Radiation Biology

PV QA 1 - Poster Viewing Q&A 1

SU_36_2360 - Development of a Method to Characterize the Effects of Radiation on Growth and Morphology of Organoids in 3D Culture

Sunday, October 21
1:15 PM - 2:45 PM
Location: Innovation Hub, Exhibit Hall 3

Development of a Method to Characterize the Effects of Radiation on Growth and Morphology of Organoids in 3D Culture
V. Venkatachalam1,2, D. R. Schmidt2,3, and M. Vander Heiden2; 1Harvard Radiation Oncology Program, Boston, MA, 2Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 3Department of Radiation Oncology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA

Purpose/Objective(s): Organoids are a powerful tool to monitor the temporal effects of radiation in vitro, offering distinct advantages over traditional 2D cell culture methods. In addition to growing into 3D structures that more closely model physiologic cellular architecture, individual organoids embedded in gel matrices can be tracked over time, making it possible to study the temporal effects of radiation on growth in clonal cell populations. However, documenting individual organoid growth in 3D culture is not straightforward in part due to the difficulties of analyzing standard bright field microscopy images of organoids. For example, compressing a Z-stack of images and counting cells can lead to under-counting of organoids that are stacked on top of one another. To overcome this challenge, our objective was to develop an analysis method that would let us track the growth and morphology of organoids in 3D culture over time using stacks of bright field images. We also validated this method by characterizing the differential effects of radiation on wild-type murine prostate organoids vs. PTEN-null prostate organoids grown either with or without enzalutamide.

Materials/Methods: Anterior prostate cells isolated from mice with the Pten gene flanked by LoxP were treated with or without adenovirus expressing Cre recombinase to yield both wild-type and PTEN-null lines. Deletion of PTEN was confirmed by PCR and immunohistochemistry in intact organoids. Organoid culture of both genotypes were treated with enzalutamide and either 0, 2, or 8 Gy gamma-radiation. Organoids were imaged using standard bright field microscopy daily between 3 and 7 days post-radiation to assess the effect of radiation on both growth rate and morphology. Stacks of images for each 3D sample were obtained by stepping through serial focal planes in the Z-direction.

Results: To overcome the challenges of quantifying data from these stacks of bright field images, we developed an algorithm to automatically segment bright field microscopy images and applied this to our dataset to generate both 3D images and histograms quantifying the number and size of organoids over time. We validated this analysis pipeline on a population of prostate cancer organoids treated with radiation and enzalutamide, showing that higher radiation dose was correlated with both a lower absolute number of organoids in culture, as well as a slower rate of organoid growth.

Conclusion: This method represents an alternative to traditional approaches to assess the biological effects of radiation on cell proliferation and survival. By tracking individual organoids growing in 3D culture over time, this method enables better visualization and quantification of the heterogeneity and temporal effects of radiation in clonal cell populations. Furthermore, our automated analysis makes it feasible to conduct chemical and genetic screens for radiation modifiers in organoid culture.

Author Disclosure: V. Venkatachalam: None. D.R. Schmidt: None. M. Vander Heiden: Consultant; Agios Pharmaceuticals, Aeglea Biotherapeutics. Stock; Agios Pharmaceuticals. Stock Options; Agios Pharmaceuticals.

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