Glucose influences endometrial receptivity to embryo implantation through O-GlcNAcylation-mediated regulation of the cytoskeleton.

Phenotypic changes to endometrial epithelial cells underpin receptivity to embryo implantation at the onset of pregnancy but the effect of hyperglycemia on these processes remains poorly understood. Here, we show that physiological levels of glucose (5 mM) abolished receptivity in the endometrial epithelial cell line, Ishikawa. However, embryo attachment was supported by 17 mM glucose as a result of glucose flux through the hexosamine biosynthetic pathway (HBP) and modulation of cell function via protein O-GlcNAcylation. Pharmacological inhibition of HBP or protein O-GlcNAcylation reduced embryo attachment in cocultures at 17 mM glucose. Mass spectrometry analysis of the O-GlcNAcylated proteome in Ishikawa cells revealed that myosin phosphatase target subunit 1 (MYPT1) is more highly O-GlcNAcylated in 17 mM glucose, correlating with loss of its target protein, phospho-myosin light chain 2, from apical cell junctions of polarized epithelium. Two-dimensional (2-D) and three-dimensional (3-D) morphologic analysis demonstrated that the higher glucose level attenuates epithelial polarity through O-GlcNAcylation. Inhibition of Rho (ras homologous)A-associated kinase (ROCK) or myosin II led to reduced polarity and enhanced receptivity in cells cultured in 5 mM glucose, consistent with data showing that MYPT1 acts downstream of ROCK signaling. These data implicate regulation of endometrial epithelial polarity through RhoA signaling upstream of actomyosin contractility in the acquisition of endometrial receptivity. Glucose levels impinge on this pathway through O-GlcNAcylation of MYPT1, which may impact endometrial receptivity to an implanting embryo in women with diabetes.NEW & NOTEWORTHY Understanding how glucose regulates endometrial function will support preconception guidance and/or the development of targeted interventions for individuals living with diabetes wishing to embark on pregnancy. We found that glucose can influence endometrial epithelial cell receptivity to embryo implantation by regulating posttranslational modification of proteins involved in the maintenance of cell polarity. Impaired or inappropriate endometrial receptivity could contribute to fertility and/or early pregnancy complications caused by poor glucose control.

Transcriptional response of endometrial cells to Insulin, cultured using microfluidics.

Obesity is a rapidly growing public health issue among women of reproductive age associated with decreased reproductive function including implantation failure. This can result from a myriad of factors including impaired gametes and endometrial dysfunction. The mechanisms of how obesity-related hyperinsulinaemia disrupts endometrial function are poorly understood. We investigated potential mechanisms by which insulin alters endometrial transcript expression. Ishikawa cells were seeded into a microfluidics device attached to a syringe pump to deliver a constant flow rate of 1uL/min of the following: 1) control 2) vehicle control (acetic acid) or, 3) Insulin (10 ng/ml) for 24 hours (n=3 biological replicates). Insulin-induced transcriptomic response of endometrial epithelial cells was determined via RNA sequencing, and DAVID and Webgestalt to identify Gene Ontology (GO) terms and signalling pathways. A Total of 29 transcripts showed differential expression levels across two comparison groups (control v vehicle control; vehicle control v insulin). Nine transcripts were differentially expressed in vehicle control v insulin comparison (p<0.05). Functional annotation analysis of transcripts altered by insulin (n=9) identified three significantly enriched GO terms: SRP-dependent cotranslational protein targeting to membrane, poly(A) binding, and RNA binding (p<0.05). Over-representation analysis found three significantly enriched signalling pathways relating to insulin-induced transcriptomic response: protein export, glutathione metabolism, and ribosome pathways (p<0.05). Transfection of siRNA for RASPN successfully knocked down expression (p<0.05) but this did not have any effect on cellular morphology. Insulin-induced dysregulation of biological functions and pathways highlight potential mechanisms by which high insulin concentrations within maternal circulation may perturb endometrial receptivity.

Injectable Colloidal Hydrogels of N-Vinylformamide Microgels Dispersed in Covalently Interlinked pH-Responsive Methacrylic Acid-Based Microgels.

