Single-cell functional and chemosensitive profiling of combinatorial colorectal therapy in zebrafish xenografts

Abstract

Cancer is as unique as the person fighting it. With the exception of a few biomarker-driven therapies, patients go through rounds of trial-and-error approaches to find the best treatment. Using patient-derived cell lines, we show that zebrafish larvae xenotransplants constitute a fast and highly sensitive in vivo model for differential therapy response, with resolution to reveal intratumor functional cancer heterogeneity. We screened international colorectal cancer therapeutic guidelines and determined distinct functional tumor behaviors (proliferation, metastasis, and angiogenesis) and differential sensitivities to standard therapy. We observed a general higher sensitivity to FOLFIRI [5-fluorouracil(FU)+irinotecan+folinic acid] than to FOLFOX (5-FU+oxaliplatin+folinic acid), not only between isogenic tumors but also within the same tumor. We directly compared zebrafish xenografts with mouse xenografts and show that relative sensitivities obtained in zebrafish are maintained in the rodent model. Our data also illustrate how KRAS mutations can provide proliferation advantages in relation to KRASWT and how chemotherapy can unbalance this advantage, selecting for a minor clone resistant to chemotherapy. Zebrafish xenografts provide remarkable resolution to measure Cetuximab sensitivity. Finally, we demonstrate the feasibility of using primary patient samples to generate zebrafish patient-derived xenografts (zPDX) and provide proof-of-concept experiments that compare response to chemotherapy and biological therapies between patients and zPDX. Altogether, our results suggest that zebrafish larvae xenografts constitute a promising fast assay for precision medicine, bridging the gap between genotype and phenotype in an in vivo setting.

Keywords: KRAS; chemotherapy functional screening; colorectal cancer; patient derived xenografts; zebrafish xenograft.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Implantation and histological analysis of human CRC zebrafish-xenografts. Human CRC cells (SW480; SW620; HCT116; Hke3 and HT29) were labeled with DiI dye (red) and injected into the PVS of 48-hpf zebrafish. At 4 dpi, the number of xenografts with a tumor implanted was quantified (A–E), and the average (AVG) implantation rate was determined from at least three independent experiments. Immunohistochemistry for Ki-67 in paraffin sections at 4-dpi xenografts (F–J′). Images were obtained using a Zeiss AxioScan Z1, generating tiled images. Note that a fine line of agarose inclusion might be detected around the xenograft due to the agarose embedding step prior to paraffin inclusion. Whole-mount immunofluorescence staining at 4 dpi, for Ki-67 (F′′–J′′). Representative images of mitotic figures in the corresponding xenografts (K–O), nuclei staining with DAPI in blue, anti-human HLA in red and EdU staining in green (P–T). Quantification of percentage of Ki-67 positive cells per xenograft (Z, ***P < 0.0001) and mitotic figures (Z′, ***P < 0.0001) in corresponding tumors (each dot represents one xenograft). Human CRC xenografts were generated in Tg(fli:eGFP) zebrafish to visualize blood vessels. Images representative of 4 dpi xenografts induced neovasculature: SW480 (U); SW620 (V); HCT116 (W); Hke3 (X) and HT29 xenografts (Y). Quantification of total vessel density (Z′′) and vessel infiltration (Z′′′); **/*** refers to comparison with HT29. HT29 tumors displayed significantly higher vessel density and infiltration than any other tumor, SW480 vs. HT29 (P = 0.0264); SW620 vs. HT29 (P = ns); HCT116 vs. HT29 (P < 0.0001); and Hke3 vs. HT29 (P < 0.0001). Infiltration potential SW480 vs. HCT116 (P = 0.0482); SW480 vs. HT29 (P = 0.0025); HCT116 vs. HT29 (P < 0.0001). Results in Z, Z′, Z′′, and Z′′′ are expressed as average (AVG) ± SEM. The number of xenografts analyzed for Ki-67; mitotic index and angiogenesis is indicated in the figures. All images in the same row are at the same magnification. *P < 0.05; **P < 0.005; ns, nonsignificant.

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 of a tumor mass at the injection site, PVS. Results are expressed as AVG ± SD of at least three independent experiments.

