SS&#39;s lab and RKS&#39;s lab are funded by grants from DBT and SERB, India. APM is a recipient of the ICMR fellowship, Government of India. DP is a recipient of the CSIR fellowship, Government of India. UN is a recipient of the DST-Inspire fellowship, Government of India. Figure 2 was generated using Biorender (https://biorender.com).

The present study was conducted with prior approval from and following the guidelines of the Institutional Animal Ethical Committee, Institute of Life Sciences, Bhubaneswar. All zebrafish maintenance and breeding were conducted at an ambient fish culture facility at 28.5 &#176;C, and the embryos were maintained in a biological oxygen demand (BOD) incubator at a temperature of 28.5 &#176;C. Here, the zebrafish AB strain was used, and the staging was carried out according to Kimmel et al.54. X-ray radiation was given at 6 hpf (shield stage), and different phenotypes were observed until 120 hpf.
1. Breeding setup and embryo collection
<ol>
	<li>Set the breeding tanks (made up of polycarbonate, capacity 1 L, see Table of Materials). Pour system water (pH, 6.8-7.5; conductivity, 500 &#181;S; and temperature, 28.5 &#176;C) into the breeding tanks covering nearly 40% of its volume. Place the divider in the tank to create two chambers, one for females and the other for males.</li>
	<li>From the parent tanks, carefully collect two healthy females and one healthy male with the help of a net, put them in their respective halves, and keep them in the dark overnight (minimum 10 h) at 28.5 &#176;C.</li>
	<li>The next morning, remove the divider and allow the fishes to mate without disturbing the breeding tanks. 
	NOTE: The females will start spawning, and the eggs will be seen lying on the bottom of the tank within 10-15 min after the fishes are allowed to mate56,57,58.</li>
	<li>Return the fishes to their tanks after spawning, collect the embryos from the breeding tank using a strainer, wash them properly with the system water, and keep the collected eggs in a Petri plate with E-3 media (4.94 mM of NaCl, 0.17 mM of KCl, 0.43 mM CaCl2, 0.85 mM of MgCl2 salts, 1% w/v of methylene blue, see Table of Materials).</li>
	<li>Observe the eggs under a dissecting microscope, remove the unfertilized or dead embryos using a Pasteur pipette, and keep the Petri plates containing fertilized eggs in the E-3 medium at 28.5 &#176;C in an incubator for their proper growth and maintenance. 
	NOTE: Unfertilized eggs can be identified with a milky white appearance with a coagulated chorion or with ruptured cells inside the chorion. Along with unfertilized eggs, eggs not undergoing cleavage and eggs with deformities like irregularities during cleavage, e.g., asymmetry, vesicle formation, or injuries of the chorion, or not actively developing, must be discarded to keep the collected embryos healthy and to keep the media clean7,56.</li>
</ol>
2. Monitoring embryos and selection for radiation experiments
<ol>
	<li>Monitor the growing embryos under the dissecting microscope, identify the proper stage7,54, and remove any dead or unhealthy embryos. Ensure adequate embryo staging as the radiation and drug doses will be given at a particular gastrulation stage. 
	NOTE: Every day, check for the level and quality of media in the culture dishes. Change the media every 24 h, along with removing dead embryos. Pasteur pipettes are preferred to be used for picking embryos or changing media.</li>
	<li>Before starting the experiment, carefully distribute the healthy embryos in the experimental plates with the help of a Pasteur pipette. For each experimental group, take 15-20 embryos. 
	&#8203;NOTE: Place only healthy embryos of the desired developmental stages in the experimental plate. Suppose the drug treatment has to be done with embryos at 6 hpf, then start seeding them in experimental plates at least 30-60 min earlier.</li>
</ol>
3. Drug treatment
<ol>
	<li>Add drugs of desired concentration to the zebrafish embryos. Prepare the drug-containing E-3 media well in advance. Ensure the stock solution of the drug has no undissolved drug before preparing the working media for treating zebrafish embryos.</li>
	<li>Before adding any drug to a medium for radiation screening, check the cytotoxic effect of the drug with the grades of concentrations of the drug. Follow the OECD guidelines to evaluate the LC 50 of the drugs under evaluation59,60,61. 
	&#8203;NOTE: Be careful while moving the plates and dishes during the irradiation or observation time. There are many chances that the plates will be disturbed during this handling, causing the media to leak out of the wells or the embryos to spill out of their respective wells, potentially contaminating nearby wells and ruining the experiment.</li>
</ol>
4. X-ray irradiation
<ol>
	<li>While setting up a radiation experiment, include a control/non-irradiated and a radiation-only group. Similarly, while performing a drug screening, include another group where the drugs will be given with the same concentration as those administered in the screening experiment along with radiation. 
	NOTE: Label both the lid and base of well plates or culture dishes so that the lids do not get misplaced.</li>
	<li>Distribute the embryos in a well plate if the radiation shields can cover and protect the extra wells from radiation while the other wells are exposed to a particular radiation dose; otherwise, use individual plates or discs to seed the embryos per radiation dose.</li>
	<li>Turn on the X-ray irradiator machine (see Table of Materials), and initiate the machine initialization and warm-up. 
	NOTE: The source to subject distance (SSD) value must be 50 cm; one can use different SSDs yet again, which requires standardization.</li>
	<li>Place the experimental plate under the irradiator inside the machine in the center, ensuring that the plate is directly below the X-ray source, and then set the dose (e.g., 5 GY) and start the X-ray. 
	NOTE: Seal the plates with paraffin film to avoid any unwanted spillage or contamination during the transportation of the plates from the incubator to the irradiator and back.</li>
	<li>After the completion of irradiation, take out the plates, shut down the machine program, switch off the machine, and check the plates under the microscope immediately after radiation. Remove the dead embryos and return the plates to the incubator at 28.5 &#176;C. Record the number of dead embryos after evaluating them under the dissecting microscope. 
	NOTE: Irradiate the different groups of embryos with designated radiation doses without much delay between individual groups as the effect of radiation may be significantly affected by the difference in the developmental stage. 
	&#8203;CAUTION: While operating the X-ray machine, take proper protective measures.</li>
</ol>
5. Data collection, imaging, and analysis
<ol>
	<li>Collect data at predetermined time intervals, such as every 24 h after the radiation is given. Record all possible observations such as survival, hatching efficiency, stage of development, heartbeat count, body and tail curvature, pericardial edema, the extension of the yolk sac, microcephaly, swim bladder development, general motility or activity, etc.62,63,64.</li>
	<li>To capture images, choose representative embryos on a clean slide, check the embryos under the microscope, orient them in a particular direction, and click on images. Rename the image files according to the group and time. 
	NOTE: The same magnification and illumination must be used while capturing pictures at different time intervals.</li>
</ol>

