 3.3. Senescent ovarian fibroblasts show altered MMPs activity and ECM remodeling

Given the disrupted Fn matrix observed in senescent fibroblasts, we next assessed the activity of MMP, using gelatin zymography. As shown in Fig. 3A-F and Supplementary Fig. S2, conditioned media from senescent fibroblasts exhibited elevated levels of active MMP-2, as well as increased levels of MMP-9 monomers, MMP-9 dimers, and MMP-9/NGAL complexes, compared to young cell-derived media. These results were further confirmed by Western blot analysis performed in parallel (Fig. 3G-I). These data indicate that senescence in ovarian fibroblasts is associated with increased activity of key ECM regulators of ECM remodeling.

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** SASP profile of senescent ovarian fibroblasts. (A-B) Cytokine levels represented as heat map (A) and bars graph (B) measured in conditioned medium of young and senescent ovarian fibroblasts. (C–D) Relative MMP-9 protein levels shown by Western blot (C) and densitometric analysis (D) in conditioned medium. Results are expressed as a mean ± SEM from three independent experiments. \* p value < 0.05; \*\* p value < 0.005. SASP, Senescence-Associated Secretory Phenotype, FBs, Fibroblasts; CM, Conditioned Medium; MMP-9, Metalloproteinase-9.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Extracellular matrix remodeling in senescent ovarian fibroblasts. (A-F) Gelatin zymography (A) analysis of young and senescent ovarian fibroblasts. (B-F) Bands representing MMPs and NGAL were quantified via densitometric analysis. (G-I) Relative NGAL and MMP-9 protein levels shown by Western blot (G) and densitometric analysis (H-I) in conditioned medium. Results are expressed as a mean ± SEM from five independent experiments. \* p value <0.05; \*\* p value <0.005. Zymogram image has been inverted (dark bands on white background). CM, Conditioned Medium; FBs, Fibroblasts; MMPs, Metalloproteinases.</figcaption></figure>

### 3.4. Senescent fibroblast secretome promotes cancer cell collective migration

To investigate whether senescent fibroblasts enhance cancer cell motility, we used an Ibi di chamber assay that mimics wound healing model by generating a defined cell-free gap. As shown in Fig. 4, wound closure occurred more rapidly in complete medium (after 48 h for IGROV1 and 18 h for SKOV3 cells). In both cell lines, the presence of senescent CM accelerated migration compared to young CM (Fig. 4A-D). To ensure that the observed effects were not due to altered proliferation, the proliferation rates of SKOV3 and IGROV1 cells were monitored over 72 h in the presence of complete medium (DMEM +10% FBS), serum-free DMEM (DMEM), or CM from young or senescent FBs. As shown in Fig. 4E and F, both IGROV1 and SKOV3 cells proliferated more rapidly in complete medium compared to serum-free DMEM or either CM. No significant differences were observed between the serum-free condition and the young or senescent CM, suggesting that the fibroblast secretome, whether from young or senescent cells, does not promote proliferation of ovarian cancer cells. These results indicate that the secretome of senescent ovarian fibroblasts while not enhancing cancer cell proliferation, enhances the migratory capacity of SKOV3 and IGROV1 ovarian cancer cells.

### 3.5. Senescent fibroblast secretome promotes apoptosis resistance and EMT

To assess whether fibroblast CM affects cell viability, we measured PARP and cleaved PARP protein levels at the 72-h time point, as cleaved PARP is a widely recognized marker of apoptosis (Carduner et al., 2014). In IGROV1 cells, both young and senescent conditioned media had no effect on total or cleaved PARP levels, with cleaved PARP predominantly detected under serum-free DMEM conditions (Fig. 5A-C). In contrast, in SKOV3 cells, cleaved PARP levels were significantly reduced in the presence of senescent fibroblast conditioned medium. We also observed that total PARP levels appeared lower in SKOV3 cells exposed to senescent conditioned media compared to other conditions (Fig. 5D-F). These results suggest that senescent secretome may promote cancer cell survival by reducing apoptosis induced by serum deprivation. Ovarian cancer metastasis involves free-floating multicellular aggregates that

