NEW YORK – Startup Cellular Analytics has adapted its circulating tumor cell (CTC) microfluidic platform to capture rare immune cells that that the firm believes could eventually be used to screen for early-stage mesothelioma and monitor for recurrence.
The Toronto-based firm's assay, dubbed MesoFind, uses an immunomagnetic capture tool to identify surface antigen biomarkers of mesothelioma precursor cells (MPCs) in the bloodstream of a patient suspected to have the disease.
Initially spun out of the University of Toronto in 2018, Cellular Analytics is commercializing a platform called CytoFind, which uses single-cell mRNA cytometry and immunomagnetic separation to identify and sensitively capture CTCs. Shana Kelley, cofounder and chief scientific officer of Cellular Analytics, said that the firm initially aims to use the platform to support drug development for lung cancer.
Mesothelioma, which is generally linked to inhaling asbestos crystals, forms in the layer of tissue covering internal organs called the mesothelium. Asbestos was widely used until the 1970s when public awareness led to banning the material in mainstream construction and fireproofing in most countries. Genetics also play a risk factor in developing mesothelioma, and clinicians usually diagnose the condition using chest X-rays and CT scans.
Kelley explained that her research team at UToronto altered the CytoFind microfluidic chip to search for and analyze MPCs — characterized by mesothelin, CD90, and CD34 — that may as diagnostic and prognostic markers for mesothelioma. While Cellular Analytics has primarily used the platform to capture CTCs, she noted that rare immune cells are also crucial for cancer detection.
"We've seen MPCs in mice lavage samples, but we've never seen them in a patient's bloodstream using flow cytometry," Kelley said. "It's a great example of how you can take a technology, program it with a few parameters and markers of interest, and [then] be able to ask the question you can't ask with other techniques."
The CytoFind process involves collecting 7.5 ml of a patient's blood sample and injecting magnetic microbeads containing antibodies that bind to biomarker-specific antigens on MPCs. The sample is then processed through the MesoFind microfluidic device under the influence of an external magnetic field. Captured cells are immunostained and separated through nanoparticle-mediated sorting for downstream identification and analysis.
"When we profile based on a marker that we're targeting with the magnetic nanoparticle, we capture the cells in the device and stain them to see them and their positions in the array to give us an expression index," Kelley explained. "We can also layer in multiplexing using immunofluorescence to get an accurate profile in one marker … but then we want to ask if the cell is [for instance] CD34-positive, EpCAM-positive, or [positive for] other markers."
Including sample processing and image analysis via immunofluorescence, MesoFind requires about four to five hours to isolate MPCs in a blood sample and provide a phenotypic profile.
"MPCs seem to correlate with the extent of damage that asbestos can create, which may identify individuals who have mesothelioma, due to their mesothelium," explained Marc De Perrot, professor of surgery and immunology at UToronto and who suggested that the team search for the MPC-linked biomarkers. "Kelley's technology allowed us to identify these MPCs, as the tool has the sensitivity to detect a number of cells in the blood."
In a validation study published earlier this month in EBioMedicine, Kelley, De Perrot, and their colleagues initially collected blood and lavage samples from mice that were injected with mesothelioma cells.
After harvesting and suspending the mouse-derived mesothelioma cells in saline, the researchers divided the samples into 0.5-ml aliquots. They then ran the samples through the MesoFind assay or through a flow cytometry instrument. To distinguish between MPC subpopulations and leukocytes, the team performed immunofluorescent staining with DAPI, CD34, CD90, and CD45.
Because of the high degree of heterogeneity in mesothelioma, the group stratified the target cells into eight different capture zones in the MesoFind microfluidic chip that corresponded to a specific expression level of cell-surface mesothelin. The group generated a mesothelin expression profile by counting the number of the cells in the different zones.
"The increasing number of MPCs in mice peritoneal lavage was similarly detected using MesoFind and flow cytometry due to the abundance of MPCs in the peritoneal cavity," the study authors noted. "[But] we were only able to visualize MPCs in the blood using MesoFind … [while] flow cytometry could not detect MPCs in the blood at any time point during tumor progression and the naïve state."
The researchers then validated the MesoFind assay on blood samples from a cohort of 56 patients, including 23 malignant pleural mesothelioma patients, 23 patients exposed to asbestos, and 10 healthy controls. In terms of the potential for screening and prognostic application, the assay had varying sensitivity (67-100 percent) and specificity (70-100 percent) depending on the different cell-surface biomarkers and mesothelioma subtypes.
