A multiplexed microfluidic system for evaluation of dynamics of immune-tumor interactions.

Recapitulation of the tumor microenvironment is critical for probing mechanisms involved in cancer, and for evaluating the tumor-killing potential of chemotherapeutic agents, targeted therapies and immunotherapies. Microfluidic devices have emerged as valuable tools for both mechanistic studies and for preclinical evaluation of therapeutic agents, due to their ability to precisely control drug concentrations and gradients of oxygen and other species in a scalable and potentially high throughput manner. Most existing in vitro microfluidic cancer models are comprised of cultured cancer cells embedded in a physiologically relevant matrix, collocated with vascular-like structures. However, the recent emergence of immune checkpoint inhibitors (ICI) as a powerful therapeutic modality against many cancers has created a need for preclinical in vitro models that accommodate interactions between tumors and immune cells, particularly for assessment of unprocessed tumor fragments harvested directly from patient biopsies. Here we report on a microfluidic model, termed EVIDENT (ex vivo immuno-oncology dynamic environment for tumor biopsies), that accommodates up to 12 separate tumor biopsy fragments interacting with flowing tumor-infiltrating lymphocytes (TILs) in a dynamic microenvironment. Flow control is achieved with a single pump in a simple and scalable configuration, and the entire system is constructed using low-sorption materials, addressing two principal concerns with existing microfluidic cancer models. The system sustains tumor fragments for multiple days, and permits real-time, high-resolution imaging of the interaction between autologous TILs and tumor fragments, enabling mapping of TIL-mediated tumor killing and testing of various ICI treatments versus tumor response. Custom image analytic algorithms based on machine learning reported here provide automated and quantitative assessment of experimental results. Initial studies indicate that the system is capable of quantifying temporal levels of TIL infiltration and tumor death, and that the EVIDENT model mimics the known in vivo tumor response to anti-PD-1 ICI treatment of flowing TILs relative to isotype control treatments for syngeneic mouse MC38 tumors.

Development of a miniaturized 384-well high throughput screen for the detection of substrates of cytochrome P450 2D6 and 3A4 metabolism.

The identification of a large number of biologically active chemical entities during high throughput screening (HTS) necessitates the incorporation of new strategies to identify compounds with druglike properties early during the lead prioritization and development process. One of the major steps in lead prioritization is the assessment of drug metabolism mediated by the cytochrome P(450) (CYP) enzymes to evaluate the potential drug-drug interactions. CYP2D6 and CYP3A4 comprise the main human CYP enzymes involved in drug metabolism. The recent availability of specific CYP cDNA expression systems and the development of specific fluorescent probes have accelerated the ability to develop robust in vitro assays in HTS format. The aim of this study was to optimize conditions for the CYP2D6 and CYP3A4 HTS assays and subsequently adapt those assays to a miniaturized 384-well format. Assay conversion to a miniaturized format presents certain difficulties, such as robustness of the signal and of compound delivery. Thus the assay optimization involved the comparison of different substrates to identify those most suitable for use in a miniaturized format. Because of current technical limitations in liquid dispensing of nanoliter volumes, assay sensitivity to organic solvents also provides a main concern during assay miniaturization. Therefore, compound activity from redissolved dry films and from DMSO stocks directly delivered into assay buffer was compared. The data indicate that compound activity was comparable in both formats. The data support the conclusion that CYP2D6 and CYP3A4 in vitro metabolism assays can be successfully performed in 384-well plate format and the substrate potencies, as evaluated by the IC(50) values, determined.

Identification of inhibitors of bacterial transcription/translation machinery utilizing a miniaturized 1536-well format screen.

