Development of a cGMP-compliant process to manufacture donor-derived, CD45RA-depleted memory CD19-CAR T cells.

Autologous chimeric antigen receptor (CAR) T cells targeting the CD19 antigen have demonstrated a high complete response rate in relapsed/refractory B-cell malignancies. However, autologous CAR T cell therapy is not an option for all patients. Here we optimized conditions for clinical-grade manufacturing of allogeneic CD19-CAR T cells using CD45RA-depleted donor memory T cells (Tm) for a planned clinical trial. Tm were activated using the MACS GMP T Cell TransAct reagent and transduced in the presence of LentiBOOST with a clinical-grade lentiviral vector that encodes a 2nd generation CD19-CAR with a 41BB.zeta endodomain. Transduced T cells were transferred to a G-Rex cell culture device for expansion and harvested on day 7 or 8 for cryopreservation. The resulting CD19-CAR(Mem) T cells expanded on average 34.2-fold, and mean CAR expression was 45.5%. The majority of T cells were CD4+ and had a central memory or effector memory phenotype, and retained viral specificity. CD19-CAR(Mem) T cells recognized and killed CD19-positive target cells in vitro and had potent antitumor activity in an ALL xenograft model. Thus we have successfully developed a current good manufacturing practice-compliant process to manufacture donor-derived CD19-CAR(Mem) T cells. Our manufacturing process could be readily adapted for CAR(Mem) T cells targeting other antigens.

Preparation of cryopreserved chimeric antigen receptor T cells for the locoreogional delivery to the neural axis.

BACKGROUND AIMS: Intracranial (IC) locoregional delivery of chimeric antigen receptor (CAR) T cells presents an attractive delivery method to central nervous system tumors. Although IC delivery is actively being employed in early-phase clinical studies, no thaw/wash methods have been published to remove the neurotoxic cryoprotectant dimethyl sulfoxide (DMSO) from CAR T-cell products before IC administration. Thus, the aim of this study was to develop and validate a simple thaw/wash procedure. METHODS: We developed a thaw/wash procedure that consist of product thaw at 37°C, equilibration for 5 min in 1 volume of preservative-free normal saline (PFNS), dilution with an additional 8 volumes of PFNS, removal of DMSO through a washing step, resuspension in 2.0 mL of PFNS and storage in a syringe at 20-25°C. Final formulated products (FPs) were assessed for quality and safety attributes and stability over 3 h from the completion of the thaw. Stability parameters included CAR T-cell viability, transgene surface expression and cytolytic activity. RESULTS: The developed procedure reduced the calculated % of DMSO to less than 0.025%. FP cell viability and recovery (versus pre-cryopreservation) were within acceptable specifications (mean viability: 85.3%, range: 83%-88%; total nucleated cell recovery mean: 76.5%, range: 65.4%-82.5%). Other prespecified quality assurance/quality control parameters including appearance/ integrity, sterility and endotoxin level (≤1.0 EU/mL), were also met by all FPs (n = 3). Three hours' post thaw/wash stability was confirmed. All products maintained cell viability greater than 70% (mean, 80.0%; range, 79%-81%), with no significant change in transgene expression or cytolytic activity of B7-H3-CAR T cells compared with thawed not diluted/washed control CAR T cells. CONCLUSIONS: We have developed a simple thaw/wash procedure to prepare B7-H3-CAR T cells for their locoregional delivery to the neural axis. While we focus here on CAR T cells, the methods could be readily adapted to other cryopreserved immune effector cell products.

Identifying the cognitive predictors of early counting and calculation skills: Evidence from a longitudinal study.

The extent to which phonological, visual-spatial short-term memory (STM), and nonsymbolic quantitative skills support the development of counting and calculation skills was examined in this 14-month longitudinal study of 125 children. Initial assessments were made when the children were 4 years 8 months old. Phonological awareness, visual-spatial STM, and nonsymbolic approximate discrimination predicted growth in early calculation skills.These results suggest that both the approximate number system and domain-general phonological and visual-spatial skills support early calculation. In contrast, only performance on a small nonsymbolic quantity discrimination task (where the presented quantities were always within the subitizing range) predicted growth in cardinal counting skills. These results suggest that the development of counting and the development of calculation are supported by different cognitive abilities.

Inhibition by chondroitin sulfate E can specify functional Wnt/ß-catenin signaling thresholds in NIH3T3 fibroblasts.

