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

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.

PTEN microdeletions in T-cell acute lymphoblastic leukemia are caused by illegitimate RAG-mediated recombination events.

Phosphatase and tensin homolog (PTEN)-inactivating mutations and/or deletions are an independent risk factor for relapse of T-cell acute lymphoblastic leukemia (T-ALL) patients treated on Dutch Childhood Oncology Group or German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia protocols. Some monoallelic mutated or PTEN wild-type patients lack PTEN protein, implying that additional PTEN inactivation mechanisms exist. We show that PTEN is inactivated by small deletions affecting a few exons in 8% of pediatric T-ALL patients. These microdeletions were clonal in 3% and subclonal in 5% of patients. Conserved deletion breakpoints are flanked by cryptic recombination signal sequences (cRSSs) and frequently have non-template-derived nucleotides inserted in between breakpoints, pointing to an illegitimate RAG recombination-driven activity. Identified cRSSs drive RAG-dependent recombination in a reporter system as efficiently as bona fide RSSs that flank gene segments of the T-cell receptor locus. Remarkably, equivalent microdeletions were detected in thymocytes of healthy individuals. Microdeletions strongly associate with the TALLMO subtype characterized by TAL1 or LMO2 rearrangements. Primary and secondary xenotransplantation of TAL1-rearranged leukemia allowed development of leukemic subclones with newly acquired PTEN microdeletions. Ongoing RAG activity may therefore actively contribute to the acquisition of preleukemic hits, clonal diversification, and disease progression.

Lymphotoxin-ß receptor in microenvironmental cells promotes the development of T-cell acute lymphoblastic leukaemia with cortical/mature immunophenotype.

Lymphotoxin-mediated activation of the lymphotoxin-ß receptor (LTßR; LTBR) has been implicated in cancer, but its role in T-cell acute lymphoblastic leukaemia (T-ALL) has remained elusive. Here we show that the genes encoding lymphotoxin (LT)-α and LTß (LTA, LTB) are expressed in T-ALL patient samples, mostly of the TAL/LMO molecular subtype, and in the TEL-JAK2 transgenic mouse model of cortical/mature T-ALL (Lta, Ltb). In these mice, expression of Lta and Ltb is elevated in early stage T-ALL. Surface LTα1 ß2 protein is expressed in primary mouse T-ALL cells, but only in the absence of microenvironmental LTßR interaction. Indeed, surface LT expression is suppressed in leukaemic cells contacting Ltbr-expressing but not Ltbr-deficient stromal cells, both in vitro and in vivo, thus indicating that dynamic surface LT expression in leukaemic cells depends on interaction with its receptor. Supporting the notion that LT signalling plays a role in T-ALL, inactivation of Ltbr results in a significant delay in TEL-JAK2-induced leukaemia onset. Moreover, young asymptomatic TEL-JAK2;Ltbr(-/-) mice present markedly less leukaemic thymocytes than age-matched TEL-JAK2;Ltbr(+/+) mice and interference with LTßR function at this early stage delayed T-ALL development. We conclude that LT expression by T-ALL cells activates LTßR signalling in thymic stromal cells, thus promoting leukaemogenesis.

Adult B-cell acute lymphoblastic leukemia cells display decreased PTEN activity and constitutive hyperactivation of PI3K/Akt pathway despite high PTEN protein levels.

Adult B-cell acute lymphoblastic leukemia remains a major therapeutic challenge, requiring a better characterization of the molecular determinants underlying disease progression and resistance to treatment. Here, using a phospho-flow cytometry approach we show that adult diagnostic B-cell acute lymphoblastic leukemia specimens display PI3K/Akt pathway hyperactivation, irrespective of their BCR-ABL status and despite paradoxically high basal expression of PTEN, the major negative regulator of the pathway. Protein kinase CK2 is known to phosphorylate PTEN thereby driving PTEN protein stabilization and concomitant PTEN functional inactivation. In agreement, we found that adult B-cell acute lymphoblastic leukemia samples show significantly higher CK2 kinase activity and lower PTEN lipid phosphatase activity than healthy controls. Moreover, the clinical-grade CK2 inhibitor CX-4945 (Silmitasertib) reversed PTEN levels in leukemia cells to those observed in healthy controls, and promoted leukemia cell death without significantly affecting normal bone marrow cells. Our studies indicate that CK2-mediated PTEN posttranslational inactivation, associated with PI3K/Akt pathway hyperactivation, are a common event in adult B-cell acute lymphoblastic leukemia and suggest that CK2 inhibition may constitute a valid, novel therapeutic tool in this malignancy.

