Cancer stem cells as a therapeutic target in 3D tumor models of human chondrosarcoma: An encouraging future for proline rich polypeptide‑1

Granger,Caroline   J.; Hoyt,Aaron   K.; Moran,Alexandra; Becker,Beatrice; Sedani,Anil; Saigh,Shannon; Conway,Sheila   A.; Brown,Jeffrey; Galoian,Karina

doi:10.3892/mmr.2020.11480

Cancer stem cells as a therapeutic target in 3D tumor models of human chondrosarcoma: An encouraging future for proline rich polypeptide‑1

Authors:
- Caroline J. Granger
- Aaron K. Hoyt
- Alexandra Moran
- Beatrice Becker
- Anil Sedani
- Shannon Saigh
- Sheila A. Conway
- Jeffrey Brown
- Karina Galoian
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Affiliations: RMSB Room 8012 (D27), Department of Orthopedic Surgery, University of Miami Miller School of Medicine, Miami, FL 33136, USA, Department of Medicine, Sylvester Comprehensive Cancer Center, Miami, FL 33136, USA
Published online on: September 2, 2020 https://doi.org/10.3892/mmr.2020.11480
Pages: 3747-3758
Copyright: © Granger et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Chondrosarcoma is a malignant bone neoplasm that is refractory to chemotherapy and radiation. With no current biological treatments, mutilating surgical resection is the only effective treatment. Proline rich polypeptide 1 (PRP‑1), which is a 15‑amino acid inhibitor of mammalian target of rapamycin complex‑1 (mTORC1), has been indicated to exert cytostatic and immunomodulatory properties in human chondrosarcoma cells in a monolayer. The aim of the present study was to evaluate the effects of PRP‑1 on an in vitro 3D chondrosarcoma tumor model, known as spheroids, and on the cancer stem cells (CSCs) which form spheroids. JJ012 cells were cultured and treated with PRP‑1. An ALDEFLUOR™ assay was conducted (with N,N‑diethylaminobenzaldehyde as the negative control) to assess aldehyde dehydrogenase (ALDH) activity (a recognized CSC marker), and bulk JJ012, ALDHhigh and PRP‑1 treated ALDHlow cells were sorted using flow cytometry. Colony formation and spheroid formation assays of cell fractions, including CSCs, were used to compare the PRP‑1‑treated groups with the control. CSCs were assessed for early apoptosis and cell death with a modified Annexin V/propidium iodide assay. Western blotting was used to identify mesenchymal stem cell markers (STRO1, CD44 and STAT3), and spheroid self‑renewal assays were also conducted. A clonogenic dose‑response assay demonstrated that 20 µg/ml PRP‑1 was the most effective dose for reducing colony formation capacity. Furthermore, CSC spheroid growth was significantly reduced with increasing doses of PRP‑1. Annexin V analysis demonstrated that PRP‑1 induced CSC cell death, and that this was not attributed to apoptosis or necrosis. Western blot analysis confirmed the expression of mesenchymal markers, and the spheroid self‑renewal assay confirmed the presence of self‑renewing CSCs. The results of the present study demonstrate that PRP‑1 eliminates anchorage independent CSC growth and spheroid formation, indicating that PRP‑1 likely inhibits tumor formation in a murine model. Additionally, a decrease in non‑CSC bulk tumor cells indicates an advantageous decline in tumor stromal cells. These findings confirm that PRP‑1 inhibits CSC proliferation in a 3D tumor model which mimics the behavior of chondrosarcoma in vivo.

