A deeper understanding of the parameters driving response and resistance to immunotherapy is needed to improve the low response rates observed in breast cancer patients. Research into immunotherapy... Show moreA deeper understanding of the parameters driving response and resistance to immunotherapy is needed to improve the low response rates observed in breast cancer patients. Research into immunotherapy response has predominantly focused on T cells, however effective immune responses require tightly regulated crosstalk between innate and adaptive immune cells. By combining profiling of blood and tumors from metastatic breast cancer patients with mechanistic studies in mouse models, we uncovered the critical role of eosinophils in immunotherapy response, and we provide proof-of-principle for eosinophil engagement to enhance immunotherapy efficacy. Focusing on resistance mechanisms to immunotherapy, we demonstrate that neoadjuvant immunotherapy triggers persistent and systemic regulatory T cell activation which blunts therapeutic efficacy against metastatic spread of breast tumors. In addition, we demonstrate that neutrophils in the tumor microenvironment pose a barrier to immunotherapy response through T cell suppression. Lastly, we demonstrate that combining the immunomodulatory agent PD1-IL2v with cisplatin is a powerful approach to induce a broad activation of systemic and intratumoral adaptive and innate immunity, resulting in effective immunotherapy responses. Overall, this work identifies several key players and their interconnectivities in anti-tumor immunity and tumor-induced immunosuppression that may be therapeutically exploited to improve immunotherapy responses for breast cancer patients. Show less
This thesis represents a comprehensive investigation into the control of cancer stemness and metastatic initiation using a combination of advanced zebrafish xenograft models and in vitro assays.... Show moreThis thesis represents a comprehensive investigation into the control of cancer stemness and metastatic initiation using a combination of advanced zebrafish xenograft models and in vitro assays. The discoveries made and the evaluation of drug efficacy provide valuable insights for the development of novel therapeutic approaches in the fight against metastatic cancer. Show less
Metastases remain the leading cause of cancer-related death worldwide. Therefore, improving the treatment efficacy against such tumors is essential to enhance patient survival. AU-011 (belzupacap... Show moreMetastases remain the leading cause of cancer-related death worldwide. Therefore, improving the treatment efficacy against such tumors is essential to enhance patient survival. AU-011 (belzupacap sarotalocan) is a new virus-like drug conjugate which is currently in clinical development for the treatment of small choroidal melanoma and high-risk indeterminate lesions in the eye. Upon light activation, AU-011 induces rapid necrotic cell death which is pro-inflammatory and pro-immunogenic, resulting in an anti-tumor immune response. As AU-011 is known to induce systemic anti-tumor immune responses, we investigated whether this combination therapy would also be effective against distant, untreated tumors, as a model for treating local and distant tumors by abscopal immune effects. We compared the efficacy of combining AU-011 with several different checkpoint blockade antibodies to identify optimal treatment regimens in an in vivo tumor model. We show that AU-011 induces immunogenic cell death through the release and exposure of damage-associated molecular patterns (DAMPs), resulting in the maturation of dendritic cells in vitro. Furthermore, we show that AU-011 accumulates in MC38 tumors over time and that ICI enhances the efficacy of AU-011 against established tumors in mice, resulting in complete responses for specific combinations in all treated animals bearing a single MC38 tumor. Finally, we show that AU-011 and anti-PD-L1/anti-LAG-3 antibody treatment was an optimal combination in an abscopal model, inducing complete responses in approximately 75% of animals. Our data show the feasibility of combining AU-011 with PD-L1 and LAG-3 antibodies for the treatment of primary and distant tumors. Show less
Kroese, T.E.; Laarhoven, H.W.M. van; Schoppman, S.F.; Deseynde, P.R.A.J.; Cutsem, E. van; Haustermans, K.; ... ; Rossum, P.S.N. van 2023
