Pancreatic ductal adenocarcinoma (PDAC) is one of the most common lethal cancer entities with hardly 10% of the patients surviving up to 5 years after diagnosis (1). Owing to the lack of early and specific symptoms, the majority of patients are diagnosed at an advanced- or even metastasized stage (2). This also implies that only 20% of the patients are eligible for resection of the primary tumor combined with adjuvant chemotherapy. However, in most cases even this curative treatment regimen only provides a temporary survival benefit, due to relapse or the development of metastases during or shortly after therapy. One reason for this poor prognosis can be seen in the evolution of resistances towards therapeutic drugs, e.g. through the activation of multidrug resistance and pro-survival pathways (3–5). Furthermore, PDAC is characterized by a pronounced inflammatory tumor stroma, which besides genetic and epigenetic alterations also contributes to the acquisition of a drug resistant phenotype in PDAC cells (6, 7).

The emergence of chemoresistance has been linked to Epithelial-Mesenchymal-Transition (EMT) in diverse cancer entities, including PDAC (8–11). Primarily, EMT is regarded as a key process in metastasis by which epithelial tumor cells acquire the capability to disconnect from the primary context and disseminate to secondary sites. Since EMT can also be seen as a dedifferentiation process, it is not surprising that EMT has been associated with the acquisition of cancer stem cell (CSC) properties. Due to their self-renewal potential and ability to undergo asymmetric cell division, CSCs are undifferentiated cells that are essential for tumor initiation and the emergence of more differentiated cell clones within the tumor. Thus, intratumor heterogeneity of PDAC might be another determinant for the response to therapeutic drugs, as particularly CSCs are highly resistant to cancer therapies (12–16).

As outlined in the review by Zhang et al. recently published in Frontiers in Oncology, the gut and tumor microbiome have emerged as a promising therapeutic target for PDAC (8, 17, 18), due to its impact on tumorigenesis and drug resistance in PDAC (19, 20). Several studies in PDAC patients demonstrated important links between the patient`s tumor microbiome and disease progression, such as correlations between patient survival and tumor microbiome diversity (21) or facilitating immune suppression (19). These findings support a link between the microbiome, disease progression and outcome of PDAC patients. Moreover, chronic inflammation associated with long-term microbial infection promotes EMT, which in turn contributes to drug resistance, cancer progression and metastasis (summarized in 8). Since EMT is linked to the acquisition of CSC properties, we postulate that the abundance and plasticity of CSCs, and thereby intratumor heterogeneity in PDAC, are critically modulated by the patient`s microbiome (of different body compartments). Considering this possible association might provide the basis for innovative therapeutic strategies targeting the microbiome.

EMT is regarded as a prerequisite for epithelial/carcinoma cells to disseminate from the primary tumor to secondary sites. Undergoing this process implies a loss of typical epithelial characteristics and a gain of mesenchymal properties, causing a fundamental functional switch from stationary to a more motile and invasive phenotype. In detail, expression of epithelial proteins like E-cadherin or occludin, both being important for epithelial cell-cell contacts, are diminished, while expression of mesenchymal markers such as N-cadherin, Vimentin, L1CAM or the transcription factor Zeb1 are enhanced (22). Accordingly, EMT is a process by which cells lose their original differentiation and function, which can be regained at secondary sites by reversion of EMT, a process called Mesenchymal-Epithelial-Transition (MET). Thus, it is not surprising that EMT coincides with the acquisition of CSC-characteristics in tumor cells (23–25). Mani et al. demonstrated that breast cancer cells that have undergone EMT acquire a stem cell-like phenotype, and subsequently these stem cell-like cells resemble cells that have undergone EMT (25).

Similar to physiological stem cells, CSCs are characterized by the ability to proliferate indefinitely and to divide asymmetrically, giving rise to both stem cells and differentiated short-lived daughter cells with limited proliferative capability (26–29). Based on these properties, CSCs - although accounting only for a small part of the entire tumor cell population - are regarded as essential for tumor initiation and progression as well as for tumor heterogeneity (27, 30–32). According to the current model, CSCs are not a fixed cell population, but that the aforementioned characteristics can be acquired and lost dependent on environmental stimuli, as CSC are highly dependent on their niche, i.e. oxygen level, surrounding stromal cells and their released factors (24, 29, 33–36). Hence, factors like oxidative, inflammatory and nutritional stress, to which tumor cells are commonly subjected to, determine the differentiation of non-CSCs into CSCs and vice versa. From an evolutionary point of view, this model implies that changes in the tissue microenvironment (e.g., inflammation and/or microbiome changes) lead to the selection of subpopulations of CSCs in a Darwinian manner. As a consequence, these CSCs develop strategies that enable them to survive the adverse conditions of the host (37). This might also provide an explanation for the marked resistance of CSCs to different therapies (8). For instance, chemotherapies aim to decrease the total number of rapidly proliferating tumor cells. However, since CSCs rarely divide and exhibit high levels of drug export molecules, this is only partially successful, as the main tumor cell population might be removed while CSCs survive and can give rise to recurrences or metastases (30, 38–42).

