Changes In Biomarker Expression Research Articles

Microneedles: A novel clinical technology for evaluating skin characteristics.

Current methods for evaluating efficacy of cosmetics have limitations because they cannot accurately measure changes in the dermis. Skin sampling using microneedles allows identification of skin-type biomarkers, monitoring treatment for skin inflammatory diseases, and evaluating efficacy of anti-aging and anti-pigmentation products. Two studies were conducted: First, 20 participants received anti-aging treatment; second, 20 participants received anti-pigmentation treatment. Non-invasive devices measured skin aging (using high-resolution 3D-imaging in the anti-aging study) or pigmentation (using spectrophotometry in the anti-pigmentation study) at weeks 0 and 4, and adverse skin reactions were monitored. Skin samples were collected with biocompatible microneedle patches. Changes in expression of biomarkers for skin aging and pigmentation were analyzed using qRT-PCR. No adverse events were reported. In the anti-aging study, after 4 weeks, skin roughness significantly improved in 17 out of 20 participants. qRT-PCR showed significantly increased expression of skin-aging related biomarkers: PINK1 in 16/20 participants, COL1A1 in 17/20 participants, and MSN in 16/20 participants. In the anti-pigmentation study, after 4 weeks, skin lightness significantly improved in 16/20 participants. qRT-PCR showed significantly increased expression of skin-pigmentation-related biomarkers: SOD1 in 15/20 participants and Vitamin D Receptor (VDR) in 15/20 participants. No significant change in TFAP2A was observed. Skin sampling and mRNA analysis for biomarkers provides a novel, objective, quantitative method for measuring changes in the dermis and evaluating the efficacy of cosmetics. This approach complements existing evaluation methods and has potential application in assessing the effectiveness of medical devices, medications, cosmeceuticals, healthy foods, and beauty devices.

Biomarker Alteration after Neoadjuvant Endocrine Therapy or Chemotherapy in Estrogen Receptor-Positive Breast Cancer.

In estrogen receptor (ER)-positive breast cancer, changes in biomarker expression after neoadjuvant therapy indicate the therapeutic response and are prognostic. However, there is limited information about the biomarker alteration caused by neoadjuvant endocrine therapy in ER-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer. We recruited ER-positive/HER2-negative breast cancer patients who received neoadjuvant chemotherapy (NCT), neoadjuvant endocrine therapy (NET), or sequential neoadjuvant endocrine-chemotherapy (NECT) at Peking University Cancer Hospital from 2015 to 2021. A total of 579 patients had paired immunohistochemistry information in both diagnostic biopsy samples and post-neoadjuvant therapy surgical samples. Through a paired comparison of the immunohistochemical information in pre-treatment and post-treatment samples, we found that progesterone receptor (PR) expression reductions were more frequent than ER expression reductions (70.8% vs. 35.2%) after neoadjuvant therapy. The percentage of patients who had a decreased Ki-67 index in the post-operative samples was similar in the three groups (79.8% vs. 79.7% vs. 78.4%). Moreover, PR losses caused by NET were related to low baseline PR expression (p = 0.001), while we did not find a significant association between PR losses and Ki-67 reductions (p = 0.428) or ER losses (p = 0.274). All three types of neoadjuvant therapies caused a reduction in ER, PR, and Ki-67 expression. In conclusion, we found that PR loss after NET was only significantly related to low baseline PR expression, and there is no significant difference in the extent of prognostic factor change including Ki-67 and ER between the PR loss and non-loss groups.

Modestly Increased Incidence of Ketosis in Caloric Restriction Does not Significantly Alter the Effects of Caloric Restriction.

Caloric restriction (CR) has been shown to slow the aging processes in a number of preclinical studies and reduces expression of aging-associated biomarkers in human trials. We hypothesized that CR would lead to increased incidence of ketosis and that ketosis in CR individuals would alter the aging-protective effects of CR or biomarkers thereof. We analyzed data from the "Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy (CALERIE, Phase 2)" Public Use Database available at calerie.duke.edu. In this study, non-obese adults between the ages of 21 and 50 were randomized to 25% CR or control (ad lib) diet groups and extensively monitored for two years. Given our focus on the effect of caloric restriction on ketosis, individuals with detectible ketones during the baseline visit (pre-randomization) and those with missing data for ketone testing were excluded from the analysis, leaving 71 control and 117 CR participants. We analyzed the incidence of ketosis as well as ketosis free survival in control and CR participants and assessed the effect of ketosis on a number of clinical lab values, functional assessments, and participant survey data related to aging biology. We report that CR was associated with modestly increased incidence of ketosis (4.4% in CR vs 1.9% in control), though CR-associated changes in T3, VO2, SUMPT-WT (weight normalized composite strength score - peak torque), physical functioning, and general health did not appear to be altered by the presence or absence of ketosis. Additional observations of interest include: 1) striking patterns of biomarker expression changes (MCP-1, TNFα, TGF-β1, GH) in both the control and CR participants between the baseline visit and the 24-month post-randomization visit and 2) pro-growth/anti-inflammatory baseline (pre-randomization) biomarker expression profile in CR individuals that later test ketone positive relative to other CR individuals. CR modestly increases the incidence of ketosis in healthy adults, yet the increase in ketosis in CR patients did not significantly affect the aging-protective effects of CR. However, given the relatively small number of participants who were ketone positive, further investigation in larger study cohorts is still required for definitive conclusions.

