1.1 Establishment of cervical organoid
Clean off the blood and bacteria on the surface of clinical surgical tissue samples, cut them into 1 mm3 sized tissue fragments, and wash them again. Tissue fragments were placed in Advanced DMEM/F12 (Gibco, Cat# 12634-010) containing 1.5 mg/mL type II collagenase (Gibco, Cat# 17101-015), type IV collagenase (Gibco, Cat# 17104-019), and 0.004% DNase, Put on the shaking bed of 37℃ incubator for 1-2h; after digestion was completed, replenish 1×DPBS (containing 2% FBS), blow continuously to disperse the cell clusters, and filter through a 70µm filter; centrifuge at 300g for 5min; after discarding the supernatant, resuspend the cells with Matrigel (Corning, Cat# 356231), and seed them in a 24-well cell culture plate to form a dome shaped structure; inverted and cured for 30 min in a 37℃ incubator; 500 µL of pre-warmed medium was added to each well. The composition of the culture medium for normal cervical organoids are listed in Table S1
In order to avoid apoptosis and difficulty in obtaining nutrients for the growing internal cells of the organoids, the organoids were passaged and frozen at a ratio of 1:2 − 1:3 every 7–14 days.
With the addition of 25 ug/ml Wnt3A (Stemgent, Cat# 04-0004-10), β-estradiol (MCE, Cat#HY-B0141 ) to the normal cervical organoid medium, we successfully cultured cervical tumoroids.
I confirm that all experiments were performed with mycoplasma-free cells.
1.2 Isolation and validation of CAFs
Attach tissue fragments onto the wall of T25 cell culture bottles, added with 2mL of CAF medium, and placed inverted into the 37℃ incubator, and the flasks were placed orthotropically after 4-6h. Cell growth was observed after 3–5 d of culture, and the elongated shuttle-forming fibrous cells were isolated to new culture flasks. The cells were cultured for a period of time with CAF characterizing markers FAP, α-SMA, Vimentin, PDGFR-α, PDGFR-β (The specific information on the antibodies is listed in Table S2) were validated by immunofluorescence staining.
1.3 H&E staining, immunohistochemistry, and immunofluorescence
The harvested organoids were wrapped with 3% agarose gel for gradient ethanol dehydration and paraffin embedding, serial sections were cut with a paraffin slicer for H&E staining and immunohistochemical staining. Primary antibody used in immunohistochemical including Ki67, MMP-9, Collagen I (The specific information on the antibodies is listed in Table S3), slides rinsed with PBS, secondary antibody incubated at 37℃ for 30 min, sections rinsed with PBS, blotted with absorbent paper.
Tissues and organoids used for immunofluorescence were dehydrated in 30% sucrose solution overnight. Sections were made on a frozen microtome and blocked at room temperature for 2 h. And the following primary antibodies were diluted with the blocking solution: CK14, CK5, P63, Ki67, CK7, CK18, PAX8 (The specific information on the antibodies is listed in Table S4). Primary antibody was incubated overnight at 4°C. Slides were washed with PBST, secondary antibody was diluted in PBST, and the secondary antibody was incubated at room temperature for 2h. Imaging was performed with a SP8 (Leica) confocal microscope.
1.4 RT-qPCR
Total RNA was extracted from harvested organoids using RNAiso Plus (Takara, Cat#9108) reagent according to the manufacturer's instructions. The isolated RNA was directly reverse-transcribed to cDNA using the Reverse Transcription Kit (Applied Biosystems, 4387406). Relative mRNA expression levels were measured by SYBR Green(Tsingke, Cat#TSE201) and calculated using the comparative threshold cycling (ΔCt) method and normalized to GAPDH as the reference gene .
1.5 Western-blot
After SDS/PAGE electrophoresis, proteins were transferred to PVDF membranes (Millipore). After being enclosed in 5% skimmed milk for 2 h, proteins were rinsed 3 times with TBST and incubated with the corresponding primary antibodies overnight in the freezer at 4°C. Primary antibody of Vimentin, Smad4, E-cad, N-cad, Sox2, CK17, Oct3/4, CK13, MMP-1, MMP-3, MMP-7, MMP-9 (The specific information on the antibodies is listed in Table S5) was used. After the membrane was rinsed three times with TBST, the secondary antibody was incubated at room temperature for 2 h, and the membrane was rinsed again three times with TBST. Incubate in developer solution for 1–2 min and develop.
1.6 Transwell
Firstly, 70 µl/well of diluted Matrigel (1:8 dilution, Corning) was applied to the upper chamber and incubated in a 37℃ CO2 incubator for 4–6 h. The supernatant was aspirated off, 200 µl/well of basal medium was added for hydration, and the cells were diluted in serum-free medium to the appropriate density. 200 µl of diluted single cell suspension was inoculated into the transwell, and 700 µl/well of medium containing 20% FBS was added to the lower chamber. After 24 h, the cells were fixed in 4% formaldehyde, stained with crystal violet for observation, and photographed under the microscope with, each sample was performed in three wells and repeated several times.
1.7 Drug sensitization test
Drugs were tested at six different concentrations using semi-log dilution method and the drugs paclitaxel, gemcitabine, olaparib, obatoclax, 5-fluorouracil, and nedaplatin (The information on the drugs is listed in Table S6) were dissolved in DMSO. Organoids that were healthy and grown for about 5 days were dissociated into single cells with TryPLExprass (Gibco, Cat#12604 054) and spread into 96-well cell culture plates (Corning) at 3000–4000 cells/well. Tumoroid medium was added and cultured for 4 days to restore growth, and the drug treatment was added for 4–5 days, and the positive control group was assayed when all the cells were killed, and ATP levels were measured using the CellTiter-Glo3D cell viability assay (G9683; Promega), and luminescence was measured using the microplate reader.