Targeting HER2 protein in individual cells using ICP-MS detection and its potential as prognostic and predictive breast cancer biomarker.

The human epidermal growth factor receptor 2 (HER2) is a transmembrane protein that has become one of the most specific prognostic and predictive biomarker of breast cancer. Its early detection is key for optimizing the patient clinical outcome. This work is focused on the detection of HER2 in individual cells using an antibody containing lutetium (Lu) as reporter group that is monitored by introducing the individual cells into the inductively coupled plasma mass spectrometer (ICP-MS). This Lu-containing antibody probe is used to label different breast cancer cell lines considered HER2 negative (MDA-MB-231) and positive (SKBR-3 and BT-474). Optimizations regarding the amount of the probe necessary to ensure complete labelling reactions are conducted in the different cell models. Concentrations in the range of 0.006 fg Lu/cell and 0.030 fg Lu/cell could be found in the HER2 negative and HER2 positive cells, respectively. In addition, the selectivity of the labelling reaction is tested by using two different metal-containing antibody probes for HER2 (containing Lu) and for transferrin receptor 1 (containing Nd), respectively, within the same cell population. Finally, the methodology is applied to the targeting of HER2 positive cells in complex cell mixtures containing variable amounts of BT-474 and MDA-MB-231 cells. The obtained results showed the excellent capabilities of the proposed strategy to discriminate among cell populations. This finding could help for scoring HER2 positive tumors improving existing technologies.

Chromatographic methods coupled to mass spectrometry for the determination of oncometabolites in biological samples-A review.

It is now well-established that dysregulation of the tricarboxylic acid (TCA) cycle enzymes succinate dehydrogenase, fumarate hydratase, and isocitrate dehydrogenase leads to the abnormal cellular accumulation of succinate, fumarate, and 2-hydroxyglutarate, respectively, which contribute to the formation and malignant progression of numerous types of cancers. Thus, these metabolites, called oncometabolites, could potentially be useful as tumour-specific biomarkers and as therapeutic targets. For this reason, the development of analytical methodologies for the accurate identification and determination of their levels in biological matrices is an important task in the field of cancer research. Currently, hyphenated gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) techniques are the most powerful analytical tools in what concerns high sensitivity and selectivity to achieve such difficult task. In this review, we first provide a brief description of the biological formation of oncometabolites and their oncogenic properties, and then we present an overview and critical assessment of the GC-MS and LC-MS based analytical approaches that are reported in the literature for the determination of oncometabolites in biological samples, such as biofluids, cells, and tissues. Advantages and drawbacks of these approaches will be comparatively discussed. We believe that the present review represents the first attempt to summarize the applications of these hyphenated techniques in the context of oncometabolite analysis, which may be useful to new and existing researchers in this field.

Sensitive determination of the human epidermal growth factor receptor 2 (HER2) by immuno-polymerase chain reaction with inductively coupled plasma-mass spectrometry detection.

Sensitive and selective analytical methods are necessary for the determination of clinical biomarkers of breast cancer. The human epidermal growth factor receptor 2 (HER2) is an important breast cancer biomarker since tumors with HER2 protein overexpression (HER2-positive tumors) turn out to be more aggressive and likely to recur. Therefore, accurate determination of serum HER2 values is critical to optimize clinical outcomes in patients with breast cancer. To gain sensitivity and selectivity in the determination of HER2, a sandwich immune assay (highly selective) has been implemented using a detection antibody labelled with a DNA marker. Further amplification of the label using the polymerase chain reaction (PCR), followed by phosphorous quantification of the PCR product (amplicon) using inductively coupled plasma mass spectrometry (ICP-MS), completes this novel assay. Considering that the concentration of the amplicon is proportional to the amount of antigen (HER2) that is recognized by the labelled detection antibody, the concentration of HER2 can be directly obtained by P-analysis. For this aim, a DNA marker of 123 base pairs has been connected to the detection antibody of a sandwich immune assay conducted in pre-coated plates containing the capture antibody of HER2. After the recognition occurred, the PCR amplification was conducted and the PCR product analysed by ICP-MS. Detection limits of 2.5 pg mL-1 of HER2 could be achieved using 35 PCR cycles (7-fold lower than the commercial ELISA method). The developed methodology has been applied to the determination of HER2 in biological samples (human serum and cell culture supernatant of breast cancer cells, MDA-MB-231) obtaining mean method recoveries of about 87% and 81%, respectively.