Tissue source

The subject, a 72-year-old man, reported no prior tobacco use, alcohol, or hepatitis B infection. His CEA level was 1.4 ng/mL (normal range: 0-5.2 ng/mL), AFP level was 4.6 U/mL (normal range: 0-5.8 U/mL), and CA 19 − 9 level was 38.3 U/mL (normal range: 0–35 U/mL; Table 1). Duodenoscopy examination indicated the presence of a tumor in the ampullary region (Fig. 1A). He did not undergo radiotherapy or chemotherapy before undergoing pancreaticoduodenectomy at Lanzhou University Second Hospital on August 29, 2022. Surgical gross views of the postoperative specimens revealed an ampulla with a 2 × 1.5 cm new organism (Fig. 1B-D). Samples were collected from the main site of infection for initial and subsequent cultivation examination.

Fig. 1

Clinical data and cell morphology. (A) Preoperative duodenoscopy findings of the patient depicting a tumor in the ampullary region (indicated by the arrow) (scale bar = 1 cm). (B–D) Gross examination of the postoperative specimen revealing a mass in the ampullary region (indicated by the arrow) (scale bar = 1 cm). (E–F) Microscopic examination showing DPC-X3 cell morphology under a light microscope (scale bar = 100 μm)

Table 1 Clinical data of the included patient

This research was sanctioned by the Medical Ethics Committee of Lanzhou University Second Hospital (2023 A-381), with the participant granting informed agreement to participate.

NXG mouse

The study utilized female NXG mice, 5–6 weeks of age, weighing 11–17 g. These rodents were obtained from Changzhou Cavens Laboratory Animal Co., Ltd. (Changzhou, China) and maintained in the specific pathogen-free facility at Lanzhou University’s Animal Experimental Center. The experimental design aimed to reduce animal distress. Prior to experimentation, mice were acclimated to laboratory settings (23℃, 12-h/12-h light/dark cycle, 50% humidity, unrestricted food, and water access) for 2 weeks. The animals were provided sterilized rodent feed without restriction, and their bedding, nourishment, and hydration were refreshed bi-daily. All protocols adhered to organizational and governmental standards for maintaining and utilizing laboratory fauna. The well-being and conduct of the subjects were monitored daily over four weeks. Mice were humanely euthanized when the xenograft tumor’s maximum diameter approached 1.5 cm or following 1 month of tumor development. Euthanasia was performed using an excessive dose of barbiturate (intravenous delivery, 150 mg/kg pentobarbital sodium), verified by the absence of heartbeat, respiration, and pupillary response.

The Medical Animal Experiment Ethics Committee of Lanzhou University Second Hospital provided ethical clearance for the animal research (D2023-318). All experiments complied with the ARRIVE guidelines and the Guidelines for the Care and Use of Laboratory Animals of China.

The techniques outlined in the following segments were comparable or equivalent to those utilized in earlier investigations [10, 16].

Primary culture, cell purification, and CL establishment

The tumor specimen was immersed in aseptic phosphate-buffered saline (PBS; Gibco, Cat#10010023) and washed 3–5 times. After cutting it to the maximum extent possible, type II collagenase (Gibco, Cat#17101015) and Dispase II (Invitrogen, Cat#17105041) were added. The tissue was digested on a shaking table at 37 °C. The liquid above was procured after the limited enzymatic breakdown of the tissue samples. This fluid was strained using a 100-mesh sieve and centrifuged at 300 ×g for 3 min. The upper liquid was removed, and the residual material was reconstituted in PBS. After centrifugation at 300 ×g for 3 min, the sediment was mixed with full growth medium (RPMI-1640(BI, Cat#c3010-0500) + 10% fetal bovine serum [FBS](BI, Cat#04-002-1 A) + 1% penicillin-streptomycin (BI, Cat#03-031-1B)). This mixture was evenly seeded onto a six-well plate (NEST, Cat#703001), and the medium was replenished after 48 h. A sterile 1-mL pipette (KIRGEN) was used to scrape off fibroblasts repeatedly under direct microscope observation to remove fibroblasts from the primary culture process. Cell proliferation was routinely monitored using an optical microscope. From the third passage forward, cells underwent subculturing at a 1:2 proportion and were kept in a serum-free quick cell cryopreservation medium (Mei5 Biotechnology, Cat#MF699-01).

