Application of multiple ultra-high-pressure homogenization to the pasteurization process of Japanese rice wine, sake.

Hiire is a pasteurization process in the production of Japanese rice wine (sake), which stabilizes the quality of product; however, it also generates the carcinogen ethyl carbamate (EC). In this study, we investigated the application of ultra-high-pressure homogenization (UHPH) as an alternative sterilization method for sake production. Microbiological analysis revealed that multiple UHPH treatments sterilized hiochi lactobacilli (Lactobacillus fructivorans, L. homohiochii, L. casei, and L. hilgardii) and Saccharomyces cerevisiae. Enzyme activity assays revealed that α-amylase, glucoamylase, and acid-carboxypeptidase activities were reduced to less than 1% of the levels in non-pasteurized sake after four-time UHPH treatment. These results show that UHPH treatment meets the two requirements of the sake sterilization process sterilization and enzyme inactivation. The UHPH-processed sake did not show any significant changes in general properties but had reduced organic acid and aromatic component contents, with ethyl caproate content showing the most significant reduction of approximately 20%. Interestingly, EC was detected in pasteurized sake but not in UHPH-processed sake. These findings indicate that the UHPH technology could be used to inactivate microorganisms and enzymes in sake without generating EC.

Synergistic Activation of Antitumor Immunity by a Particulate Therapeutic Vaccine.

Success in anticancer immune therapy relies on stimulation of tumor antigen-specific T lymphocytes and effective infiltration of the T cells into tumor tissue. Here, a therapeutic vaccine that promotes proliferation and tumor infiltration of antigen-specific T cells in both inflamed and noninflamed tumor types is described. The vaccine consists of STING agonist 2'3'-cGAMP, TLR9 ligand CpG, and tumor antigen peptides that are loaded into nanoporous microparticles (µGCVax). µGCVax is effective in inhibiting lung metastatic melanoma, primary breast cancer, and subcutaneous colorectal cancer in their respective murine models, including functional cure of HER2-positive breast cancer. Mechanistically, µGCVax potently stimulates type I interferon expression in dendritic cells, and promotes CD8+ and CD103+ dendritic cell maturation and migration to lymph nodes and other lymphatic tissues. Antitumor responses are dependent on TLR9 and interferon α/ß receptor signaling, and to a less extent on STING signaling. These results demonstrate a high potential for µGCVax in mediating antitumor immunity in personalized cancer therapy.

Attenuated Microcirculation in Small Metastatic Tumors in Murine Liver.

Metastatic cancer disease is the major cause of death in cancer patients. Because those small secondary tumors are clinically hardly detectable in their early stages, little is known about drug biodistribution and permeation into those metastatic tumors potentially contributing to insufficient clinical success against metastatic disease. Our recent studies indicated that breast cancer liver metastases may have compromised perfusion of intratumoral capillaries hindering the delivery of therapeutics for yet unknown reasons. To understand the microcirculation of small liver metastases, we have utilized computational simulations to study perfusion and oxygen concentration fields in and around the metastases smaller than 700 µm in size at the locations of portal vessels, central vein, and liver lobule acinus. Despite tumor vascularization, the results show that blood flow in those tumors can be substantially reduced indicating the presence of inadequate blood pressure gradients across tumors. A low blood pressure may contribute to the collapsed intratumoral capillary lumen limiting tumor perfusion that phenomenologically corroborates with our previously published in vivo studies. Tumors that are smaller than the liver lobule size and originating at different lobule locations may possess a different microcirculation environment and tumor perfusion. The acinus and portal vessel locations in the lobule were found to be the most beneficial to tumor growth based on tumor access to blood flow and intratumoral oxygen. These findings suggest that microcirculation states of small metastatic tumors can potentially contribute to physiological barriers preventing efficient delivery of therapeutic substances into small tumors.

Molecular and biochemical characteristics of inulosucrase InuBK from Alkalihalobacillus krulwichiae JCM 11691.

Information about the inulosucrase of nonlactic acid bacteria is scarce. We found a gene encoding inulosucrase (inuBK) in the genome of the Gram-positive bacterium Alkalihalobacillus krulwichiae JCM 11691. The inuBK open reading frame encoded a protein comprising 456 amino acids. We expressed His-tagged InuBK in culture medium using a Brevibacillus system. The optimal pH and temperature of purified InuBK were 7.0-9.0 and 50-55 °C, respectively. The findings of high-performance anion-exchange chromatography, nuclear magnetic resonance spectroscopy, and high-performance size-exclusion chromatography with multiangle laser light scattering showed that the polysaccharide produced by InuBK was an inulin with a molecular weight of 3806, a polydispersity index (PI) of 1.047, and fructosyl chain lengths with 3-27 degrees of polymerization. The size of InuBK was smaller than commercial inulins, and the PI of the inulin that it produced was lower.

