Expanding Transition Metal-Mediated Bioorthogonal Decaging to Include C-C Bond Cleavage Reactions.

The ability to control the activation of prodrugs by transition metals has been shown to have great potential for controlled drug release in cancer cells. However, the strategies developed so far promote the cleavage of C-O or C-N bonds, which limits the scope of drugs to only those that present amino or hydroxyl groups. Here, we report the decaging of an ortho-quinone prodrug, a propargylated ß-lapachone derivative, through a palladium-mediated C-C bond cleavage. The reaction's kinetic and mechanistic behavior was studied under biological conditions along with computer modeling. The results indicate that palladium (II) is the active species for the depropargylation reaction, activating the triple bond for nucleophilic attack by a water molecule before the C-C bond cleavage takes place. Palladium iodide nanoparticles were found to efficiently trigger the C-C bond cleavage reaction under biocompatible conditions. In drug activation assays in cells, the protected analogue of ß-lapachone was activated by nontoxic amounts of nanoparticles, which restored drug toxicity. The palladium-mediated ortho-quinone prodrug activation was further demonstrated in zebrafish tumor xenografts, which resulted in a significant anti-tumoral effect. This work expands the transition-metal-mediated bioorthogonal decaging toolbox to include cleavage of C-C bonds and payloads that were previously not accessible by conventional strategies.

Relapsed Ovarian Cancer Patients with Ascites and/or Pleural Effusion Still Benefit from Treatment: A Real-Life Study.

(1) Background: Relapsed HGSOC with ascites and/or pleural effusion is a poor-prognostic population and poorly represented in clinical studies. We questioned if these patients are worth treating. In other words, if these patients received the most effective treatment, would it change the course of this disease? To our knowledge this is the first real-life study to evaluate this question in this low-survival population. (2) Methods: To tackle this question we performed a retrospective, multi-centric, real-life study, that reviewed relapsed HGSOC patients with ascites and/or pleural effusion. Our rationale was to compare the OS of two groups of patients: responders, i.e., patients who had an imagological response to treatment (complete/partial response/stable disease, RECIST criteria) versus non-responders (no response/progression upon treatment). We evaluated the predictive value of clinical variables that are available in a real-life setting (e.g., staging, chemotherapy, surgery, platinum-sensitivity). Multivariate logistic regression and survival analysis was conducted. A two-step cluster analysis SPSS tool was used for subgroup analysis. Platinum sensitivity/resistance was also analyzed, as well as multivariate and cluster analysis. (3) Results: We included 57 patients, 41.4% first line responders and 59.6% non-responders. The median OS of responders was 23 months versus 8 months in non-responders (p < 0.001). This difference was verified in platinum-sensitive (mOS 28 months vs. 8 months, p < 0.001) and platinum-resistant populations (mOS 16 months vs. 7 months, p < 0.001). Thirty-one patients reached the second line, of which only 10.3% responded to treatment. Three patients out of thirty-one who did not respond in the first line of relapse, responded in the second line. In the second line, the mOS for the responders' group vs. non-responders was 31 months versus 13 months (p = 0.02). The two step cluster analysis tool found two different subgroups with different prognoses based on overall response rate, according to consolidation chemotherapy, neoadjuvant chemotherapy, FIGO staging and surgical treatment. Cluster analysis showed that even patients with standard clinical and treatment variables associated with poor prognosis might achieve treatment response (the opposite being also true). (4) Conclusions: Our data clearly show that relapsed HGSOC patients benefit from treatment. If given an effective treatment upfront, this can lead to a ~3 times increase in mOS for these patients. Moreover, this was irrespective of patient disease and treatment characteristics. Our results highlight the urgent need for a sensitivity test to tailor treatments and improve efficacy rates in a personalized manner.

Selective Inhibition of Bruton's Tyrosine Kinase by a Designed Covalent Ligand Leads to Potent Therapeutic Efficacy in Blood Cancers Relative to Clinically Used Inhibitors.

Bruton's tyrosine kinase (BTK) is a member of the TEC-family kinases and crucial for the proliferation and differentiation of B-cells. We evaluated the therapeutic potential of a covalent inhibitor (JS25) with nanomolar potency against BTK and with a more desirable selectivity and inhibitory profile compared to the FDA-approved BTK inhibitors ibrutinib and acalabrutinib. Structural prediction of the BTK/JS25 complex revealed sequestration of Tyr551 that leads to BTK's inactivation. JS25 also inhibited the proliferation of myeloid and lymphoid B-cell cancer cell lines. Its therapeutic potential was further tested against ibrutinib in preclinical models of B-cell cancers. JS25 treatment induced a more pronounced cell death in a murine xenograft model of Burkitt's lymphoma, causing a 30-40% reduction of the subcutaneous tumor and an overall reduction in the percentage of metastasis and secondary tumor formation. In a patient model of diffuse large B-cell lymphoma, the drug response of JS25 was higher than that of ibrutinib, leading to a 64% "on-target" efficacy. Finally, in zebrafish patient-derived xenografts of chronic lymphocytic leukemia, JS25 was faster and more effective in decreasing tumor burden, producing superior therapeutic effects compared to ibrutinib. We expect JS25 to become therapeutically relevant as a BTK inhibitor and to find applications in the treatment of hematological cancers and other pathologies with unmet clinical treatment.

