Cutaneous microcirculatory perfusion is commonly measured using laser Doppler flowmetry (LDF) probes, which provide a continuous, non-invasive quantification of skin blood flow (SkBF). However, inhomogeneities in the skin’s microvasculature density contribute to a decrease in reproducibility whenever an LDF probe is removed and replaced, as is the case during pre- and post-intervention or between-day measurements. Therefore, this study aimed to determine whether increasing the total number of individual LDF probes in a localized area improves the reproducibility of the measurement. Seven laser Doppler probes were secured in a custom-made acrylic holder designed to attach to the skin’s surface easily. SkBF, local skin temperature (Tsk), and blood pressure (BP) were assessed in 11 participants . SkBF and Tsk were measured from the dorsal forearm  for 5 min. Next, the multi-laser device was moved to the lateral side of the calf , and measurements were obtained for 5 min. Each arm and leg trial was cyclically repeated three times, and all trials were separated by intermissions lasting 10–15 min. The average SkBF and the cutaneous vascular conductance (CVC) from all possible LDF probe combinations were not statistically different across the three arm and leg trials. Two-way mixed-effects models with absolute agreement were used to compute the intraclass correlation coefficient (ICC) for CVC, and the minimum ICC increased with the addition of LDF probes. The ICC of the average CVC from seven LDF probes was 0.96 between the arm trials and 0.91 between the leg trials, which suggests that there is excellent reliability and little difference between trials following the removal and replacement of the device. Moreover, all individual ICC values from ≥3 LDF probe combinations were greater than 0.70 . These data suggest that SkBF measurements with multiple laser Doppler probes in a custom-made holder have excellent reproducibility after replacing the probes within the same participant. Therefore, this application could provide more reproducible assessments between repeated measurements  where the LDF probes must be removed and replaced within the same location. However, the LDF technique is not free from limitations, and the assessment of SkBF has been continuously criticized. Namely, it is highly susceptible to motion artifacts, and there is considerably large site-to-site and day-to-day signal heterogeneity. Thus, the inability to remove and replace the LDF probe significantly hinders the ability to obtain observations pre- and post-intervention without the use of normalization techniques , or converting the raw data to a percentage of “maximal” SkBF at the site). Therefore, a more broadly applied method that could achieve greater reproducibility between measurements would benefit physiology researchers.Laser light has been frequently used in various biomedical applications since its initial development in the 1960s . For dec2, yielding a sample volume of roughly ∼1 mm3 . Due to the light-scattering and absorption properties from endogenous chromophores  and other tissue components, reflected laser light becomes frequency-shifted by moving erythrocytes but remains relatively unshifted as it passes through static tissue. The broadening of the laser light, dependent on the velocity of the moving erythrocytes, is subsequently detected by photodetectors and processed by a computer. To date, LDF probes have been applied to various vascularized tissue beds  in both experiments in humans  and animy ∼1 mm3 . Thus, gy ∼1 mm3 . Due to y ∼1 mm3 , it servy ∼1 mm3 .Similar to LDF measurement, various techniques have been employed to track changes in microvascular perfusion, namely, venous occlusion plethysmography, scanning laser-Doppler imaging, laser-Doppler speckle contrast imaging, and optical coherence tomography . SeveralAlthough the skin is generally regarded as an excellent interrogation site for LDF probes, due to its non-invasive accessibility, SkBF measures are incredibly susceptible to motion artifacts and any slight movement or relocation of the LDF probe . This isEleven individuals  were invited to participate in the study. The participant characteristics are shown in As described in prior reports , blood pA custom-made holder was used to mount the seven probes in a circular and equidistant configuration . Brieflyetc.) and thermoregulatory variables  can change from day-to-day, these factors will directly affect SkBF. To decrease the possible influences from these factors, the three trials were performed in one laboratory visit.The procedures were conducted with the participant in the supine position in a room with an ambient temperature of ∼23°C. Three trials were performed with intermissions, during which participants were released from the testing table for 10–15 min. The seven probes were placed in the plastic holder. A thin thermocouple (Type T) was also placed under the probe holder. The probe holder was then placed on the participant’s dorsal forearm, approximately at the middle position between the wrist and elbow along the line between the lateral epicondyle of the humerus and the lateral side of the styloid process of radius . The posvia a data acquisition system . The mean values of the 3-min recordings of SkBF and Tsk were used as the measurement for the trial. Cutaneous vascular conductance (CVC) was calculated from the ratio of the SkBF to mean arterial pressure (MAP). The SkBF and CVC from each individual probe were calculated at first. The absolute values of BP, Tsk, SkBF, and CVC were used to examine the effects of the intervention , and comparisons between the three trials were examined using repeated measures one-way ANOVA. When appropriate, Tukey post-hoc analyses were employed.Data were sampled at 200 Hz In this test-retest reliability study, the intraclass correlation coefficient (ICC) of the CVC was used as the primary index for the reliability of the measurement. In the present study, ICC estimates and their 95% confidence intervals were calculated using SPSS statistical software  based on a mean-rating (k = 3), absolute-agreement, two-way mixed-effects model . The calculation of ICC for the CVC from each individual probe were performed at first. To test the effects of multiple probes, the mean values of each combination of the CVC from these probes were calculated . Then, the ICC for the mean CVC from all possible combinations  of the probes were calculated with ICC values ≥0.70 being considered acceptable for research .www.real-statistics.com, Charles Zaiontz) for Microsoft Excel .Statistical power analysis for ICC was performed using a publicly accessible add-in  of ICC values from the combinations of the probes were calculated and are presented with box and whiskers plots using Sigmaplot software . All values are reported as means ± SD. p < 0.05) and did not change significantly along the leg trials. HR decreased along the three arm trials and did not change significantly along the leg trials.The mean SkBF, Tsk, CVC, BPs, and HR during the three arm trials and three leg trials are shown in Tsk remained constant across all three arm trials and three leg trials. The averaged SkBF and CVC from all probes did not change significantly along the three arm trials or three leg trials . The SkBThe ICC values from the individual probes and the ICC values from the combinations of the probes in the arm trials and in the leg trials are presented with box and whiskers plots in n = 7). For the ICC for each of the seven single probes in leg, the mean statistical power was 0.993 with a range of 0.990–0.994 (n = 7). For the averaged measurements of seven probes, the mean statistical powers were 0.991 for the arm measurements and 0.994 for the leg measurements.For the ICC for each of the seven single probes in the arm, the mean statistical power was 0.993 with a range of 0.989–0.997 , commonly termed “red blood cell flux” or simply “flux,” which is defined by the manufacturer using a phantom. Specifically, flux is the product of measured red blood cell concentration and velocity; thus, flux is proportional to the velocity and concentration of erythrocytes in a local tissue and is largely impacted by changes in cutaneous vasoconstriction and dilation. SkBF signals can be isolated to capillaries with a diameter of roughly 10 microns which are located in the papillary dermis 1–2 mm below the epidermal surface . In the etc.).Similarly, with the SkBF values, the CVC values were not significantly different between trials in either the arm or leg . The CVCThis study utilized the assessment of ICC to reflect measurement reliability between trials where the LDF probes were removed and replaced within participants and on the same skin sites . Reliability reflects the extent to which measurements can be replicated in a sample of participants. Mathematically, reliability represents a ratio of true variance over true variance plus error variance . The evaThe readings from LDF probes can be significantly altered when the site is slightly moved . TherefoIn our study, the SkBF (and thus CVC) measurements were recorded from 14 individual sites . The ICC of CVC was calculated from the individual sites and the possible combinations in each trial. To ensure the reliability of the measurement, we think that the minimum value from the box and whiskers plots should be predominately considered. For the data from each single probe/site, the minimum ICC values were ∼0.60 and 0.81 at leg and arm, respectively, which indicate a moderate to good reliability. However, considering that the application of LDF will not be limited to the forearm, the SkBF/CVC measurement with a single probe has only moderate reliability. When multiple probes >3) were used, the ICC values increased with the number of the probes , 3. We s were usen = 11) as those in prior test-retests reliability studies (n = 6) and females (n = 5) or for age range , the current study was unable to compare between biological sex or various age groups. Thus, this highlights a significant area for future investigations with more subjects. Lastly, as is the case in many physiology experiments, there may be a potential limitation of the measurement device itself as manufacturing discrepancies cause variation in the absolute values from probe to probe. It cannot be overlooked that in the present study, LDF probe 3, on average, provided higher readings across all participants than that of the other probes. This is likely due to slight differences in the manufacturing of each probe. Although each probe was calibrated using the manufacturer’s calibration procedure, there may still be discrepancies between probes and connections to the light source, which is housed in a separate device. Cupping connectors that join each fiberoptic cable may also have slight variations contributing to the variable readings. However, as these readings were reproducible across all trials , the relatively high reading from probe three did not appear to detract from the original findings of this study.In the present study, there are several limitations that must be considered when interpreting these data or implementing the proposed approach for physiology research studies. First, the intermission period, which was fixed at 10–15 min, could be initially viewed as a limitation of the study since it is unclear if the measures are reproducible with longer intermissions. However, this period was necessary to establish that any observed changes in skin blood flow, if any, were from the approach itself and not a result of “real” SkBF changes that are known to fluctuate throughout the day. The possible changes in BP, central blood volume, skin and central temperature, and environmental factors such as humidity from days to weeks will actually alter SkBF. In the current study, the SkBF/CVC remained stable between all three trials in both the arm and leg. Thus, it is unlikely that these factors significantly contributed to the measurements. Second, without the use of a normalization procedure, it remains difficult to compare between various sites and individuals using the absolute SkBF and CVC values in human studies. Indeed, a prior study has shown that the absolute resting CVC values within participants were poor between days but improved with the application of heat strain . Thus, c studies . HoweverThe current study shows the addition of ≥4 LDF probes in an integrated probe holder device is sufficient for physiology research in humans. Prior to the development of this approach, our laboratory and others had been significantly limited by the LDF measurement in experiments where it is not realistic to measure SkBF continuously. Until now, protocols where the lasers had to be physically removed from the participant to complete other interventions significantly confounded the comparison of the SkBF measurement. With this new approach, various experimental designs, previously confounded by the limitation of a relatively small sample volume, may be better reliably investigated. For instance, this approach can be used in the protocols using whole-body heating with water immersion, acute exercise, or exercise training over a period of days or weeks. Moreover, the data from the multiple probes could present an “averaged” condition, effectively minimizing the effects of skin inhomogeneities. In turn, the study measuring reactive changes in blood flow, we speculate that the proposed application may also accurately track “averaged” reactive changes in SkBF and CVC. Importantly, the ability to remove and replace the LDF device allows for greater application from research in the clinical setting. As patients are routinely transferred between locations for certain diagnostic tests and procedures, the proposed approach could allow for a more reliable pre- and post-assessment of SkBF and CVC. Moreover, it is speculated that this approach could be applied to monitor the skin microcirculatory function in certain disease progressions  within the patients, as well as track the effects of medical therapy or surgical interventions  over time. It is also reasonable to speculate that this approach could be applied to animal research as well. In animal models, more invasive techniques allow for the assessment of flux from various tissues, including but not limited to the skeletal muscle. However, due to the size of the animal model, a different arrangement of probes and thus a revised integrated probe holder may be necessary to facilitate a smaller sampling area. Moreover, considering that the total sizes of the organs  in small animals  are small, the relative area measured by multiply LDF probes can be relatively much larger . Under this condition, it is possible that measurements could be used for inter-individual and inter-site comparisons, while further studies are needed to confirm this. Thus, the approach may have even greater applicability in these experiments as well.In conclusion, the present study showed that increasing the number of individual LDF probes in a custom-made holder increases the reliability of the LDF measurement during test retests. These results suggest that CVC measurements, obtained using multiple laser Doppler probes (number ≥4), have a sufficiently high degree of reproducibility after replacing the probes within the same participant. Thus, we suggest the values from multiple laser Doppler probes in a holder can be used in the intraindividual analysis for certain experiments where the LDF probes must be removed and replaced in the same participants .