This report presents the miniaturization of a HTS screen to identify inhibitors of prokaryotic transcription-translation in a 1536-well format. The in vitro assay design utilized the bacterial expression machinery to drive expression of a firefly luciferase reporter gene, which was read as an endpoint luminesence measurement. This multicomponent system permits identification of inhibitors at different steps in this pathway. Successful miniaturization required integration of homogeneous assay formats, robust liquid-handling workstations, and second-generation imaging systems. Comparison of data from a triplicate 1536-well screen of a subset of a target library that had been previously validated and followed up for hit confirmation in a 384-well plate format confirmed that triplicate screening yields data of higher confidence and quality, eliminates the time-consuming and potentially error-prone step of cherry-picking, and reduces the number of false positives and negatives. The substantial savings of reagents and reduction of the numbers of plates to process obtained in a 1536-well format as compared to a 384-well format allowed a full triplicate evaluation of the entire library of 183,000 compounds at lower cost and in less time. The triplicate-screen statistics are consistent with a highly reliable data set with a coefficient of variation of 14.8% and Z' and Z values of 0.57 and 0.25, respectively. This screen resulted in the identification of 1,149 hits (0.63% hit rate), representing a compound population at 2.5 standard deviations from the mean cutoff. Furthermore, the data demonstrate good agreement between IC(50) values derived for this assay in a 1536-well format and 384-well format.

Development of a high throughput equilibrium dialysis method.

The identification of large numbers of biologically active chemical entities during high throughput screening (HTS) necessitates the incorporation of new strategies to identify compounds with drug-like properties early during the lead prioritization and development processes. One of the major steps in lead prioritization is an assessment of compound binding to plasma proteins, because it affects both the pharmacokinetics and pharmacodynamics of the compound in vivo. Equilibrium dialysis is the preferred method to determine the free drug fraction, because it is less susceptible to experimental artifacts. However, even low-volume standard equilibrium dialysis is currently not amenable to the HTS format. Those considerations dictate the development of a high throughput equilibrium dialysis device, without compromising the analytical quality of the data. The present paper demonstrates successful development of a 96-well format equilibrium dialysis plate. Plasma protein binding of three drugs, propranolol, paroxetine, and losartan, with low, intermediate, and high binding properties, respectively, were chosen for assay validation. The data indicate that the apparent free fraction obtained by this method correlates with the published values determined by the traditional equilibrium dialysis techniques.

Analysis of protein-peptide interaction by a miniaturized fluorescence polarization assay using cyclin-dependent kinase 2/cyclin E as a model system.

As a result of the increasing size of chemical libraries, more rapid and highly sensitive strategies are needed to accelerate the process of drug discovery without increasing the cost. One means of accomplishing this is to miniaturize the assays that enter high-throughput screening (HTS). Miniaturization requires an assay design that has few steps, has a large degree of separation between the signal and background, and has a low well to well signal variation. Fluorescence polarization (FP) is an assay type that, in many cases, meets all of the above requirements. FP is a homogenous method that allows interactions between molecules to be measured directly in solution. This article demonstrates the application of FP in a miniaturized HTS format, using 1536-well plates, to measure direct binding between cyclin-dependent kinase 2/cyclin E complex (CDK2/E) and an 8-mer-peptide kinase inhibitor. The data indicate that low variability and high specificity allow rapid and precise identification of antagonist compounds affecting CDK2/E-peptide interactions.

High Throughput Quantitation of cAMP Production Mediated by Activation of Seven Transmembrane Domain Receptors.

Impairment of G protein­coupled seven-transmembrane (7 TM) receptor function has been implicated in a variety of different pathologic conditions, suggesting that the discovery of specific antagonists may lead to the development of successful therapeutic agents. The effect of different agents on receptor-ligand interaction is often measured directly in a receptor binding assay; however, this assay format can be time consuming and does not detect agents that interact at sites distal to the native ligand binding site. Cyclic adenosine monophospate (cAMP) represents a ubiquitous second messenger generated in response to ligand binding to many 7 TM receptors. The present study describes the practical adaptation of scintillation proximity methodology, using FlashPlate (NEN Life Sciences, Boston, MA) technology to evaluate cAMP production. The bioassay is based on competition between endogenously produced cAMP and exogenously added radiolabeled [125I]-cAMP. Cyclic AMP capture is mediated through an anti-cAMP antibody onto a microplate well surface. Removal of unbound radioligand is not necessary because only ligand within #20 mm of the plate surface is detected due to the proximity effect. The data indicate that the use of scintillation proximity technology allows accurate and specific evaluation of G protein­coupled receptor function as measured by cAMP production and is suitable for high throughput screening.

