# Deciphering the interaction between osteosarcoma and mesenchymal stem cells in a 3D bone-mimetic co-culture model

Giada Bassi, Mohamed Saqawa, Lorenzo Apolloni, Silvia Díaz-Prado, Emilie Ollivier, Romain Levergeois, Monica Sandri, Elisabetta Campodoni, Denis Cochonneau, Silvia Panseri, et al.

► To cite this version:

Giada Bassi, Mohamed Saqawa, Lorenzo Apolloni, Silvia Díaz-Prado, Emilie Ollivier, et al. Deciphering the interaction between osteosarcoma and mesenchymal stem cells in a 3D bone-mimetic co-culture model. Biomedicine and Pharmacotherapy, 2026, 195, pp.118956. 10.1016/j.biopha.2025.118956. inserm-05444299

HAL Id: inserm-05444299

https://inserm.hal.science/inserm-05444299v1

Submitted on 6 Jan 2026

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# Deciphering the interaction between osteosarcoma and mesenchymal stem cells in a 3D bone-mimetic co-culture model

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Giada Bassi<sup>a</sup>, Mohamed Saqawa<sup>a,b</sup>, Lorenzo Apolloni<sup>a,c</sup>, Silvia Díaz-Prado<sup>d,e,f</sup>, Emilie Ollivier<sup>f</sup>, Romain Levergeois<sup>g,h</sup>, Monica Sandri<sup>a</sup>, Elisabetta Campodoni<sup>a,b</sup>, Denis Cochonneau<sup>f,g,h,i,j</sup>, Silvia Panseri<sup>a</sup>, Dominique Heymann<sup>f,h,i,j</sup>, Monica Montesi<sup>a,*,**</sup>

## ARTICLE INFO

**Keywords:**

Osteosarcoma
Mesenchymal stem cells
3D co-culture
Tumor microenvironment
Extracellular matrix
Bone-mimetic scaffold

## ABSTRACT

Osteosarcoma (OS) is a highly heterogeneous and aggressive bone malignancy whose complexity is strongly influenced by its Tumor Microenvironment (TME). Within this niche, Mesenchymal Stem Cells (MSCs) play a pivotal role in tumor progression by undergoing phenotypic and functional reprogramming under tumor-derived cues, acquiring Cancer-Associated Fibroblast (CAF)-like features that promote proliferation, invasion, and immune evasion. The Extracellular Matrix (ECM), once regarded as a passive structural element, is now recognized as an active regulator of tumor behavior, acting as a reservoir of signaling molecules and a modulator of cell fate. However, the molecular crosstalk between OS cells, MSCs, and the ECM remains poorly understood, largely due to the limitations of conventional two-dimensional models. In this study, we established a three-dimensional (3D) bone-mimetic model of osteosarcoma (mOS-3D) by co-culturing human OS cells and MSCs within a hydroxyapatite–collagen (MgHA/Coll) scaffold that recapitulates the biochemical and structural features of native bone ECM. This in vitro platform reproduces key aspects of the OS microenvironment, enabling the investigation of tumor-stroma interactions and their impact on stemness, stromal activation, and ECM remodeling. The mOS-3D model provides a physiologically relevant and tunable system for studying the cellular mechanisms driving OS progression and offers a promising preclinical tool to explore therapeutic strategies targeting the TME.

## 1. Introduction

Osteosarcoma (OS) is a rare primary malignant bone tumor

predominantly affecting adolescents and adults over 60 years of age, with an incidence of ~3–4.5 cases per million per year [1,2].

The current therapeutic regimen, which combines neo-adjuvant and

<footnote><sup>a</sup> Institute of Science, Technology and Sustainability for Ceramics (ISSMC), National Research Council (CNR), Faenza 48018, Italy</footnote>

<footnote><sup>b</sup> Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Studies of Messina, Messina 98166, Italy</footnote>

<footnote><sup>c</sup> Department of Neuroscience, Imaging and Clinical Science, University of Studies "G. D'Annunzio", Chieti 66100, Italy</footnote>

<footnote><sup>d</sup> Grupo de Investigación en Terapia Celular y Medicina Regenerativa, Departamento de Fisioterapia, Medicina y Ciencias Biomédicas, Facultad de Ciencias de la Salud, Universidade da Coruña (UDC), Coruña, Galicia 15006 A, Spain</footnote>

<footnote><sup>e</sup> Grupo de Investigación en Terapia Celular y Medicina Regenerativa, Grupo de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC) Servizio Galego de Saúde (SERGAS), Coruña, Galicia 15006 A, Spain</footnote>

<footnote><sup>f</sup> Institut de Cancérologie de l'Ouest, Tumor Heterogeneity and Precision Medicine Laboratory, Saint-Herblain, Nantes 44805, France</footnote>

<footnote><sup>g</sup> Sorbonne Université, Inserm U933, AP-HP, Hôpital d'Enfants Armand-Trousseau, 75012 Paris cedex 12, France</footnote>

<footnote><sup>h</sup> Nantes Université, CNRS, UMR6286, US2B, Biological Sciences and Biotechnologies unit, Nantes 44322, France</footnote>

<footnote><sup>i</sup> University of Sheffield, School of Medicine and Population Health, Medical School, Beech Hill Road, Sheffield S10 2RX, UK</footnote>

<footnote>* Corresponding author at: Institut de Cancérologie de l'Ouest, Tumor Heterogeneity and Precision Medicine Laboratory, Saint-Herblain, Nantes 44805, France.</footnote>

<footnote>** Correspondence to: Institute of Science, Technology and Sustainability for Ceramics, National Research Council of Italy, via Granarolo n. 64, Faenza, RA 48018, Italy.</footnote>

<footnote>E-mail addresses: giada.bassi@issmc.cnr.it (G. Bassi), mohamed.saqawa@issmc.cnr.it (M. Saqawa), lorenzo.apolloni@issmc.cnr.it (L. Apolloni), s.diaz1@udc.es (S. Díaz-Prado), emilie.ollivier@ico.unicancer.fr (E. Ollivier), romain.levergeois@aphp.fr (R. Levergeois), monica.sandri@issmc.cnr.it (M. Sandri), elisabetta.campodoni@issmc.cnr.it (E. Campodoni), denis.cochonneau@ico.unicancer.fr (D. Cochonneau), silvia.panseri@issmc.cnr.it (S. Panseri), Dominique.Heymann@univ-nantes.fr (D. Heymann), monica.montesi@issmc.cnr.it (M. Montesi).</footnote>
adjuvant chemotherapy (i.e. cisplatin, doxorubicin, methotrexate and ifosfamide), accompanied by surgery and radiotherapy [3], has significantly improved the 5-year survival rate of patients with localized disease in 60 % of cases [4]. However, no consensual regimen is reported for OS metastasis and relapses, and long-term recovery remains a significant challenge with an overall survival rate around 20 % [5].

The limited effectiveness of treatments can be attributed to the intrinsic heterogeneity of OS. Different histological subtypes depending on the predominant type of Extracellular Matrix (ECM) [6] can be identified among OS patients and a heterogeneous set of tumor clones with distinct morphology, phenotype, genotype, metabolism, immunogenicity and, proliferative and metastatic potential coexist in a single tumor [7,8]. Mostly, tumor cells reside in a dynamic Tumour Microenvironment (TME), which consists of cellular (e.g. bone and immune cells, endothelial cells) and molecular components within a three-dimensional (3D) ECM [9]. During cancer progression, the TME emerges as a dynamic 3D milieu resulting from close interactions between genetically altered tumor cells and the host, continuously adapting, predominantly orchestrated by tumor cells, coordinating molecular and cellular events to meet the metabolic demands of tumor cells [10].

