A Novel Positive-Contrast Magnetic Resonance Imaging Line Marker for High-Dose-Rate (HDR) MRI-Assisted Radiosurgery (MARS).

Magnetic resonance imaging (MRI) can facilitate accurate organ delineation and optimal dose distributions in high-dose-rate (HDR) MRI-Assisted Radiosurgery (MARS). Its use for this purpose has been limited by the lack of positive-contrast MRI markers that can clearly delineate the lumen of the HDR applicator and precisely show the path of the HDR source on T1- and T2-weighted MRI sequences. We investigated a novel MRI positive-contrast HDR brachytherapy or interventional radiotherapy line marker, C4:S, consisting of C4 (visible on T1-weighted images) complexed with saline. Longitudinal relaxation time (T1) and transverse relaxation time (T2) for C4:S were measured on a 1.5 T MRI scanner. High-density polyethylene (HDPE) tubing filled with C4:S as an HDR brachytherapy line marker was tested for visibility on T1- and T2-weighted MRI sequences in a tissue-equivalent female ultrasound training pelvis phantom. Relaxivity measurements indicated that C4:S solution had good T1-weighted contrast (relative to oil [fat] signal intensity) and good T2-weighted contrast (relative to water signal intensity) at both room temperature (relaxivity ratio > 1; r2/r1 = 1.43) and body temperature (relaxivity ratio > 1; r2/r1 = 1.38). These measurements were verified by the positive visualization of the C4:S (C4/saline 50:50) HDPE tube HDR brachytherapy line marker on both T1- and T2-weighted MRI sequences. Orientation did not affect the relaxivity of the C4:S contrast solution. C4:S encapsulated in HDPE tubing can be visualized as a positive line marker on both T1- and T2-weighted MRI sequences. MRI-guided HDR planning may be possible with these novel line markers for HDR MARS for several types of cancer.

Targeted activation of human ether-à-go-go-related gene channels rescues electrical instability induced by the R56Q+/- long QT syndrome variant.

AIMS: Long QT syndrome type 2 (LQTS2) is associated with inherited variants in the cardiac human ether-à-go-go-related gene (hERG) K+ channel. However, the pathogenicity of hERG channel gene variants is often uncertain. Using CRISPR-Cas9 gene-edited hiPSC-derived cardiomyocytes (hiPSC-CMs), we investigated the pathogenic mechanism underlying the LQTS-associated hERG R56Q variant and its phenotypic rescue by using the Type 1 hERG activator, RPR260243. METHODS AND RESULTS: The above approaches enable characterization of the unclear causative mechanism of arrhythmia in the R56Q variant (an N-terminal PAS domain mutation that primarily accelerates channel deactivation) and translational investigation of the potential for targeted pharmacologic manipulation of hERG deactivation. Using perforated patch clamp electrophysiology of single hiPSC-CMs, programmed electrical stimulation showed that the hERG R56Q variant does not significantly alter the mean action potential duration (APD90). However, the R56Q variant increases the beat-to-beat variability in APD90 during pacing at constant cycle lengths, enhances the variance of APD90 during rate transitions, and increases the incidence of 2:1 block. During paired S1-S2 stimulations measuring electrical restitution properties, the R56Q variant was also found to increase the variability in rise time and duration of the response to premature stimulations. Application of the hERG channel activator, RPR260243, reduces the APD variance in hERG R56Q hiPSC-CMs, reduces the variability in responses to premature stimulations, and increases the post-repolarization refractoriness. CONCLUSION: Based on our findings, we propose that the hERG R56Q variant leads to heterogeneous APD dynamics, which could result in spatial dispersion of repolarization and increased risk for re-entry without significantly affecting the average APD90. Furthermore, our data highlight the antiarrhythmic potential of targeted slowing of hERG deactivation gating, which we demonstrate increases protection against premature action potentials and reduces electrical heterogeneity in hiPSC-CMs.

Machine learning-based natural language processing to extract PD-L1 expression levels from clinical notes.

