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ABSTRACT: Xanthine oxidase (XO), a rate-limiting enzyme in uric acid production, is the pivotal therapeutic target for gout and hyperuricemia. In this study, 57 peptides from α-lactalbumin and β-lactoglobulin were obtained via virtual enzymatic hydrolysis, and 10 XO inhibitory peptides were virtually screened using molecular docking. Then toxicity, allergenicity, solubility, and isoelectric point of the obtained 10 novel peptides were evaluated by in silico tools. The XO activity of these synthetic peptides was tested using an in vitro assay by high-performance liquid chromatography. Their inhibitory mechanism was further explored by molecular docking. The results showed that 4 peptides GL, PM, AL, and AM exhibited higher inhibitory activity, and their half maximal inhibitory concentration in vitro was 10.20 ± 0.89, 23.82 ± 0.94, 34.49 ± 0.89, and 40.45 ± 0.92 mM, respectively. The peptides fitted well with XO through hydrogen bond, hydrophobic interaction, and van der Waals forces, and amino acid residues Glu802, Leu873, Arg880, and Pro1076 played an important role in this process. Overall, this study indicated 4 novel peptides GL, PM, AL, and AM from whey protein exhibited XO inhibitory activity, and they might be useful and safe XO inhibitors for hyperuricemia prevention and treatment.

2′-fucosyllactose (2′-FL) is one of the most prevalent milk oligosaccharides. In this study, the anti-obesity effect of 2′-FL was explored in high-fat diet (HFD)-fed mice. Results showed that 2′-FL alleviated body weight gain, improved serum lipid profiles and increased energy expenditure. Under cold exposure, 2′-FL group showed higher body and rectal temperature, improving adaptive thermogenesis. 2′-FL alleviated lipid accumulation, increased mitochondrial DNA content, as well as upregulated the protein expression of thermogenic markers including UCP1, PRDM16, PGC-1α and the phosphorylation of AMPK in both white and brown adipose tissue. 2′-FL also increased the diversity of gut microbiota in HFD mice. The Bacteroides/Firmicutes ratio, and the Bifidobacterium abundance were increased, while Lachnospiraceae abundance was decreased after 2′-FL treatment. Moreover, 2′-FL to HFD mice altered the bile acid profiles at levels comparable to normal diet group. These results indicates that 2′-FL promotes thermogenesis and modulates gut microbiota to alleviate obesity.

ABSTRACT: The main purpose of the current study was to investigate the ameliorative effects of bovine milk osteopontin (bmOPN) on the gut dysfunction of pregnant rats fed a high-fat diet (HFD). Bovine milk osteopontin was supplemented at a dose of 6 mg/kg body weight. Bovine milk osteopontin supplementation during pregnancy reduced colonic inflammation of HFD dams, and it also increased the colonic expression of ZO-1 and claudin-4 of HFD dams. Bovine milk osteopontin significantly enriched the relative abundance of Bacteroidetes, whereas it decreased Proteobacteria, Helicobacteraceae, and Desulfovibrionaceae in feces of HFD dams. The levels of isobutyric acid and pentanoic acid in the HFD + bmOPN group were higher than that of the HFD group. Functional predication analysis of microbial genomes revealed that bmOPN supplementation to HFD pregnancies changed 4 Kyoto Encyclopedia of Genes and Genomes pathways including bile acid biosynthesis. Further, bmOPN enriched hepatic taurochenodeoxycholic acid and tauroursodeoxycholic acid plus taurohyodeoxycholic acid in the gut of HFD maternal rats. Our findings suggested that bmOPN improved the gut health of HFD pregnant rats partially through modulating bile acid biosynthesis.

