High KRT17 expression in tumour budding indicates immunologically 'hot' tumour budding and predicts good survival in patients with colorectal cancer.

Objectives: Emerging evidence has demonstrated that tumour budding (TB) is negatively associated with T-lymphocyte infiltration in CRC. Despite extensive research, the molecular characteristics of immunologically 'hot' TB remain poorly understood. Methods: We quantified the number of TB by haematoxylin-eosin (H&E) sections and the densities of CD3+ and CD8+ T-lymphocytes by immunohistochemistry in a CRC cohort of 351 cases who underwent curative resection. We analysed the differential expression and T-lymphocyte infiltration score of 37 human epithelial keratins in CRC using RNA sequencing from the TCGA dataset. In 278 TB-positive cases, KRT17 expression was evaluated in tumour centre (TC) and TB with a staining score. Patient demographic, clinicopathological features and survival rates were analysed. Results: In a CRC cohort of 351 cases, low-grade TB was associated with high CD3+ and CD8+ T-cell densities in the invasive margin (IM) but not in the TC. Of 37 human epithelial keratins, only KRT17 expression in TB had an apparent association with TB-grade and T-lymphocyte infiltration. In 278 TB-positive cases, high KRT17 expression in TB (KRT17TB) was negatively associated with low-grade TB and positively associated with high CD3+ and CD8+ T-cell densities in IM. High KRT17TB predicted early tumour grade, absence of lymph node metastasis and absence of tumour deposits. Additionally, patients with high KRT17TB had good overall survival and disease-free survival. Notably, low KRT17TB can specifically identify those patients with a poor prognosis among colorectal cancer patients with low TB and high T-lymphocyte infiltration. Conclusions: KRT17 can be employed as a new indicator for distinguishing different immunological TBs.

Mitochondrial DNA copy number plays opposing roles in T-lymphocyte infiltration of colorectal cancer based on mismatch repair status: new directions for immunotherapy?

BACKGROUND: Researchers have previously reported that mitochondrial DNA copy number (mtDNA-CN) can play different roles in microsatellite instable/mismatch repair-deficient (MSI/dMMR) and microsatellite stable/mismatch repair-proficient (MSS/pMMR) colorectal cancer (CRC). To support malignancy, dMMR CRC relies on glycolysis, while pMMR CRC favors oxidative phosphorylation. However, it is unclear whether mtDNA-CN changes are related to T cell infiltration in CRC. METHODS: The mtDNA-CN was detected by qRT-PCR in 532 patients, and the expression of CD3 and CD8 in 485 patients was detected by immunohistochemistry. The correlation between mtDNA-CN and the prognosis of CRC patients was further analyzed, and the correlation between mtDNA-CN and T lymphocyte infiltration was also analyzed. Biopsy specimens from the immune checkpoint inhibitors (ICIs) treatment cohort were obtained to verify the correlation between mtDNA-CN and the efficacy of ICIs. The effects of mtDNA-CN and MMR status on gene expression were analyzed by RNA-seq. RESULTS: Our results show that mtDNA-CN has inverse relationships to CRC prognosis in cases with different MMR statuses, potentially inducing the U-shaped association in CRC. The opposing correlations between mtDNA-CN and T lymphocyte infiltration in cases of dMMR CRC and pMMR CRC further suggest that mtDNA-CN might play an important role in CRC development. More importantly, cases of pMMR CRC with lower mtDNA-CN and of dMMR CRC with higher mtDNA-CN can benefit more dramatically from ICIs. Furthermore, RNA-seq revealed a link between the level of mtDNA-CN and T lymphocyte infiltration in CRC cases with different MMR statuses. CONCLUSION: Our study found a potential relationship between mtDNA-CN and CRC development that differs by MMR status, potentially providing a rationale for the use of mtDNA-CN as both a predictive biomarker and a therapeutic target for ICIs.

Underwater image quality assessment.

To obtain high-visual-quality underwater images by image post-processing, many underwater image restoration and enhancement methods have been proposed. Underwater image quality assessment (UIQA) methods have been developed to compare these restoration and enhancement methods. This paper comprehensively summarizes the subjective and objective UIQA methods, metrics, and datasets. Experiments are conducted on two underwater image datasets to analyze the performance of several typical UIQA metrics. Suggestions for further research directions are put forward as well.

