Thyroid cancer is the most frequent endocrine malignancy (1). The four main types are papillary thyroid cancer, follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer (2). Papillary thyroid cancer (PTC), a well-differentiated thyroid carcinoma with an incidence rate of 85%, tends to be relatively less aggressive and has a generally favourable prognosis (3). In contrast, 2% of thyroid cancer cases are comprised of anaplastic thyroid cancer (ATC) and undifferentiated thyroid carcinoma (2). Unlike PTC, ATC is highly aggressive and has a dismal prognosis (4). Despite of efforts to describe the mutation spectrum, fusion genes, copy-number modifications, and transcriptome alterations, the biology underlying thyroid cancer remains largely unknown.

Recently, microbes have been described as a characteristic hallmark of cancer (5). As commensal organisms, they are part of the human body’s normal flora. According to Globocan 2020, 19.7% of the countries in South Asia have malignancies infected with various bacteria (6). For instance, 60–90% of stomach cancer cases are linked to Helicobacter pylori (7); cervical cancer and head and neck cancers with Human Papillomaviruses (HPV) (8, 9); nasopharyngeal cancer with Epstein-Barr virus (EBV) (10); colorectal and head and neck cancer with Fusobacterium nucleatum (8, 11); Cutibacterium acnes with prostate cancer (12, 13). Pathogens, in general, play an immunomodulatory role in cancer, promoting tumour growth and generating chronic inflammation upon prolonged contact. However, the association between pathogenic microorganisms and thyroid cancer lacks systematic analysis, with sparse information about the functional association between microorganisms and their ability to control immunosuppressive behaviour.

Here, we investigate the microorganisms linked to thyroid cancer. We performed whole transcriptome sequencing on papillary thyroid cancer (n = 40) and anaplastic thyroid carcinoma (n = 30), in addition to reanalysing other malignancies with similar datasets, as reported earlier (14). Using Infectious Pathogen Detector (IPD 2.0) (14), we compared the microbial abundance of 70 thyroid cancer samples with five separate in-house cancer samples: breast cancer (n=49), tongue squamous cell carcinoma (TSCC) (n=15), cervical adenocarcinoma (n=21), and colorectal cancers (n=33). Cutibacterium acnes (or propionibacterium), a gram-positive facultative anaerobe, was significantly enriched in thyroid cancer tissue samples. Differential gene expression between thyroid cancer samples with high and low Cutibacterium loads suggests immune-modulatory pathways. In addition to analysing the immune cell fractions of both groups, we discovered a significant concentration of immunosuppressive cells in thyroid cancer samples with a higher Cutibacterium load. In the C. acnes high group, we observed an increase in immunosuppressive cytokines and a decrease in pro-inflammatory cytokines, validating our hypothesis. Cutibacterium acnes is related to late-stage illness, poor prognosis, and the formation of an immunosuppressive milieu in thyroid cancer, according to our findings.

Cutibacterium acnes is the most prevalent commensal on healthy human skin, subserous glands, mucosal surfaces, and head and neck (28, 29). These microbes maintain balanced symbiotic/commensal relationships or a ‘eubiosis’ under normal conditions. A shift in the eubiotic balance or a ‘dysbiosis’ can lead to disease, as reported in multiple cancers (30–32). Under certain conditions, Cutibacterium acnes is known to cause deep tissue infections (33–36) and promote prostate cancer tumorigenesis through interferon-1 signalling and the cGAS-STING pathway (12, 28, 37, 38). While there are no reports of Cutibacterium acnes in the thyroid gland, a dysbiosis of the gut microbiome has been linked to thyroid cancer (39, 40). In this study, we systematically analysed whole transcriptome datasets of 70 thyroid cancers along with 118 tumour samples of breast, cervical, and other cancer types. Using Infectious Pathogen Detector, IPD 2.0, we standardized the quantification of distinct cancer-associated pathogens in several tumour types. In our analysis, we observed that Cutibacterium acnes-high tumours formed a distinct subgroup of thyroid cancer. We found a 5.35-log₂ fold enrichment of the bacterium with 1.66891E-09 p value in the papillary and anaplastic thyroid tumour tissues as compared to other tumour types ( Figure 1B ; Supplementary Figure S3 ). A similar enrichment of Cutibacterium acnes was also reported earlier in prostate cancer (13). Significant enrichment of the bacterium in papillary and anaplastic thyroid carcinoma thus does underscore the association of the bacterium with the disease.