Milk fat globule membrane (MFGM), which contains abundant glycoproteins and phospholipids, exerts beneficial effects on intestinal health and immunomodulation. The aim of this study was to evaluate the protective effects and possible underlying mechanisms of MFGM on cow’s milk allergy (CMA) in a β-lactoglobulin (BLG)-induced allergic mice model. MFGM was supplemented to allergic mice induced by BLG at a dose of 400 mg/kg body weight. Results demonstrated that MFGM alleviated food allergy symptoms, decreased serum levels of lipopolysaccharide, pro-inflammatory cytokines, immunoglobulin (Ig) E, IgG1, and Th2 cytokines including interleukin (IL)-4, while increased serum levels of Th1 cytokines including interferon-γ and regulatory T cells (Tregs) cytokines including IL-10 and transforming growth factor-β. MFGM modulated gut microbiota and enhanced intestinal barrier of BLG-allergic mice, as evidenced by decreased relative abundance of Desulfobacterota, Rikenellaceae, Lachnospiraceae, and Desulfovibrionaceae, while increased relative abundance of Bacteroidetes, Lactobacillaceae and Muribaculaceae, and enhanced expressions of tight junction proteins including Occludin, Claudin-1 and zonula occludens-1. Furthermore, MFGM increased fecal short-chain fatty acids (SCFAs) levels, which elevated G protein-coupled receptor (GPR) 43 and GPR109A expressions. The increased expressions of GPR43 and GPR109A induced CD103+ dendritic cells accumulation and promoted Tregs differentiation in mesenteric lymph node to a certain extent. In summary, MFGM alleviated CMA in a BLG-induced allergic mice model through enhancing intestinal barrier and promoting Tregs differentiation, which may be correlated with SCFAs-mediated activation of GPRs. These findings suggest that MFGM may be useful as a promising functional ingredient against CMA.

Unlike T cells in other tissues, uterine T cells must balance strong immune defense against pathogens with tolerance to semiallogeneic fetus. Our previous study fully elucidated the characteristics of γδT cells in nonpregnant uterus and the mechanism modulated by estrogen. However, comprehensive knowledge of the immunological properties of αβT (including CD4+T cells and CD8+T) cells in nonpregnancy uterus has not been acquired. In this study, we fully compared the immunological properties of αβT cells between uterus and blood using mouse and human sample. It showed that most of CD4+T cells and CD8+T cells in murine uterus and human endometrium were tissue resident memory T cells which highly expressed tissue residence markers CD69 and/or CD103. In addition, both CD4+T cells and CD8+T cells in uterus highly expressed inhibitory molecular PD-1 and cytokine IFN-γ. Uterine CD4+T cells highly expressed IL-17 and modulated by transcription factor pSTAT3. Moreover, we compared the similarities and differences between human and murine uterine T cell phenotype. Together, uterine CD4+T cells and CD8+ cells exhibited a unique mixed signature of T cell dysfunction, activation, and effector function which enabled them to balance strong immune defense against pathogens with tolerance to fetus. Our study fully elucidated the unique immunologic properties of uterine CD4+T and CD8+T cells and provided a base for further investigation of functions.

Food allergy has become a significant public health problem affecting a large number of people worldwide. Maternal obesity causes inflammation and alters the immune system of offspring, which may exacerbate their food allergy. The aim of this study was to determine whether offspring mice born to obese mothers would have more serve reactions to cow’s milk protein-induced food allergy, and further investigate the underlying mechanisms. Female offspring BALB/c mice of mothers with normal and high-fat diets were sensitized with β-lactoglobulin (BLG), respectively. Maternal obesity increased the serum immunoglobulin E and mouse mast cell protease levels, though did not have significant influence on anaphylactic symptom score, core temperature and diarrhea rate of offspring mice after BLG sensitization. Furthermore, maternal obesity led to a lower level of occludin mRNA expression in BLG -sensitized mice. The mice born to obese mothers exhibited increased mRNA expression levels of GATA-3, interleukin (IL)-4 and IL-10 in jejunum after BLG sensitization, indicating maternal obesity intensified Th2-type biased immune responses. In conclusion, maternal obesity exerted exacerbating effects on the responsiveness of their offspring to cow’s milk protein sensitization.