Aberrant activation of the Wnt/ß-catenin signaling pathway is frequently associated with human disease, including cancer, and thus represents a key therapeutic target. However, Wnt/ß-catenin signaling also plays critical roles in many aspects of normal adult tissue homeostasis. The identification of mechanisms and strategies to selectively inhibit the disease-related functions of Wnt signaling, while preserving normal physiological functions, is in its infancy. Here, we report the identification of exogenous chondroitin sulfate-E (CS-E) as an inhibitor of specific molecular and biological outcomes of Wnt3a signaling in NIH3T3 fibroblasts. We demonstrate that CS-E can decrease Wnt3a signaling through the negative regulation of LRP6 receptor activation. However, this inhibitory effect of CS-E only affected Wnt3a-mediated induction, but not repression, of target gene expression. We went on to identify a critical Wnt3a signaling threshold that differentially affects target gene induction versus repression. This signaling threshold also controlled the effects of Wnt3a on proliferation and serum starvation-induced apoptosis. Limiting Wnt3a signaling to this critical threshold, either by CS-E treatment or by ligand dilution, interfered with Wnt3a-mediated stimulation of proliferation but did not impair Wnt3a-mediated reduction of serum starvation-induced apoptosis. Treatment with pharmacological inhibitors demonstrated that both induction and repression of Wnt3a target genes in NIH3T3 cells require the canonical Wnt/ß-catenin signaling cascade. Our data establish the feasibility of selective inhibition of Wnt/ß-catenin transcriptional programs and biological outcomes through the exploitation of intrinsic signaling thresholds.

The impact of the development of verbal recoding on children's early writing skills.

BACKGROUND: The spontaneous recoding of visual stimuli into a phonological code to aid short-term retention has been associated with progress in learning to read (Palmer, 2000b). AIM: This study examined whether there was a comparable association with the development of writing skills. SAMPLE: One hundred eight children (64 males) in the second year of the UK educational system (mean age 5:8 years, SD = 4 months) were recruited to the study. METHODS: The children participated in tasks to assess their general cognitive abilities, reading skills, and their predominant short-term memory (STM) strategy for retaining visually presented stimuli. On the basis of their memory profile, children were classified as either engaging in verbal recoding of the stimuli (N = 31) or not (N = 77). Writing performance was indexed as alphabet transcription, spelling, and early text production skills. RESULTS: Children classified as verbal recoders demonstrated better spelling performance and produced more individual letters, words, and T-units in their texts than did children who persisted with a visual memory strategy. In contrast, the alphabet transcription abilities of the groups did not differ. Hierarchical regression analyses revealed that variance in text production skills was associated with STM capacity and that moreover, significant independent variance in the number of words and T-units in the children's texts was predicted by individual differences in verbal recoding abilities. CONCLUSION: The results suggest that the development of verbal recoding skills in STM may play a role in children's early progress in writing, particularly their text generation skills.

Different components of working memory have different relationships with different mathematical skills.

A comprehensive working memory battery and tests of mathematical skills were administered to 90 children-41 in Year 1 (5-6 years of age) and 49 in Year 3 (7-8 years of age). Working memory could explain statistically significant variance in number writing, magnitude judgment, and single-digit arithmetic, but the different components of working memory had different relationships with the different skills. Visual-spatial sketchpad (VSSP) functioning predicted unique variance in magnitude judgments and number writing. Central executive functioning explained unique variance in the addition accuracy of Year 1 children. The unique variance explained in Year 3 multiplication explained by phonological loop functioning just missed conventional levels of significance (p=.06). The results are consistent with the VSSP having a role in the development of number writing and magnitude judgments but a lesser role in early arithmetic.

Common Trajectories of Highly Effective CD19-Specific CAR T Cells Identified by Endogenous T-cell Receptor Lineages.

