Identification of and interference with local, cancer-associated
The immune system plays a significant role during tumorigenesis with evidence for a promoting as well as a suppressing role. Indeed, the amount and location of tumor-infiltrating Th1 and cytotoxic T cells is a strong positive prognostic factor in multiple types of human cancers. However, advanced tumors have intrinsic capacities to evade immune attack (immunoediting) as well as extrinsic mechanisms including a suppressive microenvironment, which also dampens tumor-specific immunity.
The main aim of immunotherapy is to improve protective effector function of tumor-specific T cells, which relies on 3 signals: Stimulation of the TCR receptor (signal 1), costimulation (signal 2) and supportive cytokines (signal 3). In the context of cancer, naïve T cells are insufficiently primed and become progressively dysfunctional or tolerant most probably due to the low quality of these 3 signals. Although the requirements for T cell activation are well characterized, how to prevent tolerance in the context of cancer remains unclear.
Boosting antitumor responses by blocking PD-1 or CTLA-4 results in durable clinical responses only in a limited proportion of cancer patients suggesting that other pathways must be targeted to improve clinical efficacy. We therefore aim to develop an approach to prime tumor-specific CD8+ T cells, prevent induction of tolerance and achieve control of large, established tumors. We used mice that develop autochthonous prostate cancer (TRAMP mice) as well as mice carrying advanced melanoma (B16) and combined different immune interventions with adoptive transfer of tumor-specific TCR transgenic CD8+ T cells.
We found that the combination of agonistic anti-CD40 + IL-2/anti-IL-2 complexes + IL-12Fc was a distinctively effective treatment with respect to priming protective, tumor-specific immunity and eradicating tumors at advanced disease stage when given together with adoptively transferred tumor-specific T cells. We propose that improving signals 2 (costimulation) and 3 (cytokines) together with fresh tumor-specific rather than dysfunctional pre-existing memory T cells represents a potent therapy for advanced cancer (Bransi A, et al., Cancer Immunol. Res. 2015).
Currently, we are performing studies to understand the underlying mechanisms of successful immune interventions and to explore the possibility of combining immunotherapy with standard treatments including radio- and chemotherapy.
The impact of radiotherapy and of chemotherapy on immune activation
Tumor-specific immunity occurs in cancer patients but has insufficient potential to control or eliminate the tumor. Strengthening this response through immunotherapy may lead to a durable, systemic response that may also control (development of) metastases. However, many hurdles preclude the induction of protective anti-tumor immunity, including clonal deletion of high-affinity, self-reactive T cells, insufficient innate immune signals in the context of cancer and the hostility of the tumor-environment towards protective immunity resulting in compromized T cell function. It is currently not understood why a particular anti-cancer treatment works well in some patients whereas it hardly has any benefit in others and we think that the status of tumor-specific effector and regulatory immunity impacts on the patient’s responsiveness not only to immunotherapy but also to standard treatments including radio- and chemotherapy. Although our knowledge in the field of tumor-immunology is increasing, we still don’t sufficiently understand how suppressive mechanisms act and how we can target those.
We have shown in mice and humans that radiotherapy (RT) results in a plethora of local changes, most of which support tumor-specific immunity (Sharma A, et al., Clin. Cancer Res. 2013; Gupta A, et al., J. Immunol. 2012) and identified activated C3 as a crucial candidate in this context. In further experiments we identified anaphylatoxins (C3a, C5a) as crucial components to radiotherapy-induced inflammation, tumor-specific immunity and clinical effect (Surace L, et al, Immunity 2015).
Our aim is to improve the efficacy of RT by achieving a more robust immune stimulation that is sustained at the same time. We propose that that successful immunotherapy should include prolonged immune stimulation allowing in de novo priming of recent thymic emigrants, thus avoiding induction of peripheral tolerance. We will test a combination of RT with various treatments that support reactivation of existing or de novo priming of (tumor-specific) immunity. Furthermore, we perform unbiased analysis of transcripts, micro-RNAs and metabolites to understand local changes upon irradiation in order to selectively block or stimulate pathways for better therapeutic efficacy.
The role of tertiary lymphoid structures in immune defense against
The establishment of new therapeutic approaches to treat cancer is one of the major goals of biomedical research. Clinical success of blocking antibodies against CTLA4 and PD-1 has proven that modulation of immune response represents a powerful tool to tackle aggressive disease and provides a promising platform for development of novel approaches.
Tertiary lymphoid structures (TLS) in the tumor microenvironment have lately gained attention because of their significant correlation with improved survival in several tumor types. TLS were first described in autoimmune and transplant rejection conditions, where they support the infiltration and activation of adaptive immune cells. We recently observed a beneficial effect of high numbers of germinal center positive TLS on the disease free survival of non-small cell lung cancer (NSCLC) patients, and hence hypothesize that the formation of TLS in tumor microenvironment could promote anti-cancer immunity. Our knowledge about the responsible cell types and signals inducing TLS formation in tumor tissues, however, is limited.
