Clinical Validation of a Handheld Deep Learning Tool for Identification of Glaucoma Medications.

Purpose: To validate a convolutional neural network (CNN)-based smartphone application for the identification of glaucoma eye drop medications in patients with normal and impaired vision. Methods: Sixty-eight patients with visual acuity (VA) of 20/70 or worse in at least one eye who presented to an academic glaucoma clinic from January 2021 through August 2022 were included. Non-English-speaking patients were excluded. Enrolled subjects participated in an activity in which they identified a predetermined and preordered set of six topical glaucoma medications, first without the CNN and then with the CNN for a total of six sequential measurements per subject. Responses to a standardized survey were collected during and after the activity. Primary quantitative outcomes were medication identification accuracy and time. Primary qualitative outcomes were subjective ratings of ease of smartphone application use. Results: Topical glaucoma medication identification accuracy (OR = 12.005, P < 0.001) and time (OR = 0.007, P < 0.001) both independently improved with CNN use. CNN use significantly improved medication accuracy in patients with glaucoma (OR = 4.771, P = 0.036) or VA ≤ 20/70 in at least one eye (OR = 4.463, P = 0.013) and medication identification time in patients with glaucoma (OR = 0.065, P = 0.017). CNN use had a significant positive association with subject-reported ease of medication identification (X2(1) = 66.117, P < 0.001). Conclusion: Our CNN-based smartphone application is efficacious at improving glaucoma eye drop identification accuracy and time. This tool can be used in the outpatient setting to avert preventable vision loss by improving medication adherence in patients with glaucoma.

Rac1 and Nectin3 are essential for PCP-directed axon guidance in the peripheral auditory system.

Our sense of hearing is critically dependent on the spiral ganglion neurons (SGNs) that connect the sound receptors in the organ of Corti (OC) to the cochlear nuclei of the hindbrain. Type I SGNs innervate inner hair cells (IHCs) to transmit sound signals, while type II SGNs (SGNIIs) innervate outer hair cells (OHCs) to detect moderate-to-intense sound. During development, SGNII afferents make a characteristic 90-degree turn toward the base of the cochlea and innervate multiple OHCs. It has been shown that the Planar Cell Polarity (PCP) pathway acts non-autonomously to mediate environmental cues in the cochlear epithelium for SGNII afferent turning towards the base. However, the underlying mechanisms are unknown. Here, we present evidence that PCP signaling regulates multiple downstream effectors to influence cell adhesion and the cytoskeleton in cochlear supporting cells (SCs), which serve as intermediate targets of SGNII afferents. We show that the core PCP gene Vangl2 regulates the localization of the small GTPase Rac1 and the cell adhesion molecule Nectin3 at SC-SC junctions through which SGNII afferents travel. Through in vivo genetic analysis, we also show that loss of Rac1 or Nectin3 partially phenocopied SGNII peripheral afferent turning defects in Vangl2 mutants, and that Rac1 plays a non-autonomous role in this process in part by regulating PCP protein localization at the SC-SC junctions. Additionally, epistasis analysis indicates that Nectin3 and Rac1 likely act in the same genetic pathway to control SGNII afferent turning. Together, these experiments identify Nectin3 and Rac1 as novel regulators of PCP-directed SGNII axon guidance in the cochlea.

Long-Term Effects of Reproduction and Lactation on the Rat Supraspinatus Tendon and Proximal Humerus.

During pregnancy and breastfeeding, women undergo hormonal fluctuations required for fetal development, parturition, and infant growth. These changes have secondary consequences on the maternal musculoskeletal system, increasing the risk for joint pain and osteoporosis. Though hormone levels return to prepregnancy levels postpartum, women may experience lasting musculoskeletal pain. Sex disparities exist in the prevalence of musculoskeletal disorders, but it remains unclear how reproductive history may impact sex differences. Specifically, the effects of both reproductive history and sex on the rotator cuff have not been studied. Pregnancy and lactation affect bone microstructure, suggesting possible impairments at the enthesis of rotator cuff tendons, where tears commonly occur. Therefore, our objective was to evaluate how reproductive history affects sex differences of the supraspinatus tendon and proximal humerus using male, virgin female, and female rats with a history of reproduction (referred to as reproductive females). We hypothesized tendon mechanical properties and humeral bone microstructure would be inferior in reproductive females compared to virgin females. Results showed sex differences independent of reproductive history, including greater tendon midsubstance modulus but lower subchondral bone mineral density (BMD) in females. When considering reproductive history, reproductive rats exhibited reduced tendon insertion site modulus and trabecular bone micro-architecture compared to virgin females with no differences from males. Overall, our study identified long-term changes in supraspinatus tendon mechanical and humeral trabecular bone properties that result following pregnancy and lactation, highlighting the importance of considering reproductive history in investigations of sex differences in the physiology and pathology of rotator cuff injuries.