Mesenchymal Stem Cells (MSCs) specifically play a crucial role in osteosarcomagenesis[11] as well as in tumor development and progression [12–14]. They can be recruited to the TME from distant sites by cancer cells releasing cytokines (e.g. Interleukin 6 – IL-6) and “educated” into pro-tumoral mesenchymal phenotypes (T-MSCs), which dramatically accelerate the proliferation and metastatic potential of tumor cells [12]. A reciprocal, cross-destructive circuit between T-MSCs and OS cells, mediated by Interleukin-8 (IL-8), further promotes tumor progression [15]. Various mechanisms are involved in promoting the stem-like phenotype in tumor cells through MSC interaction, including cell fusion [16], the direct transformation of MSCs into Cancer Stem Cells (CSCs) [17,18] and the crosstalk mediated by cytokines and growth factors secretion [19–22]. MSCs also contribute to OS progression by differentiating into Cancer-Associated Fibroblasts (CAFs) following TME-mediated activation [23], leading to a heterogeneous population of fibroblasts-like phenotypes, with various functions and activation degree within a single OS [24–27]. However, the impact of MSCs in cancer remains controversial, and more studies involving the use of more mimetic *in vitro* models capable of mimicking the complexity of the 3D TME are needed to fully uncover the role of MSCs on tumor cells and vice versa. Significant efforts have been made over recent decades to replicate the complexity and dynamicity of the TME in a laboratory setting [28,29]. It is widely recognized the key role assumed by biomaterials as naturally derived or synthetic matrices in the form of scaffold for their ideal support in cellular behavior and, mostly, in resembling the ECM properties of the native tissue, both the normal and the diseased one [10, 30]. The nano-structural, physical, mechanical and biochemical support provided by the scaffold, in line with the required tissue functions, has a major role in actively supplying bioactive cues to the cells, regulating and stimulating specific downstream signaling pathways, improving the whole complexity of the system. Accordingly, the design of more complex *in vitro* models closely capturing the human microenvironment may enhance testing reliability and outcomes, reduce the gap between *in vitro* and *in vivo* studies, and, eventually, decrease reliance on animal models when feasible [28].

In this study, we developed a biomimetic and more predictive, *in vitro* 3D co-culture model of OS, referred to as mOS-3D. This system integrates a biomimetic scaffold composed of magnesium-doped hydroxyapatite and type I collagen (MgHA/Coll), which has been previously validated for regenerative medicine applications [31,32]. The MgHA/Coll scaffold is employed as a bone-mimetic extracellular matrix to replicate the TME. The scaffold is synthesized through a bioinspired mineralization process, and for the first time, this study explores the dynamic interactions between this biomimetic matrix and two representative cellular populations of the OS microenvironment. A

comprehensive characterization of the cellular behavior and signaling pathways of the whole co-culture system, involved in the interaction between GFP-MNNG/HOS OS cells, immortalized Bone Marrow-derived Mesenchymal Stem Cells (iBM-MSCs) and the bone-like MgHA/Coll scaffold has been investigated, compared to 3D scaffold-based monoculture and scaffold-free conditions.

This model lays the groundwork for the development of increasingly sophisticated systems that will consider the exploration of multiple cellular phenotypes in relation to a complex three-dimensional TME.

## 2. Experimental section

### 2.1. Cell culture

Human MNNG/HOS osteosarcoma cell line was purchased from American Tissue Cell Collection (USA, ref#CRL-1547). MNNG/HOS cells were stably transfected with pcDNA3-GFP vector (Invitrogen) following the manufacturer's protocol (FuGENE® HD Transfection Reagent, Promega). GFP-expressing MNNG/HOS cells (GFP-MNNG/HOS) were selected by antibiotic pressure for several weeks. Both MNNG/HOS and GFP-MNNG/HOS cells were cultured in DMEM high glucose (Lonza) supplemented with 5 % Foetal Bovin Serum (FBS) (Gibco), 1 % L-glutamine (200 mM, Gibco).

Immortalized Human Bone Marrow-derived Mesenchymal Stem cells (iBM-MSCs, referred to iMSC#9) were generated by SV40LT viral transduction as described in Piñeiro-Ramil et al. [33] and cultured in the same condition reported for GFP-MMNG HOS cells. Cells were grown in an incubator at 37°C, 5 % CO₂ and controlled humidity conditions. The cells were detached from the flask by trypsinization and centrifuged. The cell number and viability were assessed by Trypan Blue Dye Exclusion Test and all cell handling procedures were performed in sterile conditions under laminar flow hood. All cell lines used were negative for mycoplasma.

### 2.2. Synthesis and characterization of the MgHA/Coll scaffold

The MgHA/Coll scaffold was used as bone-like matrix and synthesized by biomineralization process as reported by Krishnakumar et al. [32]. Briefly, an acid aqueous suspension composed of 150 g of type I collagen gel (Typeone Biomaterials S.r.l., Calimera, LE, Italy) and phosphoric acid (2.41 g in 500 mL; H₃PO₄, 85 wt%, Sigma Aldrich) was prepared at room temperature. A basic aqueous suspension composed of 0.35 g of magnesium chloride (MgCl₂·6 H₂O, 99 wt%, Sigma Aldrich) and a calcium hydroxide suspension (2.71 g in 500 mL; Ca(OH)₂, 95 wt %, Sigma Aldrich) was prepared at room temperature. Then, the acid suspension was dropped into the basic one at 25 °C under stirring conditions; the pH neutralization induces the nucleation of hydroxyapatite nanocrystals (HA) doped with magnesium ions (MgHA) onto self-assembling collagen fibres (Coll), forming the MgHA/Coll hydrogel. The hydrogel was matured for 2 h at 25 °C, washed and freeze-dried (–40 °C and + 25 °C) for 48 h under 0.086 mbar vacuum conditions (5 Pa, LIO 3000 PLT). The scaffolds were stabilized by Dehydrothermal (DHT) crosslinking at 160 °C for 48 h under a pressure of 0.01 mbar. The scaffolds (ø: 9 mm; h:6 mm) were sterilized by 25 kGy γ-ray irradiation and used in these biological studies. In a previously published work [34], the scaffold was characterized by Scanning Electron Microscopy (SEM) (ESEM quant 200) and evaluation of its chemical-physical behavior was performed by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES), X-ray diffraction (XRD), Fourier transform infrared spectroscopy in the attenuated total reflection mode (FTIR-ATR) and Thermogravimetric analysis (TGA). Swelling and degradation behavior analysis were also reported in the published work.

### 2.3. Development of the mOS-3D model

The mOS-3D model was developed in vitro by combining the MgHA/
Coll scaffold with GFP-MNNG/HOS osteosarcoma cells and iBM-MSCs. Briefly, the MgHA/Coll scaffold (~ 0.9 mm; h: 6 mm) was pre-conditioned in cell culture media for 24 h before cell seeding. The GFP-MNNG/HOS cells and iBM-MSCs were mixed at 1:1 ratio and seeded at the total density of 50 × 10³ cells/scaffold. The model was incubated for 20 min for cellular pre-adhesion before cell media addition. Both cell types were seeded at a density of 50 × 10³ cells/scaffold to obtain the 3D scaffold-based monoculture conditions (3D iBM-MSCs monoculture and 3D GFP-MNNG/HOS monoculture). The same groups were performed under scaffold-free conditions. After 7 and 14 days of culture, the models were collected for different biological analysis.

