br In the current study we investigated the host microbiota
In the current study, we investigated the host-microbiota inter-action in lung cancer development using a genetically engi-neered mouse (GEM) model driven by an activating point muta-tion of Kras and loss of p53, which are frequently associated with human LUAD. This autochthonous model of LUAD not only faith-fully mimics the genetic and histopathological features of the hu-man disease, but also enables us to study the complex crosstalk between the commensal microbiota and host immune system in the physiological context of developing lung cancer. Using this model, we found that local microbiota associated with tumor growth could promote inflammation and cancer progression via lung-resident gd T cells. Depleting microbiota or inhibiting gd T cells or their downstream effector molecules all effectively suppressed lung cancer development. These findings provide conceptually novel insights regarding the pathogenesis of lung cancer by revealing the role of commensal microbiota in shaping the tumor-associated immune response and shed light on cellular and molecular targets for therapeutic intervention in lung cancer.
Commensal Microbiota Promote Tumor Growth in an Autochthonous GEM Model of LUAD
We have previously established a GEM model of human LUAD using conditional L-NAME of KrasLSL-G12D; p53flox/flox in mice (KP
mice) (Jackson et al., 2001). In this model, intratracheal infection with adenoviral vectors expressing Cre recombinase under the control of the Sftpc promoter activates oncogenic KrasG12D and deletes the tumor suppressor p53 in lung epithelial cells to induce lung adenoma and adenocarcinoma (DuPage et al., 2009; Sutherland et al., 2014). To evaluate the functional impor-tance of commensal bacteria in the initiation and progression of tumors in this model, we first compared tumor development be-tween germ-free (GF) KP mice and their counterparts maintained under specific pathogen-free (SPF) conditions (Figure 1A).
As shown in Figure 1A, GF mice were significantly protected against lung cancer development induced by Kras mutation and p53 deletion. Compared with age-matched SPF controls, GF KP mice exhibited substantially delayed tumor growth, with diminished tumor burden and decreased tumor numbers at both 8 and 15 weeks post-tumor initiation, and lower percent-ages of high-grade lesions (Figure 1A). At the cellular level, GF mice displayed reduced tumor cell proliferation as demonstrated by immunohistochemistry (IHC) analysis of Ki67 staining (Fig-ure 1A). To further confirm that the absence of microbiota in GF mice is responsible for their decreased tumor burden, we examined tumor development in GF KP mice exposed to the mi-crobiome via cohousing with SPF mice (referred to as ex-GF mice). Ex-GF mice displayed significantly increased tumor burden compared to their counterparts maintained in the GF conditions (Figure S1A).
To determine the effects of microbiota on different stages of lung tumor progression, we treated SPF KP mice with an anti-biotic cocktail (4Abx) of ampicillin, neomycin, metronidazole, and vancomycin at different time points after tumor initiation (2 weeks post-infection, Figure S1B; 6.5 weeks post-infection, Figure 1B). We found that this treatment robustly suppressed tu-mor growth in both early and advanced stages, resulting in sig-nificant reduction of high-grade tumors (Figure 1B). Notably, antibiotic treatment did not affect the proliferation of tumor cells in vitro (Figure S1C), arguing against the possibility of direct cyto-toxic effects of these antibiotics. Together with the findings in GF KP mice, these results demonstrate that the commensal bacteria play a profound role in promoting tumor development in the autochthonous GEM model of LUAD.
Lung Tumor Development Is Associated with Altered Local Microbiota and Increased Pro-inflammatory Cytokine Expression