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: zebrafish xenograft , colorectal cancer , chemotherapy , preclinical model , in vivo imaging
Introduction
Colorectal cancer (CRC) is the third most common cancer diagnosed worldwide, with an estimated 1.9 million new cases annually, and the third most common cause of cancer death, with an estimated 935 000 deaths annually [1]. Mortality varies widely across the globe with Western Europe and North America experiencing plateauing, while Eastern European countries, Asia and South America are witnessing an increase in both. Even so, by 2030 the global burden of CRC is expected to increase to more than 2.2 million new cases and 1.1 million deaths respectively [2].
CRC management requires a multimodal approach, heavily influenced by the stage of disease. For early-stage CRC, curative intent guides treatment strategies, typically involving surgical resection, radiotherapy, and perioperative chemotherapy. In contrast, advanced-stage CRC management primarily relies on systemic chemotherapy in combination with targeted therapy or immunotherapy [3]. Despite remarkable advancements in managing metastatic CRC, disease progression remains inevitable in most patients, necessitating multiple lines of therapy. This presents a challenge: how to select the best regime for each individual patient, thus limiting drug toxicities and avoiding potentially ineffective therapies, while increasing the chance of response.
Most therapeutic decisions are either empirical, or biomarker driven, yet difficult cases exist, where there are no proven biomarkers available to guide the treatment and there is no clear advantage between available therapies. Because of these, functional testing [i.e., direct testing of patient-derived tumor cells (PDTC)] could become an important option for such cases [4]. This could also contribute to avoiding toxicity of treatments that are not effective and improving the chance of getting a tumor response.
One of the many functional approaches includes xenografting (i.e., transplanting PDTC into a different animal host) and running different profiling tests. Most xenografts have been usually done using immunosuppressed mice and rats, but newer models using zebrafish (Danio rerio) have emerged. Unlike the murine models, using zebrafish embryos presents many advantages such as ease of manipulation, lack of an adapting immune system during the first days of development, quick grafting period and transparent tissues [5]. Also, in the refinement of preclinical models for therapeutic development, replacing murine with zebrafish xenografts offers several advantages that align with the replacement, reduction, and refinement (3R) principles of animal research [6].
Aim
The aim of this study was to establish and evaluate a zebrafish xenograft model using the HCT116 CRC cell line, in order to assess its response to clinically relevant first-line chemotherapeutic protocols (FOLFOX, FOLFIRI, and FOLFIRINOX). By monitoring primary tumor area dimensions, circulating tumor cells and metastasis in fluorescent microscopy, we aimed to determine a better modality to assess early tumor response to chemotherapy and prepare the model for PDTC xenograft for better prediction of treatment efficacy and facilitating personalized therapy strategies for CRC.
Materials and Methods
Adult housing and embryo collection
Wild-type (TU), transgenic Tg(fli1a:EGFP) and Casper adult zebrafish strains were obtained from Alexandru Ioan Cuza University of Iaşi, Romania. The adult fish were bred and housed according to international standards. They were maintained at a density of 7–8 adults per liter of water, in an environment kept at 28–29°C, with a 14/10-hour day/night cycle and were fed two times a day [7]. In the morning, after spawning the embryos were collected in Petri dishes containing E3 medium [5 mM sodium chloride (NaCl), 0.17 mM potassium chloride (KCl), 0.33 mM calcium chloride (CaCl2), 0.33 mM magnesium sulfate (MgSO4), 100 μg/mL Methylene Blue 1%] and incubated at 28.5°C for two days.
Cell line culture
HCT116 human colon cancer cell line [American Type Culture Collection (ATCC), #CCL-247) was cultured in T75 cell culture flasks in McCoy’s 5A Medium supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 100 U/mL Penicillin and 100 μg/mL Streptomycin at 37°C in a humidified incubator containing 5% carbon dioxide (CO2). Cell culture medium was changed every two to three days. At around 80% confluence, cells were harvested using 1× Trypsin/Ethylenediaminetetraacetic acid (EDTA). Afterwards, cells were centrifuged and suspended to achieve a concentration of around 100 000 cells/μL in culture medium. Viability assays were performed using Trypan Blue staining.
Fluorescent labeling
HCT116 cells were labeled using Invitrogen Vybrant™ CM-Dil according to the manufacturer’s instructions, using an adapted concentration of 7.5 μL/million cells.