Zebrafish Avatar-test forecasts clinical response to chemotherapy in patients with colorectal cancer.

Cancer patients often undergo rounds of trial-and-error to find the most effective treatment because there is no test in the clinical practice for predicting therapy response. Here, we conduct a clinical study to validate the zebrafish patient-derived xenograft model (zAvatar) as a fast predictive platform for personalized treatment in colorectal cancer. zAvatars are generated with patient tumor cells, treated exactly with the same therapy as their corresponding patient and analyzed at single-cell resolution. By individually comparing the clinical responses of 55 patients with their zAvatar-test, we develop a decision tree model integrating tumor stage, zAvatar-apoptosis, and zAvatar-metastatic potential. This model accurately forecasts patient progression with 91% accuracy. Importantly, patients with a sensitive zAvatar-test exhibit longer progression-free survival compared to those with a resistant test. We propose the zAvatar-test as a rapid approach to guide clinical decisions, optimizing treatment options and improving the survival of cancer patients.

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis.

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly visualizing the response to anti-cancer therapies (chemo, radiotherapy, and biologicals), angiogenesis and metastasis with single cell resolution, places the zebrafish xenograft model as a top choice to develop preclinical studies. The zebrafish larval xenograft assay presents several experimental advantages compared to other models, but probably the most striking is the reduction of size scale and consequently time. This reduction of scale allows single cell imaging, the use of a relatively low number of human cells (compatible with biopsies), medium-high-throughput drug screenings, but most importantly enables a significant reduction of the time of the assay. All these advantages make the zebrafish xenograft assay extremely attractive for future personalized medicine applications. Many zebrafish xenograft protocols have been developed with a wide diversity of human tumors; however, a general and standardized protocol to efficiently generate zebrafish larval xenografts is still lacking. Here we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Object detection for automatic cancer cell counting in zebrafish xenografts.

Cell counting is a frequent task in medical research studies. However, it is often performed manually; thus, it is time-consuming and prone to human error. Even so, cell counting automation can be challenging to achieve, especially when dealing with crowded scenes and overlapping cells, assuming different shapes and sizes. In this paper, we introduce a deep learning-based cell detection and quantification methodology to automate the cell counting process in the zebrafish xenograft cancer model, an innovative technique for studying tumor biology and for personalizing medicine. First, we implemented a fine-tuned architecture based on the Faster R-CNN using the Inception ResNet V2 feature extractor. Second, we performed several adjustments to optimize the process, paying attention to constraints such as the presence of overlapped cells, the high number of objects to detect, the heterogeneity of the cells' size and shape, and the small size of the data set. This method resulted in a median error of approximately 1% of the total number of cell units. These results demonstrate the potential of our novel approach for quantifying cells in poorly labeled images. Compared to traditional Faster R-CNN, our method improved the average precision from 71% to 85% on the studied data set.

Innate immune evasion revealed in a colorectal zebrafish xenograft model.

Cancer immunoediting is a dynamic process of crosstalk between tumor cells and the immune system. Herein, we explore the fast zebrafish xenograft model to investigate the innate immune contribution to this process. Using multiple breast and colorectal cancer cell lines and zAvatars, we find that some are cleared (regressors) while others engraft (progressors) in zebrafish xenografts. We focus on two human colorectal cancer cells derived from the same patient that show contrasting engraftment/clearance profiles. Using polyclonal xenografts to mimic intra-tumor heterogeneity, we demonstrate that SW620_progressors can block clearance of SW480_regressors. SW480_regressors recruit macrophages and neutrophils more efficiently than SW620_progressors; SW620_progressors however, modulate macrophages towards a pro-tumoral phenotype. Genetic and chemical suppression of myeloid cells indicates that macrophages and neutrophils play a crucial role in clearance. Single-cell-transcriptome analysis shows a fast subclonal selection, with clearance of regressor subclones associated with IFN/Notch signaling and escaper-expanded subclones with enrichment of IL10 pathway. Overall, our work opens the possibility of using zebrafish xenografts as living biomarkers of the tumor microenvironment.

