Red blood cell (RBC) distribution, RBC shape, and flow rate have all been shown to have an effect on the pulmonary diffusing capacity. Through this study, a gas diffusion model and the immersed finite element method were used to simulate the gas diffusion into deformable RBCs running in capillaries. It has been discovered that when RBCs are deformed, the CO flux across the membrane becomes nonuniform, resulting in a reduced capacity for diffusion. Additionally, when compared to RBCs that were dispersed evenly, our simulation showed that clustered RBCs had a significantly lower diffusion capability. Through the transport of blood, multicellular organisms’ cells receive nutrients, oxygen, and waste removal from the blood circulatory systems of humans. Contrary to traditional opinion, blood is actually a complex suspension of deformable cells, proteins, platelets, and plasma, as opposed to the homogeneous fluid that is typically thought of as blood. The major cause of death in developing countries is currently cardiovascular disease. Numerous arterial and vascular illnesses have poorly understood short and long-term pathways of progression, which has a direct impact on the formulation of therapeutic strategies as well as disease diagnosis and prevention. Thus, knowledge of blood composition, as well as its transportation and rheological properties, has major clinical value.2, CO, O2, etc.) in the alveolar region of the lungs has recently drawn increasing attention. The pulmonary diffusive gas transport is influenced by RBC distribution in alveolar capillaries, particularly the intake of Carbon Monoxide (CO). Diffusing capacity is a measure of the lung’s effectiveness at transferring gas. Recent research has demonstrated that while clustered distribution of RBC results in the lowest outcomes, uniform distribution produces the maximum CO diffusing capacity. The pulmonary diffusing capacity, in particular CO absorption, is impacted by differences in RBC shape [2 or CO2.The impact of blood flow on the transfer of gases , the RBCs flow in capillary is resolved. In a capillary flow, RBC motion and deformation are linked to a gas diffusion issue. Because the small domain size and number of RBCs used in the simulation, as well as other intricate biochemical elements present in real blood that are not taken into account by our cell-cell interaction model, make it difficult to make precise comparisons to the experimental results. This issue will be addressed in our future works. However, to our knowledge, this is the first research that links the microscopic mechanism of RBC aggregation to the macroscopic blood viscosity via direct 3D numerical simulation.The following is a summary of the paper. In The IFEM was created by Zhang et al.  to addreLet us look at an incompressible, three-dimensionally deformable structure in The solid domain can occupy a finite volume in the fluid domain, in contrast to the IB formulation. The union of two domains can be thought of as one incompressible continuum with a continuous velocity field since we assume that both fluid and solid are incompressible. A Eulerian fluid mesh was used in the computation since the fluid covers the entire domain; a Lagrangian solid mesh was then built on top of the Eulerian fluid mesh. When creating the momentum and continuity equations, some factors related to the coexistence of fluid and solid in s and xs , respectively, represent the material points of the structure in the initial solid domain The time-invariant position vector x represents the fluid grid in the computational fluid domain v and the pressure p are the unknown fluid field variables; the solid domain requires the computation of the nodal displacement us, which is defined as the difference between the current and the beginning coordinates: us = xs − Xs. The velocity sv is the material derivative of the displacement dus/dt. Since displacement and acceleration are not explicitly utilized in fluid calculations, they are not described under the column for the fluid domain. In the fluid calculations, the velocity σ and the external force applied to the continuum balance the inertial force of a particle in a continuum.The derivative of the Cauchy stress We can split the inertial force into two components within the solid domain, Note that we describe the fluid domain Since the entire domain Additionally, the derivative of the Cauchy stress in Equation (3) can be divided into the following parts:The fluid stress in the solid domain of Equation (7) is generally substantially smaller than the corresponding solid stress , as can Naturally, the Lagrangian description is used to determine the interaction force Thus, by adding the fluid terms and the interaction force, the governing equation for the fluid may be formed as follows:We only need to apply the incompressibility constraint once to the entire domain because we believe the entire domain to be incompressible:We added various velocity field variables Assuming that there is no traction imparted to the fluid boundary, or that The Newton–Raphson method was used to resolve the nonlinear systems. Additionally, we used the GMRES iterative algorithm to increase computing efficiency and compute residuals using matrix-free methods ,15.Note that we neglected the fluid stress within the solid domain for the sake of conciseness. The integration domain changed from We may rewrite Equation (14) using Integration by Parts and the Divergence Theorem:Again, keep in mind that the boundary integral terms on the interface between the fluid and the structure will cancel each other for both the fluid and solid domains and that, for brevity, they are not included in the corresponding weak forms.The second Piola-Kirchhoff stress Finally, the discretized form of Equation (12) in the interpolation from the fluid onto the solid grid can be represented asBy adding the velocities at the fluid nodes within the influence domain Using the solid particles in Equation (17) to interpolate the fluid velocities onto (16), the fluid was constrained to solid material locations within the solid domain. When the solid mesh is at least two times denser than the surrounding fluid mesh, not only is the no-slip boundary condition on the solid’s surface guaranteed, but the fluid is automatically stopped from penetrating the solid. Based on the numerical evidence, this heuristic criterion requires more research.(1)xs,n, and the fluid velocity vn at time step n,Given the structure configuration (2)fFSI,s,n for solid material points, using Equation (19),Evaluate the nodal interaction forces (3)fFSI,s,n to fFSI,n using the delta function as in Equation (20),Distribute the material nodal force onto the fluid mesh, from (4)vn+1 and the pressure pn+1 implicitly using Equations (21) and (22),Solve for the fluid velocities (5)vn+1 to vs,n+1 as in Equation (23), andInterpolate the velocities in the fluid domain onto the material points, i.e., from (6)us,n+1 = vs,n+1∆t and go back to step 1.Update the positions of the structure using The following is an illustration of the IFEM method using a semi-explicit temporal integration:Notably, the fluid and solid are coupled explicitly despite the fluid’s complete implicit solution. If we reformat the fluid momentum equation (just discretized in time for clarity), we getfm+1FSI,.It is obvious that the solid equations are calculated using values from the previous time step and that the interaction force is not updated during the iteration. This force must depend on the fluid velocity at the moment in order for there to be a fully implicit coupling, and the fluid equations should be linearized to include the term RBCs take on a biconcave disc form in suspension culture, unlike white blood cells, which allows them to pass through capillaries. A typical RBC’s surface to volume ratio is substantially larger than that of a sphere generated by a tensioned membrane due to the biconcave disc shape. Furthermore, the biconcave disc form implies that the membrane cytoskeleton possesses both membrane rigidity and bending. The RBC membrane is modeled with a three-dimensional finite element formulation utilizing a Lagrangian description, as seen in wn below :(25)y¯=0C1 and C2, where J1 and J2 represent the functions of the deformation gradient C’s invariants as stated in [Finally, the material behavior of the RBC membrane is represented by a Neo-Hookean strain energy function:tated in ,17. In ttated in  and Pozrtated in  also empα is the Bunsen solubility coefficient in lung tissue and plasma; D is the diffusion coefficient; P is the CO partial pressure.We assume that CO tension gradients instantly achieve steady state and that the whole CO flux is caused by CO tension gradients driving CO diffusion into RBCs. The Laplace equation describes diffuse transport:The current study does not take into account the impact of red cell migration since CO does not equilibrate with capillary blood in the duration that blood spends in the capillary. Therefore, it is anticipated that the influence of blood flow on CO diffusion will be minimal. In the process of gas exchange, according to the speed of RBC flow in capillary (0.3–0.7 mm/s) and typical length of lung capillary (about 90 um), the time scale of transport of RBC in capillary during the gas exchange within one second (0.13–0.3 s) can be obtained, while gas diffusion can reach equilibrium in ms. Thus, we can assume that the static diffusion process is true.P = Torr in the alveolar phase, 5 µm above the air–tissue interface, and P = 0 Torr at the inner membrane surface of the RBCs are the boundary conditions. We examined the full capillary length for each asymmetric distribution since RBCs might not be evenly spaced and, as a result, the capillary model might not be symmetrical. In air, tissue, and plasma, CO has distinct properties for diffusion. P is determined, the diffusive flux of CO for each element is computed as CO fluxMD of each typical region, is obtained byAP is the mean CO partial pressure at the air–tissue interface.Once the distribution of With different RBCs running within a channel, we were able to calculate the overall CO flow in this manner.The original mesh utilized in the modeling is depicted in 3·s−1·torr−1 for a set of fluid mesh with 88,200 nodes and 81,792 elements. This value is within 2% of that provided by the finest mesh, which has 172,081 nodes and 162,000 elements. Because of its reasonable accuracy and efficiency, we decided to use this type of mesh for our simulations. Our simulation, which uses a time step of 0.001 s, runs for around a minute on a computer with a 2.0 GHz CPU.Besides, four sets of fluid meshes with progressively smaller mesh sizes are used to quantify the diffusion flux of CO from the wall to the RBC surface in order to examine the impact of mesh resolution on our simulation. With nodes above 1000, the solid mesh is found to have essentially no impact on simulation outcomes; consequently, a solid mesh with 1743 nodes and 8016 elements was employed throughout our simulations. However, it is possible to obtained the total CO flow of 1.103 µmLow absorption rates result in ineffective gas transfer, and therefore the relationship between absorption rate and blood flow rate/pressure is useful for therapeutic purposes. Let us start by thinking about how RBC deformation affects the rate of CO absorption. A capillary is filled with a few evenly distributed RBCs in a circular shape. The capillary flow’s inlet velocity is set at 50 m/s. We investigated how the deformation of the RBCs affects the diffusion capacity.RBCs are deformed into a variety of asymmetric configurations under dynamic flow conditions, such as parachute-like shapes during capillary flow. M when a single RBC is bent from a circular shape into a parachute is shown in M falls from 1.18 to 1.03 throughout this deformation phase, which can be attributed to a lower absorption rate caused by the RBCs’ folded tails.The time history of DThe “breath-holding” and “stationary state” methodologies are the two main experimental methods for calculating diffusion capacity . The “brThe distribution of RBCs can also affect how much CO is taken in. The CO absorption of three different RBC distributions—uniform, random, and clustered—was investigated by Connie et al. The maximum CO uptake was observed to result from uniform distribution, whereas the worst CO uptake resulted from clustered distribution. While the actual CO uptake is carried out by 3D deformable RBCs in a dynamic process, the calculations in20 are based on circular, rigid 2D RBCs in a static condition.We looked at the uniform, random, and cluster distributions of RBCs. M, as well as the DM for each RBC. The total DM rises as the number of RBCs does, but the DM per RBC falls. This is because the total flow reduces as the local gas partial pressure is reduced when two RBCs are very close to one another.It is anticipated that the diffusion capabilities at various locations in the capillary network may vary because RBCs dynamically deform and form clusters.2 and O2 carried by red blood cells in the lungs of human body. (b) shows our experimental results, that is, the effect of RBCs on the amount of gas diffusion in different aggregation and deformation states, and it can be clearly concluded that the diffusion capacity of CO decreases when RBCs deform, and it can also be shown that RBCs in the aggregated state have significantly lower diffusion capacity.Through the use of an immersed finite element platform, we have created a computer model that connects the fluid–structure interaction problem with the gas diffusion issue. To show the viability of the created coupled model, the dynamic process of deformable RBCs flowing and gas diffusion in capillaries was investigated. Our IFEM research revealed that the RBC deformation and distribution both affect the gas diffusion capacity. The CO flux across the warped RBC membrane became nonuniform when the RBCs were deformed, which lowers the diffusion capacity. The flow of RBCs across branching capillaries will be researched in the future. We can also anticipate applying this model to other areas, such as Circulating Tumor Cells. During metastasis, CTCs are produced by primary tumors and circulate in the blood vessel . We may


In this particular case, it seems that a simple linear regression model would fit the data fairly well. It is not clear from Figure Conflict of interest: George Rakovich receives speaker fees from Medtronic® and a research grant from Baxter®.