IntroductionThe COVID-19 pandemic has posed a major burden on healthcare and economic systems across the globe for over 3 years. Even though vaccines are available, the pathogenesis is still unclear. Multiple studies have indicated heterogeneity of immune responses to SARS-CoV-2, and potentially distinct patient immune types that might be related to disease features. However, those conclusions are mainly inferred by comparing the differences of pathological features between moderate and severe patients, some immunological features may be subjectively overlooked.MethodsIn this study, the relevance scores(RS), reflecting which features play a more critical role in the decision-making process, between immunological features and the COVID-19 severity are objectively calculated through neural network, where the input features include the immune cell counts and the activation marker concentrations of particular cell, and these quantified characteristic data are robustly generated by processing flow cytometry data sets containing the peripheral blood information of COVID-19 patients through PhenoGraph algorithm.ResultsSpecifically, the RS between immune cell counts and COVID-19 severity with time indicated that the innate immune responses in severe patients are delayed at the early stage, and the continuous decrease of classical monocytes in peripherial blood is significantly associated with the severity of disease. The RS between activation marker concentrations and COVID-19 severity suggested that the down-regulation of IFN-γ in classical monocytes, Treg, CD8 T cells, and the not down-regulation of IL_17a in classical monocytes, Tregs are highly correlated with the occurrence of severe disease. Finally, a concise dynamic model of immune responses in COVID-19 patients was generalized.DiscussionThese results suggest that the delayed innate immune responses in the early stage, and the abnormal expression of IL-17a and IFN-γ in classical monocytes, Tregs, and CD8 T cells are primarily responsible for the severity of COVID-19.

BackgroundDocetaxel improves overall survival (OS) in castration-resistant prostate cancer (PCa) (CRPC) and metastatic hormone-sensitive PCa (mHSPC). However, not all patients respond due to inherent and/or acquired resistance. There remains an unmet clinical need for a robust predictive test to stratify patients for treatment. Liquid biopsy of circulating tumour cell (CTCs) is minimally invasive, can provide real-time information of the heterogeneous tumour and therefore may be a potentially ideal docetaxel response prediction biomarker.ObjectiveIn this study we investigate the potential of using CTCs and their gene expression to predict post-docetaxel tumour response, OS and progression free survival (PFS).MethodsPeripheral blood was sampled from 18 mCRPC and 43 mHSPC patients, pre-docetaxel treatment, for CTC investigation. CTCs were isolated using the epitope independent Parsortix® system and gene expression was determined by multiplex RT-qPCR. We evaluated CTC measurements for post-docetaxel outcome prediction using receiver operating characteristics and Kaplan Meier analysis.ResultsDetection of CTCs pre-docetaxel was associated with poor patient outcome post-docetaxel treatment. Combining total-CTC number with PSA and ALP predicted lack of partial response (PR) with an AUC of 0.90, p= 0.037 in mCRPC. A significantly shorter median OS was seen in mCRPC patients with positive CTC-score (12.80 vs. 37.33 months, HR= 5.08, p= 0.0005), ≥3 total-CTCs/7.5mL (12.80 vs. 37.33 months, HR= 3.84, p= 0.0053), ≥1 epithelial-CTCs/7.5mL (14.30 vs. 37.33 months, HR= 3.89, p= 0.0041) or epithelial to mesenchymal transitioning (EMTing)-CTCs/7.5mL (11.32 vs. 32.37 months, HR= 6.73, p= 0.0001). Significantly shorter PFS was observed in patients with ≥2 epithelial-CTCs/7.5mL (7.52 vs. 18.83 months, HR= 3.93, p= 0.0058). mHSPC patients with ≥5 CTCs/7.5mL had significantly shorter median OS (24.57 vs undefined months, HR= 4.14, p= 0.0097). In mHSPC patients, expression of KLK2, KLK4, ADAMTS1, ZEB1 and SNAI1 was significantly associated with shorter OS and/or PFS. Importantly, combining CTC measurements with clinical biomarkers increased sensitivity and specificity for prediction of patient outcome.ConclusionWhile it is clear that CTC numbers and gene expression were prognostic for PCa post-docetaxel treatment, and CTC subtype analysis may have additional value, their potential predictive value for docetaxel chemotherapy response needs to be further investigated in large patient cohorts.

