The role of microbiota in carcinogenic processes has not yet been elucidated and will probably be different for each tumor and their localization. However, recent data clearly indicate that the microbiota contributes to the prognosis of cancer and determines the response to treatments, particularly responses to the new immunomodulatory therapies [9, 28]. Criteria for the normal composition of lung microbiota have not yet been established, but the available data indicate that their composition in cancer patients differs considerably from that of healthy individuals [4–8]. Sampling in the respiratory system requires invasive methods, and here we have characterized for the first time, as far as we know, the respiratory microbiota surrounding central lung cancer by direct sampling of the tumor tissue by bronchoscopy. Our results reinforce the previously known particularities of the lung microbiota, which considerably differ from oral and stool microbial communities [29]. One important concern is the theoretical risk of contamination with the upper microbiota during the bronchoscopy, but our results allowed us to rule out significant contamination, as other authors had previously suggested [30–32]. Data analysis by various methodologies highlighted the particularities of the microbiota associated with cancer, but also defined the respiratory microbiota core in healthy conditions. Moreover, we considered strict inclusion criteria to avoid the possible bias of antibiotic or corticosteroid therapy, and the bacterial exchange between the anatomically separated niches such as saliva and feces. Finally, the mycobiome composition of central lung cancer was studied.
Significant differences in the lung ecosystem have been described based on health status or a lung cancer diagnosis in sputum [33], bronchoalveolar lavage [5], protected specimen brushing [6], cytological brushing [34], and surgical tissue [7, 8, 35]. The major contribution of our work is the study of the microbiome surrounding central cancer via direct sampling, but our results are not necessarily applicable to the distal airway. Interestingly, this central ecosystem was more diverse than the fecal or oral compartments. This finding contrasts, at least in part, with decreases in the microbiota biomass from upper to lower tract described in healthy people [32].
Low biodiversity is usually observed in various pathologies, including cancer, but we found significantly higher alpha-diversity values in cancer than in the control group. Other authors have published analogous [8] and opposing results [5, 6, 35]. Moreover advanced cancer stages [36] and reduced recurrence-free survival and disease-free survival [35] have been associated with higher values of alpha diversity. Eighty-eight percent of our patients were at tumor stage III or IV, and their survival was only of 198 days in the follow up. Furthermore, other factors such as environmental exposure, residence in high-population density areas [4, 36], and pack-years of tobacco smoking, can increase the biodiversity of the lung microbiota, whereas chronic bronchitis reduces it [36]. All our patients had been smokers, while most of the controls (56%) had not (Table 1).
In terms of beta diversity, the tissues involved in cancer and the contralateral bronchus had almost identical compositions, as its have been previously published [6, 34, 35], probably reflecting the environmental influence which is not restricted to the cancer area. Although the limited size of our sample prevents us from reaching solid conclusions, no differences were detected in the composition of the bronchial microbiota as a function of the histological variants of the cancer. The abundance of Firmicutes to the clear detriment of Proteobacteria was the most noticeable result in our patients, and this result was consistent in all samples. Proteobacteria dominance in health lung microbiota, especially Pseudomonas, has been also previously corroborated [5, 6, 37]. Higher concentrations of Streptococcus, Blautia, Akkermansia, and Rothia were observed in patients, but Streptococcus was consistently the major marker linked to lung cancer. This fact has been previously reported in saliva [5], sputum [33, 38], bronchoalveolar lavage [5], lung tissue [7], and protected specimen brushing [6, 34]. The exhaustive analysis of the obtained ASVs allows us to suggest that the streptococcal variants present in lung tissue are similar to those found in saliva or feces, but the low length of the 16 s rDNA amplicon sequences (460 bp) preclude us from making robust assumptions in this regard. New studies including Streptococcus cultures and molecular characterization of the species are needed to decipher whether oral lineages are different from those found in the lungs or feces and thus establish whether there are any markers truly associated with lung cancer, as occurs with Streptococcus gallolyticus subsp. gallolyticus and colorectal carcinoma [39].
There is increasing evidence of a link between Streptococcus and lung cancer. Recently, Tsay et al. [34] detected Streptococcus and Veillonella enrichment in the lower airways with ERK and PI3K pathways upregulation -an early event that contributes to cell proliferation, survival and tissue invasion- combining microbiome and transcriptomic signatures. The major question that a remains to be answered is whether the abundance of streptococci is a cause or consequence of the tumor process, as has been questioned in tumors from other localizations [40]. Streptococcus is a natural inhabitant of the oral cavity, which is connected to the lower respiratory tract by the larynx and trachea. The ASVs analysis allowed us to separate the oral streptococcal population from those found in the lung or gut, although the oral/lung bacterial exchange could occur via microaspirations [6, 34, 37].
