Cell softness regulates tumorigenicity and stemness of cancer cells.

Identifying and sorting highly tumorigenic and metastatic tumor cells from a heterogeneous cell population is a daunting challenge. Here, we show that microfluidic devices can be used to sort marker-based heterogeneous cancer stem cells (CSC) into mechanically stiff and soft subpopulations. The isolated soft tumor cells (< 400 Pa) but not the stiff ones (> 700 Pa) can form a tumor in immunocompetent mice with 100 cells per inoculation. Notably, only the soft, but not the stiff cells, isolated from CD133+ , ALDH+ , or side population CSCs, are able to form a tumor with only 100 cells in NOD-SCID or immunocompetent mice. The Wnt signaling protein BCL9L is upregulated in soft tumor cells and regulates their stemness and tumorigenicity. Clinically, BCL9L expression is correlated with a worse prognosis. Our findings suggest that the intrinsic softness is a unique marker of highly tumorigenic and metastatic tumor cells.

Identification of Candida glabrata Transcriptional Regulators That Govern Stress Resistance and Virulence.

The mechanisms by which Candida glabrata resists host defense peptides and caspofungin are incompletely understood. To identify transcriptional regulators that enable C. glabrata to withstand these classes of stressors, a library of 215 C. glabrata transcriptional regulatory deletion mutants was screened for susceptibility to both protamine and caspofungin. We identified eight mutants that had increased susceptibility to both host defense peptides and caspofungin. Of these mutants, six were deleted for genes that were predicted to specify proteins involved in histone modification. These genes were ADA2, GCN5, SPT8, HOS2, RPD3, and SPP1 Deletion of ADA2, GCN5, and RPD3 also increased susceptibility to mammalian host defense peptides. The Δada2 and Δgcn5 mutants had increased susceptibility to other stressors, such as H2O2 and SDS. In the Galleria mellonella model of disseminated infection, the Δada2 and Δgcn5 mutants had attenuated virulence, whereas in neutropenic mice, the virulence of the Δada2 and Δrpd3 mutants was decreased. Thus, histone modification plays a central role in enabling C. glabrata to survive host defense peptides and caspofungin, and Ada2 and Rpd3 are essential for the maximal virulence of this organism during disseminated infection.

Arp2/3-branched actin regulates microtubule acetylation levels and affects mitochondrial distribution.

Actin and microtubule cytoskeletons regulate cell morphology, participate in organelle trafficking and function in response to diverse environmental cues. Precise spatial-temporal coordination between these two cytoskeletons is essential for cells to live and move. Here, we report a novel crosstalk between actin and microtubules, in which the branched actin maintains microtubule organization, dynamics and stability by affecting tubulin acetylation levels. We observed that acetylated tubulin significantly decreases upon perturbation of the Arp2/3-branched actin. We subsequently discover that HDAC6 participates in this process by altering its interaction with tubulin and the Arp2/3-stabilizer cortactin. We further identify that the homeostasis of branched actin controls mitochondrial distribution via this microtubule acetylation-dependent mechanism. Our findings shed new light on the integral view of cytoskeletal networks, highlighting post-translational modification as another possible form of cytoskeletal inter-regulation, aside from the established crosstalks through structural connection or upstream signaling pathways.

Optical properties of chain inverted pyramids on silicon.

Pyramidal structures, including upright pyramids and inverted pyramids (IPs), are commonly used as light-trapping structures for silicon solar cells and silicon photodetectors. In this paper, the possible ray propagation paths in a pyramidal structure are analyzed by establishing a mathematical model in which up to seven ray paths may exist either in a regular or random pyramidal structure. To reduce the reflectivity, the proportion of the quadruple bounce should be increased because of its lower reflectivity. Therefore, a chain IP structure with a quadruple bounce proportion of 10.33% is proposed, of which the overlap value $\Delta x/w$Δx/w is 0.4. According to theoretical ray-tracing calculations, the weighted average reflectivity is reduced by 0.75% compared to that of a random IP structure. Experimentally, chain IP structures are fabricated from the surface line damage produced by the diamond wire sawing of a silicon wafer as a mask, and the reflectivity of the structures is 0.80% lower than that of a random IP structure. The theoretical analysis and experimental results both show that the chain IP structure has better optical properties than the random IP structure, indicating promising prospects for the abovementioned applications.

New signaling pathways govern the host response to C. albicans infection in various niches.