Miniaturization of a Mammalian Cell-Based Assay: Luciferase Reporter Gene Readout in a 3 Microliter 1536-Well Plate.

The combined efforts of the fields of combinatorial chemistry and genomics have significantly increased the number of compounds and therapeutic targets available for screening. The number of compounds will reach into the million range in the near future and provide vast chemical diversity for drug discovery. However, this reservoir of chemical diversity creates downstream hurdles for any screening effort. Properly examining this number of compounds increases investments dramatically, both in the number of dollars spent and amount of limited reagents depleted. Traditional HTS techniques, such as the use of 96-well microtiter plates, have paved the way for faster processing speeds, but are being rapidly overwhelmed by screening demands. Miniaturization of such assays will allow for greater throughput, while concurrently reducing cost. To date, miniaturization efforts have been most successfully applied to bacterial and soluble protein based assays. Questions about the ability to deliver microquantities of mammalian cells without disruption of the cell membrane and/or activation of stress responses have been raised. An assay has been developed in which a human T-cell screen has been adapted to a 1536-well plate format. Through the use of a luciferase reporter gene system, it is shown that a mammalian cell-based assay may be successfully performed in 3 µl and potent inhibitors of the target of interest identified.

Analysis of the site of interaction of CD28 with its counter-receptors CD80 and CD86 and correlation with function.

CD28 and CTLA-4 are homologue members of the Ig superfamily of molecules, containing a single V-like domain, transmembrane, and cytoplasmic regions. Both receptors associate with the counter-receptors CD80 and CD86, but the avidity of interaction for CD28 is about 20-fold lower than for CTLA-4. The interaction between CD28 and its cognate receptors provides a costimulatory signal for optimal T cell activation. Our previous mutational analysis of CD28 defined the highly conserved "MYPPPY" motif in the CDR3-region of the V-like domain as a key site of common and selective recognition. We have extended our analysis to cover all residues in the membrane distal loops of the V region, examining their effect on association with CD80/CD86 in cell adhesion and novel protein-based binding assays, and determining correlation between binding and functional response. Conservative F substitutions at either Y residue in the MYPPPY motif selectively reduced binding to CD86, but mutation of the three amino acids immediately C-terminal to Y 104 equivalently reduced binding to both co-receptors. The conservative F substitution of Y 26 in the CDR1-like region also reduced binding to CD80 and CD86. Other substitutions in the CDR1 loop and mutations spanning the CDR2 and DE loops had no effect. We conclude that the CDR1 and CDR3 regions contribute to a common binding site for CD80/CD86, and that the CDR3 region also carries determinants for selective recognition of these counter-receptors within the MYPPPY motif. Furthermore, for CD28, the strength of functional response, as measured by IL-2 production, directly correlates with binding avidity.

Altered major histocompatibility complex restriction in the NK1.1+Ly-6Chi autoreactive CD4+ T cell subset from class II-deficient mice.

We previously demonstrated selective enrichment of major histocompatibility complex (MHC) class II-specific autoreactive T cells in a subset of mouse CD4+ thymocytes. Here we show that a significant fraction of these autoreactive cells in the normal adult thymus expresses NK1.1 and high levels of Ly-6C and also exhibits flexibility in MHC restriction. In normal mice, this NK1.1+Ly-6Chi subfraction accounts for 10-50% of the CD4+ autoreactive subset and is enriched for MHC class II-restricted autoreactive cells as determined by mixed leukocyte reaction frequency analysis, similar to NK1.1-Ly-6C-CD4+ autoreactive cells. In contrast, in the thymus of class II-deficient littermate mice, NK1.1+Ly-6Chi cells account for most of the mature heat stable antigen (HSA)-CD4+ fraction and exhibit MHC-restricted non-class II autoreactivity. Thus, NK1.1+Ly-6ChiCD4+ T cells show flexibility in MHC class restriction, but their autoreactivity remains MHC dependent.

