This manuscript will review crystalloid  and colloid  fluids that are available for intravenous administration with a focus on their electrolyte, acid-base, colligative, and rheological effects as they relate to each solution's efficacy and safety. The goal is for the reader to better understand the differences between each fluid and the influence on plasma composition, key organ systems, and their implications when used therapeutically in animals with critical illness. Water is the body's universal solvent and the most essential nutrient of the body. Within the vessel, water is the transport medium that brings oxygen, solutes, and hormones to the interstitium and removes waste products for breakdown and excretion. Within the interstitial space, water allows movement of these substances between the capillary and the cell. Within the cell, water provides a medium for organelles that gives the cell its form. Water also functions to dissipate heat through evaporation.Restoring and maintaining fluid balance in the critically ill or injured animal can be one of the most difficult challenges of patient management. Intrinsic factors that dictate the movement of crystalloid fluids into individual body fluid compartments  include the normal distribution of total body water, and the factors involved with the movement of fluids across the capillary membrane , as well as cellular membrane and lymphatic function , 2. FluiA crystalloid solution is an aqueous solution composed of water and small solutes such as electrolytes and glucose , 5. CrysA hypotonic fluid has an effective osmolality significantly lower than that of the patient. Infusion will reduce the osmolality of the extracellular fluid resulting in redistribution of water into the intracellular compartment. Dextrose is often added to hypotonic fluids so that they are isosmotic during intravenous administration to prevent hemolysis. However, as the dextrose moves into cells and is metabolized, the fluid becomes hypotonic and they are expected to redistribute to the intracellular compartment based on total body water distribution, i.e., ~66% of the infused volume of solute-free water will move into the intracellular space.Hypotonic crystalloid solutions are used for sustaining maintenance fluid requirements, treatment of solute-free water deficits, and drug administration. Maintenance-type crystalloid fluids replace ongoing sensible  and insensible  water lost because of basic bodily functions . Solute-Dextrose containing solutions that are otherwise solute-free  can be hypo-, iso- or hyper-osmolar depending on the concentration of dextrose in the fluid. They become hypotonic as the dextrose is metabolized leaving solute-free (pure) water. An isosmotic fluid, 5% dextrose in water (D5W) is a source of solute-free water that can be used to treat a solute-free water deficit or as a diluent for drug administration. Although D5W can be used in the treatment of hypoglycemia, caution is needed to avoid iatrogenic hyponatremia. Hyperosmolar dextrose solutions containing 25–50% dextrose can be administered alone  or added to an isotonic crystalloid solution to provide dextrose supplementation without the risks of hyponatremia.An isotonic crystalloid fluid has an effective osmolality like the patient. The effective osmolality of common isotonic fluids ranges from 270 to 310 mOsm/L. Isotonic fluids have a similar sodium concentration as the extracellular fluid compartment and have minimal impact on intracellular volume. Isotonic crystalloid fluids can vary in their concentration of the electrolytes sodium, chloride, potassium, magnesium, and calcium . They main vivo SID will impact the patient's SID resulting in alteration in the patient's acid-base status. The solution 0.9% sodium chloride is the most unbalanced isotonic crystalloid, given its high, un-physiologic, chloride concentration and its 0 value SID  , 9. ThisA hypertonic fluid is a fluid that has an effective osmolality (tonicity) greater than that of the patient. Infusion will increase the osmolality of the extracellular fluid resulting in redistribution of water out of the intracellular fluid compartment, increasing extracellular volume. Common hypertonic solutions used in veterinary medicine include hypertonic saline (HTS) and mannitol. The volume effects of these solutions are short lived, since the small solutes will be redistributed and/or excreted.Hypertonic saline is available at various concentrations, with 3%, 7.2–7.5%, and 23.4% being commonly available. The 23.4% HTS is not recommended for direct infusion and should be diluted before administration. Hypertonic saline in the 3–7.5% range can be infused without dilution or in combination with isotonic crystalloids and/or colloids for hypovolemic shock resuscitation.Hypertonic saline is also used for customizing crystalloid fluid solutions to target a desired sodium concentration, most commonly for the treatment of patients with dysnatremia. In addition, mannitol and HTS can be prescribed for patients with signs of intracranial hypertension. Hypertonic saline effects include increasing plasma osmolality and sodium and chloride concentration, endogenous release of vasopressin, and immunomodulation –13. The A colloid solution contains large molecular weight particles such as proteins or hydroxyethyl starches (HES) suspended in a crystalloid solution . The larNatural colloids are protein-containing products, such as whole blood, plasma, and concentrated albumin solutions. Although any protein can contribute to COP, albumin , is most relevant because it is the smallest and most numerous of the protein particles, and, the overall negative charge of albumin attracts positive sodium molecules into its orbit, thereby increasing its osmotic capability by ~20% (the Gibbs-Donnan effect) –16. AlbuPlasma can be stored as fresh frozen plasma (FFP), frozen plasma, liquid (refrigerated) plasma, cryosupernatant (cryopoor), or stored whole blood. The concentration of albumin in these products will depend on the albumin concentration of the donor animal and is ~2.5–3 g/dL . Of the Lyophilized canine albumin is stored in a dehydrated powder form and reconstituted with 0.9% sodium chloride or 5% dextrose in water to a desired albumin concentration (5–16%). Infusion of a 16% solution  results in an ~1.2 times volume expansion. It seems well-tolerated, can increase serum albumin concentrations in dogs with septic peritonitis, and can increase albumin and COP in healthy dogs , 20.Human serum albumin (HSA) concentrates are reconstituted to contain 5–25% albumin. The high concentration of albumin and resulting high COP (200 mmHg) in 25% HSA  has the greatest capability of increasing plasma albumin and COP , howeverTo estimate the volume of a transfusion product required to increase serum albumin the following formula can be used .Albumin deficit (g) = 10 [0.3 × body weight (kg) ×  − patient albumin concentration (g/dL))].Synthetic colloids include gelatins, starches, dextrans and complex polysaccharides. Gelatins and dextrans are infrequently used. Hydroxyethyl starch is one of the most common type of synthetic colloid in use in veterinary medicine. It is a synthetic polymer of glucose (98% amylopectin), made from a waxy species of plant starch such as maize, potatoes or sorghum . It is aHydroxyethyl starch contains a polydisperse number of molecules with different MW. The MW impacts the osmotic pressure and the half-life of the product as well as the coagulation effects. Small MW molecules that are below the renal threshold (<70 kDa) will be renally excreted and the intravascular expanding effects are quickly lost. Higher MW molecules have greater impact on coagulation, and this will limit the safe daily dose of the product. Tetrastarches have a medium MW and have been shown to have less coagulation effects in comparison to an equal dose of the high MW hetastarch products , althougTables 1, 2.The molar substitution is the calculated average number of hydroxyethyl groups per glucose residue in the molecule. It can be adjusted by the degree of substitution of hydroxyl groups with hydroxyethyl groups at the C2, C3, and C6 positions on the glucose molecule . The C2:Degree of volume expansion and longevity of HES, or any colloid, in plasma is difficult to predict. It will depend not only on the degree of vascular injury, but also on the MW and dispersity, amylase concentration, elimination rate into the urine, as well as its electrical charge, shape, and effect on the endothelial glycocalyx –37. StudDespite IV fluids being commonly prescribed medications in human and veterinary medicine, the optimal fluid choice remains poorly defined. There is growing evidence in human medicine that fluid choice can have an impact on outcome, particularly in critically ill patients. Specific recommendations for the single best type of crystalloid or colloid fluid to infuse cannot be made based on current information. Patient factors to be considered when selecting an IV fluid type include volume of administration, the patient's electrolyte and acid-base status, and ongoing disease processes. Fluid factors to be considered include tonicity, electrolyte and organic anion concentration, and compatibility with drugs or other fluids that will be co-administered.Maintenance-type fluids were designed to replace solute-free water and electrolyte losses in the otherwise healthy, fasting patient. The composition of maintenance fluids was determined from research performed in healthy children, and the electrolyte composition was determined to resemble the electrolyte composition of milk , 39. TheIt has been advocated that isotonic crystalloids are given to replenish any extracellular fluid deficits and replace ongoing losses while maintenance type (hypotonic) fluids are given for daily maintenance needs. This requires two fluid types which can increase cost, and clinical experience has shown that most patients can be adequately managed with an isotonic crystalloid alone. Maintenance-type fluid therapy alone may be considered in the patient that has adequate intravascular and interstitial fluid volumes without ongoing extracellular volume losses. An example may be the cat that sustained a jaw fracture and is unable to drink water. Maintenance type fluids may also be considered in the patient that is developing hypernatremia, although adequate support of extravascular volume should be monitored. As maintenance type fluids have less effect on extracellular fluid volume than isotonic crystalloids, they have been favored in patients at risk of volume overload, such as animals with heart disease or kidney disease.Careful consideration should be taken when prescribing maintenance type fluids for the treatment of sick patients that have extracellular fluid deficits and/or