To elucidate the molecular and clinical characteristics associated with Cutibacterium acnes burden in thyroid cancer, we analysed the differential gene expression between Cutibacterium-high and -low tumours. The highly elevated genes varied dramatically between groups. Multiple inflammatory pathway genes were upregulated in Cutibacterium-rich tumours, indicating an inflamed tumour microenvironment. In addition, CytoSig analysis revealed that the Cutibacterium-high group had significantly higher levels of anti-inflammatory cytokines, including IL4, IL27, HGF, and TWEAK, while lower levels of pro-inflammatory cytokines, including IL1A, IL1B, IL6, IL12, and OSM. The apoptosis-inducing cytokine TRAIL was also discovered to be downregulated in the Cutibacterium high group. This shows that Cutibacterium acnes may play a role in thyroid cancers by inducing immunosuppression under an inflamed environment. The immunological profile of malignancies has been associated with microbial infection and bacterial dysbiosis. This suggests a possible role of Cutibacterium acnes in thyroid tumours by induction of chronic inflammation. We analysed the immune cell composition of Cutibacterium-high versus -low tumours using CIBERSORTx and detected a rise in T regulatory, or Tregs, cells. Immune cell enrichment study and gene expression analyses reveal that Cutibacterium load promotes an immunosuppressive environment, as reported in prostate cancer (41). However, Th17 and Tregs interplay and role in immunosuppression is emerging (42, 43). Th17 cells composition in combination with Tregs composition could give us more insight in immnosuppressive effect under C. acnes infection. However, CIBERSORTx analysis does not give output for Th17 cells and thus we could not determine the dual effect of Th17/Tregs in immunosuppressive environment. In addition, immunological suppression was shown by the downregulation of the Tregs inhibitory pathway and the pro-inflammatory signalling pathways, such as interleukin-1 signalling. In addition, we found that a larger load of the microorganism is associated with shorter overall survival in patients with thyroid cancer treated in-house.

There are several limitations of our study. Firstly, based on our findings, we are though unable to discern if this association (enrichment) is causal to the disease or a consequence. The establishment of causality requires additional co-culture experiments. Such experiments have the potential to provide a deeper understanding of the interactions between Cutibacterium acnes and thyroid cancer cells, shedding light on whether the bacterium actively contributes to the initiation and progression of cancer or whether its presence is a consequence of the cancerous environment. Such findings may have significant implications for thyroid cancer research and may lead to more precise diagnostic tools and individualized treatment strategies for affected individuals.

Secondly, we found that procedures like sample collection, DNA/RNA extraction, missing sampling blanks, samples/biopsies where the skin surface is traversed during biopsy collection in the FFPE production and sequencing could introduce microbial contamination, potentially affecting results. To ensure accuracy, we took strict precautions. The measures aimed to minimise contamination and enhance the reliability of our research, we used sterilised equipment, worked in a controlled environment, and followed rigorous lab protocols, including wearing sterile gloves and maintaining clean workspaces. Furthermore, in cases where tumours undergo biopsy procedures, particularly those involving percutaneous techniques, it is important the potential for microbial contamination originating from commensal bacteria that are frequently present on the skin or that predominantly inhabit sebaceous glands could not be thoroughly ruled out as described earlier (44, 45).

