This prospective observational study was conducted on patients with laryngeal or hypopharyngeal cancer who received treatment at the Beijing Friendship Hospital, Capital Medical University, between Sep 2018 and Nov 2020. This study was approved by the human research ethics committee of Beijing Friendship Hospital, Capital Medical University (Batch number: 2018-P2-198-01). The study was conducted in accordance with the Declaration of Helsinki and Chinese Law provisions and adhered to Good Clinical Practice guidelines. According to the Response Evaluation Criteria in Solid Tumors (RECIST) criteria, patients' clinical responses were assessed by experienced radiologists and physicians. Written informed consent was obtained from the patients and/or their authorized representatives. Inclusion criteria were as follows: primary or recurrent histologically confirmed LNSCC patients; aged above 18 years old; and fresh tissue available through either biopsy or surgical resection of the primary tumor site. Patients with cognitive impairment, mental health disorders, poor compliance, or allergic to chemotherapeutic agents were excluded. The CR cell viability and the clinical response were evaluated by technicians and clinicians double-blinded, respectively. All human tissue samples were obtained from diagnostic biopsies or therapeutic resections. Prior to surgery or biopsy, each patient signed written informed consent, allowing the excess tissue to be used for research studies.
Upon receipt of fresh tissue, the tissue sample was into three parts for cryopreservation, fixation, and digestion for primary cell derivation. For histology, a piece was removed and immediately fixed in formalin. The fixed tissue was processed and embedded in paraffin as described previously (Driehuis et al. 2019). For primary cell culture, tissue samples were minced and incubated at 37°C in 0.125% Trypsin (Sigma, catalog no. T1426) with high glucose DMEM (Life Technologies, catalog no. 12430-054) until digested. The tissue suspension was frequently agitated and monitored for up to 60 minutes. The suspension was strained through a 100 μm filter, centrifuged at 300 g and lysed with blood cell lysis buffer for 5 minutes. After washing twice with PBS, the resulting pellet was resuspended in Complete F medium and seeded in the CR culture system.
Mouse embryonic fibroblast cell line 3T3-J2 (RRID: CVCL_W667; purchased from Otwo Biotech, Shenzhen, China) was cultured in complete DMEM with high glucose supplemented with 10% (v/v) FBS (Life Technologies) and 100 IU/ml penicillin, and 100 mg/ml streptomycin. In the CR system, 3T3-J2 were mitotically inactivated either by irradiation or by mitomycin C-treatment (2.5 h, 4mg/ml final concentration, Sigma-Aldrich). Primary LHSCC cells were cultured in Complete F medium (Table 1) at 37℃ in a 5% CO2 humidified incubator. The medium was renewed every two days. The cell numbers of every passage were checked by a cell counter plate.
CRC derived organoid culture
The primary cells were collected from the CR culture system when the cells reached 70-80% confluence; the feeder cells were removed following trypsinization for 1-minute. The cells at an indicated count were mixed with ice-cold Matrigel and then were loaded in the center of the well of the culture plate. After polymerization by incubating at 37°C for 30 min, a prewarmed organoid medium was added to the plate. The medium was changed every alternate day. The main organoid culture methods and composition of the organoid medium were as previously described (Driehuis et al. 2019).
