Chemotherapy-induced peripheral neuropathy (CIPN) negatively affects physical function and chemotherapy dosing, yet, clinicians infrequently document CIPN assessment and/or adhere to evidence-based CIPN management in practice. The primary aims of this two-phase, pre-posttest study were to explore the impact of a CIPN clinician decision support algorithm on clinicians’ frequency of CIPN assessment documentation and adherence to evidence-based management.n = 81 usual care phase [UCP], n = 81 algorithm phase [AP]). During the AP, study staff delivered a copy of the CIPN assessment and management algorithm to clinicians (N = 53) prior to each clinic visit. Changes in clinicians’ CIPN assessment documentation  and adherence to evidence-based management at the third clinic visit were compared between the AP and UCP using Pearson’s chi-squared test.One hundred sixty-two patients receiving neurotoxic chemotherapy  answered patient-reported outcome measures on CIPN severity and interference prior to three clinic visits at breast, gastrointestinal, or multiple myeloma outpatient clinics (p = 0.31). There were no improvements in clinicians’ CIPN assessment frequency during the AP (assessment index = 0.5440) in comparison to during the UCP (assessment index = 0.6468).Clinicians’ frequency of adherence to evidence-based CIPN management was higher in the AP (29/52 [56%]) than the UCP (20/46 [43%]), but the change was not statistically significant (Implementation of a clinician-decision support algorithm did not significantly improve clinicians’ CIPN assessment documentation or adherence to evidence-based management. Further research is needed to develop theory-based implementation interventions to bolster the frequency of CIPN assessment and use of evidence-based management strategies in practice.NCT03514680. Registered 21 April 2018.ClinicalTrials.Gov,  Chemotherapy-induced peripheral neuropathy (CIPN) is a frequent dose-limiting complication of neurotoxic chemotherapy  , 2 assocTo prevent the untoward effects of CIPN, clinicians should assess patients for CIPN regularly during neurotoxic chemotherapy to identify risk and manage early CIPN symptoms. However, qualitative and quantitative evidence suggests that CIPN is under assessed and managed by clinicians. Participants in qualitative studies have reported that clinicians do not adequately follow up on CIPN concerns or provide much feedback about how to manage CIPN , 11. A mN = 25 Phase I, and N = 75 Phase II) and clinicians (N = 6)  and n = 81 for algorithm phase [AP]) and 53 clinicians were recruited. Patients were eligible if they were 1) ≥ 18 years old, 2) finished at least one infusion of neurotoxic chemotherapy  for the treatment of breast, gastrointestinal or multiple myeloma malignancies, 3) scheduled to attend at least three more clinic visits associated with neurotoxic chemotherapy receipt after consent, 4) ambulatory, 5) proficient in English, and 6) receiving care from a clinician enrolled in the study. Patients were excluded if they had a prognosis of less than 2 months or documented peripheral neuropathy due to other causes  [The study aims were conducted using a two-phase, pre and post-test design. One-hundred sixty-two patients (iabetes) , 18 prioFeasibility of CIPN algorithm implementation was evaluated based on the number of times the clinicians received the CIPN algorithm prior to a clinic visit. In addition, a two-item feasibility questionnaire prompted clinicians to state how often they used the algorithm and other CIPN educational resources to guide the assessment and management of CIPN, respectively . Clinicians’ perspectives of acceptability and satisfaction with the CIPN algorithm were evaluated using an adapted subset of eight questions from the Adapted Acceptability E – Scale . QuestioThe Patient Reported Outcomes version of the Common Terminology Criteria for Adverse Events (PRO-CTCAE™) Numbness and Tingling Severity and Interference Items ask patients to self-report the severity of numbness and tingling in the hands or feet  at its worst in the past 7 days, and how much these symptoms have interfered with activities of daily living  –22. StroStudy staff abstracted clinicians’ documentation (yes/no) of CIPN assessment  and CIPN management . Study staff also abstracted information about patients’ cancer treatment-related information, such as diagnosis, stage, chemotherapy type, pain medication use, comorbid conditions associated with increased CIPN risk  met to evaluate the appropriateness of clinicians’ CIPN management recommendations using a study-team created form. Appropriate clinician-related CIPN management was scored as yes or no . Clinicians were deemed to have provided appropriate management for moderate-severe CIPN  if a recommendation for pharmacological treatment  , dose reN = 53) participated in both the UCP and AP, but patients (N = 162) either participated in the UCP (n = 81) or AP (n = 81). Patients consented to the UCP completed the CIPN patient-reported outcome measures via iPad in the waiting room prior to seeing the clinician at three consecutive clinic visits associated with neurotoxic chemotherapy . Patients also completed a demographics questionnaire at T1. At T3, clinicians’ CIPN assessment and management documentation and patients’ cancer treatment information were abstracted from the electronic medical record by study staff (T3 must have been completed within a month of neurotoxic chemotherapy completion). Clinicians completed a demographics questionnaire upon enrollment into the study.The study consisted of two phases: the usual care phase (UCP) and algorithm phase (AP). All consented clinicians . The rate of clinicians’ CIPN assessment documentation and adherence to evidence-based management for actionable CIPN  at T3 were compared using Pearson’s chi-squared test for equality of proportions between the UCP and AP.The mean difference in QLQ-CIPN20 sensory and motor subscale and worst CIPN pain intensity score changes from T1-T3 between the UCP and AP were compared using two sample t-tests. To determine metrics related to feasibility, acceptability, and satisfaction of algorithm implementation, we described the number of times study staff provided the clinicians with the algorithm at each clinic visit and clinicians’ scores on the two-item feasibility questionnaire and Adapted Acceptability E-Scale. All analyses were conducted using data from participants who completed the measures specific to each analysis and no missing data were imputed.n = 81 UCP; n = 81 AP) and 142 patients  were evaluable for the primary or secondary analyses  years old at the time of consent and majority were female (62.3%) and White (86.8%). T3 notes of 42/53 consented clinicians (79.2%) were reviewed .Patient recruitment occurred from June 2018 to November 2019. A total of 162 patients were consented to the study . Overall, a total of 98 patients reported at least mild or greater CIPN severity at T3. The rate of appropriate CIPN management improved 12.3% from 20/46 (43.5%) patients in the UCP to 29/52 (55.8%) patients in the AP, but the change was not significant . The frequency of appropriate mild CIPN management improved 22% from 12/25 (48%) cases in the UCP to 21/30 (70%) cases in the AP, but the change was not significant . There was no improvement in the frequency of appropriate moderate-severe CIPN management documentation following algorithm implementation . Duloxetine was never prescribed to any patients reporting moderate-severe CIPN severity in either phase.Overall, numbness, tingling, or pain was documented by clinicians in 110/139 (79.1%) reviewed notes at T3 Table . The CIPn = 19) , while the lowest rated item pertained to how helpful the algorithm was in guiding CIPN assessment or management clinical interactions with patients  (n = 19)  Table . Clinici9) Table .Table 3Range = approximately 46 to 58%) [Improving CIPN assessment documentation is a two-fold challenge: 1) patients and clinicians must discuss the absence or presence of CIPN and 2) clinicians must document CIPN in the electronic medical record. Results revealed that implementation of the clinician decision support algorithm did not significantly improve clinicians’ documentation of CIPN symptom assessment. The observed CIPN assessment documentation frequency in the UCP  was considerably higher than CIPN assessment documentation frequencies reported in prior studies ( to 58%) , 13. As  to 58%) .Study results demonstrated that availability of the algorithm did not significantly improve clinicians’ management of mild or moderate-severe CIPN. Although, availability of the algorithm improved clinicians’ management of mild CIPN by 22%, mainly via increased identification and monitoring of CIPN. On the other hand, duloxetine was never prescribed for individuals with moderate-severe CIPN. It has been 7 years since the publication of the trial by Smith et al. (2013) that demonstrated the efficacy of duloxetine for chronic painful CIPN ; and 6 yThere are several explanations as to why algorithm implementation did not significantly influence clinicians’ CIPN assessment or management behaviors. Despite the high feasibility of algorithm delivery to clinicians, most clinicians “seldomly” or “sometimes” used the algorithm and/or associated educational materials to guide CIPN assessment or management. Clinicians may not have regularly used the algorithm because of a lack of time in the clinic or they found the algorithm-related assessment and management steps burdensome to implement. Moreover, the implementation plan for algorithm delivery was suboptimal. Colquhoun et al. (2017) conducted a systematic review to determine strategies for designing interventions intended to influence clinician healthcare behaviors and concluded that there were four steps critical to designing such interventions: 1) identifying barriers, 2) targeting intervention components to barriers, 3) theory selection, and 4) obtaining clinician feedback . While tDue to the clinicians’ low usage and acceptability and satisfaction ratings of the algorithm in the current study, future research should be directed towards the testing of theory-based implementation interventions to improve the assessment and management of CIPN. Derived from the Expert Recommendations for Implementing Change (identified 73 implementation strategies and associated definitions) , 41 and There are several limitations to this research. First, the generalizability of our findings is limited because the patient and clinician samples were homogenous in race and ethnicity and recruited from one academic cancer center. Second, differences in CIPN assessment and management documentation frequency between phases may be influenced by the number of notes written by particular clinicians within each phase  or the number of times each clinician received the algorithm during the study . Third, changes in CIPN assessment or management documentation by clinicians may have been a result of external factors to the study due to the lack of a true control group . Fourth, between-group changes in patients’ CIPN severity over time was confounded by the eligibility criteria . Fifth, it is possible that the methods used to measure changes in clinicians’ CIPN assessment and management documentation may not have been sensitive to the unique symptom presentations associated with CIPN due to taxanes, oxaliplatin, or bortezomib. Lastly, the frequency of CIPN assessment and appropriate management observed in this study may not have been representative of actual clinical practice because we did not audio record patient-clinician outpatient encounters and decisions regarding the appropriateness of clinicians’ CIPN management actions at T3 were made based on patients’ self-reported CIPN severity and clinicians’ CIPN documentation only.Implementation of a clinician-decision support algorithm did not significantly improve clinicians’ CIPN assessment documentation or use of evidence-based management strategies when providing care to patients receiving neurotoxic chemotherapy at breast, gastrointestinal, or multiple myeloma oncology outpatient centers. Reasons for the lack of algorithm-induced changes in clinicians’ CIPN assessment and management behaviors included that clinicians infrequently used the algorithm or associated educational materials to assess or manage CIPN and a suboptimal intervention implementation plan. Future research should be directed toward the development and testing of theory-guided implementation interventions to improve the assessment and management of CIPN in clinical practice.


