Cancer can perturb the concentration of metals in cells. For example, tumor cells often have a reduced concentration of iron. Platinum is a key common ingredient in several chemotherapeutics for cancer treatment. Platinum does not naturally occur in the body, and intracellular uptake of platinum by each cell is useful in guiding chemotherapy.
During chemotherapy, the platinum content of cells is ideally high. Relapse is indicated by the proportion of tumor cells that show low Pt uptake, indicating that the cancer has developed a “workaround” where the tumor cell can grow in the presence of cytotoxic chemotherapeutic agents. An increase in the relative population of low or nonresponders is a leading indicator of relapse, which may lead to the need for additional chemotherapy. In contrast, traditional assays of metal content measure the average over thousands of cells, including a large proportion of normal cells. This masks the response from the nonresponders or resistant cancer cells.
Table 1 – ICP/MS modules optimized for single-cell analysis of platinum uptake in chemotherapy 
I had an opportunity to discuss the assay for platinum uptake with Dr. Chady Stephan of PerkinElmer. He described the company’s Single Cell ICP-MS (SC-ICP-MS), which includes a NexION ICP-MS fitted with the Asperon spray chamber and supported by Syngistix software (Table 1). Stephan reports that early adopters of SC-ICP-MS in platinum assays are finding the data to be very useful in guiding secondary treatment of solid tumors.
PerkinElmer’s engineers redesigned the nebulizer and spray chamber of their ICP to move the large analyte droplets through the flow path without contacting the exterior walls. This interface is marketed under the Asperon brand. The lower detection limit for Pt is in the attogram per cell range.
A team at PerkinElmer used the Single Cell ICP-MS system to study the cell cycle of the nonresponders starting with ovarian cancer cell lines A2780 and A2780/CP70 (see Figure 1). CP70 cells are highly resistant to cisplatin therapy. They starved samples of both lines by washing away the nutrients, such as growth factors, in serum. Starvation arrests the cell cycle in the G1 phase. The uptake of platinum for both cells was not changed from the controls that had not been starved. This indicates that the differences in uptake are not due to cells being in different cell cycle stages.
Figure 1 – Phases of the cell cycle. G1 is the first growth phase, G2 is the second growth phase, S is the synthesis phase, and M is the mitosis phase where the cells divide in half. Serum growth medium of the cells was washed away, which arrests cells at the G1 phase in the cell cycle. The control group for each line was cells with normal serum growth medium (not starved). Cisplatin was added and the uptake kinetics were recorded. The mean intensity of Pt was measured with the Single Cell ICP-MS system. No significant difference in the rate of uptake of platinum was observed, indicating that the reason for the different uptake is not related to the cell cycle. (Figure reproduced from Ref. 1).RLS: ICPs have a reputation for measuring many metals very rapidly and with excellent detection limits. For single-cell analysis, which ones are most interesting—Na, Ca, Cu, Zn, Fe, Al …?
CS: Many elements are required in smaller amounts (microgram quantities), usually because they play a catalytic role in enzymes. Some trace mineral elements (RDA <200 mg/day) are, in alphabetical order:
- Cobalt: as a component of the vitamin B12 family of coenzymes
- Copper: a required component of many redox enzymes, including cytochrome C oxidase
- Chromium: required for sugar metabolism
- Iodine: required not only for the biosynthesis of thyroxin, but probably for other important organs such as breast, stomach, salivary glands, thymus, etc. (see iodine deficiency); for this reason, iodine is needed in larger quantities than others in this list, and is sometimes classified with the macrominerals; it can be found in ionized salt
- Iron: required for many enzymes as well as hemoglobin and some other proteins
- Manganese: required for processing of oxygen
- Molybdenum: required for xanthine oxidase and related oxidases
- Selenium: required for peroxidases (antioxidant proteins)
- Zinc: required for several enzymes such as carboxypeptidase, liver alcohol dehydrogenase, and carbonic anhydrase.
