Mike M.

Permeability is an important property of drug candidates. It is a key determinant in the absorption, distribution, metabolism and elimination of a drug. In vitro cell permeability assays allow researchers to categorize drugs by different classification strategies like the Biopharmaceutics Drug Disposition Classification System (BDCCS) and focus future studies based on their most likely routes of clearance. Monolayers of the Madine-Darby canine kidney cell line (MDCK) are commonly used for in… Show more

Permeability is an important property of drug candidates. It is a key determinant in the absorption, distribution, metabolism and elimination of a drug. In vitro cell permeability assays allow researchers to categorize drugs by different classification strategies like the Biopharmaceutics Drug Disposition Classification System (BDCCS) and focus future studies based on their most likely routes of clearance. Monolayers of the Madine-Darby canine kidney cell line (MDCK) are commonly used for in vitro permeability assay. In order to better understand the passive permeability of a compound without the confounding factor of P-glycopretein that is basally expressed in MDCK, a knockout MDCK cell line lacking the cMDR1 gene was used*. To increase assay throughput and quality of data we automated and miniaturized the assay into 96-well format from 24-well format, and integrated the workflow with RapidFire high-throughput mass spectrometry. With this approach, we achieved a significant increase in throughput, data consistency and eliminated ergonomics related issues compared to the manual assay. Here we present our method for automating the MDCK 96-well permeability assay. MDCK cells were plated on 96-well transwell plates (Corning) and grown for 5 days before the permeability assay. In separate plates, compounds were delivered by ECHO555 (Labcyte Inc.) acoustic dispensing and liquid handling was done with Biomek FxP (Beckman Coulters). Appropriately diluted compounds were first dosed at the apical side and then sampled at 0min/120min at both apical and basolateral side of the MDCK cells. Compound quantification was achieved by an eight-point standard curve using the Rapidfire mass spectrometer (Agilent technologies). Quantification data were used to calculate apparent permeability value and percentage recovery...[More] Show less

A key feature of altered metabolism in cancer cells is maladaptive lipogenic pathway activity characterized by increased de novo fatty acid synthesis. Fatty acid synthase catalyzes the biosynthesis of palmitate by condensing malony-CoA and acetyl-CoA using NADPH. Increased FASN expression has been reported in many solid and hematopoietic tumors and FASN inhibition can block tumor growth in vitro and in vivo. We evaluated the effect of a FASN inhibitor (FASNi) on the growth inhibition of 10… Show more

A key feature of altered metabolism in cancer cells is maladaptive lipogenic pathway activity characterized by increased de novo fatty acid synthesis. Fatty acid synthase catalyzes the biosynthesis of palmitate by condensing malony-CoA and acetyl-CoA using NADPH. Increased FASN expression has been reported in many solid and hematopoietic tumors and FASN inhibition can block tumor growth in vitro and in vivo. We evaluated the effect of a FASN inhibitor (FASNi) on the growth inhibition of 10 tumor cell liens including lung, colon and prostate , and ovarian lines by 5 day CellTiter-Glo luminescence viability assay...[More] Show less

Drug combination has been widely used in treating the most debilitating diseases such as cancer. The ideal drug-drug combination will broaden and/or deepen therapeutic efficacy while overcoming resistance and unwanted off-target effects. We have developed a 384-well combination compound plating method in 8×7 format (8 dilutions of Drug A and 7 dilutions of Drug B), and screened a number of compound pairs that showed synergistic effect in inhibiting tumor cell growth in either suspension or… Show more

Drug combination has been widely used in treating the most debilitating diseases such as cancer. The ideal drug-drug combination will broaden and/or deepen therapeutic efficacy while overcoming resistance and unwanted off-target effects. We have developed a 384-well combination compound plating method in 8×7 format (8 dilutions of Drug A and 7 dilutions of Drug B), and screened a number of compound pairs that showed synergistic effect in inhibiting tumor cell growth in either suspension or solid tumor lines. Compound vehicle DMSO was used as negative control and puromycin treatment as positive control for calculating% inhibition, and both HSA and Bliss independence synergy models were applied for calculating synergy scores. As 384-well 8×7 format contains one compound pair per plate, we aimed to increase the throughput by developing 384-well 5×5 format (5 dilutions of Drug A and 5 dilutions of Drug B) which contains 3 drug pairs per plate, and 1536-well 8×7 and 5×5 format which contains 4 and 12 drug pairs per plate, respectively. We compared EC50s and synergy scores generated from 384-well and 1536-well with both 8×7 and 5×5 formats, single agent EC50s within combination pairs were generally within 3 fold difference and synergy scores are largely consistent. Therefore, we have validated and enabled higher throughput drug-drug combination screen by using 384-well 5×5, 1536-well 8×7, or 5×5 format. The high-throughput method presented here can be readily adopted for combination studies in other disease areas. Show less

