## 4 Discussion  
Despite the progress in diagnostic and surgical techniques and systemic chemotherapy, the majority of patients diagnosed with primary colon cancer die due to metastasis formation, typically in the liver and peritoneum [2; 4; 5; 6]. Hematogenous metastasis is a universal pathway by which tumor cells detach from the extracellular matrix in the primary tumor and spread via the bloodstream or lymphatic system in a suspension state. The tumor cells that circulate into the bloodstream are called circulating (invasive) tumor cells [43]. The evidence from studies on metastasis has demonstrated that, once the cells detach from the primary tumor, the circulating tumor cells seem to undergo dramatic morphological changes. Some of the reported scientific data are related to the rapid rounding of cells because of cell-adhesion-mediated properties, which are probably due to a decrease in the cytoskeletal tension and the degeneration of stress filaments as well as the loss of almost all biochemical and biophysical signaling from the extracellular matrix of  
Figure 5: The effect of chitosan microparticles on the proliferative activity and viability of a colon cancer cell line incubated in a medium with pH-corrected values for 24, 48, and 72 h. The red line indicates the viability of colon cancer cells at the beginning of the experiment. Two levels of statistical significance between pH values adjusted by hydrochloric acid (* \(p<0.05\) and ** \(p<0.01\)) or lactic acid (a \(<0.05\) and b \(<0.01\)) were determined between the different applied concentrations of chitosan particles. The levels of statistical significance (# \(p<0.01\)) in the experiments with chitosan particles were determined. Figure 6: A microscopic photo of the effect of chitosan particles applied in different concentration ranges to colon cancer cells exposed to a forced acidosis cell environment for 48 h (magnification, 40\(\times\); scale bar, 50 \(\upmu\)m). the primary tumor [44; 45]. There are data that have demonstrated that the suspension state of hematogenous metastasis provides conditions conductive to the migration and re-attachment of cancer cells in vitro and metastasis formation in vivo [45; 46]. Obviously, our results from the exposure of a colon cancer cell line to a simulated acidosis environment indicate that the minimal changes in pH values could be initiators of a radical increase in cell division and could provoke cancer progression. The latter observation was confirmed with a microscopic photo showing the existence of a typical adherent colon cancer cell line in a suspension state that is not typical for those cells as well as the formation of round live cells after 48 and 72 h of incubation. The suppression of cancer cell division after 24 h of incubation in the medium with corrected pH values was shown, and at 48 and 72 h an intense increase in proliferative activity occurred. These results could be interpreted using the circadian cell cycle, which for most cells finishes after 24 h. This period could be considered an adaptation period for cancer cells to new conditions. The high concentration of viable cancer cells in the suspension state up to 72 h could be associated with an increased probability for migration and probable metastasis formation. This study provides experimental results that indicate that the induction of acidosis in solid tumors is a major and critical factor in the formation of metastasis. In addition, acidosis may also be a necessary condition to avoid the native immune response. The observed results support the \"acid-mediated tumor invasion\" hypothesis, in which the progress of tumor cells is associated with the acquirement of resistance to acid-induced toxicity during carcinogenesis because of the changed bio-chemical metabolism compared with normal cells [47]. Additionally, the research of [PERSON] et al. has convincingly demonstrated that colon tumors secrete acid into their surrounding stroma and it is necessary for their local invasion [48].