It is shown that tumour volume distributions can yield information on two aspects of cancer research: tumour induction and tumour control.From the hypothesis that the intrinsic distribution of breast cancer volumes follows an exponential distribution, firstly the probability density function of tumour growth time was deduced via a mathematical transformation of the probability density functions of tumour volumes. In a second step, the distribution of tumour volumes was used to model the variation of the clonogenic cell number between patients in order to determine tumour control probabilities for radiotherapy patients.Distribution of lag times, i.e. the time from the appearance of the first fully malignant cell until a clinically observable cancer, can be used to deduce the probability of tumour induction as a function of patient age. The integration of the volume variation with a Poisson-TCP model results in a logistic function which explains population-averaged survival data of radiotherapy patients.The inclusion of tumour volume distributions into the TCP formalism enables a direct link to be deduced between a cohort TCP model (logistic) and a TCP model for individual patients (Poisson). The TCP model can be applied to non-uniform tumour dose distributions. The estimation and analysis of tumour sizeSecondly, the distribution of cancer volumes reflects the distribution of clonogenic cell numbers (assuming a constant clonogenic cell density) and thus can be used for modelling tumour control probability (TCP) for cohorts of radiotherapy cancer patients. Currently no general analytical formulation of TCP exists which includes the population variation of tumor volumes. Often a logistic function is used to model population averaged TCP λ one can write:We assume here that the intrinsic probability density function (pdf) for the distribution of tumour volumes follows an exponential distribution.With intrinsic we mean here that the exponential probability distribution describes the distribution of all tumour volumes in a cohort of subjects, independently of a potential diagnosis. E.g., if we would select a specific age group of women, all breast tumours, including also un-observed and microscopically small ones, would be distributed exponentially. However, the mean tumour volume is assumed to change with the age of the observed cohort.g0(V) can be written as:V/a. The normalization constant was chosen such that the integral over the distribution function g0 is 1. In equation 3, a is a parameter representing a characteristic volume such that a = 0 all tumours are detected and the distribution function describes the intrinsic tumour-volume distribution. The value of a depends on the quality of the applied diagnostic procedures. As we will see later, a was decreased after mammography was employed for the detection of breast cancers.Obviously, such an intrinsic probability density function can never be observed, as tumours are only detected after they reach a certain volume. For tumour diagnosis a certain minimal tumour size is required such that they are detectable with state-of-the-art diagnostic procedures. If it is assumed that for small volumes the detection probability of a tumour is increasing linearly with volume, the actually observed tumour volume distribution a to be studied. Here we use the data of Carter et al. a was fitted separately, as the observation interval for the two data-sets was different.For model testing, breast cancer volume distributions were selected, as they include usually a large number of patients and the data are available at different times, which allows the impact of improved diagnosis on the parameter L, into a clinically observable cancer. Thus, L is the lag (time) between the first fully malignant cell and cancer. Usually the parameters of tumour induction models are determined by fitting tumour incidence data by assuming a fixed lag time usually estimated from the A-bomb survivor data Many tumour induction models gi(V) can be transformed into the pdf of the lag time f(L). Thus, knowledge of the tumour volume distribution function can yield the shape of the tumour lag time distribution and vice versa. A schematic concept of the process is provided in If a simple exponential tumour growth model is used to describe the size increase of the tumour during the lag time, the pdf which describes the tumour volume distribution b is the tumour growth factor and L the lag time. The volume VØ is the initial volume before exponential growth starts. Here VØ = 10−9 cm3, i.e., the volume of one cell, was used. The transformation between the two pdfs is a strict mathematical procedure given by g(V) from equation f(L) can be easily determined using equations 5 and 6:f(L) from L = 0 to infinity is unity. We have chosen infinity as the upper integration limit instead of a realistic maximum tumour volume, as the probability for very large tumours is practically zero. E.g., assuming an average tumor volume of 40 cm3 an integration of the exponential distribution up to a realistic upper limit of 500 cm3 yields 0.999996 instead of 1.Here an exponential tumour growth function is used to convert one probability density function into another:b = 1.05 y−1 for breast cancer taken from Talkington and Durrett For the visualization of a realistic lag time distribution we have used the tumour growth factor ρ is the number of tumour clonogens per unit volume, V is the tumour volume and S is the surviving fraction of cells after radiotherapy treatment. If the TCP curves are generated with single values of intrinsic radiosensitivity and clonogenic cell number, the experimentally obtained data cannot be fitted, as the TCP curves show a very steep gradient. This discrepancy is usually explained by the fact that TCP curves derived from populations of human tumours that have been similarly treated include different tumour volumes, different initial clonogen densities, different intrinsic radiosensitivities and different oxygenation status. However, these parameters cannot all be modeled analytically and as a consequence, they are often varied numerically  following radiotherapy. Most TCP models are based on a calculation of the Poisson probability of there being no surviving cells in a population of tumours after a fractionated treatment lly e.g. . An altelly e.g. . HoweverBy assuming that the tumour volumes are distributed according to equation 2, and assuming a constant clonogenic density we obtain:S is modelled by an exponential function using the linear quadratic model:The result of this integration yields an analytic expression for TCP in terms of a logistic model were survival ρ = 108 cm−3, 3, α = 0.36 Gy−1, β = 0.036 Gy−2, D = 30…70 Gy and df = 2 Gy, where α and β are the parameters of the LQ-model, D is the total radiotherapy dose and df is the dose per fraction.Some typical parameters have been used: a = 0.42 cm3, hence better detection efficiency, when compared to older data (a = 1.84 cm3). The average volume obtained from the fit of the exponential distribution to the two data sets is 20.7 cm3. The fitted parameters with the corresponding errors are listed in In b for breast cancer and an average tumour volume of 20.7 cm3 obtained from the comparison of the tumour size distribution with the epidemiological data was used. The average lag time for breast cancer was determined to be 22.1 years with a variance of 1.5 years. This value is consistent with the findings from the A-bomb survivors with a lag time between 10 and 30 years 3.The expression for the pdf of breast tumour lag times from equation 7 is plotted in 3, respectively. The solid lines represent the TCP calculations including an exponential distribution of tumour volumes from equation 9. Clearly, a broadening of the TCP curve is visible.In a. The data set covering diagnosed breast cancers from 1973–1998 results in a much smaller a (0.31) than the older data set . This can be explained by the clinical introduction of mammography in the late 1980s which resulted in the diagnosis of much smaller breast tumours than before a is expected for the newer data-set as only mammography-diagnosed tumours are included in the newer data In this work it was hypothesized that the intrinsic distribution of tumour volumes in a cohort is exponentially distributed. To account for the fact that the probability of the clinical detection of a tumour increases with tumour volume, the exponential distribution was modified with a sigmoidal function. The fit of this distribution function to breast volume data from literature covering two different time intervals of breast cancer diagnosis results in two different locations of the distribution maximum and consequently in different values of parameter b which we have taken from literature. The goal of this work was not to determine the absolute lag times for breast cancer, but to derive an expression for the distribution of lag times.If exponential tumour growth is used as a transformation function the probability density function for tumour volumes can be transformed into the probability density function of tumour growth or lag times, respectively. The obtained lag time distribution function can be used in the development of mechanistic models of background cancer risk. Usually, such models combine the probability of occurrence of a malignant cell per individual per year with a constant lag time in order to estimate cancer induction A limitation of this work is that tumour growth was simply modeled with an exponential model. An exponential model might be a satisfying approximation as long as the tumours are small. However, it is known that the exponential model is not universally appropriate and many other models including the Gompertz model have been proposed It was shown that the assumed exponential variation of tumour volumes can be used to derive an analytical representation for the population averaged TCP. The Poisson based TCP model for individuals was integrated by using the volume distribution as a weighting function. This resulted in the logistic function as a population averaged TCP model. The logistic function was used in the past to fit successfully observed tumour control data for radiotherapy patients without any bio-physical justification. In this work a logistic TCP model for cohorts of patients is explicitly derived from a Poisson TCP-model for individuals.The methodology presented in this work enables in principle the cohort TCP model given by equation 9 to be fitted to survival data for groups of patients. However, radiotherapy patients who have been similarly treated include not only different tumour volumes, but also different clonogen densities, different intrinsic radiosensitivites and different oxygenation, none of which were considered in this work. We are currently working on an integration of most of these factors into our analytical model. Therefore, this work should be viewed as a first step towards the development of a TCP model that can be applied equally to individuals and to populations of patients.


