Periodic Reporting for period 3 - INJURMET (Impact of tissue injury induced by diagnostic biopsies and surgery on cancer metastasis)
Berichtszeitraum: 2022-08-01 bis 2024-01-31
Background: Prostate and breast cancer belong to the most frequent tumor types occurring in the European Union with an incidence rate of 139.5 and 139.0 cases per 100,000, respectively. Needle biopsy is required for diagnosis of these cancers and primary surgery to remove the malignant lesion. Biopsies and primary surgery cause local trauma of the targeted organ with potential risk of releasing tumor cells into the surrounding tissue or blood vessels. Since the systemic spread of tumor cells from the primary lesion to distant organs is a key event in cancer progression, it is of high importance to investigate the role of tissue biopsy and surgery on the spread of cancer cells and the formation of metastasis. The lack of reliable detection and characterization assays hampered the investigation of circulating tumor cell (CTC) release caused by biopsy or surgery for many years.
Objectives: This project aims to analyze whether needle biopsies of suspicious lesions in the breast or prostate and surgical removal of the malignant lesion is accompanied by an increased release of tumor cells into the blood circulation. Moreover, we will characterize the biology of CTCs released into the blood and compare the results with the clinical outcome of the patients. Taken together, the INJURMET project will provide a comprehensive set of data on the clinically relevant question whether biopsies and surgery may have a negative side effect in some patients through the release of metastasis-competent CTCs or not.
Objectives: This project aims to analyze whether needle biopsies of suspicious lesions in the breast or prostate and surgical removal of the malignant lesion is accompanied by an increased release of tumor cells into the blood circulation. Moreover, we will characterize the biology of CTCs released into the blood and compare the results with the clinical outcome of the patients. Taken together, the INJURMET project will provide a comprehensive set of data on the clinically relevant question whether biopsies and surgery may have a negative side effect in some patients through the release of metastasis-competent CTCs or not.
Objective 1 - Monitor CTC and ctDNA release during biopsy and surgery: Most of our work in the first reporting period has been focused on the recruitment of patients with breast and prostate cancer and the monitoring of CTC release. Blood samples were taken before and after biopsy and surgery to assess the potential release of CTCs. We have applied two complementary technologies for CTC detection that are based on different enrichment principles in parallel to catch EpCAM-positive and EpCAM-negative CTCs encompassing the range of tumor cells in the spectrum epithelial to mesenchymal transition (EMT). These technologies are the CellSearch System (Fig. 1 A) and the marker independent instrument, which takes advantage of the different size and deformability of CTCs, the Parsortix (Fig. 1 B). The use of two different enrichment technologies allowed us to assess differences between the enrichment methods that are based either on biological or physical properties of the cells.
Objective 2 - Assess primary tumor biology associated with CTC release: The collection of primary tumor tissue has started in collaboration with the departments of pathology of our clinical partners, and it will be performed in the second funding period depending on the results of the monitoring and characterization of the CTCs. The analyses will include next generation sequencing and inflammation profiling.
Objective 3 - Characterize the biology of CTCs released during biopsy and surgery: We have started to capture individual CTCs and perform a phenotypic and genotypic characterization at the single cell level. This will give novel insights into the biological characteristics of CTCs in early-stage breast and prostate cancer, which is still rare because most CTC characterization studies published in the literature were focused on patients with advanced disease. We have continuously optimized our protocols for single cell CTC analysis based on new technical developments (e.g. investment of a new high-resolution microscope with scanning abilities by internal UKE funds). In view of the low concentration of CTCs in early-stage cancer patients, which poses a significant challenge to analyze in particular early-stage patients, we have discovered new CTC markers encompassing the EMT spectrum (see below, patent applications), and we will increase the volume of blood analyzed in selected patients using novel devices (e.g. nRICH device). Moreover, we have also discovered, for the first time, a new source of blood in prostate cancer which contains several log units more CTCs than the peripheral blood and might – beyond the scope of this project – allow researchers worldwide to gain more comprehensive information on CTC biology
Objective 4 - Decipher the extravasation capacity of CTCs: To assess whether CTCs released by biopsy are able to extravasate and home into the bone marrow as one of the prime metastatic sites (in particular in prostate cancer), we have set up the regulatory and organizational basis for starting receiving the bone marrow aspirates from prostate and breast cancer patients, which has become an even more challenging task during the COVID pandemic but we are optimistic that we can achieve the goal to assess the number of DTCs in the bone marrow of corresponding patients in addition to enumerate CTC counts.