Zebrafish Avatars of rectal cancer patients validate the radiosensitive effect of metformin.

Neoadjuvant chemoradiation (nCRT) followed by surgery represents the standard of care in patients with locally advanced rectal cancer. Increasing radiotherapy (RT) doses and chemotherapy cycles with 5FU have been associated with increased rates of complete response, however these strategies imply significant toxicity. In the last years, epidemiologic findings have demonstrated that metformin is associated with significantly higher rates of pathological complete response to nCRT. Also, pre-clinical studies using cell lines provide evidence for the radiosensitive effect of metformin. However, no studies have been performed using rectal cancer patient samples to test this radiosensitive effect of metformin and compared it to the standard 5FU. Here, we designed an experimental study to compare both radiosensitizers in the zebrafish xenograft model (zAvatar), using rectal cancer surgical specimens and diagnostic biopsies. Patient zAvatars confirmed that metformin has indeed a powerful in vivo radiosensitizer effect, similar to 5FU. Our work confirms that metformin constitutes a promising less toxic alternative to the standard 5FU, which could be game changing in elderly/frail patients to optimize tumor regression.

Zebrafish Patient-Derived Avatars from Digestive Cancers for Anti-cancer Therapy Screening.

Patient-derived xenografts (PDXs), also called "avatars," are generated by the implantation of human primary tumor cells or tissues into a host animal. Given the complexity and unique characteristics of each tumor, PDXs are models of choice in cancer research and precision medicine. In this context, the zebrafish PDX model (zPDX or zAvatar) has been recognized as a promising in vivo model to directly challenge patient cells with anti-cancer therapies in a personalized manner. The assay relies on the injection of tumor cells from patients into zebrafish embryos to then test and identify the best available drug combination for a particular patient. Compared to mouse PDXs, zAvatar assays take less time and do not require in vitro or in vivo cell expansion. The present article describes how to generate zAvatars from resected digestive cancer from surgeries and how to then use them for anti-cancer therapy screening. We describe the steps for tumor sample collection and cryopreservation, sample preparation and fluorescent labeling for microinjection into zebrafish embryos, drug administration, and analysis of tumor behavior by single-cell confocal imaging. We provide detailed protocols and helpful tips for performing this assay, and we address the technical challenges associated with the workflow. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Patient tumor sample collection and cryopreservation Basic Protocol 2: Generation of zAvatars and anti-cancer treatment Basic Protocol 3: Whole-mount immunofluorescence Basic Protocol 4: Confocal imaging and analysis.

Predictive and Therapeutic Implications of a Novel PLCγ1/SHP2-Driven Mechanism of Cetuximab Resistance in Metastatic Colorectal Cancer.

PURPOSE: Cetuximab is an EGFR-targeted therapy approved for the treatment of RAS wild-type (WT) metastatic colorectal cancer (mCRC). However, about 60% of these patients show innate resistance to cetuximab. To increase cetuximab efficacy, it is crucial to successfully identify responder patients, as well as to develop new therapeutic approaches to overcome cetuximab resistance. EXPERIMENTAL DESIGN: We evaluated the value of EGFR effector phospholipase C gamma 1 (PLCγ1) in predicting cetuximab responses, by analyzing progression-free survival (PFS) of a multicentric retrospective cohort of 94 treated patients with mCRC (log-rank test and Cox regression model). Furthermore, we used in vitro and zebrafish xenotransplant models to identify and target the mechanism behind PLCγ1-mediated resistance to cetuximab. RESULTS: In this study, levels of PLCγ1 were found increased in RAS WT tumors and were able to predict cetuximab responses in clinical samples and in vitro and in vivo models. Mechanistically, PLCγ1 expression was found to bypass cetuximab-dependent EGFR inhibition by activating ERK and AKT pathways. This novel resistance mechanism involves a noncatalytic role of PLCγ1 SH2 tandem domains in the propagation of downstream signaling via SH2-containing protein tyrosine phosphatase 2 (SHP2). Accordingly, SHP2 inhibition sensitizes PLCγ1-resistant cells to cetuximab. CONCLUSIONS: Our discoveries reveal the potential of PLCγ1 as a predictive biomarker for cetuximab responses and suggest an alternative therapeutic approach to circumvent PLCγ1-mediated resistance to cetuximab in patients with RAS WT mCRC. In this way, this work contributes to the development of novel strategies in the medical management and treatment of patients with mCRC.