ongoing abnormal losses. The major adverse events that may be associated with the administration of maintenance type fluids are ineffective intravascular volume support and hyponatremia. Acute, hospital acquired hyponatremia has been associated with the administration of hypotonic, maintenance type fluids in human patients , 42, 43.As When treating animals with an abnormal sodium concentration the general guideline is to avoid changing the sodium concentration rapidly . When adLactated Ringer's solution is an ideal fluid for patients with hyponatremia unless they have a sodium <120 mEq/L. In that situation, if large volumes of fluids are indicated, it may be necessary to further dilute the sodium concentration with water to make a fluid prescription that is isotonic to the patient.Balanced isotonic crystalloid solutions have a chloride concentration similar or slightly less than healthy dogs and cats while 0.9% sodium chloride and HES in 0.9% sodium chloride have a higher chloride concentration than normal dogs and cats. Human medicine has traditionally used 0.9% sodium chloride rather than balanced isotonic crystalloids for fluid therapy, and in more recent years the incidence and potential adverse effects of hyperchloremia has been a focus of many investigations.Chloride has several important physiological roles including regulation of glomerular filtration rate, blood pressure responses, gastrointestinal function, and acid-base homeostasis. The impact of hyperchloremia was demonstrated by a study in a human ICU that evaluated the effect of changing fluid therapy from a chloride-liberal approach (infusing 0.9% sodium chloride or colloids suspended in 0.9% sodium chloride) to a chloride-restricted approach  . The incPotential mechanisms for the harmful effects of administration of chloride rich fluids include altered cytokine responses, increased nitric oxide levels, hemodynamic instability and altered renal blood flow . HyperchHypochloremic metabolic alkalosis is not uncommon and is usually the result of selective gastric loss of hydrochloric acid from abnormalities such as pyloric outflow obstruction. In this case, 0.9% sodium chloride is the ideal fluid therapy as it will increase serum chloride concentration effectively and promote bicarbonate excretion by the kidney to enhance the normalization of acid-base balance.Since 0.9% sodium chloride contains no potassium, it is commonly recommended for use in patients with hyperkalemia. Potassium homeostasis is impacted by extracellular sodium concentration as well as potassium concentration with sodium-potassium exchange having an important influence. The small quantity of potassium in balanced isotonic crystalloids such as LRS or Plasma-Lyte 148 is outweighed by the benefit of the lower sodium concentration when compared to 0.9% sodium chloride. Studies in human patients have shown that the administration of 0.9% sodium chloride in patients prone to hyperkalemia is associated with a similar or higher serum potassium concentration than in patients treated with balanced isotonic crystalloids, and other adverse effects, such as hyperchloremic metabolic acidosis, were common , 57. TheNone of the commercially available isotonic crystalloids contain a substantial quantity of potassium and, as a result, addition of potassium to intravenous fluids may be necessary for patients receiving longer term fluid therapy. Potassium supplementation of IV fluids can be with potassium chloride or potassium acetate additives, either with or without potassium phosphate . PotassiHypomagnesemia occurs commonly in critically ill cats and dogs and magnesium supplementation can be challenging. Plasma-Lyte and Normosol fluids contain supplemental magnesium and may be used in patients with suspected hypomagnesemia, patients considered at risk of hypomagnesemia, and patients documented to have mild hypomagnesemia. A suggested slow replacement dose for magnesium is 0.3–0.5 mEq/kg/day . At main2), which is insufficient to treat a patient with hypocalcemia. It has been commonly recommended to avoid co-administration of LRS with blood products due to concerns that the calcium could be chelated by the citrate anticoagulant in the blood product, leading to loss of citrate activity and clotting of the blood product. Several studies have shown that the calcium concentration in LRS is not associated with increased coagulation of blood products, and LRS is safe to co-administer with blood transfusions . Crystalloid fluids tend to be acidic in nature. This is due the reaction of air with water and the process of heat sterilization. In addition, manufacturers often adjust the pH to a more acidic level for reasons such as improving stability and inhibition of microbial growth. The low pH of IV fluids has led to the suggestion that they can cause or worsen an acidemia. Although the pH of fluids can be low, the total quantity of acid (titratable acidity) is minimal and very readily buffered by the body. It will not significantly alter the acid-base balance of a patient . Plasma-As previously discussed, the administration of chloride rich fluids  can cause hyperchloremia and a metabolic acidosis , 67. TheAn ideal isotonic crystalloid would be one that matches the composition of plasma. Plasma of normal dogs and cats generally has a sodium concentration of ~140–150 mEq/L, a chloride concentration of ~95–110 mEq/L, and a bicarbonate concentration of ~20 mEq/L. A crystalloid with this composition is appealing, however, bicarbonate is difficult to keep stable in solution and needs to be stored in glass. As a result, manufacturers have added organic anions to IV fluids that are stable in solution and will act as “bicarbonate precursors” or buffers. The metabolism of these substances in the body leads to the consumption of hydrogen ions . Commonly used organic anions include lactate, acetate, and gluconate. Gluconate it excreted in the urine and not metabolized does not contribute to the acid base balance of the patient , 72.Metabolism of organic anions such as lactate and acetate can increase plasma bicarbonate concentration in a dose dependent manner and can promote the development of a metabolic alkalosis , 71. TheRapid administration of large volumes of lactate (L-lactate or a mixture of L and D-lactate) containing fluids can cause elevations in blood lactate. As lactate monitors used in clinical medicine only read L-lactate, the impact of lactate administration will be more evident with fluids containing higher levels of L-lactate. Healthy dogs receiving 180 ml/kg of LRS over an hour had a significant increase in blood lactate which was resolved 60 min after the end of the infusion . The incThe metabolism of lactate occurs primarily in the liver. Animals with significant hepatic dysfunction, such as hepatic failure or portosystemic shunt, may have limited lactate metabolism, resulting in elevations of blood lactate and lack of buffering capacity. For this reason, it is generally recommended to avoid lactate containing fluids in patients with liver dysfunction. Unlike lactate, acetate is readily metabolized by extra-hepatic tissues such as skeletal muscle and can be utilized by patients with hepatic dysfunction.The infusion of lactate has been reported to have systemic effects including immune, coagulation and hemodynamic changes. Although there are a growing number of comparison studies between lactate and acetate containing fluids, the results are inconsistent, and more research is needed to fully understand these issues .A well-cited acid-base abnormality associated with fluid administration is a dilutional acidosis. Dilutional acidosis is the result of extracellular fluid volume expansion with a non-bicarbonate containing fluid . As a reOsmolality is a measure of the total concentration of solute (number of particles) in a quantity of solvent (number of osmoles/kg solution) and osmolarity is the number of osmoles/L of solution. In most clinical scenarios the mOsm/L is equivalent to the mOsm/kg, and the terms are considered interchangeable . When prThe osmolality of an IV fluid can affect cells at the site of infusion before the fluid is diluted by the blood volume. Hyperosmolar fluids are of particular concern although very hypoosmolar fluids, such as sterile water, can also create injury. Peripheral vessels are small, and infused fluids have contact with endothelial cells in addition to erythrocytes. To avoid phlebitis, infusion of hyperosmolar fluids through a central vessel, where flow rates are faster, and the infused fluid is less likely to contact the vessel wall, is recommended. Studies suggest that a fluid osmolality >600 mOsm/L is too high for peripheral infusion , 82. A sPlasma-Lyte 148 contains sodium gluconate that is produced from fermentation by Aspergillus sp., and it has been shown that the fluid can be contaminated by small quantities of galactomannan. Galactomannan is a component of the cell wall of Aspergillus, and galactomannan assays are used as diagnostic tests to identify aspergillus. The presence of Plasma-Lyte 148 in a diagnostic sample can cause a false positive result .Any crystalloid can be used as a carrier or diluent for medications, and carrier fluids are typically either D5W or 0.9% sodium chloride, since these are not complex, are acidic, and are less likely to alter the medication it is carrying. The prescribing information of the individual drug to be infused will indicate which crystalloid carrier solution can be used. Balanced isotonic crystalloids can be co-administered with many medications, but some important drug incompatibilities have been identified. For example, Plasma-Lyte 148 is incompatible with pantoprazole, cyclosporine, and midazolam while LRS is incompatible with diazepam, ketamine, and cyclosporine , 85.The ideal balanced isotonic solutions will vary according to species. For most small animal patients, the traditionally coined “balanced” isotonic crystalloids are appropriate for fluid resuscitation, rehydration, and maintenance fluid support. In critically ill animals or patients with electrolyte or acid-base abnormalities and/or with hepatic disease, fluid choice should be based on the patient's existing acid/base and electrolyte condition, in consideration of the available information. Animals with extreme electrolyte imbalances should have a fluid prescription that corrects those imbalances without causing additional complications. Animals receiving high volumes of fluid therapy or several days of fluid therapy should be monitored for electrolyte and acid-base changes that could require a change in the fluid prescription. Hypoproteinemic animals may also benefit from infusion of a colloid solution.All authors contributed equally to the research and writing of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Obtaining