Parameters related to macrocirculation, such as the mean arterial pressure,central venous pressure, cardiac output, mixed venous saturation and centraloxygen saturation, are commonly used in the hemodynamic assessment of criticallyill patients. However, several studies have shown that there is a dissociationbetween these parameters and the state of microcirculation in this group ofpatients. Techniques that allow direct viewing of the microcirculation are notcompletely disseminated, nor are they incorporated into the clinical managementof patients in shock. The numerous techniques developed for microcirculationassessment include clinical assessment , laser Doppler flowmetry, tissue oxygen assessmentelectrodes, videomicroscopy  and nearinfrared spectroscopy. In the near future, the monitoring and optimization oftissue perfusion by direct viewing and microcirculation assessment may become agoal to be achieved in the hemodynamic resuscitation of critically illpatients. They are responsible for the exchange of oxygen and nutrientsbetween the intravascular and adjacent cells.,22 By factoring in the differentspeeds of all of the concentric blood flow rings and multiplying them by theirrespective areas, the following formula is obtained, known as Poiseuille'slaw:The microcirculation should be understood as a functional distribution system ofblood flow and thus of oxygen and nutrients to cells and tissue. Poiseuille'slaw demonstrates that if one observes the concentric rings within the vessels,by virtue of laminar flow, one can see that the flow velocity of each ring isdifferent; thus, the blood near the ring wall has a lower flow rate than themore central blood flow, mainly due to adherence of the formed blood componentsto the vascular endothelium . η, blood viscosity.where F is blood flow; ΔP, the pressure difference between the ends of thevessel; r, the radius of the vessel; L, its length; and-25 In general, blood rheology isresponsible for the amount of movement and affects not only the flow pattern butalso the functional capillary density. Vascular resistance, controlled by the endothelium,can also change regional blood flow dramatically.,24The rheological properties of fluids (viscosity is the best known of them) areextremely important to the maintenance of blood flow inmicrocirculation. and vasoactive control of therelease of substances, such as adenosine triphosphate (ATP) and nitric oxide(NO) derivatives.,21,24,25In addition to providing the tissue with oxygen by its diffusion in thearterioles and capillaries, hemoglobin has characteristics that are important tothe microcirculation, such as its direct effect on the maximum distance betweenthe place of diffusion and the mitochondria; the formation of its molecules,which have two different forms designated taut and relaxed; The convective component isdirectly related to blood flow in the microcirculation, being essentiallydetermined by the number of erythrocytes and the saturation thereof. The diffusive component, inturn, is directly related to the difference between the partial oxygen pressure(PO2) levels in the capillaries and mitochondria, the oxygendiffusion distance and the gas exchange surface.Finally, the convective and diffusive components of microcirculatory blood floware essential to the transport of oxygen to the tissue. Changes in themicrocirculation in sepsis occur due to inflammation, activation of coagulationand the complement system and damage to the capillary endothelium.Most publications on microcirculation dysfunction address patients with septicshock. It is postulated that changes in the microcirculation reduce the supplyof oxygen to the mitochondria, impairing ATP production. which produce vasoactive moleculesresponsible for regulating arteriolar contraction and help with blood pressurecontrol. Thesemolecules include vasodilating substances, such as NO and prostacyclin, andvasoconstricting substances, such as thromboxane A2, endothelin and plateletactivating factor (PAF).,29 Critically ill patients maypresent imbalances between these components, causing vasomotor instability andregional hypoperfusion.,29Vessel tonicity is regulated by the endothelial cells, Hemoglobin also has crucial importance inthis context. In anexperimental sepsis model, a significant reduction in erythrocyte deformability,contributing to microcirculatory dysfunction, has been demonstrated. Moreover, patients withsepsis suffer impairments of the convective and diffusive components ofmicrocirculatory blood flow, resulting in a heterogeneous and insufficienttissue oxygen supply.One of the major changes in the endothelium during sepsis is an increase in itspermeability, or the loss of barrier function, which leads to an imbalance inthe circulation of blood elements and tissue edema.-33 In cardiogenic shock, forexample, all microcirculatory variables undergo change, such as reductions inthe diameter of arterioles and the functional capillary density.-33 In patients with heart failure, intravenous infusionof nitroglycerin has been able to increase functional capillary density, evenwith a reduction in cardiac filling pressures, demonstrating the independence ofthe microcirculation in relation to macro-hemodynamic variables and theirdynamic character.,32The relationship between systemic and regional perfusion closely depends on thecause of circulatory shock. In an experimental model in pigs, it has beendemonstrated that, with the removal of 35% of the blood volume, rapid decreasesin the cardiac index, SvO2 and oxygen delivery (DO2)occurred, along with an increase in lactate and a reduction in tissue oxygensaturation (StO2) in skeletal muscle. Only animals that received aggressive volemicresuscitation showed an increase in StO2 values, demonstrating howthis noninvasive microcirculation measure may be relevant at thebedside. A studyconducted on patients admitted to the intensive care unit (ICU) for hemorrhagicshock demonstrated that, even in the presence of normal macrocirculatoryparameters, the sublingual microcirculation was dysfunctional for up to 3 dayspost-shock.Furthermore, the microcirculatory indices assessed in the study changed in alltrauma patients, but these changes were more pronounced in those patients withhemorrhagic shock.In hemorrhagic shock, microcirculatory changes are early and may reflect a stateof tissue perfusion with lower oxygen consumption. Among themany techniques developed to assess microcirculation are clinical assessment, laser Dopplerflowmetry, tissue oxygen assessment electrodes (PO2), videomicroscopy, sidestream dark field (SDF) orincident dark field illumination (IDF)) and near infrared spectroscopy (NIRS).By definition, any equipment that analyzes the microcirculation can do so in only thevascular bed being assessed. However, it may be considered that the area beinginvestigated is a window that reflects changes that are likely to be observedelsewhere. Furthermore, studies suggest that partialsublingual carbon dioxide pressure (PslCO2) is directly related topartial gastric carbon dioxide pressure (PgCO2), indicating that thesublingual region is a good indicator for the indirect assessment of splanchnicmicrocirculation. Itis important to note that microcirculation assessment is currently restricted toresearch protocols, and its use at the bedside as a therapeutic goal also depends ongreater scientific development in this area.,38Microcirculation assessment can be performed on different types of tissue, accordingto the technique and apparatus used. The sublingual area is often used to performvideomicroscopy, as it is easily accessible and noninvasive and is potentiallyreliable in terms of patient monitoring and management.However, cutaneous circulation has no such self-regulation, resulting in adecrease in skin perfusion and the consequent fall of regional temperaturesecondary to vasoconstriction.During circulatory failure, the vital organs demonstrate vasomotorself-regulation whereby they are able to maintain blood flow, despite thepresence of hypotension.,47 Underphysiological conditions, dTc-p shows variations between 3 - 7°C.This skin temperature drop can be assessed via peripheral-ambient (dTp-a) andcentral-peripheral (dTc-p) temperature gradients. Assuming that the ambienttemperature remains constant, the dTp-a gradient decreases while dTc-p increasesduring situations of circulatory collapse.,48 One study examined the bloodflow and temperature gradients in the forearm and fingertip in volunteerssubjected to an artificial vasodilation and vasoconstriction process. The principal findingsrevealed that differences of only 1.5°C were detected in vasoconstrictionsituations. Thus,peripheral temperature gradient analysis has demonstrated great value in termsof vasoconstriction and vasodilation, as a strong correlation between dTp-a andserum lactate levels has been demonstrated.Several studies have been conducted to assess the relationship between thetemperature gradient and vasoconstriction or vasodilation secondary to localblood flow changes. This method uses pulse oximetry and is ableto distinguish between the pulsatile (blood) and the non-pulsatile (othertissue) components and between hemoglobin and oxygenated hemoglobin. An important point is thatcalculation of the index is performed independently of the oxygen saturationvalue.,50 Peripheral perfusion indexvalues of less than or equal to 1.4 have been related to the presence of tissuehypoperfusion.Another index that may be used at the bedside to assess circulatory failuresituations is the peripheral perfusion rate. It is measured by applying firm pressure to the distalphalanx of the right and left index fingers for 15 seconds each. The time in seconds to returnto normal skin color is determined using a stopwatch. A time of 5 seconds is set as the uppernormal limit for this test, but this rate varies according to age andgender.,51 The capillary refill time maybe up to 2.9 seconds in healthy women and up to 4.5 seconds in theelderly.,51 Many studies suggest that thecorrelation between the capillary refill time and blood pressure or CO is notreliable and is a good predictor of only dehydration, reduced systolic volumeand increased serum lactate in children.Capillary refill time is useful in identifying blood hypoperfusion states inhemodynamically unstable patients. However, it is postulatedthat such changes result from skin hypoperfusion. The mottling score consists ofa semi-quantitative assessment of skin mottling, based primarily on the extentof its presence in the assessed area . The mottling score is easilyapplied at the bedside, has good correlation with tissue perfusion variables,such as lactate and urine output, and has good predictive value when assessingmortality in patients with septic shock.In the ICU, a skin assessment looking for clinical signs that may correlate withtissue hypoperfusion is the usual practice. Mottling constitutes a change inskin color, and its pathophysiology is not completely clear. For the measured flow to represent theaverage flow of at least 50 vessels, including arterioles, capillaries andvenules of various sizes, the sample volume of the current laser Dopplerapparatus should be between 0.5 and 1 