Protocol for generation of and high-throughput drug testing with patient-derived colorectal cancer organoids.

Materials and equipment Recipes Tissue collection medium Reagent Final concentration Amount Gentamicin 50 μg/mL 500 μL DMEM/F12+GLUTAMAX N/A 500 mL Total N/A 500 mL [Once prepared, keep at 4°C for up to 6 months] Colorectal cancer organoid primary culture medium Reagent Final concentration Amount Y-27632 (10 mM) 10 μM 500 μL Primocin (50 mg/mL) 100 μg/mL 1 mL Human recombinant epidermal growth factor (EGF) (100 μg/mL) 50 ng/mL 250 μL Human recombinant basic fibroblast growth factor (FGF2) (100 μg/mL) 20 ng/mL 100 μL A83-01 (10 mM) 500 nM 25 μL Nicotinamide (1 M) 10 mM 5 mL N-acetyl-L-cysteine (500 mM) 1 mM 1 mL HEPES (1 M, pH 7.4) 10 mM 5 mL Gibco GlutaMAX supplement (100X) 100X 5 mL B-27 supplement (50X) 50X 10 mL N -2 supplement (100X) 100X 5 mL Gibco Advanced DMEM/F-12 N/A 461.6 mL Total N/A 500 mL [Once prepared, keep at 4°C for up to 2 weeks or aliquot and freeze at −20°C for up to 3 months] Colorectal cancer organoid maintenance medium Reagent Final concentration Amount Y-27632 (10 mM) 10 μM 500 μL Normocin (50 mg/mL) 100 μg/mL 1 mL Penicillin-Streptomycin (10,000 U/mL) 100 U/mL and 100 μg/mL 5 mL Human recombinant epidermal growth factor (EGF) (100 μg/mL) 50 ng/mL 250 μL Human recombinant basic fibroblast growth factor (FGF2) (100 μg/mL) 20 ng/mL 100 μL A83-01 (10 mM) 500 nM 25 μL Nicotinamide (1 M) 10 mM 5 mL N-Acetyl-l-Cysteine (500 mM) 1 mM 1 mL HEPES (1 M, pH 7.4) 10 mM 5 mL Gibco GlutaMAX supplement (100X) 100X 5 mL B-27 supplement (50X) 50X 10 mL N -2 supplement (100X) 100X 5 mL Gibco Advanced DMEM/F-12 N/A 462.1 mL Total N/A 500 mL [Once prepared, keep at 4°C for up to 2 weeks or aliquot and freeze at −20°C for up to 3 months]

Step-by-step method details Preparation of tumor tissues for histological analysis, genomic analysis, and tumor organoid generation Timing: 30 min per culture This section describes how to prepare colorectal cancer tissues for PDTO generation, histological characterization and DNA extraction. 1. Transfer each tissue sample from tissue collection medium into a 50 mL centrifuge tube containing 10 mL washing PBS with clean tweezers and allow the tissue to settle down by gravity for 10 min at 20°C–22°C. CRITICAL: After each use, wash tweezers with 80% ethanol and then wipe tweezers clean with paper towel pre-wetted with 80% ethanol. Note: Tissues are kept in tissue collection medium once collected after surgical resection or biopsy. Note: Processing of the tissue within 24 hours is recommended, although tissues can be stored in tissue collection medium at 4°C for up to 72 hours. Pause point: If not processed immediately, add 4 μL 10 mM Y-27632 and 8 μL 50 mg/mL primocin to every 4 mL of tissue collection medium and stored tissues in tissue collection medium at 4°C for up to 72 hours. CRITICAL: Perform good aseptic operations. 2. Remove the supernatant by vacuum suction, add another 10–20 mL washing PBS and let the tissue settle down by gravity for 10 min at 20°C–22°C. 3. Transfer tissues with clean tweezers into petri dishes. CRITICAL: Perform good aseptic operations. After each use, wash tweezers with 80% ethanol and then wipe tweezers clean with paper towel pre-wetted with 80% ethanol. 4. Quickly weigh each tissue to avoid dryness and take photos of the tissue for record. Note: For needle biopsies, go to step 5 directly. 5. Add 2 mL DPBS to cover the tissue and prevent it from drying. 6. Cut 1 mm 3 pieces to be embedded in OCT compound for later histology analysis and DNA/RNA extraction. 7. Cut a 3 mm × 3 mm × 1 mm piece for tumor organoid generation. 8. Cut any remaining tissue into 1 mm 3 pieces and place every three pieces into 1 mL CryoStor medium for cryopreservation. Note: If available, a 1 mm 3 piece of adjacent normal colorectal tissue from surgery should be preserved in OCT compound for DNA extraction for annotation of gene mutations and identification of cross-contamination between tumor organoids from different patients. If a normal tissue is not available, buffy coat extracted from 15 mL peripheral blood sample of a patient can be used for DNA extraction. 9. Add 5 mL washing PBS into the dish and mince the 3 mm × 3 mm × 1 mm CRC tissue in the dish with scissors until the tissue suspension can pass through 1 mL tips. 10. Collect the tissue suspension into a 10 mL conical centrifuge tube.

