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Synthetic vitamin B9, also known as folic acid (FA), is an extensively used nutritional supplement as well as an adjunctive medication in cancer therapy. However, there is increasing evidence that FA can promote the progression of established colorectal cancers (CRC); therefore, great care is required in the case of its application. In order to gain knowledge about the underlying mechanisms, we analyzed the genomic and the epigenomic effect of different FA supplies on two CRC cell lines with distinct molecular backgrounds. HT-29 and SW480 cells were kept in FA-free media (0 ng/mL) or treated with 100 ng/mL and 10000 ng/mL FA for 72 hours. Firstly, cell proliferation and cell viability alterations were determined with Supforhodamine B and AlamarBlue assays; then, cell cycle analysis was performed using fluorescence-activated cell sorting (FACS). Global DNA methylation level was investigated with the pyrosequencing of long interspersed nuclear element 1 (LINE-1) retrotransposons. Micronucleus scoring performed on DAPI- and anti-γ-H2AX-stained slides, as well as Comet assay, were used to detect the impact of FA on genome integrity. Finally, we analyzed gene expression alterations with Human Transcriptome Array (HTA) 2.0. Our data revealed that 100 ng/mL FA induced significant (p≤0.05) elevation (HT-29100: 128.4±24.9%) of HT-29 cell proliferation compared to the other two circumstances (HT-290: 101.3±13.5%; HT-2910000: 86.1±20.8%). The tendency of cell viability was analogous to the results detected during cell proliferation analyses (HT-290: 91.6±13.3%; HT-29100: 115.8±30.9%; HT-2910000: 64.1±20.2%). The genomic stability of FA-supplemented HT-29 samples was improved in a significant manner (p≤0.05), based on the results of micronucleus scoring (HT-290: 0.56±0.05%; HT-29100: 0.17±0.05%; HT-2910000: 0.25±0.09%) and Comet assay (HT-290: 37.4±3.5%; HT-29100: 31.2±3.4%; HT-2910000: 20.1±3.6%). However, in SW480 cells, remarkable alterations were not detected concerning these parameters. Fundamental differences were observed between the two cell lines in the case of cell cycle (HT-29: G0/1 phase dominance; SW480: S phase dominance) and global DNA methylation analysis (HT-29: 59.1±1.0; SW480: 49.0±0.2), but exposing them to different FA doses did not lead to significant changes. Gene expression alterations were more diverse, as genes involved in carcinogenesis were either up- (HES1, SLC7A11) or downregulated (CCL2) for FA supplementation. We concluded that the effect of FA was considerably influenced by the cell type and the applied FA concentration. Thereby, translating our in vitro results to patient care, we would emphasize the importance of genetic and epigenetic investigations coupled with the choice of proper FA dose upon CRC diagnosis to achieve the best disease outcome. Citation Format: Sára Zsigrai, Alexandra Kalmár, Barbara Kinga Barták, Zsófia Brigitta Nagy, Krisztina Andrea Szigeti, Gábor Valcz, Titanilla Dankó, Anna Sebestyén, Gábor Barna, Zsolt Tulassay, Péter Igaz, István Takács, Béla Molnár. Folic acid has a cell type- and dose-dependent effect on the genome and the epigenome of colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3730.

