An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Summary

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Abstract

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time ^1-4.

The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis ^5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression ^8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes ^1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.

in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously ^1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies^15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

Protocol

1. Cell culture maintenance of dormant and metastatic tumor cell lines

1. Grow dormant (D2OR/ MCF7/K7M2-AS.46) and metastatic tumor cells (D2A1/ MDA-MB-231/ K7M2) in 10 cm culture plates containing Dulbecco's Modified Eagle's Medium (DMEM) high glucose and 10% Fetal bovine serum (FBS) and antibiotics. Once the cells reach 70-80% confluence, proceed to the following assays.

2. Cell proliferation assay of dormant (quiescent) and metastatic (proliferating) tumor cells cultured in a 3D-BME system

Culturing dormant/metastatic cells in the 3D system

1. Thaw Cultrex growth factor-reduced Bas....

Discussion

The underlying mechanisms that maintain disseminated tumor cells in a dormant state or result in their transition to metastatic growth remain largely unknown. This phenomenon has been extremely difficult to study in human patients ^4,12 and few preclinical models have been developed to address this issue. Nevertheless, some in vivo and ex-vivo model systems for tumor dormancy have been characterized (reviewed in ^1,12). However, the in vivo models for tumor dormancy can p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM high glucose	Invitrogen	11965-118
DMEM low glucose	Invitrogen	11885-092
Fetal bovine serum (FBS)	Invitrogen	10091-148
Growth factor-reduced 3-D Cultrex Basement Membrane Extract	Trevigen Inc.		Protein concentration between 14-15mg/ml
D2.0R and D2A1 cell lines			5,19
K7M2 and K7M2AS1.46 cells			20
MCF-7 and MDA-MB-231 breast cancer cells	ATCC
An 8 chamber glass slide system	Lab-Tek	177402
Cell Titer 96 AQueous One Solution cell proliferation assay kit	Promega Corp.	G3580
VECTASHIELD mounting medium with DAPI	Vector Laboratories	H-1200
Normal donkey serum	Jackson ImmunoResearch	017-000-121
Elisa Plate Reader	Bio-Tec		Record 490nm
Confocal microscope	Carl Zeiss, Inc.		Magnification x63