using a micropillar and microwell chip platform

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3D cell-based high-content screening (HCS) using a micropillar and microwell chip platform Sang-Yun Lee, Il Doh, Do-Hyun Nam, and Dong Woo Lee Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05328 • Publication Date (Web): 11 Jun 2018 Downloaded from http://pubs.acs.org on June 11, 2018

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Analytical Chemistry

3D cell-based high-content screening (HCS) using a micropillar and microwell chip platform Sang-Yun Lee1,2, Il Doh3, Do-Hyun Nam1,2,4*, Dong Woo Lee5* 1

Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and

Technology (SAIHST), Sungkyunkwan University, Seoul, 06351, Republic of Korea 2

Institute for Refractory Cancer Research, Samsung Medical Center, Sungkyunkwan University School of

Medicine, Seoul, 06351, Republic of Korea 3

Center for Medical Metrology, Korea Research Institute of Standards and Science, Daejeon, 34113, Republic

of Korea 4

Department of Neurosurgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul,

06351, Republic of Korea 5

Department of Biomedical Engineering, Konyang University, Daejeon, 35365, Republic of Korea

*

Corresponding Authors

Dong Woo Lee ([email protected]) Do-hyun Nam ([email protected])

Abstract A micropillar/microwell chip platform was applied to develop a 3D cell-based high-content screening (HCS) platform. Previously, 3D cell culture in the micropillar/microwell chip platform was only limited to cell viability measurements in a high-throughput manner. However, an HCS system could provide biological and phenotypic information which was very useful to understand complex biological functions and mechanisms of drug actions. To stain 3D cultured cells immobilized with alginate spots, we developed and optimized antibody staining procedures for 3D cultured cells. As a proof of concept, the phospho-EGFR (p-EGFR—a highly expressed receptor protein in cancer), F-actin (a protein of the actin cytoskeleton), and nuclei of 3D cultured cells were stained and analyzed after being treated with 72 different drugs. The p-EGFR inhibition of the drugs was successfully identified in the 3D cultured cells by comparing p-EGFR expression with that of F-actin and the nucleus. The p-EGFR expression levels were also measured by western blot to verify the chip data.

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Introduction High-throughput screening (HTS) was a method for scientific experimentation especially used in drug discovery and relevant fields of biology and chemistry. In cell-based assays, HTS used multi-well plates and evaluated compound efficacy and toxicity by rapidly measuring cell viability in a high-throughput manner. This cell-based HTS replaced the laborious and time-consuming manual procedures of compound efficacy and toxicity testing1– 3

. Recently, cell-based HTS was upgraded to high-content screening or analysis (HCS or

HCA) to measure biological and phenotypic information from cells as well as cell viability. HCS involved analytical methods such as automated microscopy, multi-parameter image processing, and visualization tools to extract quantitative data from cell populations. HCS typically employed fluorescence imaging of samples and reported quantitative parameters such as spatial distribution of targets and individual cell and organelle morphology which could provide a more systematic and accurate evaluation of drug candidates4, 5. Thus, HCS technology has been integrated into all aspects of drug discovery in the pharmaceutical industry and scientific research in academia6. Conventional HCS was based on 2D monolayer cells. Cells were attached to the bottom of wells, where cells could be cultured and stained easily. Unfortunately, many cells lost some of their phenotypic properties when grown in vitro as 2D monolayers7-10. The formation of tissue-like structures was inhibited in 2D monolayer cultures due to the strong affinity of cells for most artificial surfaces and the restriction to a 2D space, which severely limits intercellular contact and interactions. Compared to 2D monolayer cell cultures, many studies have reported that 3D cultured cells show different morphologies11, protein/gene expressions12, 13, and drug responses14, 15. Thus, 3D cell culture methods, such as multicellular tumor spheroids (MCTS) cultured in hydrogels, and cells grown on polymer scaffolds, on microcarrier beads, or in hanging droplets, have been widely used in biomedical and pharmaceutical research16-23. However, quantitative analysis of the cells cultured in 3D microenvironments was labor intensive due to the difficulty in manually handling hydrogels or suspended cells, as well as the numerous washing steps required for the immunofluorescence staining. The structure of cells and hydrogels could be easily damaged during media replacing, drug treating, and washing processes. To address these issues, our group had developed micropillar/microwell chip platforms culturing 3D cells on a micropillar chip as previously reported21-26. The media or reagents in the microwell chip, which was exposed to the 3D cultured cells on the micropillar chip, could