Introduction: PD-L1 expression is used to determine oncology patients' response to and eligibility for immunologic treatments; however, PD-L1 expression status often only exists in unstructured clinical notes, limiting ability to use it in population-level studies. Methods: We developed and evaluated a machine learning based natural language processing (NLP) tool to extract PD-L1 expression values from the nationwide Veterans Affairs electronic health record system. Results: The model demonstrated strong evaluation performance across multiple levels of label granularity. Mean precision of the overall PD-L1 positive label was 0.859 (sd, 0.039), recall 0.994 (sd, 0.013), and F1 0.921 (0.024). When a numeric PD-L1 value was identified, the mean absolute error of the value was 0.537 on a scale of 0 to 100. Conclusion: We presented an accurate NLP method for deriving PD-L1 status from clinical notes. By reducing the time and manual effort needed to review medical records, our work will enable future population-level studies in cancer immunotherapy.

Tumor-Derived CCL5 Recruits Cancer-Associated Fibroblasts and Promotes Tumor Cell Proliferation in Esophageal Squamous Cell Carcinoma.

Cancer-associated fibroblasts (CAF) can promote tumor growth, metastasis, and therapeutic resistance in esophageal squamous cell carcinoma (ESCC), but the mechanisms of action remain elusive. Our objective was to identify secreted factor(s) that mediate the communication between CAFs and ESCC tumor cells with the aim of identifying potential druggable targets. Through unbiased cytokine arrays, we have identified CC motif chemokine ligand 5 (CCL5) as a secreted factor that is increased upon co-culture of ESCC cells and CAFs, which we replicated in esophageal adenocarcinoma (EAC) with CAFs. Loss of tumor-cell-derived CCL5 reduces ESCC cell proliferation in vitro and in vivo and we propose this is mediated, in part, by a reduction in ERK1/2 signaling. Loss of tumor-derived CCL5 reduces the percentage of CAFs recruited to xenograft tumors in vivo. CCL5 is a ligand for the CC motif receptor 5 (CCR5), for which a clinically approved inhibitor exists, namely Maraviroc. Maraviroc treatment reduced tumor volume, CAF recruitment, and ERK1/2 signaling in vivo, thus, mimicking the effects observed with genetic loss of CCL5. High CCL5 or CCR5 expression is associated with worse prognosis in low-grade esophageal carcinomas. IMPLICATIONS: These data highlight the role of CCL5 in tumorigenesis and the therapeutic potential of targeting the CCL5-CCR5 axis in ESCC.

Water-soluble chromenylium dyes for shortwave infrared imaging in mice.

In vivo imaging using shortwave infrared light (SWIR, 1000-2000 nm) benefits from deeper penetration and higher resolution compared to using visible and near-infrared wavelengths. However, the development of biocompatible SWIR contrast agents remains challenging. Despite recent advancements, small molecule SWIR fluorophores are often hindered by their significant hydrophobicity. We report a platform for generating a panel of soluble and functional dyes for SWIR imaging by late-stage functionalization of a versatile fluorophore intermediate, affording water-soluble dyes with bright SWIR fluorescence in serum. Specifically, a tetra-sulfonate derivative enables clear video-rate imaging of vasculature with only 0.05 nmol dye, and a tetra-ammonium dye shows strong cellular retention for tracking of tumor growth. Additionally, incorporation of phosphonate functionality enables imaging of bone in awake mice. This modular design provides insights for facile derivatization of existing SWIR fluorophores to introduce both solubility and bioactivity towards in vivo bioimaging.

Kinase Inhibition by PKC412 Prevents Epithelial Sheet Damage in Autosomal Dominant Epidermolysis Bullosa Simplex through Keratin and Cell Contact Stabilization.

