3D extracellular matrix derived model of alveolar rhabdomyosarcoma

INTRODUCTION: Rhabdomyosarcoma is the most common soft tissue sarcoma in childhood, among the subtypes the Alveolar (ARMS) is the more aggressive with a higher tendency to metastasize [1]. Integrins are a class of transmembrane adhesion molecules that mediate survival, differentiation, migration and differentiation [2]. Here we investigate the role of integrins in ARMS metastatic migration in an engineered 3D scaffold. METHODS: ARMS xenografts are obtained from subcutaneous injection of RH30 cells in immunodeficient mice. Composition of the ECM is determined by proteomic analysis. The main components of the ECM are used to enrich a 3D collagen scaffold cultured in a perfusion bioreactor. Cells are analyzed by qPCR for the expression of a panel of integrins. Presence of the protein is confirmed by flow cytometry immunofluorescence. MMPs expression is evaluated by zymography. RESULTS: Verified the expression of human and ARMS marker and typical tumor morphology in xenografts, they are processed for proteomic analysis. Proteomic data analysis is currently under investigation. Preliminary data culturing RH30 cells in 3D bioreactor show upregulation of ITG5 and CXCR4 receptor compared to 2D condition. Localization and quantification at protein level will be assessed respectively by immunofluorescence and cytofluorimetry. Expression of MMP-9 and MMP-2 has been assessed by zymography comparing the expression of these MMPs in 2D vs 3D bioreactor and RH30 isolated from the xenograft. DISCUSSION & CONCLUSIONS: Preliminary data on ITG expression show that in 3D scaffold the expression of ITG5 and CXCR4 is upregulated. In parallel the active form of MMP-2 is more present in 3D models compared to 2D. Other groups reported a mechanical interaction between ITG5 and MMP-2 [3]. This interaction will be studied in a more representative engineered 3D scaffold to shed light on the complex interaction between ECM and metastatic progression

Alveolar Rhabdomyosarcoma 3D model development to mimic physiological cell-ECM interactions with focus on integrins

Introduction: Rhabdomyosarcoma (RMS) is the most common Soft Tissue Sarcoma in childhood, the two main subtypes are embryonal RMS (ERMS), associated with a better prognosis, and alveolar RMS (ARMS), more aggressive and highly metastatic. If the knowledge of RMS genomic alterations is well established, its microenvironmental characterization is still poorly defined. So far, in vitro 2D models are used to recapitulate the interactions between cancer cells and stromal cells. However, these models are not representative of the complex biological processes that happen in vivo, such as cell migration. This, in particular, depends on 3D interactions between cells and ECM via adhesion molecules, i.e. integrins. In this context, the cell-ECM and cell-cell interactions are better studied with 3D models that offer a platform where culture conditions approximate better the physiological conditions. Aim: This work aims at the development of a 3D model able to recreate the 3D complex cells-ECM interactions, with particular attention on integrins, and to represent the cell migration process taking place in physiological conditions. Material and Methods: Bioinformatic analysis was used to determine differential expression of ECM genes in ARMS end ERMS patients. Decellularization of ARMS xenogenic tumor masses employed cycles of detergents and enzymatic treatments (DET). Three different recellularization strategies were adopted: superficial seeding, microinjection and a perfusion bioreactor. Mass spectrometry (MS) analysis of ARMS tissue was performed to determine ECM protein composition. Two 3D models: 1- Ultrafoam collagen I sponge, 2- hyaluronic acid/PEG hydrogel (HA/PEG) were developed. ITGA5 role in cell motility was investigated in vitro upon siRNA transfection evaluating. Tumor growth and metastatic migration was tested in vivo. Results: 15 ECM genes were shown to be differentially expressed between ARMS from ERMS patients. Xenogenic ARMS were successfully decellularized but the three recellularization techniques tested were not optimal in terms of viability and cell distribution. MS revealed major presence of collagens (type I and type III), fibrillin, fibronectin and periostin in ARMS ECM building. With Ultrafoam collagen I sponge we obtained a tissue-like structure in 7 days of culture, higher proliferation rates (41% vs 24%), enhanced secretion of MMP-2 and overexpression of ITGA5 and CXCR4 mRNAs compared to 2D controls. HA/PEG hydrogel formed a 3D support where cultured spheroids showed no invasion. In vitro migration assay showed reduction of migrating cells upon ITGA5 siRNA silencing (24.3% vs 43.9% in the control). In the invasion assay, cells were unable to invade the Matrigel, however we reported differential cell clustering with larger multicellular strands in control cells and smaller spherical aggregates in ITGA5 silenced cells. In vivo tumor growth showed no dependence on ITGA5; conversely, extravasation rate was higher in presence of ITGA5 (30.6% vs 8.5%) in the zebrafish model. Discussion: This study highlighted the first preliminary results on ARMS cell-ECM interaction. Among the tested 3Dmodels, the direct use of the ARMS ECM evidenced reduced porosity, impacting on superficial cell seeding and prevalence of cell-cell interactions rather than cell-ECM adhesions. The use of commercially available scaffold composed of Collagen I (Ultrafoam) gave the best results in terms of interaction with the microenvironment; however, the bioreactor is inaccessible for fluorescence live imaging. In contrast, hydrogels are optically transparent and easier to enrich with other ARMS ECM specific proteins. In HA/PEG hydrogel, concentration of fibronectin has to be optimized together with the addition of other ECM proteins. In vitro results on ITGA5 expression by RH30 cells suggest that other fibronectin-binding integrins can cooperate for cell migration. Differences in cell clustering suggested an interplay between ITGA5 and cell-cell adhesion proteins. In vivo experiments imply that ITGA5 is not required for tumor growth and appeared to be functionally relevant for the extravasation process. Conclusions: This work developed three different 3D models of ARMS, each one with specific advantages and disadvantages that have to be considered depending on the investigated biological process. In the future, we foresee that deeper investigation on ARMS microenvironment could develop new prognostic or therapeutic markers to ameliorate the overall survival of the young patients

