Magnetic resonance enterography (MRE) is one of the most highly utilised tools in the assessment of patients with small bowel Crohn’s disease (CD). As a non-invasive modality, it has both patient and procedure-related advantages over ileocolonoscopy which is the current gold standard for Crohn’s disease activity assessment. MRE relies upon high-quality images to ensure accurate disease activity assessment; however, few studies have explored the impact of image quality on the accuracy of small bowel CD activity assessment. Bowel distension and motion artifacts are two key imaging parameters that impact the quality of images obtained through MRE. Multiple strategies have been employed to both minimise the effects of motion artifacts and improve bowel distension. This review discusses the definitions of bowel distension and motion artifacts within the literature with a particular focus on current strategies to improve bowel distension and limit motion artifacts in MRE. Crohn’s disease (CD) is a chronic immune-inflammatory condition that can affect the entire gastrointestinal tract (GIT), with over 40% of patients exhibiting ileal involvement . Small bThe use of MRE for assessment of small bowel CD has been recommended by international CD guidelines, with normalisation of small bowel findings, including the achievement of transmural healing, seen as a potential treatment target ,3,4. ThiTypically, MRE protocols and image sequence acquisition are shaped by local expertise and manufacturer recommendations. It remains unclear whether subtle alterations in protocol produce significant variations in image quality. For example, in the original MaRIA and sMaRIA protocols, retrograde per-rectal saline instillation of up to 2000 mL via rectal catheter and standard colonoscopy bowel preparation regimens were utilised. These have been shown to improve bowel distention and/or reduce motion artifacts in MRE, but these are not a universal part of MRE protocols due to patient tolerability ,8. The CThe measurement and integration of quality metrics in healthcare has been shown to improve disease-related outcomes in multiple disease states, including CD ,13. FurtMRE/enteroclysis protocols have rapidly evolved since inception as a validated tool for the assessment of small bowel CD . WorldwiMRE studies are usually performed on both 1.5 or 3 Tesla (T) field strengths depending on local availability. There is no consensus on which field strength is optimal for MRE . A studySequences acquired for MRE routinely include T1- and T2-weighted imaging in axial and coronal planes. T2-weighted imaging can be acquired as balanced steady-state free precession or single-shot fast spin echo sequences. Both types of sequences are rapidly acquired and depict luminal fluid well. At least one of these sequences should be acquired with fat suppression to allow better depiction of mural edema when compared to adjacent fat (suppressed) ,23. PostFasting for 4–6 h prior to the procedure is generally recommended . It is pOral contrast ingestion is used to distend normally collapsed loops of small bowel whilst replacing luminal bowel content with a uniform intraluminal material. Traditionally, 1000–1500 mL is ingested over a period of 45 to 60 min prior to the scan . This tiEnteric contrast agents are traditionally biphasic, appearing dark on T1-weighted and bright on T2-weighted images . This alMRE protocols usually include T1 sequences prior to and after intravenous (IV) gadolinium-based contrast to improve CD activity detection. Key findings in CD enhanced by IV contrast use include bowel wall enhancement, improved vasculature assessment, and the presence of lymph nodes, fistulas, and/or abscesses ,29. IV gNormal small bowel peristalsis during MRE image acquisition results in motion artifacts with subsequent degradation in image quality . Thus, aHyoscine is administered intravenously (IV) in single or split dosing with 2–3 injections. Doses ranging between 10–40 mg have been reported in various international protocols without a clear improvement in motion artifacts seen with higher doses ,19. HyosStudies have demonstrated potential benefits with intramuscular (IM) administration of both hyoscine and glucagon . One stuIn ideal scenarios, patients would be able to follow and complete all aspects of pre-MRE preparation and participate with the requirements of the scan. However, unique challenges such as claustrophobia and inability to consume the required volume of contrast can occur. In claustrophobic patients, coaching techniques to calm patients may be helpful. Anxiolytic medications can also be employed in the setting to aid the patient through the scan. In those unable to tolerate the required volume of oral contrast, optimization of imaging parameters and minimization of other artifacts is important. In our practice, careful interpretation of findings in bowel segments which are not well-distended is important to avoid over calling of findings. Reviewing acquired sequences while the patient is on the table to assess whether certain sequences require repeat imaging maybe considered on a case-by-case scenario.Artifacts frequently degrade observed image quality, with population studies showing they occur in over 35% of small bowel segments assessed on MREs . Other eMotion artifacts may relate to a patient’s body movement, breathing during imaging acquisition, or bowel peristalsis movement, the latter of which is involuntary. International consensus statements aimed at standardising the MRE imaging protocol in patients with inflammatory bowel disease have been published in an effort to improve the performance and the ability for cross-comparison of MRE ,37. DespMotion artifacts are the cumulative effect of voluntary and involuntary patient movement during the image acquisition process. It typically manifests as blurring, ghosting, and/or smearing of the bowel wall and is thought to reduce sensitivity of disease activity detection in CD. Strategies to mitigate the effect of motion artifacts in general MRI include careful pre-scan explanation and instructions to patients of the need to remain still during image acquisition, ongoing coaching of regular breathing or breath holding for the appropriate sequences, providing comfortable on-table positioning, reducing image acquisition time, and occasionally providing sedation. However, the specific technical realities of MRE present several challenges that are yet to be fully addressed ,39. The Enteroclysis is a more invasive method of intraluminal contrast delivery via nasoenteric tube to the small bowel, and its utility has been limited by poor patient tolerance and the convenience of per oral consumption of contrast. Studies have compared the impact of oral versus enteroclysis delivery of contrast in small bowel MR on motion artifacts  ,41 and aIn a pilot study of 21 patients with known (or suspected) CD, Schreyer er al found no statistically significant difference in the presence of small bowel motion artifacts when comparing MRE with MR enteroclysis . SimilarBoth studies had limitations, in particular their small sample size. Furthermore, with no clear difference in motion artifacts across either modality, the magnitude of effect of motion artifacts on CD activity assessment could not be elucidated.The use of antispasmodics in MR enterography is recommended by the European Society of Gastrointestinal and Abdominal Radiology (ESGAR) and the American College of Radiology (ACR) guidelines to reduce peristalsis during image acquisition, thus potentially reducing wall blurring artifacts ,57,58.Motion artifacts may be further reduced by refinements in antiperistalsis administration such as split dosing and IM administration of antispasmodics ,59,60. IIn a head-to-head trial, IV glucagon appeared more likely to achieve aperistalsis with a shorter time of onset compared to IV Buscopan . DespiteBreath holding is required for multiple sequences obtained during MRE with free breathing limited mostly to DWI sequences. An inability to adequately hold a breath will inevitably cause motion artifacts related to movement of the diaphragm and subsequently abdominal organs, including the small and large bowel. Whilst widely performed, studies quantifying the true impact of breath holding on motion-artifact-related degradation in MRE are lacking.Breath holding can be performed at end-expiration and inspiration with protocols developed according to local expertise. In a study relating to T1 Abdominal/Hepatic MRI, Vu et al. demonstrated a significant reduction in motion-related artifacts with end-expiration compared to end-inspiration in contrast-enhanced and unenhanced sequences . Future Current strategies to reduce motion artifacts relating to breath holding are limited to pre-MRE coaching by the MRI technologist performing the scan. These include the provision of clear instructions, practicing breath holding with patients as well as positive reinforcement during the scan. These instructions appear integral to patients remaining still for all types of MRI studies.Traditionally, MRE is performed in a prone position as the anterior abdominal wall pressure appears to reduce to the volume of the peritoneal cavity . The redAdult and paediatric MRE studies that have included motion artifact grading in the assessment of Crohn’s disease via MRE are summarised in Approaches to motion artifact grading include individual segmental scoring of motion artifact severity and combined quality grading with bowel distension see . IndividFinally, combined grading of bowel distension and motion has also been proposed. Koplay et al. used a 4-point scale incorporating both bowel distension and motion artifacts in the following manner: (1) excellent luminal distention without artifacts, (2) good luminal distention and mildly artifacted, (3) artifacted and inadequate but assessable in terms of CD, and (4) poor/nondiagnostic with CD assessment not possible . This apTo our knowledge, no consensus statements defining quality, adequacy, and specific parameters of motion artifacts in MRE have