KRT17 Promotes T-lymphocyte Infiltration Through the YTHDF2-CXCL10 Axis in Colorectal Cancer.

Poor infiltration of T lymphocytes has been regarded as a crucial mechanism of tumor immune escape. Here, we demonstrate a protective role of KRT17 in colorectal cancer, where KRT17 reversed the tumor immunosuppressive microenvironment by increasing T-lymphocyte infiltration. High-throughput RNA sequencing suggested that KRT17 was significantly upregulated in deficient mismatch repair (dMMR) tumors compared with proficient mismatch repair (pMMR) tumors. In a colorectal cancer cohort of 446 cases, KRT17 expression positively correlated with better clinical outcomes. Krt17 overexpression decreased xenograft tumor growth in immune-competent mice. T-cell depletion in a murine model showed that the presence of T lymphocytes was necessary for Krt17-mediated disruption of tumorigenesis. Mass spectrometry and coimmunoprecipitation assays suggested KRT17 caused YTHDF2 degradation through the ubiquitin-proteasome system. Through high-throughput RNA immunoprecipitation sequencing, we found that CXCL10 was the target gene of the N6-methyladenosine (m6A) "reader" YTHDF2. KRT17 synergized with anti-PD-1 for better tumor control in an immunotherapy-resistant murine model. In a cohort of patients with colorectal cancer receiving pembrolizumab, high KRT17 expression was found within the tumors of responders. Collectively, we elucidated a critical role of KRT17 in colorectal cancer to prevent immune escape. These findings present new insights into potential therapeutic strategies and effective markers of immunotherapy reactivity against pMMR tumors.

Surface acoustic wave manipulation of bioparticles.

The introduction of surface acoustic waves (SAWs) into lab-on-a-chip microfluidic systems has contributed to the development of a new cutting-edge technology-SAW-based micro/nano manipulation. Recently, the SAW technology has emerged as an important tool for manipulating micro/nano particles/cell populations by virtue of its simplicity, biocompatibility, non-invasiveness, scalability, and versatility. In custom-designed acoustic fields, this technology can be used to manipulate cells, bacteria, exosomes, and even worms precisely, and it has been used in applications such as biomedical and point-of-care diagnostic systems. In this review paper, we start by providing a comprehensive overview of the fundamental working principle and numerical simulation of SAW-based manipulation. Then, we introduce the recent advancements in the manipulation of organisms based on standing and traveling SAWs, including separation, concentration, and transport. At the end of the review, we discuss the current challenges to and future prospects of SAW-based manipulation. The conclusion is that the SAW technology will open up a new frontier in the microfluidics field and contribute significantly to the development of bioengineering research and applications.

CXCL16 inhibits epithelial regeneration and promotes fibrosis during the progression of radiation enteritis.

Radiation enteritis (RE) is a prevalent complication of radiotherapy for pelvic malignant tumors, characterized by severe intestinal epithelial destruction and progressive submucosal fibrosis. However, little is known about the pathogenesis of this disease, and so far, there is no specific targeted therapy. Here, we report that CXCL16 is upregulated in the injured intestinal tissues of RE patients and in a mouse model. Genetic deletion of Cxcl16 mitigates fibrosis and promotes intestinal stem cell-mediated epithelial regeneration after radiation injury in mice. Mechanistically, CXCL16 functions on myofibroblasts through its receptor CXCR6 and activates JAK3/STAT3 signaling to promote fibrosis and, at the same time, to transcriptionally modulate the levels of BMP4 and hepatocyte growth factor (HGF) in myofibroblasts. Moreover, we find that CXCL16 and CXCR6 auto- and cross-regulate themselves in positive feedback loops. Treatment with CXCL16 neutralizing monoclonal antibody attenuates fibrosis and improves the epithelial repair in RE mouse model. Our findings emphasize the important role of CXCL16 in the progression of RE and suggest that CXCL16 signaling could be a potential therapeutic target for RE. © 2022 The Pathological Society of Great Britain and Ireland.

Optoelectronic tweezers: a versatile toolbox for nano-/micro-manipulation.