While the consequences of nuclear DNA damage have been well studied, the exact consequences of acute and selective mitochondrial DNA (mtDNA) damage are less understood. DNA damaging chemotherapeutic drugs are known to activate p53-dependent apoptosis in response to sustained nuclear DNA damage. While it is recognized that whole-cell exposure to these drugs also damages mtDNA, the specific contribution of mtDNA damage to cellular degeneration is less clear. To examine this, we induced selective mtDNA damage in neuronal axons using microfluidic chambers that allow for the spatial and fluidic isolation of neuronal cell bodies (containing nucleus and mitochondria) from the axons (containing mitochondria). Exposure of the DNA damaging drug cisplatin selectively to only the axons induced mtDNA damage in axonal mitochondria, without nuclear damage. We found that this resulted in the selective degeneration of only the targeted axons that were exposed to DNA damage, where ROS was induced but mitochondria were not permeabilized. mtDNA damage-induced axon degeneration was not mediated by any of the three known axon degeneration pathways: apoptosis, axon pruning, and Wallerian degeneration, as Bax-deficiency, or Casp3-deficiency, or Sarm1-deficiency failed to protect the degenerating axons. Strikingly, p53, which is essential for degeneration after nuclear DNA damage, was also not required for degeneration induced with mtDNA damage. This was most evident when the p53-deficient neurons were globally exposed to cisplatin. While the cell bodies of p53-deficient neurons were protected from degeneration in this context, the axons farthest from the cell bodies still underwent degeneration. These results highlight how whole cell exposure to DNA damage activates two pathways of degeneration; a faster, p53-dependent apoptotic degeneration that is triggered in the cell bodies with nuclear DNA damage, and a slower, p53-independent degeneration that is induced with mtDNA damage. While mitochondrial DNA (mtDNA) is several orders of magnitude smaller than the nuclear genome4. These DNA-damaging events engage a number of downstream pathways depending on the degree of damage. While low levels of DNA damage can be repaired, induce cell cycle arrest, senescence, or differentiation, excessive damage can lead to cell death via apoptosis5. Specifically, nuclear DNA damage activates the classical intrinsic pathway of apoptosis that is initiated by p53, mediated by the proapoptotic protein Bax, and executed by caspases such as Caspase-3 (Casp3)7. Recently, it has become increasingly evident that exposure of cells to genotoxic drugs affects not just nuclear DNA but also mitochondrial DNA8. However, because it is challenging to separate the distinct consequences of mtDNA insult from nuclear DNA insult, it is unknown whether mtDNA damage activates the same cellular response as nuclear DNA damage.Chemotherapeutic drugs that induce DNA damage are frontline treatments for cancer therapy9, is detected and rapidly repaired by the highly effective base excision repair (BER) pathway11. While other DNA repair pathways have been reported in the mitochondria, such as single-strand and mismatch repair11, it is unclear how much these pathways functionally contribute to mtDNA repair. Notably however, mitochondria appear to completely lack the nucleotide excision repair (NER) pathway, which is typically used to repair nuclear DNA insults from chemotherapeutic drugs such as cisplatin-induced intra-strand crosslinks11.The majority of mtDNA damage, especially oxidative damage by reactive oxygen species that are generated as a byproduct of oxidative phosphorylation13. A second mouse model of mtDNA damage is one where mtDNA double-stranded breaks can be induced by the expression of a mitochondrially-targeted restriction endonuclease (Pst1)15. Even a transient induction of mtDNA double-stranded breaks is sufficient to induce an aging phenotype in these mice17. Both these mouse models underscore the importance of maintaining mtDNA fidelity and show that chronic accumulation of mtDNA mutations cause premature aging. In addition to mouse models, a few studies have also induced mtDNA damage in cell lines. Depletion of the exonuclease EXOG, which resulted in persistent single stranded brakes in mtDNA (but not nuclear DNA), induced cell death18. Interestingly, exposure of cancer cell lines to chemotherapeutic drugs that are modified to target the mitochondria can also induce cell death with markers of apoptosis21. However, whether the apoptotic pathway mediates mtDNA damage-induced cell death is not known.In contrast to our knowledge of the pathways important for mtDNA repair and during biogenesis, the pathways activated by sustained and selective mtDNA damage remain virtually unknown. Several studies have utilized the “mutator” mice, which carry a knock-in inactivating mutation in the exonuclease domain of the mitochondrial DNA polymerase POLG, to examine the outcome of mtDNA damage. As this mutant POLG lacks its proof-reading activity, the mutator mice gradually accumulate persistent mtDNA mutations and exhibit a premature aging phenotypeTo induce selective damage to mtDNA but not nuclear DNA, we have taken advantage of the unique biology of neurons where the cell body and axons are spatially distinct. While neuronal cell bodies (soma) contain both nuclear DNA and mtDNA, the only source of DNA in axons is mtDNA from the axonal mitochondria. Importantly, in order to selectively expose only the axonal mitochondria to DNA damage, we utilized compartmentalized microfluidic chambers. Our results show that exposure of axons to the DNA damaging drug cisplatin is sufficient to induce mtDNA damage which resulted in the selective degeneration of the exposed axons. Here, we examine the critical importance of the p53-depedent apoptotic pathway in mediating the mtDNA damage-induced degeneration in neurons.23. In these chambers, neurons are seeded into the soma compartment and over several days extend axons through the microfluidic grooves into the axon compartment. While the soma compartment contains nuclear DNA, as well as mtDNA , the axon compartment contains only mtDNA. Thus, these chambers allow for the exposure of only the axon chamber to DNA damaging agents to selectively damage axonal mtDNA without affecting the nuclear DNA contained in the cell bodies in the soma compartment.To induce selective damage to mtDNA but not genomic DNA, we used primary mouse neurons in microfluidic-based compartmentalized chambers Fig. . These m24. Importantly, cisplatin induces intrastrand crosslinks that are not predicted to be repaired in mitochondria, as mitochondria lack NER11. Thus, we examined whether the directed exposure of cisplatin to only the axon compartment in our neuronal microfluidic model was sufficient to induce mtDNA damage. Using an established PCR amplification assay for detecting mtDNA damage26, we found that axonal cisplatin exposure induced substantial damage to mtDNA in the axon compartment . Thus, we also examined the status of cyt c in the mitochondria of cisplatin-treated axons. Our results show that mtDNA damage did not trigger the release of cyt c from the mitochondria at any time during cisplatin-induced axon degeneration  in axons. We conducted a time course study and found that ROS levels were markedly elevated in the axons by 24 h of cisplatin exposure and remained high until overt axonal degeneration . To determine if axon pruning, or any other caspase-dependent pathway, were involved in mtDNA damage induced axon degeneration we used the pan-caspase inhibitor Q-VD-Oph (QVD). We found that QVD was unable to protect axons in response to axonal cisplatin exposure . Thus, in the previous contexts where the mass cultures of p53-deficient neurons (cell bodies and axons) were exposed to DNA damage, the status of axons had not been specifically reported. To reconcile these previous reports on p53 and our current results with directed mtDNA damage to axons, we considered two possibilities of p53 function in somas versus axons. First, that p53 is selectively important for DNA damage-induced degeneration in somas but not in axons. If so, we would predict that p53-deficient neurons, when exposed to global DNA damage (both soma and axons), would protect the soma but result in the complete degeneration of the axons. A second possibility is that exposure of entire neurons to DNA damage activates a faster, nuclear DNA damage-induced p53-dependent apoptotic degeneration while also engaging a slower, mtDNA damage-induced degeneration that is p53-independent. If so, we would predict that p53-deficient neurons exposed to DNA damage would be protected, but only transiently, and that the degeneration would occur without a distinct soma versus axon separation.Our finding that deficiency of p53 failed to prevent mtDNA damage-induced axon degeneration was surprising, as p53 deficiency has been reported to protect neurons from degeneration in response to DNA damageTo differentiate between these two possibilities, we examined the consequence of global cisplatin exposure to p53-deficient neurons and focused on the degeneration occurring in both the soma and axons. Importantly, since the microfluidic chamber model allows for the clearer differentiation of the proximal and distal axons from the soma, we conducted these experiments in microfluidic chambers where both compartments were exposed to cisplatin Fig. . We examIn this study we highlight the differential importance for p53 in governing the degenerative response to sustained nuclear versus mitochondrial DNA damage. While nuclear DNA damage is well known to activate an apoptotic cell death program, separating the distinct effects of nuclear versus mitochondrial DNA damage in cells has been experimentally challenging. Other than the few notable exceptions highlighted in the introduction, most studies investigating the molecular consequences of DNA damage in cells have been done in the context where the entire cell is exposed to DNA damage. In those situations, both nuclear and mitochondrial DNA are damaged, and the degeneration observed could be a combined effect of both nuclear and mitochondrial DNA damage pathways. The neuronal microfluidic model we utilize in this study allows for the selective induction of mtDNA damage in axons without damaging the nuclear DNA.Our key observation is that selective mtDNA damage to the mitochondria in the axons is sufficient to activate a degenerative pathway that is spatially restricted to only the exposed regions. Surprisingly, this degenerative pathway was distinct, as it did not involve the major known axon degeneration pathways . Interestingly, however, neurons deficient in p53 were partially protected from apoptosis after nuclear DNA damage but the lack of p53 did not protect distal axons from degenerating in response to mtDNA damage. Our findings here indicate that in response to DNA damage, two degenerative pathways are activated: apoptosis from nuclear DNA damage, and the degenerative response activated by mtDNA damage. Importantly, these two parallel degenerative pathways are normally not able to be differentiated in most model systems and likely occur in all contexts of global DNA damage.31. Interestingly, recent reports have indicated that p53 also plays a role in mitochondrial maintenance48. For example, p53 has been show to both bind and transcriptionally regulate TFAM, the mitochondrial transcription factor important for mtDNA transcription and mtDNA maintenance49. p53 is also known to translocate to the mitochondria in contexts of stress39, and has been shown to be important for the maintenance of mtDNA42. For example, p53 is known to directly interact with the mtDNA repair and replication machinery including the mtDNA polymerase (POLG)40, human mitochondrial single-stranded binding protein (SSDP1)50, and the BER components 8-oxo-guanine glycosylase (OGG1) and apurinic endonuclease (APE)51. Our finding that p53-deficiency does not protect axons against mtDNA damage induced degeneration suggests