Current chimeric antigen receptor-modified (CAR) T-cell products are evaluated in bulk, without assessing functional heterogeneity. We therefore generated a comprehensive single-cell gene expression and T-cell receptor (TCR) sequencing data set using pre- and postinfusion CD19-CAR T cells from blood and bone marrow samples of pediatric patients with B-cell acute lymphoblastic leukemia. We identified cytotoxic postinfusion cells with identical TCRs to a subset of preinfusion CAR T cells. These effector precursor cells exhibited a unique transcriptional profile compared with other preinfusion cells, corresponding to an unexpected surface phenotype (TIGIT+, CD62Llo, CD27-). Upon stimulation, these cells showed functional superiority and decreased expression of the exhaustion-associated transcription factor TOX. Collectively, these results demonstrate diverse effector potentials within preinfusion CAR T-cell products, which can be exploited for therapeutic applications. Furthermore, we provide an integrative experimental and analytic framework for elucidating the mechanisms underlying effector development in CAR T-cell products. SIGNIFICANCE: Utilizing clonal trajectories to define transcriptional potential, we find a unique signature of CAR T-cell effector precursors present in preinfusion cell products. Functional assessment of cells with this signature indicated early effector potential and resistance to exhaustion, consistent with postinfusion cellular patterns observed in patients. This article is highlighted in the In This Issue feature, p. 2007.

Preferential expansion of CD8+ CD19-CAR T cells postinfusion and the role of disease burden on outcome in pediatric B-ALL.

T cells expressing CD19-specific chimeric antigen receptors (CD19-CARs) have potent antileukemia activity in pediatric and adult patients with relapsed and/or refractory B-cell acute lymphoblastic leukemia (B-ALL). However, not all patients achieve a complete response (CR), and a significant percentage relapse after CD19-CAR T-cell therapy due to T-cell intrinsic and/or extrinsic mechanisms. Thus, there is a need to evaluate new CD19-CAR T-cell products in patients to improve efficacy. We developed a phase 1/2 clinical study to evaluate an institutional autologous CD19-CAR T-cell product in pediatric patients with relapsed/refractory B-ALL. Here we report the outcome of the phase 1 study participants (n = 12). Treatment was well tolerated, with a low incidence of both cytokine release syndrome (any grade, n = 6) and neurotoxicity (any grade, n = 3). Nine out of 12 patients (75%) achieved a minimal residual disease-negative CR in the bone marrow (BM). High disease burden (≥40% morphologic blasts) before CAR T-cell infusion correlated with increased side effects and lower response rate, but not with CD19-CAR T-cell expansion. After infusion, CD8+ CAR T cells had a proliferative advantage over CD4+ CAR T cells and at peak expansion, had an effector memory phenotype with evidence of antigen-driven differentiation. Patients that proceeded to allogeneic hematopoietic cell transplantation (AlloHCT) had sustained, durable responses. In summary, the initial evaluation of our institutional CD19-CAR T-cell product demonstrates safety and efficacy while highlighting the impact of pre-infusion disease burden on outcomes. This trial was registered at www.clinicaltrials.gov as #NCT03573700.

Identification-Based Multiple-Choice Assessments in Anatomy can be as Reliable and Challenging as Their Free-Response Equivalents.

Multiple-choice (MC) anatomy "spot-tests" (identification-based assessments on tagged cadaveric specimens) offer a practical alternative to traditional free-response (FR) spot-tests. Conversion of the two spot-tests in an upper limb musculoskeletal anatomy unit of study from FR to a novel MC format, where one of five tagged structures on a specimen was the answer to each question, provided a unique opportunity to assess the comparative validity and reliability of FR- and MC-formatted spot-tests and the impact on student performance following the change of test format to MC. Three successive year cohorts of health science students (n = 1,442) were each assessed by spot-tests formatted as FR (first cohort) or MC (following two cohorts). Comparative question difficulty was assessed independently by three examiners. There were more higher-order cognitive skill questions and more of the course objectives tested in the MC-formatted tests. Spot-test reliability was maintained with Cronbach's alpha reliability coefficients ≥ 0.80 and 80% of the MC items of high quality (having point-biserial correlation coefficients > 0.25). These results also demonstrated guessing was not an issue. The mean final score for the MC-formatted cohorts increased by 4.9%, but did not change for the final theory examination that was common to all three cohorts. Subgroup analysis revealed that the greatest change in spot-test marks was for the lower-performing students. In conclusion, our results indicate spot-tests formatted as MC are suitable alternatives to FR tests. The increase in mean scores for the MC-formatted spot-tests was attributed to the lower demand of the MC format.

Ybx1 fine-tunes PRC2 activities to control embryonic brain development.

