Related Experiment Video
Updated: Dec 9, 2025
A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells
Published on: January 22, 2021
Cancer stem cells as a therapeutic target in 3D tumor models of human chondrosarcoma: An encouraging future for
Caroline J Granger1, Aaron K Hoyt1, Alexandra Moran1
1RMSB Room 8012 (D27), Department of Orthopedic Surgery, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
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.
Insights
Proline rich polypeptide 1 (PRP-1) effectively inhibits chondrosarcoma cancer stem cell growth and spheroid formation in 3D models. This novel therapeutic agent shows promise for treating this bone cancer by targeting key tumor-initiating cells.
Area of Science:
- Oncology
- Biochemistry
- Cell Biology
Background:
- Chondrosarcoma is a bone cancer resistant to conventional treatments like chemotherapy and radiation.
- Current treatment options are limited, often requiring extensive surgery.
- Proline rich polypeptide 1 (PRP-1), an inhibitor of mTORC1, has shown potential in preliminary studies.
Purpose of the Study:
- To investigate the efficacy of PRP-1 against chondrosarcoma in a 3D in vitro model.
- To assess the impact of PRP-1 on cancer stem cells (CSCs) within this 3D model.
- To evaluate PRP-1's effects on CSC self-renewal and tumor-forming capabilities.
Main Methods:
- Utilized JJ012 chondrosarcoma cells cultured in 3D spheroids.
- Employed ALDEFLUOR™ assay to identify and sort aldehyde dehydrogenase (ALDH) high CSCs.
- Conducted clonogenic, spheroid formation, Annexin V/propidium iodide, and Western blot assays to analyze cell growth, death, and marker expression.
Main Results:
- A dose of 20 µg/ml PRP-1 significantly reduced colony formation capacity.
- PRP-1 dose-dependently inhibited CSC spheroid growth and anchorage-independent growth.
- PRP-1 induced CSC cell death through non-apoptotic and non-necrotic mechanisms.
- Confirmed expression of mesenchymal stem cell markers and self-renewal capacity in CSCs.
Conclusions:
- PRP-1 effectively eliminates anchorage-independent CSC growth and spheroid formation in a 3D chondrosarcoma model.
- PRP-1 demonstrates potential as a therapeutic agent to inhibit tumor formation and progression.
- The findings suggest PRP-1's utility in targeting CSCs, offering a novel biological treatment for chondrosarcoma.