Tissue digestion and generation of CRC

PDTOs in suspension culture Timing: 90–120 min per culture This section describes how to digest CRC tissues into fragments and seed the fragments to generate PDTOs. 11. Centrifuge the tissue suspension at 200 × g for 5 min. 12. Remove the supernatant by vacuum suction. 13. Resuspend the minced tissues in 2 mL tissue digestion medium. 14. Digest at 37°C incubator for 30–60 min. CRITICAL: pipette the digestion mix and tissue pieces with 1 mL tips every 10 min until the tissue pieces can pass through the tips easily. Example picture of digested tissue fragments is in Figure 1 A. 15. Centrifuge the suspension at 200 × g for 5 min. Note: Check the supernatant for remaining cells under a microscope before aspirating the supernatant. If there are still many cells floating in the supernatant, centrifuge the supernatant again at 400 × g for 5 min. 16. Remove the supernatant by vacuum suction. 17. Add 10 mL washing medium to resuspend the digested tissue. 18. Centrifuge at 200 × g for 5 min. 19. Remove the supernatant by vacuum suction. 20. Repeat steps 13–14 four times. 21. Resuspend the pellets in 1 mL colorectal cancer organoid primary culture medium (See materials and equipment setup section for recipes). 22. Take 10 μL out of suspension, dilute 1:1 with 0.4% crystal violate and count viable fragments (those not stained blue) using hemocytometer. 23. Plate 12,000–24,000 fragments in 3 mL colorectal cancer organoid primary culture medium with 150 μL Matrigel for each well of a 6-well suspension plate. 24. Incubate at 37°C in 5% CO 2 incubator. CRC PDTOs form within 1–7 days. Example picture of established CRC PDTOs is in Figure 1 B. Figure 1 Representative pictures of digested CRC tissue fragments and established PDTOs (A) Fragments from digested CRC tissues before the washing step. (B) CRC PDTOs established 24 h after tissue processing. Images were all taken using the 10X objective of Nikon eclipse TS100 microscopy. Scale bar, 200 μm. CRITICAL: Observe primary cultures carefully during the first 72 hours. Contamination by microorganisms mostly happens in the first 72 hours. If contamination happens, see Troubleshooting section, problem 1 and possible solution. CRITICAL: Some tumor organoids can adhere to the bottom of plates or tubes due to gravity and the low percentage of Matrigel. Check cultures daily. Scramble up organoids to keep healthy organoid growth if they adhere to the bottom of plates. Note: Always use filter tips that are pre-rinsed with DMEM/F12 containing GlutaMAX medium +1% BSA when resuspending organoids to avoid loss of organoids from pipetting. CRITICAL: Matrigel might get digested by enzymes secreted by some primary tumor organoid cultures. For these primary cultures, collect these into 10 mL conical centrifuge tubes, centrifuge at 200 × g for 5 min, remove supernatant by vacuum suction, add fresh colorectal cancer organoid primary culture medium containing 5% Matrigel and plate cultures into culture plates. 25. Every two to three days, refresh colorectal cancer organoid culture medium. a. Leave culture plates in a tissue culture hood for 5 min to let organoids settle down with solidified Matrigel to the plate bottom by gravity. b. Replace half of the supernatant with fresh medium by pipetting without disturbing the layer of organoids and Matrigel at the plate bottom. See Figure 2 for illustration about how the medium is changed. Figure 2 Illustration of changing medium for CRC PDTO cultures Half of the medium was removed without disturbing PDTOs and Matrigel at the bottom of the well. Fresh organoid maintenance medium was then added against the wall of the well. Created with BioRender 2024. c. Pipette the mixture of tumor organoids, Matrigel and medium approximately ten times with a 1 mL tip. CRITICAL: Two weeks after the initiation of the culture, collect the supernatant of tumor organoid cultures to check for mycoplasma status using the LookOut Mycoplasma PCR Detection Kit. Passaging of CRC PDTOs Timing: 30 min per culture This section describes maintenance of CRC PDTOs via passaging. 