that while p53 might play important roles in mtDNA replication and repair, it does not seem to play a similar role regulating survival versus degeneration like it does in response to nuclear DNA damage.Depending on the extent of DNA damage, cells can activate both repair and degenerative pathways. While we have some understanding of how this choice is made in the context of nuclear DNA damage, the pathways mediating the choice between mtDNA repair or degeneration are unknown. For nuclear DNA, p53 is important in determining the fate of the cell to repair damage or death via apoptosis in response to DNA damageOur results with p53-deficient neurons globally exposed to DNA damage have revealed that distal axons are particularly vulnerable to mtDNA damage. One interesting question from our results is why might the proximal axons be more protected than distal axons in the experiment where p53-deficient neurons were globally exposed to cisplatin? One possible explanation is that as the proximal axons are closer to the protected soma, the large number of mitochondria in the soma are more readily available to replenish mitochondria in the proximal axons. However, this capability would decrease in the distal regions of the axons that are further away from the soma, leaving the distal axons most vulnerable to mtDNA damage.55. The increased vulnerability of peripheral axons to systemic chemotherapy is likely because of the large surface area of peripheral axons and their close association with the vasculature56. CIPN has been investigated in the context of paclitaxel exposure where it induces axon degeneration via disruption of microtubules57. The exact mechanism of this axon degeneration in cisplatin-induced CIPN is unclear but damage to mtDNA has been implicated58. Interestingly, distal axons have also been found to degenerate prior to proximal axons and cell body loss in CIPN59. Adult neurons are known to engage robust mechanisms to highly restrict apoptosis and protect their cell bodies61 while allowing selective axon degeneration and remodeling via axon pruning30. Thus, the apoptotic restriction in adult neurons is comparable to the outcome of our experiments with p53-deficient neurons, where the cell body is protected but distal axons degenerate from DNA damage.The results we describe here also have broad relevance in the clinical context of chemotherapy-induced peripheral neuropathy (CIPN). CIPN is a progressive neurodegenerative condition in which the peripheral nervous system is damaged by chemotherapeutic drugs, such as cisplatin, used in cancer therapy. This condition can induce significant axon degeneration, leading to numerous neurological deficits, which are dose limiting factors for chemotherapy in cancer patients64. These side effects are surprising because neurons and cardiomyocytes are post-mitotic cells that do not replicate their DNA and are thus not expected to be preferentially selected for damage by DNA damaging drugs. However, both neurons and cardiomyocytes are highly reliant upon active metabolisms and their mitochondria for optimal function. Our results draw distinctions between the nuclear, p53-dependent versus the mitochondrial, p53-independent consequences of chemotherapeutic drugs on cells.DNA damaging chemotherapeutic drugs are generally assumed to exert their effect by damaging the nuclear DNA in rapidly dividing cells. However, it is notable that DNA damaging chemotherapeutic regiments also induce neuropathies (as discussed above) and cardiomyopathies65. Briefly, sympathetic neurons were dissected from the superior cervical ganglia (SCG) of postnatal day (P) 0-2 mice. Cells were plated directly into the microfluidic chamber device (described below) at a plating density of 10,000 cells per chamber and maintained for 5 days in vitro (DIV) in media containing NGF (AM50). Mice deficient for p53, Bax, Casp3, and Sarm1 are all from C57BL/6 backgrounds and wildtype littermates served as controls. All animal experiments were approved and conducted in compliance with the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee (IACUC).Primary sympathetic neurons were cultured as previously described30. Briefly, negative photoresist was utilized to pattern the chamber and microfluidic grooves using mylar chamber masks (Fineline Imaging). The microfluidic grooves were etched into a standard silicon wafer (Silicon Question Nation) with a groove depth of 3 μm with a groove width of 20 μm and with each groove being spaced 40 μm. The axon and soma compartments  were added on either side of the etched grooves. To generate microfluidic devices, Sylgard 184 PDMS (Dow Corning) was prepared according to manufacturer protocol and poured into the mold form. The liquid mold was placed under vacuum at room temperature to remove air bubbles from the PDMS for 30 min then placed into a 95 °C oven to cure overnight. After curing, individual PDMS chambers were cut out and trimmed then soaked ddH2O overnight, sterilized in 70% EtOH, allowed to dry, mounted onto cleaned glass coverslips  coated overnight in 100 µg/ml poly-D-lysine  and 1 µg/ml mouse laminin , after mounting the chambers were then filled with poly-D-lysine and laminin solution and allowed to incubate for 72 h. Before neurons were plated into the soma compartment of the chambers, the chambers were washed 3 times with ddH2O and allowed to dry.Microfluidic device master molds were generated using standard photolithographical procedures as previously described at the UNC CHANL facility30 and maintained in AM50 until 5 DIV. To treat entire cells (both axon and soma chambers) with cisplatin, both the axon and soma compartment were washed three times with AM50 media containing cisplatin. To treat axons alone with cisplatin, only the axon compartment was washed with AM50 containing drug while the soma compartment is maintained in untreated AM50 media. A 10 μl volume differential was established between the soma and axon compartments and was reestablished every 12–20 h to maintain fluidic isolation of the two compartments22. For drug treatments, compounds were added to AM50 media at the following concentrations: 20 μM cisplatin , 25 µM QVD-OPh . All experiments were performed with three biological replicates.Neurons were plated in microfluidic chambers as described previously66. Data was quantified from eight biological replicates.Degeneration of axons was quantified from immunofluorescent images stained for tubulin taken in the microfluidic devices. The images are analyzed in ImageJ using a published and accepted axon degeneration analysis based on axon segment continuity30. Briefly, the two compartments are stained by added solutions to the top chamber reservoirs and allowing solutions to flow through into the chamber areas. The following primary antibodies were used for staining: alpha-tubulin , Tom20 , cyt c , ɣH2AX . Nuclei were labeled with Hoechst 33258 . The following mitochondrial markers used are: Mitotracker Green FM , CM-H2DCFDA , TMRE . Quantification of cyt c release was conducted using Coloc 2 in ImageJ for colocalization using the Pearson correlation coefficient on cyt c and Tom20 stained images.Immunofluorescence staining was carried out as previously describedWhole brain lysate from wildtype and knock-out animals was analyzed by Western blot for validation of gene knock-out using the following antibodies: Bax N20 ; Caspase-3 ; Sarm1 ; beta-Actin .26. Briefly, the assay compares the amplification of a long versus short segment of mtDNA to generate an amplification ratio; when mtDNA is damaged the ratio of long:short amplification is reduced as DNA damage that blocks Taq polymerase processivity occurs with a higher frequency on longer DNA segments than on shorter fragments. The 117 bp short region used the following for amplification: forward primer: 5′-CCC AGC TAC TAC CAT CAT TCA AGT-3′, reverse primer: 5′-GAT GGT TTG GGA GAT TGG TTG ATG T-3′. The 10 kb long region used the following for amplification: forward primer: 5′-GCC AGC CTG ACC CAT AGC CAT AAT AT-3′, reverse primer: 5′-GAG AGA TTT TAT GGG TGT AAT GCG G-3′. PCR was conducted using Platinum Blue TAQ  and the conditions for amplification were followed as previously described26. The amplified mtDNA products were measured using a Picogreen DNA amplification assay as previously described using a Thermo Fluoroskan Ascent FL fluorescent plate reader25. Resulting values of the short and long mtDNA amplification fragments were then ratiometrically compared as previously reported to quantify the amount of mtDNA damage in a sample26. Experiments were conducted in biological triplicate.mtDNA was isolated by collecting axonal material from the axon compartment of treated and untreated axons using a DNeasy Blood & Tissue kit . The mtDNA was then amplified using primers specific to short (117 bp) and long (10 kb) amplicons of murine mtDNA as previously described2DCFDA, cyt c, and TMRE were imaged using ×63 oil lenses.Images were acquired on a DMI6000 inverted fluorescent microscope (Leica) using an ORCA-ER B/W CCD camera  through Metamorph software  or on a DMI8 inverted fluorescent microscope  using a Leica DFC9000 sCMOS camera through LASX software (Leica). Axons and soma were imaged using ×10 and ×20 air lenses. Images of axons showing CM-HSupplemental Figure LegendSupplemental Figure 1Supplemental Figure 2


Breast cancer is a leading disease in women. Several studies are focused to evaluate the critical role of extracellular matrix (ECM) in various biochemical and molecular aspects but also in terms of its effect on cancer cell morphology and therefore on cancer cell invasion and metastatic potential. ECM fibrillar components, such as collagen and fibronectin, affect cell behavior and properties of mammary cancer cells. The aim of this study was to investigate using the scanning electron microscopy (SEM) how the highly invasive MDA-MB-231 breast cancer cells, interplaying with ECM substrates during cell migration/invasion, modify their morphological characteristics and cytoplasmic processes in relation to their invasive potential. In particular we reproduced and analyzed how natural structural barriers to cancer cell invasion, such as the basement membrane (Matrigel) and fibrillar components of dermis (fibronectin as well as the different concentrations/array of type I collagen), could induce morphological changes in 3D cultures. Interestingly, we demonstrate that, even with different effects, all collagen concentrations/arrays lead to morphological alterations of breast cancer cells. Intriguingly, the elongated mesenchymal shaped cells were more prominent in 3D cultures with a dense and thick substrate , even though cells with different shape produced and released microvesicles and exosomes as well. It is therefore evident that the peri-tumoral collagen network may act not only as a barrier but also as a dynamic scaffold which stimulates the morphological changes of cancer cells, and modulates tumor development and metastatic potential in breast cancer.  