Epidermolysis bullosa simplex (EBS) is a severe and potentially life-threatening disorder for which no adequate therapy exists. Most cases are caused by dominant sequence variations in keratin genes K5 or K14, leading to the formation of cytoplasmic keratin aggregates, profound keratinocyte fragility, and cytolysis. We hypothesized that pharmacological reduction of keratin aggregates, which compromise keratinocyte integrity, represents a viable strategy for the treatment of EBS. In this study, we show that the multikinase inhibitor PKC412, which is currently in clinical use for acute myeloid leukemia and advanced systemic mastocytosis, reduced keratin aggregation by 40% in patient-derived K14.R125C EBS-associated keratinocytes. Using a combination of epithelial shear stress assay and real-time impedance spectroscopy, we show that PKC412 restored intercellular adhesion. Molecularly, global phosphoproteomic analysis together with immunoblots using phosphoepitope-specific antibodies revealed that PKC412 treatment altered phosphorylated sites on keratins and desmoplakin. Thus, our data provide a proof of concept to repurpose existing drugs for the targeted treatment of EBS and showcase how one broad-range kinase inhibitor reduced keratin filament aggregation in patient-derived EBS keratinocytes and the fragility of EBS cell monolayers. Our study paves the way for a clinical trial using PKC412 for systemic or local application in patients with EBS.

Di(2-ethylhexyl)phthalate impairs erythropoiesis via inducing Klotho expression and not via bioenergetic reprogramming.

Di(2-ethylhexyl)phthalate (DEHP) is the most widely used phthalate to manufacture various plastic products. However, the potential effects of DEHP on erythropoiesis have not been investigated comprehensively. Here, we aimed to investigate whether DEHP modulated the function of hematopoietic stem and progenitor cells (HSPCs) to influence erythropoiesis, and to explore the associated mechanisms. In the present study, human cell lines with a capacity to differentiate into erythroid cells and murine bone marrow cells were treated with DEHP. DEHP not only impaired HSPC function, but also suppressed erythroid differentiation in a dose-dependent manner. In addition, DEHP removal restored HSPC activity. To explore how DEHP interfered with erythroid differentiation, we focused on energy metabolism and Klotho expression. DEHP suppressed erythroid differentiation via upregulating Klotho expression, while it did not via modulating cellular bioenergetics. Therefore, our results provided a novel insight into the pathophysiological link between phthalates and dysregulated erythroid differentiation.

Rectal Radiation Dose and Clinical Outcomes in Prostate Cancer Patients Treated With Stereotactic Body Radiation Therapy With and Without Hydrogel.

Background: Patients with prostate cancer treated with stereotactic body radiation therapy (SBRT) may experience gastrointestinal (GI) toxicity. The hydrogel may mitigate this toxicity by reducing the rectal radiation dose. The purpose of this study is to compare rectal radiation dose and GI toxicity in patients receiving prostate SBRT with and without hydrogel. Methods: Consecutive patients treated with SBRT between February 2017 and January 2020 with and without hydrogel were retrospectively identified. Baseline characteristics including prostate volume, rectal diameter, body mass index (BMI), age, pretreatment prostate-specific antigen (PSA), Gleason score, T-stage, and androgen deprivation therapy (ADT) usage were compared. Dosimetric outcomes (V40Gy, V36Gy, V32Gy, V38Gy, and V20Gy), rates of acute (≤90 days) and late (>90 days) GI toxicity, and PSA outcomes were evaluated for patients with and without hydrogel. Results: A total of 92 patients were identified (51 hydrogel and 41 non-hydrogel). There were no significant differences in baseline characteristics. Rectal V38(cc) was significantly less in the hydrogel group (mean 0.44 vs. mean 1.41 cc, p = 0.0002), and the proportion of patients with V38(cc) < 2 cc was greater in the hydrogel group (92% vs. 72%, p = 0.01). Rectal dose was significantly lower for all institutional dose constraints in the hydrogel group (p < 0.001). The hydrogel group experienced significantly less acute overall GI toxicity (16% hydrogel vs. 28% non-hydrogel, p = 0.006), while the difference in late GI toxicity trended lower with hydrogel but was not statistically significant (4% hydrogel vs. 10% non-hydrogel, p = 0.219). At a median follow-up of 14.8 months, there were no biochemical recurrences in either group. Conclusion: Hydrogel reduces rectal radiation dose in patients receiving prostate SBRT and is associated with a decreased rate of acute GI toxicity.

Glucosamine Interferes With Myelopoiesis and Enhances the Immunosuppressive Activity of Myeloid-Derived Suppressor Cells.

