Comprehensive high-throughput image analysis for therapeutic efficacy of architecturally complex heterotypic organoids
Bioengineered 3D organoids with heterotypic cellular composition are gaining interest as cancer models to be used to perform drug screening and therapy development. A team of scientists from Harvard Medical School and Massachusetts General Hospital (Boston, USA) describes a comprehensive image analysis procedure for structurally complex organotypic cultures (CALYPSO) applied to fluorescence-based assays to extract multiparametric readouts of treatment effects for heterotypic tumor cultures that enables advanced pharmacological analyses.
Bioengineered 3D organoids that incorporate heterotypic cellular communication are gaining interest as cancer models since they can recapitulate key features regarding the intrinsic tumor tissues heterogeneity (Furuta et al., 2017; Griffith, L. & Swartz, 2006). However, although there is a growing appreciation for organotypic models of human disease and increasing varieties of 3D culture methods, the implementation of 3D cultures as a mainstream approach for expedited therapy screening requires the development or adaptation of quantitative analysis methods (Pampaloni et al., 2007). Indeed, there remains a relative scarcity in assays that provide functional and reliable readouts for therapeutic drug screening and assessment of treatment outcomes. The necessity for these assays is further underscored by the limitations of colorimetric toxicity assays typically used for 2D cultures (e.g., tetrazolium and trypan blue assays) that are not optimized for 3D culture models (Rizvi et al., 2010), and which simplify treatment responses by reporting a single metric that may not accurately reflect the full scope of effects (Celli et al., 2014). For 3D cultures, live/dead staining utilizing calcein AM and propidium iodide (PI)/ethidium bromide/DRAQ7/DAPI are widely used for qualitative purpose but are not frequently used as a quantitative readout (Keinzle et al., 2017; Smalley et al., 2006; Stehn et al., 2013).
A team of scientists from Department of Dermatology, Harvard Medical School and Massachusetts General Hospital (Boston, USA) addresses this issue and describes a Comprehensive Image Analysis Procedure for Structurally complex Organotypic cultures (CALYPSO) applied to fluorescence-based assays to extract multiparametric readouts of treatment effects for heterotypic tumor cultures that enables advanced assessments of treatment response. This method leverages a well-established live/dead staining protocol of the cultures that utilizes calcein AM and PI stains for live and dead cells respectively (Morris S. et al., 1990). This methodology disregards differences in treatment susceptibility between cell subpopulations within the organotypic cultures, yet considers the tumor organoids as a whole, which is comparable to how treatment responses are monitored in patients. Bulin A. et al. test the performances of CALYPSO on adherent and suspended 3D cultures of pancreatic cancer, as well as an adherent 3D culture model of micrometastatic ovarian carcinoma. The methodology can furthermore be applied to various treatment types i.e. oxaliplatin chemotherapy, X-ray radiation therapy, and photodynamic therapy. Importantly, the results in this study were obtained with commercially available materials and imaging systems that are widely available, rendering the platform highly feasible for implementation in most laboratories to facilitate high-throughput toxicological screening of pharmaceutical agents (Bulin et al., 2017).
- Celli, J. P. et al. An imaging-based platform for high-content, quantitative evaluation of therapeutic response in 3D tumour models. Sci Rep 4, 3751 (2014).
- Furuta, S. & Bissell, M. J. Pathways Involved in Formation of Mammary Organoid Architecture Have Keys to Understanding Drug Resistance and to Discovery of Druggable Targets. Cold Spring Harb Symp Quant Biol (2017).
- Griffith, L. & Swartz, M. Capturing complex 3D tissue physiology in vitro. Nat Rev Mol Cell Bio 7, 211–224 (2006).
- Kienzle et al. Dendritic Mesoporous Silica Nanoparticles for pH-Stimuli-Responsive Drug Delivery of TNF-Alpha. Adv Healthc Mater 6, 1700012 (2017).
- Morris, S. Real-time multi-wavelength fluorescence imaging of living cells. Bio. Techn 8, 296–312 (1990).
- Pampaloni, F., Reynaud, E. G. & Stelzer, E. H. K. The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Bio 8, 839–845 (2007).
- Rizvi, I. et al. Synergistic Enhancement of Carboplatin Efficacy with Photodynamic Therapy in a Three-Dimensional Model for Micrometastatic Ovarian Cancer. Cancer Res 70, 9319–9328 (2010).
- Smalley et al. Multiple signaling pathways must be targeted to overcome drug resistance in cell lines derived from melanoma metastases. Mol Cancer Ther 5, 1136–1144 (2006).
- Stehn et al. A Novel Class of Anticancer Compounds Targets the Actin Cytoskeleton in Tumor Cells. Cancer Res 73, 5169–5182 (2013).