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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - Cancer-Recurrence (Tumor cell death supports recurrence of cancer)

Teaser

Summary

The most common treatment strategy for cancer is surgical removal combined with chemotherapy. Chemotherapy is often given prior to surgery to reduce tumor size in order to improve the success rate of tumor resection and after surgery to reduce potential metastasis. Chemotherapeutic agents aim at inducing cell death, mostly (but not exclusively) by stimulating the intrinsic apoptotic pathway. However, some cells are resistant to chemotherapeutics due to their genetic or epigenetic profile, which allows them to survive the treatment and manifest later, leading to tumor recurrence. Therefore, it is of utmost importance to exactly understand the cellular and molecular mechanisms that drive the outgrowth of the few surviving cells.

It has been suggested that the outgrowth of surviving cells and recurrence of tumor growth can be (partly) induced by the chemotherapy itself. For example, in addition to killing tumor cells, doxorubicin induces myeloid cell infiltration which supports the outgrowth of surviving tumor cells, and gemcitabine influences myeloid-derived suppressor cells with subsequent growth-stimulating effects. Upon death, cells defragment and release their content in the form of apoptotic bodies. This type of extracellular vesicles (EVs) contains proteins, lipids and nucleic acids from the cell of origin. The remnants of apoptotic cells can be removed by professional phagocytes (e.g. macrophages) and non-professional phagocytes (surrounding epithelial or mesenchymal cells). If a proportion of the massive numbers of EVs (including apoptotic bodies) that are released upon chemotherapy are taken up by tumor and stromal cells, these EVs may severely affect the behavior of tumor and stromal cells and progression of the disease. For example, it has been shown that macrophages that ingest apoptotic bodies start to release TGF-Î²7. Significantly, TGF-Î² can both inhibit proliferation and induce apoptosis in epithelial cells, but can also induce epithelial-mesenchymal transition (EMT)-dependent migration. Recent data suggest that cells that acquire EMT properties also acquire stem cell properties. Since it is thought that only cells with stem cell properties have the ability to initiate tumor growth, the microenvironment created by dying cells may lead to EMT of the surviving tumor cells that enables them to migrate and regrow tumors. Thus, although induction of cell death by chemotherapy initially reduces the tumor mass dramatically, it has many unintentional and harmful side-effects on the remaining fraction of tumor cells and their microenvironment which in turn could have large consequences for the long-term outcome of cancer.

State-of-theâ€“art objectives and hypothesis:
Previous studies aimed to understand how specific chemotherapeutics, e.g. DNA damaging agents, also target stromal cells and how this influences the therapeutic outcome. Unfortunately, different chemotherapeutics will have different effects on stromal cells that precludes a generic interference strategy to prevent unwanted effects leading to tumor recurrence. However, the generic aim amongst the various therapies is to induce cell death. Therefore, we will study the unintended side-effects of cell death on the surviving tumor cells and stroma, identify the key cell types and mechanisms that mediate this effect, and test whether interference with these key cell types and mechanisms leads to reduced recurrence of tumors upon treatment. Since induction of cell death is the universal aim of therapy, interfering with unintended side-effects of tumor cell death may be a therapeutic avenue leading towards improved outcome of a wide range of therapies.

Hypothesis: The induction of tumor cell death has unintended and harmful side-effects on the remaining fraction of tumor cells and stroma, with subsequent profound consequences for the long-term outcome of cancer (Fig1).

Main aim: Gain a better understanding on the unintended side-effects of inducing tumor cell de

Work performed

Introduction
Current anti-cancer treatments are often ineffective. Whilst most patients initially benefit from anti-cancer drugs, many of them eventually experience relapse of resistant tumors throughout the body. Current clinical strategies mainly aim at inducing tumor cell death, but this induction may have serious unintentional and unwanted side effects on surviving tumor cells. We hypothesized that tumor cell death induces migration and growth of the surviving tumor cells. In this ERC project, we aim to identify the key cell types and mechanisms, such as epithelial-mesenchymal transition (EMT) and stemness, that mediate this effect, and establish whether interference with these cells (e.g. cancer stem cells) and mechanisms (e.g. EMT) can reduce recurrence of tumors after cancer therapy.