Tumors are characterized by a loss of tissue organization with abnormal and uncontrolled behavior of cells that grow independently. Cancer cells interact with the surrounding tissues by inducing extracellular matrix (ECM) changes similar to those found in wounds that never heal ,2,3,4. IAs most of cancer patient deaths are caused not by the primary tumor, but by distant metastasis, it is very important to understand why and how cancer cells gain motility and become migratory in order to penetrate into blood and lymphatic vessels and then colonize distant organs . At the Changes in tumor microenvironment play a critical role in tumor development and progression as well in drug efficacy ,13,14,15The main component of ECM is fibrillar type I collagen that alone constitutes up to 90% protein composition of connective tissues ,20. CancThe architecture and collagen fiber orientation of peri-tumoral stroma also modulate cancer cell migration and seem to be related to cancer progression ,34,35,36It is worth noticing that collagen interacts with tumor development by enhancing exosome secretion . ExosomeDifferent ECM proteins such as hyaluronan, laminin, MMPs, and fibronectin interact with collagen and affect cancer cell phenotype ,49,50,51All data previously reported motivated us to investigate using the scanning electron microscopy (SEM) how the aggressive MDA-MB-231 breast cancer cells, when interacting with major ECM components during cell migration and invasion, alter cell morphology and cytoplasmic processes in relation to their invasive potential or capability. In particular we analyzed how structural natural barriers usually opposing cancer cell invasion, such as the basement membrane (Matrigel) and the fibrillar components of dermis (fibronectin and different concentrations and array of type I collagen fibrils) could affect the morphology and invasion capability of MDA-MB-231 breast cancer cells.2. Cells were checked for the absence of mycoplasma by Venor™ GeM Mycoplasma Detection Kit . Cells at 80% of confluences were detached with Trypsin-EDTA solution and were seeded on four polystyrene flasks for 2-D control cultures, on four “Isopore Membrane Filters” with a pore size of 8.0 µm  for 3-D control cultures. To mimic structural ECM natural barriers opposing to cancer cell invasion (2.5 × 105) cells were seeded for 4 h on 20 similar filters coated with fibronectin  or Matrigel  or type I Collagen solution in different concentrations . Fibronectin, collagen, and Matrigel were diluted at the proper concentration in sterile water (pH 6) for 2 h at 37 °C. The lower chamber was filled with DMEM-High Glucose with 20% FBS. Cells were also directly seeded on four commercial lyophilized and compressed collagen membranes of tightly packed type I collagen fibers from equine Achilles tendon . The lower chamber was filled with DMEM-High Glucose with 20% FBS.MDA-MB-231 breast cancer cells (obtained from the American Type Culture Collection (ATCC) were cultured in DMEM-High Glucose , Italy.) (17.5 mM glucose) supplemented with 10% fetal bovine serum (FBS) , L-glutamine (2 mM), penicillin (100 U/mL), and streptomycin (100 µg/mL), at 37 °C in a humidified atmosphere with 5% COBreast cancer cells seeded in both 2D and 3D cultures were fixed in a Karnovsky’s solution for 30 min at 4 °C. Portions of the flasks (2D cultures) and the intact Millipore filters alone or covered by different substrates (3D cultures) with adhered cells were rinsed three times with 0.1% Sodium Cacodylate Buffer pH 7, dehydrated with increasing concentrations of ethanol, and finally dehydrated with hexamethyldisilazane  for 15 min. One sample of each Millipore group was mechanically sectioned in two portions by a razorblade. All specimens were mounted on appropriate stubs, coated with a 5 nm palladium gold film (Emitech 550 sputter-coater) to be observed under a SEM  operating in secondary-electron mode.SEM analysis of MDA-MB-231 cells cultures in 2D flasks showed different and equally distributed cell phenotypes: (a) “squid” shaped or isolated and elongated cells exhibiting lamellipodia at one end, (b) “cobblestone” shaped or flattened polygonal cells only partially showing cell–cell contacts, (c) fewer isolated globular shaped cells A. A low It is worth noticing that when MDA-MB-231 cells were cultivated on Millipore filter the flattened polygonal phenotype strongly decreased as compared to non-Millipore control cultures. On the other hand, the elongated cells with polarized lamellipodia and the globular ones were still detectable. A number of cells of all phenotypes were observed to fill and migrate through the Millipore holes A. All ceMDA-MB-231 cells cultivated in 3D cultures on Millipore filter covered by fibronectin (130 µg/mL) showed an increased cellular density probably due to a higher cell adhesion as compared to the control groups. Similarly, all cells showed more microvilli and microvesicles as compared to the same cells cultured on Millipore alone. Globular shaped cells and flattened polygonal ones were more prominent than elongated cells. Importantly, differences in the cytoplasmic protrusions were also evident, as flattened polygonal cells showed only microvilli, whereas microvesicles were observable on globular shaped cells A. Some aIt is worth noticing that when the MDA-MB-231 cells were cultured on a concentrated Matrigel layer (3.0 µg/µL) they intimately adhered to the substrate. Three cell morphologies were noted: globular shaped cells and elongated ones grown on the flattened polygonal ones. All cells exhibited both microvilli and microvesicles. Some elongated cells developing long and thin polarized filopodia at their both ends presented a clear fusiform-elongated shape A. MoreovIn order to evaluate the importance of collagen type I on the morphology of breast cancer cells and particularly how the different concentrations that form a series of arrays on Millipore filter may be critical factors for the cell functional properties and invasiveness, three different concentrations of collagen type I  were utilized. As starting point the lowest concentration was used. Although most diluted collagen solution (50 µg/mL) did not completely cover the Millipore filter, which showed an exposed rough surface, most of the Millipore holes were partially filled by intertwined collagen fibrils. When MDA-MB-231 cells grew on the Millipore filter covered by diluted type I collagen they included equally distributed and relatively isolated cells with elongated, globular, and flattened polygonal shapes A. FlatteMDA-MB-231 cells growing on a layer of higher type I collagen concentration (200 µg/mL) were mostly composed of isolated globular cells which developed many thin filopodia attaching to the collagen fibrils and radially spreading from each cell A. All ceWhen MDA-MB-231 cells grew at the highest concentration of type I collagen fibrils (3000 µg/mL) the three previously described different phenotypes were almost equally represented and all cells showed both microvilli and microvesicles. A very high cell density was observed with cells arranged in sandwich layers: the flattened polygonal cells were directly attached to the collagen fibrils, whereas both the elongated cells exhibiting a more definite fusiform shape and the globular ones grew over, but showed no cell–cell contacts. In this group many long and thin filopodia mostly originating from the elongated cells and similar to those described in concentrated Matrigel substrate were developed. Additionally, intercellular TNTs were much more observable as compared with the samples of all previous groups A,B. A nuWhen MDA-MB-231 cancer cells were seeded on a commercial lyophilized and compressed collagen membrane of tightly packed type I collagen fibers from equine Achilles tendon, isolated cells exhibited both microvilli and microvesicles on their cytoplasmic surface. They were intimately attached to the dense collagen fibrils and included few isolated flattened polygonal cells, globular and “squid” elongated ones which showed lamellipodia. Some adjacent cells developed thin intercellular connections or TNTs A,B. In aWhen a tumor mass grows, mechanical forces are transmitted to the peri-tumoral microenvironment and induce either direct or CAFs-mediated structural changes of collagen array in ECM. On the other hand, the newly formed collagen array interacts with cancer cells by inducing migratory cell phenotypes and tumor progression. In normal and pathologic conditions, cell–ECM interactions are largely mediated via integrins, a large family of cell-surface receptors which upon transferring both mechanical and chemical signaling also control cytoskeleton array and activate intracellular signaling pathways ,59,60. BBreast tumorigenesis is accompanied by new collagen crosslinking, ECM stiffening, and increased focal adhesions . The morThe presence of fibronectin on Millipore filter evoked an increased cell density probably related to higher cell adhesion to the substrate but also a slight decrease of the elongated mesenchymal cells was detectable. Cell-to-cell direct communications seemed to be improved in some cells which developed single and large composed intercellular TNTs.Epithelial basement membrane containing a thin continuous layer of collagen IV, laminin, fibronectin, and several types of proteoglycans represents the first physical barrier that migratory cancer cells have to cross when they start ECM invasion . Both maEven though the interstitial ECMs mainly consist of type I and type III collagens, but also include fibronectin and proteoglycans, all collagen substrates used to cover the Millipore filters or the collagen membrane itself contained only type I collagen, the most abundant type in ECM ,60. DespDifferently, cultures of the MDA-MB-231 cells on Millipore filter covered by a more concentrated type I collagen (200 µg/mL) mostly included isolated globular cells which showed many thin filopodia adhering to the collagen fibrils and radially spreading from each cell. Interestingly, some filopodia showed also exosomes on their cytoplasmic surface. It has been reported that fibroblasts filopodia seem to have a direct mechanical role in exosome capture and represent highways favoring exosomes penetration into the cell . HoweverWhen breast cancer cells were cultured on Millipore covered by high concentrated (3000 µg/mL) type I collagen, cells with no tight cell–cell contact and covered by many microvilli and microvesicles grew in more than one layer. It is, therefore, plausible to suggest that these cells attaching very well to the concentrated collagen substrate and developing an intense vesicles production are preparing to invade the thick collagen layer. Additionally, the many long filopodia covered by microvesicles and exosomes and originating from fusiform cells together with many TNTs are all structural changes which may suggest that an EMT process is occurring. MDA-MB-231 cells growing on a commercial lyophilized and compressed collagen membrane of tightly packed type I collagen fibers from equine Achilles tendon displayed all three cancer cell phenotypes covered by many microvesicles and short filopodia, but some globular cells rich in microvesicles seemed to try to invaginate into more exposed collagen fibrils. Several microvesicles and exosomes released by cells and attached to the collagen membrane surface demonstrated an increased vesicles production/release. It was reported that collagen interacts with tumor development by enhancing exosome secretion ; thus, cIn conclusion, cross talk between breast cancer cells observed in 3D Millipore cultures as compared to 2D flask cultures, is enhanced by fibronectin substrate which ensures an increased cell adhesion to the ground culture and promotes increasing vesicles production . When ceFinally, the commercial lyophilized and compressed collagen membrane of tightly packed type I collagen fibers from equine Achilles tendon, including randomly intertwined fibrils at the surface, does not allow MDA-MB-231 cell invasion. Nevertheless, cells exhibit fusiform (spindle-like) EMT morphological phenotype with long filopodia, intercellular TNTs, and many microvesicles and exosomes on cell surface but also shed on the collagen membrane. Even in absence of mechanical pressure loads exerted by tumor growth, breast cancer cells change their morphology into an invasive shape, thus confirming that dense collagen fibers per se physically interact with MDA-MB-231 cancer cells and stimulate their invasive potential.Regarding the role of collagen on cancer cell morphology and aggressiveness, a number of studies are more focused on collagen stiffness rather than on collagen arrangement ,76,77,78Although the analysis of the effect of ECM on breast cancer cells only at the morphological level and the use of only one cell line represents a limitation of this study, our results add important insights and knowledge, and will be helpful for the comparison between forthcoming biochemical and morphological investigations to completely explain the intriguing but dramatic interplay between collagen and cancer cells.


