Imaging tumour cell heterogeneity following cell transplantation into optically clear immune-deficient zebrafish

Abstract

Cancers contain a wide diversity of cell types that are defined by differentiation states, genetic mutations and altered epigenetic programmes that impart functional diversity to individual cells. Elevated tumour cell heterogeneity is linked with progression, therapy resistance and relapse. Yet, imaging of tumour cell heterogeneity and the hallmarks of cancer has been a technical and biological challenge. Here we develop optically clear immune-compromised rag2(E450fs) (casper) zebrafish for optimized cell transplantation and direct visualization of fluorescently labelled cancer cells at single-cell resolution. Tumour engraftment permits dynamic imaging of neovascularization, niche partitioning of tumour-propagating cells in embryonal rhabdomyosarcoma, emergence of clonal dominance in T-cell acute lymphoblastic leukaemia and tumour evolution resulting in elevated growth and metastasis in BRAF(V600E)-driven melanoma. Cell transplantation approaches using optically clear immune-compromised zebrafish provide unique opportunities to uncover biology underlying cancer and to dynamically visualize cancer processes at single-cell resolution in vivo.

Figure 1. Zebrafish cancers engraft into homozygous rag2^E450fs (casper) animals.

Donor animals shown in the left two panels while transplant recipients are to the right. (a) ZsYellow-labelled Myc-driven T-ALL from the syngeneic CG1 background, (b) EGFP-labelled neuroblastoma from AB background, (c) mCherry-labelled kRAS^G12D-driven ERMS from CG1 background, and (d) BRAF^V600E-induced melanoma arising in tp53^−/−nacre background. Tumour cells were transplanted intra-peritoneally (a,b,d) or intra-muscularly (c) into both rag2^E450fs (AB) and rag2^E450fs (casper)-recipient fish. Merged brightfield and fluorescent images are shown at 30 d.p.t. Cytospins of leukaemia cells are shown in a, whereas haematoxylin and eosin (H&E)-stained sections are shown in b–d. Scale bars equal 5 mm in whole animals images, 20 μm for cytospins shown in a, and 50 μm for histology sections shown in b–d.

Figure 2. Imaging neovascularization in rag2^E450fs (casper) zebrafish engrafted with fluorescently labelled melanoma and ERMS.

(a) GFP-labelled, amelanotic melanoma implanted into rag2^E450fs (casper) fish (n=8 animals) and imaged following intravascular injection of crimson quantum dots (Qtracker 655). Whole animal images to the left and confocal images to the right ( × 100 magnification, 100–200 μm z-stack). Quantum dot fluorescence has been pseudo-coloured white. (b) GFP-labelled, amelanotic melanoma implanted into flk1:mCherry; rag2^E450fs (casper) transgenic zebrafish (n=4 animals). (c–d) GFP-labelled ERMS engrafted into flk1:mCherry; rag2^E450fs (casper) transgenic zebrafish (n=5 animals) and serially imaged over time (c, 13 d.p.t. and d, 21 d.p.t.). White arrowheads denote the site of intra-muscular injection of tumour cells. Scale bars equal 5 mm in whole animal images and 200 μm in confocal images.

Figure 3. Resolving tumour cell heterogeneity in ERMS at single-cell resolution following engraftment into flk1:mCherry; rag2^E450fs (casper) zebrafish.

(a) Epi-fluorescent images of primary ERMS in a 32-day-old myf5:GFP; myogenin-H2b:mRFP; mylpfa:lyn-cyan triple transgenic zebrafish. (b) Epi-fluorescent images of flk1:mCherry; rag2^E450fs (casper)-recipient fish engrafted intra-muscularly with fluorescently labelled ERMS at 28 d.p.t. (n=4 animals). (c) Confocal image with mCherry-labelled vasculature outlined by white dashed lines (left, × 100 magnification). Higher magnification of boxed region (right, × 400 magnification). Myosin-expressing, differentiated cells (Diff.) and less frequent myf5-GFP+ tumour-propagating cell (TPC) denoted by arrowheads. Scale bar equals 2 mm in a, 5 mm in b, 100 μm (c, left panel) and 25 μm (c, right panel).

Figure 4. Visualizing the emergence of clonal dominance in Myc-induced T-ALL.

(a) Donor animals engrafted with monoclonal T-ALL arising in the CG1 background. (b,c) Monoclonal T-ALLs were implanted into the syngeneic CG1 strain fish and assessed for LPC frequency by limiting dilution cell transplantation (b) or latency of regrowth (c). P-values are noted within each panel. Not significant (NS). (d,e) Confocal imaging of engrafted rag2^E450fs (casper) fish at 10 d.p.t. (d) and 23 d.p.t. (e). White arrow denotes site of injection, and the location for confocal imaging. (f) Relative proportions of each fluorescent clone contained within leukaemias from individual engrafted animals (n=16 animals). Imaging was completed on the same animals at 10–13 d.p.t. and 22–24 d.p.t. The ZsYellow+ clone dominates leukaemia regrowth in animals #1–3, whereas mCherry+ dominates in animals #13–16. Scale bars equal 5 mm in whole animal images and 50 μm in confocal images.

Figure 5. Visualizing melanoma invasion and metastasis following engraftment into rag2^E450fs (casper) mutant fish.

(a) Invasion assays using retro-orbital transplantation of a BRAF^V600E, tp53^−/− pigmented melanoma. White arrow denotes the site of injection. Green arrow denotes spread to the kidney marrow that is contiguous with primary tumour growth that has arisen adjacent to the eye. Histological examination confirmed the presence of pigmented melanoma cells at the site of injection (b, left panels) and contiguous with the trunk kidney (b, right panels). (c) Metastasis assays using implantation of non-pigmented, GFP-labelled melanoma cells into the dorsal musculature of rag2^E450fs (casper)-recipient fish. White arrow denotes the site of injection. Yellow arrow denotes site of distal metastasis. (d) Quantification of metastatic growth as assessed by epi-fluorescence microscopy over time. (e) Haematoxylin and eosin-stained sections of the same animal imaged in c, confirming metastatic growth of melanoma adjacent to the thymus (top panels) and confirmed by anti-GFP immunostaining on section (bottom panels). Scale bars equal 5 mm for whole animal images, 2 mm in images of heads, 1 mm in × 40 histological images; 300 μm in × 100 histological images and 100 μm in × 400 histological images.

Figure 6. Assessing metastatic potential and the functional consequences of tumour evolution in melanoma.

(a,b) Serial imaging of engrafted GFP-labelled melanoma implanted into adult rag2^E450fs (capser) mutant fish. The 1° transplant was pigmented (a), whereas the 7° transplanted melanoma had lost pigmentation (b). Number of animals with metastatic growth is noted (*P=0.003, Fisher's exact test comparing 1° and 7° transplant). White arrow indicates the site of injection. Yellow and red arrows denote sites of distal metastases. Histological staining (H&E and anti-GFP) of distal metastasis is shown in the right panels. (c) Quantification of tumour growth at the site of initial engraftment over time. (d) Confocal imaging of micro-metastatic lesions found adjacent to the tail vasculature of flk1:mCherry; rag2^E450fs (casper)-recipient fish engrafted with 7° transplant melanoma. Yellow arrows denote micro-metastatic lesions. Scale bars equal 5 mm for whole animal images in a,b, 2 mm for histology shown in a,b and 200 μm in d.