The authors have declared no competing interests.

Zebrafish are used as valuable models in many studies, including several types of cancer research. This model provides a useful platform for large-scale drug screening67,68. Like any other toxicity evaluation method, the quantitative evaluation of the major biological changes upon radiation and/or drug treatment is the most crucial part of this protocol. In these kinds of studies, survival must not be the only criteria to observe toxicity; it needs to be supporte...

The zebrafish (Danio rerio) is a well-known animal model that has been widely used in research over the last 3 decades. It is a small freshwater fish that is easy to rear and breed under laboratory conditions. The zebrafish has been extensively used for various developmental and toxicological studies1,2,3,4,5,6,7,8. The zebrafish has high fecundity and short embryonic generation; the embryos are suitable for tracking different developmental stages, are visually transparent, and are amenable to varieties of genetic manipulation and high-throughput screening platforms9,10,11,12,13,14. Besides, the zebrafish provides in toto and live imaging for which its developmental process and different deformities in the presence of various toxic substances or factors can be easily studied using stereo or fluorescent microscopy7,15,16.
Radiotherapy is one of the major therapeutic modes used in treating cancer17,18,19,20,21,22,23,24. However, cancer radiotherapy demands potential radioprotectors to protect normal healthy cells from dying while killing malignant cells or safeguard human health during therapy involving high energy radiations25,26,27,28,29. Conversely, potent radiosensitizers are also being investigated to increase the efficiency of radiation to kill malignant cells, especially in targeted and precision therapies30,31,32,33. Therefore, to validate potent radioprotectors and sensitizers, a model suitable for semi-high-throughput drug screening and measurably exhibiting radiation effects is highly solicited. Several available models are used in radiation studies and involved in drug screening experiments. However, higher vertebrates and even the most commonly used in vivo model, mice, are unsuitable for large-scale drug screening because it is time-consuming, costly, and challenging to design such screening experiments with these models. Similarly, cell culture models are ideal for varieties of high-throughput drug screening experiments34,35. However, experiments involving cell culture are not always pragmatic, highly reproducible, or reliable as cells in culture may markedly change their behavior according to the growth conditions and kinetics. Also, varieties of cell types show differential radiation sensitization. Notably, 2D and 3D cell culture systems do not represent the whole organism scenario, and, thus, the results obtained may not recapitulate the actual level of radiotoxicity36,37. In this regard, the zebrafish provides several advantages in screening for novel radiosensitizers and radioprotectors. The ease of handling, large clutch size, short life span, rapid embryonic development, embryo transparency, and small body size make the zebrafish a suitable model for large-scale drug screening. Due to the above advantages, experiments can be readily repeated in a short time, and the effect can be observed easily under a dissecting microscope in multi-well plates. Hence, the zebrafish is gaining popularity in drug screening research involving radiation studies38,39.
The potential of zebrafish as a bonafide model to screen radiation modifiers has been demonstrated in various studies40,41,42,43,44,45. The radioprotective effect of potential radio modifiers, such as nanoparticle DF1, amifostine (WR-2721), DNA repair proteins KU80 and ATM, and transplanted hematopoietic stem cells, and the effects of radiosensitizers, such as flavopiridol and AG1478, in the zebrafish model have been reported19,41,42,43,44,45,46. Using the same system, the radioprotective effect of DF-1 (fullerene nanoparticle) was assessed both at systemic and organ-specific levels, and also the use of zebrafish embryos for radioprotector screening was further explored47. Recently, the Kelulut honey was reported as a radioprotector in zebrafish embryos and was found to increase embryo survival and prevent organ-specific damage, cellular DNA damage, and apoptosis48.