Analysis of DNA short tandem repeat sequences

DPC-X3 cells in the exponential growth stage (EGS) (P10) were collected after trypsin digestion. These cells and the original neoplastic tissue underwent short tandem repeat (STR) profiling performed by Suzhou Genetic Testing Biotechnology Company to evaluate the relationship between the cultured cells and the source tumor specimen.

Cell growth curve

DPC-X3 cells in the EGS (P25) were adjusted to a cell density of 1 × 104/mL following trypsinization. Subsequently, 0.1 mL of the cell suspension was inoculated per well onto a 96-well plate. After cell attachment, CCK-8 reagent (Dojindo, Cat#ck04) was introduced for 4 consecutive days, with a reaction period of 2 h for each instance. The absorbance value at a wavelength of 450 nm was ascertained utilizing an enzyme-linked immunosorbent assay reader, and a calibration curve was constructed. The cell population doubling periods were calculated for multiple time intervals employing the “cell calculator++”tool [V. Roth MD, Doubling Time Calculator (2006), https://doubling-time.com/compute_more.php].

Karyotyping

DPC-X3 cells in the EGS (P30) were exposed to 0.25-µg/mL colchicine for 6 h and throughout the night at 37 °C. Cells in metaphase were harvested, fixed using a methanol-acetic acid solution (3:1), stained utilizing Giemsa staining solution after trypsin digestion, and enumerated under microscopic examination. For karyotype evaluation, well-separated and adequately stained mitotic figures were chosen.

Organoid culture from DPC-X3 cells

DPC-X3 cells (P33) underwent enzymatic digestion, centrifugation, and two PBS rinses in the EGS. The cells were then suspended in a growth medium (RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin, BI). The cell suspension (1,000 cells per well) was plated onto a non-adherent six-well plate (Corning, Cat#3471), with 2 mL of culture medium added for expansion. The development and quantity of organoids were routinely examined using a phase-contrast microscope.

Scanning electron microscopy (SEM)

Cells in the logarithmic growth phase, harvested after 30 passages, were employed for SEM analysis. The cell-containing slides underwent washing with physiological saline, followed by fixation using a 4% glutaraldehyde solution (SPI-CHEM, USA). Subsequently, the samples were rinsed three times with phosphate buffer. Dehydration was conducted step-by-step using a gradient of tert-butanol (50%, 70%, 80%, 90%, and 100%) for 5 min each. The dried sample was placed into the JEOL JFD-320 freeze-drying apparatus, and upon equilibrating to ambient temperature, a conductive coating was affixed to the specimen mount utilizing a JEOL JFCC-160 ion sputtering device. The examinations were documented and micrographs were obtained employing a HITACHI Regulus 8100 scanning electron microscope.

Transmission electron microscopy (TEM)

DPC-X3 cells (P30) in the EGS were subjected to enzymatic digestion, centrifugal separation, and fixation utilizing a suitable fixative for TEM analysis. The cellular samples were maintained and shipped at 4℃ before being processed for ultrathin sectioning and staining at Wuhan Servicebio Co., Ltd. (Wuhan, China). The prepared specimens were subsequently visualized utilizing a transmission electron microscope to examine intracellular components.

