Zebrafish xenograft as a tool for the study of colorectal cancer: a review.

Colorectal cancer (CRC) is the second leading cause of cancer-related death, mostly due to metastatic disease and the fact that many patients already show signs of metastasis at the time of first diagnosis. Current CRC therapies negatively impact patients' quality of life and have little to no effect on combating the tumor once the dissemination has started. Danio rerio (zebrafish) is a popular animal model utilized in cancer research. One of its main advantages is the ease of xenograft transplantation due to the fact that zebrafish larvae lack the adaptative immune system, guaranteeing the impossibility of rejection. In this review, we have presented the many works that choose zebrafish xenograft as a tool for the study of CRC, highlighting the methods used as well as the promising new therapeutic molecules that have been identified due to this animal model.

Zebrafish ambra1b knockout reveals a novel role for Ambra1 in primordial germ cells survival, sex differentiation and reproduction.

BACKGROUND: AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population. RESULTS: We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1+/- mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads. CONCLUSIONS: By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.

Cognitive and Behavioral Development of 9-Year-Old Children After Maternal Cancer During Pregnancy: A Prospective Multicenter Cohort Study.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.This multicenter cohort study reports on the long-term effects of prenatal exposure to maternal cancer and its treatment on cognitive and behavioral outcomes in 9-year-old children. In total, 151 children (mean age, 9.3 years; range, 7.8-10.6 years) were assessed using a neurocognitive test battery and parent-report behavioral questionnaires. During pregnancy, 109 children (72.2%) were exposed to chemotherapy (only or in combination with other treatment modalities), 18 (11.9%) to surgery only, 16 (10.6%) to radiotherapy, one to trastuzumab, and 16 (10.6%) were not exposed to oncologic treatment. Mean cognitive and behavioral outcomes were within normal ranges. Gestational age at birth showed a positive association with Full Scale Intelligence Quotient (FSIQ), with the average FSIQ score increasing by 1.6 points for each week increase in gestational age (95% CI, 0.7 to 2.5; P < .001). No difference in FSIQ was found between treatment types (F[4,140] = 0.45, P = .776). In children prenatally exposed to chemotherapy, no associations were found between FSIQ and chemotherapeutic agent, exposure level, or timing during pregnancy. These results indicate a reassuring follow-up during the critical maturational period of late childhood, when complex functions develop and rely on the integrity of early brain development. However, associations were observed with preterm birth, maternal death, and maternal education.

Zebrafish ambra1b knockout reveals a novel role for Ambra1 in primordial germ cells survival, sex differentiation and reproduction

BACKGROUND: AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population. RESULTS: We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1+/- mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads. CONCLUSIONS: By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.

A zebrafish HCT116 xenograft model to predict anandamide outcomes on colorectal cancer.

Colon cancer is one of the leading causes of death worldwide. In recent years, cannabinoids have been extensively studied for their potential anticancer effects and symptom management. Several in vitro studies reported anandamide's (AEA) ability to block cancer cell proliferation and migration, but evidence from in vivo studies is still lacking. Thus, in this study, the effects of AEA exposure in zebrafish embryos transplanted with HCT116 cells were evaluated. Totally, 48 hpf xenografts were exposed to 10 nM AEA, 10 nM AM251, one of the cannabinoid 1 receptor (CB1) antagonist/inverse agonists, and to AEA + AM251, to verify the specific effect of AEA treatment. AEA efficacy was evaluated by confocal microscopy, which demonstrated that these xenografts presented a smaller tumor size, reduced tumor angiogenesis, and lacked micrometastasis formation. To gain deeper evidence into AEA action, microscopic observations were completed by molecular analyses. RNA seq performed on zebrafish transcriptome reported the downregulation of genes involved in cell proliferation, angiogenesis, and the immune system. Conversely, HCT116 cell transcripts resulted not affected by AEA treatment. In vitro HCT116 culture, in fact, confirmed that AEA exposure did not affect cell proliferation and viability, thus suggesting that the reduced tumor size mainly depends on direct effects on the fish rather than on the transplanted cancer cells. AEA reduced cell proliferation and tumor angiogenesis, as suggested by socs3 and pcnp mRNAs and Vegfc protein levels, and exerted anti-inflammatory activity, as indicated by the reduction of il-11a, mhc1uba, and csf3b mRNA. Of note, are the results obtained in groups exposed to AM251, which presence nullifies AEA's beneficial effects. In conclusion, this study promotes the efficacy of AEA in personalized cancer therapy, as suggested by its ability to drive tumor growth and metastasis, and strongly supports the use of zebrafish xenograft as an emerging model platform for cancer studies.