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be changed by replacing the microwell chip with one containing fresh media or reagents. The micropillar chip containing the 3D cultured cells could be moved to a new microwell chip filled with fresh solution. In this way, the cells were exposed to different compounds and staining solutions without damaging the cells or hydrogel, thus enabling us to perform complicated miniaturized 3D cell-based assays in a high throughput manner. Previously, we simply performed live-cell staining of the 3D cultures in the micropillar/microwell chip platform24-26 which was developed for miniaturized 3D cell-based HTS. Using the platform, viable cells on the micropillar were simply stained to measure the cell viability and to calculate the IC50 (single-parameter assay). However, this previous work lacked the ability to yield the biological and phenotypic information from 3D cultured cells which were provided by HCS to better understand complex biological functions and mechanisms of drug actions. To apply the micropillar/microwell chip platform to HCS, we developed and optimized the fixing, permeabilizing, blocking, and antibody staining procedures of 3D cultured cells on the micropillar chip. 3D cultured cells immobilized in an alginate spot could be easily moved to many different buffers for the immunofluorescence staining. However, the alginate spots attached to the micropillar detached or degraded easily during fixing, permeabilizing, and blocking, which were essential for immunofluorescence staining for HCS. Losing 3D cultured cells in the staining step was a very critical issue we had to address to apply a micropillar/microwell chip platform to HCS. To overcome this critical problem, we modified the immunofluorescence staining buffers by adding the optimum amount of calcium ions (Ca2+) to prevent degradation of alginate and keep a strong attachment of 3D cultured cells during immunofluorescence staining.

MATERIALS AND METHODS Preparation of the micropillar and microwell chips The micropillar and microwell chips manufactured by plastic injection molding were robust and flexible platforms for mammalian cell cultures, enzymatic reactions, viral infections, and compound screenings (Fig. 1). The micropillar chip made of poly(styrene-co-maleic anhydride) (PS-MA) contains 532 micropillars (with a 0.75 mm pillar diameter and 1.5 mm pillar-to-pillar distance). PS-MA provides a reactive functionality to covalently attach poly-Llysine (PLL), and ultimately to attach alginate spots by ionic interactions. In addition, the microwell chip made of polystyrene (PS) has 532 complementary microwells (with a 1.2 mm



well diameter and 1.5 mm well-to-well distance). Both the micropillar and microwell chip were similar to conventional microscopic glass slides in terms of size (75 × 25 mm). Plastic molding was performed with an injection molder (Sodic Plustech Inc., USA). Cell culture Human lung carcinoma, A549, was purchased from the Korean Cell Line Bank (Seoul, South Korea). A549 was cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS). Cell lines were maintained at 37 °C in a 5% CO2-humidified atmosphere and passaged every four days. Normally, we used the A549 cell line under 20 passages after thawing the frozen cell stock. Under 20 passages, we observed that the A549 cell line easily formed 3D cells in 0.75 % (w/w) alginate on the chip platform. Experimental procedure For drug analyses, 50 nL spots containing approximately 100 cells and 0.5% alginate (0.5 w/w) were automatically dispensed onto a micropillar chip using an ASFA™ Spotter ST (Medical & Bio Device, South Korea). The ASFA™ Spotter ST used a solenoid valve (The Lee Company, USA) to dispense 50-nL droplets of the cell–alginate mixture on the micropillar chip and 950 µL of medium or compound in the microwell chip. After dispensing the cells, as shown in Fig. 2a, the micropillar chip containing human cells in alginate was sandwiched (or “stamped”) with the microwell chip for 3D cell culture. Each microwell chip contained 532 wells, each 1.2 mm in diameter. The assembly of the micropillar/microwell chip was shown in Fig. 2b. Cells were grown for one day at 37 °C because the cells were dissociated with trypsin to make single cells. The cells need to be stabilized in the alginate spots before being treated with drugs. After stabilizing cells, we checked cell viability by staining cells. The most of cell shows alive (Green Fluorescence) as shown in Fig 2g. The micropillar chip containing the cells was then moved to a new microwell chip filled with the drugs to be tested. The inhibition time of the p-EGFR expression was dependent on the drugs. If some drugs inhibited p-EGFR and killed cells in very early time, both p-EGFR expression and cell viability in 1day or 2 day were reduced at a time. The p-EGFR inhibition inducing cell death could not be measured early. Thus, 6h or 12 h staining of p-EGFR were required. Cells exposed to the drug (Fig. 2c) for 6, 12, 24, 48, and 168 h were stained shown in Fig. 2d. The 3D cells cultured on the micropillar chip were fixed using 4% paraformaldehyde solution (PFA, Biosesang, Korea) mixed with 2.5 mM CaCl2. During fixing process using only 4%


