Purpose: In HT29 colon cancer cells, a close interplay between self-DNA-induced TLR9 signaling and autophagy response was found, with remarkable effects on cell survival and differentiation. IGF1R activation drives the development and malignant progression of colorectal cancer. IGF1R inhibition displays a controversial effect on autophagy. The interrelated roles of IGF1R inhibition and TLR9/autophagy signaling in HT29 cancer cells have not yet been clarified. In our study, we aimed to investigate the complex interplay of IGF1R inhibition and TLR9/autophagy signaling in HT29 cells.Methods: HT29 cells were incubated with tumor-originated self-DNA with or without inhibitors of IGF1R (picropodophyllin), autophagy (chloroquine), and TLR9 (ODN2088), respectively. Cell proliferation and metabolic activity measurements, direct cell counting, NanoString and Taqman gene expression analyses, immunocytochemistry, WES Simple Western blot, and transmission electron microscopy investigations were performed.Results: The concomitant use of tumor-derived self-DNA and IGF1R inhibitors displays anti-proliferative potential, which can be reversed by parallel TLR9 signaling inhibition. The distinct effects of picropodophyllin, ODN2088, and chloroquine per se or in combination on HT29 cell proliferation and autophagy suggest that either the IGF1R-associated or non-associated autophagy machinery is “Janus-faced” regarding its actions on cell proliferation. Autophagy, induced by different combinations of self-DNA and inhibitors is not sufficient to rescue HT29 cells from death but results in the survival of some CD133-positive stem-like HT29 cells.Conclusion: The creation of new types of combined IGF1R, autophagy, and/or TLR9 signaling inhibitors would play a significant role in the development of more personalized anti-tumor therapies for colorectal cancer.

The basic function of the immune system is the protection of the host against infections, along with the preservation of the individual antigenic identity. The process of self-tolerance covers the discrimination between self and foreign antigens, including proteins, nucleic acids, and larger molecules. Consequently, a broken immunological self-tolerance results in the development of autoimmune or autoinflammatory disorders. Immunocompetent cells express pattern-recognition receptors on their cell membrane and cytoplasm. The majority of endogenous DNA is located intracellularly within nuclei and mitochondria. However, extracellular, cell-free DNA (cfDNA) can also be detected in a variety of diseases, such as autoimmune disorders and malignancies, which has sparked interest in using cfDNA as a possible biomarker. In recent years, the widespread use of liquid biopsies and the increasing demand for screening, as well as monitoring disease activity and therapy response, have enabled the revival of cfDNA research. The majority of studies have mainly focused on the function of cfDNA as a biomarker. However, research regarding the immunological consequences of cfDNA, such as its potential immunomodulatory or therapeutic benefits, is still in its infancy. This article discusses the involvement of various DNA-sensing receptors (e.g., absent in melanoma-2; Toll-like receptor 9; cyclic GMP–AMP synthase/activator of interferon genes) in identifying host cfDNA as a potent danger-associated molecular pattern. Furthermore, we aim to summarize the results of the experimental studies that we recently performed and highlight the immunomodulatory capacity of cfDNA, and thus, the potential for possible therapeutic consideration.

HGFR activation drives the malignant progression of colorectal cancer, and its inhibition displays anti-autophagic activity. The interrelated role of HGFR inhibition and TLR9/autophagy signaling in HT29 cancer cells subjected to modified self-DNA treatments has not been clarified. We analyzed this complex interplay with cell metabolism and proliferation measurements, TLR9, HGFR and autophagy inhibitory assays and WES Simple Western blot-based autophagy flux measurements, gene expression analyses, immunocytochemistry, and transmission electron microscopy. The overexpression of MyD88 and caspase-3 was associated with enhanced HT29 cell proliferation, suggesting that incubation with self-DNAs could suppress the apoptosis-induced compensatory cell proliferation. HGFR inhibition blocked the proliferation-reducing effect of genomic and hypermethylated, but not that of fragmented DNA. Lowest cell proliferation was achieved with the concomitant use of genomic DNA, HGFR inhibitor, and chloroquine, when the proliferation stimulating effect of STAT3 overexpression could be outweighed by the inhibitory effect of LC3B, indicating the putative involvement of HGFR-mTOR-ULK1 molecular cascade in HGFR inhibitor-mediated autophagy. The most intense cell proliferation was caused by the co-administration of hypermethylated DNA, TLR9 and HGFR inhibitors, when decreased expression of both canonical and non-canonical HGFR signaling pathways and autophagy-related genes was present. The observed ultrastructural changes also support the context-dependent role of HGFR inhibition and autophagy on cell survival and proliferation. Further investigation of the influence of the studied signaling pathways and cellular processes can provide a basis for novel, individualized anti-cancer therapies.

Gut-associated lymphoid tissue is one of the most diverse and complex immune compartments in the human body. The subepithelial compartment of the gut consists of immune cells of innate and adaptive immunity, non-hematopoietic mesenchymal cells, and stem cells of different origins, and is organized into secondary (and even tertiary) lymphoid organs, such as Peyer’s patches, cryptopatches, and isolated lymphoid follicles. The function of isolated lymphoid follicles is multifaceted; they play a role in the development and regeneration of the large intestine and the maintenance of (immune) homeostasis. Isolated lymphoid follicles are also extensively associated with the epithelium and its conventional and non-conventional immune cells; hence, they can also function as a starting point or maintainer of pathological processes such as inflammatory bowel diseases or colorectal carcinogenesis. These relationships can significantly affect both physiological and pathological processes of the intestines. We aim to provide an overview of the latest knowledge of isolated lymphoid follicles in colonic inflammation and colorectal carcinogenesis. Further studies of these lymphoid organs will likely lead to an extended understanding of how immune responses are initiated and controlled within the large intestine, along with the possibility of creating novel mucosal vaccinations and ways to treat inflammatory bowel disease or colorectal cancer.

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