tumour-free margins is critical for avoiding re-excision and reducing local recurrence following breast-conserving surgery; however, it remains challenging. Imaging-guided surgery provides precise detection of residual lesions and assists surgical resection. Herein, we described water-soluble melanin nanoparticles (MNPs) conjugated with cyclic Arg-Gly-Asp (cRGD) peptides for breast cancer photoacoustic imaging (PAI) and surgical navigation.The cRGD-MNPs were synthesised and characterized for morphology, photoacoustic characteristics and stability. Tumour targeting and toxicity of cRGD-MNPs were determined by using either breast cancer cells, MDA-MB-231 tumour-bearing mice or the FVB/N-Tg (MMTV-PyVT) 634Mul/J mice model. PAI was used to locate the tumour and guide surgical resection in MDA-MB-231 tumour-bearing mice.The cRGD-MNPs exhibited excellent in vitro and in vivo tumour targeting with low toxicity. Intravenous administration of cRGD-MNPs to MDA-MB-231 tumour-bearing mice showed an approximately 2.1-fold enhancement in photoacoustic (PA) intensity at 2 h, and the ratio of the PA intensity at the tumour site to that in the surrounding normal tissue was 3.2 ± 0.1, which was higher than that using MNPs (1.7 ± 0.3). Similarly, the PA signal in the spontaneous breast cancer increased ~ 2.5-fold at 2 h post-injection of cRGD-MNPs in MMTV-PyVT transgenic mice. Preoperative PAI assessed tumour volume and offered three-dimensional (3D) reconstruction images for accurate surgical planning. Surgical resection following real-time PAI showed high consistency with histopathological analysis.These results highlight that cRGD-MNP-mediated PAI provide a powerful tool for breast cancer imaging and precise tumour resection. cRGD-MNPs with fine PA properties have great potential for clinical translation.The online version contains supplementary material available at 10.1007/s00259-021-05545-3. Nearly half of patients with early breast cancer undergo breast-conserving surgery (BCS) with adjuvant radiation therapy . BCS invDuring BCS, surgeons typically use visual inspection and tactile feedback to determine the tumour location and set resection margins. However, it is difficult to discriminate tumour margins from surrounding normal tissue macroscopically. Frozen section and imprint cytology are applied clinically for intraoperative margin assessment. These methods have the potential to lower the rates of positive margins, but they are labour-intensive and time-consuming and have low sensitivity due to sampling rate limitations , 5. CurrRecently, photoacoustic imaging (PAI) has been developed as a novel imaging technology for biomedical applications. PAI detects optical absorption contrast acoustically via the photoacoustic (PA) effect, a physical phenomenon that converts absorbed optical energy into acoustic energy . Based oNatural melanin is a group of biopigments with multifunctionality  . Due to vβ3 is overexpressed on activated endothelial cells during angiogenesis as well as on many tumour cells within human breast carcinomas, participating in the tumorigenesis, invasiveness, and metastasis [vβ3 has been well recognised and applied to develop the αvβ3-specific tracer [Integrin αtastasis , 21. Argc tracer . In this4OH solution (28 wt%) were purchased from Sinopharm Chemical Reagent Co., Ltd . Amine-PEG5000-amine  was obtained from Shanghai Aladdin Bio-Chem Technology Co., LTD . CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) was purchased from Promega .Melanin was obtained from Sigma-Aldrich . Sodium hydroxide, hydrochloric acid and NHThe human breast cancer cell line MDA-MB-231 and non-cancerous mammary epithelial cell line MCF-10A were purchased from Procell Life Science & Technology Co., Ltd. , and cells with passage number < 25 were used in the experiment. MDA-MB-231 cells and MCF-10A cells were cultured according to the vendors’ recommendations.All animal experiments were performed in compliance with the Guidelines for the Care and Use of Research Animals established by the Xiamen University Animal Studies Committee. Female BALB/c nude mice and BALB/c mice (4–6 weeks) were purchased from the Experimental Animal Centre of Xiamen University. FVB/N-Tg (MMTV-PyVT)634Mul/J transgenic mice were purchased from the Jackson Laboratory.2-PEG5000-NH2 aqueous solution. After vigorous stirring for 12 h, the PEG-modified MNPs were retrieved by centrifugation and washed several times to remove the unreacted NH2-PEG5000-NH2. The PEGylated MNPs  were incubated with 4-cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC) solution (1.2 mg in 36 μL of dimethyl sulfoxide) for 2 h at room temperature. The complexes were purified using a PD-10 column. The cyclic Arg-Gly-Asp-d-Phe-Cys peptide (cRGD) solution (120 μL of 5 mM in degassed water)  was added to the purified MNPs with stirring for 24 h at 4 °C. The excess cRGD peptide was removed using a PD-10 column. The final cRGD-MNPs were filtered through a 0.22-μm filter.The cRGD-MNPs were obtained as previously described . Briefly1H NMR spectra were recorded at 20 °C on a 400-MHz NMR spectrometer (Bruker) using deuterium oxide as the solvent. Zeta potentials were measured using a laser particle size analyser system . The absorption spectra were obtained using a Multiskan Spectrum Microplate Spectrophotometer .Transmission electron microscopy images were recorded using a Talos F200s transmission electron microscope  at an accelerating voltage of 100 kV. The MNP or cRGD-MNP aqueous solution was dropped onto a carbon-coated copper grid and air-dried. The The solutions of MNPs and cRGD-MNPs (120 μM) were scanned for PAI using the Vevo LAZR-X photoacoustic imaging system  at different wavelengths ranging from 680 to 970 nm  to detect the optimal excitation wavelength. Different concentrations (3.75–120 μM) of MNPs and cRGD-MNPs were dispersed in PBS and triggered by the optimal excitation wavelength to acquire the corresponding PA images. For photostability analysis, PA images of the MNP and cRGD-MNP solutions (120 μM) were obtained at different time points (0–7 days) using a 680-nm excitation.5 per well) were seeded in 6-well plates and cultured for 24 h. Then, the cells were incubated with 0.125 μM cRGD-MNPs for various times  and fixed with 4% paraformaldehyde (PFA) solution for 20 min. PAI of the MDA-MB-231 cells at different time points were performed at a wavelength of 680 nm using the Vevo LAZR-X system. For the concentration gradient experiment, MDA-MB-231 cells were incubated in medium containing the cRGD-MNPs (0.125–1 μM) for 4 h. To investigate the effect of cRGD on MNP uptake, MDA-MB-231 cells were treated for 4 h with medium containing 0.5 μM MNPs, PEG-MNPs or cRGD-MNPs, and then subjected to PAI analysis. To investigate the tumour targeting ability of cRGD-MNPs, MDA-MB-231 and MCF-10A cells (~ 3 × 105 per well) were seeded in 6-well plates and incubated with 0.5 μM cRGD-MNPs alone or with excess cRGD peptide (100 μM) for 4 h.MDA-MB-231 cells  was added to the MNPs or cRGD-MNPs solutions . After stirring at room temperature for 1 h, the solution was transferred to an ultrafiltration centrifuge tube  and centrifuged several times  to remove the free or unstable rhodamine, yielding the rhodamine-labelled MNPs (Rho-MNPs) and cRGD-MNPs (Rho-cRGD-MNPs).5 cells) were seeded in confocal dishes for 24 h. The Rho-cRGD-MNPs (0.5 μM) were added to the culture medium with or without free cRGD (100 μM) at 4 h. The cells were fixed with 4% PFA and counterstained with DAPI. The fluorescence images of the cells were collected using a confocal laser scanning microscope .MDA-MB-231 and MCF-10A cells (~ 1 × 105/well) were incubated in cell slides for 24 h and treated with Rho-cRGD-MNPs or Rho-MNPs (0.5 μM) for 4 h. The cells were fixed with 4% PFA for 10 min, and blocked with PBST containing 1% BSA and 22.52 mg/mL glycine for 30 min. The cells were incubated with anti-integrin αv antibody  at 4 ℃ overnight, following by incubation with anti-rabbit antibody  for 1 h at room temperature. Finally, the cells were stained with DAPI and observed with a fluorescence microscope .MDA-MB-231 cells (~ 1 × 103/well) were incubated in 96-well plates for 24 h. Cells were then cultured in medium supplemented with the MNPs, PEG-MNPs or cRGD-MNPs  for 72 h. The MTS assay was performed following the vendors’ recommendations. Five replicates were conducted for each group.MDA-MB-231 cells (~ 5 × 106) were inoculated subcutaneously in the right flank of nude mice. When tumour volume reached 100 mm3, the tumour-bearing mice were subjected to in vivo PAI and biodistribution studies.MDA-MB-231 cells (~ 1 × 10n = 3 each group) and intravenously injected with the MNPs or cRGD-MNPs , respectively. PA images were collected and analysed.To determine the optimal concentration of the MNPs for in vivo study, mice were randomly allocated into three groups and injected with different concentrations of cRGD-MNPs . PA images were collected at different time points  using the Vevo LAZR-X at a wavelength of 680 nm, and the average PA intensities in the tumour regions were measured. To detect the tumour targeting capability of the cRGD-MNPs, MDA-MB-231 tumour-bearing mice were randomly divided into MNPs and cRGD-MNPs groups (n = 3) and injected with cRGD-MNPs or cRGD-MNPs  with cRGD peptide (20 mg/kg). As for the blocking group, cRGD peptide was injected 15 min before cRGD-MNP injection. PA signals were collected at different time points .MDA-MB-231 tumour-bearing mice were randomly divided into two groups (each group n = 3 each group) were intravenously injected with Rho-MNPs or Rho-cRGD-MNPs . The major organs and tumours were harvested 2 h post-injection for ex vivo fluorescence imaging using the IVIS Lumina imaging system . The relative fluorescence intensities were measured.To determine the biodistribution of the nanoprobes in vivo, MDA-MB-231 tumour-bearing mice (n = 6) were intravenously injected with the cRGD-MNPs . US and PA images were obtained before injection and 2 h post-injection with excitation at 680 nm. In addition, the 4th and 5th mammary glands of the transgenic mice  with breast cancer were divided into four sections (P1–4) for in vivo and ex vivo PAI at 2 h post-injection of cRGD-MNPs. After imaging, the mammary tissues were also analysed by histology.The transgenic mouse model spontaneously develops invasive breast cancer in each mammary gland between 6 and 12 weeks of age. Mice with breast cancer , placed in the dark at 4℃ for 12–18 h. Next, the tissues were put into VG staining solution and stained for 1 min. Finally, the tissues were observed under a microscope .For surgical resection, cRGD-MNPs  were intravenously injected into MDA-MB-231 tumour-bearing mice. Tumour profiles were detected using 3D PA/US imaging 2 h post-injection under 