## 2.4. Actin and DAPI staining and scaffold colonization

The cell morphology of the 3D scaffold-based models was analyzed by Actin and DAPI staining, following the manufacturer's instructions. Briefly, the samples were fixed in 4% Paraformaldehyde (PFA) (Sigma) and permeabilized in 0.1% Triton-X 100 (Sigma) in Phosphate-Buffered Saline (PBS) 1X (Gibco), following the manufacturer's instructions. The samples were stained with Rhodamine Phalloidin Actin red 555 ready probes (Invitrogen) for F- actin filaments detection and DAPI (4',6-diamidino-2-ylindole, dihydrochloride) (Sigma) staining was used for cell nuclei counterstaining. The ability of the cells in colonizing the scaffold was investigated at day 7 by vertically sectioned the scaffold to measure the distance travelled by the cells from the seeding surface to the core of the scaffold. The NIS-Element software was used for image analysis (Nikon) and the "line" tool. For the analysis, 3 images/condition were acquired, and 4 measurements/image were performed and mediated. For the experiment, two biological replicates for each condition were performed (N = 2) and the images were acquired at the Inverted Ti-E Fluorescence Microscope using TRITC, FITC and DAPI filters for iBM-MSCs, GFP-MMNG/HOS cells and cell nuclei identification, respectively.

## 2.5. Scanning Electron Microscopy (SEM)

The cell morphology and cell-ECM interaction in the 3D scaffold-based models was also analyzed by SEM. The samples were prepared for SEM analysis as reported by Lee at al. [35] and Fradique et al. [36]. Briefly, the samples were fixed in 2.5 % (v/v) Glutaraldehyde (Merk KgaA) in 0.1 M (w/v) Sodium Cocodylate Buffer (pH 7.4, sigma) for two hours at 4°C. For the SEM analysis, the samples were washed twice in water and frozen at -20°C overnight before being freeze-dried (-40 °C and +25 °C) for 48 h under 0.086 mbar vacuum conditions (5 Pa, LIO 3000 PLT). The samples were fixed on aluminum stubs by using adhesive tape and graphite glue and sputtered coated with 20 nm thin gold film. The images were acquired under high vacuum conditions. For each condition, a biological duplicate was performed (N = 2).

## 2.6. Gene expression

The gene expression was analyzed by Real Time PCR, following the manufacturer's instructions. At each time point, the samples were homogenized in Tri Reagent (Invitrogen). For the 3D scaffold-based models, each sample was collected and homogenized in two steps of Tri Reagent by pestle-mediated breaking and vigorous stirring. For the co-culture models, both scaffold-free and 3D scaffold-based conditions, the iBM-MSCs and the osteosarcoma cells were not separated during the cellular lysis and the total RNA derived from both cell types have been analyzed by Real Time PCR and RNA sequencing, subsequently reported in the "RNA sequencing" paragraph of the experimental section. The RNA extraction and purification was performed by Direct-zol RNA MiniPrep Kit (Zymo Research), following the manufacturer's instructions. The RNA quantification and purity degree was analyzed at the Nanodrop One Microvolume UN-Vis Spectrophotometer (Thermo Fisher Scientific), following the manufacturer's instructions. The RNA was reverse transcribed into a single strand of cDNA using the High-

Capacity cDNA Reverse Transcription Kit (Applied Biosystem), following the manufacturer's instructions. The Real Time PCR was performed using TaqMan Gene Expression Assay Kits for CD105 (Endoglin, Hs00923996_m1), CD90 (Thy-1, Hs00264235_s1) and CD73 (NT5E, Hs04234686_m1) as specific MSCs markers to investigate their stem and mesenchymal phenotype. TaqMan Gene Expression Assay kits for NANOG (Hs04399610_g1), OCT-4 (Hs00999632_g1), SOX-2 (Hs01053049_s1) were used to investigate the tumor stemness of the co-culture models. TaqMan Gene Expression Assay kits for Alkaline Phosphatase (ALP, Hs01029144_m1), Bone Morphogenetic Protein 2 (BMP-2, Hs00154192_m1) and RUNX-2 (Hs00231692_m1) were used to investigate the potential osteogenic commitment of MSCs in the co-culture system, together with Hypoxia Inducible Factor 1 alpha (HIF-1α, Hs00153153_m1), Interleukin 6 (IL-6, Hs00174131_m1), Interleukin 8 (IL-8, Hs00174103_m1) and Alpha Smooth Muscle Actin (α-SMA) (ACTA2, Hs00426835_m1) as markers of the tumor microenvironment and the potential MSCs differentiation toward CAFs. The iBM-MSCs monoculture and the scaffold-free condition were used as control groups. Actin-β (ACTB, Hs01060665_g1) and GAPDH (Hs99999905_m1) were used as housekeeping gene. Two experiments were performed with a total of six biological replicates for each condition (N = 6). Each biological replicate was analyzed in three technical replicates (N = 3). Data were collected from QuantStudio 1 Real Time PCR System (Applied Biosystem) and the relative quantification of target genes was assessed by Comparative Threshold (CT) method (ΔΔCT) where relative gene expression level equals to 2^ΔΔCT [37].

## 2.7. Enzyme-Linked Immunosorbent Assay (ELISA)

The detection of Metalloproteases 2 (MMP-2) and 9 (MMP-9), together with Tissue Inhibitor of Metalloproteases - 3 (TIMP-3) and TIMP-4 has been performed on both the 3D scaffold-based and scaffold-free condition by ELISA kit (Invitrogen), following the manufacturer's instructions. Briefly, at each time point the supernatant of the samples was collected, centrifuged at 2000 g for 20 min at 4°C to remove cellular debris and stored at -80°C until the analysis. The standard curve was generated by using the curve-fitting mode of the GraphPad Prism software (version 8.0.1).

## 2.8. RNA Sequencing

RNA was extracted and purified from the different 3D scaffold-based models at 7 and 14 days of culture as reported in the "Gene expression" paragraph of the Experimental Section. RNA extraction (all RIN > 9.0), library preparation and RNAseq analyses were performed with Eurofins (Constance, Germany). Briefly, 2 µg of total RNA was isolated using the Qiagen RNeasy Mini Kit and further processed in Illumina's TruSeq Stranded mRNA Library kit. Libraries were sequenced on Illumina NextSeq 500 as paired-end 42-nt reads. The reads were aligned with the hg38 reference genome STAR [38] and the count matrix was obtained using feature counts function from R package Rsubread [39]. Genes receiving less than 10 reads on average across the compared groups were removed. The list of differentially expressed genes from DESeq2 output [40] was selected based on the adjusted p value using the Benjamini-Hochberg procedure (p value Cut off = 0.05 and q value Cut off = 0.10). Gene ontology, KEGG and Reactome pathway enrichment analyses were carried out using the open source Cytoscape software platform [41]. STRING database and MCODE Cytoscape's app were used to determine molecular complexes [42,43]. Three independent biological replicates have been analyzed for each culture condition.

## 2.9. Statistical analysis

Statistical analysis was performed by using GraphPad Prism Software (8.0.1 version). For both the 3D scaffold-based and scaffold-free condition, the results of Real Time PCR regarding the gene expression of
Endoglin, CD90, CD73, BMP-2, RUNX-2, ALP and α-SMA were plotted in the graphs as 2<sup>ΔΔCt</sup> respect to the iBM-MSCs monoculture ± Standard Error of the Mean (SEM). For the gene expression of *HIF-1α*, *IL-6* and *IL-8* in the 3D scaffold-based conditions, the results were plotted in the graphs as 2<sup>ΔΔCt</sup> respect to scaffold-free condition ± SEM. All the results of gene expression were analyzed by Two-Way ANOVA and Sidak's multiple comparisons test.

The results of ELISA were plotted in the graphs as mean ± SEM and analyzed by Two-Way ANOVA and Tukey's multiple comparisons test.

The cell colonization data was plotted in the graph as mean (μm) ± Standard Deviation and analyzed by One-Way ANOVA and Tukey's multiple comparisons test. \*p value ≤ 0.05, \*\*p value ≤ 0.01, \*\*\*p value ≤ 0.001 and \*\*\*\*p value ≤ 0.0001.