For histological examination, excised patient tissues or heterotransplanted tumors from nude mice were fixed overnight in 4% formaldehyde, dehydrated, and embedded in paraffin and followed with deparaffinization and standard hematoxylin & eosin (H&E) staining. Images were acquired on an inverted microscope (TH4-200, Olympus optical Co-Ltd, Tokyo, Japan). Cells slides were used for indirect immunofluorescence. Briefly, cells were seeded into a 24-well plate with round cover slides (a diameter of 1 cm) in the well. After reaching 60‑80% confluence, cell slides were fixed in paraformaldehyde for 15 minutes and acetone successively. After fixation, heat-induced antigen retrieval was performed using either citric acid solution in a microwave. The slides were then permeabilized with 0.1% Triton X-100 (Sigma) for 10 minutes and blocked with 1% (w/v) bovine serum albumin (BSA) for 1 hour at room temperature. Following incubation overnight at 4˚C with a primary antibody (anti-pan-keratin, proteintech, 26411-1-AP; anti-CD44, proteintech, 15675-1-AP), the cells were washed with PBS and incubated with secondary antibodies (Invitrogen) at room temperature for 1 hour. The cells were then incubated with indicated additional stains (DAPI, life technologies D1306) for 5 minutes at room temperature. The samples were analyzed using a confocal microscope (LSM880; Carl Zeiss, Germany). For immunofluorescence staining of the organoids, the whole mount staining method was performed as described previously (Hu et al. 2018). Primary antibodies used for organoids included anti-KRT5 (Santa Cruz Biotechnology; sc-32721) and anti-p63 (Abcam; ab124762). Secondary antibodies included goat anti-rabbit IgG (Alexa FluorR 594; Invitrogen; CA11012s) and goat anti-mouse IgG (Alexa FluorR 488; Invitrogen, CA11001).
Short tandem repeat(STR) Amplification Genomic DNA was extracted from cells at different passages using a DNA extraction kit (AP-MN-MS-GDNA-50; Axygen, CA, USA). A total of 21 short tandem repeat (STR) loci, including D5S818, D13S317, D7S820, D16S539, VWA, TH01, TPOX, CSF1PO, D12S391, FGA, D2S1338, D21S11, D18S51, D8S1179, D3S1358, D6S1043, PENTAE, D19S433, PENTAD, D1S1656, and Amelogenin were amplified. The fragments were amplified using PCR and separated by capillary electrophoresis using Applied Biosystems® (ABI) 3730xl Genetic Analyzer (Applied Biosystems, Foster City, CA, United States), and data were automatically analyzed with the GeneMapper Software v3.2 (Applied Biosystems, Foster City, CA, United States).
In vitro drug screening
The cells at a density of 2000 cells/well were seeded into 96-well culture plates. After 24 h, the cells were treated with different concentrations of the drugs. Control cultures received an equal amount of DMSO (0.01 to 0.1%). 72 h after treatment. The number of cell colonies was estimated by using the CCK8 assay with a slight modification. Specifically, cells treated with different concentrations of the drugs were washed twice with PBS, and then CCK8 solution (100 μL, 10 mg/mL) (Dojindo) was added into each well at 37°C for 2 hours. Following incubation, the absorbance (optical density) was measured at 450 nm using a microplate reader (Thermo Scientific Multiskan FC). The values were normalized to the vehicle (100%) and baseline control (0%). For each test, if the calculated cell viability was higher than 70% or lower than 30%, an additional screen was performed for that particular drug with an adjusted dose of the drug for the cell line. Z factor score was used as the parameter for screen quality assessment using the following equation:
Drug screens with a Z score of less than 0.3 were not used and repeated. Kill curves were generated using GraphPad® PRISM version 9.0 (Graph Pad Software, Inc., La Jolla, CA, USA), and the curves were fitted using the log (Inhibitor) vs. response -- Variable slope (four parameters)." The half-maximal inhibitory concentration (IC50), which is an essential indicator for drug sensitivity assay, was calculated by non-linear regression of the log of concentration versus the percentage of survival, implemented in GraphPad.
The cells at a density of 2000 cells/well were seeded in 96-well culture plates. After 24 h, cells were irradiated. The γ-ray irradiation was performed from a cobalt-60 source at a dose rate of 0.59 Gy/min at room temperature. A separate plate was used for each radiation dose. Plates were sealed air-tight and irradiated with a single fraction of 2, 4, 6, 8, 10 Gy. After radiation, the medium was changed. Six days later, cell viability was measured using CCK8 assay. Kill curves were graphed following the method described above.