2% isoflurane anaesthesia. For simulated tumour surgical resection, the surgery procedure was performed in four steps: PAI-guided tumour detection, partial tumour resection (P1–3), PAI detection of the residual tumour, and re-resection of the tumour bed (P4). PA images were acquired both before and after each sequential removal of tumour. Each resection was about a 5–6 mm-long and 2–3-mm-wide piece of tissue. Excised tissue was stained with H&E staining. To detect the depth of PAI, PAI of tumours were performed under various thicknesses of chicken breast .n = 15 each group). Body weight was monitored throughout the experiment. The mice were sacrificed on days 1, 7 and 30 post the injection. Blood samples and major organs  were collected at above time points. The serum biochemical parameters were analysed, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (CR). H&E staining of major organs was performed for histological analysis.BALB/c mice (4–6 weeks) were randomly divided into control group  and cRGD-MNPs group  . The two-tailed paired 1H NMR spectra    Fig. , c, d anly) Fig. , Fig. 1ely) Fig. .Fig. 1CAs seen from the overlay images and quantitative results, the PA signal intensity of MDA-MB-231 cells increased when incubated with cRGD-MNPs from 1 to 4 h, and there was no statistical difference between the 4 h and 8 h time points Fig. . As the vβ3, we determined their binding affinity using MDA-MB-231 and MCF-10A (a non-neoplastic breast cell line with low expression of αvβ3) [v , even at a high concentration up to 10 μM  . The PA  [v Fig. . These d μM Fig. .p < 0.05)  increased gradually and reached a peak 2 h post-injection Fig. . Moreove05) Fig. . In addi05) Fig. . To confWe also used fluorescence imaging to track the biodistribution of Rho-cRGD-MNPs and Rho-MNPs at 2 h post-injection Fig. . The ex To fully evaluate the PAI performance of cRGD-MNPs, we used the FVB/N-Tg(MMTV-PyVT)634Mul/J spontaneous breast cancer model that closely recapitulates human disease . The trath and 5th mammary glands of the transgenic mice were surgically divided into four segments (P1–4), and PA imaging showed complete and intense enhancement of each part of the tumour. Ex vivo tissues were imaged using the PAI system, and the tissue signal distribution  images showed the spatial distribution of the cRGD-MNPs at the tumour site, which provided the general profile of a tumour for the development of a surgical plan Fig. . From thThe in vivo biocompatibility was further evaluated in mice for prolonged durations  after intravenous administration of cRGD-MNPs. Saline served as a negative control. No considerable body weight loss was observed for the cRGD-MNPs and control groups over 30 days, indicating that cRGD-MNPs had no significant side effects in mice Fig. . The serIt remains a significant challenge in BCS to acquire clean margins during primary surgery due to the lack of precision in localising tumours and the inaccuracy of tumour excision by visual inspection and tactile feedback. Imaging-guided surgery is gaining increasing importance in the operating room. It can help detect microscopic tumours or residual lesions that are readily missed during surgery and guide intraoperative surgical margin assessment . Thus, tvβ3. Consistent with previous findings [vβ3. Moreover, mammary glands of FVB/N-Tg (MMTV-PyVT)634Mul/J transgenic mice containing carcinoma showed excellent PA intensity enhancement after the administration of cRGD-MNPs compared to the normal mammary gland tissue. These data further indicated that cRGD-MNPs could serve as a good contrast for breast cancer-specific PAI.Melanin is a naturally occurring biopolymer present in many organisms, particularly in the skin and hair. It has a good intrinsic photoacoustic property. By mimicking natural melanin, recent studies have demonstrated that melanin-based nanoparticles could serve as a multimodality nanoplatform for molecular imaging , 28, 29.findings , 31, ourMNPs have been used in diverse biomedical applications, such as imaging, controlled drug release, bioengineering and bioelectronics, antioxidant applications and theranostics . Based oIn addition to melanin nanoparticles, several exogenous contrast agents have been used for photoacoustic surgical navigation in animals, including gold nanoparticles , 35, supHowever, this study has a few limitations. First, only PAI alone was applied to surgical navigation. As previous study reported , MNPs arIn this report, we demonstrated the feasibility of PA augmented by the systemic delivery of cRGD-MNPs to provide preoperative 2D or 3D PA images of tumours for precise surgical planning, and guide initial and subsequent resections of breast cancer tumour mass. Prospectively, PAI using cRGD-MNPs is expected to be evaluated in breast cancer patients for surgical navigation in the near future.Supplementary file1 (DOCX 3845 KB)Below is the link to the electronic supplementary material.


Tumour neoantigens arising from cancer-specific mutations generate a molecular fingerprint that has a definite specificity for cancer. Although this fingerprint perfectly discriminates cancer from healthy somatic and germline cells, and is therefore therapeutically exploitable using immune checkpoint blockade, gut and extra-gut microbial species can independently produce epitopes that resemble tumour neoantigens as part of their natural gene expression programmes. Such tumour molecular mimicry is likely not only to influence the quality and strength of the body’s anti-cancer immune response, but could also explain why certain patients show favourable long-term responses to immune checkpoint blockade while others do not benefit at all from this treatment. This article outlines the requirement for tumour neoantigens in successful cancer immunotherapy and draws attention to the emerging role of microbiome-mediated tumour neoantigen mimicry in determining checkpoint immunotherapy outcome, with far-reaching implications for the future of cancer immunotherapy. However, most targeted drugs are not exclusively specific for cancer, because they usually inhibit wild-type versions of proteins, which can often be expressed in a wide range of tissues , and some small-molecule inhibitors, particularly first-generation agents, are multi-specific, thus showing additional on-target activity . Chimeric antigen receptor T cells also lack molecular specificity for cancer as their engineered affinities generally recognise wild-type antigens expressed on non-transformed cells as well as transformed cells, potentially leading to on-target, off-tumour toxicity .12One of the hallmarks of cancer is the overly high proliferative capacity of tumour cells, which can be therapeutically exploited using classical cytotoxic chemotherapy. The merit of anti-proliferative drugs in the debulking of large tumours is beyond dispute, but these agents are molecularly unselective for cancer, and invariably also damage healthy tissue such as bone marrow and the colonic mucosa.13 and well-documented evidence for infectious contributions to tumour development (e.g. human papilloma virus14 and Helicobacter pylori15), the dogma that ‘cancer is a genetic disease’ still holds true.16 The stepwise accumulation of mutations not only facilitates and exacerbates malignancy, but also generates a molecular fingerprint—comprised of tumour neoantigens—with definite specificity for cancer. While some tumour neoantigens are recurrent among cancer entities (e.g. KRAS position 12 mutations), the overall molecular fingerprint of a cancer is unique in every patient irrespective of tumour type. This collective of cancer-specific molecular aberrations, commonly termed the ‘tumour neoantigenome’, is an ideal target for cell-mediated immunity and cancer immunotherapy, with arguably the highest possible specificity for cancer considering the natural selectivity of cytotoxic, adaptive immune cells.19 Exogenously derived antigens can also be regarded as ‘tumour neoantigens’20 but, compared with endogenous neoantigens, the presence of viral or bacterial antigens is limited to relatively few cancer entities ).21 In addition, infection-related cancers still carry endogenous neoantigens,24 and some of these malignancies can be prevented with vaccines.25 This article will therefore prioritise endogenous tumour neoantigens arisen from mutations in host cells.Despite the growing appreciation of epigenetic regulationAlthough the presence of tumour neoantigens can discriminate cancer cells from ‘normal’ cells, epitopes produced by gut and extra-gut microbial species as part of their natural gene expression programmes can resemble tumour neoantigens—a phenomenon known as molecular mimicry. Molecular mimicry of tumour neoantigens by microbial species is likely to influence the host anti-cancer immune response through neoantigen-reactive T cells, and could explain why certain patients show favourable long-term responses to immune checkpoint blockade while others do not benefit at all from this treatment—tumour neoantigens are a mechanistic requirement for successful cancer immunotherapy.In this article, we will introduce the concept of microbial tumour neoantigen mimicry emanating from the huge overall amount and diversity of microbial genetic sequences in the human body, and elaborate on the potential significance of such mimicry for checkpoint immunotherapy responsiveness and outcome.20 and are excluded from central tolerance20—that is, they are not included in the mechanism used by the host to avoid attack by the immune system. Tumour neoantigens can also, in some cases, arise from the cancer-cell-specific usage of the epigenetic machinery, which can lead to the re-expression of silenced genes, the occurrence of unique splice or structural variants, and/or read-through transcription generating chimeric or intergenic products.27Tumour neoantigens mostly constitute de novo mutations that arise in transformed cells when tumour DNA is altered through non-synonymous point mutations, gene translocations, or insertions and deletions (indels)+ naive T cells (cross-priming).30 In turn, interactions between T cells with a cognate T cell receptor and the neoantigen-positive APCs induce clonal expansion and effector/memory differentiation;31 cytotoxic tumour killing by neoantigen-specific T cells results in cancer cell lysis, which might generate an ‘in situ vaccination effect’ such that beneficial epitope spreading might occur.32 In an ideal scenario, sustained anti-cancer immunity will ensue protection from cancer re-growth.Generally, immunological visibility of tumour neoantigens requires protein synthesis and a certain stability of the neoantigen product. Following the release of cytoplasmic content during cell death, for example, tumour neoantigens expressed as proteins will be taken up