image analysis in gen5 software to analyze drug response in patient-derived organoids using multiplexed live-cell imaging

Automated Kinetic Imaging for Drug Assessment in Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are rapidly emerging as a robust model system to study cancer development and therapeutic responses. PDOs are three-dimensional (3D) cell culture systems that recapitulate the complex genomic profile and architecture of the primary tumor1,2. Unlike traditional two-dimensional (2D) cultures of immortalized cancer cell lines, PDOs capture and maintain intratumoral heterogeneity3,4, making them a valuable tool for both mechanistic and translational research. Although PDOs are becoming an increasingly popular model system, commercially available reagents and analysis methods for cellular effects that are compatible with PDO cultures are limited.
The lack of robust methods to analyze subtle changes in treatment response hinders clinical translation. The gold standard cell health reagent in 3D cultures, CellTiter-Glo 3D, utilizes ATP levels as a determinant of cell viability5,6. While this reagent is useful for endpoint assays, there are several caveats, most notably the inability to use samples for other purposes after completion of the assay.
Live-cell imaging is a sophisticated form of kinetic microscopy that, when combined with fluorescent reagents, has the capacity to quantify a variety of cell health readouts within PDO models, including apoptosis7,8,9 and cytotoxicity10. Indeed, live-cell imaging has been integral to high throughput screening of compounds in 2D platforms11,12. Systems such as the Incucyte have made the technology affordable and thus accessible to research groups in a variety of settings. However, the application of these systems to analyze 3D cultures is still in its infancy.
Herein, we describe a method to assess drug response in PDO models of cancer using multiplexed live-cell imaging (Figure 1). Through analysis of bright field images, changes in PDO&#160;size and morphology can be kinetically monitored. Furthermore, cellular processes can be simultaneously quantified over time using fluorescent reagents, such as Annexin V Red Dye for apoptosis and Cytotox Green Dye for cytotoxicity. The methods presented are optimized for the Cytation 5 live-cell imaging system, but this protocol may be adapted across different live-cell imaging platforms.

Author Spotlight: Developing Multiplexed Kinetic Assays for Organoid-Based Drug Response Analysis

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

We apply patient derived organoid models, which are created from actual patient tumor specimens to examine how the tumors respond to drugs. We can also define the molecular characteristics that correlate with response. These results are then used to prioritize the best new therapies to advance in the clinical trials in the future.One of the main challenges in using patient derived organoid models is that they are a finite resource. In addition, there are very few off the shelf kits to assess drug response in organoid models that preserve the sample in a format that allows for other downstream analyses, such as transcriptomic profiling. We developed a multiplexed approach to kinetically monitor therapeutic response in organoid models.We use fluorescent dyes that are specific to different cellular processes, which provides insight into the mechanism of drug activity in each organoid model. In a single experiment we collect both functional and time course data and the samples are available for other assays. This method can be applied to assess response to a broad range of therapeutic modalities beyond small molecule inhibitors, including radiation sensitivity.We are also continuing to innovate by developing tools for other cellular phenotypes that can be kinetically measured, such as senescence.

Image Analysis in Gen5 Software to Analyze Drug Response in Patient-Derived Organoids Using Multiplexed Live-Cell Imaging

