Physical activity is emerging from epidemiologic research as a lifestyle factor that may improve survival from colorectal, breast, and prostate cancers. However, there is considerably less evidence relating physical activity to cancer recurrence and the biologic mechanisms underlying this association remain unclear. Cancer patients are surviving longer than ever before, and fear of cancer recurrence is an important concern. Herein, we provide an overview of the current epidemiologic evidence relating physical activity to cancer recurrence. We review the biologic mechanisms most commonly researched in the context of physical activity and cancer outcomes, and, using the example of colorectal cancer, we explore hypothesized mechanisms through which physical activity might intervene in the colorectal recurrence pathway. Our review highlights the importance of considering pre-diagnosis and post-diagnosis activity, as well as cancer stage and timing of recurrence, in epidemiologic studies. In addition, more epidemiologic research is needed with cancer recurrence as a consistently defined outcome studied separately from survival. Future mechanistic research using randomized controlled trials, specifically those demonstrating the exercise responsiveness of hypothesized mechanisms in early stages of carcinogenesis, are needed to inform recommendations about when to exercise and to anticipate additive or synergistic effects with other preventive behaviors or treatments. Over the past two decades, it has become clear that physical activity is associated with reduced cancer incidence. A recent pooled analysis of epidemiologic studies showed that high levels of leisure-time physical activity are associated with statistically significantly lower risks of 10 different cancers, even after adjusting for body mass index (BMI) . ConsideCancer patients are surviving longer than ever before, and fear of cancer recurrence is an important concern among cancer survivors . In the While there is strong and consistent observational evidence that increased physical activity is associated with increased overall survival in cancer survivors , as welTo our knowledge, seven observational studies have investigated the effect of pre- or post-diagnosis physical activity and breast cancer events, defined as recurrence, progression, new primary breast cancers, or breast cancer-specific deaths –24. A meclincialtrials.gov identifiers: NCT02035631, NCT02786875, NCT03091842, NCT02750826, NCT02161900, NCT02240836), prostate (NCT02252484), ovarian (NCT02529150), endometrial (NCT03095664), and other cancer survivors . Results of these RCTs will provide further evidence of a causal association between physical activity (combined with other lifestyle changes) and cancer recurrence.In addition to observational evidence, randomized controlled trials (RCTs) of exercise with recurrence outcomes are necessary for ruling out reverse causality, residual confounding, and evaluating the predictive nature of proposed biomarkers. There is emerging RCT evidence on the effect of post-diagnosis physical activity on cancer recurrence. To date, only the Supervised Trial of Aerobic versus Resistance Training (START) in breast cancer patients has published results, suggesting a protective exercise effect in patients diagnosed as human epidermal growth factor receptor 2 (HER2)-positive and patients who completed >85% of the average relative dose intensity of their originally planned chemotherapy regimen. However, the recurrence analysis from START was secondary and exploratory in nature and subgroups were based on small sample sizes . AnotherThe mechanisms whereby physical activity could lower recurrence risk in breast, colorectal, and prostate cancer patients are not well understood, although evidence has grown over the past decade. The interrelated mechanisms most often studied in relation to physical activity and cancer prognosis Fig.  include Whether physical activity influences these mechanisms independently or through reductions in adipose tissue volume and endocrine activity is difficult to discern for obesity-related cancers  since some of the same mechanisms are proposed, although some effects are becoming better understood. In postmenopausal women, for example, body fat is the primary source of endogenous estrogens, which fuel cancer progression in estrogen receptor-positive (ER+) breast cancer. There is now RCT evidence that, while exercise decreases estradiol levels, much greater decreases occur with weight loss  and exerExercise intervention trials are increasingly being conducted to demonstrate exercise modes of action in cancer patients. Structured exercise trials , ofA common limitation of these trials is the uncertainty that circulating blood biomarkers reflect downstream biological activity, whereas epigenetic and gene expression studies do not have this limitation. For instance, in colorectal cancer patients, exercise was associated with the CpG island methylator phenotype (CIMP) and mutations in TP53 and KRAS2 mutations in colon tumor tissue  and CIMPExercise during first-line and adjuvant chemotherapy might improve treatment efficacy Fig. . Shared A key consideration related to physical activity and cancer recurrence is timing. There is clinical value in understanding whether pre- or post-diagnosis physical activity (or both) prevents cancer recurrence, and whether physical activity prevents early or late recurrences (or both). Early recurrences of a slow-growing cancer could be more strongly influenced by pre-diagnosis activity whereas late recurrences could be more strongly influenced by post-diagnosis activity. Moreover, if risk of recurrence increases with cancer stage at first diagnosis , then phAPC and KRAS followed by PIK3CA, SMAD4, TP53, and others [Colorectal cancer serves as a useful example for aligning physical activity timing with risk accumulation because in colorectal cancer the early stages are observable; from field cancerization , 58 to ad others , with ond others . Recurred others  and happd others . Given td others , it is pField cancerization  refers tA number of epidemiologic studies have examined the relation between physical activity –72 or selower among individuals with higher leptin levels and there was no association with adiponectin [Physical activity might prevent adenoma recurrence by maintaining energy balance since BMI, waist circumference, and possibly weight change have been associated with higher recurrence risk –82. Corrponectin .Few epidemiologic studies have explored insulin resistance biomarkers in relation to adenoma recurrence despite insulin’s known mitogenic and anti-apoptotic properties in the colon  and exerOther epidemiologic studies investigated IGF-1 and its primary binding protein IGFBP-3, which controls IGF-1 bioavailability, in relation to adenoma recurrence since IGF-1 promotes cell proliferation and inhibits apoptosis . In someKRAS and mismatch repair gene mutations, microsatellite instability status [The mechanisms most commonly proposed for colorectal cancer recurrence, besides tumor stage, location, and cancer treatment, include y status , and MCMy status . FurtherDiabetic patients and those with increased number of metabolic syndrome conditions are at increased risk of colorectal cancer recurrence  , 97, sugTumor-promoting inflammation is an enabling characteristic of cancer  that has2-isoprostane levels after 12 months of exercise, but no change in fluorescent oxidation products or oxidized low-density lipoprotein levels [Chronic exercise might decrease recurrence risk by lowering systemic oxidative stress, resulting from immune cell overproduction of reactive oxygen species (ROS). The genetic and epigenetic changes induced by ROS may contribute to the initiation and progression of colorectal cancer . Chronicn levels .Multiple epigenetic alterations have been implicated in the development and prognosis  of colorOur review of physical activity and colorectal cancer mechanisms shows that, to date, the strongest evidence relating physical activity to colorectal cancer recurrence in humans relates to “near-diagnosis” processes , namely, weight control and insulin-related pathways. Inflammatory pathways could mediate adenoma (polyp) recurrence in some subgroups , although clarity is needed to understand whether physical activity, sedentary behavior, or body fatness is driving this mediation.Perhaps the greatest immediate opportunity for mechanistic research relates to the role of exercise in early, initiating events in colorectal cancer . Currently, there is very little evidence in humans that exercise modulates these events. Late-stage carcinogenic pathways are also not well understood, in part because epidemiologic survival studies of physical activity often exclude stage IV cases Table  to avoidPresently, there is limited epidemiologic evidence relating physical activity to cancer recurrence. Rather, benefit is inferred from cancer survival studies which often focus on post-diagnosis physical activity in breast, colorectal, and prostate cancer survivors. There may be benefit from considering pre-diagnosis activity as well as cancer stage at diagnosis and timing of recurrence (early/late) in future epidemiologic studies, to guide mechanistic research. In addition, standardized, cancer-specific definitions of recurrence are needed to build a more consistent body of epidemiologic evidence regarding recurrence. The possibility that physical activity-recurrence associations are partly explained by body fatness and sedentary behavior, or perhaps diet quality , must alThere are some indications about how physical activity might influence cancer recurrence mechanisms, although for some mechanisms  there is uncertainty regarding whether or not they are modifiable with exercise while for others , it is unclear if they are strongly predictive of recurrence. Research from RCTs that demonstrate both exercise responsiveness and clinical relevance is needed. Furthermore, studies that examine the impact of exercise on the reversibility of field cancerization or early carcinogenic events , with repeated longitudinal sampling from tissues of interest, are needed.Although our recurrence model focused on colorectal cancer, the same thought process of considering mechanisms separately for precancerous outcomes is useful for studying the role of lifestyle factors in the recurrence of other slow-growing cancers. This level of mechanistic insight is crucial for informing recommendations about when to exercise and for anticipating additive or synergistic effects with other preventive behaviors or treatments.