by tissue-patrolling antigen-presenting cells (APCs), which will then migrate to the tumour-draining lymph node and present the molecular tumour fingerprint to CD835 and microenvironmental mechanisms.36 In terms of cancer-cell-intrinsic mechanisms, downregulation of the major histocompatibility complex class I (MHC I) presentation pathway, which is essential for immune recognition and CD8+-cytotoxic-cell-mediated killing, is often observed in tumour cells, as is the increased expression of immune checkpoint molecules, which function as a ‘brake’ on the immune system, controlling the duration and extent of immune responses, maintaining self-tolerance and preventing autoimmunity.However, clinical evidence indicates that such protective anti-cancer immunity is rare among cancer patients, owing to immune evasion caused by various cancer-cell-intrinsic40 Mechanistically, ICIs neutralise negative feedback from immunological checkpoints or increase immune-stimulatory signalling, thus reinvigorating the endogenous tumour-specific T cell response. For CTLA-4-targeted immunotherapy, it has been shown that the clinical mode of action is likely to involve therapy-enhanced T cell priming, which broadens the tumour-specific CD8+ T cell response.41 Data from anti-CTLA-4-treated melanoma patients demonstrated a significant increase in the number of newly detected tumour-specific CD8+ T cell responses after treatment, whereas pre-existing virus- and tumour-specific T cell responses remained unchanged.41 Similar findings were reported for ICIs that target PD-1, thus altogether suggesting that pre-existing tumour-reactive T cells have a limited reinvigoration potential, which is outcompeted by de novo arising T cell specificities .42 The poor reinvigoration capacity of pre-existing tumour-specific T cells might indicate a partially exhausted cell state maintained independently from checkpoint signalling, which potentially limits the use of ICIs in certain patients.Immune checkpoint inhibitors (ICIs) that target checkpoint components, such as programmed death receptor (PD-1) and its ligand programmed cell death ligand 1 (PD-L1), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), or other inhibitory molecules can partly release this immune system brake and have shown impressive long-term outcomes in many cancer patients.43 and have established themselves as frontline therapeutics in a broad variety of solid tumour types.45 However, the speed of further development of ICI-based immunotherapy has started to decelerate, and significant challenges lie ahead, including the need for adapted clinical trial design and customisation of clinical safety and efficacy endpoints to more specifically account for the characteristic features of ICIs compared with classical anti-cancer drugs.46The clinical significance of ICIs is tremendous, as these agents have shown unprecedented responses even in advanced-stage tumours,53 Accordingly, tumour entities with a typically high TMB  respond significantly better to ICI-based immunotherapy than do cancers with a comparatively low TMB .54 Moreover, immunological treatments that target the tumour neoantigenome through reinvigoration (in the case of ICIs) or induction (for vaccines) of antigen-specific CD8+ T cells do not rely solely on the ‘driver’ nature of mutations, as passenger mutations can be equally immunogenic and thereby also serve as targets for immune destruction;56 by contrast, targeted treatment options require that the respective target molecule is causal for tumour growth and/or progression (‘oncogene addiction’).58The relevance of tumour neoantigens in medical oncology is highlighted by the positive association between tumour mutational burden (TMB) and the response to ICI treatment.Thus, tumour neoantigens arising from cancer-specific mutations are attractive targets for cancer immunotherapy, which has shown proven clinical relevance and important conceptual advantages over other types of cancer treatment that do not exploit the natural adaptability and versatility of the immune system.60 shown by tumours. Primary resistance mechanisms include the absence of a relevant number of immunogenic tumour antigens, defects in the antigen processing and presenting machinery, and insufficient T cell infiltration.59 Adaptive resistance to cancer immunotherapy refers to mechanisms by which cancer cells adapt to attacking immune cells, for instance through downregulation of MHC class I or upregulation of PD-L1.59 Acquired resistance refers to a clinical scenario where a patient initially responds to immunotherapy but later on progresses or relapses; oftentimes, this will involve Darwinian selection of resistant clones present already before treatment start.59Although ICIs have revolutionised the treatment of various solid tumour types and enabled unprecedented long-term remissions, a significant number of patients do not benefit from these therapies, owing to primary, adaptive, or acquired resistance mechanisms63 The time lost in treating poorly responding or refractory patients, along with the potential toxicity65 and high associated costs of ICI treatment,67 demands that better tools and predictive markers are established to discriminate patients who are likely to respond to treatment from those who are not likely to respond. More importantly, identifying the underlying mechanisms of non-responsiveness will be instrumental for the development of therapies that work in combination with ICIs to resensitise tumours to checkpoint inhibition.Only a small proportion of patients with non-small cell lung cancer (NSCLC), for example, respond favourably to ICI treatment.69 However, none of these markers is perfect and they are currently unable to predict treatment response with clinically sufficient precision.71 As an example, the predictive power of baseline tumour T cell infiltration might be reduced or lost if these T cells are exhausted or otherwise non-functional. Similarly, a high TMB indicating increased statistical odds for the presence of tumour neoantigens does not predict whether these cancer-specific antigens are immunogenic to an extent that is therapeutically relevant. Finally, the proven presence of the ICI target protein only indicates that the substrate is present to inhibit, but not whether, or to what extent, this substrate is causal for tumour growth. Therefore, novel and more reliable predictive markers are desirable to refine patient stratification and further optimise ICI treatment.Aside from TMB as a surrogate indicator for tumour neoantigens, the extent of tumour T cell infiltration at baseline and the expression of the respective ICI target (e.g. PD-1/PD-L1) at the protein level represent the most established markers for predicting the responsiveness to checkpoint blockade.Bifidobacterium72 and distinct Bacteroides species73 can promote anti-cancer immunity during ICI treatment. Accordingly, co-treatment with antibiotics reduces the clinical activity of checkpoint immunotherapy,78 whereas faecal microbiome transplantation (FMT) from ICI-responding patients to germ-free or antibiotic-treated mice partly rescues the anti-cancer effects of ICI treatment, dependent on the presence of A. muciniphila Gram-negative bacteria.79 Although these data can offer possibilities for straightforward therapeutic development , the mechanisms responsible for these observations remain elusive and a deeper understanding of the underlying biology is desirable.80Within the past decade, evidence has suggested an important role for the gut microbiome in determining the responsiveness to checkpoint immunotherapy. Two articles published in 2015 demonstrated that commensal 85 , anti-CTLA-4 treatment efficacy can be restored by oral gavage of B. fragilis, immunisation with B. fragilis-derived products or adoptive transfer of T cells specific for B. fragilis.73 It is worth mentioning in this context that a 2020 publication on cancer immunotherapy targeting CD47 (another checkpoint molecule) found that the accumulation of gastrointestinal bacteria in subcutaneously grown tumours was critical for the treatment response.86 In addition, another study showed that intratumoural accumulation of bacteria is common among cancer entities, with bacterial compositions being mostly biased towards intracellular bacteria that are capable of triggering CD8+ T cell responses.87 These results highlight the far-reaching effects of gastrointestinal microbes across organ systems/compartments and clearly emphasise the significance of the local tumour microbiome for responsiveness to cancer immunotherapy. Aside from the well-known gastrointestinal microbiome, a rich flora of microbial communities can also be found in the respiratory tract89 as well as on other various mucosal surfaces and non-mucosal surfaces of the human body.92 Thus, certain carcinomas  are likely to be influenced by a distinct tumour-associated habitat that might also shape the response to ICI therapy.A likely explanation for the functional significance of the gut microbiome in checkpoint immunotherapy responsiveness is immunological mimicry of tumour neoantigens by microbial peptide products85 Fig. . SupportConvincing evidence therefore suggests that the outcome of ICI-based cancer immunotherapy is influenced, at least partially, by the composition and functional quality of the (distant) gut- and the  tumour microbiome.