Graduate school, it turns out, is difficult—not only because one is tasked with finding and pushing the boundaries of human knowledge, but also in its rather unique ambiguity. Compared to many other professional undertakings, there is no playbook, no clearly defined set of milestones on the road to earning a Ph.D. Every graduate student’s journey is a snowflake—a scientific 'bucking bronco' ride without the cheering audience. And as many students (and their PIs) can attest, sometimes the journey ends up quite different than what was initially imagined.
This was at least partially the case for Dr. Ami Yamamoto, who recently completed her Ph.D. in the lab of Dr. Kevin Cheung in the Public Health Sciences Division at Fred Hutch. The Cheung Lab studies cancer metastasis—a process which is still not well understood but is responsible for a majority of cancer mortality in patients. Generally, cancers metastasize when cancer cells dissociate from the original tumor and enter circulation (becoming ‘circulating tumor cells’, or CTCs) before landing elsewhere in the body and establishing a new tumor. While the Cheung Lab commonly relies on ex vivo-cultured organoid models as a reasonable approximation of human tumors, the ‘holy grail’ of their work had always been to catch tumors in the process of metastasizing in vivo. As Dr. Yamamoto was to learn, however, this is a classic ‘needle in the haystack’ problem—CTCs are exceedingly rare, so purifying meaningful amounts of them out of the blood of small animals like mice is painstaking work.
Early in her PhD journey, Yamamoto and Cheung thus conceived a risky side project to establish a system to study breast cancer metastasis in vivo. Instead of mice, Yamamoto turned to rats as a model, hoping that their larger tumor and blood volumes would make isolating CTCs more tractable. Gratifyingly, this strategy proved successful—following implantation of mouse breast cancer cells, rats yielded ten times the number of CTCs and CTC clusters, concordant with a ten-fold increase in lung metastases. For the first time, the team had a reliable system to track and characterize tumor metastasis in a living animal.
With this innovative model system in hand, Yamamoto proceeded to ask a simple follow up question: after tumor cells were implanted, how long did it take for the primary tumor to start shedding cells and metastasizing? Seeding tumor cells in a cohort of rats and quantifying CTCs in circulation at systematic timepoints revealed something unexpected—CTCs were nearly undetectable in the blood until 27 days post-implantation, at which point they drastically increased, up to 50-fold! To figure out what was happening in tumors at the seemingly arbitrary 27-day timepoint, the team paired this analysis with tumor histology. With a look into the tumors themselves, Yamamoto noticed that 27 days marked a period of rapid growth—not of the tumors themselves, but of their necrotic cores—dense regions of dead and dying cells which are often seen in the center of aggressive tumors. This was a puzzle; as Yamamoto puts it, “the assumption in the field has been that metastases originate from the edge of an invading tumor, so the fact that we specifically see necrotic core growth correlating so tightly with CTC emergence in our model cast that into doubt.”
A whirlwind of follow-up experiments confirmed the finding: not only was the team able to use fluorescent microscopy to localize CTC clusters to dilated blood vessels in the necrotic core, but they used transcriptomic profiling to identify a single gene, angiopoietin-like 7 (ANGPTL7), whose loss significantly reduced necrotic core formation and lung metastases in their model. Here again, rats came to the rescue: their tumors were large enough to enable dissection of the necrotic core from the non-necrotic tumor rim. Additionally, since the team implanted mouse tumor cells into rats, they were able to computationally separate tumor-derived (mouse) from host-derived (rat) genes in their transcriptomics data. As a proof of principle for the relevance of ANGPTL7 in human cancers, Yamamoto and colleagues also demonstrated a correlation between markers of tumor necrosis and CTC abundance in a cohort of breast cancer patients using data from a recent Fred Hutch/University of Washington collaboration.
In all, this study really does reflect a journey—from the early days of high ambition set against the stark uncertainty of a new experimental system, to puzzling results which eventually coalesced into a finding challenging long-held beliefs about metastasis and nominating a potential therapeutic target. It goes to show that—whether we’re talking about a stressful cellular microenvironment and dilated blood vessels in a tumor, or steadfast perseverance and an enthusiastic embrace of the unknown in a scientist—it’s what’s on the inside that counts.
The spotlighted research was funded by the National Institutes of Health, the Department of Defense, a Komen Career Catalyst Grant, the Burroughs Wellcome Fund Career Award for Medical Scientists, the V Foundation, Seattle Translational Tumor Research, GE Healthcare, and the Doris Duke Charitable Foundation.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium members Dr. Kevin Cheung, Dr. Michael Haffner, and Dr. Carolyn Wang contributed to this study.
Yamamoto, A., Huang, Y., Krajina, B. A., McBirney, M., Doak, A. E., Qu, S., Wang, C. L., Haffner, M. C., & Cheung, K. J. (2023). Metastasis from the tumor interior and necrotic core formation are regulated by breast cancer-derived angiopoietin-like 7. Proceedings of the National Academy of Sciences, 120(10).