A Melanoma Patient-Derived Xenograft Model

Summary

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Abstract

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Introduction

Preclinical models are critical for all aspects of translational cancer research, including disease characterization, discovery of actionable vulnerabilities unique to cancer versus normal cells, and the development of efficacious therapies that exploit these vulnerabilities to increase the overall survival of patients. In the melanoma field, tens of thousands of cell line models have been heavily utilized for drug screening, with >4,000 contributed by our group alone (WMXXX series). These cell line models were derived from melanoma patients with various forms of cutaneous melanoma (i.e., acral, uveal, and superficial spreading) and diverse genotypes (i.e., BR....

Protocol

The following animal protocols follow the guidelines of The Wistar Institute’s humane ethics committee and animal care guidelines.

1. Melanoma tumor tissue collection

1. Collect tumor tissue (termed passage 0) from melanoma patients by one of the following surgery or biopsy methods.
   1. For surgical excision tissue, maintain a minimum of 1 g of tissue (resect metastases and primary lesions) in transport storage media (RPMI 1640 + 0.1% fungizone + 0.2% gentamicin) at 4 °C or on ice.
   2. For surgical biopsy tissue, maintain less than 1 g of tissue (often punch biopsies of subcutaneous [s.c.] metastases and l....

Results

Tumor tissue for melanoma PDX models can come from a variety of different sources and can also be processed per the growth dynamics of individual models and the desired use of the PDX tissue. The priority when establishing a PDX model is to have sufficient material to bank for future use and DNA for characterization (Figure 1).

Once sufficient material is banked, tumor tissue can be expanded in one of three main methods to grow enough tumor to perform a formal the.......

Discussion

We have herein described generating PDX models of melanoma with patient tissue derived from primary and metastatic tumors, core biopsies, and FNAs. When directly engrafted into NSG mice, tumors present similar morphologic, genomic, and biologic properties to those observed in the patient. In the case when only a small quantity of tissue is available to investigators, as often occurs with FNAs, the PDX technique allows for the expansion of the tumor tissue for DNA, RNA, and protein characterization, as well as for therapy.......

Materials

Name	Company	Catalog Number	Comments
1 M Hepes	SIGMA-ALDRICH CORPORATION	Cat # H0887-100ML
100x PenStrep	Invitrogen	Cat # 15140163
1x HBSS-/- (w/o Ca++ or Mg++)	MED	Cat # MT21-023-CV
2.5% Trypsin	SIGMA-ALDRICH CORPORATION	Cat # T4549-100ML	10 mL aliquots stored at –20oC
BSA	SIGMA-ALDRICH CORPORATION	Cat # A9418-500G
Chlorhexidine	Fisher Scientific	Cat# 50-118-0313
Collagenase IV (2,000 u/mL)	Worthington	Cat #4189	make up in HBSS-/- from Collagenase IV powder stock (Worthington #4189, u/mg indicated on bottle and varies with each lot); freeze 1
DMSO	SIGMA-ALDRICH CORPORATION	Cat # C6295-50ML
DNase	SIGMA-ALDRICH CORPORATION	Cat # D4527
EGTA (ethylene glycol bis(2-aminoethyl ether)-N,N,N’N’-tetraacetic acid)	Merck	Cat # 324626.25
FBS	INVITROGEN LIFE TECHNOLOGIES	Cat # 16000-044
Fungizone	INVITROGEN LIFE TECHNOLOGIES	Cat # 15290-018
Gentamicin	FISHER SCIENTIFIC	Cat # BW17518Z
Isoflurane	HENRY SCHEIN ANIMAL HEALTH	Cat # 050031
Leibovitz's L15 media	Invitrogen	Cat # 21083027
Matrigel	Corning	Cat # 354230	Artificial extracellular matrix
Meloxicam	HENRY SCHEIN ANIMAL HEALTHRequisition # ::Henry Schein	Cat # 025115	1-5mg/kg, as painkiller
NOD/SCID/IL2-receptor null (NSG) Mice	The Wistar Institute, animal facility		breeding
PVA (polyvinyl alcohol)	SIGMA-ALDRICH CORPORATION	Cat # P8136-250G
RPMI 1640 Medium (Mod.) 1X with L-Glutamine	Fisher Scientific	Cat# MT10041CM
Scalpel	Feather	Cat # 2976-22
Virkon	GALLARD-SCHLESINGER IND	Cat # 222-01-06
Wound clips	MikRon	Cat #427631