When the first- and second-line therapeutics used to treat neuropathic pain (NP) fail to induce efficient analgesia—which is estimated to relate to more than half of the patients—opioid drugs are prescribed. Still, the pathological changes following the nerve tissue injury, i.a. pronociceptive neuropeptide systems activation, oppose the analgesic effects of opiates, enforcing the use of relatively high therapeutic doses in order to obtain satisfying pain relief. In parallel, the repeated use of opioid agonists is associated with burdensome adverse effects due to compensatory mechanisms that arise thereafter. Rational design of hybrid drugs, in which opioid ligands are combined with other pharmacophores that block the antiopioid action of pronociceptive systems, delivers the opportunity to ameliorate the NP-oriented opioid treatment via addressing neuropathological mechanisms shared both by NP and repeated exposition to opioids. Therewith, the new dually acting drugs, tailored for the specificity of NP, can gain in efficacy under nerve injury conditions and have an improved safety profile as compared to selective opioid agonists. The current review presents the latest ideas on opioid-comprising hybrid drugs designed to treat painful neuropathy, with focus on their biological action, as well as limitations and challenges related to this therapeutic approach. When a noxious stimulus—let it be mechanical impact, excessive heat/cold, or exposure to harmful chemicals—is perceived, an unpleasant sensory and emotional experience arises. This universal sensation, known as pain, alerts the individual to potential or actual tissue damage and helps modify behavior to avoid the danger in the future; hence it bears vital evolutionary functions . As oppoIt is assessed that more than half of the neuropathic pain patients do not experience satisfying pharmacotherapy-induced pain relief even to a moderate extent ,7. First-line pharmacotherapy of NP includes gabapentoids, tricyclic antidepressants, and serotonin-norepinephrine reuptake inhibitors. When the response to these drugs is poor, lidocaine plasters, capsaicine patches and dually acting µ-opioid peptide (MOP) receptor agonists that concurrently inhibit the reuptake of serotonin and/or norepinephrine  are to be used as a second-line therapy. Combinations of first-line medications are also recommended at this stage of treatment ,9.Opioid drugs  are prescribed only as the third-line therapy, as they carry the risk of several negative adverse effects when administered in higher doses exacted by neuropathic pain severity. Importantly, physicians report that not all NP patients respond to exogenous opioids equally, and that some of the patients require high doses of synthetic opiates to achieve optimum analgesia, which further enhances the risk of the side effects occurrence . The mosStill, opioids can efficiently alleviate moderate to severe pain of neuropathic origin ,13, and The opioid system, with its three seven-transmembrane-spanning (7TM) G-protein coupled (Gαi–Gαo) µ-, δ- and κ-opioid peptide  receptors as well as their analgesia-inducing endogenous ligands , is intrinsically linked to pain processing in mammals, next to the profound role it plays in mood regulation, reward and addiction. Opioid receptors are present both in peripheral tissues and the central nervous system CNS, inhibiting pain transmission at different levels of the ascending and descending pain pathways ,17. The As mentioned earlier, the response to opioid treatment differs among NP patients . IndividN-methyl-D-aspartate (NMDA) receptors indol]-4-amine) is a small molecule activating nociceptin and opioid receptors dually, exhibiting a strong potency in rodent models of neuropathic pain [The promising results in basic studies led to the development of a new pharmacologic agent. Cebranopadol or GRT-6005 , belonging to the descending pain inhibitory pathway, rostral ventromedial medulla (RVM) modulating the spinal nociceptive transmission, and dorsal (sensory) root ganglia and spinal cord. Described at first as a gut hormone, it started to be associated with pain processing as soon as it was discovered in the CNS by Vanderhaeghen et al. . It was The antiopioid activity of the CCK receptor was further confirmed in studies utilizing targeted mutagenesis involving CCK receptor knock-out mice, and testing different specific antagonists of CCK receptors ,120. ImpNeuropeptide SP is one of the three products of Tac gene processing, next to neurokinin A and B. The three peptides constitute the tachykinin family and are broadly distributed in the organism, participating in vital physiological functions, such as immune, gastrointestinal and respiratory-related processes. There are three known subtypes of tachykinin receptors and SP can bind to all three of them, but it activates neurokinin-1 (NK1) receptor preferentially. 11-amino acid SP is synthesized in dorsal root ganglia and released from primary afferent fibers as a result of noxious input to transmit nociceptive signals to spinal and brainstem second-order neurons ,125 contThe selective antagonism on NK1 receptors clearly modulates hypersensitivity-related behavior in animals subjected to nerve injury, alleviating most of the observable symptoms of such hypersensitivity , but it Rewarding properties of opioids are a serious burden that can enhance the risk of misuse and opioid addiction. Importantly, NK1 receptor-containing neurons in the amygdala mediate acute opioid reward . BesidesAs opioid receptors are abundant in the gastrointestinal tract, some common and burdensome adverse events of opiate treatment result from the inhibitory effects that exogenous opioids exert on the gastrointestinal functions. Due to decreased gastric emptying that opiate therapy might cause, about 20–30% of opioid-treated patients experience nausea or vomiting. In this context, the antiemetic properties of NK1 receptor antagonists may come in useful . When it comes to the experimental evidence, analgesic doses of newly designed hybrids: opioid agonist-NK1 receptor antagonists are reported to actually produce less opioid-related adverse events, like constipation , sedatioNew ideas on the hybrid drugs designed for neuropathic pain treatment are continuously emerging. The following chapter presents some of the latest findings in the field, as well as current challenges that await the solution.The kinin system widely contributes to the sensory changes associated with neuropathic pain states; next to the already discussed NK1 receptor actions, kinin B1 (injury-induced) and B2 (constitutively expressed) receptors  play a vIt has already been mentioned that dynorphin, an inhibitory opioid neuropeptide derived from the precursor prodynorphin, is paradoxically able to promote pain and hypersensitivity via non-opioid mechanisms . It was Recently, Deekonda et al. proposed the design and synthesis of novel multifunctional ligands, where opioid enkephalin analogues were conjugated to Hoe 140, a ligand acting as a B2 receptor antagonist when tested in Guinea pig ileum. However, the group reported that the novel compounds show weak binding affinity for rat B2 receptors . Hence, Broadly distributed in the CNS, two main neurotensin (NTS1/2) receptors (GPCRs classified into two subtypes: high-affinity NTS1 and low-affinity NTS2 receptors) modulate nociception, pathological pain and stress-induced antinociception, exerting mostly inhibitory effects on neural transmission when activated . The rolThe important characteristics of NTS1 and NTS2 receptor in the context of hybrid drug design is that they mediate analgesic actions via opioid-independent mechanisms . As a reGlutamate is the most abundant excitatory neurotransmitter in the nervous system, physiologically released i.a. by sensory afferents in the spinal cord’s dorsal horn. Nerve injury is followed by the excessive release of glutamate from primary afferents and spinal interneurons, which leads to the nociception-promoting plastic changes and consecutive hyperexcitability of both lesioned and intact nerve fibers located in their close neighborhood. The glutamate-driven hypersensitivity manifests itself in evoked and spontaneous painful sensations typical for chronic neuropathic pain . To counA molecule that combines oxymorphamine (MOP agonist) and MPEP (mGlu5 receptor antagonist), MMG22, showing an exceptional analgesic potency in mouse inflammatory pain assay, was designed and synthesized by Akgün and colleagues . The groThe ultimate goal of pharmacological research is to translate a given new drug candidate into an effective therapy in the clinic. Although promising and increasingly successful thanks to the implementation of innovative in silico tools, the rational multitarget drug design approach is not devoid of tricky points. Selecting pharmacological targets that can bring desirable therapeutic outcomes when modulated simultaneously, as well as choosing high-quality basic (‘parent’) moieties to be incorporated into a hybrid molecule, is just the very first step of the process. Importantly here, all the biological characteristics of chosen targets should be carefully analyzed, to ensure that the antiopioid action of a selected system, meant to be counteracted by a novel hybrid, actually results from activating the respective pain-promoting receptors(and not from other mechanisms). For example, bradykinin receptors, targeted by dynorphins, are proposed as an adjuvant target of opioid-derived hybrid compounds, but there is evidence that pathological effects of dynorphins observed in vivo result from their ability to form pores in cell membranes . RelevanWhile designing a multifunctional drug, once the target combinations are validated, the compound must be optimized in terms of structure-activity relationships so that its desirable drug-like properties could be retained in the combined molecule. It must not be ruled out that given pharmacophores would not be characterized by the same affinity and efficacy to their target receptors once welded together, not excluding that their activity at the receptor site is eventually opposite to the starting pharmacodynamics profile . To start with, a careful in vitro analysis revealing the pharmacological characteristics of a newly synthesized hybrid particle, with the target receptor binding affinity and efficacy remaining the crucial parameters, is of great importance. The linker  type is also a factor that influences the pharmacological activity of a hybrid drug to a great extent, as it contributes to the stability of the molecule. Experimental studies show that the linker’s length and rigidity may greatly enhance the analgesic potency and longevity of the therapeutic effect provided by a given compound, likely by decreasing its susceptibility to enzymatic degradation . The nexThe translational aspect remains perhaps one of the greatest challenges related to the drug development process. Preclinical studies provide vast amount of detailed data on synergistic analgesic effects of distinct ligands, demonstrated in numerous animal models of pain and hypersensitivity. Nevertheless, the evergreen question is whether the promising results obtained in animal models of neuropathic pain would bring comparable benefits in human subjects. Some strong candidates to boost opioid effectiveness backed up by substantial basis of preclinical data did not provide similar satisfying results in patients. For example, CCK2 receptor antagonist L-365,260 failed to augment the analgesic effect of morphine when applied as a co-treatment in human subjects with chronic neuropathic pain . SimilarOpioid agonists in their prolonged action, although providing analgesia, steadily induce molecular and systemic changes that can exacerbate painful sensations in neuropathy and eventually outbalance the initial analgesic effects provided by opioid receptors activation. In parallel, opioid agonists in equivalent doses tend to be less effective in NP than in acute or inflammatory pain due to the specific antiopioid pain-promoting mechanisms that typically follow nerve tissue injury. The occurrence of these phenomena is inevitable unless the undesired effects, both resulting from the nerve tissue injury and the prolonged treatment with opioid analgesics, are pharmacologically silenced from the very beginning of the therapy. The continuously better understanding of the common features of neuropathic pain and opioid exposure creates a worthwhile opportunity to escape the additive antiopioid effects of the nerve injury and opiate treatment. As outlined in the present review, hybrid drugs approach creates some promising perspectives in this challenging attempt, as the multifunctional structures allow to enhance and prolong the opioid analgesia thanks to the simultaneous modulation of unwanted pronociceptive effects, exerted otherwise by a given neuropathy-activated system. What is more, some hybrid molecules might provide new beneficial quality as compared to the effects of their parental compounds; the prolonged analgesic action that grows up in time, observed in the case of a selected opioid agonist-MC4 receptor antagonist hybrid see  exemplifThe choice of the ancillary moiety to be incorporated into an opioid-derived hybrid compound remains a vitally important task, which once accomplished, while being backed up by well-grounded, high-quality experimental evidence delivers the possibility of designing an efficient and safe analgesic dedicated for neuropathic pain treatment.