Comparison of Proteomic Expression Profiles after Radiation Exposure across Four Different Species.

There is a need to identify biomarkers of radiation exposure for use in development of circulating biodosimeters for radiation exposure and for clinical use as markers of radiation injury. Most research approaches for biomarker discovery rely on a single animal model. The current study sought to take advantage of a novel aptamer-based proteomic assay which has been validated for use in many species to characterize changes to the blood proteome after total-body irradiation (TBI) across four different mammalian species including humans. Plasma was collected from C57BL6 mice, Sinclair minipigs, and Rhesus non-human primates (NHPs) receiving a single dose of TBI at a range of 3.3 Gy to 4.22 Gy at 24 h postirradiation. NHP and minipig models were irradiated using a 60Co source at a dose rate of 0.6 Gy/min, the C57BL6 mouse model using an X-ray source at a dose rate of 2.28 Gy/min and clinical samples from a photon source at 10 cGy/min. Plasma was collected from human patients receiving a single dose of 2 Gy TBI collected 6 h postirradiation. Plasma was screened using the aptamer-based SomaLogic SomaScan® proteomic assay technology to evaluate changes in the expression of 1,310 protein analytes. Confirmatory analysis of protein expression of biomarker HIST1H1C, was completed using plasma from C57BL6 mice receiving a 2, 3.5 or 8 Gy TBI collected at days 1, 3, and 7 postirradiation by singleplex ELISA. Summary of key pathways with altered expression after radiation exposure across all four mammalian species was determined using Ingenuity Pathway Analysis (IPA). Detectable values were obtained for all 1,310 proteins in all samples included in the SomaScan assay. A subset panel of protein biomarkers which demonstrated significant (p < 0.05) changes in expression of at least 1.3-fold after radiation exposure were characterized for each species. IPA of significantly altered proteins yielded a variety of top disease and biofunction pathways across species with the organismal injury and abnormalities pathway held in common for all four species. The HIST1H1C protein was shown to be radiation responsive within the human, NHP and murine species within the SomaScan dataset and was shown to demonstrate dose dependent upregulation at 2, 3.5 and 8 Gy at 24 h postirradiation in a separate murine cohort by ELISA. The SomaScan proteomics platform is a useful screening tool to evaluate changes in biomarker expression across multiple mammalian species. In our study, we were able to identify a novel biomarker of radiation exposure, HIST1H1C, and characterize panels of radiation responsive proteins and functional proteomic pathways altered by radiation exposure across murine, minipig, NHP and human species. Our study demonstrates the efficacy of using a multispecies approach for biomarker discovery.

The Significance of MET Expression and Strategies of Targeting MET Treatment in Advanced Gastric Cancer.

BackgroundAccurate assessment of predictive biomarker expression is critical in patient selection in clinical trials or clinical practice. However, changes in biomarker expression may occur after treatment. The aim of the present study was to evaluate the effects of chemotherapy on MET expression in gastric cancer (GC).MethodsMET expression was examined immunohistochemically before and after treatment in 122 patients with unresectable or recurrent GC, and was evaluated according to H-score or the scoring criteria used in the MetMAb trial. MET gene amplification was assessed by chromogenic in situ hybridization (CISH). The antitumor effect of MET targeted therapy was investigated in human gastric cancer cells in vitro and in vivo, and the underlying molecular mechanisms were analyzed by western blot.ResultsMET expression was associated with Lauren classification as well as tumor differentiation by either scoring system. MET amplification was not associated with clinical characteristics. Of the 71 patients who had paired pre- and post-treatment tumor tissues, 28 patients (39%) were initially positive for MET expression, and 43 (61%) were negative. Twenty-five patients (35%) showed significant changes in MET expression after treatment (P=0.007). Additionally, there was a concomitant overexpression of MET and HER2 in a subset of GC patients. MET inhibitor volitinib could significantly inhibit cell proliferation and xenograft growth in vitro and in vivo in MKN45 cells with MET and phosphorylated MET (pMET) high expressions via suppressing downstream PI3K/Akt and MAPK signaling pathways. Furthermore, combination therapy targeting both MET and HER2 demonstrated a synergistic antitumor activity.ConclusionsMET expression is altered post chemotherapy and MET status should be evaluated in real-time. Both MET and pMET expressions might need to be considered for patients suitable for volitinib treatment.