Background: Local treatment improves the outcomes for oligometastatic disease (OMD, i.e. an intermediate state between locoregional and widespread disseminated disease). However, consensus about... Show moreBackground: Local treatment improves the outcomes for oligometastatic disease (OMD, i.e. an intermediate state between locoregional and widespread disseminated disease). However, consensus about the definition, diagnosis and treatment of oligometastatic oesopha-gogastric cancer is lacking. The aim of this study was to develop a multidisciplinary European consensus statement on the definition, diagnosis and treatment of oligometastatic oesophago-gastric cancer. Methods: In total, 65 specialists in the multidisciplinary treatment for oesophagogastric cancer from 49 expert centres across 16 European countries were requested to participate in this Del-phi study. The consensus finding process consisted of a starting meeting, 2 online Delphi ques-tionnaire rounds and an online consensus meeting. Input for Delphi questionnaires consisted of (1) a systematic review on definitions of oligometastatic oesophagogastric cancer and (2) a discussion of real-life clinical cases by multidisciplinary teams. Experts were asked to score each statement on a 5-point Likert scale. The agreement was scored to be either absent/poor (<50%), fair (50%-75%) or consensus (>75%). Results: A total of 48 experts participated in the starting meeting, both Delphi rounds, and the consensus meeting (overall response rate: 71%). OMD was considered in patients with meta-static oesophagogastric cancer limited to 1 organ with <3 metastases or 1 extra-regional lymph node station (consensus). In addition, OMD was considered in patients without pro-gression at restaging after systemic therapy (consensus). For patients with synchronous or me-tachronous OMD with a disease-free interval <2 years, systemic therapy followed by restaging to consider local treatment was considered as treatment (consensus). For metachronous OMD with a disease-free interval >2 years, either upfront local treatment or systemic treatment fol-lowed by restaging was considered as treatment (fair agreement). Conclusion: The OMEC project has resulted in a multidisciplinary European consensus state -ment for the definition, diagnosis and treatment of oligometastatic oesophagogastric adeno-carcinoma and squamous cell cancer. This can be used to standardise inclusion criteria for future clinical trials. 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Show less
Jong, E. de; Quint, K.D.; Ghalbzouri, A. el; Verdijk, R.M.; Goeman, J.J.; Heidt, S.; ... ; Bavinck, J.N.B. 2023
Background: Solid organ-transplant recipients (SOTR) have an increased risk of cutaneous squamous-cell carcinoma (cSCC), metastasis and death from cSCC. In immunocompetent patients with mucosal SCC... Show moreBackground: Solid organ-transplant recipients (SOTR) have an increased risk of cutaneous squamous-cell carcinoma (cSCC), metastasis and death from cSCC. In immunocompetent patients with mucosal SCC, downregulation of HLA class I is associated with poor prognosis. Since the degree of HLA expression on tumor cells could play a role in immunogenicity and pathophysiology of cSCC metastasis, we hypothesized that decreased HLA expression is associated with an increased risk of metastasis.Methods: We compared HLA expression between primary metastasized cSCCs, their metastases, and nonmetastasized cSCCs from the same patients. Samples were stained for HLA-A, HLA-B/-C and quantified by calculating the difference in immunoreactivity score (IRS) of the primary cSCC compared with all nonmetastasized cSCCs. Results: The mean IRS score for HLA-B/C expression was 2.07 point higher in metastasized compared to nonmetastasized cSCCs (p = 0.065, 95 % CI -0.18-4.32). 83.3 % of the primary metastasized cSCCs had an IRS score of 4 or higher, compared to 42.9 % in non-metastasized cSCCs. Moderately to poorly differentiated cSCCs had more HLA class I expression compared to well-differentiated cSCCs. Conclusion: Contrary to immunocompetent patients, HLA-B/C expression tends to be upregulated in metastasized cSCC compared to non-metastasized cSCC in SOTR, suggesting that different tumor escape mechanisms play a role in SOTR compared to immunocompetent patients. Show less
Sluimer, L.M.; Bullock, E.; Rätze, M.A.K.; Enserink, L.; Overbeeke, C.; Hornsveld, M.; ... ; Tavares, S. 2023
