**2. Results**

Two mice from groups C3G-HB supplemented diet continuously before and after NNK-injection (conti.-C3G-HB) and C3G-HB supplemented diet only after NNK-injection (post-C3G-HB) were euthanized due to weight loss and eliminated from the study. Observations for symptoms of stress, i.e., changes in fur color or texture, food consumption, and behavioral abnormalities such as hunched posture, fast movements, and vocalization, were performed daily.

#### *2.1. The Composition of C3G-HB*

The nutritional composition of the C3G-HB cv. Tundra is presented in Table 1. C3G-HB is rich with proteins (68%), fat (3.3%), and fiber (8%). The C3G content of the studied haskap berry sample was 3.4%. Additionally, C3G-HB was rich in minerals, particularly manganese, magnesium, zinc, and copper (Table 1).


**Table 1.** The nutritional composition of lyophilized powder of haskap berry cv. Tundra.

All the parameters are presented in percentages unless the unit is indicated in front of the parameter in the table. ADIN, acid detergent insoluble nitrogen.

#### *2.2. The Response of Mice to C3G-HB Supplementation and NNK-Injection*

Dietary supplementation of C3G-HB and NNK injection affects the body weight of mice (Figure 1A). Body weight of naive mice was significantly higher (paired *t*-test, *p* < 0.0001) in comparison to the control and no-C3G-HB groups. For instance, at the termination, the body weight of mice given C3G-HB supplement and NNK was reduced by 3.5% (naive vs. control) and 7.6%, respectively (naive vs. no-C3G-HB). Conversely, long-term C3G-HB supplementation significantly (*p* < 0.001) increased the body weight of NNK-injected mice by 2% (no-C3G-HB vs. conti.-C3G-HB), and 3.2% (no-C3G-HB vs. post-C3G-HB), respectively (Figure 1A). In fact, the weight loss in NNK-injected, no-C3G-HB mice could be linked with their dietary intake as no-C3G-HB group had significantly lower feed intake compared to the control (paired *t*-test, *p* < 0.0035) (Figure 1B).

**Figure 1.** The effect of cyanidin-3-*O*-glucoside (C3G)-HB and 4-(methylnitrosamino)-1-(3-pyridyl)- 1-butanone (NNK) injection on the body weight of A/JCr mice. Naive mice (*n* = 5) and NNK-injected mice in group no-C3G-HB (*n* = 10) were fed a regular mouse diet. Mice in the control (*n* = 5) and NNK-injected (pre-C3G-HB, conti.-C3G-HB, and post-C3G-HB) (*n* = 10) groups were fed with the C3G-HB supplemented diet as presented in Figure 5. (**A**) Average body weight of the mice and (**B**) Average food intake over the experimental period. The effect of C3G-HB dietary supplement and NNK carcinogen injection on the body weight of mice was determined by paired *t*-test at α = 0.05. No-C3G-HB, not given C3G-HB supplemented diet; Pre-C3G-HB, C3G-HB supplemented diet only before NNK injection; Conti.-C3G-HB, C3G-HB supplemented diet continuously before and after NNK injection; Post-C3G-HB, C3G-HB supplemented diet only after NNK injection.

#### *2.3. Lung Tumorigenesis and Tumor Incidence*

PET/MRI images confirmed the presence of tumors in the lungs of NNK-injected mice (Figure 2A). The effect of C3G-HB dietary supplementation on lung tumorigenesis was determined by the number of peripheral lung tumors (Figure 2B). The group no-C3G-HB mice, injected with NNK, and fed the control diet, had an average of 14.1 ± 1.7 tumors/mouse. NNK-injected mice in group pre-, conti.-, and post-C3G-HB, that were given the C3G-HB supplement had 8.7 ± 1.4, 10.2 ± 1.2, and 9.1 ± 1.4 tumors/mouse, respectively, and a reduction of tumor multiplicity by 38.3%, 22.8%, and 35.4%, respectively, in comparison to the no-C3G-HB group (Figure 2C). The inhibition of lung

tumor multiplicity by continuous dietary supplementation of C3G-HB (conti.-C3G-HB) was not statistically significant (*p* > 0.05) from no-C3G-HB group.