Comparison of hormone-induced mRNA and protein biomarker expression changes in breast cancer cells.

Protein biomarkers estrogen receptor (ER), progesterone receptor (PR), and marker of proliferation (Ki67) are routinely assessed by immunohistochemistry to guide treatment decisions for breast cancer. Now, quantification of mRNA encoding these proteins is being adopted in the clinic. However, mRNA and protein biomarkers may be differentially regulated by fluctuations in estrogen and progesterone that occur across the menstrual cycle in premenopausal breast cancer patients. This study aimed to compare how estrogen and progesterone affect mRNA and protein biomarker expression in hormone-responsive breast cancer cells. Hormone-responsive ZR-75-1 and T-47D human breast cancer cell lines were xenografted into the mammary fat pad of BALB/c nude mice supplemented with estrogen. Progesterone or vehicle was administered prior to dissection of tumors. Protein expression of ER, PR and Ki67 was quantified by immunohistochemistry, and mRNA encoding these proteins, ESR1, PGR and KI67, respectively, was quantified by real-time PCR. mRNA expression was also quantified in breast cancer cell lines treated with estrogen and progesterone in vitro. In T-47D-xenografted tumors, estrogen and progesterone treatment reduced PGR and KI67 mRNA expression, and reduced PR and Ki67 protein positivity, compared to estrogen treatment alone. In ZR-75-1 xenografted tumors, no significant differences in protein or mRNA biomarker expression were observed. In vitro, estrogen and progesterone co-treatment significantly reduced ESR1 and PGR mRNA expression in both T-47D and ZR-75-1 cell lines. Estrogen and progesterone similarly affect mRNA and protein biomarker expression in hormone-responsive breast cancer xenografts. Further research is needed to investigate concordance between protein and mRNA biomarkers in premenopausal breast cancer.

Novel Murine Biomarkers of Radiation Exposure Using An Aptamer-Based Proteomic Technology.

Purpose: There is a need to identify new biomarkers of radiation exposure both for use in the development of biodosimetry blood diagnostics for radiation exposure and for clinical use as markers of radiation injury. In the current study, a novel high-throughput proteomics screening approach was used to identify proteomic markers of radiation exposure in the plasma of total body irradiated mice. A subset panel of significantly altered proteins was selected to build predictive models of radiation exposure and received radiation dose useful for population screening in a future radiological or nuclear event. Methods: Female C57BL6 Mice of 8–14 weeks of age received a single total body irradiation (TBI) dose of 2, 3.5, 8 Gy or sham radiation and plasma was collected by cardiac puncture at days 1, 3, and 7 post-exposure. Plasma was then screened using the aptamer-based SOMAscan proteomic assay technology, for changes in expression of 1,310 protein analytes. A subset panel of protein biomarkers which demonstrated significant changes (p < 0.05) in expression following radiation exposure were used to build predictive models of radiation exposure and radiation dose. Results: Detectable values were obtained for all 1,310 proteins included in the SOMAscan assay. For the Control vs. Radiation model, the top predictive proteins were immunoglobulin heavy constant mu (IGHM), mitogen-activated protein kinase 14 (MAPK14), ectodysplasin A2 receptor (EDA2R) and solute carrier family 25 member 18 (SLC25A18). For the Control vs. Dose model, the top predictive proteins were cyclin dependent kinase 2/cyclin A2 (CDK2. CCNA2), E-selectin (SELE), BCL2 associated agonist of cell death (BAD) and SLC25A18. Following model validation with a training set of samples, both models tested with a new sample cohort had overall predictive accuracies of 85% and 73% for the Control vs. Radiation and Control vs. Dose models respectively. Conclusion: The SOMAscan proteomics platform is a useful screening tool to evaluate changes in biomarker expression. In our study we were able to identify a novel panel of radiation responsive proteins useful for predicting whether an animal had received a radiation exposure and to what dose they had received. Such diagnostic tools are needed for future medical management of radiation exposures.

