The recent updates and revisions to the Cancer Moonshot Initiative indicate a need for better understanding cancer biomarkers on a cellular level, and techniques such as single-cell sequencing are likely to prove useful in this research area. Automated Lab Solutions’ (ALS) CellCelector is an example of an instrument that can help accelerate cancer research through its ability to efficiently and accurately isolate rare cells, such as CTCs, for proper tumor heterogeneity analysis.

In early September, the Blue Ribbon Panel released its report of recommendations for the Cancer Moonshot to the National Cancer Advisory Board. Finalized and submitted to Vice President Joe Biden on October 17, the report covers 10 suggested methods to help enable the Moonshot’s mission of accelerating 10 years of cancer research in the fields of prevention, treatment and diagnosis within the next 5 years. The report calls for revised federal funding models for cancer research in the public and private sectors, as well as enabling greater collaboration and more resources to address infrastructural and technological challenges. The White House proposed a budget of $680 million, on top of the NCI’s proposed $5.45 billion budget, for the Moonshot Initiative in FY17.

One of the challenges listed in the report is obtaining adequate technologies to address and characterize tumor heterogeneity. This refers to the challenge of analyzing the diverse tumor cells that can originate from a single tumor in a cancer patient’s body. As the report stated, NGS is useful in characterizing tumor heterogeneity, and technologies such as flow cytometry and multidimensional fluorescence microscopy enable the study of cancer cells on a single-cell basis. The concept of tumor heterogeneity has been a major step forward in cancer research, as it has indicated the constantly evolving nature of cancer cells and has raised questions on how to best treat the disease.

An important tool in understanding tumor heterogeneity on a cell-to-cell level is single-cell sequencing (SCS). According to a review entitled “Advances and Applications of Single-Cell Sequencing Techniques” by Yong Wang, PhD, and Nicholas Navin, PhD, published in the May 21, 2015 edition of Molecular Cell, SCS has advanced greatly within the last six years, especially for characterizing tumor heterogeneity and the evolution of cancerous cells in cancer research. According to Dr. Navin’s research, SCS is vital for understanding tumor heterogeneity, as SCS enables researchers to isolate and sequence a single cell genome.

There are numerous technologies that aid SCS in characterizing tumor heterogeneity, including whole-genome amplification, a method providing robust amplification of a complete genome up to a microgram level; flow cytometry using fluorescence-activated cell sorting (FACS), which enables various cells to be sorted individually based on their light scattering properties; and laser-capture microdissection (LCM), which permits the direct isolation of individual cells from heterogeneous tissues and organisms through the use of a targeted carbon dioxide laser.

However, these methods are not without their limitations. For example, whole-genome amplification can result in high allelic dropout rates (i.e., when PCR fails to amplify one or both allelic copies) and preferential amplification (i.e., when one allele is over-amplified in regards to the other); FACS is unable to sort cells that do not have an appropriate surface marker and can have low recovery rates; and surrounding nuclei can be unintentionally affected by LCM, which will result in inaccurate data. These technologies are also more efficient in isolating abundant cell populations, as opposed to rare ones. LCM is suitable for isolating rare cells, but as aforementioned, the possible UV damage to surrounding DNA/RNA cells can compromise data accuracy.

According to Dr. Navin, recently, research has begun on how to use SCS to effectively isolate and study rare cells, such as circulating tumor cells (CTCs). Successfully extracting and analyzing CTCs is an extremely important aspect to cancer research due to the fact that sequencing single CTCs will more effectively enable researchers to characterize populations, however small, of drug-resistant cells. The analysis of CTCs has been proven to advance cancer therapies, though it is not without its challenges. Not only are CTCs extremely rare, occurring at a frequency of one in one million within the blood of cancer patients, but it is also challenging to determine their molecular characterizations, due in part to the technological difficulties that arise when attempting to isolate them.

One instrument that can be used for isolating rare cells like CTCs for characterizing tumor heterogeneity in cancer research is ALS’ CellCelector. The instrument comprises an inverted microscope with a high-precision robotic arm in which various harvesting tools holding plastic/metal tips or glass capillaries can be mounted. In an interview with IBO, Constantin Nelep, PhD, marketing director at ALS, explained that the CellCelector is an automated image-based system for the selection and isolation of rare and pure cells from many sample varieties, including cells from fixed/live cell-imaging, cells in suspension/adherent cells and labeled/unlabeled cells. As Dr. Nelep indicated, the CellCelector can be used in tandem with virtually any upstream cell-enrichment method, including positive immune-magnetic enrichment, negative depletion, size-based selection, or microfluidics. By first extracting CTCs from a chip or cartridge through methods like manual pipetting or automated flushing, the cells can be scanned, detected and isolated individually by the CellCelector.

