A neuronal dysfunction based on the imbalance between excitatory and inhibitory cortical-subcortical neurotransmission seems at the basis of migraine. Intercritical neuronal abnormal excitability can culminate in the bioelectrical phenomenon of Cortical Spreading Depression (CSD) with secondary involvement of the vascular system and release of inflammatory mediators, modulating in turn neuronal activity. Neuronal dysfunction encompasses the altered connectivity between the brain areas implicated in the genesis, maintenance and chronic evolution of migraine. Advanced neuroimaging techniques allow to identify changes in functional connectivity (FC) between brain areas involved in pain processes. Through a narrative review, we re-searched case-control studies on FC in migraine, between 2015 and 2020, by inserting the words migraine, fMRI, EEG, MEG, connectivity, pain in Pubmed. Studies on FC have shown that cortical processes, in the neurolimbic pain network, are likely to be prevalent for triggering attacks, in response to predisposing factors, and that these lead to a demodulation of the subcortical areas, at the basis of migraine maintenance. The link between brain dysfunction and peripheral interactions through the inhibition of CGRP, the main mediator of sterile migraine inflammation needs to be further investigated. Preliminary evidence could suggest that peripheral nerves inference at somatic and trigeminal levels, appears to change brain FC. The 2013 Global Burden of Disease Study reports migraine to be the sixth highest cause of years lost due to disability worldwide [Nowadays, the pathophysiological perspective outlines neuronal dysfunction as a predisposing factor to migraine attack onset, with secondary involvement of the vascular system. The cortical excitability of the migraine brain is different from the not migraine one, with a critical unbalance between excitatory and inhibitory neurotransmission, fluctuating across migraine phases. A genetic predisposition to such brain dysfunction ,5, couldThis is a narrative review of recent studies on brain connectivity in migraine, focusing on fMRI and EEG/MEG studies reporting connectivity changes related to pain processing mechanisms.This review intends to provide for a mechanistic hypothesis about the possible interference on the migraine dynamic model from the novel CGRP-related peripheral approach.In this narrative review, we included studies based on PubMed search for migraine, fMRI, EEG, MEG, connectivity, pain in the 2015–2020 time window. We included only case-control studies, excluding case reports, reviews, animal models.Since migraine is primarily a disorder of brain function and excitability, functional MRI (fMRI) studies represent a powerful tool to explore migraine neural correlates. In this frame, fMRI investigations fall into two main categories, specifically the event-related and the resting state ones, exploring respectively the brain activity in response to tasks and the connectivity of specific brain areas or networks at rest. The latter relays on several methodological approaches such as seed-based analysis, independent component analysis (ICA) and graph theory-based analysis .Altogether, resting-state (RS) advanced neuroimaging techniques allow the identification of neuro-anatomical and biologically meaningful spatial brain maps organized in distributed and reproducible functional networks showing spontaneous and simultaneous fluctuations. The seed-based analysis is a model-based method looking for the linear correlation between a selected region of interest called “seed” and all the other voxels in the whole brain, thereby yielding seed-based FC maps. The seed-based analysis is a very good approach—due to its straightforwardness and interpretability—for exploring RS-fMRI functional connectivity (FC), especially when a strong “a priori” hypothesis is available. Accordingly, based on the pioneering advanced neuroimaging studies suggesting the involvement of specific brainstem areas in migraine attacks ignition, the so-called brainstem “migraine generator” has been assumed as a valuable “region of interest” in the seed-based investigations . TherefoNevertheless, the brainstem is only one of the subcortical structures that RS-fMRI investigations focused on to clarify the FC correlates underlying pathophysiological mechanisms. In this way, in the last decade, the hypothalamus has been widely investigated and its altered FC with specific brain regions involved in the regulation of sympathetic and parasympathetic functions  has been demonstrated to be helpful in explaining some of the hypothalamic-mediated autonomic symptoms that accompany or precede migraine attacks . HoweverIt is noteworthy that the thalamus constitutes the hub of central processing and integration of nociceptive inputs. In this context, several data have recently emerged on the role of altered thalamo-cortical FC patterns subtending multisensory integration abnormalities, deficits in cognitive attention, and evaluation of pain as well as in pain modulation in migraine patients. In particular, significantly weaker FC between the anterior dorsal thalamic nucleus and precuneus as well as between the ventral posterior nucleus and precuneus, inferior parietal lobule and middle frontal gyrus ,28 has bTo further complicate the complex migraine pathophysiology, very recently FC abnormalities have been identified between the cerebellum  and thalamus and other higher cortical areas  such as In the context of subcortical structures, recent observations have supported the different role of the left or right amygdala in migraine pathophysiological mechanisms, suggesting the involvement of the left amygdala in migraine genesis as well as of the right amygdala in migraine chronicization. More clearly, a stronger FC between the amygdala and both the middle cingulate cortex and precuneus of the left hemisphere has been recently observed in patients with episodic migraine compared to healthy control (HC). Contrariwise, a stronger FC between the bilateral amygdala and widespread regions of the inferior temporal lobe, prefrontal gyrus, cingulate cortex and pre- and post-central gyri of the left and right hemispheres has been ascertained in patients with chronic migraine when compared to patients with episodic migraine. In another study, the amygdala, sensitized by means of repeated episodes of aura, may strengthen its connectivity with visceroceptive areas of the neurolimbic pain network (including insular and somatosensory cortices) . The facThe RS-fMRI ICA method relies on spontaneous low frequency fluctuations emerging from spatially separated, functionally linked, continuously communicating anatomical areas encompassing the so-called RS networks. Indeed, ICA facilitates the effective extraction from the background noise of distinct RS-fMRI networks by employing mathematical algorithms to decompose the signal from whole brain voxels to spatially and temporally independent components. The insights from the investigations of networks such as the sensory-motor network (SMN), default-mode network (DMN), central executive network (CEN), salience network (SN) and visual network (VN) in migraine patients, have shed light on the obscurity of the complex pathophysiology of migraine.SMN-FC abnormalities including somatosensory  and motor  regions (extending to the supplementary motor areas) have been consistently demonstrated in migraine without aura (MwoA) patients. Specifically, migraine patients exhibited significantly weaker FC between the primary somatosensory area and several cortical brain regions involved in pain intensity and spatial discrimination as well as in trigemino-thalamus-cortical nociceptive pathways ,35. It cBy means of RS-fMRI ICA approach, a plethora of studies have been conducted to investigate the cognitive networks in order to understand the bidirectional relationship between pain experience and cognitive, affective and emotional dysregulation. The DMN is a well-known network highly relevant for self-referential cognitive processes, involved during stressful experiences, and coping strategies which are important to promote adaptation widely investigated in migraine patients, with apparently conflicting results so far ,37. IndeThe CEN is a cognitive network responsible for high-level cognitive functions and known to underlie neuronal correlates of executive function, a set of high-order cognitive processes involved in multiple aspects of individual daily living experiences such as planning for the future and adaptation . In line with neuropsychological studies, showing a slight impairment of complex executive functions including the goals maintenance, the correct allocation of attentional resources, and performance monitoring in migraine patients, a reduced FC within the CEN  has been previously reported in both MwoA and migraine with aura (MwA) when compared to HC. Interestingly, precisely CEN-FC abnormalities have been recently demonstrated to predict the development of CA, working as putative advanced neuroimaging prognostic biomarkers able to identify those patients who will develop CA over time and, therefore, those patients more prone to chronic migraine as well .The brain network that constitutes the so-called SN encompasses areas involved in the evaluation of specific inputs from external or internal behavior by assigning the appropriate relevance to stimuli for continuous processing and pain modulation. A decreased SN-FC centered in the anterior cingulate cortex, anterior insula, and parahippocampal gyrus has been consistently observed in migraine patients when compared to HC suggesting a dysfunctional mechanism leading to the excessive attribution of “salience” to normally non-salient stimuli .Altogether, the observed abnormalities of RS-FC networks subtending adaptive behavior could lead to interpret migraine through the lens of maladaptive stress responses or as a model of “allostatic overload” brain disease. In other terms, stressors trigger migraine attacks but repetitive and frequent attacks can work as stressors themselves, generating a vicious cycle able to disrupt neuro-limbic pain networks and to induce migraine chronicity .The high prevalence of photophobia during the attacks as well as the light hypersensitivity during the interictal period and the occurrence of visual stimuli as trigger factors in migraine patients have induced a growing interest in the visual pathway and, more recently on VN, in order to clarify whether FC abnormalities may be part of a constitutional pattern (likely aimed to an adaptive/defensive mechanism) or represent an acquired sensitization phenomenon in migraine patients. In this context, a significantly increased FC in the lingual gyrus within the VN in patients with MwA when compared to both patients with MwoA and HC has been found during the interictal