Hybrid Fusion FISH™ And Cell Squeezing: Non-Disruptive Delivery To Interphase Nuclei For Faster Pre-Slide Treatment In Oncology Diagnostics

Review Article: Part 4 of Series


Traditional FISH wet-lab protocols require considerable time and various reagents to prepare clinical samples for hybridization. This process is known as the pre-slide treatment or pre-hybridization steps and is critical for samples that are preserved in a multitude of fixatives (i.e. Carnoy’s, 10% neutral buffered formalin). Pre-slide treatment protocols traditionally range between 1–12 hours (depending on sample, with average ~3 hours) and is laborious for the technologist. Clinical samples can be in the form of cell suspensions (i.e. peripheral blood, urine, cervical brushings, saliva) and formalin-fixed paraffin-embedded (FFPE) solid tumor tissue blocks. In diagnostic settings, such samples can sit for lengths of time in fixation solutions which can lead to nuclear degradation and ultimately have a detrimental downstream effect on the quality of the DNA targets for FISH. More efficient and novel high-throughput methods are desired by clinical reference laboratories processing upwards of 400+ samples (slides) per day.

Hybrid Fusion FISH™ probes are “squeezed” into cells with CellSqueeze™ to preserve cellular morphology and eliminate laborious pre-slide treatment protocols.

To overcome these barriers, it is significantly more efficient to directly introduce Hybrid Fusion FISH™ probes to the cellular sample without the need for any pre-slide treatment. This eliminates time-consuming protocols for cell membrane permeabilization and disruption in addition to the steps for cytoplasm removal. Rather, cells from the clinical sample are in a homegenous mixture with Hybrid Fusion FISH™ probes and pass through a microfluidic channel (CellSqueeze™) to induce temporary cell deformation and permeabilization. Upon exit of the channel, the Hybrid Fusion FISH™ probes are trapped within the cell which allows for efficient hybridization while retaining the cellular morphology. This process is novel, cost-efficient, and saves time in the wet-lab.

Illustration of Hybrid Fusion FISH™ With CellSqueeze™

Traditional delivery of DNA probes for FISH requires complete cell membrane rupture and removal of the cytoplasm with the resulting interphase nuclei intact. Such protocols entail the use of various chemical detergents to disrupt the cell membrane and hypotonic solutions (i.e. KCl) to “burst” the cells open. Both methods yield naked nuclei which can be physically “dropped” onto microscope slides with a pipette. Cellular debris from such chemical processing requires additional steps to clean the slide for presentation. The drawbacks of such methods include a) the processing time, b) the preparations of special reagents, and c) the amount of cellular debris which results in increased “background” on the microscope slide upon analysis.

On the left, the traditional FISH protocol is presented. On the right, Hybrid Fusion FISH™ with CellSqueeze™ is illustrated.

Hybrid Fusion FISH™ with CellSqueeze™ technology allows for significantly higher DNA hybridization kinetics. The ability to introduce probes directly to the nuclei increases the rate of probe-to-target contact. This is particularly important for non-repetitive, locus-specific gene sequences. Traditional FISH hybridization kinetics are generally controlled by pushing the equilibrium towards hybridization via adjusting ribose chemistry and concentration of probe buffers. Generally, higher “sugar” (typically ribose-based, i.e. dextrose, dextran sulfate) concentrations will push sugar-based molecular probes towards hybridization, thereby increasing the rate of hybridization kinetics overall. This type of hybridization dynamics occurs irrespective of the type of hybridization product that is sought.

A more targeted, high-yield approach is to limit the amount of competing interactions that can occur during hybridization. In traditional systems, such competitive hybridization products may include bulk dimerization products (Ts:Ns, Ts:Ts), intramolecular folding of unimolecular products (Ts, Ps), non-specific binding products (Ps:Ns), and specific probe-to-target hybridization products (Ts:Ps). Hybrid Fusion FISH™ assays follow novel and unique multiplex-multi-state thermodynamics and hybridization kinetics (this will be addressed in a separate review series). For now, it is important to note that Hybrid Fusion FISH™ with CellSqueeze™ increases the rate of contact of probe-to-target via intracellular delivery of probes within geographical proximity of the nuclei while encapsulating the probes within the cell membrane.

