Supporting Information - PNAS...Imaging and Quantification. All images were obtained in a Zeiss LSM 710 confocal microscope, generally with a 5-μm interval. The number of cells was

Supporting InformationFior et al. 10.1073/pnas.1618389114SI Materials and MethodsZebrafish Xenograft Injection Details.Dil-labeled cells were injectedinto the PVS of anesthetized 48-hpf larvae (8). After injection,xenografts were transferred to 34 °C until the end of experi-ments. To reduce variability, at 24 pi, successful injected xeno-grafts were sorted into classes according to the tumor size (class1, ∼1,000 cells; class 2, ∼500 cells). Sorted classes were randomlydistributed into control and experimental groups and maintainedseparately until the end of experiment. Larvae with cells in theyolk or cellular debris were discarded.

Zebrafish Xenograft Drug Administration Details.The 24-hpi zebrafishxenografts with the same tumor size were randomly distributed inthe treatment groups: control E3 medium, FOLFIRI in E3, andFOLFOX in E3 (4.2 mM 5-FU, 0.18 mM folinic acid, 0.08 mMirinotecan, 0.08mMoxaliplatin) for three consecutive days, replaceddaily. An average of 30 xenografts were treated per condition.Using, as a reference, the maximum patient’s plasma concen-

tration of each compound (Table S2), we determined the zebra-fish maximum tolerated concentration. This concentration (10×the patient plasma concentration) showed an average mortality of21% in FOLFIRI and 19% in FOLFOX (n = 200 each condition,control 5% mortality).Cetuximab monoclonal antibody was added to the cell sus-

pension (20 μg·mL−1) at the time of injection and then, at 24 hpi,was added to the fish E3 medium at 10× the plasma concentration(100 μg·mL−1). We found that Cetuximab needs to be added directlyinto the cell suspension and in the fish water to have an effect ontumor viability. Higher concentrations of Cetuximab were toxic forthe zebrafish host. Regorafenib was at 40 mM in the fish E3medium.

Mouse Xenografts and Treatment. Animal procedures were con-ducted in accordance with the EU guidelines and approved by thePortuguese Veterinary General Board.Four-month-old nonobese diabetic (NOD)-SCID female mice

were injected with 1 × 106 HCT116 cells (right flank) and 1 × 107

Hke3 cells (left flank) mixed 1:1 with PBS (Gibco) and Matrigel(BD Bioscience), s.c., in both flanks, as previously described in ref.28. The 14-dpi tumor growth was assessed by palpation and calipers.Mice were randomly distributed into three groups (five per group):FOLFIRI, FOLFOX, and vehicle, administered by i.p. injection.FOLFIRI: 5FU (30 mg·kg−1) and calcium levofolinate (45 mg·kg−1)on days 1, 6, and 11 and irinotecan (20 mg·kg−1) on days 2, 7, and 12.FOLFOX: 5FU (30 mg·kg−1) and calcium levofolinate (45 mg·kg−1)on days 1, 6, and 11 and oxaliplatin (12.5 mg·kg−1) on days 2, 7, and12. Control mice were injected with vehicle (PBS) on the same days.Mice were screened and weighed frequently to identify possibletreatment-induced toxicity. Tumor size was evaluated twice a weekby caliper measurements, and tumor volumes were calculated ac-cording to (longest tumor diameter) × (shortest tumor diameter)2 ×0.5. When the tumors reached 1 cm3, mice were euthanized, nec-ropsy was performed, and tumors were excised and processed forroutine histopathological analysis.

Zebrafish Xenograft Implantation. The tumor implantation per-centage was calculated as follows:

% implantation=# xenografts at 4 dpi with tumor mass# xenografts at 24 hpi with tumor mass

× 100.

Cell Staining. Cells were grown to 70% confluence, washed withDulbecco’s phosphate-buffered saline (DPBS) 1× (Gibco; Life

Technologies), and stained in flask with Dil (Vybrant; MolecularProbes, Life Technologies, 4 μL/mL in DPBS 1×) for 10 min at37 °C followed by 15 min on ice in darkness. Cells were washedwith DPBS, detached with 2 mM EDTA by scraping, centrifuged4 min at 135 × g, and resuspended in DMEM. Cell viability wasassessed by the trypan blue exclusion method, and cell numberwas determined by hemocytometer counting. Cells were resus-pended in DPBS to a final concentration of 0.25 × 106 cells/μl.