There is accumulating evidence that immune dysregulation contributes to the pathophysiology of obsessive-compulsive disorder (OCD), Tourette syndrome, and Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS). The mechanistic details of this pathophysiology, however, remain unclear. Here we focus on one particular component of the immune system: microglia, the brain's resident immune cells. The role of microglia in neurodegenerative diseases has been understood in terms of classic, inflammatory activation, which may be both a consequence and a cause of neuronal damage. In OCD and Tourette syndrome, which are not characterized by frank neural degeneration, the potential role of microglial dysregulation is much less clear. Here we review the evidence for a neuroinflammatory etiology and microglial dysregulation in OCD, Tourette syndrome, and PANDAS. We also explore new hypotheses as to the potential contributions of microglial abnormalities to pathophysiology, beyond neuroinflammation, including failures in neuroprotection, lack of support for neuronal survival, and abnormalities in synaptic pruning. Recent advances in neuroimaging and animal model work are creating new opportunities to elucidate these issues. Neuroimmune interactions are increasingly appreciated as both an important regulator of normal brain development and function and a potential contributor to the pathophysiology of a range of neuropsychiatric illnesses. In particular, there is accumulating evidence that immune dysregulation can contribute to obsessive-compulsive disorder (OCD) and Tourette syndrome, at least in a subset of cases , 2. The OCD is characterized by unreasonable or excessive thoughts and fears (obsessions) and/or repetitive behaviors (compulsions) . Tourett Streptococcus -PK11195 (PK), a ligand that binds to the transporter protein (TSPO), which is expressed by activated microglia. Increased PK binding, indicative of inflammatory microglial activation, was observed in the caudate nuclei bilaterally in children with Tourette syndrome [A second recent study used in vivo positron emission tomography (PET) imaging with , which encodes the rate-limiting enzyme in the biosynthesis of histamine, as a rare but high-penetrance genetic cause of Tourette syndrome [ Hdc gene, which recapitulates this molecular abnormality, thus produces an animal model with strong etiologic validity. These mice exhibit behavioral and neurochemical abnormalities seen in patients with Tourette syndrome, further confirming the validity of the model [Tourette syndrome, like most neuropsychiatric conditions, is substantially genetic. Recent work has identified a hypomorphic mutation insyndrome . Knockouhe model , 45. Hdc-KO mice; rather, microglia from this mice exhibit reduced arborization and normal expression of inflammatory markers [We have recently described intriguing abnormalities in microglia activation in this model. We found that microglia cells are not activated in basal conditions in the striatum in markers . A simil markers . The tot markers , 17; a s Hdc-KO mice, compared with wild-type controls. This was accompanied by enhanced induction of the proinflammatory cytokines IL-1β and TNF-α. Taken together, these findings suggest that in this mouse model of Tourette syndrome there is a deficit in microglia-mediated neuroprotection, accompanied by overreactivity to environmental challenge. Such complex mechanisms cannot be appreciated in human studies and reinforce the importance of work in animal models to clarify the mechanisms of microglial dysregulation in neuropsychiatric disease.Inflammatory challenge with bacterial lipopolysaccharide (LPS) dramatically changed this pattern: microglia activation in the striatum was enhanced in Hdc-KO model and their reduced arborization at baseline [The normal number of microglia in thebaseline  contrastbaseline . It is pbaseline , 46 are baseline , 41, 46, Hoxb8 knockout mouse, described above [ Hoxb8 gene, or the consequences of its disruption, to OCD has yet to emerge. To date, there are no imaging or postmortem studies describing microglia abnormalities in patients with OCD.OCD is highly comorbid with Tourette syndrome. Some evidence suggests an immunological contribution to its pathogenesis . In aggred above , 25, has Streptococcus; this clinical syndrome is known as PANDAS, or more generally Pediatric Acute-onset Neuropsychiatric Syndrome (PANS). By analogy with the better-understood pathophysiology of rheumatic fever and Sydenham's chorea, OCD and Tourette syndrome symptoms in these cases have been hypothesized to arise from the development of autoantibodies that cross-react with proteins normally expressed in the brain; this mechanism is known as molecular mimicry. While many details of PANDAS as a clinical entity remain unclear, and some are controversial, the association of immune dysregulation with OCD and Tourette syndrome symptoms in this subset of pediatric patients is increasingly clear [Obsessive-compulsive disorder (OCD) and Tourette syndrome often strike in childhood. In a subset of cases, acute onset temporally coincides with a bout of infectious disease, particularly withly clear .The TSPO/PK PET imaging study of microglial activation described above examined both noninfectious Tourette syndrome and PANDAS . Childre+/Iba1+ activated microglia in the glomerular layer of the olfactory bulb [+ T cells, suggesting that GAS antigens could be presented to Th17 cells by local microglia.A recent study in mice examined the effects of intranasal Group A streptococcal (GAS) infection and may begin to shed light on the role of microglia in PANDAS. Repeated intranasal GAS inoculations result in increasing the number of CD68ory bulb . AbnormaThe role of microglia in neurodegenerative diseases has been understood in terms of classic, inflammatory activation, which may be both a consequence and a cause of neuronal damage. In OCD and Tourette syndrome, which are not characterized by frank neural degeneration, the nature of any contribution of microglial dysregulation to pathophysiology is much less clear. We explore a few possible mechanisms here. Hdc-KO mice display morphological abnormalities in striatal microglia that suggest quiescence, normal expression of inflammatory markers, but a reduced number of IGF-1 expressing microglia.It is increasingly clear that microglia function in both neuroprotection and neurodegeneration. We have described complex abnormalities of microglia in a mouse model of Tourette syndrome . Naïve HH2 cytokines such as IL-4, which induce a neuroprotective phenotype, at least in organotypic hippocampal cultures [+ microglia in this animal model of Tourette syndrome might lead to impaired neuroprotection and, consequently, to enhanced susceptibility to neuroinflammation after an environmental challenge , which could be associated with abnormal synaptic pruning [ Hdc-KO mice [ Hdc-KO mice, and other mouse models of Tourette syndrome, is warranted.Microglia are highly dynamic . Their cmalities . It is p pruning . CX3CR1 -KO mice . InvestiMECP2). MECP-null microglia release dramatically higher levels of glutamate, and microglia-derived glutamate has neurotoxic effects on dendrites and synapses [Increasing evidence suggests a pathophysiologically important dysregulation of glutamatergic signaling in patients with OCD . Interacsynapses . It is p Hdc-KO mouse model of Tourette syndrome [While evidence for microglial dysregulation in OCD, Tourette syndrome, and PANDAS is growing, much remains unclear. The case for microglial dysregulation is strongest in the case of Tourette syndrome; recent postmortem and PET imaging studies have produced convergent evidence for increased microglial activation in the striatum in patients –42. Thissyndrome . We have Hdc-KO model of Tourette syndrome is warranted.Mechanisms by which microglial abnormalities contribute to disease are likely to be shared across distinct etiologies and traditional diagnoses. For instance, abnormal synaptic pruning was observed both in animals inoculated with GAS (which may capture key elements of the pathophysiology of PANDAS) and in mice that develop excessive grooming after inactivation of the progranulin gene. In both cases, increased synaptic pruning cooccurs with microglia activation. In CX3CR1 knockout mice, deficient synaptic pruning accompanies alterations in neuron-microglial communication. This is accompanied by social deficits, which has been interpreted as an autism-like phenotype. Results from these models must be interpreted cautiously, as neither progranulin nor CX3CR1 has been clearly associated with any of these conditions in humans. Abnormal synaptic pruning by microglia in the more pathophysiologically groundedThese studies in animal models are intriguing and allow detailed mechanistic analysis, but their relevance for the understanding of clinical disease remains to be firmly established. Information about microglial abnormalities in patients with OCD, Tourette syndrome, and PANDAS remains very limited. This is a recently opened frontier in our understanding of the pathophysiology of these disorders. It is, however, one of great promise, which may lead to the identification of novel therapeutic targets.


Autoinflammatory diseases (AID) are characterized by seemingly unprovoked self-limited attacks of fever and systemic inflammation potentially leading to amyloidosis. Familial Mediterranean fever (FMF) is the most common AID and therefore the most studied. Besides FMF, the other main hereditary AID are tumor necrosis factor-associated periodic fever syndrome (TRAPS), mevalonate kinase deficiency (MKD), and cryopyrin-associated periodic fever syndrome (CAPS). These hereditary diseases result from a mutant gene that is involved in the regulation of inflammation, resulting in a characteristic clinical phenotype. The differential diagnosis of AID can be challenging due to a wide overlap in clinical manifestations. Moreover, a considerable proportion of patients present with autoinflammatory symptoms but without a pathogenetic variant on genetic analysis. Furthermore, non-hereditary AID, such as the periodic fever, aphthous stomatitis, pharyngitis, adenitis (PFAPA) syndrome, which is the most common AID in children worldwide, must be excluded in certain circumstances. Herein we shall review the main AID and describe a practical approach to diagnosis in a patient with a clinical suspicion of AID. The past two decades have brought a wealth of knowledge regarding the genetic background and the pathogenic mechanisms underlying many autoinflammatory diseases (AID). This newfound understanding assists us in making an earlier diagnosis and facilitates the correct diagnosis of atypical cases.The year 1997 was a seminal year for the AID. The gene for familial Mediterranean fever (FMF) was identified by two groups working independently and in parallel.4Familial Mediterranean fever is characterized by recurrent episodes of fever and serositis. Patients typically develop high-grade fever, which lasts 1–3 days, associated with peritoneal inflammation, pleural involvement, attacks of arthritis, or erysipeloid erythema.5Hence, in this age of genetic testing, our patient population includes atypical cases exhibiting, for example, only recurrent episodes of fever and exertional leg pain, patients manifesting recurrent pleuritis and pericarditis without abdominal attacks, and patients who have recurrent attacks with elevated acute-phase reactants but only rarely have an increase in body temperature.Having secured a diagnosis of FMF, whether based on a typical clinical diagnosis befitting the Tel HaShomer criteria or consisting of a somewhat atypical presentation which in turn is corroborated by genetic testing, the question of treatment comes up. In the vast majority of cases colchicine suffices. A 1–3 mg/d dose in an adult eliminates or attenuates the frequency, duration, and intensity of attacks.Initially thought to represent “severe FMF” due to more prolonged episodes of inflammation (up to 3 weeks) and dominant inheritance pattern, familial Hibernian fever was renamed tumor necrosis factor-associated periodic fever syndrome (TRAPS) when culprit mutations were localized to the p55 tumor necrosis factor (TNF) receptor.16Originally called hyperimmunoglobulinemia D syndrome (HIDS), mevalonate kinase deficiency (MKD) is a syndrome with attacks of intermediate duration (between the 3-day episodes of FMF to the prolonged 3-week episodes of TRAPS). Here, recessive mutations in the mevalonate kinase gene result in impaired mevalonate kinase activity and impaired isoprenoid biosynthesis that leads to autoinflammation, possibly through IL1 overexpression.13de novo mutations in the NLRP3 gene. The clinical syndrome of FCAS includes cold-induced fever, urticaria-like rash, and constitutional symptoms. Muckle–Wells syndrome consists of fever, urticarial rash, and arthritis episodes, unrelated to cold exposure, lasting 1–7 days with the risk of progressive sensorineural hearing loss and amyloidosis developing over time, while NOMID runs a chronic course of fever, urticaria-like rash, epiphyseal overgrowth of long bones, and chronic aseptic meningitis with all its associated ailments. Interleukin-1-blocking agents are the mainstay of therapy of cryopyrin-associated periodic fever syndrome (CAPS).13The cryopyrinopathies encompass a spectrum of disease states including, from mild to severe, familial cold autoinflammatory syndrome (FCAS), Muckle–Wells syndrome (MWS), and neonatal onset multisystem inflammatory disease (NOMID) caused by autosomal dominant or Although not a monogenic periodic fever, periodic fever, aphthous stomatitis, pharyngitis, adenitis (PFAPA) should be part of the differential diagnosis of a patient presenting with recurrent fevers. Here, patients suffer from attacks of fever, exudative pharyngitis, and aphthosis associated with enlarged cervical lymph nodes.A summary of distinguishing clinical features and response to therapy of the common AID is detailed in Having delineated the most common monogenic hereditary periodic fevers, we should now attempt to establish in whom their presence should be suspected. The following five parameters should be utilized:In the majority of cases, hereditary periodic fevers appear in early childhood and therefore pediatricians and family practitioners should be alert, especially when a positive family history or typical ethnic origin is present.In the same pediatric population, rapid appearance of fever without signs or symptoms of a respiratory or a urinary tract infection.Elevation of acute-phase reactants during attacks, with normalization of their levels during inter-critical intervals.Complete well-being between attacks.Repeated attacks and lack of seasonality: typically 4–6 attacks over an observation period of 9−12 months are needed when basing a diagnosis only on clinical manifestations—this period may be shortened by confirmatory genetic testing.http://www.printo.it/periodicfever/) is an evidence-based tool which aids general pediatricians in assessing patients with suspected periodic fevers. Patients with high diagnostic scores should be referred to specialized centers where genetic testing and therapeutic trials will be considered.The diagnostic score available on the PRINTO website (An algorithm which serves to aid in the differential diagnosis of the common periodic fevers and PFAPA is presented in In addition, the EUROFEVER diagnostic/classification criteria published by Federici et al. in 2015 highlight the prominent clinical manifestations of each and every disease.The second decade of the twenty-first century has endowed us with a heightened awareness of autoinflammatory disease. This awareness coupled with improved tools for clinical and genetic diagnosis means that these disorders are now recognized earlier. Furthermore, the understanding of the paramount role of the inflammasome in these diseases and the development of biologic drugs that block its action have opened up new possibilities in the treatment of previously refractory patients.