detach from the primary tumor and disseminate into the peritoneal cavity, where they survive in anchorage-independent conditions before attaching to the mesothelium which protects cancer cells from anoikis, a form of apoptosis triggered by loss or inappropriate cell adhesion (Carduner et al., 2014). To determine whether the senescent fibroblast secretome affects cancer cell survival under such conditions, SKOV3 cells were cultured on non-adherent surfaces to promote MCA formation and incubated for two days with complete medium, serum-free DMEM or CM from young or senescent fibroblasts. Western blot analysis was then performed on the non-adherent SKOV3 aggregates to evaluate PARP cleavage. As shown in Fig. 5G-I, cleaved PARP was detected in SKOV3 aggregates incubated with serum-free DMEM, and to a lesser extent in those treated with complete medium or young CM, whereas it was significantly reduced in aggregates exposed to senescent CM. These results, consistent with those observed under adherent conditions, suggest that the senescent fibroblast secretome promote resistance to apoptosis in ovarian cancer cells.

To further assess whether senescent fibroblasts support ovarian cancer cell survival, we examined markers associated with resistance to apoptosis, including the cadherin switch from E-cadherin to N-cadherin, a well-established hallmark of epithelial-to-mesenchymal transition in cancer cells (Araki et al., 2011; Loh et al., 2019). While E-cadherin levels appeared higher in serum-free DMEM in IGROV1 cells, no changes in N- or E-cadherin expression were observed in IGROV1 cells treated with either young or senescent conditioned medium (Fig. 6A-C). In SKOV3 cells, young conditioned medium had no effect on N- or E-cadherin expression, whereas senescent conditioned medium significantly reduced E-cadherin and increased N-cadherin levels (Fig. 6D-F), supporting the idea of an EMT-like switch. A similar trend was observed in SKOV3 multicellular aggregates, although to a lesser extent (Fig. 6G-I). Altogether, these results indicate that senescent CM promotes EMT, enhances ovarian cancer cell migration, and induces resistance to apoptosis, key steps in cancer cell dissemination.

### 3.6. Senescent fibroblast secretome enhances cancer cell invasion and dissemination across mesothelial monolayers

Invasion of ovarian cancer cells through the mesothelial layer is a key step in peritoneal metastasis and is known to be influenced by the TME
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**Fig. 4.** Senescent ovarian fibroblasts secretome promotes ovarian cancer cell migration. (A-D) Migration ability of IGROV1 (A-B) and SKOV3 (C-D) cells assessed by wound healing assay after incubation with DMEM +10 % FBS, young or senescent conditioned media. Scale bar is 100 µm. Cell migration was quantitatively evaluated by measuring the percentage of wound closure. (E-F) Proliferation analysis of IGROV1 cells cultured for 72 h (E) and SKOV3 cells cultured for 24 h (F) in the presence of DMEM +10 % FBS, young or senescent conditioned media. Results are expressed as a mean ± SEM from three independent experiments. \* p value <0.05; \*\* p value <0.005. CM, Conditioned Medium; AUC, Area Under the Curve; FBS, Fetal Bovine Serum.

(Laurent-Issartel et al., 2024). To better replicate the in vivo context and evaluate how senescent fibroblasts, as part of the TME, might promote cancer cell invasion and dissemination, we employed a physiologically relevant co-culture model in which SKOV3 spheroids were allowed to adhere to a confluent monolayer of mesothelial cells for 48 h in the presence of complete medium, young CM, or senescent CM. Spheroid disaggregation and invasion into the mesothelial layer occurred more rapidly in the presence of senescent fibroblast secretome than with young CM or complete medium (Fig. 7A, **Supplementary Video S1**). Specially, dissemination increased by 30 % at 24 h and 43 % at 48 h in senescent CM compared to DMEM, by 13 % at 48 h in young CM compared to DMEM, and by 26 % at 48 h in senescent CM compared to young CM (Fig. 7B). Collectively these data suggest that the secretome of senescent ovarian fibroblasts enhances migration, invasion and dissemination of ovarian cancer cells, thereby potentially contributing to metastasis formation and cancer dissemination.

## 4. Discussion

Over the past decades, the role of senescent stromal cells in shaping the ovarian tumor microenvironment has gained significant attention. While the contribution of senescent mesothelial cells to ovarian cancer progression is well described (Thapa et al., 2024; Mikula-Pietrasik et al., 2016; Ksiązek et al., 2009), our study provides new insights into how senescent human ovarian fibroblasts create a permissive niche that supports ovarian cancer dissemination. We show that senescent fibroblast secretome modulates ovarian cancer cell behavior through the release of pro-inflammatory SASP factors, active metalloproteinases, and altered Fn secretion, characterized by reduced levels of cellular Fn and the presence of lower molecular weight isoforms or degradation products. This altered matrix organization enhances ovarian cancer cell migration and invasion without affecting proliferation.

