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Single cell proteomics for studying circulating tumor cells and monitor disease progression on breast cancer patients

Final Report Summary - CTCPROTEOMIC (Single cell proteomics for studying circulating tumor cells and monitor disease progression on breast cancer patients)

The objective of this project is to take a novel existing technology and to develop it as a clinical assay that is capable of tracking the response of patients to medical interventions and disease progression. This will lead to more efficient and effective patient care which could on its own save millions of Euros if implemented across the EU health systems. The technology can also potentially provide information on the development of the resistance of breast cancer to endocrine therapy. The planned research is aimed at the proteomic analysis at single cell level for circulating tumour cells (CTCs), as these cells shed from the primary tumour site and are identified in transit within the bloodstream of cancer patients with metastatic degree of the disease. The proposed work represents a highly multidisciplinary approach to engage the central challenge of our proposal, combining proteomic analysis, microfluidic antibody capture chips and an ‘all optical’ platform which accounts for cell manipulation, cell lysis and protein quantification with single-molecule sensitivity. The goals of the project include carrying out analysis at single cell level; the comprehensive proteomic study of cell-to-cell variations for the expression of cancer biomarkers, which ultimately led to the heterogeneity; and complexity and evolution at phenotypic level of the disease. The technology can be extended to other types of cancer samples where an investigation of proteomic phenotype at single cell level may unravel important information hidden within the heterogeneity of the cancer population.

Analysis of single cells at protein level represent a formidable technological challenge due to the intrinsic properties of proteins in terms of their concentration, composition and dynamic status to perform their tasks and keep the cell machinery in a working state. Indeed, in the last decades the technological development and availability of new molecular assays played a pivotal role in cancer research, accounting for major advances in basic knowledge of cancer biology, drug discovery and molecular targeted therapies. However, more recent observations in the last years have forced a major re- thinking of cancer biology, and new emerging features can hold the key to drive the future direction of cancer treatment. One critical input into the field comes from the recent discovery that neoplastic cells within individual tumours originating in a particular tissue are not a homogeneous population but account for different subpopulations at both genomic and phenotypic levels. As tumour progresses, carcinoma cells liberate themselves from the primary tumour mass and begin to strike out on their own and become invasive and capable of disseminate secondary tumour sites in distant organs. This mechanism is known as metastasis and it does account for more than 90 % of tumour deaths. The field of tumour metastasis is vast and deep investigations are not addressed yet.

However, of fundamental importance for the proposed research is that the population of cancerous cells within the tumour mass is composed by multiple sub-populations with different phenotypic characteristics, namely a phenotypic heterogeneity. The latter, accounts for exploitation from cancer cells of new traits, including invasiveness, mobility, acquired resistance to drugs and immortalization. Therefore, our approach offers the opportunity to investigate the phenotypic heterogeneity for target proteins known to be involved in cancer development and progression by analysing cell by cell. Circulating tumour cells (CTCs) represent a highly valuable sample for the cancer patient at a metastatic stage of the disease since they derive directly from the solid tumour masses in the epithelial organs but can be isolated directly from the peripheral blood stream of patients. Thus, CTCs represent a direct and fresh source of information on the state of primary tumour and therefore, CTCs are an ideal candidate to carry out proteomic studies aimed at a tumour interrogation.

During the project we overcame successfully a number of difficulties related to both the nature of the sample as well as the absolute quantification of protein biomarkers at single cell level. We developed a protocol to isolate CTCs from the blood of patients in a viable state to perform further protein analysis. The protocol is based on a multi-step purification to remove the majority of blood cells followed by an enrichment step with appropriate modified micro tubes coated with specific antibodies capable to catch CTCs based on the expression of their surface antigens. The method was optimized with the use of cell lines expressing different surface biomarkers as well as in different amounts to simulate the heterogeneity of tumour cells. The protocol was found capable to recover 60 % to 90 % of the cells spiked into blood depending by the lower or higher amount of surface biomarkers expressed, respectively. We successfully isolated CTCs from the blood of breast and ovarian cancer patients with a degree of purity which allow their further analysis.