RLS: What elements show the greatest variability?
CS: Metal levels can be used to track early detection of disease, especially if monitored at the cellular level. Using nutritional metals including Zn, Cu, Fe … enables a tag-free approach to understanding cellular biology. As an example: transition metal ions (Zn, Cu, Fe …) are components of biological processes that have been implicated in many diseases, including microbial infections, cancer, and neurodegenerative disorders. As I mentioned, iron deficiency is common in cancer patients; iron, copper, and zinc are known to be elevated in neurophil of Alzheimer’s patients. Metals are also used as drugs for many diseases. Cisplatin (cis-Pt(NH3)2Cl2) is the first member of a new class of potent antitumor drugs.
RLS: How reproducible is the ICP of single cells? What about natural intercell variance versus analytical variance?
CS: The analytical variance of ICP is element-specific. One can expect a %RSD generally better than 2% and often half that. This is much smaller than the biological variance in metal content in most metal-related diseases. Our Single Cell ICP-MS is essential to understanding the intercellular variability in a cell population at the individual cell level.
RLS: What is the throughput?
CS: It all depends on how many cells you want to scan for. On average, the NexION ICP-MS Single Cell Analyzer can run a sample every 2–3 min, scanning for about 1000–2000 cells.
RLS: What about sampling technology?
CS: We have developed a specific sampling technology that enables us to introduce individual cells into the ICP-MS plasma—at the heart is the Asperon spray chamber, a linear path spray chamber that allows us to introduce intact cells into the plasma. The single-cell introduction system is backed by the Single Cell autosampler, a microfluidic autosampler that enables us to sample from various well plate sizes, ensures sample agitation prior to sampling, and consumes only 10 µL/min of analysis.
RLS: Can ICP differentiate between circulating tumor cells and regular cells?
CS: This is possible if we are to identify a specific metal difference between circulating tumor and regular cells.
RLS: Can ICP differentiate between cells in different stages of the cell cycle?
CS: Also here, if we have a specific metal or metal tag that allows us to differentiate cell stages, then yes. In this case, it appears that the chemotherapeutic agent has a mechanism of action that does not directly involve the cell cycle. Even this result is useful, however, in ruling out a possible mechanism of action.
RLS: Any use in apoptosis research?
CS: Although some micronutrients are beneficial at subtoxic levels, at moderately toxic levels, metals can induce apoptosis. For example, Cu and Mn can induce apoptosis in yeast cells. Further to that, Single Cell ICP-MS can help researchers measure the Cu and Mn in individual cells.
RLS: What about mapping metals to organelles and other subcellular locations?
CS: Yes, at a recent conference, Dr. Lauren Amable from the NIH reported on using Single Cell ICP-MS with isolated mitochondria. She was able to measure both iron and platinum levels in mitochondria. Dr. Amable also demonstrated that nuclear platinum can be measured using Single Cell ICP-MS. Thus, this technique is giving new tools to researchers to measure metals in cellular organelles.
RLS: What do you foresee as the future?
CS: Using Single Cell ICP-MS in personalized medicine—looking in vitro at the efficiency of chemical treatment by looking at the resistance level using Single Cell ICP-MS before undergoing chemotherapy treatment. Dr. Amable sees an opportunity for single-organelle ICP-MS (SO-ICP-MS); location of metals within a cell may give further insight into the etiology of diseases.
Summary
For decades, cryptologic diseases have been diagnosed by focusing on the problem cells hiding in a large excess of normal cells. Cytologists routinely rely on fluorescence activated cell sorters (FACS) and Coulter counters to separate and enumerate cells by staining and size, respectively. With SC-ICP-MS, they will be able to augment these assays by rapidly measuring the metal content of individual cells with attogram detection sensitivity.
Reference
1. https://www.perkinelmer.com/lab-solutions/resources/docs/APPNexION-2000-ICP-MS-Single-Cell-Cancer-Research-013176_01.pdf