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1	Karpik M and Reszec J: Low grade chondrosarcoma-epidemiology, diagnosis, treatment. Ortop Traumatol Rehabil. 20:65–70. 2018. View Article : Google Scholar : PubMed/NCBI
2	Boehme KA, Schleicher SB, Traub F and Rolauffs B: Chondrosarcoma: A rare misfortune in aging human cartilage? The role of stem and progenitor cells in proliferation, malignant degeneration and therapeutic resistance. Int J Mol Sci. 19:3112018. View Article : Google Scholar
3	Chow WA: Chondrosarcoma: Biology, genetics, and epigenetics. F1000Res. 7:F1000 Faculty Rev-1826. 2018. View Article : Google Scholar : PubMed/NCBI
4	Leddy LR and Holmes RE: Chondrosarcoma of bone. Cancer Treat Res. 162:117–130. 2014. View Article : Google Scholar : PubMed/NCBI
5	Mery B, Espenel S, Guy JB, Rancoule C, Vallard A, Aloy MT, Rodriguez-Lafrasse C and Magné N: Biological aspects of chondrosarcoma: Leaps and hurdles. Crit Rev Oncol Hematol. 126:32–36. 2018. View Article : Google Scholar : PubMed/NCBI
6	Andreou D, Ruppin S, Fehlberg S, Pink D, Werner M and Tunn PU: Survival and prognostic factors in chondrosarcoma: Results in 115 patients with long-term follow-up. Acta Orthop. 82:749–755. 2011. View Article : Google Scholar : PubMed/NCBI
7	Wan L, Tu C, Li S and Li Z: Regional lymph node involvement is associated with poorer survivorship in patients with chondrosarcoma: A SEER analysis. Clin Orthop Relat Res. 477:2508–2518. 2019. View Article : Google Scholar : PubMed/NCBI
8	Nie Z, Lu Q and Peng H: Prognostic factors for patients with chondrosarcoma: A survival analysis based on the Surveillance, Epidemiology, and End Results (SEER) database (1973–2012). J Bone Oncol. 13:55–61. 2018. View Article : Google Scholar : PubMed/NCBI
9	Song K, Song J, Chen F, Lin K, Ma X and Jiang J: Does resection of the primary tumor improve survival in patients with metastatic chondrosarcoma? Clin Orthop Relat Res. 477:573–583. 2019. View Article : Google Scholar : PubMed/NCBI
10	Colella G, Fazioli F, Gallo M, De Chiara A, Apice G, Ruosi C, Cimmino A and de Nigris F: Sarcoma spheroids and organoids-promising tools in the Era of personalized medicine. Int J Mol Sci. 19:6152018. View Article : Google Scholar
11	Brown HK, Tellez-Gabriel M and Heymann D: Cancer stem cells in osteosarcoma. Cancer Lett. 386:189–195. 2017. View Article : Google Scholar : PubMed/NCBI
12	Rodriguez R, Rubio R and Menendez P: Modeling sarcomagenesis using multipotent mesenchymal stem cells. Cell Res. 22:62–77. 2012. View Article : Google Scholar : PubMed/NCBI
13	Hanahan D and Weinberg RA: The hallmarks of cancer. Cell. 100:57–70. 2000. View Article : Google Scholar : PubMed/NCBI
14	Fouad YA and Aanei C: Revisiting the hallmarks of cancer. Am J Cancer Res. 7:1016–1036. 2017.PubMed/NCBI
15	Ishiguro T, Ohata H, Sato A, Yamawaki K, Enomoto T and Okamoto K: Tumor-derived spheroids: Relevance to cancer stem cells and clinical applications. Cancer Sci. 108:283–289. 2017. View Article : Google Scholar : PubMed/NCBI
16	Guo X, Chen Y, Ji W, Chen X, Li C and Ge R: Enrichment of cancer stem cells by agarose multi-well dishes and 3D spheroid culture. Cell Tissue Res. 375:397–408. 2019. View Article : Google Scholar : PubMed/NCBI
17	Mehta P, Novak C, Raghavan S, Ward M and Mehta G: Self-renewal and CSCs in vitro enrichment: Growth as floating spheres. Methods Mol Biol. 1692:61–75. 2018. View Article : Google Scholar : PubMed/NCBI
18	Ma R, Mandell J, Lu F, Heim T, Schoedel K, Duensing A, Watters RJ and Weiss KR: Do patient-derived spheroid culture models have relevance in chondrosarcoma research? Clin Orthop Relat Res. May 19–2020.doi: 10.1097/CORR.0000000000001317. (Epub ahead of print). View Article : Google Scholar
19	Fujii H, Honoki K, Tsujiuchi T, Kido A, Yoshitani K and Takakura Y: Sphere-forming stem-like cell populations with drug resistance in human sarcoma cell lines. Int J Oncol. 34:1381–1386. 2009.PubMed/NCBI
20	Gao S, Shen J, Hornicek F and Duan Z: Three-dimensional (3D) culture in sarcoma research and the clinical significance. Biofabrication. 9:0320032017. View Article : Google Scholar : PubMed/NCBI
21	Gibbs CP, Kukekov VG, Reith JD, Tchigrinova O, Suslov ON, Scott EW, Ghivizzani SC, Ignatova TN and Steindler DA: Stem-like cells in bone sarcomas: Implications for tumorigenesis. Neoplasia. 7:967–976. 2005. View Article : Google Scholar : PubMed/NCBI