period, supporting the hypothesis of an extrastriate cortex involvement in the genesis and propagation of aura phenomenon and the putatively subtending cortical spreading depression. On the other hand, even more fMRI studies support the visual cortex involvement also in migraine patients not experiencing the aura phenomenon . More spRS-FC studies have been recently conducted to identify the link between the aura phenomenon and the trigemino-vascular complex activation (known to subtend the headache phase), an unsolved conundrum about MwA pathophysiology . Among tIn addition to the abnormal intrinsic connectivity of the main RSN, a disrupted large-scale cognitive network crosstalk has been demonstrated in chronic migraine patients. More specifically, chronic migraine patients showed a significantly reduced FC between the CEN and both the DMN and the dorsal attention network (DAS) and a significantly stronger FC between the DAS and the DMN . SimilarIn a recent study, Schwedt and colleagues, looking for a putative FC migraine signature, demonstrated that migraine is associated with several highly reproducible FC alterations in different brain regions enough for constituting a brain-connectivity neuroimaging biomarker, to differentiate migraine patients from HC . More spIn the last decades, driven by the ascendancy of network science, a novel RS-fMRI methodological approach emerged to explore the structural neural substrates enabling functional communication within the brain “connectome”. According to the graph theory analysis, the brain network is investigated as a set of grey matter regions (nodes) structurally connected by white matter paths (edges). Two distinct dimensions , known by the names of ‘segregation’ and ‘integration’, provide a general framework that allows the description and the categorization of different disorders. Specially, in line with previous electroencephalographic and magnetoencephalographic observations in migraine patients, increased global efficiency and clustering of cortical areas ,50,51,52One of the most widely used methods to study brain activity in migraineurs is to send a controlled painful stimulus to the trigeminal region or to an extra-cephalic region, such as the back of the hand, and to analyze the evoked activity of the BOLD signal to the functional MRI. Both caloric stimuli and gaseous ammonia have been used in this regard. Compared to healthy subjects, the vast majority of studies carried out in migraineurs between attacks revealed an increase in BOLD activity of cortico-subcortical brain areas involved in noxious information processing and anti-nociception ,56,57,58In one study, the strength of the pain-induced BOLD signal changes did not correlate with the presence of psychopathology . MehnertUsing gaseous ammonia as painful stimulation, researchers found that the interictal reduced BOLD activation strength within the brainstem  ,58 was pThe functional BOLD activity related to the pain stimulus was also used to investigate the pathophysiology of the migraine attack and the hours immediately preceding it.In a series of patients affected by episodic migraine scanned every day for 1-month, the hypothalamus was significantly more activated during the 48 h preceding the beginning of a migraine attack . During During the attacks, sometimes compared with the interictal phase other times with the responses from healthy controls, a greater BOLD signal was detected in response to noxious stimuli within the temporal lobes , the dorIn the There are only a few papers exploring the effects of drugs used for migraine therapy on cortical processing of pain information see .Some researchers observed that 2 to 3-month treatment with the beta-blocker metoprolol did not induce any major different BOLD activation on the trigeminal pain processing of patients with migraine. However, using a more liberal and exploratory statistical threshold, the BOLD signal intensity within the hypothalamus increased during the delivery of pain stimuli compared to that under placebo suggesting a more peripheral effects of metoprolol with negligible effects  A recent fMRI study of the same group of researchers challenged this interpretation. Indeed, they explored the effects of 2-weeks treatment with Erenumab, a monoclonal antibody against the CGRP receptor, on central pain processing and found decreased BOLD activation within the thalamus contralateral to the stimulated side, of right middle temporal gyrus, right lingual gyrus, left operculum, and of both sides of the cerebellum  ThereforThe different way in which migraineurs process multimodal stimuli could be explored by methods with optimal time resolution . The higA study applied effective connectivity using nonlinear Granger Causality (GC) and brain networking analysis to 65 channels basal and visual stimulation related EEG in 19 migraine with aura (MA), 19 without aura (MO), and 11 controls and compared these findings with signal changes. A different pattern of reduced vs. increased GC respectively in MO and MA patients, as compared to controls, emerged in resting state specially in the occipital cortex, outlining a different pattern of connections and information exchange in the parietal-occipital regions, confirmed by the enhanced occipital bold signal in MA patients during visual stimulation .A more recent study confirmed that the EEG-based resting-state connectivity is different between migraine with and without aura, as patients with aura had overall lower connectivity within the theta band in the cortical areas within the Default Mode Network (DMN) and Resting State Networks (RSN) .Interestingly, EEG phase coherence fluctuated across migraine phases, as demonstrated by Cao et al.  in 50 miOne of the first MEG connectivity study in migraine described significantly increased functional connectivity in the slow wave (0.1–1 Hz) band in the frontal area as compared with controls, with migraine with aura showing increased functional connectivity in the theta (4–8 Hz) band in the occipital area. The networking structure appeared also different in migraineurs as compared to controls, indicating an abnormality of functional connectivity in both low- and high-frequency ranges .Recently, Nieboer et al. , estimatA MEG study evaluated possible correlations between individual pain sensitivity and functional connectivity at 2 to 59 Hz in pain-related cortical regions, bilateral anterior and posterior cingulate cortex, medial and lateral orbitofrontal (MOF) cortex, insula, the primary somatosensory cortex (SI), the primary motor cortex (MI) in 30 migraine patients and 27 controls. Migraine patients showed a lack of correlation between pain sensitivity and resting-state gamma oscillation, present in the control group, for a possible disruption of pain-related cortical networks .A new neurophysiological interpretation of habituation phenomena in terms of progressive synchronization and reduction of information flow within the neuronal networks activated by repetitive stimuli was suggested . FollowiA more recent study investigated modulations in effective connectivity between the sources of laser evoked potentials (LEPs) from a first to the final block of trigeminal LEPs using dynamic causal modeling (DCM) in a group of 23 migraine patients and 20 controls. The connections between the secondary somatosensory areas , insulae, anterior cingulate cortex (ACC), contralateral primary somatosensory cortex (lS1), and thalamus were investigated. Migraine patients show amplified relationships between lS1 and the thalamus, in both directions, with a lack of progressive habituation of connection strengths from the thalamus to all somatosensory areas, in line with altered thalamo-cortical network dynamics .Ren et al.  used MEGSummarizing, EEG and MEG connectivity results converge toward disrupted network connections in resting state and under painful stimulation in migraine patients, while some differences emerged within migraine phenotypes (with and without aura) and migraine phases.In conclusion, although advanced neuroimaging investigations surely represent a valuable tool to improve our knowledge of migraine pathophysiological mechanisms, a pathophysiological interpretation strongly depends on whether we would disentangle mechanisms underlying migraine attack or migraine disorder. Indeed, the lesson we have learned from Functional Connectivity studies is that subcortical structures such as the hypothalamus, thalamus or brainstem are surely involved in some moments of the migraine cycle such as the prodromal or headache phases. Moreover, migraine appears a pervasive condition, likely with a prevalence of cortical processes, leading to migraine attacks susceptibility in response to several and different predisposing factors.Indeed, changes in the activity of different cortical brain regions encompassing the neurolimbic-pain network, secondarily allowing a de-modulation of subcortical areas such as hypothalamus, amygdala and brainstem nuclei in a continuous mutual cross-talk, may represent the “neuronal background” of the migraine brain. In other terms, abnormal cortical responsivity and sensory processing may constitute the fingerprint of the migraine brain and, in the course of sensory overload and lowered energy reserve, it may ignite the major pain signaling system of the brain, the trigeminovascular system, inducing migraine attacks. In the latter, subcortical brain trigeminal and extra-trigeminal structures seem to manage the attacks propagation and maintenance.A new era for migraine treatment was opened with the use of large molecules acting on CGRP, the main mediator of sterile inflammation. The CGRP-mAbs act at the peripheral level, with an improbable and weak effect on the CNS , which wIn the present scientific scenario, growing evidence are supporting how peripheral nerves involvement at somatic and trigeminal level, could change brain functional connectivity, specially within the network devoted to pain processing ,85. The Animal experiments indicate that CGRP or activation of its receptor(s) may contribute to CSD propagation. There would be a bi-directional relationship between vascular and neuronal activities, where CSD activated changes in C-fibers function with CGRP antidromic production, while the subsequent change in vascular tone, at least in vitro, can, in turn, modulate neuronal activity, termed vascular-neuro coupling . MathemaThe complex neuronal dynamic architecture subtending migraine is worthy of an integrated approach toward this complex disease. However, future long term efficacy studies on CGRP-mAbs, coupled with neurophysiological evidence of possible interference on brain connectivity features, could possibly confirm the impression that a specific and prolonged action on the trigemino-vascular system, though limited to the periphery, has a pivotal role in migraine puzzle resolving.