26. Collect all tumor organoids into 10 mL conical centrifuge tubes. Pipette as in Step 21c. Note: Healthy tumor organoids have smooth edges. See Figure 3 A for representative images of organoids growing well. Note: Tumor organoids are ready for passaging when the cores of the tumor organoids are dark and the edges start to become rough. See Figure 4 for representative images of organoids ready for passaging. Note: Most tumor organoids are ready for passaging 7–14 days from last passage. However, for tumor organoids growing slowly, culturing for up to 3 months is required, with medium change every 2–3 days and replenishing of Matrigel every three weeks. See Figures 3 A and 3B for examples of organoids with different growth rates. Check Troubleshooting section, problem 2 and possible solutions. 27. Centrifuge at 200 × g for 5 min. 28. Remove the supernatant by vacuum suction. Note: A hazy layer of liquid Matrigel may be present above white / yellow organoid pellets after centrifugation. This will be removed by PBS washing at the next step. 29. Resuspend the pellet in 1 mL PBS and transfer to 1.5 mL autoclaved Eppendorf tubes. 30. Centrifuge at 1500 × g (Tabletop centrifuge) for 2 min. 31. Remove the supernatant by vacuum suction. 32. Resuspend the pellet in 1 mL TrypLE Express Enzyme and incubate at a 37°C incubator for 10–15 min (Larger organoids takes longer for digestion). 33. Centrifuge at 1500 × g (Tabletop centrifuge) for 2 min. 34. Remove the supernatant by vacuum suction. 35. Resuspend the pellet in 1 mL of 1% (m/v) sterile BSA in DMEM/F12 containing GlutaMAX medium. 36. Centrifuge at 1500 × g (Tabletop centrifuge) for 2 min. 37. Resuspend the pellet in 1 mL colorectal cancer organoid maintenance medium. See Figure 5 for example images of organoid fragments after digestion. Figure 5 Representative images of fragments from digested CRC PDTOs Images were all taken using the 10X objective of Nikon eclipse TS100 microscopy. Scale bar, 200 μm. 38. Dissociate organoids into small fragments by pipetting. 39. Take 10 μL out of suspension, dilute 1:1 with 0.4% crystal violate and count viable fragments (those not stained blue) using hemocytometer. 40. Resuspend 100,000–300,000 cells in 3 mL tumor organoid culture medium and 150 μL Matrigel and plate into 6-well suspension culture plates. 41. Incubate at 37°C in 5% CO 2 incubator. 42. Every two to three days, refresh tumor organoid culture medium (As Step 25). Figure 3 Representative images of CRC PDTOs at different growth rates (A) Fast-growing healthy CRC PDTOs. Images were taken one week since last passage. (B) Slow-growing CRC PDTOs. Images were taken after two months of culture. Images were all taken using the 10X objective of Nikon eclipse TS100 microscopy. Tumor organoid sizes could vary between cultures due to their different growth rates. Scale bar, 200 μm. Figure 4 Representative images of CRC PDTOs ready for passaging Images were all taken using the 10X objective of Nikon eclipse TS100 microscopy. Tumor organoid sizes could vary between cultures due to their different growth rates. Scale bar, 200 μm. Cryopreservation Timing: 15 min per culture This section describes cryopreservation of CRC PDTOs for long-term storage. 43. Collect tumor organoids into 10 mL conical centrifuge tubes. 