Single-Cell Analysis of Different Stages of Oral Cancer Carcinogenesis in a Mouse Model

Oral carcinogenesis involves the progression of the normal mucosa into potentially malignant disorders and finally into cancer. Tumors are heterogeneous, with different clusters of cells expressing different genes and exhibiting different behaviors. 4-nitroquinoline 1-oxide (4-NQO) and arecoline were used to induce oral cancer in mice, and the main factors for gene expression influencing carcinogenesis were identified through single-cell RNA sequencing analysis. Male C57BL/6J mice were divided into two groups: a control group (receiving normal drinking water) and treatment group (receiving drinking water containing 4-NQO (200 mg/L) and arecoline (500 mg/L)) to induce the malignant development of oral cancer. Mice were sacrificed at 8, 16, 20, and 29 weeks. Except for mice sacrificed at 8 weeks, all mice were treated for 16 weeks and then either sacrificed or given normal drinking water for the remaining weeks. Tongue lesions were excised, and all cells obtained from mice in the 29- and 16-week treatment groups were clustered into 17 groups by using the Louvain algorithm. Cells in subtypes 7 (stem cells) and 9 (keratinocytes) were analyzed through gene set enrichment analysis. Results indicated that their genes were associated with the MYC_targets_v1 pathway, and this finding was confirmed by the presence of cisplatin-resistant nasopharyngeal carcinoma cell lines. These cell subtype biomarkers can be applied for the detection of patients with precancerous lesions, the identification of high-risk populations, and as a treatment target.


Supplemental
. Effect of NPM1 expression on the survival of patients with head and neck cancer. Data from The Cancer Genome Atlas database was analyzed to determine the relationship between NPM1 mRNA expression and the survival rate of patients with head and neck cancer. The number of patient specimens with a high expression of NPM1 mRNA was 212. The number of patient specimens with low expression of NPM1 mRNA was 287. A high expression of NPM1 was correlated with a lower likelihood of survival. 14,16 Protocol for Generating Single-Cell 3ʹ Gene Expression Libraries from Single Cells.
First, 50% glycerol (JT Baker, 2136-01) was added to the Chromium Next GEM Chip G (Chip G; PN-1000120). Next, an appropriate amount of nuclease-free water (Thermo Fisher Scientific, R0581) and the corresponding amount of the cell suspension were added to the master mix [18.8 μl of RT Reagent B (PN2000165), 2.4 μl of Template Switch Oligo (PN3000228), 2.0 μl of Reducing Agent B (PN2000087) and 8.7 μl of RT Enzyme C (PN2000085)] to achieve a total volume of 75 μl. Subsequently, 70 μl of the combined master mix and cell suspension was added to the first row of Chip G (PN2000177). After single-cell 3' v3.1 gel beads (PN2000164) had been shaken for 30 s, 50 μl was extracted and added to the second row of Chip G, and 45 μl of partitioning oil (PN2000190) was added to the third row of Chip G. The 10X Gasket (PN370017) was then connected to the Chromium Controller and activated to coat the oil droplets. After processing, the gasket was removed and the chip holder was opened to form a 45° well. The volume of the first and second rows of Chip G was assessed, and then, 100 μl of gel beads in emulsion (GEMs) were transferred from the third row of Chip G to 8-row polymerase chain reaction (PCR) tubes. The GEMs then underwent incubation at a temperature of 53°C for 45 min and 85°C for 5 min before being maintained at 4°C.
