1Department of Biology, University of Haifa, 2Transgenic Oncogenesis and Genomics Section, Laboratory of Cancer Biology and Genetics, National Cancer Institute
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Barkan, D., Green, J. E. An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth. J. Vis. Exp. (54), e2914, doi:10.3791/2914 (2011).
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 studies15-17. 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.
1. Cell culture maintenance of dormant and metastatic tumor cell lines
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
3. Immunofluorescent staining for cell signaling molecules in dormant (quiescent) tumor cells and/or metastatic (proliferating) tumor cells
Culturing dormant/metastatic cells in the 3D system for immunfluorescence staining
*The following protocol is a modification of a 3D culture protocol published by Debnath J et al 18.
4. Representative Results:
An example of a proliferation analysis of the dormant D2.0R and metastatic D2A1 tumor cells in the 3D culture is shown in Figure 1A. D2.0R cells are dormant (quiescent) through the entire experimental 14 day culture period whereas the highly metastatic D2A1 cells remain dormant only for four to six days after which they begin to proliferate. During the initial dormant phase, many cells remain solitary in the 3-D culture (Figure 1B; day 4) whereas other non-proliferating cells form multi-cellular spheroids. The transition of D2A1 cells from a dormant to proliferative state in 3-D culture (Figure 1B; day 12) is associated with dramatic changes in cell morphology. Hence, this assay can be used to test what factor/s may trigger dormant D2.0R cells to emerge from their dormant state and what factor/s may prevent D2A1 cells to transition from their dormant state. Figure 2 is an example of an agent preventing D2A1 cells to transition from dormant to a proliferative state. As illustrated in Figure 2, treatments of D2A1 cells with a specific inhibitor of myosin light chain kinase (ML- 7) maintained D2A1 cells in a dormant state.
Cell signaling in the dormant and proliferating tumor cells cultured in the 3D system can be studied by immunofluorescence staining for cell signaling molecules. As illustrated in Figure 3 a significant increase in myosin light chain phosphorylation in D2A1 cells (red staining) followed by reorganization of the f-actin filaments forming actin stress fibers (green staining) occurs during their transition from dormancy (1-4 days) to proliferation (day 7). However, blocking myosin light chain kinase activity in D2A1 cells by shRNA or specific drug (ML-7) retains D2A1 cells in a dormant state and results in inhibition of myosin light chain phosphorylation and f-actin stress fiber organization (Figure 4).
Figure 1. in vitro model to study solitary tumor cell dormancy and the switch to metastatic growth. A) Proliferation of dormant D2.0R and metastatic D2A1 in 3-D Cultrex BME, n=8 (mean ± SE). Representative results of three experiments (* p≤0.05). B) Light microscopy images of D2.0R and D2A1 cells cultured in 3-D Cultrex BME magnification x20. Figure modified from Barkan et al 17.
Figure 2. Preventing the switch of D2A1 cells from dormancy (quiescence) to proliferation in the 3D culture system by inhibition of myosin light chain kinase (MLCK). Time course of D2A1 cell proliferation cultured in 3-D Cultrex BME , n=8 (mean ±SE). Cells were untreated (control), or treated with a specific inhibitor of MLCK (ML-7; 5 μM) for 48 hr beginning on culture day 5. Figure modified from Barkan et al 17.
Figure 3. Myosin light chain phosphorylation followed by f-actin reorganization during the switch of D2A1 cells from dormancy to proliferative growth. D2A1 cells were cultured in 3-D Cultrex BME on 8 chamber glass slide. Cells were fixed and stained with DAPI (blue) for nuclear localization, phalloidin (green) for f-actin and with an antibody against the phosphorylated form of myosin light chain (MLC-p) (red), as indicated at various time points. Merge of f-actin, and MLC-p staining (yellow). Expression of MLC-p was increased during the transition of D2A1 cells from dormancy (days1-4) to proliferative growth (day 7) followed by actin stress fiber formation (arrows). Confocal microscopy, magnification x63. White bar equals 20 microns. Figure modified from Barkan et al 17.
Figure 4. Inhibition of myosin light chain kinase (MLCK) mediated f-actin stress fiber formation in D2A1 cells. D2A1 Cells were untreated (control), or treated with inhibitor for MLCK (ML-7; 5 μM), for 48 hr beginning on culture day 5, or treated with scrambled or MLCK shRNA and stained for the phosphorylated form of myosin light chain (MLC-p) (red), f- actin (green), and nuclei (blue). Merge of f-actin, and MLC-p staining (yellow). Confocal microscopy, magnification x63. White bar equals 20 microns.
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 primarily be utilized to validate potential mechanisms regulating tumor dormancy, but are not amenable to explore in real time the biology of a single disseminated dormant tumor cell.
The 3-D system presented here models for the first time solitary tumor dormancy and the switch to metastatic growth in an in vitro model system. The proliferation assay for modeling tumor dormancy (quiescence) and the transition to proliferation in the 3D system can be followed over time. This assay can be utilized to study in a high throughput manner and in a relatively short time frame potential factors/genes that maintain dormant tumor cells in their quiescent phase or may activate them to proliferate. Furthermore, the proliferation assay can be utilized as a high throughput platform to screen for additional tumor cell lines that may have a dormant phenotype.
Studying the molecular mechanisms which governs tumor dormancy or its switch to metastatic growth in the 3D system by conventional biochemical methods (RT-PCR/ western blots) is difficult given the low amount of RNA/protein that can be extracted for biochemical studies from the dormant tumor cells. However, immunofluorescence staining of cell signaling molecules of the dormant and outbreaking tumor cells in the 3D model system, as presented in Figures 3 and 4 can be applied. Hence, the model system presented here can serve as an efficient tool to begin to explore the molecular mechanisms regulating solitary tumor cell dormancy and the transition to metastatic growth.
No conflicts of interest declared.
This research was supported in part by the Intramural Research Program of the National Cancer Institute.
|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|