Most cancers use glycolysis to harness energy for their infinite growth, invasion and metastasis8. Activation of thermogenic metabolism in brown adipose tissue (BAT) by cold and drugs instigates blood glucose uptake in adipocytes9. However, the functional effects of the global metabolic changes associated with BAT activation on tumour growth are unclear. Here we show that exposure of tumour-bearing mice to cold conditions markedly inhibits the growth of various types of solid tumours, including clinically untreatable cancers such as pancreatic cancers. Mechanistically, cold-induced BAT activation substantially decreases blood glucose and impedes the glycolysis-based metabolism in cancer cells. The removal of BAT and feeding on a high-glucose diet under cold exposure restore tumour growth, and genetic deletion of Ucp1—the key mediator for BAT-thermogenesis—ablates the cold-triggered anticancer effect. In a pilot human study, mild cold exposure activates a substantial amount of BAT in both healthy humans and a patient with cancer with mitigated glucose uptake in the tumour tissue. These findings provide a previously undescribed concept and paradigm for cancer therapy that uses a simple and effective approach. We anticipate that cold exposure and activation of BAT through any other approach, such as drugs and devices either alone or in combination with other anticancer therapeutics, will provide a general approach for the effective treatment of various cancers.Glucose uptake is essential for cancer glycolysis and is involved in non-shivering thermogenesis of adipose tissues Mild cold exposure activates a substantial amount of brown adipose tissue (BAT) in a patient with cancer, reducing tumour-associated glucose uptake, and activation of BAT in mice inhibits the growth of tumours by decreasing blood glucose and impeding glycolysis-based metabolism in cancer cells. A distinctive feature of altered metabolism commonly shared by most cancers is the high uptake of glucose that undergoes fermentation metabolism to generate lactate12. Under aerobic conditions, cancer cells use the glucose-fermentation–lactate pathway to harness energy in the form of ATP13. This cancer-associated aerobic glycolysis, that is, the Warburg effect, is an inefficient pathway to generate ATP13. Compared with mitochondrial respiration, the Warburg effect is the default pathway for energy production and only two ATP molecules are generated per glucose through aerobic glycolysis13. However, a 10–100-fold accelerated rate of glucose-based aerobic glycolysis occurs in tumour cells relative to that of glucose complete oxidation in the mitochondria of healthy cells14. Indeed, over 70% of human cancers show amplified expression of glycolytic genes17.Metabolic alterations are one of the hallmarks of cancer and malignant cells often reprogram metabolism to promote their growth, proliferation, migration and survival13. Glucose uptake is a rate-limiting step of aerobic glycolysis and is mediated by glucose transporters (GLUTs) in the cancer cell membrane19. Along with tumour growth, hypoxia becomes ostensible and hypoxia upregulates GLUT1, the key GLUT expressed in cancer cells, through hypoxia-inducible factor-1α (HIF-1α)-triggered transcriptional regulation20. Aerobic glycolysis provides abundant metabolites as intermediates for macromolecule biosynthesis, including the nucleotide substrates ribose-5-phosphate and glycine as well as the lipid substrate glycerol21. Accumulation of intracellular lactic acids facilitates their secretion through the proton-linked monocarboxylate transporter, causing a high grade of acidosis in the TME23.High rates of aerobic glycolysis results in cancer cells taking up more glucose from the tumour microenvironment (TME) and producing high amounts of lactic acids4. Cold-acclimatization-, diet- and drug-triggered sympathetic activation induces BAT activation and the conversion of white adipose tissue (WAT) to a brown-like phenotype27. Accordingly, activated BAT and browning WAT generate heat through non-shivering thermogenesis (NST)29, which is mediated by uncoupling protein 1 (UCP1) expressed in the inner membrane of the mitochondria30. Accumulating evidence shows that glucose contributes to the  BAT thermogenesis and genetic knockdown of Glut1 and Glut4 or hexokinase  markedly impairs metabolism9. BAT-mediated NST is an effective energy-expenditure mechanism for reducing body weight and improving metabolic dysfunctions in obese and diabetic animals27. As a substantial amount of BAT tissue mass exists in adult humans, it is speculated that activation of BAT thermogenesis would provide an attractive approach for treating obesity and type 2 diabetes mellitus27.BAT is a specialized tissue for energy expenditure by generating heatUcp1 restored the tumour growth rate under cold exposure. We provide mechanistic insights into NST-triggered tumour suppression. Finally, we provide preliminary findings of BAT activation in both adult healthy humans and a patient with cancer by mild cold exposure. Cold exposure also markedly reduced glucose uptake in a human tumour. Our discoveries provide a concept for cancer treatment and will offer substantial clinical benefits for patients with cancer.Here we show that BAT activation induced by cold acclimatization markedly inhibited the growth of various solid tumours, dependent on UCP1 thermogenesis. BAT removal and genetic deletion of 13. However, alterations of the TME and the host macroenvironment often engender metabolic reprogramming of cancer cells. In particular, activation of lipid metabolism may substantially reprogram cancer metabolic pathways and, as a consequence, alter tumour growth rates, metastatic potentials and drug responses34. To study the effect of thermogenesis-related metabolism in adipose tissues on tumour growth and the survival of hosts with cancer, we examined the effect of cold acclimatization, which is known to activate BAT35, on tumour growth and progression.Malignancy is considered to be a metabolic disorder and most solid tumours acquire their energy for growth and progression through accelerated glycolytic metabolism, that is, the Warburg effectSubcutaneous implantation of colorectal cancer (CRC) into immunocompetent C57BL/6 mice resulted in marked inhibition of tumour growth under 4 °C relative to those under 30 °C Fig. . The col41. Interestingly, cold exposure improved hypoxia in the CRC tumours -positive cancer-associated fibroblasts remained unchanged  to 4 °C markedly inhibited tumour growth incidence Fig. . Similarwth Fig. . Similarwth Fig. . These gApcMin/+ intestinal adenoma model largely excluded this probability, we implanted tumours into internal organs to further exclude this possibility. We chose the liver as a site for implantation because CRCs often metastasize to the liver tissue. Similar to the subcutaneous tumour model, implantation of mouse CRC into the liver resulted in marked inhibition of tumour growth under cold acclimatization  as measured in the anus was increased compared with that under the 30 °C condition + mitochondrion contents and CD31+ microvessel density were markedly increased in the BAT under cold acclimatization , and exhibits a browning phenotype18F-fluorodeoxyglucose (18F-FDG) in various organs of tumour-bearing mice. Under thermoneutral conditions, 18F-FDG distribution mainly accumulated in the tumour tissues and modest signals were found in the BAT in the xenograft and spontaneous tumour models  and 18F-FDG signals were barely detectable in tumours despite the approximately equal tumour sizes in the cold and thermoneutral groups  imaging analysis by measuring the uptake of els Fig. . Howeverups Fig. . Note thurs Fig. . These f5. To study the metabolic changes under cold exposure, thermogenic metabolism was measured in tumour-bearing mice. Despite the similar tumour size in the 4 °C and 30 °C groups, cold exposure increased the metabolism in CRC-tumour-bearing mice relative to the thermoneutral group  by RNA-sequencing (RNA-seq) showed that both glycolytic and lipid metabolisms in cold-exposed CRC tumours were attenuated Fig. . DetaileGlut1, Glut4 and Glut7, were markedly decreased in cold-exposed tumours  mice, cold exposure significantly reduced mouse body weight  were recruited for the study. Wearing very light clothing, these healthy individuals were exposed to a mildly cold ambient temperature at 16 °C for 2–6 h per day for a consecutive 14 days. PET scan analysis showed that several individuals from both the male and female groups exhibited marked activation of BAT in the bilateral areas of supraclavicular, cervical and parasternal regions Fig. . The degure Fig. .Fig. 6Ac18F-FDG  BAT was also activated by cold exposure in a patient with cancer; and (4) exposure of a patient with cancer to mild cold conditions markedly reduced glucose uptake in the tumour tissue. Together, these human data firmly link our preclinical findings to clinical relevance, although more rigorous clinical studies are needed to strengthen these conclusions.We next performed a pilot study on an 18-year-old patient with Hodgkin’s lymphoma. At the time of this study, this patient had received a combination chemotherapy of adriamycin, bleomycin, vinblastine and dacarbazine for 5 cycles. Our study was performed at the fifth cycle of chemotherapy intervals. This patient, wearing light clothing, was exposed to mild cold of 22 °C for 7 days. PET–CT scanning demonstrated significant amounts of BAT in the bilateral areas of supraclavicular, cervical and parasternal regions and became activated with heavy uptake of FDG Fig. . Howeverion Fig. . The CT-ion Fig. , which dure Fig. . These p13. Accumulating experimental evidence shows that genetic and pharmacological interference of the glycolytic pathway markedly attenuates cancer cell proliferation and tumour growth43. On the basis of this principle, therapeutic agents targeting aerobic glycolysis have been developed and entered clinical trials for treating patients with cancer44. Here we provide a therapeutic concept for cancer treatment by switching on thermogenic metabolism in BAT. Notably, exposure of tumour-bearing mice to cold results in potent inhibitory effects on tumour growth and the cold-induced antitumor activity is at least equivalently potent to most anticancer drugs. Exposure of tumour-bearing mice to mild cold 22 °C did not result in tumour suppression. Perhaps, 22 °C was not sufficiently low to fully activate BAT in our experimental models. It has been described that a few degrees difference of ambient temperature can affect tumour growth45.Cancer cells signify a metabolic machinery in which exchanges of biomolecules of sugars, lipids, amino acids, metabolites and gases vigorously occur through distinct metabolic pathways to facilitate tumour growth and metastasis. Among these metabolic pathways, cancer glycolysis—that is, the Warburg effect—is probably the best-characterized mechanism for supporting tumour growth by generating ATP as energy molecules18F-FDG uptake in the tumour tissue and simultaneous activation of BAT in a patient with cancer validate the concept of jeopardizing cancer glycolysis by cold exposure. Although it is difficult to draw conclusions from data obtained from a single patient with cancer, these preliminary findings at least demonstrate the clinical relevance of our study. Rigorously designed clinical studies are warranted for strengthening this concept. Together, our findings represent a new concept for cancer therapy that patients with cancer could potentially benefit from.This therapeutic approach is