Objective 5 - Develop novel therapeutic strategies to prevent CTC extravasation: CTCs released by biopsies and/or surgery might pose an interesting target for drugs that prevent extravasation. To develop an easy-to-use model for studying CTC release in an experimental model, we developed a novel subcutaneous prostate cancer xenograft mouse model and optimized the protocols for the analysis of CTCs and DTCs. These protocols have become already very valuable for the research community, as documented by several request from international colleagues. To further evaluate CTCs released by tissue injury and to characterize the metastatic potential of these cells, the establishment of a zebrafish xenograft model is ongoing. Recently, we established a CTC-derived, ER-positive breast cancer cell line, CTC-ITB-01 (Fig. 2). NGS analyses showed that CTC-ITB-01 cells mirror the in-situ detected CTCs and CTC-ITB-01 possesses tumorigenic and metastatic capacity in immunodeficient mice. Thus, CTC-ITB-01 will be used as an exciting new CTC model to generate our first zebrafish xenograft model.
Objective 2 - Assess primary tumor biology associated with CTC release: The collection of primary tumor tissue has started in collaboration with the departments of pathology of our clinical partners, and it will be performed in the second funding period depending on the results of the monitoring and characterization of the CTCs. The analyses will include next generation sequencing and inflammation profiling.
Objective 3 - Characterize the biology of CTCs released during biopsy and surgery: We have started to capture individual CTCs and perform a phenotypic and genotypic characterization at the single cell level. This will give novel insights into the biological characteristics of CTCs in early-stage breast and prostate cancer, which is still rare because most CTC characterization studies published in the literature were focused on patients with advanced disease. We have continuously optimized our protocols for single cell CTC analysis based on new technical developments (e.g. investment of a new high-resolution microscope with scanning abilities by internal UKE funds). In view of the low concentration of CTCs in early-stage cancer patients, which poses a significant challenge to analyze in particular early-stage patients, we have discovered new CTC markers encompassing the EMT spectrum (see below, patent applications), and we will increase the volume of blood analyzed in selected patients using novel devices (e.g. nRICH device). Moreover, we have also discovered, for the first time, a new source of blood in prostate cancer which contains several log units more CTCs than the peripheral blood and might – beyond the scope of this project – allow researchers worldwide to gain more comprehensive information on CTC biology
Objective 4 - Decipher the extravasation capacity of CTCs: To assess whether CTCs released by biopsy are able to extravasate and home into the bone marrow as one of the prime metastatic sites (in particular in prostate cancer), we have set up the regulatory and organizational basis for starting receiving the bone marrow aspirates from prostate and breast cancer patients, which has become an even more challenging task during the COVID pandemic but we are optimistic that we can achieve the goal to assess the number of DTCs in the bone marrow of corresponding patients in addition to enumerate CTC counts.
Objective 5 - Develop novel therapeutic strategies to prevent CTC extravasation: CTCs released by biopsies and/or surgery might pose an interesting target for drugs that prevent extravasation. To develop an easy-to-use model for studying CTC release in an experimental model, we developed a novel subcutaneous prostate cancer xenograft mouse model and optimized the protocols for the analysis of CTCs and DTCs. These protocols have become already very valuable for the research community, as documented by several request from international colleagues. To further evaluate CTCs released by tissue injury and to characterize the metastatic potential of these cells, the establishment of a zebrafish xenograft model is ongoing. Recently, we established a CTC-derived, ER-positive breast cancer cell line, CTC-ITB-01 (Fig. 2). NGS analyses showed that CTC-ITB-01 cells mirror the in-situ detected CTCs and CTC-ITB-01 possesses tumorigenic and metastatic capacity in immunodeficient mice. Thus, CTC-ITB-01 will be used as an exciting new CTC model to generate our first zebrafish xenograft model.
More patients from surgery will be recruited to establish a timeline of potential CTC and ctDNA release in correlation to different time points of intervention schedule. Based on clinical data obtained from the recruited cohort, the influence of different clinical parameters on the iatrogenic release of CTCs and ctDNA following clinical intervention will be analyzed. For automated enumeration of CTCs from cytospin scans of harvested cells from Parsortix experiments, it is also planned to prepare a CTC library of the picked cells for training a biased Artificial Intelligence (AI) algorithm. Complete cytospin scans of the slide sections with harvested cells will be used for unbiased training of AI algorithm in this case. Thus, together with the results that will be obtained with the zebrafish xenograft model we will be able to answer the key questions whether tissue injury indeed contributes to a significant blood-borne dissemination of viable tumor cells and to assess the clinical relevance of CTC release induced by mechanical stress. Besides, we expect to provide novel insights whether peri-biopsy or peri-surgical treatment with drugs such as Wnt or proteasome inhibitors could in principle be efficient to block CTCs from extravasation and thereby prevent metastatic colonization. These experimental studies will point to therapeutic targets that could be explored in future clinical trials to avoid the potential unwanted site effects of tumor cell release during biopsy and surgery.