Chromatin modifiers affect spatiotemporal gene expression programs that underlie organismal development. The Polycomb repressive complex 2 (PRC2) is a crucial chromatin modifier in executing neurodevelopmental programs. Here, we find that PRC2 interacts with the nucleic acid-binding protein Ybx1. In the mouse embryo in vivo, Ybx1 is required for forebrain specification and restricting mid-hindbrain growth. In neural progenitor cells (NPCs), Ybx1 controls self-renewal and neuronal differentiation. Mechanistically, Ybx1 highly overlaps PRC2 binding genome-wide, controls PRC2 distribution, and inhibits H3K27me3 levels. These functions are consistent with Ybx1-mediated promotion of genes involved in forebrain specification, cell proliferation, or neuronal differentiation. In Ybx1-knockout NPCs, H3K27me3 reduction by PRC2 enzymatic inhibitor or genetic depletion partially rescues gene expression and NPC functions. Our findings suggest that Ybx1 fine-tunes PRC2 activities to regulate spatiotemporal gene expression in embryonic neural development and uncover a crucial epigenetic mechanism balancing forebrain-hindbrain lineages and self-renewal-differentiation choices in NPCs.

Development and Optimization of a Hydrophobic Interaction Chromatography-Based Method of AAV Harvest, Capture, and Recovery.

With many ongoing clinical trials utilizing adeno-associated virus (AAV) gene therapy, it is necessary to find scalable and serotype-independent primary capture and recovery methods to allow for efficient and robust manufacturing processes. Here, we demonstrate the ability of a hydrophobic interaction chromatography membrane to capture and recover AAV1, AAV5, AAV8, and AAV "Mutant C" (a novel serotype incorporating elements of AAV3B and AAV8) particles from cell culture media and cell lysate with recoveries of 76%-100% of loaded material, depending on serotype. A simple, novel technique that integrates release and recovery of cell-associated AAV capsids is demonstrated. We show that by the addition of lyotropic salts to AAV-containing cell suspensions, AAV is released at an equivalent efficiency to mechanical lysis. The addition of the lyotropic salt also promotes a phase separation, which allows physical removal of large amounts of DNA and insoluble cellular debris from the AAV-containing aqueous fraction. The AAV is then captured and eluted from a hydrophobic interaction chromatography membrane. This integrated lysis and primary capture and recovery technique facilitates substantial removal of host-cell DNA and host-cell protein impurities.

Mitochondrial DNA sequence variation in the Boyko, Hutsul, and Lemko populations of the Carpathian highlands.

Genetic studies of the distribution of mitochondrial DNA (mtDNA) haplogroups in human populations residing within the Carpathian Mountain range have been scarce. We present an analysis of mtDNA haplogroup composition of the Boykos, Hutsuls, and Lemkos, three population groups of the Carpathian highlands. In our study Hutsuls had the highest frequency of subhaplogroup H1 in central and eastern Europe. Lemkos shared the highest frequency of haplogroup I ever reported and the highest frequency of haplogroup M(*) in the region. MtDNA haplogroup frequencies in Boykos were different from most modern European populations. We interpreted these unique mtDNA frequencies to be evidence of diverse and dynamic population histories in the Carpathian highland region.

A prospective analysis of plasma 25-hydroxyvitamin D concentrations in white and black prepubertal females in the southeastern United States.

BACKGROUND: Little is known regarding changes in vitamin D status among children living in the southern United States and whether these changes are race-dependent. OBJECTIVES: The aims were to prospectively assess plasma 25-hydroxyvitamin D [25(OH)D] concentrations in prepubertal black and white girls (n = 83) living in northeast Georgia and to determine whether 25(OH)D concentrations change with increasing age. DESIGN: Plasma samples were obtained annually over a time frame of 1-7 y, and 25(OH)D concentrations were assessed by using radioimmunoassay. Percentage body fat (%BF) and fat-free soft tissue (FFST) mass were measured by using dual-energy X-ray absorptiometry. Linear mixed-effects models were used with height, weight, body mass index percentile, %BF, FFST, pubertal stage, dietary intake, physical activity, and socioeconomic status as covariates. RESULTS: Plasma 25(OH)D values < 80 nmol/L were observed in 75% of the participants. Plasma 25(OH)D values (analyzed on the natural logarithm scale) decreased with increasing age (P = 0.02), independent of race. Plasma 25(OH)D values were higher in whites than in blacks (P < 0.0001), and the amount of this difference depended on season (P < 0.001 for all seasons). A significant negative association between FFST and 25(OH)D, beyond the effects of age, race, and season (P = 0.007), was observed. The effects of age, race, and season on 25(OH)D remained significant when dietary calcium, vitamin D, and physical activity were used as covariates; however, after adjustment for FFST, only the effects of race and season remained. CONCLUSIONS: White girls living in the southeastern United States have higher 25(OH)D concentrations than do black girls, and the magnitude of this difference depends on the season. Decreases in 25(OH)D with age are associated with increases in FFST. Whether FFST requires additional vitamin D during growth remains to be determined.