simple, cost-effective and feasible in almost all hospitals and even at home, and is most likely omnipresent for all cancer types. As adult humans have a sufficient amount of BAT that effectively takes up blood glucose after cold exposure, we reasonably speculate that a similar anticancer activity should also be seen in human patients with cancer. Our preliminary results corroborated previous findings that adult healthy male and female individuals possess a substantial amount of BAT in the supraclavicular, cervical and parasternal regions, which was activated by exposure to mild cold. The short exposure to mild cold ambient temperature of 16 °C is well tolerated by adult humans without causing any adverse effects. Marked decreases in ApcMin/+ model further excludes the anticancer effect by chilling the tumour tissue. These experimental settings provide compelling evidence to exclude the likelihood that the attenuated tumour growth rates were caused by low temperature alone. Along this line of interpretation, surgical removal of BAT reverses the cold-associated reduction in tumour growth rates, providing powerful evidence of BAT-activation-manipulated tumour growth. Similarly, genetic deletion of Ucp1 also ablates cold-induced tumour suppression. However, genetic deletion of Ucp1 has been shown to result in mitochondrial deficiency in mice46. The UCP1-dependent tumour suppression by cold may potentially reflect the mitochondrion-dependent suppression by BAT.We undertook three experimental approaches, including: (1) measuring subcutaneous temperature by a thermal probe; (2) implantation of tumours in internal organs; and (3) spontaneous tumours in genetic mouse models to exclude the possibility that direct contact of a subcutaneous tumour with low temperature deterred tumour growth. Under cold exposure, the subcutaneous temperature slightly increased rather than decreased, probably due to the robust NST in the animal body. Similarly, CBT remains unaltered during cold exposure relative to thermoneutrality. Consistent with the overall evidence of thermogenic alterations, tumours implanted into internal organs, such as the liver, also show impaired growth rates. Moreover, suppression of intestinal adenoma formation and growth in the genetic ApcMin/+ model, we showed that chronic cold exposure also potently suppressed tumour formation in addition to inhibition of tumour growth. Finally, (4) studying the effect of chronic exposure to cold on tumour suppression. These results demonstrate that activation of BAT thermogenesis is responsible for tumour inhibition.The advantages of using spontaneous tumour models of genetically propagated mouse strains encompass: (1) excluding the possibility that suppressing tumour growth by cold occurs only in fast-growing xenograft tumours, which are highly dependent on glycolysis. (2) Establishing the clinical relevance of our findings. (3) Testing the possibility of cold-triggered tumour formation. In the 18F-FDG PET–CT scanning analysis demonstrates that BAT, but not browning WAT, is the key organ responsible for glucose uptake. This finding suggests that accelerated glucose uptake by cold-activated BAT is probably the key mechanism underlying tumour suppression. However, we cannot exclude the participation of WAT, especially sWAT, in tumour suppression by cold. A recent study shows that a subpopulation of beige cells in sWAT is almost completely dependent on glucose uptake47. Perhaps extreme cold is required for triggering the WAT-mediated tumour suppression because extreme cold augments robust browning in WATs48. In addition to increases in glucose uptake, thermogenic adipose tissues also exhibit increased uptake of other nutrients and regulatory molecules for tumour growth, including succinate and branched-chain amino acids50, which may also participate in tumour suppression by cold exposure.The There are several plausible mechanisms that underlie BAT-activation-triggered tumour suppression , which may secondarily affect GLUT1 expression. The in-depth mechanism warrants further investigation.Our findings provide an example of how alteration of global metabolism substantially affects tumour growth. This concept can be generalized in other global metabolic states, which may positively or negatively affect tumour growth and even metastasis. Opposing our results, overweight and obesity accelerate tumour growth, metastasis and drug responses by providing excessive energy for cancer cellsTaken together, we show that the activation of BAT by exposure to physiologically tolerable low temperatures provides an effective approach for cancer therapy. The therapeutic effectiveness of cold exposure is at least equivalent to most available anticancer drugs.−1 penicillin and 100 μg ml−1 streptomycin . MC-38 cells were cultured and maintained in DMEM supplemented with 10% FBS, 10 mM HEPES, 0.1 mM MEM non-essential amino acid , 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. E0771 cells were cultured and maintained in Roswell Park Memorial Institute (RPMI) 1640  medium supplemented with 10% FBS, 10 mM HEPES, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. All of the cell lines used in our study were negative for mycoplasma as detected by a microplasma kit .The mouse MC-38 colon adenocarcinoma cell line was provided by R. Hernández. The mouse E0771 breast cancer cell line was purchased from CH3 BioSystems. The mouse pancreatic cancer cell line Panc02 was provided by M. Schnurr. The human colorectal carcinoma HCT 116 tumour cells, human pancreas duct epithelioid carcinoma PANC-1, mouse fibrosarcoma T241 and mouse melanoma B16-F10 were purchased from ATCC. T241, HCT 116, Panc02, PANC-1 and B16-F10 cells were cultured and maintained in a Dulbecco modified Eagle's medium  supplemented with 10% FBS, 100 U mlUcp1−/− mice in the C57BL/6 background were purchased from the Jackson Laboratory (003124). Female 6–8-week-old FVB/NJGpt-Tg(MMTV-PyMT)/Gpt mice were purchased from GemPharmatech (T004993). Male 9–10-week-old C57Bl/6J background ApcMin/+ mice (002020) were obtained from the Jackson Laboratory. Adult males and female mice aged 5–12 weeks were caged at 22 ± 3 °C under 12 h–12 h light–dark cycles before cold and warm exposure. Mice were randomly divided into groups for all of the experiments. All of the mouse studies were approved by the North Stockholm Animal Ethical Committee, Stockholm, Sweden , or by the Animal Experimental Ethical Committee of the Fudan University, Shanghai, China . No statistical methods were used to predetermine sample size. The experimenter was not blinded to the assignment of the groups and the evaluation of the results. Tumour experiments, including subcutaneous, intraorgan implantation and genetic tumour models were performed at the Karolinska Institutet, Sweden and Fudan University, China. In both locations, subcutaneous tumour volumes did not exceed 2.5 cm3 and intraorgan tumour volumes did not exceed 2.0 cm3 in any of the experiments.C57Bl/6 and SCID mice were obtained from the breeding unit at the Department of Microbiology, Tumour and Cell Biology, Karolinska Institute, Stockholm, Sweden and from the Model Animal Research Center of Nanjing University. For cold exposure, mice were housed and placed in an opened cage in the climate room at the Department of Microbiology, Tumour and Cell Biology, Karolinska Institute, Stockholm, Sweden, at the Department of Cellular and Genetic Medicine, School of Basic Medical Sciences, Fudan University, or in a climate chamber . Male and female C57Bl/6 mice aged between 5 and 10 weeks were adapted at 18 °C for 1 week before exposing to a cold temperature of 4 °C in the climate room or 5 °C in the HPP750 climate chamber or exposed to a thermoneutral temperature of 30 °C as a control, followed by tumour implantation.6 cells of T241 fibrosarcoma, E0771 breast cancer and B16 melanoma, 1–3 × 106 MC-38 tumour cells, 1.5 × 106 Panc02 cells and 5 × 106 PANC-1 cells in 100 μl phosphate-buffered saline (PBS) were subcutaneously implanted into each of C57BL/6 mouse, or immune-deficient SCID mice. For the intraliver injection model, in isoflurane-anaesthetized 7-week-old female C57BL/6 and 10-week-old male SCID mice, a subcutaneous incision was performed and the mice were injected with approximately 0.5 × 106 MC-38 or 1 × 106 HCT 116 cells in 30 μl PBS into the liver, followed by closure of the incisions with the sterile surgical suture . All surgically operated mice were subcutaneously treated with buprenorphine before and after surgical operation. Tumour sizes were measured with callipers and tumour volumes were calculated according to the standard formula (length × width2 × 0.52). For the subcutaneous tumour model, tumour-bearing mice were euthanized when the tumour size reached the indicated size and tissues including tumours, WAT depots, iBAT and liver were dissected for further assessments. Fresh tumour, WAT and BAT were immediately collected and kept on dry ice or liquid nitrogen for metabolomics and RNA-seq multi-omics analyses. Mice in the intraliver tumour models were euthanized 2–3 weeks or 6 weeks after tumour implantation. For RNA and protein extraction, fresh tissues were immediately frozen on dry ice and stored at –80 °C until further use. A portion of fresh tissues was immediately dissected, fixed with 4% paraformaldehyde (PFA)  overnight and subsequently used for histological and immunohistochemical analyses. Metabolic rates were measured when tumours reached an average size of approximately 1.0 cm3. Mice were analysed for metabolic rates, followed by euthanasia and tissue collection. Vehicle-treated mice using the same therapeutic regimens served as a control group.Approximately 1 × 1018F-FDG uptake. C57Bl/6J background ApcMin/+ male mice (aged 9 weeks) were acclimatized to 4 °C and 30 °C for 6 weeks. ApcMin/+ mice usually developed multiple intestinal adenomas between weeks 10 and 14. On week 6 after cold exposure, PET–CT analysis was performed and tumour nodules of the same size from the 4 °C- and 30 °C-exposed groups were analysed for 18F-FDG uptake.Female 10-week-old MMTV-PyMT mice were adapted at 18 °C for 24 h before exposing to 4 °C or 30 °C conditions. MMTV-PyMT mice started to develop tumours in the mammary glands at the age of 12 weeks. For BAT-removal experiments, BAT of each animal was removed at the age of 9 weeks, cold exposure started at the age of 10 weeks and continued until the termination of the experiments. At week 4 after cold exposure, PET–CT analysis was performed and tumour nodules of the same size from the 4 °C- and 30 °C-exposed groups were analysed for To measure mouse CBT, a temperature probe  was placed in the rectum of each mouse. For measuring subcutaneous body temperature, a small incision on the interscapular region of each mouse under mild isoflurane anaesthesia was surgically created and the probe was subsequently inserted into the incision for measurements. Accurate temperature was read using the MicroTherma 2T thermometer. For measuring tumour temperature, the probe was put into the tumour of the mice. All measurements were performed at room temperature.2 and 0.5% CO2. The environmental enclosure was warmed and cooled before placing the mice inside. For the whole-body energy metabolism, CO2 and O2 were measured and the data were collected every 12 min for each experimental mouse.Whole-body energy metabolism and non-shivering thermogenesis were quantitatively measured by oxygen consumption using the Oxymax CLAMS-HC comprehensive laboratory animal monitoring system with environmental enclosure (Columbus Instruments). The metabolic measurements