NO gas
10,000–100,000 ppm NO was administered from 3 L cylinders, with Nitrogen serving as its stabilizing gas (Gordon Gas and Chemical, Tel Aviv, Israel). Nitrogen supplied from 3 L cylinders served as control gases. All procedures were performed in a chemical hood. Gases were delivered via a pressure regulator through a PVC hose (International Biomedical, USA). The flow rate was set to 0.2 (for in vivo studies) -1.0 (for in vitro studies) liters per minute (LPM) using a manual flow meter.
Tumor cell lines
Mouse CT26.WT colon, 4T1 breast and LLC-1 Lewis lung carcinoma cancer cell lines were purchased from an American Type Culture Collection (ATCC) local distributor, Sartorius (Beit Haemek, Israel). CT26 and 4T1 cell lines were grown in RPMI-based media (ATCC) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Sartorius). LLC-1 cells were grown in DMEM medium (ATCC) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Sartorius).
Preparation of tumor cells
Tumor cell suspensions were prepared in a cell culture medium or Hanks’ Balanced Salt Solution (HBSS, Sartorius) at concentrations of 1 × 105 cells/ml for in vitro studies or 5.0–10.0 × 106 cells/ml for in vivo studies. Freshly prepared cells were grown to 70% confluency, harvested using trypsin (Sartorius), and counted using a hemocytometer.
In vitro studies
Cell viability was assessed following direct exposure of cancer cells to gNO in 96-well plates. Exposure of gNO at 10,000–100,000 ppm or air was delivered at 1.0 LPM in a 1.7 L box. For gNO treatment, cell culture media was removed, and cells were exposed to gNO for up to three minutes. Immediately following gas exposure, cell culture medium was added, and cells were incubated at 37 °C in 5% CO2 overnight. Cell viability was assessed using XTT (Sartorius) and Annexin V–Propidium Iodide (Miltenyi-Biotech) assays.
In vivo experiments
Study overview: CT26 cells were inoculated subcutaneously (s.c.) on the right flank of 8–10 weeks old male Balb/c mice (Envigo, Israel, and the Netherlands) at a concentration of 5.0 × 105 CT26 cells in 100 µL HBSS. Treatment was initiated once tumors reached an average volume of ~ 50 mm3 (around 10 days following tumor inoculation). Mice were evaluated for tumor volume using a digital caliper up to 14 days post-gNO treatment, and tumors were surgically removed. Seven days later (21 days post-gNO treatment of primary tumors), mice were inoculated with challenge tumor cells on the contralateral (left) flank. Primary tumor recurrence rate and challenge tumor take were evaluated up to the excision day and 75 days post-challenge inoculation, respectively.
In vivo gNO treatment
Before each treatment, mice were anesthetized by an intraperitoneal (i.p.) injection of 100 mg/kg ketamine (Zoetis) and 10–20 mg/kg xylazine hydrochloride solution (Abic). After 10 min, mice were treated by means of intratumoral delivery of 20,000 or 50,000 ppm gNO. The needle was inserted into the tumor horizontally such that it was located approximately in the tumor’s center (about half of the tumor diameter, depending on tumor size and shape). Gas was injected for 5 min using a 23G needle. The outlet pressure was set to ~ 3.5 bar using a pressure regulator connected to the cylinder. A stainless-steel PTFE coated hose was connected to the pressure regulator on one end and a manual flow meter at the other end. A PVC hose was connected to the flow controller and a 23G hypodermic needle. The treatment regimen was 20,000–50,000 ppm NO at 0.2LPM rate for 5 min using a manual flow meter. All primary tumors were resected 14 days post-gNO treatment. A week after tumor resection, all mice were re-challenged with a second dose of CT26 cells.
In vivo nitrogen treatment
Nitrogen is the balance gas for NO and was used as a control treatment. Before each treatment, mice were anesthetized by an intraperitoneal (i.p.) injection of 100 mg/kg ketamine (Zoetis) and 10–20 mg/kg xylazine hydrochloride solution (Abic). After 10 min, mice were treated with intratumor nitrogen. The needle was inserted into the tumor horizontally to place it in the tumor’s center (about half of the tumor diameter, depending on tumor size and shape). Gas was injected for 5 min using a 23G needle. The outlet pressure was set to ~ 3.5 bar using a pressure regulator connected to the cylinder. A stainless-steel PTFE-coated hose was connected to the pressure regulator on one end and a manual flow meter on the other end. A PVC hose was connected to the flow controller and a 23G hypodermic needle. The nitrogen treatment was delivered at 0.2LPM rate for 5 min using a manual flow meter. All primary tumors were resected 14 days post-nitrogen treatment. A week after tumor resection, all mice were re-challenged with a second dose of CT26 cells.