Reasons for transfer and subsequent outcomes among patients undergoing elective spine surgery at an orthopedic specialty hospital.

Objective: To evaluate the reasons for transfer as well as the 90-day outcomes of patients who were transferred from a high-volume orthopedic specialty hospital (OSH) following elective spine surgery. Materials and Methods: All patients admitted to a single OSH for elective spine surgery from 2014 to 2021 were retrospectively identified. Ninety-day complications, readmissions, revisions, and mortality events were collected and a 3:1 propensity match was conducted. Results: Thirty-five (1.5%) of 2351 spine patients were transferred, most commonly for arrhythmia (n = 7; 20%). Thirty-three transferred patients were matched to 99 who were not transferred, and groups had similar rates of complications (18.2% vs. 10.1%; P = 0.228), readmissions (3.0% vs. 4.0%; P = 1.000), and mortality (6.1% vs. 0%; P = 0.061). Conclusion: Overall, this study demonstrates a low transfer rate following spine surgery. Risk factors should continue to be optimized in order to decrease patient risks in the postoperative period at an OSH.

Predictors of Blood Transfusion in Patients Undergoing Lumbar Spinal Fusion.

OBJECTIVE: To determine risk factors for perioperative blood transfusion after lumbar fusion surgery. METHODS: After institutional review board approval, a retrospective cohort study of adult patients who underwent lumbar fusion at a single, urban tertiary academic center was retrospectively retrieved. Our primary outcome, blood transfusion, was collected via chart query. A receiver operating characteristic curve was used to evaluate the regression model. A P-value < 0.05 was considered statistically significant. RESULTS: Of the 3,842 patients, 282 (7.3%) required a blood transfusion. For patients undergoing posterolateral decompression and fusion, predictors of transfusion included age (P < 0.001) and more levels fused (P < 0.001). A higher preoperative hemoglobin level (P < 0.001) and revision surgery (P = 0.005) were protective of blood transfusion. For patients undergoing transforaminal lumbar interbody fusion, greater Elixhauser comorbidity index (P < 0.001), longer operative time (P = 0.040), and more levels fused (P = 0.030) were independent predictors of the need for blood transfusion. Patients with a higher body mass index (P = 0.012) and preoperative hemoglobin level (P < 0.001) had a reduced likelihood of receiving a transfusion. For circumferential fusion, greater age (P = 0.006) and longer operative times (P = 0.015) were independent predictors of blood transfusion, while a higher preoperative hemoglobin level (P < 0.001) and male sex (P = 0.002) were protective. CONCLUSIONS: Our analysis identified older age, lower body mass index, greater Elixhauser comorbidity index, longer operative duration, more levels fused, and lower preoperative hemoglobin levels as independent predictors of requiring a blood transfusion following lumbar spinal fusion. Different surgical approaches were not found to be associated with transfusion.

Creation of a Risk Calculator for Predicting New-Onset Cardiac Arrhythmias in Patients Undergoing Lumbar Fusion.