mm3. The method uses a confocaltechnique to measure vascular density and diameter and blood flow.,39 In the microvascular reactivity test, the upward slopeafter the occlusion is a marker of endothelial reactivity and blood rheology andcan be used as a functional microvascularization integrity parameter. The measurement can beperformed on any area of intact skin.Laser Doppler flowmetry analyzes the relative microcirculatory blood flow andreserve by means of microvascular reactivity testing.2 assessment include theaccurate assessment of tissue PO2.,53 Thesample volume analyzed by these electrodes comprises at least one hundredmicrovessels, including arterioles, capillaries, venules, interstitial and othercells, all of which contribute to the final assessed PO2value.,53 Their main use is in theindirect assessment, via PO2 levels, of perfusion and/or regionaloxygenation, especially in low-flow conditions.,43,53 One of the main problems withthe use of electrodes is the fact that it is impossible to assess microvascularperfusion directly.The potential uses of electrodes for PO2 and carbondioxide continuously and non-invasively using transcutaneous sensors. Carbon dioxide isapproximately 20 times more diffusible than oxygen, and transcutaneous oxygenmeasurement (PtCO2) is more sensitive to changes in perfusion thantranscutaneous carbon dioxide measurement. The oxygen challenge test involves temporarilyincreasing the inspired oxygen fraction (FiO2) used and monitoringthe PtcO2 response. In patients with normal lung function, increasedFiO2 is associated with a parallel increase in PtcO2since, in patients with adequate blood flow, the PtcO2 andPaO2 values are almost identical. A lack of increase in PtcO2 afteran increase in FiO2 suggests probable perfusion dysfunction andportends a worse outcome in septic shock patients.Recent advances have made it possible to measure PO,41 This technique can be appliedto organs that have a thin epithelial layer, such as the sublingual region, inwhich capillaries and venules of various sizes can be observed. The techniques used inmicrocirculation videomicroscopic assessment are OPS and SDF analyses - thelatter being the most used in recent years. An example of a microcirculatory image obtained byvideomicroscopy is shown in Videomicroscopy assesses microcirculation directly by emitting polarized greenlight, which, when absorbed, produces an image representing the red blood cells(RBCs) as black bodies. Cytocam-IDF, the only equipment able toperform this type of analysis to date, consists of a probe that incorporates IDFillumination with an array of high-resolution image projector lenses. These pictures are projectedonto a high-density sensor controlled by a computer synchronized with anilluminated unit. A recent study comparing the results of this device with SDFshowed that Cytocam-IDF could detect more capillaries (30% more) and generatebetter quality images than the SDF technique. Similar results were obtained in differentpreliminary validation studies involving neonates.This situation may change due to the recent development of a new IDF technique,described as third-generation videomicroscopy.,55There are five steps during assessment that are essential to the correct use ofvideomicroscopy: the assessment of five sites per organ, the avoidance ofpressure artifacts, the removal of secretions prior to assessment, the use ofproper focus and the calibration of contrast and recording quality.Several scores have been developed for analysis of the results obtained byvideomicroscopy.The main functional features of the microcirculation analyzed by videomicroscopyinclude vascular density (responsible for oxygen supply by diffusion), thepattern and intensity of microcirculatory blood flow (responsible for oxygensupply by convection) and flow heterogeneity . Vascular density is calculatedas the number of vessels crossing these lines, divided by the total line length.The total vascular density corresponds to the total number of vessels , while the functional capillarydensity is the number of well-perfused small vessels (< 20 µm) perunit area. Flow type isdefined as continuous, intermittent or absent. The vessels are usually separated into large(mainly venules) and small (mainly capillaries), using a cutoff value of20µm in diameter.Vessel perfusion  is defined as the proportion ofperfused vessels (PPV), calculated as the number of continuously perfusedvessels during an observation of 20 seconds, divided by the total number ofvessels of the same type.Thus, the proportion of small perfused vessels corresponds to the proportion ofwell-perfused vessels with diameters < 20 µm (mainlycapillaries). Theflow heterogeneity index is defined as the difference between the maximum andminimum PPV proportion assessed at each point of five areas, divided by its ownmean value. Each of thesemicrocirculatory parameters is obtained from an average of five video streams orpossibly more.Generally, a form with three horizontal rows and three vertical lines is placedin front of the screen while each video sequence is played.,36,42,55 In thisimage, microvascular flow is characterized as absent (0), intermittent (1) slow(2) or normal (3).,36,42,55 The mean value of these fourquadrants is reported as the microvascular flow index.Additionally, a second form, having vertical and horizontal lines, can also beplaced in front of the screen to separate the image into fourquadrants.This method proposes to divide the obtained image into four quadrants and todetermine which of these quadrants has the predominant flow type, classified asdescribed above. Inaddition, the authors added a heterogeneity index, which can be obtained bysubtracting the area of the greatest flow rate from the area with the lowestflow rate and then dividing by the mean flow velocity of all assessedareas. Normalmicrocirculation exhibits minimal blood flow heterogeneity, and there must be an adequaterelationship between perfusion and metabolism (or the supply and demand ofoxygen and nutrients) to prevent hypoxia-induced cell damage.Trzeciak et al. described a way of assessing microcirculation based on themicrovascular flow index.,58 By reducing functional capillary density and creatinga heterogeneous flow, oxygen diffusion distance increases, and as a result, poortissue oxygen extraction is observed.,40 Thus,assessment of the microcirculation is of great value, as it identifies poorperipheral perfusion conditions, even in situations with normal or increasedSvO2.Generally, tissue is better able to adapt to low-flow situations with homogeneousmicrocirculation than in heterogeneous flow situations.Recently, De Backer et al. stated that the result of videomicroscopicmicrocirculation assessment must always show the density of perfused vessels , the PPV and the microvascular flowindex for all vessels, large and small, along with the heterogeneityindex. By measuring the oxy- and deoxyhemoglobinfractions, one can calculate StO2, the total tissue hemoglobin (THb)and the absolute tissue hemoglobin index (THI); THb and THI are twomicrocirculatory blood volume indicators.,60 Themeasurements made using NIRS may be affected by the amount of adipose tissue andby the presence of edema at the assessment site.,60 Thethenar eminence region has been the most used because of the thickness of skinand because the adipose tissue covering this muscle is less affected by bodyweight variations.,60NIRS is a technique that measures the chromophores (parts or groups of atomsresponsible for the color of a molecule) of oxyhemoglobin, deoxyhemoglobin,myoglobin and cytochrome aa3 in any given tissue.2 levels. The analysis of changes inStO2 during a brief period of ischemia on the forearm, known asthe vascular occlusion test (VOT), provides a dynamic assessment ofmicrovascular reserve in just a few minutes.,61Arterial and venous vascular occlusion can be achieved when inflating asphygmomanometer positioned on the patient's arm, above the systolic bloodpressure, with the aim of inducing ischemia in the thenar muscle and causingchanges in StO2. There is still no consensus regarding the intensityand duration of VOT; two strategies have been described: the use of VOT based oninflation time, because the maximum ischemic vascular response is obtainedwithin a few minutes, andthe use of VOT based on a drop in StO2, seeking a 40% StO2target to minimize inter-individual variations in the VOT response des, %/seconds) in the thenar muscle, aftervascular obstruction, can be used to estimate this muscle's oxygenconsumption.-64 The product of the absolutevalue of Rdes and the mean THI value quantifies the amount ofhemoglobin desaturated in the tissue. After deflation of the sphygmomanometer,there is rapid restoration of blood flow, called the resaturation rate.-64 During thisreactive hyperemia, StO2 can reach higher levels than baselineStO2, indicating post-ischemic vasodilation and capillaryrecruitment.-64The desaturation rate  the use ofnitroglycerin was associated with increased microcirculatory bloodflow; (2)dobutamine infusion caused significant increases in vascular density andcapillary perfusion; and(3) the infusion of Ringer's lactate or 4% albumin solution also increased smallvessel density and perfusion. In contrast, RBC transfusion in a patient with severesepsis had no significant effect on the microcirculation. It is important to highlightthe role of individual variation in RBC transfusion. Possible causes of the lackof a transfusion effect may include changes in rheological properties, loss ofRBC deformability and reduced 2,3-diphosphoglycerate concentrations. Furthermore, RBC storage timealso showed no relationship with potential microcirculatory changes. Finally, in patients withseptic shock, norepinephrine infusion to raise MAP values above 65mmHg did notcause changes to the sublingual microcirculation pattern and did not lead to anyimprovements in parameters usually adopted in tissue perfusion monitoring, suchas arterial lactate, anion gap and the difference between the partial carbondioxide pressure of the gastric mucosa and the partial pressure of arterialcarbon dioxide.The emergence of techniques that allow direct viewing of the microcirculationhave led to studies that are focused on interventions modifying themicrocirculation of critically ill patients. The main interventions studiedinclude the use of vasodilators to obtain better microcirculatory flowhomogeneity. In patients with acute heart failure, the use of low nitroglycerindoses has resulted in an increase in functional capillary density.The isolated assessment of macro-hemodynamic parameters and global perfusion markersas shock treatment goals does not seem to be entirely appropriate, as theseparameters do not assess the state of tissue microcirculation in these patients.However, techniques that allow viewing and assessment of the microcirculation arenot yet fully developed and incorporated into clinical practice. Therefore, advancesin this area are imperative, given that the monitoring and optimization of tissueperfusion by direct viewing and microcirculation management may become an achievablegoal in the near future in the hemodynamic resuscitation of critically illpatients.


