Ice cold cell recovery solution

PDO were fixed and embedded in paraffin per a modified Trevigen, Inc. protocol.^[57
] First, Matrigel domes with PDO were washed with PBS and then fixed with 5 mL of 2% paraformaldehyde (PFA) + 0.1% glutaraldehyde (GA) in PBS at room temperature for 30 min. After washing with PBS, the domes were taken to 20% sucrose and left overnight at 4 °C until the domes sank to the bottom. Next, the solution was changed to 70% ethanol, and the domes with PDO were embedded with paraffin for sectioning and H&E‐staining in the Duke Pathology Research Histology Lab.
The CRC identity of PDO was confirmed by the maintained protein expression of CDX2 and CK20 (Figure 1C; Figure S1A,B, Supporting Information). A publsihed PDO 3D imaging protocol was followed.^[58 (link)
^] Briefly, the organoids were recovered from Matrigel using the ice‐cold cell recovery solution (Corning) and then fixed with 4% PFA at 4 °C for 45 min. After blocking, PDO were cultured with primary and then secondary antibodies for immunolabeling. Fructose–glycerol clearing solution was used for imaging the organoids under the Leica SP5 inverted confocal microscope. The following antibodies were used for immune‐fluorescence staining: CDX2 (12306S, Cell Signaling Technology), 1:200; CK20 (60183‐1‐IG, Proteintech), 1:100; Anti‐Rb IgG‐488 (ab150061, Abcam), 1:500; Anti‐Ms IgG‐594 (ab150112, Abcam) 1:500.

Sections of formalin-fixed, paraffin-embedded ascites and solid tumor patient-derived organoids were used for histopathological analyses. Organoids were recovered from BME using ice-cold cell recovery solution (Corning, New York, United States) in accordance with manufacturing protocols, fixed in phosphate-buffered 10% formalin, and embedded in 500 μL of Bio-Agar (Bio-Optica Milano Spa, Milano, ITA). Five μm sections were stained with hematoxylin and eosin (H&E) using a Leica ST5020 multistainer and 2 μm sections were cut for IHC analysis. The IHC was performed with an UltraVision LP Detection System HRP DAB kit (Thermo Scientific, Waltham, USA). Heat-induced antigen retrieval was performed using 10 mM citrate buffer pH 6.0. The following antibodies were used to characterize HGOC patients-derived organoids and parental tumors: PAX8 (ProteinTech Group, Germany, EU), WT1 (Abcam, U.K.), and CA-125 (Santacruz Biotechnology, TX).

Organoids were extracted from their BME matrix by adding ice-cold cell recovery solution (Corning) for 30–60 min. This procedure enables the dissolution of BME without damaging the organoids, and allows an optimal penetration of the antibody. Then, organoids were fixed with 4% (w/v) paraformaldehyde at 4 °C for 45 min. Throughout the protocol, samples were washed extensively to avoid background or loss of signal. Primary antibodies E-cadherin (Abcam), Vimentin (Abcam) and EpCAM (Cell Signalling) were incubated overnight at 4 °C on a horizontal shaker (40 rpm). Secondary antibodies Mouse IgG Alexa Fluor 555 (Abcam), Rabbit IgG Alexa Fluor 488 (Abcam) and the Phalloidin-iFluor 647 (Abcam) were added to each well and incubated overnight in the same conditions. Antibodies used in this process are listed in Supplementary Table 3. Optical clearing was accomplished by resuspending the organoids with homemade fructose-glycerol clearing solution. For slide preparation, a slice of double-sided tape was added on both sides of the slide to lift the coverslip, thereby maintaining the 3D structure of the organoids. The slide was then visualized by fluorescence confocal microscopy (Zeiss LSM980).