In this study, 12 flavonoid glycosides were selected based on virtual screening and the literature, and Quercimeritrin was selected as the best selective inhibitor of α-glucosidase through in vitro enzyme activity inhibition experiments. Its IC50 value for α-glucosidase was 79.88 µM, and its IC50 value for α-amylase >250 µM. As such, it could be used as a new selective inhibitor of α-glucosidase. The selective inhibition mechanism of Quercimeritrin on the two starch-digesting enzymes was further explored, and it was confirmed that Quercimeritrin had a strong binding affinity for α-glucosidase and occupied the binding pocket of α-glucosidase through non-covalent binding. Subsequently, animal experiments demonstrated that Quercimeritrin can effectively control postprandial blood glucose in vivo, with the same inhibitory effect as acarbose but without side effects. Our results, therefore, provide insights into how flavone aglycones can be used to effectively control the rate of digestion to improve postprandial blood glucose levels.

The purpose of this study was to investigate the post-heating induced changes in the textural and sensory characteristics of stirred fermented milk. The textural and rheological properties of post-heating fermented milk (55–85 °C, 25 s) with respect to viscoelastic behaviors, viscosity, textural parameters, etc., were monitored, and the friction behaviors and sensory attributes were assessed. Treatments below 65 °C/25 s increased the textural properties of fermented milk such as gel strength, firmness, and viscosity, due to the moderate aggregation and increased linkages of microgels. In this case, despite the size and amount their aggregates increased (~15–~21 μm), they exhibited similar frictional behaviors and sensory attributes. However, treatments above 65 °C/25 s degraded textural properties due to excessive aggregation (~46–~63 μm), accompanied by unacceptable grainy attributes, which could be characterized by their good correlations with tribological coefficients and particle size parameters. These findings could provide an understanding of the quality formation of post-heating fermented milk and a perspective to improve the textural defects of ambient fermented milk products.

Pre-pregnancy obesity and high-fat diet (HFD) during pregnancy and lactation are associated with neurodevelopmental delay in offspring. This study aimed to investigate whether milk fat globule membrane (MFGM) supplementation in obese dams could promote neurodevelopment in offspring. Obese female rats induced by HFD were supplemented with MFGM during pregnancy and lactation. Maternal HFD exposure significantly delayed the maturation of neurological reflexes and inhibited neurogenesis in offspring, which were significantly recovered by maternal MFGM supplementation. Gut microbiota analysis revealed that MFGM supplementation modulated the diversity and composition of gut microbiota in offspring. The abundance of pro-inflammatory bacteria such as Escherichia shigella and Enterococcus were down-regulated, and the abundance of bacteria with anti-inflammatory and anti-obesity functions, such as Akkermansia and Lactobacillus were up-regulated. Furthermore, MFGM alleviated neuroinflammation by decreasing the levels of lipopolysaccharides (LPS) and pro-inflammatory cytokines in the circulation and brain, as well as inhibiting the activation of microglia. Spearman’s correlation analysis suggested that there existed a correlation between gut microbiota and inflammation-related indexes. In conclusion, maternal MFGM supplementation promotes neurodevelopment partly via modulating gut microbiota in offspring.