Danio rerio are amenable to in vivo imaging, high-throughput drug screening, mutagenesis, and transgenesis, and they share histological and genetic similarities with Homo sapiens. The significance of zebrafish in the field of precision oncology is rapidly emerging. Indeed, modeling cancer in zebrafish has already been used to identify tumor biomarkers, define therapeutic targets and provide an in vivo platform for drug discovery. New zebrafish studies are starting to pave the way to direct individualized clinical applications. Patient-derived cancer cell xenograft models have demonstrated the feasibility of using zebrafish as a real-time avatar of prognosis and drug response to identify the most ideal therapy for an individual patient. Genetic cancer modeling in zebrafish, now facilitated by rapidly evolving genome editing techniques, represents another innovative approach to recapitulate human oncogenesis and develop individualized treatments. Utilizing zebrafish to design customizable precision therapies will improve the clinical outcome of patients afflicted with cancer.Zebrafish have proven to be a valuable model to study human cancer biology with the ultimate aim of developing new therapies.  Therefore, precision oncology seeks to identify the most effective therapy for an individual patient, based on characterization of their cancer. The development of genomic technologies and molecular diagnostics enables detection of cancer biomarkers. These relevant abnormalities associated with specific cancers lead to the identification of actionable targets. Diagnostic , prognostic (associated with disease outcome), and predictive (associated with drug response) cancer biomarkers guide clinical treatment decisions and direct the use of drugs that modulate the activity of the specific actionable target.Danio rerio) have rapidly emerged as a promising animal model of human cancer. Histological, molecular, and genetic similarities to Homo sapiens facilitate zebrafish studies of human malignancies. Zebrafish are amenable to in vivo fluorescent imaging, chemical and genetic screens, transgenesis, and high-throughput mutagenesis assays, which have brought zebrafish to the center stage of future advances in the field of precision oncology.3 A variety of attributes have contributed to the emergence of zebrafish as an attractive vertebrate model organism. Zebrafish are easy and inexpensive to maintain and breed with high fecundity, which facilitates large studies and high-throughput in vivo assays. Another advantage of working with zebrafish embryos is their conserved vertebrate features develop rapidly and genetic studies restricted to embryos can be completed in days to weeks rather than weeks to months as is often the case with mammalian models.4 However, it is important to note that zebrafish sexual maturation takes about three months, so generation studies  require a similar time frame as murine models. The small size, external development, and transparency of zebrafish embryos make them amenable to fluorescent live imaging to monitor physiological processes  and pathological phenomena . Taken together, the attractive features of the zebrafish model system underscore the reasons it has gained prominence in the study of cancer and serves as an excellent addition to other common oncology models and platforms.Zebrafish  evaluating patient prognosis in vivo and (vi) directing individualized treatments in real-time based on responses to drugs of patient cancer cell xenografts  promoter. These fish developed nevi, but required a p53M214K mutant zebrafish background for melanoma development in ~5% of zebrafish by four months.14 A crestin:EGFP reporter, recapitulating the embryonic neural crest expression patter of crestin, showed that a fate change occurs at melanoma initiation in this model, as a single melanocyte reactivates the neural crest progenitor state.15 Similarly, human oncogenic NRASQ61K expression under the control of mitfa promoter resulted in a transgenic fish that required p53 loss of function for the genesis of melanoma.16 The first p53 mutation-independent model was developed through expression of human oncogenic HRASG12V driven by the same mitfa promoter fragment. In this model, however, melanoma does not arise at a high frequency and takes several months to develop.17Instead, when HRASG12V expression is driven in melanocyte progenitor cells by the kita (c-kit in humans) promoter, melanoma occurs spontaneously by 1–3 months in ~20% of fish.18 Recently, a novel zebrafish transgenic model of uveal melanoma was created by expressing oncogenic GNAQQ209P in the melanocyte lineage using again the mitfa promoter. The corresponding p53 inactivation was also required for the malignant progression in this system.19 Importantly, zebrafish models of melanoma closely resemble human cancer, both in terms of histopathological features and molecular signatures.20Melanoma research offers many concrete examples of genetic zebrafish models used for the definition of new therapeutic targets and as an in vivo platform for drug screening. Melanoma accounts for the death of over 70% of skin cancer patients and only 14% of patients with metastatic disease survive for five years. Unlike many other tumor types, new cases and mortality of melanoma are still rising.V600E on a p53 mutant background to test genes in a recurrently amplified region on chromosome 1. The histone methyltransferase SETDB1 has been found to cooperate with BRAFV600E and accelerate melanoma. Its relevance in human malignant melanoma has also been demonstrated, and therefore, SETDB1 has been revealed as a novel oncogene in melanoma.21 RAC and RSK1, whose hyperactivation has been detected in human melanoma, have been shown in distinct studies to contribute to melanoma progression when constitutively activated in mitfa:HRASG12V and mitfa:BRAFV600E;p53−/− transgenic backgrounds, respectively.23 Lister et al.24 have used a temperature-sensitive mitfa mutant to show the oncogenic activity of Mitfa transcription factor in BRAFV600E transgenic zebrafish and the regression of BRAFV600Emitfa melanoma after Mitfa activity abrogation, thus presenting Mitfa as a promising therapeutic target. The use of zebrafish to identify novel oncogenes begins to exemplify how this model organism will be utilized to overcome tumor heterogeneity through precision oncology.These models have confirmed the role of relevant oncogenes in melanoma genesis and progression. Moreover, they have proven to be outstanding tools to test and screen for other genes that promote melanoma onset and might represent new therapeutic targets, and even, in the near future, tumor biomarkers for personalized cancer therapy. An excellent example has been reported by Ceol and colleagues. They have used transgenic zebrafish overexpressing BRAFG12V transgenic embryos.25 Moreover, a zebrafish screen of FDA-approved compounds led to the discovery of two new potential drugs cooperating with MEK inhibitors to suppress the growth of transformed melanocytes.25 Transgenic mitfa:BRAFV600E;p53−/− zebrafish embryos demonstrate a gene signature enriched for markers of multipotent neural crest cells. A chemical genetic screen was, therefore, performed to identify small molecule suppressors of the neural crest lineage. A positive result was obtained with the inhibitors of dihydroorotate dehydrogenase, whose activity as an anti-melanoma agent was then confirmed in vitro and through mouse xenograft models.26 To fully realize the utility of zebrafish in precision oncology, translating these types of drug identification and validation studies to a patient sample size of one is the ultimate goal, such that treatments can be tailored to the individual patient based on zebrafish surrogates of the individual’s tumor.The significance of zebrafish melanoma models in translational medicine and precision oncology is not limited to the discovery and characterization of potential therapeutic targets, as various studies have already shown the efficacy of zebrafish in identifying, discovering, and testing drugs for the development of new melanoma treatments. Small molecule inhibitors of MEK and PI3K/mTOR, known players in melanoma, have been validated in vivo as targeted drugs suppressing melanocyte hyperplasia phenotype in HRAS28 The focus of most zebrafish studies on brain tumors is malignant glioma, which accounts for 70% of malignant primary brain tumors, and in particular glioblastoma, the most aggressive primary brain cancer, accounting for 70% of malignant gliomas.30 Transgenic models of malignant peripheral nerve sheath tumors have also been described.35 Jung and colleagues established a transgenic zebrafish that overexpressed dominant active, human AKT1 at the ptf1a domain leading to gliomagenesis. Pharmacological tests identified AKT1/2 inhibitor as a targeted drug capable of effectively suppressing gliomagenesis, inhibiting cellular proliferation, and inducing apoptosis in established gliomas.28 The scope of available brain tumor models offers promise for using zebrafish to tailor specific treatment approaches to individual neurological cancer patients.Neurological tumors have also been modeled via transgenic expression of oncogenes, demonstrating the potential to define relevant actionable alterations driving cancer progression and to successfully test specific drugs targeting those alterations.36 Pancreatic cancer is a deadly genetic disease, with a dismal ~9% five year survival rate.37 The majority of pancreatic cancers are pancreatic ductal adenocarcinomas (PDACs) and over 90% of them carry an activating point mutation in the KRAS gene.36 Genetic models based on the Gal4/UAS system enable assessment of the effects of different KRAS mutations and the ability of other proteins to alter the response to oncogenic KRAS, potentially leading to the identification of new targets for precision oncology therapeutic strategies.39 In this regard, the involvement of a variety of core signaling pathways, including TGFβ, Wnt, Notch, and Hedgehog, in pancreatic cancer development has also been investigated using Gal4/UAS system.40The Gal4/UAS transgenic system, based on the ability of the Gal4 transcriptional activator to drive the expression of multiple transgenes under the regulation of UAS (upstream activator sequence) regulatory elements, is widely used to model KRAS-initiated pancreatic cancer in zebrafish.41 Leukemia has been modeled mainly through transgenesis. A zebrafish model of T-cell acute lymphoblastic leukemia (T-ALL), the most common type of childhood leukemia,43 was created in the early 2000s expressing a mouse c-Myc transgene fused to green fluorescent protein (GFP) under the control of a zebrafish rag2 promoter.44 Visualization of GFP+ leukemic cells has demonstrated leukemia originates in the thymus, disseminates to the gill arches and surrounding retro-orbital soft tissue, and then spreads to skeletal muscle and abdominal organs.44 Feng and colleagues have subsequently improved this model by developing conditional, heat-inducible activation of the c-Myc oncogene resulting in greater penetrance of T-ALL and increased control of disease onset.45 Similarly, another zebrafish model of T-ALL has been created by expressing the truncated human NOTCH1 protein fused to EGFP (ICN1-EGFP) under the control of the zebrafish rag2 promoter.46 While these transgenic zebrafish developed T-ALL by 5 months of age, onset of leukemia was dramatically accelerated when crossed to zebrafish overexpressing anti-apoptotic protein, Bcl2. The oncogenic synergy between NOTCH1 and Bcl2 in this model suggests