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Construction of gastric cancer patient-derived organoids and their utilization in a comparative study of clinically used paclitaxel nanoformulations | Journal of Nanobiotechnology


Study approval

All human sample collections and experiments were reviewed and approved by the Chinese PLA General Hospital Medical Ethics Committee in accordance with the 1964 Declaration of Helsinki and 1982 International Ethical Guidelines for Human Biomedical Research (approval number: S2017-010-02), and informed consent was obtained from all of the participants. The mice experiment was approved by the Institutional Animal Care and Use Committee at the Institute of Process Engineering, Chinese Academy of Sciences (Approval Number: IPEAECA2021011).

Construction, culture and passage of GC PDOs

Highly fibrotic, fatty, and severely necrotic tumor tissue should be avoided for the construction of PDOs. The clinically collected GC patient’s tumor samples should be conditioned in ice-cold PBS with 10 mM HEPES (03-025-1B, BI), 10 µM Rho kinase (ROCK) inhibitor compound Y-27632 (72302, Stemcell), and 100 U/mL penicillin-streptomycin-amphotericin B (P7630, Solarbio). The transportation time should be less than 24 h. When the tumor samples were transported to the laboratory, one part was fixed in 4% paraformaldehyde for histopathological and IHC analyses. Another part of tumor tissue, accompanied with corresponding para-carcinoma tissue, were quickly freezed in a liquid nitrogen tank for WES analysis. For construction of GC PDOs, GC patient’s tumor samples were washed with ice-cold PBS with 10 mM HEPES and 100 U/mL penicillin-streptomycin- amphotericin B at least five times. Then, we removed as much non-epithelial tissues as possible, and minced the tissues into small pieces and digested in 5 mL Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (DMEM/F12) (01-172-1ACS, BI) containing 1 mg/mL collagenase I (V900891-1, LABLEAD), II (V900892-1, LABLEAD), and IV (V900893-1, LABLEAD). During digestion, we mixed contents by shaking vigorously and by pipetting the mixture up and down using a P1000 pipette. In order to maximize cell viability, multiple-batch digestion was adopted. In detail, we monitored the digestion process under a microscope. When a certain amount of dissociated cell clusters was observed, we let the suspension stand for 2 min and collected supernatant for centrifugation (300 g, 5 min, 4 ℃) to isolate cells. The remaining undissociated sedimentation was continued to be digested and repeated the above steps. Finally, collected cells were resuspended in precooling mixture of DMEM/F12 and Matrigel (356231, Corning) at the ratio of 1:1, and seeded in a pre-heated 24-well flat bottom cell culture plate (35241, Corning) in drops of 50 µL each. After the drops have solidified, 750 µL IntestiCult Organoid Growth Medium (06010, Stemcell) was added to each well. Organoids were cultured in incubator at 37 ℃, 5% CO2. Medium was refreshed every 2–3 days.

GC PDOs were passaged with a split ratio of 1:3 to 1:2 approximately every 6–8 days. The organoids were mechanically pipetted out of Matrigel using cold DMEM/F12, and the organoids suspension was centrifuged at 300 g for 5 min at 4 ℃. Then, the organoids were dissociated by TrypLE (12604-013, Gibco), and pipetted up and down to aid disruption of the organoids. Dissociation ended when cell clusters (consisting of 2–10 cells) can be observed under microscope. Cell clusters were collected by centrifugation (300 g, 5 min, 4 ℃), re-suspended in precooling mixture of DMEM/F12 and Matrigel and then seeded as described above.

Cryopreservation and recovery of GC PDOs

The medium was removed 2–3 days after splitting. The organoids were mechanically pipetted out of Matrigel using cold DMEM/F12, and the organoids suspension was centrifuged at 300 g for 5 min at 4 ℃. Then, we aspirated the supernatant and added the cell-freezing medium (07930, Stemcell) to resuspend the organoids, and transferred the suspension to the cryogenic vial. The cryogenic vial was placed into a freezing container which was stored at − 80 ℃ for 24 h. Finally, the freezing container was transferred to the liquid nitrogen tank.