Microaspiration events are common, but their frequency is significantly increased in chronic inflammatory airway diseases [37], inducing inflammation by elevation of Th17 lymphocytes, as well as expression of inflammatory cytokines (as IL-1α, IL-1β and IL-17). The alteration of the IL-23/IL-17 axis is well known in the pathogenesis of both autoimmune diseases and tumors. Recently, it has been described that Streptococcus mitis induced IL-1β, IL-6 and IL-23 transcription and Th17 responses able of releasing the potentially proinflammatory and protumoral IL-17 [41]. S. mitis also leads to neutrophil recruitment and inflammation, macrophage chemotaxis, a higher secretion of immunological inhibitory cytokine IL-10, and an increased immune checkpoint PD-L1 expression, facilitating the cancer development and expansion [41]. Lung resident γδ T cells, and their either protective roles or pro-tumorigenic functions in cancer have been recently discovered [42]. A study has provided evidence that local lung microbiota (one of the most common genera was Streptococcus) can provoke inflammation and tumor cell proliferation, via lung resident γδ T cells activation, that release IL-17, after IL-1β and IL-23 induction in myeloid cells by these local microbiota [43]. Our results demonstrated a global streptococcal enrichment in patients with cancer that affected more than just the respiratory tract, supporting the idea that microorganisms can orchestrate the balance between tumor-promoted inflammation and anti-tumor immunity depending on the specific microenvironment [43].
Streptococcal relative abundance in bronchial biopsies was a good predictor of lung cancer, but unfortunately was not reproducible in saliva. ROC curves suggested the contralateral bronchi as the best sample (90.9% sensitivity and 83.3% specificity; AUC = 0.897). Other authors found similar results in protected specimen brushing samples (87.5% of sensitivity and 55.6% specificity, AUC = 0.693) [6]. The proportional abundance of Streptococcus should be validated in the early stages of lung cancer with subsequent follow-up to corroborate this link. Along these lines, the intestinal enrichment of Streptococcus should be exhaustively explored to identify lung cancer markers in feces.
The intestinal and the respiratory ecosystems harbor a diverse and abundant microbiota, but some particularities distinguish both ecosystems. Food intake favors a higher rate of microbial reproduction increasing the total mass that is significantly reduced after defecation. On the contrary, nutritional sources for bacteria in the airway are limited to mucus and cellular debris, while clearance is carried out by the ciliary system and the immune system, particularly by macrophages. Food is an important mode of entry of foreign microorganisms into the gut ecosystem, as is inspired air. Alien microorganisms can lead to overestimation of the real diversity of the ecosystem, but we have implemented new analytic strategies to define the lung microbiota core, which had not previously been defined. Interesting findings of our work include the elevated alpha-diversity of the bronchial microbiota in comparison with saliva or feces, and the dominance of Pseudomonas in healthy individuals. The pulmonary presence of this genus is linked to cystic fibrosis and is the major pathogen that decreases the respiratory functionality within a pathogenic colonization. However, the lack of respiratory symptoms reduces a pathogenic role of Pseudomonas in healthy individuals, although more studies are needed in that line. Predator bacteria have been classically described in environmental ecosystems as scarcely detected in human samples [44–45]. Their low representation in the total ecosystem could prevent their detection by -omic strategies. This approach is an important field of research, which could represent an ecological key to modulating the microbiota composition.
The main limitation of our work is that we cannot estimate the effect of lung cancer factors, mainly tobacco (all patients had been smokers but only the half of the controls were) and COPD (12/25 patients) on the bronchial microbiota. However, it has not been established yet if tobacco has a significant influence on lung microbiota composition, and some important studies have shown contradictory results [36, 46–47].
Whereas severe COPD has been linked with significant alterations in the lung microbiota composition [48–49], mild and moderate COPD (92% of our COPD patients) has been associated with Streptococcus enrichment [50]. This finding might explain the association of mild/moderate COPD and lung cancer [51], although further studies are needed to confirm this association.
Our study has several additional limitations, including a small number of patients, all of them from the same hospital, a lack of other -omic analyses based on genetic expression, and we cannot rule out the possibility that the differences were be influenced by tobacco exposure. On the other hand, our strengths include performing the first study of microbiota combined with mycobiome of bronchial tissue obtained directly from tumor and contralateral bronchi (not adjacent to a resected tumor), as well as performing analysis of the connected ecosystems including saliva and feces.
The mycobiome results were consistent with those obtained for bacteria. The fungal community was slightly richer and more diverse in patients than in controls, although the contralateral bronchus was more similar to controls than to the affected counterpart. The mycobiome of saliva and the affected bronchus from patients matched perfectly, but differed in controls, again suggesting that in patients with cancer the bronchial microbiota is the result of a continuous exchange with that of the oral niche. In terms of taxonomy, affected bronchi from patients had an enrichment of the Basidiomycota phylum with higher populations of Malassezia genus, whereas the enriched taxon in healthy individuals was the Ascomycota phylum and the genera Candida and Saccharomyces, as previously described [41, 52]. Although the public databases are increasing exponentially, it is important to note that a major limitation to describing the mycobiome is the lack of available taxonomic records. As far as we know, this is the first description of the lung mycobiome.