INTRODUCTION: As an increasing number of lumbar fusion procedures are being conducted at specialty hospitals and surgery centers, appropriate patient selection and risk stratification is critical to minimizing patient transfers. Postoperative cardiac arrhythmia has been linked to worse patient outcomes and is a common cause of patient transfer. Therefore, we created a risk calculator to predict a patient's likelihood of developing a new-onset postoperative cardiac arrhythmia after lumbar spinal fusion, which may improve preoperative facility selection. METHODS: A retrospective review was conducted of patients who undergoing lumbar fusion from 2017 to 2021 at a single academic center. Patients were excluded if they had any medical history of a cardiac arrhythmia. Multivariable regression was conducted to determine independent predictors of inpatient arrhythmias. The final regression was applied to a bootstrap to validate an arrhythmia prediction model. A risk calculator was created to determine a patient's risk of new-onset cardiac arrhythmia. RESULTS: A total of 1,622 patients were included, with 45 patients developing a new-onset postoperative arrhythmia. Age (OR = 1.05; 95% CI, 1.02 to 1.09; P = 0.003), history of beta-blocker use (OR = 2.01; 95% CI, 1.08 to 3.72; P = 0.027), and levels fused (OR = 1.59; 95% CI, 1.20 to 2.00; P = 0.001) were all independent predictors of having a new-onset inpatient arrhythmia. This multivariable regression produced an area under the curve of 0.742. The final regression was applied to a bootstrap prediction modeling technique to create a risk calculator including the male sex, age, body mass index, beta-blocker use, and levels fused (OR = 1.04, [CI = 1.03 to 1.06]) that produced an area under the curve of 0.733. CONCLUSION: A patient's likelihood of developing postoperative cardiac arrhythmias may be predicted by comorbid conditions and demographic factors including age, sex, body mass index, and beta-blocker use. Knowledge of these risk factors may improve appropriate selection of an outpatient surgical center or orthopaedic specialty hospital versus an inpatient hospital for lumbar fusions.

Prostate cancer extracellular vesicle digital scoring assay - a rapid noninvasive approach for quantification of disease-relevant mRNAs.

Optimizing outcomes in prostate cancer (PCa) requires precision in characterization of disease status. This effort was directed at developing a PCa extracellular vesicle (EV) Digital Scoring Assay (DSA) for detecting metastasis and monitoring progression of PCa. PCa EV DSA is comprised of an EV purification device (i.e., EV Click Chip) and reverse-transcription droplet digital PCR that quantifies 11 PCa-relevant mRNA in purified PCa-derived EVs. A Met score was computed for each plasma sample based on the expression of the 11-gene panel using the weighted Z score method. Under optimized conditions, the EV Click Chips outperformed the ultracentrifugation or precipitation method of purifying PCa-derived EVs from artificial plasma samples. Using PCa EV DSA, the Met score distinguished metastatic (n = 20) from localized PCa (n = 20) with an area under the receiver operating characteristic curve of 0.88 (95% CI:0.78-0.98). Furthermore, longitudinal analysis of three PCa patients showed the dynamics of the Met scores reflected clinical behavior even when disease was undetectable by imaging. Overall, a sensitive PCa EV DSA was developed to identify metastatic PCa and reveal dynamic disease states noninvasively. This assay may complement current imaging tools and blood-based tests for timely detection of metastatic progression that can improve care for PCa patients.

Patient-Reported Outcomes Following Anterior and Posterior Surgical Approaches for Multilevel Cervical Myelopathy.

STUDY DESIGN: Retrospective cohort. OBJECTIVE: To compare health-related quality of life (HRQoL) outcomes between approach techniques for the treatment of multilevel degenerative cervical myelopathy (DCM). SUMMARY OF BACKGROUND DATA: Both anterior and posterior approaches for the surgical treatment of cervical myelopathy are successful techniques in the treatment of myelopathy. However, the optimal treatment has yet to be determined, especially for multilevel disease, as the different approaches have separate complication profiles and potentially different impacts on HRQoL metrics. MATERIALS AND METHODS: Retrospective review of a prospectively managed single institution database of patient-reported outcome measures after 3 and 4-level anterior cervical discectomy and fusion (ACDF) and posterior cervical decompression and fusion (PCDF) for DCM. The electronic medical record was reviewed for patient baseline characteristics and surgical outcomes whereas preoperative radiographs were analyzed for baseline cervical lordosis and sagittal balance. Bivariate and multivariate statistical analyses were performed to compare the two groups. RESULTS: We identified 153 patients treated by ACDF and 43 patients treated by PCDF. Patients in the ACDF cohort were younger (60.1 ± 9.8 vs . 65.8 ± 6.9 yr; P < 0.001), had a lower overall comorbidity burden (Charlson Comorbidity Index: 2.25 ± 1.61 vs . 3.07 ± 1.64; P = 0.002), and were more likely to have a 3-level fusion (79.7% vs . 30.2%; P < 0.001), myeloradiculopathy (42.5% vs . 23.3%; P = 0.034), and cervical kyphosis (25.7% vs . 7.69%; P = 0.027). Patients undergoing an ACDF had significantly more improvement in their neck disability index after surgery (-14.28 vs . -3.02; P = 0.001), and this relationship was maintained on multivariate analysis with PCDF being independently associated with a worse neck disability index (+8.83; P = 0.025). Patients undergoing an ACDF also experienced more improvement in visual analog score neck pain after surgery (-2.94 vs . -1.47; P = 0.025) by bivariate analysis. CONCLUSIONS: Our data suggest that patients undergoing an ACDF or PCDF for multilevel DCM have similar outcomes after surgery.