The aim of this project is to investigate whether deliberate induction of TLS may represent a novel stand-alone therapeutic approach or as a means to improve the immune checkpoint blockade therapies. We will use a mouse lung cancer model to analyze the impact of TLS induction on survival and tumor-specific immune response by using state-of-the-art imaging of tumor histological sections, in vivo imaging of mouse lung cancer progression, multi-marker immunofluorescence and flow cytometry.
The role of beta-catenin/Wnt signaling in
non-melanoma skin cancer
Squamous and basal cell carcinoma (SCC, BCC) are frequent skin cancers. Wnt signaling is one of the few known molecular pathways regulating SCC initiation and progression. However, Wnt signaling is an also important regulator of normal skin homeostasis. The key downstream molecule of Wnt pathway is β-catenin, which attracts - via its N- and C-terminus - specific transcriptional co-activators to Wnt-responsive elements and thus activates Wnt-mediated transcription. In addition, β-catenin is an important component of adherens junctions. Such a structural role might be important to maintain the integrity of skin epidermis. Our focus is to determine the contribution of signaling versus structural roles of β-catenin in development and homeostasis of normal skin and skin cancer. Because this dual function complicates studying its role in both normal development and cancer, we have generated mutant mice with altered or deleted signaling but normal adherens function and established a genetic, UV-induced SCC murine model (K14-HPV8E6). We observed elevated levels of Wnt/β-catenin transcription in this SCC model indicating important role for β-catenin signaling in progression of SCC. Interestingly we also observed reduced β-catenin associated with adherens junctions, one of classical hallmarks of epithelial-mesenchymal transition. Currently we combine these SCC models with mutant strains expressing β-catenin, which is fully functional in adherens junctions but is compromised (or completely missing) in signaling outputs. Our first results indicate that in adult skin epidermis the structural role of β-catenin does not play significant role, whereas signaling outputs are essential for maintenance of hair follicles and regulating the thickness of the epidermis. Moreover, N-terminal β-catenin transcriptional co-activators are dispensable for normal development of skin and hairs, the role of C-terminal co-activators is under investigation.
We are establishing a new genetic model BCC, based on keratinocyte-specific deletion of the Ptch1 gene (K5Cre*PR1/Ptch1fl/fl), because biallelic deletion of Ptch1 in mouse epidermis results in lesions closely resembling human BCCs. BCC are thought to be caused by uncontrolled activation of the hedgehog (Hh) signaling pathway. In the majority of cases, this is due to inactivating mutations in the Hh receptor and tumor suppressor gene Ptch1. Once established, we will use this model to investigate the contribution of signaling versus structural roles of β-catenin in tumor development and progression.
We think our results may lead to development of tools that target β-catenin such that tumor progression is inhibited but leaving skin integrity intact.
The role of immune cells and platelets in metastasis
Metastasis is the major cause of death associated with solid tumors. It is becoming increasingly clear that metastases may form very early, i.e. before the primary tumor becomes clinically apparent and that such metastases may remain in a dormant state for a considerable time. Primary tumors are often curable by surgical resection or controllable by conventional therapies including radio- and chemotherapy. As the latter therapies mainly target dividing cells, dormant metastases presumably are insensitive to such treatments, whereas they can still be targeted by T lymphocytes. The interaction between the adaptive immune system and developing, early metastases is almost impossible to study in humans and most available data from preclinical models were produced using i.v. injected tumor cells as model for metastasis to the lungs. The biological relevance of such an approach, however, is unclear. Furthermore, this experimental set up does not allow a direct comparison of immune events within the primary tumor and the metastastic lesion.
We therefore established models of spontaneous metastasis: After surgical removal of the primary tumor, spontaneous metastases to different anatomical sites occur, which can be monitored and quantified by non-invasive IVIS technology. Using this method, we will specifically investigate the impact of different immune parameters on the development of metastases. Furthermore, we will test the therapeutic effect of immune stimulation on control or rejection of established metastatic lesions.
Recently, two large epidemiological studies described that chronic aspirin treatment protects against development of metastases. We will investigate whether the protective effect of aspirin is entirely due to its anti-coagulation activity or whether other activities of aspirin including impacting on myeloid cells contribute as well.
Importantly, we have started collecting paired samples from primary and metastatic tumors of various types. We will use these samples to validate the results we obtain in mice.
We think that our results will provide novel and essential knowledge on the mutual interactions between metastatic tumors and the immune system. We expect that this knowledge will contribute to developing better immunotherapies that are efficacious in controlling metastatic lesions in men.