We focus our attention on the analysis of p53 protein which is found in an altered state in more than 50 % of human cancer. We addressed successfully the optimization of a fully functional p53 assay in the microfluidic platform for the proteomic analysis of single cell was completed and is implemented routinely in the workflow. The capability of the assay were tested on a variety of cell lines in different conditions including drug challenging experiments to highlight the heterogeneous response of p53 in cell populations. The analytical assay performed with excellent sensitivity within dynamic range of from hundred to a million proteins per cell analysed. We began the discovery phase to identify a commercially available pair of antibodies for oestrogen receptor (ER) protein which is of fundamental importance in the development of breast cancer in particular where more than 70 % of breast cancer patient are ER positive. ER is also known to be involved in the resistance to endocrine therapy in breast cancer and therefore being able of reveal the ER variations at single cell may shed light on this mechanism. During the project a pair of antibodies with the desired specificity was identified but the assay did not exhibit enough sensitivity with our actual microfluidic platform for the analysis of single cell. This prompted the research to exploit the design of a new microfluidic platform to enhance the analytical capabilities down to an effective working range of protein concentrations for single cells.

The working capabilities of our p53 analytical platform prompted us to investigate further the heterogeneity of tumour samples respect to p53 expression. Despite the attempt to isolate CTCs from the blood of patients, the analysis of CTCs was limited by the lack of patients with the required characteristics to be enrolled in a pilot scheme for the project’s purpose. Therefore, we addressed our investigation to tumour samples of different origin. We successfully interrogate the p53 response in ovarian cancer cells derived from patients which have developed resistance to cisplatin. Our finding at single cell level suggest that cancer cells which evolved the resistance to cisplatin are able to reduce their level of p53 compared to their own basal level as well as to cisplatin sensitive ovarian cancer cell. This mechanism ultimately may result in an inefficient p53-mediated apoptosis and if confirmed may be potentially useful to better address the therapy of patients as well as suggest the design of new and more efficient drugs acting through different molecular target and pathways.

During the project we extend the range of application of our analytical platform to address a general hypothesis regarding the susceptibility to drug treatment of cancer cells during different phases of their cell cycle. We consider the fact that the targets of many drugs may exist in different states of conformation, concentration and modification within a cancer cell population given the heterogeneity of the sample. A major feature of cancer cells is the enhanced proliferation and mitotic duplication which naturally set the cells in different states of cell cycle phases at the time of treatment. Therefore, we decided to preliminary investigate the response in p53 protein to platinum based drugs which target DNA. We use an appropriate set of fluorescent reporters to discriminate between the different cell cycle phases prior the analysis and our first result revealed a preferential cell cycle phase for the expression of p53 protein. This is an intriguing fact and it can open new ways to treat cancer if confirmed by further evidence.

In conclusion, during the project we successfully developed a protocol for the isolation of CTCs from the blood of sample in a viable and suitable form to carry out target protein analysis in single cell. An existing technology was optimized and implemented to perform absolute protein quantification of targeted protein at single cell level and its application to a number of different problem in cancer cell analysis were addressed. We believe that our approach hold the potential to change in the future the way that cancer is managed. For instance, breast cancer accounts for over 4 % of deaths among the female population of Europe and often affects young women: over half the number of deaths occur before 65 years. This pathology is the main cause of mortality in women aged between 45 and 64 (over 12 % of deaths). In the only UK, each year more than 300.000 cases arise and they represent an important part of the use of health care resources. The incidence of mortality by breast cancer differs within the Europe and have regional trends in each country. Since there are a number of risk factors recognised as conducive to the development of breast cancer, and the geographical distribution of these cancers probably reflects an uneven spatial distribution of these, but cannot account for the mortality rates. Thus, the differences in mortality in the EU should be seen in the light of the specific national screening policies, since the seriousness of the prognosis depends mainly on how early the diagnosis is made. In this perspective, the availability of an analytical platform to address the response to treatment and monitor the development of tumours is of extreme importance to move towards a personalised approach to the real patient’s needs.