Optogenetic set-up for single cell induction.

(A) Photo-control of a protein fused to an Estrogen Receptor (ERT) is achieved by releasing the protein from its complex with cytoplasmic chaperones (CC), upon uncaging of cCYC. (B) The transgenic zebrafish line engineered to express an oncogene (KRASG12V) upon photo-activation of a CRE-recombinase fused to ERT (CRE-ERT) (as shown in A). (C) The mRNAs of Ventx-GR and mRFP (used as a marker) are injected at the one cell stage. (D) At 1dpf the embryos are mounted in channels in an agarose gel and incubated for 45 min in cCyc on a microscope stage. (E) They are washed and illuminated at 405nm on a microscope to uncage cCyc close to the otic vesicle. A diaphragm (DIA) defines an illumination zone of ∼80μm diameter (see G). Excitation (EX), Dichroic mirrors (DM) and emission (EM) filters allow for visualization of Eos and mTFP. The cell in which KRASG12V has been induced is observed within ∼1h by the fluorescence of mTFP (see G). (F) The embryos are transferred into individual wells, incubated overnight in DEX, washed at 1dpi and monitored over the next 5 days. (G) Lateral view of zebrafish at ∼1h post-activation displays a single induced cell (top: blue spot shown by arrowhead in mTFP channel) in the illumination region (middle: Eos channel) in the vicinity of the otic vesicle (white arrow) and bottom: merger of both channels. Body axes (a: anterior; p: posterior; d; dorsal; v: ventral) are shown.

Malignant transformation of a single cell triggering carcinogenesis in vivo.

(A) At 1dpf, a single cell in a zebrafish brain was photo-induced to express the oncogene KRASG12V, identified (white arrowhead) within ∼1h by the fluorescent H2B-mTFP. Membrane-bound mRFP is used as tracer. Transient (24h) DEX activation of Ventx is done following photoactivation. (B) At 1day post induction (1dpi), the activated cell has divided, giving rise to two cells (white arrowhead). After 3dpi the original cell expanded clonally (white arrowheads) by short-range dispersal within the brain. At 4dpi the brain has been colonized (left panel) by the progeny of the activated cell (white arrowheads) that display tumor growth as well as dispersal in the head or entering into the cardiovascular system (red arrowhead). At 5dpi, a tumor mass is formed. (C) Confocal microscopy of a larval head displaying tumors (arrowheads) and dispersal in the trunk (red arrowheads). (D) Histopathological sections of larval brain (dorsal view). Dashed lines (1 to 5) indicates sections showed in (E) Hematoxylin & Eosin (H&E) staining of brain at 5dpi of KR+VX-induced larva (depigmented) is compared to normal brain (Ctrl, melanocytes in brown). At 5dpi, the optic tectum (red arrows), the tegmentum (black arrow) and hypothalamus (red arrowhead) are infiltrated by a dysplasic tumor, progeny of the initial induced single cell. An asterisk (*) indicate the eye and a white arrow the otic vesicle. Scale bars and the body axes (a: anterior; p: posterior; d; dorsal; v: ventral) are shown. T=telencephalon; M=Mesencephalon; C= Cerebellum; Mc= Melanocytes.

Metastatic cells following single cell malignant transformation.

(A) A zebrafish larva, in which a single cell in the brain was photo-induced (at 1dpf) to express the oncogene KRASG12V by the blue fluorescence of the expression marker (H2b-mTFP) as in (Fig. 2), show that the progeny of the Cell of Origin of Cancer give rise to a tumor mass in the brain (white arrowhead), as well as to migrating metastatic-like cells that disseminate in the whole organism (white arrowhead), some localizing in proximity of arterial branchial arches (indicated by ζ and white arrowhead), trunk and tail fin. (B) H2b-mTFP positive cells can migrate far from the site of induction (brain) and colonize ectopic tissues located in the heart and (C) the bottom of otic vesicle (in proximity of the primary head sinus, designated by α), the digestive tract (designated by β and γ) a feature characteristic of metastatic cancer cells. Note that no anaesthetics (e.g. tricaine) or mounting media (low melting point agarose or methylcellulose) were used to block live zebrafish, during both monitoring and imaging performed on live zebrafish. An asterisk (*) indicate the eye and a white arrow the otic vesicle. Scale bars and body axes (a: anterior; p: posterior; d; dorsal; v: ventral) are indicated.