Microinjection system, cell loading and xenotransplantation
At 48 hours post fertilization (HPF) the embryos were graded [8] and those underdeveloped or with morphological abnormalities were discarded. Unhatched embryos were incubated in 1 mg/mL Pronase solution (Roche) followed by chorion detachment through vigorous pipetting and thorough rinsing in E3 medium. Prior to the microinjection procedure, 2% agarose Petri plates were prepared, and borosilicate glass capillary tubes were pulled into fine needles using a Narishige PB-7 needle puller. The microinjection needles were then backloaded using Eppendorf Microloader tips and then fastened to a needle holder (Tritech Research MINJ-4, USA) connected to a microinjector (Tritech Research MINJ-1, USA) with a pulse control module (Tritech Research MINJ-2, USA). The embryos were anesthetized using a Tricaine (Sigma Aldrich) solution (150 mg/L), positioned on agarose plates and then microinjected into the perivitelline space (PVS). After the procedure, the zebrafish were immersed in a recovery anesthetic bath for 15 minutes and then transferred to an E3 medium bath. Following this, the xenografted embryos were placed into Petri dishes and incubated at 34°C until the following day.
Drug protocols and administration
At 24 hours post injection (HPI), the xenografts were sorted based on the presence of a fluorescent signal within the PVS. To ensure the traceability of each specific embryo throughout the experiment each successful xenograft was transferred individually in a 24-well plate and randomly assigned to one of three treatment groups [FOLFOX protocol contained 0.86 mg/mL 5-Fluorouracil (5-FU), 0.14 mg/mL Folinic Acid (FA), and 0.03 mg/mL Oxaliplatin (OXA); FOLFIRI protocol contained 0.86 mg/mL 5-FU, 0.14 mg/mL FA, and 0.065 mg/mL Irinotecan (IRI); FOLFIRINOX protocol contained 0.86 mg/mL 5-FU, 0.14 mg/mL FA, 0.03 mg/mL OXA, and 0.047 mg/mL IRI], in addition to one control group. Concentrations were calculated per mL of E3 medium. The medium was refreshed daily for the rest of the experiment, with new doses of chemotherapeutics added each time.
Figure 1 shows a schematic representation of the timeline.
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
Imaging
In vivo tumor growth was monitored using a Zeiss Stereo LumarV12 fluorescent stereomicroscope equipped with an HBO100 illuminator. A QImaging Retiga 6000 camera was used to capture images of the xenografts.
Image manipulation
Images were analyzed using the Fiji software [9].
Ethics
The studies were approved by the Ethics Committees of the Alexandru Ioan Cuza University of Iaşi (Approval No. 2532/08.09.2022), Grigore T. Popa University of Medicine and Pharmacy, Iaşi (Approval No. 256/17.01.2023) and Regional Institute of Oncology, Iaşi, Romania (Approval No. 1425/16.12.2022). All embryos included in this study were sacrificed before the 120 HPF mark.
Statistical analysis
Statistical analysis was conducted using GraphPad Prism version 10. For comparisons between two groups, the Mann–Whitney test was utilized, while analysis of variance (ANOVA) was used for multiple group analyses. For categorical data, Fisher’s exact test or χ2 (chi-squared) tests were employed. All tests were two-tailed, with a significance level set at p<0.05.
Results
The fluorescent area of the primary tumor (i.e., the PVS tumor) was measured in all xenografts at day 1 and day 3 post injection. Overall, the area decreased significantly in the chemotherapy treated xenografts vs. the control group immersed only in E3 medium (p=0.0026). In Figure 2, we see representative xenografts from each group imaged at one and three days post injection (DPI). It’s noteworthy that the primary tumor tends to organize into a relatively spherical structure at 3 DPI, often protruding beyond the PVS. The biggest decrease was seen in the group treated with the FOLFIRI protocol [35.42±25.28%, mean ± standard deviation (SD)] followed by the FOLFIRINOX group (41.59±19.61%, mean ± SD) and the FOLFOX group (47.03±24.98%, mean ± SD) (Figure 3A, 3B, 3C, 3D, 3E, 3F, 3G).