Seed- and Soil-Dependent Differences in Murine Breast Tumor Microenvironments Dictate Anti-PD-L1 IgG Delivery and Therapeutic Efficacy.

We sought to determine if Stephen Paget's "seed and soil" hypothesis of organ-preference patterns of cancer metastasis can explain the development of heterogeneity in a tumor microenvironment (TME) as well as immunotherapeutic delivery and efficacy. We established single-cell-derived clones (clones 1 and 16) from parental 4T1 murine breast cancer cells to create orthotopic primary and liver metastasis models to deconvolute polyclonal complexity cancer cells and the difference in TME-derived heterogeneities. Tumor-bearing mice were treated with anti-PD-L1 IgG or a control antibody, and immunofluorescent imaging and quantification were then performed to evaluate the therapeutic efficacy on tumor growth, the delivery of therapy to tumors, the development of blood vessels, the expression of PD-L1, the accumulation of immune cells, and the amount of coagulation inside tumors. The quantification showed an inverse correlation between the amount of delivered therapy and therapeutic efficacy in parental-cell-derived tumors. In contrast, tumors originating from clone 16 cells accumulated a significantly greater amount of therapy and responded better than clone-1-derived tumors. This difference was greater when tumors grew in the liver than the primary site. A similar trend was found in PD-L1 expression and immune cell accumulation. However, the change in the number of blood vessels was not significant. In addition, the amount of coagulation was more abundant in clone-1-derived tumors when compared to others. Thus, our findings reconfirmed the seed- and soil-dependent differences in PD-L1 expression, therapeutic delivery, immune cell accumulation, and tumor coagulation, which can constitute a heterogeneous delivery and response of immunotherapy in polyclonal tumors growing in different organs.

Tumor Site-Dependent Transport Properties Determine Nanotherapeutics Delivery and Its Efficacy.

Insufficient delivery of systemically administered anticancer drugs to tumors can compromise therapeutic efficacy and develop drug delivery-based therapeutic resistance. Nanotherapeutics such as PEGylated liposomal doxorubicin (PLD) are designed to preferentially accumulate in tumors utilizing enhanced permeation and retention effect. However, their antitumor effects and resulting clinical outcomes are modest and heterogeneous among tumors. Here, we aimed to investigate whether the amount and efficacy of PLD delivered to tumors are tumor site dependent. We established orthotopic primary tumor or liver metastases models of murine breast cancer using 4 T1 cells. PLD showed significant therapeutic effects against tumors that grew in primary mammary sites but not in the liver. We found that differences in therapeutic efficacy were not because of the intrinsic biological resistance of cancer cells but rather were associated with tumor site-dependent differences in transport properties, such as the amount of PLD delivery, blood vessel function, relative vascular permeability, and mechanical pressure in tumors. Thus, transport properties in tumor is site dependent and can be used as phenotypic surrogate markers for tumor drug delivery and therapeutic efficacy.

Integrated Microfluidic System for Gene Silencing and Cell Migration.

Metastasis involves the phenotype transition of cancer cells to gain invasiveness, and the following migration at the tumor site. Here an integrated microfluidic chip to study this process is presented by combining on-chip delivery of siRNA for gene silencing and cell migration assay. The major advantage of the integrated chip is the simple input of cells and gene transfection materials, and the ultimate output of migration ability. The reverse-fishbone structure and 0.7× phosphate-buffered saline solution are the optimized parameters for improved delivery efficiency. Using the chip, it is validated that cofilin plays an essential role in regulating cancer cell migration. The integrated chip may provide a simple and effective platform for biologists to easily check the role of specific genes in metastasis.

Nanoplasmonic Quantification of Tumor-derived Extracellular Vesicles in Plasma Microsamples for Diagnosis and Treatment Monitoring.