been developed. As a result, there is significant heterogeneity of the definition of motion artifacts in studies exploring specific interventions designed to reduce motion artifacts in MRE, and the comparison of different interventions is problematic.Achieving adequate bowel distention is known to be critical to the successful execution of all small bowel cross-sectional imaging modalities. Indeed, disease activity interpretation in small bowel CD is particularly susceptible to inadequate distension given the resultant inability to detect subtle yet clinically significant lesions in this context.Real-world practice of MRE performance varies across centres according to local fine-tuning of protocols. Previous studies have examined multiple aspects of oral contrast ingestion, such as type, volume, and timing relative to the image acquisition, and will be discussed.Few studies have sought to directly assess the impact of the delivery mechanism of oral contrast on bowel distension and subsequent CD activity assessment. Theoretically, nasoenteric tube insertion (MR enteroclysis) should improve bowel distension given the capacity to administer a larger amount of contrast in a shorter duration. However, in the study by Schreyer et al., there were no significant differences in bowel distention in the terminal ileum and proximal small bowel between oral and enteroclysis methods of delivery . Furtherp = 0.13) [Similar findings were demonstrated in a larger study of 40 adult patients with CD by Negaard et al. Whilst a significant difference in bowel distension was elicited in the jejunum and proximal ileum, this was not the case in the terminal ileum ( = 0.13) . Once ag = 0.13) . More reDespite these data, MRE remains far more widely performed than enteroclysis with consensus guidelines not favouring either modality . HoweverThe role of oral contrast is both to distend and opacify the small bowel lumen to enhance distinction between the lumen and the bowel wall ,17. MultExamples of currently used biphasic contrast agents include barium sulfate, mannitol, sorbitol, polyethylene glycol (PEG), water, methylcellulose, and locust bean gum (LBG) ,54,65,66More recently, Bhatnagar et al. found jejunal distension to be significantly better with mannitol compared to PEG, though the ileum and terminal ileum were similar . In a heThe optimal volume of oral contrast ingestion to achieve adequate bowel distension on MRE is not clearly defined in the literature, yet in standard practice, volumes of 1000–1500 mL are most commonly used. Interestingly, Kinner et al. suggested that adequate small bowel distension can be achieved with merely 450 mL of oral contrast . More reA key weakness of both studies was the subjective grading of small bowel distension rather than a more objective quantification. In addition, neither study evaluated the impact of a particular ingested volume on the accuracy of disease activity assessment. Hence, there is an unmet need to investigate the relationships between oral contrast volumes, objective grading of bowel distension, and the accuracy of CD activity assessment.In summation, an individualised, flexible approach regarding the volume of oral contrast required may be preferable to achieve optimal bowel distension. This is especially relevant to patients following a total colectomy, resections of long segments of small bowel, and/or those with stoma formation. Many centres now follow a tailored approach after review of initial sequences while the patient is on table to assess whether a delay in scanning or the need for more oral contrast is required, rather than a ‘one size fits all’ approach.Timing of oral contrast in relation to image acquisition has been the subject of multiple studies ,56,66. GMore recently, in a larger cohort study of 100 patients with established CD, a shortened post-ingestion period of 45 min was superior to 60 min for distension in the stomach, duodenum, jejunum, and total small bowel when administering 1600 mL of 2% mannitol. However, there was no significant difference in proximal and terminal ileum distension across both the 45 and 60 min cohort, which is particularly relevant given the predominant distribution of small bowel CD within the terminal ileum .The individual properties of oral contrast agents may also affect the optimal timing of contrast ingestion. For instance, despite similar volumes and timing of oral ingestion, mannitol has been shown to achieve better jejunal distension than polyethylene glycol . FurtherRectal filling with water has been performed in studies assessing the ability of MRE to also incorporate colonic CD assessment ,56,65. AProkinetic agents such as erythromycin and metoclopramide have been applied in MRE protocols  in an atAdult and paediatric MRE studies that describe bowel distension grading in MR enterography/enteroclysis are summarised in Commonly observed approaches to bowel distension grading include segmental qualitative scoring with 3-, 4- and 5-point scales, with some studies incorporating the impact of diagnostic capacity in the grading . ExampleAnother qualitative approach employed by several studies is to grade distension across an entire bowel segment rather than a single anatomical point. For instance, Bekendam et al. proposed the following grading system: 0 = no distension or collapsed segment (<25% of segment adequately distended), 1 = insufficient distension (25–50% of segment adequately distended), 2 = sub-optimal distension (50–75% of segment adequately distended), 3 = optimal distension (>75% of segment adequately distended). This method appears more robust than a single point measurement, particularly when assessing long segment, small intestinal CD . AlternaTo our knowledge, no consensus statements defining quality, adequacy, and specific parameters of bowel distension in MRE have been developed. As a result, there is significant heterogeneity in the definition of bowel distension as reflected in studies examining methods to improve small bowel distension in MRE.MRE is a widely used tool for the assessment of small CD. With CD treatment targets increasingly focused on normalising previously abnormal assessment findings, high quality, reproducible images are critical to ensuring accurate CD activity assessment and hence aiding clinical decision making. Similar to the application of quality metrics in multiple aspects of healthcare, these have also been considered in the evolution of MRE utilisation for the assessment of small bowel CD. Studies have hitherto focused on strategies to minimise the detrimental effects of motion artifacts and suboptimal bowel distension, the two-quality metrics most amenable to change in cross-sectional small bowel imaging. This review highlights the significant heterogeneity in MRE protocols, the descriptors of motion artifacts and bowel distension, and the approaches to mitigating these.The main challenge to improving quality in MRE elucidated by this review is the lack of published data and/or expert consensus guidelines regarding definitions and grading of optimal versus suboptimal levels of each quality metric. As a consequence, widely disparate gradings have been applied to each metric across various studies as described in this review. Typically, therefore, the assessment of adequacy of bowel distension and motion artifacts is, at best, left to local radiologist expertise or at worst, ignored completely. Moreover, most studies utilise subjective rather than objective, reproducible analyses of image sequences which preclude the opportunity for cross-comparison of intervention strategies across different studies. Hence, it is problematic to determine the optimal type, volume, and timing of oral contrast used in MRE as well as other intervention strategies, including the use of anti-peristaltics and breath holding in addressing motion artifacts and/or bowel distension.Accurate CD assessment is a prerequisite to any modality of disease activity assessment given that inaccuracies have significant downstream implications for therapeutic decision making. This is no different with MRE, where findings relevant to CD have significant clinical implications, such as affecting decisions regarding treatment escalation and choice or referral for surgical resection. However, there are scant real-world data defining the proportion of MRE scans performed that achieve adequate image quality to accurately determine CD activity. This has clear implications on MRE-based CD activity indices which assume consistent, widespread MRE image adequacy. Hence, MRE scans with quality limitations should be excluded from studies evaluating MRE performance and should at least be clearly stated to facilitate real world comparisons.In terms of future directions, a logical stepwise approach would first involve the development of expert consensus-derived definitions of bowel distension and motion artifact grading with an objective, reproducible framework. It is important that these definitions are straightforward to apply and thus subsequently have the potential to become a routine adjunct to best practice MRE reporting. Aside from the academic benefits to comparing interventions in MRE, this would also empower clinicians with a more nuanced interpretation of an individual MRE’s suitability on which to confidently base clinical decisions.Adequate bowel distension and minimal motion artifacts are a critical prerequisite for high-quality MRE images;Optimal bowel distension can be achieved with 1000 mL of oral contrast given 45 to 60 min prior to image acquisition;Reduction of motion artifacts can be achieved through coaching patients with breath-holding strategies and through the timed use of antispasmodics;Antispasmodic properties can be manipulated to achieve minimal motion artifacts by utilising both IV and IM routes of delivery;Description of grading of both bowel distension and motion artifacts are poorly described in the literature but are a critical part of MRE reporting in CD;Consensus definitions of both bowel distension and motion artifacts in MRE are needed.