The DNA damage response (DDR) is a complex signaling network that is activated upon genotoxic stress. It determines cellular fate by either activating cell cycle arrest or initiating apoptosis and thereby ensures genomic stability. The Apoptosis Antagonizing Transcription Factor (AATF/Che-1), an RNA polymerase II-interacting transcription factor and known downstream target of major DDR kinases, affects DDR signaling by inhibiting p53-mediated transcription of pro-apoptotic genes and promoting cell cycle arrest through various pathways instead. Specifically, AATF was shown to inhibit p53 expression at the transcriptional level and repress its pro-apoptotic activity by direct binding to p53 protein and transactivation of anti-apoptotic genes. Solid and hematological tumors of various organs exploit this function by overexpressing AATF. Both copy number gains and high expression levels of AATF were associated with worse prognosis or relapse of malignant tumors. Recently, a number of studies have enabled insights into the molecular mechanisms by which AATF affects both DDR and proliferation. AATF was found to directly localize to sites of DNA damage upon laser ablation and interact with DNA repair proteins. In addition, depletion of AATF resulted in increased DNA damage and decrease of both proliferative activity and genotoxic tolerance. Interestingly, considering the role of ribosomal stress in the regulation of p53, more recent work established AATF as ribosomal RNA binding protein and enabled insights into its role as an important factor for rRNA processing and ribosome biogenesis. This Mini Review summarizes recent findings on AATF and its important role in the DDR, malignancy, and ribosome biogenesis. Upon genotoxic stress, cells activate a complex network of signaling cascades in order to promote cell cycle arrest allowing for DNA repair or programmed cell death. This signaling network is known as the DNA damage response (DDR) and contains some of the most important molecular players that mediate genomic stability –5. One oEven though many details that dictate the choice between cell cycle arrest and apoptosis remain incompletely understood, a number of different factors influencing the role of p53 regarding these two opposing outcomes have been identified in recent years. One of these factors is the Apoptosis Antagonizing Transcription Factor (AATF)/Che-1, an RNA-polymerase II-interacting transcription factor . In 2006Indeed, AATF interacts with p53 on multiple levels. A study from our group  showed tKRAS-driven lung adenocarcinoma and its connection to p53 and the DDR. Mechanistically, Welcker et al. , and provide a possible explanation for the increase in DSB and thus genomic instability  with consecutive cell death . Out of 160 bona-fide interactors a large proportion  turned out to be RNA binding proteins themselves, with 15% being ribosomal proteins . The majThese data are in line with evidence recently published by Pineiro et al. , who ideWith nucleoli being the prime location for ribosome synthesis and therefore crucially contributing to cell metabolism and proliferation , the nucSuch nucleolar-specific reactions upon DNA damage or disrupted ribosome biogenesis are known as “nucleolar or ribosomal stress” , 48. IndIn summary, AATF joins a group of proteins that are essential for ribosome biogenesis, including pivotal steps of rRNA processing and nucleolar integrity, but which also fulfill numerous extra-nucleolar functions.AATF locus negatively correlated with overall survival in p53-proficient neuroblastoma patients  targeting specific binding motifs have been shown to effectively reduce protein-RNA interactions and, for example, subsequent processing of RNA . RegardiRecent work has shed much light on the function of AATF and characterized it as a key protein both in ribosome biogenesis and DDR . Whilst RK wrote the manuscript and designed the figures. JE reviewed the manuscript and designed the figures. R-UM, KH, and FF reviewed and edited both manuscript and figures and finalized the manuscript. All authors read and approved the final version of the manuscript.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.


Embryo implantation in the uterus is an essential process for successful pregnancy in mammals. In general, the endocrine system induces sufficient embryo receptivity in the endometrium, where adhesion-promoting molecules increase and adhesion-inhibitory molecules decrease. Although the precise mechanisms remain unknown, it is widely accepted that maternal–embryo communications, including embryonic signals, improve the receptive ability of the sex steroid hormone-primed endometrium. The embryo may utilize repulsive forces produced by an Eph–ephrin system for its timely attachment to and subsequent invasion through the endometrial epithelial layer. Importantly, the embryonic signals are considered to act on maternal immune cells to induce immune tolerance. They also elicit local inflammation that promotes endometrial differentiation and maternal tissue remodeling during embryo implantation and placentation. Additional clarification of the immune control mechanisms by embryonic signals, such as human chorionic gonadotropin, pre-implantation factor, zona pellucida degradation products, and laeverin, will aid in the further development of immunotherapy to minimize implantation failure in the future. In mammals, embryo implantation in the uterus is an essential process in successful pregnancy. In general, the endocrine system regulates endometrial differentiation such that the embryo can be implanted. The initial endometrial differentiation is induced by estrogen. Then, progesterone stimulates this estrogen-primed endometrium to differentiate further to make it more suitable for embryo implantation ,2. EstroIt is widely believed that such an implantation window, which spans from a few days after ovulation to several days prior to menstruation, also exists in women . The humConcomitant with hormonal preparation, direct cross-talk between the embryo and maternal endometrium is considered necessary to achieve subsequent successful embryo implantation ,14. The PTENP1), is involved in the endometrial adhesive properties [In addition to soluble factors, microRNAs secreted from human blastocysts were proposed to be new embryonic signals that regulate adhesive properties of endometrial epithelial cells. miR-661 from nonimplanted human blastocysts was taken up by primary human endometrial epithelial cells and it reduced the attachment of trophoblast cell line spheroids to these epithelial cells . Later, operties .For human embryo implantation, several adhesion-promoting molecules, such as trophinin ,28,29, LAs a regulatory cell surface protein of adhesion-promoting molecules, we previously reported that CD9, which regulates integrin function, is specifically expressed on the endometrial luminal and glandular epithelial cells . CD9 andOn the other hand, large glycoproteins, such as MUC1, that inhibit the physiological cell-to-cell interaction were expressed on murine and human luminal epithelial layers ,53. LateThe above findings suggest that the expression of adhesion-promoting molecules increases, whereas that of adhesion-inhibiting molecules decreases according to embryonic signals during an implantation window.A sufficient period is necessary for the cross-talk between embryo and mother in order to regulate embryo attachment to the endometrium with accurate timing and placement . For exaFrom hatching to attachment, the bovine embryo floats in the uterine cavity for 4–5 days and becomes elongated prior to attachment ,59,60. TTo investigate the mechanism to prevent premature embryo attachment, we hypothesized the involvement of repulsive forces. As repulsive force-inducing molecules, we observed that human maternal endometrial epithelial cells express ephrin A1 and human blastocysts express Eph A1, leading to the proposal that the Eph–ephrin system is involved in the initial interaction between human blastocysts (Eph A1) and the endometrium (ephrin A1) . Ephrin Based on this background, we examined the time-course of mRNA expression of Eph/ephrin A in murine embryos. All subtypes of ephrin A were expressed on blastocysts. Importantly, around the hatching period, ephrin A expression transiently decreased and then increased again before attachment. Immunostaining revealed that ephrin A1 and A3 were expressed on the cell surface of the trophectoderm of blastocysts. Moreover, ephrin A3 expression on the blastocysts was weak on the opposite side of the inner cell mass, suggesting that the Eph–ephrin system acts as a regulator of the apposition site .On the other hand, Eph A1, which is a receptor of ephrin A, was expressed on the murine endometrial luminal epithelial cells before embryo attachment. After attachment, EphA1 expression markedly decreased at the implantation site, whereas its expression on the luminal epithelial cells was maintained at nonimplanted sites . AlthougAfter attachment, the human embryo initiates intraepithelial invasion. Activation of the trophectoderm is considered to be an essential process for subsequent embryo invasion through intraepithelial spaces toward the endometrial stroma . The troAs other candidates, we reported that activated leukocyte cell adhesion molecule (ALCAM) is expressed on both human blastocysts and endometrial epithelial cells . ALCAM iAfter attachment, the activated embryo initiates intraepithelial invasion in the next step and invades the maternal endometrial stromal tissues as a mass, opening the epithelial cell layer. During this process, the intercellular connection of the endometrial epithelial layer becomes reduced, maintaining the connection between the embryo-attached epithelial cells and basement membrane.In general, the epithelial layer is tightly conjugated as a barrier. Notably, immune cells migrate through the epithelial layer from the basolateral to apical membrane ,80. AdheDuring human embryo invasion, the activated trophectoderm migrates to the intercellular spaces of epithelial cells with reduced tight junctions. Through this process of trophoblast invasion, the connection between epithelial cells and the basement membrane must be maintained. Proteases degrade the epithelial structures to weaken the tight junctions among epithelial cells. However, they also reduce attachment to the basement membrane, causing detachment of the epithelial cell layer that maintains embryo attachment. In contrast to the immune system, epithelial–mesenchymal transition (EMT) was reported to be involved in opening of the epithelial layer ,85,86,87After the embryo has firmly attached to the endometrium via several adhesion molecules, luminal epithelial cells receive continuous signaling from the attached embryo. The epidermal growth factor (EGF)-receptor system was proposed to induce the reduction of tight junctions of luminal epithelial cells by reducing the local calcium concentration at the trophoblast–endometrial interface . As otheIn the porcine uterus, the expression of Eph A1, A2, A4, and A7 was strongly