Haynes-Gimore N et al. (2015), Semi-solid tumor model in Xenopus laevis/gilli ...

XB-ART-50089

Dev Biol 2015 Dec 15;4082:205-12. doi: 10.1016/j.ydbio.2015.01.003.

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Semi-solid tumor model in Xenopus laevis/gilli cloned tadpoles for intravital study of neovascularization, immune cells and melanophore infiltration.

Haynes-Gimore N , Banach M , Brown E , Dawes R , Edholm ES , Kim M , Robert J .

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Tumors have the ability to grow as a self-sustaining entity within the body. This autonomy is in part accomplished by the tumor cells ability to induce the formation of new blood vessels (angiogenesis) and by controlling cell trafficking inside the tumor mass. These abilities greatly reduce the efficacy of many cancer therapies and pose challenges for the development of more effective cancer treatments. Hence, there is a need for animal models suitable for direct microscopy observation of blood vessel formation and cell trafficking, especially during early stages of tumor establishment. Here, we have developed a reliable and cost effective tumor model system in tadpoles of the amphibian Xenopus laevis. Tadpoles are ideally suited for direct microscopy observation because of their small size and transparency. Using the thymic lymphoid tumor line 15/0 derived from, and transplantable into, the X. laevis/gilli isogenic clone LG-15, we have adapted a system that consists in transplanting 15/0 tumor cells embedded into rat collagen under the dorsal skin of LG-15 tadpole recipients. This system recapitulates many facets of mammalian tumorigenesis and permits real time visualization of the active formation of the tumor microenvironment induced by 15/0 tumor cells including neovascularization, collagen rearrangements as well as infiltration of immune cells and melanophores.

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Species referenced: Xenopus laevis
Genes referenced: gnas
GO keywords: angiogenesis [+]