HCC EV ECG score: An extracellular vesicle-based protein assay for detection of early-stage hepatocellular carcinoma.

BACKGROUND AND AIMS: The sensitivity of current surveillance methods for detecting early-stage hepatocellular carcinoma (HCC) is suboptimal. Extracellular vesicles (EVs) are promising circulating biomarkers for early cancer detection. In this study, we aim to develop an HCC EV-based surface protein assay for early detection of HCC. APPROACH AND RESULTS: Tissue microarray was used to evaluate four potential HCC-associated protein markers. An HCC EV surface protein assay, composed of covalent chemistry-mediated HCC EV purification and real-time immuno-polymerase chain reaction readouts, was developed and optimized for quantifying subpopulations of EVs. An HCC EV ECG score, calculated from the readouts of three HCC EV subpopulations ( E pCAM + CD63 + , C D147 + CD63 + , and G PC3 + CD63 + HCC EVs), was established for detecting early-stage HCC. A phase 2 biomarker study was conducted to evaluate the performance of ECG score in a training cohort ( n  = 106) and an independent validation cohort ( n  = 72).Overall, 99.7% of tissue microarray stained positive for at least one of the four HCC-associated protein markers (EpCAM, CD147, GPC3, and ASGPR1) that were subsequently validated in HCC EVs. In the training cohort, HCC EV ECG score demonstrated an area under the receiver operating curve (AUROC) of 0.95 (95% confidence interval [CI], 0.90-0.99) for distinguishing early-stage HCC from cirrhosis with a sensitivity of 91% and a specificity of 90%. The AUROCs of the HCC EV ECG score remained excellent in the validation cohort (0.93; 95% CI, 0.87-0.99) and in the subgroups by etiology (viral: 0.95; 95% CI, 0.90-1.00; nonviral: 0.94; 95% CI, 0.88-0.99). CONCLUSION: HCC EV ECG score demonstrated great potential for detecting early-stage HCC. It could augment current surveillance methods and improve patients' outcomes.

Impact of Heart Disease History on Safety of Telemedicine Cardiac Clearance Appointments.

PURPOSE: The objective of this study was to compare complication, readmission, mortality, and cancellation rates between patients who had either an in-person or telemedicine preoperative cardiac clearance visit before spine surgery. METHODS: A retrospective review was conducted on patients who underwent a spine procedure at a single tertiary academic center from February 1, 2020, to June 30, 2021. Cancellations, inpatient complications, 90-day readmissions, and inpatient and 90-day mortality rates were compared between in-person and telemedicine cardiac clearance visits. Secondary analysis included multiple logistic regression to determine independent predictors of case cancellations and complications. Alpha was set at P < 0.05. RESULTS: A total of 1,331 consecutive patients were included, with 775 patients (58.2%) having an in-person cardiac clearance visit and 556 (41.8%) having telemedicine clearance. Overall, the telemedicine cohort did not have more cancellations, complications, or readmissions. Regardless of the type of clearance, patients with a history of cardiac disease had more inpatient complications (15.8% versus 6.9%, P < 0.001) and higher 90-day mortality rates (2.3% versus 0.4%, P = 0.005). Subgroup analysis of patients with a history of cardiac disease showed that patients who had telemedicine visits had more cancellations (4.6% versus 10.9%, P = 0.036) and higher 90-day mortality rates (1.4% versus 4.4%, P = 0.045). On regression analysis, telemedicine visits were not independent predictors of preoperative cancellation rates (P = 0.173) but did predict greater preoperative cancellations among patients with cardiac history (odds ratio 2.73, P = 0.036). DISCUSSION: Patients with cardiac disease who undergo preoperative telemedicine visits have greater preoperative surgical cancellation rates and postoperative 90-day mortality rates. Although preoperative telemedicine visits may be appropriate for most patients, a history of cardiac disease should be a contraindication.