were carried out at thermoneutral and cold temperatures. The oxygen sensor was warmed up for at least 6 h before calibration with the reference gases, that is, 100% nitrogen gas and a mixture of 20.5% OMice were starved for 4–6 h during the light phase with free access to water on the day of assessment. Glucose levels in the blood samples collected from the tail vein by a 25G needle were measured using a glucometer  immediately before and at 15, 30, 60 and 120 min after oral feeding with 1.5 mg glucose with 10 μl per g body weight and intraperitoneal injection of insulin (0.5 U per kg body weight), respectively. Mice were kept at ambient temperature throughout these assays.18F-fluoro-2-deoxy-d-glucose (18F-FDG) with a radiochemical purity of more than 95% was produced by a cyclotron (Siemens CTI RDS Eclips ST) using the Explora FDG4 module. In vivo PET–CT imaging scans and image analysis were performed using an Inveon Animal-PET-CT system . Mice exposed to 4 °C or 30 °C were scanned 1 h after intravenous injection of 3.7 MBq (100 μCi) of 18F-FDG. Experimental animals were maintained with anaesthetization using 2.5% isoflurane/oxygen before and during the scanning. Three-dimensional ordered-subset expectation maximization (3D-OSEM)/maximum algorithm was used for image reconstruction. The maximal percentage-injected dose per gram was calculated and the SUV-BW of iBAT and tumour in either the subcutaneous region or liver was measured in a manually drawn region of interest. Inveon Acquisition Workplace software  was used for further analysis.Mice that were exposed to 4 °C or 30 °C were fasted for 6 h before PET–CT scanning. For human studies, CT scans were performed using the following protocols: 120 kv tube voltage, 120 mA tube current and 3.75 mm thickness of layers. For PET collection, 6–7-bed cycles were used for the whole-body scanning and each bed cycle was 2 min. Data were reconstructed using the viewpoint method.For removal of iBAT, a small incision was surgically created in each mouse under isoflurane-inhalation anaesthetization. Blood vessels in the iBAT were crushed by a pinch using sterilized forceps to prevent excess bleeding after cutting out the tissues. iBAT tissues were carefully dissected using autoclaved surgical scissors, followed by the closure of the incision with the sterile surgical suture. Sham-operated mice were used as controls. All of the mice were allowed to recover for a week at ambient temperature before further analyses.3) was the criterium to euthanize each mouse. This end point and body condition score BCS-1 (moribund state) were not exceeded in any of the experiments.Survival studies of tumour-bearing mice were performed at Fudan University, Shanghai, China according to the ethical permit in which the humane end point . The quality of RNA samples was assessed by agarose gel electrophoresis and using the Agilent 2100 Bioanalyzer (Agilent Technologies). All of the samples displayed a 260/280 ratio of around 2.0. cDNA libraries were constructed and sequenced by Majorbio Biotech. In brief, 200 ng RNA for each group was used for the library construction using the Truseq RNA sample prep Kit (Illumina). The constructed DNA was enriched by PCR amplification and then purified by Certified Low Range Ultra Agarose (Bio-Rad) gel electrophoresis. Clone clusters were generated on the Illumina cBot, using the Truseq PE Cluster Kit v3-cBot-HS, and high-throughput sequencing was performed on an Illumina Miseq sequencer, using Truseq SBS Kit v3-HS (200 cycles). Aligned reads were summarized using featureCounts (v.2.0.0) based on the Gencode vM19 annotation. Differential expression analysis between three 30 °C and two 4 °C samples was performed using the R (v.4.0.3) package DESeq2 (v.1.30.0). GSEA was performed with GSEA (v.4.1.0) using the GSEAPreranked tool, whereby genes were preranked on the basis of their g for 5 min at 4 °C to remove debris. Subsequently, 300 μl of the aqueous fraction was centrifugally filtered through a 5 kDa cut-off filter using an Ultrafree MC-PLHCC filter . The filtrate was dried by a dryer for 180 min and dissolved in 25 μl of both Milli-Q water-containing reference compounds . The amount of all anion- and cation-charged metabolites in the samples was measured by CE−TOFMS. For analysis of cationic metabolites, a fused silica capillary  with a cation buffer solution  as the electrolyte was used. The sample was injected at a pressure of 50 mbar for 10s. To determine anionic metabolites, a chemically coated cationic polymer capillary  was used with an anion buffer solution  as the electrolyte. The sample was injected at a pressure of 50 mbar for 25 s. For measuring cation- and anion-charged metabolites, the CE capillary was preconditioned by flushing the running electrolyte for 3 or 4 min before sample injection. Each sample was inserted with a pressure injection of 50 mbar for 10 s under 20 °C of the capillary temperature and 27 kV and 30 kV applied voltage for measurement of cation- and anion-charged metabolites, respectively. Sheath liquid  was delivered at 10 µl min−1 to the CE interface by a pump using the Agilent 1200 series pump equipped with a sheath flow splitter. Electrospray ionization–TOFMS was conducted in positive-ion mode with 4 kV capillary voltages for cationic metabolites and was set in the negative-ion mode with 3.5 kV for anionic metabolites.Metabolomics analysis was performed in house or with the assistance from Shanghai Biotree Biotech. Metabolites in MC-38 tumours and adipose tissues were measured by capillary electrophoresis time of flight mass spectrometry (CE–TOFMS) using the Agilent 7100 CE Capillary Electrophoresis system equipped with an Agilent 6230 Time-of-Flight mass spectrometer (Agilent Technologies) according to previously reported methods. In brief, snap-frozen tumour and adipose tissue samples (approximately 100 mg for tumour and 30 mg for adipose tissues) were homogenized with 500 μl of methanol containing internal standards  and 500 μl of chloroform and 200 μl of ultrapure water (50% v/v) were subsequently added into the sample solution. The solution was centrifuged at 2,300m/z 50 to 1,000 at 1.5 cycles per second during separation and detection. An automatic recalibration of each acquired spectrum was performed using the masses of reference standards. For the CE–TOFMS system control and data acquisition, we used an Agilent MassHunter software for TOFMS (Agilent Technologies). All target metabolites were identified by matching their m/z values and migration times with the normalized m/z values and migration times of corresponding authentic standard compounds. Data were quantified by comparing the ratio of analyte peak area/internal standard peak area of sample solution to that of the reference standard mixture. The quantitative formula was as follows: X nmol mg−1 = (sample relative area)/(standard relative area) ×  × (amount of methanol for metabolite isolation (500 µl)) × 1/(sample weight) × (concentration of metabolite in the reference standard mixture (50 µM))/). The sample relative area = (metabolite peak area in the sample solution)/. The standard relative area = (metabolite peak area in the reference standard mixture)/. Chromatograms of each metabolite were extracted and quantified using the Agilent MassHunter Qualitative Analysis software (Agilent Technologies). For lipidomics analysis, 50 μl mouse serum sample was diluted into extraction buffer : n-hexane  = 2:3 (v:v) with internal standards . Samples were sonicated in an ice-water bath for 5 min, then centrifuged at 4 °C for 15 min at 12,000 rpm. Then, 400 μl of supernatant was dried in a speed vac, stored under nitrogen and reconstituted in 160 μl of n-hexane. After centrifugation at 12,000 rpm for 5 min, the supernatant was projected for gas chromatography–mass spectrometry  for further analysis.The spectrometer was scanned from −1 in PBS. After 21 days of treatment, the mice were euthanized for further analysis.CL-316,243 disodium salt  was administered systemically by daily intraperitoneal injection at a dose of 1 mg kgMice in thermoneutral and cold environments were anaesthetized and kept at room temperature for 5 min for imaging. Thermal images on the back of mice were captured using an infrared thermal imager . Setting environmental temperature as the baseline, thermal images of mice were acquired and were further analysed using Fotric AnalyzIR.The paraffin-embedded tissue sections of 5 μm thickness were incubated at 60 °C for approximately 2 h to melt the covered paraffin. After the incubation, the warm slides were put in Tissue-Clear  for deparaffinization and dehydrated in serial steps using 99%, 95% and 70% ethanol. For haematoxylin and eosin (H&E) staining, the dehydrated slides were stained with H&E. For immunohistochemical staining, tissue slides were boiled by microwave for 20 min in an unmasking solution  and subsequently blocked with 4% either goat or donkey serum. To visualize cells in the adipose tissues and TME, we stained the WAT, BAT and tumours with antibodies listed below. For the TME, the slices of tumour tissue were stained with rabbit anti-mouse cleaved-caspase-3 , rabbit anti-mouse Ki-67 , rabbit anti-mouse FSP1 , rabbit anti-mouse IBA1  or rabbit anti-mouse CD45  antibodies, followed by staining with species-matched secondary antibodies as follows: Alexa Fluor 555-labelled goat anti-rabbit  or Alexa Fluor 488-labelled donkey anti-rabbit  antibodies. For detection of hypoxia in the tumour, the slides were stained with a rabbit anti-CA9 antibodies  for primary staining and Alexa Fluor 488-labelled donkey anti-rabbit or Alexa Fluor 555-labelled goat anti-rabbit antibodies for the secondary staining. For adipose tissues, the slide with the samples was stained with rabbit anti-mouse UCP1 , rabbit anti-mouse COX4 , and guinea pig anti-mouse perilipin  antibodies, followed by dyeing with Alexa Fluor 555 goat anti-rabbit and Alexa Fluor 647 goat anti-guinea pig  secondary antibodies. Positive signals were detected using a fluorescence microscope equipped with a camera  using NIS-Elements D3.2 (Nikon). For H&E staining, the image was captured using a light microscope (Nikon Eclipse TS100) with the camera  using NIS-Elements F v.3.0 software (Nikon). Images were analysed using Adobe Photoshop CS5 extended software and ImageJ.