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

CAROLINE J. GRANGER, AARON K. HOYT, ALEXANDRA MORAN, BEATRICE BECKER, ANIL SEDANI, SHANNON SAIGH, SHEILA A. CONWAY, JEFFREY BROWN, KARINA GALOIAN

Research output: Contribution to journalArticlepeer-review

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 (mTORC 1), 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 ALDE FLUOR ™ assay was conducted (with N,N-diethylaminobenzaldehyde as the negative control) to assess aldehyde dehydrogenase (ALD H) activity (a recognized CSC marker), and bulk JJ012, ALD Hhigh and PRP-1 treated ALD Hlow 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 (STRO 1, CD 44 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-C SC 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.

Original languageEnglish (US)
Pages (from-to)3747-3758
Number of pages12
JournalMolecular medicine reports
Volume22
Issue number5
DOIs
StatePublished - Nov 2020

Keywords

  • Cancer stem cells
  • Chondrosarcoma
  • Proline rich polypeptide 1
  • Proline rich polypeptide-1
  • Spheroids

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Oncology
  • Cancer Research

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