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.

Establishment of Pancreatobiliary Cancer Zebrafish Avatars for Chemotherapy Screening.

BACKGROUND: Cancers of the pancreas and biliary tree remain one of the most aggressive oncological malignancies, with most patients relying on systemic chemotherapy. However, effective biomarkers to predict the best therapy option for each patient are still lacking. In this context, an assay able to evaluate individual responses prior to treatment would be of great value for clinical decisions. Here we aimed to develop such a model using zebrafish xenografts to directly challenge pancreatic cancer cells to the available chemotherapies. METHODS: Zebrafish xenografts were generated from a Panc-1 cell line to optimize the pancreatic setting. Pancreatic surgical resected samples, without in vitro expansion, were used to establish zebrafish patient-derived xenografts (zAvatars). Upon chemotherapy exposure, zAvatars were analyzed by single-cell confocal microscopy. RESULTS: We show that Panc-1 zebrafish xenografts are able to reveal tumor responses to both FOLFIRINOX and gemcitabine plus nanoparticle albumin-bound (nab)-paclitaxel in just 4 days. Moreover, we established pancreatic and ampullary zAvatars with patient-derived tumors representative of different histological types. CONCLUSION: Altogether, we provide a short report showing the feasibility of generating and analyzing with single-cell resolution zAvatars from pancreatic and ampullary cancers, with potential use for future preclinical studies and personalized treatment.

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.

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.

Using Zebrafish Larvae as a Xenotransplantation Model to Study Ewing Sarcoma.

Tumor models allowing for the in vivo investigation of molecular mechanisms driving tumor progression and metastasis are important to develop novel strategies for cancer treatment. Unfortunately, for Ewing sarcoma no adequate genetic animal models are currently available. Mouse xenograft models are the state of the art to model Ewing sarcoma in vivo. Here, we describe an alternative Ewing sarcoma xenograft model in embryonic and larval zebrafish. This xenograft model offers live imaging and easy compound testing opportunities hereby complementing mouse xenograft models. In this chapter, we provide a detailed protocol how to xenograft Ewing sarcoma cells (shSK-E17T) into 2-day-old zebrafish and how xenografted zebrafish can be imaged and analyzed over consecutive days to study tumor proliferation.

Zebrafish Xenografts Unveil Sensitivity to Olaparib beyond BRCA Status.

Poly (ADP-ribose) polymerase (PARP) inhibition in BRCA-mutated cells results in an incapacity to repair DNA damage, leading to cell death caused by synthetic lethality. Within the treatment options for advanced triple negative breast cancer, the PARP inhibitor olaparib is only given to patients with BRCA1/2 mutations. However, these patients may show resistance to this drug and BRCA1/2 wild-type tumors can show a striking sensitivity, making BRCA status a poor biomarker for treatment choice. Aiming to investigate if the zebrafish model can discriminate sensitivities to olaparib, we developed zebrafish xenografts with different BRCA status and measured tumor response to treatment, as well as its impact on angiogenesis and metastasis. When challenged with olaparib, xenografts revealed sensitivity phenotypes independent of BRCA. Moreover, its combination with ionizing radiation increased the cytotoxic effects, showing potential as a combinatorial regimen. In conclusion, we show that the zebrafish xenograft model may be used as a sensitivity profiling platform for olaparib in monotherapy or in combinatorial regimens. Hence, this model presents as a promising option for the future establishment of patient-derived xenografts for personalized medicine approaches beyond BRCA status.

Zebrafish xenografts as a fast screening platform for bevacizumab cancer therapy.

Despite promising preclinical results, average response rates to anti-VEGF therapies, such as bevacizumab, are reduced for most cancers, while incurring in remarkable costs and side effects. Currently, there are no biomarkers available to select patients that can benefit from this therapy. Depending on the individual tumor, anti-VEGF therapies can either block or promote metastasis. In this context, an assay able to predict individual responses prior to treatment, including the impact on metastasis would prove of great value to guide treatment options. Here we show that zebrafish xenografts are able to reveal different responses to bevacizumab in just 4 days, evaluating not only individual tumor responses but also the impact on angiogenesis and micrometastasis. Importantly, we perform proof-of-concept experiments where clinical responses in patients were compared with their matching zebrafish Patient-Derived Xenografts - zAvatars, opening the possibility of using the zebrafish model to screen bevacizumab therapy in a personalized manner.

Platinum-Triggered Bond-Cleavage of Pentynoyl Amide and N-Propargyl Handles for Drug-Activation.