Similarly, the radioprotective effects of polymers generated via Hantzsch&#39;s reaction were checked on zebrafish embryos in a high-throughput screening, and the protection was mainly conferred by protecting cells from DNA damage49. In one of the previous studies, the lipophilic statin fluvastatin was found as a potential radiosensitizer using the zebrafish model with this approach50. Similarly, gold nanoparticles are considered to be an ideal radiosensitizer and have been used in many studies51,52.
The embryonic development in zebrafish involves cleavage in the initial 3 h in which a single-celled zygote divides to form 2 cells, 4 cells, 8 cells, 16 cells, 32 cells, and 64 cells that are easily identified with a stereomicroscope. Then, it attains the blastula stage with 128 cells (2.25 h post-fertilization, hpf), where the cells double every 15 min and proceed through these following stages: 256 cells (2.5 hpf), 512 cells (2.75 hpf), and reaching 1,000+ cells in just 3 h (Figure 1). At 4 h, the egg attains the sphere stage, followed by the formation of a dome shape in the embryonic mass7,53,54. The gastrulation in zebrafish starts from 5.25 hpf54, where it reaches the shield stage. The shield clearly indicates the rapid convergence movement of the cells to one side of the germ ring (Figure 1) and is a prominent and distinct phase of gastrulating embryos that can be easily identified53,54. Although radiation exposure to embryos could be done at any stage of their development, radiation exposure during gastrulation might have more distinct morphological changes facilitating better readouts of radiation-induced toxicities55; similarly, administration of drugs to embryos can be started as early as 2 hpf54.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6 Well plates</td>
			<td>Corning</td>
			<td>CLS3335</td>
			<td>Polystyrene</td>
		</tr>
		<tr>
			<td>B.O.D Incubator</td>
			<td>Oswald</td>
			<td>JRIC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>10101-41-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>External Tank for the 1.0 L Breeding Tank</td>
			<td>Tecniplast</td>
			<td>ZB10BTE</td>
			<td>Polycarbonate</td>
		</tr>
		<tr>
			<td>Glass petriplates</td>
			<td>Borosil</td>
			<td>3165A75</td>
			<td>Glass</td>
		</tr>
		<tr>
			<td>GraphpadPrism</td>
			<td>GraphPad Software, Inc.</td>
			<td></td>
			<td>Version 5.01</td>
		</tr>
		<tr>
			<td>Kline concavity slides</td>
			<td>Himedia</td>
			<td>GW092-1PK</td>
			<td>Glass</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene blue hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>66720-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Tarsons</td>
			<td>380020</td>
			<td>Paraffin film</td>
		</tr>
		<tr>
			<td>Pasteur pipettes</td>
			<td>Himedia</td>
			<td>PW1212-1X500NO</td>
			<td>Polyethylene plastic</td>
		</tr>
		<tr>
			<td>Perforated Internal Tank for the 1.0 L Breeding Tank</td>
			<td>Tecniplast</td>
			<td>ZB10BTI</td>
			<td>Polycarbonate</td>
		</tr>
		<tr>
			<td>Polycarbonate Divider for the 1.0 L Breeding Tank</td>
			<td>Tecniplast</td>
			<td>ZB10BTD</td>
			<td>Polycarbonate</td>
		</tr>
		<tr>
			<td>Polycarbonate Lid for the 1.0 L Breeding Tank</td>
			<td>Tecniplast</td>
			<td>ZB10BTL</td>
			<td>Polycarbonate</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide pellet</td>
			<td>SRL</td>
			<td>1949181</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope Leica M205FA</td>
			<td>Leica</td>
			<td>Model/PN MDG35/10 450 125</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Rad 225 Precision X-Ray</td>
			<td>Precision X-Ray</td>
			<td>X-RAD 225XL</td>
			<td></td>
		</tr>
	</tbody>
</table>

