The current approaches for diagnosing and staging CRC are not able to noninvasively define tumour malignant potential that is directly correlated with therapy selection and prognosis. The controversial results regarding the relation between MnSOD expression and tumour malignant potential in CRC reported in clinical researches, and some results concerning MEMRI of neoplasms from recent studies [27–38], inspired us to explore the application of MEMRI in noninvasive evaluation of CRC metastatic potential. In addition, the attempt to correlate MEMRI appearance with MnSOD expression in CRC cells was made.
Our invasion assay has verified the differential metastatic potential of seven CRC cells, which was consistent with the results reported in previous studies. These researches documented that SW620 and LoVo cells were derived from metastatic lymph nodes of colorectal cancer, the tumourigenicity, growth kinetics and metastatic potential of these cells were proved to be more aggressive than in SW480, DLD-1, HCT15 and Caco-2 that were derived from primary tumours [19,20,21,22,23,24]. Our study confirmed that SW620, LoVo and DLD-1, HCT15 have shown the highest and lowest Invasion Index, respectively.
As one of the most important antioxidant enzymes, MnSOD protein has been considered downregulated in malignant neoplasms in the earliest studies, though subsequent researchers have found increased MnSOD expression in malignant mesothelioma and some neoplasms arising from thyroid, kidney and central nervous system cells as well as in CRC in most clinical studies [14]. This was verified in our in vitro experiment. However, the relation between MnSOD expression and malignant potential is more complicated. Some clinical studies have shown that MnSOD expression is positively correlated with CRC malignant potential [8,9,10,11,12]. However, our in vitro study found that the cells with the highest MnSOD expression had low metastatic potential, whereas cells with a relatively low expression encompassed both high and low metastatic potentials. This indicates that the expression of MnSOD in CRC cells was not correlated to malignant potential. Because the expression of MnSOD is a response to oxidative stress [39, 40], the microenvironmental redox status of tumours influences its expression. The different redox status in human body and cell culture environments might contribute to the discrepancy between the present study and previous clinical researches.
In this study, both MEMRI studies in cells and xenografts have shown significant difference in T1 shortening between CRCs with different metastatic potential: highly aggressive cells/tumours showed two to three times greater T1 shortening than less aggressive ones, with high statistical significance. Similar findings were reported by Nofiele et al. in human breast cancer cells [36]. The author observed that the Mn induced a longitudinal relaxation rate (R1) in the most aggressive cells significantly higher (two times) than that in less aggressive ones. Based on these results, it could be postulated that MEMRI might be used to distinguish aggressiveness in cancers.
Mn2 + is a well-known intracellular contrast agent for MRI. It is taken up and accumulated by cells and shortens the T1 relaxation time of adjacent water thanks to its strong paramagnetic effect. Compared with extracellular agents (such as Gd-based contrast agents), its enhancement effect is more dependent on cell density than on tumour vascularity. At a biochemical level, Mn is involved in mitochondrial function, and the greater the mitochondrial density, the higher the level of Mn uptake. Hence, it could be speculated that tumours with higher cell density and/or mitochondria would enhance more markedly on MEMRI. This was reflected in our study by the fact that the xenografts that were significantly enhanced with Mn administration have invariably shown histological features of packed tumour cells though only sparse microvessels were found.
At molecular level, several factors involving Mn2+ uptake in cells have been reported such as voltage-gated Ca2+-channel activity, anti-N-methyl-D-aspartate, astroglia and microglia activity/cellularity, and metal transporters [41]. However, the mechanism involved in Mn2+ uptake in tumour cells is not fully understood. In a study of human breast cancer the overexpression of calcium-sensing receptor (CaSR) in tumour was suggested to be involved in better Mn-enhancement [29]. However, another study attempting to link CaSR expression to Mn-enhancement in human breast cancer cells did not find this correlation [36]. Nevertheless, the authors found that the most aggressive cells that showed the lowest level of CaSR expression enhanced the most markedly whereas the less aggressive cells with higher levels of CaSR expression enhanced only minimally; furthermore, the higher the concentration of MnCl2 administration, the greater the enhancement of both cells. This indicates that, although CaSR plays a role in Mn uptake in tumour cells via its ion-channel regulation, the T1 shortening effect of Mn2+ on MRI could not be explained solely by ion-channel activity.