The rapid development of micromanipulation technologies has opened exciting new opportunities for the actuation, selection and assembly of a variety of non-biological and biological nano/micro-objects for applications ranging from microfabrication, cell analysis, tissue engineering, biochemical sensing, to nano/micro-machines. To date, a variety of precise, flexible and high-throughput manipulation techniques have been developed based on different physical fields. Among them, optoelectronic tweezers (OET) is a state-of-art technique that combines light stimuli with electric field together by leveraging the photoconductive effect of semiconductor materials. Herein, the behavior of micro-objects can be directly controlled by inducing the change of electric fields on demand in an optical manner. Relying on this light-induced electrokinetic effect, OET offers tremendous advantages in micromanipulation such as programmability, flexibility, versatility, high-throughput and ease of integration with other characterization systems, thus showing impressive performance compared to those of many other manipulation techniques. A lot of research on OET have been reported in recent years and the technology has developed rapidly in various fields of science and engineering. This work provides a comprehensive review of the OET technology, including its working mechanisms, experimental setups, applications in non-biological and biological scenarios, technology commercialization and future perspectives.

Label-free purification and characterization of optogenetically engineered cells using optically-induced dielectrophoresis.

Optogenetically engineered cell population obtained by heterogeneous gene expression plays a vital role in life science, medicine, and biohybrid robotics, and purification and characterization are essential to enhance its application performance. However, the existing cell purification methods suffer from complex sample preparation or inevitable damage and pollution. The efficient and nondestructive label-free purification and characterization of the optogenetically engineered cells, HEK293-ChR2 cells, is provided here using an optically-induced dielectrophoresis (ODEP)-based approach. The distinctive crossover frequencies of the engineered cells and the unmodified cells enable effective separation due to the opposite DEP forces on them. The ODEP-based approach can greatly improve the purity of the separated cell population and especially, the ratio of the engineered cells in the separated cell population can be enhanced by 275% at a low transfection rate. The size and the membrane capacitance of the separated cell population decreases and increases, respectively, as the ratio of the engineered cells grows in the cell population, indicating that successful expression of ChR2 in a single HEK293 cell makes its size and membrane capacitance smaller and larger, respectively. The results of biohybrid imaging with the optogenetically engineered cells demonstrated that cell purification can improve the imaging quality. This work proves that the separation and purification of engineered cells are of great significance for their application in practice.

Accurate and Automatic Extraction of Cell Self-Rotation Speed in an ODEP Field Using an Area Change Algorithm.

Cells are complex biological units that can sense physicochemical stimuli from their surroundings and respond positively to them through characterization of the cell behavior. Thus, understanding the motions of cells is important for investigating their intrinsic properties and reflecting their various states. Computer-vision-based methods for elucidating cell behavior offer a novel approach to accurately extract cell motions. Here, we propose an algorithm based on area change to automatically extract the self-rotation of cells in an optically induced dielectrophoresis field. To obtain a clear and complete outline of the cell structure, dark corner removal and contrast stretching techniques are used in the pre-processing stage. The self-rotation speed is calculated by determining the frequency of the cell area changes in all of the captured images. The algorithm is suitable for calculating in-plane and out-of-plane rotations, while addressing the problem of identical images at different rotation angles when dealing with rotations of spherical and flat cells. In addition, the algorithm can be used to determine the motion trajectory of cells. The experimental results show that the algorithm can efficiently and accurately calculate cell rotation speeds of up to ~155 rpm. Potential applications of the proposed algorithm include cell morphology extraction, cell classification, and characterization of the cell mechanical properties. The algorithm can be very helpful for those who are interested in using computer vision and artificial-intelligence-based ideology in single-cell studies, drug treatment, and other bio-related fields.

The dynamic cellular and molecular features during the development of radiation proctitis revealed by transcriptomic profiling in mice.

