DOI: 10.5061/dryad.w3r228128
title: Targeting cell-matrix induced chemoresistance with regorafenib in a 3D model of osteosarcoma

description: Over the past four decades, there has been little advancement in treatment
 strategies for osteosarcoma (OS), the predominant primary bone tumor in
 the pediatric patient population. Current therapy involves multiple rounds
 of chemotherapy and surgical resection, which are associated with
 significant morbidity and suboptimal survival rates. A key challenge in
 developing new treatments is the difficulty in replicating the OS tumor
 microenvironment, particularly cell interactions with the extracellular
 matrix (ECM). This study uses an in vitro model of OS to investigate the
 cell response to collagen type I, the primary component of the
 osteosarcoma ECM. After seven days of culture within three-dimensional
 collagen hydrogels, OS cells displayed a more elongated cellular
 morphology and reduced sensitivity to the standard chemotherapy used for
 OS treatment compared to cells grown on two-dimensional substrates. To
 test whether this model could be used to study treatment strategies used
 for high-risk OS patients, we applied a metronomic regimen combining
 regorafenib, a multi-tyrosine kinase inhibitor, with front-line
 chemotherapy to overcome cell-matrix-induced chemoresistance. We
 identified overexpression of the ATP-binding cassette transporter ABCG2, a
 drug efflux pump, as a potential mechanism of resistance in 3D culture.
 Regorafenib’s inhibitory effect on ABCG2 suggests a mechanistic basis for
 its ability to restore chemosensitivity in 3D culture. Altogether, these
 findings highlight the importance of cell-matrix interactions in in vitro
 OS models, provide valuable insights into a matrix-induced mechanism of OS
 chemoresistance, and suggest an approach to its treatment.
description: # Targeting cell-matrix induced chemoresistance with regorafenib in a 3D
 model of osteosarcoma Dataset DOI:
 [10.5061/dryad.w3r228128](10.5061/dryad.w3r228128) ## Description of the
 data and file structure All raw data files are compressed into the .zip
 file - R_Rao_et_al_JBMR-A-25-0496.zip Final Figures and Supplemental Data,
 and Tables are provided in the Zenodo Supplemental Information.  Figure 1
 - Osteosarcoma cell behavior across 2D and 3D culture. (b) These data
 depict the microarchitecture of collagen (COL) hydrogels in 2D and 3D
 configurations obtained through second harmonic generation imaging. (c)
 Quantification of mesh size from second harmonic generation images. (d)
 Cell viability images (green = live, red = dead) in the 2D tissue culture
 plastic (TCP), 2D COL, and 3D COL conditions. (e) Quantification of cell
 count through seven days of culture. (f) Cell morphology by F-actin
 staining (green) and nuclear (blue) visualization. (g) Quantification of
 cell density from the cell morphology images.  Figure 2 - Increased
 expression of ALP and activation of the PI3K/AKT pathway in osteosarcoma
 cultures within 3D COL matrices. (a) Alkaline phosphatase (ALP) activity
 of cells cultured in 2D TCP, 2D COL, and 3D COL over a 7-day culture
 period. Data is normalized to DNA content. (b) Gene expression was
 analyzed through RT-PCR of AKT/Protein Kinase B expression. (c) Protein
 expression through Western blot of phosphorylated-AKT (active) and total
 AKT. (d) Quantification of Western blot. Figure 3 - Decreased
 chemosensitivity in OS cultures within 3D COL matrices. (a-c) Dose
 response data after 24 hours of treatment with methotrexate, doxorubicin,
 and cisplatin in the three culture conditions. (d-f) Extrapolation of
 half-maximal inhibitor concentrations (IC50) to each drug. Figure 4 -
 Analysis of diffusivity, mesh size at day 7 of culture, and drug
 penetration in COL matrices. (a) Fluorescence recovery after