Ever since the emergence of novel coronavirus disease 2019 (COVID-19) in Wuhan, China, it has impacted mankind globally. WHO declared it a public health emergency since March 11, 2020 [Delays during oncological treatment have been classified as primary, secondary, and tertiary . A signiFactors that might delay treatment can be grouped into two categories  & ventilators• Manpower shortageSocial containment and travel restrictions during the pandemic, imposes difficulties for patients to attend the hospital for treatment. Also, the financial issues faced by poor strata are a big challenge amongst cancer patients, especially in LMIC. Furthermore, some patients come from far off place for treatment, propelling them to look for accommodation and food facilities. Amidst the lockdown, arranging for food and shelter is another biggest hurdle faced by these patients. Allocation of in-patient facilities to the COVID patients, due to the huge case burden, further aggravates this situation.With the increase in surge of COVID-19 patients, hospitals are forced to allocate resources and oncology surgeries to be delayed. Of late, due to the rising trend of this pandemic there has been an acute shortage of ventilators as well as PPE, causing further surgical delays. Also, a shortage of staff members dealing with oncology treatment execution leads to unwanted delays.Some positive effects of treatment delays during the crisis phase could be:(i) Vulnerable patients, ie, elderlies and those with comorbidities, can be kept out of hospitals and treatment facilities, as they are prone to get infected.(ii) Immune-dampening effects of radiotherapy (RT) and/or systemic therapy can be curtailed.These can be grouped into three categories  patients:• Delay in detecting treatment response• Delay in the diagnosis of recurrenceImpact of delay in definitive treatment: Surgery, RT, and chemotherapy (CT) either alone or in combination forms the definitive modality of treatment for most of the malignancies. Delay in the initiation of radical treatment might lead to a decrease in locoregional control and overall survival (OS). Chen et al. in their study inferred that an increase in waiting time for RT is generally associated with deterioration in local control rates and OS [(i) s and OS . Similars and OS ,5. HencePsychological impact: Fear of disease progression or recurrence is quite common amongst patients awaiting oncology treatment. Elevated levels of fear of progression can affect patients’ well-being, QOL, and social functioning [(ii) ctioning . TreatmeRT and CT are usually given at intervals to allow sufficient time for normal cells to undergo repair of the sub-lethal damage. Surviving tumor cells have the propensity to proliferate during treatment breaks. Chances of accelerated repopulation of tumor cells increases in patients with delays in ongoing CT/RT ,8. PatieAlso, patients with terminal illness requiring hospice admission and strong opioids during palliative care might land up with aggravations of symptoms, in case of delays in palliative care treatment, if any. Treatment interruptions, thus, can lead to progressive symptoms and worsened survival chances.FU is an integral part of the management of oncology patients. It is essential for assessing clinical response, late effects of treatment, detecting residual/recurrences, second malignancy, and symptomatic and supportive treatment if required . TreatmeThe oncology clinicians have a very important role to play during the crisis (Box 3Identifying tumor type and risk stratification approach:• Appropriate rationalized treatmentCounseling:• Improve clinician-patient relationAmendments in treatment protocols:• Altered fractionationTelemedicine approach:• Especially for follow-upAs different tumor histologies vary in their reproductive capability, it is the rapidly proliferating tumor cells that are of utmost concern. Hence, a risk stratification approach according to tumor subtype can be followed. In a study published by Kutikov. A et al., a risk-based conceptual approach regarding oncology treatment during the COVID-19 pandemic has been highlighted .An important way to reduce the mental stress amongst the oncology patients is by a good clinician-patient relationship. An effective counseling is thus, of immense importance for these patients. Mostly, patients diagnosed with benign conditions, early-stage or low-grade malignancy, and post-surgery patients requiring only FU are usually benefitted the most.To reduce overall treatment time to shorten hospital visits, altered fractionation regimen, especially hypofractionated or accelerated radiotherapy regimens, wherever feasible, may be followed.Telemedicine has emerged as a boon amidst this crisis. All queries of patients and attendants could be adequately addressed through a telemedicine approach. Especially, those patients on regular FU, may be managed by this approach and made to defer unnecessary visits to hospitals. Only if, deemed necessary they should be asked to visit the hospital during the crisis.Although factors causing treatment delays, might have a negative impact on the survival and QOL in oncology patients, advantages associated with it makes it paramount to carefully weigh the risk of treatment delays vs benefits of continuing therapy during the crisis. A detailed discussion and counseling of patients regarding the possible outcomes of treatment interruption on disease control and its effect on survival might help in overcoming the associated stress. Optimal patient care should be targeted by identifying tumor type and appropriate risk stratification approach. Furthermore, all possible measures to curtail the crisis such as triaging to identify the critical cases, amendments in treatment protocols, and telemedicine facilities should be sought.


Cancer comprises a collection of highly proliferative and heterogeneous cells growing within an adaptive and evolving tumour microenvironment. Cancer survival rates have significantly improved following decades of cancer research. However, many experimental and preclinical studies do not translate to the bedside, reflecting the challenges of modelling the complexities and multicellular basis of human disease. Organoids are novel, complex, three-dimensional ex vivo tissue cultures that are derived from embryonic stem cells, induced pluripotent stem cells or tissue-resident progenitor cells, and represent a near-physiological model for studying cancer. Organoids develop by self-organisation, and can accurately represent the diverse genetic, cellular and pathophysiological hallmarks of cancer. In addition, co-culture methods and the ability to genetically manipulate these organoids have widened their utility in cancer research. Organoids thus offer a new and exciting platform for studying cancer and directing personalised therapies. This review aims to highlight how organoids are shaping the future of cancer research. In spite of this success, however, cancer is the most common cause of death in the United Kingdom, and is expected to continue to remain as such, with 212,546 cancer deaths predicted for 2035.3Cancer mortality rates have significantly declined by ~26% over the past two decades,5 an observation that is thought to reflect the challenges of modelling the complexities and multicellular basis of human disease.6 Despite these challenges, several pivotal systems, such as two-dimensional (2D) cell cultures, explants, organ-on-a-chip system and animal models, will continue to be essential to understand the biology of cancer. Nevertheless, novel and innovative model systems can improve the translational success of preclinical studies, and the methodology for tumour-derived organoid cultures has consequently emerged  or well-characterised immortalised cell lines—have been extensively used to study cancer. Although primary cell lines have a limited lifespan and are slow-growing, they are advantageous because they maintain some donor-cell characteristics and can be linked to clinicopathological data. By contrast, artificial manipulation or natural genetic mutations confer on immortalised cell lines the ability to proliferate indefinitely, making them a more convenient and well-established preclinical model, but rendering them less representative of the original tumour; furthermore, serial passages induce genotypic and phenotypic changes that might confound experimental results.12Tissue slices and organ cultures derived from tissue explants provide the architecture, morphology and cellular composition that 2D cell cultures lack. However, these models have a short lifespan  and are expensive and difficult to maintain.13 This system allows more accurate modelling of organ-system physiology: for example, it facilitates the establishment of tissue–tissue interfaces, has separate vascular, extracellular and parenchymal compartments and allows for physiologically representative co-culture with microbes and immune cells.14 In cancer research, this technology has been used to study interactions between tumour cells and the extracellular milieu, cancer-associated epithelial–mesenchymal transition, angiogenesis, tumour invasion, cell migration and metastasis. Despite this impressive resume, this model does have disadvantages: for example, organ-on-a-chip platform commonly uses cell lines, and there is often significant variation and inconsistency between different chips, making experimental replication difficult.Organ-on-a-chip technology refers to a multichannel microfluidic perfusion culture system, made from glass, plastic or a flexible polymer, that is lined with living human cells.20 Tumour growth in vivo can be induced by chemicals , viruses  or radiation . Genetically engineered animals are popular because tumours can be induced to develop in transgenic mice  or knockout mice , and key genes can be conditionally manipulated.21Preclinical animal models continue to be a key aspect of cancer research, and these have been reviewed elsewhere.21 Animal models are expensive, require extensive resources, and the data from many promising preclinical animal studies are often not validated in human models, or do not proceed in drug development towards clinical application,22 reflecting the different genetic, cellular and immunological characteristics in animals compared with humans. Steps taken to overcome these issues include transplanting human cancer tissue or cell lines into humanised rodents.24 These xenografts can be orthotopic  or heterotopic .Animal models are fundamental for translational cancer research—both for biological studies of pathogenesis and functional drug studies —and continue to be one of the cornerstone experimental approaches in the cancer research field. However, they do have limitations.7 reported that single-cell-sorted Lgr5+ stem cells located at the bottom of intestinal crypts can initiate crypt–villus organoids when embedded in Matrigel , and that these intestinal organoids contained differentiated cell types that are present in the original tissue.A critical player in organoid cultures is the stem cell, a self-renewing cell that can give rise to many different cell types within a tissue. Stem cells display unique markers such as leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) in the intestine. In 2009, Sato et al.25 Hence, although spheroid culture is a useful 3D culture methodology, offering a bridge between traditional 2D culture and costly in vivo animal studies, organoid-based 3D culture methodology offers several advantages to spheroids owing to enhanced architectural and physiological functions.It is important to distinguish organoids from spheroids: both are cultured in a 3D format, but spheroids are simpler, homogeneous, 3D structures that lack the multiple cell types seen in organoids. Spheroids typically represent free-floating cell aggregates with no matrix component—they usually depend on cell–cell adhesion for viability. Spheroids can be generated from immortalised cell lines, primary cells or fragments of tissue and, as such, their viability is limited as they do not contain a progenitor phenotype. Spheroids develop a necrotic core as they grow in size, and possess no or limited tissue structure and a less representative tissue architecture .26 Although the tissue for organoid culture is most commonly derived from surgical resection specimens,28 organoid cultures have been