Evaluating and comparing immunostaining and computational methods for spatial profiling of drug response in patient-derived explants

AbstractPatient-derived explants (PDEs) represent the direct culture of fragments of freshly-resected tumour tissue under conditions that retain the original architecture of the tumour. PDEs have advantages over other preclinical cancer models as platforms for predicting patient-relevant drug responses in that they preserve the tumour microenvironment and tumour heterogeneity. At endpoint, PDEs may either be processed for generation of histological sections or homogenised and processed for ʻomic' evaluation of biomarker expression. A significant advantage of spatial profiling is the ability to co-register drug responses with tumour pathology, tumour heterogeneity and changes in the tumour microenvironment. Spatial profiling of PDEs relies on the utilisation of robust immunostaining approaches for validated biomarkers and incorporation of appropriate image analysis methods to quantitatively and qualitatively monitor changes in biomarker expression in response to anti-cancer drugs. Automation of immunostaining and image analysis would provide a significant advantage for the drug discovery pipeline and therefore, here, we have sought to optimise digital pathology approaches. We compare three image analysis software platforms (QuPath, ImmunoRatio and VisioPharm) for evaluating Ki67 as a marker for proliferation, cleaved PARP (cPARP) as a marker for apoptosis and pan-cytokeratin (CK) as a marker for tumour areas and find that all three generate comparable data to the views of a histomorphometrist. We also show that Virtual Double Staining of sequential sections by immunohistochemistry results in imperfect section alignment such that CK-stained tumour areas are over-estimated. Finally, we demonstrate that multi-immunofluorescence combined with digital image analysis is a superior method for monitoring multiple biomarkers simultaneously in tumour and stromal areas in PDEs.This paper reports the optimisation of digital pathology approaches for evaluating drug responses in patient-derived explants at endpoint. The authors compare three image analysis software platforms and find that all three generate comparable data to that of a histomorphometrist. They also demonstrate the power of multi-immunofluorescence staining for monitoring multiple biomarkers simultaneously compared to virtual double staining of sequential sections by immunohistochemistry.

Multiplexed Nanobiosensors: Current Trends in Early Diagnostics.

The ever-growing demand for fast, cheap, and reliable diagnostic tools for personalised medicine is encouraging scientists to improve existing technology platforms and to create new methods for the detection and quantification of biomarkers of clinical significance. Simultaneous detection of multiple analytes allows more accurate assessment of changes in biomarker expression and offers the possibility of disease diagnosis at the earliest stages. The concept of multiplexing, where multiple analytes can be detected in a single sample, can be tackled using several types of nanomaterial-based biosensors. Quantum dots are widely used photoluminescent nanoparticles and represent one of the most frequent choices for different multiplex systems. However, nanoparticles that incorporate gold, silver, and rare earth metals with their unique optical properties are an emerging perspective in the multiplexing field. In this review, we summarise progress in various nanoparticle applications for multiplexed biomarkers.

Individual-specific functional connectome biomarkers predict schizophrenia positive symptoms during adolescent brain maturation.

Even with an overarching functional dysconnectivity model of adolescent‐onset schizophrenia (AOS), there have been no functional connectome (FC) biomarkers identified for predicting patients' specific symptom domains. Adolescence is a period of dramatic brain maturation, with substantial interindividual variability in brain anatomy. However, existing group‐level hypotheses of AOS lack precision in terms of neuroanatomical boundaries. This study aimed to identify individual‐specific FC biomarkers associated with schizophrenic symptom manifestation during adolescent brain maturation. We used a reliable individual‐level cortical parcellation approach to map functional brain regions in each subject, that were then used to identify FC biomarkers for predicting dimension‐specific psychotic symptoms in 30 antipsychotic‐naïve first‐episode AOS patients (recruited sample of 39). Age‐related changes in biomarker expression were compared between these patients and 31 healthy controls. Moreover, 29 antipsychotic‐naïve first‐episode AOS patients (analyzed sample of 25) were recruited from another center to test the generalizability of the prediction model. Individual‐specific FC biomarkers could significantly and better predict AOS positive‐dimension symptoms with a relatively stronger generalizability than at the group level. Specifically, positive symptom domains were estimated based on connections between the frontoparietal control network (FPN) and salience network and within FPN. Consistent with the neurodevelopmental hypothesis of schizophrenia, the FPN–SN connection exhibited aberrant age‐associated alteration in AOS. The individual‐level findings reveal reproducible FPN‐based FC biomarkers associated with AOS positive symptom domains, and highlight the importance of accounting for individual variation in the study of adolescent‐onset disorders.

Delivering Robust Candidates to the Drug Pipeline through Computational Analysis of Arrayed CRISPR Screens.

Genome-wide arrayed CRISPR screening is a powerful method for drug target identification as it enables exploration of the effect of individual gene perturbations using diverse highly multiplexed functional and phenotypic assays. Using high-content imaging, we can measure changes in biomarker expression, intracellular localization, and cell morphology. Here we present the computational pipeline we have developed to support the analysis and interpretation of arrayed CRISPR screens. This includes evaluating the quality of guide RNA libraries, performing image analysis, evaluating assay results quality, data processing, hit identification, ranking, visualization, and biological interpretation.