High expression of the non-receptor tyrosine kinase FER is an independent prognostic factor that correlates with poor survival in breast cancer patients. To investigate whether the kinase activity... Show moreHigh expression of the non-receptor tyrosine kinase FER is an independent prognostic factor that correlates with poor survival in breast cancer patients. To investigate whether the kinase activity of FER is essential for its oncogenic properties, we developed an ATP analogue-sensitive knock-in allele (FERASKI). Specific FER kinase inhibition in MDA-MB-231 cells reduces migration and invasion, as well as metastasis when xenografted into a mouse model of breast cancer. Using the FERASKI system, we identified Ski family transcriptional corepressor 1 (SKOR1) as a direct FER kinase substrate. SKOR1 loss phenocopies FER inhibition, leading to impaired proliferation, migration and invasion, and inhibition of breast cancer growth and metastasis formation in mice. We show that SKOR1 Y234, a candidate FER phosphorylation site, is essential for FER-dependent tumor progression. Finally, our work suggests that the SKOR1 Y234 residue promotes Smad2/3 signaling through SKOR1 binding to Smad3. Our study thus identifies SKOR1 as a mediator of FER-dependent progression of high-risk breast cancers. Show less
The immune system plays a dual role in cancer development. Besides the potential to eliminate cancer cells, immunoregulatory mechanisms exist that counteract anti-tumor immunity.Research in this... Show moreThe immune system plays a dual role in cancer development. Besides the potential to eliminate cancer cells, immunoregulatory mechanisms exist that counteract anti-tumor immunity.Research in this thesis focusses on the role of regulatory T cells (Tregs), a type of adaptive immune cell that plays a major role in tumor-associated immunosuppression. Specifically, the role of Tregs was investigated during the development of primary- and metastatic breast cancer, and in the context of novel immunotherapeutics. This was done by using advanced genetically engineered mouse models that recapitulate human breast cancer.The results in this thesis describe that breast tumors are, already early in their development, able to mobilise Tregs in the tumor-draining lymph nodes, thereby creating a local immunosuppressive niche leading to increased lymph node metastasis. In addition, it was found that the immunotherapeutic treatments anti-PD1 and anti-CTLA4 inadvertently activate Tregs, resulting in a diminished efficacy of this treatment in mice bearing breast tumors. Finally, we describe a mechanism by which intratumoral macrophages are critical promote the intratumoral accumulation of Tregs in breast tumors.Insights from this thesis may eventually contribute to the development of therapeutic applications that are aimed at overcoming immunoregulatory mechanisms in breast cancer. Show less
Local coagulation activation has been shown to impact both primary tumor growth and metastasis in mice. It is well known that components of the blood clotting cascade such as tissue factor and... Show moreLocal coagulation activation has been shown to impact both primary tumor growth and metastasis in mice. It is well known that components of the blood clotting cascade such as tissue factor and thrombin play a role in tumor progression by activating cellular receptors and local formation of fibrin. However, whether venous thromboembolism (VTE) or a hypercoagulable state has a direct impact on cancer progression is unknown. Here we have combined an orthotopic murine breast cancer model, using female Nod-SCID mice, with siRNA-mediated silencing of antithrombin (siAT) leading to the induction of a systemic hypercoagulable state. We show that, compared to control siRNA-treated (not experiencing a hypercoagulable state) tumor-bearing mice, siAT treated tumor-bearing mice do not show enhanced tumor growth nor enhanced metastasis. We conclude that, in this murine model for hypercoagulability, induction of a hypercoagulable state does not contribute to breast cancer progression. Show less
Piaggio, F.; Croce, M.; Reggiani, F.; Monti, P.; Bernardi, C.; Ambrosio, M.; ... ; Amaro, A. 2022
Background and aim of the study: Mutations in the G alpha-genes GNAQ and GNA11 are found in 85-90% of uveal melanomas (UM). Aim of the study is to understand whether the mutations in both genes... Show moreBackground and aim of the study: Mutations in the G alpha-genes GNAQ and GNA11 are found in 85-90% of uveal melanomas (UM). Aim of the study is to understand whether the mutations in both genes differentially affect tumor characteristics and outcome and if so, to identify potential mechanisms. Methods: We analyzed the association between GNAQ and GNA11 mutations with disease specific survival, gene expression profiles, and cytogenetic alterations in 219 UMs. We used tandem-affinity-purification, mass spectrometry and immunoprecipitation to identify protein interaction partners of the two G-proteins and analyzed their impact on DNA-methylation. Results: GNA11 mutation was associated with: i) an increased frequency of loss of BRCA1- associated protein 1 (BAP1) expression (p = 