Before thawing of cryopreserved organoids, we prepared a 15-mL conical tub with 10 mL of DMEM/F12 at room temperature. Then, we removed the cryogenic vial from the liquid nitrogen tank, and incubated the cryogenic vial in a water bath at 37 ℃. The cryogenic vial was removed from the water bath when there was a small clump of ice in it. Finally, we transferred the suspension from the cryogenic vial to the previously prepared 15-mL conical tub. The organoids suspension was centrifuged at 300 g for 5 min at 4 ℃, and re-suspended in precooling mixture of DMEM/F12 and Matrigel and then seeded as described above.

HE and IHC staining of GC PDOs and primary tumor tissues

Tumor samples and PDOs pellets were fixed in 4% paraformaldegyde at 4 ℃ for 24 h and 30 min, respectively, and then followed by dehydration, paraffin embedding, sectioning and standard HE staining. IHC were performed for LGR5 (1:100, Affinity DF2816), E-cadherin (1:2000, proteintech 20874-1-AP), Ki-67 (1:10000, proteintech 27309-1-AP), and CEA (1:200, Bioss bs-0060R). Briefly, paraffin sections were deparaffinized in xylene and rehydrated through a graded ethanol series. After heat-mediated antigen retrieval with a sodium citrate buffer (10 mM, PH 6.0), the sections were blocked at room temperature for 30 min. The primary antibodies were diluted in 3% bull serum albumin (BSA), and staining was performed overnight at 4 ℃. Sections were then incubated with secondary antibodies at room temperature for 60 min. HE and IHC images were viewed and captured using an automatic multispectral imaging system (PerkinElmer Vectra II, USA).

WES analysis

Genomic DNA was fragmented using NEBNext dsDNA Fragmentase (NEB, Ipswich, MA, USA) following by DNA ends repairing. End-repaired DNA fragments were dA-tailed and ligated with the NEBNext adaptor (NEB, Ipswich, MA, USA). Biotinylated RNA library baits and magnetic beads were mixed with the barcoded library for targeted regions selection using the SureSelect Human All Exon V6 Kit (AgilentTechnologies, Palo Alto, Calif.). The captured sequences were further amplified for 150 bp paired-end sequencing in Illumina X-ten system (Gene Denovo Biotechnology Co. China). To identify somatic SNV, the Burrows-Wheeler Aligner (BWA) was used to align the clean reads from each sample against the human reference genome (GRCh38). Somatic CNV was identified using VarScan 2 with the following parameter: phred base quality ≥ 20, minimum coverage ≥ 20. Mutational signatures were deciphered by using a non-negative matrix factorization method.

RNA-sequencing analysis

Total RNA was extracted using Trizol method. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo (dT) beads, while prokaryotic mRNA was enriched by removing rRNA by Ribo-Zero™ Magnetic Kit (Epicentre, Madison, WI, USA). Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit (Qiagen, Venlo, The Netherlands), end repaired, poly (A) added. The transcriptome sequencing was performed using Illumina HiSeq2500 (Gene Denovo Biotechnology Co. China). RNA expression levels were determined using the fragments per kilobase of transcript per million mapped reads method. The fold-change method was used to identify RNAs that were differentially expressed after castration using the R package DEGseq (R-3.6.2). Genes with a log2 fold change > 2 and an adjusted P < 0.05 were deemed to be significantly differentially expressed.

Characterization of PTX nanoformulations

Images of PTX nanoformulations were obtained using a JEM-1400 TEM (JEOL, Japan) with an accelerating voltage of 120 KV. The size distribution and zeta potential of PTX nanoformulations were analyzed by DLS system (Malvern ZEN 3600 Zeta sizer, UK).

Drug testing on GC PDOs

Single cells dissociated by TrypLE from the last passage PDOs were filtered through a 70-µm cell strainer. After cell counting, we resuspended these cells in IntestiCult Organoid Growth Medium containing 5% (vol/vol) Matrigel and then dispensed them in ultra-low attachment 96-well plate (3474, Corning) at 2000 cells per well. Organoids grew in 96-well plate for 3 days and then were treated with PTX nanoformulations (Albu-PTX and Lipo-PTX) at concentrations of 10, 2.5, 0.625, 0.16, 0.04, 0.01, 0.0025, 0.0006, and 0.00015 µM. The two PTX nanoformulations were commercial drugs used in the clinic, and we referred to the drug instructions to determine the content of PTX in nanoformulations (1000 mg Albu-PTX contains 100 mg of PTX, 1200 mg Lipo-PTX contains 30 mg of PTX). 5 µM staurosporin (HY-15141, MCE) treated wells were set as a positive control, and PBS treated wells were set as a negative control. Organoids cell viability was evaluated after 5 days drugs treatment. In detail, after adding CellTiter-Glo 3D Reagent (G9683, Promega) to the drug-screening plates, we performed the readout by measuring the luminescence intensity of each well. Then, we calculated the average luminescence value of the negative and positive control wells. Furthermore, we set the positive control to 0% viability and the negative control as 100%, and calculated the viability for each well according to the following formula:

