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Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells
Published on: June 16, 2019
TRAIL-mediated apoptosis in breast cancer cells cultured as 3D spheroids
Siddarth Chandrasekaran1, Jocelyn R Marshall1, James A Messing1
1Department of Biomedical Engineering, Cornell University, Ithaca, New York, United States of America.
Abstract:
TNF-alpha-related-apoptosis-inducing-ligand (TRAIL) has been explored as a therapeutic drug to kill cancer cells. Cancer cells in the circulation are subjected to apoptosis-inducing factors. Despite the presence of these factors, cells are able to extravasate and metastasize. The homotypic and heterotypic cell-cell interactions in a tumor are known to play a crucial role in bestowing important characteristics to cancer cells that leave the primary site. Spheroid cell culture has been extensively used to mimic these physiologically relevant interactions. In this work, we show that the breast cancer cell lines BT20 and MCF7, cultured as 3D tumor spheroids, are more resistant to TRAIL-mediated apoptosis by downregulating the expression of death receptors (DR4 and DR5) that initiate TRAIL-mediated apoptosis. For comparison, we also investigated the effect of TRAIL on cells cultured as a 2D monolayer. Our results indicate that tumor spheroids are enriched for CD44hiCD24loALDH1hi cells, a phenotype that is predominantly known to be a marker for breast cancer stem cells. Furthermore, we attribute the TRAIL-resistance and cancer stem cell phenotype observed in tumor spheroids to the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE₂) pathway. We show that inhibition of the COX-2/PGE₂ pathway by treating tumor spheroids with NS-398, a selective COX-2 inhibitor, reverses the TRAIL-resistance and decreases the incidence of a CD44hiCD24lo population. Additionally, we show that siRNA mediated knockdown of COX-2 expression in MCF7 cells render them sensitive to TRAIL by increasing the expression of DR4 and DR5. Collectively, our results show the effect of the third-dimension on the response of breast cancer cells to TRAIL and suggest a therapeutic target to overcome TRAIL-resistance.
Insights
Three-dimensional breast cancer spheroids resist tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) therapy by downregulating death receptors. Targeting the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE₂) pathway overcomes this resistance, offering a potential therapeutic strategy.
Area of Science:
- Oncology
- Cell Biology
- Cancer Therapeutics
Background:
- Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) shows promise for cancer therapy by inducing apoptosis.
- Cancer cells exhibit resistance to apoptosis, facilitating metastasis.
- Tumor microenvironment interactions, particularly cell-cell contacts, influence cancer cell survival and characteristics.
Purpose of the Study:
- To investigate the impact of three-dimensional (3D) spheroid culture on breast cancer cell sensitivity to TRAIL-induced apoptosis.
- To identify mechanisms underlying TRAIL resistance in 3D breast cancer models.
- To explore the potential of targeting the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE₂) pathway to overcome TRAIL resistance.
Main Methods:
- Breast cancer cell lines (BT20, MCF7) were cultured as 2D monolayers and 3D spheroids.
- Cells were treated with TRAIL, and apoptosis, death receptor expression (DR4, DR5), and cancer stem cell markers (CD44, CD24, ALDH1) were assessed.
- The role of the COX-2/PGE₂ pathway was investigated using the COX-2 inhibitor NS-398 and siRNA-mediated COX-2 knockdown.
- Changes in TRAIL sensitivity, death receptor expression, and cancer stem cell populations were analyzed.
Main Results:
- 3D tumor spheroids exhibited increased resistance to TRAIL-mediated apoptosis compared to 2D cultures.
- TRAIL-resistant spheroids showed downregulated expression of death receptors DR4 and DR5.
- Tumor spheroids were enriched for CD44hiCD24loALDH1hi cells, a breast cancer stem cell phenotype.
- Upregulation of the COX-2/PGE₂ pathway was associated with TRAIL resistance and the cancer stem cell phenotype in spheroids.
- Inhibition of COX-2/PGE₂ pathway with NS-398 reversed TRAIL resistance and reduced the CD44hiCD24lo population.
- COX-2 knockdown via siRNA sensitized MCF7 cells to TRAIL by increasing DR4 and DR5 expression.
Conclusions:
- The three-dimensional architecture of breast cancer spheroids confers resistance to TRAIL therapy.
- The COX-2/PGE₂ pathway plays a critical role in mediating TRAIL resistance and promoting cancer stem cell characteristics in 3D cultures.
- Targeting the COX-2/PGE₂ pathway represents a promising therapeutic strategy to enhance the efficacy of TRAIL-based cancer treatments.