Fig. S2.

Histological analysis of human CRC zebrafish xenografts. Hematoxylin eosin staining (A–E′) and immunohistochemistry for CK20 (F–J′) were performed in histological sections, and corresponding images were obtained using a Zeiss AxioScan Z1, generating tiled images. Note that a fine line of agarose inclusion might be detected around the xenograft due to the agarose embedding step prior to paraffin inclusion. Whole-mount imunofluorescence staining for 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 with DAPI is in blue. Dashed boxes indicate areas of zoom. All images are anterior to the left, posterior to right, dorsal up, and ventral down.

Fig. 2.

Human CRC cells show different metastatic potential. At 4 dpi, it is possible to detect human tumor cells throughout the zebrafish body, in the brain (A), eye (B), gills (C), muscle (D), and CHT (E). Immunofluorescence for Ki-67 (F–J) and anti-human HLA (K–O) in the CHT region at 4 dpi in the indicated xenografts. To distinguish between early and late metastatic steps, tumor cells were injected into the PVS only (group_a) or in the PVS and directly into circulation (group_b) (P). Quantification of Early (EMP) and Late (LMP) Metastatic Potential (Q) and percentage of Ki-67 positive cells in the CHT micrometastasis (R); each dot represents one xenograft. Results are averages from at least three independent experiments. The number of xenografts analyzed for Ki-67 is indicated in the images. The number of xenografts analyzed for EMP and LMP are as follows: SW480 (EMP, n = 62; LMP, n = 66); SW620 (EMP, n = 50; LMP, n = 69); HCT116 (EMP, n = 73; LMP, n = 57); Hke3 (EMP, n = 74; LMP, n = 250); HT29 (EMP, n = 31; LMP, n = 94) (Q). Results in Q and R are expressed as AVG ± SEM. Nuclei staining with DAPI is in blue. All pictures in the same row (F–O) are at the same magnification. All images are anterior to the left, posterior to right, dorsal up, and ventral down. *P < 0.05; **P < 0.005; ***P < 0.0001.

Fig. 3.

CRC xenografts show different sensitivities to standard chemotherapy. Human CRC zebrafish xenografts were treated in vivo with FOLFOX (F–J) and FOLFIRI (K–O) compared with nontreated controls (A–E). Zebrafish were killed and fixed at 4 dpi, 3 days posttreatment (3 dpT). Mitotic index (P) (DAPI in blue) and cell death by apoptosis (Q) (activated caspase3 in green) were analyzed and quantified. Average tumor size (number of DAPI cells), normalized to respective controls, was also quantified to compare between different xenografts in different conditions (R). All pictures are at the same magnification (A–O). Results in P–R are expressed as AVG ± SEM. Results are averages from at least three independent experiments, and the total number of xenografts analyzed is indicated in the images; ns, nonsignificant; P values are indicated in the text, ***P < 0.001. HCT116 and Hke3 polyclonal xenografts (1:1) were generated and randomly treated with FOLFIRI (T) and compared with untreated controls (S). Xenografts were fixed at 4 dpi, 3 dpT. The percentages of each clone (U), cell death by apoptosis (V) (activated caspase3), mitotic index (W), and the size of each clone per xenograft (X), were analyzed and quantified. Each dot represents a xenograft, Hke3_caspase3 **P = 0.041, Hke3_mitosis **P = 0.006, remaining P values are indicated in the text, and *P < 0.05; **P < 0.005; ***P < 0.001. The total number of xenografts analyzed is indicated in the images. HCT116 was labeled with DiI (red) and Hke3 with DeepRed (green, false color).

Fig. 4.

HCT116 and Hke3 mouse xenografts validate zebrafish chemosensitive profile. HCT116 and Hke3 double mouse xenografts were generated and randomly treated with FOLFOX (n = 5) and FOLFIRI (n = 5) and compared with PBS-treated controls (n = 5). H&E (A–F) staining, as well as immunofluorescence to detect apoptotic cells (activated caspase3) (A′–F′), was performed in paraffin sections. Mitotic index (G) (DAPI in blue) and cell death by apoptosis (H) (activated caspase3 in green) were quantified in fields distant from the necrotic center of the tumor. Quantification of tumor area (cm2) was also determined (I); ns, nonsignificant; P values are indicated in the text, and *P < 0.05; **P < 0.005; ***P < 0.001. Results in G and H are expressed as AVG ± SEM.