Alveolar Rhabdomyosarcoma 3D model development to mimic physiological cell-ECM interactions with focus on integrins.

Introduzione: Rabdomiosarcoma (RMS) \ue8 il sarcoma dei tessuti molli pi\uf9 frequente in et\ue0 pediatrica, le due sottoclassi principali sono quella embrionale (ERMS), associata ad una prognosi favorevole, e quella alveolare (ARMS) altamente metastatica e con prognosi sfavorevole. Se da un lato la conoscenza del profilo genetico di RMS \ue8 ben approfondita, l\u2019aspetto della caratterizzazione del suo microambiente \ue8 ancor\u2019oggi poco definita. Finora, le interazioni tra cellule tumorali e stromali sono state indagate con modelli in vitro 2D. Tuttavia, questi modelli non sono rappresentativi dei complessi processi biologici che avvengono in vivo, tra i quali la migrazione cellulare. Questa dipende dalle interazioni 3D tra cellule e la matrice extracellulare (ECM) attraverso molecole di adesione come le integrine. Difatti, le interazioni cellula-cellula e cellula-ECM sono ben rappresentate in modelli 3D, che mimano meglio la condizione fisiologica. Scopo: Lo scopo di questo lavoro \ue8 lo sviluppo di un modello 3D di RMS, in grado di ricreare le interazioni cellula-matrice, con particolare attenzione sulle integrine, e di rappresentare i processi di migrazione cellulare che avvengono in condizioni fisiologiche. Materiali e Metodi: Sono state eseguite analisi bioinformatiche sull\u2019espressione di geni della ECM di pazienti affetti da ARMS e da ERMS. Per il primo modello 3D, le masse di ARMS sono state decellularizzate mediante trattamento detergente-enzimatico. Tre differenti strategie di ricellularizzazione sono state testate: semina superficiale, microiniezione e bioreattore a perfusione. L\u2019analisi proteomica del tessuto di ARMS \ue8 stata eseguita per determinare la composizione proteica della ECM. Sono stati sviluppati due ulteriori modelli 3D basati su: Ultrafoam e hydrogels di acido ialuronico/PEG (HA/PEG). Il ruolo di ITGA5 nella motilit\ue0 cellulare \ue8 stato investigato in vitro a seguito di trasfezione con siRNA. La crescita tumorale e la migrazione metastatica sono stati testati in vivo. Risultati: 15 geni correlati alla ECM risultano differenzialmente espressi tra pazienti affetti di ARMS ed ERMS. Per il primo modello 3D, le masse di ARMS sono state decellularizzate ma le tecniche di ricellularizzazione non hanno garantito un buon risultato in termini di distribuzione e vitalit\ue0 cellulare. La composizione della matrice riporta collageni (tipo I e III), fibrillina, fibronectina e periostina. Tra i due ulteriori modelli 3D, Ultrafoam fornisce una struttura simile al tessuto con: superiore proliferazione (41% contro 24%), secrezione di MMP-2 e sovra espressione dei geni ITGA5 e CXCR4 rispetto ai controlli 2D. Gli hydrogels di HA/PEG formano un supporto 3D dove gli sferoidi non dimostrano invasivit\ue0. L\u2019inibizione in vitro di ITGA5 risulta in una ridotta abilit\ue0 migratoria (24.3% contro 43.9% nel controllo). Nel test di invasivit\ue0 entrambe le cellule risultano incapaci di invadere il Matrigel, tuttavia \ue8 stata osservata una differente organizzazione cellulare tra cellule di controllo e silenziare. La crescita tumorale in vivo non mostra associazione con la presenza di ITGA5; tuttavia, la frequenza di extravasazione risulta maggiore in presenza di ITGA5 (30.6% contro 8.5%). Discussione: Tra i modelli 3D testati, l\u2019utilizzo diretto della matrice decellularizzata ha evidenziato una ridotta porosit\ue0 del supporto, risultando in una distribuzione di cellule superficiale e la prevalenza di interazioni cellula-cellula piuttosto che cellula-ECM. Il supporto di Ultrafoam ha prodotto i migliori risultati in termini di interazioni cellula-microambiente; tuttavia, il bioreattore \ue8 inaccessibile per la visualizzazione al microscopio. D\u2019altra parte, l\u2019hydrogel \ue8 otticamente trasparente e pi\uf9 direttamente funzionalizzabile con altre proteine di matrice specifiche di ARMS. Negli hydrogels di HA/PEG, la concentrazione di fibronectina verr\ue0 ottimizzata assieme all\u2019aggiunta di altre proteine di matrice. I risultati in vitro evidenziano che altre proteine cooperano nella migrazione cellulare nelle cellule di ARMS. La differente organizzazione in Matrigel suggerisce un cross-talk tra ITGA5 e proteine di adesione cellula-cellula. I risultati in vivo indicano che ITGA5 non \ue8 necessaria per la crescita tumorale, tuttavia sembra avere un ruolo funzionale nel processo di extravasazione. Conclusioni: Questo lavoro ha sviluppato tre differenti modelli 3D per lo studio di ARMS, ciascuno con vantaggi e svantaggi che devono essere considerati a seconda del processo biologico da investigare. In futuro prevediamo che un\u2019analisi pi\uf9 dettagliata del microambiente di ARMS potr\ue0 portare alla luce di nuovi marcatori prognostici e terapeutici per migliorare la sopravvivenza dei giovani pazienti