detected in endometrial epithelial cells during early pregnancy. Ephrin A1 stimulated the proliferation of endometrial luminal epithelial cells via the activation of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling proteins, suggesting that ephrin A1 is involved in the interactions between porcine blastocysts and endometrial luminal epithelial cells by activating PI3K and MAPK signal transduction pathways .Notably, we observed that both EPH A1 and ephrin A4 signals promoted the invasion of a human choriocarcinoma-derived cell line, JEG-3 cells, without affecting cell proliferation . It was Based on these findings, we propose that the ephrin A signal induces intercellular dissociation and adhesion to the basement membrane in endometrial epithelial cells as embryo signals and suggest that this signal facilitates subsequent trophoblast invasion .It is widely accepted that cytokine networks function in the establishment of endometrial receptivity, suggesting that the immune system plays an important role in embryo–maternal cross-talk ,99,100. On the other hand, we found that the intravenous administration of splenocytes from early pregnancy induced successful implantation in pseudo-pregnant day 2 recipient mice by promoting endometrial differentiation, which is suitable for embryo implantation . In the The next question is how the maternal immune system recognizes the presence of a developing embryo before implantation and distinguishes the developing embryo from nonfertilized eggs and/or other organisms in the female genital tract . To achiImportantly, early pregnancy factor (EPF) is not species- or embryo-specific, whereas PIF is not species-specific to the maternal immune system. Consequently, as a new candidate for the embryonic signal before implantation, we paid attention to the zona pellucida (ZP) because it functions in the species- and oocyte-specific binding of spermatozoa . As deveAs described above, hCG, a species- and embryo-specific glycoprotein, was proposed to directly affect the endometrial function as an embryonic signal after hatching ,20,21,23In contrast to the above lectin–glycan interaction, hCG induces regulatory T-cells to migrate to the trophoblast through LH/hCG receptors that are expressed on regulatory T-cells . SubsequIn invasion assays using the murine embryo and BeWo cells, peripheral blood mononuclear cells (PBMCs) derived from women in early pregnancy promoted the murine trophectoderm and BeWo cell invasion more than those obtained from nonpregnant women. Importantly, when PBMCs from nonpregnant women were incubated with hCG, hCG-treated PBMCs promoted invasion more than nontreated PBMCs by soluble chemoattractive factors derived from PBMCs, suggesting that hCG alters PBMC functions to facilitate embryo implantation ,133. LatBased on these findings, we propose that hCG stimulates maternal immune cells at the implantation site through lectin–glycan interaction, which in turn, promotes embryo attachment and invasion based on cooperation between the endocrine and immune systems .During human placental formation, the cytotrophoblast differentiates into EVT in the anchoring villi and invades the endometrial stromal tissues as a single cell, reconstructing maternal spiral arteries. The reduction of arterial contractility caused by arterial reconstruction facilitates adequate maternal blood flow into the intervillous spaces . FailureAs a representative embryonic signal during human trophoblast invasion, hCG was initially reported to directly reduce trophoblast invasion by inhibiting the enzyme activity of urokinase-plasminogen activator . HoweverAs a novel embryonic signal candidate, we identified a new cell surface aminopeptidase, initially named ‘laeverin’, which belongs to the M1 peptidase family , and wasAt present, implantation failure is one of the most important clinical problems in IVF treatment. It should be noted that IVF therapy skips a large part of the maternal immune recognition process when the developing embryo is present in the female genital tract. Consequently, to achieve successful implantation, the precise assessment and accurate control of an active state of maternal immune tolerance mediated by regulatory T-cells is considered to be necessary . RecentlIn contrast to immunosuppressive therapy, we previously developed a novel therapy using autologous PBMCs. Briefly, PBMCs are isolated from patients and incubated with hCG, an embryonic signal, in order to sensitize them. The activated PBMCs are administered into the uterine cavity to induce adequate endometrial differentiation three days before blastocyst transfer. This treatment effectively improved the pregnancy and implantation rates in patients with four or more repeated IVF therapy failures . The intEmbryonic signals elicit two different effects: induction of immune tolerance and adequate local inflammation; the former protects against immune rejection, whereas the latter contributes to endometrial differentiation and maternal tissue remodeling during embryo implantation and placentation ,109,110 In conclusion, we propose the involvement of repulsive molecules, the Eph–ephrin system, in endometrial receptivity for embryo implantation, together with adhesion-promoting and adhesion-inhibiting molecules. This Eph–ephrin system may contribute to opening tight junctions among endometrial epithelial cells to induce embryo invasion toward endometrial stroma tissues, suggesting that Eph–ephrin molecules are new candidates for embryonic signals that induce suitable local inflammation for embryo invasion. Embryonic signals also elicit two different effects from the immune system: induction of immune tolerance that protects against immune rejection, and sufficient local inflammation that functions in endometrial differentiation and remodeling during embryo implantation and placentation. Further clarification of the precise roles and possible clinical usage of embryonic signal candidates, such as PIF, ZP degradation products, or laeverin, will help improve immunotherapy to minimize implantation failure in the future.


Thyroid cancer is one of the most common endocrine cancers, with an increasing trend in the last few decades. Although papillary thyroid cancer is the most frequent subtype compared with follicular or anaplastic thyroid cancer, it can dedifferentiate to a more aggressive phenotype, and the recurrence rate is high. The cells of follicular adenomas and follicular carcinomas appear identical in cytology, making the preoperative diagnosis difficult. On the other hand, anaplastic thyroid cancer poses a significant clinical challenge due to its aggressive nature with no effective therapeutic options. In the past several years, the roles of genetic alterations of thyroid tumors have been documented, with a remarkable correlation between genotype and phenotype, indicating that distinct molecular changes are associated with a multistep tumorigenic process. Besides mRNA expression profiles, small noncoding microRNA (miRNA) expression also showed critical functions for cell differentiation, proliferation, angiogenesis, and resistance to apoptosis and finally activating invasion and metastasis in cancer. Several high-throughput sequencing studies demonstrate that miRNA expression signatures contribute clinically relevant information including types of thyroid cancer, tumor grade, and prognosis. This review summarizes recent findings on miRNA signatures in thyroid cancer subtypes. Thyroid cancer is the most common type of endocrine malignancy, and incidence has increased in the last few decades –4. AlthoWHO Classification of Tumors of Endocrine Organs, FTC is defined as a malignant epithelial tumor showing follicular cell differentiation and lacking the diagnostic nuclear features of PTC. Because of the difficulty of definitively diagnosing FTC, there has been an overdiagnosis of the follicular variant of papillary thyroid cancer [Besides PTC, follicular thyroid cancer (FTC) is the other differentiated thyroid cancer , 12. Thed cancer . Since Fd cancer . Accordid cancer , 15; theAnaplastic thyroid cancer (ATC) is another type of thyroid cancer with extremely poor prognosis –18. ATC Lastly, medullary thyroid cancer (MTC) occurs in two forms: hereditary (HMTC) and sporadic (SMTC). Out of all, 75% of MTCs are sporadic and typically present at a late stage with nodal metastasis present at the time of diagnosis and a relatively poor prognosis.In the past several years, knowledge of the genetic alterations of thyroid tumors has greatly increased. There is a remarkable correlation between genotype and phenotype, indicating that distinct molecular changes are associated with stages in a multistep tumorigenic process. Alterations of genes or gene pathways are linked to specific histologic features. This genotype/phenotype correlation provides the rationale for the numerous studies on gene expression profiling. The analysis of mRNA expression features may represent a useful tool for the preoperative diagnosis of thyroid nodules .The translation from basic science to the potential diagnostic application also extends to recent studies on microRNAs (miRNAs) , 22. TheWhen miRNAs were discovered over 30 years ago, it was believed that they were conventional protein-coding genes. Yet after experimentation, Hammond made the remarkable discovery that instead of coding for protein, they represented a new class of long regulatory noncoding RNA which was later called miRNA . miRNAs Differential miRNA expression profiles have been reported in tumors and normal tissues; such a phenomenon suggests the key role of miRNA in tumorigenesis. A single miRNA can have multiple targets with varying binding strengths. In addition to the amount and availability of the binding site, the binding strength determines whether miRNAs inhibit gene production by inhibiting translation or stimulating degradation of mRNA. Weak binding strength tends to inhibit the gene expression by targeting translation. mRNA instability and degradation most likely result when the corresponding miRNA sequence is completely complimentary, leading to tight binding and activation of the RNA-mediated interference (RNAi) pathway .There have been many studies that have identified the links between changes in miRNA expression from normal to cancer cells and tumor growth. With these findings and various tumors, miRNAs are currently being tested as potential diagnostic or prognostic biomarkers –41. The Numerous studies are going on to use miRNAs as potential biomarkers for human thyroid cancer diagnosis, prognosis, and therapeutic targets; these need further validation using a large cohort of samples. miRNAs are highly specific for a given cell and disease, relatively stable due to its shorter length (24 nucleotides) and therefore can get detected and reliably measured in various biological materials, including archived formalin-fixed and paraffin embedded samples or serum. With the availability of high throughput next-generation sequencing, miRNAs can be detected more abundantly and differentially from control compared with other miRNA