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Sensitivity of osteosarcoma cell lines to autophagy inhibition as determined by pharmacologic and genetic
Daniel L Gustafson1,2,3,4, Lindsey O Viola3, Christina G Towers5
1School of Biomedical Engineering, Colorado State University, Fort Collins, Colorado, USA.
Abstract:
Pharmacologic inhibition of autophagy can be achieved using lysosomotropic agents such as hydroxychloroquine (HCQ) that interfere with fusion of the autophagosome to the lysosome thus preventing completion of the recycling process. The goal of the present study is to determine the sensitivity of eight canine (cOSA) and four human (hOSA) osteosarcoma tumour cell lines to antiproliferative and cytotoxic effects of lysosomal autophagy inhibitors, and to compare these results to the autophagy-dependence measured using a CRISPR/Cas9 live-cell imaging assay in OSA and other tumour cell lines. Antiproliferative and cytotoxic response to HCQ and Lys05 was determined using live cell imaging and YOYO-1 staining. CRISPR/Cas9 live cell imaging screen was done using species specific guide RNA's and transfection of reagents into cells. Response to autophagy core genes was compared to response to an essential (PCNA) and non-essential (FOXO3A) gene. cOSA and hOSA cell lines showed similar antiproliferative and cytotoxic responses to HCQ and Lys05 with median lethal dose (Dm ) values ranging from 4.6-15.8 μM and 2.1-5.1 μM for measures of anti-proliferative response, respectively. A relationship was observed between antiproliferative responses to HCQ and Lys05 and VPS34 CRISPR score with Dm values correlating with VPS34 response (r = 0.968 and 0.887) in a species independent manner. The results show that a subset of cOSA and hOSA cell lines are autophagy-dependent and sensitive to HCQ at pharmacologically-relevant exposures.
Insights
Lysosomal autophagy inhibitors like hydroxychloroquine (HCQ) show antiproliferative effects on canine and human osteosarcoma cell lines. Autophagy-dependence, particularly involving VPS34, correlates with sensitivity to these inhibitors.
Area of Science:
- Oncology
- Cell Biology
- Pharmacology
Background:
- Autophagy is a cellular recycling process that can be pharmacologically inhibited using lysosomotropic agents.
- Hydroxychloroquine (HCQ) is a lysosomotropic agent that disrupts autophagosome-lysosome fusion, inhibiting autophagy completion.
- Osteosarcoma (OSA) is a primary bone cancer with limited treatment options, and understanding its response to autophagy inhibition is crucial.
Purpose of the Study:
- To evaluate the antiproliferative and cytotoxic effects of lysosomal autophagy inhibitors on canine (cOSA) and human (hOSA) osteosarcoma cell lines.
- To compare the sensitivity of these cell lines to autophagy inhibitors with their measured autophagy-dependence using a CRISPR/Cas9 live-cell imaging assay.
- To investigate the relationship between drug response and the role of core autophagy genes, specifically VPS34.
Main Methods:
- Sensitivity to hydroxychloroquine (HCQ) and Lys05 was assessed using live-cell imaging and YOYO-1 staining to determine antiproliferative and cytotoxic responses.
- Autophagy-dependence was measured via a CRISPR/Cas9 live-cell imaging screen utilizing species-specific guide RNAs.
- Drug response was correlated with CRISPR scores for autophagy core genes (VPS34) and control genes (PCNA, FOXO3A).
Main Results:
- Both cOSA and hOSA cell lines exhibited similar antiproliferative and cytotoxic responses to HCQ and Lys05.
- Median lethal dose (Dm) values for anti-proliferative effects ranged from 4.6–15.8 μM for HCQ and 2.1–5.1 μM for Lys05.
- A strong, species-independent correlation was observed between antiproliferative responses to HCQ/Lys05 and VPS34 CRISPR scores (r = 0.968 and 0.887).
Conclusions:
- A subset of canine and human osteosarcoma cell lines are dependent on autophagy and sensitive to hydroxychloroquine (HCQ) at pharmacologically relevant concentrations.
- VPS34 activity is a key determinant of sensitivity to lysosomal autophagy inhibitors in osteosarcoma.
- These findings suggest that autophagy inhibition could be a viable therapeutic strategy for a subset of osteosarcoma patients.