Glucosamine (GlcN) is the most widely consumed dietary supplement and exhibits anti-inflammatory effects. However, the influence of GlcN on immune cell generation and function is largely unclear. In this study, GlcN was delivered into mice to examine its biological function in hematopoiesis. We found that GlcN promoted the production of immature myeloid cells, known as myeloid-derived suppressor cells (MDSCs), both in vivo and in vitro. Additionally, GlcN upregulated the expression of glucose transporter 1 in hematopoietic stem and progenitor cells (HSPCs), influenced HSPC functions, and downregulated key genes involved in myelopoiesis. Furthermore, GlcN increased the expression of arginase 1 and inducible nitric oxide synthase to produce high levels of reactive oxygen species, which was regulated by the STAT3 and ERK1/2 pathways, to increase the immunosuppressive ability of MDSCs. We revealed a novel role for GlcN in myelopoiesis and MDSC activity involving a potential link between GlcN and immune system, as well as the new therapeutic benefit.

Patient-derived organoids as a platform for modeling a patient's response to chemoradiotherapy in esophageal cancer.

3D patient-derived organoids (PDOs) have been utilized to evaluate potential therapies for patients with different cancers. However, the use of PDOs created from treatment-naive patient biopsies for prediction of clinical outcomes in patients with esophageal cancer has not yet been reported. Herein we describe a pilot prospective observational study with the goal of determining whether esophageal cancer PDOs created from treatment naive patients can model or predict clinical outcomes. Endoscopic biopsies of treatment-naive patients at a single tertiary care center were used to generate esophageal cancer PDOs, which were treated with standard-of-care chemotherapy, gamma-irradiation, and newer non-standard approaches, such as proton beam therapy or two small molecule inhibitors. Clinical outcomes of patients following neoadjuvant treatment were compared to their in vitro PDO responses, demonstrating the PDO's ability to mirror clinical response, suggesting the value of PDOs in prediction of clinical response to new therapeutic approaches. Future prospective clinical trials should test the use of pre-treatment PDOs to identify specific, targeted therapies for individual patients with esophageal adenocarcinoma.

Liquid marble clearance and restoration via gas bubble insertion and bursting.

There is hitherto a lack of a simple way to disrupt the coating of particles from liquid marbles in order to introduce additional reagents. Here, a 40 µL liquid marble, created on a superhydrophobic substrate with a 2 mm hole, forms an overhead and overhanging liquid component from which a single gas bubble of up to 28 µL volume could be introduced via the latter. This caused a localized clearing of the particle shell at the apical region of the overhead component because the particles could not be energetically sustained at the thin film region of the bubble. The subsequent dispensation of 5 µL of an external liquid directly onto the shell-free apex of the liquid marble allowed the coalescence of the two liquid bodies, bubble rupture, and restoration of complete particle shell encapsulation. The addition of the liquid via the overhanging component was alternatively found incapable of increasing the size of the overhead drop component. The localized bubble-actuated transient shell clearance at the apex of the liquid marble to allow the addition of reagents shown here portends new vistas for liquid marbles to be used in biomedical applications.

Caspase-8 loss radiosensitizes head and neck squamous cell carcinoma to SMAC mimetic-induced necroptosis.

Caspase-8 (CASP8) is one of the most frequently mutated genes in head and neck squamous carcinomas (HNSCCs), and CASP8 mutations are associated with poor survival. The distribution of these mutations in HNSCCs suggests that they are likely to be inactivating. Inhibition of CASP8 has been reported to sensitize cancer cells to necroptosis, a regulated cell death mechanism. Here, we show that knockdown of CASP8 renders HNSCCs susceptible to necroptosis by a second mitochondria-derived activator of caspase (SMAC) mimetic, birinapant, in combination with pan-caspase inhibitors Z-VAD-FMK or emricasan and radiation. In a syngeneic mouse model of oral cancer, birinapant, particularly when combined with radiation, delayed tumor growth and enhanced survival under CASP8 loss. Exploration of molecular underpinnings of necroptosis sensitivity confirmed that the level of functional receptor-interacting serine/threonine protein kinase 3 (RIP3) determines susceptibility to this mode of death. Although an in vitro screen revealed that low RIP3 levels rendered many HNSCC cell lines resistant to necroptosis, patient tumors maintained RIP3 expression and should therefore remain sensitive. Collectively, these results suggest that targeting the necroptosis pathway with SMAC mimetics, especially in combination with radiation, may be relevant therapeutically in HNSCC with compromised CASP8 status, provided that RIP3 function is maintained.