genetic modifier screens may reveal other genes that interact with NOTCH1 to promote T-ALL.46 All of these rag2 promoter-driven transgenic zebrafish models are amenable to drug and genetic screening to identify individualized treatment strategies for leukemia and lymphoma patients. Indeed, a zebrafish screen for therapeutics that alter the number of hematopoietic stem cells (HSCs) has led to the development of Prohema, a derivative of prostaglandin E2 (PGE2), currently in Phase II clinical trials for use in leukemia and lymphoma patients receiving blood transplantations.48Leukemia, the ninth most common cancer type is a cancer of blood-forming tissues usually involving dysfunction of white blood cells.V600E) in thyroid epithelial cells has recently been shown to induce thyroid cancer in adult zebrafish. Combinatorial treatment with BRAF and MEK inhibitors rescue normal follicular architecture, restore thyroid hormone production, and reduce epithelial mesenchymal transition stimulated by BRAFV600E. The model has demonstrated in vivo the genetic requirement for Twist expression downstream of BRAFV600E, as ablation of twist3 by CRISPR-Cas9 suppressed BRAF-mediated oncogenesis.49 The in vivo validation of targeted molecular therapies for the treatment of thyroid cancer demonstrates the applicability of the zebrafish system to precision oncology approaches.Stable transgenic expression of oncogenic BRAF , including extensive contributions by Dr. Gong’s group.55 As a recent example, they developed a transgenic system for a liver-specific, mifepristone-inducible expression of oncogenic krasV12 via permanent genomic recombination mediated by the Cre-loxP system,55 which will facilitate the study of liver tumors that originate from a single cell or a small number of precursor cells through clonal expansion. In all, 20–40% of HCC are defined by an activating mutation in the gene encoding β-catenin. Evason and colleagues created a transgenic zebrafish expressing hepatocyte-specific activated β-catenin. They used the model to screen for druggable pathways that mediate β-catenin-induced liver growth and identified two c-Jun N-terminal kinase (JNK) inhibitors and two anti-depressants as potential targeted therapeutics.56 As is true in other tumor types discussed in this section, zebrafish have contributed to drug development to treat liver cancer.Liver cancer is the second leading cause of cancer-related death. Hepatocellular carcinoma (HCC) accounts for 90–95% of liver cancer cases.57 xenotransplantation of human cells into zebrafish represent a young frontier in zebrafish cancer modeling. However, the field has evolved rapidly, and xenograft zebrafish models utilizing various injection sites, developmental stages, and transplanted specimens  have been developed59  in injection sites such as blastodisc, yolk sac, bloodstream, perivitelline space, and orthotopic sites, including the hindbrain ventricle and vitreous cavity.62 The transplanted cells can be studied for up to 21 days post fertilization (dpf), at which point the zebrafish has developed a fully functional innate and adaptive system.59 While embryonic xenotransplantation offers numerous advantages, a limitation is that many of the tumor types being modeled occur predominantly in adults.With the first experiment reported in 2005,59 Table . Engraft63 The study of cancer cell transplantation in adult fish requires immune suppression by irradiation or dexamethasone pre-conditioning, and the use of transparent transgenic zebrafish allows the rapid identification of the transplanted cells.59 Casper fish, a cross between the nacre and roy mutant lines, lacking all types of pigments, are commonly used for this purpose.64 More recently, Tang et al.65 developed an optically clear immunocompromised transgenic mutant zebrafish line for optimized cell transplantation and direct visualization of fluorescently labeled cancer cells in the adult fish. Although juvenile and adult zebrafish transplantation models more closely match the developmental state and age of humans afflicted with cancer, the requirement for immune-deficient zebrafish represents the downside.Xenotransplantation in juvenile and adult zebrafish seeks to overcome the limitation of translating embryonic zebrafish models to mature human cancer patients. Transplantation of human cancer cells in 30 dpf zebrafish has been established by Stoletov and colleagues in 2007 by injecting cells into the peritoneal cavity and treating the fish with dexamethasone to prevent rejection.66 Other xenograft zebrafish models have been used to explore relevant pathways in melanoma, such as Nodal in cellular plasticity and tumorigenicity,67 and TGF-β in cellular resistance to MEK inhibitors.68 Recently, a drug treatment system for the long-term administration of anti-melanoma drugs in adult casper zebrafish transplanted with a zebrafish melanoma cell line has been developed. The adult environment may be relevant to test and identify new promising treatments.69Both embryonic and adult zebrafish have been used as transplantation models to study drug efficacy in melanoma. van der Ent and colleagues injected different human uveal melanoma cell lines generated from primary tumors and metastases into the yolk of 48 hpf zebrafish embryos. They have shown that targeted inhibition of known pathways by specific drugs proves effective in counteracting cancer cells migration and proliferation, thus demonstrating the applicability of the zebrafish xenograft model for drug screening and discovery.70). However, several studies have transplanted cells from mouse models of pediatric brain tumors and vestibular Schwannoma.72 A glioblastoma xenograft model has been proposed as a system for high-throughput screening of anti-angiogenic compounds. Transplanted cancer cells have been shown to be capable of inducing angiogenesis, which was enhanced by TGF-β1 and inhibited by targeted drugs, such as JNK, ERK, and PI3K inhibitors.73 Another zebrafish glioma xenograft model has been used to evaluate the in vivo efficacy of a novel small molecule radiation sensitizer identified through an in vitro drug screening in human glioma cells.74 The biological behavior of glioma cancer stem cells (GSCs) has also been explored in a zebrafish xenograft model. GSCs induced angiogenesis, which was inhibited by several anti-angiogenic agents. Moreover, the model revealed the in vivo activity of a synthetic compound, Nordy, previously found to promote GSCs differentiation in vitro. Nordy suppressed angiogenesis, tumor invasion, and proliferation of the zebrafish GSC xenograft.76 Taken together, zebrafish glioblastoma and glioma xenograft models have proven to be valuable systems for testing various molecularly targeted therapies.A variety of zebrafish neurological tumor xenograft models have been established, primarily through xenotransplantation of different human malignant glioma cells  or a tyrosine kinase inhibitor.71 When feasible, orthotopic xenograft models in zebrafish brain tissue offer the advantage of more faithfully replicating human disease by utilizing the same anatomical tumor microenvironment.Many brain tumor xenograft models have been obtained by orthotopically implanting cancer cells in embryonic or even larvae and juvenile brain.77 These results suggest this model could be used to identify new therapies for pancreatic cancer.Xenotransplantation of pancreatic cancer cells in zebrafish has also been proposed for the screening of new anti-cancer compounds. Guo and colleagues established a pancreatic adenocarcinoma xenograft model in zebrafish embryos and adults and found that a known small molecule inhibitor, U0126, targeting the KRAS signaling pathway, represses proliferation and migration of the transplanted cancer cells in zebrafish larvae.62 Injection of retinoblastoma cells into the vitreous cavity of the zebrafish embryo has permitted quantitative analysis of the tumor cells’ proliferative potential and the anti-cancer effect of systemically administered drugs. This model offers a potential screening platform for retinoblastoma anti-cancer drugs.62Two studies have shown an orthotopic transplantation zebrafish model may represent a powerful tool for the development of specific drugs for the treatment of retinoblastoma, the most common intraocular childhood cancer, which often invades the brain and metastasizes.8 the rapidly increasing number of zebrafish xenograft cancer models suggests that zebrafish xenografts are on the road to a clinical application in precision oncology.Xenotransplantation of human cancer cells directly derived from individual patients  represents a fascinating and forthcoming opportunity for the development of zebrafish-based patient-specific clinical approaches for cancer treatment. Such patient-derived xenografts in zebrafish offer a platform for real-time in vivo evaluation of patient prognosis and drug responses, aimed at identifying the most appropriate individualized therapy Fig. , right. 9Welker and colleagues standardized a patient-derived orthotopic zebrafish xenograft model of glioblastoma. They transplanted two patient-derived glioblastoma cell lines, serum-grown adherent and neurospheres, into the midbrain region of embryonic zebrafish. In vivo tumor growth and cancer cell proliferation, migration, and differentiation were described, with different characteristics in adherent and neurosphere glioblastoma cell lines. Furthermore, currently used glioblastoma therapeutics decreased xenotransplant tumor burden and significantly rescued survival. These results provide proof of principle for the use of the model as a platform for drug screening.5 The authors previously tested the in vitro sensitivity of three T-ALL cell lines, with specific mutations in PTEN and NOTCH1 genes, to three different inhibitors  and demonstrated that the same cell lines were sensitive to the same drugs upon xenotransplantation in the zebrafish embryos. The relevance of the zebrafish xenotransplantation model as a preclinical platform for a personalized therapy was demonstrated by xenotransplanting two primary patient-derived bone marrow samples into zebrafish embryos and treating with the three inhibitors. One patient sample responded drastically to NOTCH1 inhibitor  within 1 week following biopsy, highlights how the zebrafish xenotransplantation response can direct personalized therapy in real-time.5A preclinical human cancer xenotransplantation platform has been recently developed in zebrafish to inform therapeutic decisions in T-ALL patients Fig. .5 The autor Fig. , suggest78 Metastases are associated with poor prognosis due to the difficulty of treating such a complex and diffuse process. Moreover, unlike most other cancer processes, such as tumor initiation, proliferation, apoptosis, invasion etc., metastasis cannot be well modeled in vitro and the development and utilization of in vivo models of the dynamic sequence of steps from the local invasion to the distant colonization remains challenging. The transparency and ease of genetic manipulation of zebrafish embryos, coupled with the emerging opportunities offered by the xenograft models, represents an exceptional frontier to model and visualize the entire process of metastasis at single-cell resolution.Most cancer deaths are caused by metastasis, as opposed to primary tumors. Metastases result from the spread of cancer from the primary site to distant organs where new tumors form. Metastatic cancers have acquired the capacity to escape the primary malignant lesion site through intravasation into the bloodstream, migration, extravasation, and colonization