Some patients develop drug resistance to programmed cell death protein 1/programmed death-ligand 1(PD-1/PD-L1) therapy but the mechanism is unclear. Therefore, the study of drug resistance to PD-1 therapy is quite important. In this sense, we obtained B16F10-R tumours resistant to anti-PD-1 therapy through multiple rounds of drug resistance screening in vitro. We found that COX2 expression was significantly elevated and COX2 inhibitors in combination with anti-PD-1 monoclonal antibodies (mAbs) could reverse this resistance phenomenon. Knockout of the ptgs2 gene in B16F10-R tumours also restored tumour sensitivity to anti-PD-1 therapy. Therefore, we believe that the combination of COX2 inhibitors and anti-PD-1 mAbs may become a new choice for the drug resistance of anti-PD-1 therapy in the future.Immunotherapy is an effective method for tumour treatment. Anti-programmed cell death protein 1 (PD-1) and anti-programmed death-ligand 1 (PD-L1) monoclonal antibodies play a significant role in immunotherapy of most tumours; however, some patients develop drug resistance to PD-1/PD-L1 therapy. Cyclooxygenase-2 (COX2) is expressed in various solid tumours, and prostaglandin E2 (PGE2) drives the development of malignant tumours. We developed a drug-resistant B16F10 (B16F10-R) tumour mouse model through four rounds of selection in vivo. Subsequently, we investigated changes in PD-L1 expression and lymphocyte infiltration in B16F10-NR and B16F10-R tumours. Additionally, we explored the role of COX2 in acquired resistance to pembrolizumab, an anti-PD-1 treatment. Immune cell infiltration was significantly decreased in resistant tumours compared to B16F10-NR tumours; however, ptgs2 gene expression was significantly elevated in resistant tumours. Aspirin or celecoxib combined with pembrolizumab can effectively reverse tumour drug resistance. In addition, ptgs2 knockout or the use of the EP4 inhibitor E7046 abrogated drug resistance to anti-PD-1 treatment in B16F10-R tumour cells. Our study showed that inhibition of the COX2/PGE2/EP4 axis could increase the number of immune cells infiltrating the tumour microenvironment and recover drug-resistant tumour sensitivity to pembrolizumab. Thus, we highlight COX2 inhibition as a promising therapeutic target for drug-resistant tumours for future consideration. In recent years, tumour immunotherapy has made major strides in cancer treatment . The PD-Anti-PD-1 therapy can increase the expression of inflammatory cytokines, which may counteract its therapeutic effects ,14. CyclThis study investigated the function of COX2 in anti-PD-1 acquired resistance by developing a drug-resistant B16F10 tumour mouse model. Our study showed that COX2 derived from tumours plays an essential role in adaptive tumour resistance. Inhibiting the COX2/PGE2/EP4 axis could increase the number of infiltrating T and NK cells in the tumour microenvironment (TME) and recover drug-resistant tumour sensitivity to pembrolizumab.Pdcd1 transgenic mice were housed in a pathogen-free environment at the Laboratory Animal Research Centre, Tongji University, as previously described [Human escribed . All aniptgs2 melanoma cells were generated using the CRISPR-Cas9 system (Cas9-2hitKO). The guide RNA sequences targeting ptgs2 were sgRNA-1, 5′-GCTTTACAGACTTAAAAGCA-3′ and sgRNA-2, 5′-TTCAAGACAGATCATAAGCG-3′. All cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 1% penicillin/streptomycin  and 10% foetal bovine serum (Gibco) at 37 °C and 5% CO2.B16F10 melanoma cells were purchased from the American Type Culture Collection . B16F10-NR and B16F10-R melanoma cell lines were generated by four rounds of selection for anti-PD-1 resistance. B16F10-R-knockout (KO) ptgs2 cells (1 × 105 cells) were resuspended in phosphate-buffered saline PBS  and injected subcutaneously into the flanks of 6–8-week-old female human Pdcd1 transgenic mice. Mice were randomised into groups, each comprising 6–8 mice. When tumours grew to 50 mm3, mice were administered pembrolizumab  or the control isotype for ophthalmic intravenous injection twice a week, and aspirin , SC560 , celecoxib , and E7046  for intraperitoneal injection three times a week. The longest dimension (length) and longest perpendicular dimension (width) were measured every two days using a calliper. Tumour volume (mm3) = (length × width × width)/2. For in vivo B16F10-R cell selection, B16F10 tumours were digested with trypsin and collagenase until reaching 1500 mm3, which was the humane endpoint. CO2 inhalation was used to euthanise the mice. The cells were resuspended in DMEM and cultured for two weeks. The tumour cells were re-injected subcutaneously into another mouse for subsequent rounds of in vivo selection.B16F10, B16F10-NR, B16F10-R, and B16F10-R-knockout Ptgs1/ptgs2 transcript levels were measured using an RT-PCR SYBR Green I kit (Solarbio). RT-qPCR was performed on a ROCHE LightCycler® 96  in a 96-well plate, and each sample was prepared in triplicates. The following primer pairs were used: GAPDH, forward primer 5′-TGGCCTTCCGTGTTCCTAC-3′ and reverse primer 5′-GAGTTGCTGTTGAAGTCGCA-3′; mouse COX1, forward primer 5′-TTACTATCCGTGCCAGAACCA-3′ and reverse primer 5′-CCCGTGCGAGTACAATCACA-3′; mouse COX2, forward primer 5′-AGCAAATCCTTGCTGTTCCAA-3′ and reverse primer 5′-GCAGTAATTTGATTCTTGTC-3′. Total RNA was extracted using the Animal Tissue/Cell Total RNA Extraction Kit  and cDNA was acquired using a reverse transcription kit . Tumours were cut and digested with trypsin and collagenase. A 40 µm mesh filter  was used to filter the single-cell suspension . The cells were blocked with purified rat anti-mouse CD16/CD32 , and dead cells were stained with BD Horizon Fixable Viability Stain 510 . Cell surface staining was performed using the following antibodies: APC-Cy™7 mouse anti-mouse CD45.2 , PE hamster anti-mouse CD3e , PerCP-Cy™5.5 rat anti-mouse CD4 , FITC rat anti-mouse CD8a , APC anti-mouse NK-1.1 antibody , FITC anti-mouse CD49b (pan-NK cells) antibody , PE/Cyanine7 anti-mouse TCR β chain antibody , and PE anti-mouse CD274  antibody . The following antibodies were used as isotype controls: APC-Cy™7 Mouse IgG2a, κ Isotype Control , PE Hamster IgG1 κ Isotype Control , PerCP-Cy™5.5 Rat IgG2a, κ Isotype Control , FITC Rat IgG2a, κ Isotype Control , APC Mouse IgG2a, κ Isotype Control , FITC Rat IgM, κ Isotype Control , PE Rat IgG2b, κ Isotype Control . All analyses were performed using a CytoFLEX LX .6 cells/mL in 96-well plates at 37 °C in the absence or presence of 100 mL of conditioned medium from tumour cells plus or minus LPS (10 to 100 ng/mL) in a total volume of 200 mL. After overnight culture, PGE2 concentration in the supernatant was determined by ELISA.Cells were plated at 0.5–1 × 10Cell lysis was performed with RIPA buffer  supplemented with protease  and phosphatase inhibitors (Selleck). The BCA Protein Assay Kit  was used to measure protein concentrations. The samples were boiled at 100 °C and centrifuged to obtain the supernatant, after which 100 µg of protein was loaded onto 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels . The proteins were transferred to 0.45 µm polyvinylidene fluoride (PVDF) membranes , and the membranes were blocked in a Western blocking buffer  for 2 h at 15 °C. The sections were then incubated with primary antibodies (1:2000) overnight at 4 °C and with the relevant secondary antibody  for 2 h at 15 °C. Bands were visualised using BeyoECL Plus . Protein bands were quantified relative to the loading control (GAPDH). The following antibodies were used for Western blotting: anti-GAPDH , anti-rabbit-IgG-HRP , and anti-COX2 .p < 0.05. Survival analysis was based on the following criteria: tumour volume, tumour necrosis, and pathological death. Survival analysis was performed using the log-rank test.Statistical analyses were performed using GraphPad Prism version 6.0 . Statistical differences were determined using unpaired two-tailed t-tests, one-way ANOVA, or two-way ANOVA. The results are shown as the mean ± SEM, and the significance was set at Pdcd1 transgenic mice to acquire resistance to anti-PD-1 treatment. In the human Pdcd1 transgenic mice, the B16F10 tumour cell was sensitive to pembrolizumab treatment. We treated mice with 10 mg/kg pembrolizumab and obtained an anti-PD-1-resistant cell line (B16F10-R) after four rounds of in vivo selection (To acquire a cell line resistant to anti-PD-1 (pembrolizumab) treatment, we established an in vivo B16F10 tumour model using human election A. Analogelection A. With aelection B. Moreovelection .This B16F10-NR cell line was very sensitive to