0.0005), ii) monosomy of chromosome 3 (p < 0.001), iii) amplification of chr8q (p = 0.038), iv) the combination of the latter two (p = 0.0002), and inversely with v) chr6p gain (p = 0.003). Our analysis also showed a shorter disease-specific survival of GNA11-mutated cases as compared to those carrying a GNAQ mutation (HR = 1.97 [95%CI 1.12-3.46], p = 0.02). GNAQ and GNA11 encoded G-proteins have different protein interaction partners. Specifically, the Tet Methylcytosine Dioxygenase 2 (TET2), a protein that is involved in DNA demethylation, physically interacts with the GNAQ protein but not with GNA11, as confirmed by immunoprecipitation analyses. High risk UM cases show a clearly different DNA-methylation pattern, suggesting that a different regulation of DNA methylation by the two G-proteins might convey a different risk of progression. Conclusions: GNA11 mutated uveal melanoma has worse prognosis and is associated with high risk cytogenetic, mutational and molecular tumor characteristics that might be determined at least in part by differential DNA-methylation. (C) 2022 Elsevier Ltd. All rights reserved. Show less
Background: In patients with active cancer and atrial fibrillation (AF) anticoagulation, thrombotic and bleeding risk still entail uncertainty.Aim: We explored the results of an international... Show moreBackground: In patients with active cancer and atrial fibrillation (AF) anticoagulation, thrombotic and bleeding risk still entail uncertainty.Aim: We explored the results of an international survey examining the knowledge and behaviours of a large group of physicians.Methods and results: A web-based survey was completed by 960 physicians (82.4% cardiologists, 75.5% from Europe). Among the currently available anticoagulants for stroke prevention in patients with active cancer, direct oral anticoagulants (DOACs) were preferred by 62.6%, with lower values for low molecular weight heparin (LMWH) (24.1%) and for warfarin (only 7.3%). About 46% of respondents considered that DOACs should be used in all types of cancers except in non-operable gastrointestinal cancers. The lack of controlled studies on bleeding risk (33.5% of respondents) and the risk of drug interactions (31.5%) were perceived as problematic issues associated with use of anticoagulants in cancer. The decision on anticoagulation involved a cardiologist in 27.8% of cases, a cardiologist and an oncologist in 41.1%, and a team approach in 21.6%. The patient also was involved in decision-making, according to similar to 60% of the respondents. For risk stratification, use of CHA2DS2-VASc and HAS-BLED scores was considered appropriate, although not specifically validated in cancer patients, by 66.7% and 56.4%, respectively.Conclusion: This survey highlights that management of anticoagulation in patients with AF and active cancer is challenging, with substantial heterogeneity in therapeutic choices. Direct oral anticoagulants seems having an emerging role but still the use of LMWH remains substantial, despite the absence of long-term data on thromboprophylaxis in AF. Show less
Tumors are complex ecosystems containing not just cancer cells, but a large variety of cell types, including immune cells. Moreover, tumors have a systemic influence: they can signal long distances... Show moreTumors are complex ecosystems containing not just cancer cells, but a large variety of cell types, including immune cells. Moreover, tumors have a systemic influence: they can signal long distances using soluble molecules and hijack non-neoplastic cells (such as immune cells) in distant organs for their own benefit, thus maximising their metastatic potential. The phenotype of immune cells in tumors and in systemic environments is therefore a key determinant of cancer progression and response to therapy.This thesis aims to understand what governs the tumor-immune ecosystem. We argue that cancer-intrinsic genetic aberrations have a dominant role in determining the tumor immune contexture, as well as systemic inflammatory activation. Understanding the intricate connection between the genetics of breast cancer and anti-tumor immune responses will help develop personalised immune intervention strategies for cancer, tailored to the genetic makeup of a patient’s tumor. Furthermore, we examine in detail the role of neutrophils in cancer-induced systemic inflammation, and how they influence the progression and spread of breast cancer. While tumors can be highly heterogeneous in nature, we show that neutrophils themselves also have a tremendous phenotypic diversity. Mapping this heterogeneity in neutrophil phenotypes may help to utilise these cells in cancer immunotherapy. Show less
