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Experimental evidence of stem cells for metastasis

For the first time, scientists from Heidelberg have characterised cancer cells that initiate metastasis in the blood of breast cancer patients using an in-vivo xenograft mouse model. These cells have the properties of cancer stem cells and are characterised by three surface molecules that can be used as biomarkers for disease progression.

The original tumour (primary tumour) is not the main cause of cancer-related deaths, but rather the development of metastases in other organs. Metastases develop as individual cancer cells that break away from the primary tumour, circulate through the bloodstream or the lymph (circulating tumour cells, CTCs) and form a secondary tumour in a different organ. After a rest, which can last months or even years, the tiny micrometastases develop into large secondary tumours, which often are untreatable and lead to the death of the patient.

Even small primary tumours can metastasise; for example, one in five breast cancers of a centimetre in diameter already contain metastases, which frequently spread to the bones. The reason why certain tumour types prefer specific organs is as yet poorly understood.

Circulating tumour cells and cancer stem cells

Stem cells in a nitrogen tank. © HI-STEM

Cancer cells that circulate in a patient’s blood can be detected with specific biomarkers. Studies have shown that breast cancer patients with a large number of CTCs have a poor prognosis. However, up until now, experimental evidence was lacking as to whether CTCs can develop into metastases. Many breast cancer patients do not develop metastases even though they have CTCs circulating around their blood.

“We were convinced that only very few of the various circulating cancer cells are capable of forming a secondary tumour in a different organ,” says Prof. Dr. Andreas Trumpp, head of the DKFZ’s Department of Stem Cells and Cancer, director of the Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH) and coordinator of the metastasis stem cell project. The researchers were therefore aiming to find this small subpopulation of metastasis-initiating cells (MICs) which are resistant to therapy, very mobile and therefore also known as metastasis stem cells.

Dormant stem cells are resistant to therapy

The internationally renowned stem cell researcher Trumpp has over the last ten years made a significant contribution towards a breakthrough in cancer stem cell theory. Theory holds that tumours are hierarchically organised and only a few cancer stem cells are responsible for the growth of the tumour.

Such cancer stem cells, which were initially identified in leukaemia, but have since also been demonstrated for many other tumour types, hide in a special microenvironment (the so-called stem cell niche, often in the bone marrow) like normal stem cells. This niche contains dormant cells that divide relatively rarely and are therefore resistant to antiproliferative chemotherapies. This also explains why chemotherapies are initially successful and primary tumours, whose large cell mass is only slightly or not at all resistant to treatment, shrink. However, months or even years later, single surviving cancer stem cells might become reactivated and cause tumour relapse. These secondary tumours are largely resistant to chemotherapy drugs (see link on the right-hand side: "Metastasis-inducing cancer stem cells", 11th May 2009).

A transplantation test for the detection of MICs

Dr. Irène Baccelli © HI-STEM

Trumpp and his colleague Klaus Pantel from the Institute of Tumour Biology at the Hamburg University Hospital have developed a novel functional xenograft model system for testing circulating cancer cells for their ability to metastasise. With this test, specifically labelled cancer cells from the blood of breast cancer patients can be transplanted directly into the bone marrow of immunodeficient mice. Human metastases that form at a later stage can then be detected in this in-vivo animal model using a non-invasive monitoring approach. 

However, before the researchers were able to do this, CTCs with potential MICs had to be isolated from the blood of breast cancer patients. Dr. Irène Baccelli from Trumpp’s team analysed the blood of more than 350 breast cancer patients. She isolated circulating tumour cells using specific surface molecules and directly transplanted them into the femoral bone marrow of the mice with defective immune systems. Patient samples and clinical data were provided by Professor Dr. Andreas Schneeweiss and his team from the National Center for Tumour Diseases (NCT) in Heidelberg and histological analyses were carried out by Wilko Weichert from the Institute of Pathology at the Heidelberg University Hospital.

After many transplantations, metastases actually started forming in the bones, lungs and livers of some of the animals. Immunohistochemistry involving human-specific antibodies showed that the tumour cells actually originated from the breast cancer patients. This proved that CTCs contained metastasis stem cells, although with a low frequency. In subsequent experiments, Baccelli and her colleagues analysed the surface molecules of those CTCs where cell transplantation had led to metastases (MICs) in order to characterise their molecular properties.

Prognostic biomarkers

Circulating tumour cells isolated from the blood of breast cancer patients, which formed a metastasis in the bone marrow of the mice. The stem cell marker CD44 is stained red. © Irène Baccelli, DKFZ

CD44 is a typical protein on the surface of breast cancer stem cells. This protein helps cells to settle in the bone marrow. Baccelli first isolated CD44-positive cells from the patients’ CTCs and then screened this cell population for specific surface markers which help the cells to survive in foreign tissue. These include, for example, a signalling molecule that protects the cells from attacks by the immune system (CD47) and a surface receptor that enhances the cancer cells’ migratory and invasive capabilities (MET).

Using a state-of-the-art fluorescence-activated cell sorter (FACS®), the researchers were able to isolate those CTC subpopulations that exhibited all three characteristics (CD44, CD47, MET) simultaneously. Another round of transplantation tests showed that these triple-positive cells were the cells from which the metastases originated (MICs). 

New therapeutic approaches

Prof. Dr. Andreas Trumpp © HI-STEM

Clinical data confirmed the results. In a small patient group, the researchers observed that as the disease advances, the number of triple-positive cells increases, but the total number of CTCs does not. In addition, patients with very high numbers of triple-positive cells had particularly high numbers of metastases and a much poorer prognosis than women in whom only few of these metastasis-inducing cells were detected. 

It seems that the triple-positive MICs have “a substantially higher biological relevance for disease progression than previously studied CTCs,” Andreas Schneeweiss explains. The researchers plan to confirm the results in a large clinical study.

The triple-positive cells are also a prospect for potential new therapeutic approaches for treating advanced breast cancer. Therapeutic antibodies targeting CD47 to inhibit its functions are already being developed. A substance inhibiting the activity of the MET receptor has already been approved and shows a good level of effectiveness in the treatment of advanced non-small-cell lung cancer. The substance may also help breast cancer patients with detectable metastasis-initiating cells. 

“The triple-positive cells we have found turn out to be not only a promising biomarker of disease progression in breast cancer but also a prospect for potential new therapeutic approaches for treating advanced breast cancer,” says Andreas Trumpp, summarising the promising results obtained from metastasis-initiating cells, which were published in the internationally renowned journal Nature Biotechnology in April 2013.

Baccelli I, Schneeweiss A, Riethdorf S, Stenzinger A, Schillert A, Vogel V, Klein C, Saini M, Bäuerle T, Wallwiener M, Holland-Letz T, Höfner T, Sprick M, Scharpff M, Marmé F, Sinn HP, Pantel K, Weichert W, Trumpp A: Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograph assay (2013). Nature Biotechnology 2013, Jun; 31(6):539-44.

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