Tumour-derived extracellular vesicles (EVs) are of increasing interest as a resource of diagnostic biomarkers. However, most EV assays require large samples, are time-consuming, low-throughput and costly, and thus impractical for clinical use. Here, we describe a rapid, ultrasensitive and inexpensive nanoplasmon-enhanced scattering (nPES) assay that directly quantifies tumor-derived EVs from as little as 1 µL of plasma. The assay uses the binding of antibody-conjugated gold nanospheres and nanorods to EVs captured by EV-specific antibodies on a sensor chip to produce a local plasmon effect that enhances tumour-derived EV detection sensitivity and specificity. We identified a pancreatic cancer EV biomarker, ephrin type-A receptor 2 (EphA2), and demonstrate that an nPES assay for EphA2-EVs distinguishes pancreatic cancer patients from pancreatitis patients and healthy subjects. EphA2-EVs were also informative in staging tumour progression and in detecting early responses to neoadjuvant therapy, with better performance than a conventional enzyme-linked immunosorbent assay. The nPES assay can be easily refined for clinical use, and readily adapted for diagnosis and monitoring of other conditions with disease-specific EV biomarkers.

Gemcitabine enhances the transport of nanovector-albumin-bound paclitaxel in gemcitabine-resistant pancreatic ductal adenocarcinoma.

The mechanism for improved therapeutic efficacy of the combination therapy with nanoparticle albumin-bound paclitaxel (nAb-PTX) and gemcitabine (gem) for pancreatic ductal adenocarcinoma (PDAC) has been ascribed to enhanced gem transport by nAb-PTX. Here, we used an orthotopic mouse model of gem-resistant human PDAC in which increasing gem transport would not improve the efficacy, thus revealing the importance of nAb-PTX transport. We aimed to evaluate therapeutic outcomes and transport of nAb-PTX to PDAC as a result of (1) encapsulating nAb-PTX in multistage nanovectors (MSV); (2) effect of gem on caveolin-1 expression. Treatment with MSV/nAb-PTX + gem was highly efficient in prolonging animal survival in comparison to other therapeutic regimens. MSV/nAb-PTX + gem also caused a substantial increase in tumor PTX accumulation, significantly reduced tumor growth and tumor cell proliferation, and increased apoptosis. Moreover, gem enhanced caveolin-1 expression in vitro and in vivo, thereby improving transport of nAb-PTX to PDAC. This data was confirmed by analysis of PDACs from patients who received gem-based neo-adjuvant chemotherapy. In conclusion, we found that nAb-PTX treatment of gem-resistant PDAC can be enhanced by (1) gem through up-regulation of caveolin-1 and (2) MSV through increasing accumulation of nAb-PTX in the tumor.

Macrophage Polarization Contributes to the Anti-Tumoral Efficacy of Mesoporous Nanovectors Loaded with Albumin-Bound Paclitaxel.

Therapies targeted to the immune system, such as immunotherapy, are currently shaping a new, rapidly developing branch of promising cancer treatments, offering the potential to change the prognosis of previously non-responding patients. Macrophages comprise the most abundant population of immune cells in the tumor microenvironment (TME) and can undergo differentiation into functional phenotypes depending on the local tissue environment. Based on these functional phenotypes, tumor-associated macrophages (TAMs) can either aid tumor progression (M2 phenotype) or inhibit it (M1 phenotype). Presence of M2 macrophages and a high ratio of M2/M1 macrophages in the TME are clinically associated with poor prognosis in many types of cancers. Herein, we evaluate the effect of macrophage phenotype on the transport and anti-cancer efficacy of albumin-bound paclitaxel (nAb-PTX) loaded into porous silicon multistage nanovectors (MSV). Studies in a coculture of breast cancer cells (3D-spheroid) with macrophages and in vivo models were conducted to evaluate the therapeutic efficacy of MSV-nAb-PTX as a function of macrophage phenotype. Association with MSV increased drug accumulation within the macrophages and the tumor spheroids, shifting the inflammation state of the TME toward the pro-inflammatory, anti-tumorigenic milieu. Additionally, the treatment increased macrophage motility toward cancer cells, promoting the active transport of therapeutic nanovectors into the tumor lesion. Consequently, apoptosis of cancer cells was increased and proliferation decreased in the MSV-nAb-PTX-treated group as compared to controls. The results also confirmed that the tested system shifts the macrophage differentiation toward an M1 phenotype, possessing an anti-proliferative effect toward the breast cancer cells. These factors were further incorporated into a mathematical model to help analyze the synergistic effect of the macrophage polarization state on the efficacy of MSV-nAb-PTX in alleviating hypovascularized tumor lesions. In conclusion, the ability of MSV-nAb-PTX to polarize TAM to the M1 phenotype, causing (1) enhanced penetration of the drug-carrying macrophages to the center of the tumor lesion and (2) increased toxicity to tumor cells may explain the increased anti-cancer efficacy of the system in comparison to nAb-PTX and other controls.