Many types of human cells self-destruct to maintain biological homeostasis and defend the body against pathogenic substances. This process, called regulated cell death (RCD), is important for various biological activities, including the clearance of aberrant cells. Thus, RCD pathways represented by apoptosis have increased in importance as a target for the development of cancer medications in recent years. However, because tumor cells show avoidance to apoptosis, which causes treatment resistance and recurrence, numerous studies have been devoted to alternative cancer cell mortality processes, namely necroptosis, pyroptosis, ferroptosis, and cuproptosis; these RCD modalities have been extensively studied and shown to be crucial to cancer therapy effectiveness. Furthermore, evidence suggests that tumor cells undergoing regulated death may alter the immunogenicity of the tumor microenvironment (TME) to some extent, rendering it more suitable for inhibiting cancer progression and metastasis. In addition, other types of cells and components in the TME undergo the abovementioned forms of death and induce immune attacks on tumor cells, resulting in enhanced antitumor responses. Hence, this review discusses the molecular processes and features of necroptosis, pyroptosis, ferroptosis, and cuproptosis and the effects of these novel RCD modalities on tumor cell proliferation and cancer metastasis. Importantly, it introduces the complex effects of novel forms of tumor cell death on the TME and the regulated death of other cells in the TME that affect tumor biology. It also summarizes the potential agents and nanoparticles that induce or inhibit novel RCD pathways and their therapeutic effects on cancer based on evidence from in vivo and in vitro studies and reports clinical trials in which RCD inducers have been evaluated as treatments for cancer patients. Lastly, we also summarized the impact of modulating the RCD processes on cancer drug resistance and the advantages of adding RCD modulators to cancer treatment over conventional treatments. Cell death  plays a fundamental role in maintaining physiological homeostasis by removing damaged cells, and it may also be an aberrant pathological reaction to damaging stimuli . The NomOur review outlines the molecular mechanisms and processes of four different types of RCD, necroptosis, pyroptosis, ferroptosis, and the newly discovered cuproptosis, as well as their different roles in the initiation and progression of cancer. We specifically focus on RCD processes that influence the TME and the latest advancements in targeting necroptosis, pyroptosis, ferroptosis, and cuproptosis for cancer therapy. We describe the mechanisms of the various cancer therapies currently available, showing that they mainly depend on different RCD modalities. A reasonable assumption is that these novel RCD modalities constitute a mechanism of defense against tumor progression and migration. Furthermore, the significance and prevalence of RCDs in combating cancer drug resistance have been included, demonstrating that the use of conventional therapy in conjunction with RCD modulators might hold significant potential for cancer treatment. Hopefully, this information will lead to improved guidance for approaches to tumor therapy.The most extensively studied RCD modality is apoptosis, which leads to immunogenicity or induces no immunogenic response in different contexts , 19. TheNecroptosis is a regulated form of necrosis that depends on the phosphorylation of mixed-lineage kinase-like (MLKL) by receptor interacting kinase-1 (RIPK1) and RIPK3 . The necPyroptosis, a proinflammatory RCD pathway used in diverse types of cells, is triggered by human caspase-1, -3, -4, -5 (mouse caspase-11), -6, -8, and -9 and activated by a number of inflammatory bodies, including NLRP3 Fig.  35–40].–40.35–40GSDMD has been reported to be cleaved by caspase-1, -4, -5, or -11, which is a hallmark of the canonical pyroptosis pathway . PAMPs a−, comprising subunits SLC3A2 and SLC7A11), leading to decreased GSH biosynthesis and inactivation of GPX4 . M. M200]. However, cancer cells that undergo ferroptotic death are also associated with the release of PGE2, a significant immunosuppressant that disrupts the anticancer activities of NK cells, DCs, and cytotoxic T cells , 203. DeCuproptosis is associated with immune cell infiltration. As shown in a recent study, melanoma patients with higher expression of CRGs experienced a longer OS. One prominent CRG is LIPT1, whose expression is positively linked to PD-L1 expression and negatively correlated with regulatory T cell infiltration Fig.  207]. I. I207]. A 2016 study revealed that alanine released from stroma-associated pancreatic stellate cells by autophagy was a substitute carbon source that fueled the TCA cycle in pancreatic ductal adenocarcinoma. This change in fuel source reduced the tumor cell reliance on glucose and nutrients obtained from serum, which are limited in the pancreatic TME . Based o6-adenosine methylation significantly inhibits TRAF5-induced necroptosis both in vitro and in vivo . . 217]. TSince cuproptosis is a novel RCD, determining whether it occurs among noncancerous TME cells is a challenge. However, upon Cu stimulation, exosomes secreted by MΦs increase angiogenesis mediated by endothelial cells in vitro and in vivo . Ryuhei As we previously discussed, tumor cells and other cells in the TME that undergo necroptosis, pyroptosis, ferroptosis, or cuproptosis possibly contribute to strong antitumor immunity. Additionally, mechanisms for bypassing the apoptosis signaling pathways that cause the death of cancer cells have attracted considerable attention for their use in anticancer therapy . TherefoTargeting necroptosis, pyroptosis, and ferroptosis to develop new anticancer medications for therapeutic use has been a long process, and recently, compounds inducing cuproptosis have shown promise as anticancer strategies , 226. InAccording to recent investigations, many clinically approved medications exert potent antitumor effects by inducing (or inhibiting) inflammatory RCD modalities in preclinical studies . CQ has In addition, metformin inhibits cancer cell proliferation by inducing mitochondrial dysfunction to cause pyroptotic cell death . SpecifiC−, thus promoting ferroptosis by inhibiting GSH production [Sorafenib is an FDA-approved anticancer drug for the treatment of HCC, RCC, and thyroid cancer . Sorafenoduction . Additiooduction . Additiooduction . Etoposioduction . In the oduction .Peter Tsvetkov and colleagues identified that the Cu ionophore elesclomol induces cuproptosis by inducing lethal proteotoxic stress in various types of cancer cells  in the TME within 72 h of treatment to accelerate the Fenton reaction and efficiently induce ferroptosis in various cancer cell lines . AnalogoThe administration of NPs reverses cisplatin resistance in cancer cells by inducing cuproptosis. Exogenous platinum is widely presumed to cause drug resistance induced by high concentrations of GSH in cancer cells. According to Lu et al., the diethyldithiocarbamate-Cu complex effectively induces cuproptosis in A549/DDP cell lines by downregulating FDX1 expression. Most of the administered diethyldithiocarbamate-Cu complex maintained a stable chemical structure when mixed with GSH in solvent, suggesting that it potentially combats cisplatin-resistant cancer cells . AccordiAn increasing number of small compounds are being tested to target the necroptotic cell death pathway. For instance, Zhou et al. revealed that emodin, an anthraquinone compound purified from various Chinese medicinal herbs, induces necroptosis in glioma cell lines by enhancing TNF/RIPK1/RIPK3 pathway activation and thus inhibits U251 cell proliferation . AdditioDobrin et al. found that treatment of triple-negative breast cancer cells with ivermectin induces pyroptosis by activating the P2X4/P2X7-gated pannexin-1 channel . Also, bRecently, an increasing number of investigations have demonstrated that small-molecule compounds play essential roles in inducing ferroptosis in tumor cells. Zhang et al. discovered that the benzopyran derivative IMCA significantly downregulates SLC7A11 expression and reduces the contents of cysteine and GSH in cells, resulting in lipid ROS accumulation and ferroptosis in human CRC cell lines . In addiWan et al. documented that radiation therapy (RT) causes tumor cells to release microparticles with broad antitumor effects and thus abrogates immunogenicity primarily via ferroptotic cell death . MechaniAdditionally, human umbilical cord mesenchymal stem cells (hUCMSCs) were recently identified as a viable cancer therapy option. For example, these cells prevent NSCLC and HCC cells from migrating . AdditioThe growth of schwannomas is proposed to be inhibited via a unique approach based on both the introduction of an adeno-associated virus (AAV-1) and treatment with the GSDMD PFD. This combination was created using an AAV-1-based vector encoding the mouse GSDMD N-terminus under the control of the promoter P0, which is unique to Schwann cells. This gene did not cause neurotoxicity to surrounding tissues following an intratumor injection and inhibited the development of the NF2 and HEI-193 schwannoma cell lines through GSDMD-mediated pyroptosis . The intNecroptosis occurs in cancer cells, and the TME is partially protumorigenic because the inflammation underlying necroptosis may trigger tumor development by promoting cell proliferation, genomic instability, angiogenesis, and metastasis . Liu et The utilization of pyroptosis inhibitors has significant research promise because of the dual role that pyroptosis plays in cancer. The study in 2019 revealed that delivering the specific anti-GSDMB antibody in biocompatible nanocapsules significantly inhibited the metastasis and drug resistance of HER2 breast cancer cells . In addiCurrent evidence suggests that ferroptosis induced by doxorubicin (DOX) was proved to contribute to the side effect of cancer therapy, including cardiotoxicity . The DOXFinally, the use of cuproptosis inhibitors, the most recent kind of cell death, in cancer has yet to be revealed. GSH was found to inhibit cuproptosis in cells, but this can lead to cisplatin resistance in tumor cells . FurtherDespite the fact that a variety of reports on novel RCD activators and inhibitors have been published lately, clinical trials evaluating the effects of modulators of novel RCDs are still in their infancy. In this section, we summarize the clinical trials to date in primary outcome measures or interventions that have involved the investigation of relevant biomarkers of novel RCD measurement and list them in Table One clinical study aimed to investigate whether the immediate necroptosis induced by the nonablative cryosurgical freezing could be beneficial to the subsequent injection of immunotherapeutic drugs (NCT04739618). This study recruited 32 participants with metastatic solid cancer who are first treated by nonablative cryosurgical freezing and then receive multiplex immunotherapy  and evaluate overall response rate of radiographic changes. In addition, another study posted in 2018 sought for the efficacy of RIPK1 inhibitor GSK3145095 alone and in combination with pembrolizumab included 8 participants. The serious adverse event rate of this study is 50% and it was terminated following an internal review of the company (NCT03681951).Another phase II clinical study aims to evaluate the efficacy and safety of ferroptosis inhibitor MIT-001 for the prevention of oral mucositis in patients with lymphoma or multiple myeloma receiving conditioning chemotherapy with autologous hematopoietic stem cell transplantation (NCT05493800). This research was launched on August 9, 2022, and we shall keep track of its progress and other messages about the relationship between ferroptosis and inflammatory side effect of cancer therapy.So far, we have found only the modulators of these two forms of RCDs, necroptosis and ferroptosis, in clinical trials and the results of these cancer therapy methods remain to be discovered. As high-quality articles on cell death modalities continue to emerge, more clinical trials will be conducted with the research