In Vivo Identification of Genetic Requirements for the Generation and Colonization of Circulating Tumor Cells

Metastasis is the spread of cancer cells from a primary tumor to distant organs within the body, which accounts for more than 90% of all cancer-related deaths worldwide. The fact that only very few anti-metastatic treatments are available highlights the complexity of this multistep process and evidently shows our current lack of understanding of metastasis-associated molecular and genetic mechanisms. Cancer cells that detach from a primary tumor and are transported through the bloodstream to distant organs are called circulating tumor cells (CTCs). CTCs travel either as single cells or as CTC clusters and it has been shown that CTC clusters have a strongly increased metastatic potential compared to single CTCs, and their occurrence in patients is associated with a poor clinical outcome. Considering that in most cases the number of CTCs is substantially higher than the number of metastatic lesions, it is pivotal to discover the characteristics of successful CTCs that survive and are able to form metastasis. Although CTC clusters are critical mediators of metastasis, the pattern of affected organs is remarkably variable depending on the tumor of origin, indicating that intrinsic cancer cell traits are important determinants of distant metastasis. Cancer cells that are endowed with distinct metastatic traits are selected from a genetically- and epigenetically-heterogeneous tumor cell population making them prime metastatic precursors. Altogether, the current state of research indicates that CTCs emerge as highly effective metastatic effectors, combining the advantages of intra- and intercellular interactions making them prime targets for the development of metastasis-suppressive treatments.
The goal of my PhD thesis was to identify genes that are required for (i) primary tumor growth, (ii) single CTCs and CTC clusters to generate from a primary tumor lesion and intravasate into the host blood stream and (iii) CTCs to colonize specific organs including the brain and the bone. Through the conduction of an in vivo genome-wide loss-of-function screen in a human breast CTC-derived xenograft model (CDX), unique in its property to spontaneously mirror the metastatic pattern from the patient of origin, we were able to pinpoint genes that are required for the individual steps of the metastatic cascade as well as for organo-tropism in breast cancer. Individual knockout of our candidate genes indeed reduced the number of CTC clusters and thus the extend of metastasis without altering the primary tumor load compared to the non-targeting control, underlining their role in CTC cluster formation. Additionally, we found that breast CTCs are dependent on a kinase to be able to intravasate into the host blood stream and its knockout results in significantly decreased CTC numbers and formation of metastases. Furthermore, we were able to identify metastatic gene signatures correlating with worse metastasis-free survival (MFS) in breast cancer patients. The identification of specific dependencies of individual steps of breast cancer progression may enable a better understanding of the biology of metastasis as well as the development of new cancer therapies.
During the time of my PhD, colleagues and I developed an integrated workflow to identify phenotypic and molecular features enabling detailed characterization of CTCs at single cell resolution. The combination of live immunostaining and robotic micromanipulation of primary single and clustered CTCs allowed the ex vivo assessment of their proliferative and survival capabilities as well as an in vivo metastasis-formation assay. Additionally, a protocol is provided to achieve the dissociation of CTC clusters into individual cells facilitating intra-cluster heterogeneity investigation. Applying our workflow resulted, for instance, in precise quantification of the proliferation and survival potential of single CTCs and individual cells within CTC clusters, showing that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.
Simultaneously, I worked on a project aiming at the development of a microfluidic device enabling isolation, culture and drug screening of CTCs and DCCs from blood and ascites samples derived from metastatic breast and ovarian cancer patients to provide a platform enabling data-driven treatment decision-making for refractory cancer. We developed the MyCTC Chip, comprising a cancer cell isolation and culture chamber as well as six drug screening chambers enabling simultaneous testing of five different anti-cancer agents and one control. With this novel technology we were able to isolate CTCs that were spiked in whole blood with high capture efficiencies for both single CTCs and CTC clusters. We were able to isolate and culture cancer cells from breast and ovarian cancer patient ascites samples. Subsequently, we transferred these cells into the individual drug screening chambers enabling drug testing on patient-specific cancer cells. Currently, experiments are conducted to optimize drug application on chip. This novel technology will provide a valuable tool for treatment-decision making for cancer patients in daily clinical practice.