Colony formation assay
The cells were seeded into 6-well culture plates immediately after exposure to 0 Gy and 4 Gy of γ-ray irradiation. After 7 days of incubation, the colonies were fixed in paraformaldehyde and stained with crystal violet solution. Colonies containing more than 50 cells were counted; the relative colony-forming efficiency was calculated and plotted.
Whole-exome sequencing and bioinformatics analysis
Whole-exome sequencing data were mapped against human reference genome GRCh37, and variants were called using the IAP pipeline (https://github.com/UMCUGenetics/IAP). To identify somatic genomic variants associated with laryngeal and hypopharyngeal cancer, WES was conducted on 3 paired normal/tumor CR cell lines following the protocol previously described (Li et al. 2019). We filtered out somatic single-nucleotide variations (SNVs), somatic InDels and copy number variants of tumor cells with evidence in their corresponding normal controls. The following genes were detected as cancer-associated genes and screened for all detected somatic genomic variants including, ABL1, ADAMTS12, AKT1, ALK, APC, ATM, ATR, BRAF, CASP8, CCND1, CDH1, CDH12, CDKN2A, COL1A2, COL22A1, CSF1R, CSMD3, CTNNB1, DICER1, EGFR, ERBB2, ERBB4, ESR1, EZH2, FAT1, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, GRM8, HNF1A, HRAS, IDH1, IDH2, IRF6, JAK2, JAK3, KDR, KIT, KRAS, MDM2, MED1, MET, MLH1, MLL2, MPL, NAV3, NOTCH1, NOTCH2, NOTCH3, NPM1, NRAS, OR4C15, PDGFRA, PIK3CA, PKHD1L1, PRB4, PRDM9, PTEN, PTPN11, RB1, RET, RIMS2, RIPK4, SI, SLC2A13, SMAD4, SMARCB1, SMO, SRC, STK11, SYNE1, SYNE2, TP53, TP63, USH2A, and VHL.
Heterotransplantation in nude mice and in vivo treatment studies
Animal experiments were approved by the Institutional Animal Care and Use Committee of Beijing Institute of Biotechnology (ethics approval code IACUC-DWZX-2019-517, approved in May 2019). Four to five-week-old female BALB/C-nude mice were procured from SPF (Beijing) Biotechnology Co., Ltd. Mice were cared for in accordance with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. The mice were housed under specific pathogen-free conditions, at a temperature of 24 °C with a relative humidity of 50% – 60%, under a 12-h-light/12-h-dark schedule. Animals were provided ad libitum access to standard rodent food and tap water. Mice were subcutaneously injected with 5 × 106 of primary cancer cells in the right flank (0.2 mL cell suspension per mouse). Six weeks after tumor cell inoculation, tumors were removed, and tumor tissues were fixed in 4% formaldehyde, embedded in paraffin, and subjected to an H&E staining procedure.
For in vivo treatment assay, tumor-bearing mice were established following the above-mentioned method. When tumors reached approximately 200 mm3, the mice were randomized into three groups (n ≥3/group) according to tumor volumes and body weights. The treatments included vehicle control, 4 mg/kg cisplatin by intraperitoneal (i.p.) injection twice a week, 5Fu (100 mg/kg/week; i.p.), and paclitaxel (30 mg/kg/week; i.p.). Tumor volumes were measured using an electronic Vernier caliper and calculated with the formula . On the 28th day after the first treatments, mice were weighed and then euthanized with CO2 asphyxia. Subsequently, the tumors were harvested, weighed, and photographed.
Statistical analysis was performed using GraphPad Prism 9.0 (GraphPad Software, Inc). All in vitro experiments were performed in triplicate and repeated three times. Data were expressed as mean ± standard deviation (SD). A two-tailed Student t-test was used to analyze differences between two groups. One-way analysis of variance (ANOVA) with Bonferroni correction was used to analyze multiple groups. The mean ± standard deviation was presented in all graphs, and raw data points were indicated. P values of <0.05 were considered statistically significant.