anti-PD-1 treatment, but anti-PD-1 therapy did not effectively control B16F10-R tumour growth after four selection rounds C. In addThus, the B16F10-R tumour model showed acquired resistance to anti-PD-1 treatment. The persistence of this phenotype in B16F10-R tumour cells after several consecutive in vitro cell cultures suggested that the acquired resistance was caused by genetic changes in the tumour cells. To understand the difference between drug-resistant and non-drug-resistant tumours, we hypothesised that there would be an alteration in PD-L1 expression on the tumour surface. Flow cytometry analysis showed that PD-L1 expression on the surface of B16F10-R tumour cells was slightly increased A. This pPdcd1 transgenic C57BL/6J mice were injected with aspirin, pembrolizumab, or PBS. Notably, tumour growth in the group treated with pembrolizumab alone was not affected compared to that in the PBS group. In addition, aspirin alone did not inhibit tumour growth. However, the combination of aspirin and pembrolizumab considerably inhibited tumour growth in B16F10-R tumour cells compared with that in the PBS group (To further confirm whether tumour-acquired resistance to anti-PD-1 therapy was due to decreased immune cell infiltration, we investigated the effects of inhibitors that increased immune cell infiltration. Aspirin is a non-selective inhibitor of COX1/COX2 which can inhibit the expression of COX in tumour cells and then inhibit the production of PGE2, decreasing the infiltration of immune cells into tumours . Next, wBS group A.Next, we examined the TME using flow cytometry analysis, which showed that the infiltration of CD3+, CD4+, and CD8+ T cells was markedly increased in the two aspirin groups with or without pembrolizumab . In addiSince aspirin is a non-selective inhibitor that inhibits both COX1 and COX2, we wanted to determine which, if any, played a more important role in tumour-acquired resistance. Thus, we verified the difference in the expression of COX1/COX2 in B16F10-R and B16F10-NR tumour tissues via RT-qPCR to estimate the relative abundance of COX1/COX2 mRNA. B16F10-R tumours exhibited significantly higher mRNA levels of both COX1 and COX2 than B16F10-NR tumours C,D.To determine whether COX1 or COX2 was more vital, the selective COX1 inhibitor SC560 and selective COX2 inhibitor celecoxib were used in combination with pembrolizumab. We found that SC560 combined with pembrolizumab did not affect tumour growth A, whereaTo further verify the role of COX2 in the development of acquired resistance to anti-PD-1 treatment, we knocked out COX2 in B16F10-R tumour cells , and we Most of the functions of PGE2 are mediated by four PGE2 receptors, EP1, EP2, EP3, and EP4 . PGE2 inMalignant tumours can lead to the inactivation of cytotoxic T cells after PD-1 binds to its ligand via the upregulation of PD-L1 expression. Therefore, anti-PD1 or anti-PD-L1 monoclonal antibodies can restore the immune inhibition of tumour growth by blocking the PD-1/PD-L1 pathway. In several cancer types, pembrolizumab has considerably increased patient survival through its therapeutic inhibition of PD-1 . AlthougIn this study, we established a melanoma mouse model of B16F10 tumour resistance to anti-PD-1 treatment using four rounds of pembrolizumab therapy and selection in vivo. Additionally, human Pdcd1 transgenic C57BL/6J mice were used to study secondary drug resistance. After four rounds of treatment, B16F10 tumours became resistant to pembrolizumab in vivo. This resistance persisted in cultured B16F10 cells in vitro, suggesting that the acquired resistance was caused by hereditary changes in tumour cells.The detection of PD-L1 expression on the tumour surface ruled out the possibility that a decrease in its expression led to immune escape of the B16F10-R tumour. Flow cytometry analysis of the TME revealed that the number of infiltrating immune cells, including CD3+, CD4+, and CD8+ T cells, as well as NK cells, was considerably reduced in B16F10-R tumours compared to that in B16F10-NR tumours. We speculated whether the reduction in immune cell infiltration in B16F10-R tumours resulted in tumour immune escape against anti-PD-1 therapy.The COX2-PGE2 pathway has been reported to be involved in the infiltration of NK and T cells in tumours . In addiptgs2 gene in drug-resistant tumour cells to verify the function of COX2 in tumour drug resistance. After knockout, the sensitivity of drug-resistant tumours to pembrolizumab changed significantly. In addition, infiltrating lymphocytes in the TME increased dozens of times. These results illustrate that COX2 expression is highly correlated with tumour drug resistance.RT-qPCR results showed that the expression of COX1/COX2 was significantly higher in drug-resistant tumours than in wild-type tumours. The combination of selective inhibitors of COX1/COX2 with pembrolizumab indicated that COX2 may be the main reason for the development of drug resistance. Therefore, we knocked out the Subsequently, we targeted EP4 receptors, which were one of downstream receptors of PGE2, and found that EP4 was associated with drug resistance in some tumours. Moreover, we found that E7046, an inhibitor of EP4, combined with pembrolizumab, inhibited the growth of drug-resistant tumours and restored lymphocyte infiltration in the TME. This result also revealed the crucial function of the COX2/PGE2/EP4 pathway in tumour drug resistance.In all the animal experiments related to drug-resistant tumours, we observed two common phenomena. First, the use of a single inhibitor or pembrolizumab alone did not affect tumour growth, and only a combination of therapies inhibited tumour growth. Second, as long as COX2-related inhibitors were used, the number of infiltrating immune cells in the tumours increased significantly. To explain these two phenomena, we postulated that after the tumour acquired drug resistance, the expression of PD-L1 on the tumour cell surface did not decrease, but the lymphocytes in the tumour were significantly reduced. When only pembrolizumab was used, no effect was observed, probably due to the extreme lack of immune cells. When only COX2-related inhibitors were used, the lymphocytes in the tumour recovered, but the tumour still expressed PD-L1, and immune escape was carried out through the PD-1/PD-L1 pathway; hence, there was no overall effect. Tumour growth can only be effectively inhibited by simultaneous blocking of the PD-1/PD-L1 pathway and restoration of lymphocyte infiltration in the TME with COX2 inhibitors.In summary, upregulation of COX2 might be the reason for tumour-acquired resistance to anti-PD-1 therapy. Our work demonstrated that COX2 inhibitors could promote pembrolizumab efficacy and inhibit tumour growth in a drug-resistant model. Mechanistically, celecoxib inhibited the COX2 effect and suppressed PGE2 production, which in turn elevated the infiltration of CD3+, CD4+, and CD8+ T cells, as well as NK cells, resulting in the suppression of tumour growth. Tumour drug resistance is a complex process caused by a range of situations. The results of our study may only reveal one of the causes of tumour-acquired resistance to anti-PD-1 therapy, and further research, including clinical tests, is needed to verify their applicability. Nonetheless, our findings further our understanding of the mechanisms underlying drug resistance in tumours. Furthermore, we identified a new role for the COX2 signalling pathway in anti-PD-1-resistant tumours. Our study suggests that elevated COX2 expression is a potential biomarker of poor immunotherapy response in anti-PD-1-resistant tumours. The dual targeting of PD-1 and COX2 in tumours may enhance the efficacy of immune checkpoint therapy and overcome drug resistance to anti-PD-1 therapy. This is an important addition to our current understanding of tumour-acquired resistance to anti-PD-1 therapy and provides research directions for developing a clinical treatment for anti-PD-1-resistant tumours and immunotherapy.In conclusion, we developed an anti-PD-1-resistant B16F10 tumour model using human Pdcd1 transgenic mice. Lymphocyte infiltration was significantly reduced and COX2 gene expression was increased in B16F10-R tumours compared with B16F10-NR tumours. Moreover, COX2 inhibitors could restore the immune cell infiltration in tumour, and the combination with pembrolizumab could inhibit tumour growth again, lifting the limitation of drug resistance. Therefore, the combination of COX2 inhibitors and anti-PD-1 mAbs may become a new choice for the drug resistance of anti-PD-1 therapy in the future.


