1. A bioengineering method for modeling alveolar Rhabdomyosarcoma and assessing chemotherapy responses
- Author
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David L. Perrin, Ehsan Samiei, Mohammad Esmaeillou, Arya Emami, Saeid Ghavami, Mohsen Akbari, Joseph W. Gordon, Kiarash Roustai Geraylow, Mahboubeh Kavoosi, Evan Stefanek, and Milad Ashrafizadeh
- Subjects
DFS, Disease-free survival ,Clinical Biochemistry ,Apoptosis ,MYOD1, Myogenic muscle differentiation transcription factor 1 ,Biofabrication ,Extracellular matrix ,DNA, Deoxyribonucleic acid ,0302 clinical medicine ,Rhabdomyosarcoma ,Medicine ,RPMI, Roswell Park Memorial Institute ,PDMS, Polydimethylsiloxane ,RGD, Arginylglycylaspartic acid ,0303 health sciences ,IgG, Immunoglobulin G ,RMS, Rhabdomyosarcoma ,DAPI, 4’,6-Diami- dino-2-Phenylindole, Dihydrochloride ,EDTA, Ethylenediaminetetraacetic acid ,Primary tumor ,EM, Engineered model ,PAX, Paired box gene ,AKT, Protein Kinase B ,3. Good health ,Medical Laboratory Technology ,HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) ,030220 oncology & carcinogenesis ,Alveolar rhabdomyosarcoma ,Protocol Article ,Cell death ,EthD-1, Ethidium homodimer-1 ,Programmed cell death ,3D, Three-dimensional ,Science ,dECM, Decellularized extracellular matrix ,2D, Two-dimensional ,MEK, Mitogen-activated extracellular signal-regulated kinase ,FOXO1, Forkhead box protein O1 ,03 medical and health sciences ,BSA, Bovine serum albumin ,ECM, Extracellular matrix ,In vivo ,RT, Room temperature ,DMEM, Dulbecco's phosphate buffered saline ,Autophagy ,030304 developmental biology ,business.industry ,ICC, Immunocytochemistry ,LC3, Microtubule associated protein 1A/1B-light chain 3 ,PNIPAAm, Poly-N-isopropylacrylamide ,medicine.disease ,Cell culture ,TMZ, Temozolomide ,FBS, Fetal bovine serum ,Cancer research ,business - Abstract
Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue malignant tumor. Treatment of RMS usually includes primary tumor resection along with systemic chemotherapy. Two-dimensional (2D) cell culture systems and animal models have been extensively used for investigating the potential efficacy of new RMS treatments. However, RMS cells behave differently in 2D culture than in vivo, which has recently inspired the adoption of three-dimensional (3D) culture environments. In the current paper, we will describe the detailed methodology we have developed for fabricating a 3D engineered model to study alveolar RMS (ARMS) in vitro. This model consists of a thermally cross-linked collagen disk laden with RMS cells that mimics the structural and bio-chemical aspects of the tumor extracellular matrix (ECM). This process is highly reproducible and produces a 3D engineered model that can be used to analyze the cytotoxicity and autophagy induction of drugs on ARMS cells. The most improtant bullet points are as following:•We fabricated 3D model of ARMS.•The current ARMS 3D model can be used for screening of chemotherapy drugs.•We developed methods to detect apoptosis and autophagy in ARMS 3D model to detect the mechansims of chemotherapy agents., Graphical abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue malignant tumor. Despite numerous clinical trials that have investigated the efficacy of multidrug chemotherapy regimens, outcomes for high-risk RMS patients are still poor which has inspired research into new targeted therapies that molecularly target malignant RMS cells. In order to evaluate such therapies in vitro in a realistic tumor microenvironment, we have bioengineered a cell-laden 3D tissue-engineered model with uniform geometry and an uncomplicated and reproducible biofabrication process. The transparent collagen-I hydrogel promotes cell attachment and growth and allows for analysis of the cellular response to drugs through brightfield microscopy, immunocytochemistry, and live/dead viability assays.Image, graphical abstract
- Published
- 2021