Despite the improvements in drug screening, high levels of drug attrition persist. Although high-throughput screening platforms permit the testing of compound libraries, poor compound efficacy or unexpected organ toxicity are major causes of attrition. Part of the reason for drug failure resides in the models employed, most of which are not representative of normal organ biology. This same problem affects all the major organs during drug development. Hepatotoxicity and cardiotoxicity are two interesting examples of organ disease and can present in the late stages of drug development, resulting in major cost and increased risk to the patient. Currently, cell-based systems used within industry rely on immortalized or primary cell lines from donated tissue. These models possess significant advantages and disadvantages, but in general display limited relevance to the organ of interest. Recently, stem cell technology has shown promise in drug development and has been proposed as an alternative to current industrial systems. These offerings will provide the field with exciting new models to study human organ biology at scale and in detail. We believe that the recent advances in production of stem cell-derived hepatocytes and cardiomyocytes combined with cutting-edge engineering technologies make them an attractive alternative to current screening models for drug discovery. This will lead to fast failing of poor drugs earlier in the process, delivering safer and more efficacious medicines for the patient. Despite improvements in drug screening, there is still a high percentage of drug attrition during development. This presents in either in pre-clinical modeling, clinical trials or after drug approval, with greater expense incurred the further along the pipeline the compounds are removed. Therefore, fast failing is key to improving the success and the cost of human drug development. The percentage of drug failure at phase II and phase III is high and the main reasons for failure are the lack of efficacy, 48% in phase II and 55% in phase III, and safety, 25% in phase II and 14% in phase III ). Key toCurrent cell-based models used within industry rely heavily on immortalized cell lines, usually derived from human tumors. These models have advantages, such as cost-effective scale up and well to well consistency. Additionally, these cell lines are amenable to genetic engineering, permitting gain and loss of function analysis. While these models demonstrate advantages, they offer limited biological relevance when compared to the intact organ and primary cell types. Currently, primary cells and tissue slices are the gold standard for drug discovery, as they exhibit greater resemblance to the organ of interest. There are however drawbacks with these resources. The main disadvantages of using primary cell types or tissue slices are their labor intensive isolation from diseased organs, the scarcity of donor tissue, the rapid loss of cell phenotype, and significant batch to batch variation .Stem cell technology has shown promise in drug screening ,6 and haThe liver plays a central role in drug disposition; it is responsible for drug uptake, metabolism and excretion. Several factors are involved in the off-target effects of drugs with differential metabolism playing a key role. Cytochrome P450 enzymes play an important roles during drug metabolism, with five family members  responsible for the metabolism of approximately 90% of marketed drugs. Genetic polymorphisms in the CYP450 family members affects drug metabolism, efficacy and safety ,14. PolyDetection of potential cardiotoxic drugs is also important due to the percentage of heart disease that exists in the population . Drug meThe factors discussed above are important considerations for drug development. One major consideration is the diversity of the population, which is not captured by many in vitro models. This requires the development of more sophisticated systems for human drug development. We believe that stem cell-based technologies have the power to capture variability observed in the population. Stem cell-derived hepatocytes and cardiomyocytes have been used successfully to study drug metabolism on defined genetic backgrounds, providing critical proof of concept that pluripotent stem cell-derived cell types are enabling for human drug development . Hepatocyte differentiation attempts to recreate aspects of human liver development using growth factors and small molecules ,12,39–50Hepatocyte monolayer differentiation systems usually consist of stagewise approach where the stem cell populations are driven to definitive endoderm using growth factors such as, activin A and Wnt3a ,51 Fig.. This isRecent advances in the last decade have demonstrated the potential of HLCs as a tool to model human diseases and drug exposure and some examples follow. Rashid et al.  and Cayo2 > 0.7); HLCs and PHHs displayed similar CYP2C9-mediated metabolism for the hepatotoxic substrate benzbromarone; and the impact of CYP2D6 polymorphic variants on CYP2D6 activity was comparable between HLCs and the originating PHHs. The authors noted that future studies should examine whether HLCs differentiated from other cell can also recapitulate interindividual differences in drug metabolism.In addition to the above, collections of iPSC-derived cell lines from multiple donors would facilitate studies aimed to determine whether Bdonor specific^ factors modify the pharmacological profile of drug candidates. For example, panels of human iPSC-derived HLCs with distinct CYP450 genotypes would be useful to uncover potentially significant sources of interindividual variability in drug metabolism. However, widespread implementation of this attractive paradigm is limited by difficulties inherent to the production and characterization of relatively large numbers of iPSC-derived cell lines . FurtherRandomly distributed 2D cultures/co-cultures containing primary human hepatocytes (PHHs) and HLCs are straight-forward to create but do not allow control over homotypic/ heterotypic cell-cell interactions and cell-ECM signaling that are known to affect liver functions in vivo. In contrast, several engineering tools, such as cellular microarrays, protein micropatterning, microfluidics, biomaterial scaffolds, and bioprinting, now allow precise control over the cellular microenvironment to enhance hepatocellular function. Long-term (4+ weeks) stabilization of function typically requires co-cultivation with liver- or non-liver-derived non-parenchymal cell types . For insIn addition to the MPCC platform, PHHs , stem ceThe engineering approaches described above improve HLC function, but do not always elucidate the microenvironmental signals (and their combinations) underlying the observed responses. On the other hand, cellular microarrays, in which viable cells are seeded onto printed spots of materials/biomolecules, represent a powerful approach for precisely defining the optimal microenvironment of cells –75. CellIn 2006 and 2007, Takahashi and Yamanaka demonstrated that the introduction of specific factors into differentiated fibroblasts, of fetal and adult origin, induce cellular reprogramming into PSCs ,83. The 2+ handling properties, and response to beta adrenergic stimulation , contractile force, Camulation . Severalmulation . Exploit2 + indicators, small molecule voltage probes, patch clamp, multi-electrode arrays, impedance measurements, and bright field microscopy.iPSC-derived cardiomyocytes are an attractive model for pharmaceutical research applications. The use of iPSC-derived cardiomyocytes in the context of pharmaceutical applications can be divided in two broad categories: (1) screening of drug candidates to identify compounds with cardiotoxic potential, and (2) in vitro modeling of cardiac diseases for the identification and validation of pharmacological targets. In general, cultures of iPSC cardiomyocytes are suitable for medium to high-throughput assays and high content imaging. There are diverse functional screening platforms that provide a range of information derived from the analysis of either single cells or multiple cells per well (for a review please see ). CellulThe iPSC-derived cardiomyocyte model has become a popular platform for evaluating the impact of drugs on various cellular parameters relevant to cardiac physiology. Some examples include early work by Tanaka et al. investigating the effects of ion channel inhibitors and beta-adrenergic agonists on electrophysiological properties  of human iPSC-cardiomyocytes . Work byDespite the advances, further refinement is required to better model the physiology of the organ of interest. Doing so will increase specificity and sensitivity of the screening models, thereby reducing the potential for off target drug events and failure. The ability to move beyond the current limitations requires interdisciplinary collaboration. By combining the best stem cell models with chemistry, physics, and engineering, new automated screening assays with improved function and physiology can be developed. Current areas of promise are discussed in the final section.Biomaterials and recombinant extracellular matrices have been shown to improve cell phenotype and cell maturity ,94 in boFor screening proposes, stem cell differentiation procedures must be fully reproducible, display low levels of variation between wells and plates and be amenable to high content analysis and multiplexing. Automation of the protocols and a high-throughput assay development is pivotal for this; automated liquid handling systems allows the generation of large quantities of cells with reduced variation making them suitable for testing large compound libraries. Combining this with multiparametric profiling assays such as automated microscopy ,100 or hWhile hepatotoxicity and cardiotoxicity are critical issues to address during pharmaceutical drug development, drugs can also cause toxicity to other organ systems resulting in adverse outcomes. Thus, culture platforms that can adequately mimic organ-organ interaction upon drug exposure are also required. By combining microfluidics, micropatterning, and 3D cultures, it is now possible to model organ microenvironment and organ-organ interaction ,106. SucWe believe that with the recent advances in stem cell differentiation, scale up, and performance, it is possible to create more accurate human tissue models for drug development. Advances in stem cell biology over the last decade has provided the field with more accurate human cell-based models that recapitulate key aspects of human drug metabolism, with better precision than cancer lines. These systems also provide comparable activity to primary cells. While this is encouraging, further improvements are necessary to improve predictive power. Tissue engineering has already played an important role in this space, with organ-on-chip devices now available via several commercial sources. Future efforts in the field should focus on developing high-throughput multi-organ systems, capable of real-time monitoring and multiplexing to reduce costs and improve the quality of data output.


