4-nitroquinoline 1-oxide-induced oral epithelial lesions exhibit time- and stage-dependent changes in the tumor immune microenvironment

Abstract

Oral tongue squamous cell carcinoma (OTSCC) is the most common cancer of the oral cavity and is associated with high morbidity due to local invasion and lymph node metastasis. Tumor infiltrating lymphocytes (TILs) are associated with good prognosis in oral cancer patients and dictate response to treatment. Ectopic sites for immune activation in tumors, known as tertiary lymphoid structures (TLS), and tumor-associated high-endothelial venules (TA-HEVs), which are specialized lymphocyte recruiting vessels, are associated with a favorable prognosis in OSCC. Why only some tumors support the development of TLS and HEVs is poorly understood. In the current study we explored the infiltration of lymphocyte subsets and the development of TLS and HEVs in oral epithelial lesions using the 4-nitroquinoline 1-oxide (4NQO)-induced mouse model of oral carcinogenesis. We found that the immune response to 4NQO-induced oral epithelial lesions was dominated by T cell subsets. The number of T cells (CD4+, FoxP3+, and CD8+), B cells (B220+) and PNAd+ HEVs increased from the earliest to the latest endpoints. All the immune markers increased with the severity of the dysplasia, while the number of HEVs and B cells further increased in SCCs. HEVs were present already in early-stage lesions, while TLS did not develop at any timepoint. This suggests that the 4NQO model is applicable to study the dynamics of the tumor immune microenvironment at early phases of oral cancer development, including the regulation of TA-HEVs in OTSCC.

Keywords: 4-nitroquinoline 1-oxide (4NQO); high endothelial venules (HEVs); oral carcinogenesis; tertiary lymphoid structures (TLS); tumor infiltrating lymphocytes (TILs); tumor microenvironment.

Figure 1

Histopathological evaluation in the 4NQO mouse model. (A) Forty-eight mice were given 4NQO-water (n=30) or regular drinking water (n=18) for 16 weeks, followed by 12 weeks observation where the mice were sacrificed at different timepoints. (B) Each tongue was assigned a score corresponding to the lesion with the most severe histopathological grade: normal/hyperplasia, low-grade dysplasia, high-grade dysplasia, or SCC. Scale bar indicates 100 µm. (C) The severity of epithelial lesions increased in mice sacrificed at later endpoints.

Figure 2

Distribution of immune cells and HEVs in relation to epithelial lesions on the tongue of 4NQO-exposed mice. (A) Four µm thick sagittal tongue sections (scale bar indicates 400µm) were stained with H&E and divided into seven sectors (1–7) to (B) map the location of epithelial lesions of the tongue following 4NQO-exposure. Data is presented as percentage, and n represents the number of sectors that were examined across all 4NQO-exposed mice. (C-G) Total counts of each of the immune cells and HEVs within the seven sectors of the tongue in 4NQO-exposed mice (n=30) and healthy controls (n=18). Data is log-transformed (log2(y+1)), and error bars indicate median with IQR. (H) Total counts of each of the immune cells and HEVs in the seven sectors for all 4NQO-exposed mice. The range in the counts for the respective markers are shown to the right where the white is the lowest count and dark purple is the highest count.

Figure 3

Distribution of immune cell- and HEV markers in the tongue mucosa. (A) Shown are representative images for the distribution of CD4-, FoxP3-, CD8-, and B220 cells, and PNAd positive vessels in areas of the tongue graded as normal/hyperplasia, low-grade dysplasia, high-grade dysplasia, and SCC. For CD4, FoxP3, and B220, the same SCC is shown, but the images for FoxP3 and B220 only show parts of the SCC that represented the staining patterns. Scale bar indicates 100µm. (B) The number of positively stained cells or vessels for all the markers (CD4, FoxP3, CD8, B220, and PNAd) were significantly higher (P<0.0001) in mice exposed to 4NQO (n=30) compared to healthy controls (n=18). Error bars indicate median with interquartile range (IQR).

Figure 4

Immune cell and HEV count by endpoint and histopathological grade. (A) Shown are the total counts of CD4+, FoxP3+, CD8+ and B220+ cells and HEVs in the tongues of 4NQO-exposed mice (n=30), and the total counts of each of the markers in the tongues graded as (B) normal/hyperplasia (n=1), (C) low-grade dysplasia (n=3), (D) high-grade dysplasia (n=19), and (E) SCC (n=7). (F–J) The total counts of all the immune cell markers and HEVs in the tongues of 4NQO-exposed mice sacrificed at endpoint weeks ≤20 (n=12), endpoint weeks 21-24 (n=9), and endpoint weeks 25-28 (n=9). (K–O) The total counts of immune cells and HEVs in the tongues of 4NQO-exposed mice plotted against the most severe histopathological lesion. Data is log transformed (log2(y-+)). Scale bar indicates median with IQR (A–E, K–O) or mean with standard deviation (SD) (F–J).

Figure 5

In vivo PET/MRI results of mice following i.v. injection of [18F]FDG. (A) PET/MRI image of a representative animal (animal e4) representing uptake of [18F]FGD in the hyperintense lesion (left and right). The T2-weightes (middle) shows the lesion on the lateral side of the tongue marked by the crosshair. Overlapping PET and MRI image is shown to the left, while the middle and right shows MRI and PET image, respectively. (B) Standardized uptake values (SUV) of the whole lesion (round dots) and leg muscle (square dots, reference tissue), (C) lesion-to-muscle ratio in the four carcinogen-exposed mice (e1-e4), and (D) volumes of the hyperintense lesions of each animal (e1-e4) derived from T1-weighted MRI. (E) Representative images of Ki-67 staining in the (dorsal) tongue of healthy controls and 4NQO-exposed mice. Scale bar indicates 50µm. (F) SUV of lymph nodes of 4NQO-exposed (n=4, e1-e4, upward pointing arrowheads) and control mice (n=2, c1-c2, downward pointing arrowheads). Error bars indicate mean with SD. (G) PET/MRI results showing SUV of the lymph nodes indicated by arrows/labels in the four 4NQO-exposed mice. Representative images of (H) H&E- and (I) Ki-67-stained sections of cervical lymph nodes in 4NQO-exposed mice and healthy controls. Detailed images of plasma cells and Ki-67+ cells from outlined areas (black box) are shown in H&E- and Ki-67-stained sections, right panels of (H, I), respectively. Scale bars indicate 200µm (H, I) lymph node overview), 50µm (E, H) outlined area control and 4NQO, (I) outlined area 4NQO) or 20µm (I) outlined area control).