The biguanide drug metformin used for treating Type 2 diabetes has anticancer properties and affects many pathways involving glucose metabolism, energy balance, and cell survival. A number of retrospective clinical studies have indicated a reduced risk of cancer and improved cancer outcomes in Type 2 diabetic patients taking metformin. Several of its effects are mediated through the induction of cellular stress and subsequent activation of AMP kinase, but many other mechanisms act independently of AMP kinase activation. Metformin has been shown to inhibit the effects of tumor necrosis factor (TNF)-alpha. TNF-alpha interferes with insulin signaling to produce insulin resistance in the insulin signaling pathway and promotes apoptosis through NF-KB in the apoptosis pathway. In addition, metformin reduces cellular proliferation by decreasing the amount of available insulin or by directly affecting the mammalian target of rapamycin complex involved with regulating protein synthesis. It can prevent tumors from acquiring stem cell-like properties, upregulate apoptotic pathways, and bolster the immune system’s fight against cancer. Gaining a greater understanding of metformin’s various mechanisms of action will continue to elucidate metformin’s role as an effective treatment for cancer. In reducing hepatic glucose production, which in T2DM leads to lower fasting plasma glucose concentrations, it is widely thought to indirectly activate AMP-protein kinase (AMPK) through inhibition of Complex I of the electron transport chain in the mitochondria which increases the intracellular AMP: ATP ratio. Metformin acts mainly through AMPK in downregulating expression of several gluconeogenic genes and affects lipid metabolism leading to decreased steatosis. Although AMPK is thought to be the preferred mechanism for metformin action, other AMPK independent mechanisms impacting glucose and lipid metabolism have also been demonstrated.–5Since the introduction of biguanides in the 1950s, metformin has become one of the most commonly used drugs to treat hyperglycemia in Type 2 diabetes mellitus (T2DM)patients. It primarily works by increasing insulin receptor sensitivity, increasing cellular glucose uptake, and decreasing hepatic glucose production. More recent retrospective studies have shown increased overall survival rates, fewer recurrences, and longer periods of remission in T2DM patients suffering from colon, lung, gastroesophageal, thyroid, and prostate cancers.–9 Given metformin’s impact on insulin signaling and glucose and lipid metabolism, it is of great interest to researchers to investigate the possibility of repurposing an old T2DM drug into a potential anticancer adjuvant. This review examines a few of the molecular targets and pathways that may play a role in metformin’s antineoplastic mechanisms of action.In 2005, an epidemiological study proposed a connection between T2DM patients’ use of metformin as an antidiabetic medication and a reduced risk of cancer. Insulin is a mild growth factor regulating cell division and inhibiting apoptosis, but its real risk for promoting cancer is its ability to potentiate the mitogenic actions of other growth factors. Insulin can activate farnesyltransferase to farnesylate RAS to send it to the plasma membrane and prime it for activation by other growth factors. In the context of hyperinsulinemia, the excessive activation of farnesyltransferase leading to more membrane attached RAS enhances the mitogenicity of insulin and other growth factors. Too much insulin also causes insulin-like growth factor 1 (IGF-1) to be displaced from its binding proteins and decreases IGF binding protein production. IGF-1 is a more powerful growth factor than insulin that can also bind to insulin receptors and can stimulate the RAS-RAF-MEK-ERK or the PI3K/AKT/mammalian target of rapamycin (mTOR) pathways while also inhibiting the antiapoptotic phosphatase and tensin homolog (PTEN). A study examining the role of tumor necrosis factor (TNF)-alpha in insulin resistance demonstrated that the insulin signaling and apoptosis pathways are intimately connected through PTEN phosphatase, TNF-alpha, and insulin. PTEN inhibits PI3K signaling of the insulin pathway producing insulin resistance. In contrast, PTEN, stimulated by TNF-alpha, promotes apoptosis through its actions on NF-KB.,15 In addition to decreasing circulating insulin levels through increased insulin receptor sensitivity, metformin has been shown to decrease IGF-1 receptor and insulin receptor expression in endometrial cancer cell lines. The lower levels of circulating insulin and IGF-1 reduce the amount of binding to the insulin receptor, especially to isoform A in cancer cells, leading to less tumor growth.Insulin has been shown to promote cancer growth by increasing glucose uptake and utilization through the phosphatidylinositol-3-kinase (PI3K) pathway and cell proliferation by mitogen-activated protein kinase activation. AMPK phosphorylates tuberous sclerosis complex 2 leading to an inhibition of mTOR in combination with tuberous sclerosis complex 1. AMPK can also directly phosphorylate raptor, a regulatory protein associated with the mTOR complex, to prevent mTOR activation. Metformin also inhibits Rag GTPases, a family of proteins usually sensitive to amino acid levels, which are needed to translocate mTOR to the nucleus so it can complex with RAS-homolog enriched in brain to be activated. Similar to amino acid withdrawal, metformin induces a cytosolic withdrawal and inactivation of mTOR from the nucleus. Another group using phosphoproteomics have found that certain mTOR substrates are still positively regulated in cancer cells following metformin treatment and proposed that metformin does not inhibit mTOR but rather rewires its signaling. In their study, metformin-treated cancer cells were less sensitive to growth following IGF-1 stimulation and displayed increased activity in PP2A phosphatase which deactivates p70S6K, a kinase downstream of mTOR signaling. In pancreatic cancer cell lines, potential crosstalk exists between the insulin/IGF-1 stimulated PI3K/AKT/mTOR pathway and the GqPCR stimulated mTOR pathway. Addition of insulin potentiates phosphatidylinositol 4,5-bisphosphate hydrolysis and increases intracellular Ca2+ concentration activated by GqPCR agonists such as bradykinin and angiotensin II. This occurred in a mTOR dependent manner as treatment with rapamycin negated the potentiating effects of the insulin. Administration of metformin reversed the insulin-induced Ca2+ mobilization following the addition of GqPCR agonists leading to a decrease in DNA synthesis.While having an indirect effect by lowering insulin levels, much of metformin’s other antineoplastic activity has been attributed to AMPK activation. AMPK has been shown to inhibit mTOR which regulates protein synthesis and cell proliferation. A population of pancreatic cells survives after ablation of the KRAS oncogene, and they retain tumorigenic potential. These surviving cells are termed cancer stem cells (CSCs) and tend to be resistant to conventional chemotherapy due to their slower growth rate and a number of anti-apoptotic mechanisms. Hypoxia-inducible factor 1-alpha , activated in a hypoxic tumor microenvironment, drives expression of pluripotent genes Oct4, Sox3, and KLF-4 which helps influence the phenotype of CSCs. Metformin activated AMPK inhibits HIF1-alpha which negatively impacts the Warburg effect and suppresses tumor growth due to decreased aerobic glycolysis and downregulation of Oct4.,27 The Warburg effect is a phenomenon in which cancer cells preferentially use aerobic glycolysis which yields one mole of ATP per mole of glucose compared to 38 moles of ATP per mole of glucose in oxidative phosphorylation through the citric acid cycle. These CSCs have higher oxygen consumption and higher expression of voltage-dependent anion channel (VDAC), a mitochondrial marker, and addition of oligomycin, a mitochondrial stressor, did not increase their rate of glycolysis. The CSCs’ lack of compensation to a mitochondrial stressor may indicate that a compound targeting oxidative phosphorylation (such as metformin in a diabetic patient) in combination with targeted treatment for an oncogenic pathway may be a more effective therapy to eliminate pancreatic tumors and prevent their relapse.In many cancers, reprogramming of metabolic pathways occurs to support rapid tumor growth and cellular immortality. In pancreatic ductal adenocarcinoma, the KRAS oncogene is responsible for >90% of the cases. Cyclin D1 is overexpressed in many cancers including cancers of the breast, esophagus, lung, and bladder due to a defect in post-translational regulation. Hence, drugs that impact Cyclin D1 expression are of great therapeutic interest. Metformin has been shown in three prostate cancer cell lines to block cell cycle progression from G0/G1 to S as seen by a decrease in Cyclin D1, E2F1, and Rb phosphorylation. It is known that phosphorylation of Cyclin D1 at Thr286 induces its degradation in the proteasome, but it is unclear if metformin leads to its phosphorylation. On the transcriptional level, protein regulated in development and DNA damage response 1 (REDD1) expression is increased after metformin administration. Metformin works through p53 to increase REDD1 expression and decrease Cyclin D1 expression. The net effect is to induce cell cycle arrest and apoptosis.Metformin has been shown to affect apoptosis and encourage cell cycle arrest. Activated AMPK phosphorylates MDMX, a protein that regulates P53 ubiquitination and turnover, leading to its stabilization and activation in promoting apoptosis or cellular senescence.,33 Metformin blocks the Hexokinase II phosphorylating activity uncompetitively after the enzyme-substrate complex is formed and binds in the region that glucose-6-phosphate normally occupies. On metformin binding, Hexokinase II preferentially localizes to the cytosol and does not bind to the outer mitochondrial membrane. Previous studies have shown that Hexokinase II inhibition and disassociation from the mitochondrial outer membrane have a potent anti-tumor effect through increased mitochondrial membrane permeability and release of apoptotic proteins into the cytosol.,35 Furthermore, AKT phosphorylates Hexokinase II at Thr473 which enables its association with the mitochondrial membrane to perform its enzymatic function and protect the cell from leakage of apoptotic proteins. Metformin can block AMPK dependent AKT phosphorylation which is normally induced by IGF-1. This additional indirect mechanism of Hexokinase II perturbance further complements metformin’s antineoplastic efficacy as a glucose metabolism disrupter to promote mitochondrial membrane permeability and subsequent apoptosis.In addition to inhibition of Complex I of the electron transport