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paraformaldehyde solution, the cross-linking of alginate easily degraded and the alginate spots were detached from micropillars and the encapsulated cells were release in the solution. Divalent cations (e.g, Ca2+) were well-known to make cross-linking with alginate27. Thus, Ca2+ was used to prevent degradation of alginate in cell fixing process. At low CaCl2 concentrations, the alginate spots were degraded and detached from the micropillar chip. At high CaCl2 concentrations, the 3D cultured cells shrunk. While moving the micropillar chip, we checked the microwell chips and staining dish with a microscope and could not find any cells. Cells on the micropillar were also tracked through staining process and cell lose did not observed as shown in Fig.2g. Thus, we confirmed the cells on the alginate spot were not lost in the fixation process. After fixation, the micropillars were moved into a permeabilizing and blocking solution (1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) containing 0.3% Triton-X) for 1 h. Following this, each micropillar chip was incubated overnight at 4°C with the antibody staining solution. The antibody staining solution was prepared by adding Anti-EGFR (200:1, Abcam, UK, phosphor Y1092, Alexa Fluor 488, green fluorescent dye with excitation suited to 488-nm lasers), Hoechst 33342 (1000:1, Thermofisher Scientific, Korea, Hoechst 33342, blue fluorescent dye that could be excited using a 358-nm laser), and F-actin phalloidin (400:1, Thermofisher Scientific, Korea, Alexa Fluor 594, red fluorescent dye that could be excited using 561-nm or 594-nm lasers) to the permeabilizing and blocking solution. The stained chip was washed for 15 min. In the staining buffer solution (MBD-STA500, Medical & Bio Device, South Korea) and then dried completely in a dark environment. To measure the p-EGFR, nucleus, and cytosol intensities, stained cells on the micropillar were scanned using an optical scanner (ASFA™ Scanner HE, Medical & Bio Device, Korea) shown in Fig. 2e. Scanned images (Fig. 2f) were evaluated using an image analysis software (ASFA™ Ez SW, Medical & Bio Device, Korea). The layouts of the chips were shown in Fig. 3. Cells/alginate spots were dispensed onto the micropillar chip (Fig. 3a). To evaluate the efficacy and p-EGFR inhibition of the 72 drugs using a single chip, we designed a high-dose drug heat map model that dispenses 20 µM of the drugs into the microwell shown in Fig. 3b. With this layout, each 72-drug model had 6 replicates. For a detailed analysis of the drugs, we designed a dose response curve (DRC). The microwell chip was divided into 12 regions shown in Fig. 3c. Each region was composed of a 6 × 6 microwell array corresponding to six different drug doses (including one control) and their six replicas.