successfully established from other tissue sources, for example, endoscopic biopsy from Barrett’s oesophagus,27 needle biopsy for hepatocellular carcinoma,29 endoscopic ultrasound-scan-guided fine-needle biopsy for pancreatic ductal adenocarcinoma30 and ascitic fluid for both pancreatic and ovarian cancers.31 Successful tumour organoid cultures can therefore be generated from a small amount of biological material and from cancers that are difficult to access in the clinical setting. Previously, the interval between specimen collection and successful establishment of organoid culture has been dictated by the viability of fresh samples, which has limited the time, location and demographics from which a patient sample can be taken, but Tsai et al.32 published a robust method in 2018 to cryopreserve fresh human biopsy tissue and later thaw the specimen to generate gastrointestinal organoid cultures, thus overcoming this limitation. The diverse methods for tissue acquisition and the ability to cryopreserve specimens highlight the functional utility of organoid culture across a variety of cancer types and clinical situations.The epithelial compartments of many tissues, including normal, premalignant tissues and tumours, have been modelled using organoids.34 and histological assessment of these organoids demonstrated that the original tumour architecture and protein expression profile was maintained.33 This faithful representation has been reported across a variety of other tumour types, including, but not limited to, lung, ovarian, uterine, colorectal, bladder, liver, breast and biliary tract cancers.38 There is also evidence that epigenetic signatures in organoids appear to be reflective of those found in the primary lesion, indicating that the biology of the tumour is broadly represented.39Organoid cultures have been well-characterised in the literature, and this has provided a robust evidence base to validate the use of these models. For example, oesophageal adenocarcinoma organoids derived from oesophagectomy tissue specimens recapitulate the diverse genomic and transcriptomic landscape of the primary tumour,35 However, the sensitivity of organoids to growth factors can be exploited to establish many tumour organoid cultures. For example, normal colonic organoids require the ligand Wnt3a for survival, whereas the majority of colonic cancer cells demonstrate hyperactivation of the Wnt/β-catenin pathway independent of Wnt3a.40 Therefore, the selective removal of Wnt from organoid media prevents normal colonic organoids from outcompeting colonic cancer organoids.35 Not all colonic tumour cells display aberrant Wnt signalling though, and therefore, it is important to explore the implications of selecting tumour cells by their requirement(s) for specific factors.42 In future, it might be informative to use growth factor requirements to characterise, rather than select, tumour organoids. Nonetheless, it is important to remember that organoid function can be influenced by altering its media conditions, and it is therefore important to characterise organoid cultures before experimentation.Once organoid cultures have been established, they require a complex and individualised combination of growth factors for survival and maintenance. It is essential to use optimised media formulations to ensure that experiments are reliable and reproducible. Organoid cultures from different tissues will have unique media requirements. Subtle changes to these cocktails can have marked consequences—for example, normal colonic organoids will outcompete colonic cancer organoids when cultured in media optimised for normal colonic organoids, potentially owing to apoptosis resulting from genomic instability in the tumour organoids.33 and some groups reporting success beyond 1 year.43 Patient- and disease-specific characteristics are retained well over several passages.43 There is evidence that mutations do accumulate over time33 although this is perhaps unsurprising, given the known evolution of cancer in vivo,45 and this is consistent with tumour evolution in vitro.36 Tumour organoids also possess distinct organoid signatures that reflect real-life inter-patient variability.46 However, such inter-patient variability increases the sample-size requirements for robust power calculation-based experiments, which can be expensive. Ultimately, though, the expense must be weighed against the ability of this model to more accurately represent human disease.Long-term organoid culture is possible, with most groups reporting successful culture up to 6 months,Our ability to manipulate tumour organoids further improves the utility of this culture system in cancer research. Several experimental approaches can be used to reveal novel insights into cancer pathogenesis Fig. .Fig. 3Th47 High-throughput microscopy can be performed in 2D-organoid-derived monolayer cultures, which would be difficult to perform in the 3D equivalent.As discussed above, 2D cell cultures constitute a key experimental platform in laboratory research—a myriad of validated experiments can be performed using these simple and inexpensive cultures. Consequently, the ability to establish 2D monolayers from epithelial-derived organoids allows functional experiments to be carried out, such as wound healing and transepithelial electrical resistance assays to measure functional permeability, while maintaining the unique characteristics of ex vivo organoid cultures, such as molecular identity to the original tissue and presence of a number of different epithelial cell types, such as parietal, chief and mucous neck surface mucosal cells from the stomach Fig. .47 High-48 In addition, the efficiency of gene silencing using small-interfering (si)RNA is significantly reduced in the presence of serum in 2D cultures, whereas serum improves knockdown efficacy in organoid cultures by promoting the internalisation of siRNA.49However, given the increased complexity of organoid models, there are some considerations for applying traditional 2D-based assays to 3D cultures. For example, organoid-derived monolayers cannot be easily passaged or propagated in 2D; instead, they achieve a homoeostatic state with balanced proliferation, differentiation and apoptosis.50 As an example, in human cerebral organoids, CRISPR/Cas9 technology has been used to facilitate the expression of an oncogenic HRasG12V construct by homologous recombination into the TP53 locus, thereby simultaneously disrupting this tumour-suppressor locus.51 This approach enables putative-initiating genetic mutations to be recreated, and the natural history of tumour initiation of human gliomas to be followed. CRISPR/Cas9 has also been used to study the origin of mutational signatures in cancer by the selective deletion of critical DNA-repair genes.52Genome editing has been used to improve the use of organoid models and, as such, normal/non-cancerous organoid cultures can be genetically manipulated to undergo malignant transformation. The prokaryotic clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has revolutionised how we approach genome editing in the laboratory, allowing precise and consistent in vitro genome editing.53 Due to the ability of organoids to maintain tumour cell heterogenicity, this genetic manipulation allowed researchers to compare quiescent cells from glioblastoma tumours with their proliferative counterparts, which has revealed novel insights into the pathophysiology of glioblastoma. Indeed, quiescent cells were reported to differentially express genes involved in cell-cycle control, metabolic adaptation, interaction with the extracellular matrix and mesenchymal transition, and showed higher resistance to therapy compared with proliferative cells. Both hypoxia and transforming growth factor-β were identified as potential niche factors that promoted quiescence. This use of organoid technology has therefore laid the foundations for developing new therapeutic strategies for glioblastoma by uncovering novel mechanisms of recurrence for this tumour.53By inserting an inducible histone 2B-green fluorescent protein (GFP) reporter into patient-derived organoid cells, CRISPR/Cas9 technology has been used to track quiescent versus proliferative cells during glioblastoma recurrence.54 Following genetic manipulation, the resulting requirement for growth factors in the stem cell niche was exploited to select for mutant organoids, and subsequent colonoscopy-guided orthotopic injection of organoids into mice was performed to investigate the contribution of specific mutations to carcinogenesis. The resultant tumours arising in vivo were reflective of human disease. This study demonstrates how genetically modified organoids can be used to create novel orthotopic models of cancer.To model the well-defined progression from adenoma to carcinoma that occurs during colorectal carcinogenesis, CRISPR/Cas9 has been used to genetically engineer sequential mutations associated with stem cell niche regulation, senescence and DNA mismatch repair in colonic organoids from mice to mirror the molecular pathogenesis of serrated colorectal cancer.56 Through the use of CRISPR/Cas9 technology, patient biopsy-derived Barrett’s organoids that lack APC have been generated and used to demonstrate a fundamental role of aberrant Wnt/β-catenin signalling in the neoplastic progression of Barrett’s-associated oesophagus.57Genome editing in organoids has also revealed novel insights into the pathophysiology of previously hard-to-model diseases. Barrett’s oesophagus describes distal oesophageal columnar metaplasia with malignant potential. The disease is difficult to study because available cell lines and animal models are poorly representative of the underlying biology—cell lines lack the phenotypic diversity seen in the oesophageal submucosal glands, and mouse models do not accurately model neoplastic progression.58 and magnetic nanoparticle viral transduction of gastrointestinal organoids for further study in in vitro assays or in vivo functional analyses in mice.59Alternative, less costly and time-consuming methods of genomic manipulation have also been reported in the context of gene editing. Such examples include lentiviral transduction of prostate epithelial organoids to demonstrate that genetic alterations that are commonly found in human prostate cancer can be modelled in human organoid culture,60 added supplements known to promote lung development, which resulted in formation of bronchial cells; by passage 4, budding tubule-like organoids had emerged. Haematoxylin and eosin staining demonstrated that the organoids had pseudostratified epithelium, comprising basal cells and luminal cells that resembled normal bronchial mucosa. Interestingly, the non-neoplastic bronchial organoids had motile cilia found in large bundles, whereas lung cancer organoids had one single primary cilium per cell, indicating that, under the correct conditions, organoids can represent the structural organisation of their tissue of origin. In creating an organoid biobank from non-neoplastic and different subtypes of lung cancer tissue, the architectural, protein expression profile and molecular profiling was maintained from the tissue of origin.Organoids can also recapitulate the spatial arrangement of their tissue of origin. When non-neoplastic bronchial mucosa failed to maintain its organoid ‘status’ during subculturing, Kim et al.61 This is a unique feature of organoid culture that reflects human anatomy and physiology, and it allows straightforward access to the basolateral surface via the culture media. Access to the luminal surface, however, is more difficult and, given the importance of antigens, microbiota and cell signalling receptors at the luminal surface of the epithelial barrier in colorectal carcinogenesis, there has been a focussed effort to improve luminal accessibility in organoid models. Breakdown of the epithelial barrier, with the consequent translocation of bacteria and luminal antigens through the colonic mucosa, is believed to be an important initiating event in colorectal carcinogenesis,62 and a protocol for the microinjection of fluorescently labelled dextran into patient-derived human intestinal organoids establishes a platform to study epithelial barrier dynamics in vitro.61 Breakdown of the epithelial barrier will result in increased permeability and translocation of fluorescently labelled dextran