Appropriate fluid management is essential in the treatment of critically ill trauma patients. Both insufficient and excessive fluid volume can be associated with worse outcomes. Intensive fluid resuscitation is a crucial element of early resuscitation in trauma; however, excessive fluid infusion may lead to fluid accumulation and consequent complications such as pulmonary edema, cardiac failure, impaired bowel function, and delayed wound healing. The aim of this study was to examine the volumes of fluids infused in critically ill trauma patients during the first hours and days of treatment and their relationship to survival and outcomes.We retrospectively screened records of all consecutive patients admitted to the intensive care unit (ICU) from the beginning of 2019 to the end of 2020. All adults who were admitted to ICU after trauma and were hospitalized for a minimum of 2 days were included in the study. We used multivariate regression analysis models to assess a relationship between volume of infused fluid or fluid balance, age, ISS or APACHE II score, and mortality. We also compared volumes of fluids in survivors and non-survivors including additional analyses in subgroups depending on disease severity , blood loss, and age.A total of 52 patients met the inclusion criteria for the study. The volume of infused fluids and fluid balance were positively correlated with mortality, complication rate, time on mechanical ventilation, length of stay in the ICU, INR, and APTT. Fluid volumes were significantly higher in non-survivors than in survivors at the end of the second day of ICU stay (2.77 vs. 2.14 ml/kg/h) and non-survivors had a highly positive fluid balance (6.21 compared with 2.48 L in survivors).In critically ill trauma patients, worse outcomes were associated with higher volumes of infusion fluids and a more positive fluid balance. Although fluid resuscitation is lifesaving, especially in the first hours after trauma, fluid infusion should be limited to a necessary minimum to avoid fluid overload and its negative consequences. Appropriate fluid management is essential in the treatment of critically ill trauma patients. Recent studies have highlighted that both insufficient and excessive volumes are associated with increased mortality –3. FluidMany study authors recently suggested that restrictive fluid therapy (RFT) may be better for patients –12. TherAfter obtaining a consent of the Jagiellonian University Ethics Committee, we screened records of all consecutive patients admitted to intensive care unit (ICU) from the beginning of 2019 to the end of 2020. Adults who were admitted to ICU after trauma were included in the study. The exclusion criterion was death before the end of day 2 after admission to the ICU. We retrospectively extracted clinical and preclinical data from electronic and traditional (paper) records of the patients. Data included pre-hospital medical records, emergency department (ED), operating theater (OT), and ICU records. All data was collected and analyzed anonymously.We extracted the following patients’ data: age, weight, Injury Severity Score (ISS), APACHE II score, Glasgow Coma Scale (GCS), Revised Trauma Score (RTS) volume of fluids infused, volume of blood products transfused, fluid loss, blood loss, fluid balance, mortality, ventilator free days, total number of complications, time until ready for discharge from ICU, time of continuous renal replacement therapy (CRRT), lactate level, hematocrit level, platelet count, activated partial thromboplastin time (APTT), international normalized ratio (INR), pH, base excess. ISS, APACHE II, and RTS scores were obtained with the use of online calculators , 19. TheThe study was carried out according to the STROBE  guidelines .t-test, Mann–Whitney and chi-square test was used to compare groups of survivors and non-survivors. The calculation of the minimum sample size was based on patients’ data of the patients in the study by van Mourik et al.  or fluid balance (liters), age, ISS or APACHE II score, and mortality. The results of the logistic regression model were presented as odd ratios (ORs) and 95% confidence intervals (CI). The Poisson regression model was used to assess the relationships between cumulative fluid volume or fluid balance and ventilator free days, total number of complications, time until ready for discharge from ICU, time of CRRT, volume of blood products transfused, APTT and INR. The results of Poisson regression were presented as incidence rate ratio (IRR) and 95% CI. Spearman correlation was used asses the relationships between numerical variables. For the calculation purposed in the regression and correlation analysis, the time until ready for discharge was established at a value of 28 both for patients who were hospitalized in the ICU for more than 28 days and for patients who died before day 28, to reflect the real situation, as these patients have never left the ICU. The Kolmogorov-Smirnov test was used to verify the normality of the data distribution. The student’s k et al. , where tA total of 1,628 patients were admitted to ICU from the beginning of 2019 until the end of 2020. Of this group, 61 patients were admitted after severe trauma. Nine patients died before the end of the second day of stay in the ICU and 52 patients were included in the study. Patient flow diagram is presented in In a multivariable logistic regression analysis volume of the infused fluids and the fluid balance were positively associated with mortality  vs. 2.14 (0.61) ml/kg/h and had significantly more positive fluid balance (6.21 vs. 2.48 L). Analyses in subgroups depending on the severity of the disease , blood loss, and age also showed significant differences in majority of subgroups .In our study, we demonstrated an association between fluid volumes received by critically ill trauma patients during the first hours and days of treatment and treatment outcomes, including mortality, mechanical ventilation, and length of ICU stay. Additionally, non-survivors received much more fluid than survivors and had substantially higher positive fluid balance. Intensive fluid resuscitation is essential for patients in hemorrhagic shock after trauma, however, it can lead to fluid overload, which in consequence, can worsen the condition and lead to serious complications, including pulmonary edema, cardiac failure, delayed wound healing, tissue breakdown and impaired bowel function. Endothelial glycocalyx shading and increased vascular permeability caused by hemorrhagic shock and systemic inflammatory response syndrome (SIRS) associated with trauma can be further aggravated by aggressive fluid resuscitation . Thus, fOur findings are in correspondence with other studies. For non-emergency surgeries, many authors showed that liberal (excessive) fluid therapy in perioperative setting decreases survival of patients , 35, 36.Our study refers not only to pre-hospital or perioperative periods, but also to the entire time from trauma to the end of the second day of stay in the ICU. We analyzed data until the end of the second day of stay in the ICU, since this is the time of resuscitation, stabilization, and optimization phase according to the ROSE concept of fluid therapy. On day 3 some patients already reach the de-escalation phase, which requires different fluid approaches . In ICU In our study, we observed that patients with higher cumulative fluid volume or more positive fluid balance had lower number of days without mechanical ventilation and patients with higher fluid balance had additionally higher total number of complications and longer time until ready for discharge from ICU. This fact was already noticed in other studies in ICU setting , but notOur study has many limitations, the most important of which are a retrospective design and a relatively small number of patients; therefore, some correlations could not have been detected and the results might be subject to bias. Although we included possible confounders in our analysis, we can only be sure of an association between worse outcomes and higher volumes of fluids infused; however, we cannot be sure whether excessive fluid administration actually deteriorated the treatment results. Additionally, we did not address the issue of the type of fluid used and, yet, differences may exist between colloids and crystalloids.In our study, we demonstrated that in critically ill trauma patients, worse outcomes were associated with higher volumes of infusion fluids and more positive fluid balance. Even though fluid resuscitation is lifesaving, especially in the first hours after trauma, fluid infusion should be limited to a necessary minimum to avoid fluid overload and its negative consequences. Our findings emphasize the importance of avoiding unnecessary fluid overload in critically ill trauma patients; however, larger, prospective, randomized clinical trials are needed to address this problem. A well-structured and individualized approach is needed to guide physicians in fluid therapy in this clinically challenging situation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Jagiellonian University Ethics Committee. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.AW, JG, TD, WS, and JD collected the patient data and imputed it into the database. AW, JG, TD, MP, JWoro, JWord, and RZ analyzed and interpreted the patient data. MP, TD, and AW performed the statistical analyzes. AW, TD, JG, JWoro, JWord, and RZ were the main contributors. All authors read and approved the final manuscript.


Fluid challenges are widely adopted in critically ill patients to reverse haemodynamic instability. We reviewed the literature to appraise fluid challenge characteristics in intensive care unit (ICU) patients receiving haemodynamic monitoring and considered two decades: 2000–2010 and 2011–2021.We assessed research studies and collected data regarding study setting, patient population, fluid challenge characteristics, and monitoring. MEDLINE, Embase, and Cochrane search engines were used. A fluid challenge was defined as an infusion of a definite quantity of fluid (expressed as a volume in mL or ml/kg) in a fixed time (expressed in minutes), whose outcome was defined as a change in predefined haemodynamic variables above a predetermined threshold.We included 124 studies, 32 (25.8%) published in 2000–2010 and 92 (74.2%) in 2011–2021, overall enrolling 6,086 patients, who presented sepsis/septic shock in 50.6% of cases. The fluid challenge usually consisted of 500 mL (76.6%) of crystalloids (56.6%) infused with a rate of 25 mL/min. Fluid responsiveness was usually defined by a cardiac output/index (CO/CI) increase ≥ 15% (70.9%). The infusion time was quicker (15 min vs 30 min), and crystalloids were more frequent in the 2011–2021 compared to the 2000–2010 period.In the literature, fluid challenges are usually performed by infusing 500 mL of crystalloids bolus in less than 20 min. A positive fluid challenge response, reported in 52% of ICU patients, is generally defined by a CO/CI increase ≥ 15%. Compared to the 2000–2010 decade, in 2011–2021 the infusion time of the fluid challenge was shorter, and crystalloids were more frequently used.The online version contains supplementary material available at 10.1186/s13054-022-04056-3. Fluid administration in the intensive care unit (ICU) is one of the most common and disputed interventions triggered at the bedside by several clinical variables , 2.Fluid therapy aims to increase stroke volume (SV) and cardiac output (CO) to optimise systemic blood flow and tissue perfusion. As with any therapeutic intervention, the final clinical effect elicited may vary because of a complex interplay between the patient's intrinsic conditions and the therapy itself.Fluid responsiveness can occur only if both ventricles work on the ascending, steep part of the Frank–Starling curve, i.e. in cases where CO is preload dependent , 4. PrelSeveral variables defining the characteristics of the fluid challenge have been further investigated in studies adopting continuous haemodynamic monitoring, showing that the amount of fluids given, the rate of administration, and the threshold adopted to define fluid responsiveness impact the outcome of a fluid challenge –12. MoreHowever, neither the nature, mode of administration, and method to assess the effectiveness of the fluid challenge are standardised in current clinical practice, and the definition of fluid challenge responsiveness is also variable among studies –18.Whether or not these findings have modified the modalities of fluid challenge and the definition of fluid responsiveness in published studies is uncertain. To address this issue, we systematically reviewed existing literature from the year 2000. We appraised the characteristics of fluid challenges in critically ill patients  enrolled in research studies receiving continuous haemodynamic monitoring and assessed the relationship between the reported fluid responsiveness and predefined independent variables. Secondarily, we compared data from studies published in 2011–2021 versus those published in 2000–2010.Preferred Reporting Items for Systematic Reviews and Meta-Analysis-Protocols (PRISMA-P) guidelines  in November 2021 (CRD42021284761).We adhered to the A systematic literature search was performed, including the following databases: PUBMED®, EMBASE®, and the Cochrane Controlled Clinical trials register. The following keywords and their related MeSh terms were used: “fluid challenge”, “fluid responsiveness”, “stroke volume variation”, “pulse pressure variation”, “dynamic indices OR indexes”, “passive leg raising”, OR “passive leg raising test”, “functional haemodynamic test OR tests”. Included papers were also examined to identify other studies of interest missed during the primary search.Articles enrolling at least 20 adult critically ill patients, written in English and published from 1st January 2000 