purpose stated as understanding these four cell death modalities; therefore, we believe that in the near future, better use of necroptosis, pyroptosis, ferroptosis, pyroptosis, and other mechanisms will optimize anticancer treatments.The data from Wang et al. have revealed that the epigenetic repression of RIPK3 allows NSCLC cell lines to escape from necroptosis, which subsequently increases resistance to chemotherapy . Xu ZhaoNowadays, immunotherapy represented by ICIs has become a major breakthrough in cancer treatment and has achieved considerable success in clinical treatment of some solid tumors –335. HowBy creating vaccine viruses that loaded MLKL expression, Hoecke et al. directly delivered the necroptosis mediator MLKL to tumor cells, which successfully promoted necroptotic death and enhanced antitumor immunity. The potent antitumor immunity is attributed to the increased immunity directly against neo-epitopes . AdditioPyroptosis is the main host defensive mechanism, and it boosts the tumor-killing activity of immune cells . Wang etIt has been reported that lipid peroxides caused by ferroptosis can be utilized as a signal to facilitate the recognition and processing of tumor antigens by DCs and present them to CD8 + T cells, activating cytotoxic T lymphocytes to enhance tumor immunotherapy . Thus, tSince cuproptosis is a novel mode of cell death reported this year, its role in immunotherapy is more focused on research in bioinformatics. The cuproptosis-related modification patterns developed by Zhiyong Cai and colleagues were demonstrated to be employed in prediction of immune cell infiltration in TME and evaluation of an individual’s sensitivity to ICIs . It is hApproaches targeting novel RCD modalities hold promise as novel treatments for cancer, and considerable efforts are devoted to translating novel regulators to the clinic. Thus, we complemented the review of approved drugs that modulate novel RCD pathways with descriptions of some newly developed beneficial small-molecule compounds and nanomaterials and clinical trials which intend to explore the changes in the expression levels of four novel biomarkers of RCD. Finally, RCDs can also make tumors more responsive to immunotherapy by regulating tumor immunogenicity and enhancing lymphocyte infiltration in the TME.However, in spite of the discovery of many compounds and agents that induce or modulate novel RCD programming and that exert strong antitumor effects, many studies reported opposite outcomes. For instance, Chao-Chieh Lin and colleagues discovered that the expression of the key necroptosis mediator RIPK3 in recurrent tumor cells contributed to clonogenic cell growth, causing p53 destabilization and promoting the activities of the YAP/TAZ pathways . Yee et


Health system change can increase the reach of evidence-based smoking cessation treatments. Proactive electronic health record (EHR)-enabled, closed-loop referral  to state tobacco quitlines increases the rates at which patients who smoke accept cessation treatment. Implementing such system change poses many challenges, however, and adaptations to system contexts are often required, but are understudied. This retrospective case study identified adaptations to eReferral EHR tools and implementation strategies in two healthcare systems.In a large clustered randomized controlled trial  conducted in 2016–2017, 11 primary care clinics in two healthcare systems implemented quitline eReferral, starting with 1 pilot clinic per system followed by 2 phases of implementation . Adaptations were informed by stakeholder input from live trainings, follow-up calls and meetings in the first month after eReferral launch, emails, direct observation by researchers, and clinic staff survey responses. Retrospective, descriptive analysis characterized implementation strategy modifications and adaptations using the Framework for Reporting Adaptations and Modifications to Evidence-based Implementation Strategies (FRAME-IS). A pre- and post-implementation survey assessed staff ratings of eReferral acceptability and implementation barriers and facilitators.Major modifications to closed-loop eReferral implementation strategies included aligning the eReferral initiative with other high-priority health system objectives, modifying eReferral user interfaces and training in their use, modifying eReferral workflows and associated training, and maintaining and enhancing interoperability and clinician feedback functions. The two health systems both used Epic EHRs but used different approaches to interfacing with the quitline vendor and integrating eReferral into clinician workflows. Both health systems engaged in iterative refinement of the EHR alert prompting eReferral, the eReferral order, trainings, and workflows. Staff survey comments suggested moderate acceptability of eReferral processes and identified possible targets for future modifications in eReferral, including reducing clinician burden related to EHR documentation and addressing clinicians’ negative beliefs about patient receptivity to cessation treatment.System-wide implementation of tobacco quitline eReferral in primary care outpatient clinics is feasible but requires extensive coordination across stakeholders, tailoring to local health system EHR configurations, and sensitivity to system- and clinic-specific workflows.NCT02735382. Registered on 12 August 2016.www.clinicaltrials.gov, The online version contains supplementary material available at 10.1186/s43058-022-00357-4. Contributions to the literatureThis case study applies a recent adaptation framework to characterize modifications to closed-loop electronic referral to a state tobacco quitline  implemented in two health systems.We demonstrate that eReferral was an efficient and acceptable way to extend tobacco use treatment in primary care that required tailoring to health system needs and contexts.Descriptive analysis identified facilitators and barriers to eReferral implementation that may influence the frequency and quality of referrals.Many individuals attempt to quit smoking, but few use the recommended combination of evidence-based counseling and medication . To incrElectronic health record (EHR)-enabled closed-loop referral  can facilitate the referral of primary care patients who smoke to a state tobacco quitline . The eReAdaptations and refinements are often critical to effective implementation  and therThe present study examines modifications to EHR tools and implementation strategies supporting tobacco quitline eReferral in Wisconsin, USA, before, during, and after a clustered randomized controlled trial (C-RCT) of eReferral in primary care outpatient clinics across two health systems, System A and System B . PreviouThe present study focuses on modifications to implementation strategies to support eReferral (rather than quitline interventions) that may affect the population health impact of the intervention , as specified by the RE-AIM framework . The curFRAME-IS guides the systematic documentation of modifications to intervention implementation strategies. This analysis focuses on adaptations and modifications to implementation strategies supporting eReferral. Neither the evidence-based-practice (quitline-provided smoking cessation counseling and nicotine replacement therapy) nor the foundational functions of eReferral  changed over the course of the project, i.e., the core components remained unchanged. Instead, modifications and adaptations focused on the mechanisms of eReferral, not treatment content , with thData for this retrospective case study were derived from a 2-group C-RCT that compared eReferral versus paper faxed referral (F2Q) of adult primary care outpatients to the Wisconsin Tobacco Quit Line (WTQL) . The WTQHealth systems A and B are regional not-for-profit healthcare systems in Wisconsin, and both use the Epic EHR. Details regarding populations served by each health system, clinic eligibility, randomization, masking, workflows, and results are reported in a previously published study . Health The research team played the role of a coordinating center and facilitator of eReferral implementation with health system and WTQL stakeholder partners. This coordination involved helping negotiate agreements between health systems and the WTQL for two-way secure data sharing and interoperability. The research team also provided financial resources to cover health system information technology (HIT) costs to ensure that HIT development would be prioritized and would occur in a timely manner to ensure completion of the C-RCT during the grant funding period. Once the HIT teams were assembled, the research team coordinated the development and functional testing of eReferral with the WTQL.This case study focuses on WTQL eReferral and not fax referral. Figure The WTQL proactively called referred patients to offer services and automatically returned results regarding the referral outcome  to the patient’s record in the EHR and the referring provider’s inbox. In System A, any medications dispensed by the WTQL were also automatically added to the patient medication list in the EHR, in addition to being returned to the provider inbox and patient record as part of the referral result report. System B did not adopt automatic updating of patient medication lists.Although specific settings, processes, and implementation strategies were modified, the core workflow steps and eReferral functions were maintained throughout the study.Figure In all clinics, the eReferral launch was immediately preceded by a 30–60-min live training for clinic staff in the eReferral workflow. A telephone check-in with the clinic manager occurred approximately 1 week later and a second 30–60-min in-person, group feedback session and training refresher with clinic staff occurred approximately 1 month after launch (timing varied across clinics). The WTQL e-mailed monthly summary reports to clinic managers to provide performance feedback and track progress at each clinic.Throughout implementation, but particularly during HIT development and pilot testing, the research team facilitated frequent communication among stakeholders to identify challenges to eReferral and to implement relevant adaptations (planned efforts to tailor and improve implementation strategies) . Stakehostrongly disagree, 7 = strongly agree). Quantitative survey responses were treated as cross-sectional because we could not link respondents across waves. Survey responses from Health System A and Health System B were combined for analyses of acceptability and implementer perceptions.Just before and 6 months after the launch of eReferral in each clinic, the stakeholders who implemented eReferral  were e-mailed invitations to complete a brief (5-min) anonymous online survey. The survey was preceded by an online consent form. Proceeding to the survey was considered evidence of consent to participate. Fifteen survey items addressed the perceived value of addressing tobacco use, self-efficacy regarding addressing patient tobacco use, and satisfaction with the referral process. The items are shown in Table In addition, the 6-month post-implementation survey invited implementing staff to identify facilitators and barriers of eReferral and share information about modifications they made to implementation strategies . Open-ended items asked respondents to identify barriers to and facilitators of addressing patients’ tobacco use, and what they typically said to patients to introduce the WTQL.This retrospective case study followed the standards for reporting qualitative research, outlined in Table SAcceptability and staff perceptions were analyzed via descriptive statistics and ANOVA tests of pre-post differences in the 15 quantitative staff survey items using R 3.6.2. Free responses on the staff survey were treated as qualitative data. Illustrative individual responses and common themes were summarized after independent extraction and discussion by two authors (JK and DM).Table SAdaptations to align eReferral to healthcare system goals and policies began in the health system recruitment process and continued throughout implementation planning and pilot testing. During the planning phase in System B, stakeholders