Human Tissue Processing.All samples used for zPDX establishmentwere obtained from Champalimaud Hospital or Prof. DoutorFernando Fonseca Hospital with written informed consent. Thestudy was approved by the Ethics Committees of both Hospitals.Neoplastic colonic tissues were obtained from surgically resectedspecimens.The samples were kept in collectionmedia containing amixture

of antibiotics and antifungals until processing (Table S4) at 4 °C.Fresh tumors were washed seven times in ice-cold PBS andchopped into small pieces and cryopreserved in 90% FBS 10%DMSO and stored in liquid nitrogen. Cryopreserved humanprimary tumor tissue was defrosted, and tumor tissue was furtherminced in mix1 (Table S4). Subsequently, tissue was digestedwith Liberase (Roche) for 10 min at 37 °C with vigorous pipet-ting. Tumor cell suspension was passed through a 70-μm cellstrainer and centrifuged at 1,200 rpm for 4 min. For cell labeling,tumor cells were incubated with the fluorescent cell tracker DiI(10 μL·mL−1) in mix2 (Table S4), for 15 min at 37 °C and thenfor 5 min on ice. Tumor cells were checked for viability withtrypan blue dye exclusion. Cells were resuspended in mix1 sup-plemented with human EGF (50 ng·mL−1, Peprotech) (TableS4) to a final concentration of 0.25 × 106 cells per milliliter forinjection. Samples with high bacterial load or poor quality withextensive necrosis which resulted in few viable cells and/or failedto show tumor masses at 24 hpi were discarded.

Histological Evaluation of Primary, Xenografts, and PDX Tumors.Mouse tumor xenografts, zPDX, and primary human tumorswere formalin-fixed, paraffin-embedded, sectioned at 3 μm, andstained with Hematoxylin and Eosin for routine histopathologi-cal analysis. Microphotographs were captured in a Leica DM2000microscope coupled to a Leica MC170 camera.

Immunofluorescence. Zebrafish xenografts were fixed in 4% form-aldehyde and stored in methanol at −20 °C. Primary antibodiesused: anti-Ki-67 (mouse Novocastra clone MM1), anti-caspase3(rabbit CST), anti-human HLA (rabbit former MHC-class IABCAM, ab52922), anti–β-catenin (mouse Novocastra clone17C2), anti-CK20 (mouse Novocastra clone PW31), anti-laminin(rabbit polyclonal Sigma), anti-human mitochondria (mouse Mil-ipore clone113-1), in general 1:100. Secondary antibodies Alexagoat anti-mouse 488 (1:400) and Alexa goat anti-rabbit 647(1:400), nuclei were counterstained with DAPI. Xenografts weremounted with Dako media and stored at 4 °C. For mouse im-munofluorescence in paraffin sections, slides were incubated withanti-caspase3 (rabbit CST 1:100) after antigen retrieval in Tris-EDTA 10 mM pH9 (20 min at 70 °C).

Immunohistochemistry.Xenografts were embedded in 2% agaroseto facilitate paraffin embedding. Then 3-μm sections were per-formed and stained with H&E every five slides. Heat-inducedepitope retrieval was performed at ∼100 °C using Citrate Buffer(∼pH 6, anti-CK20 mouse Novocastra) or EDTA Buffer (∼pH 9,

Fior et al. www.pnas.org/cgi/content/short/1618389114 1 of 12

www.pnas.org/cgi/content/short/1618389114

anti-Ki-67 mouse, Novocastra) for 20 min before incubation withprimary antibodies.

EdU Staining. Two hours prior to fixation, 4-dpi xenografts wereplaced in 500 mM EdU in E3 medium. Xenografts were fixed in4% FA and stored in 100% methanol. EdU detection was per-formed according to manufacture recommendations (EdU Click-iT- Alexa 488 Invitrogen).