**Figure 2.** The effect of C3G-rich haskap berry supplement (C3G-HB) on NNK-induced lung tumorigenesis in A/JCr mice. Saline was injected as a sham for mice in naive and control groups. A single intraperitoneal injection of NNK (100 mg/kg body weight) was used to induce lung tumors in the rest of the mouse groups (pre-C3G-HB, conti.-C3G-HB, and post-C3G-HB). Naive mice were fed a regular mouse diet. Mice in control and NNK-injected groups were fed the C3G-HB supplemented diet, as presented in Figure 5. (**A**) The presence of lung tumors was confirmed by PET/MRI scan and a representative comparison between naive and no-C3G-HB groups (*n* = 3). (**B**) The number of peripheral tumors was counted in each lung under a dissecting microscope (*n* = 5 for naive and control groups, and *n* = 10 for NNK-injected groups). (**C**) The effect of C3G-HB dietary supplement on lung tumor multiplicity was analyzed by one-way ANOVA with Dunnett's test at α = 0.05. No-C3G-HB, not given C3G-HB supplemented diet; Pre-C3G-HB, C3G-HB supplemented diet only before NNK injection; Conti.-C3G-HB, C3G-HB supplemented diet continuously before and after NNK injection; Post-C3G-HB, C3G-HB supplemented diet only after NNK injection. \* Indicate statistical difference at *p* ≤ 0 05 with mean ± SD. NS, Results do not significantly different.

The tumor incidence was not affected by the consumption of the C3G-HB dietary supplement. The tumor incidence of NNK-injected mice was 100% (10/10 and 9/9). Untreated mice (saline-injected) in naive group; 2 out of 5 mice (0.4 ± 0.2) and control; 1 out of 5 mice (0.2 ± 0.2) showed one "spontaneous" tumor on their lungs.

#### *2.4. Lung Tumor Area*

The lung tumor area was measured in three consecutive lung sections, representing three depths (top, middle, and bottom) of the lungs (Figure 3). The H and E-stained sections revealed the internal tumor area and tumor characteristics. Tumor lesions were less differentiated and composed of cells with higher nuclear crowding and cytological atypia. The H and E-stained sections indicated a significantly higher (*p* < 0.0001) tumor area in the no-C3G-HB group that received NNK and the control diet. The tumor area in each section was calculated using ImageJ software. The tumor burden in NNK-injected mice was 21.6 ± 4.1. Tumor area was significantly reduced in NNK-treated mice that received the C3G-HB-supplemented diet; 7.6 ± 2.8 (pre-C3G-HB), 7.1 ± 0.6 (conti.-C3G-HB), and 6.9 ± 0.6 (post-C3G-HB), and accordingly reduced by 64.7%, 67.3%, and 68.1%, respectively.

**Figure 3.** The effect of C3G-HB dietary supplementation on lung tumor area in A/JCr mice. Lung tumors were induced by a single intraperitoneal injection of NNK (100 mg/kg body weight). Saline was injected as a sham for the mice in naive and control groups. Naive mice were fed a regular mouse diet. Mice in control and NNK-injected groups were fed the C3G-HB supplemented diet, except for the mice in no-C3G-HB group (refer to Figure 5). Formalin-fixed lung sections were stained with H and E (3 sections/mouse). (**A**) The whole lung area was imaged, and the tumor area was measured by ImageJ software. (**B**) Representative H and E-stained sections, 200× magnification. (**C**) One-way ANOVA with Dunnett's test at α = 0.05 was used for data analysis to compare the treatment effect. No-C3G-HB, not given C3G-HB supplemented diet; Pre-C3G-HB, C3G-HB supplemented diet only before NNK injection; Conti.-C3G-HB, C3G-HB supplemented diet continuously before and after NNK injection; Post-C3G-HB, C3G-HB supplemented diet only after NNK injection. \* Indicate statistical difference at *p* ≤ 0 05 with mean ± SD. NS, Results do not significantly different.