BACKGROUND: Radiation proctitis (RP) is the most common complication of radiotherapy for pelvic tumor. Currently there is a lack of effective clinical treatment and its underlying mechanism is poorly understood. In this study, we aimed to dynamically reveal the mechanism of RP progression from the perspective of RNomics using a mouse model, so as to help develop reasonable therapeutic strategies for RP. RESULTS: Mice were delivered a single dose of 25 Gy rectal irradiation, and the rectal tissues were removed at 4 h, 1 day, 3 days, 2 weeks and 8 weeks post-irradiation (PI) for both histopathological assessment and RNA-seq analysis. According to the histopathological characteristics, we divided the development process of our RP animal model into three stages: acute (4 h, 1 day and 3 days PI), subacute (2 weeks PI) and chronic (8 weeks PI), which could recapitulate the features of different stages of human RP. Bioinformatics analysis of the RNA-seq data showed that in the acute injury period after radiation, the altered genes were mainly enriched in DNA damage response, p53 signaling pathway and metabolic changes; while in the subacute and chronic stages of tissue reconstruction, genes involved in the biological processes of vessel development, extracellular matrix organization, inflammatory and immune responses were dysregulated. We further identified the hub genes in the most significant biological process at each time point using protein-protein interaction analysis and verified the differential expression of these genes by quantitative real-time-PCR analysis. CONCLUSIONS: Our study reveals the molecular events sequentially occurred during the course of RP development and might provide molecular basis for designing drugs targeting different stages of RP development.

Transanal total mesorectal excision combined with intersphincteric resection has similar long-term oncological outcomes to laparoscopic abdominoperineal resection in low rectal cancer: a propensity score-matched cohort study.

Background: Transanal total mesorectal excision (taTME) or intersphincteric resection (ISR) has recently proven to be a valid and safe surgical procedure for low rectal cancer. However, studies focusing on the combination of these two technologies are limited. This study aimed to evaluate perioperative results, long-term oncologic outcomes, and anorectal functions of patients with low rectal cancer undergoing taTME combined with ISR, by comparing with those of patients undergoing laparoscopic abdominoperineal resection (laAPR). Methods: After 1:1 propensity score matching, 200 patients with low rectal cancer who underwent laAPR (n = 100) or taTME combined with ISR (n = 100) between September 2013 and November 2019 were included. Patient demographics, clinicopathological characteristics, oncological outcomes, and anal functional results were analysed. Results: Patients in the taTME-combined-with-ISR group had less intraoperative blood loss (79.6 ± 72.6 vs 107.3 ± 65.1 mL, P = 0.005) and a lower rate of post-operative complications (22.0% vs 44.0%, P < 0.001) than those in the laAPR group. The overall local recurrence rates were 7.0% in both groups within 3 years after surgery. The 3-year disease-free survival rates were 86.3% in the taTME-combined-with-ISR group and 75.1% in the laAPR group (P = 0.056), while the 3-year overall survival rates were 96.7% and 94.2%, respectively (P = 0.319). There were 39 patients (45.3%) in the taTME-combined-with-ISR group who developed major low anterior resection syndrome, whereas 61 patients (70.9%) had good post-operative anal function (Wexner incontinence score ≤ 10). Conclusion: We found similar long-term oncological outcomes for patients with low rectal cancer undergoing laAPR and those undergoing taTME combined with ISR. Patients receiving taTME combined with ISR had acceptable post-operative anorectal function.

Complete secretion of recombinant Bacillus subtilis levansucrase in Pichia pastoris for production of high molecular weight levan.

Three signal peptides from α-mating factor (α-MF), inulinase (INU) and native levansucrase (LS) were compared for secretion efficiency of Bacillus subtilis levansucrase SacB-T305A in Pichia pastoris GS115. The first complete secretion of bacterial levansucrase in yeasts under methanol induction was achieved while using α-MF signal. The secreted recombinant Lev(α-MF) proved to be glycosylated by combination of NanoLC-MS/MS and Endo H digestion. Interestingly, glycosylation not only improved significantly the polymerase thermostability, but also reversed the products profiles to favor synthesis of high molecular weight (HMW) levan which accounted for approximately 73 % to total levan-type polysaccharides. It indicated for the first time that the glycosylation of recombinant B. subtilis levansucrase affected significantly the products molecular weight distribution. It also provided a promising enzymatic way to effectively product HMW levan from sucrose resources.

Three-year outcomes of transanal total mesorectal excision versus standard laparoscopic total mesorectal excision for mid and low rectal cancer.