On the left, the results from traditional FISH protocols are presented. On the right, Hybrid Fusion FISH™ with CellSqueeze™ technology is illustrated.

In the above example, the pre-hybridization states are shown for both the traditional FISH method and Hybrid Fusion FISH™ with CellSqueeze™. In the traditional method, probes are applied directly onto the slide, and this increases the background (low signal:noise) of the diagnostic test. However, Hybrid Fusion FISH™ with cell squeezing is able to contain the probes inside of the cells prior to hybridization. This significantly removes slide background (noise) for a clean and robust analysis by an automated imaging system and the pathologist. Note the amount of unused probe in the traditional method vs. Hybrid Fusion FISH™: more probe is retained within the cell (right) as compared to random distribution and “chance” entry (left). The probe:cell contact efficiency is significantly greater with Hybrid Fusion FISH™ with CellSqueeze™. Finally, the morphology of the cells are fully retained (right), whereas there is a mixture of whole cells and naked nuclei in the traditional method (left).

Hybrid Fusion FISH™ probes are maximally utilized for hybridization (post-cell squeezing) while subsequently minimizing the rate of low-quality hybridizations. High-quality hybridizations are defined as maximizing the Ts:Ps hybridization products. In theory, the rate of such products increases with a) the geographical proximity of probes to nucleus and b) the buffer chemistry to push the equilibrium towards hybridization. Hybrid Fusion FISH™ with CellSqueeze™ minimizes probe wastage and maximizes overall hybridization efficiency in addition to reducing technician wet-lab time and reagent costs.

The clinical applications for Hybrid Fusion FISH™ with CellSqueeze™ are best suited for samples that are difficult to process under chemical conditions. Such samples may include urine (bladder cells and dislodged prostate cells after digital rectal massages), cervical samples (cervical brushings of the endocervical canal), and blood (peripheral blood and bone marrow). Such samples vary in overall quality and are characteristic of inter-patient variability. In traditional FISH, sample-specific adjustments in wet-lab protocols are implemented to account for the variability — this is time-consuming. With Hybrid Fusion FISH™ and CellSqueeze™, the probes are directly introduced inside of cells prior to hybridization regardless of the sample quality. The standardization of such a wet-lab protocol that can be implemented with a variety of samples is highly desired by large-volume clinical reference laboratories.

Hybrid Fusion FISH™ and CellSqueeze™ microfluidics are emerging into the clinical diagnostics marketplace to increase the workflow efficiency while reducing the associated costs of such tests in clinical labs. Hybrid Fusion FISH™ cancer screening assays can be implemented on a large global scale if the workflow efficiencies are economically feasible. Introduction of novel protocols (such as cell squeezing ) to address the lack of standardization of current FISH wet-lab protocols are desired. While common cell squeezing technologies are typically used for the introduction of therapeutic agents, Hybrid Fusion FISH™ with CellSqueeze™ is enabling such microfluidic applications for the introduction of diagnostic reagents in oncology reference laboratories.

LOOKING FORWARD

DISCLAIMER: CellSqueeze™ is a trademark and property of SQZ Biotech Inc. This article is part 4 of a series of articles dissecting, researching and evaluating Hybrid Fusion FISH™ applications within the clinical diagnostics and research environments. Prevnos Inc. is a cancer diagnostics company that is rapidly evolving the current cytogenetics and molecular cancer genetics markets. The company is the inventor of the world’s first Hybrid Fusion FISH™ tests (consumables) and the world’s most economical fluorescence microscope termed the Retina™ FISH scope (capital equipment). The company is also the inventor of GEN+ Connect (software), which enables digital pathology for molecular cytogeneticists around the world. Furthermore, the company explores clinical research with pharmaceutical companies for companion diagnostics. Prevnos engages in various forward-thinking R&D projects including, but not limited to, targeted cell-enrichment with subsequent FISH, CTC FISH™, non-disruptive FISH probe delivery vehicles, single-cell isolative technology for FISH, tyrosine kinase inhibitor master FISH assays for lung cancer, molecular characterization for stratification of prostate cancer patients, and COMET (chromatin organization mediated electrophoretic transfer) FISH™ assays for assessing DNA viability.

To learn more, visit Prevnos at www.prevnos.com