in HIT alerted the research and implementation planning team to potential misalignment between the proposed interface mechanism with the WTQL and System B interoperability capabilities and preferences. Based on this, the nature of the interface supporting interoperability was adapted to the System B HIT context, while preserving core interoperability functions and HIPAA compliance.In addition, during the pilot testing phase in System A, clinical and HIT stakeholders suggested ways to redesign the eReferral alert so it would meet the criteria for Meaningful Use of the EHR  by documExtensive stakeholder input was gathered and used to adapt the eReferral alert and order in the EHR throughout the planning, piloting, and implementation study phases , which caused the alert to present prematurely. In System A, where a “hard-stop” firing rule was in place, clinicians selected “patient declined,” without ever engaging the patient, to conclude their pre-charting . Selecting “patient declined” during pre-charting suppressed the appearance of the alert during the patient-facing part of the encounter and reduced opportunities for clinicians to discuss eReferral with patients. To accommodate pre-charting, a “Defer” option was added to the alert. A “Defer” option allowed clinicians to satisfy the “hard-stop” alert and close the chart after pre-charting, while also allowing the alert to fire again when the encounter was reopened when the clinician was with the patient. Clinic and system stakeholders in System A suggested and implemented this solution and trained clinic leaders and clinicians in this added functionality in the alert. Health System B, on the other hand, did not implement a “hard-stop” rule and did not need to add a “Defer” option to facilitate chart closure during pre-charting and so offered only “Order” and “Refused” options in the alert.To affect clinical care, EHR tools like eReferral must be integrated into sustainable workflows. In both systems, prior to WTQL eReferral implementation, assessing and addressing tobacco use was often assigned to medical assistants or nurses. In this study, eReferral was designed to involve clinicians/providers because the literature points to the efficacy of brief clinician counseling regarding quitting . In addiStakeholder input also led to the inclusion of automated messages regarding quitline services to after-visit summaries provided to all patients for whom the eReferral alert fired. These messages were tailored to clinician eReferral alert response. For patients who accepted eReferral, a congratulatory message encouraged them to accept the call from the WTQL that would come in a few days. For all others , the visit summary described WTQL services and provided the toll-free quitline number.Stakeholder input also identified why some clinicians addressed the eReferral alert at low rates. When medical assistants or other staff who conducted rooming activities secured the EHR session at the workstation in the clinic room with the encounter open (rather than logging out fully), this altered the appearance of the eReferral alert making it less prominent for clinicians. To address this, clinic managers provided additional training to clinic staff who conducted rooming activities to log out fully of the workstation (rather than just secure it).Post-implementation staff surveys indicated that some primary care teams continued to shift smoking treatment activities to medical assistants or nurses even after adopting eReferral. After data collection ended, one system shifted primary responsibility for eReferral back to medical assistants  while the other retained the clinician-centered workflow when eReferral was adopted as the standard of care system-wide.Interfaces between the WTQL and the EHR were disrupted by updates and changes at either entity . In pilot and mid-implementation phases, clinicians and clinic managers reported concerns regarding the length of time it took to receive patient outcomes from the WTQL. Tweaking of interoperability and WTQL feedback reports was required to ensure that feedback provided to referring clinicians was timely, complete, and clear  post-implementation survey respondents completed open-ended survey questions about their perceptions of WTQL referral facilitators and barriers. Many responses identified lack of time or time pressure as a barrier to addressing tobacco use well. One respondent wrote “With everything else that is being thrown at primary care, I find I have less and less time to address even important things like smoking cessation at office visits.” Another described the following barrier: “Increasing demands on time in regard to satisfying electronic medical records and organizational requirements for best practice protocol. This results in much less time with patients and much more time in front of the computer.”Several clinicians, medical assistants, and nurses cited patient lack of interest in quitting and/or resistance to treatment as eReferral barriers. For example, one medical assistant noted: “… most of all it is the willingness of the patient. Often when they say they are interested they lose all of their interest when walking out the door.” Some clinicians reported modifying the eReferral offer language by first assessing patients’ readiness to quit before referring to the quitline. Clinician and staff respondents in eReferral clinics mentioned barriers related to low WTQL connection rates. One noted: “Referral process to the tobacco quit line was okay, but it seemed like the majority of time they were unable to reach my patients for some reason. It ended up being a waste of time, so I stopped doing it.”Many open-ended responses, especially from medical assistants, endorsed the effectiveness of clinic eReferral protocols in engaging patients in tobacco cessation treatment, but some responses suggested that EHR and workflow steps could be more efficient. For example, one respondent noted that the BPA alert fires at inopportune times in their workflow: “The BPA fires before even entering the room and then again as I try to exit the room, so often times it is an after-thought.” Another respondent wrote: “It would be nice to have better flow within our EHR. Currently we have the tobacco quitline initially, then a social history section in one place, best practice advisories in another place, and no clear way of pulling documentation into our notes.”In addition, consistent with stakeholder input before and during implementation Tables S and 2, mImplementation of eReferral to a state tobacco quitline in primary care clinics in two regional healthcare systems required extensive stakeholder engagement in implementation monitoring and adaptation and achieved moderate acceptability ratings among implementing clinic teams. Customizing EHR tools, workflows, and training for healthcare systems requires engaging diverse stakeholders at multiple stages of planning, piloting, and implementation. Key implementation strategies modified to enhance eReferral implementation included aligning eReferral with health system priorities; refining EHR eReferral displays, workflows, and training; and maintaining interoperability and closed-loop eReferral functionality. Surveys of clinic staff indicated that the adapted approach had moderate acceptability in terms of clarity, ease, efficiency, and effectiveness. Survey responses also suggested that limited time to address tobacco use and lack of positive feedback regarding client acceptance of quitline services are barriers to eReferral.Applying the FRAME-IS framework to the analysis of meeting notes, email chains, training documents, and open-ended clinician and clinic staff survey responses regarding eReferral implementation identified several planned, system-wide adaptations. These adaptations were purposive to increase adoption, feasibility, acceptability, and sustainment of tobacco quitline eReferral in adult primary care, and/or to increase reach among patients so more were effectively connected with evidence-base quitline interventions. Diverse healthcare system stakeholders  helped to identify challenges and develop adaptations to address these challenges. Although the research team played a coordinating role in this process, most of the problems and solutions were generated by healthcare system personnel.In addition to planned, system-wide adaptations, other, more local modifications were identified in the present analysis. Check-ins with front-line clinic staff during site visits and refresher trainings, and calls with clinic managers mid-implementation, helped to identify specific workflow practices that posed challenges . Some of these challenges were addressed by system-wide solutions , while others involved more local solutions . These local modifications and solutions suggest that even for presumptively straightforward EHR-based strategies, stakeholder input on specific clinic practices and contexts should be collected during implementation planning.Post-implementation staff survey responses identified moderate levels of agreement that eReferral is clear, easy, efficient, and effective. Survey responses identified barriers to eReferral, including limited time and insufficient or negative feedback about patient results. Open-ended responses among some staff underscored concerns about eReferral workflow efficiency  and effectiveness . Open-ended responses also captured variability in the way eReferral was implemented at the individual staff level . Assessing the implementation perspectives and practices of a broad range of clinicians and staff earlier in implementation may provide a rich source of possible adaptation targets   and straSurvey respondents identified challenges to addressing tobacco use in primary care that are consistent with other evidence on barriers to and facilitators of smoking treatment implementation in healthcare , 24–26. The present results should be interpreted with the following limitations in mind. First, our methods of assessing adaptations may have captured more centralized, planned adaptations than local and/or unplanned modifications. As such, our modification list is likely not exhaustive or fully representative of modifications in quitline eReferral. Second, staff survey responses were modest overall and lower at the 6-month post-implementation wave than at the pre-implementation wave. As such, the comparability of samples across waves is unclear and the representativeness of the respondent sample is unknown. Because these surveys were anonymous and treated as cross-sectional rather than longitudinal, we do not know if these changes across the waves reflect within-person changes or sampling differences . The primary purpose of the quantitative survey analyses was to assess the acceptability of eReferral processes after its implementation, rather than change in attitudes over time. The pre-implementation ratings are reported to provide context rather than comparison with post-implementation and adaptation and results. Third, adaptations were intended to improve the adoption, feasibility, acceptability, and reach of eReferral. Because this was a retrospective case study, we were not able to directly assess the impacts of specific adaptations on these outcomes. In future research, proactive application of other frameworks, such as the Model for Adaptation Design and Impact (MADI) , can guiWidely adopted EHR platforms, including Epic, offer tools to support eReferral to low-barrier, no-cost evidence-based smoking cessation treatment offered at no cost to health systems or patients by state tobacco quitlines. Engaging health system and quitline stakeholders in planned, centralized adaptation of implementation strategies may help to align quitline eReferral with broader priorities and objectives, engage stakeholders in interface and workflow refinement, and maintain interoperability and feedback loops in ways that are acceptable and sustainable in adult primary care settings.Additional file 1: Table S1. Standards for Reporting Qualitative Research (SRQR). Table S2. Modifications and adaptations to eReferral strategies before, during, and after eReferral implementation in two healthcare systems (HS).