Transplantation of single cell(s) from hyperplasic tissue reveals cancer-initiating potential.

(A) Following KRASG12V plus Ventx activation, hyperplasic tissues (blue fluorescent) are observed at 6dpf, top figure. The cells of the hyperplasia were dissociated and isolated blue (KRAS expressing) cells were transplanted (≈ 1 cell per host) at 2dpf in a Nacre (mitf -/-) zebrafish line for a better tracking of the transplanted cells. The transplanted H2b-mTFP positive (blue) cell (red arrow) can be visualized as early as 3 hours post transplantation (3hpt) in the yolk of the host, close to the duct of Cuvier. White arrow indicates the head/eye (lateral view). (B) At 3 dpt the blue cell(s) from the hyperplasic tissue of the donor have colonized the host Nacre zebrafish larvae. Tumors in the brain, digestive tract and intestine, are observed and characterized by the blue fluorescence of the donor KRAS expressing cells (red arrows; n=31 out of 52 host individuals). In the bottom, immunofluorescence (IF) analysis of representative host zebrafish larvae with specific high level of phosphorylated ERK activity (pERK, red arrows) in the brain, intestine and digestive tract. (C) A high number of exogenous blue fluorescent cells are here observed to migrate in the tail (red arrows). These observations indicate that the transplanted founder cell has both migratory, colonizing behavior, as well as survival growth advantage in the host to form tumors, and thus to re-initiate carcinogenesis. Scale bars and body axes (a: anterior; p: posterior; d; dorsal; v: ventral) are shown; T=telencephalon; M=Mesencephalon; C= Cerebellum; N=notochord; VF= ventral fin.

A two-steps “Vogelgram” model of deterministic and irreversible single-cell malignant transformation in vivo.

Within a healthy tissue, a normal cell (in light grey) acquires (step 1) genetic mutation(s) in driver oncogene(s) (Mut-Drivers such as kRASG12V). Such a mutant cell (in blue, referred as a preprocancer41 cell) can (step 2) aberrantly activate the expression of Epi-Drivers involved in pluripotency/reprogramming (e.g. VENTX/NANOG, POU5/OCT4) thus undergoing deterministic and irreversible malignant cell transformation (red cell, the Cell of Origin of Cancer); (step 3) in situ short-range dispersal of the early malignant cells and (step 4) further progression to cancer mass and to the appearance of metastatic cells. Inversely, the mutant (preprocancer) cell (in blue) can maintain its physiological functions and be eventually eliminated from the healthy tissue.

Characterization of the double transgenic line Tg(β−actin:loxP-EOS-stop-loxP-KRASG12V-T2A-H2B-mTFP; ubi:Cre-ERT; myl7:EGFP).

(A) A transgenic fish at 6dpf displays green EOS fluorescence throughout the body and a strong green fluorescence in the heart due to the additive expression of EGFP under a heart specific (myl7) promotor, red arrowhead. (B) Upon global illumination (at 1dpf) with a ∼365nm UV lamp in presence of cCYC, cyclofen is uncaged which releases CRE-ERT from its complex with cytoplasmic chaperones and results in floxing of EOS and ubiquitous expression of KRASG12V and H2B-mTFP. The embryos thus display blue fluorescence in the nuclei shown here at 1h post induction (hpi). (C) Stable and ubiquitous expression of the blue H2B-mTFP fluorescent protein in zebrafish late embryos (3dpf) and larvae (6dpf). Note that in all pictures the asterisk (*) indicate the eye. Scale bars and body axes (a: anterior; p: posterior; d; dorsal; v: ventral) are shown.

Transient activation of Ventx reprogramming factor.

(A) top: At one cell stage Ventx-GR mRNA is injected together with mRFP mRNA (used as a tracer), which expression throughout the embryo (here shown at 1dpf) is a proxy for Ventx-GR expression. Bottom: Immunofluorescence analysis show that Ventx protein can be visualized in fixed embryos with the help of an antibody (Ab) directed against the HA tag linking Ventx and GR (and visualized by a second Ab linked to a green fluorescent marker). Upon addition of DEX at 1dpf embryos, Ventx is released from its complex with cytoplasmic chaperones and diffuses into the cell nucleus, resulting in a pointillist image of the nuclei (middle). At 2dpf, after washing out DEX, Ventx protein is no longer detected and the pointillist image is lost (right). Note that in all pictures the asterisk (*) indicate the eye. Scale bars and body axes (a: anterior; p: posterior; d; dorsal; v: ventral; l: left; r: right) are shown.