### 3. Results and discussion

The inherent heterogeneity of the OS poses a significant challenge in understanding the pathology and developing effective treatments. The TME, composed of a dynamic ECM, various cell types and fluctuating environmental conditions, plays a pivotal role in OS progression and contributes to drug resistance [9,44]. Among cell types, MSCs are well-established contributors to both the initiation and progression of OS [13,19,21]. However, many aspects of the reciprocal crosstalk between MSCs and OS cells remain unclear, and only a limited number of studies have explored this interaction in advanced *in vitro* 3D models.

The recognized importance of creating a model integrating a tissue-like ECM has been crucial to provide mimetic environments whose signaling, nutrient gradients and mechanical stimuli can affect the multiple cellular components of the TME [29]. Developing more predictive *in vitro* platforms which ensure a greater capture of the cellular complexity of OS is essential for generating reliable data. Addressing these challenges, our study presents a biomimetic 3D co-culture model of OS, here referred as mOS-3D, incorporating multiple cell types and the MgHA/Coll scaffold as bone-like ECM. The widely validated scaffold [34,45] was synthesized by mimicking the biomineralization process that naturally occurs during bone formation, providing environmental signals and a cell-friendly nanostructure [31,46]. Although the MgHA/Coll scaffold was originally developed for regenerative medicine applications, its use here in a co-culture system of osteosarcoma and stromal cells represents a novel approach, enabling the study of tumor-stroma interactions and the stem cell niche within a 3D environment. The scaffold was seeded with GFP-MNNG/HOS cells and iBM-MSCs (iMSC#9) at 1:1 ratio. After 7 and 14 days of culture, the mOS-3D models were investigated to uncover the cellular behavior of

the complex system in terms of reciprocal interaction between the cell types and between cells and ECM, compared to 3D scaffold-based monocultures and scaffold-free conditions.

#### 3.1. The key role of the bone-like MgHA/Coll scaffold

A customized 3D bone-like ECM, here referred as MgHA/Coll, was used in this study as a bioinspired bone mimetic scaffold (Fig. 1). The synthesized scaffold has been widely characterized as a biomaterial for regenerative medicine applications and successfully validated for the development of Tumor Engineered models of OS cancer stem cell niche in our previous works [34,45]. A bioinspired, pH-driven and eco-sustainable biomineralization process was employed to synthesize the highly biomimetic and biocompatible hybrid scaffold [46]. Specifically, the pH neutralization alone was exploited to obtain Magnesium-doped Hydroxyapatite nanocrystals (MgHA) directly nucleated on the self-assembling collagen fibrils (MgHA/Coll), which acted as a defined template to finely control particle size, morphology and crystallinity, yielding a bioactive phase closely resembling native hard tissue. The freeze-drying process enabled the macro- and micro-porosity of the scaffold to be tailored, achieving high reproducibility and anatomical fidelity. A reagent-free dehydrothermal treatment (DHT) was used as a biocompatible cross-linking strategy to enhance the structural stability of the material under cell culture conditions [34,45]. The resulting scaffold was fully characterized in terms of its morphology, physicochemical and mechanical properties, degradation and swelling behavior in a recently published work [34]. It showed an isotropic structure with an 80% of randomly distributed and interconnected macro- and microporosity and a total 60 wt% of MgHA mineral phase that fully embedded and homogenously distributed on the collagen fibers. This closely mimics the biochemical composition and morphology of natural bone tissue [47]. The hydrophilic behavior of the scaffold and a degradability less than 2 wt% up to 28 days, make it a robust platform suitable for the modelling of *in vitro* 3D cell cultures and for supporting the cellular adhesion, growing and, cell-cell and cell-matrix interactions together with a precise control of the microenvironment, thereby enhancing the whole model functionality with promising implications for both basic research and drug discovery [48, 49]. This scaffold-based system provides unparalleled insights into disease modelling by promoting a more predictive understanding of the interactions between different cellular phenotypes and the ECM, thereby clarifying the biological mechanisms that underline tumor progression.

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Schematic representation of the design, synthesis and properties of the MgHA/Coll scaffold.</figcaption></figure>
## 3.2. The cellular morphology and colonization of the mOS-3D model

The results successfully reported a strong interaction between the cells and the bone-like scaffold as well as between the two cell types, showing overall healthy round-shaped cell nuclei and elongated cytoskeleton, together with a huge scaffold colonization starting from 7 days of culture in all 3D scaffold-based conditions (Fig. 2). Direct interaction between the OS cell and the iBM-MSC was observed by actin and DAPI staining at both time points (Fig. 2 A and S1), while a dense cellular network was reported in all 3D scaffold-based conditions by SEM analysis (Fig. 2B). These data confirmed that both cell types were able to adhere, grow and interact with the matrix and with each other colonizing the scaffold in all directions. These results also confirm the cytocompatibility of the MgHA/Coll scaffold and that its cell-suitable interconnected macro- and microporosity favors cellular adhesion and colonization, thanks to its naturally inspired biochemical composition and synthesis process [31,32].

The cellular ability in colonizing the scaffold was also quantitatively analyzed at 7 days of culture to investigate if the cellular migration and invasion into the matrix were differently affected by co-culture conditions, compared to monocultures. The increased migration and invasion ability is a key player in the tumor progression and metastatic process [21,50], however, few studies explored this aspect by investigating the cellular behavior across a 3D bone-like matrix with sustained mechanical properties. In addition, emerging evidence has demonstrated the metastasis-promoting effects on OS by MSCs [13,21,22]. In this study, after DAPI staining, each 3D scaffold-based condition was sectioned by scalpel, and the migrating cells from the seeding surface to the core of

the scaffold were identified at the fluorescent microscope by cell nuclei identification (Figure S2). The distance travelled by the cells has been calculated in µm and graphically reported (Fig. 2C), showing that the tumor cells have a significantly higher migratory potential compared to iBM-MSCs alone (p value ≤ 0.001), according to the well-recognized metastatic potential of OS cells [50]. Interestingly, the co-culture condition in the mOS-3D model exhibited migratory and invasive behavior that was intermediate between the two monoculture conditions in 3D. Specifically, the co-culture showed a significantly higher migratory activity compared to iBM-MSCs monoculture (p value ≤ 0.05), although still lower than that observed in the 3D monoculture of OS cells. The behavior observed in the mOS-3D model may be the result of two concurrent phenomena: on the one hand, the potential role of MSCs in limiting tumor invasiveness and metastatic spread [51], and on the other, the changes in MSCs behavior when co-cultured with tumor cells, possibly due to the well-documented phenomenon of MSC "education" into tumor-associated MSCs (T-MSCs) or cancer-associated fibroblasts (CAFs) - that are known to acquire enhanced migratory and invasive capabilities [26,52].

## 3.3. Gene expression profile of iBM-MSCs and the TME

The gene expression profile of the monocultures and co-culture was investigated in both 3D scaffold-based and scaffold-free conditions to uncover the overall cellular behavior of the co-culture and the nature of the interaction between the two cell types and with the ECM. Gene expression, RNA sequencing, and the evaluation of MMPs and their inhibitors were performed on the entire co-culture system without

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Cell morphology and colonization of the mOS-3D model. (A) Actin and DAPI staining of the 3D scaffold-based conditions at 7 and 14 days of culture. Actin Red555 ReadyProbes Reagent was used to stain F-actin filaments and distinguish iBM-MSCs from GFP-MNNG/HOS cells. GFP-MNNG/HOS cells in green, iBM-MSCs in red and cell nuclei in blue. Scale bars 100 µm; (B) SEM analysis of the 3D scaffold-based conditions at 7 and 14 days of culture. Red asterisks indicate cells "embedded" in the scaffold matrix; (C) Colonization distance travelled by the cells from the seeding surface to the core of the scaffold at day 7 of culture. The data are graphically plotted as mean ± SEM. *p* value ≤ 0.05, ***p* value ≤ 0.001.</figcaption></figure>
separating the two cell populations. Due to the technical limitations associated with this specific biomaterial, cell recovery after culture did not provide a sufficient yield of viable cells to enable reliable separation or further analyses. Therefore, comparisons were made between the co-culture systems and the respective monocultures grown within the same 3D scaffolds, allowing assessment of the overall molecular modulation induced by the multicellular environment.