In our study, the responses differed between SKOV3 and IGROV1 cells, with IGROV1 showing reduced effects. These differences likely reflect the distinct origins and phenotypes of the two cell lines with
<figure><img src="image_13.png" /><figcaption>**Fig. 5.** Senescent fibroblasts secretome promotes resistance to apoptosis.

(A-I) PARP and cleaved PARP protein levels shown by Western blot and quantified via densitometric analysis in adherent IGROV1 (A-C), SKOV3 (D-F), and MCA SKOV3 (G-I) cells incubated with DMEM +10 % FBS, DMEM, young or senescent conditioned media. Results are expressed as a mean ± SEM from three independent experiments. \* p value <0.05; \*\* p value <0.005. CM, Conditioned Medium; FBS, Fetal Bovine Serum; MCA: Multicellular Aggregates.</figcaption></figure>

SKOV3, derived from adenocarcinomas in ascites, displays a more mesenchymal-like phenotype, whereas IGROV1, derived from a solid primary tumor, retains a more epithelial-like profile (Lopez et al., 2023). Pazos et al. also reported that, compared to SKOV3, IGROV1 cells express low levels of TWIST, an inducer of N-cadherin, and show no change in SNAIL, a repressor of E-cadherin, after TGF-β treatment (Pazos et al., 2017). Consistent with this, a recent study from our group demonstrated that IGROV1 cells migration was slower than that of SKOV3 cells in the presence of TGF-β and TNF-α (Boché et al., 2024), both molecules that can be found in the SASP and known to promote migration, in line with our present findings. Furthermore, SKOV3 cells are p53-null, a feature that may increase their plasticity and responsiveness to EMT signals. Indeed, p53 activity has been shown to suppress EMT in cancer cells through the p53-miR-200c pathway (Chang et al., 2011). Together, these studies suggest that SKOV3 cells are more sensitive to EMT-promoting cues than IGROV1. Aging, a major risk factor for ovarian cancer, is associated with the accumulation of senescent cells that alter the tissue landscape in ways that favor tumor progression. A higher burden of senescent cells correlates with increased malignant

ascites in patients, highlighting their potential role in disease severity (Veenstra et al., 2022; Uruski et al., 2021). However, experimental evidence is just beginning to emerge about how the SASP promotes dissemination a in ovarian cancer. In this study, we analyzed the secretome of senescent fibroblasts, known for their abundant ECM production and presence both in the deeper layers of the peritoneum and at primary ovarian tumor sites. Mapping the SASP of senescent ovarian fibroblasts revealed the overexpression of several cytokines. Well-known senescence-associated factors such as RAGE, IL-6, and GDF-15, previously implicated in EMT and cancer cell migration in ovarian and other cancers were found to be upregulated, alongside additional markers (Veenstra et al., 2022; Lin et al., 2022; Guo et al., 2019). Interestingly, we also identified novel cytokines, including CHI3L1 and FGF-7, which are not yet established as senescence markers, but whose overexpression has been associated with chemoresistance and ovarian cancer metastasis (Mortan et al., 2024; Chiang et al., 2015). Surprisingly, IL-8 and MCP-1 were found downregulated in our analysis. Although frequently associated with tumor-promoting activities, both cytokines have context-dependent dual roles and can also contribute to
<figure><img src="image_14.png" /><figcaption>**Fig. 6.** Senescent fibroblasts secretome promotes epithelial-mesenchymal transition. (A-I) N-cadherin and E-cadherin protein levels shown by Western blot and quantified via densitometric analysis in adherent IGROV1 (A-C), SKOV3 (D-F), and MCA SKOV3 (G-I) cells incubated with DMEM +10% FBS, DMEM, young or senescent conditioned media. Results are expressed as a mean ± SEM from three independent experiments. \* p value <0.05; \*\* p value <0.005; \*\*\* p value <0.001. CM, Conditioned Medium; FBS, Fetal Bovine Serum; MCA: Multicellular Aggregates.</figcaption></figure>