Polyvinyl chloride (PVC) plastic fragments release Pb additives that are bioavailable in zebrafish

Plastic polymers such as polyvinyl chloride (PVC) may contain chemical additives, such as lead (Pb), that are leachable in aqueous solution. The fragmentation into microplastics (MPs) of plastics such as PVC may facilitate desorption of chemical additives and increase exposure of aquatic animals. In this study, the role of chemical additives in the aqueous toxicity of PVC, high-density polyethylene (HDPE) and polyethylene terephthalate (PET) MPs were investigated in early-life stage zebrafish (Danio rerio) by assessment of changes in expression of biomarkers. Exposure of zebrafish larvae to PVC for 24 h increased expression of metallothionein 2 (mt2), a metal-binding protein, but no changes in expression of biomarkers of estrogenic (vtg1) or organic (cyp1a) contaminants were observed. HDPE and PET caused no changes in expression of any biomarkers. A filtered leachate of the PVC also caused a significant increase in expression of mt2 and indicated that a desorbed metal additive likely elicited the response in zebrafish. Metal release was confirmed by acid-washing the MPs which mitigated the response in mt2. Metal analysis showed Pb leached from PVC into water during exposures; at 500 mg PVC L−1 in water, 84.3 ± 8.7 μg Pb L−1 was measured after 24 h. Exposure to a Pb-salt at this concentration caused a comparable mt2 increase in zebrafish as observed in exposures to PVC. These data indicated that PVC MPs elicited a response in zebrafish but the effect was indirect and mediated through desorption of Pb from PVC into the exposure water. Data also indicated that PVC MPs may act as longer-term environmental reservoirs of Pb for exposure of aquatic animals; the Pb leached from PVC in 24 h in freshwater equated to 2.52% of total Pb in MPs leachable by the acid-wash. Studies of MPs should consider the potential role of chemical additives in toxicity observed.

DOP17 Identification of biomarkers and mechanistic insight for upadacitinib in ulcerative colitis: Analysis of serum inflammatory mediators in the phase 2b U-ACHIEVE study

Abstract Background The U-ACHIEVE trial evaluated upadacitinib (UPA), an oral JAK1 selective inhibitor, in patients with moderately to severely active ulcerative colitis (UC). Patient-reported and endoscopic outcomes improved after UPA treatment. This analysis used pharmacodynamic profiling to link changes in serum biomarkers to changes in UC disease activity, and to assess the UPA mechanism of action in UC. Methods U-ACHIEVE (NCT02819635) was a randomised, double-blind, placebo (PBO)-controlled phase 2b clinical trial. Adults with an inadequate response, loss of response, or intolerance to corticosteroids, immunosuppressants, or biologic therapies were randomised to receive 7.5, 15, 30, or 45 mg UPA once daily or PBO for 8 weeks (weeks). Serum samples (baseline [BL], weeks 2, 4, and 8) were analysed by OLINK® inflammation panel (92 proteins) and by Singulex immunoassay for interleukin-1b (IL-1b), IL-17A, IL-17F, and IL-22. Protein-level changes were analysed by a mixed-effect model; BL protein level was adjusted as a covariate; treatment group, time point, and their interaction were included as fixed effects. Spearman rank-correlation coefficients were used to determine the relationship between changes of serum biomarker levels and improvements in adapted Mayo scores and endoscopic subscores. Multiplicity adjusted P values were calculated using 1000 runs of random permutations. Results Paired BL and week 8 serum samples were available from 114 patients (PBO, n = 17; UPA 7.5 mg, n = 21; UPA 15 mg, n = 21; UPA 30 mg, n = 29; UPA 45 mg, n = 26). UPA treatment reduced expression of pro-inflammatory mediators associated with immune cell migration, type I/II IFN responses, T-cell responses, macrophage and dendritic cell activity and increased expression of biomarkers associated with haematopoiesis, neuroprotection and mucosal repair in a dose-dependent manner. Improvements in adapted Mayo score, endoscopic subscore, and stool frequency correlated with increases in CX3CL1, DNER and FLt3L (p &amp;lt; 0.05 for all). Endoscopic improvements correlated with reductions in OSM, and improvements in fatigue correlated with increases in CCL25 and NT-3. There was a substantial overlap in biomarkers modulated by UPA in patients with UC and Crohn’s disease (Figure). Conclusion UPA modulated expression of serum pro-inflammatory mediators found in pathways associated with the pathogenesis of UC, including immune cell migration, type I/II IFN responses, T-cell responses, macrophage and dendritic cell activity, haematopoiesis, neuroprotection, and mucosal repair. Consistent correlations were observed between changes in biomarker expression and improvements in disease activity and symptoms of UC.

