Pm 100

Quantitative evaluation of cell viability was performed using the Cell Proliferation Kit II (XTT) (Roche Molecular Biochemicals, Mannheim, Germany), according to the manufacturer’s protocol. In cancer cell-targeted NIR-PIT, cancer cells were seeded in 96-well plate at 3.0 × 10³ cells per well and incubated for 24 h. The medium was replaced with fresh medium containing 20 μg/mL of Pan- or Tra-IR700 and incubated for 6 h at 37 °C. In dual-targeted NIR-PIT, cancer cells and FEF3 cells were seeded in 96-well plates at a density of 3 × 10³ cells/well total and co-cultured for 24 h. To stimulate FEF3, the medium was changed to CM/cancer and co-cultured for another 72 h. The cells were treated with the following APCs: (1) no treatment (control); (2) 20 μg/mL of Pan- or Tra-IR700; (3) 20 μg/mL of FAP-IR700; (4) 20 μg/mL of Pan- or Tra-IR700 and FAP-IR700, and incubated for 6 h at 37 °C. In all wells, the medium was replaced with fresh medium after the antibody reaction. Cells were irradiated with a red light-emitting diode (LED), which emits light at a wavelength of 670–710 nm (L700-05AU 700 nm; Epitex Co, Kyoto, Japan), and a power density of 15 or 25 mW/cm², as measured with an optical power meter (PM 100, Thorlabs, Inc., NJ, USA). Cell viabilities were evaluated after incubation for 1 h at 37 °C after irradiation.

Tumor cells (TE4: 1 × 10⁴/well, OE19: 6 × 10³/well) were plated (in 96-well microplates) and stimulated with CM from CAFs (CM/CAF^TE4 or CM/CAF^OE19) or normal medium for 2 days. FEF3 cells (2 × 10³/well) were plated (in 96-well microplates) and stimulated with CM from tumor cells (CM/TE4 or CM/OE19) for 4 days. After stimulation, FAP-IR700 was added to the culture medium at 20 μg/mL and incubated for 6 h at 37 °C. After washing cells with PBS, the medium was replaced with normal medium, and NIR light was administrated to the cells with a red light-emitting diode (LED) at 20 J/cm² or the indicated intensity (L700-05AU 700 nm, Epitex Co, Kyoto, Japan) with a power density of 15 mW/cm² as measured using an optical power meter (PM 100, Thorlabs, Inc., Newton, NJ). After exposure to NIR light, cell proliferation was immediately measured by using WST-1 assays, as described above.

Cells were seeded on 35‐mm cell culture dishes and incubated for 48 hours at 37°C. The medium was replaced with fresh phenol red‐free RPMI 1640 containing TROP2‐IR700 (10 µg/mL). Cells were incubated for another 24 hours at 37°C, washed with PBS, and added fresh phenol red‐free RPMI 1640. Cells were irradiated with NIR light using a 690‐nm continuous wave laser (ML6540‐690; Modulight, Inc). Power density of 29.5 mW/cm² was measured with an optical power meter (PM 100; Thorlabs). The doses of irradiation for each dish were 0, 1, 2, 4, 8, and 16 J/cm², respectively. After irradiation, cells were collected and resuspended with PBS, followed by LIVE/DEAD® cytotoxicity assay (Life Technologies) which can detect damaged cellular membranes.