Miniring approach for high-throughput drug screenings in 3D tumor models

There is increasing interest in developing 3D tumor organoid models for drug development and personalized medicine approaches. While tumor organoids are in principle amenable to high-throughput drug screenings, progress has been hampered by technical constraints and extensive manipulations required by current approaches. Here, we introduce a miniaturized, fully automatable, flexible high-throughput method using a simplified geometry to establish 3D organoids from cell lines and primary tumors and robustly assay drug responses.

effective, potentially hard for drugs to efficiently penetrate and difficult to dissolve fully at the end of the experiment 17 .
In other applications, organoids are first formed and then transferred to different plates for drug treatment or final readout which can result in the tumor spheres sticking to plastic or breaking 8,16 . In other studies, assays are performed by disrupting the organoids to single cell suspensions at the end of the experiment [12][13] . All these manipulations introduce significant variability limiting applicability in screening efforts 6 .
To circumvent these issues, we optimized an assay system for 3D organoid high-throughput drug screenings that is low cost, simple, robust, requires few cells and is easily automated. Our strategy takes advantage of a specific geometry and can be performed using regular plates, without any need to transfer the samples at any time or dissociating the pre-formed tumor organoids. Single cell suspensions are pre-mixed with cold Matrigel (3:4 ratio) and 10 µl of this mixture is plated in a ring shape around the rim of the wells of a 96 well plate (Fig. 1a). The Matrigel rapidly solidifies upon short incubation at 37°C (Fig. 1a). The combination of small volume and surface tension holds the cells in place until the Matrigel solidifies and prevents 2D growth at the center of the wells. As such, further media removal, changes of conditions or treatment addition can be easily performed pipetting in the center of the well, preventing any disruption of the gel or need to pipette organoids. Cancer cell lines grown in the miniring format give rise to organized tumor organoids that recapitulate features of the original histology (Fig 1b and S1; Table S1). The miniring approach is also suitable to establish patient-derived tumor organoids (PDTOs). Primary patient samples grow and maintain the heterogeneity of the original tumor as expected. As an example, Patient #1 PDTOs recapitulate features of high-grade serous carcinoma (HGSC) as well as clear cell tumor (Fig. 1b). Fewer than 5000 cells per well are sufficient to provide a quantifiable readout (Fig. 1b).
Next, we optimized a treatment protocol and readouts for the miniring approach. Our standardized paradigm includes: seeding cells on day 0, establishing organoids for 2-3 days followed by two consecutive daily treatments, each performed by complete medium change (Fig. 1c). Three drugs (ReACp53 12 , Staurosporine and Doxorubicin) were tested at five concentrations in triplicates ( Fig. 1d-g). We optimized different readouts so that the final assay can be adapted to the specific research question or instrument availability. After seeding cells in standard white plates, we performed a luminescence-based ATP assay to obtain a metabolic readout of cell status, calculate EC 50 and identify cell-specific sensitivities (Fig. 1, S2 and S3). Results show how the Matrigel in the miniring setup is thin enough to allow penetration not only of small molecules but also of higher molecular weight biologics such as peptides 12 . We performed two consecutive treatments which allows the drugs to not only penetrate the gel but also reach organoids that may be bulky 12 . However, the assay is flexible and can be easily adapted to single drug treatments followed by longer incubations, multiple consecutive recurring treatments, multi-drug combinations or other screening strategies ( Fig. S3).
We also implemented assays to quantify drug response by measuring cell viability after staining of live organoids with specific dyes followed by imaging. We optimized a calcein release assay coupled to propidium iodide (PI) staining and a caspase 3/7 cleavage assay ( Fig. 1e-g and S4). Both are performed upon seeding the cells in standard black plates. Tumor organoids are stained with the reagents after dispase release and neutralization. After a 30-45 minutes incubation, organoids are imaged with a Celigo S cell imager. Images are then segmented and quantified ( Fig. 1e-g   and S4). As the organoids are assayed in the same well in which they are seeded, it is important to determine which assay/plate to use beforehand. Although the assays are testing different biological events, results are concordant across the methods for the three molecules we tested (Fig. 1, S4 and S5).
Most importantly, the miniring approach offers the possibility to perform multiple assays on the same plate/set of samples. For example, we coupled the ATP metabolic assay to 3D tumor count and total area measurement. We did so while testing suitability of the approach to identify drug susceptibilities of primary ovarian cancer samples obtained from the operating room. We used one patient-derived cell line, S1 GODL 18 , to optimize conditions ( Fig. S5) and two ovarian cancer patient samples (Table S1 and Fig. 2). We optimized the initial seeding cell number and selected 5000 cells/well in order to obtain a low enough number of organoids for size distribution analysis but sufficient to measure an ATP signal by CaspaseGlo 3D. We prepared six 96 well plates and tested 252 different kinase inhibitors at two different concentrations for each patient (120 nM and 1 µM). We used the same experimental paradigm optimized above. All steps (media change, drug treatment) were automated and performed in less than 2 minutes/plate using a Beckman Coulter Biomek FX integrated into a Thermo Spinnaker robotic system. At the end of the experiment, PDTOs were first imaged in brightfield mode for organoid count/size distribution analysis followed by ATP assay. We observed a high degree of equivalence between outcomes from the two methods ( Fig. 2a and c).
Results highlight individual sensitivities to different drugs ( Fig. 2a-g). The three samples tested showed minimal overlap in their response to kinase inhibitors (Fig. 2g).
Cells obtained from Patient #1 at the time of cytoreductive surgery 18 were chemo-naïve, and the heterogeneous nature of this clear cell/HGSC tumor was fully recapitulated in the PDTOs (Fig. 1b). The organoids were sensitive to 16/252 molecules tested and responded mostly to a variety of cyclin-dependent kinase (CDK) inhibitors with a stronger response to inhibitors hitting CDK1/2 in combination with CDK 4/6 or CDK 5/9 ( Fig. 2a-c