Firstly, the expression of specific mesenchymal stem cell surface markers was analyzed to investigate potential changes to the mesenchymal phenotype under different culture conditions, and the relative expression of these markers was reported compared to the iBM-MSC monoculture (Fig. 3 A-A3). The reported results showed that all the MSCs surface markers (Endoglin, CD90 and CD73) were significantly inhibited in the co-culture condition, in both the scaffold-free model (Fig. 3 A1, A3) and the mOS-3D model (Fig. 3 A, A2) at both time points (Fig. 3 A–A3).

In detail, a significant downregulation of *Endoglin* (p value ≤ 0.05 at day 7 and p value ≤ 0.0001 at day 14), as well as *CD90* (p value ≤ 0.001 at day 7 and p value ≤ 0.0001 at day 14) and *CD73* (p value ≤ 0.01 at day 7 and p value ≤ 0.0001 at day 14) was reported in the co-culture and this downregulation is consistent over time in the mOS-3D (Fig. 3 A, A2). By contrast, the downregulation trend in scaffold-free conditions was statistically significant only for *Endoglin* (p value ≤ 0.01) and *CD90* (p value ≤ 0.0001) after 7 days of co-culture. (Fig. 3 A1, A3). Since these three markers are specifically associated with MSCs, the results could suggest an alteration of the mesenchymal phenotype in the presence of tumor cells, which becomes more structured over time and is more pronounced when the scaffold is present. Indeed, based on the minimal

criteria established by the International Society for Cellular Therapy (iSCT), human MSCs are identified by the combined expression of *Endoglin*, *CD73* and *CD90* cell-surface markers, which must be expressed by ≥ 95 % of the cell population [53]. Although *Endoglin*, or *CD105*, is a type I membrane glycoprotein whose expression has been shown to vary depending upon MSC source and culture time *in vitro*, it is established that its absence identifies differentiated MSCs [54]. The *CD73* is an ecto-5'-nucleotidase that converts extracellular adenosine monophosphate to adenosine, and it is expressed by MSCs in a different form compared to that one expressed by other cell types (i.e. endothelial cells, epithelial cells), making it a unique and specific marker for MSCs identification [55]. The *CD90*, also known as *Thy1*, is a glycosylphosphatidylinositol-linked protein involved in cell-cell and cell-matrix interactions that is highly expressed in all MSCs, irrespective of the source, and its expression has been related to the undifferentiated status of MSCs [56].

The expression of the three transcription factors *OCT-4*, *SOX-2* and *NANOG* as essential markers of stemness, especially related to the malignant aspect of the self-renewal and pluripotency of both tumor cells and iBM-MSCs in the TME [57], were investigated in this study (Fig. 3 B-B3). Our results highlighted the significant upregulation of *SOX-2* in the co-culture condition at both time points in the mOS-3D (p value ≤ 0.0001), compared to iBM-MSCs monoculture (Fig. 3 B, B2). The trend was also preserved in scaffold-free conditions, reporting a significant upregulation of *SOX-2* in the co-culture at 14 days (p value ≤ 0.0001), compared to iBM-MSCs monoculture (Fig. 3 B1, B3). An increasing trend of *NANOG* expression has been observed after 7 days of culture in scaffold-free conditions, even if no significant differences have been

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Profile of gene expression (qPCR). (A-A3) Gene expression of the cell-surface markers in the co-culture as fold-change expression relative to iBM-MSCs monoculture in the mOS-3D model (A, A2) and in scaffold-free condition (A1, A3); (B-B3) gene expression of stemness markers in the co-culture as fold-change expression relative to iBM-MSCs monoculture in the mOS-3D model (B, B2) and in scaffold-free condition (B1, B3); (C-C3) gene expression of the osteogenic commitment's markers in the co-culture as fold-change expression relative to iBM-MSCs monoculture in the mOS-3D model (C, C2) and in scaffold-free condition (C1, C3). The graphs show the mean ± SEM. \*p value ≤ 0.05, \*\*p value ≤ 0.01, \*\*\*p value ≤ 0.001, \*\*\*\*p value ≤ 0.0001.</figcaption></figure>
reported (Fig. 3 B1). Similarly, no significant differences were observed for *OCT-4* expression in co-culture at both time-points in scaffold-free conditions and in the mOS-3D (Fig. 3 B-B3).

In line with the supportive role of MSCs in osteosarcoma progression, our results could indicate that co-culturing MSCs with OS cells leads to the formation of a TME characterized by the upregulation of stemness-associated transcription factors, notably *SOX-2* and *NANOG*. These markers are integral to maintaining self-renewal and pluripotency in CSCs and have been identified as predictive biomarkers for poor prognosis in various cancers, including OS [58–63]. Various studies have reported that CSCs may originate not only from tumor cells but also from other cell types, such as MSCs [19,64,65]. The observed increase in *SOX-2* and *NANOG* expression in our mOS-3D could imply a potential malignant transformation of one of the two cell types, MSCs or OS, towards CSCs, possibly driven by their interaction with each other's bone-like ECM. In the case of MSCs, this transformation may be facilitated by mechanisms such as cell fusion events, where MSCs and tumor cells merge, leading to nuclear reprogramming and the acquisition of CSC-like properties [59,66–68]. Furthermore, the TME, including factors secreted by OS cells, may contribute to the reprogramming of MSCs, enhancing their stemness and promoting a pro-tumorigenic phenotype [69]. On the other hand, it is well-established that MSCs can enhance the stemness properties of tumor cells within the TME through the secretion of soluble cytokines and small molecules [19,69,70]. These interactions underscore the complexity of the tumor stroma and its role in cancer progression. Since our results clearly demonstrate an alteration of the mesenchymal phenotype, alongside the stable expression of *OCT-4* and *NANOG*, or even their increase under scaffold-free conditions, and the upregulation of *SOX-2*, these findings may be associated with the induction of a pro-tumoral phenotype in MSCs, characterized by enhanced 'malignant' stemness properties overriding their typical mesenchymal traits.

To better understand the fate of the cells in our models, the gene expression of *RUNX-2*, *BMP-2* and *ALP* was analyzed, elucidating if the changes in the mesenchymal phenotype could be related to the potential osteogenic commitment (Fig. 3 C-C3). Our results reported the whole absence of osteogenic induction in iBM-MSCs grown in the presence of tumor cells up to 14 days, with a marked downregulation of the three markers in the mOS-3D model (Fig. 3C, C2), compared to scaffold-free conditions (Fig. 3C1, C3). In detail, while *RUNX-2* expression did not show significant changes in the scaffold-free co-culture at both time points with respect to iBM-MSCs monoculture (Fig. 3 C1, C3), a significant decrease of its expression was observed after 14 days in the mOS-3D model (p value ≤ 0.0001) (Fig. 3 C, C2). A significant downregulation of *BMP-2* and *ALP* was also reported compared to iBM-MSCs monoculture, especially in the mOS-3D model at both time points (p value ≤ 0.05 for *BMP-2* and undetermined for *ALP* at day 7; p value ≤ 0.0001 for both at day 14) (Fig. 3 C, C2) while the scaffold-free condition showed a slight significance only after 7 days of culture (p value ≤ 0.05 for both). The undetermined *ALP* mRNA levels observed at day 7 in the scaffold-based cultures, both in co-culture and in iBM-MSC monoculture, are consistent with reports showing that osteogenic marker expression, including *ALP*, can be delayed in 3D scaffold systems compared to 2D, reflecting the longer adaptation required within a complex tridimensional environment [71–73].