Neuroimaging appearance of hypothalamic hamartomas in monozygotic twins with Pallister-Hall syndrome: case report and review of the literature.

BACKGROUND: Pallister-Hall syndrome (OMIM #146510) is a rare autosomal dominant condition caused by a mutation in the GLI3 gene. The cardinal feature of Pallister-Hall syndrome is the presence of hypothalamic hamartomas, which may manifest with seizures, panhypopituitarism and visual impairment. In Pallister-Hall syndrome, dysplastic histogenetic processes responsible for hypothalamic hamartomas are thought to disrupt early craniofacial development. The clinical presentation of Pallister-Hall syndrome may include: characteristic facies (low-set and posteriorly angulated ears, short nose with flat nasal bridge), cleft palate and uvula, bifid epiglottis and laryngotracheal cleft, limb anomalies (e.g., polysyndactyly, short limbs and nail dysplasia), anal atresia, genitourinary abnormalities and congenital heart defects. CASE PRESENTATION: We report the case of two monochorionic diamniotic twins diagnosed with Pallister-Hall syndrome during the neonatal period, after the identification of a hypothalamic hamartoma on day 1 by cerebral ultrasound scan, later confirmed by brain magnetic resonance imaging. Cerebral ultrasound and magnetic resonance imaging presentations were identical in both twins. DISCUSSION AND CONCLUSIONS: We review previously published cases (four reports) of hypothalamic hamartomas identified via cerebral ultrasound and compare reported ultrasonographic features. Main differential diagnoses based on cerebral ultrasound findings are discussed. Full description of typical magnetic resonance imaging appearance is also provided. This is the first case reported in the literature of monochorionic diamniotic twins affected by genetically confirmed Pallister-Hall syndrome with identical hypothalamic hamartomas at cerebral ultrasound and magnetic resonance imaging. Moreover, this paper adds to the existing literature on the sonographic appearance of hypothalamic hamartomas. Considering the consistency in hypothalamic hamartomas' sonographic appearance, we support the use of cerebral ultrasound as a first-line neuroimaging modality in case of clinical suspicion of Pallister-Hall syndrome.

[Formula: see text]Long-term neurodevelopmental outcome after prenatal exposure to maternal hematological malignancies with or without cytotoxic treatment.

Data on the long-term neurodevelopmental outcomes of children exposed to hematological maternal cancer with or without treatment during pregnancy are lacking. A total of 57 children, of whom 33 males and 24 females, prenatally exposed to hematological malignancies and its treatment, were invited for neuropsychological and physical examinations at 18 months, 36 months, 6, 9, 12, 15 and 18 years of age. Oncological, obstetrical, neonatal and follow-up data of these children were collected. Parents were asked to complete questionnaires on their child's general health, school performances, social situation, behavioral development, executive functioning, and if their child receives supportive care. Non-Hodgkin lymphoma was diagnosed in 35.1%, Hodgkin lymphoma in 28.1%, acute myeloid leukemia in 15.8%, chronic myeloid leukemia in 12.3%, and acute lymphoblastic leukemia in 8.8%. Cognitive development at a median age of 10.7 years was within the normal range. In subgroup analyses of children in early childhood, the gestational age at birth was correlated with the cognitive outcome at a median age of 1.7 years. Scores for language development, intelligence, attention, memory and behavior, as well as clinical neurological and general pediatric examinations were within normal ranges. In subgroup analyses, the need for supportive care in the child was associated with the loss of the mother. Prenatal exposure to hematological maternal malignancies with or without treatment did not affect the neurodevelopment of the child in the long term. Yet, caution is indicated and surveillance of the emotional development of the child is needed, especially when the mother is deceased to cancer.

First Case Report of Pregnancy on Alectinib in a Woman With Metastatic ALK-Rearranged Lung Cancer: A Case Report.

This is the first case report of a patient with ALK-rearranged metastatic lung adenocarcinoma who became pregnant during treatment with alectinib. A multidisciplinary team of gynecologists, neonatologists, oncologists, psychologists, and pharmacologists was set up to handle the case. According to patient's preference, the study drug was continued throughout pregnancy and the woman delivered a healthy baby girl at 35 weeks and 5 days of gestation. Fetal parameters remained normal during pregnancy. At birth, alectinib levels were 14 times higher in maternal plasma than in the fetus (259 versus 18 ng/mL). The average concentration of alectinib in the placenta was 562 ng/g. The baby was followed during her first 20 months, and no developmental anomalies were observed. After 32 months from diagnosis, the mother is well and in partial remission.

