PMRENE > ERE LARNER» URN / BFA, RESEARCH ARTICLE creck tor updates ADVANCED. THERAPEUTICS, ‘wuradvtherap.com In Vitro Study on Al-PRS Enabled Precision Cocktail Drugs Design for Treating Human Colorectal Carcinoma Hsin-Yu Yang, Venkanagouda S. Goudar, Yi-Chi Hung, Chih-Hsuan Ouyang, Masturah Bte Mohd Abdul Rashid, Liang-Yi uo, Jen-Kuei Wu, Ya-Wen Cheng, Po-Li Wei, Huey-En Tzeng, Jeng-Kai Jiang, Edward Kai-Hua Chow, Chih-Yung Yang, Chih-Ming Ho, Yun Yen, and Fan-Gang Tseng* ‘Colorectal cancer (CRC) is currently the third most common cancer in the ‘world. Due to the development of treatment resistance, the efficacy of current ‘chemotherapeutic agents against CRC has reached a plateau. Drug activity depends on the entire physiological response; therefore, drugdose parameters cannot be designed efficiently by using conventional prediction-based methodologies. In this work, the APRS (arificial intelligence-based phenotypic response surface) platform is successfully applied to find optimal drug-dose combinations in vitro from a pool of ten approved drugs. The ALPRS platform optimizes effective drug-dose ‘combinations without reference to molecular pathways or drug interaction data, With the aid of APRS platform, efficient one, two, three, and four paring to gemcitabine (6.8 months)-treated groups!"! However, the efficiency ofthe regimen and its usage in CRC treatment are still under debate.! On the other hand, these current conven tuonal chemotherapeutic agents were scheduled with a combina- tion of targeted drugs, such as monoclonal antibodies and drugs Pol Wei Division f Colorectal Surgery Department of Surgery Taipei Medical Univeraty Hospital Taipei Medical University Tapes 10301, Tatwan, ROC Pal Wei Department of Surgery Schoo! af Meeieine College of Medicine Tipe: Medical University “pei M0801, Tarwan, ROC Pal Wei Graduate Inctute of Cancer Bilogy and Drug Discovery Tipe’ Medical University Tpei 110301, Tatwan, ROC HAE. Taeng Deparment of Internal Medicine Schoo af Medicine Tupes Medical University Tape’ 1031, Taiwan, ROC HAE. Teen Department of Internal Medicine Division of Hematology/Oncology Tipet Medical Univers |osptal Taipei M031, Taiwan, ROC J. jong Division of Colon & Rectal Surgery Deparment of Surgery Tipe’ Veterans General Hospital pel T1217, Taiwan, ROC Je. fang Department of Medicine National Yang Ming Chiao Tung University Taipei City 1112, Teiwan, ROC EACH. Chow Cancer Science Insitute of Singapore National Universi of Singapore Singapore 117389, singapore E.KH, Chow The N.1 Institute for Heath National Unversiy of Singapore Singapore 117455, Singapore The institute for Digital Medicine (WisDM) Yong Loe Lin Schoal of Medicine National University of Singapore Singapore 117587, Singapore EK, Chow [NUS Centre for Cancer Reseorch (N2CR) Yong loo Lin School of Medicine National Unversiy of Singapore Singapore 117587, Singapore EACH, Chow Lepartment ot Pnarmacology Yong Loo Lin Schoo! of Medicine National University of Singapore Singapore 117597, Singapore ‘Abu Theo, 203, 2200098 2200298 (2 of 1) ARYANGE, THERAPEUTICS ‘wurwadvtherap.com that target vascular endothelial growth factor (e., bevacizumab and ramucirumab) or epithelial growth factor receptor (EGER, ¢¢g.,cetuximab and panitamumab).”! Additionally, these conven tional combinatorial regimens were used in combination with regoraferub (multiknase inhibitor) asa third-hne therapy o en ie the treatment efficacy. These recently developed therapies potentially overcome chemotherapy-induced cytotoxicity and en 1c patient response in certain populations with CRC."! De. spite significant improvements that have been made inefficient regimen development for CRC over the last few years, the 5-year survival rte of patients with advanced CRC has remained at <8% due to the development of treatment resistance"! In this regard, a key point to contemplate is that drug- — £(C,t) “The experimental design allowed for minimum number ofdeug com: binations to be tested for factor screening and model fitng. The ex penimental datapoints were Re nto = second-order quadratic regression ode, as described in previous paped'®9 and second-order polynomial model wa aka + Dkn ++ De Be? Dl? 2 where y represents the desired output, is the mh drug dosage, by is the intercept term, by isthe single drug cefclent ofthe mth dr, Pw ‘the interaction coefciert between the mth and mth drugs and by. isthe (quadkate coefficient forthe mth drug. ‘After investigating the IC5O values of each drug inthe ell ines, the C10 and 1C20 values of each drug were calculated in ten drug search spaces. Hence, the drugs were mentioned with respective concentrations, For eraple, the IC10 of cetuximab was annotated as C1, and IC20 was snnotated a5 C2, The results ofthe APRS analyse were correlated into 8 second-order quadratic eerie, a8 previously described by Rech et For in vito experiments, the relative viability of the eels was used as an ‘experimental aata pol for Ring. Patients and Specimen Cllecion: This study was approved bythe in- stituional review board (IRB) of Taipei Veterans General Hospital (IRB number. 