to 31st December 2021 in indexed scientific journals, were considered. Editorials, commentaries, letters to the editor, opinion articles, reviews, and meeting abstracts were excluded. Studies enrolling paediatric or obstetric populations were excluded. References of selected papers, review articles, commentaries, and editorials on this topic were also reviewed to identify other studies of interest missed during the primary search. When multiple publications of the same research group/centre described potentially overlapping cohorts, the most recent publications were selected.A fluid challenge was defined as an infusion of a definite quantity of fluid (expressed as a volume in mL or ml/kg) in a fixed time (expressed in minutes), whose outcome was defined as a change in one of the following haemodynamic variables above a predetermined threshold: CO, cardiac index (CI), SV, SV index (SVI), or surrogate of SV, i.e., velocity–time integral (VTI) in the left ventricular outflow tract and aortic blood flow (ABF), as assessed by transthoracic, transoesophageal echocardiography or oesophageal Doppler. We included studies adopting both a specific  and a broad definition  of the fluid used for the fluid challenge. Studies adopting changes in systemic arterial pressure to define fluid responsiveness were excluded. Finally, we considered the predefined clinical reasons and triggers to start fluid challenge infusion.Three couples of examiners independently evaluated titles and abstracts. The articles were then subdivided into three subgroups: “included” and “excluded” (if the two examiners agreed with the selection) or “uncertain” (in case of disagreement). In the case of “uncertain” classification, discrepancies were resolved by further examination performed by one of the three expert authors . We used a standardised electronic spreadsheet  to extract data from all included studies, recording: the study setting , patient characteristics , criteria for haemodynamic instability, fluid challenge characteristics, pre- and post-fluid challenge haemodynamic variables. When necessary, the corresponding authors of the included studies were contacted to obtain missing data related to trial demographics, methods, and outcomes  and, therefore, the statistical unit of observation for all the selected variables was the single study and not the patient. Due to the discrepancy between the overall patients enrolled in the trials over the two considered decaders, the comparisons were not weighted for study size.Fluid challenge was the exposure variable, and clinical and haemodynamic characteristics were considered outcome variables. Descriptive statistics of individual studies used different statistical indicators for central tendency and variability, such as means and standard deviations , whereas absolute and relative frequencies were adopted for qualitative variables. To show one indicator for the quantitative variables, we collected means with standard deviations (SD) or medians and inter-quartile ranges (IQR).Student's t test or Mann–Whitney test in case of parametric or nonparametric distributions, respectively, were used to assess the difference in mean values between responders and non-responders.p values < 0.05 significant.Statistical analyses were conducted using GraphPad PRISM® 8  and STATA®15 . For all comparisons, we considered The electronic search identified 3,963 potentially relevant studies. Figure The general characteristics of the patients are reported in Table p = 0.39), in the percentage of fluid responders , in the percentage of studies adopting crystalloids over colloids  used transpulmonary thermodilution/dye dilution calibrated haemodynamic monitoring; 22 (17.7%) studies adopted the pulmonary artery catheter monitoring. Echocardiography  was used in 31 (25.0)% of studies, and 5 (4.0%) used oesophageal doppler monitoring. Uncalibrated pulse wave analysis monitoring was used in the other 14 (11.2)% studies  was used in 68 (62.4)% of studies. Oliguria (i.e. a drop in urine output below 0.5 mL/h for 2 or 3 consecutive hours) was used in 54 (49.5)% studies, skin mottling or peripheral hypoperfusion in 47 (43.1)% studies, tachycardia (i.e. an increase in heart rate above 100–110 beats/min) in 43 (39.4)%, the need for initiating the infusion or reducing the dose of vasoactive drugs in 41 (37.6)% studies, an increase in blood lactate in 34 (31.2)% studies, a diagnosis of sepsis/septic shock in 12 (11.0)% studies, and renal or hepatic dysfunction in seven (6.4)% studies. Fifteen studies (12.1%) did not report any trigger to start fluid challenge administration.p = 0.03). Crystalloids were used in 61.9% of studies published between 2011–2021 and 34.3% in the 2000–2010 decade (p = 0.007) . The threshold adopted was an increase in CO or surrogates ≥ 15% in 67.4% of the studies of the 2011–2021 decade and in 81.2% of the studies published in 2000–2010 (p = 0.17)  fluid challenge is usually performed infusing a bolus of 500 mL of fluid, most often a crystalloid, in less than 20 min; 2) the response to fluid challenge is usually defined as a CI or CO increase ≥ 15% as compared to baseline; 3) positive response to fluid challenge is reported in about 50% of ICU patients; 4) the most common trigger for fluid challenge administration is usually the occurrence of hypotension, followed by oliguria and clinical signs of hypoperfusion; 5) the comparison between the 2000–2010 and 2011–2021 decades of publication showed no difference in the percentage of fluid responders (52% on average for both the decades), the volume infused (500 ml), and the criteria defining fluid responsiveness. On the contrary, compared to the 2000–2010 decade, in the period 2011–2021, the fluid challenge infusion time was lower, and crystalloids were more frequently used.Among the included studies, the fluid challenge usually consisted of a median volume of 500 mL administered over a 20-min period and defined as a positive response by an increase ≥ 15% of CO or surrogate. These characteristics and responsiveness definition are to be considered good practice, for the response of CO to a fluid bolus is poorly followed by the simultaneous changes in arterial pressure , 29 or h, increase in CVP) dynamically indicate fluid non-responsiveness are rarely used [Interestingly, 500 mL was also the median volume fluid challenge used in the FENICE study  , whereasely used . Since fely used , smallerp = 0.03), indicating a trend towards the increase in the infusion rate in more recent studies. This global inception cohort study evaluated the clinical use of the fluid challenge in daily practice, whereas our review considered only research papers adopting the fluid challenge as a part of a protocol, limiting the comparison with the results of the FENICE. Moreover, in contrast with a previous metanalysis, including ICU studies up to 2014 [Moreover, the FENICE study reported a median of 24 min of infusion time and a rate of 17 mL/min . Hence,  to 2014 , crystal to 2014 , 14.Limitations of our review have to be considered when extrapolating the results to clinical practice. First, the present study does not report any outcome endpoints. A recent large randomised-controlled trial showed no difference in mortality rate among ICU patients receiving different fluid bolus infusion rates . HoweverAnother potential source of bias is related to the different haemodynamic monitorings used to assess fluid challenge responsiveness. When considering the median cut-off value identifying responders from non-responders, the accuracy of measurement of the changes in CO, or its surrogates, is undoubtedly relevant. Additionally, the reliability of different monitorings in tracking the dynamic trends of CO may not be consistent and may be below the boundaries of accuracy and precision of the Critchley–Critchley criteria . Hence, We excluded studies in which the fluid challenge response has been assessed without haemodynamic monitoring and, hence assessing changes in systemic arterial pressures, potentially limiting the whole comparability of the technique in the two considered decades. Finally, the overall number of patients enrolled in the trials of the two considered decades was considerably different. This could bias the comparisons between the two groups if weighted for study size.This systematic review, including research studies on fluid challenge use in critically ill adult patients receiving haemodynamic monitoring, showed a positive response in 52% of the patients. This test was usually performed infusing a bolus of 500 mL fluid, more often a crystalloid, in less than 20 min, and fluid responsiveness was generally indicated as a CI or CO increase ≥ 15% compared to baseline. Fluid challenge administration is usually triggered by hypotension. In the 2011–2021, the infusion time was shorter, and crystalloids were more frequently used than in the 2000–2010 decade.Additional file 1. Table S1. PRISMA-DTA checklist. Table S2. Extracted data in each study assessed for eligibility. Table S3. Full-text articles excluded, not fitting eligibility criteria. Table S4. Studies on functional haemodynamic tests or dynamic indexes of fluid responsiveness. Figure S1. Characteristics of fluid challenge administration and monitoring along the considered years.


High-Grade Serous Ovarian Carcinoma (HGSOC) is the most prevalent and lethal subtype of ovarian cancer, but has a paucity of clinically-actionable biomarkers due to high degrees of multi-level heterogeneity. Radiogenomics markers have the potential to improve prediction of patient outcome and treatment response, but require accurate multimodal spatial registration between radiological imaging and histopathological tissue samples. Previously published co-registration work has not taken into account the anatomical, biological and clinical diversity of ovarian tumours.In this work, we developed a research pathway and an automated computational pipeline to produce lesion-specific three-dimensional (3D) printed moulds based on preoperative cross-sectional CT or MRI of pelvic lesions. Moulds were designed to allow tumour slicing in the anatomical axial plane to facilitate detailed spatial correlation of imaging and tissue-derived data. Code and design adaptations were made following each pilot case through an iterative refinement process.3) and compositions (cystic and solid proportions). The pilot cases informed innovations to improve specimen and subsequent slice orientation, through the use of 3D-printed tumour replicas and incorporation of a slice orientation slit in the mould design, respectively. The overall research pathway was compatible with implementation within the clinically determined timeframe and treatment pathway for each case, involving multidisciplinary clinical professionals from Radiology, Surgery, Oncology and Histopathology Departments.Five patients with confirmed or suspected HGSOC who underwent debulking surgery between April and December 2021 were included in this prospective study. Tumour moulds were designed and 3D-printed for seven pelvic lesions, covering a range of tumour volumes (7 to 133 cmWe developed and refined a computational pipeline that can model lesion-specific 3D-printed moulds from preoperative imaging for a variety of pelvic tumours. This framework can be used to guide comprehensive multi-sampling of tumour resection specimens. While hBiomarkers that integrate routinely collected radiological data with molecular features may improve prediction of patient outcome and treatment response , 4. Howewithin radiomic habitats  or magnetic resonance imaging (MRI) to inform lesion-specific mould printing prior to planned surgical resection. Our method is specifically aimed to allow for detailed spatial correspondence between imaging and tissue-derived data. Multidisciplinary work of this kind is inherently operationally complex – to maximise the reproducibility of our work, we provide a detailed overview of case selection and evaluation, computational modelling, tissue processing and critical considerations around the research pathway development.22.1Five patients with confirmed or suspected HGSOC undergoing debulking surgery between April and December 2021 at Addenbrooke’s Hospital  were enrolled in this prospective pilot study. Inclusion required informed written consent to the CTCR-OV04 observational study .Representative cases of the two major treatment pathways in HGSOC , i.e. im2.2Preoperative CT and MRI scans were anonymised prior to downloading from the hospital PACS system. This included removal of all directly identifiable information from the images themselves, and deletion of all directly identifiable information from the DICOM headers in the accompanying image metadata. De-identified images were then downloaded in DICOM format from the hospital PACS system and separated into different series using OsiriX DICOM Viewer .via its plugin to XNAT, hosted at the local node of the repository established by the CRUK National Cancer Imaging Translational Accelerator   using Microsoft Advanced Medical Image Labeler  or the Open Health Imaging Foundation viewer  .org.uk) . Challen.org.uk) Figure 12.3https://github.com/mariadeor/3d-moulds-for-ovarian-cancer.A computational pipeline was implemented to generate a mould to specifically hold and slice the segmented lesion in the axial plane, using the input DICOM images and the DICOM-RT file containing the segmented ROIs. A series of tunable values  allows the user to customise the final design to case-specific requirements. The pipeline runs on Python with an interface to OpenSCAD for building the mould structures. All code is available on 2.3.1The first step of the pipeline re-slices and interpolates the input images to a standardised isotropic voxel size of 1×1×1 mm using zero-order spline interpolation to homogenise the process independently of the reconstructed slice thickness of the