MicroRNAs and siRNAs, both of which are AGO-bound small RNAs, are essential for mammalian spermatogenesis. Although their precise germline roles remain largely uncharacterized, recent discoveries suggest that they function in mechanisms beyond microRNA-mediated post-transcriptional control, playing roles in DNA repair and transcriptional regulation within the nucleus. Here, we discuss the latest findings regarding roles for AGO proteins and their associated small RNAs in the male germline. We integrate genetic, clinical and genomics data, and draw upon findings from non-mammalian models, to examine potential roles for AGO-bound small RNAs during spermatogenesis. Finally, we evaluate the emerging and differing roles for AGOs and AGO-bound small RNAs in the male and female germlines, suggesting potential reasons for these sexual dimorphisms. Summary: This Review article summarizes the latest findings regarding roles for AGO proteins and their associated small RNAs in the male germline, with a particular focus on spermatogenesis. The process of spermatogenesis involves complex, dynamic patterns of gene regulation and chromatin rearrangement. In early embryogenesis A, mammalRecent studies have revealed that small RNAs play essential roles during many of the events that occur during spermatogenesis. Small RNAs are short non-coding RNAs that function by guiding a crucial co-factor – an AGO or PIWI protein of the Argonaute family – to target RNAs. Through this ability to guide regulatory complexes to RNA transcripts in a sequence-specific manner, small RNAs comprise an elegant system of gene regulation. Since their discovery , it has de novo methylation of transposon-encoding genes, thus protecting the germline genome from detrimental transposon accumulation , endogenous small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs)  – all thmulation . Althougmulation , roles fmulation .Beyond their piRNA populations, male germ cells also express a developmentally dynamic landscape of miRNAs and siRNAs . CompareHere, we review the emerging roles of AGO-bound small RNAs, which include miRNAs and siRNAs, during spermatogenesis. We begin by providing an overview of Argonaute proteins, focusing on the AGO subclade and their associated small RNAs. We highlight the mechanisms by which these AGO-bound small RNAs are generated, and how they function to regulate their targets. We next summarize what is known about the identities of miRNAs required for spermatogenesis, and we also describe the characterization of germline siRNAs, which currently are not thought to play an essential role in male germ cell development. We then discuss recent studies that have identified roles for AGO-bound small RNAs in the male germline, integrating recent findings in mammalian somatic cells with those in the germline to speculate on the mechanisms by which small RNAs might function, including novel nuclear roles in heterochromatin formation and DNA damage repair. Finally, we discuss the differing roles for AGO-bound RNAs in the male and female germlines, suggesting potential reasons for why this sexual dimorphism might exist.Schizosaccharomycespombe, possess only a single Argonaute, many have multiple Argonautes, which are often functionally specialized; in Drosophilamelanogaster there are five, Homosapiens eight, Arabidopsisthaliana ten, and Caenorhabditiselegans twenty seven , they often involve extensive base-pairing and thus can lead to transcript cleavage . CleavagDgcr8, Drosha and Dicer germline cKOs are largely congruent, and include infertility (or subfertility), decreased sperm count and disrupted sperm morphology. Although DGCR8, DROSHA and DICER are all small RNA biogenesis factors, they also possess additional, non-overlapping roles in the cell  10 impedes PGC proliferation  3, results in defective progression from the leptotene/zygotene stage of meiotic prophase I to the pachytene stage  mouse models in which the small RNA biogenesis factors DGCR8, DROSHA or DICER were disrupted specifically in the male germline were the foundational experiments that revealed essential roles for AGO-bound small RNAs in spermatogenesis B. The phthe cell . Howeverenotypes B. For exferation  and sperferation . By contne stage , ultimatne stage . Finallyongation . TogetheDicer, Dgcr8 and Drosha cKOs imply that disruption of miRNAs is causative, whereas a defect specific to Dicer cKO animals suggests that siRNAs are crucial for the process. Indeed, comparative studies revealed similar defects in Dicer and Dgcr8, as well as Dicer and Drosha, cKOs during meiotic prophase I and spermatid elongation, thus implicating miRNAs, and not siRNAs, as factors essential to both processes , resulting in a network of downstream effects. The difficulties in identifying the misregulated processes are reflected by the wide variety of explanations for infertility in miRNA-deficient germlines, including upregulation of gene expression from the X and Y chromosomes , an incrDgcr8, Drosha and Dicer cKOs. These studies have highlighted essential roles for the miR-34 family in spermatogenesis in mice, and suggest that members of the miR-17-92 cluster (Mirc1) might also perform important functions.Multiple approaches have been employed to tease out the identities of specific miRNAs that, when disrupted, lead to the germ cell phenotypes observed in Dgcr8, Drosha and Dicer cKOs, suggesting that loss of the miR-34 family in these cKO animals probably contributes to many of the observed defects. Notably, it was observed that PGCs appear normal in miR-34 family knockout mice  had previously been postulated to have a role in the testis, largely owing to its preferential and strong expression in the male germline . Expressout mice . This reDdx4 Cre, resulted in reduced testis size and weight, with many tubules containing only Sertoli cells, although the mice are fertile  – might contribute to early spermatogonial differentiation. Germline conditional loss of the miR-17-92 cluster, driven by  fertile . However fertile . Taken tAnalysis of the expression patterns of miRNAs, together with clinical studies investigating expression changes in infertile men, have identified many other miRNAs with potential roles in spermatogenesis. Although high expression of a miRNA does not necessarily imply functionality, only highly expressed miRNAs are likely to have an impact on target expression . The mosTogether, miRNA knockout mouse studies have demonstrated that the miR-34 family is required for spermatogenesis. Given that multiple miRNA families are dynamically expressed at various stages during spermatogenesis, it is likely that many other miRNA families are also essential. There is strong evidence that at least one of these miRNA families belongs to the miR-17-92 cluster. Finally, miRNA profiling experiments have identified many miRNAs, including miR-103 and miR-17, as being highly expressed during certain stages of gametogenesis, indicating that they might also play an important role in the regulation of male germ cell gene expression.Dicer but not Drosha  clade of Argonautes, acts in the germline to guide small RNAs into the nucleus to target nascent pre-mRNAs and recruit transcriptional silencing co-factors  pathway . For insDDR foci . These dormation . A similormation  and flieormation . Furtherormation . AlthougDicer- and Dgcr8-deficient mouse spermatocytes, which results from fusions of the X and Y chromosomes to autosomes and one another (Dicer- and Dgcr8-deficient spermatocytes, including ATM, a key regulator of the repair response, which is regulated by multiple germline miRNAs. Thus, chromosomal instability in miRNA-deficient germlines may simply derive from a loss of canonical post-transcriptional regulation of DSB repair machinery. To test whether small RNAs directly participate in DNA damage repair during male meiosis (Given the crucial role that DSB repair plays in homologous recombination in germ cells, it is intriguing to consider whether non-canonical regulation of DDR foci by small RNAs occurs during mammalian meiosis. As male mammalian germ cells enter prophase I of meiosis, the protein SPO11 creates multiple DSBs across the chromosomes, activating DNA damage repair at those sites via homologous recombination . The onl another . However meiosis , it will meiosis , and thiAlthough mammalian male and female gametes differ from one another in their morphology and cellular composition, both are generated from the highly regulated differentiation of PGCs into cells that undergo meiosis to produce gametes. Thus, perhaps small RNA-mediated gene regulation would be expected to be similar between males and females, especially before the first meiotic division. Instead, there is considerable sexual dimorphism in the requirement for small RNAs in gametogenesis . FurtherAgo2 is also essential for proper mouse oocyte maturation (Ago2 for male gametogenesis. It is unknown why the miRNA pathway is downregulated in murine oocytes. One explanation is that in females, transposon-silencing siRNAs would have to compete with miRNAs for available AGO proteins, whereas in males the usage of AGO-independent small RNAs, namely piRNAs, avoids such competition. Together, these observations have led to the dogma that in all mammals, siRNAs are the required class of small RNA for oogenesis, and miRNAs and piRNAs the required small RNAs for spermatogenesis.Unlike in male mice, in which miRNAs appear to play important roles throughout spermatogenesis, miRNAs are dispensable for the later stages of oogenesis , includituration ; female Dicer gene, leading to the expression of a unique, truncated isoform of DICER in mouse oocytes that more effectively generates siRNAs from dsRNAs (Muridae family, which includes mice and rats. Therefore, if siRNAs play an important role in the repression of transposons in mouse oocytes, but are only effectively produced by a form of Dicer unique to the Muridae family, how are the deleterious effects of transposons kept at bay in other mammals that lack this unique Dicer isoform? It has recently been revealed that PIWI proteins and piRNAs are dynamically expressed in human, macaque and bovine ovaries, suggesting that piRNAs could repress transposons in oocytes (Dicer isoform in the Muridae family has kindled speculation that in mice and rats AGO-bound siRNAs have supplanted a role typically performed by PIWIL3-bound piRNAs in other eutherian oocytes (Piwil3, as well as other PIWI genes, in mammalian organisms other than mice would determine whether piRNA pathways play essential roles in oogenesis in non-Muridae mammals.Growing evidence, however, suggests this paradigm, with only siRNAs required for oogenesis, might be the exception rather than the norm in mammals. In mice, a retrotransposon that becomes activated in oocytes is found within the m dsRNAs . This mim dsRNAs . However oocytes . Mice (a oocytes , suggest oocytes . DisruptDicer at early stages of oogenesis and reported no major defects prior to oocyte maturation, approximately