A zebrafish HCT116 xenograft model to predict anandamide outcomes on colorectal cancer

Effects of experimental treatments on tumor angiogenesis

The use of Tg(fli1:EGFP)^y1 transgenic xenografts allows the visualization of fish vascularization (Fig. ​(Fig.2A).2A). In particular, in these xenografts, a focus was made on blood vessels close to the tumor area, directly involved in its support and growth. In fish exposed to AEA, angiogenesis around the tumor (Fig. ​(Fig.2B)2B) resulted less developed than in the other experimental groups. In particular, the intensity of the signal associated with blood vessels that generate from the subintestinal area was lower than in Ctrl. At the same time, no differences were found between Ctrl and the groups receiving AM251 alone or in combination with AEA. In addition, a deeper investigation obtained using the Tg(mpeg1:EGFP)^gl22 transgenic xenografts provided evidence that the reduction of vascularization is not caused by different recruitment of macrophages around the tumor area since a similar number was found in all experimental groups (Supplementary Fig. 2).

Evaluation of HCT116 cell metastatic potential in xenografts

These results set the basis for a more detailed analysis of the effect of AEA on the occurrence of metastasis. Therefore HCT116 tumor cells were directly injected into the Cuvier’s vein, thus forcing the metastasis appearance. Since evidence regarding the specificity of the AEA effect was gained by performing experiments with its inhibitor AM251, for this experiment only two experimental groups were considered: Ctrl and AEA. Moreover, since the precise control of the number of HCT116 cells injected in the Cuvier’s vein results sometimes very challenging, 3 h after the injection (3 hpi), the animals were divided into two sub-groups on the basis of the number of circulating cells injected: small and large (Fig. ​(Fig.3A).3A). Xenograft from each group was randomly divided between the two experimental conditions, Ctrl and AEA treated. At 3 dpi, the number of metastatic cells present in the animal (as the quantity of red fluorescence detectable). As shown in Fig. ​Fig.3C3C and D, AEA treatment reduces metastasis growth when the tumor cells are directly placed into blood circulation.

Zebrafish and HCT116 RNA-seq and RT-PCR Validation

Starting from the results above described, obtained by confocal microscopy investigation, RNA sequencing analysis was performed to unveil molecular markers involved in tumor cell proliferation, angiogenesis, and immune response. Since a strong variability among replicates was observed, one replicate per experimental group was discarded. The choice of the samples to discard was based on the overall clustering of all the samples after principal component analysis and not only on the clustering in specific comparisons.

Considering the Danio rerio genome, with respect to Ctrl, 38 differentially expressed genes (DEGs) were found in AEA larvae, 8 upregulated and 30 downregulated and 6 DEGs with respect to AEA + AM251 larvae, 2 upregulated and 4 downregulated. No DEGs were found between AM251 and Ctrl larvae (see Heatmaps, Fig. ​Fig.44).

Regarding the Homo sapiens genome, no DEGs were found among different experimental groups. RNAseq data can be accessed through NCBI Bioproject—ID PRJNA911178

Nevertheless, the accuracy and reliability of RNA seq results were supported by a series of Real-Time PCRs on either DEGs, false-discovery rate (FDR) < 0.05, or not statistically significant genes (FDR ≥ 0.05) selected considering their role in cell proliferation, angiogenesis, and immune system (vegfaa, vegfab, vegfd, mep1b, socs3, csf3b, il11a, mhcIuba, pcnp, casp3). Details regarding the description of the selected genes are reported in Supplementary Table 1.

PCR results among different experimental groups are reported in Supplementary materials. As shown in Supplementary Figs. 3 and 4, the relative abundance in a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was consistent with RNA-Seq results, suggesting that the transcript identification and quantification were highly consistent between the two techniques. Genes selected from RNASeq analysis were in good accordance with the expression intensities by RT-qPCR.

Molecular analysis of biomarkers involved in tumor cell survival/growth

Western blot analysis on WT xenografts of Lc3A/B and Casp3 did not show any significant changes among experimental groups. On the contrary, Vegf-C and Il6 protein analysis revealed a significant reduction in xenograft exposed to AEA. No significant changes were induced in xenografts exposed to AM251 alone or in combination with AEA (Fig. ​(Fig.55).

In vitro AEA effect on HCT116 cell proliferation

To investigate the effects of AEA exposure on cell viability and proliferation, HCT116 cells were reared with five different AEA concentrations in the range from 0.005 to 5 μM. In vitro tests were carried out in low serum concentrations (i.e., 0.5% serum) to limit cellular growth and increase cell responsiveness to the compound. Indeed, the effect of cannabinoids is cell context-dependent, being modulated by the presence of growth factors [17]. Cell viability was evaluated using the CCK-8 assay after 72 h of treatment but in contrast to what was previously observed in animal models, all AEA concentrations had no anti-proliferative activity (Supplementary Fig. 5).