Synergy between a reprogramming factor and KRASG12V oncogene efficiently induces tumors in zebrafish larvae.

(A) Images of control zebrafish larvae at 6dpf: without any treatment (left), with transient incubation in cCYC (without UV, middle) and with incubation in DEX + cCYC (without UV, right). Note that zebrafish does show neither morphological anomalies nor mortality. (B) Photoactivation (via cCYC uncaging and Cre-ERT activation) of KRASG12V (blue nuclei) in 1dpf zebrafish without (left) or with subsequent transient activation (right) of Ventx-GR by Dexamethasone (DEX) (described in Fig. S2). These larvae (labelled as KR+VX) develop hyperplasic tissues within 6-9 dpf (dorsal view, red arrows indicate hyperplasia). Photoactivation (via cCYC uncaging and Cre-ERT activation) of KRASG12V (blue nuclei) in 1dpf zebrafish with subsequent transient activation of NANOG-GR or POU5/OCT4-GR by Dexamethasone (DEX) (set-up described in A). These larvae (labelled as KR+NANOG or KR+POU5/OCT4) develop hyperplasic tissues within 6-9 dpf (dorsal view, red arrows indicate hyperplasia). (C) Quantification of zebrafish developing abnormal hyperplasia upon the indicated treatments. Note that only the synergy between reprogramming factors (i.e. VENTX, NANOG or POU5/OCT4) and the KRASG12V oncogene reproducibly and efficiently induce tumors in zebrafish larvae. The total number of zebrafish larvae analyzed are indicated above the bars. φ indicates no event (0%). Note that in all pictures the asterisk (*) indicate the eye. Scale bars and body axes (a: anterior; p: posterior; d; dorsal; v: ventral; l: left; r: right) are shown.

Synergy between the Ventx reprogramming factor and the KRASG12V oncogene induces tumors in zebrafish larvae.

(A) Phosphorylated ERK (pERK) immunofluorescence at 6dpf in non-activated larvae (Ctrl), in larvea in which only KRASG12V was activated (KR) and in larvae in which both KRASG12V and VENTX were activated (KR+VX). Notice in the last one the strong pERK activation in the hyperplasic abdominal outgrowth. The digestive tract is shown between the red arrows. (B) Confocal microscopy of KR+VX zebrafish larva at 6dpf (ventro-lateral view) displays the immunofluorescence of active phosphorylated ERK (pERK, in green and indicated by red arrowheads) in the abdomen of a DAPI labelled zebrafish larva. (C) Histopathological analysis by Hematoxylin & Eosin (H&E) staining of hyperplasic tissues in zebrafish larvae (6dpf) upon activation of KRASG12V and Ventx (KR+VX) is compared with normal tissues (Ctrl). Larvae overexpressing KR+VX develop hyperplasic and dysplastic cancer-like features and tissue outgrowth in the gut/intestine (red arrow), pancreas (blue arrow) and liver. (D) Such cancer-like features specifically and exclusively reduce the survival rate to 20% two weeks after induction.

KRASG12V plus Ventx induces a cancer-like gene expression signature.

Both KRASG12V and Ventx-GR (KR+VX) were activated in zebrafish at 1dpf. The larvae (n=24) were collected at 6 dpf and processed for RT-qPCR analysis. Compared to the controls (Ctrl ; Ctrl+ cCyc ; Ctrl + cCYC + Dex) and larvae in which only KRASG12V (+cCyc+UV; +cCyc+UV+ Dex) or Ventx (Ventx+Dex) were activated, the co-activation of KRASG12V + Ventx (+cCyc+UV+VentX+Dex) displayed significant changes in gene expression, with over-expression of genes involved in pluripotency/reprogramming (nanog, pou5f3/oct4, lin28) and stemness (aldh1a, tert) and under-expression of oncosuppressor genes (foxo3, lats1,spop) and genes involved in epigenetic memory (tet3, dnmt).