To begin, image the fluorescently-labeled PDOs using the Cytation 5 live cell imaging system. Then, launch the Gen5 software. Navigate to Experiments and click Open in the Task Manager.Open the desired experiment and select Plate, followed by View in the toolbar. Then, switch the data dropdown menu to Z Projection. Double-click on a chosen well.Select Analyze. Click, I want to set up a new image analysis data reduction step, and click OK.In Analysis settings, set type to Cellular Analysis and detection channel to Z Projection transformed TSF Brightfield. Click Options to open the primary mask and count page.Check Auto in the threshold box, and click OK to close the popup window. Under Object Selection, define minimum and maximum object sizes to filter out cellular debris or single cells, and select other features as desired. Next, click Apply and the masked objects will be highlighted.To set up a subpopulation analysis in the cellular analysis toolbar, click on Calculated Metrics. Then, click Select or create object level metrics of interest at the bottom-right corner. From available object metrics, choose desired metrics, such as Circularity, and click Insert.Click OK to confirm. Next, select Subpopulation Analysis in the cellular analysis toolbar, and click Add to create a new subpopulation. Enter a name for the subpopulation.In the object metrics, click Add Condition to add a chosen metric. Input desired parameters in the Edit Condition window, and click OK.Select the desired results in the table at the bottom of the window and click OK, followed by Apply. Under Object Details, select the designated subpopulation to view masking.If satisfied, click OK in the Cellular Analysis window, and click Add Step to apply the analysis to all wells.

image-analysis-gen5-software-to-analyze-drug-response-patient-derived

Research

JoVE Journal

Cancer Research

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...

Plating Intact Patient-Derived Organoids and Setting Up Imaging Parameters for Multiplexed Live-Cell Imaging

To begin, take a tube with patient-derived organoids or PDO suspension in an equal amount of basement membrane extracts, or BME. Using a 20 microliter pipette, seed five microliter domes into the center of each well of a pre-warmed 96 well plate. After seeding all the wells, place the lid on the plate and gently invert it.Incubate the inverted plate at 37 degrees Celsius for 20 minutes in the tissue culture incubator to allow the domes to solidify. Flip the plate over so that the lid is facing upwards. Then incubate again.After adding fluorescent dyes and drugs to the plates containing the PDOs. Place the plate in citation five. Start the Gen5 software and in the task manager window, select imager manual mode and click capture now.In the settings, specify the objective, filter, microplate format, and vessel type, select use lid and use slower carrier speed and click okay. Then choose a well of interest. Select the brightfield channel, click on auto expose and adjust the settings as required.Next to set up the Z stack, expand the imaging mode tab, select Z stack, and adjust the focus using the course adjustment arrows until all PDOs are out of focus. Establish this point as the bottom of the Z stack and repeat in reverse to set the top. Afterward, set up the fluorescent channels by opening the edit imaging step.Under channels, select the desired number of channels, designate one channel for the brightfield and additional channels for each fluorescent channel. Close the window by clicking okay, change the dropdown menu to the GFP to set the exposure for the first fluorescent channel, click auto expose and adjust the exposure settings under the exposure tab to reduce the background signal. To copy exposure settings to the image mode tab, click the copy icon and then click edit imaging step to open a new window.In the GFP channel, click the clipboard icon in the exposure line to add settings for illumination, integration time, and camera gain to the channel. Now click on the camera icon and choose image pre-processing under add processing step. On the brightfield tab, uncheck the apply image pre-processing option.For each fluorescent channel tab, ensure apply image pre-processing is checked. Deselect, use same options as channel one, and then click okay to close the window. Now click on Z projection under the add processing step, adjust the slice range if necessary, and close the window by clicking okay.To set up the protocol for kinetic imaging, in the toolbar click image set, and from the dropdown menu, choose create experiment from this image set. Click on options under the other heading and check the discontinuous kinetic procedure box. Under estimated total time, enter the runtime for the experiment.Close the window by clicking okay. Additionally, set plate layout and temperature gradient at this time. Validate the data reduction steps by clicking through the windows.Then navigate to file and click save protocol as in the toolbar. Select a save location. Enter a file name and click save.To begin kinetic imaging, load the protocol into the bio spa on demand scheduling software and choose an available slot. In the procedure info tab, select user from the menu. Next to the protocol slot, click select and choose add a new entry.Now click select next to the protocol slot. Click open to navigate and import the protocol into the scheduling software. Enter the required imaging time for the plate and click okay to close the window.Under interval, input the desired frequency of imaging, select schedule, plate, or vessel to initiate the plate validation sequence, click yes to accept this schedule.