from the organoid lumen to the extracellular space. In addition to barrier integrity, the contribution of host–microbiota interactions to colorectal carcinogenesis can be studied by microinjection of live bacteria into the centre of colorectal organoids.63 Microinjection can be arduous and time consuming in the case of a large number of organoids. High-throughput microinjection of organoids is a solution to this problem, and can be achieved in the laboratory with semi-automated microinjection, microfabricated cell culture devices and computer vision systems (CVis).64 This technique could be applied to organoid cultures derived from various tumours, and could facilitate the injection not only of bacteria but also of chemical compounds, biological molecules, siRNA and other microorganisms.Recreating structural morphology is especially important for cancers that arise from tissues with high structural organisation, such as the colon and rectum. Colorectal organoids self-organise to maintain apicobasal polarity with a hollow central lumen.65 This approach allows tissue-specific spatial and chemical factors to be considered in organoid cultures and, furthermore, can be adapted for use in cancer research—for example, to provide insights into factors that contribute to the loss of breast tissue organisation during carcinogenesis.One of the hallmarks of cancer is a loss of tissue organisation. Developments in microfabrication technology now enable organoids to be integrated into an extracellular matrix that contains specific biomolecules. For example, one study using normal breast organoid cultures created a defined chemoattractant gradient, using growth factors such as epidermal growth factor, to direct the formation of epithelial branches.68 Co-culture of mouse tumour organoids with adipocytes has provided novel insights into colon cancer. For example, Wen et al.69 demonstrate that adipocytes promote the proliferation and dedifferentiation  of colon cancer organoids. The authors further suggest that adipocytes function as a metabolic regulator and energy provider to promote the growth of colon cancer cells, which offers a potential mechanism to help explain the relationship between obesity and colorectal cancer.Many laboratory findings fail to translate to the clinic because cell cultures do not accurately recapitulate cell behaviour and function within the wider tumour microenvironment, which includes the extracellular matrix, blood vessels, signalling components and other cell types. Consequently, organoids offer a biologically relevant platform to improve translatability. Co-cultures are not a new concept in the laboratory, as they are often used to study interactions between epithelial cells and other important cell populations, such as lymphocytes, neurones and blood vessels. The successful co-culture of epithelial cancer organoids with immune cells has revealed important insights into the pathogenesis of many cancers, and the ability to genetically manipulate such organoids with or without immune cells provides a specific and relevant model for studying carcinogenesis.70 Co-culture experiments can overcome this. For example, established pancreatic ductal adenocarcinoma organoids normally develop ductal and basement membrane structures, but this organisation is lost following co-culture with pancreatic stellate cells in a collagen matrix, coincident with basement membrane destruction and increased invasion into the collagen matrix.71 Furthermore, co-culturing pancreatic cancer organoids with both stromal and immune cells leads to the activation of myofibroblast-like cancer-associated fibroblasts, an observation that was not apparent in 2D culture models.25 A model system that allows interaction between cancer cells, stromal cells and immune cells is therefore important for studying the pathogenesis of cancer.The extracellular matrix is not a passive bystander in cancer biology; however, the biological consequences of this are often not explored or adjusted for in traditional laboratory experiments.72 There is also evidence that adding primary prostate stromal cells to 3D cultures of human prostate epithelial cells increases the formation of prostate organoids and non-random architectural organisation in the form of branching.73As well as making organoid cultures more representative of the in vivo scenario, co-culture can improve the differentiation yield. For example, co-culture of human-induced pluripotent stem cells with human adipose microvascular endothelial cells leads to an increased yield of hepatocyte-like clusters and the generation of hepatocyte-like organoids that resemble mature tissue rather than cell cultures.74 describe a method for generating extracts of mammary extracellular matrix that can spontaneously gel to form hydrogels. Importantly, these hydrogels retain biological signalling responses that are different between cancer and normal epithelial organoid cultures.74Techniques to improve organoid cultures continue to emerge, such as the use of self-generating hydrogels comprising extracellular matrix derived from human tissue instead of Matrigel. For example, Mollica et al.75 Usui et al.76 successfully developed air–liquid interface organoid models from normal and tumour colorectal tissues of human patients, and were able to demonstrate the presence of epithelial, goblet and fibroblast cells in normal colonic tissue, and epithelial, goblet, myofibroblast and cancer stem cells in colorectal cancer tissue, as well as to show that colorectal tumour organoids were more resistant to chemotherapeutic agents than colorectal cancer cell lines. Similarly, when investigating the effect of resistance to gemcitabine treatment, co-culture of pancreatic ductal adenocarcinoma organoids with cancer-associated fibroblasts resulted in an increased IC50 when compared with organoids cultured alone,25 indicating that organoid co-culture models can also offer novel insights into treatment responses. Ensuring a representative tissue microenvironment is therefore an important consideration for organoid culture in cancer research.Air–liquid interface systems, in which the basal surface of stem cells is in contact with the media and the apical surface is exposed to air, have also attracted interest. This set-up can more accurately reflect the conditions of the tumour microenvironment in certain cancers, such as the luminal surface of colorectal cancer.77 Orthotopic transplantation is important to consider because subcutaneous xenografts often do not accurately recapitulate cancer invasion or metastasis.78 Orthotopic models of colorectal cancer have been developed from organoids and seen to produce uniform tumours that grow and metastasise reliably, depending on the metastatic potential of the cancer cells.78 One key example is the immunocompetent mouse model of colorectal cancer that recapitulates the well-defined human adenoma–carcinoma–metastasis sequence following orthotopic transplantation of colonic organoids.79 This approach can be applied to native or genetically modified human or mouse organoids: progression to adenocarcinoma occurs over 6 weeks, and spontaneous metastasis takes >20 weeks. Similar protocols use colonoscopy-guided mucosal injection and transplantation of organoids into the caecal mucosa of the mouse colon.81Patient-derived xenografts involve the implantation of human tissue or cells into humanised or immunodeficient rodents. This approach has provided invaluable insights into cancer invasion and metastasis; however, it can be improved further by the transplantation of organoids.82 transplanted patient-derived rectal cancer organoids into mice, resulting in the generation of an invasive rectal carcinoma that metastasises to the liver and lung, as expected. Furthermore, the engrafted tumours display heterogeneous responses to chemotherapy, as also expected from clinical data.A similar case exists in rectal cancer, for which there is a lack of anatomically relevant endoluminal rectal cancer mouse models. Ganesh et al.83 Such an approach could be used to generate the entire spectrum of cancer genotypes involved in carcinogenesis and metastasis,84 creating a biological library that can be used to investigate downstream phenotype changes, as has been reported.86 The insertion of a GFP tag by lentiviral transduction facilitates the straightforward detection of metastatic dissemination in such models.87Orthotopic transplantation can also take place after phenotypic and/or genotypic characterisation and/or manipulation of the tumour. For example, the CRISPR/Cas9 technology can be used to investigate the contribution of driver mutations in colorectal cancer.88 Orthotopic transplantation overcomes many problems associated with other mouse models of colorectal cancer, such as a high tumour burden and tumours arising in the small intestine rather than the colorectum; as such, the use of organoids compared with cell culture clearly improves the translational ability of orthotopic transplant models.Cancers that develop from an orthotopically transplanted breast cancer organoid in mouse models not only reflect the morphology of the tumour of origin, but also the drug sensitivities, thereby rendering the ability to genetically modify such tumour-derived organoids invaluable in the study of drug resistance.89 have described a novel metastasis-on-a-chip system, in which colorectal cancer cells within a cancer organoid reside in a single microfluidic chamber that is connected to downstream chambers containing liver, lung and endothelial constructs in order to assess the metastatic preference of colorectal cancer cells. Other examples include the development of breast cancer-associated bone metastasis model90 and a multi-organ chip-based model of lung metastases with cell compartments representing bone, liver and brain.91 Organoids can therefore improve current disease models while helping to meet the international agenda outlined by the 3Rs.Replacing, reducing and refining (3Rs) the use of animals in research is an international priority, and patient-derived organoid cultures represent an exciting platform to facilitate this principle. There are still limitations, such as the inability to mirror system-level interactions, multi-tissue interactions, multidirectional immune system interactions and explorations of drug pharmacokinetics and pharmacodynamics. However, these alternative organoid-based methods are evolving to study primary tumours ex vivo, and are adapting in complexity to overcome some of these limitations. As an example, metastasis adds a whole new dimension, and this process is difficult to study without the use of animal models. As discussed above, to overcome this, organ-on-a-chip technology constitutes an excellent animal-free model system for studying cancer metastasis, and can be improved by using organoid cultures. Aleman and Skardai92 Animal-free alternatives, such as hydrogels made from alginates, do exist and have been used in novel model systems of the tissue microenvironment, such as a 3D bioprinted multicellular construct of breast tissue containing breast cancer cells and adipocytes,93 or utilising hyaluronic acid and collagen in a novel immersion bioprinting technique to allow organoid culture in 96-well plates for high-throughput drug screening, validated with patient-derived glioblastoma and sarcoma organoids.94Matrigel, which is currently widely used in the synthesis of organoids, is a basement membrane matrix with biological activity derived from Engelbreth–Holm–Swarm murine sarcomas.98 This knowledge could be especially useful when there are a lack of robust data from large randomised control trials, which is often the case for rare and metastatic cancers. For example, this approach has been explored using organoids from appendiceal,99 neuroendocrine prostate100 and sarcoma cancers94 to test the efficacy of various chemotherapeutic agents. Researchers in the Netherlands have also established colorectal cancer organoids from ascitic fluid and peritoneal metastasis and used them to assess sensitivity to chemotherapy agents in an in vitro hyperthermic intraperitoneal chemotherapy model.101 Consistent with variable clinical outcomes following hyperthermic intraperitoneal chemotherapy, the authors reported inter-patient variability in response to commonly used chemotherapeutics, suggesting that organoids could