half of the germ cells at these earlier stages failed to undergo Dicer excision (The discrepancies among mammals regarding which classes of small RNAs are required for female gametogenesis highlight the evolutionary plasticity of germline biology. Furthermore, such discrepancies caution against building a paradigm of mammalian germline regulation based on observations from a single model organism. The apparent diversity in AGO-bound small RNA regulatory strategies in females prompts the question of whether those in males are similarly diverse. It is unclear whether siRNAs are essential for male gametogenesis in mice, and even less is known about their role in other mammals. As for miRNAs, the miR-34 family is highly expressed in the mature testis of many other mammals, including pig, rhesus monkey and cow , and itsexcision . As miRNDgcr8 and Drosha germline cKO mice is caused by the loss of miRNA families in addition to miR-34 will be most efficiently answered by the generation of additional miR-family knockouts, with highly expressed miRNAs demonstrating unique germline expression patterns or those correlating with infertility being the most promising candidates. Such candidates include members of the miR-17-92 cluster, which appear to play a role in the early stages of spermatogenesis, during spermatogonia differentiation. As illustrated by early attempts to study the miR-34 family, redundantly functioning miRNAs are encoded in multiple genomic locations, necessitating a complex knockout strategy. The advent of the CRISPR system greatly simplifies the process of creating multi-loci knockouts, allowing for comprehensive disruptions of miRNA families in order to study their role in spermatogenesis. Given the essential roles many miRNAs play in other tissues, cKO strategies will also be needed in cases where whole-animal knockouts are lethal.The studies reviewed here reveal that AGO proteins and AGO-bound RNAs are increasingly implicated in many of the events occurring during spermatogenesis, although it is clear that further studies are needed to fully define their functions in this context. Notably, of the thousands of AGO-bound small RNAs identified in the male germline, only the miR-34 family has been definitively shown to be essential for spermatogenesis. Thus, roles for the majority of small RNAs in the germline remain to be determined. Whether the infertility of Dicer cKOs mask the phenotypes that are due to nuclear AGO function. In addition, the inability to maintain male germ cells in culture (in vitro differentiation of ESCs into functional spermatozoa (Perhaps the major outstanding question relating to mammalian germline small RNAs regards the existence of their roles beyond post-transcriptional gene regulation. Given that AGO proteins are present in the nucleus of male germ cells, and that examples of nuclear roles for AGO proteins in other organisms or other cell types are plentiful, it seems probable that they function in a manner beyond post-transcriptional gene regulation during spermatogenesis. Why then has so little robust evidence for nuclear AGO function in the male germline been found? One possibility is that such alternative functions do exist, but that the dramatic phenotypes that result from loss of miRNA regulation of mRNAs in  culture  or to earmatozoa  overcomermatozoa . The devC. elegans, which ensure long-term silencing of transposable elements? Could interactions exist between the miRNA and piRNA pathways? Answering these questions will help to explain how male mammalian germ cells are able to coordinate the dramatic chromatin rearrangements, genetic reprogramming and cellular morphogenesis that drive spermatogenesis. The answers to these questions are also likely to shed light on events occurring in somatic cells.Beyond the identities and roles of AGO-bound small RNAs lies a more complex question, which the male germline – home to all three classes of small RNAs – is uniquely poised to answer: how do the functions and pathways mediated by the three classes of small RNAs interconnect? If siRNAs do play a role in the germline, might they interact with the piRNA pathway in a manner similar to WAGO-associated small RNAs in


Mortality remains alarmingly high for patients diagnosed with pancreatic ductal adenocarcinoma (PDAC), with 93% succumbing to the disease within five years. The vast majority of PDAC cases are driven by activating mutations in the proto-oncogene KRAS, which results in constitutive proliferation and survival signaling. As efforts to target RAS and its downstream effectors continue, parallel research aimed at identifying novel targets is also needed in order to improve therapeutic options and efficacy. Recent studies demonstrate that self-renewing cancer stem cells (CSCs) contribute to metastatic dissemination and therapy failure, the causes of mortality from PDAC. Here, we discuss current challenges in PDAC therapeutics, highlight the contribution of mesenchymal/CSC plasticity to PDAC pathogenesis, and propose that targeting the drivers of plasticity will prove beneficial. Increasingly, intrinsic oncogenic and extrinsic pro-growth/survival signaling emanating from the tumor microenvironment (TME) are being implicated in the de novo generation of CSC and regulation of tumor cell plasticity. An improved understanding of key regulators of PDAC plasticity is providing new potential avenues for targeting the properties associated with CSC . Finally, we describe the growing field of therapeutics directed at cancer stem cells and cancer cell plasticity in order to improve the lives of patients with PDAC. Malignancies of the pancreas can be subdivided into two main categories, those arising from exocrine cells, which produce digestive enzymes, and those from endocrine cells, which produce and release hormones such as insulin and glucagon . UpwardsPDAC develops from normal pancreatic epithelium, which transitions first through a non-malignant, neoplastic state referred to as pancreatic intraepithelial neoplasm (PanIN) before culminating in a fully transformed state, and this transformation relies heavily on the early mutation of the oncogene KRAS, with ~90% of PDACs possessing activating RAS mutations . HyperacAs in many other cancer types, research is uncovering how PDAC cells adapt to varying stimuli  by changing cell state. PDAC cells that have an ability to undergo reprograming as the tumor microenvironment (TME) changes are said to have cellular plasticity. The most prominent example of cellular plasticity occurs when an epithelial cancer cell transitions into a migratory, invasive, mesenchymal cell, in a process called epithelial–mesenchymal transition (EMT). This transition involves passing through a series of intermediate states , with soObviously, not all cells are able to respond as fluidly to a changing environment (low plasticity), but those cells that do appear to be an important driving force behind metastatic dissemination and therapy failure. As new insights into the dynamic changes in PDAC cell states and the signaling pathways that contribute to plasticity are defined, new opportunities for targeting the deadly nature of PDAC will emerge. Here, we discuss how tumor cell intrinsic and extrinsic signals from the tumor microenvironment contribute to E–M/CSC plasticity and delve into how identifying novel factors that contribute to E–M/CSC plasticity can lead to the next generation of therapeutics aimed at extending patient survival.In the last 40 years, survival rates for patients with PDAC have remained unchanged, while patients with nearly every other cancer type have seen remarkable increases in survival. This is due, in part, to a lack of early diagnostic measures and the absence of efficient therapeutic options following diagnosis. Only 25% of patients have localized tumors eligible for surgery; the vast majority present with advanced, metastatic disease that cannot be treated with surgery, leaving these patients to receive radiation or chemotherapy. To date, the mainstay therapy for PDAC remains single-agent treatment with gemcitabine, a deoxycytidine analog. Recently, FOLFIRINOX, the combination of 5-fluorouracil (5-FU), leucovorin, irinotecan, and oxaliplatin, has proven beneficial, despite complicated side effects of neutropenia and thrombocytopenia . In the Not surprisingly given the prevalence of RAS mutations in PDAC, early drug development efforts focused on targeting mutant KRAS and downstream RAS effectors, including Rapidly Accelerated Fibrosarcoma (RAF)/Mitogen Activated Protein Kinase (MEK)/Extracellular Signal-Regulated Kinases (ERK) and Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Protein Kinase B (AKT) signaling responsible for driving cancer cell proliferation and survival . HoweverOne strategy that is proving effective is to block the interactions between RAS and binding partners responsible for transmitting signals. A notable clinical trial for advanced pancreatic cancers is assessing rigosertib, which blocks RAS from associating with the RAS-binding domain of RAF, thereby preventing RAF/MEK/ERK signaling . AdditioWhile new drugs continue to be developed and improved upon, one of the major hurdles to be addressed in pancreatic cancer therapeutics is the inaccessibility of the tumors to the administered drugs. A fundamental characteristic of PDAC is the presence of desmoplasia, an alteration and remodeling of the surrounding tumor stroma . This deApproaches to overcome this hurdle by targeting the desmoplasia and fibrosis involve the use of adhesion kinase inhibitors that disrupt tumor cell–stroma interactions. In preclinical testing, Focal Adhesion Kinas (FAK) inhibitors significantly reduced desmoplasia and increased immune surveillance , resultiMetastatic dissemination and therapeutic resistance leading to tumor recurrence are the main reasons behind mortality associated with PDAC. The observation that most patients present with metastatic disease at diagnosis, coupled with studies demonstrating metastatic dissemination can precede clinically evident malignancy, emphasizes the importance of understanding pathways that contribute the metastatic cascade in PDAC ,44. MetaCirculating tumor cells (CTCs) have elevated levels of the key transcription factors ZEB1, SNAI1, and TWIST and their downstream targets . In patiIn PDAC, the ability to survive therapy, engage in self-renewal, and initiate tumor formation with limited cell numbers are hallmarks of the stem cell state ,61,62. AEarly hypotheses purported that CSCs arose from transformed tissue stem cells . ConventG12D/p53R172HKPC mouse model [E–M/CSC plasticity allows cells to switch between cell states . These transitions are often manifested by multiple factors, including but not limited to extrinsic secreted factors in the tumor microenvironment, metabolic stress such as hypoxia, or tumor-intrinsic drivers. Krebs et al. demonstrated a significant reduction in E–M/CSCs following ZEB1 knock-out resulted in the suppression