To form Dynabeads, 125 μl of Recovery Agent (PN220016) was added to each sample, and the samples were kept at room temperature for 2 min. Next, 125 μl of the combined Recovery Agent and partitioning oil was extracted from the bottom of the tube, and a Dynabeads cleanup mix was prepared [182 μl of Cleanup Buffer (PN2000088), 8 μl of Dynabeads MyOne SILANE (PN2000048), 5 μl of Reducing Agent B (PN2000087), and 5 μl of nuclease-free water (Thermo Fisher Scientific, R0581)]. Each sample was supplemented with 200 μl of the cleanup mix and maintained at room temperature for 10 min. The samples were then placed in the 10X Magnetic Separator (PN120250) in the high position (10X Magnetic Separator-High) until the liquid was clear and then removed. Next, 300 μl of 80% alcohol was added to each sample. After 30 s, the alcohol was removed and 200 μl of Post Library Construction QC 50 min 80% alcohol was added to each sample. After 30 s, the alcohol was removed again, and then, the sample was removed from the magnetic holder and centrifuged and placed in the 10X Magnetic Separator in the low position (10X Magnetic Separator-Low) to remove alcohol and air dry for 1 min. The samples were removed from the magnetic base, immediately supplemented with 35.5 μl of Elution Solution I [98 μl of elastic buffer (EB) (Qiagen, 19086), 1 μl of 10% Tween 20 (Sigma, P9416-50ML) and 1 μl of Reducing Agent B (PN2000087)], and then maintained at room temperature for 2 min. They were then returned to the magnetic base in the 10X Magnetic Separator-Low until the liquid was clear, and 35 μl of the samples were transferred to a new 8-row PCR tube. After preparation of a cDNA amplification mix [50 μl of Amp Mix (PN2000047) and 15 μl of cDNA primers (PN2000089)], 35 μl of it was added to each sample (35 μl). A reaction was induced as follows: the temperature was set at 98°C for 3 min, 98°C for 15 sec, 63°C for 20 sec and 72°C for 1 min for 1 cycle and then 63°C for 20 sec and 72°C for 1 min for 11 cycles before being set at 72°C for 1 min and then maintained at 4°C. cDNA Cleanup-SPRIselect with shaking the SPRIselect reagent (Beckman Coulter, B23318); 60 μl (0.6X) was added to each sample, and then, the samples were maintained at room temperature for 5 min before being placed in the 10X Magnetic Separator-High until the liquid was clear. The supernatant was removed, and 200 μl of 80% alcohol was added to each sample. After 30 s, the alcohol was removed, and 200 μl of 80% alcohol was added to each sample. After 30 s, the alcohol was again removed. The sample was then removed from the magnetic stand, centrifuged and placed in the 10X Magnetic Separator-Low to remove the remaining alcohol and air dry for 2 min. The sample was then removed from the magnetic base, supplemented with 40.5 μl of EB (Qiagen,19086) and maintained at room temperature for 2 min. Finally, 40 μl of the sample was transferred to a new 8-row PCR tube for measurement of the cDNA concentration. Fragmentation, end repair, and A-tailing were performed with a fragmentation mix [5 μl of Fragmentation Buffer (PN2000091) and 10 μl of Fragmentation Enzyme (PN2000090)] on ice. After 10 μl of cDNA was placed in a new 8-row PCR tube, 25 μl of EB (Qiagen, 19086) was added to each sample, and then, 15 μl of the fragmentation mix was added to each sample. The sample was placed in a thermal cycler under the following conditions: precool block at 4°C, fragmentation at 32°C for 5 min, end repair and A-tailing at 65°C for 30 min and finally maintenance at 4°C. Each sample was supplemented with 30 μl (0.6X) of shaken SPRIselect reagent and maintained at room temperature for 5 min before being placed in the 10X Magnetic Separator-High until the liquid was clear. After 75 μl of the supernatant had been placed in a new 8-row PCR tube, each sample was supplemented with 10 μl (0.8X) of shaken SPRIselect reagent (Beckman Coulter, B23318), maintained at room temperature for 5 min and then placed in the 10X Magnetic Separator-High until the liquid was clear. Next, 80 μl of the supernatant was removed, and 125 μl of 80% alcohol was added to each sample. After 30 s, the alcohol was removed and replaced with another 125 μl of 80% alcohol. After 30 s, the alcohol was again removed; samples were removed from the magnetic stand and then centrifuged and placed in the 10X Magnetic Separator-Low until the liquid was clear. The remaining alcohol was then removed, and samples were removed from the magnetic stand and centrifuged, supplemented with 50.5 μl of EB (Qiagen,19086) and maintained at room temperature for 2 min. Next, samples were placed in the 10X Magnetic Separator-Low until the liquid was clear, and 50 μl of the samples were placed in a new 8-row PCR tube.
Adaptor ligation was performed using an adaptor ligation mix [20 μl of Ligation Buffer (PN2000092), 10 μl of DNA Ligase (PN220110) and 20 μl of Adaptor Oligos (PN2000094)]. After the addition of 50 μl of the adaptor ligation mix to each 50-μl sample, a temperature reaction was induced as follows: the temperature was set at 20°C 15 min and then maintained at 4°C. Each sample was supplemented with 80 μl (0.8X) of shaken SPRIselect reagent and maintained at room temperature for 5 min before being placed in the 10X Magnetic Separator-High until the liquid was clear. The supernatant was removed, and 200 μl of 80% alcohol was added to each sample. After 30 s, the alcohol was removed and replaced with 200 μl of 80% alcohol. After 30 s, the alcohol was again removed. Samples were taken out of the magnetic holder, centrifuged and placed in the 10X Magnetic Separator-Low. The remaining alcohol was removed, and the samples were air dried. After 2 min, the samples were removed from the magnetic holder and centrifuged. After being supplemented with 30.5 μl of EB, the samples were maintained at room temperature for 2 min and then placed in the 10X Magnetic Separator-Low until the liquid was clear. Next, 30 μl of the samples were placed in new 8-row PCR tubes.