−1 proteinase K  in a 10 mM Tris-HCl buffer (pH 7.4) for 5 min and blocked with 3% skim milk, followed by staining overnight at 4 °C with goat anti-mouse CD31 antibodies . After rigorous rinsing with PBS, blood vessels were detected with donkey anti-goat Alexa Fluor 555-labelled secondary antibodies , mounted in Vectashield mounting medium (Vector Laboratories), and stored at −20 °C in the dark before microscopy examination using the Nikon C1 confocal microscope (Nikon). The images were recorded using the camera of a Nikon C1 confocal microscope using the EZ-C1 v.3.91 software (Nikon). Captured images were further analysed using Adobe Photoshop CS5 extended software.Paraformaldehyde-fixed tumours, sWAT and iBAT tissue samples were digested with 20 μg ml6 MC-38 GFP tumour cells in 100 μl PBS were subcutaneously implanted into each C57BL/6 mouse. Tumour tissues were collected around 0.5 cm3 in size, and the fresh tumour tissues were cut into small pieces, followed by collagenase digestion. In brief, the minced tumours were enzymatically digested with 0.15% collagenase I  and 0.15% collagenase II  in PBS for 40 min at 37 °C, and centrifuged at 1,500 rpm at 4 °C for 10 min. The pellets were resuspended with PBS containing 1% FBS. Single-cell pellets were obtained by filtration using 70 μm cell strainers followed by centrifugation at 1,300 rpm at 4 °C for 10 min. The pellets were fixed using 1% PFA in PBS for 15 min at room temperature and washed in PBS. The cell pellets were further resuspended in PBS, and cold 70% ethanol was added dropwise to the pellets while vortexing. The samples were stored at 4 °C for further analysis. To the DNA content, fixed single-cell suspensions in PBS were incubated with 20 mg ml−1 propidium iodide (PI)  and 100 mg ml−1 RNase  final on ice for 30 min. PI signals in GFP-positive cells were immediately analysed on the BD FACSCalibur Flow Cytometer (BD Bioscience), and data analysis was performed using the CellQuest Pro software . Gating strategies are shown in Supplementary Fig. Approximately 3 × 10Total RNA was extracted from cells and tissues using the TRIzol  and GeneJET RNA Purification Kits  according to the manufacturer’s instructions. The total RNA was reverse-transcribed and cDNAs were used for PCR and qPCR analyses using the primers listed in Supplementary Table Freshly isolated mouse CRC tumours were washed and immersed with the assay buffer, and then homogenized using a Dounce homogenizer. Tissue lysates were applied to the LDH activity assay using the LDH assay kit  according to the manufacturer’s instructions.Tissue lysates from tumours in different mouse models were extracted using a lysis buffer  with a mixture of proteinase inhibitors  and a phosphatase inhibitor cocktail  by a homogenizer . Protein concentrations in tissue lysates were measured using the BCA assay. An equal amount of protein from each experimental group and a protein ladder  was placed onto an SDS–PAGE gel  or a 10% SDS–PAGE gel . The separated protein on the gel was transferred onto a polyvinylidene difluoride membrane , followed by blocking using 5% bovine serum albumin  in Tris borate EDTA (TBE) and staining with primary antibodies at 4 °C overnight. The membranes were subsequently stained with specific species-matched secondary antibodies conjugated with IRDye dissolved in 3% BSA in TBE for 1 h at room temperature. For visualization of positive signals, the Odyssey CLx system (LI-COR) was used. The positive signals were quantified using an Image Studio v.3.1 (LI-COR). β-Actin was used as the loading control for all blots. Primary and secondary antibodies were as follows: PI3K p85 , phosphorylated PI3K p85 , AKT , phosphorylated AKT , mTOR , phosphorylated mTOR , β-actin , rabbit anti-mouse beta-actin polyclonal , donkey anti-rabbit IRDye 680RD  and donkey anti-mouse IRDye 800CW  antibodies. For some of the samples, primary antibodies including rabbit anti-PI3K p85 alpha , rabbit anti-phosphorylated PI3K p85 alpha , rabbit anti-AKT , rabbit anti-phosphorylated AKT , rabbit anti-GLUT1  and mouse anti-GAPDH  antibodies were used. Secondary antibodies including goat anti-mouse HRP-conjugated IgG  and goat anti-rabbit HRP-conjugated IgG  antibodies were used. Target proteins were visualized using the EZ ECL pico luminescence reagent  with the Molecular Imager ChemiDoc XRS System (Bio-Rad). Full blot gels are provided in Supplementary Fig. −2) and 3 women  aged between 22 and 25 years were recruited for the cold-exposure studies. Before cold exposure, all of the volunteers were fasted overnight and kept at 28 °C for 1 h. After 1 h, 18F-FDG at a dose of 0.1 mCi kg−1 was intravenously injected into each of the volunteers, followed by a PET scan . The healthy male and female individuals with T-shirts and shorts were exposed to a mildly cold ambient temperature of 16 °C for 2–6 h per day for consecutive 14 days. On day 13, all of the volunteers were fasted overnight and continued cold exposure for 2 h the next morning. PET scans were performed while the individuals intermittently soaked their feet in ice water. PET scan images were collected.All of the human studies were approved by the Ethical Review Committee in the Qilu Hospital, Shandong University, Shandong Province, China (KYLL-202011-011-02), and informed consent for human research and the publication of the PET–CT images was obtained from all of the participants. Healthy volunteers including 3 men . Data are presented as the means of determinants ± s.e.m. For RNA-seq, Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-022-05030-3.Supplementary Figs. 1 and 2Supplementary Fig. 1: FACS gating strategy for Extended Data Fig. 1c, d. Supplementary Fig. 2: full scan gels for western blotting analysis for Fig. 4e, Fig. 5c and Extended Data Fig. 5l.Reporting SummarySupplementary Table 1A list of nucleotide sequences of primers used for qPCR in this study.


Pancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed at a late stage and becomes resistant to several treatments. Significant clinical effects have been reported for cancer immunotherapies on a subset of patients diagnosed with epithelial cancers. Cancer organoid co-culture with autologous peripheral blood lymphocytes offers an innovative immunotherapeutic approach that is increasingly being tested, although there is a lack of cutting-edge platforms enabling the investigation of cancer-T cell interactions for individual patients. In this study, a pancreatic cancer organoid culture from a genetically engineered pancreatic cancer murine model was established and co-cultured with autologous peripheral blood lymphocytes to induce a tumour-specific T cell response to pancreatic cancer. Co-culturing autologous peripheral blood lymphocytes with cancer organoids can be an effective strategy to enrich tumour-reactive T cells from the peripheral blood of murine models; this approach could potentially be transferred to humans. Co-culture of peripheral blood lymphocytes and cancer organoids could provide an unbiased approach to evaluating the sensitivity of tumour cells to T cell-mediated priming on an individual patient level. •Pancreatic cancer organoids co-cultured with PBMCs can establish an individualized ex-vivo model system to support T cell-based therapies.•Granzyme B intracellular staining showed increased tumour reactivity in T cells co-cultured with cancer organoids compared to healthy controls.•CD137 staining showed that the T cell responses induced by co-culture were specific to the cancer organoids. Only 10The numerous strategies that cancers use to elude the immune system make it challenging to predict whether a patient will benefit from immunotherapy agents, elucidate any underlying resistance mechanism and determine the best immunotherapy approach for overcoming any resistance. T cell repertoire and cancer mutation profile are unique to every patient. Consequently, a more effective therapeutic strategy to exploit the patient's anti-tumour immunity requires a personalized experimental evaluation of immune responses that should be applicable within the clinical timescale .Ex vivo strategies to assess T cell-tumour interaction have mainly focused on melanoma due to the number of available patients, the ease of tumour biopsy and the availability of techniques for the expansion of tumour infiltrating lymphocytes (TILs) . With thIn this study, pancreatic cancer organoids were established from mouse models and were co-cultured with peripheral blood mononuclear cells (PBMCs). Tumour organoids are three-dimensional tumour cell cultures retaining the mutational burden and histological features of the primary tumour. Organoids can be obtained from needle biopsies or surgical tumour resection. The aim was to establish whether tumour-reactive T cells can be established by co-culturing PBMCs with tumour organoids and also whether T cell responses induced by organoid co-culture are tumour-specific or should be considered artefacts. Differentiation of PBMCs into T cells provides an easily accessible alternative to peripheral isolated lymphocytes. This approach could be helpful towards achieving patient-specific medicine; in theory, tumour-derived organoids could be used to establish personalized ex vivo model systems to support T cell-based therapies. These organoids could also shed light on the interactions between malignant cells and T cells.A proof of concept is provided that co-culturing tumour organoids with PBMCs is a sound and unbiased approach to obtaining tumour-reactive T cells that could potentially suppress tumour cells. More broadly, the findings corroborate the idea that this strategy can affect the priming of T cells against tumour cells towards personalized immunotherapy.22.1G12D Trp53lox/+ Pdx1-Cre (KPC)) were brought from Nihon University School of Medicine of Tokyo (Japan) and were harvested at Yamaguchi University's mouse facility. All animal studies were conducted in compliance with the international guidelines for the care and use of laboratory animals and were approved by the Yamaguchi University Institutional Review Board.Two pairs  of C57BL/6 genetically engineered mouse models (GEMMs) of pancreatic cancer /F12 medium (GIBCO) plus 100 mg/mL collagenase II (Sigma-Aldrich), 10 μM Y-27632 (Sigma-Aldrich) and 0.06 mg/mL DNase I (Sigma-Aldrich).One male GEMM mouse was sacrificed by cranial dislocation after six months; the sample collection time point was collectively agreed once the mouse developed a clear and touchable mass in the abdomen and was no longer amenable to movement. The pancreatic tumoral mass was extracted and immediately minced into 10 mm2.34 cells per 10 uL medium) and 20 uL per well of Matrigel resuspension was added in a 48-well culture plate. The plate was placed into a 37°C incubator for 30 min to allow the Matrigel to solidify and 250 mL of organoid medium was then added and the nascent organoids were incubated at 37°C with 5% CO2. Pancreatic organoid medium consisted of adDMEM/F-12 medium (Gibco) supplemented with 50% Wnt3a medium (Protocol 1)  supplemented with 150 U/mL IL-2 (Novartis) and incubated overnight at 37°C with a slightly opened lid wrapped in Parafilm to enable gas exchange.24 h before the co-culture experiment, tumour organoids were stimulated overnight with 200 ng/mL INFγ to enhance antigen presentation. Meanwhile, a 96-well U-bottom plate was coated with 5 μg/mL anti-CD28 (50 μL per well) to provide co-stimulatory signals during co-culture. The 96-well U-bottom plate was subsequently wrapped in Parafilm and incubated for 24 h at 4°C. Furthermore, 2-3 x 102.84 cells per mL in T cell culture medium. Meanwhile, PBMCs were counted using an automated cell counter, washed in PBS and resuspended at 1 × 106 cells per mL in T cell culture medium supplemented with 300 U/mL IL-2 (Novartis) (2x concentrated). Equal volumes (100 μL) of PBMCs and dissociated organoids were mixed for a PBMC:tumour cell ratio of 20:1.200 μL per well of PBMC-dissociated organoid suspension mix was then plated and incubated at 37°C for 10 days. The co-culture medium was refreshed every 2–3 days.Organoids incubated with INFγ were centrifuged , resuspended in 1 mL TrypLE (Gibco) and incubated for 15 min at 37°C. Dissociated organoids were centrifuged and washed in PBS and then resuspended in T cell culture medium. The resulting single-cell organoid suspension was counted using a hemocytometer and resuspended at 5 × 102.9Along with the expansion of tumour organoids, a healthy pancreatic organoid cell culture was established. A mouse with the same genetic background (C57BL/6) was sacrificed, the pancreatic tissue was removed and healthy pancreatic organoids were expanded using the same protocol as used for pancreatic cancer organoids.2.10Following co-culture, cells were immediately evaluated for tumour reactivity using Granzyme B and CD137 expression as read-out. As a positive control, an equal volume and