potentially allow treatment regimens to be individualised to improve prognosis and reduce rates of recurrence.Organoids could be a future tool to facilitate decisions regarding patient management. As an example, patient-derived tumour organoids could help determine whether a particular patient will be sensitive or resistant to specific treatments for many cancer types in a personalised medicine approach.103 For example, rimonabant, a cannabinoid receptor 1 antagonist previously used in the management of obesity, which inactivates Wnt signalling and might therefore modulate cancer stemness in colorectal cancer, was shown to be selectively toxic towards colorectal cancer organoids but not healthy colonic cells, highlighting the potential use of rimonabant as a candidate for the treatment of colorectal cancer.104 The use of organoids for assessing response and toxicity to therapy is not restricted to chemical compounds; however, patient-derived rectal cancer organoids irradiated ex vivo were seen to display heterogeneous sensitivities that correlate with the patient’s clinical response to radiotherapy.82 Furthermore, Nagle et al. used organoids to demonstrate that proton irradiation carried out in a magnetic field did not impact biological responses.105In addition to providing insights into individualised treatment responses, organoids could also help inform on drug toxicity.106In addition to helping to select the correct therapy for patients, organoid models could also prevent cancer patients from receiving ineffective treatments. For example, in metastatic colorectal cancer, Ooft et al. demonstrated the use of patient-derived organoids in preventing patients from receiving ineffective irinotecan-based chemotherapy; interestingly, however, these patient-derived organoids were unable to predict the outcome for treatment with 5-fluorouracil plus oxaliplatin.33 Success rates could be improved by employing tissue-quality evaluation protocols before culturing; this approach entails preparing and histologically examining an aliquot of cell suspension to select for epithelial cell-prominent samples following tissue dissociation.60This potential approach to personalised medicine has limitations. There can be a low success rate of generating some organoid cultures, probably dependent on tissue type. For example, Li et al. report an efficiency rate of 31% for generating oesophageal adenocarcinoma organoids.107 High-throughput sequencing of organoid cultures is difficult, given that these cells are not likely to proliferate fast enough to generate the cells necessary for a very large screen. However, a bespoke, clinically relevant, drug screen could be performed on the resistant organoid culture to identify effective chemotherapy agents, and this approach appears more feasible at present. In addition, new methods to overcome this issue for high-throughput applications are emerging, including the use of bioprinting with alternative support matrix combinations to allow organoid culture in small-well culture plates, for example 96- or 384-well plates.94Patient-derived organoid culture could be useful in the study of chemotherapy resistance. When a patient becomes resistant to therapy, it might be possible to select for resistant cancer cells in culture by manipulating niche factor requirements, thereby facilitating screening for drugs that are effective against the resistant cells.108 This biobank includes most gastric cancer subtypes, and whole-exome and transcriptome analysis data are available for these cultures. Analysis has uncovered new understanding of cancer biology and disease pathogenesis. In addition, the utility of this organoid biobank is reflected in the results of the drug-sensitivity screen as this highlights the potential impact of drugs at a very early stage of development. A breast cancer organoid biobank of >100 primary and metastatic cancers,38 a lung cancer biobank60 and an ovarian cancer biobank of 56 organoids109 have generated similar results when used for in vitro drug screening. Across these studies, the organoid tumour biobanks remarkably maintain disease-specific subtype characteristics, such as morphology, transcriptomic profile and genomic mutational analysis to the native tumour even after long-term culture.109 Accordingly, they offer an exciting and realistic tool for precision medicine for use after the identification of a patient’s cancer subtype through histopathological identification with validated tumour biomarkers.Individualised cancer therapy based on ex vivo experiments using a patient’s own cancer organoids represents an ambitious goal for personalised medicine, and this approach is currently not achievable and is too expensive for most healthcare systems. However, organoid biobanks could represent a realistic intermediate step towards this goal. A gastric organoid biobank has been established and comprises 64 normal, dysplastic, cancer and lymph node metastasis organoids from 34 patients.110 These T cells are able to recognise and kill autologous tumour organoids but, remarkably, ignore healthy autologous organoids. This method enables us to investigate the mechanisms that underlie patient responses to immunotherapy. Furthermore, this approach could facilitate the generation of T-cell populations that could be used for autologous T-cell transfer therapy.The immune response to tumours differs according to the biology of the underlying cancer. Autologous co-culture of tumour organoids derived from patients with colorectal cancer or non-small-cell lung cancer with peripheral lymphocytes leads to the expansion and enrichment of tumour-reactive T cells.111 This is an exciting but underdeveloped concept that could have therapeutic benefits—for example, to help re-epithelialise areas affected by radiotherapy.More direct potential therapeutic applications of organoids exist. Schwartz et al. have demonstrated that airbrush spraying of intestinal organoids onto a decellularised native extracellular matrix leads to the formation of an epithelial monolayer that resembles the intestinal surface.112 The lympho-organoids became fully integrated into the endogenous lymphatic system and restored lymphatic drainage. Furthermore, upon immunisation, the lympho-organoids were able to support antigen-specific endogenous immune responses. Therefore, therapeutic injection of lympho-organoids could become a novel therapeutic strategy for patients following radiotherapy or lymph node dissection for breast cancer.Radical surgery for breast cancer is often accompanied by radiotherapy or lymph node dissection. These adjuvant or neoadjuvant therapies are lifesaving, but increase the risk of life-long complications such as lymphoedema. In 2019, Lenti et al. investigated the transplantation of lympho-organoids into the region of dissected lymph nodes in mice.Tumour-derived organoids are emerging as a tissue culture model that has exciting translational potential in the era of precision medicine. Tumour-derived organoids accurately represent the diverse genetic, molecular, morphological, architectural and functional pathophysiological hallmarks of cancer. Established cultures demonstrate intra-tumour and inter-patient heterogenicity, and can be further modified by genome editing, co-culture and orthotopic transplantation into rodents. In the clinic, tumour-derived organoids could be used to inform decisions on cancer treatment. In the wider setting, however, the translational impact of organoids is dependent on infrastructure, appropriate skill set and funding and, whilst the use of organoids is limited for now, there is future potential for this methodology and application to translate into clinical practice Fig. . Overall


Dysfunction of the immune T cell compartment occurs in many hematopoietic as well as solid cancers and hampers successful application of new immunotherapeutic approaches. A complete understanding of T cell dysfunction might improve the outcome of such therapies, but an overview in the various cancers is still lacking. We aim to map areas of similarities and differences in solid versus hematopoietic malignancies, providing a high-level rather than a detailed perspective on T cell dysfunction in those tumors.Cancer cells escape, suppress and exploit the host immune system to sustain themselves, and the tumor microenvironment (TME) actively dampens T cell function by various mechanisms. Over the last years, new immunotherapeutic approaches, such as adoptive chimeric antigen receptor (CAR) T cell therapy and immune checkpoint inhibitors, have been successfully applied for refractory malignancies that could only be treated in a palliative manner previously. Engaging the anti-tumor activity of the immune system, including CAR T cell therapy to target the CD19 B cell antigen, proved to be effective in acute lymphocytic leukemia. In low-grade hematopoietic B cell malignancies, such as chronic lymphocytic leukemia, clinical outcomes have been tempered by cancer-induced T cell dysfunction characterized in part by a state of metabolic lethargy. In multiple myeloma, novel antigens such as BCMA and CD38 are being explored for CAR T cells. In solid cancers, T cell-based immunotherapies have been applied successfully to melanoma and lung cancers, whereas application in e.g., breast cancer lags behind and is modestly effective as yet. The main hurdles for CAR T cell immunotherapy in solid tumors are the lack of suitable antigens, anatomical inaccessibility, and T cell anergy due to immunosuppressive TME. Given the wide range of success and failure of immunotherapies in various cancer types, it is crucial to comprehend the underlying similarities and distinctions in T cell dysfunction. Hence, this review aims at comparing selected, distinct B cell-derived versus solid cancer types and at describing means by which malignant cells and TME might dampen T cell anti-tumor activity, with special focus on immunometabolism. Drawing a meaningful parallel between the efficacy of immunotherapy and the extent of T cell dysfunction will shed light on areas where we can improve immune function to battle cancer. Roughly a decade ago the interplay between malignant and immune cells has been recognized as an emergent hallmark of cancer . We are The full repertoire of innate and adaptive immune cells plays crucial roles in the pathogenesis of neoplastic diseases, with T cells being considered special players in cancer progression  due to tCancer cells and the tumor niche aid T cell tolerance: immune cells can be subverted to a tolerogenic, inert and dysfunctional state by cancer cells at every level of the cycle, by means of physical interactions and soluble mediators in the immunosuppressive tumor microenvironment (TME), .Before continuing, in order to better understand how cancer cells impact on each step of the process, it is beneficial to briefly recap what must happen to elicit a successful immune response to cancer. First of all, the genetic aberration(s) leading to oncogenesis generate neoantigens for tumor-specific T cell responses . In ordeT cell development and differentiation is shaped and affected by the tumor . Naïve CAs a reflection of the events regulating anticancer immunity, immunotherapy can use at least three sites for intervention: promoting priming, shifting the balance to anti-tumor T cell responses, and counteracting immunosuppression in the TME . ImmunotVaccination strategies will not be discussed here as we focus on T cells, except to state that we are currently witnessing a revival of cancer vaccines, especially due to increasing knowledge of neoantigens . While iIn general, the mechanisms of resistance to T cell based immunotherapy are overlapping with those used at first by cancers to evade immune destruction and initiate tumor progression . As a coIn order to explore T cell dysfunction in cancer, we will address first the efficacy of T cell-based therapies, such as checkpoint blockade and transfer of cellular immune effectors into patients. We aim at mapping areas of similarities and differences in solid and hematopoietic tumors, with the premise that immunotherapy efficacy depends on the state of T cells in cancer patients. Here, T lymphocytes are characterized by features of cancer-induced anergy, senescence and/or exhaustion. Anergy is caused by insufficient co-stimulatory signals during priming; senescence is growth arrest