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).

Figure 4. Primary output parameters obtained through CALYPSO, including viability heatmaps as well as normalized total area, fractional live area and normalized viability, demonstrate the ability to report treatment response dynamics on spheroid and non-spheroid organoids of MIA PaCa-2 cells grown with primary dermal fibroblasts following treatment with PDT and oxaliplatin chemotherapy. (A) Live/dead images of calcein and PI fluorescence were superimposed in ImageJ and depicted side-by-side with the corresponding viability heatmaps, providing spatial information on the viability distributions throughout the tumor nodules. Depicted are untreated adherent cocultures of MIA PaCa-2 cells and primary dermal fibroblasts, either untreated or treated with 25 J/cm2 BPD-PDT or 1 mM oxaliplatin (72 h). Dose response correlations between the PDT radiant exposure and the (B) median total area (mean ± SEM), (C) median fractional live area (mean ± st. dev.), and (D) the median viability of the tumor organoids (mean ± st. dev.). Data represents the mean of the median value per image (N = 12–24). (E–G) Distributions of residual total area (E), fractional live area (F) and viability (G) of the individual tumor organoids following treatment with BPD-PDT at a radiant exposure of 25 J/cm2
(green bars) or 500 μM oxaliplatin (blue bars) in comparison to the no treatment control group (black bars). (Bulin A. et al., Comprehensive high-throughput image analysis for therapeutic efficacy of architecturally complex heterotypic organoids. Nature Scientific Reports 7, 16645, 2017).

References

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