Cell culture and labeling

HCT116 cells were selected considering their dedifferentiated phenotype, invasive and metastatic potential, and clinical significance [73]. Cells were tested for mycoplasma (VenorGeM Classic, Minerva Biolabs GmbH, # 11-1025) and cultured in RPMI Medium 1640 with GlutaMAX-I (Thermo Fisher Scientific, Waltham, USA, # 61870036,) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, USA, #10270106) in a humidified atmosphere containing 5% of CO₂ at 37 °C. For xenotransplantation, cells were washed, trypsinized, and re-suspended in RPMI medium containing Dil Vybrant Red Fluorescent dye (Thermo Fisher Scientific, Waltham, USA, #V22885) for 20 min at 37 °C. Cells were then centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and cells were washed two times with PBS. Finally, cells were re-suspended in PBS for injection in the zebrafish yolk.

Xenotransplantation

Wild type (WT), Tg(fli1:EGFP)^y1, and Tg(mpeg1:EGFP)^gl22 zebrafish embryos at 48 hpf were immobilized with 0.04% tricaine (Merck KGaA, Darmstad, Germany, #E10521) and positioned on a thick film of 2% agarose in fish water. To investigate the tumor volume and interaction with the host and the therapeutic molecules, approximately 200–400 Dil-labeled (VybrantTM DiI) HCT116 cells were injected into the inferior section of the yolk sac (Supplementary Fig. 1). To investigate the metastatic potential of the tumor, the same number of cells were injected directly into the Cuvier’s vein. After injection, xenografts were maintained at 35 °C until the end of the experiments. At 3 hpi, unsuccessfully injected xenografts were discarded [74].

In vivo and in vitro drug administration

Arachidonylethanolamide (Anandamide—AEA) (Merck KGaA, Darmstad, Germany, #94421-68-8) and 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM251) (Merck KGaA, Darmstad, Germany, # 183232-66-8), were dissolved in Ethanol 100% and further diluted in fish water and used at 10 nM as previously described in [75, 76].

In vivo exposure

At 6 hpi zebrafish xenografts were randomly divided into four experimental groups:

• Controls (Ctrl): exposed to Ethanol (EtOH)
• AEA: exposed to 10 nM AEA
• AM251: exposed to 10 nM AM251
• AEA + AM251: exposed 10 nM AEA + 10 nM AM251

Chemicals were daily replaced till 4 dpf. The control group received the same amount of EtOH used to dissolve chemicals.

The sample size for in vivo experiments is shown in figure legends throughout the manuscript. The size of the samples used in zebrafish experiments was determined by the alpha error of 0.05 and the statistical power of 95%. The minimum number of 6 controls and 6 mutated/treated samples was implied by the sample population’s required size (Sample Size Calculator, ClinCalc.com). The number of individual embryos that could be made available for each experiment after fluorescence screening also had an impact on sample size.

Gross congenital abnormalities, which typically affect 5–10% of an egg clutch, were excluded from the analysis. The purpose of the data exclusion is only to collect biologically meaningful phenotypes resulting from the specific treatment and not from sporadic changes that are frequently seen in wild animals.

In vitro exposure

Cell viability was determined using the cell counting Kit-8 colorimetric assay (CCK-8, Bimake, USA, # B34304). HCT 116 cells were seeded in 96-well plates with RPMI 1640 (Thermo Fisher Scientific, Waltham, USA, # 61870010) and 10% fetal bovine serum (FBS) at a density of 5 × 10³ cells/well. After 24 h of incubation at 37 °C, the medium was removed and a new culture medium containing 0.5% FBS and AEA were added. AEA, in EtOH, was directly dissolved in cell culture medium to 50 µM solution and then serially diluted to give a final test concentration of 5, 10, 100 nM, and 1 and 5 µM. Concentrations were selected based on previous in vitro studies evaluating the anti-carcinogenic AEA properties in different cell lines [77, 78]. Cells were treated at 37 °C for 72 h changing the medium every day, after which cell viability was measured. Briefly, 10 µl of CCK-8 reagent was added to each well and incubated for 30 min at 37 °C, then absorbance at 450 nm was measured using a microplate reader (Infinite F200 PRO, Tecan, Männedorf, Switzerland). The absorbance in the control group was regarded as 100% cell viability. The percentage of viability was calculated using the formula: “[OD (optical density) of treated cells background absorbance/OD of untreated cells (control)—background absorbance] × 100”. All controls and samples were measured by four independent experiments with five replicates for each concentration. Values are expressed as the mean ± SD.

Live imaging

Totally, 3 dpi WT, Tg(fli1:EGFP)^y1 and Tg(mpeg1:EGFP)^gl22 xenografts were anesthetized with 0.04% tricaine, embedded in 1% low-melting agarose, placed on a depression slide and analyzed by confocal microscopy. The Nikon C2 confocal system (Nikon, Minato, Japan), along with the software NIS ELEMENTS, was used to record images. At 3 dpi, WT xenografts injected in the Cuvier’s vein were anesthetized with 0.04% tricaine, embedded in 1% low-melting agarose, placed on a depression slide and analyzed with Leica DMR fluorescent microscope (Leica, Wetzlar, Germany) and the Nikon DS-Fi2 digital camera.