Clonogenic survival assay (CSA) is an adherent-dependent assay that measures the ability of single cells to proliferate and form colonies after treatment with chemotherapy or targeted therapies. Along with anchorage-independent assays performed in soft agar, CSA is a reliable method for investigating KRAS oncogene addiction of cancer cell lines. Cell lines addicted to the RAS pathway have impaired growth in CSA upon KRAS knockdown or downstream pathway inhibition, but are resistant in standard… Show more

Clonogenic survival assay (CSA) is an adherent-dependent assay that measures the ability of single cells to proliferate and form colonies after treatment with chemotherapy or targeted therapies. Along with anchorage-independent assays performed in soft agar, CSA is a reliable method for investigating KRAS oncogene addiction of cancer cell lines. Cell lines addicted to the RAS pathway have impaired growth in CSA upon KRAS knockdown or downstream pathway inhibition, but are resistant in standard proliferation/viability assays. In this study, we first established a 12-well CSA assay with a panel of KRAS-dependent and -independent cell lines and measured their response to three compounds (GILD-1-3) that target the RAS pathway plus puromycin (GILD-4) as a control. As expected, KRAS-dependent cell lines had greater sensitivity to GILD-1 and selumetinib (GILD-2) than KRAS-independent cell lines. No difference was seen in sensitivity to GILD-3 or puromycin (GILD-4). In order to increase the throughput of screening to identify additional compounds that would discriminate between RAS-dependent and -independent cell lines, we aimed to miniaturize CSA to 384-well plate format. Two KRAS-dependent (SK-CO-1 and LS-180) and two KRAS-independent (NCI-H460 and NCI-H1703) cell lines were chosen for further optimization and miniaturization. EC50 values of GILD-1 through 4 were measured at four seeding densities based on CSA growth rate. Interestingly, EC50 values varied with different cell seeding densities with the two lower cell seeding densities recapitulated the EC50 values obtained in the 12-well assay. This was in contrast to standard proliferation/viability assay in which EC50 values remain largely unchanged regardless different cell seeding densities. Since the lowest cell seeding density gave rise to higher standard deviation, we selected the second lowest cell seeding density that still generated a similar EC50 to the 12-well CSA assay as a 384-well cell seeding density ... Show less

Receptor tyrosine kinase-like orphan receptor (ROR1) is expressed on chronic lymphocytic leukemia (CLL) and other cancers, but not on normal post-partum tissues, except for a small subset of precursor B cells known as hematogones. Because of its restricted expression on cancer cells, ROR1 is an attractive target for developing novel anti-cancer therapies.

In this study, we designed several different peptides, corresponding to distinct epitopes in the extracellular domain of ROR1. Each… Show more

Receptor tyrosine kinase-like orphan receptor (ROR1) is expressed on chronic lymphocytic leukemia (CLL) and other cancers, but not on normal post-partum tissues, except for a small subset of precursor B cells known as hematogones. Because of its restricted expression on cancer cells, ROR1 is an attractive target for developing novel anti-cancer therapies.

In this study, we designed several different peptides, corresponding to distinct epitopes in the extracellular domain of ROR1. Each peptides was conjugated with keyhole-limpet hemocyanin (KLH) to generate a peptide-KLH vaccine, which we used to immunize C57BL/6 mice, first in complete Freund’s adjuvant (CFA) and then again 2 weeks later in incomplete Freund’s adjuvant (IFA). Two weeks following the second injection the sera from immunized mice were tested for binding activity for recombinant ROR1 protein via immunoblot analyses or ELISA or for cell-surface ROR1 by flow cytometry. We identified one peptide-KLH vaccine (R22-KLH) that induced high-titer anti-ROR1 antisera specific for ROR1-expressing cells that did not react with normal human or mouse tissues, which did not express this protein. The antisera generated by mice immunized with R22-KLH were capable of directing specific complement-dependent cytotoxicity (CDC) against neoplastic cells that expressed ROR1 (e.g. human CLL cells, EW36, or JeKo-1), but not against cells that did not express ROR1 (e.g. Jurkat). Show less