This study advances the development of oral carcinogenesis GEMM model, which relies on defects in the TGF-β and PI3K/Akt signaling pathways which are commonly altered in human HNSCCs (35). TGF-β provides strong growth inhibitory signals in epithelial cells, and alterations in TGF-β signaling promotes tumorigenesis in some human cancers. Previous reports have shown that deletion of Tgfbr1 alone is not sufficient for HNSCCs oncogenesis in mice (27). It is likely that cooperation of the TGF-β pathway with other pro-oncogenic pathways is required for malignant transformation in HNSCC. Decreased expression of Tgfbr1 in mice was associated with activation of the PI3K/Akt pathway leading to increase cell proliferation and survival (26, 27). Similarly, activation of the PI3K/Akt pathway by genetic deletion the negative regulator Pten is not sufficient to cause HNSCC due to the induction of premature senescence induced by phosphorylated Akt in the presence of the tumor suppressor TGF-β (35). Therefore, this model relies on the combined deletion of both Tgfbr1 and Pten to activate the PI3K/Akt pathway and escape premature senescence caused by TGF-β signaling, leading to the development of HNSCC.
Previous work on the Tgfβr1/Pten 2cKO mouse model administered tamoxifen for conditional gene deletion via application to the oral cavity, leading to tumor development at different sites within and around the oral cavity (including the ear, skin, and paw) at varying degrees in individual mice. (17, 36). We therefore developed several intralingual injection protocols to explore the possible variations in cancer development between different tamoxifen injection regimens. Our selection of the tongue as a representative organ for experimental HNSCC using this model allows for easy assessment of tumor progression and efficacy of chemopreventive and therapeutic interventions. Further, lesions and tumors in the tongue occur in many cases of human HNSCCs as well as in carcinogen induced mouse models of HNSCC (9, 10). We observed no major differences between the three tamoxifen injection regimens, which led us to proceed with three intralingual injections of 500 µg in 30ul corn oil every other day for 1 week for subsequent experiments.
HNSCC tumors derived from this protocol were very similar pathologically to human HNSCCs and showed similar molecular alterations. The epidermal growth factor receptor (EGFR) is overexpressed in more than 90% of HNSCC cases and in most in vivo models (37). Similar upregulation in the proliferation marker Ki-67, the pro-survival markers bcl-2 and bcl-xl, the metalloproteinase 9 (MMP9), and the transcription factor nuclear factor-kappa B (Nf-κb)(38–40), which are common HNSCC cancer progression and associated markers were found to be upregulated in our model.. Furthermore, the transcriptional repressor Id1 was found to be upregulated in our injection protocol tumors. Id1 is negatively regulated downstream of TGF-β signaling, thus deletion of Tgfβr1 can cause the overexpression of this gene which inactivates the tumor suppressor TP53 (41). TP53 has been found to mediate senescence based on constitutive Akt activity due to PTEN deletion, which we confirmed with high levels of phospho-Akt in the tumors of our mice (42). Senescence in these carcinomas can also be subverted by the inactivation of any member of the p19-p53-p21 pathway, which also work on suppressing the activity of TP53 (43, 44). Our data further confirms the important role Id1 plays in the escape from senescence, which ultimately result in HNSCC tumor development. However, we did not observe a significant upregulation of cyclin D1 (CCND1), which is commonly found to be increased in human and in other mouse models of HNSCC (17, 45). Further exploration of this observation is needed, although other mechanisms associated with the NF-κB signaling pathway may promote HNSCC development and progression in this model(46).
