**1. Introduction**

Colorectal cancer (CRC) is the third leading cause of cancer death worldwide and the second leading cause of cancer mortality in Europe [1,2]. Although CRC incidence and mortality rates have decreased over the past decades, global trends have shown that the incidence among young adults aged 20–49 has increased [3]. While the prognosis of patients with early-stage disease is excellent, 40% of patients across all disease stages ultimately die from their disease within five years [4]. In the last decade, it became clear that differences in oncological outcome can be partly explained by differences in tumor biology. CRC is a highly heterogeneous group of tumors, and the pathogenesis of CRC is a complex and multifactorial process involving the accumulation of various genetic and epigenetic alterations [5]. Beyond these alterations, it is widely accepted that tumor location (right- and left-sided colon cancer, rectal cancer) is a crucial factor involved in disease progression as well as prognosis and influences disease management [6–8]. Right-sided colon cancer (RSCC) occurs within the cecum, ascending colon, hepatic flexure and transverse colon, while leftsided colon cancer (LSCC) arises in the splenic flexure, descending colon and sigmoid colon. The right- and left-sided colon have distinct embryological origins, developing from the mid- and hindgut [6]. Moreover, differences in clinical and molecular characteristics have been observed. In particular, RSCC presents with microsatellite instability, BRAF mutations, high immunogenicity and a worse prognosis [9,10]. To date, colon cancer (CC) and rectal cancer (RC) are synonymously termed CRC. However, based on experimental, translational and clinical research, there is more and more evidence to divide CC and RC as self-standing tumor entities [11].

Epidemiologic studies have identified a number of environmental factors that affect the risk of CRC carcinogenesis, including lifestyle, nutritional factors and the microbiome [12,13]. Microbes have been linked to cancer in 10–20% of cases [14–16]. Novel data have demonstrated that locations previously considered sterile, such as the liver, pancreas and even tumor tissue, harbor their own site-specific microbiome [17–19]. The human intestinal microbiome primarily comprises *Firmicutes, Bacteroidetes, Actinobacteria*, and *Proteobacteria* [16]. After rapid changes in the first years of life, the gut microbiome remains relatively stable for decades and displays gradual changes with advancing age. High diversity might be a key feature of a healthy microbiome [20–24]. Dysbiosis refers to an abnormality in the composition and/or function of the host's symbiotic microbial ecosystem that exceeds its constitutive capacity and, as a result, has adverse effects on the host [25]. The microbiome can impact cancer initiation, progression and response to therapy [26–28]. In 2022, the microbiome is mentioned as a distinctive enabling characteristic for the acquisition of hallmarks of cancer capabilities [16,29]. The hallmarks of cancer, first published in 2000 by Hanahan and Weinberg and updated in 2011, are defined as a core set of functional capabilities acquired by human cells through their way to form malignant tumors [29,30]. Nevertheless, uncertainty remains regarding the direct and indirect effects of the microbiome in cancer [31–33]. Sequencing and association studies have demonstrated changes in microbial composition and ecology in patients with CRC, specifically, a decrease in commensal bacterial species (e.g., butyrate-producing bacteria) and the enrichment of opportunistic pathogens (e.g., proinflammatory). Moreover, there is strong evidence that the gut microbiome influences the efficacy of immune checkpoint inhibitors (ICIs) in CRC and other types of cancers [34–39]. However, recent studies have revealed that the ileal microbiota also determines the prognostic and predictive features and therapeutic responses of CC [40,41].

Altogether, most microbiome studies in CRC have focused on the analysis of fecal rather than tumor or mucosal samples. Furthermore, most studies have considered colon and rectal cancers as one disease entity, CRC. As mentioned before, obvious differences exist in tumor biology, molecular carcinogenesis, treatment and response to therapy. In surgical oncology, for decades researchers have been becoming aware that CC and RC are different diseases, based on multimodal treatments, surgical techniques, complication rates and relapse patterns. To overcome the heterogeneous nature of the microbiome, research should concentrate on separately describing results for CC and RC.

To address these aspects, we focused our attention on mapping associations between the microbiota and clinicopathologic features of tumor tissue and healthy tissue of the ileum, colon or rectum as well as fecal samples collected before and after surgery from patients with primarily untreated CC. The results provide a deeper understanding of the complex microbiome landscape in patients with colon cancer.