number of PBMCs were incubated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin (Sigma-Aldrich) solution at 37° for 4 h. As a negative control, an equal volume and number of PBMCs were incubated with healthy organoids at 37° for 10 days and 24 h for Granzyme B and CD137 assays, respectively.2.10.16 cells per mL in 400 μL T cell medium and split into two aliquots (200 μL each). Cells were washed twice in FACS buffer and stained with the following antibodies (extracellular staining): FITC-CD8a (1:80)(Peprotech), Per55-I-A/I-E (1:200) (Peprotech), APC-TCRb (1:40) (Peprotech), at 4°C for 20 min. Cells were then washed twice in FACS buffer, fixed and stained (intracellular staining) for Granzyme B (PE-conjugated anti-GrB) (Peprotech), using the Cytofix/Cytoperm kit (BD), according to the manufacturer's instructions. PBMCs co-cultured with healthy organoids served as a negative control whereas PBMCs stimulated with 50 ng/mL PMA (Sigma-Aldrich) and 1 μg/mL Ionomycin (Sigma-Aldrich) served as a positive control; half of the contents of each co-culture well was incubated with PMA/Ionomycin solution for 5 h on ice.After 10 days of incubation, each co-culture well was resuspended at 2 × 102.10.26 cells per mL, washed twice in FACS buffer and stained with the following antibodies: PE-anti-CD137 (1:30) (Peprotech), FITC-CD8a (1:80) (Peprotech), Per55-I-A/I-E (1:200) (Peprotech), APC-TCRb (1:40) (Peprotech). PBMCs co-cultured with healthy organoids served as a negative control whereas PBMCs stimulated with 50 ng/mL PMA (Sigma-Aldrich) and 1 μg/mL Ionomycin (Sigma-Aldrich) served as a positive control.In a parallel co-culture experiment, after 24 h incubation, co-cultured T cells were resuspended at 1 × 1033.1G12D Trp53lox/+ Pdx1-Cre mice developed a well differentiated pancreatic cancer  silences the expression of mutant KrasG12D whereas expression of Trp53 is maintained on both alleles. When Cre is expressed in the pancreas, the STOP cassette is excised and the mutant KrasG12D alleles are expressed and Trp53 is knocked out on a single allele  staining, histological analysis revealed that the tumours in this study were carcinomas, predominantly ductal adenocarcinomas (PDAC), defined by the presence of neoplastic glandular  cells in a dense fibrous stroma . As a co3.3Cancerous cells from pancreatic cancer mouse models, and cells from normal pancreatic tissue, embedded in Matrigel and organoid medium, started developing organoids - defined as a three-dimensional (3D) cellular cluster derived exclusively from primary tissue - after 3–5 days of incubation , Fig. 6.3.4+ T cells was evaluated after 10 days of co-culture with cancer organoids by staining for granzyme B  were added to provide co-stimulation and to support T cell proliferation, respectively. The tumour reactivity of PBMCs-turned-CD8anzyme B A. As a nanzyme B C and staanzyme B B. We obsanzyme B , Fig. 8.3.5It was next evaluated whether the T cell response induced by organoid co-culture was tumour-specific or should be considered an artefact of IFNγ treatment or organoid culture. Towards this goal, PBMC expression of the activation marker CD137 was compared upon stimulation with tumour organoids A. To mor4G12D Trp53lox/+ Pdx1-Cre engineered mouse models were used to investigate pancreatic cancer. These genetically engineered mouse models (GEMMs) developed pancreatic cancer with a six-month latency – cancer growth is expected between four and eight months. Although this mouse model does not represent the most common GEMM for pancreatic cancer, it is known for the development of a well differentiated pancreatic ductal adenocarcinoma (PDAC) with a strong stromal desmoplastic reaction and no metastasis [LSL-Krastastasis . The usetastasis .After collection of the tumour sample, organoids were generated from both healthy pancreatic tissue and malignant pancreatic tissue. PBMCs were extracted from the blood of both a healthy mouse and a pancreatic cancer GEMM. Pancreatic cancer organoids were then co-cultured with PBMCs in order to establish a potential individualized ex vivo model system to support T cell-based therapies. This is consistent with recent recommendations to explore new models to facilitate the development of patient-specific treatments .T cell-mediated tumour killing is unpredictable due to the heterogeneity of T cell receptors (TCRs) and human leukocyte antigen (HLA), and the unique nature of neo-antigens expressed in human cancers . It is tDijkstra et al. recently established a transferable approach based on colorectal cancer organoids co-cultured with T cells isolated from patients ; using tHere, a murine pancreatic cancer organoid culture and a parallel culture of healthy pancreas organoids were established. Co-culture of peripheral blood lymphocytes with pancreatic cancer organoids resulted in significantly increased induction of tumour reactivity in T cells compared to healthy controls, as assessed by intracellular staining of granzyme B. Staining for expression of the activation marker CD137 showed that the T cell responses induced by co-culture were specific to the cancer organoids.Although the use of cancer co-culture with immune cells is not novel per se, we believe that our approach is methodologically solid, elegant and could be leveraged as a platform for different applications. One possible application is to exploit the co-culture of autologous cancer organoids and T cells as model systems for developing patient-specific immune-oncological treatment regimens and for the investigation of tumour immune networks. Given that tumour organoids include stromal cells, thus re-creating the original tumour microenvironment, they can be considered a sound approach for accurately predicting drug response and resistance as compared to tumour cell lines. Also, establishing tumour organoid cultures from a limited quantity of cancer cells , coupled with the potential expansion of tumour-reactive T cells, could enable minimally invasive tumour treatment at multiple time points. For a patient who initially responded and subsequently relapsed, for instance, establishing a co-culture of tumour organoids with autologous T cells from blood might shed light on the underlying cause of relapse. A recent investigation by Zaretsky et al. observed that the paired biopsy strategy is effective in a small cohort of patients with melanoma who relapsed due to the acquisition of JAK1/2 mutations . Another5G12D. The molecular specificity of any identified tumour-reactive T cells will remain enigmatic without a deeper understanding of cancer target antigens. Ultimately, further evidence could be provided for the specificity of tumour cell killing activity by autologous T cells by performing a cytotoxicity assay using a green fluorescent apoptosis probe targeting active caspase-3/7.In terms of limitations, firstly we used genetically engineered murine models which, we believe, do not perfectly re-capitulate the pancreatic cancer tumour microenvironment in humans. Organoid model systems need refinement to better mimic cancers, for example by incorporating components of the tumour microenvironment such as fibroblasts and blood vessels. Second, in our study only a single pancreatic cancer GEMM mouse was used. A larger sample size is needed to determine the success rate of this approach. Third, in this study we focus on a pancreatic tumour type with a known and severe mutational load; the possibility of extending this approach to poorly immunogenic tumours requires further testing. Fourth, a more rigorous approach for the validation of pancreatic cancer organoid cells at the molecular level will also need to be considered; for instance, genome sequencing of pancreatic cancer organoids would allow confirmation of the presence of the expected mutations e.g. Kras6In this proof-of-concept study, T cells isolated from murine blood were co-cultured with autologous pancreatic cancer organoids from GEMM, demonstrating the feasibility of obtaining tumour-reactive T cells by co-culturing murine PBMCs with matched murine tumour organoids.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The goal of this study was to investigate the extent of the alveolar-capillary membrane porosity in patients with severe re-expansion pulmonary oedema. The biochemistry of airway fluid of two patients who died of re-expansion oedema was compared to their blood biochemistry. The airway fluid was comparable to plasma, while no blood cells were observed across the alveolar-capillary membrane. The membrane was linked to a fishnet that traps cells on one side, while plasma sieved through. Re-expansion pulmonary oedema is a rare form of acute lung injury, occurring as a complication of sudden lung re-expansion after pleural drainage [1]. Re-expansion pulmonary oedema is a rare form of acute lung injury, occurring as a complication of sudden lung re-expansion after pleural drainage . This poA 44-year-old patient presented with a loculated right empyema thoracis from transdiaphragmatic rupture of a right subphrenic abscess. After failed thoracentesis coupled with a demonstrable visceral pleural cortex on chest computed tomography scan, a single-stage pleural drainage and lung decortication were done via thoracotomy. About 3000 ml of thick pus was drained from both the chest and abscess, and the lung re-expanded fully after decortication. Large amounts of frothy serosanguineous fluid were noted in the patient’s endotracheal tube. Bronchoscopy revealed large amounts of the fluid pouring from left and right bronchial trees. Despite being on a ventilator, 400 ml of airway fluid was suctioned in about 15 min and even more thereafter. Ineffective ventilation due to build-up of fluid in the endotracheal tube resulted in hypoxic cardiac arrest and death (Fig. A 31-year-old male patient presented with a massive right pleural effusion. A chest drain was inserted, draining 800 ml of straw-coloured fluid. The drain was clamped with the aim of draining smaller amounts in a controlled fashion. Unfortunately, the clamp slipped off resulting in sudden drainage of 1500 ml, causing pulmonary oedema that required ventilation. The endotracheal tube rapidly filled up with large amounts of serosanguineous fluid, again resulting in death. The airway fluid and blood biochemical properties were compared (Table First described in 1853 by Pinault . ReperfuDirect comparison between airway fluid and blood biochemical components in our cases demonstrated a massive protein leak through the capillary alveolar barrier. Total protein, albumin and lactate dehydrogenase levels were comparable to serum Table . Both smet al. [Hydrostatic mechanism was previously demonstrated in 5 out of 7 patients by Sue et al. . Using tAs shown in our study, ineffective ventilation due to fluid accumulation in the airway is fatal and extracorporeal membrane oxygenation should be considered.Our study demonstrated an alveolar-capillary membrane that is permeable to plasma, but not to blood cells, in some patients with severe re-expansion pulmonary oedema. Although larger studies have been previously reported, this study adds some understanding to the potentially complex pathophysiological mechanisms of this condition.Written informed consent was obtained from both patients relatives for publication.Conflict of interest: none declared. The data underlying this report will be shared upon request to the corresponding author.Interactive CardioVascular and Thoracic Surgery thanks Paola Ciriaco, Francoise Le Pimpec-Barthes and the other anonymous reviewers for their contribution to the peer review process of this article.