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
An important characteristic of cancer is the ability of the cells to intravasate blood vessels and colonize distant tissues. To quantify this event xenografts were screened for circulating tumor cells (i.e., the presence in circulation or in the tissues of CM-Dil labeled cells that do not form clusters, excluding any PVS and intravitelline space cells) and for micrometastasis (MM) (i.e., CM-Dil labeled cells in the tissue that form clusters, excluding any PVS and intravitelline space clusters). Interesting enough, most xenografts (84%) presented circulating cells (CC) at 1 DPI and less than half (40%) presented MM. In Figure 4, we can see the difference between CC and a MM in fluorescent microscopy (note the cluster of cells present in A9 embryo as opposed to the single dispersed cells in B9 embryo). While the full clearance of CC was not statistically significant (absence of any CM-Dil labeled cells in circulation) (Table 1), the total number of CC cells tended to decrease in all groups during the three days with the biggest difference observed in the FOLFIRI group, followed by FOLFIRINOX and FOLFOX groups (Figure 2). Moreover, FOLFIRI protocol seemed to perform better than the FOLFOX protocol at clearing the CC (p=0.0224) (Figure 5A, 5B, 5C, 5D, 5E, 5F, 5G).
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
Table 1.
Total number of xenografts by group presenting circulating cells or metastases
|
Control |
FOLFIRI |
FOLFIRINOX |
FOLFOX |
Significance |
|
|
n (%) |
12 (100%) |
15 (100%) |
13 (100%) |
19 (100%) |
NS (p=0.9972) |
|
CC (day 1), n (%) |
11 (92%) |
12 (80%) |
9 (69%) |
18 (94%) |
|
|
CC (day 3), n (%) |
11 (92%) |
11 (73%) |
8 (61%) |
12 (63%) |
|
|
MTS (day 1), n (%) |
3 (25%) |
6 (50%) |
3 (23%) |
12 (63%) |
NS (p=0.6966) |
|
MTS (day 3), n (%) |
3 (25%) |
3 (20%) |
1 (8%) |
12 (63%) |
CC: Xenografts with circulating cells; 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; MTS: Xenografts with metastases; n: No. of xenografts in each group; NS: Not significant
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
Metastases were also observed primarily in the tail region of the embryos that often distorted the local anatomy of the tail by extending beyond its natural alignment and enlarging the dimensions of the caudal vein (Figure 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H).
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
We also observed complete metastasis clearance in three FOLFIRI and two FOLFIRINOX xenografts and no clearance in the FOLFOX or the control groups. While this alone is not statistically significant (Table 1), if we consider the metastases dimensions measurements, the chemotherapy subgroups had lower overall MM at day 3 than the control (p=0.0176) and the biggest difference was observed in the FOLFIRINOX subgroup. When comparing the efficacy of each regimen, the only statistically significant difference was observed between the FOLFOX and FOLFIRINOX regimens, with FOLFIRINOX showing superior performance (p=0.0088), although this was based on only three xenografts (Figure 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H). Taken together, from the data presented we can conclude that HCT116 zebrafish xenografts are more susceptible to regimes that include IRI as opposed to OXA since the CC and MM responses were better, and the best response of the primary tumor was observed in the FOLFIRI subgroup. This modulation of response to chemotherapy suggests that monitoring the CC and MM in embryos at 1 DPI vs. 3 DPI can be used as a marker for sensibility to a certain line of treatment and probably the grading of response.
To analyze the angiogenic capacity of CRC using the zebrafish model, we utilized transgenic Tg(fli1a:EGFP) embryos [10], which express enhanced green fluorescent protein (eGFP) specifically in vascular endothelial cells. The PVS where tumor cells were grafted is naturally avascular, therefore any green fluorescent signal (507 nm wavelength) detected in this space indicates the formation of new endothelial-lined vessels, confirming active angiogenesis. Our observations revealed that eGFP-positive signals were primarily detected in and around larger xenografts, suggesting that while neovascularization is an early event in tumor development, it requires the tumor to reach a critical size before becoming detectable through fluorescence microscopy (Figure 4; Supplementary material #1). The presence and extent of neovascularization may serve as a potential parameter for assessing tumor aggressiveness and predicting response to adjuvant therapy in future patient-derived xenograft (PDX) studies.
Discussions
In this study, we have shown a zebrafish colorectal xenograft model using HCT116 cell line in which we were able to grade a tumor response to first line chemotherapeutic protocols commonly used to treat CRC in a clinical setting. All protocols reduced the primary tumor size, number of circulating tumor cells and MM, making these parameters valuable markers in the follow up of xenografts. The adoption of paired measurements through sequential imaging of individual embryos, rather than pooled statistical analysis of group-housed specimens, offers two significant advantages: it addresses the inherent variability in xenografted material, and suggests that fewer experimental subjects may be needed to achieve clinical relevance.