INTRODUCTION: Since transanal total mesorectal excision (taTME) was introduced, it has become an important topic in rectal cancer treatment. Many previous studies reported positive relevant short-term results, histopathological results, and associated complications. Recently, concerns regarding the oncological safety of taTME have been raised due to reports showing high local recurrences (LR) rates. Therefore, this study aimed to compare the 3-year outcomes between taTME and laparoscopic total mesorectal excision (laTME) for mid-low rectal cancer. METHODS: A total of 104 patients who underwent taTME were matched with 208 patients treated by laTME. The primary endpoint was 3-year LR rate; secondary endpoints in this matched-cohort study included the perioperative outcomes and histopathological outcomes. RESULTS: taTME was associated with lower permanent ostomy rate (1% vs 13.5%) and lower conversion rate (0% vs 3.4%) compared to laTME. A similar quality of resected specimens was detected for each group. In both groups, the local recurrence rate was 3.8%. Within 3 years after surgery, the disease-free survival (DFS) rates were 78.8% in the taTME group and 76.9% in the laTME group (P = 0.640), while the overall survival (OS) rates were 93.3% in the taTME group and 89.9% in the laTME group (P = 0.327). CONCLUSION: No significant differences regarding 3-year local recurrence rate (3.8%) were observed in the taTME group compared to laTME group.

Non-invasive acquisition of mechanical properties of cells via passive microfluidic mechanisms: A review.

The demand to understand the mechanical properties of cells from biomedical, bioengineering, and clinical diagnostic fields has given rise to a variety of research studies. In this context, how to use lab-on-a-chip devices to achieve accurate, high-throughput, and non-invasive acquisition of the mechanical properties of cells has become the focus of many studies. Accordingly, we present a comprehensive review of the development of the measurement of mechanical properties of cells using passive microfluidic mechanisms, including constriction channel-based, fluid-induced, and micropipette aspiration-based mechanisms. This review discusses how these mechanisms work to determine the mechanical properties of the cell as well as their advantages and disadvantages. A detailed discussion is also presented on a series of typical applications of these three mechanisms to measure the mechanical properties of cells. At the end of this article, the current challenges and future prospects of these mechanisms are demonstrated, which will help guide researchers who are interested to get into this area of research. Our conclusion is that these passive microfluidic mechanisms will offer more preferences for the development of lab-on-a-chip technologies and hold great potential for advancing biomedical and bioengineering research studies.

Enhancing cancer-associated fibroblast fatty acid catabolism within a metabolically challenging tumor microenvironment drives colon cancer peritoneal metastasis.

Most cancer-related deaths result from the progressive growth of metastases. Patients with peritoneal metastatic (PM) colorectal cancer have reduced overall survival. Currently, it is still unclear why colorectal cancer (CRC) cells home to and proliferate inside the peritoneal cavity, and there is no effective consolidation therapy for improved survival. Using a proteomic approach, we found that key enzymes of fatty acid oxidation (FAO) were decreased in patients with PM colorectal cancer. Furthermore, we confirmed that carnitine palmitoyltransferase IA (CPT1A), a rate-limiting enzyme of FAO, was expressed at significantly low levels in patients with PM colorectal cancer, as determined by RT-qPCR, IHC, and GEO dataset analysis. However, lipidomics revealed no difference in FFA levels between PM and non-PM primary tumors. Here, we showed that cancer-associated fibroblasts (CAFs) promote the proliferation, migration, and invasion of colon cancer cells via upregulating CPT1A to actively oxidize FAs and conduct minimal glycolysis. In addition, coculture-induced glycolysis increased in cancer cells while fatty acid catabolism decreased with lower adiponectin levels. Importantly, inhibition of glycolysis significantly reduced the survival of CRC cells after incubation with conditioned medium from CAFsCPT1A-OE in vitro and impaired the survival and growth of organoids derived from CRC-PM. Finally, we found that directly blocking FAO in CAFsCPT1A-OE with etomoxir inhibits migration and invasion in vitro and decreases tumor growth and intraperitoneal dissemination in vivo, revealing a role for CAF CPT1A in promoting tumor growth and invasion. In conclusion, our results suggest the possibility of testing FAO inhibition as a novel approach and clinical strategy against CAF-induced colorectal cancer with peritoneal dissemination/metastases.

Recent advances in AFM-based biological characterization and applications at multiple levels.