Imaging and Quantification. All images were obtained in a ZeissLSM 710 confocal microscope, generally with a 5-μm interval.The number of cells was quantified with ImageJ software Cellcounter plugin.The number of total DAPI or Ki-67 cells = AVG (3 slices

Zfirst, Zmidle, Zlast) × total number slices/1.5.

Metastatic Potential Quantification. To distinguish between earlyand late metastatic events, human CRC cell lines were injectedinto the PVS only (group_a) or PVS plus circulation (group_b).At 1 hpi, we further confirmed and sorted xenografts into thesetwo groups. At 4 dpi, we analyzed and quantified the number ofxenografts that had tumor masses away from the injection site—inthe CHT.For tumor cells in group_a to efficiently establish metastasis,

they would have to go through all of the metastatic steps, whereascells in group_b only must go through the later-stage ones. Thus,considering that maximum metastatic efficiency is achieved whencells are placed in circulation (group_b), the reduction of colo-nization in group_a would reflect the effort to undergo the early

metastatic steps. The raw percentage of CHT colonization wasconverted into EMP and LMP, calculated as follows:

EMP=% CHT colonization in groupa% CHT colonization in groupb

× 100

LMP=% CHT colonization in group b.

Imaging and Angiogenesis Quantification.All images were obtainedin a Zeiss LSM 710 Zeiss confocal microscope at 40× objective,with a 3-μm interval.

total vessel densityðTVDÞ=GFPareaðfli:eGFP labeled vesselsÞtumor area

TVD=GFPareaðfli:eGFP labeled vesselsÞ

center of the tumor excluding superficial slices.

TVD and VI were quantified in maximum z projections of corre-sponding images, using Image J. This method revealed a 1% rel-ative error in relation to quantification of each slice (n =35 xenografts compared). Movies were created using ImageJ.

Statistical Analysis. Prism software was used for statistical analysis.All datasets were challenged by a normality test. Datasets with aGaussian distribution were analyzed by unpaired t test. Datasetsthat did not pass the normality test were analyzed by the Mann−Whitney test. Differences were considered significant at P < 0.05.

SW480 SW620 HCT116 Hke3 HT290

20

40

60

80

100

% im

plan

tatio

n

Fig. S1. Implantation score of human CRC zebrafish xenografts. Human CRC cells (SW480, SW620, HCT116, Hke3, and HT29) were labeled with the DiI dye(red), injected into the PVS, and imaged at 4 dpi. The average implantation was quantified at 4 dpi, i.e., percentage of xenografts that showed the presence ofa tumor mass at the injection site, PVS. Results are expressed as AVG ± SD of at least three independent experiments.




-catenin

-catenin

laminin

CK

20H

&E

A B

F

K O

P

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G H I

L M N

Q S T

J

R

Fig. S2. Histological analysis of human CRC zebrafish xenografts. Hematoxylin eosin staining (A–E′) and immunohistochemistry for CK20 (F–J′) were per-formed in histological sections, and corresponding images were obtained using a Zeiss AxioScan Z1, generating tiled images. Note that a fine line of agaroseinclusion might be detected around the xenograft due to the agarose embedding step prior to paraffin inclusion. Whole-mount imunofluorescence stainingfor laminin (K–O) and β-catenin (P–T′). All imunofluorescence images are at the same magnification. (Scale bar, 50 μm.) DiI is in red, and nuclei staining withDAPI is in blue. Dashed boxes indicate areas of zoom. All images are anterior to the left, posterior to right, dorsal up, and ventral down.



contro

l

cetuxim

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SW62

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CONTROL cetuximab cetuxi + FIFOLFIRI

Act. caspase3 DAPI 4dpi

A B C D

N=12 N=9N=16 N=12

E F G

Fig. S3. Cetuximab treatment has no significant effect in SW620 KRASG12V tumors. SW620 (A–D) xenografts were treated for three consecutive days, withCetuximab (B), FOLFIRI (C), and Cetuximab in combination with FOLFIRI (cetuxi + FI) (D). Cell death by apoptosis (F) (activated caspase3, in green), mitotic index(E), and AVG tumor size (G) (number of DAPI cells per tumor) were analyzed and quantified at 4 dpi and 3 dpT. Results in E–G are expressed as AVG ± SEM.Total number of xenografts analyzed is indicated in the images; ns, nonsignificant; P values are indicated in the text, ***P < 0.001. Datasets with a Gaussiandistribution were analyzed by unpaired t test, otherwise by Mann−Whitney for non-Gaussian distribution. All pictures are at the same magnification.