Image processing and data analysis Nucleus, p-EGFR and F-actin of 3D cultured cells in alginate were stained with different color fluorescents (Blue, Green and Red). Red fluorescent was used for filamentous actin (Factin), which is composed of a cell cytoskeleton, to check cell viability after fixing the 3D cultured cells because the cytoskeleton degraded when the 3D cultured cells were affected by the drugs. Green fluorescent dye was used for p-EGFR in the membrane of the cell. Blue fluorescent dye was used for the nucleus of the cell. An automatic optical fluorescence scanner (ASFA™ Scanner ST, Medical & Bio Device, South Korea) was used to measure the red, green, and blue fluorescence intensities using an 8-bit code among the RGB codes (0– 255), and the 3D cultured cells were identified according to intensity thresholds ranging from 20–40. p-EGFR expressions were calculated by dividing the size of the green area (p-EGFR expression) by the size of the blue area (Nucleus of cell). The cell viability was calculated by the size of red area (F-actin expression). The relative F-actin and p-EGFR expression values were normalized to their corresponding controls (no drug treatment). The dose response curves were obtained by plotting the expression values according to the dose of the drugs in GraphPad Prism 5. The area under the curve (AUC) values were calculated automatically in the XY analysis completed with the Graphpad Prism software. Western blot assay Total cell lysates were prepared using a cOmplete™ Lysis-M buffer solution (Roche Life Science, Germany) from the A549 lung cancer cell lines. Protein extracts were resolved using 4–20% Mini-PROTEAN TGX™ Precast Protein Gels (Bio-Rad, Hercules, CA) and transferred onto iBlot® PVDF gel Transfer Stack membranes (Thermofisher Scientific, Korea). After blocking non-specific binding sites for 1 h in 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 (TBS-T), the membranes were incubated overnight at 4°C with specific primary antibodies. The antibodies were Anti-EGFR (phospho Y1092) antibody (1:1000, Abcam, Cambridge, UK) and anti-beta actin (1:2000, Abcam). These were used in accordance with the manufacturers’ instructions.

Results and Discussion Multi-parameter heat map of 72 drugs in a single chip We selected clinical cancer drugs from current clinical trials and standard target oncology


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drugs. From the drug library, we selected 70 drugs whose targets were well-known, such as EGFR, phosphoinositide 3-kinase (PI3K), mechanistic target of rapamycin (mTOR), vascular endothelial growth factor (VEGFR), MET gene, tyrosine-protein kinase Met (c-Met), fibroblast growth factor receptors (FGFR), etc. These drugs were in phase III or IV trials or were approved oncology drugs from public data provided by the US Food and Drug Administration (FDA). The 72 drugs selected (including two controls) were dispensed into microwells. The p-EGFR inhibition times were dependent on the cells and drugs. Thus, after the drug treatment, the 3D cultured cells on the micropillar chips were stained at 6, 24, 48, and 168 h for p-EGFR, F-actin, and a nuclear stain. To check the p-EGFR inhibition by the drugs, we calculated the relative p-EGFR expression and compared it with that of the control treatment. p-EGFR inhibition was confirmed when the p-value between the relative p-EGFR expressions of the drug and control was less than 0.05, and the cell viability calculated by Factin expression was less than 50%. The relative p-EGFR expression levels were measured at 6, 24, 48, and 168 h. Over time, the 3D cultured cells were affected by the drugs and subsequently died, which would reduce p-EGFR expression. Thus, p-EGFR expression differed according to the exposure time. Therefore, for each drug, we selected the lowest pEGFR expression level as the representative to indicate the level of p-EGFR inhibition by that drug (Table 1). From these criteria, 41 of the 70 drugs exhibited p-EGFR inhibition. Ten drugs targeting p-EGFR and 30 drugs targeting different biomolecules exhibited p-EGFR inhibition in the proposed platform. Fig. 4 shows the p-EGFR expression relative to cell viability after 168 h of drug treatment. We divided this into four regions based on 50% pEGFR expression and 50% cell viability. Region #1 (< 50% p-EGFR and < 50% F-actin) showed highly effective drugs in the A549 cell line. The 11 drugs in this region inhibited pEGFR and killed 3D cultured cells. In region #2, 15 drugs showed a high efficacy for killing 3D cultured cells; however, the drugs did not reliably inhibit p-EGFR expression (> 50% pEGFR). Other pathways might be involved in the death of the 3D cultured cells. No effective drugs were found in region #3 (there was no p-EGFR inhibition or cell death). In Region #4, the drugs reliably inhibited p-EGFR expression, but the cells overcame this and survived (> 50% F-actin). In this case, an alternative signal pathway in the A549 cell line ensured the survival of the 3D cultured cells. In a detailed study of individual drugs, the F-actin and pEGFR expressions were plotted against time. Of the 11 EGFR targeting drugs, AEE778 showed a high p-EGFR inhibition and drug efficacy in the 3D cultured A549 cells. In Fig. 4a, p-EGFR was blocked at 6 h, and the A549 cells started to die implying a high efficacy.