2021-10-0118CF). Patents with CRC were included inthe study tnd provided written informed consent to patcipate inthis study. For patients enrolled, clinical profiles, including inital cance staging, med. leation information, treatment responses, progression fre survival, and overall survival, were recorded, Blood samples were collected afer pa- tients gave ther consent to participate. Patients were foloned up at 3 ‘months interval ater bod collection, with clinical response to weatment talusted using computer tomoaraphy imaging and Response Evaluation Gira Said Tumors (RECIST] version 11 Immunefurescence Staining The immunofluotescence staining of TCs was confirmed by antiPan Cytokeratin (lone: AEIJAE3, Thermo Fisher) and antiCD4S antibody (clone: HI30, Stem cell technologies, Car v= fot dint ‘Ae Thro. 2023, 200298 Ny x Xo +) mich +) yuck + re 2200298 (9 of 11) ADVANCED: THERMPEUTIO’ ‘wunnadvtherap.com bridge, MA, USA), according to a previously described method!" In bie, the cells were fied 10min with paraformaldetyde and washed with ‘hosphate-buffered saline (PBS). CTCs were incubated in 0.1% Tton 100 in PBS for permeabilization. After being treated Triton X-100, antibody an Gtokeratin and CD45 were used o analyze CTCs and cll nuclei were counterstained using DAPL Images were examined and captured by Tuo rescence microscopy (CTC Enrichment and Expansion: Perigheral venous blood (30 mt) was coleced fom stage IVA colon cancer patient in vacated ethylenedi aminetetreacetic acid (EDTA) tubes (BD Biosciences, San Jose, CA, USA), and blood samples were subjected to Ficoll aque (Merck, Kenilworth, N) USA) to isolate peripheral blood mononuclear cells (PBMCs) according twa previously described method!" In bi, blood was add toa Leu coSep tube with FicolhPaque Plus and centrifuged to obtain the PBMC fraction that contained CTCs. The pellet was resuspended in PBS contain ing PE bovine suns albumin, 210 w EDTA, and CTCeniced by the RoseteSep CTC Enrichment Cocktail Kit (Stem Call Technologies, Crm bridge, MA. USA) The enriched cells were obtained and suspended in DMEM /F12 medium conteining CGT, FCT, 027 supplement, and platelet Iysate (Thermo Fisher Scientif, In, Waltham, MA, USA) Calls were Soo Efficacy = ma t 0 seeded onto a substrate called binary callidal estals (BCC). On the ‘other hand, presence of CTCs was confirmed by staining wth EpCAMCFITC fd nuclear staining (Hoechst. CTC spheroige were cultured for 15 days, CelterGlo Luminescent Cell Viability Assay (promega) was treated and cequlbrated the plate and its contents st room temperature for 2 min rd recorded luminescence. The luminescence was above 15% 10° RLU. represented CTC growth wel. Here, the luminescence of ease one was 1870400 RLU an cove to was 1147600 RLU. ‘Drug Screening and Gell Vabilty: CTC spheroids were cultured for 20 ays, after which spheroids were harvested and seeded in 6-well culture plates for drug sensitivity assays. Cell number of case one was 773 calls per wel and ease to was 4557 cells per wel. The eels were treated us Ing st drug combinations including SFU gemcitabine cegorafeib, and cetuximab, at diferent doses (C15, 1CZ0, and IC30}.AferWeatment, the calls were incubated with each testing drug for 72h. The analysis of cell ability using a Nuorescence-based vabiity assay was performed using 8 LIVE/DEAD viabilty/erotoxcty kit according tothe manufacturer’ i Structions (Thermo Fisher) and according toa previous papet!™l The el ative cell viability was caleulated as the percentage of ells teated with Grugs compare to untreated contr cell Potent: The international application published under the patent cooperation treaty at World Intllectusl Propery Organation Inter rational Bureau, interational publication number 11201505579T/SC, WO2014/11374A1, and 10603380/US. ‘Supporting Information Supporting Information i avalble fom the Wiley Online Library o* fom the author Acknowledgements ‘Authors thank Taipei Veterans General Hospital Dr. iang's team to assist te collet blood sample and many thing. Authors thank Redemia Sinica (© 202 Wie. vou cmbADVANCED ‘SCIENCE NEWS wwwadvancedsciencenews.com for providing faites, This research was funded by the Ministry of Sci- ence and Technology (MOST) (project number. MOST110-2221-£007 D17-MY3) and Shakelton project (reject number: MOST108.2638-€-007 -001-MY2) and UCLA-Robert Benson Funds (R4744 AZ}. Conflict of Interest ‘The authors declare no confi of interest ‘Author Contributions HWY, VSG. and Y-CH. ae contbuted equaly to this work, Concep- tualzstion: FLC.T, CoM. and YY; methodology: C:H.0,, MMAR, LY. and CY software: MBMAR: validation: ¥-CH. HX. and VSG) resources: FCT, CMH, WY, JeKJ, EKC, PLW, HET, J Kl, and YW data curation H.V%. and ¥-GH cwiting orginal deat preparation: Y-CH. and H.V¥: writing review and editing: FT. and C-MH, supervised CTC, PDX and cll ine experiments, respectively FGI funding acquisition: F-G.T, YY, and CMM. 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