Yak milk casein (YMC) is the main protein in the yak milk. Peptides released from Yak milk casein (YMC) have multiple bioactivities, including anti-inflammation and immune-regulation, suggesting that these peptides might be able to inhibit cancer theoretically. However, the anti-cancer peptides from YMC have only been sparsely studied. Breast carcinoma is the most common carcinoma in women worldwide. Thus, the paper herein was to identify yak milk casein (YMC)-derived anti-breast cancer peptides via gel filtration, reversed phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) for the first time. The inhibitory effects of the hydrolysates on the cell viabilities, cell cycles and apoptosis of breast cancer cells were evaluated with a cck8 kit and a flow cytometry. The result showed that YMC hydrolysates (YMCH) obtained by united hydrolyzation with trypsin (3 h) and alkaline protease (3 h) displayed the highest cell viability inhibition rate for MCF7 (20.74 ± 1.39%) and MDA-MB-231 (26.73 ± 2.87%) cells. Three peptides were identified in the RP-HPLC subfraction F3-4, and a nonapeptide (TPVVVPPFL) showed the most potent inhibitory effects on both cancer cells and displayed good gastrointestinal stability. TPVVVPPFL could induce G2-M cell cycle arrest in MCF7 cells and S cell arrest in MDA-MB-231 cells and induce apoptosis in both cancer cells. Moreover, in silico analysis indicated that the peptide had non-toxic and no inhibitory roles on P4502D6-enzyme. Together, this study shows that YMC is a good source of anti-breast cancer cells peptides.

The effect of bovine α-lactalbumin hydrolysates (α-LAH) on attenuating high-fat diet (HFD) induced nonalcoholic fatty liver disease (NAFLD) was investigated. C57BL/6J mice were fed with HFD for 8 weeks to mimic the clinical features of NAFLD, and then were fed with HFD and bovine α-LAH at different doses (100, 200 and 400 mg/kg b.w.) for another 12 weeks to investigate the beneficial effects of bovine α-LAH on NAFLD. Our data showed that bovine α-LAH markedly reversed the increase of body weight gain, tissue weight, serum lipids, liver injury markers, and serum pro-inflammatory cytokines induced by HFD in C57BL/6J mice. Bovine α-LAH supplementation also ameliorated fat accumulation and oxidative stress, down-regulated PPARγ associated gene expression, and up-regulated PPARα associated gene expression in the liver of mice fed HFD. These findings suggested that bovine α-LAH is effective in attenuating HFD induced NAFLD in C57BL/6J mice.

Castration-resistant prostate cancer (CRPC) is the major cause of death from prostate cancer. Biomarkers to improve early detection and prediction of CRPC especially using non-invasive liquid biopsies could improve outcomes. Therefore, we investigated the plasma exosomal miRNAs associated with CRPC and their potential for development into non-invasive early detection biomarkers for resistance to treatment. RNA-sequencing, which generated approximately five million reads per patient, was performed to identify differentially expressed plasma exosomal miRNAs in 24 treatment-naive prostate cancer and 24 CRPC patients. RT-qPCR was used to confirm the differential expressions of six exosomal miRNAs, miR-423-3p, miR-320a, miR-99a-5p, miR-320d, miR-320b, and miR-150-5p (p = 7.3 × 10−8, 0.0020, 0.018, 0.0028, 0.0013, and 0.0058, respectively) firstly in a validation cohort of 108 treatment-naive prostate cancer and 42 CRPC patients. The most significant differentially expressed miRNA, miR-423-3p, was shown to be associated with CRPC with area under the ROC curve (AUC) = 0.784. Combining miR-423-3p with prostate-specific antigen (PSA) enhanced the prediction of CRPC (AUC = 0.908). A separate research center validation with 30 treatment-naive and 30 CRPC patients also confirmed the differential expression of miR-423-3p (p = 0.016). Finally, plasma exosomal miR-423-3p expression in CRPC patients was compared to 36 non-CRPC patients under androgen depletion therapy, which showed significantly higher expression in CRPC than treated non-CRPC patients (p < 0.0001) with AUC = 0.879 to predict CRPC with no difference between treatment-naive and treated non-CRPC patients. Therefore, our findings demonstrate that a number of plasma exosomal miRNAs are associated with CRPC and miR-423-3p may serve as a biomarker for early detection/prediction of castration-resistance.