44. Centrifuge at 200 × g (Benchtop centrifuge) for 5 min. 45. Remove the supernatant by vacuum suction. Note: A hazy layer of liquid Matrigel may be present after centrifugation. This will be removed by PBS washing at the next step. 46. Resuspend the pellet with 3 mL PBS. 47. Centrifuge at 200 × g (Benchtop centrifuge) for 5 min. 48. Remove the supernatant by vacuum suction. CRITICAL: For mature tumor organoids (those cultured for more than one week), go to step 45; For tumor organoids cultured for 3–5 days since last passage, go to step 51 directly. 49. Add 1 mL TryPLE to resuspend the pellet and incubate at a 37°C incubator for 10 min. 50. Centrifuge at 200 × g (Benchtop centrifuge) for 5 min. 51. Remove the supernatant by vacuum suction. 52. Add 1 mL of 1% BSA in DMEM/F12 containing GlutaMAX medium to stop digestion. 53. Centrifuge at 200 × g (Benchtop centrifuge) for 5 min. 54. Remove the supernatant by vacuum suction. 55. Resuspend pellets in 1 mL CryoStor and transfer to a cryotube. Note: Tumor organoids should be cryopreserved at a density of 10,000 organoids or 2 million viable cells per vial. 56. Put cryotubes in CoolCell LX Cell Freezing Container. Keep the container in −80°C for more than 8 h before transferring frozen organoid stocks to liquid nitrogen facility. CRITICAL: Store frozen organoid stocks in vapor phase liquid nitrogen for long-term storage and avoidance of cross-contamination. Thawing Timing: 15 min This section describes how to revive cryopreserved CRC PDTOs. 57. Add 4 mL colorectal cancer organoid maintenance medium into a 10 mL centrifuge tube. 58. Warm frozen tumor organoid stocks at 37°C water bath to thaw rapidly. 59. Gently transfer the thawed organoid stock to a 10 mL conical centrifuge tube. 60. Centrifuge at 200 × g for 5 min. 61. Remove supernatant by vacuum suction. 62. Resuspend pellets with 3 mL colorectal cancer organoid maintenance medium (See recipes) containing 5% Matrigel and plate them into 6 well suspension culture plates. 63. Incubate at 37°C in 5% CO 2 in a humidified incubator. Preparation for drug screening Timing: 60–90 min This section describes how to prepare single-cell suspension of CRC PDTOs to be seeded into 384-well plates for drug screening. 64. Collect PDTOs ready for drug assay (2–4 weeks since last time CRC PDTOs were passaged) into 15 mL tubes. 65. Centrifuge at 200 × g for 5 min and then remove supernatant. 66. Resuspend PDTOs with 1 mL PBS. 67. Centrifuge at 200 × g for 5 min and then remove supernatant. 68. Resuspend PDTOs with 1 mL TrypLE Express enzyme and incubate at 37°C for 10–15 min. 69. Centrifuge at 200 × g for 5 min and then remove supernatant. 70. Resuspend PDTOs with 5 mL DMEM/F12 containing GlutaMAX medium with 1% bovine serum albumin (BSA). 71. Centrifuge at 200 × g for 5 min and then remove supernatant. 72. Resuspend PDTOs with 1 mL colorectal cancer maintenance medium. 73. Dissociate PDTOs into single cells with 26G needles and 1 mL syringes and filter through 70-μm cell strainers into a Falcon centrifuge tube to obtain single cell suspension. 74. Mix 10 μL of single cell suspension with 10 μL 0.4% trypan blue solution and count live cells (cells not stained blue) using hemocytometers. Note: Cell viability should be more than 90%, which means that less than 10% of the cells in the hemocytometer should be stained blue. 75. Resuspend single cells in colorectal cancer organoid maintenance medium containing 3% Matrigel. Note: For assay on one 384-well plate, suspend 750,000 cells in 15 mL colorectal cancer organoid maintenance medium containing 450 μL Matrigel. Multiply the cell number and medium volume by the number of assay plates if needed. CRITICAL: Wells of the outside two columns and two rows are not used for drug screening and instead topped up with 100 μL PBS per well to minimize the impact of edge effects on assay results. Therefore, only the 12 × 20 = 240 wells in the center of a 384-well assay plate are plated with cells for drug screening, which requires at least 240 × 3000 = 720,000 cells in 240 × 60 = 14,400 μL colorectal cancer organoid maintenance medium containing 3% Matrigel. Prepare extra amount of cells and medium to account for dead volume during cell plating. 