The hERG channel activator, RPR260243, enhances protective IKr current early in the refractory period reducing arrhythmogenicity in zebrafish hearts.

Human ether-à-go-go related gene (hERG) K+ channels are important in cardiac repolarization, and their dysfunction causes prolongation of the ventricular action potential, long QT syndrome, and arrhythmia. As such, approaches to augment hERG channel function, such as activator compounds, have been of significant interest due to their marked therapeutic potential. Activator compounds that hinder channel inactivation abbreviate action potential duration (APD) but carry risk of overcorrection leading to short QT syndrome. Enhanced risk by overcorrection of the APD may be tempered by activator-induced increased refractoriness; however, investigation of the cumulative effect of hERG activator compounds on the balance of these effects in whole organ systems is lacking. Here, we have investigated the antiarrhythmic capability of a hERG activator, RPR260243, which primarily augments channel function by slowing deactivation kinetics in ex vivo zebrafish whole hearts. We show that RPR260243 abbreviates the ventricular APD, reduces triangulation, and steepens the slope of the electrical restitution curve. In addition, RPR260243 increases the post-repolarization refractory period. We provide evidence that this latter effect arises from RPR260243-induced enhancement of hERG channel-protective currents flowing early in the refractory period. Finally, the cumulative effect of RPR260243 on arrhythmogenicity in whole organ zebrafish hearts is demonstrated by the restoration of normal rhythm in hearts presenting dofetilide-induced arrhythmia. These findings in a whole organ model demonstrate the antiarrhythmic benefit of hERG activator compounds that modify both APD and refractoriness. Furthermore, our results demonstrate that targeted slowing of hERG channel deactivation and enhancement of protective currents may provide an effective antiarrhythmic approach.NEW & NOTEWORTHY hERG channel dysfunction causes long QT syndrome and arrhythmia. Activator compounds have been of significant interest due to their therapeutic potential. We used the whole organ zebrafish heart model to demonstrate the antiarrhythmic benefit of the hERG activator, RPR260243. The activator abbreviated APD and increased refractoriness, the combined effect of which rescued induced ventricular arrhythmia. Our findings show that the targeted slowing of hERG channel deactivation and enhancement of protective currents caused by the RPR260243 activator may provide an effective antiarrhythmic approach.

Variation in RARG increases susceptibility to doxorubicin-induced cardiotoxicity in patient specific induced pluripotent stem cell-derived cardiomyocytes.

Doxorubicin is a potent anticancer drug used to treat a variety of cancer types. However, its use is limited by doxorubicin-induced cardiotoxicity (DIC). A missense variant in the RARG gene (S427L; rs2229774) has been implicated in susceptibility to DIC in a genome wide association study. The goal of this study was to investigate the functional role of this RARG variant in DIC. We used induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) from patients treated with doxorubicin. iPSC-CMs from individuals who experienced DIC (cases) showed significantly greater sensitivity to doxorubicin compared to iPSC-CMs from doxorubicin-treated individuals who did not develop DIC (controls) in cell viability and optical mapping experiments. Using CRISPR/Cas9, we generated isogenic cell lines that differed only at the RARG locus. Genetic correction of RARG-S427L to wild type resulted in reduced doxorubicin-induced double stranded DNA breaks, reactive oxygen species production, and cell death. Conversely, introduction of RARG-S427L increased susceptibility to doxorubicin. Finally, genetic disruption of the RARG gene resulted in protection from cell death due to doxorubicin treatment. Our findings suggest that the presence of RARG-S427L increases sensitivity to DIC, establishing a direct, causal role for this variant in DIC.

Proton and photon radiosensitization effects of niraparib, a PARP-1/-2 inhibitor, on human head and neck cancer cells.