The Effect of Online Prescription Drug Monitoring on Opioid Prescription Habits After Elective Single-level Lumbar Fusion.

INTRODUCTION: The United States opioid epidemic is a well-documented crisis stemming from increased prescriptions of narcotics. Online prescription drug monitoring programs (PDMPs) are a potential resource to mitigate narcotic misuse by tracking controlled substance prescriptions. Therefore, the purpose of this study was to evaluate opioid prescription trends after implementation of an online PDMP in patients who underwent single-level lumbar fusion. METHODS: Patients who underwent a single-level lumbar fusion between August 27, 2017, and August 31, 2020, were identified and placed categorically into one of two cohorts: an "early adoption" cohort, September 1, 2017, to August 31, 2018, and a "late adoption" cohort, September 1, 2019, to August 31, 2020. This allowed for a 1-year washout period after Pennsylvania PDMP implementation on August 26, 2016. Opioid use data were obtained by searching for each patient in the state government's online PDMP and recording data from the year before and the year after the patient's procedure. RESULTS: No significant difference was observed in preoperative opioid prescriptions between the early and late adoption cohorts. The late adoption group independently predicted decreased postoperative opioid prescriptions (ß, 0.78; 95% confidence interval [CI], 0.65 to 0.93; P = 0.007), opioid prescribers (ß, 0.81; 95% CI, 0.72 to 0.90; P < 0.001), pharmacies used (ß, 0.90; 95% CI, 0.83 to 0.97; P = 0.006), opioid pills (ß, 0.61; 95% CI, 0.50 to 0.74; P < 0.001), days of opioid prescription (ß, 0.57; 95% CI, 0.45 to 0.72; P < 0.001), and morphine milligram equivalents prescribed (ß, 0.53; 95% CI, 0.43 to 0.66; P < 0.001). CONCLUSIONS: PDMP implementation was associated with decreased postoperative opioid prescription patterns but not preoperative opioid prescribing behaviors. LEVELS OF EVIDENCE: 4.

Circulating Tumor Cell-Based Messenger RNA Scoring System for Prognostication of Hepatocellular Carcinoma: Translating Tissue-Based Messenger RNA Profiling Into a Noninvasive Setting.

Numerous studies in hepatocellular carcinoma (HCC) have proposed tissue-based gene signatures for individualized prognostic assessments. Here, we develop a novel circulating tumor cell (CTC)-based transcriptomic profiling assay to translate tissue-based messenger RNA (mRNA) signatures into a liquid biopsy setting for noninvasive HCC prognostication. The HCC-CTC mRNA scoring system combines the NanoVelcro CTC Assay for enriching HCC CTCs and the NanoString nCounter platform for quantifying the HCC-CTC Risk Score (RS) panel in enriched HCC CTCs. The prognostic role of the HCC-CTC RS was assessed in The Cancer Genome Atlas (TCGA) HCC cohort (n = 362) and validated in an independent clinical CTC cohort (n = 40). The HCC-CTC RS panel was developed through our integrated data analysis framework of 8 HCC tissue-based gene signatures and identified the top 10 prognostic genes (discoidin domain receptor tyrosine kinase 1 [DDR1], enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase [EHHADH], androgen receptor [AR], lumican [LUM], hydroxysteroid 17-beta dehydrogenase 6[HSD17B6], prostate transmembrane protein, androgen induced 1 [PMEPA1], tsukushi, small leucine rich proteoglycan [TSKU], N-terminal EF-hand calcium binding protein 2 [NECAB2], ladinin 1 [LAD1], solute carrier family 27 member 5 [SLC27A5]) highly expressed in HCC with low expressions in white blood cells. The panel accurately discriminated overall survival in TCGA HCC cohort (hazard ratio [HR], 2.0; 95% confidence interval [CI], 1.4-2.9). The combined use of the scoring system and HCC-CTC RS panel successfully distinguished artificial blood samples spiked with an aggressive HCC cell type, SNU-387, from those spiked with PLC/PRF/5 cells (P = 0.02). In the CTC validation cohort (n = 40), HCC-CTC RS remained an independent predictor of survival (HR, 5.7; 95% CI, 1.5-21.3; P = 0.009) after controlling for Model for End-Stage Liver Disease score, Barcelona Clinic Liver Cancer stage, and CTC enumeration count. Our study demonstrates a novel interdisciplinary approach to translate tissue-based gene signatures into a liquid biopsy setting. This noninvasive approach will allow real-time disease profiling and dynamic prognostication of HCC.