Major achievements
So far, we have been very successful and have published 22 papers in high-impact journals such as Nature, Cell, and Cell Reports. As proposed, we have developed mouse models, intravital imaging tools, and single cell sequencing approaches to study whether surviving cancer cells undergo a phenotypic change (e.g. undergo epithelial-to-mesenchymal transition (EMT) or acquire stemness). The techniques we have developed to follow EMT in living mice have been published in Cell Reports (Beerling et al, Cell Reports 2016). The method to study stemness in breast tissue has been published in Nature (Scheele et al, Nature 2017) and Cell (Hannenzo, Scheele et al, Cell 2017). For the remainder of the project, these tools will be utilized to test how (cancer) stem cell properties and EMT properties in surviving tumor cells are affected by tumor cell death of surrounding cells.

Dying tumor cells that undergo programmed cells death (apoptosis) disintegrate into small extracellular vesicles (EVs) called apoptotic bodies. In research line 3, we proposed to identify tumor cells that take up EVs released from dying cells. For this, we have developed a cre-reporter system based on the Cre/LoxP system. In this system, reporter cells change color (red to green) upon the uptake of EVs release from cells that express Cre. For our experiments, we have utilized this Cre-Lox system, where apoptotic cancer cells express Cre and tumor and/or stromal cells a Cre-reporter. We have published this methodology in Nature Protocols (Zomer et al, Nat Prot 2016). We have performed all proposed experiments; both in vitro and in vivo experiments illustrate that although cells can take up EVs, however the vast majority of cells the cargo of apoptotic bodies is not released in the cytoplasm of the recipient cells. This indicates that communication of tumor cells through release of EVs is mostly mediated by living cells.

Since most EV-comminication happens between living cells, we next focused on the content of EVs released by living cancer cells. In research line 5, we proposed to characterize EVs and cells using genomic and proteomic techniques. We have successfully isolated and identified the cargo of EVs using RNA sequencing and mass spectrometry. For the initial analysis, we have compared EVs and cells of more-benign and more-malignant cells. Interestingly, in both populations of EVs we found the presence of a network of RNA and protein molecules involved in migration and metastasis. However, the number of RNAâ€™s and proteins involved in migration is larger in EVs from the more-malignant than in EVs from the more-benign cells. The manuscript that describes this data is submitted.

To test our hypothesis that tumor cell death has non-intended side-effects that stimulate surviving cells to migrate and regrow tumors, we have isolated tumor cells from mammary tumors that spontaneously develop in the following FVB mice: MMTV-PyMT; MMTV-Cre; R26-LoxP-STOP-LoxP-DTR mice. These tumor cells express the diphtheria toxin receptor (DTR). The idea was that administration of diphtheria toxin (DT) will induce apoptosis of DTR-expressing mammary tumor cells. In a

Final results

Cancer therapeutics target the â€œaverageâ€ differentiated tumor cell, while tumors are actually very heterogeneous. Therefore many therapies fail to target â€œnon-averageâ€ populations of cells with a genetic or epigenetic profile that makes them resistant to the chemotherapeutics. Although important knowledge on how these resistant cells regrow tumors has been obtained using traditional techniques including histochemistry, (q)PCR and Western blotting, these approaches provide snapshots of large populations of cells and therefore fail to provide crucial information about the history of individual cells (e.g. the few surviving cells amongst the majority of dying cells in a regressing tumor). Over the years, we have pioneered high resolution intravital imaging techniques and combined these with high-end genetic fluorescent mouse models in order to visualize the behavior of individual tumor cells in living mice. Using our state-of-the-art intravital imaging and fluorescent genetic tumor mouse models, we were able to study the plasticity of the behavior of individual cells during tissue development and homeostasis, tumor progression, and therapy responses. In this ERC project, we have used our unique imaging tools and fluorescent mouse models to study the potential non-intended side-effects of tumor cell death on surrounding and distant surviving tumor cells and stromal cells. We have and will further identify key cell types and mechanisms that mediate non-intended side-effects of tumor cell death on surviving tumor cells, and we will establish whether interference with these key cell types and mechanisms leads to reduced recurrence of tumors after chemotherapy. Since induction of cell death is the most important characteristic of all chemotherapeutics, our study is and will contribute significantly to fundamental knowledge on detailed mechanisms of tumor recurrence that hold true for almost all chemotherapeutics. Therefore, with the use of the state-of-the-art intravital imaging technology and advanced mouse models, this project is likely to identify new drug targets that help to design co-targeting strategies to improve clinical strategies.