Effects of in-utero exposure to chemotherapy on fetal brain growth.

OBJECTIVE: Children exposed to chemotherapy in the prenatal period demonstrate normal neurocognitive development at 3 years but concerns regarding fetal brain growth remain high considering its vulnerability to external stimuli. Our aim was to evaluate the impact of in-utero chemotherapy exposure on brain growth and its effects on neurodevelopmental outcome. METHODS: The protocol was approved by the local ethics committee. Brain regional volumes at term postmenstrual age were measured by MRI in children exposed to in-utero chemotherapy and compared with normal MRI controls. Brain segmentation was performed by Advanced Normalization Tools (ANTs)-based transformations of the Neonatal Brain Atlas (ALBERT). Neurodevelopmental assessment (Bayley-III scales) was performed at 18 months corrected age in both exposed infants and in a group of healthy controls. Multiple linear regressions and false discovery rate correction for multiple comparisons were performed. RESULTS: Twenty-one newborns prenatally exposed to chemotherapy (epirubicin administered in 81% of mothers) were enrolled in the study: the mean gestational age was 36.4±2.4 weeks and the mean birthweight was 2,753±622 g. Brain MRI was performed at mean postmenstrual age of 41.1±1.4 weeks. No statistically significant differences were identified between the children exposed to chemotherapy and controls in both the total (398±55 cm3 vs 427±56 cm3, respectively) and regional brain volumes. Exposed children showed normal Bayley-III scores (cognitive 110.2±14.5, language 99.1±11.3, and motor 102.6±7.3), and no significant correlation was identified between the brain volumes and neurodevelopmental outcome. CONCLUSION: Prenatal exposure to anthracycline/cyclophosphamide-based chemotherapy does not impact fetal brain growth, thus supporting the idea that oncological treatment in pregnant women seems to be feasible and safe for the fetus.

The epg5 knockout zebrafish line: a model to study Vici syndrome.

The EPG5 protein is a RAB7A effector involved in fusion specificity between autophagosomes and late endosomes or lysosomes during macroautophagy/autophagy. Mutations in the human EPG5 gene cause a rare and severe multisystem disorder called Vici syndrome. In this work, we show that zebrafish epg5-/- mutants from both heterozygous and incrossed homozygous matings are viable and can develop to the age of sexual maturity without conspicuous defects in external appearance. In agreement with the dysfunctional autophagy of Vici syndrome, western blot revealed higher levels of the Lc3-II autophagy marker in epg5-/- mutants with respect to wild type controls. Moreover, starvation elicited higher accumulation of Lc3-II in epg5-/- than in wild type larvae, together with a significant reduction of skeletal muscle birefringence. Accordingly, muscle ultrastructural analysis revealed accumulation of degradation-defective autolysosomes in starved epg5-/- mutants. By aging, epg5-/- mutants showed impaired motility and muscle thinning, together with accumulation of non-degradative autophagic vacuoles. Furthermore, epg5-/- adults displayed morphological alterations in gonads and heart. These findings point at the zebrafish epg5 mutant as a valuable model for EPG5-related disorders, thus providing a new tool for dissecting the contribution of EPG5 on the onset and progression of Vici syndrome as well as for the screening of autophagy-stimulating drugs. Abbreviations: ATG: autophagy related; cDNA: complementary DNA; DIG: digoxigenin; dpf: days post-fertilization; EGFP: enhanced green fluorescent protein; EPG: ectopic P granules; GFP: green fluorescent protein; hpf: hours post-fertilization; IL1B: interleukin 1 beta; Lc3-II: lipidated Lc3; mpf: months post-fertilization; mRNA: messenger RNA; NMD: nonsense-mediated mRNA decay; PCR: polymerase chain reaction; qPCR: real time-polymerase chain reaction; RAB7A/RAB7: RAB7a, member RAS oncogene family; RACE: rapid amplification of cDNA ends; RFP: red fluorescent protein; RT-PCR: reverse transcriptase-polymerase chain reaction; SEM: standard error of the mean; sgRNA: guide RNA; UTR: untranslated region; WMISH: whole mount in situ hybridization; WT: wild type.