Experimental lung metastasis models

To investigate the metastatic colonization ability of tumor cells in Atgl^ΔMCs or Hilpda^ΔMCs mice, the mice were orthotopically implanted with tumor cells (2 × 10⁵ cells for each mouse). On day 10, the tumor-bearing mice were intravenously (IV) injected with luciferase-labeled tumor cells (1 × 10⁶ cells for each mouse). Lung tissues were harvested on day 24 for ex vivo bioluminescence imaging (BLI).

To investigate the metastasis-initiating potentials of lung MCs-educated tumor cells, the luciferase labelled AT3 (AT3-Luc) cells were cultured with conditioned medium from WT or Atgl^ΔMCs lung CD140a⁺ MCs. Then the tumor cells were digested, washed, and IV injected into NSG mice (1 × 10⁵ cells for each mouse). On day 7, the mice were anesthetized and intraperitoneally (IP) injected with D-luciferin (150 mg/kg) for the determination of in vivo BLI signal.

To investigate chemoresistant capacities of tumor cells after implanted into WT or Atgl^ΔMCs mice, naïve mice were first IV injected with AT3-Luc cells (1 × 10⁶ cells for each mouse) and 7 days later, the established lung metastasis was examined by in vivo BLI before treatment. Then the mice were IV injected with DOX (5 mg/kg). 2 days after treatment, the lung metastasis was examined with in vivo BLI to determine the survival of metastatic tumor cells.

To investigate the effect of lipid-laden NK cells in controlling lung metastasis, NSG mice were first IV injected with AT3-Luc cells (1 × 10⁵ cells for each mouse), and 24 hours later, the mice were IV injected with primary lung NK cells (5 × 10⁵ cells for each mouse) isolated from naïve WT or Atgl^ΔMCs mice. On day 8, the mice were anesthetized and IP injected with D-luciferin (150 mg/kg) for the determination of in vivo BLI signal. All mice received a maintenance dose of 500 ng of IL-15 daily (IP, from day 1 to day 7) to sustain NK cell survival.

To investigate the therapeutic efficiency of anti-IL-1β in controlling lung metastasis, the mice were orthotopically implanted with AT3 cells (2 × 10⁵ cells for each mouse). On day 8, mice were IP injected with anti-IL-1β or isotype control (2.5 mg/kg) every 3 days for total 6 times. On day 14, the mice were IV injected with AT3-Luc cells (1 × 10⁶ cells for each mouse), and at the end point (day 25), the lung tissues were harvested for ex vivo BLI.

To investigate the combined therapeutic efficacy of anti-IL-1β and adoptive therapy of NK cells in controlling lung metastatic colonization by AT3-Luc cells, NSG mice were first orthotopically implanted with AT3 tumor cells (2 × 10⁵ cells for each mouse). On day 15, the AT3-Luc cells were IV injected (5 × 10⁴ cells for each mouse) into the mice. From day 17 to day 26, the mice were IP injected with anti-IL-1β or isotype control every 3 days for 4 times. On day 20 and day 26, mice were IV injected with NK cells (1 × 10⁶ cells for each mouse). At the end point (day 28), mice were euthanized, and the lung tissues were rapidly harvested for ex vivo BLI. All mice received a maintenance dose of 500 ng of IL-15 daily (IP, from day 20 to day 27) to sustain NK cell survival.

To investigate the combined therapeutic efficacy of anti-IL-1β and adoptive therapy of NK-92 cell lines in controlling lung metastatic colonization by MDA-4175-Luc cells, NSG mice were first orthotopically implanted with MDA-4175 tumor cells (2 × 10⁵ cells for each mouse). On day 25, the luciferase labelled MDA-4175-Luc cells were IV injected (5 × 10⁴ cells for each mouse) into the mice. From day 28 to day 37, the mice were IP injected with anti-IL-1β or isotype control every 3 days for 4 times. On day 31 and day 37, mice were IV injected with NK-92 cells (2 × 10⁶ cells for each mouse). At the end point (day 40), mice were euthanized, and the lung tissues were rapidly harvested for ex vivo BLI.

To investigate the effect of LY294002 in controlling lung metastasis, the mice were orthotopically implanted with AT3 cells (2 × 10⁵ cells for each mouse). On day 10, mice were IP injected with vehicle or LY294002 (10 mg/kg) every 2 days for total 8 times. On day 14, the mice were IV injected with AT3-Luc cells (1 × 10⁶ cells for each mouse), and at the end point (day 25), the lung tissues were harvested for ex vivo BLI.

To investigate the effect of anti-IL-1β in controlling lung metastasis under NK cell deficiency, the mice were orthotopically implanted with AT3 cells (2 × 10⁵ cells for each mouse). On day 8 mice were IP injected with anti-IL-1β (2.5 mg/kg), anti-NK1.1 (1.25 mg/kg) or isotype control every 3 days for total 6 times. On day 14, the mice were IV injected with AT3-Luc cells (5 × 10⁵ cells for each mouse), and at the end point (day 25), the lung tissues were harvested for ex vivo BLI.