Candida albicans, the major invasive fungal pathogen of humans, can cause both debilitating mucosal infections and fatal invasive infections. Understanding the complex nature of the host-pathogen interaction in each of these contexts is essential to developing desperately needed therapies to treat fungal infections. RNA-seq enables a systems-level understanding of infection by facilitating comprehensive analysis of transcriptomes from multiple species (e.g., host and pathogen) simultaneously. We used RNA-seq to characterize the transcriptomes of both C. albicans and human endothelial cells or oral epithelial cells during in vitro infection. Network analysis of the differentially expressed genes identified the activation of several signaling pathways that have not previously been associated with the host response to fungal pathogens. Using an siRNA knockdown approach, we demonstrate that two of these pathways-platelet-derived growth factor BB (PDGF BB) and neural precursor-cell-expressed developmentally down-regulated protein 9 (NEDD9)-govern the host-pathogen interaction by regulating the uptake of C. albicans by host cells. Using RNA-seq analysis of a mouse model of hematogenously disseminated candidiasis (HDC) and episodes of vulvovaginal candidiasis (VVC) in humans, we found evidence that many of the same signaling pathways are activated during mucosal (VVC) and/or disseminated (HDC) infections in vivo. Our analyses have uncovered several signaling pathways at the interface between C. albicans and host cells in various contexts of infection, and suggest that PDGF BB and NEDD9 play important roles in this interaction. In addition, these data provide a valuable community resource for better understanding host-fungal pathogen interactions.

Raman-Activated Droplet Sorting (RADS) for Label-Free High-Throughput Screening of Microalgal Single-Cells.

Raman-activated cell sorting (RACS) has attracted increasing interest, yet throughput remains one major factor limiting its broader application. Here we present an integrated Raman-activated droplet sorting (RADS) microfluidic system for functional screening of live cells in a label-free and high-throughput manner, by employing AXT-synthetic industrial microalga Haematococcus pluvialis (H. pluvialis) as a model. Raman microspectroscopy analysis of individual cells is carried out prior to their microdroplet encapsulation, which is then directly coupled to DEP-based droplet sorting. To validate the system, H. pluvialis cells containing different levels of AXT were mixed and underwent RADS. Those AXT-hyperproducing cells were sorted with an accuracy of 98.3%, an enrichment ratio of eight folds, and a throughput of ∼260 cells/min. Of the RADS-sorted cells, 92.7% remained alive and able to proliferate, which is equivalent to the unsorted cells. Thus, the RADS achieves a much higher throughput than existing RACS systems, preserves the vitality of cells, and facilitates seamless coupling with downstream manipulations such as single-cell sequencing and cultivation.

Role of retrograde trafficking in stress response, host cell interactions, and virulence of Candida albicans.

In Saccharomyces cerevisiae, the vacuolar protein sorting complexes Vps51/52/53/54 and Vps15/30/34/38 are essential for efficient endosome-to-Golgi complex retrograde transport. Here we investigated the function of Vps15 and Vps51, representative members of these complexes, in the stress resistance, host cell interactions, and virulence of Candida albicans. We found that C. albicans vps15Δ/Δ and vps51Δ/Δ mutants had abnormal vacuolar morphology, impaired retrograde protein trafficking, and dramatically increased susceptibility to a variety of stressors. These mutants also had reduced capacity to invade and damage oral epithelial cells in vitro and attenuated virulence in the mouse model of oropharyngeal candidiasis. Proteomic analysis of the cell wall of the vps51Δ/Δ mutant revealed increased levels of the Crh11 and Utr2 transglycosylases, which are targets of the calcineurin signaling pathway. The transcript levels of the calcineurin pathway members CHR11, UTR2, CRZ1, CNA1, and CNA2 were elevated in the vps15Δ/Δ and vps51Δ/Δ mutants. Furthermore, these strains were highly sensitive to the calcineurin-specific inhibitor FK506. Also, deletion of CHR11 and UTR2 further increased the stress susceptibility of these mutants. In contrast, overexpression of CRH11 and UTR2 partially rescued their defects in stress resistance, but not host cell interactions. Therefore, intact retrograde trafficking in C. albicans is essential for stress resistance, host cell interactions, and virulence. Aberrant retrograde trafficking stimulates the calcineurin signaling pathway, leading to the increased expression of Chr11 and Utr2, which enables C. albicans to withstand environmental stress.