The highly metastatic and immunosuppressive microenvironment of ovarian cancers is a major determinant of the aggressive nature and therapeutic resistance of ovarian cancer. Therefore, we believe that a thorough understanding of the mechanisms that regulate the composition and function of the tumor microenvironment is critical for the development of a more effective course of treatment for this devastating malignancy. This review summarizes the recent literature on the major metabolic pathways affecting macrophage immune metabolism and its impact on phenotypic and functional changes in macrophages in the ovarian tumor microenvironment.The interaction between tumor cells and macrophages in the tumor microenvironment plays an essential role in metabolic changes in macrophages and reprograms them towards a pro-tumorigenic phenotype. Increasing evidence indicates that macrophage metabolism is a highly complex process and may not be as simple as previously thought. Pro-inflammatory stimuli switch macrophages towards an M1-like phenotype and rely mainly on aerobic glycolysis and fatty acid synthesis, whereas anti-inflammatory stimuli switch macrophages towards an M2-like phenotype. M2-like macrophages depend more on oxidative phosphorylation (OXPHOS) and fatty acid oxidation. However, this metabolically reprogrammed phenotypic switch in macrophages remained a mystery for a while. Therefore, through this review, we tend to describe how macrophage immunometabolism determines macrophage phenotypes and functions in tumor microenvironments (TMEs). Furthermore, we have discussed how metabolic reprogramming in TAM can be used for therapeutic intervention and drug resistance in ovarian cancer. Ovarian tumor development is a complex, heterogeneous process influenced by several epigenetic and microenvironmental factors ,2. A pooRecent evidence suggests that macrophages are highly reprogrammed in TMEs ,16. LikeTumor-infiltrating macrophages or macrophages residing in ascites may alter their metabolism in the tumor microenvironment . TAMs wiGlycolysis is linked with TAM recruitment and tumorigenesis . TAMs, iHypoxic TAMs show a markedly higher glycolysis rate and an increase in growth factors, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), which aid the progressiveness of cancer by enhancing tumor growth, angiogenesis, and metastasis . In otheHence, TAMs are known for establishing metabolic coordination with cancer cells and creating a pro-invasive TME. Recently, eight glycolysis-related genes  were also identified, which are known as prognostic genes as they are the signature survival genes in ovarian cancer .Despite these results, more research is needed to ascertain the role of TAM glycolysis in immune and stromal cell recruitment and function. The high glycolytic rate of tumor cells increases lactic acid production. Tumor cell-derived lactic acid induces hypoxia-inducible factor (HIF)-1α-dependent peritumoral polarization of TAMs, and hypoxia is associated with the accumulation of peritumoral TAMs . Lactic Altogether, the acidic TME results from high glycolysis of tumor cells, and poor perfusion plays a pivotal role in tumor progression. Acidity (independent of lactic acid) augments the pro-tumoral polarization of TAMs in prostate cancer . In TAMsApart from glucose metabolism, lipids are a crucial metabolic hallmark of cancer. TAMs exhibit alterations in lipid metabolism, including increased fatty acid production, absorption, and storage, which have been linked to functional reprogramming, but the underlying processes remain largely not understood in ovarian cancer .In the tumor microenvironment, adipocytes are the prime source of fatty acids. In the TME, adipocyte-derived lipids, including fatty acids, affect cancer cells and various peripheral cells, such as cancer-associated fibroblasts, dendritic cells, macrophages, and other immune cells . Cancer Lipid metabolism plays a crucial role in TAMs differentiation and function. Recent reports suggested that macrophages show more lipid accumulation and dependency on fatty acid oxidation (FAO) compared to glycolysis in TMEs. Inhibition of FAO reduced the number of TAMs in the TME . In addiOvarian cancer cells are also responsible for the depletion of lipids rafts and the efflux of cholesterol from macrophages which promotes IL4-mediated reprogramming of macrophages by inhibiting the expression of IFNγ induced genes, hence promoting the tumorigenic functions of TAMs. Inhibiting the cholesterol efflux by deleting the ABC transporter reduces tumor progression and can be a novel therapeutic strategy .Lipogenesis and lipid transport mediated decreased infiltration of M1 macrophages are the reason for the high metastatic rate in obese people . It has Ovarian tumor cells are also found to release metabolites such as 5-lipoxygenase (5-LOX), a member of the lipoxygenase gene family, which is a crucial enzyme assisting in the conversion of arachidonic acid to 5-HETE and leukotrienes. Increased 5-LOX metabolites from hypoxic ovarian cancer cells promoted migration and invasion of macrophages, which is mediated by the upregulation of matrix metalloproteinase (MMP)-7 expression through the p38 pathway . This inIn ovarian cancer, TAMs regulate metabolic function through PPARβ/δ and some signature genes  associated with immune regulation and tumor progression that correlate with short relapse-free survival in serous ovarian cancer ,48Several studies have shown that targeting lipid metabolism significantly reduces tumor burden. Studies have shown that targeting cholesterol metabolism using a simvastatin-based nanomedicine strategy reverses epithelial–mesenchymal transition and repolarizes TAM to treat drug-resistant cancer cells .Several studies explored the potential consequences of amino acid metabolism on the functional reprogramming of TAMs. However, most of this research is observational, with only a few providing mechanistic insight. Arginine depletion in culture medium was the first proof of this theory in the 1970s, where it was demonstrated that macrophages could inhibit lymphocyte activity by depleting arginine in media . The comGlutaminolysis, a process by which cells convert glutamine into the TCA cycle, has been correlated with ovarian cancer aggressiveness . RecentlApart from this, some amino acids such as aspartic acid, asparagine, branched-chain amino acids , and some 1 carbon (1C) metabolites  also play a role in ovarian cancer metabolic reprogramming. However, specific mechanisms have yet to be explored in the context of TAM reprogramming.So far, we have discussed that metabolic reprogramming is a common feature of macrophages and tumor cells that function symbiotically. Moreover, macrophages directly ‘fuel’ tumors by exporting different metabolites preferentially used by adjacent tumor cells. Recently, Goossens et al. showed that cancer cells promote the efflux of cholesterol from macrophages, which are associated with increased IL4 expression, while decreased IFNγ-related gene expression results in reprogramming pro-tumorigenic TAMs .It has also been reported that extracellular vesicles (EVs) released by tumor cells and macrophages participate in the metabolite shuttling in the tumor microenvironment . HoweverThe most effective metabolite in EVs that regulate metabolic reprogramming has not been identified yet.EVs’ content greatly resembles the cells of their origin; however, it is essential to note that EVs’ metabolite cargo is greatly affected by current cell culture conditions that sometimes generate EVs with distinctively different metabolites than their parent cells .It is unclear whether genes or EVs induce differences in metabolic profile in particular cells from other cells.There are still technical obstacles in isolating and purifying EVs from multiple body fluids for metabolite characterization .Despite the various advancement in EVs-related research in the past few years, the question of the significance of EVs-mediated metabolic reprogramming in ovarian cancer is still unsolved. The following factors limit this study:In addition to these factors, the metabolite composition of EVs is highly affected by pre-analytical factors that include storage, handling, and quantification . Enzyme However, the lack of a consensual approach to the separation of EVs is a major obstacle to the advancement of EV research. Thus, further research should focus on developing standard methods for the isolation and purification of EVs and exploring the possibility of targeting the bioactive molecules in EVs to elucidate further the activities and interactions of EVs and the immunotherapeutic roles they may be able to play in the metabolic reprogramming of the TME.The persisting challenge in clinical oncology is to improve cancer treatments’ efficacy. At the same time, the tendency of cancer cells to become resistant to the majority of treatments limits complete pathological regression . A tumorTAMs promote tumor development in solid tumors, increase angiogenesis, induce lymph angiogenesis, remodel the stroma, metastasize, and inhibit the immune system by modulating and releasing numerous pro-angiogenic factors, including VEGF-A, tumor necrosis factor (TNF), FGF, thymidine phosphorylase, urokinase plasminogen activator, and adrenomedullin (ADM). They also promote stromal remodeling, tumor cell invasion, and metastasis by releasing many enzymes, including plasmin, MMPs, cathepsin B, PDGF, and TGF-β1. TAMs have been shown to exhibit tumor-supporting properties in various cancers .However, a growing number of experimental and clinical data suggest that TAMs play a dual function in tumor growth and survival, and their anti-cancer activity has been partially discovered . HoweverSo far, it has been shown that tumor cell-derived stromal cells (which may be found in the tumor microenvironment) help the cancer cells proliferate, invade, and metastasize ,71,72. TCurrent TAM therapies fall into three categories: (1) suppressing pro-tumor TAMs, which includes TAM recruitment inhibition and depletion; (2) activating anti-tumor TAMs, which refers to converting pro-tumorigenic macrophages to anti-tumorigenic macrophages; and (3) reprogramming of TAMs ,75. In aOne therapeutic approach based on macrophages is converting TAMs into M1 type macrophages. In this regard, OXPHOS pathways play a vital role in the M1 transition of M2 TAMs. Inhibition of mitochondrial OXPHOS is known for preventing the reprogramming of pro-inflammatory M1 macrophages to the anti-inflammatory M2 phenotype, reducing tumor burden. This was further supported by a study where metformin and IACS010759-mediated inhibition of OXPHOS has reduced the tumor burden . InteresTherefore, it is worth exploring the possibility that tumors with SDH deficient mutations and SDH subtypes can be therapeutically targeted by exploiting their metabolic state. Recently, succinate dehydrogenase overexpression in ovarian cancer was targeted using the anti-metabolic compound shikonin, which demonstrated potent anti-tumor efficacy .The recent advancements in ovarian cancer research suggest the unresponsiveness of the current therapeutic approaches in reducing mortality rates. It is becoming more evident that the metabolic reprogramming of the TAM and TME plays a crucial role in the tumor. According to recent studies, TAM enhances the progression of ovarian tumors, but because of its high degree of plasticity, it can also be used to suppress tumor growth by remodeling the M2-like phenotype into an M1-like phenotype, and the plasticity of TAMs can be harnessed in ovarian cancer therapeutics.Exploring the different states of TAMs using high throughput next-generation techniques such as single-cell sequencing and metabolomics research will improve the current understanding of ovarian TMEs and provide specific information about them. So far, the mechanistic insights gained from the molecular pathways required for these alterations have been limited, and thus current treatments that seek to modify metabolic pathways will be insufficient unless all therapeutic targets are explored. Considering TAMs as a key player will improve current regimen decision-making and open new avenues for precision immuno-oncology. In addition, macrophages can help with tumor therapy and the prevention and management of adverse effects in ovarian cancer treatment.