of a distant site.80 Zebrafish xenograft models of human cancer cell invasion, metastasis, and responsiveness to pharmacological or genetic intervention have been correlated to tumorigenicity of analogous human tumor cells in mouse xenograft models.81 Furthermore, Tulotta et al.82 demonstrated cross communication between zebrafish and human ligands and receptors, which enables the study of the interactions between human cancer cells and host microenvironment during the metastatic processes.The xenotransplantion procedure is well optimized and automated quantitative assays are available to study invasion and metastasis of cancer cells.83 used real-time intravital imaging to study the dynamic process of intravascular locomotion and extravasation of fluorescent human cancer cells injected into the pericardium of 48 hpf zebrafish embryos, thus providing new insights into the underlying molecular regulation, which involves β1 integrin, Twist and VEGFA. Au et al.10 elegantly showed the migration dynamics of clusters of circulating tumor cells isolated from the blood of breast cancer and melanoma patients. The zebrafish xenograft metastasis model represents a valuable model to test the metastatic potential of human precision oncology target genes. We recently adapted Stoletov’s model to demonstrate neuropilin-2 promotes extravasation and metastasis of human pancreatic cancer and renal cell carcinoma cells in zebrafish , promotes extravasation of A549 human lung cancer cells.85 In conjunction with studies of tumor transcriptomes, survival data, and prevalence of lymph node metastases in human lung cancer patients, we demonstrated that NME2 decreases metastatic potential through transcriptional repression of focal adhesion factor vinculin.85 In a study highlighting the mechanism by which T-lymphoblastic lymphoma progresses to the metastatic T-ALL, Feng et al.86 focused on the intravasation process as one of the first steps in metastasis, identifying the role of Sphingosine-1-phosphate receptor 1 (S1P1) and intracellular adhesion molecule 1 (ICAM1) in counteracting cancer cells from entering the vasculature. In a xenograft model of PDAC metastasis, the calcium binding protein S100P was shown to facilitate cancer cell intravasation and extravasation.87 Thus, various stages of cancer metastasis have been accurately modeled in zebrafish transplantation settings.Outstanding examples of zebrafish metastasis models have recently emerged Table . Stoleto88 A zebrafish xenograft model was also used to demonstrate the anti-metastatic activity of the small molecule VPC-18005, targeting the DNA-binding ETS domain of ERG, against prostate cancer cells aberrantly expressing ERG.89 Liekens et al.90 proposed the FDA-approved anti-DNA virus agent cidofovir as an anti-metastatic agent based on its ability to inhibit metastasis of FGF2-driven tumor cells in zebrafish embryos and mice. Amplification of ADP-ribosylation factor 1 (ARF1) is associated with poor outcomes of patients with breast cancer. A zebrafish xenograft model demonstrated that specific inhibition of Arf1 by small molecule LM11 impairs metastatic capability of breast cancer cells. LM11 may, therefore, represent a potential precision oncology treatment for patients with ARF1 amplification.91 Similarly, a zebrafish xenograft model was used to show the anti-metastatic effect of novel compounds designed to antagonize P2 × 7 receptor, previously reported as a key mediator in cancer metastasis. Significant inhibition of the invasion of MDA-MB-231 triple-negative breast cancer cells has been reported, thus identifying new potential drugs for individualized therapy.92 SIRT1 and CXCR4 were identified as novel pro-metastatic players, respectively, in Ewing sarcoma and triple-negative breast cancer, and the anti-metastasis effect of pharmacologically antagonizing these targets was demonstrated in zebrafish.93 Continued development of these zebrafish metastasis models, as well as establishment of new zebrafish-based systems, will accelerate their implementation in precision oncology applications.Zebrafish metastasis models have emerged as a promising system and continued development is likely necessary for applications in personalized medicine. Nevertheless, these models have already demonstrated their usefulness. They facilitate the investigation of new molecules shown to regulate various aspects of the metastasis process and aid in the discovery of targeted drugs that will potentiate our capacity to counteract metastasis in specific pathological conditions. For instance, Ghotra and colleagues have identified SYK as a candidate kinase target for the treatment of advanced prostate cancer. SYK was found to be upregulated in human prostate cancer and associated with malignant progression. They then used a zebrafish xenograft model to show that pharmacologic inhibitors of SYK kinase, currently in phase I–II trials for other indications, prevent metastatic dissemination of prostate cancer cells.6 by transplanting direct explants from gastrointestinal human tumors into zebrafish embryos and larvae, into the yolk sac and organotopically in the liver, respectively. They showed the zebrafish model permits rapid analysis (micrometastasis formed within 24 h after transplantation) of primary human tumor specimen metastasis. Weiss and colleagues similarly transplanted pancreatic carcinoma cells and resected specimens of human pancreatic carcinoma into zebrafish embryos. The model was used to demonstrate the anti-metastatic in vivo activity of retinoid acid receptor antagonists, following the identification of the retinoid acid target miR-10A as a key mediator of metastasis in pancreatic cancer.8 The possibility of a near future application of the zebrafish model in precision oncology emerges from the study of Mercatali et al.7 They injected zebrafish embryos with a primary culture of bone metastasis derived from a 67 years old patient with breast cancer and compared its metastatic potential with that of established cancer cell lines. Importantly, primary cell behavior reflected the clinical course of the patient’s medical history, underscoring the noteworthy benefits that such an approach might signify for the evaluation of the patient prognosis and the identification of the most appropriate individualized therapy.Zebrafish are amenable to modeling the metastatic potential of cancer cells derived from patient specimens and transplanted in the embryo. Initial progress was demonstrated by Marques et al.94 Similarly, the CRISPR/Cas9 system has been shown to be a simple, quick, and scalable technique for in vivo editing of zebrafish genes.95 Thus, the genome editing toolbox offers strategies for recreating oncogenic mutations and perhaps even mimicking chromosomal rearrangements or fusions through knock-in technology. Genome editing in zebrafish can be achieved in a matter of months, which makes it possible to create a genome edited zebrafish that faithfully replicates key drivers of an individual cancer patient’s tumor, in sufficient time to use the zebrafish to identify the ideal individualized treatment strategy. A similar precision medicine approach using genome edited zebrafish has been proposed to treat cardiovascular disease.96 Taken together, genome editing in zebrafish offers an efficient strategy for modeling the key molecular characteristics driving tumor progression in a specific patient and screening drugs to identify the ideal treatment approach in real-time.The advent of genome editing presents another promising strategy by which zebrafish will likely be utilized to tailor cancer therapies to individual patients. Whole-genome sequencing is commonly used to determine the molecular and genetic signature of a specific patient’s tumor. Upon identification of actionable mutations and alterations, transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated systems (Cas) genome editing tools could be used to mimic key oncogenic mutations/alternations in a zebrafish avatar of an individual patient’s disease. Ekker and colleagues have used TALENs to achieve precise locus-specific DNA breaks in somatic and germline tissue of zebrafish as well as in vivo targeted knock-ins through homology directed repair.86 to overcome these concerns, but additional time and optimization is necessary for adult xenograft models to become as widely used and standardized as embryonic xenograft platforms.The majority of zebrafish xenotransplantation studies require the injection of cancer cells into embryos. While this methodology has advantages as described , shortcomings of this approach must also be considered. The modeled tumors typically initiate during in adulthood naturally, and the embryonic environment presents undeniable differences that may impact cancer biology. For instance, embryonic development relies on the activation of specific programs and signaling pathways that are not active in adult organs under physiological conditions, but can become aberrantly activated during pathological stress, such as tumor initiation, progression, and metastasis. Various studies have utilized xenografts in adult zebrafish59 This is also a drawback of human cancer cell xenotransplantation using adult zebrafish, which requires previous immunosuppression or the use of genetically immunocompromised zebrafish lines.86 A few attempts to develop immunocompetent xenograft systems have been reported. Cancers arising in syngeneic donors can be directly transplanted into a sibling recipient without the need of irradiation or dexamethasone pre-conditioning. However, only tumors made in clonal zebrafish lines can be transplanted into related recipients, and syngeneic fish are difficult to rear and not widely available.97 The impact of limitations that arise from using immunocompromised zebrafish models can be lessened by validating results in immunocompetent genetic cancer models established in adult zebrafish or other model organisms.Another limitation of modeling human cancer in zebrafish embryos is that such studies ignore the contribution of the immune system on cancer cell behavior, as embryos have not yet developed a functional immune system.97Long-term study of transplanted tumor cell behavior and drug response is particularly challenging. Embryo xenograft models are used in short-term assays, as long-term approaches compromise host viability. Xenotransplantation in adult fish after irradiation also prevents long-term studies because the immune system recovers within 20 days of irradiation. Dexamethasone-mediated chemical ablation of the immune system is a solution effective for solid tumor transplantation but not for leukemia. Nevertheless, mutant zebrafish lines harboring mutations that eliminate the immune response are emerging and represent a promising evolution toward the goal of long-term cancer biology and precision oncology studies in adult zebrafish.97A recent report described a novel protocol for the long-term orthotopic transplantation of zebrafish brain tumor tissue into immunocompetent recipients. The method is based on the injection of zebrafish brain tumor cells into the fourth ventricle of a 48 hpf embryo. This allows tumors to grow in immunocompetent animals over the life of the zebrafish, enabling long-term monitoring of tumor cell behavior and drug response, including re-transplantation of tumors over many generations for potential studies on tumor evolution or drug relapse mechanisms.Lastly, a minor but intrinsic limitation of zebrafish xenografts of human tumor cells is the contrasting temperatures of the host (human cancer cells) and receipt (zebrafish). Zebrafish