Tumor progression effects on drug vector access to tumor-associated capillary bed.

Over the last decade, the benefits of drug vectors to treat cancer have been well recognized. However, drug delivery and vector distribution differences in tumor-associated capillary bed at different stages of disease progression are not well understood. To obtain further insights into drug vector distribution changes in vasculature during tumor progression, we combined intra-vital imaging of metastatic tumors in mice, microfluidics-based artificial tumor capillary models, and Computational Fluid Dynamics (CFD) modeling. Microfluidic and CFD circulation models were designed to mimic tumor progression by escalating flow complexity and chaoticity. We examined flow of 0.5 and 2µm spherical particles, and tested the effects of hematocrit on particle local accessibility to flow area of capillary beds by co-circulating red blood cells (RBC). Results showed that tumor progression modulated drug vector distribution in tumor-associated capillaries. Both particles shared 80-90% common flow area, while 0.5 and 2µm particles had 2-9% and 1-2% specific flow area, respectively. Interestingly, the effects of hematocrit on specific circulation area was opposite for 0.5 and 2µm particles. Dysfunctional capillaries with no flow, a result of tumor progression, limited access to all particles, while diffusion was shown to be the only prevailing transport mechanism. In view of drug vector distribution in tumors, independent of formulation and other pharmacokinetic aspects, our results suggest that the evolution of tumor vasculature during progression may influence drug delivery efficiency. Therefore, optimized drug vectors will need to consider primary vs metastatic tumor setting, or early vs late stage metastatic disease, when undergoing vector design.

Aureolysin of Staphylococcus warneri M accelerates its proteolytic cascade, and participates in biofilm formation.

The aureolysin (Aur) gene of S. warneri M (aurWM) was cloned and sequenced. Analyses of the aurWM-inactivated mutant (S. warneri Mau) suggested that AurWM was probably associated with efficient processing of the PROM protease (homolog of V8/SspA serine protease), whereas considerable amount of mature-PROC protease (homolog of SspB cysteine protease) accumulated without AurWM. Additionally, AurWM appeared to affect biofilm formation in an uncertain suppressive way.

Enhanced performance of macrophage-encapsulated nanoparticle albumin-bound-paclitaxel in hypo-perfused cancer lesions.

Hypovascularization in tumors such as liver metastases originating from breast and other organs correlates with poor chemotherapeutic response and higher mortality. Poor prognosis is linked to impaired transport of both low- and high-molecular weight drugs into the lesions and to high washout rate. Nanoparticle albumin-bound-paclitaxel (nAb-PTX) has demonstrated benefits in clinical trials when compared to paclitaxel and docetaxel. However, its therapeutic efficacy for breast cancer liver metastasis is disappointing. As macrophages are the most abundant cells in the liver tumor microenvironment, we design a multistage system employing macrophages to deliver drugs into hypovascularized metastatic lesions, and perform in vitro, in vivo, and in silico evaluation. The system encapsulates nAb-PTX into nanoporous biocompatible and biodegradable multistage vectors (MSV), thus promoting nAb-PTX retention in macrophages. We develop a 3D in vitro model to simulate clinically observed hypo-perfused tumor lesions surrounded by macrophages. This model enables evaluation of nAb-PTX and MSV-nab PTX efficacy as a function of transport barriers. Addition of macrophages to this system significantly increases MSV-nAb-PTX efficacy, revealing the role of macrophages in drug transport. In the in vivo model, a significant increase in macrophage number, as compared to unaffected liver, is observed in mice, confirming the in vitro findings. Further, a mathematical model linking drug release and retention from macrophages is implemented to project MSV-nAb-PTX efficacy in a clinical setting. Based on macrophage presence detected via liver tumor imaging and biopsy, the proposed experimental/computational approach could enable prediction of MSV-nab PTX performance to treat metastatic cancer in the liver.

Redirecting Transport of Nanoparticle Albumin-Bound Paclitaxel to Macrophages Enhances Therapeutic Efficacy against Liver Metastases.