KRASG12V plus Ventx induces a cancer-like gene expression signature.

Some of the data shown in Fig.S5 are here displayed with their error bars. Compared to the controls (Ctrl in light grey; Ctrl+ cCyc in grey; Ctrl + cCYC + DEX in dark grey) and larvae in which only KRASG12V (KRASG12V in light blue; KRASG12V + DEX in blue) or Ventx (VENTX in green) were activated, the co-activation of KRASG12V + VENTX (in red) displayed significant changes in gene expression. (A) Pluripotency/reprogramming factors like nanog, pou5f3/oct4 and lin28, as well as stemness markers like aldh1a2 and telomerase (tert) are significantly up-regulated (p<0.05, compared to Ctrl) by KR+VX but not by KRASG12V (KR) or Ventx (VX) alone. (B) Conversely, onco-suppressors such as foxo3, lats1 and spop are significantly down-regulated (p<0.05) by KR+VX but not by KRASG12V (KR) or Ventx (VX) alone when compared to control. For all qPCR graphs, error bars represent s.e.m. values. For statistical analyses, samples were compared with the respective control by Unpaired Student’s t-test. * p<0.05, ** p<0.005. *** p<0.0001.

Metastatic spreading following activation of KRASG12V and Ventx (KR+VX).

(A) Schematic representation and confocal microscopy of zebrafish head showing the early malignant cells (H2B-mTFP + blue cells; white arrowheads) in the brain. Green EOS fluorescence has been used to visualize the whole zebrafish head. (B) Short range dispersal in the brain of the early progeny of the initial malignant cell (H2B-mTFP + blue cells; red arrowheads). An asterisk (*) indicate the eye and a white arrow the otic vesicle. (C) Schematic representation of the zebrafish larva trunk (left, dotted square), where H2B-mTFP+ cells (white arrowheads in center panel) can localize in KR+VX larvae, both as isolated cells (as in the dotted square α, 44,5µm of mean distance) or as small clusters (as in the dotted square β, r=22.7µm). (D) Schematic representation of zebrafish larva digestive tract (left, dotted square), and confocal microscopy showing H2b-mTFP+ cells (white arrowheads, center and right panel) localized in the gut. Green EOS fluorescence has been used to visualize the digestive tract. Red arrows indicate the bulb, the green arrows point to the mid-gut, the blue arrows the distal-gut and the blue asterisk (*) indicate to the gut lumen. Scale bars and body axes (a: anterior; p: posterior; d; dorsal; v: ventral) are shown.

Single-cell activation of the KRASG12V oncogene is not sufficient to initiate carcinogenesis.

(A) At 1dpf, a single cell in the brain was photo-induced to express the oncogene kRasG12V and identified (white arrowhead) within ∼30 min by the blue fluorescence of the expression marker (H2b-mTFP). The otic vesicle (indicated by white arrow) is used as a spatial reference. At 5 days post induction (5 dpi), the activated cell has disappeared. Note in the graph that, whereas KRASG12V plus VENTX (KR+VX) efficiently induces cancer (100%), KRASG12V alone (KR) is not sufficient (0%, indicated by φ). (B) Expression of kRasG12V was activated in 1 dpf embryos (without subsequent activation of Ventx). The cells of the larvae were dissociated and isolated cells (kRas expressing, H2b-mTFP+ blue cells) were transplanted (≈ 1 cell per host) at 2dpf in a Nacre (mitf -/-) zebrafish line for a better tracking of the transplanted cells. The transplanted H2b-mTFP+ blue cell (red arrow) can be visualized as early as 3 hours post transplantation (3hpt) in the yolk of the host. At 6 dpt the blue cell has disappeared in host Nacre zebrafish larvae. Note in the graph that, whereas the transplanted cell experiencing KRASG12V plus VENTX (KR+VX) activation efficiently give rise to cancer (60%), KRASG12V alone (KR) is not sufficient to initiate cancer in host zebrafish (0%, indicated by φ). In all figures, the otic vesicle (white arrow) is indicated as well as the eye (white asterisk: *). Scale bars and body axes (a: anterior; p: posterior; d; dorsal; v: ventral; l: left; r: right) are shown.