Introduction: In the progression of colorectal cancer, metastasis is a critical step in the development of systemic disease. Early diagnosis of metastatic progression, however, is not available. We present a novel approach to detect metastatic predisposition early, from patient blood. Background: Metastasis is disseminated by cancer stem cells. They rapidly adapt to hypoxia, they prefer hypoxic metabolism, which is also a major factor in clinical therapy-resistance and dissemination. By studying cancer stem cell differentiation we found that they readily form spheroids (small multicellular clusters or organoids) with hypoxic centers. Hypothesis: We propose that cancer stem cells develop hypoxic spheroids to provide a protected hypoxic micro-niche for survival. Based on the fact that spheroid formation is universally found in vitro, we asked whether cancer stem cells may also form spheroids in vivo, when they enter circulation. Our hypothesis is that in vivo circulating tumorspheres carry cancer stem cells and contribute to metastatic dissemination. To investigate the possibility of circulating cancer spheroids in colorectal and other cancers, we worked out the methodology to detect and characterize them from patient blood. Experimental Procedures: Blood samples were collected from 20 colorectal and 86 mixed cancer cases. 16 of the 20 colorectal and 62 of the 86 mixed cancers were metastatic, respectively. We also tested 44 controls. The samples were analyzed by flow cytometry using forward scatter and SYTO16 fluorescent labels. For spheroid capture we used simple nylon mesh filters (10 um and 20 um pore size). From the collected spheroids RNA was isolated and gene expression assays were performed for stem cell, hypoxia and epithelial markers. We also studied the role of inflammation and immune check point mechanisms in dissemination. Results: We found that spheroids with epithelial lineage develop hypoxia and carry stem cells. By using Partec and Beckman-Coulter flow cytometers we detected circulating spheroids in patient blood. In the majority of colorectal cancer cases (56%) spheroids in blood indicated metastasis, while the controls were all negative. Furthermore, in half of the mixed cancer cases spheroids in blood also correlated with metastasis. All healthy samples and non-metastatic cases were negative. A subset of spheroids was positive for immune checkpoint and platelet markers suggesting immune/inflammation mechanisms in dissemination. Follow-up clinical studies will investigate the prospective significance of the results. Conclusions: We showed that cancer stem cells form spheroid clusters in vivo and can be detected in circulation. We found significant correlation between spheroids in blood (spheremia) and metastasis in colorectal and other cancers. The most critical new finding of our studies is that systemic dissemination of cancer involves a novel spheroid mechanism. Based on this observation we developed a simple blood test that may detect metastatic predisposition early in colon cancer progression. Citation Format: Viktoria Denes, Laszlo Graf, Gabor Barna, Balint Tegze, Pal Jakso, Peter Geck. Blood test of metastatic predisposition in colon cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B03.

INTRODUCTION Metastasis is the turning point in the progression of most cancers. The prognosis of systemic, disseminated cancer is usually negative, but methods for early metastasis detection are not available in the clinical practice. We present a novel approach that may have the potential to detect metastatic predisposition early from patient blood. BACKGROUND It is almost universally accepted that metastases are disseminated by cancer stem cells. It is also known that hypoxia in a cancer is a major factor in therapy-resistance and dissemination. The findings are consistent with other observations that stem cells rapidly adapt to hypoxia and prefer hypoxic metabolism. We studied cancer stem cell differentiation and found that most stem cells (cancer and normal) readily form spheroids in suspension culture in vitro. Further studies showed that these spheroids develop hypoxic centers and are positive for stem cell markers. HYPOTHESIS These observations suggest that in vitro spheroid formation may reflect a fundamental biological feature of cancer stem cells and they may find a perfect niche in the hypoxic microenvironments in spheroids. The fact that spheroid formation is universally found in vitro raised the possibility that stem cells may also form spheroids in vivo, and by providing a “portable” hypoxic milieu, circulating tumorspheres may be critical in metastatic dissemination. We investigated, therefore, whether cancer spheroids can enter circulation and worked out the methodology to detect them in blood. EXPERIMENTAL PROCEDURES Blood samples were collected from 106 patients, 62 metastatic and 44 controls. The samples were analyzed by flow cytometry using forward scatter and fluorescent labels. For spheroid capture we used simple nylon mesh filters (10 um and 20 um pore size). From the collected spheroids total RNA was isolated and gene expression studies were performed for stem cell, hypoxia and other markers. Both the collected spheroids and in vitro spheroid models were analyzed by immunohistochemistry. RESULTS We found that spheroids show complex metabolic architecture with hypoxia, stem and other markers. We tested several flow cytometers and found that the wide scatter of spheroids was not optimal in some instruments, but performed well in Partec and Beckman-Coulter systems. In our pool almost half of the metastatic samples were spheroid positive, while all healthy samples were negative. Follow up clinical studies will investigate the prospective significance of the results. CONCLUSIONS Our data demonstrate significant correlation between cancer spheroid positivity in blood and metastasis. The results suggest that circulating in vivo spheroids may be involved in the dissemination of metastatic cancer stem cells and can be used as a new prognostic/diagnostic approach. Citation Format: Viktoria Denes, Laszlo Graf, Gabor Barna, Balint Tegze, Pal Jakso, Eva Pallinger, Peter Geck. Metastasis blood test by in vivo tumorsphere detection. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1545.