Fig. 5.

Differential sensitivity to Cetuximab in human CRC in zebrafish-xenografts. HCT116 (A–D) and Hke3 (E–H) xenografts were treated for three consecutive days, with Cetuximab (B and F), FOLFIRI (C and G) and Cetuximab in combination with FOLFIRI (cetuxi + FI) (D and H) and compared with control nontreated xenografts (A and E). Mitotic index (I) (DAPI in blue), cell death by apoptosis (J) (activated caspase3 in green), and AVG tumor size (K) (number of DAPI cells per tumor) were analyzed and quantified at 4 dpi and 3 dpT. Average tumor size and the percentage of activated caspase3 were normalized to respective controls to compare between different xenografts. Results are expressed as AVG ± SEM. *P < 0.05; **P < 0.005; ***P < 0.0001; ns, nonsignificant.

Fig. S3.

Cetuximab treatment has no significant effect in SW620 KRASG12V tumors. SW620 (A–D) xenografts were treated for three consecutive days, with Cetuximab (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 Gaussian distribution were analyzed by unpaired t test, otherwise by Mann−Whitney for non-Gaussian distribution. All pictures are at the same magnification.

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. Mitotic index (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.

Fig. 6.

The zPDX can be efficiently established using human CRC primary samples. Cell suspensions derived from surgically resected human colon tumors were labeled with the lipophilic DiI dye (red) and injected into the PVS of 48-hpf wt or Tg(fli:EGFP). At 4 dpi, the number of zebrafish with an implanted tumor was quantified (A–F) (each dot represents the implantation percentage of each experiment). Representative confocal images of 4-dpi zPDX showing neovasculature in Tg(fli:EGFP) (A′–E′) and tumor masses with high cytomorphologic and architectural diversity (DAPI) (A′′–E′′). The number of nuclei (tumor size) (G) and mitotic figures (H) in these tumor masses was quantified; each dot represents one xenograft. Representative images of mitotic figures (A′′′–E′′′) (red arrows) and corresponding quantification (H). HLA and human mitochondria-immunostained cells at 24 hpi (I) and 4 dpi (J). Tubular structures with luminal CEA staining (K and L). (Scale bar, 50 μm.) *P < 0.05; **P < 0.005; ***P < 0.0001; ns, nonsignificant.

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 delineate mucin 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.

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 arrows depict 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 the xenograft due to the agarose embedding step prior to paraffin inclusion.

Fig. 7.

The zPDX treatment response may predict relapse and correlate with known genomic biomarkers of Cetuximab resistance. Five zPDX, corresponding to patients subjected to curative surgery and postoperative FOLFOX adjuvant treatment, were treated with FOLFOX for 3 days and processed for immunofluorescence. Cell death by apoptosis (A) (activated caspase3) was analyzed and quantified. The zPDX#7 control vs. FOLFOX P = 0.037; zPDX#9 control vs. FOLFOX P = 0.016. (B) Relapse and CEA levels information for the five patients analyzed. (C) Confusion matrix displays the number of patients with actual and predicted responses in zPDX, i.e., responders are patients that did not relapse (R), and patients that relapse are the nonresponders (NR). (D) Three zPDX were treated with FOLFIRI and with FOLFIRI in combination with Cetuximab, and cell death by apoptosis (activated caspase3) was analyzed. The zPDX#5 control vs. FOLFIRI P = 0.0043, and control vs. FOLFIRI+Cetuximab P = 0.0084; zPDX#9 control vs. FOLFIRI P = 0.001, and control vs. FOLFIRI+Cetuximab P = 0.012. (E) Genomic information of the analyzed patients. (F) Confusion matrix displays the number of patients with mutations predicted of resistance with predicted responses in zPDX. *P < 0.05; **P < 0.005; ***P < 0.0001; ns, nonsignificant.

Fig. S7.

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