Injectable hydrogels offer great potential to augment damaged or degenerated soft tissues. A key criterion for such gels is that their modulus is as close as possible to that of the target tissue. The majority of synthetic hydrogels have used low molecular weight polymer chains which may cause problems if they diffuse away from the injection site and/or increase the local osmotic pressure. We previously introduced a different approach of injecting preformed ultra-high molecular weight pH-responsive microgels (MGs) that interlink to form hydrogels. MGs are crosslinked polymer colloid particles that swell when the pH approaches the particle pKa. These colloidal hydrogels are termed doubly crosslinked microgels (DX MGs). The gel moduli of previous DX MGs were much greater than that reported for human nucleus pulposus (NP) tissue of the spinal intervertebral disk. Here, we replace some of the pH-responsive poly(ethyl acrylate-co-methacrylic acid) (PEA-MAA) MGs with hydrophilic non-ionic MGs based on poly(N-vinylformamide) (NVF). We investigate the morphology and mechanical properties of these new injectable composite DX MGs and show that the mechanical properties can be tuned by systematically varying the NVF MG content. Using this approach, the gel moduli close to that for NP tissue are achieved. These injectable new pH-responsive gels exhibit low cytotoxicity. Our work provides a potential new system for minimally invasive intervertebral disk augmentation.

Protein O-GlcNAcylation Promotes Trophoblast Differentiation at Implantation.

Embryo implantation begins with blastocyst trophectoderm (TE) attachment to the endometrial epithelium, followed by the breaching of this barrier by TE-derived trophoblast. Dynamic protein modification with O-linked ß-N-acetylglucosamine (O-GlcNAcylation) is mediated by O-GlcNAc transferase and O-GlcNAcase (OGA), and couples cellular metabolism to stress adaptation. O-GlcNAcylation is essential for blastocyst formation, but whether there is a role for this system at implantation remains unexplored. Here, we used OGA inhibitor thiamet g (TMG) to induce raised levels of O-GlcNAcylation in mouse blastocysts and human trophoblast cells. In an in vitro embryo implantation model, TMG promoted mouse blastocyst breaching of the endometrial epithelium. TMG reduced expression of TE transcription factors Cdx2, Gata2 and Gata3, suggesting that O-GlcNAcylation stimulated TE differentiation to invasive trophoblast. TMG upregulated transcription factors OVOL1 and GCM1, and cell fusion gene ERVFRD1, in a cell line model of syncytiotrophoblast differentiation from human TE at implantation. Therefore O-GlcNAcylation is a conserved pathway capable of driving trophoblast differentiation. TE and trophoblast are sensitive to physical, chemical and nutritive stress, which can occur as a consequence of maternal pathophysiology or during assisted reproduction, and may lead to adverse neonatal outcomes and associated adult health risks. Further investigation of how O-GlcNAcylation regulates trophoblast populations arising at implantation is required to understand how peri-implantation stress affects reproductive outcomes.

Self-curing super-stretchable polymer/microgel complex coacervate gels without covalent bond formation.

Elastic physical gels are highly desirable because they can be conveniently prepared and readily shaped. Unfortunately, many elastic physical gels prepared in water require in situ free-radical polymerization during the gel formation stage. In contrast, complex coacervate gels are physical gels that can be prepared by simply mixing two pre-formed oppositely-charged polyelectrolytes. However, as far as we are aware, highly elastic complex coacervate gels have not yet been reported. Herein, we combine polyanionic microgel particles with a well-known commercially-available cationic polyelectrolyte to prepare polymer/microgel complex coacervate (PMCC) physical gels. This new family of gels requires annealing at only 37 °C and behaves like a covalent gel but does not form covalent bonds. Thermal reconfiguration of the dynamic ionic bonds transforms the shapeable pre-gel into a highly elastic gel that is super-stretchable, adhesive, self-healing, highly swellable and can be further toughened using Ca2+ as an ionic crosslinker. Our PMCC gels have excellent potential for applications as engineering gels and structural biomaterials, as well as for wound healing and water purification.

Core-Shell-Shell Nanoparticles for NIR Fluorescence Imaging and NRET Swelling Reporting of Injectable or Implantable Gels.