High-glucose-induced structural changes in the heparan sulfate proteoglycan, perlecan, of cultured human aortic endothelial cells.

Hyperglycemia is an independent risk factor for diabetes-associated cardiovascular disease. One potential mechanism involves hyperglycemia-induced changes in arterial wall extracellular matrix components leading to increased atherosclerosis susceptibility. A decrease in heparan sulfate (HS) glycosaminoglycans (GAG) has been reported in diabetic arteries. The present studies examined the effects of high glucose on in vitro production of proteoglycans (PG) by aortic endothelial cells. Exposure of cells to high glucose (30 vs. 5 mM glucose) resulted in decreased [(35)S] sodium sulfate incorporation specifically into secreted HSPG. Differences were not due to hyperosmolar effects and no changes were observed in CS/DSPG. Enzymatic procedures, immunoprecipitation and Western analyses demonstrated that high glucose induced changes specifically in the HSPG, perlecan. In double-label experiments, lower sulfate incorporation in high-glucose-treated cells was accompanied by lower [(3)H] glucosamine incorporation into GAG but not lower [(3)H] serine incorporation into PG core proteins. Size exclusion chromatography demonstrated that GAG size was unchanged and GAG sulfation was not reduced. These results indicate that the level of regulation of perlecan by high glucose is posttranslational, involving a modification in molecular structure, possibly a decrease in the number of HS GAG chains on the core protein.

High glucose-induced alterations in subendothelial matrix perlecan leads to increased monocyte binding.

OBJECTIVE: Hyperglycemia is an independent risk factor for cardiovascular disease in diabetic patients, although the link between the two is unknown. These studies were designed to model effects of high glucose on an early event in atherogenesis: the binding of monocytes to subendothelial matrix (SEM). METHODS AND RESULTS: SEM was prepared from human aortic endothelial cells (HAECs) and bovine aortic endothelial cells (BAECs) cultured in the presence of low (5 mmol/L) or high (30 mmol/L) glucose for 3 to 5 days. Monocyte binding was significantly higher (P<0.05) to the SEM of both HAEC and BAEC exposed to high glucose. This increase was a result of changes in SEM heparan sulfate proteoglycans (HSPGs). Metabolic radiolabeling of BAEC demonstrated a 24% decrease in [35S]sulfate incorporation into SEM HSPG produced by cells incubated in 30 mmol/L versus 5 mmol/L glucose, whereas no glucose-associated differences were measured in [35S]methionine incorporation into proteoglycans (PGs) or non-PG proteins. Autoradiography revealed 2 high-molecular weight SEM HSPGs. One was a hybrid PG that contained both heparan sulfate and chondroitin sulfate/dermatan sulfate chains. Both PGs were identified by Western blotting as perlecan. CONCLUSIONS: These results illustrate that hyperglycemia-induced structural changes in perlecan may result in a SEM that is more favorable to retention of monocytes.

Isolating pure populations of monocytes from the blood of pregnant women: comparison of flotation in iodixanol with elutriation.

Observations that the innate arm of the immune system is upregulated in pregnancy have highlighted the need for methods of isolating pure populations of monocytes for studies into pregnancy and pre-eclampsia without activating them during the isolation process. Density gradient centrifugation using iodixanol is a useful method for isolating relatively pure populations of unactivated monocytes from human blood but has not been validated in pregnant subjects. We compared the ability of monocytes isolated from pregnant women by density gradient centrifugation using iodixanol (n=6) with monocytes isolated by countercurrent centrifugal elutriation (n=6) in terms of their ability to produce interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) under basal conditions and after stimulation with bacterial lipopolysaccharide (LPS). Under basal conditions, monocytes isolated by density gradient centrifugation produced low amounts of IL-6 and MCP-1. Production of IL-6 and MCP-1 after stimulation of the monocytes with LPS was much greater (p<0.01). There was no statistically significant difference between the two methods in terms of stimulated levels of either cytokine.