Potentilla discolor Bunge, as a traditional Chinese medicine, exhibits many phytochemical activities. The aim of the present study was to investigate the effects of Potentilla discolor Bunge water extract (PDBW) and its underlying mechanisms on gluconeogenesis and glycogen synthesis in high-fat diet/streptozotocin (HFD/STZ)-induced type 2 diabetic mice. LC-MS/MS analyses of PDBW identified 6 major compounds including apigenin-7-O-β-D-glucoside, epicatechin, quercetin 3-O-β-D-glucuronide, kaempferol-3-O-β-D-glucopyranoside, scutellarin, and quercitrin. In the study, a mouse model of type 2 diabetes was induced by 4-week HFD combined with STZ (40 mg/kg body weight) for 5 days. After oral administration of PDBW at 400 mg/kg body weight daily for 8 weeks, the mice with type 2 diabetes showed significant decrease in the levels of fasting blood glucose and glycated hemoglobin A1c (HbA1c), and increase in the insulin level. PDBW improved the glucose tolerance, insulin sensitivity and lipid profiles. Furthermore, PDBW inhibited the mRNA levels of key gluconeogenic enzymes [phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase)] in liver. PDBW also promoted glycogen synthesis by raising the liver glycogen content, decreasing the phosphorylation of glycogen synthase (GS) and increasing the phosphorylation of glycogen synthase kinase3β (GSK3β). Besides, PDBW induced the activation of protein kinase B (Akt) and AMP-activated protein kinase (AMPK), which might explain changes in the phosphorylation of above enzymes. In summary, PDBW supplementation ameliorates metabolic disorders in a HFD/STZ diabetic mouse model, suggesting the potential application of PDBW in prevention and amelioration of type 2 diabetes.

Gut microbiota plays an important role in the development of obesity and related metabolic endotoxemia. The aim of this study was to evaluate whether milk fat globule membrane (MFGM) could alter the composition of gut microbiota and exert a beneficial impact on combating high-fat diet-induced metabolic endotoxemia. Results showed that MFGM improved the gut microbiota dysbiosis induced by high-fat diet in C57BL/6J mice, including increasing the Bacteroidetes/Firmicutes ratios and the relative abundance of S24-7. Spearman correlation analysis indicated that there existed a correlation between gut microbiota and obesity-related indexes. MFGM also alleviated high-fat diet-induced intestinal inflammation by decreasing the levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), toll-like receptor 4 (TLR4) and increasing the expression of tight junction proteins including zonulin-1 (ZO-1) and occludin. Moreover, MFGM significantly decreased the levels of lipopolysaccharides (LPS), IL-6 and TNF-α in serum of high-fat diet-induced mice. These findings demonstrated that MFGM supplementation ameliorated obesity-related inflammation and endotoxemia partly via modulating the composition of gut microbiota in mice challenged with a high-fat diet.

Prostate cancer (PrCa) involves a large heritable genetic component. Here, the authors perform multivariate fine-mapping of known PrCa GWAS loci, identifying variants enriched for biological function, explaining more familial relative risk, and with potential application in clinical risk profiling.

Abstract Prostate cancer is the most common cancer among western men, with a significant mortality and morbidity reported for advanced metastatic disease. Current understanding of metastatic disease is limited due to difficulty of sampling as prostate cancer mainly metastasizes to bone. By analysing prostate cancer bone metastases using high density microarrays, we found a common genomic copy number loss at 6q16.1–16.2, containing the FBXL4 gene, which was confirmed in larger series of bone metastases by fluorescence in situ hybridisation (FISH). Loss of FBXL4 was also detected in primary tumours and it was highly associated with prognostic factors including high Gleason score, clinical stage, prostate-specific antigen (PSA) and extent of disease, as well as poor patient survival, suggesting that FBXL4 loss contributes to prostate cancer progression. We also demonstrated that FBXL4 deletion is detectable in circulating tumour cells (CTCs), making it a potential prognostic biomarker by ‘liquid biopsy’. In vitro analysis showed that FBXL4 plays a role in regulating the migration and invasion of prostate cancer cells. FBXL4 potentially controls cancer metastasis through regulation of ERLEC1 levels. Therefore, FBXL4 could be a potential novel prostate cancer suppressor gene, which may prevent cancer progression and metastasis through controlling cell invasion.

