ACEA Biosciences
This image shows scientists at ACEA Biosciences examining an electronic microtiter plate. When the plate is placed in the company's xCELLigence instrument, microelectronic sensors integrated onto the plate's underside enable the detection of changes in cell morphology and viability. Migrating cells contact and adhere to the sensors, causing changes in electrical impedence. These changes permit label-free, real-time monitoring of processes such as the immune cell–mediated killing of cancer cells.

April 1, 2016 (Vol. 36, No. 7)

 

Within the past decade or so, a revolutionary idea has emerged in the minds of scientists, physicians, and medical experts. Instead of using man-made chemicals to treat cancer, let us instead unleash the power of our own bodies upon the malignancy.

This idea is the inspiration behind cancer immunotherapy, which is, according to most experts, a therapeutic approach that involves training the immune system to fight off cancer. In the words of one expert, cancer immunotherapy means “taking the immune system’s inherent properties and turbo charging those to fight cancer.”

Cancer immunotherapy technologies are being developed to accomplish
several tasks:

  • Enhance the molecular targeting of cancer cells
  • Report the rate of killing by specific immune agents
  • Direct immune cells toward tumor destruction.

Since its inception, the field has evolved, and it continues to do so. It began with in vivo investigations of tumor growth and development, and it progressed through laboratory investigations of cellular morphology and survival curves. And now it is adopting pathway analysis to guide therapeutic development and improve patient care.

To begin to understand cancer immunotherapy, one must understand how the immune system targets tumor cells. One of the prominent adaptive components of the immune system is the T cell, which responds to perceived threats through the massive increase in clonal T cells targeted in some way toward the diseased cell or pathogen.

The T-Cell Repertoire

In most cells in the body, the DNA sequence is exactly the same. “However, that is not true for T cells and B cells,” says Harlan Robins Ph.D., co-founder and chief science officer at Seattle-based Adaptive Biotechnologies. “They are the only cells in your body where DNA actually rearranges dynamically.”

Immunologists call this process VJD rearrangement. It happens during T-lymphocyte development and affects three gene regions, the variable (V), the diversity (D), and the joining (J) regions. This rearrangement of the genetic code allow for the structural diversity in T-cell receptors responsible for antigenic specificity including antigenic targets on tumor cells. In the case of cancer, specificity is complicated because the tumor is actually part of the body itself, one of the reasons cancers naturally evade detection.

The specificity problem would always hinder attempts to goad the immune system into attacking cancer, scientists realized, unless technologies emerged that could efficiently track the clonal diversity of T cells inside patients. Existing technologies, such as spectratyping, were inadequate.  In 2007, when Dr. Robins and his collaborators began developing the technology, only 10,000 T-cell receptor sequences had been reported in all the literature using older methodologies.

“The immunology field of the time had no connection with high-throughput sequencing,” notes Dr. Robins, recalling his days as a computational biologist for the Fred Hutchinson Cancer Research Center. “It became clear that instead of using this old technology to look at T-cell receptors, we could just directly sequence them—if we could amplify them correctly.”

With its first experiment, Dr. Robins’ team ended up with six million T-cell receptor sequences. “Our approach,” Dr. Robins modestly suggests, “kind of changed the scale of what we were able to do.” The team went on to develop advanced multiplex sequencing technology, doing work that essentially started the field of immune sequencing. “Previously,” maintains Dr. Robins, “no one had ever been able to quantitatively do a multiplex PCR.”

Adaptive Biotechnologies’ immunoSEQ Assay, a high-throughput research platform for immune system profiling, is designed to generate sequencer-ready libraries using highly optimized primer sets in a multiplex PCR format that targets T- and B-cell receptor genes. This image depicts how the assay’s two-step PCR process can be used to quantify the clonal diversity of immune cells.

Adaptive Biotechnologies’ product, the ImmunoSEQ® assay, uses several hundred primer pairs to quantify the clonal diversity of T cells. Using this technology, researchers and clinicians can focus on T-cell clones that are expanded specifically in or near a tumor or that are circulating in the blood stream.

“You obviously can’t get a serial sample of the tumor,” explains Dr. Robins, “but you can get serial samples of blood,” allowing for immune cell repertoire tracking during the progression of a disease. The technology is already being used to assess leukemias in the clinic, directly tracking the leukemia itself based on the massive clonal expansion of a single cancerous B or T cell.

Eventually, Dr. Robins’ team hopes to monitor serial changes in T cell clones before, during, and after therapeutic intervention. The team has even developed a tumor infiltrating lymphocyte (TIL) assay to examine clones that are attracted to tumors.