N=11 N=10

E FFE

N=19 N=13

Hke

3H

T29

CONTROL

N=41 N=16

Act. caspase3 fli:eGFP DiI

A

Regorafenib

Act. caspase3 fli:eGFP DiI

C D

N=42 N=16

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0

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Fig. S4. Regorafenib third line treatment on Hke3, HT29, and SW620 tumors. Hke3 (A–B′), HT29 (C–D′), and SW620 (E–F′) xenografts were generated in Tg(fli:eGFP)zebrafish to visualize blood vessels. At 24 hpi, xenografts were randomly distributed into control and regorafenib, and treated for three consecutive days. Mitoticindex (G) (DAPI in blue), cell death by apoptosis (H) (activated caspase3 in green), and AVG tumor size (I) (number of DAPI cells per tumor) were quantified at 4 dpi.Total vessel density (J) was also quantified. Results are expressed as AVG ± SEM. All pictures are at the same magnification. (Scale bar, 50 μm.) **P < 0.005;***P < 0.0001; ns, nonsignificant.



primary zPDX 4dpi

Patie

nt #

2Pa

tient

#4

Patie

nt #

5

C

A1

A2

B

Fig. S5. The zPDX conserve basic histological features of the original tumors. Representative microphotographs of parental tumors: patient #2 (A1 and A2),patient#4 (B), patient#5 (C), and their matching zebrafish PDX (A′–A′′′, B′ and B′′, and C′ and C′′). A′, B′, and C′ are a low magnification of a representative zPDX,showing localization of the tumor mass in the abdominal cavity. A′′, A′′′, B′′, and C′′ are higher magnifications of different tumors. Black dashed lines delineatemucin lakes, and red dashed lines denote glandular structures. Mucin and necrotic debris are pointed with red and black arrows, respectively. (Scale bar, 50 μm.)Note that a fine line of agarose inclusion might be detected around the xenograft due to the agarose embedding step prior to paraffin inclusion.



zPDX5zPDX2

PAS

+ D

zPDX 4dpi

A B

Fig. S6. PAS+D staining of zPDX sections. Representative microphotographs zPDX tumors derived from patient#2 (A) and patient#5 (B) at 4 dpi; red arrowsdepict mucin content within glandular structures by PAS+D staining. (Scale bar, 50 μm.) Note that a fine line of agarose inclusion might be detected around thexenograft due to the agarose embedding step prior to paraffin inclusion.

FOLF

OX

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zPDX#9 zPDX#10


C E

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BRAFV600E KRAS A146


zPDX#11

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N=4

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D

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N=10

Fig. S7. zPDX treatments. Five zPDX were treated with FOLFOX for 3 days and processed for activated caspase3 immunofluorescence (A–E′). Three zPDX weretreated with FOLFIRI and FOLFIRI plus Cetuximab, activated caspase3 was detected by immunofluorescence (F–H′′). Number of zPDX analyzed for each con-dition is indicated in the figure. (Scale bar, 50 μm.)



Table S1. Origin and critical mutations of the selected CRC cell lines, adapted from ref. 45


Origin Primary tumor Lymph node metastasis(same patient as SW480)

Primary tumor Generated fromHCT116 by HR*

Primary tumor

Disease CRC adenocarcinoma CRC adenocarcinoma CRC carcinoma CRC carcinoma CRC adenocarcinomaKRAS G12V G12V G13D wt wtPI3K wt wt H1047R H1047R P449TPTEN wt wt wt wt wtBRAF wt wt wt wt V600ETP53 R273H R273H wt wt R273H

P309S P309S

*Homologous recombination.

Table S2. Standard chemotherapy compounds used in advanced CRC, mode of action, and corresponding patientplasma concentration

Chemotherapy compounds Mode of actionPatient plasma

concentration, mM Ref.

