In vivo efficacy study

Preparation of animal tumor model of NSCLC

1. Prepare a suspension of 344SQ-green fluorescent protein (GFP)/fLuc cells in 50% (v/v) Hanks’ Balanced Salt Solution and 50% Matrigel
2. Anesthetize the mice with Ketamine (80 mg/kg) + Xylazine (8 mg/kg) + Acepromazine (1 mg/kg) and lay it in right lateral decubitus position
3. Clean the skin surface with alcohol swab and make an incision between ribs 10 and 11 by placing the blade parallel to the rib cage
4. Transfer the cell suspension to a 1-mL tuberculin syringe and inject 50uL of 2.5 x 10³ - 5 x 10³ cells into the left lung parenchyma in the lateral dorsal axillary line
5. Close the incision using wound closure clips and place the animal in the left lateral decubitus position until recovery

Note: Day 1 is defined as the day of tumor inoculation 

Bioluminescence imaging

1. Inject 150 mg/kg of 15 mg/mL (in 1x DPBS) D-luciferin solution intraperitoneally and start imaging after 15 minutes
2. Anesthetize the mice by placing them in an inhalation induction chamber comprising 2-3% isoflurane in oxygen.
3. Place the anesthetized animals in the sample stage of the imaging chamber of IVIS lumina optical imaging system in supine position 
   Note: Bioluminescence imaging (BLI) is done once prior to the commencement of the treatments to obtain baseline bioluminescence to confirm the presence of tumor. Following the start of treatments (Day 8), BLI is performed once every week to monitor tumor growth.

Chemotherapy in conjunction with chemosensitizers

1. Randomize mice into 4 groups of 10 mice each
2. Administer a total of 4 doses (on days 8, 12, 15 and 19) of either of the formulations intravenously (unless indicated otherwise):
   1. Normal saline
   2. C₆CP/AZD7762 PM (POx micelles coloaded with C₆CP and AZD7762 at a weight ratio of 10/20, yielding a final dose of 10 mg/kg of C₆CP and 20 mg/kg of AZD7762)
   3. C₆CP/VE-822 PM (POx micelles coloaded with C₆CP and VE-822 at a weight ratio of 10/10, yielding a final dose of 10 mg/kg of C₆CP and 10 mg/kg of VE-822)
   4. anti–PD-1 antibody (250 μg per mouse; administered intraperitoneally)

Immunotherapy alone and in combination with immune checkpoint blockade

1. Randomize mice into 4 groups of 13 mice each
2. Administer a total of 4 doses (on days 8, 12, 15 and 19) of either of the formulations intravenously (unless indicated otherwise):
   1. Normal saline
   2. Resiquimod PM (5 mg/kg)
   3. anti–PD-1 antibody (250 μg per mouse; administered intraperitoneally and continued after day 17 for four more times – days 22, 26, 29 and 33）
      Note: Since the mice in this group reached the study endpoint before day 33, they did not receive the fourth dose 
   4. Resiquimod PM (5 mg/kg) + anti–PD-1 (250 μg per mouse; administered intraperitoneally and continued after day 17 for four more times – days 22, 26, 29, and 33)

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