Injectable gels that support load are desirable for restoring the mechanical properties of degenerated load-bearing tissue. As these gels become increasingly sophisticated, the need to remotely image them and monitor their swelling increases. However, imaging such gels and monitoring their swelling using noninvasive means is challenging. Here, we use a very low concentration of near-infrared (NIR) core-shell-shell (CSS) reporter nanoparticles to both image and monitor swelling changes of two load-supporting gels. The load-supporting injectable gel consisted of covalently interlinked pH-responsive microgel (MG) particles. The latter gel was not cytotoxic and is termed a doubly cross-linked microgel (DX MG). Inclusion of a complementary fluorescent dye enabled ratiometric monitoring of gel swelling changes in response to pH via nonradiative resonance energy transfer (NRET). In addition, changes in the CSS nanoparticle emission intensity provided a NIR-only method that could also be used to monitor gel swelling. The gel was able to be imaged using NIR light, after being subcutaneously injected into a tissue model. To demonstrate versatility of our approach, CSS and the dye were included within a model implantable gel (poly(acrylamide/acrylic acid)) and fluorescent detection of swelling investigated. Because the concentrations of the reporting species were too low to affect the mechanical properties, our approach to remote gel imaging and swelling monitoring has good potential for application in injectable gels and implants.

Highly compressive and stretchable poly(ethylene glycol) based hydrogels synthesised using pH-responsive nanogels without free-radical chemistry.

Poly(ethylene glycol) (PEG) based hydrogels are amongst the most studied synthetic hydrogels. However, reports on PEG-based hydrogels with high mechanical strength are limited. Herein, a class of novel, well-defined PEG-based nanocomposite hydrogels with tunable mechanical strength are synthesised via ring-opening reactions of diglycidyl ethers with carboxylate ions. The pH responsive crosslinked polyacid nanogels (NG) in the dispersed phase act as high functionality crosslinkers which covalently bond to the poly(ethylene glycol) diglycidyl ethers (PEGDGE) as the continuous matrix. A series of NG-x-PEG-y-z gels are prepared where x, y and z are concentrations of NGs, PEGDGE and the PEGDGE molecular weight, respectively. The hydrogel compositions and nano-structural homogeneity of the NGs have strong impact on the enhancement of mechanical properties which enables property tuning. Based on this design, a highly compressive PEG-based nanocomposite hydrogel (NG-13-PEG-20-6000) exhibits a compressive stress of 24.2 MPa, compressive fracture strain greater than 98% and a fracture energy density as high as 1.88 MJ m-3. The tensile fracture strain is 230%. This is amongst one of the most compressive PEG-based hydrogels reported to-date. Our chemically crosslinked gels are resilient and show highly recoverable dissipative energy. The cytotoxicity test shows that human nucleus pulposus (NP) cells remained viable after 8 days of culture time. The overall results highlight their potential for applications as replacements for intervertebral discs or articular cartilages.

Do the properties of gels constructed by interlinking triply-responsive microgels follow from those of the building blocks?

Microgels (MGs) are swellable crosslinked polymer colloids. They can also be used as the only building block to construct nanostructured hydrogels which are denoted as doubly crosslinked microgels (DX MGs). Here, new triply responsive DX MGs comprised of interlinked MGs of oligo(ethylene glycol)methacrylate (OEGMA), 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA), methacrylic acid (MAA) and a o-nitrobenzyl-based UV photocleavable crosslinker are investigated. The MGs swelled or collapsed in response to temperature and pH changes. These behaviours were rationalised with a generic model using Monte Carlo simulations. The MGs also degraded when UV irradiated due to photocleavage of nPh. DX MGs were assembled from the MGs to give injectable gels that were not cytotoxic to nucleus pulposus cells. Comparison of the responsive properties of the DX MGs and MGs showed that the temperature and pH responses of the former were mostly governed by the latter. However, two key differences were found. Firstly, whilst increasing the crosslinker mol% in the MG building blocks (x) did not change MG particle swelling, the compression modulus (E) and swelling of the DX MG gels were strongly affected by x. The E value for the gels was tuneable using x which is a potentially useful new observation for DX MGs. Secondly, UV irradiation of the DX MGs enhanced gel mechanical photostability in contrast to the behaviour of the MGs. We find that the properties of the DX MGs do not simply follow those of the parent MGs and propose mechanisms to account for the differences. The new family of multi-responsive DX MGs presented in this study have potential application for soft tissue repair as injectable gels or as gel implants which report sterilisation.

Plasmonic and colloidal stability behaviours of Au-acrylic core-shell nanoparticles with thin pH-responsive shells.