Two distinct non-T helper type 2 interleukin-4+ cell subsets in mice as revealed by single-cell cytokine analysis.

We have previously defined four murine CD4+ peripheral T cell subsets, fractions (Fr.) I-IV, based on expression of the 6C10 and 3G11 determinants (Hayakawa, K. and Hardy, R. R., J. Exp. Med. 1988. 168: 1825). These subsets also show distinctive levels of other cell surface markers: the two minor subsets, Fr. III and Fr. IV, are both CD45RBlow/-, L-selectin (Mel-14)- and CD44hi, characteristic of secondary T cells. The patterns and levels of cytokine production by individual cells in each subset were determined by bioassay for interleukin (IL)-2/IL-4 or IL-4/interferon (IFN)-gamma production after anti-CD3 stimulation. Our data revealed that these four phenotypically defined subsets largely coincide with clusters of cells showing uniform distinctive cytokine profiles, i.e. IL-2+/IFN-gamma-/IL-4- (Fr. I and Fr. II, L-selectin+), IL2+/IFN-gamma +/IL-4+ (Fr. III, L-selectin-), and IL-2-/IFN-gamma low/-/IL-4+ (Fr. IV, L-selectin-). Besides these subsets, an L-selectin-negative cell subfraction within Fr. II appears to represent a transitional population between the IL-2+/IFN-gamma-/IL-4- stage and the IL-2+/IFN-gamma +/IL-4+ stage. Taken together, these results demonstrate the presence of two IL-4+ secondary T cell subsets with distinct cytokine production patterns, and show that the majority of IL-4+ cells found in healthy adult laboratory mice co-produce IFN-gamma, and thus are not typical T helper type 2 cells.

Selective enrichment of major histocompatibility complex class II-specific autoreactive T cells in the thymic Thy0 subset.

We show here a unique enrichment of autoreactive T cells in the CD4+ mouse thymic subset, Thy0. A single- and 10-cell AMLR (autologous mixed leukocyte reaction) assay demonstrates that more than 30% (one cell per well) and almost all (10 cells per well) Thy0 cultures from normal mice exhibit reactivity specific to autologous cells, resulting in induction of interleukin 3 secretion. In contrast, no other mature thymic or splenic CD4+ T cell subsets showed such a high frequency. The majority of this AMLR reactivity in the Thy0 subset is accounted for by reactivity with self-major histocompatibility complex class II. Furthermore, antigenic selection in generating Thy0 subset is suggested by studies with T cell hybrids from a T cell receptor (TCR) V beta transgenic mouse line, 2B4 beta EH. TCR V-gene analysis of T cell hybrids revealed that those from Thy0, half of which responded to self-class II, consisted predominantly of cells that expressed endogenous TCR V beta s alone (without the transgene), unlike hybrids generated from peripheral naive T cells. Thus, we suggest that the presence of Thy0 results from selective stimulation of cells expressing TCR with sufficient affinity for autoantigens in the thymic CD4+ T cell repertoire.

Age- and strain-related differences in murine spleen cell responses to different activation signals.

The effect of age on the response of splenocytes to activation with anti-CD3 mAb and a combination of anti-CD3 mAb and TPA, as evidenced by interleukin-2 (IL-2) and interleukin-4 (IL-4) production and cell proliferation, was examined in the C57BL/6 and DBA/2 murine strains. Depending on the mode of activation, there were age and strain differences in IL-2 and IL-4 production. With all modes of activation, cells from the old C57BL/6 mice produced less IL-2 than their young counterparts. In DBA/2 mice there was no age-related difference in IL-2 production with anti-CD3 mAb activation alone, whereas when the same cell population was activated with anti-CD3 mAb and TPA an age-associated decrease in IL-2 production occurred. In both strains, there was an age-related increase in IL-4 production with anti-CD3 mAb activation. After addition of TPA, however, there was an age-related decrease in IL-4 production. An age-related decline in the proliferation occurred with all modes of activation in both mouse strains. There were also strain-related differences in proliferation after the addition of forskolin, an inhibitor of Th1-cell function. While forskolin inhibited the proliferation of cells from the young C57BL/6 mice only, in the DBA/2 mice proliferation of cells was inhibited in both age groups. There were no strain-related differences in inhibition by anti-transferrin receptor (TrfR) mAb, although cells from the old mice were slightly more sensitive to this inhibition.