5-FU Blocks thymidylic acid formation; DNA biosynthesis 426.2 46Oxaliplatin DNA crossliking agent; preventing DNA replication

and transcription8.1 47

Folinic acid or leucoverin Stabilizes binding of 5-FU to thymidylate synthase 18.5 48Irinotecan Analog of camptothecin, inhibits topoisomerase

activity, blocking DNA replication, and transcription8 49

Table S3. Histopathology primary tumor information

Patient sample Primary tumor location pTNM Observations RELAPSE

P1 Right colon T1N0 Conventional —

P2 Left colon T3N2 FO adjuvant YesP3 Right colon T3N2a Mucinous —

P4 Right colon T3N1b Mucinous —

P5 Right colon T3N1a Conventional —

P7 Left colon pT4N0 FO adjuvant NoP8 Right colon pT4N0 Neoadjuvant FOLFOXIRI +

FOLFOX adjuvantNo

P9 Rectum pT3N1c FO adjuvant NoP10 Right colon pT3N2a FO adjuvant YesP11 Rectum pT1N0 No treatment —

List of surgically resected human CRC tumors samples. Tumor node metastasis status: T1, tumor invades sub-mucosa; T3, growth into the outermost layers of the colon. pN0, no malignant lymph nodes; pN1, cancer cells arefound in one to three lymph nodes; pN1a, cancer cells in one regional lymph node; pN1b, cancer cells in two tothree regional lymph nodes; pN2, cancer cells in four or more regional lymph nodes; pN2a, cancer cells are foundin four to six lymph nodes. Relapse after 3 mo to 6 mo postsurgery.



Table S4. Reagents/solutions for patient samples processing

Reagent Supplier Cat. Number Final concentration

Collection medium Advanced DMEM/F-12 Gibco 12634010 —

Penicillin-Streptomycin Sigma-Aldrich P4333 100 U/mLAmphotericin B solution Sigma-Aldrich A2942 100 μg/mLKanamycin solution Sigma-Aldrich K0254 100 μg/mLGentamicin solution Sigma-Aldrich G1397 500 μg/mLNystatin Suspension Sigma-Aldrich N1638 2400 U/mLAmoxicillin/Clavulanic acid Clavepen 12634010 220 μg/mLMetronidazol Braun P4333 80 μg/mL

Mix1 DMEM/F-12 Gibco 11320–074 —

FBS Gibco 10270106 60%Y-27632 Cliniscience A11001 10 μMPrimocin Invivogen ANT-PM-2 100 μg/mLPutrescin Sigma-Aldrich P5780 10 μg/mLNicotinamide Sigma-Aldrich N3376 10 mM

Mix2 DMEM/F-12 Gibco 11320–074 —

Y-27632 Cliniscience A11001 10 μMPrimocin Invivogen ANT-PM-2 100 μg/mLPutrescin Sigma-Aldrich P5780 10 μg/mLNicotinamide Sigma-Aldrich N3376 10 mMDNase I Fermentas EN0525 5 U/mL

Movie S1. A 3D projection of 4-dpi SW480 xenograft in Tg(fli1:eGFP) fish.

Movie S1


http://movie-usa.glencoesoftware.com/video/10.1073/pnas.1618389114/video-1


Movie S2. A 3D projection of 4-dpi SW620 xenograft in Tg(fli1:eGFP) fish.

Movie S2

Movie S3. A 3D projection of 4-dpi HCT116 xenograft in Tg(fli1:eGFP) fish.

Movie S3





Movie S4. A 3D projection of 4-dpi Hke3 xenograft in Tg(fli1:eGFP) fish.

Movie S4

Movie S5. A 3D projection of 4-dpi HT29 xenograft in Tg(fli1:eGFP) fish.

Movie S5





Movie S6. Z stack of 4-dpi HT29 xenograft in Tg(fli1:eGFP) fish.

Movie S6

Movie S7. Migration and intravasation/extravasation of CRC cells in the CHT of 24-hpi SW620 xenografts.

Movie S7





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Supporting Information - PNAS...Imaging and Quantification. All images were obtained in a Zeiss LSM 710 confocal microscope, generally with a 5-μm interval. The number of cells was