Lung cancer is a malignant tumor with the highest morbidity and mortality worldwide.¹ It has been widely deemed a complicated disease caused by interactions between host and environmental factors. However, the mechanisms by which environmental risk factors and other tumor-extrinsic factors control lung carcinogenesis are still poorly understood. Bacterial infections are prevalent in lung cancer patients. Up to 50%–70% of lung cancer cases are complicated by pulmonary infections throughout the disease.² In such diseases and treatments, there must be a process of microbiota dysbiosis and recovery.

With extensive research on the mechanism of lung cancer occurrence, studies have discovered that the development and progression of cancer are associated with the failure of immune surveillance. Therefore, new treatments have emerged as a practical option for patients diagnosed with advanced lung cancer. An immune checkpoint inhibitor (ICI) is capable of relieving the immune escape. ICIs impede tumors from activating inhibitory checkpoint pathways on immune cells, thereby unleashing the host's immune response. However, inter-individual responses to ICIs are often heterogeneous. Multiple mechanisms of ICI resistance were implicated in the poor response rates, including low tumor mutational burden (TMB), low programmed death-ligand 1 (PD-L1) expression, poor antigenicity of tumor cells, local immunosuppression, functional exhaustion of tumor-infiltrating lymphocytes, and the absence of priming or defective antigen presentation during priming.³

Additionally, increasing evidence has revealed that the commensal microbiota is an essential biological factor contributing to inter-individual heterogeneity of response to ICIs. Clinical and/or preclinical studies have indicated that the gut microbiota composition can significantly influence antitumor immunity and immunotherapy efficacy.⁴ Compared with other tumor biomarkers, including PD-L1 and TMB, the commensal microbiota is plastic. Evidence has shown that anti-PD-1 response could be improved by increasing the α diversity or enhancing the abundance of specific microbes. However, most published data on the impact of microbiota in regulating the efficacy of different types of drugs were obtained by studying the gut bacteria. Given an expanding knowledge of the microbiome's effect (and specific microbes) on normal immune system development and drug action, the hypothesis that the microbiome influences therapeutic tumor responses should be further investigated. The recent discovery of the microbiome's impact on the efficacy of checkpoint inhibitors and the subsequent work to elucidate the immunological mechanisms driving these effects has revolutionized microbiome research in oncology.

Although recent studies in the field focused on the interplay of microbiota and immunity in the intestine, interactions between the gut microbiota and other microbiota communities with extra-intestinal organ immunity are gaining increased attention. Particularly, lung cancer-related microbiota has expanded and refined from the systemic influence of intestinal flora to the local regulation of respiratory or tumor flora. With the development of many studies, it was discovered that different bacterial parts or bacterial sources play different or even opposite roles in disease regulation. The relationship between the respiratory microbiota and the response to ICIs in lung cancer has not been widely explored, although the respiratory microbiota was found to provoke inflammation associated with lung cancer.⁵

The existing data, obtained by comparing the lung microbiota of healthy persons versus patients with different lung diseases, revealed significant differences in lung commensal composition, even in lung cancer patients. Although the microbiota identified in lung cancer may vary from one study to another depending on the sample type, sampling method, and patient cohort, the findings are consistent that lung cancer is associated with a dysregulated local microbiome, featured by increased total bacterial abundance, reduced α-diversity, and altered bacterial composition.⁶ Although there are some usual mechanisms (eg, the release of free radicals) that cause damage to DNA and other proteins, there are other complex molecular mechanisms showing the role of lung microbiota in inducing carcinogenesis.⁶ The microbiome in tumor evolution has confirmed that although the microbiome can influence cancer cells themselves, it can also modulate the cancer-immune environment. Because of the extensive exposure of the lung to external environment, it is a critical site of immune–microbiota interaction and exists in homeostasis maintained by lung-resident immune cells.