 

Circulating Tumor Cells

“Years ago, they were just interested in what was happening in the tumor,” says Daniel Adams, senior research scientist at Creatv MicroTech. “Now people have realized that the immune system is reacting to the tumor.”

Scientists such as Adams have been tracking tumor cells and tumor-modified stromal cells, as well as components of the non-adaptive immune system, directly within the bloodstream to examine changes that occur over time.

“We can’t go back in to re-biopsy the patient every year, or every time there is a recurrence,” says Adams, “It’s just not feasible.”

That is why Creatv MicroTech, with locations in Maryland and New Jersey, has developed the CellSieve, a mechanical cell filter. The CellSieve, which improves on older technology through better polymers and engineering, isolates circulating tumor cells (CTCs) and stromal cells in order to capture them for further clinical analysis.

“As a patient goes through therapy, the patient’s resistance builds, and the cancer recurs in different subpopulations,” states Adams. “And after a few years, the original tumor mass is no longer applicable to what is growing in the patient farther down the road.”

Although CTCs are exceedingly rare in the bloodstream, with just one or two in every 5 to 10 mL of blood, and although these cells have a very low viability, the surviving CTCs have a high prognostic value.

“We looked at 30 to 40 breast cancer patients over two years,” reports Adams. “And we showed that if you have a dividing CTC, you have a 90% chance of dying in two years and a 100% chance of dying within two and half years.”

Furthermore, the immune system response can be tracked, says Adams, by examining stromal cells, which can also be collected with the CellSieve filtration device. That is, these cells can be collected serially. Much recent evidence supports the conclusion that stromal cells in the tumor environment co-evolve with the tumor, suggesting that stromal marker changes reflect tumor changes.

“There is this plethora of stromal cells and tumor cells out there in the circulation for you to look at,” declares Adams. “Once the cells are isolated, you can subject them to pathological approaches, biomarker approaches, or molecular approaches—or all of the above.”

A MicroTech Creatv study published in the Royal Society of Chemistry showed the efficacy of following up CTC isolation with techniques such as fluorescence in situ hybridization (FISH), histopathological analysis, and cell culture.


Isolation, culture and expansion of cells isolated on CellSieve™. (A) MCF-7 cells spiked into vacutainers, isolated by filtration and cultured on CellSieve for 2-3 weeks. A 3 dimensional cluster attributed to this cell line is seen on the filter. (green=anti-cytokeratin, blue=DAPI) (B) PANC-1 cells spiked into vacutainers, isolated by filtration and grown on CellSieve for 2-3 weeks. PANC-1 is seen growing as a monolayer on the filter. (C) SKBR3 cells are spiked into blood, filtered by CellSieve. The CTCs are identified by presence of anti-cytokeratin and anti-EpCAM, and absence of anti-CD45. After CTCs are counted, cells are subtyped by HER2 FISH. (D) SKBR3 cells are spiked into vacutainers, isolated by filtration and grown on CellSieve for 2-3 weeks. Expanded colonies were directly analyzed as a whole colony and as individual cells, molecularly by HER2/CR17 FISH probes. (E) Circulating stromal cell, e.g. a 70 µm giant cancer associated macrophage can be identified for clinical use, myeloid marker in red. (F) A cell of interest can be identified and restained with immunotherapeutic biomarkers, e.g. PD-L1 (green) and PD-1 (purple). (G) After filtration, cells were identified with histopathological stains (e.g. H&E) for cytological analysis. (H) After H&E, external cell structures were analyzed by SEM. [Creatv MicroTech].

Cancer-Killing Assays

Diverse mechanisms are at play in cancer biology. Our understanding of these mechanisms contributes to a couple of virtuous cycles. It strengthens and is strengthened by diagnostic approaches, such as immune- and tumor-cell monitoring. The same could be said of therapeutic approaches. Cancer biology will inform and be informed by cancer immunotherapies such as adoptive cell transfer. To maintain the virtuous cycle, however, it will be necessary to conduct in vitro testing.

“There is no doubt that immunotherapy is going to play a major role in the treatment of cancer,” says Brandon Lamarche, Ph.D., technical communicator and scientist at ACEA Biosciences. “Regardless of what the route is, what is going to have to happen in terms of the research area is that you need an effective cell-killing assay.”