The localised surface plasmon resonance (LSPR) of Au nanoparticles (NPs) as well as its interaction with nearby entities provides a wealth of fundamental and practical information at the nanometre scale. A number of studies have investigated core-shell NPs with Au cores and polymer shells that are temperature-responsive. However, there are very few studies of pH-responsive Au-polymer NP shells. Precipitation polymerisation is a scalable method and here we establish such a method to synthesise pH-responsive Au-poly(methyl methacrylate) copolymer core-shell NPs without the need for pre-functionalisation. The comonomers used were methacrylic acid (MAA) or 2-carboxyethyl acrylate (CEA) and the shells were crosslinked with ethylene glycol dimethacrylate. A series of five core-shell systems with collapsed shell thicknesses less than 30 nm are studied. The shell-thicknesses for the CEA-based core-shell NPs are relatively thin (≤5 nm) compared to related Au-polymer core-shell NPs prepared using precipitation polymerisation. The LSPR properties of the core-shell NPs were dependent on the shell thickness and were successfully simulated using finite difference time domain (FDTD) calculations. Two systems are considered further as exemplars. The MAA-based core-shell system with the thickest shell exhibited enhanced colloidal stability to added electrolyte. The CEA-based core-shell dispersion with the thinnest shells displayed reversible pH-triggered aggregation and was cytocompatible for HeLa cells. Proof-of-concept data are presented that demonstrate intracellular pH reporting.

Core-shell-shell cytocompatible polymer dot-based particles with near-infrared emission and enhanced dispersion stability.

Polymer dots (PDs) are promising fluorescent probes for biomaterials applications. Here, novel cytocompatible composite PD particles have been synthesised with a core-shell-shell morphology. The particles show near-infrared emission, improved fluorescent brightness and enhanced colloidal stability compared to pure PDs. The particles also show non-radiative resonance energy transfer (NRET) with a model dye.

Vitamin D attenuates sphingosine-1-phosphate (S1P)-mediated inhibition of extravillous trophoblast migration.

INTRODUCTION: Failure of trophoblast invasion and remodelling of maternal blood vessels leads to the pregnancy complication pre-eclampsia (PE). In other systems, the sphingolipid, sphingosine-1-phosphate (S1P), controls cell migration therefore this study determined its effect on extravillous trophoblast (EVT) function. METHODS: A transwell migration system was used to assess the behaviour of three trophoblast cell lines, Swan-71, SGHPL-4, and JEG3, and primary human trophoblasts in the presence or absence of S1P, S1P pathway inhibitors and 1,25(OH)2D3. QPCR and immunolocalisation were used to demonstrate EVT S1P receptor expression. RESULTS: EVTs express S1P receptors 1, 2 and 3. S1P inhibited EVT migration. This effect was abolished in the presence of the specific S1PR2 inhibitor, JTE-013 (p < 0.05 versus S1P alone) whereas treatment with the S1R1/3 inhibitor, FTY720, had no effect. In other cell types S1PR2 is regulated by vitamin D; here we found that treatment with 1,25(OH)2D3 for 48 or 72 h reduces S1PR2 (4-fold; <0.05), but not R1 and R3, expression. Moreover, S1P did not inhibit the migration of cells exposed to 1,25(OH)2D3 (p < 0.05). DISCUSSION: This study demonstrates that although EVT express three S1P receptor isoforms, S1P predominantly signals through S1PR2/Gα12/13 to activate Rho and thereby acts as potent inhibitor of EVT migration. Importantly, expression of S1PR2, and therefore S1P function, can be down-regulated by vitamin D. Our data suggest that vitamin D deficiency, which is known to be associated with PE, may contribute to the impaired trophoblast migration that underlies this condition.

Synthesis of polyacid nanogels: pH-responsive sub-100 nm particles for functionalisation and fluorescent hydrogel assembly.

Nanogels are crosslinked polymer particles with a swollen size between 1 and 100 nm. They are of major interest for advanced surface coatings, drug delivery, diagnostics and biomaterials. Synthesising polyacid nanogels that show triggered swelling using a scalable approach is a key objective of polymer colloid chemistry. Inspired by the ability of polar surfaces to enhance nanoparticle stabilisation, we report the first examples of pH-responsive polyacid nanogels containing high -COOH contents prepared by a simple, scalable, aqueous method. To demonstrate their functionalisation potential, glycidyl methacrylate was reacted with the -COOH chemical handles and the nanogels were converted to macro-crosslinkers. The concentrated (functionalised) nanogel dispersions retained their pH-responsiveness, were shear-thinning and formed physical gels at pH 7.4. The nanogels were covalently interlinked via free-radical coupling at 37 °C to form transparent, ductile, hydrogels. Mixing of the functionalised nanogels with polymer dots enabled covalent assembly of fluorescent hydrogels.

Responsive Nanogel Probe for Ratiometric Fluorescent Sensing of pH and Strain in Hydrogels.