Tivozanib, targeting Vascular Endothelial Growth Factor (VEGFR), also showed a high pEGFR inhibition and efficacy shown in Fig. 4b. Interestingly, we found that Ibrutinib targeting Bruton's tyrosine kinase (BTK) and Bosutinib targeting dual Sarcoma/Abelson (Src/Abl) severely inhibited p-EGFR expression, whereas the cell viabilities calculated by F-actin were not reduced by these drugs. As shown in Fig. 4c, the p-EGFR expression was highly inhibited, but the A549 cell line overcame the drugs, survived, and formed colonies over time. In Fig. 4d, XL147 from region #2 exhibited high cell death followed by a relative reduction in p-EGFR expression. Thus, we could confirm that XL147 did not inhibit p-EGFR or cause the death of the 3D cultured cells. Multi-parameter dose response curves of 12 drugs Of the 41 drugs inhibiting p-EGFR, 11 drugs exhibited a high efficacy (cell viability < 50%). Interestingly, Tivozanib (AV-951), Regorafenib, Cabozantinib (XL184), Foretinib (XL880), Dovitinib (TKI-258), Dasatinib (BMS-354825), AUY922 (NVP-AUY922), Ruxolitinib, Dabrafenib, and Vemurafenib, all of which target different biomolecules, exhibited high pEGFR inhibition and drug efficacy in the A549 cell line. Among the ten p-EGFR targeting drugs, AEE788 (NVP-AEE788) was effective in the A549 cell lines. From the heat map in Fig. 4, we selected eight drugs that highly inhibited p-EGFR expression and four drugs that did not. The eight drugs included three EGFR-targeting drugs (AEE78828, Afatinib29 and CI103330). For further study of the selected 12 drugs, we dispensed six doses of each drug into the microwell chip (Fig. 3c). 3D cultured cells exposed to the 12 drugs were stained 6 h after the drug treatment shown in Fig. 5a. From the immunofluorescence staining, the dose response curves (DRCs) based on the p-EGFR and F-actin expression were drawn as shown in Fig. 5b. The DRC of each mini-block (6 × 6 array) was drawn. In all cases, F-actin, which indicates cell viability, was not reduced (due to a very short exposure time), whereas the pEGFR expression levels were reduced. Thus, we accurately measured p-EGFR inhibition. To quantify the p-EGFR inhibition, AUCs (area under curve) were calculated for the p-EGFR and F-actin curves. An AUC ratio between p-EGFR and F-actin under 100% indicated pEGFR inhibition. As a result, we found that eight drugs (AEE788, Afatinib, CI-1033, Cabozantinib, Bosutinib, Tivozanib, Dasatinib and Ibrutinib) showed p-EGFR inhibition, while the four other drugs (Everolimus, Raloxifene, Astemizole, Fenretinde) did not. In previous reports, four non-EGFR targeting drugs (Cabozantinib31, Bosutinib32, Dasatinib32 and Ibrutinib33) affected EGFR signaling. However, Tibozanib had not been reported in


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relation to EGFR signaling which indicates a new finding from this work. Of the eight drugs, AEE788, Bosutinib, Dasatinib and Ibrutinib showed high p-EGFR inhibition (p-EGFR/Factin less than 90%, hereafter termed positive drugs). In Fig. 5, Everolimus, Raloxifene, Astemizole, and Fenretinde (negative drugs) showed no p-EGFR inhibition. To verify the p-EGFR inhibition of these eight drugs, the p-EGFR expression levels of 3D cells exposed to the drugs were measured by western blot. As shown in Fig. 6, the four positive drugs exhibited high inhibition of p-EGFR (less than 50%) while the four negative drugs showed no inhibition by western blot. Bosutinib, Dasatinib and Ibrutinib, the highest pEGFR inhibiting drugs among the non-EGFR targeting drugs in Fig. 5, showed low p-EGFR levels by western blot. AEE788, the highest p-EGFR inhibiting drug among the three EGFR targeting drugs in Fig. 5, also showed low p-EGFR levels which was correlated with the measurement of the chips (Fig. 5)