Recent studies point toward the potential of locally entrenched lung microbiota to impact pulmonary immunity. It was recently demonstrated that microbiota-immune cross talk can activate tissue-resident lymphocytes to establish a protumorigenic microenvironment. The increased local bacterial diversity and altered composition of lung microbiota stimulated interleukin (IL)-1β and IL-23 production from myeloid cells. These cytokines promoted the activation and proliferation of Vy6+Vδ1+γδ T cells.⁵ An opposite dynamic was observed in pulmonary melanoma metastases models, where antibiotics impaired a functional γδ T–cell induction and IL-17 production, leading to an accelerated pulmonary metastases, emphasizing the highly context-specific microbiome-immune interaction.⁷ Additionally, in a murine model, the administration of aerosolized antibiotics, which significantly decreased bacterial biomass, was linked to an enhancement of antitumoral immune response via T cell and natural killer (NK) cell activation and reduction of immunosuppressive regulatory T cells, and tumor metastases were reduced.⁸ Exploring the roles of the lung microbiome in impacting immunity in lung cancer require more mechanistic studies. Indeed, the bidirectional relationship between microbiome perturbation and immune dysregulation is still limited to research based on animal models. However, it should be highlighted that mice carry intrinsic biases that might represent confounding factors during the interpretation of the results. Although it was confirmed that the germ-free mice have a similar level of B and T cells, conventional and CD103 + dendritic cells, and Plasmacytoid Dendritic Cells compared to normal mice, the lack of microbiota regulatory with the correct progress of the immune system.⁹ Therefore, alterations in the immune response in these mice also testify that they do not respond to different types of immunotherapy.

On this basis, data were sequenced based on high throughput in our own 30 advanced lung cancer patients. The lung tumor samples from our study were obtained at the time of surgery, from which a section of tumor was removed. The removed section was placed in a sterile vessel. The tissues were snap-frozen in liquid nitrogen and placed at −80°C for long-term storage until DNA extraction. We also explored the relationship and potential mechanisms of propagation of lung microbes and the local immune microenvironment of lung cancer among the Chinese population, taking into account the high heterogeneity of the flora. To delineate the immunological landscape in our samples, we analyzed our gene expression profiles by single-sample Gene Set Enrichment Analysis (ssGSEA), which classifies gene sets with common physiological regulation, chromosomal localization, and biological functions.¹⁰ Gene markers of 28 immune cells were obtained from a previous study.¹¹ We used correlation analysis to identify associations between the lung microbiome and tumor-infiltrating immune cells. Similar conclusions were obtained as described above. According to the results of the joint analysis, it was confirmed that the diversity of lung flora is associated with multiple immune cell immersion (Figure 1a). The above mentioned γδT cells, although they may not be statistically meaningful because of the small sample size, are a good reflection of trends consistent with the content of the article (Figure 1b). The infiltration of CD56 bright NK cells in tumor tissues is a close negative correlation with the α diversity of the lung flora (Figure 1c). To some extent, the conclusions reached in animal models were confirmed from the dimensions of the patient sample. Theimportance of the local tumor microbiota to the tumor immune microenvironment in situ has been further confirmed. It is also essential for the interaction of local flora in the study of the systematic role of intestinal flora. Therefore, multi-location, multi-factor tumor flora research is imperative. However, the study of the lung microbiome and the interplay between commensal microbial communities and pulmonary immunity is only in its infancy, with many more mechanistic insights to be revealed in future studies.

Finally, there is a growing understanding that tumors host microbes, with variations dependent on each specific location of the tumor. In Science, Nejman et al.¹² synthetically analyzed the tumor microbiome and revealed its reasons and potential effects in different tumors.¹² This research provides a strong foundation and new direction for future research on intratumor microbiology.

Thecausal role of microbiome in tumor development has always been controversial. However, whether or not bacteria play a causal role in tumorigenesis, it is necessary to explore the influence of intratumor bacteria on the different cancer phenotypes, immune system, and its interactions with tumor cells. Therefore, with the establishment of more extensive studies, both in animal models and patient lung tissues, it is still essential to validate these findings and to gain a more profound knowledge of the composition of lung microbiota and their roles in lung cancer. Such research could lead to novel therapeutic targets and potentially better treatment and disease outcomes in lung cancer patients.