In this study a new pH-responsive nanogel probe containing a complementary nonradiative resonance energy transfer (NRET) fluorophore pair is investigated and its ability to act as a versatile probe of network-related changes in three hydrogels demonstrated. Fluorescent sensing using NRET is a powerful method for studying relationships between Angstrom length-scale structure and macroscopic properties of soft matter. Unfortunately, inclusion of NRET fluorophores into such materials requires material-specific chemistry. Here, low concentrations of preformed nanogel probes were included into hydrogel hosts. Ratiometric photoluminescence (PL) data for the gels labeled with the nanogel probes enabled pH-triggered swelling and deswelling to be studied as well as Ca2+-triggered collapse and solute release. PL measurements during compression of a nanogel probe-labeled nanocomposite gel demonstrated mechanochromic behavior and strain sensing. The new nanogel probes have excellent potential for investigating the internal structures of gels and provide a versatile ratiometric fluorescent platform for studying pH and strain.

Hydrogel Composites Containing Sacrificial Collapsed Hollow Particles as Dual Action pH-Responsive Biomaterials.

In this study hydrogel composites are investigated that contain sacrificial pH-responsive collapsed hollow particles (CHPs) entrapped within a poly(acrylamide) (PAAm) network. The CHPs were prepared using a scalable (mainly) water-based method and had a bowl-like morphology that was comparable to that of red blood cells. The CHPs were constructed from poly(methyl methacrylate-co-methacrylic acid), which is a pH-responsive copolymer. The PAAm/CHP composite morphology was probed with optical microscopy, CLSM and SEM. These data showed the CHPs were dispersed throughout the PAAm network. Inclusion of the CHPs within the gel composites increased the modulus in a tunable manner. The CHPs fragmented at pH values greater than the pKa of the particles, and this process decreased the gel modulus to values similar to that of the parent PAAm hydrogel. CHPs containing a model drug were used to demonstrate pH-triggered release from PAAm/CHP and the release kinetics obeyed Fickian diffusion. The composite gels had low cytotoxicity as evidenced by Live/Dead and MTT assays. The hydrogel composites showed dual action pH-triggered softening with simultaneous drug release which occurred without a volume increase. The hydrogel composites may have potential application as enteric gels or for intra-articular drug delivery.

A study of physical and covalent hydrogels containing pH-responsive microgel particles and graphene oxide.

In this study we mixed low concentrations of graphene oxide (GO) with microgel (MG) particles and formed composite doubly cross-linked microgels (DX MG/GO) gels. The MG particles comprised poly(ethyl acrylate-co-methacrylic acid-co-1,4-butanediol diacrylate) with pendant glycidyl methacrylate units. The MG/GO mixed dispersions formed physical gels of singly cross-linked MGs (termed SX MG/GO), which were subsequently heated to produce DX MG/GO gels by free-radical reaction. The influence of the GO concentration on the mechanical properties of the SX MG/GO and DX MG/GO gels was investigated using dynamic rheology and static compression measurements. The SX MG/GO physical gels were injectable and moldable. The moduli for the DX MG/GO gels increased by a factor of 4-6 when only ca. 1.0 wt % of GO was included. The isostrain model was used to describe the variation of modulus with DX MG/GO composition. Inclusion of GO dramatically altered the stress dissipation and yielding mechanisms for the gels. GO acted as a high surface area, high modulus filler and played an increasing role in load distribution as the GO concentration increased. It is proposed that MG domains were dispersed within a percolated GO network. Comparison of the modulus data with those published for GO-free DX MGs showed that inclusion of GO provided an unprecedented rate of modulus increase with network volume fraction for this family of colloid gels. Furthermore, the DX MG/GO gels were biocompatible and the results imply that there may be future applications of these new systems as injectable load supporting gels for soft tissue repair.

Injectable biocompatible and biodegradable pH-responsive hollow particle gels containing poly(acrylic acid): the effect of copolymer composition on gel properties.