Discovery and characterization of circulating tumor cell clusters in neuroendocrine tumor patients using nanosubstrate-embedded microchips.

Circulating tumor cell (CTC) clusters are present in cancer patients with severe metastasis, resulting in poor clinical outcomes. However, CTC clusters have not been studied as extensively as single CTCs, and the clinical utility of CTC clusters remains largely unknown. In this study, we aim sought to explore the feasibility of NanoVelcro Chips to simultaneously detect both single CTCs and CTC clusters with negligible perturbation to their intrinsic properties in neuroendocrine tumors (NETs). We discovered frequent CTC clusters in patients with advanced NETs and examined their potential roles, together with single NET CTCs, as novel biomarkers of patient response following peptide receptor radionuclide therapy (PRRT). We observed dynamic changes in both total NET CTCs and NET CTC cluster counts in NET patients undergoing PRRT which correlated with clinical outcome. These preliminary findings suggest that CTC clusters, along with single CTCs, offer a potential non-invasive option to monitor the treatment response in NET patients undergoing PRRT.

SARS-CoV-2-specific T cell responses and immune regulation in infected pregnant women.

We studied the T cell response to SARS-CoV-2 spike and non-spike peptide epitopes in eight convalescent pregnant women together with the immune monitoring that included innate tolerogenic dendritic cell populations important to maintain the immunological mother/fetus interface to address a potential risk for the antiviral cellular response in the outcome of pregnancy. Four subjects had pre-existing chronic inflammatory conditions that could have potentially affected the SARS-CoV-2-specific T cell response. Seven of eight subjects responded to SARS-CoV-2 peptides with differences within CD4+ T helper (Th) and CD8+ cytotoxic T cells (CTL). SARS-CoV-2-specific inducible regulatory T cells (iTreg) were numerous in circulation. CD4+ T cell memory included central memory T cells (TCM) and effector memory (TEM). As far as the CD8+ memory repertoire, TCM and TEM were very low or absent in eight of eight subjects and only effector cells that revert to CD45RA+, defined as TEMRA were measurable in circulation. T cells were in the normal range in all subjects regardless of pre-existing inflammatory conditions. The immune phenotype indicated the expansion and activation of tolerogenic myeloid dendritic cells including CD14+ cDC2 and CD4+ ILT-4+ tmDC. In summary, SARS-CoV-2 infection induced a physiological anti-viral T cell response in pregnant women that included SARS-CoV-2-specific iTreg with no negative effects on the tolerogenic innate dendritic cell repertoire relevant to the immune homeostasis of the maternal-fetal interface. All eight subjects studied delivered full-term, healthy infants.

Filling gaps in trustworthy development of AI.

Incident sharing, auditing, and other concrete mechanisms could help verify the trustworthiness of actors.

Circulating trophoblast cell clusters for early detection of placenta accreta spectrum disorders.

Placenta accreta spectrum (PAS) is a high-risk obstetrical condition associated with significant morbidity and mortality. Current clinical screening modalities for PAS are not always conclusive. Here, we report a nanostructure-embedded microchip that efficiently enriches both single and clustered circulating trophoblasts (cTBs) from maternal blood for detecting PAS. We discover a uniquely high prevalence of cTB-clusters in PAS and subsequently optimize the device to preserve the intactness of these clusters. Our feasibility study on the enumeration of cTBs and cTB-clusters from 168 pregnant women demonstrates excellent diagnostic performance for distinguishing PAS from non-PAS. A logistic regression model is constructed using a training cohort and then cross-validated and tested using an independent cohort. The combined cTB assay achieves an Area Under ROC Curve of 0.942 (throughout gestation) and 0.924 (early gestation) for distinguishing PAS from non-PAS. Our assay holds the potential to improve current diagnostic modalities for the early detection of PAS.

