Microbiota Diversity in Non-Small Cell Lung Cancer Gut and Mouth Cavity Microbiota Diversity in Non-Small Cell Lung Cancer Patients

Altogether, 141 NSCLC patients seeking care at the Department of Respiratory Diseases, University Hospital Olomouc, were recruited for the study (Table I). However, some of the patients diagnosed to have a lung lesion on X-ray and thus suspected to suffer lung cancer were also sampled at the time of yet unclear diagnosis to ensure the culture results not to be biased by any healthcare interventions assumed to follow. Most of them turned out to carry a metastasis of an extrapulmonary cancer or lymphoma. These patients were included as a quasi-control group to support the correct interpretation of the differences observed when comparing the microbiota of adenocarcinoma versus squamous cell carcinoma patients. Patients were recruited from April 2019 to October 2022, regardless of disease stage, age, smoking status, or gender.

Rectal swabs were collected using the Transystem™ 116C Traditional Bacteriology Transport Swabs (COPAN Diagnostics Inc., USA); 10 ml of sterile saline was moved into the mouth cavity and collected into a sterile tube. On the same day, both samples were delivered to the laboratory. Petri plates with Columbia Blood Agar (CBA), MacConkey Agar (MCA), Brain Heart Infusion (BHI) agar with 10% sheep blood, Schaedler Agar (SChA), and nalidixic acid and sulfamethazine (NAS) (Waite et al. 2012) agar plates were inoculated for bacterial cultivation. Concerning the purpose of different plates, CBA was inoculated to grow a broad spectrum of aerobic bacteria, and MCA to grow the subset of non-fastidious gram-negative bacteria to enable better differentiation of their colony morphology compared to CBA. BHI and Schaedler Agar were inoculated to grow anaerobic bacteria, and the semi-selective NAS agar was inoculated and cultured under microaerophilic conditions to facilitate the growth of streptococci, Streptococcus anginosus group (SAG), in particular. Sabouraud Glucose Agar with chloramphenicol (SGA) was inoculated for fungal cultivation (all from Oxoid, UK). Plates were incubated at 37°C in ambient air supplemented with 5% carbon dioxide (CBA, MCA), at 37°C in an anaerobic gas mixture (BHI, SChA, NAS; 80% nitrogen, 10% carbon dioxide, 10% hydrogen), and 30°C in ambient air (SGA). All aerobic plates were kept in a moist chamber during incubation to protect agar from drying and secure the best growth of microbes. Bacterial plates were incubated for 3 days; SGA was incubated for 5 days.

All colonial morphotypes were subcultured for 24 hours on appropriate media and conditions to verify pure culture and achieve the best results following identification; the cultivation period was appropriately prolonged in slowly growing isolates to collect sufficient cells for identification. Bacterial isolates were subcultured on Columbia Blood Agar, and toothpick sampling was used to prepare a direct smear on MALDI TOF target plate, which was then overlaid by 1 μl of 70% formic acid, air dried, and overlaid by 1 μl of matrix solution (α-cyano-4-hydroxycinnamic acid solution, HCCA; Sigma-Aldrich, USA). A MALDI Microflex^® LT instrument (Bruker Corporation, USA) was used for identification in automated mode by the manufacturers’ instructions (MALDI Biotyper^® 3.1 User manual revision 1). In cases when the confidence score values dropped below 2.0, user-targeted laser shots were performed additionally to improve the score. Because of the limited resolution power in the case of the Streptococcus mitis group (S. mitis, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pseudopneumoniae), members of this group are referred to as one complex species.

Multiple Correspondence Analysis was applied to the microbiome data to assess the relationships between all the observed variables. Both the TNM level and the site were – independently – used as supplementary variables in the multivariate model so that they would not affect the construction of the model and allow the determination of the overall dependence of microbiome composition on those parameters. The chi-squared test was applied for further bivariate assessments. Kaplan-Meier survival curves were plotted for all the categorical variables and Peto and Peto modification of the Gehan-Wilcoxon test was used to assess the differences between the curves. All the statistical analyses and visualizations were performed in the R environment (R Core Team 2021). The multivariate assessment was performed using the R package FactoMineR (Lê et al. 2008); the survival analysis was performed using sur-vminer (Kassambara et al. 2021) and survival R packages (Therneau et al. 2023). The chi-square test or Fisher’s exact test was applied appropriately to evaluate the statistical significance of differences in the frequency of each identified species between ADC and SCC.