microarrays, northern blots, TaqMan microRNA Assays Human panel, and confirmed by qRT-PCR assays. The miRNA expression profile presents a significant variability between different kinds of thyroid cancers, even if they originate from the same type of thyroid cells , 49. ForIn this review, we focus on miRNA expression in thyroid cancer to evaluate histological type-specific miRNA signatures, functional interactions with mRNAs, and miRNAs with the best potential as prognostic or diagnostic markers. Our goal is to provide a resource to strategize what future studies need to be performed. Although there are other review papers about miRNA and thyroid cancer subtype, such as review articles by Marini et al. and Celano et al. , 51, ourAs papillary thyroid carcinoma (PTC) is the most common thyroid cancer, most miRNA expression research has been done in PTC cell lines or patient samples , 52–64. Ab Mutalib et al. found miR-146b expression is significantly higher in PTC patients with lymph node metastasis positive (PTC LNM-P) or PTC patients with lymph node-negative (PTC LNN) fold change 6.0 and 4.7, respectively, when compared with adjacent normal thyroid tissue . AdditioAcibucu et al. and Yip et al. also found upregulation of miRNA 221 and miRNA 222 in PTC compared with normal thyroid tissue and in PTC with an increased risk of recurrence , 57. TheCantara et al. demonstrated that miR-95 and miR-190 are accurate and sensitive diagnostic markers for PTC in Caucasians . Their rAnother study  showed tBesides PTC, follicular thyroid cancer (FTC) is the other type of differentiated thyroid cancer. In contrast to papillary thyroid cancer, follicular adenomas and carcinomas share the same tumorigenic pathway. Many studies have shown that the mRNA/miRNA expression profiles of follicular neoplasms differ from those of papillary carcinoma ; howeverIn a meta-analysis study, miR-637, miR-181c-3p, miR-206, and miR-7-5p were revealed to be de novo potential FTC biomarkers; especially miR-7-5p showed the potential to distinguish between benign and malignant thyroid tissue in several published datasets . StokowyBesides the miRNAs reported above, one study found that miR-146b, miR-183, and miR-221 (which are observed to be deregulated in PTC) are upregulated and miR-199b was downregulated in FTC versus normal thyroid cells .Follicular variant of papillary thyroid carcinoma is the most common variant of PTC and is diagnosed in about 15–30% of PTC, sharing features of PTC and FTC on clinical, morphological, and genetic levels. It is important to distinguish between FTC and PTC with the follicular variant in clinical practice. Although miRNA deregulation was extensively studied in PTCs and FTCs, limited information is available for PTC with the follicular variant. Despite the high similarity in miRNA expression between PTC with the follicular variant and classical PTC, Dettmer et al. demonstrated that miR-375 is highly upregulated in PTC with the follicular variant and to a lesser extent in classic PTC, but not in FTC, hyperplastic nodule, or normal thyroid [Anaplastic thyroid carcinoma (ATC) is a highly malignant tumor. ATC is not beneficial in the radioactive scan but present with high mitotic activity in histologic appearance. miRNA profile showed significant downregulation of miR-30d, miR-30a-5p, miR-125b, and miR-26a expression. Out of these, a possible pathogenic role is suspected for miR-125b and miR-26a . As seenβ2, thus it is suggested to interfere with metastasis by sustaining an epithelial character of tissues [miR-30a and miR-200, two tumor suppressor miRNAs, are downregulated in ATC compared with the differentiated thyroid tumors and normal thyroid tissue , 94. The tissues –97.Similar to miR-30a, miR-125b which is also a tumor suppressor miRNA, is downregulated in ATC. Another study suggests that the downregulation of miR-12b leads to PIK3CD upregulation, which is known to be involved in cell survival, growth, division, and glucose homeostasis , 98.Family genes of let-7 are located on different chromosomes and are abundantly expressed in the normal thyroid gland  . DeregulFassina et al.  found thIn a miRNA microarray expression profile of a primary cohort of 12 SMTC and 7 HMTC samples, a number of miRNAs were identified to be differentially expressed , 107. ItChu et al. studied miR-21 and concluded that overexpression of miR-21 and MALAT1 regulate MTC progression and miR-21 inhibition reduced cell proliferation . As mentmiRNAs play a critical role in the regulation of various diseases, including thyroid cancer. Because the dysregulation of miRNA can be seen in many diseases, the possibility of using these as therapeutic agents has increased. According to Christopher et al., different laboratories have conducted recent studies in humans and animals. Their study shows us that miRNAs could become established as a new kind of drug . Charact50 values  and the lack of information on direct targets.Another method that can be used to approach the therapeutic targeting of miRNAs is by expression vectors or miRNA sponges. This vector-based plan of action consists of introducing an mRNA that contains many binding sites for the specific miRNA that is dysregulated. Hence why it is considered a sponge. By overexpressing these mRNA-specific sponges, the miRNA that is causing the oncogenic or tumor-suppressive role binds to the specific sites in the sponge, thus liberating the target mRNAs and allowing their expression. Although many studies of this approach have been performed in vitro, only transgenic animals have been used for studies in vivo. The last approach taken in the therapeutic targeting of miRNAs is the usage of small-molecule inhibitors. According to Li and Rana these approaches utilize reporter-based assay systems for compound library screening, and they have identified small molecules that could specifically inhibit miRNA expression . Instead+/I− symporter (NIS). It was discovered that the overexpression of miR-339 and miR-146b reduced the expression NIS in thyroid tumors. In contrast, the inhibition of these miRNAs allowed thyroid cancer cells to increase the uptake of radioactive iodine.According to Wojcicka et al., no study has reported the clinical use of miRNAs for thyroid cancer treatment, although its strong potential has been mentioned in many functional and preclinical studies . Most ofVarious biotech and pharmaceutical companies have already started to develop products that use miRNAs therapeutics. However, they are focusing on miRNA mimics and antagomiRs. We have already established that miRNA mimics are used to re-establish the concentration of the specific miRNA that is downregulated and that is causing the pathology. In change, antagomiRs are used to repress those miRNAs that are upregulated and that are causing the pathology. According to Bonneau et al., there are two challenges that these companies have faced that must be managed for the success of this novel project . Those aIn conclusion, we have summarized our literature search data in a Venn diagram to show the upregulated and downregulated gene pulls in subtypes of thyroid cancer Figures . We haveWith miR-146 being one of the most studied and most promising miRNAs involved with thyroid cancer, there is much interest in it as a gene target in hopes to reveal new prognostic biomarkers in PTC, especially those responsible for lymph node metastasis. Currently, more work is being done to profile miRNA in serum to find potential prognostic and diagnostic biomarkers. We suggest the need for more studies in vivo and multiple miRNA classifiers. Different studies have reported that high-throughput sequencing yielded more insight and better results in clarifying the relationship between miRNA and thyroid cancer , 24, 33.The limitation of our review article is that we did not perform meta-analysis; therefore, this lacks quantitative or qualitative analyses due to the limited number of literature on miRNA for each subtype of thyroid cancer and thus could not reveal the biases, strengths, and weaknesses of existing studies cited here. We also did not provide the mechanism of up and downregulation of miRNA-clusters and their pathways linked to each subtype-specific mRNA target. That would lead to another extended review on each subtype-specific pathway analysis, which awaits further ongoing extensive research.Future studies are needed not only to identify novel miRNAs, but also their biological functions, roles in tumorigenesis, and ability to predict thyroid cancer prognosis, diagnosis, and treatment. In order to plan promising therapeutic strategies, it is necessary to investigate both upregulated and downregulated pulls of miRNA signatures and their mRNA targets in each of the thyroid cancer subtypes.Recently, our lab focused on the miRNA signature in different ethnicities. However, in the literature search, we found that very few studies have been conducted to demonstrate the ethnic differences in miRNA expression in thyroid cancer health disparities , 74, 117


Living cells genetically engineered to execute nucleic acid nanotechnology programs thus have outstanding potential to significantly extend the current limits of synthetic biology. This perspective discusses the recent developments and future challenges in the field of synthetic bionanotechnology. Thus far, researchers in this emerging area have implemented dozens of programmable RNA nanodevices that provide precise control over gene expression at the transcriptional and translational levels and through CRISPR/Cas effectors. Moreover, they have employed synthetic self-assembling RNA networks in engineered bacteria to carry out computations featuring up to a dozen inputs and to substantially enhance the rate of chemical synthesis. Continued advancement of the field will benefit from improved in vivo strategies for streamlining nucleic acid network synthesis and new approaches for enhancing network function. As the field matures and the complexity gap between in vitro and in vivo systems narrows, synthetic bionanotechnology promises to have diverse potential applications ranging from intracellular circuits that detect and treat disease to synthetic enzymatic pathways that efficiently produce novel drug molecules.Enabled by its central role in the molecular networks that govern cell function, RNA has been widely used for constructing components used in biological circuits for synthetic biology. Nucleic acid nanotechnology, which exploits predictable nucleic acid interactions to implement programmable molecular systems, has seen remarkable advances in  RNA plays a pivotal role in the molecular networks that govern cellular function ,2, serviin vivo RNA networks.In parallel with these developments, the field of nucleic acid nanotechnology, which exploits the predictable base-pairing properties of DNA and RNA for implementing molecular systems, has seen remarkable advances in nanoscale self-assembly and molecular computation. Using DNA-based self-assembly methods, it is now possible to construct three-dimensional structures of virtually any geometry with nanometer-scale