Cell culture
MKN-7 (gastric tubular adenocarcinoma) and MKN-45 (gastric adenocarcinoma) cells were obtained from the Japanese Collection of Research Bioresources Cell Bank and American Type Culture Collection, respectively. HSC44PE (gastric signet ring cell adenocarcinoma) (Yanagihara et al. 2004), MKN-7 and MKN-45 cells were cultured in RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA) medium supplemented with 10% fetal bovine serum (FBS; Life Technologies, New York, NY, USA) and 1% penicillin-streptomycin (P/S) solution (Sigma-Aldrich). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.
Fibroblast culture
Cancer associated fibroblasts (CAFs) were isolated from human gastric cancer tissue based on a previous report (Ishii T et al. 2021). CAFs were cultured in α-minimum essential medium (α-MEM; Life Technologies), supplemented with 10% FBS and 1% P/S solution, in a humidified atmosphere containing 5% CO2 at 37°C. Fibroblast culture study was approved by the Institutional Review Board of the National Cancer Center (IRB number: 2005-043 and 2018 − 309).
Fluorescence labeling of gastric cancer cell lines
Lentiviruses were produced using 293T cells transfected with pCAG-HIV, pCMV-VSV-G-RSV-Rev, and CSII-CMV-mRFP1 vector (Riken BioResource Center, Japan), using Lipofectamine 2000 reagent (Invitrogen, NY, USA). Virus-containing medium was filtered through a 0.45 µm filter, and 8 µg/mL (final concentration) of polybrene (Santa Cruz, Dallas, TX, USA) was added for target cell transduction, as previously reported (Yamazaki et al. 2018). The mRFP-positive fractions of the gastric cancer cell lines were sorted by a FACS AriaII (BD Bioscience) (Fig. 1A) and were further cultured. We confirmed that over 90% of the cultured cells were mRFP-positive (Fig. 1B).
Lifetime extension of CAFs
Transduction was performed with human telomerase reverse transcriptase (hTERT) and mutant forms of cyclin-dependent kinase 4 (CDK4R24C) in combination (hTERT/CDK4R24C). hTERT and CDK4R24C cDNAs were kindly gifted by Dr. Masutomi K (Division of Cancer Stem Cells, National Cancer Center Research Institute) and Dr. Kiyono T (Division of Carcinogenesis and Cancer Prevention, National Cancer Center Research Institute), respectively. Using the packaging constructs PCAG-HIV, pCMV-VSV-G-RSV-Rev (RIKEN BioResource Center), CSII-CMV-hTERT-IRES2-Venus, and CSII-CMV-CDK4R24C-IRES2-Venus, infection was achieved as previously described (Hashimoto et al. 2017). The venus-positive fractions of CAFs were sorted and cultured further (Fig. 1C, left and middle). We confirmed increased levels of both hTERT and CDK4 in CAFs (CAFs-hTERT/CDK4R24C) by qRT-PCR analysis (Fig. 1C, right). Primers used are listed in Supplementary Table 1.
Two-dimensional (2D) collagen invasion assay (Supplementary Fig. 1)
The 2D collagen invasion assay was performed as previously described (Miyashita et al. 2019). Briefly, CAFs and cancer cells was plated on a collagen (Cellmatrix Type Ⅰ-A; Nitta Gelatin)-coated (0.3% type I collagen gel) 96-well plates (Incucyte® Imagelock 96-well Plate: Sartorius, Germany) at a density of 1.15×10⁵ cells/well (6.5×10⁴ cancer cells + 5.0×10⁴ CAFs). After 1 h incubation, we made a wound in the cell layer using 96-well WoundMaker (Sartorius), and embedded cells in type I collagen gel. Scratched field images were captured using the IncuCyte Live-Cell Imaging System (Sartorius).
Three-dimensional (3D) collagen invasion assay
To measure the invasiveness of cancer cells and CAFs in 3-dimensional (3D) culture mimicking the human TME, we first generated 3D hybrid cancer spheroids (Supplementary Fig. 2A) (Nomura et al. 2022). We prepared a cell mixture of 0.1 × 10⁴ gastric cancer cells (HSC44PE and MKN-7) and 0.9 × 10⁴ CAFs with extended lifespan. 1.0 × 10⁴ cells from this mixture was seeded onto 96-well low attachment plates (Sumitomo Bakelite, Tokyo, Japan) and incubated overnight at 37°C. The medium was removed, and the cell aggregates were embedded in 50 µL collagen (Cellmatrix Type I-A; Nitta Gelatin, Oosaka, Japan) and incubated for 30 min at 37°C. After confirmation of polymerization of collagen, 100 µL of mixed medium (RPMI-1640: α-MEM = 1:1) was added. Time-lapse imaging analysis was performed using Incucyte (Sartorius) during incubation, and the medium was changed daily.
Evaluation of the number of cancer cells and CAFs invading the collagen gel discontinuously from the main spheroid
This evaluation was performed according to a previously described protocol (Nomura et al. 2022). We encircled the edge of the main spheroid on day 3 of culture on the bright-field image (orange solid line in Supplementary Fig. 2B, left), then, calculated the average distance between the center of gravity and edge of the cell cluster (the average of the center of gravity distance). We drew a circle with the center of gravity of the cell cluster as the center and a radius of four times the average of the center of gravity distance (orange dotted circle in Supplementary Fig. 2B, left). The circle on the bright-field image was reflected in the fluorescent image (orange dotted circle in Supplementary Fig. 2B, right). The number of cancer cells and CAFs invading the collagen gel discontinuously from the main spheroid in the circle were measured by WinROOF 2021 image analysis software (MITANI Corporation, Fukui, Japan).
Overexpression and knockdown of podoplanin of CAFs
To generate podoplanin or mutant podoplanin-overexpressing CAFs, lentiviruses were produced using 293T cells transfected with PCAG-HIV, pCMV-VSV-G-RSV-Rev, and either wild-type podoplanin (PDPN-WT) (kindly provided by Dr. N. Fujita, Japanese Foundation for Cancer Research), or mutant podoplanin lacking the cytoplasmic region (PDPN-Del.IC) (Supplementary Fig. 3A) (Martin-Villar et al. 2006, Ito et al. 2012).
To generate podoplanin-knockdown CAFs, two different short-hairpin RNA (shRNA) expression constructs, sh‐podoplanin1 and sh‐podoplanin 3, were created as previously described (Hoshino et al. 2011). An shRNA specific for the luciferase gene (sh‐luciferase) was used as a control (Supplementary Fig. 3B).
Podoplanin expression analysis by flow cytometry
After incubation with an APC-conjugated anti-podoplanin antibody (clone NC-08, BioLegend, CA, USA), FACS analysis was performed using FACS Accuri C6 Plus (BD Biosciences)(Supplementary Fig. 3C and D).
Real-time reverse-transcriptase polymerase chain reaction (RT-PCR)
Total RNA was purified using the Nucleo Spin RNA Plus Kit (TaKaRa Bio, Osaka, Japan), and cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara Bio). RT-PCR was carried out on a Smart Cycler System (Takara Bio) with SYBR Premix Ex Taq (Takara Bio) and RT-PCR primers. Information regarding the primers used is provided in Supplementary Table S1. Genes with CT values greater than 29 cycles (vimentin and ZEB2) were excluded from the analysis.
Statistical analysis
The significance of the differences between the two groups was evaluated using the Welch t-test. P-values were determined using two-sided analyses, and the statistical significance level was set at P < 0.05.