These results could demonstrate that the downregulation of the specific cell-surface markers observed in iBM-MSCs within the mOS-3D model cannot be attributed to their osteogenic differentiation, even up to 14 days. On the other hand, even if *RUNX-2*, *BMP-2* and *ALP* are commonly expressed by osteosarcoma cells, there is evidence in the literature that MSCs presence in the TME could downregulate these markers, suggesting a potential alteration of the differentiated status of OS cells towards a more aggressive tumoral phenotype [74,75]. These data are consistent with the results of osteosarcoma (OS) cells in scaffold-free 3D cultures with MSCs, and with the more aggressive phenotype observed in murine models when OS cells are co-inoculated

with MSCs [76]. Most interestingly, the co-culture with tumor cells appears to decrease the well-documented osteoinductive effect of the MgHA/Coll scaffold [77,78]. Therefore, the simultaneous presence of tumor cells, iBM-MSCs and the bone-mimetic ECM created a more malignant TME with suggested alteration of the cellular phenotypes.

The 3D cellular organization has a significant impact on the morphogenesis, phenotype and gene expression profile of cells [79]. The role of the matrix, along with its constituents and structure, is widely recognized in modulating cell interaction with the microenvironment and triggering specific cellular behaviors, such as adhesion, differentiation, migration and invasion. [80] Considering this, the gene expression profile of several TME-associated markers was examined in this study, comparing the cellular response of all the mono- and co-cultures grown in 3D with respect to scaffold-free conditions at two time points, 7 and 14 days (Fig. 4).

Our results reported an increase in *HIF-1α* mRNA level in all culture conditions on day 7, even if no statistically significant differences were reported. However, after 14 days, a significant downregulation in the co-culture was reported (p value ≤ 0.001) (Fig. 4B2). Tumor hypoxia ranges from minimal to mild to severe, with distinct regulatory mechanisms operating at each level, highlighting the dynamic interplay between transient and chronic hypoxic cells [81,82]. The time-dependent regulation of the hypoxic marker observed in our study can reflect this specific aspect of the OS condition in vivo.

Looking on the interleukin's levels, especially *IL-6* and *IL-8* for their multiple biological activities in cancer [83], the results showed that both *IL-6* and *IL-8* were significantly upregulated in iBM-MSCs monoculture grown in 3D at day 7 (p value ≤ 0.001 for *IL-6*; p value ≤ 0.0001 for *IL-8*) (Figure 4 A1), while no significant differences were observed in GFP-MNNG/HOS cells (Fig. 4A), compared to scaffold-free condition. The observed cytokines upregulation in iBM-MSCs monoculture could depend on various factors such as their source and microenvironmental cues, emphasizing a context-dependent regulation of cytokines production [83] associated with the relevant role assumed by the MgHA/-Coll scaffold. Interestingly, the gene expression profile of the co-culture reported a significant downregulation of both *IL-6* (p value ≤ 0.0001) and *IL-8* (p value ≤ 0.001) in the mOS-3D model, compared to the scaffold-free condition at day 7 (Figure 4A2). This trend is further highlighted after 14 days of culture (p value ≤ 0.0001 for both cytokines) (Figure 4B2).

These results are consistent with the well-established notion that cytokines involved in the pro-cancer TME exhibit temporal specificity during tumor progression. Different cell populations may secrete distinct pathogenic cytokines at various stages of the disease, highlighting a time-dependent pattern of cytokine expression and production [83,84]. Moreover, the observed malignant transformation of MSCs, induced by the presence of tumor cells and the bone-mimetic scaffold, could be considered one of the potential causes of the time-dependent reduction in the expression of these cytokines. It is important to emphasize that this effect should not be attributed to the scaffold itself. Although the MgHA/Coll biomaterial was originally developed for regenerative medicine applications [71,85,86], in this context it acts with the coexisting osteosarcoma and stromal cell populations. The pathological signals exchanged within this multicellular system, together with the biomimetic properties of the scaffold, appear to drive MSCs toward malignant-like reprogramming rather than reflecting an intrinsic tumorigenic effect of the material.

It has been reported that in OS, there is an extensive production of incompletely mineralized matrix, named osteoid matrix [87]. The significant collapse of *IL-6* and *IL-8* mRNA levels observed in our results, especially at 14 days in the 3D GFP-MNNG/HOS monoculture and in the mOS-3D model, may reflect the potential development of a TME which favors ECM production by tumor cells over bone resorption.

One of the recognized markers of CAFs, *Alpha-Smooth Muscle Actin* (*α-SMA*), was also investigated to assess if the observed alteration of the mesenchymal phenotype of MSCs was potentially related to the
<figure><img src="image_6.png" /><figcaption>Fig. 4. Gene expression of TME-associated markers. Gene expression of GFP-MMNG/HOS monoculture (A, B), iBM-MSCs monoculture (A1, B1) and the co-culture (A2, B2) in the 3D scaffold-based condition at 7 (A-A2) and 14 days (B-B2) as fold-change expression relative to scaffold-free condition. The graphs show the mean ± SEM. \*p value ≤ 0.05, \*\*p value ≤ 0.01, \*\*\*p value ≤ 0.001, \*\*\*\*p value ≤ 0.0001.</figcaption></figure>

differentiation of iBM-MSCs into CAFs following tumor cells' education. The results reported no significant differences between the 3D and the scaffold-free condition in both the iBM-MSCs monoculture and the co-culture at 7 days (Figure 4A1, A2), while a significant reduction of α-SMA was reported at 14 days in the mOS-3D model (p value ≤ 0.0001), compared to scaffold-free condition (Figure 4B2). These findings, consistent with the dysregulation of interleukin expression, support our hypothesis that MSCs could potentially undergo malignant transformation without differentiating into CAF within our 3D OS model. While various studies have reported the differentiation of MSCs into CAFs under the influence of tumor cells via increased levels of IL-6 and IL-8 in TME [15,22,88], our results suggest an alternative pathway leading to a different malignant phenotype. In fact, based on our gene expression results, it appears more accurate to interpret the malignant transformation observed in this model as a transition of MSCs toward a

CSC-like phenotype rather than toward CAFs. This interpretation is consistent with the marked overexpression of stemness-related genes, such as NANOG and SOX2, previously discussed.

### 3.4. Evaluation of MMPs and TIMPs

Considering the critical role of the matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) in regulating extracellular matrix remodeling and maintaining tissue homeostasis as well as the well-documented dysregulation of this balance in cancer progression [89,90], the expression levels of MMP-2 and MMP-9, along with TIMP-3 and TIMP-4, were quantitatively assessed in both 3D scaffold-based and scaffold-free cell culture models (Fig. 5 and Table 1).

In the 3D scaffold-based condition, a significantly higher level of MMP-2 was reported in iBM-MSCs monoculture with respect to mOS-3D

<figure><img src="image_7.png" /><figcaption>Fig. 5. MMPs and TIMPs analysis. MMP-2 (A, B), MMP-9 (A1, B1), TIMP-3 (A2, B2) and TIMP-4 (A3, B3) in both 3D scaffold-based and scaffold-free condition. The data are reported in the graphs as mean ± SEM. \*p value ≤ 0.05, \*\*p value ≤ 0.01, \*\*\*p value ≤ 0.001, \*\*\*\*p value ≤ 0.0001.</figcaption></figure>
model after 7 days of culture (p value ≤ 0.0001), and to tumor cells at both time points (p value ≤ 0.0001 at day 7; p value ≤ 0.01 at day 14); MMP-2 was significantly higher expressed in the mOS-3D model with respect to tumor cells at both time points (p value ≤ 0.05 for both 7 and 14 days) (Fig. 5A). This trend was also observed in scaffold-free conditions, where MMP-2 was significantly higher in iBM-MSCs monoculture, compared to co-culture and tumor cells (p value ≤ 0.0001) and a significant difference between the co-culture and tumor cells was observed (p value ≤ 0.01) at 14 days (Fig. 5B).