Thirdly, relating to computational microbial quantification protocol, a recent work by Gihavi et al. underscores the false positive detection of microbes from the metagenomic studies due to misclassification and batch effects (46). In our current study, we used two computational approaches (IPD 2.0 and Kraken2), working on distinct approaches, to quantify microbes from samples. IPD 2.0 uses stringent two step approach to negate human-microbe misalignment in the analysed samples. In the primary alignment step the sequence traces are first aligned to human genome and later un-aligned sequences are considered further. In the secondary alignment, IPD 2.0 further confirms the sequence uniqueness towards genera by ruling out any sequences which align significantly to multiple organisms (including human contigs) using a conventional alignment method Blast+ (47). We thus implement rigorous steps to address false positive detection of microbial traces in our dataset. Further, in the PCR validation step as described in methodology, we have QC’ed our primers and we ensure the identification of C. acnes based on melt curve analysis, negative samples and non-template controls where we did not see the amplification. Further, we classified thyroid tumours having the top 10% pathogen load as Cutibacterium -high and with no expression as Cutibacterium -negative.

Overall, the research identifies a correlation between the presence of Cutibacterium acnes and an immunosuppressive environment in cases of thyroid cancer, without establishing a causal relationship between these factors. Of note, the disparities in Cutibacterium acnes concentrations observed between thyroid cancer and other types of cancer may be attributed to inherent fluctuations in the microbiome, rather than a direct influence on the progression of thyroid cancer. In the same way, the increase in Cutibacterium acnes presence in thyroid cancer may be attributed to alterations in the tumour microenvironment. It is worth noting that the existence of microorganisms as well as the severity of cancer may as well be impacted by patient-related factors such as overall health and genetic predisposition. Additionally, it is crucial to emphasise that the study is based on retrospective data obtained from a singular region, which may introduce inherent biases and restrict the extent to which the findings may be generalised. Similarly, the potential association between Cutibacterium acnes and a more unfavourable outcome may be fortuitous, possibly attributable to unidentified confounding factors. Conversely, it is plausible that the microorganism could exert an impact on the response to treatment, rather than directly facilitating the progression of aggressive tumours. Taken together, these aforementioned limitations highlight the necessity for additional experiments in order to elucidate the intricate connections between Cutibacterium acnes, the tumour microenvironment, and the advancement of thyroid cancer.

Our study presents a comprehensive landscape of microorganisms found from the transcriptome dataset of Indian origin across six distinct tumour types. We identified a subpopulation of thyroid cancers with a poor prognosis that may be driven predominantly by inflammation caused by Cutibacterium and other microorganisms. We further demonstrate that Cutibacterium is associated with advanced disease and poor overall and progression-free survival in thyroid cancer patients. This study could be a basis for designing more studies to understand the diversity in treatment response, disease recurrence and resistance, and adoption of additional measures such as using antibiotics for similar cancers of specific origin.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The genomic datasets can be accessed from the Array Express database under the accession numbers E-MTAB-12479, E-MTAB-12476, E-MTAB-9766, E-MTAB-6619, E-MTAB-4653, E-MTAB-8801, E-MTAB-11404, E-MTAB-9281, and E-MTAB-11407. The colorectal cancer data have been deposited at the European Genome-phenome Archive, under accession number EGAS00001005970.

The studies involving humans were approved by The Institutional Ethics Committee (IEC) of Tata Memorial Centre approved the study with wavier of consent (IEC number 900744). The studies were conducted in accordance with the local legislation and institutional requirements. The ethics committee/institutional review board waived the requirement of written informed consent for participation from the participants or the participants’ legal guardians/next of kin because The study was conducted on retrospective samples.

VT, AD, and KP each participated in the conceptualization and design of the study. Additionally, AD and KP participated in Project administration, Supervision, Writing (review & editing), and fund acquisition. VT participated in Data curation, Formal analysis (performing experiments and data analysis), Investigation, Software (programming codes), Validation (qPCR-based validation), Visualization, and Writing-original draft. SD, DP, VN, RM, VP, AB, and AC participated in the formal analysis. PS, LV, PG, VV participated in methodology (transcriptome library preparation and data sharing), MB, NMi, SR, SK, SB, PaC, PrC, NMe, and AS provided the resources for the study. All authors contributed to manuscript revision, read, and approved the submitted version.