[Clinical value of the fetal echocardiography routine inspection in grass-roots hospitals].

OBJECTIVE: To estimate the significance and clinical value of fetal echocardiography in the prenatal test of the grass-roots hospital. METHODS: 22 570 pregnant women were reviewed, newborns echocardiography were carried out in 19 978 cases. As for the abnormal cases in prenatal screening, the result was compared with the superior hospital diagnosis, also newborns echocardiography was executed to identify the result. RESULTS: 140 cases with fetal cardiac abnormalities had been diagnosed, of which 130 cases confirmed. The diagnosis coincidence rate was 92.9% (130/140) and prenatal positive detection rate was 0.6% (130/22 570). Omission diagnostic rate was 6.2% (8/130). Misdiagnosis rate was 3.8% (5/130). 3 cases were lost to follow-up. 174 cases of newborns cardiac anomalies were diagnosed in postpartum echocardiography. CONCLUSIONS: it is of importance to carry out the fetal echocardiography routine inspection in grass-roots hospitals, of which will facilitated the early detection and diagnosis of congenital heart abnormalities.

[Significance of ultrasonic detection of muscle layer thickness of cesarean section scar in late pregnancy].

OBJECTIVE: To explore the clinical value of high frequency ultrasound in monitoring muscle layer thickness of cesarean section scar in late pregnancy. METHODS: A total of 131 patients re-conceived after cesarean section were selected randomly from our hospital.High frequency ultrasound was used for monitoring muscle layer thickness of cesarean section scar. And ultrasonic results and operative findings were compared. RESULTS: Based upon the ultrasonic results, they were divided into 2 groups: group 1 ( ≤ 2 mm, n = 61), there were confirmed (n = 19) and misdiagnosed (n = 11) uterine rupture; group 2 ( > 2 mm, n = 70), confirmed (n = 5) and misdiagnosed (n = 5) uterine rupture. CONCLUSION: Monitoring muscle layer thickness of cesarean section scar is of great significance for preventing uterine rupture in late pregnancy.

SR-like RNA-binding protein Slr1 affects Candida albicans filamentation and virulence.

Candida albicans causes both mucosal and disseminated infections, and its capacity to grow as both yeast and hyphae is a key virulence factor. Hyphal formation is a type of polarized growth, and members of the SR (serine-arginine) family of RNA-binding proteins influence polarized growth of both Saccharomyces cerevisiae and Aspergillus nidulans. Therefore, we investigated whether SR-like proteins affect filamentous growth and virulence of C. albicans. BLAST searches with S. cerevisiae SR-like protein Npl3 (ScNpl3) identified two C. albicans proteins: CaNpl3, an apparent ScNpl3 ortholog, and Slr1, another SR-like RNA-binding protein with no close S. cerevisiae ortholog. Whereas ScNpl3 was critical for growth, deletion of NPL3 in C. albicans resulted in few phenotypic changes. In contrast, the slr1Δ/Δ mutant had a reduced growth rate in vitro, decreased filamentation, and impaired capacity to damage epithelial and endothelial cells in vitro. Mice infected intravenously with the slr1Δ/Δ mutant strain had significantly prolonged survival compared to that of mice infected with the wild-type or slr1Δ/Δ mutant complemented with SLR1 (slr1Δ/Δ+SLR1) strain, without a concomitant decrease in kidney fungal burden. Histopathology, however, revealed differential localization of slr1Δ/Δ hyphal and yeast morphologies within the kidney. Mice infected with slr1Δ/Δ cells also had an increased brain fungal burden, which correlated with increased invasion of brain, but not umbilical vein, endothelial cells in vitro. The enhanced brain endothelial cell invasion was likely due to the increased surface exposure of the Als3 adhesin on slr1Δ/Δ cells. Our results indicate that Slr1 is an SR-like protein that influences C. albicans growth, filamentation, host cell interactions, and virulence.

Investigation of the function of Candida albicans Als3 by heterologous expression in Candida glabrata.