Conversely, the MMP-9 showed undetectable levels in both the 3D scaffold-based and scaffold-free conditions, except for a statistically significant level in GFP-MNNG/HOS monoculture at 14 days, compared to iBM-MSCs monoculture and co-culture in scaffold-free conditions (p value ≤ 0.0001) (Fig. 5A1, B1). These results suggest the presence of an imbalance in proteolytic activity in favor of MMP-2 over MMP-9 (Table 1), in line with the literature reflecting this overbalance in poor response to therapy in OS patients [91,92].

Looking at the TIMPs level, our results showed that in the presence of the scaffold, the GFP-MNNG/HOS monoculture expressed the highest level of TIMP-3, with a significant difference with respect to iBM-MSCs at both time points (p value ≤ 0.05 at day 7 and p value ≤ 0.0001 at day 14) and to the mOS-3D at 14 days (p value ≤ 0.001) (Fig. 5A2). Moreover, a significantly higher level of TIMP-3 was reported in the co-culture at 14 days in the absence of scaffold with respect to the iBM-MSCs monoculture (p value ≤ 0.0001) (Figure 5B2), suggesting a prevalent inhibitory activity. The highest levels of TIMP-4 were reported in iBM-MSCs monoculture in both 3D scaffold-based and scaffold-free conditions (Figure 5A3, B3). In both conditions, a significantly higher level of TIMP-4 was reported at 14 days in the iBM-MSCs monoculture with respect to tumor cells and the co-culture (p value ≤ 0.05 for both in the mOS-3D model and p value ≤ 0.0001 for both in the scaffold-free condition) (Figure 5 A3, B3). However, the balance between TIMPs regulation of the MMPs activity needs to be considered. It is well-known that in normal bone tissue TMPs regulate MMPs by forming a strong noncovalent complex with a 1:1 stoichiometry, while in the tumoral tissue, this balance is broken [93]. The MMP/TIMP ratio analysis confirmed an overall significant imbalance in enzymatic activity favoring inhibitory action (Table 1). Specifically, in both 3D scaffold-based and scaffold-free conditions, the MMP-9/TIMP-3 and MMP-2/TIMP-4 ratios were close to zero at both time points. The only exception indicating a shift towards MMP activity was observed in the MMP-2/TIMP-3 ratio. In the mOS-3D model, this ratio demonstrated a balanced enzymatic activity at both time points (0.783 and 1.033). Notably, iBM-MSC monocultures exhibited a significantly higher MMP-2 activity relative to its inhibitor. In scaffold-based conditions, the MMP-2/TIMP-3 ratios were markedly elevated at both time points (6.404 and 2.174). In scaffold-free conditions, the ratio was approximately balanced on day 7 and shifted towards increased MMP-2 activity by day 14 (10.118) (Table 1).

Overall, the results indicate that our models can create a TME which disfavors excessive matrix degradation by MMPs in favor of the inhibitory activity of TIMPs and, as a result, the production of new matrix by tumor cells. This is also in line with the previously reported down-regulation of cytokines (i.e. IL-6 and IL-8) observed in the 3D scaffold-based condition by gene expression analysis, both favoring bone deposition over bone resorption and suggesting the development of a “non-destructive” microenvironment.

### 3.5. Differential transcriptomic profiles of the mOS-3D Model

To better characterize the functional impact of iBM-MSCs on GFP-MNNG/HOS cells, we analyzed the differential transcriptomic profile of each cell type cultured alone and in co-culture in a 3D scaffold-based condition at 7 and 14 days using a comparative RNA-sequencing (RNA-seq) approach (Figs. 6 and 7). The addition of iBM-MSCs to GFP-MNNG/HOS cells induced a reproducible effect on all biological replicates. Hierarchical clustering confirmed this observation (|log2FC= > 1, p value adjusted <0.05), reporting numerous genes with significantly different expression at 14 days of culture, compared to day 7. Moreover, mOS-3D model showed a significantly different expression of numerous genes compared to monocultures (Fig. 6A, B).

In detail, a total of 357 and 675 genes were downregulated and overexpressed, respectively, in 3D GFP-MNNG/HOS monoculture from 7 to 14 days (Table S1). Similarly, in the 3D iBM-MSC monoculture, 250 and 204 genes were downregulated and overexpressed, respectively, while 407 and 292 downregulated and overexpressed genes, respectively, were reported in the mOS-3D Model (Tables S1).

The Protein network was determined by String database (Fig. 7A), and the functional analysis of these genes was carried out using GO and KEGG databases (Fig. 7B and Table S2). As expected, HIF-1 was the main affected pathway in the 3D GFP-MNNG/HOS monoculture. Indeed, HIF-1 is a transcription factor that functions as a master regulator of oxygen homeostasis [94] and the presence of the ECM-like scaffolds strongly affects the diffusion of oxygen in the cell culture leading to the formation of hypoxic foci [95]. HIF is also involved in various critical processes of tumor development by regulating angiogenesis, glucose metabolism, immune response and drug resistance [96]. In this context, FoxO signaling pathway, glycolysis/glycogenolysis and fructose/mannose metabolism are also affected according to our results as shown in Fig. 7B.

These data were confirmed by GO database analysis (Table S2). After 2 weeks of culture, the ossification signaling pathway was modulated as shown in Table S2 confirming that GFP-MNNG/HOS cells are cancer cells originated from MSCs differentiated toward osteoblastic lineage [97].

The 3D iBM-MSCs monoculture exhibits immune regulator functions (Fig. 7B, Table S1 and Table S2). Indeed, numerous chemokines playing a role in myeloid cell recruitment and activation were downregulated (Table S2). In addition, TNF and IL-17 signaling pathways were among the most regulated pathways in 3D iBM-MSCs monoculture (Fig. 7B), in line with the literature reporting MSCs as key regulators of adaptive and innate immune responses [98,99].

The modulated genes of mOS-3D model showed a differential transcriptomic profile, which is almost exclusively related to metabolic modulation (Fig. 7B and Table S1), and which is confirmed by GO database analysis (Table S2). Overall, in line with the gene expression results, these data demonstrated that in the mOS-3D model some metabolic pathways are altered, supporting the potential fuel of the OS