Abstract LB-003: Intracellular delivery of SOCS3 suppresses solid tumor progression

Abstract Introduction Suppressor of cytokine signaling 3 (SOCS3) functions as a negative-feedback regulator of JAK/STAT signaling that suppresses JAK kinase activity and promotes degradation of the activated cytokine receptor complex. Loss of SOCS3 enhances the growth and survival of some solid tumors; therefore, methods to replenish intracellular levels of the protein may provide an effective therapy against solid tumors dependent on JAK/STAT signaling for growth or survival. Objective The present study utilized advanced macromolecule transduction domain (aMTD) sequences to develop improved cell-permeable (iCP-) SOCS3 as a protein-based biotherapeutic against solid tumors including gastric-, colorectal, lung-, breast-cancer and glioblastoma. Methods Cell-permeable SOCS3 proteins, with (iCP-SOCS3) and without (Non-CP-SOCS3) a 12-amino acid aMTD sequence or containing an earlier generation of MTD based on the signal peptide of FGF4, termed membrane-translocating motif (MTM) were expressed and purified from E. coli. The proteins also contained a solubilization domain (SD) to enhance the solubility of the recombinant proteins in physiological buffers. The therapeutic potential of iCP-SOCS3 as anti-cancer agent for solid tumors was examined both in cultured cells and in human-mouse xenograft models. Results Compared to CP-SOCS3, iCP-SOCS3 fused to aMTD exhibited higher solubility and yield (25- and 300-folds higher, respectively) that are qualified to be developed for clinical use. Moreover, it exhibited cell-/tissue-permeability along with strong biological activity that was shown by significantly suppressed IFN-γ-induced phosphorylation of STAT1 and STAT3. In vitro study conducted with various cancer cell lines demonstrated that iCP-SOCS3 significantly suppresses cancer-associated phenotypes (e.g., &amp;gt;80% inhibition of proliferation in lung cancer and glioblastoma, &amp;gt;70% induction of apoptosis in gastric cancer, &amp;gt;65% inhibition of migration/invasion in gastric cancer) and induces changes in biomarker expression (e.g., p21, cleavage caspase-3 in breast cancer). In addition, iCP-SOCS3 significantly suppressed the tumor growth of human solid tumor xenografts (65% inhibition in gastric-, 79% in colorectal-, 69% in lung-, 63% in breast-cancer and 78% in glioblastoma CDXs, respectively). Conclusion In conclusion, these results provide further evidence that SOCS3 can function as a tumor suppressor and intracellular delivery of SOCS3 with iCP-SOCS3 may provide novel protein therapy against solid tumors. Citation Format: Seulmee Shin. Intracellular delivery of SOCS3 suppresses solid tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-003.

Effects of supplemental calcium and vitamin D on tight-junction proteins and mucin-12 expression in the normal rectal mucosa of colorectal adenoma patients.

The physical gut barrier, comprised of a thick mucus layer and the epithelium, plays an important role in defense against microbes and foreign antigens. Calcium and vitamin D may be involved in maintaining the integrity of the intestinal mucosal barrier, the dysfunction of which may lead to endotoxemia and inflammation, and contribute to colorectal carcinogenesis. We investigated supplemental calcium (1200 mg, daily) and/or vitamin D3 (1000 IU daily) effects on intestinal barrier function-related biomarkers in a subset of 105 participants from a large colorectal adenoma recurrence chemoprevention clinical trial. We assessed expression of the tight junction proteins claudin-1 (CLDN1), occludin (OCLD), and mucin-12 (MUC12) in the normal-appearing colorectal mucosa using standardized, automated immunohistochemistry and quantitative image analysis. Following 1 year of treatment, in the calcium relative to the no calcium group, the CLDN1, OCLD, and MUC12 expression increased by 14% (P = 0.17), 23% (P = 0.11), and 22% (P = 0.07), respectively. In secondary analyses, the estimated calcium treatment effects were greater among participants with baseline serum 25-OH-vitamin D concentrations below the median value of 22.69 ng/mL (CLDN1: 29%, P = 0.04; OCLD: 36%, P = 0.06; MUC12: 35%, P = 0.05). There were no biomarker expression changes in the vitamin D3 alone group; however, modest increases were found in the combined calcium/vitamin D3 group. At baseline, obesity, history of a sessile-serrated adenoma, colorectal MIB-1/Ki-67 expression, and a family history of colorectal cancer were associated with CLDN1, OCLD, and MUC12 expression. Our study supports continued investigation of factors that could affect intestinal mucosal barrier integrity relevant to colorectal carcinogenesis.