R. aeria was the species showing the highest overrepresentation in ADC versus SCC patients; however, it was found in very few cases, which limits the informativeness of the result. Similarly, the second-ranked S. constellatus, a streptococcal species causing pyogenic infections in various body sites, showed low numbers without statistical significance. However, it is worth mentioning its association (together with S. anginosus, see further in the text), with early gastric cancer (Zhou et al. 2022). Whereas little is known about the pathogenic or pro-oncogenic potential of N. maccacae ranked 3, the No. 4 S. mutants has been described to promote tumor progression in oral squamous cell carcinoma (Tsai et al. 2022). S. sanguinis has no apparent link to any tumor. However, it is recognized to be associated with infective endocarditis, and the elevation of pro-inflammatory genes has been observed in mouse models (Hashizume-Takizawa et al. 2019).

The picture is brighter in the case of S. anginosus, which appears to be directly linked to the development of esophageal, gastric, pharyngeal, and oral cancer (Zhang et al. 2019). Although other members of oral microbiota (Porphyromonas gingivalis, Fusobacterium nucleatum, Treponema denticola) have also been linked to oral cancer, none of them is known to cause infection of the respiratory pathways, whereas S. anginosus was described to do so in at least two chronic respiratory diseases, namely cystic fibrosis, and chronic obstructive pulmonary disease (Parkins et al. 2008; Navratilova et al. 2016). S. anginosus, when present in the oral microbiota, may thus be capable of affecting respiratory pathways and increase the risk of ADC much more than SCC, which is strongly linked to smoking. On the other hand, the difference is not as distinct and unequivocal (53.5% in ADC versus 34.6% in SCC, p = 0,028) as it might be in the case of a straightforward mechanism.

Therefore, S. anginosus interstrain differences in pro-inflammatory action described earlier. Interestingly, the association between S. anginosus presence in microbiota and risk of ADC was also confirmed by rectal swab culture (49.4% in ADC versus 22.6% in SCC, p = 0,002). This suggests that this bacterium is perfectly suited to colonize both the mouth cavity and the gut, with no preference for one of these environments over the other. We can only speculate about possible other mechanisms of S. anginosus pro-oncogenic activity beyond the general pro-inflammatory action. To mention at least one of them, the production of hydrogen sulfide (H₂S) can be found in a long list of S. anginosus virulence factors (Asam and Spellerberg, 2014) and H₂S has also been described to create a favorable immune microenvironment for colon cancer (Yue et al. 2023).

Regarding the species underrepresented in ADC versus SCC, the probiotic species L. fermentum, which is widely known for its anti-inflammatory properties, was significantly underrepresented in ADC. The commensal yeast species Candida tropicalis was found in low numbers and the difference was not significant.

When the data from the ADC versus SCC mouth cavity microbiota relation were compared to the quasicontrol group, the significance was convincingly confirmed in the case of the overrepresentation of S. mutans and S. anginosus and underrepresentation of L. fermentum, but questioned in N. macacae and S. sanguinis, where any hints about a possible role in tumorigenesis are also lacking in the available literature.

Not surprisingly, less pronounced differences between ADC and SCC patients were found in the complex culturable gut microbiota sampled by rectal swabs. K. oxytoca was the species showing the highest overrepresentation in ADC versus SCC patients. However, it was found in very few cases, which limits the informativeness of the result. S. anginosus was the only one that met the criterion of statistical significance among the other species found to be overrepresented in ADC versus SCC; the same was true for C. aurimucosum in the species underrepresented in ADC versus SCC. Interestingly, C. aurimucosum was also found in individuals previously less responsive to NSCLC targeted therapy (Routy et al. 2018; Sędzikowska and Szablewski 2021).

When the data from the ADC versus SCC rectal swab microbiota relation were compared to the quasicontrol group to guarantee a more reliable interpretation, the significance of S. anginosus overrepresentation (p = 0,109) was not convincingly confirmed, whereas C. aurimucosum underrepresentation (p = 0,024) was.

Taken together, the considerable overrepresentation of S. anginosus in both the mouth wash and rectal swabs of ADC versus SCC patients is the most noticeable discovery of our study, which has yet to be reported in any microbiome-based study. Because ADC represents one of the most frequent forms of NSCLC, our finding of relative overrepresentation of streptococcal species in the mouth wash of ADC patients (S. anginosus, S. constellatus, S. mutans and S. sanguinis) is well in accordance with the results of (Tsay et al. 2018) who described the increased abundance of streptococcal species in lower respiratory pathways of lung cancer patients compared to controls.