Assays

A distinguishing feature of the HMS LINCS Center is its use of a wide range of measurement technologies ranging from direct assays of drug-kinase interaction in cell extracts, to multiplex biochemical assays of cell signaling proteins, to imaging assays, to assays of transcriptional response (in collaboration with the Broad LINCS Center) and cell viability. Although the individual assays used in the HMS LINCS Center are quite common in conventional cell-biological studies, our goal is to collect “systematic” data sets in which the assays are performed in a quantitative and reproducible way on many samples, with many perturbing small molecules (or other perturbagens) applied at multiple doses, and in many cell lines. HMS LINCS assays continue to evolve and improve over time. Because most HMS LINCS datasets use multiple types of assays, we provide information on specific assay protocols with each dataset in the HMS LINCS Database.

As explained in the description of the LINCS approach, it is not possible to collect all types of data for all perturbations and cell lines; instead we follow an adaptive data collection strategy in which exploratory studies are used to focus subsequent experiments on areas of of cell-perturbation-measurement in space in which we believe that informative signatures can be identified.

HMS LINCS assays and dataset types

1. KINOME scan and KiNativ Assays measure interactions of kinase inhibitors with kinases

HMS LINCS collects drug-target interaction data on the binding of small molecule drugs to recombinant kinases (KINOMEscan assays) or to kinases present in cell extracts (KiNativ assays and other mass-spec assays). Even clinical-grade small molecule kinase inhibitors are not mono-specific and kinases having similar ATP binding pockets often exhibit drug cross-reactivity, even if the kinases have very different biological roles.To better understand which proteins are bound by kinase inhibitors, we are creating a set of profiles based on experiments performed in vitro using commercially available methods that are in widespread use in industry. We are also developing new mass spec methods in-house to monitor kinase inhibitor binding to kinases in cell extracts: see McAllister, F. E. et al. Mass Spectrometry Based Method to Increase Throughput for Kinome Analyses Using ATP Probes. Anal. Chem. 85, 4666–4674 (2013). doi:10.1021/ac303478g PMID:23607489. The commercial profiling methods we are employing include the KINOMEscan assay from DiscoveRx and the KiNativ assay from ActivX.  KINOMEscan is based on measuring drug binding with a panel of over 400 purified kinases. KiNativ assays measure the competition of an inhibitor for the ATP binding pocket of kinases in cell lysates. Looking forward, biochemical profiling of inhibitors in-house will allow for more in-depth analysis of drug interactions in multiple cell types.

KINOMEscan and KiNativ assay data on HMS LINCS compounds, and more details about the assay protocols, can be found in the HMS LINCS Database.

2. Biochemical responses

Biochemical response data on the abundance and state of modification of 20-100 signaling proteins is carried out using micro-plate-based ELISA assays, bead-based ELISA (xMAP) assays, Western blotting and reverse phase lysate arrays (RPLAs).

Reverse phase lysate microarray technology (Wolf Yadlin et al. 2009 doi 10.1016/j.cbpa.2009.06.027). A: Experimental design. Cells are grown in 96 well plates and treated with perturbagens. Each well is then lysed to generate ~30ul of cleared lysate that is spotted onto dupicate nitrocellulose-coated slides, creating multiple copies of the array. Each array is probed with one or two antibodies with fluorescent readout. B: Example data. In this experiment cells were serum starved, then treated with one concentration of one small molecule drug (from a library of 84), plus EGF. Lysates were prepared 1hr later and probed with validated antibodies to phosphoepitopes in the EGF pathway. Red is degree of inhibition (loss of signal normalized to no drug control), green is degree of stimulation.

3. Microscopy and imaging assays

In microscopy and imaging assays, after treatment with perturbagens, cells are fixed and stained with fluorescent dyes and/or immunostained in order to monitor various cell properties (shape, size, nuclear area) or protein levels (in the immunofluorescence experiments). This allows measurement of protein modification and cellular localization, and scoring of  cell cycle state, cell growth or apoptosis over time. We are collecting both fixed and live cell imaging data on responses to perturbagens. Live-cell assays involve either white-light microscopy or fluorescent reporter proteins. Fixed cell imaging involves the use of dyes, stains, reporter proteins, and immunofluorescence. Many HMS LINCS fixed cell assays are analyzed using ImageRail software.

ImageRail screenshot

Responses of SkBR3 breast cancer cells to the ErbB1 inhibitor gefitinib or the Mek inhibitor PD0325901 in the presence of varying concentrations of EGF. Measurements were performed using immunofluorescence with anti-phospho Erk antibodies. The left hand panel is a screen shot from ImageRail, software we have developed for high throughput analysis of cells in 96 and 384 well plates. The scatter plots show data from six wells, which each point representing a single cells, and the histograms data from 1000 cells in 60 different wells.

4. MGH (CMT) growth inhibition assay

The Center for Molecular Therapeutics (CMT) at Massachusetts General Hospital has established a screening platform to collect data on the responses of a large collections of tumor cell lines (currently over 800 lines) to small molecule drugs. Typically a single measure of response, cell viability at three days, is collected at multiple drug doses. For example, three concentrations of each compound might be tested in a ten-fold dilution series, or nine concentrations tested in a two-fold dilution series. Cells are fixed with 4% formaldehyde and stained with the fluorescent nucleic acid stain Syto60 from Invitrogen. Fluorescent signal intensity is quantified using a plate reader. MGH growth inhibition assay data and more detail about the assay protocol can be found in the HMS LINCS Database. CMT also collaborates with the Wellcome Trust Sanger Institute on a larger drug response project, Genomics of Drug Sensitivity in Cancer, that includes extensive exome sequencing and bioinformatics analysis. Data collected at the Sanger Institute and publicly released are made available to the LINCS project

5. Transcriptional responses

Transcriptional response data on the abundance of 1000 landmark mRNAs in perturbed cells (collected in collaboration with Broad LINCS Center). In collaboration with the Broad LINCS Center we are collecting 1000-gene transcriptional signatures in parallel with data from HMS LINCS biochemical and phenotypic assays. This should make it possible to link the events in immediate early signal transduction to the transcription they control.