of metastatic outgrowth in the murine KRASse model . Furtherse model , high exse model . Both huse model ,81. FAM8se model . Importase model ,84. FAM8se model .In addition to cancer cell-intrinsic genetic alterations, the TME can influence tumor development and progression, although little remains known about the intricacies of the stromal-tumor cell crosstalk during PDAC development . In expeAberrant TME resulting from increased desmoplasia has been linked to aberrant metabolism and chemotherapy resistance in PDAC. Tumor cells make intrinsic adjustments to survive in the nutrient- and oxygen-deprived niche by altering glucose metabolism, utilizing glutamine instead of depending on glucose and upregulating scavenger pathways of macroautophagy . RecentlWhile the concept of cellular plasticity is fairly new and targeting drivers of this process holds significant promise, further studies to identify and target drivers of Mes/CSC plasticity are still needed. However, since therapeutic resistance has been heavily contributed to the CSC phenotype the attempt at targeting pathways which pre-existing or inherent CSC are dependent on have been an avenue of study for some time ,102,103.Streptomyces albus, selectively targets tumorsphere initiation and growth in CD133+ PDAC CSCs. When combined, salinomycin and gemcitabine drastically reduced the human pancreatic xenograft model’s outgrowth compared to either agent alone [While novel oncogenic signaling is promising, other targets utilizing small molecules inhibitors of molecular targets other than kinases have shown promise. CD133+ CD44+ pancreatic CSCs were shown to be less reliant on glycolysis and sensitive to inhibitors of oxidative phosphorylation such as oligomycin that reduced spherogenic potential and regressed tumors in mice . Whereasnt alone . Interesnt alone . A recennt alone ,120. Metnt alone . Metabolnt alone ,123. Simnt alone .Others have harnessed the immune system in PDAC. For instance, Chimeric Antigen Receptor (CAR) T cells engineered to target a native protein mesothelin overexpressed on pancreatic cancer cells show significant anti-tumor activity independent of chemotherapy or radiation . HarnessWhile a lot of effort has been put into targeting CSC populations, these novel therapies may only be effective at killing pre-existing CSC and not cells undergoing cellular plasticity. A better understanding of how epithelial cancer cells are reprogrammed into mesenchymal/CSC is needed, as more avenues of targeting their generation and maintenance must be pursued. It is evident however, that targeting Mes/CSCs poses new challenges with respect to the timing and specific target needed to alter this cellular plasticity. There is also an urgent need to improve on experimental model systems to better represent the PDAC tumor microenvironment  to identify drivers of Mes/CSC plasticity. Recently, dendritic cells primed with pancreatic CSC lysates were shown to have increased anti-tumor activity, which may serve as a feasible strategy for targeting these difficult to kill cells. The future may envision chimeric antigen receptor therapy targeting the circulating and intra-tumoral Mes/CSC populations. It will be of utmost importance to identify targets of Mes/CSC plasticity exclusive to cancer tissues so as to increase the therapeutic index and maintain the patient’s quality of life.


Drug-induced organ damage stands as a prevalent yet much-neglected issue globally. Keeping in view it’s rising frequency, health care providers stand obliged to be well versed with the de-merits of the agents they prescribe. Drug therapies causing damage present with a non-specific clinical presentation, histological findings or radiology, which further elaborates on the necessity of a conscientious diagnosis. Pulmonary architecture ranging from the airways, lung parenchyma, mediastinum, pleura, pulmonary vasculature or the neuromuscular system, all can fall victim to the dreaded outcomes of this menace. In order to establish successful diagnosis, the definite temporal relation between initiation of drug therapy and the development of the respiratory symptoms needs to be drawn. The most common form of pharmacologically arising lung toxicity is drug-induced pneumonitis or interstitial lung disease. Unfortunately, there is no adequate data available to review the extensiveness of this medication-associated risk in Pakistan which further highlights the necessity of carefully monitoring this overlooked yet assessable malady. Furthermore, identification and surveillance of this drug attributed peril shall help diminish burden on healthcare resources of the country. We present three recent cases of different types of drug-induced lung damage under treatment at our University Hospital. Drug-induced organ damage presents as a common dilemma globally. As per a Swedish study, adverse drug reactions stand as the seventh most common cause of death in Sweden alone  whereas,The lung is one of the vital organs of the human body and because of its large surface area falls victim to damage from various etiological agents, namely infectious diseases, allergens, neoplastic growth, traumatic injuries, pollutants and pharmacological agents. Lung damage due to drug therapy has a non-specific clinical presentation, histological findings, and radiology, thus setting strict demands for appropriate and prompt diagnosis. Around 400 medications are known to cause drug-induced respiratory diseases alone with the true frequency being unknown [Drug-induced lung damage can involve the airways, lung parenchyma, mediastinum, pleura, pulmonary vasculature or the neuromuscular system. The most common form of pharmacologically arising lung toxicity is drug-induced pneumonitis or drug-induced interstitial lung disease (DILD). Though the literature covering such a perilous subject is, unfortunately, scarce, it points out that 10% of patients receiving chemotherapeutic agents develop fibrosis in their lungs . Drug-inIn June 2014, a 60-year-old postmenopausal female was presented to the outpatient clinic with a high-risk triple negative invasive ductal carcinoma of the breast. She began adjuvant treatment with Paclitaxel 80 mg/min, 126.4 mg IV over one hour, Doxorubicin 60 mg (97.2 mg IV over 15mins) and cyclophosphamide 600 mg/min (97.2mg IV over 30mins) every week. After the third and fourth cycles of chemotherapy, she developed progressive fatigue and a transient shortness of breath (SOB) lasting a few days. On auscultation, there were bilateral basal velcro type crackles with the peak flow of 400 ml pulmonary function test (PFT). The computed tomography (CT) scan of the chest showed diffuse ground-glass opacities bilaterally possible representative of hypersensitivity pneumonitis as shown in  with shortness of breath (SOB), dry cough and undocumented fever since one month. Two months earlier, the patient had been admitted for acute SOB and palpitations for a day. The patient was diagnosed with Atrial fibrillation on electrocardiogram (ECG). Amiodarone infusions were administered, along with a prescription for a 200 mg dosage of amiodarone to be taken every day. Four months later the patient presented to pulmonology clinic with SOB and cough for over a month. On physical examination, bilateral lower lung crepitations were heard. On further examinations, the chest X-radiation (X-ray) showed bilateral lower lobe infiltrates consistent with non-cardiac pulmonary edema as shown in  therapy  for every two weeks. After the third cycle, the patient developed shortness of breath with the mild dry cough. She presented to the Pulmonology clinic with worsening shortness of breath over a month accompanied by a mild dry cough. On auscultation, there were end-inspiratory crackles and harsh vesicular breathing. Pulmonary function tests revealed reduced functional residual capacity and a reduction in diffusing capacity from 99% to 61%. The forced expiratory volume was decreased to around 50% with forced expiratory volume/forced vital capacity (FEV/FVC) of about more than 90% confirming a restrictive lung damage. The high-resolution computed tomography (HRCT) showed basal atelectasis and bilateral fibrosis as shown in  depends upon establishing a definite temporal relation between exposure to the causative agent and the development of the respiratory symptoms and signs. It is vital that all other causes of lung damage be excluded to ensure accurate diagnosis of this condition .Various different mechanisms have been postulated to explain the mechanism of drug-induced lung damage. The first one is direct damage to the pneumocytes either via chemical injury to normal lung tissue  or in thPaclitaxel, Doxorubicin, and cyclophosphamide are common adjuvants to breast cancer therapy . CyclophAmiodarone is a bi-iodinated derivative of benzofuran . It is cFour forms of pulmonary toxicity have been reported: Chronic Interstitial pneumonitis which is most common; organizing pneumonia with or without Bronchiolitis obliterans; acute respiratory distress syndrome (ARDS) though this occurs rarely and lastly, a solid pulmonary mass . AmiodarThe incidence of Amiodarone-induced pulmonary toxicity varies from five percent to 15% and is correlated with dosage, the age of the patient, and preexisting lung disease . IndividBleomycin associated lung damage, as seen in one of our cases, is the most frequently reported drug-induced lung disease with Bleomycin-induced pneumonitis (BIP) occurring in zero percent to 46% of the patients receiving the drug for chemotherapy , 21. BleAs stated above, drug-induced lung disease is a diagnosis reached after excluding other causes, and an objective assessment of the patient’s baseline pulmonary status as well as treatment history is vital to differentiate drug-induced disease from a primary pathology. The diagnostic criteria set up by Irey, et al. to diagnose drug reactions is very helpful in assessing the patient  which isThe physical findings of drug-induced lung damage are non-specific and can reveal crackles and digital clubbing  while thThere are no firm guidelines for the treatment of drug-associated interstitial lung disease and therapy tends to be on an empirical basis . WithdraIn conclusion, drug-induced lung disease is an important but frequently overlooked differential in clinical practice. It is of cardinal importance to keep this differential in mind for patients on chemotherapy since they are very prone to develop this condition. Halting the offending drug, instituting supportive therapy and corticosteroids, regular follow-up in the clinic and radiological monitoring to assess the status of the patient’s condition is the mainstay in managing drug-induced lung disease. Through our case series, we aim to highlight and emphasize the importance of this disorder and hope those other institutions will share their experiences with this condition to further raise awareness about such a disease which is particularly prevalent but sadly overlooked in the developing world.