Drug sensitivity test

Utilizing logarithmically growing DPC-X3 cells at passage 35 (P35), a single-cell suspension was prepared following trypsin digestion. Subsequently, 10,000 cells per well were inoculated and seeded into a 96-well plate. Upon cell adherence, the experimental group received varying concentrations of antitumor drugs: gemcitabine (600µmol/L, 150µmol/L, 30µmol/L, 6µmol/L, 1.5µmol/L, 0.3µmol/L, 0.06µmol/L, 0.015µmol/L, 0.003µmol/L, 0.0006µmol/L. plasma peak concentration: 19.01 µmol/L [17]), paclitaxel (50µmol/L, 25µmol/L, 12.5µmol/L, 2.5µmol/L, 0.5µmol/L, 0.1µmol/L, 0.025µmol/L, 0.005µmol/L, 0.001µmol/L. plasma peak concentration: 16.2 µmol/L [18]), oxaliplatin (400µmol/L, 200µmol/L, 100µmol/L, 40µmol/L, 20µmol/L, 10µmol/L, 5µmol/L, 2.5µmol/L, 1.25µmol/L, 0.625µmol/L, 0.3125µmol/L, 0.15625µmol/L. plasma peak concentration: 11.33 µmol/L [19]), and 5-fluorouracil [5-FU] (3840µmol/L, 960µmol/L, 240µmol/L, 60µmol/L, 15µmol/L, 3µmol/L, 0.6µmol/L, 0.15µmol/L, 0.03µmol/L, 0.006µmol/L. plasma peak concentration: 76.92 µmol/L [20]), while the control group received the corresponding drug solution. Following 72 h of drug treatment, the complete growth medium was substituted with 100 µL of a serum-free medium comprising 10% (v/v) CCK-8. Following a 2-hour incubation, the OD value was measured at 450 nm.

Transplantation tumor formation experiment

Employing cells in the EGS at passage 33 (P33), the cellular concentration was regulated to 1 × 107/mL following trypsin treatment and thoroughly homogenized. A pair of NXG mice were administered an inoculation of these cells (0.1 mL per animal) on the right scapular region and dorsal area. On the subsequent day, neoplasm development in the athymic rodents was monitored and documented. After 28 days, the animals were humanely sacrificed and examined to assess the progression of the xenografted tumor.

IHC staining

Cells from the 42nd passage were enzymatically dissociated and plated on sanitized microscope slides for cultivation. Following a 48-hour incubation period, the adherent cells underwent a washing process with phosphate-buffered saline, immobilized using 4% paraformaldehyde solution for 15 min, allowed to dry in ambient air, and permeabilized with 0.5% Triton X-100 for 20 min. Sections of xenograft tumors and original neoplastic tissues were embedded in paraffin and subjected to overnight heating at 60 ℃.

The tissue samples underwent dewaxing, gradient alcohol hydration, and antigen retrieval utilizing the Autostainer Link 48 device from Dako. Afterwards, 3% hydrogen peroxide solution was added, followed by incubation at 37 ℃ for 15 min to inhibit peroxidase activity. Then, 100 µL of normal goat serum was dispensed dropwise, succeeded by a 15-minute incubation at 37 ℃ for blocking purposes.

Primary antibody (Fuzhou Maixin ready-to-use antibodies [CK7, Cat#MAB-0828; CK20, Cat#MAB-0834; CDX2, Cat#MAB-1056; Ki-67, Cat#MAB-0672; E-cadherin, Cat#MAB-0738; Vimentin, Cat#MAB-0735; CEA, Cat#MAB-0852; CA19-9, Cat#MAB-0778]) was introduced to the cells and kept at 37 ℃ for 1 h. The DAB staining kit (Dako, Cat#K3468) was utilized for chromogenic development, succeeded by washing the cells under flowing water for 5 min. After counterstaining with hematoxylin, the samples underwent dehydration and xylene clearing, and neutral resin was employed to seal the coverslip. Subsequently, the slides were examined under a microscope.

Whole-genome resequencing (WGS) of DPC-X3 cells

Sequencing and data analysis were conducted at OE Biotech (Shanghai OE Biotech Co., Ltd). In short, genomic DNA was extracted from the DPC-X3 sample, and after passing the electrophoresis detection, the library was constructed. After the library construction was qualified, used a sequencer for dual end sequencing. After obtaining raw sequencing data from the sequencing machine, bioinformatics analysis was performed.

Statistical analyses

All statistical analyses were performed using the SPSS 26.0 software (IBM, Armonk, NY, USA). Data were expressed as mean ± standard deviation (SD). Student’s t-tests and Analysis of Variance (ANOVA) were used for two-group and multi-group comparisons, respectively. A p-value less than 0.05 was considered statistically significant.