Verschoor, A.J.; Speetjens, F.M.; Dijkstra, P.D.S.; Fiocco, M.; Sande, M.A.J. van de; Bovee, J.V.M.G.; Gelderblom, H. 2019
Paxillin is a well-known multidomain scaffold protein that is involved in the regulation of cell-matrix adhesiondynamics, a process required for the tumor cell migration and invasion.... Show morePaxillin is a well-known multidomain scaffold protein that is involved in the regulation of cell-matrix adhesiondynamics, a process required for the tumor cell migration and invasion. Phosphorylation of the serine residue 178requires c-Jun NH2-terminal kinase (JNK) activation, which occurs downstream of epidermal growth factor receptor (EGFR)-mediated signaling and drives cell migration. In this study, we investigated the significance of paxillin Ser178 phosphorylation in breast cancer progression.We employed the rat mammary carcinoma MTLn3 cell line with which we established stabile variants of both wild type and mutant GFP-paxillin constructs. With those, we next performed several in vitro assays including cell proliferation, migration and focal adhesion dynamics. Finally, we monitored the metastatic spread of both cell line variants in an othrotopic mouse model for breast cancer.Here we show that expression of the phospho-defective mutant paxillinS178A in the metastatic mammary adenocarcinoma MTLn3 cell-line significantly decreased EGF-induced cell migration, which was correlated with impaired focal adhesion dynamics. Moreover, paxillinS178A attenuated lung metastasis formation in an orthotopic in vivo mammary gland tumor/metastasis model, demonstrating the importance of JNK-mediated paxillin phosphorylation in breast cancer progression. Expression of paxillinS178A caused a decrease in EGFR expression while re-expression of EGFR in MTLn3-paxillinS178A cells fully restored EGF-driven cell motility and focal adhesion dynamics. Furthermore, re-expression of EGFR in MTLn3-paxillinS178A rescued spontaneous metastasis from breast to lung.Overall our data show an important role for JNK-mediated paxillin Ser178 phosphorylation in the regulation of EGFR expression and thereby, in EGF-driven cell migration and metastasis formation. Show less
Smolle, M.A.; Praag, V.M. van; Posch, F.; Bergovec, M.; Leitner, L.; Friesenbichler, J.; ... ; Leithner, A. 2019
This thesis describes i) the function of an alternatively spliced coagulation factor in hemostasis, ii) the contribution of coagulation factors on cancer progression, and iii) expands our view... Show moreThis thesis describes i) the function of an alternatively spliced coagulation factor in hemostasis, ii) the contribution of coagulation factors on cancer progression, and iii) expands our view on cancer-associated thrombosis. Inhibition of Tissue Factor (TF) signaling with the antibody (Mab-10H10) resulted in decreased tumor initiating capacity and metastasis in a triple negative breast cancer (TNBC) cell line. Since this is a tumor type that is difficult to treat, and has high relapse-rates, it would be of interest to target TF signaling. Dual treatment of TNBC with conventional chemotherapy and Mab-10H10 could result in a positive treatment strategy as both highly proliferative and cancer stem cells are targeted. Furthermore, we provided a proof-of-principle study to search for novel biomarkers in patients with cancer-associated thrombosis in an unbiased manner. Up till now it is challenging to accurately predict those cancer patients with elevated risk of thrombosis. Furthermore, patients with cancer-associated thrombosis have poorer survival. Expansion of this study to validation cohorts and other tumor types will give insights in the underlying molecular mechanism of cancer-associated thrombosis. Eventually, this will aid a better prediction model to select those cancer patients with high risk of thrombosis and those who might benefit from thromboprophylaxis. Show less
This thesis describes the respective contribution of the expression of Tissue factor isoforms full length Tissue Factor (flTF) and alternatively spliced Tissue Factor (asTF) as well as Factor... Show moreThis thesis describes the respective contribution of the expression of Tissue factor isoforms full length Tissue Factor (flTF) and alternatively spliced Tissue Factor (asTF) as well as Factor VII by tumor cells to promote cancer progression. Cohorts of breast, colon, and bone cancer specimens and a multitude of in vitro and in vivo models were used to explore the mechanism behind enhanced cell proliferation and metastasis in in vitro and in vivo models, as well as decreased patient survival associated with TF and FVII expression in cancer patients. Show less