Well viability =\(\frac{{{\text{Well}}\,{\text{value}} – {\text{Average positive control}}}}{{{\text{Average negative control}} – {\text{Average positive control}}}}*100\%\)  

Finally, we transferred the well viability to the GraphPad Prism 9.0.0 software, and chose the option “log (inhibitor) vs normalized response-variable slope” to create a dose-response kill curve.

Live-Dead staining analysis

Live-Dead staining (C2015S, Beyotime) was carried out for GC PDOs after treatment with Albu-PTX or Lipo-PTX at a PTX concentration of 0.04 µM. Organoids were incubated for 30 min in a solution of calcein-AM and PI, according to the manufacturer’s instructions. Live cells (green fluorescence, 488 nm) and dead cells (red fluorescence, 550 nm) were simultaneously detected under a CLSM (Nikon A1R, Japan).

Intra-PDO distributions of PTX nanoformulations

We labeled the PTX nanoformulations with fluorescent dyes and monitored their distributions in PDOs by CLSM. In detail, Albu-PTX was incubated with Cy5-SE (GC35771, GLPBIO), and Lipo-PTX was labeled with DiD (D22031, LABLEAD) at 37 ℃ for 2 h. After complete removal of free dye, the fluorescence intensities of Cy5-labeled Albu-PTX and DiD-labeled Lipo-PTX were detected to confirm the comparative fluorescence signal intensity (Excitation: 638 nm, Emission: 670 nm). Then the two kinds of fluorescent-labeled nanoformulations were separately added to the culture medium at a PTX concentration of 0.04 µM. Before monitoring the distributions of PTX nanoformulations in PDOs by a CLSM at the indicated time point (6 h, 12 h, 24 h, 48 h, 72 h, and 96 h after adding the PTX nanoformulations), the PDO cell nucleus was labeled with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. The same image acquisition parameters (include pinhole size, detector gain, amplifier offset/gain, scan speed/average, zoom, and laser intensity) were used when photographing different samples with CLSM (Nikon A1R, Japan). The fluorescence colocalization analysis between nanoformulations and PDO cells as well as a series of Z-stack images and corresponding 3D reconstruction data were acquired by the software of CLSM.

Drug testing on GC PDX

Fresh GC patient’s tumor samples were conditioned in ice-cold PBS with 10 mM HEPES, 10 µM ROCK inhibitor compound Y-27632, and 100 U/mL penicillin-streptomycin-amphotericin B. When the tumor samples were transported to the laboratory, they were cut into 25–50 mm3 pieces and then transplanted subcutaneously into NPG mice (female, 6–8 weeks, Beijing Vital River Laboratory, China) to establish the PDX model. After the engraftment for three passages, the tumor samples were transplanted into the armpits of NPG mice for drug testing experiment. When the tumor volumes reached about 180 mm3, tumor-bearing NPG mice were allocated randomly to three groups and received four round intratumoral injection of PBS, Albu-PTX or Lipo-PTX. Albu-PTX was diluted in 0.9% Sodium Chloride Injection and Lipo-PTX was diluted in 5% Glucose Injection. For each injection, the PTX dose was 5 mg/kg, and the total volume was 100 µL. The tumor burden was monitored with a digital caliper every 2 days. The tumor volume was calculated using the following formula: Volume (mm3) = length × width2 / 2. Mice were considered dead when the tumor volume reached 1500 mm3.

Statistical analyses

Statistical analyses were performed using GraphPad Prism 9.0.0 software. A two-tailed Student’s t test was used to compare two groups, one-way ANOVA with Tukey post-hoc test was used for the multi-group comparison, and log-rank test was used for the survival comparison. P < 0.05 was considered significant; significant values were indicated as **P < 0.01, ***P < 0.001, and ****P < 0.0001.


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