ACEA Biosciences, a San Diego-based company, has developed a microtiter plate that is coated with gold electrodes across 75% of the well bottoms. When the microtiter plates are placed in the company’s xCELLigence plate reader, the electrodes enable the detection of changes in cell morphology and viability through electrical impedance.

“The instrument provides a weak electric potential to the electrodes on the plate, so you get electrons flowing between these electrodes,” explains Dr. Lamarche. Researchers can then apply reagents or non-adherent immune cell suspensions to adherent cancer cells and examine the effect.

Dr. Lamarche asserts that the xCELLigence system overcomes problems that bedevil competing cell-killing assays. These problems include leaky and radioactive labels, such as chromium 51, and assays that can only provide users with an endpoint for cell killing. “With xCELLigence,” he insists, “you’re getting the full spectrum of what’s happening, and there’s all kinds of subtleties in the cell-killing curves that are very informative in terms of the biology.”

ACEA would like to see the xCELLigence system become the new standard in cell-killing assays from standard research to clinical testing on patient tumors. Dr. Lamarche envisions a day when patient tumor cells are quickly screened with therapeutic scenarios to determine the most efficacious killing option. “xCELLigence technology,” he suggests, “enables you to quickly sample a broad spectrum of conditions with a very simple workflow.”

Bioinformatics of Immuno-Oncology

From monitoring to treatment modalities, the field of cancer immunotherapy is aided by bioinformatics-minded data-mining experts, such as the analysts at Thompson-Reuters who are compiling data archives and applying advanced analytics to find new targets. “Essentially,” says Richard Harrison Ph.D., the company’s chief scientific officer for the life science division, “for every stage within pharmaceutical drug development, we have a database associated with that.”

The analysts at Thompson-Reuters curate and compile databases such as MedaCore and Cortellis, which they provide to their clients to help them with their research and clinical studies. “We can take customer data, and using our tools and our pathway maps, we can help them understand what their data is telling them,” explains Dr. Harrison.

Matt Wampole, Ph.D., a solutions scientist at Thompson-Reuters, spends his days reaching out and working with customers to help them understand and better use the company’s products. “Bench researchers,” he points out, “don’t necessarily know what is upstream of whatever expression change might be leading to a particular change in regulation.” Dr. Wampole indicates that he is part of a “solution team” that aids clients in determining important signaling cascades, regulators, and so on.

“We have a group of individuals who are very ‘skilling’ experts in the field,” Dr. Wampole continues, “including experts in the areas such as biostatistics, data curation, and data analytics. These experts help clients identify models, stratify patients, understand mechanisms, and look into disease mechanisms.”

Dr. Harrison sums up the Thompson-Reuters approach as follows: “We look for master regulators that can serve as both targets and biomarkers.” By examining the gene signatures from both the patient and from curated datasets, in the case of cancer immunotherapy, they hope to segregate patients according to what drugs will work best for them.

“We are working with a number of pharmaceutical companies to put our approach into practice for clinical trials,” informs Dr. Harrison. The approach has already been applied in several studies, including one that used data analysis of cell lines to help predict drug response in patients. Another study helped stratify glioblastoma patients.

Tumor-Targeted Delivery Platform

PsiOxus Therapeutics, which is focused on immune therapeutics in oncology, has developed a patented platform for tumor-targeted delivery based on its oncolytic vaccine, Enadenotucirev (EnAd), which can be delivered systemically via intravenous administration.

According to company officials, EnAd’s anti-cancer scope can be expanded by adding new genes, thereby enabling the creation of a broad range of unique immuno-oncology therapeutics. In a recent study conducted at the University of Oxford, researchers led by Philip G. Jakeman, Ph.D., sought to improve the models for evaluating cancer therapeutics by introducing ex vivo methodologies for research into colorectal cancer.

The ex vivo approach utilized was able to exploit a major advantage by preserving the three-dimensional architecture of the tumor and its associated compartments, including immune cells. The study, which was presented at the International Summit on Oncolytic Viral Therapeutics in Quebec, showed the tissue slice model can provide a novel means to assessing an oncolytic vaccine in a system that more accurately recapitulates human tumors, provide a more stringent test for oncolytic viruses, such as EnAd, and allow study of the human immune cells within the tumor 3D context.

By maintaining the components of the tumor immune microenvironment, this new methodology could become useful in analyzing anti-viral responses within tumors, or even in evaluating therapeutics that target immunosuppressive tumor micro-environments, noted the Oxford team.

Ian Clift Ph.D. is a Scientific Communications Consultant, Biomedical Associates and Clinical Assistant Professor, Indiana University

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