and S5b).
Interestingly, CDK inhibitors have found limited applicability in ovarian cancer therapy so far 19 .
Based on the profiles of the CDK inhibitors tested and on the response observed ( Fig. S5b-c), we selected four untested molecules to assay. We anticipated that Patient #1 should not respond to Palbociclib (targeting only CDK4/6) and THZ1 (CDK7) while expecting a response to JNJ-7706621 (CDK1/2/3/4/6) and AZD54338 (CDK1/2/9; Fig. S5bc). However, we observed a strong response to THZ1 but no response to JNJ-7706621 (Fig. 2h). Interestingly, both THZ1 and BS-181 HCl specifically target CDK7, however Patient #1 PDTOs showed a strong response to the former but no response to the latter which could be attributed to the different activity of the two as recently observed in breast cancer 20 .
Cells were obtained from Patient #2, a heavily pre-treated patient diagnosed with progressive, platinum-resistant HGSC (Table S1). PDTOs only showed a strong response to 3/252 drugs tested, with sensitivity to two of these 4 (BGT226, a PI3K/mTOR inhibitor and Degrasyn, a deubiquitinases inhibitor) shared with all other tested samples (Fig.   2c, 2f and S5a). Moderate responses (50-60% residual cell viability at 1 µM) were observed for EGFR inhibitors and we could detect high expression of EGFR at the plasma membrane of the tumor cells (Fig. S5e). Remarkably, Patient #2 PDTOs showed a very moderate response to our positive control, Staurosporine, a pan-kinase inhibitor with very broad activity 21 . The significant lack of response to multiple therapies observed for Patient #2 could be due to overexpression of efflux membrane proteins. Indeed, the PDTOs showed a high level of expression of ABCB1 (Fig. 2i).
High-expression of the ATP-dependent detox protein ABCB1 is frequently found in chemoresistant ovarian cancer cells and recurrent ovarian patients' samples and has been correlated with poor prognosis 22,23 .
In conclusion, the miniring approach can be a robust tool to standardize precision medicine efforts, given its ease of applicability to many different systems and drug screening protocols as well as limited cell requirement which allows testing of samples as obtained from biopsies/surgical specimens without the need for expansion. As demonstrated above, the method rapidly allowed to pinpoint drug sensitivities in tumor samples and allowed us to identify a tumor "fingerprint", with multiple inhibitors converging on a given pathway. Interestingly, many of the drugs identified in our screening do not have a specific, unequivocal biomarker or genomic signature predictive of response. Thus, patients may greatly benefit from PDTO testing prior to therapy selection 6,8,24 .
Our strategy can be successfully used to test samples that do not easily grow as patient-derived xenografts (PDX) in vivo. In fact, Patient #1 cells injected in NSG mice (500K/mouse, 12 mice) did not give rise to detectable tumor masses over six weeks (data not shown). Complete automation, scalability to 384 well plates, and flexibility to use different supports beside Matrigel can further extend applicability of the miniring approach.   warmed PrEGM to each well using an EpMotion (Eppendorf). Two days after seeding, medium is removed and replaced with fresh PrEGM containing the indicated drugs. The same procedure is repeated twice in two consecutive days. 24h after the last treatments, media is removed and wells are washed with 100 µl of pre-warmed PBS.
Organoids are then released from Matrigel for downstream experiments by 40 minutes of incubation in 50 µl of 5mg/mL dispase (Life Technologies #17105-041). All steps are performed with the EpMotion for small scale experiments and medium is removed/added from the center of the wells. For the high-throughput kinase screening experiment, we utilized a Beckman Coulter Biomek FX system with 96 channel head integrated into a Thermo Spinnaker robotic system with Momentum scheduling software. In short, an intermediary dilution plate (Axygen P-96-450V-C-S) was filled with 100 µl/well of media and pre-warmed to 37°C. Using pre-sterilized p50 tips, 1 µl of drug is transferred from a library compound plate to the intermediary media plate and thoroughly mixed. Next, the robot gently removed 100 µl of media from the matrigel/cell plate. The liquid handler was set up to hit the dead center of well with no contact to the Matrigel miniring. As a last step, the robot transferred 100 µl from the intermediary plate (media+drug) to the matrigel/cell plate. Media was easily dispensed without touching or disrupting the Matrigel miniring. The total process time outside of the CO 2 incubator was less than 2 so that the temperature was controlled throughout. (2) residual cell viability at 1 µM is ≤ 25%. For Patient #2, partial hits are defined as drugs giving response comparable to Staurosporine (50-60% residual viability at 1 µM).