Due to the vital factor of enzymes in biological systems, enzyme-activated fluorophores, which could turn “on” the fluorescence signal from an “off” state, offer non-invasive and effective potential for the accurate bioimaging of particular cells, tissues, or bacteria. Comparing with the traditional imaging probes, enzyme-activated organic small fluorophores can visualize living cells within small animals with high sensitivity, high imaging resolution, non-invasiveness, and real-time feedback. In this mini review, well-designed enzyme-activated organic fluorescent probes with multiple functions are exclusively reviewed through the latest development and progress, focusing on probe design strategy, fluorescence property, enzyme activation process, and bioimaging applications. It is worth noting that multi-enzyme-activated strategies, which could avoid the production of “false-positive” signals in complex biological systems, effectively provide high selective and real-time bioimaging, indicating the exciting potential of intraoperative fluorescence imaging and diagnosis tools.Bioimaging techniques are of increasing importance in clinical and related fields, which also have been successfully applied in the  GGT is  imaging . More im imaging  covalent imaging 628/I582) was used for monitoring Casp3 activity and apoptosis imaging in HeLa cells. By the introduction of lipophilic nitroaromatic moieties, fluorogenic probes  and the carrier protein bovine serum (BSA). The BH-NO2 part has a diphenylxanthene group (electron donor) and a dioxaborininochromenone group (electron acceptor), and an aromatic nitro group on the donor side of strong electron-withdrawing capability is the fluorescence quencher, as well as the NTR-recognition moiety. With the carrier bovine serum affording water dispersibility and biocompatibility, BH-NO2@BSA could be readily taken up by the liver. In hepatic tumor cells, BH-NO2@BSA would be activated by the highly expressed NTR-reducing aromatic nitro group to diphenylamino group, endowing the activated probe with AIE feature. The activated probe (BH-NH2) exhibits an enhanced fluorescence at around 791 nm or 923 nm as well as a strong photoasorber for PA imaging. With dual-modal imaging NTR-activated probe, orthotopic liver tumors could preoperatively be precisely located by PA imaging, while tumor margins could accurately be defined by real-time intraoperative fluorescence imaging in the mice models  imaging, targeting lactamase BlaC and decaprenylphosphoryl-β-d-ribose 2′-epimerase (DprE1), with the emission of 520 nm  was developed for f 520 nm . BlaC isf 520 nm . The duaTo further certificate the AND-gate strategy, the homologous probe targeted other enzymes was designed. Similar to Casp3, FAPα is highly expressed in the tumor microenvironment, with higher proteolytic activity in a broad range of tumor types. New probe FAP-Cat targeted FAPα and Cats was synthesized with the corresponding substrates of FAPα and Cats according to the AND-gate strategy, resulting in a more concentrated signal at the outer margins of the tumor compared with previous results of DEATH-Cat-2 in a 4T1 mouse model of breast cancer. Meanwhile, the DEATH-Cat-2 probe produced a significantly brighter signal in a metastatic lung cancer model.Robotic fluorescence-guided surgery with improved AND-gate probes DEATH-Cat-FNIR for a Vinci Surgical System was taken to resect of a primary subcutaneous mammary tumor and subsequently used probe fluorescence to assess the remaining subcutaneous tumor bed. Analysis of the H&E-stained tissues by a board-certified pathologist confirmed accurate detection of residual tumor cells by the AND-gate probes DEATH-Cat-FNIR after excision of a bulk tumor. After another robotic fluorescence-guided surgery to resect small metastatic cancer lesions in a metastatic lung cancer model, the resected lung tissues were imaged using the LiCor Pearl imaging system to confirm the probe selectivity of AND-gate probes DEATH-Cat-FNIR for metastatic lesions. The fluorescence signal of the resected lung section from the AND-gate probe also matched the location of tumor cells as determined by adjacent H&E-stained slides, indicating the ability of the AND-gate probe as a diagnostic indicator. Combining the selectivity of different enzymes, multivariate AND-gate imaging probes demonstrate a versatile platform for real-time intraoperative fluorescence imaging and tumor diagnosis.in vivo is also summarized to further evaluate the promising clinical applications.Multifunction enzyme-activated fluorescent probes for bioimaging have been summarized in this mini review. Design strategies of multifunction fluorescence probes are sorted to single-enzyme-activated probes with more functions and multi-enzyme-activated probes listing the representative recent research work, involving fluorescence enhancement, enzyme immobilizing, activation reaction, multiple imaging modality, and multi-enzyme activation. The fluorescence imaging in cell or in situ detection and long-term tracking of enzyme-activated fluorescent probes is influenced. Moreover, this type of fluorescent probes is prone to the aggregation fluorescence quenching (ACQ) phenomenon in high concentration in biological systems. However, it still has broad application scenarios of this strategy, as relatively simple in design and easy to implement. On the other hand, AIE fluorescent materials have very low fluorescence efficiency in dilute solvents and can achieve stable and highly efficient luminescence under aggregation or solid-state conditions, enabling in situ detection and long-term tracking of a particular target. Water-soluble AIE fluorescent probes avoid single-molecule diffusion and excretion during biological detection, providing an effective in situ detection.The construction of enzyme-activated fluorescent probes based on the different strategies should be noted. Quenched fluorescence probes are activated by cleaving the quenching group (containing substrates) by the selected enzyme, and the remaining part of the fluorescence probes have the disadvantage of easy diffusion to other sites and excretion by the biological system. Therefore, the ability of Numerous examples of enzyme-activated fluorescence probes have been reported in the literature for selective visualization of specific markers in cells or tissues, but there is still room for improvement. A single-enzyme-activated probe may also show weak fluorescence in normal tissue, leading to instability, uncertainty, and diagnostic inaccuracy. There is a hard choice of the probe size because small molecule probes provide quick diffusion and deep penetration into living cells, while these probes and their enzyme-processed products suffer from easily diffusing ability across the cell membrane, leading to their limitation of real-time imaging and retention time. Multifunction enzyme-activated fluorescence probes provide possible solutions. Considering bioimaging requirement, there is a major trade-off between accessibility and wavelength. Longer wavelengths are better for tissue penetration and minimize tissue damage. Multimodality PA/fluorescence probes provide an additional solution to this problem. A series of overexpressed enzymes in tumor tissues have been utilized for enzyme-activated fluorescent probes. The successful design of a dual-enzyme strategy can significantly improve selectivity and accuracy to identify the location and size of specific tumor tissues, demonstrating fluorescence-guided efficient tumor resection, therapeutics, and even aiding the development of biomedicine.Previous advance of enzyme-activated fluorescence bioimaging research lays the versatile and reliable foundation to broad applications in clinic-related fields. Enzyme-activated fluorescence probes are used to aid the diagnostic bioimaging, the fluorescence-guide surgery, and the evaluation of cancer treatment, as well as the research and development of anticancer and antibiotics drugs. It is hoped that this perspective will provide an insight into the enzymatic activated organic fluorescent probes for invasive, selective, and real-time bioimaging in cells or in model animals. There are a number of directions to explore in the future that could improve the performance of enzyme-activated fluorescent probes with more functions.