Atomic force microscopy (AFM) has found a wide range of bio-applications in the past few decades due to its ability to measure biological samples in natural environments at a high spatial resolution. AFM has become a key platform in biomedical, bioengineering and drug research fields, enabling mechanical and morphological characterization of live biological systems. Hence, we provide a comprehensive review on recent advances in the use of AFM for characterizing the biomechanical properties of multi-scale biological samples, ranging from molecule, cell to tissue levels. First, we present the fundamental principles of AFM and two AFM-based models for the characterization of biomechanical properties of biological samples, covering key AFM devices and AFM bioimaging as well as theoretical models for characterizing the elasticity and viscosity of biomaterials. Then, we elaborate on a series of new experimental findings through analysis of biomechanics. Finally, we discuss the future directions and challenges. It is envisioned that the AFM technique will enable many remarkable discoveries, and will have far-reaching impacts on bio-related studies and applications in the future.

4D Printing: A Review on Recent Progresses.

Since the late 1980s, additive manufacturing (AM), commonly known as three-dimensional (3D) printing, has been gradually popularized. However, the microstructures fabricated using 3D printing is static. To overcome this challenge, four-dimensional (4D) printing which defined as fabricating a complex spontaneous structure that changes with time respond in an intended manner to external stimuli. 4D printing originates in 3D printing, but beyond 3D printing. Although 4D printing is mainly based on 3D printing and become an branch of additive manufacturing, the fabricated objects are no longer static and can be transformed into complex structures by changing the size, shape, property and functionality under external stimuli, which makes 3D printing alive. Herein, recent major progresses in 4D printing are reviewed, including AM technologies for 4D printing, stimulation method, materials and applications. In addition, the current challenges and future prospects of 4D printing were highlighted.

Detection and isolation of free cancer cells from ascites and peritoneal lavages using optically induced electrokinetics (OEK).

Detection of free gastric cancer cells in peritoneal lavages and ascites plays a vital role in gastric cancer. However, due to the low content of cancer cells in patients' peritoneal lavages, traditional detection methods lack sensitivity and cannot satisfy clinical demand. In this study, we used an optically induced electrokinetics (OEK) microfluidic method for label-free separation and characterization of patient gastric cancer cells. This method showed high effectiveness and sensitivity. We successfully separated cancer cells from a simulated peritoneal lavage mixture of gastric cancer cell lines and peritoneal lavage cells in a ratio of 1:1000. We further separated gastric cancer cells from six patients' ascites with purity up to 71%. In addition, we measured the cell membrane capacitances, which may be used as a biomarker for gastric cancer cells. Thus, our method can be used to effectively and rapidly detect peritoneal metastasis and to acquire cellular electrical information.

Dynamically directing cell organization via micro-hump structure patterned cell-adhered interfaces.

Cell adhesion plays an important role in cell communication, organ formation and tissue maintenance. Spatial microstructure patterning has the capability to regulate cell functions such as cell adhesion and cell proliferation as well as cellular mechanical properties. In this study, we present a simple method to fabricate micro-hump patterned interfaces based on electrohydrodynamic jet (E-jet) printing to control and direct cell organization. Micro-hump structures were rapidly fabricated by E-jet printing and arbitrary cell patterns can be achieved by selective cell adhesion induced by this surface topography. Furthermore, cellular mechanical properties were regulated by changing the density of microstructures. The technique we proposed could dynamically direct cell organization in a controlled manner, providing help for exploring the fundamental mechanism of cell adhesion and sensing.

Determination of Dielectric Properties of Cells using AC Electrokinetic-based Microfluidic Platform: A Review of Recent Advances.

Cell dielectric properties, a type of intrinsic property of cells, can be used as electrophysiological biomarkers that offer a label-free way to characterize cell phenotypes and states, purify clinical samples, and identify target cancer cells. Here, we present a review of the determination of cell dielectric properties using alternating current (AC) electrokinetic-based microfluidic mechanisms, including electro-rotation (ROT) and dielectrophoresis (DEP). The review covers theoretically how ROT and DEP work to extract cell dielectric properties. We also dive into the details of differently structured ROT chips, followed by a discussion on the determination of cell dielectric properties and the use of these properties in bio-related applications. Additionally, the review offers a look at the future challenges facing the AC electrokinetic-based microfluidic platform in terms of acquiring cell dielectric parameters. Our conclusion is that this platform will bring biomedical and bioengineering sciences to the next level and ultimately achieve the shift from lab-oriented research to real-world applications.