+ T cells. Molecularly, these cells recognise tumour neoantigens typically as 8–11-mer peptides94 bound to MHC class I on the tumour cell surface. Random 11-mer permutation of the 20 proteinogenic amino acids in humans  can generate more than 1014 theoretical combinations, although the number of possible tumour neoantigens is correspondingly smaller considering that many of these sequence combinations will already be part of the host genome.Cancer immune surveillance is, to a large extent, carried out by CD896 and viruses can harness the host cell translational machinery to exclusively use the endogenous amino acid repertoire. It is believed that the human gastrointestinal tract harbours approximately 1014 microbes, which, together, equate to roughly 1000 times the number of cells and 10000 times the DNA content of the human body.97 Exact mathematical modelling is beyond the scope of this article, but it is with definite statistical likelihood that numerous individual tumour neoantigens are immunologically mimicked by peptide products of gut and extra-gut microbial species: identical peptide mimics comprise the same amino acid sequence as the corresponding tumour neoantigens and result in identical T cell epitopes, whereas ‘notably similar’ peptide mimics contain the same epitope core but differ in their flanking region or length, thus altering antigen presentation efficiency and/or generating T cell epitopes with nuance-like specificity alterations.99 Importantly, the actual likelihood of mimicry might be smaller than theoretically possible considering that microbial genomes encode functional proteins whose sequences might not be randomly structured which might lower the number of actually possible sequence permutations. In addition, not every neoantigen-mimicking microbial peptide will reach a concentration that is high enough for immunological recognition and modulation of the anti-cancer immune response.Many microbes that colonise the human body can synthesise all 20 proteinogenic amino acids of humans,101 Such microbial-antigen-containing APCs can also cross-present their phagocytosed cargo via MHC class I to induce cellular immune responses.103 Furthermore, intracellular microbes present in both cancer and immune cells87 can trigger direct CD8+ T cell responses in the tumour microenvironment (TME). Tumour neoantigen mimicry by microbial species is therefore likely to produce T cell clones of both the CD8 and CD4 lineage that are tumour-reactive despite never having previously seen actual tumour antigens. Such independent emergence of T cell populations with overlapping specificity can lead to functional immunological crosstalk that shapes overall anti-cancer immunity and influences the outcome of checkpoint-based immunotherapy.84The abundance of class-switched IgA antibodies with high affinities for microbial species in the gut microenvironment provides clear evidence for APC-dependent, humoral responses elicited and matured in gut-associated lymphoid tissue and/or regional lymph nodes.105 as well as the default unresponsiveness to food proteins in healthy individuals .107 However, it is unknown how compartment-specific microbial tolerance influences neoantigen-directed tumour immune surveillance (even more so during perturbations such as dysbiosis or infection). Although immune tolerance to commensals and their tumour neoantigen mimics might intuitively be more associated with reduced anti-cancer immunity, evidence has shown that T cells that are specific for certain gut commensals serve immune-stimulatory functions during checkpoint-based immunotherapy.73 These data suggest that the ‘tolerogenic’ environment of the gut, including tolerance to neoantigen-mimicking commensals, can nevertheless support anti-cancer immunity based on yet-to-be-determined mechanisms; conceivable means include compartment-specific effects as well as temporary disruptions of epithelial barrier function leading to translocation into the systemic circulation and subsequent failure of tolerance.Currently, the net effect of such immune interaction is unclear. Generally, the gastrointestinal tract and other body surfaces are considered a rather tolerogenic environment, an idea that is consistent with the notion of productive interplay with commensals under homoeostatic conditions81 In these patients, immunogenic neoantigens can be identified using a quality fitness model that takes into account differential presentation and homology to microbial-derived peptides.81 Of note, ‘high-quality’ neoantigens are selectively lost upon metastatic disease progression, suggesting immunoediting and, ultimately, immunological escape of clinically relevant tumour neoantigens.81In further support of clinically relevant tumour neoantigen mimicry, long-term survivors of pancreatic cancer show tumour-neoantigen-specific T cell clones with predicted cross-reactivity to microbial epitopes.109 and inosine,110 are known to have immune-modulatory activity and to influence cancer immunotherapy. Short-chain fatty acids are differentially expressed among immunotherapy responders and non-responders, and a high faecal abundance of acetic, propionic, butyric and valeric acid correlates with prolonged progression-free survival.109 The underlying mechanisms are yet to be fully elucidated, but are likely to involve altered T cell homoeostasis and differentiation as well as modulation of APC functions109 brought about by these short-chain fatty acids. Inosine as produced by Bifidobacterium spp. in the gut facilitates enhanced ICI treatment efficacy through a mechanism that involves the increased systemic translocation (owing to ICI-induced decreased gut barrier function) of inosine and subsequent activation of anti-cancer T cells.110 Considering the basic principles of immune activation and differentiation, it is very much conceivable that T cells, as well as their correspondingly induced cytokines, that recognise mimicked tumour neoantigens are subject to regulation by microbial metabolites.The microbiome-derived metabolome refers to the aggregate of individual microbial metabolites in a given compartment, several of which, including short-chain fatty acidsAt this stage, no conclusions on the role of microbial tumour neoantigen mimicry can be drawn; mechanistic studies in mice as well as consolidation in the clinical setting are warranted. Below we provide insight into how to experimentally address microbial tumour neoantigen mimicry to better understand responsiveness to checkpoint immunotherapy and optimise patient stratification. Molecular and immunological tools are available to define the contribution of gut microbes to the responsiveness of checkpoint immunotherapy and to systematically assess whether microbial tumour neoantigen mimicry acts to foster or impede protective anti-cancer immunity.Bifidobacterium, Bacteroides and Escherichia spp.) before the genetically modified bacteria are introduced into the gastrointestinal tract of immunocompetent mice using oral gavage; sustained bacterial engraftment might involve antibiotic conditioning.111 Tumour neoantigen mimicry can be enforced by transplanting tumour cells into bacteria-reconstituted mice, and the impact of such mimicry on anti-cancer immunity and immunotherapy responsiveness can be investigated , provided that limitations with adaptive immune responses can be overcome in humanised mice.116Modern genetic engineering facilitates the expression of almost any gene of interest in prokaryotic and eukaryotic cells and is key to mechanistically dissecting the function of genes. Using this reverse genetics approach to introduce tumour neoantigens into gut commensals, mouse models of immunological tumour mimicry can be experimentally generated. To achieve this, the genetic sequences encoding tumour neoantigens need to be cloned into suitable commensals ,124 they are largely artificial and can have limited predictive and translational value.126 Conversely, tumour neoantigens from mutagen-induced  or genetically-engineered tumours  might be more relevant because they have evolved ‘naturally’ and gone through immune selection and immune-editing processes.135 Such engineered/autochthonous models will produce genetically heterogeneous tumours with a comparatively high frequency of protein-altering mutations and copy number variations.136 From such models, clonal tumour cell lines can be established, and the tumour neoantigens can be identified using next-generation sequencing at the DNA level and compared with autologous reference tissue/blood.18 RNA sequencing can determine the fraction of neoantigens putatively expressed, and peptide–MHC binding algorithms such as NetMHCpan137 can predict neoantigen immunogenicity.The mimicked (neo-)antigens can include surrogate tumour antigens such as minigene-encoded epitopes of viral glycoproteins140 The enzyme-linked immunospot assay can probe the neoantigen-specific reactivity of ex vivo stimulated T cell populations142 and T cell receptor sequencing can characterise the clonality and overall repertoire of intratumoural T cell subsets.143To investigate the functional significance of immunological tumour neoantigen mimicry for ICI responsiveness and outcome, readouts addressing the quality and strength of the anti-cancer immune response as well as changes in tumour growth are required. Flow cytometry/fluorescence-activated cell sorting can help to delineate general immune (activation) patterns in tumour tissue, blood and lymph nodes, and can further facilitate neoantigen-specific T cell tracking and purification, making use of peptide–MHC multimer technology.144 and, third, ‘metastatic’ growth in a relevant compartment.119 These scenarios will allow various clinical settings, including those of high tumour burden, minimal residual disease and oligometastatic disease, respectively, to be experimentally explored. System perturbations such as co-treatment with antibiotics to reduce or abrogate microbial tumour neoantigen mimicry could be envisaged for control purposes; here, the particular timing of antibiotic treatment should be carefully selected considering treatment duration as well as lagging phase and washout effects.75Complementing these important immune-targeted readouts, the effects on functional tumour growth need to be assessed, and a number of scenarios should be explored: first, ‘continuous’ tumour growth resulting from a high dose of transplanted tumour cells; second, tumour engraftment emanating from a critically low dose of transplanted tumour cells;18 and the gastrointestinal microbiome composition needs to be characterised using metagenomic shotgun sequencing of stool samples.145 Dedicated bioinformatics interrogation can uncover the overlapping tumour neoantigen and microbial sequences, and provide an estimate for the overall ‘tumour antigenic similarity’. Correlating the degree of this similarity with clinical endpoints such as survival, treatment response and immune-related adverse events, a potential proxy for treatment efficacy,65 will demonstrate how microbial tumour neoantigen mimicry affects patient outcomes under ICI therapy  potential. By contrast, the composition of the gut microbiome is more dynamically regulated149 might conceivably be responsible for modulating both natural81 and ICI-induced84 anti-cancer immunity; however, evidence suggests that molecular mimicry of tumour neoantigens by microbial species is likely to be an important factor. The net effect of tumour neoantigen mimicry on ICI treatment efficacy currently remains unknown and future mechanistic and clinical studies are clearly warranted. Nonetheless, the results of various studies that have investigated microbial composition in the context of checkpoint immunotherapy responsiveness support a model in which mimicry has favourable effects on anti-cancer immunity and ICI-related treatment outcome  is associated with immune activation and long-term survival of pancreatic cancer patients151 as well as a memory T cell signature and favourable response to PD-1-targeted immunotherapy in patients with NSCLC.152 Along these lines, the beneficial effects of therapeutic FMT from ICI-responding patients79 might be at least partially attributable