scans. From there, the tumour segmentation is orientated such that it complies with (a) the imposed base of the mould location and (b) the slicing direction, in the anatomical axial plane Figure 12.3.2Once the tumour segmentation is appropriately oriented in the WCS, the 3D tumour volume is reconstructed from the 2D segmentations. To minimise the layered appearance of the stack of 2D segmentations and increase the resemblance to the actual specimen, two algorithms are sequentially applied. First, the 3D surface mesh of the tumour volume is extracted using the Lorensen and Cline marching cubes algorithm . Next, t2.3.3The mould is designed to aid tumour slicing in the axial plane, which is the standard view for radiological assessment of the abdomen and pelvis. The final mould is an ensemble of three structures: (i) the mould cavity, (ii) the slicing guide and (iii) the orientation guide Figure 3First, the cavity of the mould is carved around the convex hull of the tumour replica Figure 2Next, the slicing guide is modelled: it covers the whole length of the tumour on the x axis  and it has the number of slicing slits resultant from dividing the tumour length by the slice width (tunable). The central slicing slit is aligned with the centre of the modelled tumour, and further slicing slits are placed at set intervals on either side of this. The slicing slits have tunable width and are projected to the mould cavity in order to guide the tissue knife all the way through the tumour upon slicing. In parallel to this process, the slicing slits positions are used to extract the 2D outlines of each tissue slice based on the segmentation. These can be printed in real size, have overlaid the base of the mould and the position of the “orientation incision” –explained below– and are fundamental for allowing the co-registration between the tissue and the imaging slices .The last structures to be built are the two orientation guides, which are single-slit guide walls located perpendicularly to the x axis at each end of the tumour. They have the purpose of determining the course of the “orientation incision” across the top part of the tumour, to ensure the correct orientation of the slices when removed from the mould. This is key for the registration of histopathological samples with imaging .Finally, the structures are assembled onto a common baseplate. In order not to increase the printing time and minimise the waste of printing material, the baseplate is not a solid block but a tunable offset to the mould cavity and the walls attached.2.4All moulds and tumour replicas were 3D-printed using the Prusa i3 MMU2S MK3S  printer loaded with PLA filament. Preceding 3D model slicing and print preparation was done with PrusaSlicer software , setting the infill value to 20% and the layer height to 0.3 mm.2.5Upon resection by a Specialist Gynae-Oncology Surgeon , tissue specimens were immediately checked visually against their respective tumour replicas and placed within their respective moulds in the operating theatre. As an additional layer of confirmation of correct orientation, orientation sutures were placed by the surgeon prior to resection for the third and subsequent cases. Following confirmation of stability, specimen-bearing moulds were placed in designated clinical specimen containers and transferred to the Cambridge Human Research Tissue Bank (HRTB) following routine tissue transfer procedures.At the HRTB, all specimens were reviewed and processed by a Consultant Histopathologist specialising in gynaecological cancers (MJ-L) according to standard procedures. Tumours were sliced within their corresponding moulds. Frozen sections were taken for cellularity assessment or to evaluate or confirm likely diagnosis in suspected or confirmed HGSOC, respectively. Benign pathology precluded further tissue processing for research purposes. Malignant tissue sampling for research use was restricted to tissue regions not required for diagnostic purposes in the patient’s clinical care.33.1Five patients were included in this pilot study Table 1.3.2For two cases undergoing neoadjuvant chemotherapy (NACT) following histopathological confirmation of HGSOC (Cases 1 and 5), routine NACT response assessment images were used for segmenting tumour regions corresponding to expected specimens from DPS. For the remaining three cases undergoing IPS, baseline diagnostic imaging was used.Tumours were delineated using CT for the majority of cases . For one patient  who underwent CT for NACT response assessment, as well as additional MRI prior to surgery, the MRI was used as it was acquired closer to the time of surgery  Figure 4Segmentations were performed between two and six days before surgery (median five days prior). Segmentation times varied between cases depending on complexity, taking up to one hour for particularly complex cases.3.33) and compositions (cystic and solid proportions)  Table 2.The computational pipeline for mould design took under two minutes to run in each case. 3D-printing took a median of 4 hours and 41 minutes (range 2-10 h) for the lesion-specific moulds and 2 hours and 32 minutes for the tumour replicas (32 min-7 h).3.3.1The main aims for the first case were: (a) to build a local research pathway that can be implemented without altering existing standard of care pathways (see Section 3.4); (b) to determine which anatomical structures to include in the mould design; and (c) to test if the meshing and smoothing of the image segmentations produced moulds and tumour replicas that matched the actual size and shape of the resected specimen.A post-NACT HGSOC case with a sufficiently large lesion to span at least three specimen slicing positions (with a slice width of 10 mm) was selected. Specimen collapse following tissue slicing was expected due to the cystic nature of the lesion of interest, and sufficient tissue for sampling was not anticipated based on imaging features. Instead, overall mould design alone (rather than sampling) was the focus of this first pilot case.A lesion-specific (left ovary only) mould was 3D-printed and used . Despite the fact that manual rotation on the axial plane was successful, the subsequent transformation to the WCS was unsuccessful, implying that the slicing axis was not aligned to the axial plane. This highlighted the importance of ensuring accurate transformation to the WCS as per enabling specimen slicing along the desired anatomical plane.3.3.2To test specimen slicing and slice orientation, a suspected malignant right ovarian tumour for IPS resection was selected on the basis of predominantly solid tumour composition. While frozen section assessment and subsequent review of diagnostic formalin-fixed paraffin-embedded (FFPE) blocks confirmed benign tumour pathology and thereby precluded tissue analysis for research purposes, the case provided key learning to improve the mould creation process.We successfully incorporated our learning from Case 1, including: specimen orientation using the tumour replica, which closely resembled the resection specimen , similarly to the case above, the slice overspill issue recurred. This demonstrated that the mould height would need to be increased perpendicularly upwards (rather than following the narrowing tumour contour) from the widest xy dimension of the tumour Figure 7Secondly, the loss of slice orientation in relation to each other on removal from the mould was addressed by placing a partial-depth perpendicular incision across the tissue slices while remaining in the mould – we refer to this as the “orientation incision” Figure 3Thirdly, accurate rotation and transformation to the WCS to orient the mould in relation to the intended slice alignment  was once again hampered by the lack of anatomical landmarks or distinctive tumour shape features. An extra ROI drawn during segmentation, marking the optimal base location, was planned for the following case to automate accurate tumour rotation and transformation to WCS.Additionally, this case presented an opportunity to test pathway compatibility with bilateral disease, with doubled modelling and 3D-printing time. The two moulds and replicas were completed in time for the planned surgery, and tumour replicas closely resembled the corresponding resection specimens as with previous cases . From a technical perspective, the transition from CT to MRI was seamless and did not require any alterations to the pipeline, as the inputs from both modalities are a DICOM set of images and a DICOM-RT file containing the segmented ROIs.Both lesions were of small size and approximately ovoidal in shape, with their longest axis along the craniocaudal axis (both below 4 cm). This meant that only a limited number of slices with a high thickness-to-diameter ratio would result from using the original slice thickness of 10mm, which would be unideal for multisampling within tissue slices. To enable the option of increasing tissue sampling resolution, the impact of reducing the intervals between slits was explored in preparation for Case 5. A test mould of the previous case was re-modelled and 3D-printed with reduced slice thickness from 10 mm to 5 mm, which confirmed that mould sturdiness was retained.The two moulds and corresponding tumour replicas for this case were successfully printed in time for the resection procedure  coordinated case selection and all communication with clinical teams as a single point of contact, disseminating required case information and confirming availability for participation by each team. Case confirmation triggered the research pathway which interfaces with the clinical care pathway across two phases: from the point of specimen retrieval intraoperatively through to tissue sampling in Histopathology, both coordinated by the same clinical fellow for consistency. The surgical and histopathology teams were briefed at their convenience on the day of surgery with no additional workload or delay.To achieve this, we developed a research pathway that fits entirely The interval between case confirmation and surgery ranged between 3 and 16 days, and the shortest window for segmentation, modelling and 3D-printing was two and a half days. We demonstrated that all elements of the pathway required for producing moulds and tumour replicas in time for surgery could be completed for every clinical timeline encountered in this pilot study  of the pilot study, we could not establish a meaningful volumetric tolerance to quantitatively study the suitability of a lesion for our mould design. However, other lesion characteristics beyond volume are crucial for the mould design. For instance, the length of the tumour in the axial plane (slicing direction) highlights the importance of the presence of tunable parameters including slice thickness, in order to allow the granularity of tissue sampling to be adjusted on a case-by-case basis and ensure that a sufficient number of slices can be acquired for meaningful tissue multisampling. We have shown that the structural integrity of moulds was retained when halving slice thickness from 10 mm to 5 mm while increasing knife slits from 0.5 mm to 1 mm. This was of particular importance in Case 5, in which the post-NACT tumour was found to have negligible tumour cellularity below the threshold required for fresh tissue-based genomic analysis. By reducing the slice thickness, all slices could be placed in standard tissue cassettes to be made into FFPE blocks, which can instead be sectioned and subjected to other analyses such as computational pathology approaches.From a clinical perspective, the main challenge in implementing 3D mould-based sampling of resection specimens in HGSOC is that an increasing proportion of patients are treated with NACT followed by DPS . While tAccurate image-tissue co-registration relies on two further technical properties of mould design, namely stability and orientation of tissue slices while within the mould. Firstly, as shown by Cases 2 and 3, the accuracy of specimen slicing is dependent on the stability and support afforded by its mould, which requires both the robustness of the mould itself, as well as a sufficient mould height to provide lateral support. To avoid specimen overspill, we therefore do not constrain mould height to the point at which the widest xy dimension of the tumour occurs , but ratSecondly, without a method to mark tumour slices within the mould, their orientation to each other and to the radiological imaging is immediately lost upon removal from the mould, particularly for ovoid tissues lacking obvious shape or substructure characteristics Figure 9In practice, a research pathway of this kind must be highly adaptable to the requirements of a patient’s individual treatment pathway and tumour characteristics, and be implemented efficiently with minimal to no impact on resources and professional workloads within the wider healthcare setting. While previous work has used multiparametric MRI and PET/CT , 29, we Our approach is based on clinical standard-of-care imaging and did not include any dedicated imaging sessions for optimising mould design. This was the likely reason for the mismatch between the segmented tumour and actual specimen volume in Case 4, who had the longest elapsed time between imaging and surgery. Of note, in most routine clinical settings, cross-sectional imaging is undertaken in prior preparation for but not on the day of surgery, and the cost of further imaging or risk of radiation exposure is not justified without unique clinical indications. An additional scan, ideally an MRI including T2-weighted images, close to the day of surgery could easily solve this problem in cases with a long delay between the last preoperative standard-of-care scan and surgery. However, we found that all other segmentations generated tumour replicas which closely resembled the resected specimen, including that of a post-NACT case whose imaging was performed 