Champalimaud Foundation

Colorectal Zebrafish Xenograft Model

This research, conducted at the Champalimaud Foundation in Lisbon, is focused on the immune response after implantation of human cancer cells in zebrafish. The cells in the implanted tumors were characterized using SORT-seq. Here, we explain how SORT-seq contributed to this remarkable study.

Xenograft Zebrafish Single Cell Sequencing, at Single Cell Discoveries

The problem

Cancer immunoediting is a process in which the immune system can constrain and promote tumor development. Either the immune system eliminates the developing tumor (immune surveillance), or the immune system drives further growth of cancer cells (tumor promotion).

This study aimed to investigate the contribution of innate immunity to cancer immunoediting by using a zebrafish embryo xenograft. The zebrafish xenograft is an excellent model in this case since its adaptive immunity is not fully developed until 2 to 3 weeks post-fertilization.

This time-efficient assay is based on injecting human tumor cells into the zebrafish embryo and accessing tumor behavior and response to anti-cancer therapy after four days.

The first author, Vanda Póvoa, explains the results of engrafting the tumor cells in the zebrafish.

“We found that some tumors engraft very well in the fish, while others get cleared in just four days. Moreover, we found not only with cell lines but also with patient-derived tumors that sometimes the administration of chemotherapy results in a strange phenotype: more tumors in the zebrafish.”

“So, we start to think maybe these results are caused by the immune system. The immune system may eliminate some tumor cells by causing fish immune suppression with chemotherapy, which results in more tumors. So, we decided to focus on a pair of cell lines derived from the same patient: a primary and a metastasis cell line with contrasting engraftment rates. We defined the primary tumor as a regressor (poorly engrafted) and the metastasis as a progressor (efficiently engrafted)”.

Experiments with this pair of cell lines generated more results that pointed towards the innate immune system and immunoediting, Vanda explains.

“Bulk RNA sequencing data* shed some light on the immune system, so we started with polyclonal (mixed tumors) experiments. We thought, maybe if we mix the cell lines, progressors can protect the regressors from getting cleared by the immune system. And indeed, when we mix the cell lines (1:1), we get something in between.”

These results suggested that regressors were triggering the activation of the innate immune system and progressors providing an immunosuppressive environment, which the following experiments further proved.

“We started with the characterization of the immune system. Since the zebrafish don’t have an adaptive immune system yet, we can focus on the innate immune system. On the first day post-injection, the regressors recruit a lot more neutrophils than the progressors. The same was true for the macrophages. When looking at the mixture, having progressors in the mix blocks recruitment of neutrophils and macrophages into the tumor.”

The recruitment of neutrophils and macrophages by the regressors probably causes poor engraftment in the zebrafish. Vanda Póvoa explains how this was confirmed:

“As a proof of concept, we used mutants that transiently deplete neutrophils and macrophages. The progressors engrafted the same, but we saw many more tumors in the case of regressors at the end of the experiment. Besides the mutants, we also depleted the immune cells with a drug. Then we also saw more and bigger regressor tumors.”

And finally, the authors showed that cancer immunoediting is present in the zebrafish xenografts.

“The regressor tumor cells that were able to evade the immune system in the zebrafish the first time were injected for a second round. We found bigger tumors and less detection by macrophages. So, this was proof that immunoediting takes place.”

The Fior Lab Team

The solution: SORT-seq

Although this sounds like a complete and solid study, some questions remained about immunoediting for the authors and the paper's reviewers.

“To show the community that cancer immunoediting is happening, we needed single-cell sequencing to prove that we have different clones subjected to this change.”

SORT-seq was performed on the zebrafish xenografts on days one and four to identify the clearance or expansion of specific subclones in the regressors.

“We wanted to compare the clones we expanded between days one and four and the ones that we cleared. So, we used SORT-seq, dissected the tumors, and sorted the GFP+ cancer cells and the GFP- fish immune cells. We only needed five plates for this experiment.”

The SORT-seq experiment provided the authors with many data that helped them identify the human cancer subclones that were expanded, cleared, or remained after the engraftment of the regressors.

“We identified six different clusters and found that two clusters disappeared after four days. Other clusters were expanded or remained the same. Single-cell sequencing enabled us to make this classification. For example, a subclone with IL-10 signaling, so immune-suppressing, was expanded. A subclone with IFN signaling, so inflammatory, was cleared.”

The results of the GFP-fish immune cells were not included in the paper but gave essential implications for future studies.

“We also wanted to know if, with single-cell sequencing, we could identify the zebrafish immune system. So, we filled the plates with 80% human dissected tumors and the other 20% GFP-, so cells from the zebrafish. So, when Mauro analyzed the data, he saw that these cells were not human. It was cool that it was possible to analyze both species within the same sample. We will probably move forward on that.”

With SORT-seq, the authors could do more than identify the subclones, Vanda explains.

“My experience with SORT-seq is great. Even with a few cells, we detected many changes. We could identify the subclones and the origin of individual cells. We could even identify the origin of the patient-derived tumor cells from the crypt or the villus in the intestine based on the Wnt expression. We were really happy.”

It was the group’s first experience with SORT-seq. Vanda explains how Single Cell Discoveries assisted them in the process:

“We tried a different type of fixation during the sample processing. You allowed us to try this and gave us a lot of information about the problems we might encounter with our samples. It was nice to work with a team like this that gives you feedback, not only in the bioinformatics part but also in processing the samples. So, it was a valuable experience working with you. If needed, there was always someone to talk to. We also had many meetings with your experts in the lab. Working with you was reliable and saved us money and time. It gave us a lot of confidence because we got so much feedback.”

The result

Shortly after we generated the single-cell sequencing data, the authors published the paper in Nature Communications.

“The results gave our paper a lot more impact and allowed us to answer to the demands of the reviewers at Nature Communication. Without SORT-seq, we would not have published in Nature Communications. The reviewers required Single-cell RNA sequencing to move forward. If we didn’t do single-cell RNA sequencing, we could not publish because the impact would be too low.

Vanda Póvoa explains the impact of the paper on different fields.

For the cancer community, we showed that there is immunoediting happening here. And second, for the zebrafish community, we can use zebrafish as a vessel for cancer and study transcriptional changes within just one week.

The study allows the group to move forward with the zebrafish xenografts.

“For the future, we will probably not work with this pair of cell lines anymore but move on to patients. Patients are our main goal. With single-cell sequencing, we want to try to identify different profiles before and after chemotherapy. Or maybe identify immune cell populations in zebrafish that can read human cancer cells. But regarding the paper and this story, we are now done. The idea is that now we can use the zebrafish to read the cancers. So, we will work more with Single Cell Discoveries for sure.”

Read the full paper here. You can find a “behind the paper” on Nature here.

*Bulk RNA sequencing data was not generated by Single Cell Discoveries

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