Associations of chemo- and radio-resistant phenotypes with the gap junction, adhesion and extracellular matrix in a three-dimensional culture model of soft sarcoma

Abstract

Background: Three-dimensional (3D) culture models are considered to recapitulate the cell microenvironment in solid tumors, including the extracellular matrix (ECM), cell-cell interactions, and signal transduction. These functions are highly correlated with cellular behaviors and contribute to resistances against chemo- and radio-therapies. However, the biochemical effects and mechanisms remain unknown in soft sarcoma. Therefore, we developed an in vitro 3D model of sarcoma to analyze the reasons of the chemo- and radio-resistance in therapies.

Methods: Four soft sarcoma cell lines, HT1080, RD, SW872, and human osteosarcoma cell line 1 (HOSS1), a cell line established from a patient-derived xenograft, were applied to 3D culture and treated with growth factors in methylcellulose-containing medium. Spheroids were examined morphologically and by western blotting, RT-qPCR, and immunofluorescence staining to analyze cell adhesion, gap junctions, ECM genes, and related factors. Proliferation and colony formation assays were performed to assess chemo- and radio-resistances between 3D and two-dimensional (2D) cell cultures. Annexin V and Propidium Iodide staining was used to detect early apoptotic sarcoma cells treated with Doxorubicin, Gemcitabine, and Docetaxel in the 3D model.

Results: The four soft sarcoma cell lines formed spheres in vitro by culture in modified condition medium. Compared with 2D cell culture, expression of ECM genes and proteins, including COL1A1, LOX, SED1, FN1, and LAMA4, was significantly increased in 3D culture. Analysis of cadherin and gap junction molecules showed significant changes in the gene and protein expression profiles under 3D conditions. These changes affected cell-cell communication and were mainly associated with biological processes such as cell proliferation and apoptosis related to chemo- and radio-resistances.

Conclusions: Our findings revealed significant differences between 3D and 2D cell culture systems, and indicated that cellular responsiveness to external stress such as radiation and chemotherapeutics is influenced by differential expression of genes and proteins involved in regulation of the ECM, cell adhesion, and gap junction signaling.

Fig. 1

Effects of spheroid formation and growth. a: HT1080, RD, SW872 and HOSS1 soft sarcoma cell lines grown in 3D culture at day 8. HOSS1 cells established from an osteosarcoma PDX were also grown in special medium without FBS. b: Cell counts in 3D cultures at various time points. c: Cell counts in 3D spheres between weeks 1 and 2 were not significantly different (range: 40–65 %). d Stable proliferation of the four cell lines in 3D culture. HT1080 cells required 4 days, whereas the other cell lines needed 8 days. The four cell lines underwent stable growth after these initial periods. e, f: Cell numbers and growth time in 2D cultures at various times. In contrast to 3D cultures, cells did not lose stability until 10 days in 2D culture. Shaded areas indicate stable proliferation. g: Doubling times of 2D and 3D cell cultures at day 8 after plating. Results are the mean ± SD (n = 6). N.S., not significant, Bar = 100 μm

Fig. 2

Differences in adhesion and gap junction molecule expression of HOSS1 cells in 3D and 2D cultures. a: Immunofluorescence staining of Cx26, Cx43, and Cx45 in 3D (upper) and 2D (lower) cultures of HOSS1 cells. Nuclei were counterstained with DAPI. Bar = 20 μm. Magnification, ×400. b, c: RT-qPCR and western blot analyses revealed up-regulation of the mRNA and protein expression of connexins in 3D cultures, respectively. d, e: mRNA expression of N-cadherin (d) and E-cadherin (e) in HOSS1 cells as determined by RT-qPCR. f: Western blotting showed up-regulation of E-cadherin in a time-dependent manner in 3D cultures, whereas N-cadherin expression reached a peak day 10 and decreased thereafter. Results represent the mean ± SD of three independent experiments with Student’s t-test

Fig. 3

ECM-related gene expressions of HOSS1 cells in 3D and 2D cultures. a: RT-qPCR analysis of the expression of ECM-related genes in 3D and 2D cultures of HOSS1 cells at day 8. Data are presented as fold differences relative to 2D-cultured cells for each gene, which was defined as 1 (calibrator). Data are mean ± SD of 3 independent experiments with Student’s t-test. Error bars indicate SD. b: Western blotting of ECM-related protein expression in 3D and 2D cultures at day 8

Fig. 4

Increases in chemo-resistances against anticancer agents and radio-resistances in 3D cultures of HOSS1 cells. a: RT-qPCR analysis of drug resistance-related genes (ATP-binding cassette transporters) in 3D and 2D cultures. Values in 2D cultures for each gene were defined as 1. Results are the mean ± SD (n = 3) with Student’s-t test. b: Western blot analysis of HOSS1 cells in 3D and 2D cultures. c, d: Clonogenic survival of 3D- and 2D-cultured cells exposed to various concentrations of Doxorubicin (10, 100 nM, 1 μM, and 10 μM), Gemcitabine (1, 10, 100, and 1000 μM), and Docetaxel (1, 10, 100, and 1000 μM) (c) or X-ray doses (2, 4, 6, and 8 Gy) (d) at 24 hours after plating. ^* P < 0.05; ^** P < 0.01 vs. respective 2D-cultured cells. Results of a representative independent experiment of three are shown

Fig. 5

Apoptosis of soft sarcoma cells treated with anticancer agents in 3D and 2D cultures. a: Flow cytometric analysis of apoptotic HOSS1 cells in 2D (upper) and 3D (lower) cultures treated with 0.02 μM Doxorubicin, 2 μM Gemcitabine, or 2 μM Docetaxel (Annexin V positive: apoptotic cells; PI positive: dead cells.) About 2–3 fold fewer HOSS1 cells were apoptotic in 3D cultures than in 2D cultures. b, c, d: HT1080, RD, SW872, and HOSS1 cells were exposed to Doxorubicin (b), Gemcitabine (c), or Docetaxel (d) in 3D and 2D cultures. Apoptosis ratios were calculated from three independent experiments for all four cell lines. ^* P < 0.05 vs respective 2D-cultured cells