The latter form the essence of what can be defined as the community effect, implying that the growth characteristics of the tumor are dictated by non-cognate intercellular interactions. The generation of this type of functional tumor heterogeneity was previously ascribed to cell non-autonomous effects, though the underlying cellular processes of the phenomena remained incompletely identified . New finThe fulcrum of this activity is the unfolded protein response (UPR), a phylogenetically conserved adaptive response to ER stress. The UPR is mediated by three initiator/sensor ER transmembrane molecules: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6), which are repressed through association with the chaperone 78 kDa glucose-regulated protein (GRP78) during homeostatic proteostasis. When client proteins accumulate in the ER, GRP78 dissociates from these transmembrane proteins, activating downstream signaling to normalize protein folding and secretion through the activation of ER stress responsive genes. Notable concomitant effects also consist in the selective inhibition of translation to reduce ER client protein burden and facilitate a homeostatic balance of cellular functions. Thus, the UPR orchestrates the cellular response to exogenous  and endogenous  stressors that may be native to the tumor microenvironment (TME) . Not surUpon ER stress transmission, receiver cancer cells were found to develop tolerance to subsequent metabolic, pharmacological, and genotoxic stressors, with survival gains observed both in vitro and in vivo. Receiver cells also underwent several other adaptive changes including the transcriptional upregulation of pro-inflammatory cytokine genes, the activation of Wnt signaling, and the cytoplasmic enrichment of telomerase reverse transcriptase (TERT), features which have been independently implicated in driving chemoresistance and tumorigenicity , 5.vs. apoptosis). Strikingly, receiver cancer cells also displayed increased GRP78 expression intracellularly and at the cell surface, which persisted for over one week. Given the wealth of reports linking GRP78 to tumor cell survival [The UPR machinery appeared to be durably altered in receiver cells, with particular effects on the PERK pathway and GRP78. Upon ER stress transmission, receiver cancer cells decreased expression of both PERK and its downstream effector ATF4, during homeostatic as well as stress conditions. Since PERK activation leads to the transcription and translation of ATF4, which drives expression of the pro-apoptotic target CHOP , we concsurvival , cytoproThese studies demonstrate that UPR-based intercellular communication could drive functionally-heterogeneous tumor cell clones. Differential expression of UPR genes and proteins  may account for differential clonal survival in the same tumor. However, no clinical studies exist to confirm the conclusion that, independent of driver mutations, tumors cells have differential expression of the UPR. Conceptually, intercellular communication through UPR-based signaling represents a new mechanism to regulate population dynamics and contribute to intra-tumor heterogeneity, allowing neighboring cells to acquire growth advantage when selective pressures  emerge.What are the therapeutic implications of these findings? The dynamics and function of different cell types in the TME pose considerable challenges for cancer therapy, including chemotherapy and immunotherapy. Earlier experiments indicate that transmissible ER stress drives direct immunomodulation of innate immune cells, macrophages and dendritic cells, and indirect modulation of T cells during their activation from naïve to effector stage . TherefoAssessing the multiple effects, and associated mechanisms of action, on different cellular targets in the tumor microenvironment in response to intercellular communication of ER stress may be key to future therapeutic interventions. For this to be effective, attention should be paid to specific signaling targets in relation to tumor type and history, immune phenotype of infiltrating cells, genomic heterogeneity, and growth dynamics. Targeting intercellular ER stress transmission, and broadly the UPR, may become part of a future interventions in association with chemotherapy and immunotherapy.


Editorial on the Research TopicMonitoring Pathophysiology in the Injured BrainBrain injuries can be caused by, for instance, spontaneous hemorrhages, thromboembolic incidents or traumatic events, and are considerable sources of morbidity and mortality . Brain iIn this Research Topic, a number of authors from several key centers of excellence worldwide have shared their knowledge on the monitoring of acute brain injuries. These contributions provide updated knowledge of the pathophysiology of TBI and other acute brain injuries, as well as of refining patient management strategies. The overall goal of improving patient outcomes by the detection of deleterious secondary injury processes occurring in the injured brain.Stovell et al. reviewed the monitoring of brain metabolism using magnetic resonance spectroscopy (MRS) in acute TBI. While this technique is still in its infancy, it holds much promise as increasing evidence suggests that deranged metabolism is a major exacerbating factor following brain injury, especially in relation to mitochondrial dysfunction. The MRS technique shows potential for the evaluation of specific brain regions where tissue fate may be analyzed granularly in both acute and chronic phases of brain injury.Rostami et al. evaluated the correlation between cerebral blood flow and metabolism in patients following SAH monitored using Xenon-CT in combination with cerebral microdialysis (CMD). They observed that reduced global blood flow the first 3 days post-SAH was significantly associated with brain metabolism monitored using CMD, specifically high levels of lactate indicating anaerobic metabolism. These results suggest that monitoring of cerebral blood flow and brain neurochemistry could aid physicians and guide treatments, such as optimizing cerebral perfusion pressure (CPP) and adjusting oxygen and glucose substrate levels in SAH patients.Lindblad et al. evaluated multiple electrode aggregometry (Multiplate®) used to assess platelet functions in NICU-treated TBI patients. Although platelet count may be normal, several studies have shown that they do not function properly following TBI. Thus, the primary hemostasis may be altered, specifically by an impaired response of the arachidonic acid (ASPI) receptor. The authors could show that 65% of the included TBI patients had abnormally low ASPI values. This was associated with a more unfavorable outcome, although interestingly not with hemorrhagic progression. These results highlight the need for future research and validated methods of platelet assessment for patients not on platelet anti-aggregation therapies.Marklund evaluated the current role of the neurological wake-up test (NWT) in neurocritical care monitoring, reviewing the available literature. While intracranial pressure (ICP) and CPP monitoring as well as serum markers indicate that the NWT could induce a mild stress response including increased ICP and the release of stress-related hormones, the consequences of these are not fully understood. While qualitative prospective studies concerning its safety are lacking, the NWT remains an important monitoring tool in selected TBI and SAH patients, preferably in combination with multimodal monitoring.Carteron et al. report on how CMD has allowed them to individualize neurocritical care therapy in TBI and SAH patients. Specifically, CMD may help guide optimization of CPP, blood transfusions, glucose infusions, and brain tissue oxygenation targets. Newer markers include electrolytes, markers of oxidative stress, or endothelial proteins, which may also be measured by CMD in order to better predict outcome or guide treatment.Helbok et al. conducted a systematic review on the clinical use of CMD in patients with aneurysmal SAH, focusing on secondary brain injury and clinical outcome. They found that the metabolic changes detected by CMD were associated with early and delayed secondary brain injuries and suggested that CMD be used in conjuncture with other monitoring modalities. In summary, while CMD in SAH is less studied than in TBI, it is an emerging area that might establish itself as a future standard monitor of SAH patients, awaiting additional investigations and multi-center trials.Ercole et al. review and summarize several different technologies to monitor molecular signals in TBI. Mass spectroscopy as well as optical spectroscopy also hold a potential to unravel the in vivo molecular signatures in TBI patients. Altogether, by introducing an “-omics” approach in TBI, it will be possible to gain further understanding of the systems biology involved.Posti et al. reviewed the field of metabolomic monitoring as a diagnostic tool in severe TBI, by investigating different metabolites , and how they are associated with injury severity and clinical outcome. This is a promising field although larger prospective trials are necessary to establish better thresholds and specific metabolic patterns associated with brain injury and patient outcome.Thelin et al. analyzed the current literature, focusing on some of the most commonly studied biomarkers including S100B, neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), and neurofilament light (NF-L) in order to review serum level dynamics of these proteins following TBI. In aggregate, shorter half-lives were seen for S100B and UCH-L1 in contrast to NSE, GFAP, and especially NF-L that exhibit increased levels for prolonged periods of time post-injury. These differences should be taken into account when assessing the capability of these biomarkers to predict outcome and monitor secondary pathologies in TBI patients.Tsitsopoulos et al. reviewed possibilities for short- and long-term monitoring of axonal injury in TBI. Biomarkers, specifically NF-L, is mentioned as a potential diagnostic candidate, as it has been found enriched in myelinated axons and seen to be elevated in serum in patients with radiologically verified DAI. Novel MRI tools are increasingly used for the detection and progress of axonal injury.Zeiler et al. reviewed the literature on CMD and CSF cytokines in both TBI and SAH patients. They could conclude that there is an association with elevated levels of certain cytokines, among others, interleukin-6 and tumor necrosis factor alpha, and long-term functional outcome. In summary, cytokines as mediators of inflammatory activity can be monitored in the extracellular fluid by CMD and in CSF and appear to be associated with secondary injury pathophysiology and clinical outcome in both TBI and SAH.Thelin et al. reviewed several modalities that are used to monitor the neuroinflammatory response following acute brain injury, focusing on TBI and SAH. In summary, the authors suggest a multimodal monitoring approach in order to improve the understanding of the neuroinflammatory response following acute brain injury to determine its role for tissue as well as patient outcome.Vink et al. summarize the role of Substance P in the secondary pathophysiology of TBI, reviewing its role in the neurogenic inflammation and association to ICP increases as observed in several of their studies. In summary, the role of substance P in neurogenic inflammation following TBI is increasingly understood and opens up potential future therapeutic targets for patients suffering from acute brain injury.Zoerle et al. address in their review how to rethink the concept of neuroprotection. They suggest that different aspects of TBI care need to be refined and related to TBI pathophysiology, thus requiring individualized care in part guided by advanced monitoring approaches. These monitoring tools could prompt the clinicians to earlier detection of signs that may need treatment. By using these improved neurocritical care interventions, more adequate neuroprotection might be achieved.Nordström et al. have written a summary on aspects of the physiological and biochemical foundations