resolution using hundreds or even thousands of programmed DNA strands –15. The in vivo could be used to construct synthetic organelles that localize enzymes and constrain reactant flux for highly efficient synthesis of valuable chemicals. Moreover, genetically encoded neural networks could lead to single cells with the computing power of multiple neurons and the ability to respond intelligently to changes in environmental conditions. Such information-processing systems also have the long-term potential for upload into human cells to passively scan the transcriptome and intervene only if cellular dysfunction is detected by producing a drug or silencing a pathway.The fact that these impressive feats of molecular design, assembly, and computation have made use of biomolecules suggests that cells themselves can be genetically engineered to take advantage of nucleic acid nanotechnology and significantly expand the existing capabilities of synthetic biology. For instance, nucleic acid nanostructures assembled in vivo. Thus, systems implemented in live cells have mostly relied on RNA molecular networks, which have distinct properties from their in vitro DNA counterparts. Second, nucleic acid nanotechnology systems often employ strands with well-defined 5′ and 3′ ends and partially double-stranded complexes. In vivo systems implemented with transcribed RNA, however, often require specific sequence elements at their ends, such as terminator hairpins and 5′ untranslated regions. Partially double-stranded complexes between two RNAs are also not guaranteed to assemble in the cell. Third, most self-assembled nucleic acid nanostructures are formed using annealing procedures [in vivo systems are limited to isothermal nucleic acid reactions. They may also need to integrate cotranscriptional folding pathways, which have been harnessed for in vitro assembly of RNA origami nanostructures [in vivo nucleic acid circuitry must be coupled to an observable cellular output that is genetically encoded, such as a reporter protein, enzyme, or aptamer, to assess circuit function and alter cell behavior. Most in vitro circuits rely on DNAs with chemically synthesized fluorophore/quencher pairs [Realizing these synthetic bionanotechnology systems, however, requires addressing multiple technical challenges. First, the bulk of work in nucleic acid nanotechnology has focused on DNA molecules with exposed single-stranded domains to initiate reactions ,24. DNA ocedures –15, wherructures . Fourth,er pairs  and in oer pairs  for outpin vivo. They found that the expression of optimized RNA scaffolds was associated with an increase in the rate of hydrogen production in a two-enzyme pathway by 48-fold in engineered E. coli cells. This work has since been extended for scaffolding a four-enzyme pathway [E. coli [One of the earliest major works using genetically engineered cells for RNA nanostructure self-assembly was reported by the Silver laboratory and employed RNA as a programmable scaffold for proteins . In this pathway  and the [E. coli .Beyond structural applications, our laboratory and others have demonstrated the use of synthetic bionanotechnology for sensing and logic in living cells. Much of this work has been enabled by the sequence design package NUPACK developed for nucleic acid nanotechnology systems ,31. NUPAde-novo-designed RNA-based prokaryotic translational regulator called the toehold switch [E. coli with stronger thermodynamics and improved kinetics, helping them achieve changes in gene expression above 600-fold, a level similar to protein-based regulators and over an order of magnitude improvement compared with previous riboregulators. The design of the toehold switch allowed the sensors to detect virtually any trigger sequence, which we exploited to detect endogenous transcripts and construct libraries of dozens of sensors with low cross-talk. Using toehold-mediated designs, the Lucks group has also demonstrated that nucleic acid nanotechnology principles can be applied to transcriptional regulation. Computer-designed Small Transcription Activating RNAs (STARs) employ toehold-mediated interactions to prevent formation of a transcriptional terminator, enabling programmable control of downstream transcription (Using NUPACK, we developed a fully d switch  inspiredcription . These dE. coli [E. coli and mammalian cells.The ability of CRISPR/Cas enzymes to regulate gene expression with a wide dynamic range in hosts ranging from bacteria to humans has made them an important focus area for synthetic bionanotechnology. Recently NUPACK has been used for generating several conditional guide RNA implementations for the enzymes Cas9 and Cas12a that respond to binding of a cognate trigger RNA. Siu and Chen demonstrated toehold-mediated activatable guide RNAs for Cas9 that operated in E. coli , while OE. coli  (Figure E. coli . These sE. coli and are considerably more genetically compact than equivalent protein-based circuits. These ribocomputing circuits employ toehold switches, strand-displacement, and RNA self-assembly to encode AND, OR, and NOT logic functions that operate successfully in the cytoplasm (E. coli [Having an established set of orthogonal, genetically encoded systems to detect transcripts in live cells has opened up new possibilities for cellular computing systems. Recently, we have reported RNA-based logic circuits  that canytoplasm . NOT logytoplasm . A similytoplasm . In addi(E. coli .Outside of the cell, toehold switches have also been incorporated into portable diagnostic devices for detection of infectious disease. These tests make use of cell-free transcription–translation systems embedded into paper substrates to detect amplified transcripts using genetically encoded pathogen-specific toehold switches . The resin vitro compared with those realized in living cells. For DNA nanostructures, tile approaches have been used to assemble nanostructures consisting of 66 unique tiles at physiological 37°C temperatures in vitro [E. coli [in vitro [E. coli consisted of 12 potential input RNAs and a single gate RNA, which combined five toehold switch modules and GFP for processing and output [Despite recent progress in synthetic bionanotechnology, there remains a substantial gap between the complexity of nucleic acid systems implemented in vitro . In cont[E. coli . For molin vitro . This syd output  (Figure in vitro and in vivo systems provides a clear roadmap marking the capabilities required to extend the limits of synthetic bionanotechnology. Some critical future advances and research directions include:Fortunately, the gap between Efficient RNA circuit synthesis: More complex circuits will require larger numbers of unique RNA species to be transcribed in vivo. Given the short length of DNA/RNA species employed in the most complex in vitro circuits, the use of promoter/terminator pairs to encode each species is an inefficient means of genetic encoding. Accordingly, future circuits will benefit from the use of efficient self-cleaving ribozymes [in vivo RNA systems could employ alternative strategies where multiple processing elements are incorporated into a single transcript, analogous to the gate RNAs in ribocomputing devices [ibozymes  or progribozymes  to divid devices ,39. Alon devices ,46 can b devices .Enhanced signal processing: DNA components that implement signal amplification, rectification, thresholding, and annihilation are important elements in neural network circuits [in vivo will need to be developed to implement circuits that operate with comparable complexity in living cells. RNA signal amplification systems in which an input RNA is used catalytically are also valuable for detection of low-copy transcripts (E. coli [circuits ,18. Paranscripts  and have(E. coli . Such caRNase mitigation strategies: Genetically encoded in vivo RNA circuits have generally been limited to a single strand-displacement step [in vitro networks can require five or more sequential strand-displacement steps [ent step , whereasnt steps , which rnt steps , self- cnt steps , circulant steps , or compnt steps  to reducEnhanced specificity and bandwidth: In principle, the use of RNA for in vivo signal processing could enable networks with hundreds or even thousands of unique RNAs to function simultaneously in the cell. Implementing systems with maximum bandwidth will require RNA circuitry with specificity down to the single-nucleotide level. In vitro toehold exchange probes and related systems have demonstrated impressive specificity [cificity –54. IntrExpanded RNA/peptide binding libraries and enclosed nanostructures: Genetically encoded RNA nanostructures that assemble in vivo can act as programmable scaffolds directing the assembly of proteins and enzymes within the cell to establish new enzymatic pathways (pathways . As the in vivo to construct nanostructures that lead to enhanced enzymatic activity, sensors that detect diverse transcripts, and logic devices that process up to a dozen different input molecules. In the coming years, it is likely that future major developments will arise from RNA circuitry powered by sequential strand-displacement reactions, which will enable critical signal amplification and rectification capabilities, and related systems that will interface directly with endogenous transcripts to monitor and manipulate cell state. Such systems can be deployed by bacteria in the environment for monitoring, reporting, and remediation efforts, or even those housed in the gut where they can respond to gastrointestinal ailments or changes in nutrition. Increasingly sophisticated in vivo nucleic acid assembly methods will be harnessed to position larger numbers of enzymes and to fabricate synthetic organelles that replicate the compartmentalization afforded by natural systems. In the absence of effective compartmentalization schemes, however, efforts should be directed toward pathways in which reactants cannot be lost to diffusion. Possible targets include signaling proteins [In conclusion, nucleic acid nanotechnology is providing synthetic biologists with a powerful set of molecular tools that can be harnessed to provide living cells with new functions. Genetically encoded nucleic acids have thus far been used proteins  along wiproteins , which cNucleic acid nanotechnology is providing synthetic biologists with a powerful set of molecular tools that can be harnessed to provide living cells with new functions.in vivo to construct nanostructures to scaffold enzymes, sensors that detect diverse transcripts, and logic devices that process up to a dozen different input molecules.Genetically encoded nucleic acids have been used in vivo nucleic acid assembly methods, and further development of circuitry for mammalian cells.It is likely future major developments will arise from RNA networks powered by sequential strand-displacement reactions, increasingly sophisticated These future efforts will require multiple new capabilities including improved methods for expressing nucleic acid networks and novel techniques for enhancing network stability, sensitivity, specificity, and function.