Tumor volume calculation
Local tumor growth was determined by measuring 3 mutually orthogonal tumor dimensions 2–3 times per week, according to the following formula:
$${\text{Tumor Volume }} = \frac{\pi }{6} \times \left[ {{\text{Diameter 1}} \times {\text{ Diameter 2}} \times {\text{ Diameter 3}}} \right]$$
Primary tumor excision
Fourteen days after NO treatment of CT26 tumor-bearing mice, all remaining tumors were excised. The tumor of one mouse in the 20,000 ppm NO group was excised 10 days post-treatment due to tumor size. This mouse was excluded from the average primary CT26 tumor volume graph but included in challenge assay graphs. Briefly, all surgery tools (Vet Market, Israel) were washed with Septal Scrub (Teva, Israel) and heat disinfected, followed by Ethanol 70% (Romical, Israel) disinfection. Mice were anesthetized by an i.p injection of 100 mg/kg ketamine and 20 mg/kg xylazine hydrochloride solution. Local anesthesia using 0.5% Lidocaine cream (Vet Market, Israel) was applied to the tumor surface. After 10 min, the tumor was disinfected by gauze pads (Life, Israel) saturated with Ethanol 70%. The tumor was stabilized using a sterile tweezer and detached from the skin using sterile scissors. The operated tumor site was disinfected via local administration of Polydine (Teva, Israel) and Ethanol 70% (Romical, Israel). The skin was fused using medical clips (Bar Naor, Israel). After treatment, all mice were closely monitored until complete recovery. Dipyrone (Teva, Israel) was administered in drinking water for 3 days (0.4 g/200 ml drinking water).
Challenge tumor inoculation
On day 21 post-gNO treatment of the primary tumor, the appropriate cancer cell suspensions were prepared, and s.c. cell inoculation was repeated on the contralateral (left) flank. The appearance of a second induced tumor (challenge tumor) was monitored 2–3 times a week by visual and palpable observation. Naïve or nitrogen-treated mice inoculated with tumor cells served as controls.
Conditions for terminating the participation of a particular animal in the experiment
Animals found in a moribund condition or showing severe pain and enduring signs of severe distress were humanely euthanized. The health condition of the animals was assessed via the below mouse distress scoring:
-
1.
Appearance: normal—0, coat staring, ocular or nasal discharge-1, piloerection—2, hunched up—3.
-
2.
Hydration status: normal—0, skin tents when pinched quickly recovers—1, skin tents when pinched slowly recovers—2, skin remains tented severe dehydration -3.
-
3.
Natural Behavior: normal, i.e., active—0, less mobile and alert—1, isolated—2, restless/shivering/very still—3
-
4.
Body weight: not different from controls—0, weight loss of 0–10%—1, weight loss of 10–20%, weight loss over 20% -3.
-
5.
Tumor volume (of all tumors): 500–1,000 mm3—1; 1,000–1,500 mm3—2; > 1,500 mm3—animals with a > 1,500 mm3 tumor were humanely euthanized immediately.
When the total score is ≥ 7, the mouse was assessed 1–2 times a day, and wet food was placed at the bottom of the cage. When the total score was 10 or the total tumor burden exceeded 1,500 mm3, the mouse was euthanized by cervical dislocation. Briefly, the mouse was first anesthetized using Ketamine and Xylazine anesthetic mix as previously described, followed by cervical dislocation. When animals were euthanized for humane reasons or found dead, the time of death was recorded as precisely as possible. The conditions for animal sacrifice described above are reflected as the endpoint for the survival experiment.
Histology and immunohistochemistry
Colon tumor samples from mice were harvested, fixed in 4% formaldehyde, sent to a Contract Research Organization (CRO), and kept in the fixative for 48 h. The tissues were processed for paraffin embedding using one cassette per animal. 4 μm paraffin sections were cut, placed on glass slides, and stained with Hematoxylin & Eosin (H&E) for general histopathology and immunohistochemistry (IHC) using the following antibodies: BD pharmigen, Cat#550286), CD3 (T-lymphocytes, Abcam Cat#Ab16669), CD4 (helper T-lymphocytes, Abcam, Cat#: 183685), CD8 (effector/cytotoxic T-lymphocytes, Novusbio, Cat#NBP2-29475), and CD11b (dendrocytes, Proteintech, Cat#17342).