 photobleaching images. (b) Quantification of the diffusion coefficient
 from each fluorescently labeled particle within 3D COL hydrogels. The
 associated MATLAB script accompanies the data. (c) Second harmonic
 generation images of osteosarcoma cells cultured on 2D COL and 3D COL
 after 7 days of culture, with quantification of mesh size. (d) Qualitative
 assessment of drug penetration of doxorubicin through 2D TCP, 2D COL, and
 3D COL culture conditions. Figure 5 - Pre-treatment with regorafenib
 (REG), a tyrosine kinase inhibitor used in metastatic and relapsed
 osteosarcoma patients. (a) Live/Dead images and (b) quantification of cell
 viability. (c) Second harmonic generation images of cell-seeded 3D COL
 hydrogels after 7 days of culture with and without REG. (d) Quantification
 of the mesh size of the hydrogel. (e) Dose response data with and without
 REG pre-treatment after 24 hours of treatment with doxorubicin. (f) Dose
 response data in 2D TCP, 2D COL, and 3D COL with REG pre-treatment. (g)
 Extrapolation of IC50 values with and without REG pre-treatment. Figure 6
 - Elevated ABCG2, a drug efflux pump, expression in osteosarcoma cells
 cultured in 3D COL matrices. (a) Gene expression data of ABCG2 in the
 three culture conditions. (b) Protein expression of ABCG2 through Western
 blot. (c) Quantification of Western blot data.  Figure S1 -
 Characterization of COL matrix properties. (a) Rheology data of a
 temperature ramp over 3 minutes. (b) Frequency sweep of a fully formed COL
 hydrogel.  Figure S2 - Second harmonic generation imaging of acellular and
 cell-laden 2D COL and 3D COL hydrogels. Images of acellular cells (blue =
 nuclei, green = F-actin) in 2D and 3D COL culture. Quantification of mesh
 size.  Figure S3 - Quantification of cell viability on day 7 of culture in
 2D TCP, 2D COL, and 3D COL.  Figure S4 - Images of cell viability of
 osteosarcoma cells on days 1, 3, and 7 in 3D COL. Green = live, red =
 dead.  Figure S5 - Montage of z-stack of cells within 3D COL. Green =
 live, red = dead.  Figure S6 - Quantification of COL mesh size from second
 harmonic generation images over a 7-day culture period.  Figure S7 -
 Quantification of metabolic activity on day 7 culture after REG
 pre-treatment (0 μM, 1 μM, and 5 μM).  Figure S8 - Impact of REG
 pre-treatment on osteosarcoma cells cultured on 2D TCP and 2D COL. (a)
 Live/Dead images (green = live, red = dead) with and without REG
 pre-treatment. (b) Cell viability quantification with and without REG
 pre-treatment.  Figure S9 - ABC transporter gene expression.
 Quantification of gene expression of (a) ABCC1 and (b) ABCB1.  ### Files
 and variables All data is organized in the "Raw Data" folder and
 separated into folders corresponding to the Figures of the
 manuscript. Files are organized as raw Excel, raw images, and analyzed
 images.  Excel files are tabulated as "Raw" and
 "Analyzed" sheets to allow for reproducibility in data analysis.
 The "Raw" sheets are obtained from the source of data curation
 (spectrophotometer or imaging apparatus). "Analyzed" data are
 labeled with conditions and relevant formulas to allow for reanalysis. 
 Images are labeled in folders labeled as "Final" - indicating
 these are the images used for the publication - and "Raw" -
 indicating these images were used for data analysis.  File types  * Images
 as .tif, .tiff, and .PNG files * Data sheets as .xlsx Abbreviations * 2D =
 two-dimensional * 3D = three-dimensional * COL = collagen * SHG = second
 harmonic generation * ALP = alkaline phosphatase * AKT = protein kinase B
 * pAKT = phosphorylated AKT (active) * FRAP = fluorescence after
 photobleaching * REG = regorafenib * ABC = ATP-Binding Cassette  ##
 Code/software Open software via MATLAB script used for Figure 4 FRAP data,
 referenced in the Methods section ## Access information NA