chain, metformin binds directly to Hexokinase II to induce apoptosis. Hexokinase II has been previously shown to be upregulated in cancers and binds to its mitochondrial outer membrane receptor VDAC to phosphorylate glucose using ATP produced in the mitochondria. It plays a role in the immune system’s fight against cancer by strengthening the memory T cell response following administration of an experimental anticancer vaccine by regulating genes involved in fatty acid oxidation. Furthermore, metformin was shown to increase the efficacy of tumor-infiltrating T lymphocytes by inducing a phenotype switch from a central memory T cell to an effector memory T cell resulting in increased production of tumor-fighting cytokines and decreased T cell apoptosis. Another gene regulatory effect of metformin is activation of p38 to phosphorylate and destabilize specificity protein-1 (SP-1). This downregulates SP-1 targeted genes including pyruvate kinase. Metformin downregulated the Stat3-Bcl2 pathway to inhibit cell growth in esophageal squamous cell carcinoma by increasing autophagy and apoptosis. Finally, metformin’s antineoplastic action can induce RNA degradation of C-myc through increased expression of miR-33a, a miRNA which binds to C-myc RNA following upregulation of the DICER enzyme.Despite the many beneficial effects of metformin being mediated by AMPK, metformin has been shown to have other potential anticancer mechanisms independent of AMPK activation. Metformin blocks NF-KB preventing transcription of pro-inflammatory cytokines such as TNF-alpha needed to promote tumor growth in prostate cancer cells. Beyond its direct anticancer effects, some through AMPK, researchers are continuing to discover a myriad of actions related to glucose and lipid metabolism, energy balance, and genomic regulation, all of which may contribute to metformin’s reduction in cancer risk among T2DM patients. Ongoing research will result in a better understanding of metformin’s mechanisms and will hopefully enable physicians to optimize care in concert with other antineoplastic treatments. In the future, we anticipate metformin or a derivative drug may be used in cancer therapy independent of a patient’s diabetic status.The recent revelation of metformin’s ability to reduce cancer risk in T2DM patients has generated even greater interest in its potential impact on both cancer and T2DM. These effects may or may not be primarily regulated by AMP kinase. Given that T2DM patients are already at a greater risk of developing cancer, the ability to adapt one of the most common diabetic drugs to cancer treatment could have tremendous implications clinically.


The Arp2/3 complex orchestrates the formation of branched actin networks at the interface between the cytoplasm and membranes. Although it is widely appreciated that these networks are useful for scaffolding, creating pushing forces and delineating zones at the membrane interface, it has only recently come to light that branched actin networks are mechanosensitive, giving them special properties. Here, we discuss recent advances in our understanding of how Arp2/3-generated actin networks respond to load forces and thus allow cells to create pushing forces in responsive and tuneable ways to effect cellular processes such as migration, invasion, phagocytosis, adhesion and even nuclear and DNA damage repair. Acanthamoeba and mammalian cells as a 7-protein complex that nucleates actin filaments and localises to dynamic actin structures [The Arp2/3 complex represents a unique molecular machine that enables the formation of branched actin filaments at the surfaces of lipid membranes. The Arp2/3 complex was first described in ructures ,2. Uniquructures ,5, some ructures , but theThe Arp2/3 complex is an inefficient nucleator of actin by itself and requires a nucleation promoting factor to activate it. The most well understood family of nucleation promoting factors is the WASP family, which consists of WASP/N-WASP, Scar/WAVE1-3, WASH, JMY and WHAMM proteins. WASP/N-WASP and Scar/WAVE proteins act at the plasma membrane, with WASP and N-WASP also triggering branched actin formation during membrane trafficking. WASH, JMY and WHAMM all act on internal membranes including endocytic vesicles, the Golgi complex and autophagosomes. Nucleation promoting factors help to specify not only the activity status of the Arp2/3 complex, but also the location where it will be activated. The WASP family of nucleation promoting factors have been extensively reviewed elsewhere ,7 and arCaenorhabditis elegans anchor cell that are characterised by a highly dynamic actin-comet-like appearance [Box 1Actin-nucleating proteinsProtein complexes that catalyse actin polymerisation serving as initiation sites for new actin filaments.Dendritic nucleationThe Arp2/3 complex nucleates a new (‘daughter’) filament from the side of the pre-existing (‘mother’) filament. Monomers add to barbed ends of both filaments, until capping by a barbed-end capping protein. This activity allows formation of branched actin networks.Nucleation promoting factorProteins that direct nucleation of actin filaments by recruiting actin monomers to pre-existing actin filaments and directly binding to and activating the Arp2/3 complex.LamellipodiaThin, sheet-like membrane protrusions typically found in the leading edge of motile cells in 2D.HaptotaxisDirectional motility of cells in response to a gradient of cellular adhesion sites or surface-bound proteins or chemoattractants.InvadopodiaDynamic actin-rich structures of the plasma membrane that are typically found in cancer cells and are involved with ECM degradation during invasion.PodosomesDynamic actin-rich and ECM-degrading cellular protrusions typically found in endothelial, immune and other physiological cell types.Capping proteinsProteins that regulate actin polymerisation by binding to barbed ends of growing actin filaments and blocking further exchange of subunits at these ends.PseudopodiaFinger-like projections of the cytoplasm of eukaryotic cells commonly observed in cells invading 3D or fibrillar ECM.Focal adhesionsAdhesive contacts between cells and the ECM mediated through the interaction of transmembrane proteins such as integrins to ECM ligand sites as well as intracellular protein complexes that are connected to actin filaments.Adherens junctionsCell-to-cell junction whose cytoplasmic face is directly linked to actin filaments. They are typically composed of cadherin receptors and cytoplasmic adaptor proteins (catenins).Alt-text: Box 1The best understood function of the Arp2/3 complex is to nucleate actin underneath the plasma membrane to effect protrusion of the cell edge. These protrusions are known as lamellipodia and form membrane sheets that can either remain adherent to the substratum or ruffle back in a wave form toward the cell body . Formation of endocytic vesicles requires dynamic changes in the plasma membrane. Through a stepwise process of pulling, sculpting and pushing, plasma membrane mechanics allow endocytic vesicle invagination, maturation and ultimately scission. Thus, this process involves increased membrane tension over time and spatially, and these forces are mediated through branched actin networks. Akamatsu et al.  and Sun Cell surface receptors such as integrins are the main mediators of biological and mechanical signals derived from the surrounding microenvironment. Just underneath the plasma membrane, focal adhesions are both signalling and mechanical hubs that directly connect the ECM–integrin interface to the cytoskeleton. Leading edge protrusions appear to depend on integrin activation and signalling components of focal complexes, including FAK or paxillin ,26. The in vivo is still unknown. Recent evidence suggested that loss of the ArpC4 subunit in skin keratinocytes results in a psoriasis-like disease in mice [in vivo might reveal the contexts where Arp2/3 mechanosensitive pushing forces are important for tissue maintenance and homeostasis.Cells organised in tissues respond to external forces collectively, as a coherent unit. This is usually mediated through adherens junctions that transmit forces and other signals across larger fields. Adherens junctions mediate tissue integrity and give epithelial and endothelial tissues mechanical strength and resilience. While branched actin structures have been described in these junctions before, two recent studies highlight the role of the Arp2/3 complex in generating mechanoresponsive branched actin networks that provide pushing force in response to pulling by myosin-II generated tension inside the cell and by external pressures ,34. Epit in mice , and Arp in mice . FurtherBranched actin networks have also recently been implicated in providing strength and support for cells under stress due to migration in confined spaces. Cell migration in the interstitial spaces of the body can involve squeezing through tight spaces, experiencing high levels of pressure or confinement. These stresses can damage the cell membrane and the nucleus and even cause DNA damage ,38. Two in vitro model shows that Arp2/3 and myosin contractile activity form spontaneous contractile waves in metaphase Xenopus egg extracts observed in cell-sized confinement chambers with natural phospholipids [Another case where nuclear integrity needs to be compromised is during nuclear envelope breakdown before cell division. In some mammalian cells, microtubules tear the nuclear membrane using force generated by the motor dynein, but a recent study in starfish oocytes showed that the nuclear membrane is ruptured by actin polymerisation driven by the Arp2/3 complex at the surface of the nuclear envelope . Dendritholipids . Tissue C. elegans anchor cell provides an elegant physiological model of normal developmental invasion during vulval development. Kelley et al. [Generation of branched actin networks in invadopodia or pseudopods creates a pushing force that helps cells to breach basement membranes and invade through the ECM. Such protrusions recruit mitochondria in close proximity to the dynamic branched actin networks. It is becoming clear that the cell regulates connections between dynamic actin turnover and production of ATP locally to power cytoskeletal dynamics. We and others observed links between invasive behaviour and mitochondrial recruitment to sites where force generation by branched actin are driving invasive protrusion ,44. The y et al.  found thy et al. . Thus, aIt is becoming apparent that Arp2/3-dependent branched actin filaments lie at the cell–microenvironment interface and thus they establish a route for transmission of biological, chemical and mechanical signals from the surrounding environment to the cell core and vice versa . This alVP and LMM drafted and edited the manuscript together. VP drew the Figures.Nothing declared.


