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. 2017 Nov 30;7(1):16645.
doi: 10.1038/s41598-017-16622-9.

Comprehensive high-throughput image analysis for therapeutic efficacy of architecturally complex heterotypic organoids

Anne-Laure Bulin  1 , Mans Broekgaarden  1 , Tayyaba Hasan  2
Affiliations

Comprehensive high-throughput image analysis for therapeutic efficacy of architecturally complex heterotypic organoids

Anne-Laure Bulin et al. Sci Rep. .

Abstract

Bioengineered three-dimensional (3D) tumor models that incorporate heterotypic cellular communication are gaining interest as they can recapitulate key features regarding the intrinsic heterogeneity of cancer tissues. However, the architectural complexity and heterogeneous contents associated with these models pose a challenge for toxicological assays to accurately report treatment outcomes. To address this issue, we describe 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 analyses. The capacity of this approach is exemplified on various 3D models including adherent/suspension, mono-/heterocellular cultures and several disease types. The subsequent analysis revealed specific morphological effects of oxaliplatin chemotherapy, radiotherapy, and photodynamic therapy. The procedure can be readily implemented in most laboratories to facilitate high-throughput toxicological screening of pharmaceutical agents and treatment regimens on organotypic cultures of human disease to expedite drug and therapy development.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Heterotypic 3D cultures generate a heterogeneous mix of tumors with architecturally complex geometries. (A) A conventional monoculture of PDAC cells (MIA PaCa-2 cell line) develop into asymmetrical spheroids over 11 days. (B) When MIA PaCa-2 cells are cocultured with fibroblasts (primary dermal fibroblast cell line), a heterogeneous mix of non-spheroidal organoids are formed. Images were taken using darkfield microscopy. (Scale bar = 500 µm).
Figure 2
Figure 2
Schematic representation of the image analysis workflow following live/dead staining and confocal fluorescence imaging of 3D cultures. Following acquisition of the live and dead fluorescence and brightfield images, a mask is created from the brightfield images and individual objects (i.e., organoids) are indexed. The mask is then applied to both fluorescence images, after which the intensities per object are extracted for subsequent viability calculations. The masked fluorescence images are then thresholded to extract the live and dead area for every object. This method links all readouts are directly linked to the object index.
Figure 3
Figure 3
Treatment outcomes of PDAC 3D models grown as adherent monocultures (MIA-PaCa2) subjected to PDT (25 J/cm2), oxaliplatin chemotherapy (1 mM) and radiotherapy (10 Gy). (A) Brightfield and live/dead fluorescence images, obtained using a confocal microscope, as well as viability heatmaps and live area maps derived from subsequent CALYPSO analysis are given for each group. (B) Boxplot depicting the spread in normalized organoid total areas as quantified from the brightfield images depicted in panel A. (C) Boxplot depicting the spread in viability of every individual organoid within the treatment and control groups. (D) Boxplot depicting the spread in fractional live area of every individual organoid within the treatment and control groups. All boxplots depict median, 25th and 75th percentile, and the 90% confidence interval.
Figure 4
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).
Figure 5
Figure 5
Readouts from CALYPSO are paired and allow advanced analysis of treatment response to investigate correlations between total residual area, residual viable area, and viability for every individual tumor organoid. Depicted here are results obtained on organoids made of MIA PaCa-2 cocultured with human dermal fibroblasts. Organoids were either untreated (black) or treated with either PDT at a radiant exposure of 25 J/cm2 (green) or 500 µM oxaliplatin (blue). Panels A, B and C represent 3D scatter plots of the paired data (organoid total area, organoid live are and viability) measured on cultures from (A) the no treatment control group, (B) the PDT (25 J/cm2) group, and (C) the 500 μM oxaliplatin group (72 h). Panels D, E and F represent linear regression performed on series of paired data allowing a deeper comparison of the effect of PDT and oxaliplatin chemotherapy regarding the no treatment control group. (D) Linear regression analysis between organoid viability and organoid size (area), displaying that PDT is more efficient in eradicating smaller organoids, whereas no size-related effects regarding viability are observed for oxaliplatin chemotherapy. (E) Linear regression analysis between fractional organoid live area and total organoid area, demonstrating that PDT reduces the relative live area more effectively for smaller organoids compared to larger organoids. (F) Linear regression analysis between organoid viability and fractional live area, demonstrating that PDT treatment leaves behind large viable nodules with high viability and small viable nodules with low viability. A similar trend was observed for oxaliplatin chemotherapy, although the steepness of the correlation suggests a much more homogeneous dispersion of viability and viable organoid size.
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