to an increase in the overall microbial diversity, similar to the results seen for FMT in patients with inflammatory bowel disease.154Several mechanisms, including discrepancies in the levels of some metabolites,ome Fig. . First, 85 Importantly, the gut microbiome engages in crosstalk with the tumour microbiome such that therapeutic FMT will automatically also modulate the tumour microbial composition.151 Clinical research will show whether such interventions are sufficient to reinstate relevant tumour neoantigen mimicry or whether targeted engineering of commensals based on the tumour neoantigen landscape would be required. Such personalised, molecularly informed approaches are technically feasible, and could be achieved within a clinically reasonable time frame (approximately 6–8 weeks), provided that trial data can overcome safety concerns regarding the use of genetically-modified live bacteria.Assuming a protective role for microbial tumour neoantigen mimicry, interventions aimed at supplementing probiotics and/or increasing microbial diversity represent rational therapeutic approaches that warrant clinical investigation.141 Given the clarification of the definite role of tumour neoantigen mimicry in ICI responsiveness, this knowledge can help to complement MHC class I binding prediction137 and assist in the consideration of which neoantigens to choose for vaccination. In this way, the anti-cancer immune response could be (re-)directed specifically to molecularly mimicked neoantigens, provided that mimicry indeed shows beneficial effects on ICI treatment efficacy.Another promising anti-cancer strategy is the use of ICIs together with tumour-neoantigen-specific vaccines.155—by the process of immunoediting. Immunoediting of tumour neoantigens has been reported, and is associated with metastatic progression in pancreatic cancer.81 Other prominent mechanisms of cancer immune escape include somatic loss of HLA class I molecules (loss of heterozygosity (LOH))156 and defects in the MHC class I antigen processing and presenting machinery.157 Importantly, HLA class I LOH predicts poor overall survival in a subgroup of ICI-treated NSCLC patients and can be used as a complementary marker for checkpoint immunotherapy responsiveness to refine TMB-based stratification.158 In the context of tumour neoantigen mimicry and associated therapeutic interventions, HLA class I LOH needs to be considered as a potential confounder for tumour-microbiome cross-reactive T cell responses. While HLA class I LOH occurs spontaneously and is not preventable per se, any neoantigen-directed therapeutic intervention should ensure coverage of several HLA class I alleles to avoid dependence on a single genetic locus.Immunogenic cancer cells adapt to the selective pressure of the immune system, conceptually traversing through three successive phases—elimination, equilibrium and escapeThis review article has highlighted the crucial importance of tumour neoantigens for cancer immunotherapy and provided impetus for investigating the significance of microbial tumour neoantigen mimicry for checkpoint immunotherapy responsiveness and outcome. Immunologically informed considerations on ICI therapeutic use will optimise their clinical performance and set the stage for microbiome-targeted interventions to boost response rates and prolong survival.


To describe fluid resuscitation practices in Brazilian intensive care units and to compare them with those of other countries participating in the Fluid-TRIPS.This was a prospective, international, cross-sectional, observational study in a convenience sample of intensive care units in 27 countries (including Brazil) using the Fluid-TRIPS database compiled in 2014. We described the patterns of fluid resuscitation use in Brazil compared with those in other countries and identified the factors associated with fluid choice.versus other countries: 56.4%, p < 0.001). In Brazil, the percentage of patients receiving crystalloid solutions was higher , and 0.9% sodium chloride was the most commonly used crystalloid . The multivariable analysis suggested that the albumin levels were associated with the use of both crystalloids and colloids, whereas the type of fluid prescriber was associated with crystalloid use only.On the study day, 3,214 patients in Brazil and 3,493 patients in other countries were included, of whom 16.1% and 26.8% (p < 0.001) received fluids, respectively. The main indication for fluid resuscitation was impaired perfusion and/or low cardiac output  was the crystalloid most commonly prescribed. Serum albumin levels and the type of fluid prescriber were the factors associated with the choice of crystalloids or colloids for fluid resuscitation. It is one of the most common interventions in critically ill patients. Despite being a frequent intervention, fluid resuscitation lacks a clear definition. The choice of fluid to be administered as well as the dose and speed are not well determined, leading to differences in bedside practices.-12 Even with recent published guidelines including new evidences,,14 delays and failures with translating recommendations into practice are common, leading to variability in care.,16 The Saline versus Albumin Fluid Evaluation - Translation of Research Into Practice Study (SAFE-TRIPS), a cross-sectional study conducted in 2007 including 391 intensive care units (ICUs) across 25 countries, reported that resuscitation practices varied significantly. Although colloid solutions were more expensive and may possibly be harmful in some patients, they were administered to more patients and during more resuscitation episodes than crystalloids.In the last 15 years, multiple randomized controlled trials and subsequent meta-analyses have shown that the type of fluid used for resuscitation, particularly hydroxyethyl starch (HES), may negatively affect outcomes. This study demonstrated an important change in clinical practice, with a preferential use of crystalloids, specifically buffered salt solutions, over colloids. Another interesting finding of this study was that fluid choice was determined by local practice rather than by any identifiable patient characteristic.Recently, the same group conducted a similar observational study in a convenience sample of ICUs: the Fluid-TRIPS.The number of contributing ICUs from Brazil in the Fluid-TRIPS was just over half of all participating units, allowing the unique opportunity to separately analyze Brazilian data. Our hypothesis was that Brazilian ICUs would have different standards for fluid resuscitation, mainly regarding the choice of crystalloids.Thus, the objective of this study was to describe current practices on fluid resuscitation in Brazilian ICUs and to compare Brazil with the other countries participating in the study.This secondary analysis of a prospective, international, cross-sectional, observational study was carried out in a convenience sample of ICUs in 27 countries using the Fluid-TRIPS database, compiled in 2014.Universidade Federal de São Paulo, and the institution’s Ethics and Research Committee approved the study protocol under the number CAAE 36093314.4.1001.5505 with a waiver for Informed Consent considering the observational nature of the study.In Brazil, we recruited participating sites at critical care meetings through the Brazilian Research in Critical Care network (BRICNet) website and contacts and personal contacts with key opinion leaders. Participation was voluntary, and any hospital willing to join the study was considered eligible, with no exclusion criteria. The coordinating center was the th and 11th 2014. Methodological details were previously published. Briefly, the study day was defined as a 24-hour period. The investigators included all patients over 16 years old who required one or more fluid resuscitation episodes during the study period. There were no exclusion criteria. The total number of patients being treated in the ICUs on the study day was also recorded. We defined a fluid resuscitation episode as an hour during which a patient received a specifically prescribed intravenous fluid bolus of any crystalloid or colloid solution, a continuous infusion of 5mL/kg/hour or greater of crystalloid and/or any dose of colloid by continuous infusion.In Brazil, the sites collected data on any single day between December 9We recorded information on fluid availability in the participating ICUs as well as data related to patients, including demographic data, illness severity scores, admission diagnosis, laboratory test data, clinical data on the study day, predefined subgroup characteristics , and information on the type and volume of fluids for resuscitation. The reason for fluid resuscitation and the prescriber characteristics were also recorded. We defined specialist or assistant physician as the board-certified intensivist or the physician responsible for the ICU on the study day. We defined senior resident or fellow as graduated students or residents in the last years of their residency, and we defined residents as those in the first years of their residency regardless of the specialty as it is usual in Brazil to have residents of different specialties in training.Instituto D’Or de Ensino e Pesquisa, Rio de Janeiro, Brazil.We collected all data using an electronic data capture system , hosted at Continuous variables are expressed as the mean ± standard deviation - SD or the median [interquartile range]. Categorical variables are expressed as counts (percentages). The comparison of the data between Brazil and other countries and between the administration of colloids and crystalloids in Brazilian patients were performed using a t-test or Wilcoxon rank-sum test for continuous data or Pearson’s chi-squared test for categorical data, as appropriate. Differences in the proportions of crystalloid and colloid episodes were tested using generalized estimating equations (GEEs), accounting for clustering at the patient level. multivariable analyses using GEEs accounting for clustering at the patient level were conducted to determine associations between patient demographics, clinical characteristics and the type of fluid administered. We used 2 binary outcomes in the analysis: 1) crystalloid episode Yes versus crystalloid episode No, and 2) colloid episode Yes versus colloid episode No. The denominators of these two outcomes were the total number of fluid episodes; thus, as a given patient could have received crystalloids as well as colloids within the same hour (the same fluid episode), the total number of fluid episodes was higher than the sum of crystalloid episodes and colloid episodes. As these outcomes were analyzed separately, two different sets of odds ratios (ORs) were generated for each variable. Variables meeting a predetermined level of statistical