Staphylococcus aureus and worse clinical outcomes. Neutrophil extracellular traps (NETs) are elevated in adults with ARDS. Whether pediatric ARDS (PARDS) is similarly associated with altered neutrophil expression of ISGs and neutrophil extracellular trap release is not known. Tracheal aspirate fluid and cells were collected within 72 h from seventy-seven intubated children. Primary airway neutrophils were analyzed for differential ISG expression by PCR, STAT1 phosphorylation and markers of degranulation and activation by flow cytometry. Airway fluid was analyzed for the release of NETs by myeloperoxidase-DNA complexes using an ELISA. Higher STAT1 phosphorylation, markers of neutrophil degranulation, activation and NET release were found in children with versus without PARDS. Higher NETs were detected in the airways of children with ventilator-free days less than 20 days. Increased airway cell IFN signaling, neutrophil activation, and NET production is associated with PARDS. Higher levels of airway NETs are associated with fewer ventilator-free days.Acute respiratory distress syndrome (ARDS) is a heterogeneous condition characterized by the recruitment of large numbers of neutrophils into the lungs. Neutrophils isolated from the blood of adults with ARDS have elevated expression of interferon (IFN) stimulated genes (ISGs) associated with decreased capacity of neutrophils to kill  While the overall mortality rate of PARDS is around 15%, the mortality rate is 35% in children with severe PARDS1. To date, little is known about the biological mechanisms associated with PARDS, as it remains a heterogeneous syndrome with pathology initiated by a number of diverse triggers, including pulmonary or systemic infection (sepsis), trauma, aspiration, near-drowning, and cardiac arrest3. Regardless of the trigger, neutrophils are recruited into the airways of children with PARDS where they not only combat infectious organisms and viruses, but also contribute to a hostile airway environment that may inflict further lung damage4. For example, in adults with acute respiratory distress syndrome (ARDS), worse clinical outcomes are associated with increased levels of both circulating and airway fluid neutrophil-related products and in dysregulated neutrophil functions7.Pediatric acute respiratory distress syndrome (PARDS) affects nearly 7% of intubated and mechanically ventilated children for acute respiratory failure8. However, the mechanisms underlying these observations are still unclear. Respiratory viruses have been shown to induce type I interferon (IFN) (IFNα/β) responses that then upregulate expression of interferon-stimulated genes (ISGs) such as MX1, ISG15, and IFIT110, which influence neutrophil behavior and function6. Since viral lower respiratory tract infections are a primary trigger for PARDS, we questioned whether differential ISG expression was associated with neutrophil responses in intubated children at risk or with PARDS. We hypothesized that children with PARDS would have a greater type I IFN response resulting in more neutrophil extracellular trap (NET) release.We have previously demonstrated that neutrophils exposed to airway fluid from mechanically ventilated children with virally-induced respiratory failure and bacterial coinfection have decreased respiratory burst and bacterial killing capacityThis prospective, observational study was performed in the thirty-six-bed academic medical/surgical Pediatric Intensive Care Unit (PICU) at Emory University/Children’s Healthcare of Atlanta at Egleston from January 2018 through February 2020. The study was approved by the Institutional Review Board at Emory University (IRB 00034236 and IRB 00113035) and all methods were carried out in accordance with relevant guidelines and regulations in the Declaration of Helsinki. Informed consent was obtained from the parents of all subjects prior to collection and use of their samples.11. Children had to have lung injury within 7 days of a known clinical insult, new infiltrate(s) consistent with acute pulmonary parenchymal disease on chest imaging and be receiving oxygen delivered either non-invasively or invasively to maintain an oxygen saturation between 88 and 97%. Children were excluded if they had any peri-natal related lung disease, respiratory failure fully explained by cardiac failure or fluid overload, chronic respiratory failure with mechanical ventilation via a tracheostomy or RAM cannula, confirmed immunodeficiency disorder, immunosuppression from chemotherapy for an oncologic process, chronic immunosuppression in a bone marrow transplant or solid organ transplant recipient, no parent or legal guardian present to provide written informed consent, or the attending physician did not wish the patient to participate in the study. Children were enrolled as controls if they were endotracheally intubated for airway protection and without lung pathology or signs of systemic infection or inflammation. For example, children selected as controls were electively intubated to facilitate radiologic imaging, for non-airway or cardiothoracic surgeries, and for airway protection following an acute ingestion or altered mental status without suspicion for infection or systemic inflammation.All patients admitted to the PICU who were greater than 2 days, with a corrected gestational age of at least 40 weeks, and were younger than 18 years old that met criteria for being at risk or having PARDS, as defined by the Pediatric Acute Lung Injury Consensus Conference (PALICC), were screened for eligibility13. Need for mechanical ventilation to 28-days was monitored to calculate ventilator-free days14. Lung injury severity was categorized according to PALICC criteria11.Clinical data were abstracted from the medical record onto a standardized form. Variables included demographics; fraction inspired oxygen, mean airway pressure, arterial oxygen saturation or arterial oxygen pressure used to calculate an oxygen saturation index (OSI) or oxygenation index (OI), respectively; laboratory and microbiology results; length of mechanical ventilation and need for reintubation; length of PICU stay, use of high frequency oscillatory ventilation (HFOV) or extracorporeal life support (ECLS), and vital status. Severity of illness was determined by the Pediatric Risk of Mortality (PRISM)-III and pediatric Logistic Organ Dysfunction (PELOD) scores were calculated within 24 h of intubation8. Children who were mechanically ventilated with HFOV were suctioned only if clinically indicated and approved by the attending physician. Tracheal aspirate samples were immediately placed on ice for transport to the laboratory for processing.Tracheal aspirates were obtained from patients on conventional mechanical ventilation by instilling 1–5 mL of sterile saline through the inline Ballard suction catheter and into a sterile Luken’s trap as part of routine suctioning per published protocolsg to generate a cell pellet and a fluid fraction. The fluid fraction was spun at 3000×g to generate cell-free airway supernatant (ASN), aliquoted, and stored at − 80 °C8. Airway cells were resuspended in PBS-EDTA and cell density was quantified using a Countess hemocytometer using trypan blue exclusion to determine cell viability. Cell purity was also assessed by cytospin preparations and Diff-Quik staining of airway cells. Cell viability was also assessed using a Live/Dead Aqua stain in the surface staining flow cytometry panel.Tracheal aspirate was gently dissociated using repeated passage through an 18G needle after the addition of 6 ml of PBS-EDTA. Dissociated tracheal aspirate was then centrifuged at 800×(−ΔΔCt) method was used to calculate the relative differences in gene expression. The data are reported as the mean ± SEM of individual patient’s airway cells.RNA was isolated from airway cells stored at − 80  C in RNA Later using the NucleoSpin RNA II kit with on-column genomic DNA digestion according to the manufacturer’s protocol . RNA was quantified using a NanoDrop Fluorospectrometer (Therma Scientific). RNA integrity (RIN) was measured at the Emory Integrated Genomics Core on an Agilent 2100 bioanalyzer , gating and analysis were performed with FlowJo v.10 8. Single cells were separated from doublet cells by gating on forward scatter area versus side scatter area profiles8. Live cells were then selected by exclusion of dead events positive for the dye Live/Dead Aqua, and platelets or platelet/leukocyte aggregates were excluded by staining for CD41a+ events8. CD66b+ neutrophils were then confirmed by gating on CD66b versus side scatter area8. Mean fluorescence intensities of surface markers are reported for CD66b+ neutrophils. To determine the background mean fluorescence intensity signal in the reporter channels , we report background mean fluorescent intensities in CD66b+ neutrophils using fluorescent minus controls that were only stained for CD41a, Live/Dead Aqua, CD66b, and CD16  for the fluorescent-minus reporter channels are for PB: 562 , AF488: 3949 , and for PE: 378  and are below the 10th percentile value of the No PARDS and Yes PARDS data. Due to limited numbers of cells in some tracheal aspirate samples, not every sample had a fluorescence minus control performed. For some samples, airway cells were fixed, permeabilized, and stained intracellularly with STAT1-PE (Clone: D1K9Y) and Phosphorylated (Tyr701)-STAT1-AF488  and then analyzed with by flow cytometry . Expression analysis was performed using FlowJo version 10 software .Airway cells collected from human subjects were preincubated with human TruStain FcX receptor blocking solution  and Live/Dead Aqua (Thermo Fisher) for 10 min on ice in the dark followed by surface staining with CD63-Pacific Blue , CD41a-BV510 , S1PR3-AF488 , CD66b-PerCPCy5.5 , Arg1-PE , and CD16-APCCy7  for 30 min on ice in the dark. Samples were treated with BD PhosFlow Lyse/Fix Buffer , washed twice with PBS-EDTA, and stored at 4 °C in the dark until acquisition on a CytoFLEX flow cytometer 15. For the capture antibody, Human MPO ELISA kit  was used according to the manufacturer’s instructions. We added 200 µL of airway fluid to the wells and incubated for 1 h. After washing 3 times with 300 µL of wash buffer, 100 µL incubation buffer containing a peroxidase-labeled anti-DNA mAb  was used and expressed as relative fluorescence units (RFU). Each sample’s RFU value was normalized to the mean and standard deviation (SD) for each experiment using the standardization formula: /SD experiment value) for each experiment.To quantify NETs from the airway fluid, a capture ELISA myeloperoxidase (MPO) associated with DNA was performed as previously describedp value less than 0.05.Statistical analyses were performed using JMP Pro 14  and GraphPad Prism 8 for Windows. Unless otherwise stated, comparisons between samples of children with versus without PARDS were made using a two-tailed Mann–Whitney U test for nonparametric data. For PCR studies, a ROUT outlier test using a 1% false discovery rate was performed prior to a two-tailed Mann–Whitney U test. Statistical significance was defined as a Seventy-seven patients were enrolled into the study within 72 h of intubation and mechanical ventilation. Of these 77 children, 42 (54.6%) had PARDS and 35 (45.4%) did not have PARDS. Table + neutrophils , an enzyme stored in the primary and tertiary granules of human neutrophils  signaling pathway was activated by measuring the phosphorylation