CD4+ and CD8+ T and NK cells, are the major effector cells in anti-tumoral cell mediated immunity. CD8 + T-cell and NK cell infiltration into the HNSCC tumor microenvironment are essential to promote cancer cell cytotoxicity and are crucial to the success of immune checkpoint inhibitors currently used in HNSCC treatment (47). Our immunological analysis of tumor draining lymph nodes from tumor bearing Tgfβr1/Pten 2cKO mice showed CD8 + T-cells expressing higher levels of IFNγ, TNF-α, and the cytotoxic enzyme perforin, while expressing lower levels of the immuno suppressive cytokine IL-10 (48). We also observed a greater level of TNF-α producing CD4 + T-cells, suggesting a Th1 antitumoral phenotype in response to HNSCC tumors (49). Furthermore, increased levels of CTLA-4 expressing T-cells (likely Tregs) were observed in draining lymph nodes of tumor bearing Tgfβr1/Pten 2cKO mice which suggests the development of tumor immune escape mechanisms that are typical in patients with HNSCC. The immune response changes were observed in the tumor draining lymph nodes rather than the spleen, indicating a localized immune response to these tumors.
To further demonstrate the utilization of the Tgfβr1/Pten 2cKO model for HNSCC chemoprevention studies, we explored mechanisms of HNSCC inhibition by chemopreventive agents in BRB. We previously showed that BRBs modulate Treg activity, and proinflammatory, apoptotic, angiogenic and cell cycle related pathways during HNSCCs chemoprevention, using the 4NQO carcinogen induced model (31, 50–52). Others have also demonstrated similar mechanisms of BRB mediated chemoprevention in other cancers, including esophageal, colorectal, and cervical cancers(53–55). Tgfβr1/Pten 2cKO mice fed BRB supplemented diet displayed lower tumor volumes and tongue widths compared to mice receiving a control diet, further supporting chemopreventive ability of BRBs against HNSCCs. BRBs contain numerous bioactive compounds, and the immunological, cellular and biochemical mechanisms underlying BRB mediated HNSCC chemoprevention are not completely understood. Increased recruitment of tumor infiltrating lymphocytes (TILs) to HNSCC tumor microenvironments often correlate with better prognosis and response to treatment (56). BRB diet administration increased the recruitment of TILs to the HNSCC tumor microenvironment. BRB also increased the frequency of IFN-γ and IL-2 producing CD4+ T-cells in HNSCC tumors, cytokines which promote T cell expansion, macrophage activation and cytotoxic CD8+ T-cell activation, which are essential to creating an antitumoral microenvironment (57–59). Increased TNF- α production by CD8+ T-cells of Tgfβr1/Pten 2cKO mice fed BRB diet promotes tumor cell cytotoxicity (60), and increased survival outcomes in HNSCC(61, 62). Similar to observations in 4NQO induced mouse model of HNSCC, we demonstrated that that dietary administration of BRB promotes granzyme B production by CD8 + T-cells in the tumor microenvironment of Tgfβr1/Pten 2cKO mice(31). These results along with previous reports by our group, indicate an important antitumoral immunomodulatory role of BRB bioactive compounds in HNSCC tumors driven by deregulated PI3K/Akt and TGF-β signaling.
In conclusion, we have developed an optimized protocol for development of easily quantifiable HNSCC tumors using Tgfβr1/Pten 2cKO mice. The intralingual tamoxifen injection regimen facilitates the localization of HNSCC tumors, allows for easier quantification, and significantly shortens the experimental timeline. Our analysis of HNSCCs associated biomarkers demonstrates that this approach recapitulates the pathological and molecular characteristics of human HNSCC and other in vivo HNSCC models. We observed the presence of lymph node metastasis and characterized the immune response associated with HNSCC tumors associated with defective PI3K/Akt and TGF-β signaling. Furthermore, using this model, we showed that dietary BRB administration promotes an antitumoral microenvironment characterized by IFN-γ and IL-2 producing CD4+ T-cells, and TNF- α and Granzyme B producing CD8+ T cells in HNSCC tumors. Future studies will utilize this model to characterize mechanisms of cancer inhibition by chemopreventive or immunotherapeutic agents in HNSCCs initiated by alterations in the TGF-β and PI3K/Akt pathways.