zebrafish larvae as a model to evaluate potential radiosensitizers or protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

The overall layout of the protocol is depicted in Figure 2. The effect of radiation and the characterization in a dose-dependent manner was evaluated with the following analyses.
Assessment of X-ray-induced toxicities 
Using a stereomicroscope, the following abnormalities were assessed and characterized after the drug treatment and/or radiation. As per the OECD guidelines61, for toxicity evaluation in ...

Watch this Scientific Journal Video about Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors at JoVE.com

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

zebrafish-larvae-as-model-to-evaluate-potential-radiosensitizers-or

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Cancer Research

1.4K Views.  Institute of Life Sciences. The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation. 

Video: Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e.g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed.

Here we explain a protocol for modelling the biophysical microenvironment where crosslinking and increased stiffness of the basement membrane (BM) induced by advanced glycation endproducts (AGEs) has pathological relevance.

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the starting point to achieve reproducible and reliable results. All samples should be treated in the same way from blood collection to storage. Depending on the analyses to be performed, whole blood and serum samples should be stored at -20 &#176;C or -80 &#176;C until use. Blood/serum samples should also be aliquoted to avoid multiple freeze-thawing. Another important issue is the sample conditions during shipment from one laboratory to another. If dry ice is not available or the shipment takes longer than a few days, alternative approaches are needed. One option is to use filter paper for blood collection. Here, we propose a method for blood and serum sample collection that takes advantage of dried blood spots (DBS) and dried serum spots (DSS). We developed the procedure to extract DNA from DBS for the downstream evaluation of some single nucleotide polymorphisms (SNPs) by real time PCR. We also optimized an ELISA assay starting from proteins eluted from DSS. This method can be used with other ELISA assays or procedures evaluating proteins.

This protocol describes a simple and useful method to store peripheral blood and serum/plasma for downstream analyses such as single nucleotide polymorphism (SNP) evaluation and ELISA assay.

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the ...

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.