Pain is encoded by the spatiotemporal neural communication that represents the integration of cognitive, emotional-affective, and sensorimotor aspects of pain. Therefore, future studies should seek to identify the pathological oscillatory brain communication in chronic pain as a therapeutic target for tACS. In conclusion, tACS could be effective for re-establishing oscillatory brain activity and assisting social interaction, and it might help develop novel approaches for pain control.Developing effective tools and strategies to relieve chronic pain is a high-priority scientific and clinical goal. In particular, the brain regions related to pain processing have been investigated as potential targets to relieve pain by non-invasive brain stimulation (NIBS). In addition to elucidating the relationship between pain and oscillatory brain activity, transcranial alternating current stimulation (tACS), which can non-invasively entrain oscillatory brain activity and modulate oscillatory brain communication, has attracted scientific attention as a possible technique to control pain. This review focuses on the use of tACS to relieve pain through the manipulation of oscillatory brain activity and its potential clinical applications. Several studies have reported that tACS on a single brain reduces pain by normalizing abnormal oscillatory brain activity in patients with chronic pain. Interpersonal tACS approaches based on inter-brain synchrony to manipulate inter-brain communication may result in pain relief  The brain regions involved in pain processing include the primary and secondary somatosensory cortices; primary motor and supplementary motor cortices; insular cortex; anterior cingulate cortex; thalamus; regions within the prefrontal and parietal cortices; and regions involved in emotion, memory, and fear processing in the amygdala, hippocampus, and subcortical structures, including the basal ganglia . Among tvia the application of a weak oscillatory current to the brain through the scalp  and magnetoencephalography (MEG) have demonstrated that neural oscillatory frequencies corresponding to the theta (4–7 Hz), alpha (8–12 Hz), beta (13–29 Hz), and gamma bands (30–100 Hz) are associated with pain processing . The eluhe scalp . It shifhe scalp .As a mechanism of the brain’s complex processes based on neural communication across distant brain areas, synchronization of oscillatory brain activity among networks is hypothesized to facilitate information transfer across brain regions by temporally aligning neural processing . Therefovia prosocial effects. This concurrent manipulation of brain activities helps elucidate the role of inter-brain oscillatory communication in pain processing via interpersonal interaction. However, the application of dual-brain stimulation to modulate the clinician’s brain activity in clinical settings is unrealistic. Therefore, this review discusses the potential of using brain-to-brain interfaces, which allow two brains to mutually exchange decoded neural information for analgesia via interpersonal interaction. Finally, to inform future pain control research, the altered functional connectivity among large-scale distributed brain networks is reviewed and the potential of tACS approaches to modulate network-level abnormalities of oscillatory communication in chronic pain is discussed.This review explores the potential for applying tACS techniques to achieve pain control by modulating oscillatory brain activity. First, this review focuses on tACS studies that target a single brain for pain control by normalizing oscillatory brain activity. Next, inter-brain synchrony related to pain processing in the context of social interaction revealed by hyperscanning, which is a neuroimaging technique used to measure the activity of multiple brains simultaneously, is reviewed. Then, this review discusses a potential dual-brain approach using tACS to reduce pain Accumulating evidence shows that chronic pain is closely associated with altered neural oscillations . The mosA literature search regarding original research using tACS for pain control was carried out in PubMed, Scopus, and Web of Science using the following search terms:  AND . Searches were limited to papers published in English before April 2022. No inclusion or exclusion criteria were established because there were only five studies that modulated experimental and chronic pain using tACS as a result of a literature search. The effects of tACS on experimental pressure pain in healthy individuals were first investigated by Few tACS studies have adapted conditions other than somatosensory alpha oscillations to modulate pain. A recent study investigated whether gamma-tACS over bilateral prefrontal cortices modulates experimental heat pain . Gamma oThus, alpha-tACS over the sensorimotor cortex may represent a potential approach to relieve chronic pain. However, tACS studies for pain control are limited, and this hypothesis is strongly influenced by publication and reporting biases. Moreover, the alpha-tACS effect on experimental pain in healthy individuals is inconsistent. The personalized frequency-location parameters might reduce variability in the tACS response. Moreover, important insights into the neural mechanisms of chronic pain could be yielded using appropriate tACS parameters considering pathological factors, such as disease and duration of onset. So far, I have discussed tACS approaches targeting a single brain. Next, the focus will shift to tACS based on inter-brain communication between two brains in the context of interpersonal interactions.via interpersonal interaction, a neuroimaging method that allows simultaneous investigation in multiple persons is useful. Studies measuring the activity of multiple brains simultaneously, termed hyperscanning, have revealed that various interpersonal factors can be reflected in inter-brain communication between individuals engaging in joint actions and communication (It is known that interpersonal interactions relieve pain . To elucnication . HyperscPain empathy enables one to understand or recognize others’ pain perception by observing them experiencing pain, inducing emotional sharing associated with pain reduction . Peng etvia movement synchrony between the clinician and patient, providing a good foundation for facilitating pain therapy.Neural coupling during interpersonal interaction may either cause or be caused by behavioral entrainment, emotional sharing, and common understanding, and these factors may work jointly to promote analgesia . Therefovia prosocial interaction. However, applying NIBS to the clinician’s brain is not realistic in the clinical field because of ethical issues regarding the manipulation of healthy brains. Thus, rather than being a therapeutic tool, hyper-tACS is useful as a technique to investigate the role of frequency and site-specific brain-to-brain communication in analgesia via interpersonal interaction, which can complement hyperscanning and single-brain stimulation studies. After clarifying the relationship between inter-brain oscillatory communication and analgesia, it is desirable to use the brain-to-brain interface, which could induce inter-brain synchrony by modulating the patient’s brain activity using tACS and adjusting it to the clinician’s brain activity. A brain-to-brain interface enables the mutual exchange of decoded neural information between two brains through a brain-computer interface. This interface receives the sender’s neural information and transmits it to the receiver’s brain via electrical stimulation (As aforementioned, alpha-band inter-brain synchrony between dyads is involved in pain empathy and interpersonal touch processing. Although it remains unclear whether the specific frequency and site of inter-brain communication are associated with the clinician-patient relationship, it is well known that the alpha–mu band in centroparietal areas comprises the most robust oscillations of inter-brain synchrony during social interaction . Therefomulation . Direct Pain is a complex, multifaceted experience that has physiological, emotional-affective, and cognitive dimensions, indicating that chronic pain progression might be influenced by altered integration between sensory and contextual process networks . ConsistFurther investigation with regard to tACS approaches is warranted in future studies. Networks, such as the DMN and SN, comprise deep brain sites that are difficult to stimulate with conventional NIBS. As a new tACS technique, transcranial temporal interference stimulation may be able to solve this problem . This teIn conclusion, despite the translational potential of tACS based on oscillatory brain activity for pain control, the methodology remains in its infancy and poses several issues that require greater innovation. Neuroscientific knowledge of intra- and inter-brain communication related to pain processing is instrumental in developing optimal tACS approaches as non-pharmaceutical alternatives for the treatment of chronic pain.The author confirms being the sole contributor of this work and has approved it for publication.