Importantly, the researchers found that all MPC subpopulations showed a much higher mesothelin expression in mesothelioma patients compared to healthy donors. They therefore believe that MPCs could be helpful to refine screening in asbestos-exposed individuals by focusing on patients at high risk for mesothelioma.
Kelley and her team also found that that circulating mesothelin-positive, CD90-positive, CD45- MPC subtypes increased with pleural damage related to asbestos and continued to rise during the early development of mesothelioma. Meanwhile, mesothelin-positive, CD34-positive, CD45-negative subtypes began to rise with established malignant pleural mesothelioma and were associated with more advanced mesothelioma.
The study has several limitations, according to one expert. Lucian Chirieac, an associate professor of pathology at Brigham & Women's Hospital who was not involved with the research, raised multiple issues with the study's patient cohort, including the lack of comparison between benign and malignant pleural conditions.
"There are a lot of conditions that could mimic mesothelioma, such as pleurisy, where liquid fills in the chest cavity, and we have to be careful, since some conditions could be benign or inflammatory and some are cancerous," Chirieac said. "This is incredibly important, as we on the pathology side sometimes cannot tell between benign inflammatory conditions and mesothelioma."
Chirieac believes that the paper would "generate more value" if the study authors had included another population of patients that were sick but were diagnosed with a different pleural disease.
Because the study's patient cohort was relatively small, Kelley and her team are now scaling up the cohort in prospective trials to see if they can monitor patients through mesothelioma development and recurrence.
In addition, Chirieac noted that he did not spot any correlation between the patient's cancer stage and higher counts of MPC in the study.
"Paradoxically, the tumors with worse prognosis, [including] desmoplastic, sarcomatoid, and biphasic, have the lowest MPC counts," Chirieac said. "[That] should be another good point to make, and [the study authors] do indeed try to make this point of value between circulating cells and the extent of the disease."
Chirieac also questioned the utility of a test that does not show clear physical proof that the patient has mesothelioma.
"If we have an early diagnosis, like with this test, where the diagnosis turns out to be positive … what are you going to do with the patient?" Chirieac asked. "How are you going to tell the patient that they will develop malignant mesothelioma when there's nothing palpable there?"
In a follow-up email, Kelley responded by pointing out that the MesoFind assay would not serve as an ultimate diagnostic assay, but instead a screening assay that clinicians could potentially use prior to or alongside CT scans and other standard methods for detecting mesothelioma. She also noted that all mesothelioma patients in the study had previously been diagnosed by a pathologist.
"MesoFind allows us to look at individuals that have no pleural damage on CT scan, but are at risk of developing aggressive mesothelioma, or even a genetic abnormality of mesothelioma," De Perrot added. "If the blood analysis from MesoFind is abnormal, we could do a biopsy and may potentiality be able to identify genetic abnormalities in the pleura."
While Chirieac argued that the UToronto researchers will need to widen the study's scope to cover different patient populations, he believes the study's results are promising and that the group has introduced a new concept (MPCs) to make sense of predicting mesothelioma development in real time.
As the team pursues future studies, Cellular Analytics is now considering the best avenue to build a product around MesoFind. Kelley believes the plans will include either commercializing assay using the MesoFind microfluidic chip, or building "instrumentation" to sell to clinics that could screen for mesothelioma so "they could run the analysis themselves."
Kelley said that Cellular Analytics is licensing the MesoFind technology from UToronto under the same patents it originally filed for the CytoFind platform.
In addition to fleshing out a product based on MesoFind, Cellular Analytics is also commercializing the CytoFind assay to establish drug sensitivity for oncology drug development, starting with lung cancer. The firm aims to partner with pharma companies to help target certain patient populations for drug trials, in addition to potential cell-therapy applications.
"We've looked at proteins that are therapeutic targets, such as [the tumor suppressor gene] BRCA2, and want to show that we could profile them in CTCs," Kelley said. "So, therapeutic decision making is another application, by giving doctors therapy guidance."
Cellular Analytics has raised an undisclosed amount through seed funding and has also launched a Series A financing round. However, Kelley declined to provide further details about the round besides that the firm will use the proceeds to further develop CytoFind.
"The idea is to make a piece of instrumentation for CytoFind so that it can be rolled out across the field to researchers," Kelley said. "We want people to use it as an open platform, [where] you can put any set of biomarkers on the platform for profiling."