During hematogenously disseminated infection, blood-borne Candida albicans invades the endothelial cell lining of the vasculature to invade the deep tissues. Although the C. albicans Als3 invasin is critical for invasion and damage of endothelial cells in vitro, a C. albicans als3Δ/Δ mutant has normal virulence in the mouse model of disseminated infection. We hypothesized that the contribution of Als3 to virulence is obscured by the presence of additional C. albicans invasins. To elucidate the in vivo function of Als3, we heterologously expressed C. albicans ALS3 in Candida glabrata, a yeast that lacks a close ALS3 ortholog and has low virulence in mice. We found that following intravenous inoculation into mice, the ALS3-expressing strain preferentially trafficked to the brain, where it induced significantly elevated levels of myeloperoxidase, tumor necrosis factor, monocyte chemoattractant protein 1, and gamma interferon. Also, the ALS3-expressing strain had enhanced adherence to and invasion of human brain microvascular endothelial cells in vitro, demonstrating a potential mechanism for ALS3-mediated neurotropism. In addition, upon initiation of infection, the ALS3-expressing strain had increased trafficking to the cortex of the kidneys. With prolonged infection, this strain persisted in the kidneys at significantly higher levels than the control strain but did not induce an elevated inflammatory response. Finally, the ALS3-expressing strain had increased resistance to neutrophil killing in vitro. These results indicate that during disseminated infection, Als3 mediates initial trafficking to the brain and renal cortex and contributes to fungal persistence in the kidneys.

Mechanisms of Candida albicans trafficking to the brain.

During hematogenously disseminated disease, Candida albicans infects most organs, including the brain. We discovered that a C. albicans vps51Δ/Δ mutant had significantly increased tropism for the brain in the mouse model of disseminated disease. To investigate the mechanisms of this enhanced trafficking to the brain, we studied the interactions of wild-type C. albicans and the vps51Δ/Δ mutant with brain microvascular endothelial cells in vitro. These studies revealed that C. albicans invasion of brain endothelial cells is mediated by the fungal invasins, Als3 and Ssa1. Als3 binds to the gp96 heat shock protein, which is expressed on the surface of brain endothelial cells, but not human umbilical vein endothelial cells, whereas Ssa1 binds to a brain endothelial cell receptor other than gp96. The vps51Δ/Δ mutant has increased surface expression of Als3, which is a major cause of the increased capacity of this mutant to both invade brain endothelial cells in vitro and traffic to the brain in mice. Therefore, during disseminated disease, C. albicans traffics to and infects the brain by binding to gp96, a unique receptor that is expressed specifically on the surface of brain endothelial cells.

Electrochemical performances and volume variation of nano-textured silicon thin films as anodes for lithium-ion batteries.

Electrochemical behaviors of nano-textured silicon thin film (NTSTF) coated with Al2O3 or Cu layers as anodes for lithium-ion batteries have been investigated. The cyclic performance of NTSTF electrodes is superior to dense Si thin films. The NTSTF with a 5 nm thick Cu coating layer shows superior cyclic performance and rate performance to other NTSTF samples. The volume changes of NTSTF electrodes after the first cycle and the tenth cycle have been investigated. This series of electrodes shows an anisotropic volume variation: the height does not change but the diameter does expand. This finding shows the feasibility of dealing with the vertical expansion and contraction of Si-based powder electrodes in Li-ion batteries.

Antisense RNA associated with biological regulation of a restriction-modification system.

Restriction-modification systems consist of a modification enzyme that methylates a specific DNA sequence and a restriction endonuclease that cleaves DNA lacking this epigenetic signature. Their gene expression should be finely regulated because their potential to attack the host bacterial genome needs to be controlled. In the EcoRI system, where the restriction gene is located upstream of the modification gene in the same orientation, we previously identified intragenic reverse promoters affecting gene expression. In the present work, we identified a small (88 nt) antisense RNA (Rna0) transcribed from a reverse promoter (P(REV0)) at the 3' end of the restriction gene. Its antisense transcription, as measured by transcriptional gene fusion, appeared to be terminated by the P(M1,M2) promoter. P(M1,M2) promoter-initiated transcription, in turn, appeared to be inhibited by P(REV0). Mutational inactivation of P(REV0) increased expression of the restriction gene. The biological significance of this antisense transcription is 2-fold. First, a mutation in P(REV0) increased restriction of incoming DNA. Second, the presence of the antisense RNA gene (ecoRIA) in trans alleviated cell killing after loss of the EcoRI plasmid (post-segregational killing). Taken together, these results strongly suggested the involvement of an antisense RNA in the biological regulation of this restriction-modification system.

Surface Engineering in SnO2/Si for High-Performance Broadband Photodetectors.