Conclusions A micropillar/microwell chip platform was successfully applied to HCS (High Content Screening). Immunofluorescence staining of 3D culture cells on the micropillar chip was developed and optimized. As a proof of concept, p-EGFR, F-actin, and the nucleus were measured in a 3D-cultured A549 cell line. In the heat map of 72 drugs, 11 drugs showed high p-EGFR inhibition and low viability, indicating that these were high efficacy drugs targeting p-EGFR. we drew dose response curves based on p-EGFR as well as cell viability according to the doses of the 12 drugs. By calculating the AUCs, we quantitatively measured p-EGFR inhibition without the effect of cell death. This was verified by western blot and by previous reports. With this study, we demonstrate that the micropillar/microwell chip platform could be applied to the studies of mechanism of drug action of the 3D cultured cells. In the future, for medical applications, patient-derived cells (PDCs) may be screened and matched with gene data through big data analysis. In addition, real-time imaging reagents may be adapted to our 3D HCS platform, and the mechanism of drug action could be analyzed in real time, which will enable a deeper understanding of PDC-drug-gene relationships.

Acknowledgements This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of



Health & Welfare, Republic of Korea (HI14C3418). This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (No. 2018R1C1B5045068).


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Figure 1. Schematic view of the micropillar/microwell chip platform for 3D cell-based highcontent screening (HCS). Single pillar chip contains 532 test spots on the pillars, and each spot was stained with three colors for p-EGFR (Green), F-actin (Red), and the nucleus (Blue).



Figure 2. Experimental procedures for multi-color staining of 3D cultured cells using the

micropillar chip. (a) Cell and alginate mixture dispensed on the micropillar. (b) 1 day incubation for stabilizing the cells by dipping the micropillar chip into the microwell chip filled with growth media. (c) Drug exposure by replacing the microwell chip with one filled with drugs. (d) Antibody staining of the micropillar chip through sequential staining steps. (e) Multicolor image scanning after drying the alginate spots. (f) Multi-color image analysis. (g) Tracking image of cells on micropillar while immunofluorescence staining


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Figure 3. Chip layout for the 72 drug heat map and multi-parameter dose response curve.

(a) Micropillar chip layout for cell spot loading. (b) Microwell chip layout for 72 drugs with 7 replicates. (c) Microwell chip layout for 12 drugs with 6 different doses and their 6 replicates.



Figure 4. The multi-parameter heat map of 72 drugs. p-EGFR expressions were measured

at 6, 24, 48, and 168 h (7 day) after drug treatment. Among them, the minimum p-EGFR expressions were selected for each drug and compared with the 7 day F-actin expression. pEGFR and F-actin expressions over time were for (a) AEE788, (b) Tivozanib, (c) Bosutinib and (d) XL147.


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Figure 5. The multi-parameter dose response curve (DRC) of 12 drugs. p-EGFR and F-

actin expressions were measured at 6 h after drug treatment. The area-under-curves (AUCs) were calculated to compare the expression level according to the dose of the drugs. (a) Full Factin and p-EGFR images of the sample micropillar chip. (b) F-actin (Red dots) and p-EGFR (Green dots) expressions according to the doses of the 12 drugs. (c) Area-Under-Curves (AUCs) were calculated from (b). Shading means p-EGFR/F-actin expression less than 100%)



Figure 6. p-EGFR expressions in (a) stained immunofluorescence images and (b) the

western blot assay using the same Anti-EGFR (phospho Y1092) antibody.