Identifying DPP-IV inhibitory peptides from dietary protein has attracted increased attention. In the present study, bovine α-lactalbumin hydrolysates were generated by alcalase for various hydrolysis times, and DPP-IV inhibitory activity of these hydrolysates was determined. The 4 h hydrolysates displayed the most potent DPP-IV inhibitory activity, with DPP-IV inhibition rate of 82.30 ± 1.39% at concentration of 1.0 mg/mL. DPP-IV inhibitory peptides were isolated from the 4 h-hydrolysates with gel filtration chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC). Using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS), two DPP-IV inhibitory peptides were identified, and their amino acid sequences were Glu-Leu-Lys-Asp-Leu-Lys-Gly-Tyr (ELKDLKGY) and Ile-Leu-Asp-Lys-Val-Gly-Ile-Asn-Tyr (ILDKVGINY), respectively. Furthermore, molecular docking analysis showed that peptides ELKDLKGY and ILDKVGINY could form hydrogen bonds, pi-cation interactions, and salt bridges with DPP-IV. These findings indicated that bovine α-lactalbumin may be a potential source of natural DPP-IV inhibitor.

Isolation of circulating tumor cells (CTCs) from peripheral blood has the potential to provide a far easier "liquid biopsy" than tumor tissue biopsies, to monitor tumor cell populations during disease progression and in response to therapies. Many CTC isolation technologies have been developed. We optimized the Parsortix system, an epitope independent, size and compressibility-based platform for CTCs isolation, making it possible to harvest CTCs at the speed and sample volume comparable to standard CellSearch system. We captured more than half of cancer cells from different cancer cell lines spiked in blood samples from healthy donors using this system. Cell loss during immunostaining of cells transferred and fixed on the slides is a major problem for analyzing rare cell samples. We developed a novel cell transfer and fixation method to retain >90% of cells on the slide after the immunofluorescence process without affecting signal strength and specificity. Using this optimized method, we evaluated the Parsortix system for CTC harvest in prostate cancer patients in comparison to immunobead based CTC isolation systems IsoFlux and CellSearch. We harvested a similar number (p = 0.33) of cytokeratin (CK) positive CTCs using Parsortix and IsoFlux from 7.5 mL blood samples of 10 prostate cancer patients (an average of 33.8 and 37.6 respectively). The purity of the CTCs harvested by Parsortix at 3.1% was significantly higher than IsoFlux at 1.0% (p = 0.02). Parsortix harvested significantly more CK positive CTCs than CellSearch (p = 0.04) in seven prostate cancer patient samples, where both systems were utilized (an average of 32.1 and 10.1 respectively). We also captured CTC clusters using Parsortix. Using four-color immunofluorescence we found that 85.8% of PC3 cells expressed EpCAM, 91.7% expressed CK and 2.5% cells lacked both epithelial markers. Interestingly, 95.6% of PC3 cells expressed Vimentin, including those cells that lacked both epithelial marker expression, indicating epithelial-to-mesenchymal transition. CK-positive/Vimentin-positive/CD45-negative, and CK-negative/Vimentin-positive/CD45-negative cells were also observed in four of five prostate cancer patients but rarely in three healthy controls, indicating that Parsortix harvests CTCs with both epithelial and mesenchymal features. We also demonstrated using PC3 and DU145 spiking experiment that Parsortix harvested cells were viable for cell culture.