Silicon-based photodetectors are important optoelectronic devices in many fields. Many investigations have been conducted to improve the performance of silicon-based photodetectors, such as spectral responsivity and sensitivity in the ultraviolet band. In this study, we combine the surface structure engineering of silicon with wide-bandgap semiconductor SnO2 films to realize textured Si-based heterojunction photodetectors. The obtained SnO2/T-Si photodetectors exhibit high responsivity ranging from ultraviolet to near-infrared light. Under a bias voltage of 1 V, SnO2/T-Si photodetectors (PDs) with an inverted pyramid texture show the best performance, and the typical responsivities to ultraviolet, visible, and near-infrared light are 0.512, 0.538, 1.88 (800 nm, 67.7 µW/cm2) A/W@1 V, respectively. The photodetectors exhibit short rise and decay times of 18.07 and 29.16 ms, respectively. Our results demonstrate that SnO2/T-Si can serve as a high-performance broadband photodetector.

Enhanced YOLOv5 network-based object detection (BALFilter Reader) promotes PERFECT filter-enabled liquid biopsy of lung cancer from bronchoalveolar lavage fluid (BALF).

Liquid biopsy of cancers, detecting tumor-related information from liquid samples, has attracted wide attentions as an emerging technology. Our previously reported large-area PERFECT (Precise-Efficient-Robust-Flexible-Easy-Controllable-Thin) filter has demonstrated competitive sensitivity in recovering rare tumor cells from clinical samples. However, it is time-consuming and easily biased to manually inspect rare target cells among numerous background cells distributed in a large area (Φ ≥ 13 mm). This puts forward an urgent demand for rapid and bias-free inspection. Hereby, this paper implemented deep learning-based object detection for the inspection of rare tumor cells from large-field images of PERFECT filters with hematoxylin-eosin (HE)-stained cells recovered from bronchoalveolar lavage fluid (BALF). CenterNet, EfficientDet, and YOLOv5 were trained and validated with 240 and 60 image blocks containing tumor and/or background cells, respectively. YOLOv5 was selected as the basic network given the highest mAP@0.5 of 92.1%, compared to those of CenterNet and EfficientDet at 85.2% and 91.6%, respectively. Then, tricks including CIoU loss, image flip, mosaic, HSV augmentation and TTA were applied to enhance the performance of the YOLOv5 network, improving mAP@0.5 to 96.2%. This enhanced YOLOv5 network-based object detection, named as BALFilter Reader, was tested and cross-validated on 24 clinical cases. The overall diagnosis performance (~2 min) with sensitivity@66.7% ± 16.7%, specificity@100.0% ± 0.0% and accuracy@75.0% ± 12.5% was superior to that from two experienced pathologists (10-30 min) with sensitivity@61.1%, specificity@16.7% and accuracy@50.0%, with the histopathological result as the gold standard. The AUC of the BALFilter Reader is 0.84 ± 0.08. Moreover, a customized Web was developed for a user-friendly interface and the promotion of wide applications. The current results revealed that the developed BALFilter Reader is a rapid, bias-free and easily accessible AI-enabled tool to promote the transplantation of the BALFilter technique. This work can easily expand to other cytopathological diagnoses and improve the application value of micro/nanotechnology-based liquid biopsy in the era of intelligent pathology.

Nanostructure formation and passivation of large-area black silicon for solar cell applications.

Nanoscale textured silicon and its passivation are explored by simple low-cost metal-assisted chemical etching and thermal oxidation, and large-area black silicon was fabricated both on single-crystalline Si and multicrystalline Si for solar cell applications. When the Si surface was etched by HF/AgNO(3) solution for 4 or 5 min, nanopores formed in the Si surface, 50-100 nm in diameter and 200-300 nm deep. The nanoscale textured silicon surface turns into an effective medium with a gradually varying refractive index, which leads to the low reflectivity and black appearance of the samples. Mean reflectance was reduced to as low as 2% for crystalline Si and 4% for multicrystalline Si from 300 to 1000 nm, with no antireflective (AR) coating. A black-etched multicrystalline-Si of 156 mm × 156 mm was used to fabricate a primary solar cell with no surface passivation or AR coating. Its conversion efficiency (η) was 11.5%. The cell conversion efficiency was increased greatly by using surface passivation process, which proved very useful in suppressing excess carrier recombination on the nanostructured surface. Finally, a black m-Si cell with efficiency of 15.8% was achieved by using SiO(2) and SiN(X) bilayer passivation structure, indicating that passivation plays a key role in large-scale manufacture of black silicon solar cells.