In summary, CSCs contribute to tumorigenicity, tumor progression, metastasis, recurrence as well as therapy resistance in PDAC (26, 42, 43). Given the fact that EMT as well as the interconversion from non-CSC to CSC are both processes defining tumor cell plasticity and heterogeneity, and either process is highly dependent on the inflammatory/stress level of the surrounding microenvironment, it is reasonable to postulate that the microbiome is another important determinant for defining evolution of CSC. Confirming the contribution of the microbiome to tumor cell plasticity might provide additional mechanistic insight into tumorigenesis and the survival of PDAC patients.

As summarized above and in the recently published review by Zhang et al. in this journal (8), the microbiome composition (in different body compartments) is considerably altered in PDAC patients compared to healthy individuals. This altered diversity may be a consequence of tumorigenesis, as the evolution of an inflammatory tumor microenvironment might promote bacterial translocation from the gut into the pancreas (8, 17, 21, 57). Besides, there is growing evidence that the microbiome is an important determinant of PDAC development and therapy response (8, 17, 21, 67–69). One mechanism by which the microbiome composition seems to drive PDAC progression and therapy resistance is promoting EMT. Importantly, EMT induction has been linked to the acquisition of CSC properties, and both EMT cells and CSC are characterized by profound drug resistance (30, 38–40). Considering these well-established interrelationships, it is reasonable to speculate that the abundance and plasticity of CSCs, and thereby intratumor heterogeneity in PDAC and patient´s outcome, are essentially influenced by the patient`s microbiome ( Figure 1 ). This hypothesis is in line with studies supporting the fact that CSC properties can be gained or lost depending on the tumor microenvironment (24, 33–36). Since it is well known that an inflammatory microenvironment impacts the phenotype and genotype of tumor cells, it can be assumed that the altered composition of the gut as well as the tumor microbiome contribute to the inflammatory processes and thereby to the switch from a physiological (tumor suppressive) into an inflammatory (tumor promoting) tumor microenvironment. This in turn may induce EMT and CSC-properties in PDAC cells, e.g. by elevated levels of EMT/CSC inducing factors such as Transforming Growth Factor-beta1 or Tumor Necrosis Factor-alpha. Further, it cannot be ruled out that bacteria and their released factors directly induce EMT, as it could be demonstrated for F. nucleatum, and also promote the gain of CSC properties (56, 57, 61). A high abundance of CSCs in PDAC tissues could be related to PDAC dissemination, and with this progression and resistance to therapy (26, 31, 39, 41, 70–72). Hermann et al. (73) demonstrated that different CSC populations exist in PDAC and exhibit distinct functional capabilities. Thus, CD133+CXCR4+ CSCs were found to be particularly responsible for metastasis (73). Adding to the view of CSC heterogeneity in PDAC, own unpublished data indicate that PDAC cells can exhibit different CSC phenotypes that are characterized by distinct CSC marker expression (high Sox2 or high Nestin expressing CSCs) along with different migratory and invasive abilities. As a consequence, different metastasis patterns can be observed in a preclinical PDAC metastasis model (unpublished data). In line with this, Nestin was found to be upregulated in various human malignancies (74, 75) including PDAC, where it associated with an elevated liver metastatic potential of CSCs (31, 75). Considering this profound knowledge, we postulate that a more diverse microbiome composition, which was detected in LTS PDAC patients (21), might act in favor of a host defense by controlling the number and phenotype of CSCs in PDAC, resulting in a lower metastatic potential and less resistance towards chemotherapy ( Figure 1A ).

Accordingly, future studies are urgently needed to explore whether- and how a certain microbiome composition (e.g., those of LTS patients or Fusobacteria) influences intratumor heterogeneity through the gain and loss of CSC phenotypes, and in turn determines disease progression, therapy responses and survival of PDAC patients. Furthermore, since it is known that certain bacteria can increase the efficacy of therapy (18), the potential of microbiome modulation as an integral part of anti-cancer therapy needs to be further investigated. Given the fact that CSCs are mandatory for tumor initiation, novel therapeutic concepts aimed at their complete eradication. However, since the CSC pool can be constantly regenerated by conversion of non-CSC into CSCs, these strategies will likely ultimately fail. Instead, therapeutic strategies aiming to prevent or control CSCs may be more effective. Thus, the therapeutic enrichment of certain bacteria and/or restoring (physiological) microbiome diversity might be a promising strategy to effectively suppress the appearance, heterogeneity and survival of CSCs, thereby controlling disease progression and increasing the efficacy of therapeutics.

Publicly available datasets were analyzed in this study. This data can be found here: https://pubmed.ncbi.nlm.nih.gov.

Conceptualization, MB and L-MP. Supervision, SS and JB. Visualization, L-MP. Writing – original draft, MB, L-MP, and SS. Writing – review and editing, MB, L-MP, JB, and SS. All authors contributed to the article and approved the submitted version.

This project and its publication were supported by the Research and Training Group 2501 on Translational Evolutionary Research (RTG 2501 TransEvo) funded by the Deutsche Forschungsgemeinschaft.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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