Age-related changes in release of soluble interleukin-2 receptor by murine lymphocytes.

The purpose of this study was to investigate whether the presence of soluble interleukin-2 receptors (sIL-2R) in the supernatants of activated splenocytes correlates with age progression in C57BL/6 mice. In addition, the relationship between IL-2R cell surface expression and release of sIL-2R was examined. The results indicated splenocytes from young (3-4-month-old) mice release higher levels of sIL-2R than those from intermediate (18-19-month-old) or old (24-25-month-old) mice. While there was no statistical difference between sIL-2R levels in intermediate and old mice in this study, the old mice did have a slightly higher release of sIL-2R than the intermediate mice. There was a correlation between IL-2R cell surface expression and the level of sIL-2R in the young and the intermediate age groups. In old mice there was no correlation between these two parameters.

Evaluation of electrocardiogram in emergency room as a decision-making tool.

The contribution of the electrocardiogram to the clinical judgment used by the physician in the emergency room to determine the necessity for hospitalizing patients was evaluated. Thirty-five percent of all 1,578 patients with presumed myocardial infarction referred to the Chaim Sheba Medical Center, Tel Hashomer, Israel, for a one-year period had subsequently diagnosed myocardial infarctions. The ECG in the emergency room detected only 65 percent of these. The physician's clinical judgment was impressive in his decision to admit to the hospital almost all of the remaining 35 percent, while not admitting very many of the patients who did not have subsequently diagnosed myocardial infarctions. When the myocardial infarction was not evident on the ECG and the abnormalities on the tracings were identical for patients with subsequent myocardial infarctions and those without, again the physician made the right choice more often than the wrong. The follow-up ECG also attested to the good judgment of the physician in the emergency room. Of the emergency room ECGs of patients without subsequent myocardial infarctions who were admitted to the hospital, 17 percent showed myocardial infarction by follow-up, while this happened to only 2 percent of those denied admission.

[Hypoplasia of the right ventricular myocardium (Uhl's disease). Report of a case with review of the literature (author's transl)]. / L'ipoplasia del miocardio ventricolare destro (malattia di Uhl). Caso clinico e rivisione della letteratura

A rare case of congenital hypoplasia of the right ventricular myocardium ("Parchment Right Ventricle" or Uhl's disease) diagnosed by angiocardiography is presented. The predominant clinical feature was recurrent paroxysms of severe arrhythmias which could be controlled only by electric shock. Right heart failure was also present. After a follow-up period of 8 years, the patient, now 27 years old, on diuretic and antiarrhythmic treatment, is well. The literature on Uhl's disease is reviewed and classified into two clinical types; a fatal infantile type in which extreme hypoplasia is present, and a milder adult type in which the anatomical lesion is usually more limited. In the infantile type intractable heart failure was invariably present; in the adult type severe arrhythmias often constituted the major clinical problem. Some of the difficulties in diagnosis and treatment are emphasized.

Disposition of presumed coronary patients from an emergency room. A follow-up study.

All patients with presumed coronary problems seen at the Chaim Sheba Medical Center during a one-year period were followed up. The fate of those who were not hospitalized and the factors contributing to the two types of erroneous decisions, ie, refusing hospitalization to those needing it and unnecessary hospitalization of others, were evaluated. Approximately 50% of the patients were not admitted. Myocardial infarctions were later diagnosed in 6% of these patients. Another 8% were eventually categorized as other cardiac emergencies. Ten percent of all patients subsequently diagnosed as having myocardial infarctions were not admitted. On the other hand, 56% of the patients whose cases were later not considered to have been emergencies were hospitalized unnecessarily. Previous hospitalization for cardiac disease played a major role in making an error of both types. Other factors influencing the physician's decision regarding the patients' disposition included their age, sex, ethnic origin, and the findings from the emergency room electrocardiogram.