The inflammation and lymphocyte infiltration levels were histologically assessed by an experienced veterinary pathologist (Pathologica, Israel). Semi-quantitative analysis of pathological changes in H&E-stained sections was performed using a scoring system for the presence and severity of pathological changes in 10 non-overlapping fields, as follows: Immune cells level: 0: no positive cells, 1: < 5 cells positive cells, 2: Very mild staining (5 to < 15 cells), 3: Mild staining (15 to < 25 cells), 4: Moderate staining (25 to < 50 cells), 5: Marked staining (≥ 50 cells). Inflammation was scored as: 0: Scant or absence of inflammatory cells, 1: Inflammatory cells present but markedly less than tumor cells, 2: Inflammatory cell number roughly equal to tumor cells, 3: Predominantly inflammatory cells. Lymphocyte infiltration was scored as: 0: Absence of lymphocytes, 1: < 5 lymphocytes per field, 2: 5 to < 20 lymphocytes per field, 3: ≥ 20 lymphocytes per field.
Continuous outcomes were analyzed by analysis of variance (ANOVA). The between-group difference was derived from ANOVA with treatment as a factor.
Flow cytometry analysis
CT26 tumor-bearing mice were treated with 50,000 ppm gNO, 20,000 ppm gNO or nitrogen. Fourteen days post gas treatment all tumors were resected. Mouse spleens were dissociated at day 21 post gas treatment with the GentleMACS Octo (Miltenyi-Biotech). Mouse blood samples were processed 21 days post gas treatment using RBC Lysis Solution (cat#130-094-183, Miltenyi-Biotech). Extracellular labeling of T-cells was performed with FITC anti-CD3 (cat#130-119-758, Miltenyi-Biotech), VioGreen anti-CD4 (cat#130-118-693, Miltenyi-Biotech), and APC-Vio770 anti-CD8 (cat#130–120-806, Miltenyi-Biotech) antibodies. Extracellular labeling of B-cells was performed with PE-Vio615 anti-CD19 (cat#130-111-890, Miltenyi-Biotech) antibodies. Extracellular labeling of polymorphonuclear myeloid-derived suppressor cells (MDSCs) was performed with FITC anti-CD3 (cat#130-119-758, Miltenyi-Biotech) and PE-Vio615 anti-CD19 (cat#130-111-890, Miltenyi-Biotech) for negative staining, VioGreen anti-CD11 (cat#130-113-811, Miltenyi-Biotech), PE Ly-6G (cat#130-123-780, Miltenyi-Biotech) antibodies for positive staining and VioBlue anti-Ly6C (cat#130-111-921, Miltenyi-Biotech) for low staining. Samples were analyzed using MacsQuant™ 16 Flow cytometer.
IFNγ assay
Mouse spleens were dissociated with GentleMACS Octo (Miltenyi-Biotech). T-cells were activated overnight in TexMACS + 10% FBS medium. 2 µL/mL Cell stimulation cocktail (cat#TNB-4970-UL100, Tonbo-Biosciences) was added for 2 h at 37 °C. 1µL/mL Brefeldin A solution (cat# TNB-4506-L001, Tonbo-Biosciences) was then added for 2 h at 37 °C. Extracellular labeling of T-cells was performed with FITC anti-CD3 (cat#130-119-758, Miltenyi-Biotech), VioGreen anti-CD4 (cat#130-118-693, Miltenyi-Biotech), and APC-Vio770 anti-CD8 (cat#130-120-806, Miltenyi-Biotech) antibodies. After fixation and permeabilization of cells, intracellular labeling of T-cells was performed with APC anti-IFNγ antibody (cat#130-123-283, Miltenyi-Biotech). Samples were analyzed using MacsQuant™ 16 Flow cytometer.
Statistical analysis
Statistical analysis was performed using Excel (Microsoft, USA) or GraphPad Prism 9.3.1 (GraphPad Software, USA) with P < 0.05 considered statistically significant unless stated otherwise.