Phase contrast light microscope

Methods for the sample preparation and 3′ tag‐based scRNA‐seq of eight tumour tissue samples have been previously reported.¹⁴ For scRNA‐seq in seven MPE samples, the cells were resuspended gently with 1 ml phosphate‐buffered saline (PBS), and the contaminated red blood cells were further removed using RBC lysis buffer (Roche, Cat. No. #11814389001). The cells were centrifuged and washed twice with cold PBS. After staining with trypan blue (Bio‐RAD, Cat. No.#1450013), the cell cellular viability was checked under a phase contrast light microscope (Nikon, Japan). Single‐cell suspension with a concentration of 800–1200 cells/μl (90–95% viability) was loaded onto a 10× Chromium a Chip to capture a total of 8000–10 000 cells for each sample. After mixing with the barcoded gel beads on a Chromium Controller (10×Genomics), 5′‐tag‐based reverse transcription reaction was performed. After the droplets broke, the barcoded‐cDNA was purified using DynaBeads, followed by cDNA amplification for 14 cycles. After partial cDNA fragmentation and splicing, mRNA sequencing libraries of scRNA‐seq suitable for the Illumina sequencing platform were constructed. Part of the individual 5′‐tag‐based cDNA library product from MPE samples was used as templates for the construction of T cell receptor (TCR) joining (VDJ) sequencing library following the manufacturer's instructions (10×Genomics).

All tissues were fixed overnight in formalin solution, dehydrated in ethanol, embedded in paraffin and sectioned at 5 mm. To remove the paraffin, specimens were treated with xylene and ethanol. Slides were blocked with 5% normal goat serum and incubated overnight at 4°C with anti-YPEL3 (Proteintech). After washing with PBS, slides were incubated with goat anti-rabbit horseradish peroxidase for 30 min at room temperature. Immunohistochemical (IHC) reactions were detected using the DAB kit. Slides were examined under a phase-contrast light microscope (Nikon).

Cell cultures from each animal were analyzed every week with a phase-contrast light microscope (Nikon) and computer program for image capturing (NIS-Elements) by placing the 12-well plates under the microscope. This was done to ensure that the cells developed properly and formed adherent stromal layers. Moreover, the cultures were checked to ensure the absence of contamination, which is seen as small unadhered particles in the medium.

Fresh tumor lesions were stored in GEXSCOPETM tissue preservation solution (Singleron Bio Com, Nanjing, China) and processed on ice after the surgery within 30 mins. The specimens were washed with Hanks Balanced Salt Solution (HBSS) three times and minced into 1–2 mm pieces. Then, the tissue pieces were digested with 2 mL of GEXSCOPETM tissue dissociation solution (Singleron) at 37 °C for 15 min with sustained agitation. After digestion, the samples were filtered through 40-µm sterile strainers and centrifuged at 800 × g for 5 min. Subsequently, the supernatants were discarded, and the cell pellets were suspended in 1 mL phosphate-buffered saline (PBS; HyClone, United States). To remove red blood cells, 2 mL of GEXSCOPETM red blood cell lysis buffer (Singleron) was added, and cells were incubated at 25 °C for 10 min. The solution was then centrifuged at 500 × g for 5 min and resuspended in PBS. The samples were stained with trypan blue (Sigma, United States) and the cellular viability was evaluated under the phase contrast light microscope (Nikon, Japan).