Etichettatura di cancro al seno xenotrapianti derivati da pazienti con tracciabili Reporter per crescita tumorale e metastasi Studi
Summary
We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.
Abstract
The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients’ tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.
Introduction
Lo sviluppo di xenotrapianti derivati da pazienti tumorali (PDXs), in cui i campioni di tumore chirurgicamente asportati vengono innestati direttamente in topi immuno-compromessi, offre diversi vantaggi rispetto ai modelli di xenotrapianto di cellule-linea standard e rappresenta un importante passo avanti nella ricerca sul cancro 1,2. PDXs possono essere mantenuti e ampliati con passaggi successivi con minima alterazione delle caratteristiche genetiche e biologiche del tumore coltivate nel primo passaggio; e riflettere più accuratamente l'eterogeneità del tumore di xenotrapianti derivate da linee cellulari tumorali umane 3-8. Questi modelli sono ora ampiamente utilizzate come piattaforma per la personalizzazione cancro terapeutica 9,10, come piattaforma preclinico nello sviluppo di farmaci 6,11 e come uno strumento sperimentale per lo studio della biologia del cancro 4,12.
La maggior parte dei PDXs vengono impiantati e propagate per via sottocutanea, che permette di tenerne la misurazione della crescita tumorale nel tempo utilizzando pinze. però, La malattia metastatica è stato più difficile da modellare con PDXs. In particolare per il cancro al seno, xenotrapianti con capacità metastatica a diversi organi sono stati descritti 3,5,13, ma la frequenza di diffusione spontanea di siti metastatici è estremamente bassa. Dove riportato, l'identificazione e la quantificazione degli oneri si basa metastatico in esame istologico laboriosa degli organi bersaglio post-mortem. linee cellulari di cancro che esprimono bioluminescente (luciferasi, Luc) o fluorescente (Green Fluorescent Protein, GFP) reporter gene sono comunemente utilizzati in modelli sperimentali di metastasi del cancro al seno a cervello, polmoni, ossa e il fegato dopo intracardiaca, la coda-venosa, intrafemoral e iniezione milza 14-16. Anche se questi modelli bypassare diffusione dei tumori primari, che sono preziosi per studiare i meccanismi di tropismo d'organo e di colonizzazione metastatica. Tuttavia, le cellule derivate da tumori di pazienti primari e PDXs possono avere bassi tassi di trasfezione o trasduzione using procedure standard. Una alternativa è quella di stabilire linee cellulari derivate PDX-17 in vitro, che può essere poi etichettati utilizzando protocolli di coltura dei tessuti convenzionali. Questo approccio tuttavia, non è adatto per l'etichettatura maggior PDXs, per cui linea cellulare derivazione è difficile e può modificare il fenotipo delle cellule. Qui vi presentiamo un protocollo per la trasduzione di cellule tumorali PDX-dissociate con vettori lentivirali adatti per l'imaging in vivo. Inoltre, descriviamo metastasi sperimentale con iniezione intracardiaca delle cellule dissociate PDX luc-GFP etichettati in topi immunocompromessi.
Un protocollo di base per la trasduzione di organoidi PDX-dissociate con gene reporter che esprime lentivirus è stato descritto in precedenza 18. Nel protocollo corrente si descrivono metodi aggiuntivi per arricchire per le cellule tumorali umane ed ottenere vicino efficienza di trasduzione 100%, così come l'uso di PDXs etichettati per rilevare il cancro al seno sperimentalemetastasi. Questo protocollo può essere adattato per l'etichettatura più tipi di cancro di PDXs con vari marcatori luminescenti e fluorescenti e la modulazione dell'espressione genica (cioè, shRNA knockdown di geni di interesse).
Protocol
Representative Results
Discussion
Critical steps within the protocol:
The use of high titer lentiviral particles (>108 TU/ml) is a critical step in the success of this protocol, as allows careful control of the media composition during in vitro transduction. While multiple methods for production of high-titer viral particles have been well described18,19; this protocol uses lentiviral particles produced as described in detail at www.kottonla…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors thank Dr. Darrel Kotton at Boston University for providing the pHAGE-EF1aL-dsRed-UBC-GFP-W vector and protocols for high titer lentiviral production used in these studies. This work was funded by DOD BCRP W81XWH-11-1-0101 (DMC), ACS IRG # 57-001-53 (DMC), NCI K22CA181250 (DMC) and R01 CA140985 (CAS).NCI P30CA046934 Center grant supported in vivo imaging and tissue culture cores at University of Colorado AMC.
Materials
| DMEM/F12 (1:1) | Hyclone | SH30023.01 | |
| bFGF | BD Biosciences | 354060 | |
| EGF | BD Biosciences | 354001 | |
| Heparin | Sigma | H4784 | |
| B27 | Gibco/Thermo Fisher | 17504-44 | |
| Anti-fungi-antibiotics | Hyclone | SV30010 | |
| Accumax | Innovative Cell Technologies | AM-105-500 | Digestion Buffer |
| FBS | Atlanta Biologicals | S11550 | |
| HBSS Red Ca++/Mg++ free | Hyclone | SH30031.02 | |
| Hepes | |||
| 10X PBS | Hyclone | SH30258.01 | |
| Cultrex | Cultrex | 3433-005-01 | Basement Matrix Extract (BME) |
| 30C shaker | NewBrunswick Scientific CO. INC | Series 25 Incubator Shaker | |
| 70um filters | Falcon | 7352350 | |
| scalpels | Fisher | 22079690 | |
| Clorhexidine disinfectant | Durvet | NDC# 30798-624-35 | |
| Red blood cell lysis reagent | Sigma | R7757 | |
| Neuraminidase | Sigma | N7885-1UN | |
| EpCAM (CD326+) microbeads* | Miltenyil Biotec | 130-061-101 | |
| Lineage cell depletion Kit, mouse* | Miltenyil Biotec | 130-090-858 | |
| MiniMACS Separator | Miltenyil Biotec | 130-042-102 | |
| Mini MACS Magnetic Stand | Miltenyil Biotec | 130-042-303 | |
| MS Columns | Miltenyil Biotec | 130-042-201 | MS or LS columns can be used, adjust to number of cells. |
| Illumatool Tunable light system | Lightools research | Various | For in vivo fluorescence imaging |
| Xenogen IVIS200 imaging device | Xenogen | Various | For in vivo luminiscence imaging |
| Human Cytokeratin Clone MNF116 Monoclonal antibody | DAKO | M0821 | Pan-cytokeratin |
| Epidermal Growth factor receptor antibody | Cell signaling | 4267S | EGFR |
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