76. Seed cells into 384-well PDTO assay plates at the density of 3,000 cells per 60 μL per well using a MANTIS liquid handling robot. Note: If fast-growing PDTOs become too confluent during organoid drug assay, repeat organoid assay with 2,000 cells per well. 77. Use Microclime lids to minimize edge effect. 78. Culture plated cells for 3 days at 37°C in 5% CO 2 in a humidified cell culture incubator. Drug stock plate preparation and drug addition Timing: 60–90 min This section describes preparation of drug stock plates for adding drug treatments to CRC PDTO drug testing plates. 79. To prepare drug stock plates at 200X (for single-agent screen) or 600X (for FOLFOX or FOLFIRI combination matrix screen) stock concentrations, add 15 μL DMSO (negative control) to corresponding positions on drug stock plates. Drug stock plate designs are shown in Figure 6 . Figure 6 Illustration of compound plate design (A) Design of the compound plate for single-agent screen. (B) Design of the compound plates for FOLFIRI/FOLFOX screen. Drug titration directions are shown by fading colors. The darkest colors indicate the locations of drugs with the highest concentration. Bort, positive control (Bortezomib); DMSO, negative control; 5FU, 5-fluorouracil; SN38, SN-38; Oxali, oxaliplatin; Rego, regorafenib; TAS, TAS-102; Gem, gemcitabine; Peme, pemetrexed; Temo, temozolomide; Erloti, Erlotinib. Note: Use one drug stock plate for single-agent screen but use two drug stock plates for combination matrix screen due to the two-drug matrix design. 80. Add 15 μL Bortezomib (positive control, 300 μM for single-agent screen and 200 μM on stock plates for combination matrix screen) to corresponding positions on drug stock plates. Drug stock plate designs are shown in Figure 6 . 81. Add 15 μL drugs at the highest concentrations of titration series to corresponding positions on drug stock plates. Drug stock plate designs are shown in Figure 6 . Note: For single-agent screen, the highest drug concentrations on stock plates are 10 mM for 5-fluorouracil, 100 μM for SN-38, 15 mM for oxaliplatin, 10 mM for regorafenib, 10 mM for TAS-102, 3 mM for gemcitabine, 2 mM for pemetrexed, 150 mM for temozolomide and 10 mM for erlotinib. Note: For FOLFOX or FOLFIRI combination matrix screen, the highest drug concentrations on stock plates are 30 mM for 5-fluorouracil, 300 μM for SN-38, 45 mM for oxaliplatin. 82. Use JANUS liquid handling robot to titrate drugs from highest to lowest concentrations (9-point, 4-fold dilution series on stock plates for single-agent screen, 6-point 4-fold dilution series on stock plates for FOLFOX/FOLFIRI matrix screen) according to the drug stock plate design ( Figure 6 ). Note: For 4-fold dilution series, add 3 μL drugs into 9 μL DMSO and mix well for each dilution. 83. Add drugs into 384-well assay plates from stock plates using a JANUS liquid handling robot with PinTool addition accessory. Note: PinTool adds 100 nL drugs per addition. Thus, for single-agent screen, perform 3 PinTool addition from single-agent drug stock plates into assay plates to add 300 nL of the 200X stock drugs into 60 μL medium in each well. For FOLFOX or FOLFIRI combination matrix screen, perform 1 PinTool addition from each combination matrix screen stock plate into assay plates to add 100 nL of the 600X stock drugs into 60 μL medium in each well. 84. Switch the Microclime plate lids to transparent assay lids for Nunc 384-well plates. 