40 days prior to surgery.A second limitation is that these pilot cases did not yield HGSOC tissue for multi-site genomic profiling. This highlights the inherent challenges of case selection in the context of standard treatment pathways for advanced pelvic ovarian tumours. For cases undergoing IPS, tumours are likely to be of greater volume and cellularity for sampling, but histological diagnosis is often lacking preoperatively. An IPS case may be benign despite significantly elevated CA-125 and suspicious qualitative radiological features (as we found with Cases 2 and 3), however a significant proportion of tumour will be required for diagnostic purposes and may leave a small proportion for research purposes even if malignancy is confirmed. DPS cases are more likely to have limited remaining tumour tissue for sampling (Case 5), or have greater cystic components with or without sufficient tissue for sampling as a feature of NACT response (Case 1). With regard to tumours containing cystic components, we have shown that moulds can be modelled for a discrete solid component, and this can be extended to multiple distinct areas of solid disease. The majority of cases for which tumours are predominantly cystic with minimal or no residual tumour tissue after NACT will be identifiable at the preoperative imaging stage. The manual base placement step allows the base to be placed by the surface with the largest proportion of solid components to maximise stability – however specimen stability would remain challenging in very complex masses containing a large number of discrete cystic and solid areas. Future case selection will be informed by these clinical factors, and may benefit from focusing on IPS cases with prior tissue diagnosis from laparoscopic biopsy or DPS cases with predominantly solid residual disease post-NACT.This work was motivated by the need to guide comprehensive multi-sampling of pelvic tumour resected specimens for correlation of imaging and genomic features. A key strength of our work is that the pipeline facilitates direct co-registration of tissue coordinates and imaging slices on the same transverse axis by constraining tissue slicing to the axial plane. Our pipeline is thereby highly suited to implementing systematic grid-based sampling, by aligning a grid-based system with the orientation cut of each tissue slice in order to directly map sample coordinates onto spatial coordinates of the corresponding image slice. Such a coordinate-based system will be well-suited to larger-scale prospective biological validation of previously described radiomic ‘habitats’ .5We developed a computational pipeline for modelling lesion-specific 3D-printed moulds to guide slicing and multi-sampling of solid pelvic tumour resection specimens. This work provides a framework for obtaining spatially co-registered imaging and multi-sampled tissue data, thus aiming to perform detailed multi-level characterisation of intratumoural heterogeneity of pelvic gynaecological tumours. We highlight specific challenges pertaining to ovarian tumours, propose a highly tunable design that is adaptable to the specific requirements of a given case, and provide recommendations for pathway implementation. This pipeline can be implemented alongside clinical treatment pathways for patients with newly diagnosed HGSOC and other ovarian tumours.es220@medschl.cam.ac.uk.The datasets presented in this article are not readily available because of ethical restrictions due to the presence of patient data. Requests to access the datasets should be directed to Prof Evis Sala, The studies involving human participants were reviewed and approved by Cambridgeshire and Hertfordshire Research Ethics Committee approval reference 08/H0306/61. The patients/participants provided their written informed consent to participate in this study.Conceptualisation: MR, MD-O, CM, VB, RW, LR, MJ-L, MC-O, JB, LES and ES. Methodology: MR, MD-O, CM, VB, RW, LR, MJ-L, MC-O, JB, LES and ES. Software: MD-O , AG, MG and MC-O (previous developments). Validation: MD-O, MR, CM, VB, RW, LR, MJ-L, MC-O, LES and ES. Investigation: MR, MD-O, CM, VB, LES, RW, MJ-L, JB and ES. Resources: HB, KH, PP, MJ-L, JB and ES. Data curation: MD-O, MR and LES. Visualisation: MR, MD-O and CM. Writing—original draft: MD-O and MR. Writing—review and editing: MR, MD-O, LES, CM, VB, RW, LR, AG, MJ-L, MG, SU, HB, KH, PP, MC-O, JDB and ES. Supervision: ES, LES, MC-O, JB and RW. Project administration: MR, JB and ES. Funding acquisition: JB and ES. All authors contributed to the article and approved the submitted version.


Photoacoustic imaging (PAI), an emerging biomedical imaging technology, capitalizes on a wide range of endogenous chromophores and exogenous contrast agents to offer detailed information related to the functional and molecular content of diseased biological tissues. Compared with traditional imaging technologies, PAI offers outstanding advantages, such as a higher spatial resolution, deeper penetrability in biological tissues, and improved imaging contrast. Based on nanomaterials and small molecular organic dyes, a huge number of contrast agents have recently been developed as PAI probes for disease diagnosis and treatment. Herein, we report the recent advances in the development of nanomaterials and organic dye-based PAI probes. The current challenges in the field and future research directions for the designing and fabrication of PAI probes are proposed. The advancement of non-invasive imaging technologies, including ultrasound imaging (UI), computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), Raman imaging (RI) and fluorescence imaging (FI), is contributing significantly in biomedical research and clinical investigation . Among tThe first photoacoustic (PA) effect was described by Alexander Graham Bell in 1880 . In follIt is unavoidable that with the increasing depth of biological tissues, the light intensity and PA signal-to-noise ratio show a dramatic attenuation because of the light scattering effect. One of the efficient ways to overcome this problem is to use high performance exogenous chromophores to enhance the imaging contrast ,12. As sFunctional nanomaterials with NIR light absorption and thermal expansion properties have been widely used as the probes for PAI in biological and medical research. To date, several nanomaterials, including gold nanomaterials, metal  based nanomaterials, carbon and its combined metallic nanomaterials, and semiconducting polymer nanoparticles, have been developed as the PAI exogenous contrast agents for biomedical research and (pre)clinical diagnosis and treatment, which are summarized in this section.Gold nanomaterials, including gold nanoparticles (GNPs), gold nanorods (GNRs), gold nanocages (GNCs), gold nanostars (GNSts), gold nanoflowers (GNFs) and gold nanodisks (GNDs) , have be111In) labelled GNRs (111In-Tra-GNRs) . Su. Su- detrageenan . The ima2O2), superoxide anion (O2•−), and hypochlorous acid (HOCl), are normal by-products of numerous cellular processes, are often associated with cancer cell homeostasis [ROS, such as hydrogen peroxide  mouse models, a strong PA signal at 770 nm was observed because of the high level of ClO−. Meanwhile, the injection of lipoic acid to regenerate GSH in ALI models attributed to the recovery of DiOH-BDP and led to the decrease in PA signal at 770 nm. As a result, PA signals at 770 nm can be explored for detecting ClO−/GSH by using DiOH-BDP in vitro and vivo [Rao’s group has also reported several polymer-based nanoprobes for PA detection and imaging ,85,86,87and vivo .2OH to generate PA signals after reacting with GSH in vivo. In another work, Li et al. developed an activatable hydrophilic NCTy probe for Cys detection in vivo [The PA signal of “off-on” probes is absent at normal tissues and it will be activated and generate signals when interacting with targets. Companion diagnostics (CDx) is a new emerging technology to detect biomarkers that are highly related to activating the drug to prognosticate whether this specific drug is able to benefit a patient . Such in in vivo . The maiA unique donor-acceptor-donor (D-A-D) structure-based “off-on” PA probe (PS-NO) for nitric oxide (NO) detection was developed by Wang et al. through coupling benzothiadiazole as the chromophore and diphenylamine as the NO recognition unit . Comparep < 0.001) from that at the normal site. This result coincided with data from the PH-sensitivity study in vitro, suggesting the good ability of CTN as a NIR II-responsive pH-sensitive nanoprobe for tumour PAI. After one year, Wang and co-workers optimised CTN nanoprobe and named stimuli-activatable nanotheranostics (SHT) for tumour detection [2O2-activated/acid-facilitated PA signal changes in both vitro and vivo.Charge-transfer complex (CTC) materials contain charge-donor and charge-acceptor that possess excellent properties, including anisotropic conductivity and photoconductivity. Wang et al. fabricated a NIR II-responsive and pH-sensitive self-assembled charge-transfer (CTN) nanoprobe for PAI guided therapy . Four hoetection . SHT demPorphyrin is a conjugated aromatic macrocycle that has high extinction coefficients and biocompatibility, but the application of porphyrin in molecular imaging is limited due to the low absorption (below 700 nm) and insufficient photostability. In 2019, Wu et al. synthesized a new porphyrin derivative with the absorption in NIR region, porphyrin-diketopyrrolopyrrole (Por-DPP) as the small molecule for the preparation of organic nanoparticles theranostic applications . In aque“Always on” PA probes are usually applied to the detection of biomarkers that have different concentrations in diseased and normal sites. The PA intensities of those probes keep “on” regardless of whether they succeed in interacting with the molecular of intertest or not . The aza-) and hypochlorous acid (HOCl). The cleavage of IR755′s C=C bond led to the bleach of the PA820, while the PA700 (derived from SP1 polymer nanoparticle) was retained, allowing ratiometric PAI of ROS in mice.The structure transformation without changing the major conjugated framework is the main feature of ratiometric PA probes. After interacting with target analytes, those probes possess a high sensitivity and show significant alteration of PA intensity . In 2014+ in vivo [+ to convert into hCy7′ for switching on PA signals and LP contributed to the cellular internalization process of liposoluble MeHg+. The PA signal intensities from the control zebrafish group (with LP-hCy7 and without MeHg+) were strong at 690 nm and weak at 860 nm. Nevertheless, mice injected with MeHg+ experienced a remarkable decrease and increase in PA signals at 690 nm and 860 nm, respectively suggesting that LP-hCy7 can be applied to detect MeHg+ in vivo through recording the ratiometric PA signal (PA860/PA690)  that had two key elements, liposome (LP) and cyanine dye (hCy7) for the detection of MeHg in vivo . The mai0/PA690) .2O2 selective NIR PA probe which demonstrated great hydrophilicity and biocompatibility and was highly applicable for PAI in mouse models with the injection of H2O2 [2O2 and hydrophilic OEG segment for enhancing water-solubility. The reaction of H2O2 with boronic acid pinacol ester led to the increase in PA825, while the decrease in PA720, resulting in ratiometric PAI of H2O2 in vitro and in A549 xenograft tumor of nude mice.In 2018, Lu et al. reported an H of H2O2 . The proIn light of the great potential of PAI in biomedical imaging, a number of PAI probes have been developed in recent years. These probes mainly include nanomaterials and small organic dyes and corresponding small molecular dye-based nanoparticles. Among these agents, the small organic dyes exhibited excellent proprieties, such as good excretion ability and easily conjugating with target groups. Importantly, compared with nanomaterials, porphyrins and cyanine PA probes possess better application potential in clinical use due to their low toxicity. The long circulation time, capability in targeting diseases, and high PA signals of nanomaterials also allowed the PA nanoprobes to be used for imaging and the imaging-guided therapy of different diseases . Furthermore, nanoparticles are also able to carry some other anticancer drugs, such as DOX and cisplatin for combination therapy, providing a unique platform for theranostic applications. This review article highlighted the recent advances of PAI probes for theranostic applications. The inorganic nanomaterials, polymer nanoparticles, and small-molecules and corresponding organic nanoparticles were discussed.With the continuous development of PAI, more PA probes should be designed to address increasing biomedical issues by overcoming several unavoidable shortcomings. As of the endogenous contrast agents, some PA probes have high biological and low signal background ratios (S/N) during PAI in vivo . TherefoAlthough PAI has been widely used in clinical diagnosis, the use of exogenous PAI probes remains limited. This is mainly because of the uncertain safety issues of the probes, including nanomaterials and small molecules that were summarized in this work. Therefore, the long-term safety investigations of these probes could be one of the key points for the translation of the exogenous probes in clinical diagnosis. Moreover, although the acoustic waves are highly penetrable in body, the shallow penetration of the incident light hampers the clinical application of PAI in deep tissue (over 2–3 cm deep) . It has Overall, as the development of PA probes and the PAI technique are progressing gradually, we are convinced that more and more exogenous PA probes with excellent sensing and imaging capability would be developed to promoting the PAI technique in the not-too-distance future, and such technology advances would significantly benefit to future diseases early diagnosis, treatment and treatment monitoring.


