Ex vivo-behandling Reaktion fra primære tumorer og / eller associerede Metastaser til præklinisk og klinisk udvikling af Therapeutics
Summary
Etablerede cancer cellelinjer og xenografter har været grundpillen i kræftforskning i de sidste mange årtier. Seneste oplysninger tyder dog på, at terapeutiske reaktion i høj grad er påvirket af tumorcellens mikromiljø. Derfor har vi udviklet en ex vivo analyse af primær tumor prøver for lægemiddeludvikling formål.
Abstract
The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.
Introduction
Udvikling af effektive kræftlægemidler har vist sig at være ekstremt udfordrende. Cancercellelinjer og tumor eksplantater – samt xenotransplantater er blevet anvendt i kræftforskning i over et halvt 1,2,3 århundrede. Til dato er uundværlig den molekylære analyse af narkotika følsomhed og modstand i både etablerede cancer cellelinjer og patient-afledte xenografter (PDX). Men afprøvning af forbindelser i etablerede cancercellelinier er ikke ofte prædiktive for effektivitet in vivo, og den tilsvarende in vivo undersøgelser i dyr, især i PDX modeller er meget dyrt og tidskrævende. Begrænsningerne af disse modelsystemer, nemlig manglende evne til at informere om indflydelsen af det native mikromiljø i tumorudvikling og respons på terapeutiske strategier har ført forskningsområde at udvikle yderligere metoder til at komplementere disse analyser. Af de seneste, forstærkes opmærksomhed mod ex vivo analyse af patientens tumeller eksplantater 4, 5 som følge af større forståelse for, at kræft terapeutisk respons er ikke eksklusivt til iboende molekylære sammensætning af cancerceller, men snarere er stærkt præget af tumorcellens mikromiljø 6, 7 en funktion, der ikke kan blive gentaget ved traditionelle dyrkningsmetoder og / eller PDX. Ex vivo analyse i ovennævnte sammenhæng (dvs.., påvirkning af tilstødende omgivende tumor celle mikromiljø) indebærer vurdering af levedygtige primære tumor / metastase sektioner, snarere end ex vivo analyse af cellulære isolater 8, 9.
Vi rapporterer her om en ex vivo teknik (dvs.., Præcision-skiver friske vævssnit) af både patientens primære tumorer og tilhørende metastaser (dvs. lymfeknuder), der trofast informerer om respons (IC50), off-target effekter og muliggør molekylær Analyse af resistens og feedbackmekanismer. Derudover en korrelativ analyse af therapeutic følsomhed / resistens mod biomarkør og genekspression profil kan udføres i et forsøg på at identificere patienter mere tilbøjelige til at reagere på det eksperimentelle stof af interesse (dvs.. høj lægemiddelrespons matcher patient med særlige biologiske profil). Anvendelse af ex vivo teknik og vurdering i en multi-parameter mode er bevægelse i retning af patientens valg og en generel forbedring af de kliniske resultater.
Ex vivo-behandling respons analyse kunne blive et standard værktøj i præklinisk og klinisk udvikling af kræftmedicin og er planlagt som et skridt i retning af en skræddersyet medicin tilgang i terapeutiske udviklingsstrategier.
Protocol
Representative Results
Discussion
Kræft biologer står over for betydelige udfordringer, når de forsøger at udvikle effektive terapeutiske strategier. Test narkotika i udviklingen på etablerede cancercellelinjer kan ikke præcist afspejler in vivo respons og in vivo forsøg på PDX modeller er arbejdskrævende og meget dyrt. I betragtning af ovenstående, er anvendelsen af ex vivo-teknikker til primær patient tumorer 14, 15 er nu placeret ved siden af den molekylære analyse af etablerede kræft cellelinjer og …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank the MSKCC Tissue Procurement Service Team (TPS), specifically, Maria Corazon Mariano, Priscilla McNeil, Anas Idelbi, Daniel Navarrete and Katrina Allen, in all of their efforts in the successful pursuit of this project and funding from the following sources: 5 R21 CA158609-02 and the Conquer Cancer Foundation and the Breast Cancer Research Foundation. In addition, the authors would like to thank Eric Cottington PhD, Vice President of the Office of Research and Project Administration, the Office of Technology Development, Research Outreach and Compliance and RTM Information Systems Support, in the support of the submission of this manuscript.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Vibratome Leica VT1000s | Leica | 14047235613 | |
| UltraPure agarose | Invitrogen | 16500500 | Prepare 4% and 6% before use |
| Injector blade | Ted Pella | 121-4 | |
| MEM with Penicillin + Streptomycin | Media Core Facilities (MSKCC) | The media is prepared at Memorial Sloan Kettering Cancer Center | |
| Scalpel no. 10 | Thermo Scientific | 31-200-32 | |
| Disposable forceps | Cole-Parmer | 84011182 | |
| Embedding mold | Electron Microscopy Science | 70181 | |
| FBS (heat inactivated) | Gemini | 100106 | |
| 24 well plates | Corning | 3524 | |
| Formalin (10%) | Sigma Diagnostics | SDHT501128 | |
| 16% Formaldehyde solution | Thermo Scientific | 28908 | |
| Embedding microsettes | Simport | M503-2 | |
| Ethanol (70%) | Fisher Scientific | A405P-4 | |
| Waterbath | Fisher Scientific | 15-462-2SQ | |
| Microwave | General Electric | ModelJES2051DNBB | |
| Adhesive (Ethyl Cyanoacrylate) | Sigma-Aldrich | E1505-5G | |
| 10mm dishes | BD Falcon | 353003 | |
| 15ml tubes | BD Falcon | 352096 |
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