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Georgetown Team Sees Potential for Novel CTC Culture Method to Study Metastatic Cancer

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NEW YORK – A team led by researchers at Georgetown University has developed an unbiased method to isolate and propagate circulating tumor cells (CTCs) and has demonstrated its use in metastatic breast cancer.

The researchers have filed a patent for the workflow, which they say could be used to identify genetic and molecular markers of metastasis, personalize cancer treatment, and develop targeted therapeutics.

Seema Agarwal, senior author and associate professor of pathology at Georgetown University, explained that her team sought to develop an unbiased collection technique that minimized any stress to CTCs, which tend to be fragile, during isolation.

After collecting a patient's blood sample, the team removes red blood cells (RBCs) and granulocytes using gradient centrifugation. They then harvest the rest of the cells, including CTCs, and put them into a custom cell culture growth medium for culture, followed by downstream analysis. 

In a proof-of-concept study, published last month in Cancers, Agarwal's team collected blood samples from 12 metastatic breast cancer patients representing all three major subtypes — HER2 positive, hormone receptor positive, and triple negative — and samples from five healthy donors.

After removing RBCs from the blood samples, the researchers harvested all remaining cells, including CTCs, in the plasma and placed them into culture. After a month of growing the cells, they isolated RNA from all 12 cultures.

To see whether they had indeed cultured CTCs, the group then tested for the presence of epithelial, mesenchymal, and breast tissue markers to establish the cells' tissue of origin using reverse-transcriptase quantitative PCR. They selected cytokeratin 5 and 8 and mammaglobin and successfully identified all three biomarkers in all 12 cultures.

Agarwal's team then used RNA-seq on the six samples that yielded sufficient RNA and could be cultured for more than 30 days in order to characterize their gene expression. These six samples all contained CD45+ leukocytes, they noted, which have previously been shown to support CTC survival.

Overall, the researchers identified 7,234 genes that were significantly differentially expressed in the CTCs-containing samples compared to the healthy donors. Increased expression of multiple genes was correlated with a significant drop in overall patient survival, they found.

Using multiple bioinformatics tools to confirm that the CTCs in the samples originated from the cancers, the team identified 52 significantly enriched key cancer pathways and 21 enriched genes that are important to breast cancer progression and metastasis.

To determine the identity of leukocytes in the cell cultures, Agarwal's team then applied two computational tools to the RNA-seq data. The group mainly found enriched CD8 T cells, neutrophils, and macrophages in the CTC cultures and noted that the isolation technique appeared to favor the survival of macrophages and neutrophils.

"There are reports coming out that neutrophils and macrophages do help in the longevity of CTCs in patients … and [those] patients with CD45+ cells have [a] poorer prognosis," Agarwal said. "We are now finding that wherever we had CD45+ cells, we had a much better survival and expansion of cells."

However, Agarwal acknowledged that her team dealt with multiple limitations while performing the study. Because of the small number of CTCs that could be extracted via a blood sample, the group was unable to directly compare the technique with other methods for isolating and propagating CTCs. They also pointed out that they will need to pursue further studies to demonstrate the suitability of the method across different cancers.

Daniel Hayes, a breast cancer researcher at the University of Michigan who is developing a wearable CTC microfluidic chip, said the 100 percent CTC culture success rate was "quite unusual" for a breast cancer cohort. He speculated whether the high success rate is related to the specific breast cancer types Agarwal's team looked at in the study.

"Traditionally, we've been able to get estrogen receptor negative [cells] to grow much faster than estrogen receptor positive, grade 1 breast cancer [cells]," Hayes said. "Maybe their cell culture is more sensitive at detecting cancer cells than [Menarini Silicon Biosystems'] CellSearch … but they will need to prove it."

Agarwal and her colleagues now plan to validate the technique in a larger cohort of metastatic breast cancer patients. In addition, she hopes to apply the method to other solid tumors, including lung, colon, and pancreatic cancer.

With her team's success of short-term CTC propagation in cell culture, Agarwal envisions the technique eventually being used for multiple clinical applications, such as creating patient-derived xenografts, cancer drug discovery, and targeted drug therapy.

"We could take DNA and RNA from cultured CTCs and [do] a detailed genomic analysis that can be done very quickly in a more meaningful way, as prior to expansion, there are very few CTCs in the patient's blood," Agarwal said. "We don't know whether the cultured cells represent the heterogeneity of the CTCs, but as we move forward, we hope to characterize the heterogeneity of the cells."

While some companies have found moderate success in propagating extracted CTCs in culture, Hayes noted that the clinical relevance behind the extracted CTCs still remains to be seen.

"Part of the problem is that the cells you grow in culture don't necessarily represent the cells left inside the patient's body," Hayes explained. "Going from biopsy to culture probably kills some cells that are important, and the cells that survive may not relate to ones back inside [the body]."

Hayes, who previously received royalties from Menarini Silicon Biosystems for his efforts on developing the CellSearch technology, said that researchers currently don’t know whether CTCs or other identified cells are the source of the disease or simply represent cells from the tissue that is sending out the metastatic cells into a patient's bloodstream.

At the same time, Hayes said, he is looking forward to more extensive trials by Agarwal's team to determine the clinical significance of the study's findings.

Agarwal's group has filed for IP related to the isolation and propagation method with the US Patent and Trademark Office. However, she said it is still too early to decide whether the group will commercialize the technique, as it will need to identify preclinical variables and other potential hurdles.

"We need to expand the study and see what is unique to our tech compared to other methods out there," Agarwal said. "We will also need to see the usefulness for non-metastatic breast cancer patients, as we could perform a molecular profile and see if the patient is tumorigenic or not … and decide if there is a need for a patient to be treated aggressively."

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