of NCC treatment strategies. Here, they address the basic concepts of NCC with focus on the key aspects that form the base for the Lund concept, including intracapillary hydrostatic pressure modulation, fluid therapies, and the implementation of brain tissue oxygen- and CMD monitoring.Grände critically evaluates the Lund concept, 25 years after its introduction. He focuses his review on temperature, ventilation, nutrition, osmotherapy, decompressive craniectomy, and sedation management. Following the introduction of more advanced monitoring techniques such as CMD, brain tissue oxygen monitoring, and other ICP- and CPP-guiding protocols, many current NCC strategies and targets have gradually approached several of those comprising the Lund concept.Figaji reviewed how treatment and monitoring of TBI and spinal cord injury differs between adults and children. As especially younger children have different anatomy and physiology than adults, there are several aspects, which need to be considered by the treating physician. One of the main findings of this literature review is the lack of suitable studies focusing primarily on the pediatric CNS following acute brain and spinal injury. This lack of knowledge could lead to adult thresholds and ranges for intracranial monitoring parameters being used in many pediatric situations, which might be inappropriate or even harmful to this patient population.In this research topic, many aspects of the current knowledge and possible future direction of multimodal monitoring in TBI patients are reviewed. Commonly, only limited aspects of the underlying pathophysiology in acute brain injury can and are monitored in the clinical setting. Introducing additional monitoring modalities including parameters such as inflammation markers, multiple metabolites, and a more granular evaluation of structural injuries, a more comprehensive understanding of the evolution of TBI pathology and ongoing secondary injury processes may emerge in the near future. This will require continued large research efforts, but is expected to facilitate development of novel therapeutic options, personalized medicine, and treatment strategies for patients suffering from acute brain injury.ET, DN, AH, and NM confirm being the sole contributors of this work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite tremendous research efforts focused on diagnosis and treatment, pancreatic ductal adenocarcinoma remains the third leading cause of cancer-related death in the United States, with a 5-year overall survival rate of less than 5%. Although resistance is rather complex, emerging evidence has demonstrated that epigenetic alterations (e.g. miRNA) have important roles in PDAC progression as well as resistance to therapy. Certain miRNAs have been identified as potential prognostic biomarkers in PDAC. In this review, we summarize the recent developments in miRNA research related to PDAC therapeutic resistance mechanisms and the potential of miRNAs as prognostic biomarkers for future clinical management of PDAC. Pancreatic ductal adenocarcinoma (PDAC) is the third deadliest cancer in the United States . It is cIn addition to patients presenting with advanced disease, many patients also experience early appearance of post-operative recurrence . TherefoBased on a large body of growing evidence, we know PDAC resistance is regulated, at least in part, by epigenetic alterations including miRNA. miRNAs are small non-coding RNAs 18–22 nucleotides in length that have been identified to be associated with tumorigenesis, cell cycle control, apoptosis, proliferation, chemoresistance, invasion and metastasis . In PDACIn terms of resistance of PDAC to chemotherapeutic treatment, miR-21 is one of the most investigated oncogenic miRNAs related to gemcitabine resistance. Elevated expression of miR-21 inhibits the anti-tumor activity of gemcitabine, and is significantly associated with shorter survival time . GiovannPrevious studies have demonstrated that the miR-34 family  is associated with p53 and p38-MAPK pathways in response to DNA damage . Down-reZhang et al. suggested that miR-214 enhances chemoresistance to gemcitabine by down-regulating the tumor suppressor gene ING4, while miR-15a can suppress the growth of chemoresistant PDAC cells via targeting WNT3A and FGF7, contributing to progression and proliferation through the phosphorylation of the kinases ERK and AKT . MoreovemiR-155 expression has been shown to induce gemcitabine resistance. Prolonged exposure to gemcitabine leads to increased miR-155 expression, which inhibits apoptosis and increases exosome production, resulting in gemcitabine resistance . Li et aBased on the poor prognosis of PDAC, the development of early detection methods, more effective treatment options and better prognostic biomarkers are of critical significance. Besides the significance of miRNAs for early detection and diagnosis, accumulating evidence suggests that miRNAs have great potential as prognostic biomarkers , 34. DilBeyond miRNA, other types of noncoding RNAs  have also been implicated in cancer resistance and prognosis –42. It hIt is clear that PDAC utilizes a variety of mechanisms to maintain a highly resistant phenotype. The highly plastic nature of PDAC resistance is mediated by genetic and epigenetic alterations. The epigenetic controls such as miRNAs allow cells to quickly adapt to the genotoxic stress environment caused by chemotherapy. miRNAs can quickly modulate mRNA translation in PDAC cells in response to chemotherapeutic treatment. As a result, a number of miRNAs have shown great potential as prognostic biomarkers in PDAC. Hopefully these biomarker miRNAs will form a solid foundation to better manage clinical treatment strategies to enhance survival benefits and avoid toxicity. Beyond miRNAs as prognostic biomarkers, as miRNAs are multi-targeted entities that suppress a number of key targets and pathways, some of these miRNAs will be good candidates to develop as novel therapeutics for overcoming PDAC resistance.


Although the initiation of injection drug use has been well characterized among at-risk youth, factors that support or impede cessation of injection drug use have received less attention. We sought to identify socioeconomic factors associated with cessation of injection drug use among street-involved youth.From September 2005 to May 2015, data were collected from the At-Risk Youth Study (ARYS), a prospective cohort study of street-involved youth in Vancouver, Canada. Multivariate extended Cox regression was utilized to identify socioeconomic factors associated with cessation of injection drug use for six months or longer among youth who were actively injecting.Among 383 participants, 171 (44.6%) youth reported having ceased injection  at some point during study follow-up. Youth who had recently dealt drugs , engaged in prohibited street-based income generation , and engaged in illegal income generating activities  were significantly less likely to report cessation of injection drug use.Our findings suggest that socioeconomic factors, in particular engagement in prohibited street-based and illegal income generating activities, may pose barriers to ceasing injection drug use among this population. Effort to improve access to stable and secure income, as well as employment opportunities may assist youth in transitioning away from injection drug use.Our study is not a randomized controlled trial; thus the trial registration is not applicable. Youth who are street-involved, defined as being homeless or using services for homeless youth, experience excess morbidity and mortality relative to the general population of adolescents and young adults , 2. AlthAmong adult populations of people who inject drugs, numerous factors have been associated with cessation of injection drug use including stable housing, use of supervised injection facilities and engagement with addiction treatment , 6. AmonThe At-Risk Youth Study (ARYS) is an ongoing prospective cohort study of street-involved youth in Vancouver, Canada. This study has been described in detail previously . In brieAll participants who completed a baseline survey and were seen for study follow-up between September 2005 and May 2015 were eligible for the study. The present analysis was restricted to participants who reported active injection  and who returned for at least one additional follow-up visit to assess for cessation of injection drug use. The primary outcome of interest was self-reported cessation of injection drug use during the preceding six months at any follow-up visit. Specifically, participants were asked, “In the last six months, have you used a needle to chip, fix, or muscle even once (yes vs. no)?”Socioeconomic factors that we hypothesized might be associated with cessation of injection drug use included: homelessness; living with family; eviction from housing; living in the Downtown Eastside neighborhood (Vancouver’s drug use epicenter); employment ; loss of income assistance (being cut off or denied income assistance); health care access ; incarceration ; sex work involvement ; drug dealing; engaging in prohibited street-based income generating activities ; and engaging in illegal income generating activities . All socioeconomic factors were time-updated measures based on activities or situations in the preceding six months time period. To protect against reverse causation whereby reported socioeconomic factors were a consequence of injection cessation, measures were taken from the study follow-up visit that preceded the visit at which a participant reported cessation of injection.The following mental health related factors which we hypothesized might influence socioeconomic status were also considered: self-reported history of mental illness ; childhood physical or sexual abuse ; and depression at study baseline . Mental health related measures were not time-updated.We also considered sociodemographic and drug-use related factors that we hypothesized, based on a review of the prior available literature, might potentially confound the relationship between socioeconomic factors and injection cessation , 11. The, no further person-time at risk was contributed by that participant), but if they reported resuming injection drug use at a later visit, they reentered the cohort of individuals at risk; participants who did not report any cessation were right-censored at the time of their last follow-up visit. We also used an extended Cox proportional hazards regression model with time-updated variables to examine bivariate associations between each of the sociodemographic and socioeconomic factors, and time to cessation of injection drug use. The extended Cox model has been validated  21–24) year, 248 (64.8%) were male and 276 (72.1%) were white. An additional 151 youth reported injection drug use at study enrollment but did not return or were not yet eligible to return for a study visit due to the nature of an open cohort study. The 383 youth who completed a study follow-up visit were similar to the 151 who did not with regard to all study variables at baseline  and other prohibited and illegal street-based income sources (82%) , 19. YouOur study contributes to the understanding that stable and safe income sources are critical for the health and well-being of street-involved youth , 20, 21.There are several limitations to this study. First, the ARYS cohort is not a random sample. Our snowball sampling methods may have reduced heterogeneity and validity of the findings, although it is noteworthy that the characteristics of the ARYS sample are similar to those from other cohorts of street-involved youth , 11. AnoIn sum, our study suggests that economic vulnerability characterized by resorting to risky income generation strategies including drug dealing, prohibited street-based and other illegal activities, may pose barriers for street-involved youth to cease injection drug use. These findings underscore the potential for social interventions that provide stable and secured income sources to influence drug use trajectories and reduce drug related harm. Further study in this area is warranted.