after extensive proliferation; exhaustion is characterized by expression of inhibitory signals after an excessive activating signal due to chronic antigen stimulation . ReversiThe success of T cell based immunotherapy in solid tumors is limited to checkpoint blockade therapies, with adoptive cell therapy lagging behind ,19. AntiLoss of immunogenicity is the first mechanism of escape that cancer cells adopt against an immune response. In general, a higher number of identified neoantigens together with more CD8+ tumor infiltrating lymphocytes (TILs) correlate with increased patient survival . Since nLoss of immunogenicity has obvious negative consequences for adoptive CAR T cell therapy as well: the search for tumor specific antigens is still ongoing in solid tumors. Unlike B cell malignancies in which the tumor cells express the B-cell marker CD19, solid tumors rarely express one tumor specific antigen . They arAnother hurdle faced by immunotherapy in solid cancers is tumor infiltration. Physical barriers can prevent TILs from infiltrating the TME  and thisWhen the cancer is immunogenic and T lymphocytes manage to infiltrate the tumor, ICIs seem still the most promising therapy and it is achieving remission in patients that a few years ago were deemed as incurable. Indeed, studies reported that in solid tumors ICIs are more efficient in patients showing TIL infiltrates . An intrAdoptive immunotherapy using TILs or CAR T cells seems to require further improvements, for several reasons. Tumor infiltrating lymphocytes are dysfunctional and exhausted, due to chronic stimulation and their coping mechanisms to survive the microenvironment at the tumor site. Thus, the obvious limitation to TIL adoptive cell therapy is the expansion of sufficient numbers of reactive T cells in vitro, because TILs might be already terminally differentiated due to chronic stimulation in the TME. T cell phenotypes within the tumor do not mirror the immune composition in peripheral blood , where TEven when the generation of a CAR T cell infusion product is successful and able to reach the tumor site, those cells have to face the TME. The TME is inhabited by stromal cells and suppressive immune cells including myeloid-derived suppressor cells (MDSCs), tumor associated macrophages with M2 phenotype and Tregs. Together with malignant cells, those cell types express ligands for inhibitory receptors on T cells and secrete soluble factors impacting T cell priming, survival and function, which we will discuss later. Microenvironmental clues also matter in hematopoietic tumors.Hematopoietic malignancies develop in the same niche in which immune responses are generated, suggesting that these tumors fail to trigger immune sensing mechanisms, or effectively impair anti-tumor immune responses when they do take place . T cell ®  and Yescarta®  targeting the B cell antigen CD19 to treat B-acute lymphocytic leukemia (B-ALL) and DLBCL, respectively. CAR T cell products showed high efficacy in high-grade malignancies, but not in low-grade hematopoietic cancers. The pathogenesis of those two categories of tumors is different: high-grade tumors, such as B-ALL, are fast growing and characterized by immature precursors, while low-grade tumors, like CLL, show a slower growth of mature B cells. This subtle and persistent growth induces chronic stimulation, a state of cancer-induced T cell dysfunction and acquisition of a terminally differentiated phenotype. T cell defects in CLL due to disease and/or therapy impair ex vivo expansion and response to CAR T cells. Previous therapies for CLL, including alkylators and fludarabine, have a profound negative effect on T cell function, exacerbating the T cell defect in CLL. The Bruton tyrosine kinase (BTK) inhibitor Ibrutinib, instead, may potentially improve T cell immunity [To date, the major successes of CAR T cell therapies have been recorded in hematological tumors ,46,47. Timmunity  and may immunity . A recenimmunity , most liimmunity  showed tIn order to compare the hurdles of adoptive T cell therapy in hematopoietic cancers with the ones described for solid tumors, we will first discuss antigen selection and immunosuppressive nature of TME. While finding an optimal CAR T cell target is still a challenge in solid cancers, CD19 appears as a highly suitable target in B cell hematologic malignancies for several reasons: its expression is ubiquitous on malignant and normal B cells. Loss of normal B cells can be overcome by immunoglobulin replacement therapy . A more Regarding the immunosuppressive feature of the tumor niche, hematopoietic cancers can be compared to solid tumors when considering that the solid component is still significant in leukemia, lymphoma or myeloma, where secondary or primary lymphoid organs function as the TME. MM is homing in the bone marrow and like in solid tumors, T cells at the tumor site are more severely impaired than the ones in peripheral blood . Those cCompared to solid cancers, hematopoietic malignancies show more profound abnormalities in T cell development and differentiation, which are affecting also the blood compartment. Multiple myeloma patients show quantitative and functional T cell abnormalities , with a Immune dysregulation is also prominent feature of CLL from its early stage and alterations within the T cell compartment become more apparent with disease progression or after treatment . The absThere are few studies regarding the impact of T cell dysfunction in B-ALL development and relapse after (CAR T cell) therapy, and these focused mostly on the bone marrow microenvironment ,74, whicAs hypothesized by Curran et al. , tumor sCo-stimulation and co-inhibition is another process in T cell activation which is often impaired in cancer. As previously discussed, the expression of inhibitory receptors  on T cells represents a physiological mechanism aimed at mitigating tissue damage and autoimmunity  Figure . This meAnother molecular mechanism subverted by cancer cells is the very last step of the cancer-immunity cycle, namely the killing of tumor cells by T cells. A functional CRISPR/Cas9-based genome-wide knockout screen performed in B-ALL identified the death receptor signaling pathway as a resistance mechanism to CD19 CAR T-induced cell death , suggestBy-stander cells in TME can be subverted by cancer cells to suppress T cell function: it is the case of immunosuppressive M2 macrophages, Tregs and MDSCs. The increased fraction of Tregs identified in both solid and hematopoietic cancers inhibits anti-tumor immunity by various mechanisms, such as the constitutive expression of CTLA-4, inhibiting CD80/86 co-stimulatory signals on APC  or direcFor reason of limited space, we indicated excellent reviews on bystander cells, extensively discussing the role of Tregs , macrophIn the context of the cancer-immunity cycle, soluble signals such as cytokines represent signal 3 to balance TCR signaling either to a potent immune response  or a tolerogenic state , . Other tInflammatory cytokines such as TNF-α, IL-6, TGF-β, and IL-10, have been shown to participate in cancer initiation and progression , dependiIL-10 is a potent anti-inflammatory cytokine, secreted by cancer and immune cells ,105. IL-Another pro-inflammatory cytokine with a typical pro-tumorigenic and immunosuppressive effect is IL-6. Elevated IL-6 levels have been detected in serum of patients with systemic cancers. IL-6 has a role in multiple myeloma development, where malignant cells induces senescence in bone marrow stromal cells  and cytoCytokines and soluble factors in general play a relevant role in shaping the immune composition in solid cancer as well. To give an example, CD8+ T cells infiltrating the lung TME and the pleural effusion are dysfunctional: they are unresponsive or poorly responsive to any T cell activating stimulus and functionally impaired . SurprisBy-stander cells in the TME are also educated by cancer cells to secrete soluble factors negatively affecting T cell immunity. Besides the above mentioned cytokines, MDSCs and M2 macrophages release nitric oxide (NO) and reactive oxygen species (ROS)  that impThe tumor microenvironment is poor in nutrients due to the high metabolic demand of malignant cells; solid cancers are characterized by gradients of nutrients, hypoxia and by-products which can act as immunosuppressive metabolites. To give some examples, adenosine is known to affect priming  and funcWe will expand in the next section on why metabolism is important in cancer immunity (dys)function.Metabolic reprogramming is inherently linked to T cell development, activation, function, differentiation, and survival ,121. AftGlycolysis is required to obtain energy, biomass and precursors essential to initiate the differentiation program towards dividing cells and is also crucial for the production of effector molecules like IFN-γ, IL-17 and Granzyme B in T cells ,127,128.Rewiring of cellular metabolism is also a well-known feature of cancer cells , which aThe Warburg effect indeed describes tumor metabolism only partially , and theStudies correlating cancer metabolism and immune dysfunction are flourishing. Interestingly, cytokine secretion by tumor cells is regulated by external stimuli and stressors such as glucose deprivation, as shown in a model of lung cancer  and thisAs shown by experiments conducted in murine models of melanoma and sarcoma, in solid tumors the mechanisms underlying T cell metabolic dysfunction can be rooted in metabolic competition for glucose ,138, or In addition to melanoma and sarcoma murine models, in CLL patients T cells have defects in activation, accompanied by the inability to properly upregulate glucose uptake and glycolytic metabolism . Immune In addition, metabolic plasticity also regulates differentiation and skewing of T cells, with terminally exhausted effector cells showing a glycolytic metabolism, in contrast to fatty acid β-oxidation characterizing memory subsets , Figure. Thus, tIn the last decade we witnessed the rise of the immunometabolism field and studies on the topic have increased exponentially. Reports describing the alterations in T cell metabolism can have profound implications in the optimization of immunotherapeutic strategies. In view of those findings, researchers are making efforts in the direction of manipulating the metabolism of either TILs or CAR T cells to more efficiently utilize nutrients, with the aim of maximizing their performance and prolong their survival, eventually improving the efficacy of cancer immunotherapy. The design of CAR constructs can be improved by ‘armoring’ T cells with enhanced metabolic functions. On the other hand, this knowledge could also be beneficial in improving ICIs therapy when combined with drugs targeting and fine-tuning metabolism as adjuvant therapy.In summary, hematopoietic and solid cancers are showing different defect in central versus peripheral tolerance, with B-cell lineage malignancies displaying a more unique defect in TCR selection and priming, and solid cancers presenting adequate immune response initiation which is then dampened at the tumor site.Many trials are testing combinatorial approaches of immunotherapy with other treatments. A strategy could be optimizing the schedule of immunotherapy administration, for example after surgery to remove tumor cells inducing T cell dysfunction, taking care not to exacerbate it via chemotherapy. An early use of immunotherapy to treat patients with early-stage cancers could lead to an improved efficacy. To date, immunotherapy has primarily been used in patients with advanced stage or relapsed cancers. However, multiple trials are addressing the benefit of the use of immunotherapy in early-stage tumors. Hopefully, those ongoing trials will reveal even better results than those obtained in advanced settings.Studies reporting the importance of metabolism in cancer immunotherapy are flourishing, but still a complete overview of metabolic interactions between malignant and immune cells in the various cancer types is lacking. A deeper investigation of immunometabolism should indeed lead to a profound improvement of immunotherapeutic strategies: ICIs approaches could benefit from the administration of adjuvant therapies targeting metabolism and nutrient utilization, and CAR T cell persistence could be boosted by armoring the chimeric TCR with genes involved in the regulation of cellular metabolism.