A sample index PCR mix [50 μl of Amp Mix (PN2000047) and 10 μl of SI Primer (PN2000095)] (Supplemental Table 2) was prepared and used to perform sample index PCR. Each 30-μl sample was supplemented with 60 μl of the sample index PCR mix and 10 μl of the individual Chromiμm i7 Sample Index and then subjected to a temperature reaction under the following conditions: temperature set at 98°C for 45 sec, 98°C for 20 sec, 54°C for 30 sec, 72°C for 20 sec and 98°C for 20 sec for 16 cycles and then set at 72°C for 1 min before being maintained at 4°C.
Post Sample Index PCR Double Sided Size Selection-SPRIselect Each sample was supplemented with 60 μl (0.6X) of shaken SPRIselect reagent and maintained at room temperature for 5 min. The samples were then placed in the 10X Magnetic Separator-High until the liquid was clear, and 150 μl of the supernatant was transferred to a new 8-row PCR tube. Each sample was supplemented with 20 μl of (0.8X) shaken SPRIselect reagent and maintained at room temperature for 5 min before being placed in the 10X Magnetic Separator-High until the liquid was clear; 165 μl of the supernatant was then removed, and 200 μl of 80% alcohol was added to each sample. After 30 s, the alcohol was removed, replaced with 200 μl of 80% alcohol and then removed again after 30 s. The samples were removed from the magnetic base and centrifuged, supplemented with 35.5 μl of EB and maintained at room temperature for 2 min before being placed in the 10X Magnetic Separator-Low until the liquid was clear. Finally, 35 μl of the samples was transferred to a new 8-row PCR tube for assessment of their concentration after establishment of a gene expression library.

NPM1-RTR TTCACTGGCGCTTTTTCTTCA
RNA extraction: Mouse tongues were placed in a 1.5-ml microcentrifuge tube containing 1 ml of TRI reagent (Zymo, R2050-1-200). The tissues were cut with scissors, ground using a micropestle and then placed on ice for 5 min. After the addition of 200 μl of chloroform (PanReac AppliChem, A3691, 1000), it was shaken upside down to achieve an even distribution, placed on ice for 5 min and then centrifuged at 12 000 rpm at a temperature of 4°C for 15 min. The supernatant was transferred to a new 1.5-ml microcentrifuge tube and supplemented with an equal volume of isopropanol (PanReac AppliChem, A3928, 0500PE). The mixture was shaken upside down and mixed well. After resting on ice for 10 min, it was then centrifuged at 12 000 rpm at a temperature of 4°C for 10 min to precipitate RNA. The supernatant was removed, leaving behind a white RNA pellet. After the addition of 1 ml of 70% alcohol (ECHO, 484000011107A-69EC) and a mixture of 5 diethyl pyrocarbonate (VWR, 71003-388) and water, the RNA pellet was washed and centrifuged at 7500 rpm at a temperature of 4°C for 5 min to remove the supernatant. The microcentrifuge tube was left open on a sterile operating table to air dry. After the alcohol had completely evaporated, 30 μl of diethyl pyrocarbonate water was added; RNA was purified, and its quality and concentration were assessed using a NanoDrop spectrophotometer before it was finally stored in a refrigerator at −80°C.
Reverse-transcription PCR (RT-PCR): in a PCR tube, 4 μl of 5X cDNA Synthesis Mix (PCR Biosystems, PB30.31-10) was combined with 1 μl of ΜltraScript 2.0 for cDNA synthesis (PCR Biosystems, PB30.31-10) and 2 μg of RNA with enough nuclease-free water (Thermo Fisher Scientific, R0581) to yield a total volume of 20 μl. The samples were placed in a PCR machine for a cDNA synthesis reaction with the temperature set at 55°C for 30 min and 95°C for 10 min before being maintained at 12°C. Subsequently, the cDNA underwent RT-PCR analysis in 8-row PCR tubes containing 10 μl of 2X qPCRBIO SyGreen Blue Mix (PCR Biosystems, PB20.16-01), 0.8 μl (10 μM) of forward primer, 0.8 μl (10 μM) of reverse primer, 1 μl of cDNA and 7.4 μl of nuclease-free water (Thermo Fisher Scientific, R0581) (Supplemental Table 3). The aforementioned q-PCR reaction mixture was placed in a real-time PCR apparatus, and real-time quantification was performed under the following conditions: temperature set at 95°C for 2 min, 95°C for 5 sec, 65°C for 30 sec and 95°C for 5 sec for 40 cycles and then 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec to melt the curve.