Abstract LB-010: Intracellular delivery of SOCS3 suppresses solid tumor progression

Abstract Introduction Suppressor of cytokine signaling 3 (SOCS3) functions as a negative-feedback regulator of JAK/STAT signaling that suppresses JAK kinase activity and promotes degradation of the activated cytokine receptor complex. Loss of SOCS3 enhances the growth and survival of some solid tumors; therefore, methods to replenish intracellular levels of the protein may provide an effective therapy against solid tumors dependent on JAK/STAT signaling for growth or survival. Objective The present study utilized advanced macromolecule transduction domain (aMTD) sequences to develop improved cell-permeable (iCP-) SOCS3 as a protein-based biotherapeutic against solid tumors including gastric-, colorectal, lung-, breast-cancer and glioblastoma. Methods Cell-permeable SOCS3 proteins, with (iCP-SOCS3) and without (Non-CP-SOCS3) a 12-amino acid aMTD sequence or containing an earlier generation of MTD based on the signal peptide of FGF4, termed membrane-translocating motif (MTM) were expressed and purified from E. coli. The proteins also contained a solubilization domain (SD) to enhance the solubility of the recombinant proteins in physiological buffers. The therapeutic potential of iCP-SOCS3 as anti-cancer agent for solid tumors was examined both in cultured cells and in human-mouse xenograft models. Results Compared to CP-SOCS3, iCP-SOCS3 fused to aMTD exhibited higher solubility and yield (25- and 300-folds higher, respectively) that are sufficient to be developed for clinical use. Moreover, it exhibited cell-/tissue-permeability along with strong biological activity that was shown by significantly suppressed IFN-γ-induced phosphorylation of STAT1 and STAT3. In vitro study conducted with various cancer cell lines demonstrated that iCP-SOCS3 significantly suppresses cancer-associated phenotypes (e.g., &amp;gt;80% inhibition of proliferation in lung cancer and glioblastoma, &amp;gt;70% induction of apoptosis in gastric cancer, &amp;gt;65% inhibition of migration/invasion in gastric cancer) and induces changes in biomarker expression (e.g., p21, cleavage caspase-3 in breast cancer). In addition, iCP-SOCS3 also significantly suppressed the tumor growth of human solid tumor xenografts (65% inhibition in gastric-, 79% in colorectal-, 69% in lung-, 63% in breast-cancer and 78% in glioblastoma CDXs, respectively). Conclusion Taken together, these results provide further evidence that SOCS3 can function as a tumor suppressor and intracellular delivery of SOCS3 with iCP-SOCS3 may provide novel protein therapy against solid tumors. Citation Format: Youngsil Choi, Minyong Jung, Seulmee Shin, Kuysook Lee, Daewoong Jo. Intracellular delivery of SOCS3 suppresses solid tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-010.

Abstract LB-009: Intracellular delivery with SOCS3 suppresses angiogenesis in tumors

Abstract Introduction: Suppressor of cytokine signaling 3 (SOCS3) is a negative feedback regulator in the Janus kinase/signal transducer and activator of transduction (JAK/STAT) signaling pathway, in which it suppresses inflammation and cancer. The JAK/STAT pathway is activated in some tumors thus enhancing the expression of genes involved in proliferation and angiogenesis, notably, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietin-1 and phospoinositide 3-kinase (PI3K)-protein kinase B (AKT). Objective: The aim of this study is to develop an improved cell-permeable (iCP)-SOCS3 and determine its applicability as an anti-angiogenic agent to treat cancer. Methods: The iCP-SOCS3 was expressed in E. coli as a 37 kDa recombinant protein fused to an advanced macromolecule transduction domain (aMTD) and humanized solubilization domain (SD). Uptake of a fluorescently tagged iCP-SOCS3 by cells and tissues was monitored by flow cytometry and confocal microscopy. Biological and biochemical activities of iCP-SOCS3 were determined by in vitro angiogenesis assays and changes in biomarker expression and anti-tumor activities were analyzed in mice bearing human tumor xenografts. Results: The iCP-SOCS3 improved on an earlier generation of CP-SOCS3 fused to FGF4-derived membrane translocating motif (MTM) with regard to solubility, yield, delivery into cells and tissues and biological activity. The iCP-SOCS3 suppressed proliferation of human umbilical vein endothelial cells (HUVECs) by 60% and migration by 54% via inhibition of AKT/mTOR signaling pathway. Moreover, iCP-SOCS3 inhibited invasion of EC cells in PANC-1 and U87MG conditioned medium by 38% and 95%, respectively. In tube formation assay, iCP-SOCS3 reduced the number of branch points by 79% and tube length by 60% compared to the vehicle. In chick chorioallantoic membrane (CAM) assay, iCP-SOCS3 significantly inhibited angiogenesis by 83% in dose dependent manner compared to the reference drug, retinoic acid. The anti-angiogenic activity of iCP-SOCS3 was also shown in matrigel plug assay. The new vessel formation induced by U87-MG glioblastoma cells was significantly reduced by the treatment of iCP-SOCS3. In PANC-1, U87-MG and HepG2 cell-derived xenograft models, iCP-SOCS3 suppressed expression of pro-angiogenic factors including VEGF, ERK and angiopoietin 1. Conclusion: These results validate iCP-SOCS3 targets in promoting angiogenesis and suggest ways to reduce angiogenesis in various types of tumors. Citation Format: Yukdong Jung, Kuysook Lee, Youngsil Choi, Daewoong Jo. Intracellular delivery with SOCS3 suppresses angiogenesis in tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-009.

