Poster Presentation 37th Lorne Cancer Conference 2025

Investigating colorectal cancer metastasis using a novel zebrafish xenotransplantation model (#249)

Amna Shoaib Siddiqui 1 , Amardeep Dhillon 1 2 , Faiza Basheer 1 2
  1. Deakin University, School of Medicine, Geelong, VIC, Australia
  2. Institute of Mental and Physical Health and Clinical Translation, Geelong, VIC, Australia

Background:
Colorectal cancer (CRC) causes over 900,000 deaths each year, primarily due to metastasis1. At diagnosis, 25% of patients already present with metastasis, and approximately 50% of all CRC patients experience relapse after treatment. Metastasis requires CRC cells to detach from the primary tumor, navigate through physiological barriers, and successfully colonize distant organs. However, understanding the mechanisms that drive the outgrowth of micro-metastatic lesions and why some dormant lesions relapse remains a challenge.

Zebrafish embryos provide a useful model to study metastasis2, but current models are best suited to studying early stages of metastasis as they develop adaptive immunity by 5 days post-fertilization (dpf), preventing further study of the fate of micro-metastatic lesions that develop after xenotransplantation of cancer cells. To address these limitations, we have established a novel zebrafish xenotransplantation model using immune-deficient Casper SCID zebrafish (Casper il2rgc.a-/-)3. This model allows for longer-term investigation of micro-metastatic lesions and their potential to develop into larger tumors.

Materials & Methods:
Human colorectal cancer cell lines were labelled with cm-Dil dye and injected into the circulation and perivitelline space of 2-day-old Casper SCID zebrafish embryos. These embryos were imaged using confocal microscopy at 5-, 11-, and 18-days post-injection (dpi). This model enabled long-term tracking of micro-metastatic lesions and allowed for the investigation of key metastatic processes, including intravasation, extravasation, and angiogenesis.

Results:
In standard Casper zebrafish, immune rejection of cancer cells was evident after 5 dpi, limiting long-term analysis. By 18 dpi, only 10% (n=29) of the Casper zebrafish retained small micro-metastases, indicating effective clearance of cancer cells from circulation. In contrast, the Casper SCID model permitted sustained tracking of colorectal cancer (CRC) cells. At 18 dpi, 76% (n=17) of the Casper SCID zebrafish exhibited distinct metastatic phenotypes, reflecting human metastasis patterns. Among these, 35.5% displayed both micro-metastases and evidence of metastatic colonization, while 41% presented with micro-metastases alone.

Conclusion:
The Casper SCID zebrafish model offers a unique opportunity to study the later stages of metastasis, overcoming the limitations of immune rejection seen in standard models. This provides a potential platform for investigating the key pathways involved in metastatic colonization and tumor progression.

  1. Sung et al., 2021. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. A Cancer Journal for Clinicians, 71(3), 209–249.
  2. Martinez-Lopez M, Póvoa V, Fior R. Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis. J Vis Exp. 2021 Jun 19;(172). doi: 10.3791/62373. PMID: 34223839.
  3. Sertori et al., 2022. Generation and Characterization of a Zebrafish IL-2Rγc SCID Model. International Journal of Molecular Sciences, 23(4)