Arterial heparan sulfate is negatively associated with hyperglycemia and atherosclerosis in diabetic monkeys.

BACKGROUND: Arterial proteoglycans are implicated in the pathogenesis of atherosclerosis by their ability to trap plasma lipoproteins in the arterial wall and by their influence on cellular migration, adhesion and proliferation. In addition, data have suggested an anti-atherogenic role for heparan sulfate proteoglycans and a pro-atherogenic role for dermatan sulfate proteoglycans. Using a non-human primate model for human diabetes, studies examined diabetes-induced changes in arterial proteoglycans that may increase susceptibility to atherosclerosis. METHODS: Control (n = 7) and streptozotocin-induced diabetic (n = 8) cynomolgous monkeys were assessed for hyperglycemia by measurement of plasma glycated hemoglobin (GHb). Thoracic aortas obtained at necropsy, were extracted with 4 M guanidine HCL and proteoglycans were measured as hexuronic acid. Atherosclerosis was measured by enzymatic analysis of extracted tissue cholesterol. Glycosaminoglycan chains of arterial proteoglycans were released with papain, separated by agarose electrophoresis and analysed by scanning densitometry. RESULTS: Tissue cholesterol was positively associated with hexuronic acid content in diabetic arteries (r = .82, p < .025) but not in control arteries. Glycosaminoglycan chain analysis demonstrated that dermatan sulfate was associated with increased tissue cholesterol in both control (r = .8, p < 0.05) and diabetic (r = .8, p < .025) arteries, whereas a negative relationship was observed between heparan sulfate and tissue cholesterol in diabetic arteries only (r = -.7, p < .05). GHb, which was significantly higher in diabetic animals (8.2 +/- 0.9 vs 3.8 +/- 0.2%, p < .0005) was negatively associated with heparan sulfate in diabetic arteries (r = -.7, p < .05). CONCLUSIONS: These data implicate hyperglycemia induced modifications in arterial proteoglycans that may promote atherosclerosis.

Phonological short-term memory contributions to sentence processing in young children.

Two experiments investigated the contribution of phonological short-term memory to the processing of spoken sentences by 4- and 5-year-old children. In Experiment 1, sentences contained either short or longer words, and varied in syntactic structure. Overall, repetition but not comprehension of the sentences was significantly influenced by word length. In Experiment 2, children selected on the basis of their high phonological short-term memory ability were founded to be superior at repeating sentences to children of lower phonological short-term memory ability, although the two groups did not differ in their comprehension accuracy for the same sentences. In both experiments, comprehension and repetition performance were differently influenced by particular sentence structures. It is proposed that sentence repetition in children is constrained by phonological memory capacity, and is therefore directly influenced by memory-related factors that include the length and number of words in sentences, and individual differences in memory skills.

Chondroitin sulfate-E is a negative regulator of a pro-tumorigenic Wnt/beta-catenin-Collagen 1 axis in breast cancer cells.

Expression of the glycosaminoglycan chondroitin sulfate-E (CS-E) is misregulated in many human cancers, including breast cancer. Cell-surface associated CS-E has been shown to have pro-tumorigenic functions, and pharmacological treatment with exogenous CS-E has been proposed to interfere with tumor progression mediated by endogenous CS-E. However, the effects of exogenous CS-E on breast cancer cell behavior, and the molecular mechanisms deployed by CS-E are not well understood. We show here that treatment with CS-E, but not other chondroitin forms, could interfere with the invasive protrusion formation and migration of breast cancer cells in three-dimensional organotypic cultures. Microarray analysis identified transcriptional programs controlled by CS-E in these cells. Importantly, negative regulation of the pro-metastatic extracellular matrix gene Col1a1 was required for the anti-migratory effects of exogenous CS-E. Knock-down of Col1a1 gene expression mimics the effects of CS-E treatment, while exposing cells to a preformed collagen I matrix interfered with the anti-migratory effects of CS-E. In addition, CS-E specifically interfered with Wnt/beta-catenin signaling, a known pro-tumorigenic pathway. Lastly, we demonstrate that Col1a1 is a positively regulated target gene of the Wnt/beta-catenin pathway in breast cancer cells. Together, our data identify treatment with exogenous CS-E as negative regulatory mechanism of breast cancer cell motility through interference with a pro-tumorigenic Wnt/beta-catenin-Collagen I axis.