NEW YORK – Italian sample prep firm Tethis has developed a novel liquid biopsy method that uses a nanomaterial-coated slide to isolate and capture circulating tumor cells (CTCs) and CTC clusters at the site of collection to maintain cellular and integrity and clinical informativity.
The Milan-based company believes the method can help downstream assays detect early-stage breast cancer in patients to minimize repeated mammography screening prior to curative surgery.
Roberta Carbone, CSO of Tethis, explained that the firm was founded in 2004 as a spinout from the University of Milan by physics professor Paolo Milani to commercialize a proprietary film composed of nanostructured titanium oxide. The film mimics the porosity and adsorption properties of extracellular matrix structures and is capable of spontaneously immobilizing all cell types at room temperature.
Tethis initially used the technology to produce gas sensors for environmental control and biological applications. However, the firm recently expanded the method to the liquid biopsy space by building slides called "Smart BioSurface" (SBS) slides coated with the nanomaterial to capture a monolayer of cells in a patient's blood sample.
"If you take any living cell and leave it touching the slide … without any shear stress or manipulation, it will attach to the surface and stay there," Carbone explained. "You can hand pipet the individual cells off the slides and prepare them for downstream applications."
Carbone said the SBS slides can be used to collect different kinds of cellular information, including morphological and genetic analysis, from a fresh blood sample at the point of collection. The method does not require any sort of chemical preservative for blood collection that other sample prep methods may need to ship blood samples for analysis.
To standardize slide preparation, Carbone's team built a liquid handling platform called CellSeed to ensure automated cell seeding and fixation on the SBS slides. CellSeed allows users to perform blood processing immediately after extracting a patient's blood sample, and can seed cells from about 10 ml of blood onto 20 slides in two to three hours.
After the cells have been seeded on the SBS slides, Carbone said that users can apply downstream methods — including cytology and immunocytochemistry, immunofluorescence, fluorescence in situ hybridization (FISH), and single-cell molecular analysis — to detect different morphological, genetic, and other molecular biomarkers.
In a proof-of-concept study published this week in the British Journal of Cancer, Carbone and colleagues led by researchers at the University of Basel in Switzerland, applied the SBS slides and CellSeed instrument to detect breast cancer-derived CTCs in blood samples from patients prior to curative surgery.
Collecting 10-ml blood samples from 28 stage I-II breast cancer patients (21 luminal-like, 4 triple-negative (TNBC), and 3 HER-2 positive) and 30 healthy controls, the team removed red blood cells through lysis and centrifugation. Remaining white blood cell fraction was then counted and dispensed on SBS slides using CellSeed and shipped to Tethis' lab.
Carbone's team then stained the slides with pan-CK and CD45 antibodies and scanned them using a microscope to identify CK-positive cells. The group classified CTCs as cells with CK-positivity, CD45-negativity, and cancer-related morphological features such as irregular cell size, shape, and a large nucleolus.
Overall, the researchers found that the SBS CellSeed workflow produced a clinical specificity of about 97 percent and sensitivity of 75 percent for all subtypes (81 percent when excluding the TNBC-positive samples).
While the group found single CTCs in 21 breast cancer patients, Carbone noted that her team was surprised to identify clustered CTCs in five patients with early-stage breast cancer. However, her team will need to determine the CTC clusters' ability to represent a risk factor for disease progression in larger clinical studies.
"We were able to identify CTC clusters that were previously described only in metastatic sections," Carbone said. "This indicates that CTC clusters in early-stage breast cancers are a prognostic index for disease progression."
The group has launched prospective trials to validate the workflow's prognostic potential in various breast cancer subtypes. Carbone's team is also trying to identify whether biomarkers expressed in TNBC tumors are present in CTCs using the method.
While the researchers demonstrated SBS-CTC CellSeed's ability to help detect CTC and CTC clusters in early-stage breast cancer in the study, Carbone said that Tethis is speaking with clinicians about potential clinical applications for the tool, including testing to minimize mammography screening.
"We've got an indication that [CellSeed] would be a good test to see if we can analyze [a blood sample] after a mammography when the results are unclear, or if a patient is at risk for a BRCA mutation," Carbone said. Such patients could also be followed over time using a simple blood draw, she added.
Tethis currently holds five patents covering SBS and its different applications and has submitted applications for other patents related to CellSeed with the US Patent and Trademark Office and European Patent Office.
Tethis soon plans to apply for a CE-IVD mark to offer the SBS CellSeed method clinically in the European market. While the firm is still deciding the exact regulatory path for the US, Carbone anticipates that it will commercialize the platform as a laboratory-developed test.
Despite declining to say how much funding Tethis has raised since its founding, Carbone noted that her team expects to raise additional financing for large validation studies as part of its regulatory approval plans.
Sample prep competitors
As Tethis gears up to commercialize the SBS CellSeed platform, other liquid biopsy firms are developing or offer assays for CTC isolation and purification from patient blood samples.
Menarini Silicon Biosystem's CellSearch assay, which identifies epithelial biomarkers to enrich and enumerate CTCs, was recently found to help predict breast cancer prognosis and treatment response. The Bologna, Italy-based firm's assay helps manage patients with metastatic breast, prostate, and colorectal cancers.
UK-based Angle recently filed a full de novo submission to the FDA for its Parsortix/HyCEAD molecular cell-sorting workflow, which uses a disposable cassette to harvest CTCs from blood based on their less-deformable nature and larger size compared to other blood components.
Bio-Rad Laboratories subsidiary Celsee offers its Genesis system, which allows users to enumerate and analyze single cells such as CTCs for downstream applications, including cytometry, transcriptomics, multi-omics, rare-cell enrichment and enumeration, and immune monitoring.
NRichDx, which launched its magnetic-based sample prep Revolution system in 2019, is also developing the platform to collect cell-free genetic material, exosomes, and CTCs.
Ann Arbor-based startup Akadeum Life Sciences is developing a microbubble technology to isolate and identify CTCs and other molecules for downstream applications. The method uses low-density glass-shell, air-filled bubbles with surface antibodies that functionalize to cellular targets.
Carbone highlighted that the SBS CellSeed platform stands out from other sample prep methods because it can analyze fresh blood samples immediately collected from the patient. In addition, the slides can be stored at room temperature, minimizing any damage that the cells might face during transportation.
"This instrument can be placed at the point of blood collection and [directly] process blood," Carbone said. "This is what makes the difference between us and tests that other companies are making, unless they can analyze blood without shipping it to their labs."
Tethis is now working on a "sample preparator" that will serve as an automated version of the SBS CellSeed workflow. Carbone envisions the platform being installed for preanalytical standardization without requiring a trained operator, but she declined to provide more details about the instrument at this time.
"We believe that our project addresses … [the] pre-analytical standardization in sample preparation … [and] understanding of real needs in diagnostics will be the way to bring our solution into the clinics … to finally improve peoples' quality of life," Carbone said.
Despite initially focusing on breast cancer diagnostics, Carbone said her team also eventually plans to evaluate the method's use for detecting prostate and lung cancer-linked biomarkers in blood samples.
The firm is working with informatics firms and building an international network of pathologists to evaluate the ability of SBS CellSeed, in combination with digital pathology, to identify CTCs using automated imaging analysis.
"We plan to evaluate other clinical contests … where CTC identification in early settings is still not clearly demonstrated but will be of paramount importance for the optimal management of these cancer patients," Carbone said. "My vision is to build a 'library' of data regarding cell features to teach AI algorithms to be able to detect and recognize putative cancer cells by either morphology, cytological staining, and biomarker expression."