Materials and Reagents

Alcohol swab (B.D. Biosciences)
Nair hair removal cream
Cotton swab
Sterile gauze pads
Tuberculin syringe (B.D. Biosciences)
344SQ-green fluorescent protein/Firefly luciferase (GFP/fLuc) cells (source: J. Kurie, MD Anderson Cancer Center)
Hanks' Balanced Salt solution (Gibco)
Matrigel (Corning)
D-luciferin (PerkinElmer)
1× DPBS (Gibco)
Isofluorane (VetOne)
Ketamine (Vedco)
Xylazine (Acorn animal health)
Acepromazine (Vedco)
Anesthetic cocktail (see Recipes)

Note: Refer to Vinod et al. (2020a) (protocol based exclusively on Poly(2-oxazoline) preparation for reagents for Sections C and D.

Equipment

Surgical instruments
1. Autoclip Wound Closing System – Staples + Applier (Braintree Scientific, Inc)
2. Iris Surgical Scissors, 4½ inch, curved (Fisher Scientific)
3. Delicate Specialty Dissection Forceps, Serrated, 5" (Fisher Scientific)
IVIS-Lumina II optical imaging system (PerkinElmer Inc., Hopkinton, MA)
Small Animal induction chamber

Software

Living Image^® software

Procedure

Preparation of animal tumor model of NSCLC

Prepare a suspension of 344SQ-green fluorescent protein (GFP)/fLuc cells in 50% Hanks' Balanced Salt Solution and 50% Matrigel (v/v).
Anesthetize the mice by injecting the anesthetizing cocktail intraperitoneally and lay it in the right lateral decubitus position.
Apply hair removal cream using cotton swab in the chest area and leave for 1 min (be sure not to exceed 1 min to avoid giving the mice a chemical burn). Wipe away with dry sterile gauze pad. Use damp sterile gauze pad to remove any residual cream.
Clean the skin surface with an alcohol swab and make an incision between ribs 10 and 11 by placing the blade parallel to the rib cage (see Figure 1A).
Transfer the cell suspension to a 1-ml tuberculin syringe and inject 50 μl of 2.5 × 10³-5 × 10³ cells into the left lung parenchyma in the lateral dorsal axillary line (see Figure 1B).
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.

Figure 1. Schematic showing the site of incision (A) and the site of orthotopic injection (B)

Bioluminescence imaging

Inject 150 mg/kg of 15 mg/ml (in 1× DPBS) D-luciferin solution intraperitoneally and start imaging after 15 min.
Anesthetize the mice by placing them in an inhalation induction chamber (for ~5 min) comprising 2-3% isoflurane in oxygen.
Place the anesthetized animals in the sample stage of the imaging chamber of the IVIS lumina optical imaging system in the supine position.
Acquire luminescent images using the following acquisition settings in the Living Image^® software:
1. Pixel Width: 1
2. Pixel Height: 1
3. Image units: counts
4. Luminescent exposure (seconds): 60
5. Field of view: 24
6. Emission filter: open
7. Subject size: 1.5
  Note: Bioluminescence imaging (B.L.I.) is done once prior to the commencement of the treatments to obtain baseline bioluminescence to confirm the presence of the tumor. Following the start of treatments (Day 8), B.L.I. is performed once every week to monitor tumor growth.

Chemotherapy in conjunction with chemosensitizers

Randomize mice into four groups of 10 mice each
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 (volume: 100-200 μl).
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

Randomize mice into four groups of 13 mice each.
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 (volume: 100-200 μl).
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).

Data analysis

Analyze the B.L.I. data by specifying a region of interest outlining the tumor and quantify the total radiance using Living Image Software to measure the luciferase activity, which is representative of the tumor burden. Refer to Vinod et al. (2020b) for representative BLI images.

Recipes

Anesthetic cocktail
To prepare the anesthetic cocktail, dilute stock solutions of Ketamine, Xylazine and Acepromazine in 1× DPBS and administer 100 μl of the cocktail per 10 g of mouse body weight. Use the following doses of each anesthetic:
Ketamine: 80 mg/kg (stock conc. 100 mg/ml)
Xylazine: 8 mg/kg (stock conc. 100 mg/ml)
Acepromazine: 1 mg/kg (stock conc. 10 mg/ml)

Acknowledgments

The original work (Vinod et al., 2020b) was funded by the National Cancer Institute (NCI) Alliance for Nanotechnology in Cancer (U54CA198999, Carolina Center of Cancer Nanotechnology Excellence).

Competing interests

A.V.K. is co-inventor on patents pertinent to the subject matter of the present contribution and A.V.K. and M.S.P. have co-founders' interest in DelAqua Pharmaceuticals Inc. having intent of commercial development of POx based drug formulations. The other authors have no competing interests to report.

Ethics

The protocol (IACUC# 18-174) was approved by The University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee. The validity period of IACUC# 18-174 is of 3 years.

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