Obviously, differential biological behavior of tumours and Mn2+ concentration are also involved in Mn-enhancement. The fact that our study employed identical Mn2+ concentration for different cells and tumours either in in-vitro or in in-vivo studies, and that different aggressive malignancies still showed differential Mn-enhancement, further underscores the important role of neoplastic cell biological properties in MEMRI appearance. Actually, two researches have documented the relation between the biological properties of tumour cells and the Mn-enhancement effect in MRI. Braun et al. [30] found that some tumour cells with a higher proliferation rate demonstrated more Mn uptake and greater T1 shortening. Saito et al. [41] detected early cell alteration that was identified by cell-cycle and proliferation changes after irradiation exposure both in vitro and in vivo by using MEMRI. Therefore, it seems reasonable to attribute the greater Mn-enhancement in more aggressive CRC to a higher cell proliferation rate.
Apart from Mn uptake, the subcellular location of intracellular Mn2+ may also contribute to the enhancement effect. Free Mn2+ in cytosol has greater access to water to induce more T1 shortening compared with mitochondrial Mn2+. Because of the more severe functional impairment of mitochondria in more malignant tumour cells, the Mn2+ entering the mitochondria might be less than that in less aggressive tumour cells, resulting in more Mn2+ accumulating freely in cytosol, thus causing greater T1 shortening. Finally, the contribution from Mn2+ in the extracellular compartment should probably also not be overlooked [42].
In addition, studies in malignant mesothelioma [34, 43] favoured the hypothesis that greater Mn-enhancement is related to higher MnSOD expression in tumours. However, in our study the relatively low MnSOD expression cells has clearly shown greater Mn-enhancement than in cells with higher expression. Given that MnSOD is located in the mitochondria and needs binding Mn2+ to activate it, it was speculated that cells with a lower MnSOD expression have a lower intracellular Mn2+ concentration, which leaves more space for further Mn2+ uptake. As a consequence, its T1 shortening was greater than in cells with high MnSOD expression. The contraindicated observations indicate that this relation is not consistent in different cancer types.
In summary, the tumour-enhancement effect of Mn appears to be influenced mainly by the following factors: tumour cellularity, ion-channel regulation, cell-cycle and proliferation rate, tumour aggressiveness, subcellular location of Mn2+ as well as MRI technical issues such as agent concentration. However, to what extent each single factor contributes to MEMRI appearance under certain imaging condition still needs studies to clarify.
Manganese toxicity is the main obstacle to translate this MEMRI study into clinical application. Finding the appropriate dosage that generates a sufficient enhancement effect with minimum adverse effects would be the prerequisite to translate animal study into clinical use. However, even the most widely used gadolinium-based MRI contrast agents are not free of adverse effects. Recently, they have been found to induce nephrogenic systemic fibrosis in the presence of renal failure [44] and the problem of gadolinium deposition in various organs is of increasing concern [45, 46]. On the other hand, an orally administered Mn agent, such as CMC-001, has shown promising results in terms of reducing toxic effects and improving the detection of liver metastases in both in-vitro studies and clinical trials [3, 47]. Newly developed Mn-based magnetic nanoparticles also have the potential to reduce toxicity and increase the specificity to target tumours [47]. Given that a significant portion of CRC patients developed remote metastasis, either at first diagnosis or after surgery [5,6,7], MEMRI approaches evaluating tumour metastatic potential prior to the occurrence of remote metastatic disease could provide valuable information for prognosis and therapy planning. This technique might be also applied to other neoplasms other than CRC.
In conclusion, MEMRI showed the potential to noninvasively distinguish high- from low-metastatic-potential CRC by revealing a greater T1 shortening in the former. However, the MnSOD expression in CRC cells did not correlate with malignant potential.