Current treatments for liver metastases arising from primary breast and lung cancers are minimally effective. One reason for this unfavorable outcome is that liver metastases are poorly vascularized, limiting the ability to deliver therapeutics from the systemic circulation to lesions. Seeking to enhance transport of agents into the tumor microenvironment, we designed a system in which nanoparticle albumin-bound paclitaxel (nAb-PTX) is loaded into a nanoporous solid multistage nanovector (MSV) to enable the passage of the drug through the tumor vessel wall and enhance its interaction with liver macrophages. MSV enablement increased nAb-PTX efficacy and survival in mouse models of breast and lung liver metastasis. MSV-nAb-PTX also augmented the accumulation of paclitaxel and MSV in the liver, specifically in macrophages, whereas paclitaxel levels in the blood were unchanged after administering MSV-nAb-PTX or nAb-PTX. In vitro studies demonstrated that macrophages treated with MSV-nAb-PTX remained viable and were able to internalize, retain, and release significantly higher quantities of paclitaxel compared with treatment with nAb-PTX. The cytotoxic potency of the released paclitaxel was also confirmed in tumor cells cultured with the supernatants of macrophage treated with MSV-nAB-PTX. Collectively, our findings showed how redirecting nAb-PTX to liver macrophages within the tumor microenvironment can elicit a greater therapeutic response in patients with metastatic liver cancer, without increasing systemic side effects.

A specifically designed nanoconstruct associates, internalizes, traffics in cardiovascular cells, and accumulates in failing myocardium: a new strategy for heart failure diagnostics and therapeutics.

AIMS: Ongoing inflammation and endothelial dysfunction occurs within the local microenvironment of heart failure, creating an appropriate scenario for successful use and delivery of nanovectors. This study sought to investigate whether cardiovascular cells associate, internalize, and traffic a nanoplatform called mesoporous silicon vector (MSV), and determine its intravenous accumulation in cardiac tissue in a murine model of heart failure. METHODS AND RESULTS: In vitro cellular uptake and intracellular trafficking of MSVs was examined by scanning electron microscopy, confocal microscopy, time-lapse microscopy, and flow cytometry in cardiac myocytes, fibroblasts, smooth muscle cells, and endothelial cells. The MSVs were internalized within the first hours, and trafficked to perinuclear regions in all the cell lines. Cytotoxicity was investigated by annexin V and cell cycle assays. No significant evidence of toxicity was found. In vivo intravenous cardiac accumulation of MSVs was examined by high content fluorescence and confocal microscopy, with results showing increased accumulation of particles in failing hearts compared with normal hearts. Similar to observations in vitro, MSVs were able to associate, internalize, and traffic to the perinuclear region of cardiomyocytes in vivo. CONCLUSIONS: Results show that MSVs associate, internalize, and traffic in cardiovascular cells without any significant toxicity. Furthermore, MSVs accumulate in failing myocardium after intravenous administration, reaching intracellular regions of the cardiomyocytes. These findings represent a novel avenue to develop nanotechnology-based therapeutics and diagnostics in heart failure.

Computational analysis of drug transport in tumor microenvironment as a critical compartment for nanotherapeutic pharmacokinetics.

Over the last decade, nanotherapeutics gained increasingly important role in drug delivery because of their frequently beneficial pharmacokinetics (PK) and lower toxicity when compared to classical systemic drug delivery. In view of therapeutic payload delivery, convective transport is crucial for systemic distribution via circulatory system, but the target domain is tissue outside vessels where transport is governed by diffusion. Here, we have computationally investigated the understudied interplay of physical transports to characterize PK of payload of nanotherapeutics. The analysis of human vasculature tree showed that convective transport is still 5 times more efficient than diffusion suggesting that circulating and payload releasing drug vectors can contribute mostly to systemic delivery. By comparing payload delivery using systemic circulation and drug vectors to microenvironment, internalized vectors were the most efficient and showed Area under the Curve almost 100 higher than in systemic delivery. The newly introduced zone of influence parameter indicated that vectors, especially internalized, lead to the largest tissue fraction covered with therapeutically significant payload concentration. The internalization to microenvironment minimizes effects of plasma domain on payload extravasation from nanotherapeutics. The computed results showed that classical PK, which mostly relies on concentration profiles in plasma, sometimes might be inadequate or not sufficient in explaining therapeutic efficacy of nanotherapeutics. These results provide a deeper look into PK of drug vectors and can help in the design of better drug delivery strategies.

The protein kinase promiscuities in the cancer-preventive mechanisms of NSAIDs.