The potential of various pH-responsive alkyl (meth)acrylate ester- and (meth)acrylic acid-based copolymers, including poly(methyl methacrylate-co-acrylic acid) (PMMA-AA) and poly(n-butyl acrylate-co-methacrylic acid) (PBA-MAA), to form pH-sensitive biocompatible and biodegradable hollow particle gel scaffolds for use in non-load-bearing soft tissue regeneration have been explored. The optimal copolymer design criteria for preparation of these materials have been established. Physical gels which are both pH- and redox-sensitive were formed only from PMMA-AA copolymers. MMA is the optimal hydrophobic monomer, whereas the use of various COOH-containing monomers, e.g., MAA and AA, will always induce a pH-triggered physical gelation. The PMMA-AA gels were prepared at physiological pH range from concentrated dispersions of swollen, hollow, polymer-based particles cross-linked with either cystamine (CYS) or 3,3'-dithiodipropionic acid dihydrazide (DTP). A linear relationship between particle swelling ratios, gel elasticity, and ductility was observed. The PMMA-AA gels with lower AA contents feature lower swelling ratios, mechanical strengths, and ductilities. Increasing the swelling ratio (e.g., through increasing AA content) decreased the intraparticle elasticity; however, intershell contact and gel elasticity were found to increase. The mechanical properties and performance of the gels were tuneable upon varying the copolymers' compositions and the structure of the cross-linker. Compared to PMMA-AA/CYS, the PMMA-AA/DTP gels were more elastic and ductile. The biodegradability and cytotoxicity of the new hollow particle gels were tested for the first time and related to their composition, mechanical properties, and morphology. The new PMMA-AA/CYS and PMMA-AA/DTP gels have shown good biocompatibility, biodegradability, strength, and interconnected porosity and therefore have good potential as a tissue repair agent.

Injectable doubly cross-linked microgels for improving the mechanical properties of degenerated intervertebral discs.

The use of injectable pH-responsive doubly cross-linked microgels (DX microgels) to improve the mechanical properties of degenerated intervertebral discs is demonstrated for the first time. The microgel comprised methyl methacrylate (MMA), methacrylic acid (MAA), ethyleneglycol dimethacrylate (EGD) and glycidyl methacrylate (GM) and was poly(MMA/MAA/EGD)-GM. The GM facilitated covalent interparticle cross-linking. The DX microgels are shown to have tunable mechanical properties. Degeneration of model bovine intervertebral discs (IVDs) was induced using collagenase. When injected into degenerated IVDs the DX microgels were shown to improve the strain, modulus, toughness and resilience. The extent of mechanical property improvement was an increasing function of DX microgel concentration, suggesting tunability. Cytotoxicity studies showed that the DX microgel was biocompatible under the conditions investigated. The results of this study imply that injectable DX microgels have good potential as a future regenerative medicine strategy for restoring the mechanical properties of degenerated load-bearing soft tissue, such as IVDs.

A multiwell electrochemical biosensor for real-time monitoring of the behavioural changes of cells in vitro.

We report the development of a multiwell biosensor for detecting changes in the electrochemical open circuit potential (OCP) generated by viable human cells in vitro. The instrument features eight culture wells; each containing three gold sensors around a common silver/silver chloride reference electrode, prepared using screen-printed conductive inks. The potential applications of the device were demonstrated by monitoring rheumatoid synovial fibroblasts (RSF) and HepG2 hepatocarcinoma cells in response to chemical and biological treatments. This technology could provide an alternative to conventional end-point assays used in the fields of chemotherapy, toxicology and drug discovery.

Electrochemical monitoring of rat mammary adenocarcinoma cells: an in vitro assay for anticancer drug selection.

We report the application of an electrochemical biosensor (Oncoprobe; Marks & Clerk, Manchester, UK) to determine whether changes in the open circuit potential (OCP) of rat mammary adenocarcinoma cells (MTLn3) treated in vitro with four cytotoxic anticancer drugs could predict their effects in vivo. MTLn3 cells were seeded onto sensors, then exposed to each anticancer compound (cisplatin, doxorubicin, paclitaxel, or vinblastine), and monitored for 44 hours. Electrochemical monitoring in vitro detected OCP responses to all four drugs, with cisplatin and doxorubicin producing greater changes over a shorter period than vinblastine and paclitaxel. Syngeneic MTLn3 cells were used to generate palpable tumors in 50 female Fischer 344 rats. Animals were divided into five equal groups; on day 12 four of the groups received an anticancer drug, and one received a saline control. Fourteen days later the animals were killed, and primary tumor weights were determined. Tumors from cisplatin- and doxorubicin-treated rats were significantly reduced in weight compared to the control, paclitaxel-, and vinblastine-treated groups. The anticancer drug-induced changes observed through real-time electrochemical monitoring of MTLn3 cells in vitro correlated well with the in vivo animal model, unlike the conventional end-point assays of lactate dehydrogenase release and Alamar Blue.