Characterization of the physical properties of tumor-derived spheroids reveals critical insights for pre-clinical studies.

Three-dimensional spheroids are widely used as cancer models to study tumor cell proliferation and to evaluate new anticancer drugs. Growth-induced stress (i.e., stress that persists in tumors after external loads removal) influences tumor growth and resistance to treatment. However, it is not clear whether spheroids recapitulate the tumor physical properties. Here, we demonstrated experimentally and with the support of mathematical models that, like tumors, spheroids accumulate growth-induced stress. Moreover, we found that this stress is lower in spheroids made of 5,000 cancer cells and grown for 2 days than in spheroids made of 500 cancer cells and grown for 6 days. These two culture conditions associated with different growth-induced stress levels also had different effects on the spheroid shape (using light sheet microscopy) and surface topography and stiffness (using scanning electron microscopy and atomic force microscopy). Finally, the response to irinotecan was different in the two spheroid types. Taken together, our findings bring new insights into the relationship between the spheroid physical properties and their resistance to antitumor treatment that should be taken into account by the experimenters when assessing new therapeutic agents using in vitro 3D models or when comparing studies from different laboratories.

FitEllipsoid: a fast supervised ellipsoid segmentation plugin.

BACKGROUND: The segmentation of a 3D image is a task that can hardly be automatized in certain situations, notably when the contrast is low and/or the distance between elements is small. The existing supervised methods require a high amount of user input, e.g. delineating the domain in all planar sections. RESULTS: We present FitEllipsoid, a supervised segmentation code that allows fitting ellipsoids to 3D images with a minimal amount of interactions: the user clicks on a few points on the boundary of the object on 3 orthogonal views. The quantitative geometric results of the segmentation of ellipsoids can be exported as a csv file or as a binary image. The core of the code is based on an original computational approach to fit ellipsoids to point clouds in an affine invariant manner. The plugin is validated by segmenting a large number of 3D nuclei in tumor spheroids, allowing to analyze the distribution of their shapes. User experiments show that large collections of nuclei can be segmented with a high accuracy much faster than with more traditional 2D slice by slice delineation approaches. CONCLUSIONS: We designed a user-friendly software FitEllipsoid allowing to segment hundreds of ellipsoidal shapes in a supervised manner. It may be used directly to analyze biological samples, or to generate segmentation databases necessary to train learning algorithms. The algorithm is distributed as an open-source plugin to be used within the image analysis software Icy. We also provide a Matlab toolbox available with GitHub.

Impact of physical confinement on nuclei geometry and cell division dynamics in 3D spheroids.

Multicellular tumour spheroids are used as a culture model to reproduce the 3D architecture, proliferation gradient and cell interactions of a tumour micro-domain. However, their 3D characterization at the cell scale remains challenging due to size and cell density issues. In this study, we developed a methodology based on 3D light sheet fluorescence microscopy (LSFM) image analysis and convex hull calculation that allows characterizing the 3D shape and orientation of cell nuclei relative to the spheroid surface. By using this technique and optically cleared spheroids, we found that in freely growing spheroids, nuclei display an elongated shape and are preferentially oriented parallel to the spheroid surface. This geometry is lost when spheroids are grown in conditions of physical confinement. Live 3D LSFM analysis of cell division revealed that confined growth also altered the preferential cell division axis orientation parallel to the spheroid surface and induced prometaphase delay. These results provide key information and parameters that help understanding the impact of physical confinement on cell proliferation within tumour micro-domains.

Experimental estimation of stored stress within spherical microtissues : What can and cannot be inferred from cutting experiments.

Biological tissues accumulate mechanical stress during their growth. The mere measurement of the stored stress is not an easy task. We address here the spherical case and our experiments consist in performing an incision of a spherical microtissue (tumor spheroid) grown in vitro. On the theoretical part we derive a compatibility condition on the stored stress in spherical symmetry, which imposes a relation between the circumferential and radial stored stress. The numerical implementation uses the hyperelastic model of Ciarlet and Geymonat. A parametric study is performed to assess the influence of each parameter on the shape of the domain after the incision. As a conclusion, the total radial stored stress can be confidently estimated from the measurement of the opening after incision. We validate the approach with experimental data.

Oxygen Partial Pressure Is a Rate-Limiting Parameter for Cell Proliferation in 3D Spheroids Grown in Physioxic Culture Condition.

The in situ oxygen partial pressure in normal and tumor tissues is in the range of a few percent. Therefore, when studying cell growth in 3D culture systems, it is essential to consider how the physiological oxygen concentration, rather than the one in the ambient air, influences the proliferation parameters. Here, we investigated the effect of reducing oxygen partial pressure from 21% to 5% on cell proliferation rate and regionalization in a 3D tumor spheroid model. We found that 5% oxygen concentration strongly inhibited spheroid growth, changed the proliferation gradient and reduced the 50% In Depth Proliferation index (IDP50), compared with culture at 21% oxygen. We then modeled the oxygen partial pressure profiles using the experimental data generated by culturing spheroids in physioxic and normoxic conditions. Although hypoxia occurred at similar depth in spheroids grown in the two conditions, oxygen partial pressure was a major rate-limiting factor with a critical effect on cell proliferation rate and regionalization only in spheroids grown in physioxic condition and not in spheroids grown at atmospheric normoxia. Our findings strengthen the need to consider conducting experiment in physioxic conditions (i.e., tissue normoxia) for proper understanding of cancer cell biology and the evaluation of anticancer drugs in 3D culture systems.