ALS developed the MagnetPick slide holder to allow for rapid scanning as well as extraction of immuno-magnetically enriched single cells, ensuring a processing time for a single sample (including isolation of up to 30 cells) of under one hour. Dr. Nelep explained that once the sample has been scanned and the target cells have been detected, the time for single cell recovery is less than 30 seconds per cell, totaling in an automated isolation time of 96 cells in approximately 45 minutes.

The CellCelector is similar in principle to Silicon Biosystems’ DEPArray, in that they are both image-based single-cell isolation systems. The DEPArray instead uses dielectrophoresis to capture and move cells within an electronic cartridge. However, as Dr. Nelep noted, there is a longer single-cell recovery time with the DEPArray and a limited number of single-cell recoveries than with the CellCelector. The number of cells that can be sorted within the electronic cartridge in the DEPArray is limited to approximately 16,000 cells, whereas the CellCelector screens cells based on the size of the dish or slide that is being used for cell seeding, making it useful for sorting up to even a million cells. Also, the DEPArray has a restricted electrode size of 40 µm, resulting in a limited size of circulating tumor microemboli (CTM) clusters in CTC analysis. CTM are important cancer biomarkers that indicate the collective migration of tumor cells, and are useful in cancer research and understanding tumor heterogeneity.

Dr. Nelep explained that the CellCelector utilizes a gentle process that ensures that live cells can be cultivated after the sorting and isolation process. Users can choose between fully automated cell detection, in which the sample is scanned, then studied through image analysis and target cell detection; manual cell selection, which is beneficial for sampling a few dozen cells; and the intermediate case, in which the user can pick and choose the region of interest in the sample to analyze.

The CellCelector has some basic technical differences from technologies such as LCM and flow cytometry. Unlike LCM, the CellCelector uses mechanical aspiration which does not stress cells and RNA, explained Dr. Nelep. “When picking directly from tissue, CellCelector technology can be considered laser-free microdissection,” Dr. Nelep said. “[Here], the area isolated during one pick corresponds to the internal diameter of the glass capillary used (e.g., 20 µm for single-cell isolation) or up to 220 µm (or even more if necessary) when one wants to isolate a homogeneous areas of tumor tissue.”

The state of the sample is also different for each technology. “LCM usually needs completely dried samples and has problems when the tissue is humid, while the CellCelector technology always needs the presence of liquid,” said Dr. Nelep. “The sample needs to be (at least locally) humidified to create a liquid junction between the capillary and the cell [in order] to be aspirated.”

In comparison to flow cytometry, Dr. Nelep stated that the two technologies play in different leagues. “Flow cytometry usually works with samples having hundreds of thousands or millions of cells, and it’s inefficient for a low number of target cells within the sample (e.g., few hundreds or less and when one wishes to recover the majority of them),” he explained. “So in terms of rarity, the CellCelector can handle samples with much rarer cell populations.“ There is also no intrinsic cell loss with the CellCelector, as every cell can be recovered, Dr. Nelep noted, which is especially important when researching rare cells like CTCs.

Dr. Nelep described the CellCelector as “image-based plate cytometery.” In this way, the instrument differs from flow cytometery in a basic sense. “The CellCelector uses imaging to detect cells, and one can select cells based on the morphology or even on some intercellular parameters (it’s very important for CTC identification or, for example, in the application where one needs to select only double-nucleated cells),” he explained. “That is not possible with flow cytometry.”

Flow cytometry is useful when sorting a large number of single cells, as doing so with the CellCelector may take many hours. However, if research requires for a couple dozens of single cells to be isolated, the CellCelector offers a quicker and simpler alternative, especially if the absolute number of target cells is low or only a few hundred of cells per sample are needed to be isolated.

For the purposes of characterizing tumor heterogeneity by isolating rare cells, such as CTCs, through SCS, the CellCelector has ample pertinent capabilities, and the technology is constantly advancing. “There might be some difficulties when trying to perform single-cell RNA analysis on some difficult samples (like FFPE tissue), but it depends on the upstream sample preparation as well as on the downstream analysis protocol,” noted Dr. Nelep. “We are working with several customers and partners to test, improve and standardize such protocols.”