Proton NMR Characterization Of Primary And Metastatic Melanoma Cancer Cells In 2D & 3D Cultures

Cancer is a group of 200 diseases responsible for the death of millions of lives every year. Cancer can be treated and cured with significant success rate if diagnosed at an early stage. Diagnosing cancer at advanced or later stages has proven to be very fatal. Hence, diagnosing cancer at an earlier stage is critical in mitigating cancer deaths.

It is vital to develop better, easy, and rapid diagnostic methods for diagnosing cancer. One possible way is using nuclear magnetic resonance (NMR) to characterize and categorize various cancers. This is usually done by subjecting the intact cells to a magnetic field using proton NMR spectroscopy. The signals or markers that are obtained from this analysis usually represent the cell membrane composition. The success or results of this methodology can be translated into a clinical diagnostic platform with the help of magnetic resonance imaging coupled with mass spectrometry. (MRI-MS) as both these instruments share the common principle.

Researchers had already used NMR to identify the lipid signals (as cell membrane is made of phospholipids and embedded proteins) that differentiate cancer cells and healthy cells. But the major lacuna in this research area is the specificity of this method, which is very important in the arena of diagnostics. This study sheds light on the specificity of NMR based cancer characterizing methods.

The key feature of this study is the selection of the cell lines for the analysis. We have selected two melanoma cell lines that were derived from a common donor. These two cell lines vary in their cancer stages. One cell lines are derived from a primary tumor whereas the other cell line is derived from the site of metastasis. Thus the two cell lines we selected will have an identical genetic background but may differ in their gene expression profiles that were responsible for differentiating them into primary melanoma cell line and metastatic melanoma cell line. Our hypothesis is that NMR based methodologies are apt for cancer characterization if proton NMR spectrometry can differentiate between these closely similar cell lines. We employed two types of cell culturing methods namely monolayer culture (known as two-dimensional flat culture system) and spheroidal cell culture (known as a three-dimensional culture) in order assess the effect of space constraints (if any) in these cell lines.

In this study, we are concerned only about the differential NMR signals or markers that are qualitative in nature as these markers are highly relevant in clinical settings and make the characterization more conclusive than quantitative markers.

The proton NMR spectrum of the primary and metastatic melanoma cells in the monolayer culture was very similar and varied only in their intensity (quantitative markers) expect the marker at 5.86 ppm. In the spheroid culture, a total of nine NMR markers differentiates between the primary and metastatic melanoma spheroids. Among those nine markers eight (3.58 ppm, 3.60 ppm, 3.67 ppm, 3.75 ppm, 4.12 ppm, 4.39 ppm, 6.16 ppm, 8.07 ppm) markers were characteristic of primary melanoma whereas, one characteristic marker (8.25 ppm) was observed in metastatic melanoma spheroids. From this study, it is more certain than before NMR methodology would be an apt modality for characterizing cancer cells. Thus proving the specificity of this method in characterizing cancer cell lines. The results suggested that the glucose accumulation, protein, phospholipid composition vary significantly between the primary and metastatic stages of melanoma. These results also suggest that space constraints do play a greater role in the cell membrane biochemistry and possibly mitigate the biological identity of cells. This observation encouraged us to analyze the differences in a particular cell line with respect to the cell culturing method.

As expected melanoma cells under investigation express differential NMR markers with respect to the cell culture methods. Total of 15 NMR markers differentiates primary melanoma cells cultured as monolayer and as spheroids. Among them, 10 markers were characteristically observed in spheroids. On the other hand, a total of 5 NMR markers differentiates metastatic melanoma cells with respect to the cell culture methods. Among these 5 markers, 2 markers were observed characteristically in spheroids. These observations suggested that the method of cell culture has a significant effect on the phospholipid composition (cell membrane biochemistry) of the cells.

This study sheds light on the possible avenues for the development of reliable, better and rapid cancer diagnostic methods using magnetic resonance and importantly motivates further researches on understanding the effects of phospholipid and glucose accumulation in cancer development, progression, and invasion.

These findings are described in the article entitled Proton NMR characterization of intact primary and metastatic melanoma cells in 2D & 3D cultures, published in the journal Biological Research. This work was led by Gokula Krishnan Ramachandran from the National University of Singapore.

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