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	Fig. 1. In vivo growth of 15/0 semi-solid tumor grafts. (A) Representative images of collagen alone (I) and 15/0 tumor grafts (dashed white line) at day 3, 10, 16, 24 and 30 following engraftment (II–VI). Note the accumulation of melanophores in the tumor grafts at later time points. (B) Average increase in area ±SEM of tumor or collagen only grafts determined over time relative to collagen only graft at day 0. Values are expressed as the mean±SEM from 15 animals in 3 independent experiments. P values calculated using students TTEST. LG-15, stage 54 (2 week old) tadpoles were subcutaneously grafted with 500,000 cells of 15/0 tumor while control animals were grafted with equal volume of collagen alone. Animals were imaged on a Nikon SMZ1500 stereomicroscope.
	Fig. 2. Characterization of 15/0 tumor cells within the semi-solid tumor graft. (A) Semi-solid tumors containing 500,000 15/0 tumor cells were either grown for 9 days in vitro (I and IV) or grafted subcutaneously in vivo in LG-15 tadpoles (II and V). Control collagen mass without tumor cells was maintained in vitro (III and VI). Semi-solid tumors were then stained with 0.1 μg/ml DAPI (II, IV, and VI) and cells visualized using a Leica DMIRB inverted fluorescence microscope. (B) H&E staining of cryosections of tumor collagen grafts 3, 10, 16 and 30 days post-transplantation (I–IV). Magnified images of boxed section from above panel (V–VIII). Slides were imaged using an Axiovert 200 inverted microscope. (C) Gömöri trichrome staining of cryosections of tumor collagen grafts 3, 10, 16 and 30 days post-transplantation (I–IV) Magnified images of boxed section from above panel (V–VIII). Slides were visualized using an Axiovert 200 inverted microscope.
	Fig. 3. Neo-angiogenesis in 15/0 collagen grafts. LG-6 or LG-15 tadpoles were grafted with tumor grafts containing 500,000 15/0 tumor cells and animals were intracardiacally injected with either Texas red 70 KDa or TMR 2000 KDa dextran and imaged. (A) Ex vivo images of vascularized grafts from LG-15 tadpoles at day 7 post-transplantation. PKH-labeled 15/0 cells (I) with Texas red-dextran filled vasculature (II). Vascularized tumors were imaged ex vivo using an Olympus BX40 conventional fluorescence microscope. (B) Intravital imaging of vasculature in LG-6 15/0 collagen grafted animals. Bright field image of vasculature entering tumor mass (I). Texas-red dextral filled vasculature entering semi-solid tumor (dashed line indicates tumor boundary) (II). Texas-red dextral filled vasculature within tumor mass (III). Black cells are melanophores within the tumor graft. Animals were imaged using a Nikon TE2000-U epifluoresence microscope. (C) TMR loaded vasculature within 15/0-collagen tumors in LG-6 tadpoles showing SHG signal (I), CFSE labeled tumor (II) and TMR loaded vessels (III) from 15/0-collagen tumor bearing animals. Animals were imaged using an Olympus two-photon laser-scanning microscope. Images from representative animals shown.
	Fig. 4. Accumulation of melanophores in growing 15/0 tumor graft. (A) 15/0 tumor graft heavily infiltrated by host melanophores at 30 days post-transplantation. (B) Ex vivo bright field imaging of a tumor graft showing melanophore (black arrow) accumulation within the 15/0 WT tumor (⁎; delimited by a dashed white line) compared to normal skin melanophores (blue arrow). (C) One week in vitro culture of tadpole melanophores (m) derived from a semi-solid 15/0 tumor grafted into a LG-15 tadpole recipient that was invaded by melanophores as shown in A. Not the presence of numerous of other cells including 15/0 tumor cells (black arrows) and tumor infiltrating leukocytes (red arrows) (D) Melanophore (m) enriched three weeks in vitro culture of 15/0 tumor graft-derived melanophores. Note that only a few fibroblast (f) besides melanophores. Animals were imaged using the Nikon SMZ1500 stereomicroscope and cells with an Olympus BX40 microscope.
	Supplementary data: Fig. S1: Immobilization of tumor cells on a collagen matrix. A collagen solution was made by adding setting solution Type I rat tail collagen in order to initiate the pH dependent polymerization. Tumor cells were then mixed with the collagen solution at a concentration of 500,000 cells per 10 μL graft. Collagen/tumor mix was then pipetted into individual wells of 6 well plates and incubated at 27 °C for 30 min to allow collagen polymerization. Two milliliters of media, was added to tumor collagen grafts. Plates containing the grafts were then incubated at 27 °C in chambers containing a blood gas mix (5% CO2, 21% O2, 74% N) until use. Small subcutaneous incisions were made on anterior left or right region of tadpoles and the collagen tumor grafts were inserted subcutaneously within the incisions.
	Fig. S2: Intra vital imaging of semi-solid 15/0 tumor cells in LG-6 tadpoles. Semi-solid grafts containing 15/0 tumor cells labeled with 40 μM CFSE labeled were grafted into LG-6 tadpoles. Animals were intra vitally imaged one-week post-transplantation using a two-photon microscope. SHG of collagen (I) and CFSE fluorescence (II) within LG-6 tadpoles. Arrow indicates melanocyte.
	Fig. S4: Ex vivo neovasculature in 15/0 WT collagen grafts. Images of vascularized tumor grafts at day 5 (A) and day 7 (B-C) post-transplantation. PKH- labeled 15/0 cells (I, IV and VII) with Texas red-dextran filled vasculature (II, V and VII). Collagen grafts containing PHK-labeled 15/0 tumor cells were subcutaneously implanted into LG-15 tadpoles. At varying times post-engraftment animals were intracardiacally injected with Texas red 70 KDa dextran. Ten minutes Texas red post-injection, grafts were surgically removed, mounted on slides and imaged for fluorescence. Vascularized tumors were imaged ex vivo using an Olympus BX40 conventional fluorescence microscope.
	Fig. S3: Schematic of tumor cells embedding into a collagen matrix. A collagen solution was made by adding drop-wise a setting buffer to Type I rat-tail collagen in order to initiate the pH dependent polymerization. Tumor cells were then mixed with the collagen solution at a concentration of 500,000 cells per 10μL graft. Collagen/tumor mix was then pipetted into individual wells of 6 well plates and incubated at 27°C for 30 min to allow collagen polymerization. Two milliliters of media, was added to tumor collagen grafts. Plates containing the grafts were then incubated at 27°C in chambers containing a blood gas mix (5% CO2, 21% O2, 74% N) until use. Small subcutaneous incisions were made on anterior left or right region of tadpoles and the collagen tumor grafts were inserted subcutaneously within the incisions.

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