85. Image each well of PDTO assay plates with an automated Nikon Eclipse Ti2 Inverted Microscope System (NIS-Elements AR software version 5.21.03, 64-bit) with a 4X bright-field objective in a 25 μm Z-stack over a 500-μm range every 24 h for 7 consecutive days. Organoid drug sensitivity analysis Timing: 60–90 min This section describes image analysis of organoid viability changes under drug treatment to generate drug dose-response curves. 86. Measure size changes of PDTOs treated with different drug doses/combinations via ImageJ. 7 a. Open Nikon ND2 file. >run("Bio-Formats Macro Extensions") >idnd2 = "FileName.nd2" //path to ND2 file >EDOFFile = "Output.tif" //path to tiff output >s = 1 //Select well within the nd2 file Note: Need to install ImageJ plugins: Bio-Formats and Extended Depth of Field ( http://bigwww.epfl.ch/demo/edf/ ). b. Extract z-stack and flatten image. >run("Bio-Formats Importer", "open=["+idnd2+"] color_mode=Composite split_channels view=Hyperstack stack_order=XYCZT series_"+s) >run("EDF Easy mode","quality='1' topology='1' show-topology='off' show-view='off'") >run("8-bit") >saveAs("tiff", EDOFFile) c. Generating files including organoid area text file and a PNG image overlaying identified organoids over original tiff image. >tifFile = "Output.tif" //path to tiff input from Step 1 >outPNG = "Output_overlay.png" //path overlayed output image >outMeas = "Output_measurements.csv" //path organoid measurement d. Open tiff image. >open(tifFile) >name = getTitle >mainTitle=getTitle() >run("Duplicate…", "title=originalimage") >selectWindow(mainTitle) e. Subtract background and enhance contrast. >run("Subtract Background...", "rolling=10 light sliding") >run("Enhance Local Contrast (CLAHE)", "blocksize=127 histogram=256 maximum=3 mask=∗None∗ fast_(less_accurate)") >setAutoThreshold("MaxEntropy") >setOption("BlackBackground", true) f. Identify organoids. >run("Convert to Mask") >run("Analyze Particles...", "size=200-Infinity show=Masks") >run("Invert LUT") >run("Fill Holes") >run("Watershed") >run("Set Measurements...", "area mean standard modal min centroid center perimeter bounding fit shape feret's integrated median skewness kurtosis area_fraction display redirect=originalimage decimal=3") >run("Analyze Particles...", "size=200-12000 circularity=0.20-1.00 show=Outlines display exclude clear summarize") g. Output measurements and PNG files >rename("mask_of_organoids") >run("Convert to Mask") >run("Merge Channels…", "c1 = [mask_of_organoids] c4 = originalimage create") >saveAs("PNG", outPNG) >saveAs("Measurements", outMeas) 87. For single-agent screen, fit drug curves using a four-parameter log-logistic function of the drc R library for full models with normalization to negative controls (DMSO) and positive controls (Bortezomib) to determine effective concentration metrics (EC). Quantify drug response as area under the dose response curve (AUC). 88. For FOLFOX/FOLFIRI matrix screen, calculate pan-matrix relative activity score by averaging the inhibition of PDTO viability over the matrix. 89. Calculate the robust Z′ score and CV% of negative control to confirm the assay quality. Robust Z′ factor: RZ’ = 1–3 X (m.a.d.(Cpos)+m.a.d.(Cneg))/abs(median(Cpos)-mediam(Cneg)). Coefficient of variation: CV = s.d. / mean X 100%. CRITICAL: Tumor organoids treated with vehicle and positive controls should exhibit distinct differences in sizes, which reflect changes in viability. 9 Robust Z′ factor is a measure that is sensitive to any change in assay conditions and an indicator of assay quality in a high-throughput screen. Plates run through the PDTO platform should have CVs of DMSO control wells lower than 20%, and robust Z′ factors higher than 0.5.

Materials availability

This study did not generate new unique reagents.