<table><caption>**Table 1**
Ratio of MMP/TIMP is reported in the table.</caption><thead><tr><th rowspan="2">MMP/TIMP ratio</th><th rowspan="2"></th><th colspan="2">MMP-9/TIMP-3</th><th colspan="2">MMP-2/TIMP-3</th><th colspan="2">MMP-2/TIMP-4</th><th colspan="2">MMP-9/MMP-2</th></tr><tr><th>Day 7</th><th>Day 14</th><th>Day 7</th><th>Day 14</th><th>Day 7</th><th>Day 14</th><th>Day 7</th><th>Day 14</th></tr></thead><tbody><tr><td rowspan="3">3D scaffold-based condition</td><td>mOS-3D model</td><td>0.001</td><td>0.004</td><td>0.783</td><td>1.033</td><td>0.049</td><td>0.102</td><td>0.002</td><td>0.004</td></tr><tr><td>iBM-MSCs monoculture</td><td>0.000</td><td>0.000</td><td>6.404</td><td>2.174</td><td>0.112</td><td>0.072</td><td>0.000</td><td>0.000</td></tr><tr><td>GFP-MNNG HOS monoculture</td><td>0.000</td><td>0.000</td><td>0.128</td><td>0.293</td><td>0.012</td><td>0.064</td><td>0.000</td><td>0.001</td></tr><tr><td rowspan="3">Scaffold-free condition</td><td>co-culture</td><td>0.000</td><td>0.001</td><td>0.264</td><td>0.385</td><td>0.088</td><td>0.127</td><td>0.000</td><td>0.003</td></tr><tr><td>iBM-MSCs monoculture</td><td>0.000</td><td>0.000</td><td>0.873</td><td>10.118</td><td>0.103</td><td>0.132</td><td>0.000</td><td>0.000</td></tr><tr><td>GFP-MNNG HOS monoculture</td><td>0.000</td><td>0.002</td><td>0.076</td><td>0.104</td><td>0.026</td><td>0.066</td><td>0.000</td><td>0.024</td></tr></tbody></table>
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** mOS-3D model exhibits differential transcriptomic profile. (A) Heatmap visualization of the z-scored expression of genes that were differentially expressed at 7 and 14 days in the different 3D scaffold-based cultures (|log2FC= > 1, p value adjusted <0.05); (B) Volcano plot analysis of differentially expressed genes in mOS-3D model at 14 days compared to day 7. The genes 2-fold overexpressed or downregulated appear in green.</figcaption></figure>

growth and of its drug sensitivity/resistance.

4. Conclusions

This study reports the successful development and characterization
of an *in vitro* mOS-3D model combining the bone-like MgHA/Coll scaf-
fold with multicellular phenotypes, investigating the cell/cell and cell/
ECM interactions. The model demonstrated effective cellular in-
teractions and colonization of the MgHA/Coll scaffold. The potential
alteration of the cellular phenotypes in the co-culture models, together
with the disruption of MMPs levels in favor of the inhibitors, were
revealed, highlighting the relevant role of the bone-like matrix in
affecting cellular response and suggesting the successful development of
a “non-destructive” tumor microenvironment favoring the production of
tumoral matrix. MSCs are important cellular constituents of tumor
stroma that contribute to the regulation of local immunity, tumor
metabolism and drug response [100]. Indeed, MSCs appear as cancer
promoters and their role in OS is suspected not only in the proliferation
of cancer cells [14] but also in the preparation of the tumor niche by IL-6
containing extra-vesicles release [101]. A reciprocal crosstalk has also
been demonstrated, and OS-derived extracellular vesicles induce a
tumor-like phenotype of a fibroblastic cell line [102]. RNA sequencing
data confirmed the modulation of transcriptomic profiles in the mOS-3D
model compared to 3D monocultures and revealed major modulations of
metabolic pathways. Interestingly, the mOS-3D model appears to exhibit
a more “neutral” transcriptomic profile compared to both

mono-cultures, in favor of major crosstalk between the two cell types. This model represents a promising approach to enhancing heterogeneity in *in vitro* platforms and allows, for the first time, investigation of the cellular responses in such a complex system, not only through morphological analysis but also via deeper molecular biology studies. These findings lay the groundwork for future translational applications, including the development of patient-specific 3D *in vitro* models that could advance personalized medicine by providing more predictive, patient-tailored platforms. However, the MgHA/Coll scaffold is highly mimetic of the healthy bone tissue [31,32] and improvements in the pathological composition and structure of the material are needed to resemble the diseased matrix. Functionalization of the scaffold with tumor-specific molecules and the compartmentalization of cells into distinct scaffold areas will be considered for future studies to enhance the triggering of tumoral pathways within the microenvironment.

CRediT authorship contribution statement

**Monica Montesi:** Writing – review & editing, Writing – original draft, Visualization, Validation, Funding acquisition, Data curation, Conceptualization. **Giada Bassi:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis. **Dominique Heymann:** Writing – review & editing, Writing – original draft, Validation, Supervision, Funding acquisition, Data curation, Conceptualization. **Lorenzo Apolloni:** Writing – review & editing, Investigation. **Mohamed Saqawa:** Writing – review &
<figure><img src="image_9.png" /><figcaption>**Fig. 7.** mOS-3D model is characterized by differential profiles of protein networks and related functional pathways. (A) Protein network generated with String database. Each point represents one protein, and each line represents one protein interaction; (B) Main functional KEGG pathways related to the genes most significantly modulated in the 3D GFP-MNNG/HOS monoculture, 3D iBM-MSCs monoculture and mOS-3D model at 14 days compared to day 7.</figcaption></figure>

editing, Investigation. **Silvia Díaz-Prado:** Writing – review & editing, Resources. **Romain Levergeois:** Writing – review & editing, Investigation, Formal analysis. **Emilie Ollivier:** Writing – review & editing, Visualization, Investigation, Formal analysis. **Elisabetta Campodoni:** Writing – review & editing, Visualization, Resources. **Monica Sandri:** Writing – review & editing, Resources. **Silvia Panseri:** Writing – review & editing, Methodology, Investigation. **Denis Cochonneau:** Writing – review & editing, Methodology, Investigation.

## Funding

This work was funded by grants from the European Commission (Grant N. 101079372 – STRIKE, HORIZON-MSCA-2021-DN – 01–01 and Grant N. 101079372 – PREDICTOS, HORIZON-WIDERA-2021-AC-CESS-03) and the Italian Ministry of University and Research (PRIN2022PNRR MERCURY Grant N. 2017C8RYSS and PNRR project ECS_00000033_ECOSISTER).

## Supporting Information

The **supporting information** is available free of charge at:

Representative panel of scaffold colonization analysis of the 3D scaffold-based conditions by DAPI staining (Figure S1); list of the 50 most significantly modulated genes between day 7 and 14 in 3D monocultures and mOS-3D model (Table S1); main functional pathways related to the most significantly modulated genes between day 7 and 14 in the 3D mono-cultures and mOS-3D model (Table S2) (PDF).

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biopha.2025.118956.

## Data availability

Data will be made available on request.

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GLOSSARY

**α-SMA**: alpha-Smooth Muscle Actin

**ALPL**: Alkaline Phosphatase

**BMP**: Bone Morphogenetic Protein

**CAF**: Cancer-Associated Fibroblasts

**CSC**: Cancer Stem Cells

**DAPI**: 4',6-diamidino-2-ylindole dihydrochloride

**DHT**: Dehydrothermal

**DMEM**: Dulbecco's Modified Eagle Medium

**ECM**: Extracellular Matrix

**FBS**: Foetal Bovine Serum

**FTIR-ATR**: Fourier Transform Infrared Spectroscopy in the Attenuated Total Reflection mode

**GFP**: Green Fluorescent Protein

**HIF**: Hypoxia Inducible Factor

**ICP-OES**: Inductively Coupled Plasma-Optical Emission Spectrometry

**IL-6**: Interleukin 6

**IL-8**: Interleukin 8

**iBM-MSCs**: Immortalized Bone Marrow-derived Mesenchymal Stem Cells

**MgHA/Col**: Magnesium-substituted Hydroxyapatite/Collagen

**MMP**: Matrix Metalloproteinases

**MSC**: Mesenchymal Stem Cell

**OS**: Osteosarcoma

**PFA**: Paraformaldehyde

**RUNX2**: Runt-related Transcription Factor 2

**SEM**: Scanning Electron Microscopy

**TGA**: Thermogravimetric Analysis

**TIMP**: Tissue Inhibitor of Metalloproteinases

**TME**: Tumor Microenvironment

**T-MSCs**: Tumor-associated Mesenchymal Stem Cells

**XRD**: X-ray Diffraction