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Cancer Research

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Published: September 30th, 2016

DOI:

10.3791/54393

1Medicine, University of Colorado Denver Anschutz Medical Campus

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Colorectal cancer (CRC) is a significant contributor to cancer deaths in the United States. In 2015, there were an estimated 132,700 new cases of CRC with 49,700 deaths 1. Although the prognosis in patients with localized disease is excellent, patients with advanced disease have poor outcomes, making this a major priority in the development of novel therapies. Despite standard of care chemotherapeutic regimens and newer biologics that are deployed against this disease, there has been only an incremental increase in overall survival. Accordingly, there is a significant effort in understanding the driver pathways involved in facilitating tumor growth in this ....

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Ethics Statement: Patient-derived colorectal adenocarcinoma tumor specimens were obtained from consenting patients at the University of Colorado Hospital in accordance with a protocol approved by the Colorado Multiple Institutional Review Board (08-0439). All animal work was performed under animal protocols approved by the University of Colorado Denver Institutional Animal Care and Use Committee (IACUC, Protocol # 51412(06)1E and 96813(04)1E).

1. Receiving and Preparing Patient Blood

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Similarities of Common Mutations in the CRC PDTX Models and the TCGA

We investigated whether the percentage of common mutations (KRAS, NRAS, BRAF, PIK3CA, APC, CTNNB1 and TP53) in the CRC PDTX bank were representative to the mutation frequency seen in the CRC patient population. As shown in Figure 2A (TCGA) and B (CRC PDTX bank), the frequency of mutations in these gene.......

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The PDTX drug discovery platform offers an improved model to the shortcomings of other preclinical models that are unreliable in predicting clinical activity of novel compounds. Importantly, tumors in this model are biologically stable, retain metastatic potential, and exhibit similar drug responsiveness from generation to generation. In this model, patient derived tumors are injected into athymic nude mice, passaged, and subsequently used in therapeutic evaluation. There are several critical steps for a successful PDTX .......

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This work was supported by grant 1R01CA152303-01.

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Name Company Catalog Number Comments
RPMI or DMEM Corning 10-040-CV
Penicillin-Streptomycin Corning 30-002-CI
Non-essential Amino Acids Corning 25-025-CI
Fetal Bovine Serum Corning 35-010-CV Thaw in -4 °C, then activate for 30 minutes at 60 °C water bath
CPT blood tube BD vacutainer 362761
Microcentrifuge tube Surelock A-7002
Phospate-Buffered Saline Corning 21-040-CV
Cyrogenic vials Cyroking C0732901
Plastic tumor cutting dish Trueline TR4001
Scissors Roboz RS-5881
Forceps Roboz RS-5135
Matrigel (gelatinous protein mixture) Corning 354234 Store at -20 or -80 °C, then thaw on ice, do not leave at room temperature
10% Formalin cups Protocol 032-059
Liquid Nitrogen Dewar Storage Thermolyne CY50900
Portable liquid nitrogen dewar Nalgene 4150-2000
Dimethyl Sulfoxide Fischer 67-68-5
Freezing container: Mr Frosty Nalgene 5100-0001
Isopropyl Alcohol Decon 64-17-5
Trocars Innovative Research of America MP-182
Anesthesia machine Patterson Veterinary none
Anesthesia box Patterson Veterinary none
Isoflurane Vet one 1038005
F-Air Canister Bickford Omnicon 80120
Meloxicam Vet one 5182-90C
Calipers Fowler 54-100-167
Weight scale Ohaus Scout Pro SP601

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