Cancer chemotherapeutic agents are developed and approved largely on the back of studies that demonstrate an average efficacy and safety across possibly thousands of patients. Even for emerging biomarker-driven treatments, there’s still no guarantee that every matched tumor will respond in the same way to the same therapy. The situation becomes even more complicated when treating metastatic tumors, for which therapeutic guidelines may indicate that there are a number of possible approaches, each with similar average response rates. The upshot is that individual patients will often have to undergo multiple rounds of treatment on a trial-and-error basis.
Ideally, it would be possible to screen individual tumors for response to different treatments so that the best therapeutic approach can be identified in advance for each patient. The current gold standard for personalized screening involves directly transplanting primary tumors into immunocompromised mice, waiting for the tumors to grow, and using the resulting patient-derived xenografts (PDX) as a model for testing tumor responses to cancer therapy. However, this approach requires a significant amount of tumor sample, and it takes at least a month for the transplanted tumors to engraft and grow in mice so that the effects of therapy can be observed at different levels. As a result, the approach is impractical for screening in a clinical setting.
Researchers in Portugal have now shown that zebrafish larvae represent a much faster, and highly sensitive in vivo model for assessing how individual tumors respond to cancer therapy. Rita Fior, Miguel Godinho Ferreira, and colleagues at the Champalimaud Centre for the Unknown (CCU), in Lisbon transplanted cells from human colorectal cancer (CRC) cell lines into zebrafish larvae.
Within just a few days, the resulting xenografts could be used to investigate differences in tumor cell proliferation rates and metastatic and angiogenic potential. The researchers could also investigate—with single-cell resolution—cellular changes within the same tumor. The zebrafish xenografts also represented an accurate measure of sensitivity to the colorectal cancer drug cetuximab, and exhibited treatment responses that mirrored those of xenografts in mice.
Zebrafish Xenografts Mirror Human Tumor Responses to Cancer Therapy
For their proof-of-concept studies, the team then injected zebrafish larvae with cancer cells taken directly from primary patient CRC tumor samples, without first growing the cells in the laboratory. They found that the human tumor xenografts (zPDX) that developed in the larvae responded to cancer drugs that were used to treat the patients in exactly the same way that the patients’ tumors responded.
The researchers suggest that the zebrafish larvae xenografts represent a “promising in vivo screening platform for precision medicine.” Their studies are reported in the early online edition of Proceedings of the National Academy of Sciences (PNAS), in a paper entitled, “Single-cell Functional and Chemosensitive Profiling of Combinatorial Colorectal Therapy in Zebrafish Xenografts.”
Combined Expertise in Developmental Biology, Zebrafish, and Tumor Evolution
The potential use of zebrafish larvae bearing human tumor xenografts to recapitulate therapeutic response was conceptualized independently by Dr. Fior—whose background is not in cancer, but in developmental biology and zebrafish, she told GEN —and by Dr. Ferreira, whose expertise and interests are in the heterogeneous nature and evolution of tumors. It’s this heterogeneity between tumors and changes within tumors as the disease progresses that make it so difficult to know how to select the best chemotherapy. “In some cases, the efficacy rate of chemotherapies can be low, sometimes around 35%,” Ferreira noted. “This means that some patients risk taking inadequate drugs that weaken them—and without a proper test, there is no way to know who will benefit and who won't.”
“I developed a particular interest in cancer therapy when a family member was diagnosed with cancer in 2009,” Dr Fior explains to GEN. “At the time, I didn’t realize that clinicians select a treatment based on international guidelines, and that because there may be more than one therapeutic option that apparently has equivalent response rates, there is no way of knowing which treatment will work best. My background in developmental biology and zebrafish sparked the idea of using the fish larvae as a screening platform; like an ‘antibiogram’ to test treatments on tumor xenografts derived from patient’s tumor samples. Miguel was working along the same lines, and we teamed up for the reported studies.”
The researchers’ initial experiments confirmed that when cancer cells from different human CRC cell lines were injected into the zebrafish larvae, the cells implanted and proliferated into xenografts that exhibited tumor-specific cellular and angiogenic charateristics, as well as the ability to form metastases. “What was particularly encouraging was the very good engraftment rates,” Dr. Fior tells GEN.