significance (p < 0.1) with the administration of crystalloids or colloids in univariate models were included in the final multivariable model. Associations were considered statistically significant if p < 0.01. The results of the multivariable analysis are presented as adjusted ORs and 95% confidence intervals (95%CI). Details regarding the handling of missing data are provided in the main paper. All analyses were carried out using the R statistical software package, version 3.1.0 (2014-04-10).As in the main study, During the 24-hour study period, 3,214 patients were included in Brazil, of whom 519 (16.1%) received fluids. Almost half of the patients received fluids within the first two days of ICU admission (46%). The baseline characteristics of patients in Brazil and those of patients in the other countries are shown in In Brazil, 217 ICUs participated in the study (participating centers are listed at the end of this manuscript). The overall summary of FLUID-TRIPS data is shown in Supplementary material). The total volume of resuscitation fluid received and net fluid balance on the survey day were higher in Brazil than in other countries , and the main indication for fluid resuscitation was impaired perfusion and/or low cardiac output (71.7%)  , despite the availability of different fluids at the participating ICUs . In Brazil and other countries, the most commonly used balanced crystalloid solution was Ringer’s lactate. Plasma Lyte was used more frequently in other countries than in Brazil . The percentage of patients receiving crystalloid or colloid solutions or the number of crystalloid or colloid episodes were not modified in the presence of trauma, TBI, sepsis or ARDS. These conditions did not lead to significant changes in the total volume of resuscitation fluid received on the survey day. However, patients with sepsis and ARDS had a higher net fluid balance on the survey day .Compared to other countries, crystalloid solutions were more frequently used than colloid solutions in Brazil . In BrazWe analyzed the factors associated with the choice of crystalloids or colloids for fluid resuscitation episodes. The multivariable analysis  suggesteAmong the patients who received crystalloids, the odds of having an albumin level ≥ 2g/dL were 9.4 times (OR = 8.6 [0.8 - 89.8]) that of having an albumin level < 27g/dL.Supplementary material).There was also a higher chance of having unknown/missing values for albumin  than having an albumin level < 27g/dL. Similarly, among those who received colloids, the odds of having an albumin level ≥ 27g/dL was one-fiftieth (OR = 0.2 [0.0 - 0.9]) that of having levels < 27g/dL. In addition, for patients receiving crystalloids, the odds of them being prescribed by a senior resident/fellow was 9.9 times higher  than that of them being prescribed by a specialist/assistant physician. For patients receiving colloids, there was no clear association with fluid prescriber. The univariate analysis is available in table 7S  was the most prescribed crystalloid in Brazil, despite the availability of balanced solutions. In other countries, balanced solutions were the preferred crystalloids. The availability of serum levels and the current albumin level were the factors associated with the choice of crystalloids or colloids for fluid resuscitation. In addition, the type of fluid prescriber was significantly associated with crystalloid use. Aligned with these principles, the main indications for fluid administration in Brazilian ICUs were similar to those found in the main study and in other studies addressing this issue.,19 Our results also showed a reduction in the use of colloid solutions.-20 The evidence of harm from recent randomized clinical trials (RCTs) with synthetic colloids such as HES (3-12) could explain the preference for crystalloid solutions in Brazil and in other countries. It is interesting to note that the higher proportion of the use of colloids in other countries is represented by the use of albumin. As albumin is expensive, the costs may have limited its use in Brazil, a middle-income country.The results in Brazil are consistent with more recent studies regarding fluid resuscitation practices. Fluid resuscitation aims at improving tissue perfusion by restoring the perfusion pressure of vital organs and ensuring adequate cardiac output.-24 We believe our findings are potentially useful for hypothesis generation, and further studies are necessary to better evaluate potential factors associated with this choice.Another aspect that differentiates Brazil from other countries was the use of 0.9% sodium chloride as the crystalloid solution of choice. Although Plasmalyte is a high-cost balanced solution in Brazil, there are low-cost balanced solutions available . Our study was not designed to assess the potential reasons for this difference between Brazil and other countries. It is possible that this was influenced by the variation in availability among the sites and countries, which would bias any further analysis. The relatively small number of patients and variables in our database might also compromise the reliability of eventual findings. Another possible explanation is a cultural preference derived from years of using saline potentially associated with a reduced awareness of the potential adverse effects of hyperchloremic solutions, as the controversy around balanced vs. unbalanced crystalloids was not as intense as it is currently. However, in Brazilian ICUs, albumin serum levels had a clear role in guiding the choice of fluid. This preference is not supported by the available evidence. The results from high-quality RCTs suggest that intravenous albumin administration does not reduce the mortality rate in mixed populations of critically ill patients, including those who have hypoalbuminemia. Even albumin supplementation in addition to crystalloids targeting serum concentrations higher than 30g per liter in septic patients did not improve survival at 28 and 90 days. Thus, we believe that this finding probably reflects local practice patterns rather than solid evidence. It is worth mentioning that senior residents and fellows were more likely to prescribe crystalloid fluids to patients than specialists, probably suggesting that academic exposure to scientific evidence promotes changes in practice behaviors. Another potential explanation is the generation difference. The specialists were previously exposed to a cultural environment in which colloids were heavily used based on their potential better effect on oncotic pressure. In contrast, the new generation, composed of residents and fellows, was exposed to scientific evidence of harm with colloid use. This also suggests that continuous training, even for specialists, is important to ensure better quality of care.Sepsis, ARDS, trauma and TBI did not influence the choice between colloids and crystalloids. The uncertainty about the ideal fluid for these specific diseases could explain this finding. This is the first study to describe resuscitation fluid practices in a large sample of Brazilian ICUs. The use of standard case report forms and definitions across all countries and detailed information on clinical factors that may potentially influence the choice of fluid for resuscitation at the time of the fluid episode allowed not only comparisons with other countries but also analyses of national practice patterns. Among the limitations of the study, it is important to mention the generalizability of the results. Even with a large sample of ICUs, the use of convenience sampling might have not reflected practices adopted in all Brazilian ICUs. Another limitation is the definition of fluid resuscitation episodes. Finally, the interpretation of fluid administration practices in specific patient populations, such as those with sepsis, requires caution due to relatively small patient numbers.This study has strengths and some limitations, some of which were mentioned in the main study.Crystalloids were more frequently used than colloids for fluid resuscitation in Brazilian intensive care units. Sodium chloride (0.9%) was the most prescribed crystalloid in Brazil, despite the availability of balanced solutions. The availability of serum levels and the low albumin level were the factors that influenced the choice between crystalloid or colloid for fluid resuscitation. In addition, senior residents/fellows were more likely to prescribe crystalloid fluids to patients than specialists.


Even during hydrodynamically stable fluid displacement where a more viscous fluid displaces a less viscous fluid in porous media or in Hele-Shaw cells, the growing interface fluctuates slightly. This fluctuation is attributed to thermodynamic conditions, which can be categorized as the following systems: fully miscible, partially miscible, and immiscible. The dynamics of these three systems differ significantly. Here, we analyze interfacial fluctuations under the three systems using Family–Vicsek scaling and calculate the scaling indexes. We discovered that the roughness exponent, The displacement of one fluid by another is an important process, not only in industrial and environmental fields, such as chromatography, enhanced oil recovery, and CO They performed hydrodynamically stable displacements in a radial Hele-Shaw cell and observed interfacial deformation only in the partially miscible system [When a more viscous fluid displaces a less viscous fluid in porous media or in a Hele-Shaw cell, which is comprised of two parallel plates with a thin gap between them, the interface between the two fluids becomes stable. However, in the reverse situation, where a less viscous fluid displaces a more viscous fluid, the interface becomes hydrodynamically unstable and forms finger-like patterns. This phenomenon is known as the Saffman–Taylor instability  or viscoreported ,10,11,12reported ,14,15,16e system . They pre system ,18 due te system . It is de system , and is e system .Interfacial fluctuations are ubiquitous in nature at various lengths and time scales. They include the propagation of flame fronts, as well as deposition processes, such as falling snow, atomic deposition, and bacterial growth . In geneing laws  as it exZ theory . In two l models . An expel models . They dia et al. , who obsi et al.  demonstrThe roughness of the growth interface for various phenomena was analyzed via scaling. Fluid displacement occurs frequently in industrial and environmental improvements ,27,28; h2SO4 solution system as fully miscible, a 36.5 wt% PEG–20 wt% Na2SO4 solution system as partially miscible, and a phase L–phase H (explained below) as immiscible  in a hydrodynamically stable displacement of liquids using Family–Vicsek scaling. We confirmed that the fluctuations in all systems with steady values of uid flow ,34,35. H