CTNS gene encoding cystinosin, and leads to end-stage renal disease within the second decade. The cornerstone of management relies on cysteamine therapy to decrease lysosomal cystine accumulation in target organs. However, despite cysteamine therapy, patients display severe bone symptoms, and the concept of “cystinosis metabolic bone disease” is currently emerging. Even though its exact pathophysiology remains unclear, at least five distinct but complementary entities can explain bone impairment in addition to CKD-MBD: long-term consequences of renal Fanconi syndrome, malnutrition and copper deficiency, hormonal disturbances, myopathy, and intrinsic/iatrogenic bone defects. Direct effects of both CTNS mutation and cysteamine on osteoblasts and osteoclasts are described. Thus, the main objective of this manuscript is not only to provide a clinical update on bone disease in cystinosis, but also to summarize the current experimental evidence demonstrating a functional impairment of bone cells in this disease and to discuss new working hypotheses that deserve future research in the field.Patients with chronic kidney disease (CKD) display significant mineral and bone disorders (CKD-MBD) that induce significant cardiovascular, growth and bone comorbidities. Nephropathic cystinosis is an inherited metabolic disorder caused by the lysosomal accumulation of cystine due to mutations in the  Pediatric patients with chronic kidney disease (CKD) display significant abnormalities in bone and mineral metabolism, also known as CKD-MBD, for mineral and bone disorders associated with CKD. The pathophysiology of CKD-MBD is complex and multifactorial, multiple factors were identified such as abnormalities in calcium and phosphate metabolism, resistance to growth hormone (GH), modifications of the GH-insulin such as growth factor type 1 (IGF1) axis, hypogonadism, malnutrition, and drug toxicity (corticosteroids) ,3: 1) abMoreover, in addition to “conventional” ROD and CKD-MBD, there is now evidence that some genetic renal diseases may also have a direct negative impact on bone per se, notably in primary hyperoxaluria, nephropathic cystinosis, autosomal dominant polycystic kidney disease, and Pierson syndrome ,8,9,10. CTNS gene encoding cystinosin, a 367 amino acids lysosomal cystine transporter with seven transmembrane domains. To date, over 140 pathogenic mutations have been reported. The most frequent one is a large deletion of 57 kb involving the first nine exons of the CTNS gene and part of exon 10, detected in approximately 50% of mutant alleles of patients of north European and North American origin. Other mutations include deletions, small insertions, duplications, missense, nonsense, splice site and promoter sequence mutations as well as genomic rearrangements. Severe truncating mutations affected both alleles are generally associated with infantile nephropathic cystinosis, the most frequent and severe form of the disease [Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disease caused by the lysosomal accumulation of cystine because of mutations in the  disease . Cystino disease .Cysteamine therapy is a mainstay of its management, decreasing lysosomal cystine accumulation in target organs . The use2 vitamin D deficiency) together with iatrogenic effects of its supportive management, 2) deficiency in nutrition and micro-nutrition, and notably copper deficiency [CTNS mutation and cysteamine on osteoblasts and osteoclasts [Indeed, this concept of CMBD is currently emerging, with the description in vivo of bone fragility and symptoms ,19, and ficiency , 3) hormeoclasts .CTNS gene in certain strains of mice is associated neither with renal phosphate wasting nor with renal failure, but causes severe growth retardation and osteopenia with decreased mineralization and cortical thickness [Osteogenesis Imperfecta type VI and cystinosis as a contiguous gene syndrome had also been described [In animal models, the invalidation of the hickness , thus rahickness ,26,27; oescribed .We first reported in 2016 three cases of teenagers with cystinosis displaying a severe bone phenotype who underwent bone biopsies at the time of orthopedic surgery [Ctns-/- [We therefore performed a pilot observational study in 10 patients  using biomarkers , Dual X-ray absorptiometry  at the ultra-distal tibia . At a meIn the meantime, a North American team performed bone and mineral evaluations of 30 patients  with cystinosis at a mean age of 20 (range 5–44) years, including history and physical examination, biochemicals, DXA , vertebral fracture assessment, skeletal radiographs, and renal ultrasounds . HistomoWith these data in mind, international guidelines were published in 2019 to guide the diagnosis of CMBD and clinical management of these specific patients . BrieflyCtns knockout (-/-Ctns) mice (of C57BL/6 background) reveals a decrease in bone mineral density (BMD), trabecular volume (TV) and number/thickness of bone trabeculae compared to control animals. Incidentally, these mice did not display any sign of Fanconi syndrome. Therefore these changes are not secondary to altered kidney functions and are associated with a reduction in the number of osteoclasts and osteoblasts within bone tissue in 1 month-old mice. Ctns-/- mice show a decrease in plasma markers of bone turnover, and notably procollagen type 1 amino-terminal propeptide and tartrate-resistant acid phosphatase [The study of bone phenotype in one month-old sphatase .Bone remodeling is a tightly regulated process involving bone resorption of old bone and bone formation to maintain a healthy skeleton. These two activities are coupled, through a complex crosstalk between osteoblasts and osteoclasts . In addiConforti et al. showed in 2015 that mesenchymal stem cells (MSCs) isolated from a patient with cystinosis display a reduced ability to differentiate into osteoblasts, which can be reverted after cysteamine treatment . Therefo-/- mice [-/- mice displayed increased cystine content and a decreased number of alkaline phosphatase positive cells, with a decreased expression of the main genes involved in osteoblastic differentiation and activity . In vitro mineralization assays also demonstrated a reduced ability to release mineralized nodules from cystinotic osteoblasts as compared to control cells, although this study did not address the in vivo therapeutic effect of cysteamine on bone phenotype [Battafarano et al. also recently described osteoblastic dysfunction in Ctns-/- mice : the exphenotype .CTNS gene expression during osteoclastogenesis, with a peak observed at Day 6 on mature osteoclasts following the same pattern that cathepsin K transcripts a late marker of osteoclast differentiation which is essential for bone resorption [Second, in human osteoclasts differentiated from peripheral blood mononuclear cells (PBMCs), we showed that CTNS is required for proper osteoclastic differentiation and resorption activity, with a progressive increase of sorption . These eWe therefore evaluated resorption capacity of osteoclasts derived from cystinotic patients; in contrast to what we observed for differentiation, in vitro bone resorption is decreased in osteoclasts from patients as compared to controls; similarly, we also found that cathepsin K transcript levels are decreased in patients in comparison to controls. Overall, in vitro, cystinosin deficiency increases osteoclastogenesis but impairs bone resorption activity of osteoclasts derived from human PBMCs.The analysis of cysteamine treatment on both osteoclastic differentiation and resorption: cysteamine reveals no effect on osteoclastic differentiation whatever the origin of the donors and the doses tested. However, when examining the resorptive activity of cysteamine-treated osteoclasts both in controls and patients, unexpectedly, a beneficial anti-resorptive effect is observed in controls, showing a 50% inhibition of the resorption activity with 50 µM. In contrast, resorption by patient-derived osteoclasts is not changed at this concentration, the decreased osteoclastic activity is detectable and significant only at 200 µM of cysteamine . To summCtns gene expression was observed during osteoclastogenesis. In contrast, after differentiation, a lower number of mature osteoclasts was observed in Ctns-/- cultures compared with wild-type cells. This discrepancy with regards to human osteoclast cultures could be due to the fact that murine osteoclasts, in this case, are derived from the bone marrow of mice with no renal failure. Furthermore, a reduced ability to resorb bone was also seen in Ctns-/- osteoclasts [It is noteworthy that Battafarano et al. observed almost similar results in murine cells: a progressive increase of the eoclasts .TCIRG1, ClCN7, OSTM1, SLC29A3, Cathepsin K and MMP9, all of them leading to an osteopetrotic or osteosclerotic bone phenotype due to impaired osteoclast mediated resorption, and bone hardening with increased bone mineral density [Lysosomes, vesicular trafficking and exocytosis are essential and tightly regulated in mature osteoclasts in order to achieve an efficient resorption . Among h density . Thus, d-/-Ctns defects neither at the differentiation nor at the resorption level. Our study reveals that cystinosin holds a function in osteoclasts that is independent of lysosomal cystine efflux. In conclusion, cystinosin deficiency induces an intrinsic defect both in osteoblasts and osteoclasts; moreover, cysteamine dosage exerts dual effects on bone cells: in vitro low concentration of cysteamine have beneficial anti-resorptive effects on healthy human derived osteoclasts, but cysteamine treatment does not rescue -/-CTNS MSCs [On the contrary, low doses of cysteamine stimulate osteoblastic differentiation and mineralization, with an inhibitory effect at higher doses, likely explaining, at least partly, the bone toxicity observed in patients receiving high doses of cysteamine , and the-/- MSCs . Figure Recent findings demonstrated that lysosomal dysfunction in cystinosis is associated with defective autophagic mitochondria clearance in epithelial cells from the proximal tubule. These defects lead to increased oxidative stress that disorganizes tight-junction and might explain the epithelial defects in cystinosis . In CTNS-/-Ctns osteoclasts might not rely on cystine efflux but on cystinosin itself or its interaction with other pathways, as recently discussed in a general review of new perspectives in cystinosis [The study of proximal tubular kidney cells obtained from healthy donors and patients with cystinosis reveals that cystinosin deficiency is associated with altered mammalian target of rapamycin complex 1 (mTORC1) signaling, characterized by abnormal lysosomal retention of mTORC1 during starvation followed by delayed reactivation of mTORC1 . Alteratstinosis . In this-/-Ctns mice [Andrzejewska et al. showed that the mTORC1 pathway is down-regulated in proximal tubular cell lines derived from -/- mice . A simil-/- mice . CTNS gene have various functional consequences, linked to their structural impact on cystinosin. Extensive deletions (such as the 57-kb deletion) cause the absence of protein while severe truncating mutations lead to the synthesis of an inactive variant [CTNS mutations impair the interaction between the Ragulator-Rag complex and mTORC1, preventing its activation. On the other hand, mutations of more limited structural impact might allow, in some extent, to maintain an efflux activity as well as the interaction between mTORC1 and the lysosomal membrane-attached Ragulator-Rag complex.DNA mutations in the  variant . Both thIt could be argued that the down-regulation of mTORC1 is due to the accumulation of cystine. However, as shown by Andrzejewska et al., decrease of lysosomal cystine levels by cysteamine did not rescue mTORC1 activation in proximal tubular cells, thus suggesting that the down-regulation of mTORC1 is due to the absence of cystinosin rather than to the accumulation of cystine . Even though this remains a working hypothesis that deserves to be confirmed, CTNS mutations and/or cysteamine therapy on bone cell biology and the molecular mechanisms behind these differential effects should lead to more targeted therapeutic strategies.Bone impairment in cystinosis is now recognized as a late-onset complication of a multi-systemic disease. It is important that physicians take these potential complications into account in the management of patients with nephropathic cystinosis, as they can considerably affect their quality of life and even require invasive procedures. International guidelines were recently published to improve the diagnosis and management of CMBD, although the underlying pathophysiology remains to be fully elucidated. In the future, understanding the differential impact of the different