Essentially, the cells behaved in the zebrafish larvae exactly as they would do in a native tumor. The zebrafish larva xenograft model also demonstrated sufficient resolution, “at the allele level,” Ferreira claims, to detect different treatment requirements in tumors that are genetically very similar.
In subsequent proof-of-concept studies, the researchers confirmed that in four out of five cases, zebrafish avatar zPDX xenografts derived directly from primary patient tumor samples responded to chemotherapy in the same way as did the tumors in the patients. “For two of the patients, the tumors transplanted into the larvae did not respond to the chosen chemotherapy,” comments Rita Fior. “And in fact, consistent with our results, a short time afterward, those patients relapsed.” On the other hand, two other patients whose avatars responded to the treatment “are still doing well as far as we know,” Dr. Ferreira adds.
Interestingly, when the researchers sequenced the tumors for which the matching zebrafish larvae xenografts didn’t respond to cetuximab, they found that in each case the tumor carried mutations that were highly linked with cetuximab resistance, “corroborating our zPDX results with genomic data,” the team writes.
Promising Results with Additional Tumor Types
“These are very encouraging results, but they are really early findings,” Dr. Fior stressed to GEN. “We have confirmed that the system works with the monoclonal antibody cetuximab, and since publication of the PNAS paper we have had promising results with triple negative breast cancer clinical samples. In parallel we have also started gathering liver metastasis samples to test in the zebrafish larva model. Pancreatic cancer is another tumor type we would like to study, and potentially also acute myeloid leukemia.”
The team will have to validate the system in hundreds of patients to confirm the predictive power of zebrafish larvae xenografts for multiple tumor types and therapies. “If everything goes well, we will be able to inform oncologists on the result of the different therapies in the avatars; they will always have the final word in terms of deciding which therapy to choose, but they will be able to base themselves on individual tests,” Dr. Ferreira concludes.
“Ideally, the screening system will be used more routinely when clinical guidelines give clinicians options for equivalent therapies, so that they can predict which therapy will work most effectively for that particular patient,” Dr. Fior says. “It takes about 4–5 days to grow the xenografts and about 2–3 weeks to complete all the immunohistochemistry, imaging and analysis, so results are generated quickly. It’s a lot faster than the mouse model, in which we need to grow palpable tumor masses. In the zebrafish larva model, small xenografts grow quickly and features such as angiogenesis can be investigated directly under the microscope.”
Although the zebrafish larva system does need some optimization, it represents an ideal model in terms of availability and cost of larvae, Dr. Fior noted. “We were particularly encouraged at how well the cells engrafted, which makes it easy to generate potentially more than a hundred avatars for each patient, if there is sufficient sample.”
The results of many years of multidisciplinary research and genetic manipulation in zebrafish indicate that even complex systems and behaviors are remarkably conserved between human and the zebrafish, Dr. Fior suggested to GEN. “It’s really striking. The zebrafish won’t completely recapitulate the human system, and there will always be caveats, but so far it appears to be a very promising model.”
Ideally it would be possible to use zebrafish larvae in combination with the mouse xenograft model, the authors suggest in their published paper. Zebrafish larvae allow for visualization of single cells and response to treatment in multiple xenografts, which improves statistical power. Mouse PDX models involve studying the behaviors of large, palpable tumors in response to longer treatment time. This has the advantage of enabling the study of tumor evolution, emergence of resistance clones, and overall disease progression. “It’s also a model which has been used for studying cancer for many years, so there’s a huge amount of knowledge already about tumor growth and behavior in experimental mice, and we can manipulate the mouse model as required,” Dr. Fior pointed out. Nevertheless, the authors conclude, “we envisage that zebrafish and mouse xengraft models may complement each other; zebrafish as a fast screening platform, and mouse xenografts to accompany tumor evolution and relapse.”
A Potential Model for Drug Discovery
While the researchers are currently focused on using the zebrafish avatars as a model system for screening tumors for therapeutic response, Dr. Fior is separately overseeing a project that is harnessing the zebrafish system to study innate immunity, potentially to help discover new immune-system modulators for cancer therapy. “We have already seen that in some instances, the zebrafish larvae will reject primary tumor cells, but not a metastasis, and if we mix the cells, then the tumor is protected. Further study in this system may find clues that can point us in the right direction to identify potential interactions between the tumor and immune cells, to identify new therapeutic approaches.”