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Metastases, rather than primary tumors, are responsible for over 90% of cancer patient deaths. Understanding how tumors acquire the ability to invade and metastasize is essential for the identification of new targets and development of therapies against these processes.
Metastasis is a multistep process which involves the ability of cancer cells to migrate (cell motility), invade into surrounding tissues, intravasate in blood vessels or lymphatics, extravasate and grow at new sites (See figure 1). Because not all cells within the primary tumor acquire these abilities, metastasis is a very inefficient process and therefore all the different steps must be successfully completed(1-3).
1.1 How can we study metastasis?
Over the past decades studies of metastasis have provided us with a lot of useful information, but details of the processes involved in metastasis were mainly unclear. As mentioned before, for metastasis, cells need to migrate and invade into surrounding tissues. These two processes are thought to be the key steps in systemic spread of cancer and this migration continues after the primary tumor is removed. Understanding the mechanisms behind migration and invasion will provide new information that is needed to prevent the formation of metastases elsewhere in the body. To study this typical behavior of metastasizing tumor cells a technique called intravital imaging can be used. With this technique it is possible to image tumor cells in vivo in living mice (See figure 2) (4-7).
What is causing the behavior of metastasizing tumor cells? It has been suggested that the tumor microenvironment plays a very important role. Stimuli from tumors affect components of the extracellular matrix (EMC; such as collagen) and cells present in the environment of the tumor (such as T cells, macrophages and endothelial cells). These EMC components and cells have regulatory functions mediated by proteolytic enzymes, cytokines, growth factors and angiogenesis-promoting factors (8-10). It is not possible for a cancer cells to develop into an invasive cancer without interactions with these cells and components present in the tumor environment (11).
How does the immune system play a role in during cancer development and progression? How does an organism react to cancer cells. The main function of the mammalian immune system is to monitor tissue homeostasis, to protect against invading of infectious pathogens and to eliminate damaged cells. It is therefore surprising that cancer occurs with such a high frequency in humans (9).
To study the tumor microenvironment during breast cancer metastasis experimental mouse models (such as BALBcc and FVB/ola) are available in which mouse mammary tumor cells (fluorescent labeled) are injected into the mammary gland.
It has been suggested that the tumor microenvironment plays an important role during these steps.
To study the tumor microenvironment during breast cancer metastasis we use mouse models (BALB/c and FVB/Ola) in which we inject mouse mammary tumor cells into the mammary gland. Because injection of non-self tumor cells (e.g. human or rat) activates an immune response, injected cells are eliminated by immune cells. To prevent such a response, SCID (no T and B cells) or nude (no T cells) mice are used. However, mice lacking an immune response cannot be used to study the influence of the microenvironment.
For this reason we want to create a new mouse model in which the immune response is suppressed. To suppress the immune response we want to inject cells that are in a senescence-like state (cells are unable to divide without loss of cell viability). This way the immune system will hopefully recognize the injected cells as 'self' instead of 'non-self' (reference to the paper where they show this for rats).
Having a mouse model that is 'compatible' to a (breast) cancer cell line we want to study, allows us to study any cell line we want, especially breast cancer cell lines from the clinic (patient).
Conclusion: Having a mouse model in which all the immune players are intact allows us to study the first steps of metastasis and hopefully helps us to identify the key cells that play a role in the processes of migration and invasion.
How can we study the tumor microenvironment?
As mentioned before intravital imaging is a technique used to image tumor cells in vivo in living mice. The tumor microenvironment plays an important role during the first steps of metastasis, especially the immune response during these steps (immunosurveillance).
To determine which immune cells are involved, we would like to visualize them in vivo and in vitro.
In vivo visualization of immune cells requires genetic manipulation, which is time consuming.
In vitro visualization of multiple immune cells is difficult (time consuming and material consuming) due to lack of colors.
For this reason we want to explore a technique called fluorescence lifetime imaging microscopy (FLIM), which should enable us to visualize multiple cell types simultaneously in vivo or in vitro.
Explain definition Lifetime
It has been shown that endogenous molecules that are present in every cell are fluorescentïƒ autofluorescence, and these molecules have different lifetimes
The molar ratio of these molecules is different among various tissues, therefore the average lifetime of different tissues varies (see ref ….)
Other studies have already shown that this technique could be useful to distinguish between normal en tumor tissues. We want to explore this further by investigating if we can employ this technique to distinguish between different cell types.
Can we induce a senescence-like state in rat mammary tumor cells by a chemical, irradiation or molecular approach?
Is it possible to distinguish between normal and tumor cells in vitro and in vivo based on (auto) fluorescence lifetime?
2. Materials and methods
Cell culture and treatment
Kep1-11 and Kep1-11 Dendra-2 cells (mouse mammary tumor cells derived from K14cre;Cdh1F/F;Trp53F/F mice(22)) were cultured in DMEM-F12 (Gibco) supplemented with 5% heat-inactivated bovine calf serum (FCS), 100 IU/ml penicillin, 100 µg/µl streptomycin, 5 ng/ml insulin and 5 ng/ml epidermal growth factor (EGF, Sigma). MTLn3-wt cells (rat mammary carcinoma cells) were cultured in MEM-alpha (Gibco) supplemented with 5% FCS, 100 IU/ml penicillin and 100 µg/µl streptomycin. HUVEC cells (Human endothelial cells from the umbilical vein) were cultured in endothelial basal medium (Clonetics EBM-2, Lonza) supplemented with 10 ml FBS, 0.2 ml Hydrocortisone, 2 ml hFGF-B, 0.5 ml VEGF, 0.5 ml R3-IGF-1, 0.5 ml ascorbic acid, 0.5 ml hEGF, 0.5 ml GA-1000 and 0.5 ml heparin in a total of 500 ml. MDCK cells (Madin-Darby Canine Kidney Cells) were cultured in DMEM supplemented with 10% FCS, 100 IU/ml penicillin and 100 µg/µl streptomycin.
For experimental purposes, Kep1-11 cells were cultured together with HUVEC imaged in L15-medium (Leibovitz) supplemented with L-glutamine, but no phenol red (Gibco).
To induce a senescence-like phenotype or cell cycle arrest cells were treated with different concentrations of doxorubicin, methotrexate or irradiated with low doses of Grey.
The fluorescence lifetime