Kambayashi et al.; Nakamura (a)]. Importantly, although many predictive biomarkers for both ICi tumor response and irAEs have been identified, no single biomarker has shown to be predictive on its own; therefore, a combination of multiple biomarkers should be utilized . Furthermore, irAEs and tumor response can be interconnected, but often are not; thus, irAEs or biomarkers for irAEs cannot be reliably used as biomarker for tumor response . Further studies are needed to improve biomarkers for predicting the efficacy of ICi treatment . To this end, Kümpers et al., show that PD-L1 expression on primary tumors and melanoma metastases is not associated with the clinical response of anti-PD1 antibodies, while several previous studies suggest an association of PD-L1 status and response to anti-PD1 antibodies . Another research paper in this Research Topic suggests that baseline serum levels of CXCL5, which have been reported previously as a biomarker for autoimmune disease, could be a predictive marker for the efficacy of anti-PD1 antibodies . While ICi were initially only used for definitive therapy, the field has quickly moved to adjuvant and, more recently, to neoadjuvant therapy (Nakamura (a)]. Fujimura, Kambayashi et al. show in a case report that N+I combination therapy for BRAF-mutated advanced melanoma before primary tumor resection strikingly increased CD8+ cytotoxic T cells in the primary tumor, leading to induced anti-melanoma immune response in metastases in six different organs, but also induced serious AEs after administration of N+I combination therapy . In this Research Topic, we focus on the latter. The most commonly used ICi are monoclonal antibodies targeting cytotoxic T-lymphocyte-associated protein 4 , programmed cell death 1  and, more recently, programmed death-ligand 1 . In addition to multiple described resistance mechanisms to ICi , EGFR inhibitors and ICi for cutaneous squamous cell carcinoma (cSCC), HER2 antagonists for extramammary Paget's disease (EMPD), ICi for Merkel cell carcinoma (MCC), and experimental approaches for skin adnexal carcinomas. They conclude that, emerging molecular targeting therapies are not necessarily effective for all NMSC patients. Development of further treatment options for NMSC is required, especially for rare forms of NMSC, such as skin adnexal carcinomas . A retrospective case series by Hiura et al. of 13 patients with unresectable cSCC demonstrates the potential advantage of continued chemotherapy after concurrent chemoradiotherapy (CCRT), which will be validated in a future study. Hidaka et al. discuss in a thought-provoking review the role that Aryl Hydrocarbon Receptor (AHR) plays in carcinogenesis and maintenance of skin cancers. AHR is a key modulator of UVR- and carcinogenic chemical-induced skin carcinogenesis and is also associated with the efficacy of MAPKi and ICi in melanoma . Thus, the authors propose that the AHR system may provide a putative target for prevention and therapy of skin cancer . Oka and Miyagaki review novel and future therapies for advanced Mycosis fungoides and Sézary syndrome covering a wide range of different drug classes. Most approaches showed limited efficacy. Thus, the authors recommend personalized therapy and call for creatively designed international clinical trials (Oka and Miyagaki).On the heels of the success of systemic therapies for melanoma, similar approaches are being tested in non-melanoma skin cancers (NMSC). Kato et al. review the role of dermoscopy in the diagnosis of melanoma and non-melanoma skin cancers including BCC, sebaceous carcinoma, actinic keratosis, Bowen's disease, cSCC, MCC, EMPD, and angiosarcoma. Oh et al. take skin imaging further by discussing the utility of ultrasound imaging, optical coherence tomography, confocal microscopy, and two-photon microscopy as diagnostic tools. Especially a combination of tools is advised to allow for highest resolution and highest imaging depth, which are usually reciprocal . The principle of these devices is to analyze signals reflected or scattered from the skin. Indeed, the fact that autofluorescent structures within the skin  can be co-imaged with highly crystalline triple-helix structures  utilizing the second harmonic generation phenomenon, which then can be quantified  for skin tumor diagnosis. They conclude that AI classifiers have dramatically improved over the last years and still keep improving and thus may gain sufficient sensitivity and specificity to bear the screening burden for detecting malignant skin tumors . Importantly, they emphasize that the advent of AI-based skin cancer diagnostic should be considered a useful assistance, rather than a threat to dermatologists .Dermoscopy has become an indispensable diagnostic tool for pigmented and unpigmented cutaneous lesions . Kato etantified . Oh et aNakamura (b) discusses the new role of sentinel lymph node biopsy (SLNB) for invasive melanoma post DeCOG-SLT and MSLT–II, in the context of modern adjuvant and neoadjuvant therapy approaches.Fujii and Kanekura review the methods for diagnosis of early stage T-Cell Lymphoma. They demonstrate that next-generation sequencing not only detects TCR clonality with superior sensitivity over conventional methods and is therefore better to diagnose early Mycosis fungoides, but also allows for temporal tracking of specific TCR clones and therefore better for assessment of progression or recurrence (Fujii and Kanekura).TF and NH wrote the manuscript. All authors provided intellectual input to the editorial.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.


Concussion or mild traumatic brain injury (mTBI) in athletes can cause persistent symptoms, known as post-concussion syndrome (PCS), and repeated injuries may increase the long-term risk for an athlete to develop neurodegenerative diseases such as chronic traumatic encephalopathy (CTE), and Alzheimer’s disease (AD). The Center for Disease Control estimates that up to 3.8 million sport-related mTBI are reported each year in the United States. Despite the magnitude of the phenomenon, there is a current lack of comprehensive prognostic indicators and research has shown that available monitoring tools are moderately sensitive to short-term concussion effects but less sensitive to long-term consequences. The overall aim of this review is to discuss novel, quantitative, and objective measurements that can predict long-term outcomes following repeated sports-related mTBIs. The specific objectives were (1) to provide an overview of the current clinical and biomechanical tools available to health practitioners to ensure recovery after mTBIs, (2) to synthesize potential biological mechanisms in animal models underlying the long-term adverse consequences of mTBIs, (3) to discuss the possible link between repeated mTBI and neurodegenerative diseases, and (4) to discuss the current knowledge about fluid biomarkers for mTBIs with a focus on novel exosomal biomarkers. The conclusions from this review are that current post-concussion clinical tests are not sufficiently sensitive to injury and do not accurately quantify post-concussion alterations associated with repeated mTBIs. In the current review, it is proposed that current practices should be amended to include a repeated symptom inventory, a cognitive assessment of executive function and impulse control, an instrumented assessment of balance, vestibulo-ocular assessments, and an improved panel of blood or exosome biomarkers. Up to 3.8 million sports-related concussions  are diagnosed annually in the United States; however, it is estimated that only 50% of mTBIs are reported . A potenDemographic studies have identified several risk factors for sustaining sports-related mTBIs. The greatest risk of sustaining an mTBI exists among males playing American football or Australian rugby, females playing soccer , and botResearch on the typical neurocognitive sequelae of concussions suggests there is little uniformity in the presentation and course of PCS . DespiteThe accurate assessment of mTBI and post-concussive symptoms is crucial to athlete safety and is often at the center of return-to-play (RTP) decisions for healthcare providers and sports medicine staff . ImportaThe objective of this review is to provide an overview of: (1) clinical and biomechanical tools available to sport medicine practitioners to ensure recovery after mTBIs before RTP, (2) potential biological mechanisms in animal models underlying long-term adverse consequences of mTBIs, (3) the link between repeated mTBI and neurodegenerative diseases, and (4) current knowledge of fluid biomarkers for mTBIs, with an emphasis on blood biomarkers and on emerging exosomal biomarkers. More research into specific mechanisms of injury is needed to improve the prognostics for sport-related mTBIs and a better understanding of the neuronal consequences reflected by plasma or exosome biomarkers is also necessary.General guidance for the management of sports-related mTBIs includes an assessment of symptoms and disposition after first addressing acute trauma . Injury The diagnosis of concussion has historically been based on self-reported symptoms  includinThe Concussion in Sport Group (CISG) first introduced the Sport Concussion Assessment Tool (SCAT) 1 to provide a multifaceted standardized assessment of concussion which has since evolved into the SCAT2, SCAT3, and SCAT5 . The diaTM Core Battery  and detriments in the vestibulo-ocular system can lead to dizziness, nausea, and balance impairment. In a battery of vestibular-based laboratory tests, post-concussion deficits can be identified using the Dynamic Visual Acuity Test DVAT; , and theThe call for more sensitive concussion assessment and the growing awareness of CNS compensatory function has increased the utility of balance testing. Compensatory CNS function is observed when, for example, an “eyes closed” balance task results in instability. In that case, vision can be said to compensate for deficits in the vestibular system . In factThe Balance Error Scoring System BESS;  is an obMeasuring dynamic balance  may be a better indicator of athlete readiness to RTP. However, clinicians are currently limited in their ability to measure dynamic balance as those methods are still being optimized. A slower normal gait speed  has beenIn sum, there is a growing base of concussion research that confirms our understanding of concussion as a multi-faceted injury, affecting both cognitive, and balance systems. To date, the measurement of mTBI-related balance and cognitive sequelae as required by NCAA regulations is moderately sensitive to short-term concussion effects, but less sensitive for long-term PCS.the most physiologically relevant models are those that utilize pneumatically-driven or electromagnetic impact devices to deliver single or repetitive closed head injuries to mice or rats. Recent studies have begun to use un-anesthetized animals and animals in which the head is free to move in response to the impact rather than being immobilized. These models may ultimately prove to replicate the dynamic injury of mTBI more accurately than those using anesthetized and immobilized animals; however, they are not without some level of controversy.Most biological mechanisms for long-term effects of mTBIs have been discovered via animal models. There have been several thorough reviews published previously describing the relative pros and cons of various mouse and rat models of TBI . TherefoSeveral models of repetitive mTBIs have demonstrated persistent multi-focal axonal injury using electron microscopy, silver staining, and diffusion tensor imaging . Axonal Although neither of the specific Tau pathologies of AD or CTE have been precisely replicated in mouse models of mTBI, marked alterations in Tau phosphorylation, and processing do occur in mice following TBIs. For instance, a single unilateral impact in un-anesthetized mice induced rapid tauopathy within 24 h of injury, which persisted and progressed to the contralateral uninjured cortex by 5.5 months post-injury . Severalbona fide NFTs is not efficiently induced by this trauma/gene interaction paradigm, at least not in mouse models. To our knowledge, only one laboratory has described a mouse model which seems to meet (most of) these criteria. This model induces a tauopathy marked by hyperphosphorylation which was detectable in cortical axons ipsilateral to a cortical impact injury by 24 h post-injury and in neuronal soma by 2 weeks post-injury , repetitive mTBI induced extensive telencephalic neurofibrillary tangles (NFTs) in only one of twelve transgenic mice . In a set-injury . Moreoved spread , providiIt is well known that recovery from neurotrauma, such as a stroke or TBI, can be confounded by the brain’s ability to perpetually activate microglia. Reactive gliosis following neural injury can lead to chronic inflammation and neurodegeneration . UndoubtIn two distinct models of Aβ precursor protein (APP) mutagenesis, the Tg2576 mouse and the APP/PS1 transgenic mouse, mTBI enhanced Aβ aggregation, and precipitated cognitive impairment of pre-symptomatic AD mice . In contIn conclusion, these previous studies suggest that a genetic propensity for AD pathology is exacerbated by repeated mTBIs, thus providing a potential biological mechanism for the fact that not everyone with multiple mTBIs, either resulting from active duty or from high impact sports, succumb to neurodegenerative diseases including CTE when they get older. Animal studies are valuable in that they can dissect long-term effects in a relatively short time period, and researchers can modify specific genetic factors to explore their impact on long-term brain health.Although several sources conclude no connection between exposure to a single mTBI and incidence of neurodegenerative disease , other spost mortem study, investigators found evidence of CTE in the brains of teenage athletes following repeated mTBIs  and amyotrophic lateral sclerosis ALS; , althougThe relationship between multiple mTBIs and neurodegenerative diseases is a controversial issue that is currently being debated in several lawsuits. Because of the inherent difficulty in providing tangible evidence of long-term risk after multiple mTBIs in professional players, prognostic biomarkers are needed to provide confirmation of damage and association with neurodegenerative conditions. Fluid biomarker research has gained increasing attention over the last decade for its promise to help quantify injury severity, determine when it is safe for athletes to return to play, and to track the long-term consequences of mTBIs for brain health. That research also highlights the underlying biological mechanisms of mTBI and can be used to evaluate the effectiveness of therapeutic interventions . HoweverIn addition, current biomarkers are not necessarily adequate to examine the long-term risk associated with mTBIs. While other potential biomarkers exist such as Positron Emission Tomography (PET), Magnetic Resonance Imaging (MRI) modalities, and computerized electroencephalogram (EEG), this review focuses solely on fluid biomarkers including exosomes. Existing fluid biomarkers have often shown inconsistent results across published studies. A number of fluid biomarkers have been examined for their ability to assess the severity of mTBI  but a paN-terminal fragment of αII spectrin (SNTF), Tau, neurofilament-light (NF-light), ubiquitin C-terminal hydrolase-L1 (UCH-L1), and brain-derived neurotrophic factor (BDNF). Glial activation markers include S100 calcium binding protein B (S100B) and glial fibrillary acidic protein (GFAP). Lastly, inflammatory cytokines have been measured both in CSF and blood and include the pro-inflammatory cytokines interleukins-1, -12, and -18 , tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ). Over the last few years, much attention has been brought to the development of blood biomarkers that can accurately diagnose mTBI acutely or predict the long-term changes in the brain associated with one or repeated mTBIs  neuronal/axonal damage, (2) activation of glial cells, and (3) release of inflammatory cytokines that occur due to neuronal injury. Neuronal biomarkers that have been examined include neuron-specific enolase (NSE), calpain-derived ed mTBIs , and earThe first neuron-specific biomarker to be explored for mTBI was NSE . NSE is SNTF  has been identified as a protein that also accumulates preferentially in damaged axons, as a result of neuronal stretch injury (Neurofilament-light (NF-light) is another biomarker for axonal damage that can be measured in serum. NF-light levels are increased in ice hockey players with PCS  is an enzyme involved both in the processing of ubiquitin precursors and of ubiquitinated proteins and is highly expressed in the neuronal cytoplasm  is a neurotrophic factor highly involved in brain health, with serum levels increasing after physical exercise or cognitive training  and glial fibrillary acidic protein (GFAP) are proteins located in the cytoplasm of astrocytes and have received a lot of attention as markers of glial activation, particularly following mTBIs  and may also increase the risk for long-term neurodegenerative conditions including CTE, AD, or other neurodegenerative or neuropsychiatric conditions. Ongoing research suggests that current RTP protocols should evolve to include quantitative assessments of reported symptoms, cognition, vestibulo-ocular function, dynamic balance, and exosomal biomarkers. The expansion of that battery will confer a direct benefit to the athletic community and increase the safety of RTP protocols. It will also contribute to concussion management education and awareness as well as earlier detection of symptoms that may be related to long-term neurodegenerative disease.Several studies have suggested that a battery including self-reported symptoms as well as a neurocognitive and balance tasks have a sensitivity to concussion exceeding 90%. However, most studies have not considered long-term PCS changes in the brain that may impact brain health. Therefore, it appears important to obtain and bank baseline data on all athletes. That includes individualized testing for balance, cognitive performance, and biomarkers. Our experience from a cohort consisting of more than 300 NCAA Division I athletes in high-impact sports clearly shows a need for expanded testing paradigms including instrumented balance measurements and better cognitive batteries that capture long-term changes in executive function, impulse control, and decision making, for example supplementing the ImPACT battery with portions of the ANAM test battery.in vitro studies can reveal biological mechanisms involved in disease progress long-term, as discussed above. Based on previous and current findings in the field, a model is emerging in which diffuse axonal injury, Tau pathology, and neuroinflammation incrementally contribute to long-term changes in the brain that eventually lead to increased risk for tauopathies such as AD or CTE with aging.Although more research is needed to determine the most sensitive and specific biomarkers for long-term brain health, it seems appropriate to include a panel of biomarkers in mTBI protocols in cases where PCS appears . FurtherA-CG, AL, DL, KG, and BD contributed to the conception of the review, coordinated writing efforts, and edited final article version. A-CG, AL, and KG wrote the Introduction. MP and BD wrote the balance assessment part. KG prepared the cognitive assessment part. AL and A-CG wrote the biomarker and exosome portions. DL, LK, and HF worked on the animal studies section. All authors collaborated with or were part of the DU Concussion research group and contributed to critical revisions of the manuscript, read, and approved the submitted version.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.


A major obstacle in the fight against cancer is the development of drug resistance in response to therapy. Development of resistance can be facilitated by a small population of drug-tolerant cancer cells characterized by an increasead capacity of adaptation to various stresses. Here, we describe features and vulnerabilities of these drug-tolerant persister cells that can be exploited to prevent the development of drug resistance.Designing specific therapies for drug-resistant cancers is arguably the ultimate challenge in cancer therapy. While much emphasis has been put on the study of genetic alterations that give rise to drug resistance, much less is known about the non-genetic adaptation mechanisms that operate during the early stages of drug resistance development. Drug-tolerant persister cells have been suggested to be key players in this process. These cells are thought to have undergone non-genetic adaptations that enable survival in the presence of a drug, from which full-blown resistant cells may emerge. Such initial adaptations often involve engagement of stress response programs to maintain cancer cell viability. In this review, we discuss the nature of drug-tolerant cancer phenotypes, as well as the non-genetic adaptations involved. We also discuss how malignant cells employ homeostatic stress response pathways to mitigate the intrinsic costs of such adaptations. Lastly, we discuss which vulnerabilities are introduced by these adaptations and how these might be exploited therapeutically.  In the last decades, targeted therapies, as well as immunotherapeutic approaches, have been developed with the promise of revolutionizing cancer care. However, despite the undeniable improvement in patient prognosis delivered by these new therapies, the development of drug resistance remains a major obstacle in cancer treatment. Drug resistance can be categorized as either primary resistance, when the treatment has no objective effect on cancer cells from the start, or as secondary resistance, in which unresponsiveness follows an initial response. The development of acquired drug resistance is intimately linked to intratumor heterogeneity in terms of genetic, epigenetic, transcriptional and metabolic state. Intratumor genetic heterogeneity has been demonstrated extensively by genome sequencing efforts in solid and hematological tumors, shedding light on the complex clonal composition and dynamics of the tumor . HoweverAcquired drug resistance has been attributed largely to the selection or de novo acquisition of specific gene mutations that confer proliferative and/or survival advantage to escape the therapeutic pressure A. HoweveGenetic and non-genetic adaptations during the acquisition of drug resistance are not mutually exclusive C. It hasOperationally, these cells are defined as drug-tolerant persister (DTP) cells. They represent a rare and noncycling or slowly cycling population of cells in the presence of the drug, characterized by altered metabolism, distinct epigenetic features and transcriptional program. In vitro, they can be generated by exposing tumor cells to cytotoxic concentrations of therapeutically relevant drugs . The vasEGFR-mutant non-small cell lung cancer (NSCLC) cells treated with gefitinib. DTPs generated by gefitinib treatment appeared at a very low frequency (about 0.3%) and were characterized by extensive epigenetic alterations rather