Temporal characterization of lymphatic metastasis in an orthotopic mouse model of oral cancer

Abstract

Background: The overall mortality rate in cases of head and neck squamous cell carcinoma (HNSCC) has not improved over the past 30 years, mostly because of the high treatment failure rate among patients with regionally metastatic disease. To better understand the pathobiologic processes leading to lymphatic metastasis development, there is an urgent need for relevant animal models.

Methods: HNSCC cell lines were implanted into the tongues of athymic nude mice. Histology, immunohistochemistry, and ex vivo 2-photon microscopy were used to evaluate tumor progress and spread.

Results: Orthotopic xenografts of different HNSCC cell lines produced distinct patterns of survival, tumor histology, disease progression rate, and lymph node metastasis development. Remarkably, all injected cell types reached the lymph nodes within 24 hours after injection, but not all developed metastasis.

Conclusion: This orthotopic xenograft model closely mimics several characteristics of human cancer and could be extremely valuable for translational studies focusing on lymphatic metastasis development and pathobiology.

Keywords: head and neck squamous cell carcinoma; lymph node metastasis; oral cancer; orthotopic xenograft mouse model; physiologic characterization.

Figure 1

Orthotopic xenografts of different HNSCC cell lines produce distinct survival patterns in nude mouse model. 1×10⁶ cells were implanted per animal. Mice were sacrificed once weight loss exceeded 20% body weight. (A) Kaplan-Meier survival curve for nude mice implanted with cancer cells. Data shown are from one representative experiment out of four independent experiments. (n=5 for 011-, 012-, and 019-cell injected mice; n=4 for OKT- and NSC-injected mice - 23 mice total) (B) Mean survival times of nude mice implanted with cancer cells. Pooled data from four independent experiments. (n=5, 12, 10, 4, and 4 for 011-, 012-, 019-, OKT- and NSC-injected mice, respectively – 35 mice total) 011, JHU-SCC-011 cells; 012, JHU-SCC-012 cells; 019, JHU-SCC-019 cells; OKT, OKF4/TERT-1 cells; NSC, normal saline control. *P<.05; **P<.01; ***P<.0001

Figure 2

JHU-SCC-011 cell xenografts form well differentiated keratinizing squamous cell carcinomas in the tongue. Representative histological sections of tongue xenografts in nude mice (n=24) were stained by hematoxylin/eosin (H&E) and immunohistochemistry for cytokeratin 5 confirming epithelial origin of labeled cells. Black square denotes the area visualized by higher magnification.

Figure 3

JHU-SCC-012 cell xenografts produce moderately differentiated squamous cell carcinomas in the tongue and cervical lymph nodes. Representative histological sections of (A) tongue xenografts and (B) metastatic cancer cells in the cervical lymph nodes of nude mice (n=6) are shown. Tissue sections were stained by hematoxylin/eosin (H&E) and immunohistochemistry for cytokeratin 5 confirming epithelial origin of labeled cells. Black square denotes the area visualized by higher magnification.

Figure 4

JHU-SCC-019 cell xenografts produce poorly differentiated squamous cell carcinomas in the tongue and cervical lymph nodes. Representative histological sections of (A) tongue xenografts and (B) metastatic cancer cells in the cervical lymph nodes of nude mice (n=25) are shown. Tissue sections were stained by hematoxylin/eosin (H&E) and immunohistochemistry for cytokeratin 5 confirming epithelial origin of labeled cells. Black square denotes the area visualized by higher magnification.

Figure 5

Orthotopically implanted cells reach the cervical lymph nodes within 24 hours. In vitro growth curves of GFP-expressing (A) 011G and (B) 019G cells compared to their parent cell lines. Absorbance measurements are in linear correlation with cell numbers. Error bars represent SD (n=3). Representative microscopy images of lymph nodes from mice after tongue injection with GFP-expressing (C) JHU-SCC-011 and (D) JHU-SCC-019 cells. Grayscale image at low magnification (10× objective, 1200 × 1200 um field-of-view) of a lymph node demonstrating the distribution of GFP signal. Insets are specific regions-of-interest imaged by two-photon microscopy using 40× objective, showing individual cells within the subcapsular and cortical space. (E) GFP-positive cell counts obtained from two-photon micrographs in regions-of-interest (ROI) in the lymph nodes at day 1, 7, and 14 post-injection. White and grey bars represent cells in the subcapsular and cortical area, respectively. For each cell line two animals per time point were analyzed (18 animals total). Error bars represent SD for ROIs (n≥5 for all time points). *P<.05, 011G/019G/OKTG, stably GFP-transfected JHU-SCC-011/JHU-SCC-019/ OKF4/TERT-1 cells, respectively. JHU-011, JHU-SCC-011 cells; JHU-019, JHU-SCC-019 cells.