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Table 1. Information on the 72 drugs including the measured Minimum p-EGFR and F-actin expressions. (Shading means p-EGFR expression less than 50%) Drugs

Target

1_DMSO

-

2_AEE788 3_Afatinib 4_BMS-599626

EGFR

Minimum F-actin p-EGFR [%] [%]

Drug

Target

Minimum F-actin p-EGFR [%] [%]

100

100

37_AZD4547

FGFR1/2/3

76

1

0 0 68

38_BGJ398 39_Dovitinib 40_Bosutinib

FGFR1/2/3

EGFR

30 50 48

100 38 9

97 0 111

5_Erlotinib HCl

HER1/EGFR

67

65

41_Dasatinib

Bcr-Abl

25

30

6_Dacomitinib

EGFR

68

0

42_Nilotinib

Bcr-Abl

73

91

7_Gefitinib

EGFR

59

4

43_AZD6244

MEK1

78

2

8_Lapatinib 9_Neratinib 10_CI-1033

EGFR

74 76 82

44_Trametinib 45_Bortezomib 46_Carfilzomib

MEK1/2

EGFR, HER2

64 60 49

64 80 100

33 111 0

11_CO-1686

EGFR

62

15

47_ABT-199

Bcl-2

73

17

12_BKM120 13_BYL719 14_XL147

PI3K

4 10 8

48_ABT-888 49_AUY922 50_Dabrafenib

PARP

PI3K

100 100 65

100 28 29

84 6 25

15_Everolimus

mTOR

100

75

51_Ibrutinib

16_AZD2014 17_PF-05212384

mTOR

73 100

24 8

52_LDE225 53_LDK378

Smoothened

18_XL765 19_BEZ235

P3k/mTOR

100 100

42 21

54_LGK-974 55_Olaparib

PORCN

20_AZD5363

Akt1/2/3

21_Axitinib

EGFR

EGFR

PI3K

P3k/mTOR

P3k/mTOR

VEGFR1/2/3, PDGFRβ and cKit

22_Cediranib

VEGFR, Flt

23_Imatinib

v-Abl, c-Kit and PDGFR

24_Pazopanib HCl

VEGFR1/2/3, PDGFR, FGFR,

Flt3, c-Kit, FGFR1/3, VEGFR1/2/3, PDGFRα/β dual Src/Abl

Proteasome Proteasome

HSP (e.g. HSP90) BRAFV600

Btk, modestly potent to Bmx, CSK, FGR, BRK, HCK

ALK

PARP1/2

12

80

100 100

84 0

100 100

65 53

69

29

56_Panobinostat

HDAC

63

0

100

71

57_PF-04449913

HSP90

100

83

100

102

58_Ruxolitinib

JAK1/2

45

89

59_Sotrastaurin

PKC

30

11

100

95

89

80

60_Vemurafenib

B-RafV600E

35

3

25_Sunitinib Malate

VEGFR2 and PDGFRβ

100

0

61_Vismodegib

Hedgehog/smothen

100

100

26_Tandutinib

FLT3 ,PDGFR, and KIT

100

13

62_PHA-665752

c-Met inhibitor

100

92

27_Tivozanib

VEGFR, c-Kit, PDGFR

20

4

63_TMZ

alkylating agent

88

106

21

2

64_Amorolfine

morpholine antifungal drug

88

108

53

5

65_Mevastatin

HMG-CoA reductase inhibitor

29

49

66_Amiodarone

antiarrhythmic medication

27

0

67_Fluvastatin Na

28_Regorafenib 29_Vandetanib 30_Cabozantinib 31_Foretinib

c-Kit

VEGFR1/2/3, PDGFRβ, Kit, RET and Raf-1 VEGFR2 VEGFR2,c-Met, Ret, Kit, Flt1/3/4, Tie2, and AXL

HGFR and VEGFR, mostly for Met and KDR

100

74

75

108

Anticholesterol agent. HMG-CoA inhibitor

100

102

32_Crizotinib

Met, ALK

70

0

68_Mycophenolic acid

Inosine-5’-monophosphate dehydrogenase inhibitor

100

42

33_INCB28060

Met

62

98

69_Raloxifene HCl

Estrogen receptor inhibitor

100

109

34_LEE011 35_PD 0332991 36_LY2835219

CDK4/6

100 100 67

74 9 18

70_Astemizole 71_Fenretinide 72_DMSO

Histamine receptor ligand

100 100 100

110 100 100

CDK4/6 CDK4/6

Retinoic acid receptor ligand -



152x162mm (150 x 150 DPI)


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using a micropillar and microwell chip platform

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