Individuals with diabetes face higher risks for macro- and microvascular complications than their non-diabetic counterparts. The concept of precision medicine in diabetes aims to optimise treatment decisions for individual patients to reduce the risk of major diabetic complications, including cardiovascular outcomes, retinopathy, nephropathy, neuropathy and overall mortality. In this context, prognostic models can be used to estimate an individual’s risk for relevant complications based on individual risk profiles. This review aims to place the concept of prediction modelling into the context of precision prognostics. As opposed to identification of diabetes subsets, the development of prediction models, including the selection of predictors based on their longitudinal association with the outcome of interest and their discriminatory ability, allows estimation of an individual’s absolute risk of complications. As a consequence, such models provide information about potential patient subgroups and their treatment needs. This review provides insight into the methodological issues specifically related to the development and validation of prediction models for diabetes complications. We summarise existing prediction models for macro- and microvascular complications, commonly included predictors, and examples of available validation studies. The review also discusses the potential of non-classical risk markers and omics-based predictors. Finally, it gives insight into the requirements and challenges related to the clinical applications and implementation of developed predictions models to optimise medical decision making.The online version contains peer-reviewed but unedited supplementary material including a slideset of the figures for download, which is available to authorised users at 10.1007/s00125-022-05731-4. Precision medicine in diabetes emphasises tailoring diagnostics or therapeutics to subgroups of populations sharing similar characteristics, thereby minimising error and risk while maximising efficacy . One focWhile individuals with diabetes are at higher risk for macro- and microvascular diseases than those without diabetes, the risk is likely to differ substantially from person to person. Diabetes evolves from a variety of pathophysiological constellations, and the presence of other risk factors beyond hyperglycaemia is likely to differ. Precise prognosis of an individual’s likelihood of developing complications would identify those at highest risk, prompting more intensive medical treatment to control risk factors and prevent complications. Precise prognostics allow an individual to be matched to others with a similar complications risk and, through knowledge of treatment efficacy, enable optimal choice of therapy . PrecisiImportantly, precision prognostics differs from attempts to identify subsets of individuals based on physiological variables alone without the use of event information in the process of classification. To illustrate, recent attempts to identify subclasses of diabetes in newly diagnosed individuals , 6 allowFor prediction models to qualify for implementation into routine care, they should undergo different stages: model development; model evaluation in terms of prognostic performance ; translation to clinical decision support; and evaluation of the clinical implementation –10. In tC index ranging between 0.5  and 1.0 (perfect discrimination) −2 decrease in eGFR, either alone or in combination  criteria), standardisation appears less common for microvascular complications. Some studies aimed at addressing this issue have derived models for different diagnosis definitions or differently composed endpoints. For instance, the Risk Equations for Complications of Type 2 Diabetes (RECODe) models predicts nephropathy as microalbuminuria, macroalbuminuria, renal failure or end-stage renal disease, doubling of serum creatinine, or >20 ml minbination . Still, Second, the pathophysiological interconnection of diabetes-related secondary diseases complicates the prediction of diabetic complications in type 2 diabetes –18 and tThird, the composition of the study sample used for model development is important. The baseline risk and the estimated weights for individual risk factors incorporated into prognostic models are average-based and depend on the derivation cohort. This potentially conflicts with the concept of precision prevention, as the ‘average’ may not accurately reflect the risk in minorities or subgroups in particularly heterogeneous study samples. One may, for example, argue that separate prognostic models for diabetes complications are needed for the different diabetes clusters  rather tStatistical models for predicting macro- and microvascular complications are widely available. While some models were developed in individuals with diabetes, others , the estimate was imprecise  ) .Several models have been developed to predict all-cause mortality as the ultimate complication of diabetes. Models that have been externally validated include the RECODe model, the model by Chang et al and the ENFORCE model , 60, 61.It is worth noting the development of different models within single studies predicting multiple diabetes-related complications, including macro- and microvascular complications and/or overall mortality, namely the RECODe models , the UKPOverall, a wide variety of models applicable in clinical practice for the prediction of microvascular complications, and in particular macrovascular complications, as well as mortality is available. Rather than developing new models, future research should focus on external validation and comparison of existing models in target populations, with the aim of providing information about appropriate model choices and implementation.As already discussed, conventional prediction models for macro- and microvascular complications and mortality include a limited set of clinical characteristics and biomarkers based on their availability in routine care. However, information on biomarkers not routinely collected may also be predictive, although their usefulness depends on the extent to which they provide information for prediction not already provided by established risk factors. Thus, novel predictors not only need to be associated with endpoints but also need to demonstrate improvements in risk prediction as evaluated by discrimination, calibration and reclassification statistics.Investigations of predictive biomarkers can either be hypothesis-driven or exploratory. Particularly, methodological developments aimed at identifying, characterising and quantifying biological molecules do now support the screening of high numbers of potentially predictive biomarkers related to the genome, transcriptome, proteome or metabolome Fig. . NumerouFigure Specific biomarkers or biomarker combinations can lead to improvements in risk prediction of CVD in diabetes, beyond classical CVD risk factors. As reviewed elsewhere in more detail , N-termip=0.04). Combining genetic risk scores for several complication-related traits to give a multi-polygenetic risk score using a total of ~600 variants yielded moderate discriminative abilities for major macrovascular (C statistic 0.68) and microvascular events (C statistic 0.67) in ADVANCE [Genetic risk scores, combining large numbers of individual gene variants, have been evaluated in recent years in terms of predicting the risk of diabetes complications. In the Action to Control Cardiovascular Risk in Diabetes (ACCORD) and Outcome Reduction With Initial Glargine Intervention (ORIGIN) studies, a polygenetic risk score for coronary artery disease, combining information on 204 variants from genome-wide association studies, had poor discriminative ability for major cardiovascular events (C statistic 0.57) . Still,  ADVANCE . This riBesides classical risk factors and novel biochemical and genetic markers, prediction models for diabetes-related complications have also included other individual characteristics relating to current treatment, comorbidities or the presence of complications other than those predicted , 77. Fur1c and systolic BP. As a consequence, the model provides information not only on the need for treatment initiation based on the individual risk but also the requirements regarding treatment intensity and combination. Still, very few studies have directly evaluated precision prognostic treatment approaches vs standard care. In the Early detection of diabetic kidney disease by urinary proteomics and subsequent intervention with spironolactone to delay progression (PRIORITY) trial, the urinary proteomic CKD273 score was used to quantify the risk for developing microalbuminuria. Participants who were classified as high risk were entered into an RCT to test whether progression to microalbuminuria could be prevented with the mineralocorticoid receptor antagonist spironolactone. However, development of microalbuminuria was not significantly different from that seen with placebo [For precision prognostics to be successful, it should allow clinicians to match a patient to others with a similar complication risk and optimise therapies for this patient to result in an extended complication-free life. Thus, precision prognostic models are not useful by themselves, but rather they have a positive impact on medical decision making. The availability of validated prognostic models that accurately predict risk is an important first step towards this goal. One major obstacle preventing application of precision prognostic models into care is the largely unknown clinical benefit. Reporting discrimination and calibration will always be important for a prediction model but if the model is to be used for making clinical decisions, decision-analytical measures should be reported. For example, decision-curve analysis, plotting the net benefit of a prognostic model across different threshold probabilities, allows the definition of a single probability threshold that can be used to categorise individuals as positive or negative while weighting false-positive and false-negative classifications . Further placebo .Cost-effectiveness analysis should, in addition to treatment effects, be informative for identifying optimal thresholds of predicted risk to target treatments based on precision prediction models, as has been demonstrated for diabetes prevention interventions . In thisStatistical models to calculate absolute risks need to be ‘translated’ into test instruments for their practical use. In this context, effective strategies to communicate absolute risks and risk limits or classifications are necessary to enable clinicians and patients to make treatment decisions. As an example, the Joint Asia Diabetes Evaluation platform, a web-based data collection and decision support system, provides personalised risk categorisation and absolute risk estimation for CVD and retinopathy. Individuals with diabetes enrolled in these integrated care programmes experienced lower rates of major complications than those in routine care . HoweverGiven the largely unknown clinical benefit of precision prognostics, it is no surprise that there is currently limited reference to prognostic models in medical guidelines for the treatment of diabetes. The ADA recommends the use of the Pooled Cohort Equation CVD risk model, although this model was not developed specifically in individuals with diabetes . Still, Although an increasing number of prognostic models have been developed and validated to predict diabetes complications, the concept of precision prognosis as a component of precision medicine is still in its infancy. Epidemiological and clinical research could inform its further development (Text box 2).ESM Table 1(XLSX 39 kb)Slideset of figures(PPTX 329 kb)