22	Tang QL, Zhao ZQ, Li JC, Liang Y, Yin JQ, Zou CY, Xie XB, Zeng YX, Shen JN, Kang T and Wang J: Salinomycin inhibits osteosarcoma by targeting its tumor stem cells. Cancer Lett. 311:113–121. 2011. View Article : Google Scholar : PubMed/NCBI
23	Galoyan AA, Shakhlamov VA, Aghajanov MI and Vahradyan HG: Hypothalamic proline-rich polypeptide protects brain neurons in aluminum neurotoxicosis. Neurochem Res. 29:1349–1357. 2004. View Article : Google Scholar : PubMed/NCBI
24	Galoian K, Abrahamyan S, Chailyan G, Qureshi A, Patel P, Metser G, Moran A, Sahakyan I, Tumasyan N, Lee A, et al: Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma. Int J Oncol. 52:139–154. 2018.PubMed/NCBI
25	Galoian K, Luo S, Qureshi A, Patel P, Price R, Morse AS, Chailyan G, Abrahamyan S and Temple HT: Effect of cytostatic proline rich polypeptide-1 on tumor suppressors of inflammation pathway signaling in chondrosarcoma. Mol Clin Oncol. 5:618–624. 2016. View Article : Google Scholar : PubMed/NCBI
26	Galoian KA, Temple TH and Galoyan A: Cytostatic effect of novel mTOR inhibitor, PRP-1 (galarmin) in MDA 231 (ER-) breast carcinoma cell line. PRP-1 inhibits mesenchymal tumors. Tumour Biol. 32:745–751. 2011. View Article : Google Scholar : PubMed/NCBI
27	Galoian K, Scully S and Galoyan A: Myc-oncogene inactivating effect by proline rich polypeptide (PRP-1) in chondrosarcoma JJ012 cells. Neurochem Res. 34:379–385. 2009. View Article : Google Scholar : PubMed/NCBI
28	Galoian K, Scully S, McNamara G, Flynn P and Galoyan A: Antitumorigenic effect of brain proline rich polypeptide-1 in human chondrosarcoma. Neurochem Res. 34:2117–2121. 2009. View Article : Google Scholar : PubMed/NCBI
29	Galoian K, Qureshi A, Wideroff G and Temple HT: Restoration of desmosomal junction protein expression and inhibition of H3K9-specific histone demethylase activity by cytostatic proline-rich polypeptide-1 leads to suppression of tumorigenic potential in human chondrosarcoma cells. Mol Clin Oncol. 3:171–178. 2015. View Article : Google Scholar : PubMed/NCBI
30	Hoyt AK, Moran A, Granger C, Sedani A, Saigh S, Brown J and Galoian KA: PRP1 significantly decreases the ALDHhigh cancer stem cell population and regulates the aberrant Wnt/β-catenin pathway in human chondrosarcoma JJ012 cells. Oncol Rep. 42:103–114. 2019.PubMed/NCBI
31	Horibata S, Vo TV, Subramanian V, Thompson PR and Coonrod SA: Utilization of the soft agar colony formation assay to identify inhibitors of tumorigenicity in breast cancer cells. J Vis Exp. e527272015.PubMed/NCBI
32	Cesarz Z and Tamama K: Spheroid culture of mesenchymal stem cells. Stem Cells Int. 2016:91763572016. View Article : Google Scholar : PubMed/NCBI
33	Dong G, Wang S, Ge Y, Deng Q, Cao Q, Wang Q, Shang Z, OuYang W, Li J, Liu C, et al: Serum-free culture system for spontaneous human mesenchymal stem cell spheroid formation. Stem Cells Int. 2019:60418162019. View Article : Google Scholar : PubMed/NCBI
34	Rieger AM, Nelson KL, Konowalchuk JD and Barreda DR: Modified annexin V/propidium iodide apoptosis assay for accurate assessment of cell death. J Vis Exp. 25972011.PubMed/NCBI
35	Whelan JS and Davis LE: Osteosarcoma, chondrosarcoma, and chordoma. J Clin Oncol. 36:188–193. 2018. View Article : Google Scholar : PubMed/NCBI
36	Mas A, Prusinski L, Yang Q, Diaz-Gimeno P, Stone L, Diamond MP, Simón C and Al-Hendy A: Role of Stro1⁺/CD44⁺ stem cells in myometrial physiology and uterine remodeling during pregnancy. Biol Reprod. 96:70–80. 2017. View Article : Google Scholar : PubMed/NCBI
37	Galoczova M, Coates P and Vojtesek B: STAT3, stem cells, cancer stem cells and p63. Cell Mol Biol Lett. 23:122018. View Article : Google Scholar : PubMed/NCBI
38	Fang Y and Eglen RM: Three-dimensional cell cultures in drug discovery and development. SLAS Discov. 22:456–472. 2017.PubMed/NCBI
39	Song L, Yuan X, Jones Z, Zhou Y, Ma T and Li Y: Assembly of human stem cell-derived cortical spheroids and vascular spheroids to model 3-D brain-like tissues. Sci Rep. 9:59772019. View Article : Google Scholar : PubMed/NCBI
40	Leek R, Grimes DR, Harris AL and McIntyre A: Methods: Using three-dimensional culture (Spheroids) as an in vitro model of tumour hypoxia. Adv Exp Med Biol. 899:167–196. 2016. View Article : Google Scholar : PubMed/NCBI
41	Huang G, Ye S, Zhou X, Liu D and Ying QL: Molecular basis of embryonic stem cell self-renewal: From signaling pathways to pluripotency network. Cell Mol Life Sci. 72:1741–1757. 2015. View Article : Google Scholar : PubMed/NCBI