immune surveillance (David et al., 2016; Deshmane et al., 2009; Takasugi et al., 2023). In pancreatic cancer, patients with higher circulating levels of MCP-1 showed improved survival. In the same study, MCP-1 was also shown to mediate antiproliferative and proapoptotic effects of monocytes on pancreatic cancer cells (Monti et al., 2003). In our context, the downregulation of IL-8 and MCP-1 could reflect a mechanism by which senescent fibroblasts reduce immune surveillance, thereby favoring ovarian cancer dissemination. These findings highlight promising new candidates for further investigation of the role of senescent fibroblasts and their potential contribution to cancer dissemination. Importantly, while most ovarian cancers disseminate through peritoneal spread, approximately 10% of cases metastasize via hematogenous routes (Yeung et al., 2015). The presence of a senescent

fibroblast population may facilitate both peritoneal and non-peritoneal metastasis, as these cells are present in deeper layers of the peritoneum and within ovarian connective tissue. By altering ECM properties and promoting invasion, senescent fibroblasts could act as key enablers of systemic dissemination. However, their specific role in non-peritoneal metastatic niches remains largely under-explored.

Recent studies highlight that ECM remodeling observed in cancer is largely driven by stromal cell-derived matrix alterations rather than by the cancer cells themselves (Puttock et al., 2023; Gopal et al., 2017). These stromal changes influence both malignant and non-malignant cells within the tumor microenvironment, promoting a niche that supports tumor progression. Senescent mesothelial cells have been shown to express an ECM enriched in Fibronectin, Laminins, and Hyaluronan,
<figure><img src="image_15.png" /><figcaption>Fig. 7. Senescent fibroblasts secretome promotes ovarian cancer cell dissemination.</figcaption></figure>

(A) Representative images of SKOV3 ovarian cancer spheroids deposited on Met-5A monolayer and incubated in DMEM, young or senescent conditioned medium for 48 h. (B) Areas (µm²) of spheroid disaggregation calculated with the software ImageJ. Scale bar is 200 µm. Results are expressed as a mean ± SEM from three independent experiments. \* p value <0.05; \*\* p value <0.005; \*\*\* p value <0.001. CM, Conditioned Medium.

generating a senescence-associated matrisomal phenotype. This altered peritoneal barrier enhances the colonization of invading ovarian cancer cells and contributes to the metastatic burden of the disease (Thapa et al., 2024). Oxidative stress has been found to induce senescence in human peritoneal mesothelial cells derived from the omentum, a tissue frequently colonized by ovarian tumors, leading to increased Fn production and thereby facilitating the adhesion and dissemination of ovarian cancer cells (Kenny et al., 2014). We previously demonstrated the presence of Fn isoforms in patient samples (Carduner et al., 2013), which could originate from senescent stromal cells such as mesothelial cells or fibroblasts. The senescent phenotype observed in ovarian fibroblasts is characterized by a marked disorganization of Fn architecture, despite elevated intracellular Fn levels. This discrepancy between Fn production and matrix incorporation is consistent with our previous findings in aging skin fibroblasts, where increased Fn mRNA and protein levels did not result in proper ECM integration (Perié et al., 2024). In line with these earlier observations, our current in vitro model shows that senescent ovarian fibroblasts produce and accumulate Fn but fail to assemble it into a structured fibrillar network. Additionally, several Fn isoforms with lower molecular weight were detected both intracellularly and into the ECM. As observed in our previous study on skin fibroblasts (Perié et al., 2024), these results in ovarian fibroblasts suggest that altered Fn fibril formation and the presence of Fn fragments contribute to the onset of cellular senescence, which is associated with increased metalloproteinase activity and enhanced SASP release. Norris et al. demonstrated that Fn fragments can induce fibroblast expression of matrix metalloproteinases and may even act as proteolytic enzymes themselves. Notably, a 90-kDa plasmin-generated Fn fragment shares homology with streptokinase (Norris et al., 1982). Fn fragments produced by endogenous proteases have also been shown to act as potent chemoattractant for human peripheral blood monocytes, in contrast to intact Fn, which lacks this activity. Fn fragments were also found to significantly enhance the migration of HT1080 fibrosarcoma cells compared to intact Fn, particularly in the context of periodontal disease and chronic foot wounds in diabetes (Stanley et al., 2008). This migratory effect may explain how the senescent secretome enhances SKOV3 and IGROV1 ovarian cancer cell migration in our study. We previously showed that Fn amyloid-like aggregation impairs its ECM incorporation and promotes SKOV3 cell migration, emphasizing the role

of altered Fn in ovarian cancer dissemination (Bascetin et al., 2018).