than any genetic alteration [V600EBRAF melanoma model, treatment with vemurafenib was shown to upregulate EGFR expression to overcome the mitogen-activated protein kinase (MAPK) pathway inhibition in response to drug treatment. However, when the drug is removed, the supraphysiological level of MAPK activation induces a state of oncogene-induced senescence, and cells expressing high levels of EGFR are counter selected. This, in turn, leads to the restoration of vemurafenib sensitivity [V600EBRAF colon cancer patients [In vitro, after drug withdrawal, DTPs can resume proliferation, but their progeny remains equally sensitive to the initial therapy, indicative of a transient and not an inheritable resistance mechanism ,6. The fteration . Similarsitivity . In linepatients ,9. Clinical evidence also indicates that, after initial progression under treatment, patients can respond to the rechallenge with the same therapy following a “drug holiday” . Such diThere is still no clear consensus about characterization, markers or even the origin of the different slowly proliferating drug-tolerant phenotypes. Nonetheless, they share common features such as a slow cell cycle, altered metabolism, altered epigenetic features, resistance to apoptosis and immune evasion. Underlying all these features is the increased cellular plasticity and heterogeneity that provides cells with multiple possibilities to rewiring cellular signaling to evade cytotoxic therapies . Below, Multiple definitions of cell cycle-restricted drug-tolerant cancer cells are used, associated with different contexts. For instance, dormant cancer cells are defined as rare cells that can disseminate early and stay latent in distant niches before being reactivated and cause disease relapse after successful treatment of the primary tumor . TherefoSince DTPs are characterized by a reversible restriction of proliferation, they resemble quiescent cells. Quiescence is a physiological cell cycle state (G0) prevalent in adult stem cells from tissues with low turnover but also a characteristic of cancer stem cells (CSCs). Even though the relationship between CSCs and DTPs is still not completely clarified, some similarities become evident. For instance, increased tolerance to therapies is also a common characteristic of CSCs (reviewed in ), while Perhaps at the end of the “spectrum” of cell cycle-restricted phenotypes, senescence is defined as a stable cell cycle arrest. Senescence induction is the physiological cellular response to telomeres shortening, DNA damage, excessive oncogenic signaling or a variety of other stress signals . InteresVery recently, two groups identified a state of drug adaptation that resembles “diapause”, a delay in embryonic development that can occur under stress conditions ,23. One In these diapause-like DTP models, the suppression of MYC activity was sufficient to induce the state of drug tolerance, pointing toward a close link between proliferation state and drug tolerance. Consistent with this, it has been proposed previously that the slow proliferation rates common to the phenotypes described above provide a survival advantage in the presence of cytotoxic drugs ,26. MoreG12DKRAS pancreatic ductal adenocarcinoma (PDAC) [V600EBRAF melanoma [Cellular plasticity allowing changes in chromatin state and transcription modulation also impact the overall metabolism of slow-proliferating drug-tolerant phenotypes. DTP cells have been described to slow down their metabolism, becoming less dependent on glycolysis and increasingly dependent on mitochondrial oxidative phosphorylation (OXPHOS). The upregulation of this pathway has been shown in models of a (PDAC) , BRAFV60melanoma , as wellmelanoma  and Chromelanoma , upon trmelanoma ,31. Intemelanoma . Moreovemelanoma ,33. It imelanoma .V600EBRAF melanoma cells to survive MAPK inhibition before the development of drug resistance [Another common feature of DTP cells is the ability to sustain their metabolism with alternative sources of nutrients such as autophagy or fatty acid oxidation (FAO) pathways ,35,36. Asistance ,38. In tsistance . EGFR-mutated NSCLC can survive gefitinib and osimertinib treatments by upregulating the anti-apoptotic protein MCL1 via the mTORC1-mediated post-transcriptional regulation of mRNA translation [Overall metabolism and protein production in human cells are mostly regulated according to proliferation states . Consistnslation . Understnslation .It is still understood poorly whether persister cells are also at the basis of adaptive resistance to immunotherapies. However, a recent study described the emergence of a discrete subpopulation of cells resistant to an immune-checkpoint inhibitor (anti-PD1 antibody) in a murine organotypic spheroid ex vivo model . These cIn response to adoptive T cell transfer (ACT), the tumor microenvironment becomes rich in inflammatory mediators. In particular, TNFɑ has been identified to induce the reversible loss of antigens in melanoma cells, preventing their killing ,50. SimiTherapeutic interventions can also indirectly favor DTPs immune evasion. In response to T cell therapy, the inflamed stromal cells secrete hepatocyte growth factor (HGF) and favor the recruitment of immune-suppressive neutrophils that restrain T cell expansion and function . The recMoreover, multiple reports, both in solid and hematological cancers, highlighted the fundamental role of DTP cell plasticity in favoring immune evasion. EGFR-mutated NSCLC upon tyrosine-kinase inhibition (TKI). Mesenchymal cells from NSCLC biopsies have been used as surrogate of persister cells to identify FGFR inhibition as a promising combination therapy to prevent DTP survival and expansion [EGFR-mutant lung adenocarcinoma in response to erlotinib results in reduced expression of MHC-I complex and loss of tumor antigen presentation [The enhanced cellular plasticity of cancer cells facilitates the non-genetic adaptations underlying drug tolerance reviewed above. Such plasticity involves changes in the transcriptional program of the cells, with various intracellular pathways potentially merging into epigenetic changes and modulation of transcriptional factors to dictate cell behavior ,25. For xpansion . In tripxpansion . Changesxpansion ,58. Cellentation . Longitudinal scRNAseq analysis of lung cancer patient biopsies, before and during target therapy, identified, at MRD, cells expressing an alveolar-regenerative signature, suggesting a therapy-induced cell plasticity . RemarkaGenetic and epigenetic alterations leading to oncogenic signaling confer onto cancer cells the capabilities summarized as the hallmarks of cancer , as wellThe specific triggers and molecular mechanisms by which slow-proliferating drug-tolerant cancer cells engage these stress pathways are variable and so are the vulnerabilities associated with the different DTPs. For instance, autophagy has been shown to tame excessive oxidative stress, supporting the survival of dormant metastatic breast cancer cells by “eating” damaged mitochondria, with little effect in proliferating metastatic cells. Autophagy inhibition prevents the dormancy-to-growth switch and induces apoptosis in this model . A stem The rewiring of mitogenic and survival pathways has also been proposed to support the viability of cells having slow-proliferating drug-tolerant phenotypes, creating additional targetable vulnerabilities. Using EGFR-driven NSCLC models, Sharma and colleagues showed that treatment with gefitinib or cisplatin results in a population of DTPs that have a repressed chromatin state induced by an upregulated IGF-1R signaling, whose inhibition is lethal to these cells . The IGFGiven the dynamic nature of the slow-proliferating drug-tolerant phenotypes, the timing of targeting may be critical. This adds one more layer to the already complex nature of the interplay between oncogenic prosurvival signaling and stress response pathways. While some alterations in prosurvival signaling or stress response pathways may be transient and related to the slow-proliferating drug-tolerant phenotype, others can be maintained after full-blown resistance is established. Ideally, vulnerabilities of DTPs should persist during full-blown drug resistance, as it would increase the window during which the vulnerability could be exploited. Such a “collateral sensitivity” approach has been successfully used to target drug-tolerant persister cells  as well BRAF-mutated melanoma cells initially respond to BRAF inhibition but rapidly become tolerant when in close contact with stromal cells. Mechanistically, melanoma-associated fibroblasts respond to BRAFi with an augmented fibronectin production that enhances FAK signaling in melanoma cells. The coinhibition of BRAF and FAK prevented the reactivation of MAPK and resulted in better control of tumor growth [Different strategies can be conceived to target DTPs. Targeting epigenetic modifiers, such as the lysine demethylase KDM5, has been shown to decrease the number of persister cells in melanoma, colon and breast cancer models . Likewisr growth . The altr growth ,30. In ar growth . An alternative approach could be to redirect DTPs to a permanent and more homogeneous dormant state. Ideally, DTPs should remain dormant for a long period of time so they can be eradicated by the immune system or by targeting specific vulnerabilities. In particular, drug-induced cancer senescence and the consequent SASP activation have been shown to promote immune-mediated clearance of tumor cells in mouse models of lung and pancreatic cancer ,88. FurtThe most straightforward therapeutic approach to deal with DTP generation would be to simultaneously target both the oncogenic signaling and the stress response pathways that help cancer cells deal with the oncogenic activity. Because the increased dependence of cancer cells on stress response pathways is intimately linked to the oncogenic activity, perturbation of oncogenic signaling with drugs can improve the efficacy of targeting stress pathways. Experimental data pointing in this direction can be found in the recent literature. For instance, therapy resistance of AML stem cells was reversed by cytokine stimulation, sensitizing these cells, but not normal hematopoietic stem cells, to chemotherapy . It is nThe data reviewed here suggest that a better conceptual and mechanistic understanding of the “push and pull” dynamic between oncogenic signaling and stress response pathways may provide a wealth of innovative targeting opportunities to forestall drug tolerance and resistance. In spite of the fact that the interest in the biology of DTPs is relatively recent, a large number of studies have been performed to characterize chemo- and targeted therapy-tolerant persister cells. However, further studies will be required in particular to investigate DTPs in vivo in their crosstalk with the microenvironment and cells of the immune system.To investigate the vulnerabilities of slow-proliferating drug-tolerant cells, it might be worthwhile to perform unbiased loss of function genetic screens to identify genes essential for their survival. This could lead to the identification of compounds that can be used to kill drug-tolerant cells before they give rise to fully resistant tumors. However, the experimental set-up should take into consideration the low frequency of these cells, which may vary according to tumor type and drug treatment. Most likely, genome-wide screens will be not feasible, and focused libraries should be used for these studies. The fact that different drug-resistant clones can arise from a DTP population may be an additional complicating factor ,95. Notw