The Core-Clock Gene NR1D1 Impacts Cell Motility In Vitro and Invasiveness in A Zebrafish Xenograft Colon Cancer Model.

Malfunctions of circadian clock trigger abnormal cellular processes and influence tumorigenesis. Using an in vitro and in vivo xenograft model, we show that circadian clock disruption via the downregulation of the core-clock genes BMAL1, PER2, and NR1D1 impacts the circadian phenotype of MYC, WEE1, and TP53, and affects proliferation, apoptosis, and cell migration. In particular, both our in vitro and in vivo results suggest an impairment of cell motility and a reduction in micrometastasis formation upon knockdown of NR1D1, accompanied by altered expression levels of SNAI1 and CD44. Interestingly we show that differential proliferation and reduced tumour growth in vivo may be due to the additional influence of the host-clock and/or to the 3D tumour architecture. Our results raise new questions concerning host-tumour interaction and show that core-clock genes are involved in key cancer properties, including the regulation of cell migration and invasion by NR1D1 in zebrafish xenografts.

Developments in zebrafish avatars as radiotherapy sensitivity reporters - towards personalized medicine.

BACKGROUND: Whereas the role of neoadjuvant radiotherapy in rectal cancer is well-established, the ability to discriminate between radioresistant and radiosensitive tumors before starting treatment is still a crucial unmet need. Here we aimed to develop an in vivo test to directly challenge living cancer cells to radiotherapy, using zebrafish xenografts. METHODS: We generated zebrafish xenografts using colorectal cancer cell lines and patient biopsies without in vitro passaging, and developed a fast radiotherapy protocol consisting of a single dose of 25 Gy. As readouts of the impact of radiotherapy we analyzed proliferation, apoptosis, tumor size and DNA damage. FINDINGS: By directly comparing isogenic cells that only differ in the KRASG13D allele, we show that it is possible to distinguish radiosensitive from radioresistant tumors in zebrafish xenografts, even in polyclonal tumors, in just 4 days. Most importantly, we performed proof-of-concept experiments using primary rectum biopsies, where clinical response to neoadjuvant chemoradiotherapy correlates with induction of apoptosis in their matching zebrafish Patient-Derived Xenografts-Avatars. INTERPRETATION: Our work opens the possibility to predict tumor responses to radiotherapy using the zebrafish Avatar model, sparing valuable therapeutic time and unnecessary toxicity.

microRNAs regulate TAL1 expression in T-cell acute lymphoblastic leukemia.

The transcription factor TAL1 is a proto-oncogene whose aberrant expression in committed T-cell precursors is associated with the development of T-cell acute lymphoblastic leukemia (T-ALL). The mechanisms leading to aberrant activation of TAL1 in T-ALL patients who lack chromosomal rearrangements involving the TAL1 locus remain largely unknown. We hypothesized that TAL1 levels decrease during normal T-cell development at least in part due to miRNA-dependent silencing, in which case TAL1 over-expression in some T-ALL cases could be the consequence of deregulated miRNA expression. By performing computational prediction of miRNAs that bind to the human TAL1 mRNA we compiled a list of miRNAs that are candidates to regulate TAL1. Using a luciferase reporter system and mutagenesis assays we confirmed the miRNA-TAL1 mRNA interactions and selected candidate miRNAs: miR-101, miR-520d-5p, miR-140-5p, miR-448 and miR-485-5p. Over-expression of these microRNAs in different T-ALL cell lines consistently resulted in the down-regulation of TAL1 protein. In accordance, inhibition of miR-101 and miR-520d-5p promoted TAL1 protein expression. Importantly, we found that miR-101, miR-140-5p, miR-448 and miR-485-5p were down-regulated in T-ALL patient specimens and T-ALL cell lines. Our results show for the first time the existence of epigenetic regulation of TAL1 by specific miRNAs which may contribute, at least in part, to the ectopic expression of TAL1 in some T-ALL cases.