Orthotopic non-metastatic and metastatic oral cancer mouse models

Abstract

Oral cancer is characterized by high morbidity and mortality with a predisposition to metastasize to different tissues, including lung, liver, and bone. Despite progress in the understanding of mutational profiles and deregulated pathways in oral cancer, patient survival has not significantly improved over the past decades. Therefore, there is a need to establish in vivo models that recapitulate human oral cancer metastasis to evaluate therapeutic potential of novel drugs. Here we report orthotopic tongue cancer nude mouse models to study oral cancer growth and metastasis using human metastatic (UMSCC2) and non-metastatic (CAL27) cell lines, respectively. Transduction of these cell lines with lentivirus expressing red fluorescent protein (DsRed) followed by injection into tongues of immunodeficient mice generated orthotopic tongue tumors that could be monitored for growth and metastasis by fluorescence measurement with an in vivo Imaging System (IVIS 200). The growth rates of CAL27-DsRed induced tumors were higher than UMSCC2-DsRed tumors after day 15, while UMSCC2-DsRed tumors revealed metastasis beginning on day 21. Importantly, UMSCC2 tumors metastasized to a number of tissues including the submandibular gland, lung, kidney, liver, and bone. Further, immunohistochemical analyses of tongue tumors induced by CAL27 and UMSCC2 cells revealed elevated expression of components of protumorigenic pathways deregulated in human cancers, including Cyclin D1, PCNA, Ki-67, LSD1, LOXL2, MT-MMP1, DPAGT1, E-cadherin, OCT4A, and H3K4me1/2. These orthotopic mouse models are likely to be useful tools for gaining insights into the activity and mechanisms of novel oral cancer drug candidates.

Keywords: Mouse models; Oral cancer; Orthotopic tumors metastasis.

Fig. 1

Orthotopic injection of CAL27 cell induces tongue tumor in nude mice. Orthotopic tongue tumors were generated by injecting CAL27 cells expressing DsRed into the tongue of nude mice (n = 5), and imaged by an in vivo Live imaging system (IVIS 200) at regular intervals starting at day 7 to day 31. Control mice (n = 5) were injected with vehicle. (A) In vivo imaging of CAL27-DsRed injected mice shows primary tongue tumor growth 24 days post injection; lane A1 represents nude mice injected with vehicle whereas lanes A2–A5 show mice injected with CAL27-DsRed cells (T represents region of interest of tongue for quantification of fluorescence intensity); (B) in vivo imaging of CAL27-DsRed cells injected into the tongue shows tumor growth 31 days post injection; lane B1 represents nude mice injected with vehicle only whereas lane B2–B5 shows mice injected with CAL27-DsRed cells (T represents region of interest of tongue for quantification of fluorescence intensity); (C) caliper measurements at different internals show growth of tongue tumors, but not in vehicle-injected mice (n = 5; ^*P < 0.05, ^*P < 0.01; 2-way ANOVA) and (D) quantification and normalization of fluorescence intensity data for IVIS imaging at day 24 and day 31 (n = 5; *P < 0.01; 2-way ANOVA).

Fig. 2

Orthotopic injection of UMSCC2 cells induces primary tumor and spontaneous metastasis in nude mice. Orthotopic tongue tumors were generated by injecting UMSCC2 cells expressing DsRed into the tongues of nude mice (n = 5) and imaged with an in vivo Live Imaging System (IVIS 200) at regular interval starting at day 7 to day 31. Control mice (n = 5) were injected with vehicle. (A) In vivo imaging of UMSCC2-DsRed cells injected tongues shows primary tumor growth 24 days post injection; the lane B1 represents nude mice injected with vehicle only and lanes B2–B5 show mice injected with CAL27-DsRed cells (T and M represents region of interest of tongue and metastasis of other organs for quantification of fluorescence intensity, respectively); (B) in vivo imaging of UMSCC2-DsRed cells injected shows primary tongue tumor growth 31 days post injection; lane C1 represents nude mice injected with vehicle only whereas lanes C2–C5 show mice injected with UMSCC2-DsRed cells (T and M represents region of interest of tongue and metastasis of other organs for quantification of fluorescence intensity, respectively); (C) fluorescence imaging of internal organs after necropsy shows the presence of UMSCC2 cells (T = tongue; SL = sublingual tissue including salivary gland; LG = lung; H = heart; K = kidneys; LV = Liver; I = Intestine; M = Mandible and FT = femur and tibia); (D) caliper measurements at different internals shows growth of tongue tumors, but not in vehicle-injected mice (n = 5; ^*P < 0.01; 2-way ANOVA); (E) quantification and normalization of fluorescence intensity data for IVIS imaging at day 24 and day 31 (n = 5; ^*P < 0.01; 2-way ANOVA) and (F) quantification of metastasis by fluorescence intensity data for IVIS imaging at day 24 and day 31 (n = 5; ^*P < 0.01; 2-way ANOVA).

Fig. 3

Comparison of CAL27 and UMSCC2 induced primary tumors. (A) Quantification of fluorescence imaging shows differences in intensity of Cal 27 and UMSCC2 induced tumors (n = 5; ^*P < 0.05, ^*P < 0.01; 2-way ANOVA) and (B) western blot analysis and quantification of, Cyclin D1, PCNA, LSD1 and H3K4Me1/2 compared to respective β-actin from tissue extracts (n = 3; ^*P < 0.05, ^*P < 0.01; 2-way ANOVA) and (C) H & E staining (100X) from tissue sections injected with UMSCC2 and CAL27 compared to vehicle are shown. immunostaining with (D) non-immune IgG control (100X and 400X), (E) anti-Ki-67 antibody (100X and 400X) and (F) anti-LSD1 antibody (100X and 400X) of tongue tumors sections in vehicle, CAL27 and UMSCC2 injection groups; scale bar = 1 mm for 100X and 0.1 mm for 400X.

Fig. 4

Characterization of CAL27 and UMSCC2 induced primary tumors by immunostaining with OSCC related protein expression. Immunostaining with (A) E-cadherin antibody (100X and 400X), (B) DPAGT1 antibody (100X and 400X), (C) anti-OCT4A antibody (100X and 400X), (D) anti-LOXL2 antibody (100X and 400X) and (E) MT-MMP1 antibody (100X and 400X) of tongue tumors sections in vehicle, CAL27 and UMSCC2 injection groups; scale bar = 1 mm for 100X and 0.1 mm for 400X.