In vivo studies with zebrafish xenograft seem to be valuable for clinical application. The most significant changes in the primary tumor area observed in the FOLFIRI subgroup followed by FOLFIRINOX and FOLFOX protocols. In addition, the number of circulating tumor cells and the metastasis dimensions also decreased more in the IRI-based regimes than in the OXA groups, altogether suggesting that HCT116 zebrafish xenografts are more sensitive to IRI than to OXA in vivo. Interesting enough, in classic cell culture conditions, HCT116 cell line is more sensitive to OXA than to IRI [11], and in more complex cultures like organoids the sensitivity to OXA is heavily influenced by the extracellular matrix and even by the cell culture medium used [12, 13]. Nonetheless, our data aligns with other studies in the literature [14] where greater tumor reduction has been reported in zebrafish xenografts treated with FOLFIRI compared to those treated with FOLFOX. This highlights the fact that each chemotherapy profiling model has its own inherent limitations and challenges but also its own advantages such as high throughput drug screening, genetic manipulation, fast expansion in case of organoids and better stromal interaction, a function vascular system and innate immunity in case of zebrafish embryo xenografts [15, 16].
In our model, we have also observed neovascularization occurring at the primary tumor site, as well as circulating tumor cells and MM. These processes necessitate a living model for proper observation. CRC is known to induce the formation of new blood vessels and specifically HCT116 cell line was shown both in mice xenografts [17] and cell culture to be highly angiogenic [18]. Additionally, other studies in the literature suggest that specific cells tend to have a consistent pattern of metastasis localization, implying that the phenotype of the grafted cells is maintained in the zebrafish model [19]. This could hold significant clinical implications since it could provide an important prognostic factor in patients who have a high-risk disease (poorly differentiated carcinoma, vascular or perineural invasion, etc.) but are candidates for curative surgery. Such profiling may help the decision of adjuvant/neoadjuvant chemo/ radiation therapy and zebrafish PDX (zPDX) might identify tumors with aggressive clones that might respond better to a certain chemotherapeutic adjuvant treatment.
Functional cancer models such as mouse and zebrafish xenografts or three-dimensional (3D) cell cultures are becoming increasingly important in cancer research and precision medicine. With the advancements in treatments and modern technologies that are expected to increase the pace of drug development and discovery [20, 21] more and more treatment options will make it to clinicians.
Nowadays treatments are guided by biomarkers which although especially useful, still have limitations in terms of accessibility, tumor heterogeneity [22] and even availability since most of the traditional chemotherapeutics do not have proven biomarkers [23]. Moreover, biomarkers are highly specific in their use (diagnostic, therapeutic, prognostic). Unlike them, functional testing with a patient’s own tumor cells could provide a more complete picture of the disease by allowing a wide range of chemotherapeutics tests and thus testing multiple pathways simultaneously.
zPDX have been shown to correlate with the treatment outcome [24, 25, 26], lymph node involvement, and even in a small cohort poor response of the xenograft to treatment with Carboplatin was a predictor of relapse [27]. The largest zPDX study to date that included 55 different CRC patients found that a chemosensitive xenograft was a predictor for longer progression free survival irrespective of the disease stage. It remains to be seen if this translates into a longer overall survival, but the data is highly encouraging since zPDX could be used to filter the available treatment options when many are available and provide important prognostic information [28]. zPDX will become more important as time goes on, they are able to bridge the gaps of knowledge in edge cases where there is no clear distinction between available treatment options and provide an informed decision based on the options available.
Conclusions
Our research demonstrates the effectiveness of zebrafish xenografts as a translational platform for evaluating chemotherapy response. The methodology of tracking individual xenografts through paired experiments proved particularly valuable, enabling precise comparative analysis of multiple parameters: tumor dimensions, metastatic burden, circulating tumor cell populations, and neovascularization. These combined metrics provide a comprehensive framework for assessing therapeutic response. The model’s ability to reveal distinct treatment outcomes within days makes it especially promising for clinical applications. We believe this is an important stepping stone towards developing personalized PDX that could hold significant clinical applications.
Conflict of interests
The authors declare that they have no conflict of interests.
Source of funding
This research was funded by the PhD scholarship of Grigore T. Popa University of Medicine and Pharmacy, Iaşi, Romania.
Statement about animal rights
All fish have been treated in accordance with EU Directive 2010/63, and appropriate measures were taken to minimize pain or discomfort. All embryos included in the study have been sacrificed before the 120 hours post fertilization.
Supplementary materials
Supplementary movies are available for this paper at: https://doi.org/10.7910/DVN/EELGHS.
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