Subtype and Functional Biomarker Changes of NK Cells in Peripheral Blood of Patients with Myelodysplastic Syndrome

To analyze the subtype and functional biomarker expression changes of natural kill cells(NK) in peripheral blood of patients with myelodysplastic syndrome(MDS) and normal people, so as to evaluate the relationships between these changes and hematopoietic functions and to explore the role of NK cells in the pathogenesis of MDS. The quantity of NK cells and the expression of biomarkers(NKp30,NKp46,NKG2A) on NK cells were detected by flow cytometry in 35 MDS patients from 2015 to 2016 in our hospital and 34 normal controls. The correlation between these changes and hematopoietic functions, including the percentages of neutrophil(ANC), hemoglobin in peripheral blood and the hematopoietic function in bone marrow(CD34+%) were evaluated. The percentage and quantity of NK cells and CD56dim NK cells in MDS patients were significantly lower than those in normal controls(P<0.05); the percentage of CD56bright NK cells was higher than that in controls. The percentage of CD56dim NK cells in NK cells of MDS patients was significantly lower than that of controls; the percentage of CD56bright NK cells in NK cells of MDS patients was significantly higher than that of controls. The expression of NKp30 and NKp46 of MDS patients was significantly lower than that of controls. In MDS group, the percentage of NK cells and CD56dim NK cells of peripheral blood lymphocytes in high risk MDS group was significantly lower than that in low risk MDS group. The percentage of NK and CD56dim NK cells negatively correlated with that of CD34+% in bone marrow, but positively correlated with ANC and Hb. The CD34+% in bone marrow negatively correlated with expression of NKp46, but positively correlated with expression of NKG2A. The decrease of NK number and function may cause the immune surveillance and lead to disease progression.

A Two-Stage Dissociation System for Multilayer Imaging of Cancer Biomarker-Synergic Networks in Single Cells.

The monitoring of cancer biomarkers is crucial to the early detection of cancer. However, a limiting factor in biomarker analysis is the ability to obtain the multilayered information of various biomarker molecules located at different parts of cells from the plasma membrane to the cytoplasm. A two-stage dissociation nanoparticle system based on multifunctionalized polydopamine-coated gold nanoparticles (Au@PDA NPs) is reported, which allows for the two-stage imaging of cancer biomarkers in single cells. We demonstrate the feasibility of this strategy on sialic acids (SAs), p53 protein, and microRNA-21 (miRNA-21) in MCF-7 breast cancer cells by two custom-built probes. Furthermore, the multicolor fluorescence information extracted is used for the monitoring of biomarker expression changes under different drug combinations, which allows us to investigate the complex interactions between various cancer biomarkers and to describe the cancer biomarker-synergic networks in single cells.

A Two‐Stage Dissociation System for Multilayer Imaging of Cancer Biomarker‐Synergic Networks in Single Cells

AbstractThe monitoring of cancer biomarkers is crucial to the early detection of cancer. However, a limiting factor in biomarker analysis is the ability to obtain the multilayered information of various biomarker molecules located at different parts of cells from the plasma membrane to the cytoplasm. A two‐stage dissociation nanoparticle system based on multifunctionalized polydopamine‐coated gold nanoparticles (Au@PDA NPs) is reported, which allows for the two‐stage imaging of cancer biomarkers in single cells. We demonstrate the feasibility of this strategy on sialic acids (SAs), p53 protein, and microRNA‐21 (miRNA‐21) in MCF‐7 breast cancer cells by two custom‐built probes. Furthermore, the multicolor fluorescence information extracted is used for the monitoring of biomarker expression changes under different drug combinations, which allows us to investigate the complex interactions between various cancer biomarkers and to describe the cancer biomarker‐synergic networks in single cells.