Alveolar Rhabdomyosarcoma Decellularization

In cancer research, it is an urgent need in the obtainment of a simple and reproducible model that mimics in all the complexity the pathological microenvironment. Specifically, the will to improve the overall survival of young patients affected by rhabdomyosarcoma compels researchers to develop new models resembling the multifaceted environment of the pathology to deeply study the disease under novel and different aspects. To this end, we developed a decellularization protocol for alveolar rhabdomyosarcoma (ARMS) able to maintain the three-dimensional structure. The attained extracellular matrix (ECM) can be used as 3D in vitro model suitable to both recapitulate the in vivo cancer microenvironment, and also for drug testing. Here, we first describe a detergent-enzymatic method and then we analyze the decellularization efficiency and the scaffold proteins

A perfusion-based three-dimensional cell culture system to model alveolar rhabdomyosarcoma pathological features

Abstract Although a rare disease, rhabdomyosarcoma (RMS) is one of the most common cancers in children the more aggressive and metastatic subtype is the alveolar RMS (ARMS). Survival outcomes with metastatic disease remain dismal and the need for new models that recapitulate key pathological features, including cell-extracellular matrix (ECM) interactions, is warranted. Here, we report an organotypic model that captures cellular and molecular determinants of invasive ARMS. We cultured the ARMS cell line RH30 on a collagen sponge in a perfusion-based bioreactor (U-CUP), obtaining after 7 days a 3D construct with homogeneous cell distribution. Compared to static culture, perfusion flow induced higher cell proliferation rates (20% vs. 5%), enhanced secretion of active MMP-2, and upregulation of the Rho pathway, associated with cancer cell dissemination. Consistently, the ECM genes LAMA1 and LAMA2, the antiapoptotic gene HSP90, identified in patient databases as hallmarks of invasive ARMS, were higher under perfusion flow at mRNA and protein level. Our advanced ARMS organotypic model mimics (1) the interactions cells-ECM, (2) the cell growth maintenance, and (3) the expression of proteins that characterize tumor expansion and aggressiveness. In the future, the perfusion-based model could be used with primary patient-derived cell subtypes to create a personalized ARMS chemotherapy screening system