Analysis of a Preliminary microRNA Expression Signature in a Human Telangiectatic Osteogenic Sarcoma Cancer Cell Line

Abstract

Telangiectatic osteosarcoma (TOS) is an aggressive variant of osteosarcoma (OS) with distinctive radiographic, gross, microscopic features, and prognostic implications. Despite several studies on OS, we are still far from understanding the molecular mechanisms of TOS. In recent years, many studies have demonstrated not only that microRNAs (miRNAs) are involved in OS tumorigenesis, development, and metastasis, but also that the presence in high-grade types of OS of cancer stem cells (CSCs) plays an important role in tumor progression. Despite these findings, nothing has been described previously about the expression of miRNAs and the presence of CSCs in human TOS. Therefore, we have isolated/characterized a putative CSC cell line from human TOS (TOS-CSCs) and evaluated the expression levels of several miRNAs in TOS-CSCs using real-time quantitative assays. We show, for the first time, the existence of CSCs in human TOS, highlighting the in vitro establishment of this unique stabilized cell line and an identification of a preliminary expression of the miRNA profile, characteristic of TOS-CSCs. These findings represent an important step in the study of the biology of one of the most aggressive variants of OS and the role of miRNAs in TOS-CSC behavior.

Keywords: cancer stem cells; microRNAs; telangiectatic osteosarcoma.

Figure 1

Biopsy specimen and primary cell culture of TOS. (A) Biopsy specimen of TOS by needle aspiration. Observation in phase contrast of primary cell culture of TOS after 24 h from enzymatical and mechanical treatment of the biopsy (B) and after three days (C). Original magnification 10×.

Figure 2

Sarcosphere formation assay on TOS and sarcosphere adhesion. Sarcosphere formation under non-adherent conditions in TOS1 cell line after 24 h (A), 7 days (B), 14 days (C), and 21 days (D,E). Sarcospheres from TOS1 cell line showed adherent expansion by reintroduction and reculturing in monolayer, adherent conditions at 48 h from the isolation (F) and after 7 days (G). Observation in phase contrast. Original magnification 10×-20-40×.

Figure 3

Adipogenic differentiation assay and expression of adipogenic gene markers. Adipogenic differentiation at 0 days (A) and after 14 days (B) of induction by cytochemical staining with OIL Red O. Red shows the lipidic vesicles, violet shows the nuclei counterstained by hematoxylin. Observation is in brightfield. Original magnifications: 10×. (C) RT-PCR show the expression of PPARγ (left) and of LPL (right) in TOS1-CSCs after 14 days of adipogenic induction.

Figure 4

Osteogenic differentiation assay_ALP and HA. Osteogenic differentiation_ALP at 0 days (A) and after 10 days (B) of induction by cytochemical staining for ALP with Fast Blue BB. Blue shows the ALP + cells; in orange/red the nucleus counterstained by Propidium Iodide. Composite observation in brightfield and fluorescence. Osteogenic differentiation_HA at 0 days (C) and after 20 days (D) of induction by cytochemical staining for Hydroxyapatite (HA) with Alizarin Red S. The cells only are contrasted in blue/grey, and the grainy deposits of HA are stained in red. Observation in phase contrast. Original magnification: 20×.

Figure 5

ALDH activity assay. The ALDH colorimetric assay detected high levels of ALDH activity in TOS1-CSCs cell line. However, the assay detected the absence of this activity in the finite differentiated cell line of fibroblasts, FIB48. Error bars: SD. *: p < 0.05 vs. FIB48.

Figure 6

Chondrogenic differentiation assay and expression of hyaline-chondrogenic gene markers. Histological evaluation of TOS1-CSC condrogenic 3Dimensional cell pellet at 21 days (A). Alcian Blue stain demonstrates positive glycosaminoglycan production (B). Stereomicroscopy images. Original magnification: 16× and 40×. (C) RT-PCR show the expression of COLXA1, DCN, BGN, and ACAN in TOS1-CSCs (left) compared to their expression in a primary cell line of human articular chondrocytes (ACs) (control) (right) after 21 days of chondrogenic induction.

Figure 7

Immunofluorescence staining of Mesenchymal Stem Cell (MSC) and of Hematopoietic surface markers. Immunofluorescence staining of CD44 (A), CD105 (C), CD90 (E) and of CD45 (G) of the TOS1-CSC cell line. Respectively, CD44 (B), CD105 (D), CD90 (F) and of CD45 (H) in a human fibroblast cell line (negative control). LSCM in conventional colors: green for MSC markers and blue for nuclei. Original magnification: 10×. Bar size: 100 µm.

Figure 8

Immunofluorescence staining of embryonic stem cell (ESC) markers. Immunofluorescence staining of Nanog (A), of KLF4 (C), of Sox2 (E) and of POU5F1 (G) of TOS1-CSCs line and of HCT8 cell line (B,D,F,H) (negative control). LSCM in conventional colors: green for ESC nuclear/perinuclear markers and red for cytoskeleton. Original magnification: 10×.

Figure 9

Immunofluorescence staining of neoplastic markers. Immunofluorescence staining of c-Kit (A) and of Nestin (C) of TOS1-CSC cell line. Immunofluorescence of c-Kit (B) and of Nestin (D) of human fibroblast cell line (negative control). LSCM in conventional colors: green for c-Kit and Nestin and blue for nuclei. Original magnifications: 10×. Bar size: 100 µm.

Figure 10

Gene expression in isolated RT-PCR showed the expression of SATB2 and PROM1 gene in TOS1 primary cell culture and in the isolated TOS1-CSC cell line. In addition to this, RT-PCR also showed the expression of the ESC marker genes and of EZR, AXL and MYC only in the TOS1-CSC cell line.

Figure 11

Expression levels of ESC gene markers. The relative POU5F1 (A), KLF4 (B), Nanog (C), SOX2 (D) and LIN28A (E) mRNA expression levels in TOS1-CSC cell line was determined by RT-qPCR. The results are expressed relative to GAPDH mRNA levels. Data represent the mean with standard deviation (n = 3). * p < 0.05 versus TOS1 cell line.

Figure 12

Expression levels of 18 miRNAs in TOS1-CSCs line. RT-qPCR analysis of expression of Let-7c (A), Let-7d (B), Let-7e (C), Let-7f (D), miR-135 (E), miR-184 (F), miR-221 (G), miR-18a (H), miR-200 (I), miR-1 (L), miR-744 (M), miR-296 (N), miR-9 (O), miR-199a-3p (P), miR-320 (Q), miR-654 (R), miR-141 (S), and miR-369-3p (T) in TOS1 and in TOS1-CSC cell lines. Data represent the mean with standard deviation (n = 4); * p < 0.001 as compared to TOS1 cell line.

Figure 13

Expression levels of four miRNAs in the TOS1-CSC line. RT-qPCR analysis of expression of Let-7a (A), miR-25 (B), miR-423-5p (C), miR-500 (D), miR-365 (E) and Let-7g (F) were examined by real-time RT-PCR analysis in both TOS1 cell line and isolated TOS1-CSC cell line. Data represent the mean with standard deviation (n = 4); * p < 0.001 as compared to TOS1 cell line.