Covalent Chemistry-Mediated Multimarker Purification of Circulating Tumor Cells Enables Noninvasive Detection of Molecular Signatures of Hepatocellular Carcinoma.

Transcriptomic profiling of tumor tissues introduces a large database, which has led to improvements in the ability of cancer diagnosis, treatment, and prevention. However, performing tumor transcriptomic profiling in the clinical setting is very challenging since the procurement of tumor tissues is inherently limited by invasive sampling procedures. Here, we demonstrated the feasibility of purifying hepatocellular carcinoma (HCC) circulating tumor cells (CTCs) from clinical patient samples with improved molecular integrity using Click Chips in conjunction with a multimarker antibody cocktail. The purified CTCs were then subjected to mRNA profiling by NanoString nCounter platform, targeting 64 HCC-specific genes, which were generated from an integrated data analysis framework with 8 tissue-based prognostic gene signatures from 7 publicly available HCC transcriptomic studies. After bioinformatics analysis and comparison, the HCC CTC-derived gene signatures showed high concordance with HCC tissue-derived gene signatures from TCGA database, suggesting that HCC CTCs purified by Click Chips could enable the translation of HCC tissue molecular profiling into a noninvasive setting.

Retinal organoids on-a-chip: a micro-millifluidic bioreactor for long-term organoid maintenance.

Retinal degeneration is a leading cause of vision impairment and blindness worldwide and medical care for advanced disease does not exist. Stem cell-derived retinal organoids (RtOgs) became an emerging tool for tissue replacement therapy. However, existing RtOg production methods are highly heterogeneous. Controlled and predictable methodology and tools are needed to standardize RtOg production and maintenance. In this study, we designed a shear stress-free micro-millifluidic bioreactor for nearly labor-free retinal organoid maintenance. We used a stereolithography (SLA) 3D printer to fabricate a mold from which Polydimethylsiloxane (PDMS) was cast. We optimized the chip design using in silico simulations and in vitro evaluation to optimize mass transfer efficiency and concentration uniformity in each culture chamber. We successfully cultured RtOgs at three different differentiation stages (day 41, 88, and 128) on an optimized bioreactor chip for more than 1 month. We used different quantitative and qualitative techniques to fully characterize the RtOgs produced by static dish culture and bioreactor culture methods. By analyzing the results from phase contrast microscopy, single-cell RNA sequencing (scRNA seq), quantitative polymerase chain reaction (qPCR), immunohistology, and electron microscopy, we found that bioreactor-cultured RtOgs developed cell types and morphology comparable to static cultured ones and exhibited similar retinal genes expression levels. We also evaluated the metabolic activity of RtOgs in both groups using fluorescence lifetime imaging (FLIM), and found that the outer surface region of bioreactor cultured RtOgs had a comparable free/bound NADH ratio and overall lower long lifetime species (LLS) ratio than static cultured RtOgs during imaging. To summarize, we validated an automated micro-millifluidic device with significantly reduced shear stress to produce RtOgs of comparable quality to those maintained in conventional static culture.

The Role of Extracellular Vesicles in Disease Progression and Detection of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and one of the leading causes of cancer-related death worldwide. Despite the improvements in surveillance and treatment, the prognosis of HCC remains poor. Extracellular vesicles (EVs) are a heterogeneous group of phospholipid bilayer-enclosed particles circulating in the bloodstream and mediating intercellular communication. Emerging studies have shown that EVs play a crucial role in regulating the proliferation, immune escape, and metastasis of HCC. In addition, because EVs are present in the circulation at relatively early stages of disease, they are getting attention as an attractive biomarker for HCC detection. Over the past decade, dedicated efforts have been made to isolate EVs more efficiently and make them useful tools in different clinical settings. In this review article, we provide an overview of the EVs isolation methods and highlight the role of EVs as mediators in the pathogenesis and progression of HCC. Lastly, we summarize the potential applications of EVs in early-stage HCC detection.