NSAIDs have been observed to have cancer-preventive properties, but the actual mechanism is elusive. We hypothesize that NSAIDs might have an effect through common pathways and targets of anticancer drugs by exploiting promiscuities of anticancer drug targets. Here, we have explored NSAIDs by their structural and pharmacophoric similarities with small anticancer molecules. In-silico analyses have shown a strong similarity between NSAIDs and protein kinase (PK) inhibitors. The calculated affinities of NSAIDs were found to be lower than the affinities of anticancer drugs, but higher than the affinities of compounds that are not specific to PKs. The competitive inhibition model suggests that PK might be inhibited by around 10%, which was confirmed by biochemical screening of some NSAIDs against PKs. NSAIDs did not affect all PKs universally, but had specificities for certain sets of PKs, which differed according to the NSAID. The study revealed potentially new features and mechanisms of NSAIDs that are useful in explaining their role in cancer prevention, which might lead to clinically significant breakthroughs in the future.

Capillary collagen as the physical transport barrier in drug delivery to tumor microenvironment.

The capillary wall is among the most important barriers that controls mass exchange between tumor microenvironment and systemic circulation. There are numerous studies on endothelial cells role in this mass exchange, but the role of capillary collagen of Type-IV in transport of small molecules and nanotherapeutics is less known. Our recent study revealed that the capillary wall collagen modulates the drug transport across the wall, and that it can be taken as a biophysical marker for drug transport. In our in vivo investigations with the 3LL and 4T1 tumors we noticed the differences in the collagen content in capillary walls. The imaging analysis and transport computational model of the capillary microenvironment showed that the penetration of doxorubicin (DOX) and pegylated liposomal doxorubicin (PLD) is substantially reduced by larger collagen content in the capillaries of the 3LL tumors. The results pointed to the importance of transport oncophysics, which opens a new avenue with respect to classical biology in understanding and improving drug delivery by nanotherapeutics, and aims to better explain the therapeutic resistance.

Liposomal doxorubicin extravasation controlled by phenotype-specific transport properties of tumor microenvironment and vascular barrier.

Although nanotherapeutics can be advantageous over free chemotherapy, the benefits of drug vectors can vary from patient to patient based on differences in tumor microenvironments. Although systemic pharmacokinetics (PK) of drugs is considered as the major determinant of its efficacy in clinics, recent clinical and basic research indicates that tumor-based PK can provide better representation of therapeutic efficacy. Here, we have studied the role of the tumor extravascular tissue in the extravasation kinetics of doxorubicin (DOX), delivered by pegylated liposomes (PLD), to murine lung (3LL) and breast (4T1) tumors. We found that phenotypically different 3LL and 4T1 tumors shared the similar systemic PK, but DOX extravasation in the tumor extravascular tissue was substantially different. Liquid chromatography-mass spectrometry (LC-MS) measurements showed that DOX fluorescence imaged by fluorescence microscopy could be used as a marker to study tumor microenvironment PK, providing an excellent match to DOX kinetics in tumor tissues. Our results also suggest that therapeutic responses can be closely related to the interplay of concentration levels and exposure times in extravascular tissue of tumors. Finally, the computational model of capillary drug transport showed that internalization of drug vectors was critical and could lead to 2-3 orders of magnitude more efficient drug delivery into the extravascular tissue, compared to non-internalized localization of drug vectors, and explaining the differences in therapeutic efficacy between the 3LL and 4T1 tumors. These results show that drug transport and partitioning characteristics can be phenotype- and microenvironment-dependent and are highly important in drug delivery and therapeutic efficacy.

Microfluidic cytometric analysis of cancer cell transportability and invasiveness.

The extensive phenotypic and functional heterogeneity of cancer cells plays an important role in tumor progression and therapeutic resistance. Characterizing this heterogeneity and identifying invasive phenotype may provide possibility to improve chemotherapy treatment. By mimicking cancer cell perfusion through circulatory system in metastasis, we develop a unique microfluidic cytometry (MC) platform to separate cancer cells at high throughput, and further derive a physical parameter 'transportability' to characterize the ability to pass through micro-constrictions. The transportability is determined by cell stiffness and cell-surface frictional property, and can be used to probe tumor heterogeneity, discriminate more invasive phenotypes and correlate with biomarker expressions in breast cancer cells. Decreased cell stiffness and cell-surface frictional force leads to an increase in transportability and may be a feature of invasive cancer cells by promoting cell perfusion through narrow spaces in circulatory system. The MC-Chip provides a promising microfluidic platform for studying cell mechanics and transportability could be used as a novel marker for probing tumor heterogeneity and determining invasive phenotypes.