The ability to create ways to control drug activation at specific tissues while sparing healthy tissues remains a major challenge. The administration of exogenous target-specific triggers offers the potential for traceless release of active drugs on tumor sites from antibody-drug conjugates (ADCs) and caged prodrugs. We have developed a metal-mediated bond-cleavage reaction that uses platinum complexes [K2PtCl4 or Cisplatin (CisPt)] for drug activation. Key to the success of the reaction is a water-promoted activation process that triggers the reactivity of the platinum complexes. Under these conditions, the decaging of pentynoyl tertiary amides and N-propargyls occurs rapidly in aqueous systems. In cells, the protected analogues of cytotoxic drugs 5-fluorouracil (5-FU) and monomethyl auristatin E (MMAE) are partially activated by nontoxic amounts of platinum salts. Additionally, a noninternalizing ADC built with a pentynoyl traceless linker that features a tertiary amide protected MMAE was also decaged in the presence of platinum salts for extracellular drug release in cancer cells. Finally, CisPt-mediated prodrug activation of a propargyl derivative of 5-FU was shown in a colorectal zebrafish xenograft model that led to significant reductions in tumor size. Overall, our results reveal a new metal-based cleavable reaction that expands the application of platinum complexes beyond those in catalysis and cancer therapy.

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.

The histone acetyltransferase hMOF promotes vascular invasion in hepatocellular carcinoma.

BACKGROUND & AIMS: Vascular invasion is a major prognostic factor in hepatocellular carcinoma (HCC). We previously identified histone H4 acetylated at lysine 16 (H4K16ac), a histone modification involved in transcription activation, as a biomarker of microvascular invasion (mVI) in HCC. This study aimed to investigate the role of hMOF, the histone acetyltransferase responsible for H4K16 acetylation, in the process of vascular invasion in HCC. METHODS: hMOF expression was assessed by RT-qPCR and immunohistochemistry in a retrospective series of HCC surgical samples, and correlated with the presence of mVI. The functional role of hMOF in HCC vascular invasion was investigated in vitro in HCC cell lines using siRNA, transcriptomic analysis and transwell invasion assay, and in vivo using a Zebrafish embryo xenograft model. RESULTS: We found that hMOF was significantly upregulated at the protein level in HCC with mVI, compared with HCC without mVI (P < .01). Transcriptomic analysis showed that hMOF downregulation in HCC cell line lead to significant downregulation of key genes and pathways involved in vascular invasion. These results were confirmed by transwell invasion assay, where hMOF downregulation significantly reduced HCC cells invasion. Finally, hMOF downregulation significantly reduced tumour cell intravasation and metastasis in vivo. CONCLUSIONS: Altogether, these results underpin a critical role for hMOF in vascular invasion in HCC, via transcription activation of key genes involved in this process. These data confirm the major role of epigenetic alterations in HCC progression, and pave the way for future therapies targeting hMOF in HCC.

Zebrafish Avatars towards Personalized Medicine-A Comparative Review between Avatar Models.

Cancer frequency and prevalence have been increasing in the past decades, with devastating impacts on patients and their families. Despite the great advances in targeted approaches, there is still a lack of methods to predict individual patient responses, and therefore treatments are tailored according to average response rates. "Omics" approaches are used for patient stratification and choice of therapeutic options towards a more precise medicine. These methods, however, do not consider all genetic and non-genetic dynamic interactions that occur upon drug treatment. Therefore, the need to directly challenge patient cells in a personalized manner remains. The present review addresses the state of the art of patient-derived invitro and invivo models, from organoids to mouse and zebrafish Avatars. The predictive power of each model based on the retrospective correlation with the patient clinical outcome will be considered. Finally, the review is focused on the emerging zebrafish Avatars and their unique characteristics allowing a fast analysis of local and systemic effects of drug treatments at the single-cell level. We also address the technical challenges that the field has yet to overcome.

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.

Fine-tuning of fgf8a expression through alternative polyadenylation has a selective impact on Fgf-associated developmental processes.

The formation of distinct 3'UTRs through alternative polyadenylation is a mechanism of gene expression regulation that has been implicated in many physiological and pathological processes. However, its functions in the context of vertebrate embryonic development have been largely unaddressed, in particular with a gene-specific focus. Here we show that the most abundant 3'UTR for the zebrafish fgf8a gene in the developing embryo mediates a strong translational repression, when compared to a more sparsely used alternative 3'UTR, which supports a higher translational efficiency. By inducing a shift in the selection efficiency of the associated polyadenylation sites, we show a temporally and spatially specific impact of fgf8a 3'UTR usage on embryogenesis, in particular at late stages during sensory system development. In addition, we identified a previously undescribed role for Fgf signalling in the initial stages of superficial retinal vascularization. These results reveal a critical functional importance of gene-specific alternative 3'UTRs in vertebrate embryonic development.