BACKGROUND: Combining photon or proton radiotherapy with targeted therapy shows promise for head and neck cancer (HNSCC). The poly (adenosine diphosphate [ADP]-ribose) polymerase-1/2 inhibitor niraparib targets DNA damage repair (DDR). We evaluated the effects of niraparib in combination with photons or protons, and its effects on the relative biological effectiveness (RBE) of protons, in human HNSCC cell lines. METHODS: Radiosensitivity was assessed and RBE was calculated with clonogenic survival assays; unrepaired DNA double-strand breaks were evaluated using immunocytochemical analysis of 53BP1 foci. RESULTS: Niraparib reduced colony formation in two of the four cell lines tested (P < .05), enhanced radiosensitivity in all four cell lines, delayed DDR (P < .05), and increased proton vs photon RBE. CONCLUSION: Niraparib enhanced the sensitivity of four HNSCC cell lines to both photons and protons and increased the RBE of protons, possibly by inhibiting DDR. Niraparib may enhance the effectiveness of both photon and proton radiotherapy for patients with HNSCC.

Expansion of Circulating Tumor Cells from Patients with Locally Advanced Pancreatic Cancer Enable Patient Derived Xenografts and Functional Studies for Personalized Medicine.

Improvement in pancreatic cancer treatment represents an urgent medical goal that has been hampered by the lack of predictive biomarkers. Circulating Tumor Cells (CTCs) may be able to overcome this issue by allowing the monitoring of therapeutic response and tumor aggressiveness through ex vivo expansion. The successful expansion of CTCs is challenging, due to their low numbers in blood and the high abundance of blood cells. Here, we explored the utility of pancreatic CTC cultures as a preclinical model for treatment response. CTCs were isolated from ten patients with locally advanced pancreatic cancer using the Labyrinth, a biomarker independent, size based, inertial microfluidic separation device. Three patient-derived CTC samples were successfully expanded in adherent and spheroid cultures. Molecular and functional characterization was performed on the expanded CTC lines. CTC lines exhibited KRAS mutations, consistent with pancreatic cancers. Additionally, we evaluated take rate and metastatic potential in vivo and examined the utility of CTC lines for cytotoxicity assays. Patient derived expanded CTCs successfully generated patient derived xenograft (PDX) models with a 100% take rate. Our results demonstrate that CTC cultures are possible and provide a valuable resource for translational pancreatic cancer research, while also providing meaningful insight into the development of distant metastasis, as well as treatment resistance.

The extracellular signal-regulated kinase 1/2 modulates the intracellular localization of DNA methyltransferase 3A to regulate erythrocytic differentiation.

DNA methylation, catalyzed by DNA methyltransferases (DNMTs), is a heritable epigenetic mark, participating in numerous physiological processes. DNMT3A is of particular relevance to hematopoietic differentiation, because DNMT3A mutations are strongly related to hematopoietic malignancies. Additionally, DNMT3A deficiency has been reported to increase the hematopoietic stem cell pool by limiting their differentiation. Our previous study demonstrated that complete loss of DNMT3A resulted in anemia, while DNMT3A haploinsufficiency caused an elevated population of erythrocytes in the content of oncogenic KRAS. Since erythropoiesis is tightly regulated via the erythropoietin (EPO)-mediated RAS-RAF-MEK-ERK1/2 pathway, the question arises whether DNMT3A cooperates with RAS signaling to modulate erythropoiesis. Human leukemia cell lines were used, with differentiation capabilities towards megakaryocyte and erythroid lineages. Overexpression of DNMT3A was found to enhance erythrocytic differentiation of K562 cells, while DNMT3A knockdown suppressed differentiation. Furthermore, higher DNMT3A expression was detected in late-stage mouse erythroblasts along with the DNMT3A translocation to the nucleus. Further studies demonstrated that both ERK1/2-DNMT3A interaction and serine-255 phosphorylation in DNMT3A led to DNMT3A translocation into the nucleus, and modulated erythrocytic differentiation. Our results not only explore the critical role of DNMT3A in erythropoiesis, but also provide a new insight into ERK1/2-DNMT3A interaction in the hematopoietic system.