Host cell invasion and virulence mediated by Candida albicans Ssa1.

Candida albicans Ssa1 and Ssa2 are members of the HSP70 family of heat shock proteins that are expressed on the cell surface and function as receptors for antimicrobial peptides such as histatins. We investigated the role of Ssa1 and Ssa2 in mediating pathogenic host cell interactions and virulence. A C. albicans ssa1Δ/Δ mutant had attenuated virulence in murine models of disseminated and oropharyngeal candidiasis, whereas an ssa2Δ/Δ mutant did not. In vitro studies revealed that the ssa1Δ/Δ mutant caused markedly less damage to endothelial cells and oral epithelial cell lines. Also, the ssa1Δ/Δ mutant had defective binding to endothelial cell N-cadherin and epithelial cell E-cadherin, receptors that mediate host cell endocytosis of C. albicans. As a result, this mutant had impaired capacity to induce its own endocytosis by endothelial cells and oral epithelial cells. Latex beads coated with recombinant Ssa1 were avidly endocytosed by both endothelial cells and oral epithelial cells, demonstrating that Ssa1 is sufficient to induce host cell endocytosis. These results indicate that Ssa1 is a novel invasin that binds to host cell cadherins, induces host cell endocytosis, and is critical for C. albicans to cause maximal damage to host cells and induce disseminated and oropharyngeal disease.

Candida albicans Als3, a multifunctional adhesin and invasin.

Candida albicans is part of the normal human flora, and it grows on mucosal surfaces in healthy individuals. In susceptible hosts, this organism can cause both mucosal and hematogenously disseminated disease. For C. albicans to persist in the host and induce disease, it must be able to adhere to biotic and abiotic surfaces, invade host cells, and obtain iron. The C. albicans hypha-specific surface protein Als3 is a member of the agglutinin-like sequence (Als) family of proteins and is important in all of these processes. Functioning as an adhesin, Als3 mediates attachment to epithelial cells, endothelial cells, and extracellular matrix proteins. It also plays an important role in biofilm formation on prosthetic surfaces, both alone and in mixed infection with Streptococcus gordonii. Als3 is one of two known C. albicans invasins. It binds to host cell receptors such as E-cadherin and N-cadherin and thereby induces host cells to endocytose the organism. Als3 also binds to host cell ferritin and enables C. albicans to utilize this protein as a source of iron. Because of its multiple functions and its high expression level in vivo, Als3 is a promising target for vaccines that induce protective cell-mediated and antibody responses. This review will summarize the multiple functions of this interesting and multifunctional protein.

Multi-Modal Microfluidics (M3) for Sample Preparation of Liquid Biopsy: Bridging the Gap between Proof-of-Concept Demonstrations and Practical Applications.

Liquid biopsy, the technique used to shed light on diseases via liquid samples, has displayed various advantages, including minimal invasiveness, low risk, and ease of multiple sampling for dynamic monitoring, and has drawn extensive attention from multidisciplinary fields in the past decade. With the rapid development of microfluidics, it has been possible to manipulate targets of interest including cells, microorganisms, and exosomes at a single number level, which dramatically promotes the characterization and analysis of disease-related markers, and thus improves the capability of liquid biopsy. However, when lab-ready techniques transfer into hospital-applicable tools, they still face a big challenge in processing raw clinical specimens, which are usually of a large volume and consist of rare targets drowned in complex backgrounds. Efforts toward the sample preparation of clinical specimens (i.e., recovering/concentrating the rare targets among complex backgrounds from large-volume liquids) are required to bridge the gap between the proof-of-concept demonstrations and practical applications. The throughput, sensitivity, and purity (TSP performance criteria) in sample preparation, i.e., the volume speed in processing liquid samples and the efficiencies of recovering rare targets and depleting the backgrounds, are three key factors requiring careful consideration when implementing microfluidic-based liquid biopsy for clinical practices. Platforms based on a single microfluidic module (single-modal microfluidics) can hardly fulfill all the aforementioned TSP performance criteria in clinical practices, which puts forward an urgent need to combine/couple multiple microfluidic modules into one working system (i.e., multi-modal microfluidics, M3) to realize practically applicable techniques for the sample preparation of liquid biopsy. This perspective briefly summarizes the typical microfluidic-based liquid biopsy techniques and discusses potential strategies to develop M3 systems for clinical practices of liquid biopsy from the aspect of sample preparation.