Senescent fibroblasts in our model exhibit a pronounced secretory phenotype, characterized by high levels of MMP-9, a key enzyme involved in ECM degradation commonly associated with aging and tumor invasion across various cancers (Jabłońska-Trypuć et al., 2016; Panwar et al., 2018; Huang, 2018). We also observed upregulation of neutrophil gelatinase-associated lipocalin (NGAL), which binds and stabilizes MMP-9, prolonging its extracellular activity (Yan et al., 2001). The NGAL/MMP-9 complex has been described in other cancers and is associated with poor prognosis (Candido et al., 2016). NGAL has also emerged as an early screening biomarker in ovarian cancer, with higher expression reported in borderline and grade 1 tumors, further suggesting its relevance as a molecular target for early diagnosis (Lim et al., 2007). NGAL has been linked to the downregulation of E-cadherin and upregulation of Vimentin, hallmarks of EMT (Li et al., 2016).

To our knowledge, this is the first report of NGAL and MMP-9 being co-secreted by senescent ovarian fibroblasts. Their upregulation supports a key role for senescent fibroblasts in shaping the ovarian tumor microenvironment and promoting EMT and cancer cell invasion.

Our findings reveal two complementary effects of senescence-induced ECM remodeling, impaired Fn assembly disrupts matrix structure and signaling, while NGAL-stabilized MMP-9 enhances ECM stiffness and degradation. Together, these changes generate a microenvironment that favors cancer cell survival and invasion (Lopez et al., 2023; Carduner et al., 2014; Carreiras et al., 2002) as evidenced by migration and invasion assays. Furthermore, the upregulation of N-cadherin and downregulation of E-cadherin confirmed the invasive phenotype of ovarian cancer cells by EMT. This cadherin switch also promotes resistance to anoikis (Kim et al., 2012), which was observed in our study when SKOV3 cells were treated with the senescent fibroblast secretome. Altogether, these data highlight the role of the senescent ovarian fibroblast secretome in promoting ovarian cancer cell disseniation and underscore the complexity of a multiscale and multicellular tumor microenvironment. This complexity presents a challenge for current therapies but also opens new avenues for intervention, such as targeting senescent cells or their secretome using senolytic strategies (Chaib et al., 2022).
## 5. Conclusion

These findings provide strong evidence that senescent fibroblasts are potent modulators of the tumor microenvironment, especially in ovarian cancer. Altered Fibronectin organization and increased MMPs activities may underlie these effects by disrupting extracellular matrix integrity, linking age-related stromal changes to disease progression. Future functional studies modulating Fn levels, as well as investigating the impact of Fn fragmentation and the newly identified SASP factors in senescent fibroblasts, will be essential to assess more precisely their contribution to ovarian cancer dynamics. By uncovering the dual role of senescent fibroblasts in ECM remodeling and EMT induction, our study adds a new dimension to the understanding of stromal influences in ovarian cancer.

### Abbreviations

<table><tr><td>ECM</td><td>Extracellular matrix</td></tr><tr><td>EMT</td><td>Epithelial-Mesenchymal Transition</td></tr><tr><td>TME</td><td>Tumor Microenvironment</td></tr><tr><td>Fn</td><td>Fibronectin</td></tr><tr><td>MMPs</td><td>Metalloproteinases</td></tr><tr><td>SASP</td><td>Senescence-associated secretory phenotype</td></tr><tr><td>NGAL</td><td>Neutrophil Gelatinase-Associated Lipocalin</td></tr><tr><td>FBS</td><td>Fetal Bovine Serum</td></tr><tr><td>FBs</td><td>Fibroblasts</td></tr><tr><td>CM</td><td>conditioned medium</td></tr></table>

Supplementary data to this article can be found online at https://doi.org/10.1016/j.exger.2025.112909.

### CRediT authorship contribution statement

**Luce Perié:** Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. **Alexandra Landras:** Investigation. **Mickaëla Bignard:** Investigation. **Marion Theuret:** Data curation. **Mélanie Briand:** Resources. **Franck Carreiras:** Data curation. **Cédric R. Picot:** Writing – review & editing, Validation, Supervision, Project administration, Investigation, Funding acquisition, Data curation, Conceptualization.

### Funding sources

This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 945380, from the MENRT (CY Cergy Paris Université) and from CY Initiative Horizon Programme.

### Declaration of competing interest

The authors declare that there is no conflict of interest.

### Data availability

Data will be made available on request.

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