A zebrafish colorectal xenograft model for chemotherapy response

Abstract

Colorectal cancer (CRC) is the third most diagnosed cancer globally and the third leading cause of cancer-related deaths. Early-stage CRC treatment consists of a combination of surgery, radiotherapy, and chemotherapy, while advanced CRC remains difficult to manage, most patients will experience disease progression and require multiple lines of systemic therapy. Choosing the right therapy is often a challenge since most chemotherapeutics lack distinctive biomarkers, thus functional testing has emerged as a promising strategy to personalize therapy and minimize unnecessary toxicity. In this study, we present our zebrafish (Danio rerio) xenograft model to evaluate the response to first-line chemotherapeutic protocols commonly used in clinical practice. We demonstrate that after a short course of chemotherapy there is an evident reduction in primary tumor size, circulating tumor cells, and metastasis area during the follow-up period. These changes were more pronounced in subgroups treated with Irinotecan, indicating the xenografts sensitivity to these protocols. We believe this model has significant potential for both fundamental cancer research and translational applications.

Keywords: chemotherapy; colorectal cancer; in vivo imaging; preclinical model; zebrafish xenograft.

Figure 1

Graphical depiction of the timeline of the experiment. DPI: Days post injection; FOLFIRI: Chemotherapy protocol consisting of 5-Fluorouracil (5-FU), Folinic Acid (FA), and Irinotecan (IRI); FOLFIRINOX: Chemotherapy protocol consisting of 5-FU, FA, Oxaliplatin (OXA), and IRI; FOLFOX: Chemotherapy protocol consisting of 5-FU, FA, and OXA; HPF: Hours post fertilization. Image created with Biorender.com

Figure 2

Representative zebrafish xenografts from each group. Individual dynamic follow-up of each embryo at 1 DPI (left column) and 3 DPI (right column). Each xenograft was imaged in two channels (bright field and orange) and then the pictures were merged. The scale bars represent 1000 μm

Figure 3

(A–G) Relative tumor size is calculated as the area of perivitelline space tumor at 3 DPI divided by 1 DPI and expressed as a percentage; all groups are then individually compared. *: p<0.05, **: p<0.01, ns: p>0.05

Figure 4

Comparative overview of two Tg(fli:EGFP)/Casper perivitelline tumor, CC and metastases burden. The scale bars represent 1000 μm (A1–A3 and B1–B3). Zoomed in view of the perivitelline space using fluorescent microscopy captures superimposed images of embryo vasculature and HCT116 (A4) with arrowheads indicating the endothelium in the green spectrum within the tumor area (A5) and tumor cells in the orange spectrum (A6). Zoomed in view of the tail region using fluorescent microscopy captures superimposed images of embryo vasculature and HCT116 (A7) with arrowheads highlighting highly deformed blood vessels and the caudal vein, while the arrow points to micrometastasis. Smaller primary tumor with no green signal within the mass (B4–B6). Tail view of the xenograft with arrowheads pointing to the vasculature and arrows indicating CC (B7). Note the straight caudal vein and the absence of secondary disseminations. Green (A8 and B8) and orange channels (A9 and B9) were used for better visualization of the microanatomy. The scale bars represent 100 μm (A4–A9 and B4–B9). CC: Circulating cells

Figure 5

(A–G) Circulating cells (CC) change is calculated as the number of CC at 3 DPI divided by the number of CC at 1 DPI expressed as a percentage. *: p <0.05, **: p<0.01, ***: p<0.001, ns: p>0.05

Figure 6

(A–G) Metastasis area change calculated as the area at 3 DPI divided by the area at 1 DPI expressed as a percentage; (H) Caudal vein dimension increase in tail metastases. *: p<0.05, **: p<0.01, ***: p<0.001, ns: p>0.05