Protein energy malnutrition is recognized as a leading cause of morbidity and mortality in dialysis patients. Protein–energy-wasting process is observed in about 45% of the dialysis population using common biomarkers worldwide. Although several factors are implicated in protein energy wasting, inflammation and oxidative stress mechanisms play a central role in this pathogenic process. In this in-depth review, we analyzed the implication of sodium and water accumulation, as well as the role of fluid overload and fluid management, as major contributors to protein–energy-wasting process. Fluid overload and fluid depletion mimic a tide up and down phenomenon that contributes to inducing hypercatabolism and stimulates oxidation phosphorylation mechanisms at the cellular level in particular muscles. This endogenous metabolic water production may contribute to hyponatremia. In addition, salt tissue accumulation likely contributes to hypercatabolic state through locally inflammatory and immune-mediated mechanisms but also contributes to the perturbation of hormone receptors . It is time to act more precisely on sodium and fluid imbalance to mitigate both nutritional and cardiovascular risks. Personalized management of sodium and fluid, using available tools including sodium management tool, has the potential to more adequately restore sodium and water homeostasis and to improve nutritional status and outcomes of dialysis patients. Malnutrition is a common disorder in dialysis patients ,2,3. ProProtein–energy malnutrition in dialysis patients is most often characterized by inflammation and oxidative stress mechanisms ,16,17,18Protein energy malnutrition is now a well-recognized leading cause of morbidity and mortality in dialysis patients ,21. In tProtein–energy malnutrition has multiple causes in dialysis patients that may act individually but most likely tend to act in combination either simultaneously or sequentially ,27. For Patient-related factors depend mainly on age, gender, life and diet habits, socio-economic conditions, cultural assets, past medical history (long standing history of disease and treatment), and comorbid conditions . Most of them are fixed factors out of medical reach ,28.Uremic-related factors consist of all disorders associated with kidney failure that impact negatively nutritional status. Uremia is a complex disorder involving multiple factors that act synergistically to impede nutritional status . To faciDialysis-related factors are superimposed conditions to uremia with dual and opposite action. On one hand, hemodialysis has a positive action by controlling uremic disorders and restoring internal milieu homeostasis through periodic reduction of nitrogenous waste products and correction of electrolytic and fluid volume imbalance. On the other hand, hemodialysis has a negative action by creating unphysiological cyclic shifts and by triggering directly protein muscle degradation to compensate for amino acid, peptides, and nutrient losses . DialysiIntercurrent-illness-related factors occur more frequently in dialysis patients as markers of vulnerability and contributing to their morbidity. They should be considered additional stressors and will not be discussed further. For example, they include various episodes of infection, cardiac event, vascular access problem, and surgical or any interventional acts.In this context, sodium and fluid imbalance, as well as their management, are emerging factors that contribute to protein–energy malnutrition. This is the main focus of this review to highlight this overlooked problem in dialysis patients. To facilitate description of the link between fluid disorders and protein energy malnutrition, we split this pathway into two parts, fitting with tides phenomena of intermittent hemodialysis. In brief, the tides up phenomenon reflects fluid and sodium accumulation occurring during the interdialytic period , while the tides down phenomenon reflects fluid and sodium depletion resulting from dialytic treatment (as a result of ultrafiltration and sodium depletion). Interestingly, both conditions are associated with the protein–energy-wasting process.Chronic fluid overload (FO), a surrogate marker of water bound sodium excess, is quite common in hemodialysis patients. According to the method used to assess fluid status in dialysis patients , FO prevalence may differ substantially. Using a non-invasive objective tool, such as multifrequency bioimpedance, to estimate fluid status, it was determined that FO is present in 40 to 60% of dialysis patients. In this context, it is interesting to note that FO prevalence is almost similar in hemodialysis and peritoneal dialysis patients, as shown in recent prospective international studies using the same MF-BIA tool ,45,46,47Tissue sodium accumulation, as a surrogate marker of water free sodium stored in skin and muscle, is another common feature of kidney disease and dialysis patients recently identified as an additional vital risk factor . Tissue Hyponatremia, a well-known marker of poor outcome in hemodialysis patients  has beenClinical evidence linking fluid imbalance in dialysis patients with poor outcomes has been briefly summarized in this paragraph. Interestingly, all pathophysiologic pathways involved in these deleterious mechanisms are mediated by inflammation and associated with the protein–energy-wasting process .While FO, inflammation, and malnutrition are independent risk factors for mortality ,49,50,64Rapid sodium removal and fluid depletion as summarized by high ultrafiltration rate (>10 mL/h/kg) may lead to poor outcomes with increased cardiac risks ,78. ThisTissue sodium accumulation  is associated with various pathophysiologic findings involving on one side the cardiovascular system  independently from pressure level and mechanical consequences but on the other side various metabolic pathways that have direct effects on nutritional status  ,56,84. THyponatremia is difficult to explain in an anuric dialysis patient in which dialysis is the main source of exchange (sodium and water) with external milieu . SeveralIn brief, these tides up and down phenomena, reflecting interdialytic fluid and sodium accumulation and intradialytic fluid and sodium depletion, respectively, are likely involved in the protein–energy malnutrition of hemodialysis patients . During Optimal fluid and sodium management constitute a cornerstone component of dialysis adequacy in reducing cardiovascular burden in this fragile population. This is summarized in the dry weight probing approach ,90. UnfoClinical management is a global process that permits the attainment and maintenance of “dry weight” in dialysis patients . HoweverRestoration of fluid and sodium imbalance relies on three main components: The first component is dietary counseling and educating patients to reduce sodium and fluid intakes with the support of specialized dieticians; the second component is based on the preservation of residual renal function. This aspect is fundamental but unfortunately will concern a limited number of patients over a short period of time; the third component relates to the treatment schedule program and dialysis prescription. For space reasons, only dialysis-related aspects will be discussed in this paragraph. Three items may be initiated to achieve this target: Firstly, weekly treatment time; secondly, ultrafiltration volume and rate; thirdly, dialysate sodium prescription.Weekly dialysis treatment time is the main factor that conditions total ultrafiltered volume and ultrafiltration rate per session. Several studies have documented that an hourly ultrafiltration rate greater than 13 mL/kg was associated with an increased risk of mortality of almost 50% ,100. PreThe prescription of dialysate sodium concentration is another important component in the prescription of dialysis that has been neglected ,103. TheAs a brief summary, it is time to take fluid balance and nutrition seriously, particularly in dialysis patients, with appropriate measures to reduce protein–energy wasting burden in this fragile population .As highlighted in this in-depth narrative review, sodium and water imbalance and fluid management in dialysis patients tend to be strongly associated with the protein–energy-wasting process. Fluid and sodium imbalance in dialysis patients are superimposed factors that tend to precipitate highly malnutritional exposed patients. Inflammation, and its related pathways, is the main source of protein energy wasting. Tissue sodium accumulation and depletion, as well as their metabolic consequences, tend to worsen nutritional conditions. Hyponatremia, a condition associated with poor outcomes, is strongly associated with mixed water and sodium disorders. We hypothesize that hyponatremia reflects intense hypercatabolism, resulting in metabolic water production from cellular oxidation phosphorylation processes. It is time to act more precisely on sodium and fluid imbalance to mitigate both cardiovascular and nutritional risks and to improve patient-reported outcomes. Personalized management of sodium and fluid using available tools, including sodium management tools, has the potential to more adequately restore sodium and water homeostasis and to improve the nutritional status of dialysis patients. In brief, fluid and sodium imbalance must be considered to be strong determinants of the protein–energy-wasting process requiring more precise approaches. This new hypothesis deserves further interventional clinical and nutritional studies to be validated.