High-Throughput Label-Free Isolation of Heterogeneous Circulating Tumor Cells and CTC Clusters from Non-Small-Cell Lung Cancer Patients.

(1) Background: Circulating tumor cell (CTC) clusters are emerging as clinically significant harbingers of metastases in solid organ cancers. Prior to engaging these CTC clusters in animal models of metastases, it is imperative for technology to identify them with high sensitivity. These clusters often present heterogeneous surface markers and current methods for isolation of clusters may fall short. (2) Methods: We applied an inertial microfluidic Labyrinth device for high-throughput, biomarker-independent, size-based isolation of CTCs/CTC clusters from patients with metastatic non-small-cell lung cancer (NSCLC). (3) Results: Using Labyrinth, CTCs (PanCK+/DAPI+/CD45-) were isolated from patients (n = 25). Heterogeneous CTC populations, including CTCs expressing epithelial (EpCAM), mesenchymal (Vimentin) or both markers were detected. CTCs were isolated from 100% of patients (417 ± 1023 CTCs/mL). EpCAM- CTCs were significantly greater than EpCAM+ CTCs. Cell clusters of ≥2 CTCs were observed in 96% of patients-of which, 75% were EpCAM-. CTCs revealed identical genetic aberrations as the primary tumor for RET, ROS1, and ALK genes using fluorescence in situ hybridization (FISH) analysis. (4) Conclusions: The Labyrinth device recovered heterogeneous CTCs in 100% and CTC clusters in 96% of patients with metastatic NSCLC. The majority of recovered CTCs/clusters were EpCAM-, suggesting that these would have been missed using traditional antibody-based capture methods.

Patterns of protein expression in human head and neck cancer cell lines differ after proton vs photon radiotherapy.

BACKGROUND: Proton radiotherapy (PRT) may be a less toxic alternative to photon radiotherapy (XRT) for patients with head and neck squamous cell carcinoma (HNSCC). However, the molecular responses of HNSCC cells to PRT vs XRT are unclear. METHODS: Proteomics analyses of protein expression profiles by reverse-phase protein arrays were done for two human papillomavirus [HPV]-negative and two HPV+ cell lines. Expression patterns of 175 proteins involved in several signaling pathways were tested. RESULTS: Compared with PRT, XRT tended to induce lower expression of DNA damage repair-and cell cycle arrest-related proteins and higher expression of cell survival- and proliferation-related proteins. CONCLUSIONS: Under these experimental conditions, PRT and XRT induced different protein expression and activation profiles. Further preclinical verification is needed, as are studies of tumor pathway mutations as biomarkers for choice of treatment or as radiosensitization targets to improve the response of HNSCC to PRT or XRT.

New Labyrinth Microfluidic Device Detects Circulating Tumor Cells Expressing Cancer Stem Cell Marker and Circulating Tumor Microemboli in Hepatocellular Carcinoma.

Hepatocellular Carcinoma (HCC) is one of the most lethal cancers with a high mortality and recurrence rate. Circulating tumor cell (CTC) detection offers various opportunities to advance early detection and monitoring of HCC tumors which is crucial for improving patient outcome. We developed and optimized a novel Labyrinth microfluidic device to efficiently isolate CTCs from peripheral blood of HCC patients. CTCs were identified in 88.1% of the HCC patients over different tumor stages. The CTC positivity rate was significantly higher in patients with more advanced HCC stages. In addition, 71.4% of the HCC patients demonstrated CTCs positive for cancer stem cell marker, CD44, suggesting that the major population of CTCs could possess stemness properties to facilitate tumor cell survival and dissemination. Furthermore, 55% of the patients had the presence of circulating tumor microemboli (CTM) which also correlated with advanced HCC stage, indicating the association of CTM with tumor progression. Our results show effective CTC capture from HCC patients, presenting a new method for future noninvasive screening and surveillance strategies. Importantly, the detection of CTCs with stemness markers and CTM provides unique insights into the biology of CTCs and their mechanisms influencing metastasis, recurrence and therapeutic resistance.