are typically reared at 28–29 °C, while human cells thrive at 37 °C. The compromise usually adopted is to raise zebrafish embryos at 33–35 °C following xenotransplantation of human cancer cells. Importantly, a possible impact of such temperature difference on the physiology of the fish and the biology of the tumor cells cannot be ruled out.Given the value of zebrafish as a disease model, its power as a tool in precision oncology is becoming increasingly appreciated and applications are dawning. This advancement has been fueled by studies utilizing zebrafish cancer models to define molecular players of carcinogenesis, identify actionable alterations, and develop targeted therapies. Results to date provide a basis for individualized medicine research directions as well as platforms, workflows, and systems for applications in precision oncology Table .As the number of zebrafish cancer models being developed continues to grow with a greater focus on translation studies, we anticipate zebrafish models to increasingly contribute to personalized medicine. Indeed, studies have already presented zebrafish models of human cancer with specific genetic alterations and have demonstrated models’ abilities to test and identify targeted drugs that inhibit tumor growth and metastatic potential by acting on the specific molecules and pathways responsible for the tumorigenic phenotype. One can easily visualize this approach extending to wide potentialities for defining individualized treatments for oncology patients. Given a specific tumor, driven by different mutations/alterations in different patients, which, therefore, present different responses to cancer treatment, we might imagine a tumor-specific panel of zebrafish mutants/transgenics reproducing the array of the known oncogenic alterations found in patients harboring that tumor type. A future effort to consolidate and standardize the existing models, enriching the panel with models of other significant alterations found in patients, could lead to a complete in vivo platform used to identify the best available drug for each molecular signature. Concurrently, the panel could be used to screen drugs libraries for the identification of new active compounds. Ultimately, such a strategy will define a personalized treatment plan for each patient based on the molecular, genetic, and clinical characteristics of their cancer.5 suggest a precision oncology future in which primary specimens from patients diagnosed with cancer could be xenotransplanted in zebrafish embryos to test the responses of the patient cancer cells to various available drugs. The output of the test, obtainable in days, will dictate the most effective treatment for an individual cancer patient.A more extensive use of patient-derived xenografts in zebrafish will also represent in the near future a powerful complementary approach to develop individualized treatments and to define the most appropriate therapeutic strategy for specific alterations found in patients. First insights into the possibility of a direct, real-time application of zebrafish xenograft models in the clinicTaken together, the existing and developing array of zebrafish models within the collective toolbox of the zebrafish and precision oncology research community create a promising future where zebrafish may emerge alongside current clinical applications, such genomic technologies and molecular diagnostics, to improve our ability to precisely tailor individualized cancer therapies and positively impact the clinical outcome of each patient afflicted with cancer.Data sharing is not applicable to this article as no data sets were generated.


Macacine alphaherpesvirus 1; BV) occurs naturally in macaques of the genus Macaca, which includes rhesus and long-tailed (cynomolgus) monkeys that are widely used in biomedical research. BV is closely related to the human herpes simplex viruses (HSV), and BV infections in its natural macaque host are quite similar to HSV infections in humans. Zoonotic BV is extremely rare, having been diagnosed in only a handful of North American facilities with the last documented case occurring in 1998. However, BV is notorious for its neurovirulence since zoonotic infections are serious, usually involving the central nervous system, and are frequently fatal. Little is known about factors underlying the extreme neurovirulence of BV in humans. Here we review what is actually known about the molecular biology of BV and viral factors affecting its neurovirulence. Based on what is known about related herpesviruses, areas for future research that may elucidate mechanisms underlying the neurovirulence of this intriguing virus are also reviewed.Monkey B virus ( Macacine alphaherpesvirus 1; BV), an alphaherpesvirus enzootic in macaques of the genus Macaca. Though exceptionally rare, zoonotic BV infection following exposure to macaques has a mortality rate of ~80%. Here we review what is known about this relatively neglected virus with regard to its infamous neurovirulence.Herpesviruses are ubiquitous viruses, found in a wide variety of species including mammals, birds, and reptiles. Three subfamilies of the Herpesviridae family  are found in the order Primates. Of these, alphaherpesviruses typically infect and remain within the peripheral sensory nervous system for the life of their host as part of their natural life cycle. The close and prolonged association of these viruses with their host over its entire lifetime with only rare impairment of nervous system function implies an exquisite degree of host-virus coadaptation. On occasion stability of this commensal symbiotic host-virus relationship can be altered, resulting in severe or even fatal disease often involving the central nervous system (CNS). In cases where an alphaherpesvirus infects a host of another species, the result can be, but is not always, catastrophic. The most notorious example of this is monkey B virus  being used as biomedical research models for nearly a century [Herpesvirus simiae, monkey B virus, Herpes B, and Cercopithecine herpesvirus 1), BV is currently designated as Macacine alphaherpesvirus 1 by the International Committee on Taxonomy of Viruses.Monkey B virus (BV) occurs naturally in all 17 species of macaque monkeys that comprise the genus century . AlthougFrom a biological standpoint, in macaques BV is very much like herpes simplex virus (HSV) in humans. Both HSV and BV are normally transmitted horizontally via direct contact and exchange of bodily secretions , 5–7. ThAs with HSV in humans, most primary BV infections occur in mucosal epithelium and do not usually produce overt clinical signs, although lesions are sometimes visible on close inspection . As the As is typical of other alphaherpesviruses, BV can periodically reactivate from the latent state in response to various stressful stimuli, resulting in shedding of infectious virus. While lesions may be apparent, most recurrences do not produce clinically apparent lesions; rather, infectious virus is shed asymptomatically. Like HSV in humans, the frequency of BV shedding appears to be fairly low (2-3%) –37. StreIn many ways, BV is the macaque equivalent of human HSV: BV and its macaque hosts have coevolved, resulting in an exquisitely fine-tuned interaction with one another that results in perpetuation of the virus within the host's nervous system with minimal adverse effects on the host but also with occasional shedding of infectious virus that can be transmitted to a naïve host, thereby ensuring perpetuation of the virus. The notorious neurovirulence of BV is therefore not evident in its natural macaque hosts; the neurovirulence of BV only becomes apparent when BV infects other species, particularly humans.In 1932 a young physician, William Brebner, performing poliovirus research with rhesus macaques was bitten on the finger –42. He dWhile the exact number is not available, less than 60 additional cases of pathogenic zoonotic BV infection have occurred sporadically over the last 85 years, all resulting from exposure to laboratory or captive macaques or macaque tissues –50. WhilWhile the clinical course of zoonotic BV infections can vary, initial symptoms usually develop within 1–3 weeks of an exposure incident . The natThough most persons known to be infected with BV die, some do survive and some survivors can periodically shed virus after recovery , 52–54. A puzzling aspect of zoonotic BV infections is the notable lack of any fatal or even clinically evident BV infections in Asia where BV-positive macaques and humans have copious close interactions, and exposure to macaque bodily fluids through bites, scratches, and mucosal splashes is common , 57–59. In addition to the hundreds of monkey temples across Asia, tens of thousands of macaques are free-ranging in urban areas such as Singapore, Hong Kong, Delhi, and the famous wild monkey parks of Japan. Macaques are also commonly found as pets, and a centuries-old tradition of keeping and training performing monkeys continues in China and Japan. Finally, it should be noted that Indonesia, Thailand, Malaysia, Singapore, Vietnam, Cambodia, Laos, Philippines, China, and Japan all have active biomedical research programs and/or primate breeding research centers which collectively employ thousands of workers and involve tens of thousands of macaques each year. Many of these breeding facilities operate under conditions of extreme animal overcrowding with husbandry and handling protocols that are substandard . Despite frequent contact between humans and free-ranging, temple, pet, or urban macaques in Asia, fatal cases of BV have only occurred in the US and Canada following contact with captive macaques. This geographic restriction of zoonotic BV infections has long been and remains a puzzle. de novo response will occur to antigens specific to the second virus. Thus, detection of BV-specific antibodies is a difficult problem, as the BV-specific antibody response develops more slowly and may be overshadowed by the immediate and strong response to cross-reactive antigens. Consequently, persons infected with HSV who had experienced an asymptomatic BV infection would be expected to have higher levels of antibodies directed against antigens shared by all primate alphaherpesviruses than would be present in sera of persons only infected with HSV.One immediate question is whether or not BV is even zoonotically transmitted in Asia. There is no question that individuals in communities living near macaques or who work in SE Asian monkey forests have a history of extensive macaque contact and injury . If suchLimited testing compared the relative reactivity of sera from persons working in monkey forests with that of persons having no known contact with monkeys (negative controls) and patients that died of zoonotic BV infection (positive controls) . When seWhen assessing the neurovirulence of BV, it is important to recognize that, within Asia where hundreds of thousands of macaques come into daily contact with millions of people, there is no conclusive evidence of zoonotic infections, neurological or otherwise. Genetic differences in human subjects (Asian versus non-Asian background) would seem an unlikely explanation for the lack of fatal BV infections in Asia since many non-Asian tourists visiting monkey forests in Asia and non-Asian military troops serving in Asia have experienced bites and scratches from macaques without any resulting zoonotic BV infections , 61, 64.Perhaps a more likely explanation lies in the monkeys, that is, captive versus free-ranging. Do captive macaques shed BV more frequently as a result