of signal transducer and activator of transcription 1 (STAT1) by intracellular staining of fixed airway samples. Airway cells from children with PARDS had increased expression of phosphorylated-Y701 STAT1 (P-STAT1) and STAT1 compared with children who did not meet PARDS criteria Fig. . Cells wRDS Fig. . Our res18. Relative units of NETs were significantly higher in the airway fluid of children with versus without PARDS , a lower proportion of children with ventilator-free days over 20 days . Children with PARDS had fewer ventilator-free days  compared with children without PARDS . The proportion of children with PARDS had fewer ventilator-free days over 20 days compared with children without PARDS .Airway NET release was quantified in airway fluid by (MPO)-DNA complexesRDS Fig. A. Higherays Fig. B, and a 1. Viral and bacterial infections activate type I IFN signaling pathways resulting in an increase in antiviral ISG expression and proinflammatory responses critical for host defense19. A finely tuned type I IFN response is crucial to a host as an excessive or prolonged type I IFN response may lead to impaired gas exchange in the lung due to cellular and tissue destruction20. IFNs prime mature neutrophils for NET release upon stimulation with a second stimulatory signal21. Although antimicrobial proteins expressed on NETs can inactivate pathogens and prevent viral spread to neighboring cells, the lung is vulnerable to the damage that histones and proteolytic enzymes contained within NETs can inflict to host tissue. Therefore, excessive and prolonged type I IFN signaling may contribute to the production of NETs which fill the alveolar spaces, lead to increased inflammation, and result in impaired lung function, which are the hallmarks of PARDS. Our study assessed neutrophil activation, the differential expression of ISGs and activation of the STAT1 signaling pathway, and NETosis in the airways of intubated children with acute respiratory failure due to lower respiratory tract infections. We found increased neutrophil degranulation markers, phosphorylation of STAT1 (Y701), and NETs, as measured by MPO-DNA complexes, in the airways of children with PARDS compared with children without PARDS. Higher levels of airway NETs were associated with fewer ventilator-free days. Our findings are summarized in Fig. Acute LRTI are the trigger for the majority of PARDS23. At higher levels, ISGs contribute to dysregulated lung inflammation, disease progression, and are linked to worse clinical outcomes20. For example, in a mouse model of SARS-CoV infection, the delayed expression of high levels of type I IFN in the presence of high viral titers resulted in lethal pneumonia22; however, early treatment with type I IFN within the first six hours of SARS-CoV infection was protective. In Influenza A virus, disease severity and progression are associated with overshooting the IFN-driven inflammatory response whereby exogenous supplementation with type I IFN correlated with increased morbidity and mortality25. RSV also induces high levels of pro-inflammatory cytokines directly related to type I IFN, and mice that lack IFNAR have less proinflammatory cytokine release resulting in a less severe disease course following RSV infection26. By contrast, there was no effect of IFNAR deletion on pathogenicity of mice infected with SARS-CoV; however, STAT1 deficient mice showed increased susceptibility, prolonged viral shedding and mortality27.The timing and magnitude of the type I IFN response are important in modulating the immune response to viral infection6. For example, high expression of a panel of ISGs  from circulating neutrophils from a subgroup of adults with ARDS compared with normal ISG expressing neutrophils had reduced migration toward the neutrophil chemokine interleukin-8 (IL-8), decreased p38 MAP kinase phosphorylation, superoxide anion release, IL-8 release, and a shift from necrotic to apoptotic cell death that was associated with a diminished capacity to kill Staphylococcus aureus, but not Pseudomonas5. Subsequent hierarchal clustering analysis of the aforementioned ISG panel from circulating neutrophils of ARDS patients within 72 h of initiation of mechanical ventilation showed that both High- and Low-range expression had fewer 28-day ventilator-free and ICU-free days and higher 90-day mortality compared with Mid-range ISG expressing patients6. These data suggest that a targeted middle ground for IFN levels exists to benefit the host. An IFN signal below a lower threshold would result in an ineffective antiviral host response, while an IFN signal above an upper threshold would trigger a detrimental inflammatory host response. IFN signaling outside the target zone would increase lung inflammation and ARDS severity20. By contrast, our findings are from airway cells with a neutrophil predominance rather than from circulating neutrophils for adults with ARDS6. Additionally, we assessed each ISG, IFIT1, ISG15, and Mx1, individually rather than in aggregate, and we note that ISG expression is highly variable in children with versus without PARDS. Finally, the release of NETs was not assessed in the aforementioned studies.Differential type I IFN and ISG expression are associated with neutrophil dysfunction and worse clinical outcomes29. NETs play prominent roles in bacterial pneumonia32, RSV bronchiolitis34, influenza pneumonia36, sepsis40, SARS-CoV241, small-vessel vasculitis15, systemic lupus erythematosus43, and transfusion-related acute lung injury44. NETs induce dose-dependent cytotoxic effects on human alveolar epithelial cells due to histone and myeloperoxidase induced damage to alveolar epithelial and endothelial cells46. NETs also influence the macrophage function47, T cell proliferation48, and amplify IFN production by plasmacytoid dendritic cells42. Conversely, interferons influence the process of NETosis in mature neutrophils49.Excessive NET formation has been reported in the serum and bronchoalveolar lavage fluid from adults with ARDS; however conclusions with respect to clinically relevant outcomes are difficult to draw due to the heterogeneity in study design50. Interferonopathies, autoimmune diseases, and tumor-associated neutrophils are all examples where high IFN/ISG levels influence neutrophil activation and function51. Priming of neutrophils with IFN-α and subsequent stimulation with C5a resulted in increased STAT1 phosphorylation at tyrosine 701 and NET production in mature neutrophils21. Activation of neutrophils by type I IFNs led to increased NETosis that triggered biofilm formation by Pseudomonas aeruginosa and persistence in the lung50. NET production, like IFN signaling, exists in a balance. Excessive NET release can lead to the systemic spread of inflammation through platelet interactions and result in multiple organ dysfunction52; however, depletion or defective NET production can lead to the spread of infection early in the course of disease40.High IFN/ISG expression polarizes neutrophils to an activated “N1” phenotype that can lead to increased NET production, degranulation, and influence neutrophil interactions with other immune and airway epithelial cells56. NETosis is driven by NET-specific kinases that regulate transcription initiation in neutrophils. For example, ERK was shown to differentially regulate NOX-dependent NETosis54. Interestingly, Khan and colleagues also noted that STAT1 was a transcription factor that was upregulated in the NOX-dependent NETosis pathway54. We were not able to study the relative importance of NOX-dependent versus calcium influx (NOX-independent) mechanisms on NET formation given that NETosis has already occurred by the time of tracheal aspirate sampling in our patients. Additionally, we did not quantify the relative amounts of neutrophils undergoing NETosis versus apoptosis. Quantifying neutrophil apoptosis should be part of future studies as NOX-dependent NETosis is dependent on the activation of the kinase Akt to suppress apoptosis and switch to NETosis57.Several mechanisms regulating the formation of NETs are known and include NADPH oxidase (NOX2) dependent and calcium channel NOX-independent NETosis60. Second, there is heterogeneity in the identity of viral and bacterial pathogens infecting study participants precluding any conclusions regarding the influence of organism on study results. We did not stratify the patients based on bacterial growth in an endotracheal respiratory culture as there is controversy regarding whether this is a true bacterial coinfection versus merely codetection of bacterial and viral organisms61. Thirdly, we are not powered to detect differences in mortality associated with higher ISG gene expression as seen in the adult ARDS cohort; however, we did detect a significant difference in ventilator-free days in children with a higher airway NET burden6. We are limited in the ability to study the mechanisms regulating airway NET release as NETosis has already occurred in patients at the time of sample collection’ however, network analysis of transcription factor signaling pathways from recovered airway neutrophils could be performed as previously described54. Additionally, future experiments using blood-derived neutrophils from healthy donors could be incubated in patient airway fluid to simulate the airway environment with PMA and calcium-ionophore stimulation, and appropriate NOX inhibition, to study mechanistic regulation of NET formation. Finally, we did not explore the role of other signaling pathways, such as that initiated through the sphingolipid receptor or other kinase signaling cascades, as mechanisms involved in the pathogenesis of NETs or PARDS severity.While we detected higher type I IFN signaling and NET production in the airways of children with versus without PARDS, due to the clinical nature of our study, we are not able to attribute causation of neutrophil dysfunction to high ISG expression in study participants. There are several additional limitations to our study. First, this is a single-center study with a limited sample size; however, despite the limited number of children studied, we were able to detect differences in neutrophil type I IFN signaling pathway phosphorylation and function in intubated children with versus without PARDS. The majority of children in this study were at risk for developing PARDS, with the remaining children being equally distributed amongst mild, moderate and severe PARDS categories; however, the degree of PARDS severity was not associated with neutrophil dysfunction due to heterogeneity in neutrophil function and a limited sample size. We also excluded immunocompromised patients from our study which is a major risk factor associated with PARDS-related mortalityIn summary, we describe the differential type I IFN signaling based on the presence or absence of PARDS and show that airway levels of NETs are higher in children with versus without PARDS. Higher levels of airway NETs are associated with fewer ventilator-free days. Future work will explore the influence of type I IFN signaling, NETs and the PARDS airway environment on macrophage and T-cell activation and function. Defining underlying mechanistic differences in children with acute respiratory failure due to lower respiratory tract infections is needed to move beyond supportive care and toward targeted personalized therapies for pediatric patients with moderate/severe PARDS.Supplementary Information.