Tumor volume, angiogenesis, and metastatic potential quantification

All images of WT, Tg(fli1:EGFP)^y1, and Tg(mpeg1:EGFP)^gl22 xenografts obtained with the Nikon C2 confocal system were analyzed using Fiji imaging software (https://imagej.net/software/fiji/). The tumor size was obtained by calculating each Z stack’s tumor area (red fluorescent structure), while the angiogenesis quantification was performed using the image calculator process. To identify only endothelial cells, HCT116 fluorescence signals were subtracted from fli1-eGFP signals. Total signal intensity was calculated according to previous work [79]. Macrophage analyses using the Tg(mpeg1:EGFP)^gl22 xenografts were performed by counting the number of macrophages present in the area surrounding the tumor. An equal area was selected for all the samples. The percentage of animals with micrometastases was calculated by counting the number of 3-dpi larvae with micrometastases in all the different treatment conditions, despite being injected exclusively in the yolk. The total fluorescence of the metastasis of animals injected directly in the Cuvier’s vein was analyzed with the imaging software Fiji, to observe the effect of therapeutic molecules on the proliferation of metastases.

RNA extraction, cDNA synthesis, and Real-time PCR

Total RNA was extracted from 5 pools of ±20 larvae for each experimental group, using RNAzol RT (Merck KGaA, Darmstad, Germany, # R4533). Genomic DNA was removed by DNase I digestion (Merck KGaA, Darmstad, Germany, # AMPD1). RNA concentrations were determined by nanophotometer P330 (Implen, München, Germany) and integrity was assayed with Bioanalyzer (Agilent, CA, USA). Part of the total RNA was used for cDNA synthesis, and part was to create a library for RNA-seq. The reverse transcription was conducted from 1 μg total RNA with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, Massachusetts, USA). The qRT-PCRs were performed with the SYBR green method in a CFX thermal cycler (Bio-rad, Milan, Italy) as previously described [80]. For each experimental group, replicates (N = 5) were run in duplicate. The final primer concentration was 10 pmol/μL. Ribosomal protein 13 (rpl13) and ribosomal protein 0 (rplp0) mRNAs were used concomitantly to normalize target genes expression levels analyzed by CFX Manager Software version 3.1 (Bio-Rad), including GeneEx Macro Conversion and GenEx Macro files and results are represented by bar-plots along with the standard deviation. Specific primer pairs for target genes (Supplementary Table 1) were designed with Primer-Blast.

RNA-seq analysis and differential expression analysis of Homo sapiens/D. rerio xenografts

The starting dataset included RNA-seq reads from 12 samples belonging to the four experimental groups, i.e., Ctrl, AEA, AM251, and AEA + AM251. The quality of the reads was assessed with the software FASTQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) then a trimming step was performed in order to remove adapters and low-quality bases from the reads. The following parameters were used: the minimum length was set to 35 bp and the quality score to 25. The software TRIMMOMATIC [81] was used for this scope. On average, 140 million filtered reads were obtained per sample.

The high-quality reads were aligned against the Homo sapiens genome (GRCh38–104) and D. rerio genome (GRCz11–105) with STAR aligner (version 2.7.9a) [82]. On average, 0.74% of the total reads could be mapped uniquely on the human genome and 81.76% of the reads could be mapped uniquely on the zebrafish genome. In order to perform disambiguation between the two species, the software disambiguate was used. FeatureCounts (version 2.0.0) [83] was used to calculate gene expression values as raw fragment counts. In addition, a normalization was applied to the raw fragment counts by using the trimmed mean of M-values and the fragments per kilobase million normalizations.

Western blot analysis

Whole larval homogenates from 3 different pools of 15–20 larvae per group, were electrophoresed and transferred to PVDF as previously described [84]. Briefly, 7 mg of each protein sample was separated using 4% stacking and 10% separating sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto a filter using a mini trans-blot electrophoretic transfer cell (Bio-Rad, Milan, Italy). The transfer was carried out for 30 min using Trans-Blot TurboTM Transfer System. After blocking in 2% bovine serum albumin (BSA; Merck KGaA, Darmstad, Germany, # A2153) in PBS. The following primary antibody was used: LC3A/B (Cell Signaling, Beverly, MA, USA, #BK4108S), Caspase 3 (Cell Signaling, Beverly, MA, USA, # BK9661S), IL6 (Abcam, Cambridge, UK, #ab208113,) and VEGF-C (Cell Signaling, Beverly, MA, USA, # BK2445S,) antibodies were diluted 1:1000 in a solution containing 2% BSA and 0.1% TWEEN 20 in PBS and incubated overnight at 4 °C. β-actin antibody (Cell Signaling, Beverly, MA, USA, # BK4967S) was used as internal standard. The reaction was visualized with ECL-PLUS (GE Healthcare, Milano, Italy) chemiluminescent reagent for Western blotting. Densitometric analysis was performed using Fiji software for Windows.

The author wishes to thank Dr. Riccardo Aiese Cigliano from Sequentia Biotech (www.sequentiabiotech.com) for his contribution to statistical analyses and the technical staff of the Zebrafish Facility at the Department of Biology, UniPD.