of some husbandry practices? When they are shedding do they shed more virus? Do free-ranging monkeys preferentially shed virus (or more virus) genitally rather than orally, while captive monkeys shed more orally? All these are questions that have been examined only superficially or not at all. It is particularly intriguing that amongst the dozens of primate breeding facilities that have operated in Asia for decades, some of which contain up to 10,000 macaques, there have been no reported cases of zoonotic BV. The macaques housed in these facilities are exported and are a source of animals used in biomedical research in North America. Other possibilities such as recombinant BV in captive monkeys that arose through past practices of cohousing of different species during capture and shipping have also been raised. The lack of BV isolates from free-ranging macaques for comparison to BV isolates from captive macaques and isolates recovered from zoonotic cases will be necessary to address these and many other aspects of BV neurovirulence.Rabbits have historically been used as an animal model for BV infections. Until recently, all testing of antivirals for anti-BV activity was conducted using rabbits –70. Rabb (M. fascicularis) were found to have similar LD50 values of approximately 104 PFU [ (M. nemestrina) and lion-tailed macaques (M. silenus) were not lethal (at 106 PFU) despite producing clinical signs of neurological involvement. This is interesting when one considers that most if not all cases of zoonotic BV have been associated with exposure to rhesus or long-tailed macaques rather than pigtail macaques .In this mouse model, all BV isolates from rhesus macaques and an isolate from a long-tailed macaque 104 PFU . In cont (Papio spp.) and SA8 of vervets (Cercopithecus aethiops), have been reported (with one probable exception [ (Colobus guereza) [As mentioned above, the neurovirulence of BV actually relates to its neurovirulence in nonnatural host species. In this regard, it is interesting that no zoonotic infections due to the two viruses most closely related to BV, HVP2 of baboonsxception ). Both bxception , 80 and guereza) . Given tguereza) , 76.From the time of its original isolation in 1932, extensive antigenic cross-reactivity between BV and HSV has been noted, indicating that these viruses are closely related , 82–85. BV has the typical virion structure of alphaherpesviruses, the genome being enclosed within an icosahedral capsid that is embedded in an amorphous protein tegument and surrounded by a lipid membrane envelope , 94. LikL and RS, resp.) of the genome that do not encode proteins, miRNAs, or small RNAs. Within these areas, there are islands of reiterated sequences. While the primary sequence of these reiterated repeat units and the number of iterations are not conserved among all BV isolates, the positions of these repeat islands are conserved suggesting they likely serve some unknown function.In 1971, an isolate of BV from a rhesus macaque was adapted to replicate in primary rabbit cells, and this strain E2490) now serves as the “standard” or “laboratory” BV strain , 96. The90 now seγ34.5) and ORF P genes in the simian viruses [The genome of BV (as well as HVP2 and SA8) is very similar to that of HSV2 and ChHV in its genetic organization, with homologs of almost every HSV gene being present in the same order and orientation in BV , 106. Th viruses , 106. ThBased on DNA sequence data, variation in predicted amino acid (AA) sequence identity of homologous BV and HSV2 proteins averages 62.5% . In cont50 of BV in mice [The highly conserved nature of most HSV, ChHV, and simian virus proteins argues for the homologous proteins of each virus having similar functions. Certainly this is true for structural capsid proteins  and viral enzymes. However, a number of BV proteins have regions of dissimilarity relative to other simian viruses and even among different genotypes of BV. Given the interaction of viral immediate early (IE) proteins with host cell proteins to facilitate expression of the viral DNA and initiate the lytic replication cycle, it might be expected that the IE proteins would be highly conserved. However, these regulatory IE proteins are actually some of the least conserved, both among BV genotypes and between BV and other primate viruses. By virtue of their expression on the surface of virions and infected cells, glycoproteins and other membrane associated proteins are another group of proteins that likely interact with elements of host cells. While some are strongly conserved (>90% AA sequence identity), others are very poorly conserved (<62% AA identity). Consistent with this, a number of glycoproteins have some degree of virus-specific antigenicity. The gB (UL27) and gD (US6) glycoproteins are major immunogens of BV, and while both are structurally conserved and have many cross-reactive epitopes, each also has some degree of antigenic BV-specificity , 117. Bo in mice .While BV encodes homologs of all the various HSV proteins mentioned above, little is known about these BV proteins regarding functional equivalency to their HSV counterparts. BV glycoproteins gC (UL44) and gD (US6) as well as VHS (UL41) have been shown to have similar structural and functional properties to their HSV homologs , 120–1226 PFU but does induce an adaptive immune response. In contrast, isolates of the second subtype (HVP2nv) are extremely neurovirulent in mice, having an LD50 of ~104 PFU (like BV). This dichotomous mouse-specific neurovirulence phenotype of HVP2 isolates allowed mapping of the neurovirulence locus. Recombinant HVP2ap/nv viruses were constructed and tested for neurovirulence in mice. Correlation of the neurovirulence phenotype with genome sequence analyses (defining which parts of the genome were derived from HVP2ap versus HVP2nv) identified a limited region of 3-4 genes that was associated with the neurovirulence phenotype. Based on this, the UL39 gene was the most likely candidate for the “neurovirulence gene,” and this was ultimately confirmed by construction and testing of UL39 ORF-specific recombinant viruses [Although not examined in BV, the UL39 gene is interesting with regard to host-specific neurovirulence. Despite the lack of any clinical differences in its natural baboon host, phylogenetic analyses and testing in mice place HVP2 isolates into two distinct subtypes , 80, 124 viruses . Thus, rThe UL39 gene encodes the large subunit of the ribonucleotide reductase protein (R1). UL39 has been associated with neurovirulence in HSV (the ability to replicate in nondividing nerve cells) and is one of several genes deleted from prototype HSV gene delivery vectors . While tThe HSV R1 protein is involved in evasion of cell death by preventing necroptosis in human but not mouse cells –131. In γ34.5) gene encoding the ICP34.5 (RL1) protein and the ORF P gene (on the opposite strand and overlapping the RL1 gene) have been shown to be primary determinants of neurovirulence in mice [In HSV both the RL1 , no differences were detectable between the parental wild-type virus and the E90ΔRL1 mutant. In primary mouse skin fibroblasts infected at a low multiplicity of infection , wild-type BV forms small plaques by 24 hrs  PI and subsequently spreads to adjacent cells involving the entire cell monolayer by 48 hrs PI . Thus, the RL1 region of BV does not appear to encode all the same functions ascribed to the γ34.5 protein of HSV and is not a major determinant of BV neurovirulence.The E90ΔRL1 mutant was also tested in mice using the skin scarification model to assess what effect this region has on neurovirulence. As mentioned above, HSV mutants lacking the RL1 gene are not neurovirulent in mice and fail to spread within the nervous system. In contrast, the neurovirulence of the E90ΔRL1 mutant was actually slightly increased relative to that of the parental wild-type virus, the mutant having an LDOne possibility that has been raised to explain the restriction of zoonotic BV cases to North America is that some BV isolates circulating in these captive macaques are potentially recombinant viruses, the recombinant viruses having arisen due to past practices of cocaging monkeys of different species during importation. Sequencing of multiple BV isolates from different US rhesus breeding colonies failed to detect any such recombinants . HoweverIn assessing the host-specific nature of BV neurovirulence, differences that occur in how the virus behaves in the natural versus nonnatural host obviously need to be examined. There are really two critical points where differences in host-virus interactions in humans and macaques would most probably affect neurovirulence/clinical disease. The first is the ability of BV to invade the nervous system. In the normal course of pathogenesis, viral replication in epithelial tissue at the site of inoculation is needed to produce sufficient levels of infectious progeny virus to allow invasion of sensory neurons innervating the site  –151. SecReplication at the Site of Infection. The occurrence of herpetic lesions at injury sites in zoonotic BV patients suggests that BV can replicate effectively in human epithelial tissue. However, the rarity of zoonotic BV infections relative to the number of annual exposure incidents that occur and the apparent lack or rarity of asymptomatic zoonotic infections could indicate that zoonotic transmission of BV does not always lead to an active or clinically apparent infection in many or even most cases. This could be due to any number of things including (1) monkeys not shedding virus at the time of contact; (2) too low levels of virus transmitted to overcome preexisting (cross-reactive) immunity to HSV; or (3) innate host resistance resulting in inefficient or abortive replication of BV in human epithelial cells relative to its ability to replicate in macaque cells. While it has been shown that HSV replication in rhesus cells can be very inefficient or abortive [abortive –154, sucRecent studies have begun to investigate the comparative replication of BV in macaque versus human cells. It has been shown that while the HSV gD glycoprotein binds host cell proteins HVEM, nectin 1 and nectin 2 to facilitate viral entry, the BV gD glycoprotein only binds nectin 1 and nectin 2 in both human and macaque cells , 155. WhThe HSV IE protein ICP47 (US12) mediates downregulation of MHC expression on the surface of infected cells, thus likely evading activation of host antiviral T cells and altering susceptibility of infected cells to killing by natural killer (NK) cells . The BV Replication, Latency, and Reactivation in Neurons. Virtually nothing is known regarding details of the establishment of BV latency or reactivation from latency. However, based on what is known about HSV, some predictions can be made about what might occur differently in these aspects of BV in its natural versus nonnatural host. When HSV infects a neuron, the viral envelope fuses with the cell plasma membrane, releasing the nucleocapsid and tegument proteins into the cytoplasm. The nucleocapsid  is transported to the nucleus where the viral DNA is immediately coated with histones and cellular repressor proteins to prevent viral IE gene expression and entry into the lytic replication cycle, resulting in latency [ latency , 160. To latency . At the  latency , 161. ThAs mentioned previously, there is evidence supporting the ability of BV to establish latent infections in humans and to reactivate at later times just as occurs in macaques. Even so, slight alterations in the most basic molecular aspects of these processes could have a radical effect on the ultimate outcome of BV infections in humans. It may be that in human neurons BV usually overcomes the innate preemptive response of the neuron to effectively repress viral IE gene expression and establish latency. This would result in lytic replication of BV in human neurons with production of progeny virus and subsequent spread of the virus to other neurons and/or accessory cells. Given the apparently central role played by the multifunctional ICP0 protein in the establishment of latency , 161, vaWhen reactivated from latency, progeny virions (or virion components) are transported anterograde down the axon where the virus exits the neuron and undergoes lytic replication in epithelial tissue , 162. WhSince reactivation from latency must involve replication of the virus in the neuron, it has been hypothesized that replication and production of progeny virus in neurons is much less efficient than in epithelial cells, thereby sparing widespread destruction of host neurons . This coA similar possibility is that while BV may become latent in humans, it is reactivated much more readily in humans than in macaques (days as opposed to months or years). This would be consistent with the variable time  between exposure incidents and the first appearance of clinical symptoms in zoonotic BV patients , 46, 51.Another possibility is that in humans BV is more readily transported down dendrites than in macaques, resulting in more efficient spread of the virus within the human nervous system. Interestingly, in pseudorabies virus , there does appear to be some virus-specificity in attachment of virions to dynein motor proteins  versus kinesin motor proteins  . BindingBV is ubiquitous in populations of captive and free-ranging macaques and despite many exposure incidents every year; zoonotic infections are extremely rare and have only been documented in North America. Notwithstanding, BV is notorious for its extreme neurovirulence in the handful of humans who have been infected. Biological aspects of BV infection in its natural macaque host are very similar to that of HSV in humans, including primary replication in epithelial tissue, invasion of sensory neurons, establishment of latency in sensory ganglia, and periodic reactivation from latency in response to stress allowing transmission of the virus to a new host. Phylogenetic analysis of the primate alphaherpesviruses suggests that these viruses have likely coevolved with their hosts, not surprising given the exquisite details that must be involved in virus-host interactions to maintain the virus within the nervous system for the lifespan of its host without serious adverse consequences while still allowing transmission and perpetuation of the virus within the host species. Given that nonhuman primates are our closest phylogenetic relatives, it is not surprising that BV should be very much like HSV with regard to the virus-host interactions and mechanisms involved in maintaining the virus-host relationship in a balanced state. The orthologous nature of the BV and HSV genomes and similarity of encoded proteins support this. Based on comparative sequence analyses, it also appears that BV encodes LATs, miRNAs, and small RNAs that would be involved in the intricate regulation of viral latency in neurons as in HSV.γ34.5) and ORF P genes in BV. Despite lacking these genes, this region of the simian virus genome remains nearly the same size as in HSV. Although both the RL1 and ORF P genes are important determinants of HSV neurovirulence in mice, deletion of this region of the BV genome has little effect on its neurovirulence in mice.Due to the hazardous and restrictive nature of performing research with infectious BV, very little experimental or molecular research has been done on this intriguing virus. To date the only genetic characteristic that differs dramatically between BV and HSV is the lack of homologs of the RL1 ( in vitro in human versus macaque cells have been identified that might account for the divergent neurovirulence of BV in these two species. Given the delicate balance that exists in neurons between repression of gene expression to establish latency and lytic replication, even slight differences in any of the multitude of host-virus interactions affecting this balance could ultimately alter the pathogenesis of BV in a nonnatural host species, resulting in very different outcomes of infection in macaques versus humans. Considerably more research into molecular aspects of host-virus interactions in macaque versus human cells, both epithelial and neural, needs to be pursued to better understand the extreme neurovirulence exhibited by BV in humans.BV has attained its reputation as having extreme neurovirulence due to the high mortality associated with the approximately 60 zoonotic cases that have occurred since its identification 85 years ago. However, for comparison HSV causes ~500 cases of encephalitis each year in the US, and without treatment is ~70% fatal with only ~5% of patients fully recovering , charact


For over four decades the thoracic aortic ring model has become one of the most widely used methods to study vascular reactivity and electromechanical coupling. A question that is rarely asked, however, is what function does a drug-mediated relaxation (or contraction) in this model serve in the intact system? The physiological significance of adenosine relaxation in rings isolated from large elastic conduit arteries from a wide range of species remains largely unknown. We propose that adenosine relaxation increases aortic compliance in acute stress states and facilitates ventricular-arterial (VA) coupling, and thereby links compliance and coronary artery perfusion to myocardial energy metabolism. In 1963 Berne argued that adenosine acts as a local negative feedback regulator between oxygen supply and demand in the heart during hypoxic/ischemic stress. The adenosine VA coupling hypothesis extends and enhances Berne's “adenosine hypothesis” from a local regulatory scheme in the heart to include conduit arterial function. In multicellular organisms, evolution may have selected adenosine, nitric oxide, and other vascular mediators, to modulate VA coupling for optimal transfer of oxygen (and nutrients) from the lung, heart, large conduit arteries, arterioles and capillaries to respiring mitochondria. Finally, a discussion of the potential clinical significance of adenosine modulation of VA coupling is extended to vascular aging and disease, including hypertension, diabetes, obesity, coronary artery disease and heart failure. From these studies emerged the idea that a labile substance was released from the heart  when the oxygen supply became inadequate for the oxygen needs of the heart, and the labile substance dilated the coronary-resistance vessels, thereby increasing coronary blood flow and restoring the balance between oxygen need and supply.Robert M. Berne (originator of the “Adenosine Hypothesis”). Quoted from Berne, , p141, A2A, A2B, and A3 , adenosine is formed in the myocyte from the breakdown of adenosine 5′ monophosphate (AMP), diffuses across the interstitial space to the coronary artery and causes vasodilation  , increase cytosolic Ca2+, activate endothelial NO synthase and thereby produce NO. At the same time, increased cytosolic Ca2+ may activate small- and intermediate-conductance activated K+ channels to produce endothelium-dependent membrane hyperpolarization (EDH)  (Figure 3 receptor subtype than in a muscular tail artery, and a significantly lower abundance of A1 and A2A receptors in the conduit (Leal et al., Perhaps the most compelling set of human data supporting a role for adenosine in VA coupling comes from Wong and colleagues who reported a relationship between increased aortic stiffness, lower VA coupling and lower coronary blood flow in patients with stable angina undergoing an adenosine stress test (Wong et al., Finally, since the heart and elastic and geometric properties of conduit arteries are important predictors of aging and disease progression, the adenosine VA hypothesis may offer a new conceptual scheme for future research and therapeutic intervention. Questions include whether adenosine levels change in large conduit arteries and along their length in healthy vs. older patients with hypertension, diabetes mellitus, obesity, coronary artery disease and/or heart failure (AlGhatrif and Lakatta, All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This article offers a detailed review of the current approaches to anticancertherapy that target the death receptors of malignant cells. Here, we provide acomprehensive overview of the structure and function of death receptors andtheir ligands, describe the current and latest trends in the development ofdeath receptor agonists, and perform their comparative analysis. In addition,we discuss the DR4 and DR5 agonistic antibodies that are being evaluated atvarious stages of clinical trials. Finally, we conclude by stating that deathreceptor agonists may be improved through increasing their stability,solubility, and elimination half-life, as well as by overcoming the resistanceof tumor cells. What’s more, effective application of these antibodiesrequires a more detailed study of their use in combination with otheranticancer agents. The major current approaches to cancer therapy are based on a combination ofchemotherapy and surgery. But, because of the lack of cancer specificity, theyare often associated with a variety of severe side-effects and complications.For this reason, the design of highly specific drugs, such as monoclonalantibodies, for a targeted inhibition of cancer cells appears to be a verypromising direction . A betteA fundamentally different approach to anticancer therapy is the search fortumor necrosis factor receptor superfamily (TNFRSF) agonists. So-called deathreceptors containing a death domain comprise a separate group of thesuperfamily. These include the tumor necrosis factor receptor 1 (TNFR1), tumornecrosis factor receptor 6 , death receptor 4 (DR4), deathreceptor 5 (DR5), etc. Receptors DR4 and DR5 are the most promising candidatesfor targeted therapy of tumor diseases, because their expression levels aresignificantly higher in cancer cells than in normal ones.By using phage display, a group of researchers selected a YCKVILTHRCY peptidethat was able to bind specifically to DR5. Tyr residues were added to the endsof the peptide to increase its solubility. The peptide properties wereinvestigated both in the monomeric and dimeric forms. Both forms were demonstrated to interact with DR5 and induce apoptosisin tumor cells of the Colo205 line. The effectiveness of the monomer may beassociated with the fact that the peptide contains numerous hydrophobicresidues and, at high concentrations, may aggregate in an aqueous medium [68].Another research group also used phage display to select a GRVCLTLCSRLT peptidewith high affinity for DR5 (ICCurrently, there exist many approaches for affecting tumor cells, in particularthrough apoptotic pathways. Unfortunately, many of these approaches remaininappropriate due to cell resistance, as well as the inefficiency andinstability of therapeutic agents. Other agents offer new opportunities for thetreatment of tumor diseases. A more detailed investigation of the complexmechanism involving death receptor signaling pathways will boost thedevelopment of new agents that could be capable of overcoming the resistanceand selectively affect cancer cells. On the other hand, effective use ofexisting death receptor antibodies requires a more detailed investigation oftheir application in combination therapy.