WEAVE project “Common targetable biomarkers in canine hemangiosarcoma and human angiosarcoma”

Lead partner: UNG (Ario de Marco). Other partners: Dr. Hilde de Rooster (Ghent University, Belgium) and Dr. Antonio Cosma (Luxembourg Institute of Health)

Ario de Marco bibliography

Duration: Nov 2023 - Oct 2026

UNG group members: Claudia D’Ercole, Matteo De March, Xinyu Hu, Klara Kropivšek

Topic:

The higher incidence of some malignancies in dogs can represent an invaluable option for studying rare pathologies in humans. Dogs represent an excellent translational model since they develop spontaneous tumors that share many clinical and epidemiologic features with human cancers, including histologic morphology, tumor genetics, molecular targets, biological behavior, and response to conventional therapies. In contrast to rodents bearing induced tumors that do not represent the structural and genetic complexity of spontaneous tumors, cancer in pet dogs develops naturally in the context of an intact immune system, which is characterized by tumor growth over long periods of time developing recurrence and metastasis to relevant distant sites.

Angiosarcoma (AS) is a rare (1 case/1 million people, corresponding to roughly 1% of all yearly diagnosed sarcomas), genomically complex and aggressive vascular sarcoma. The limited availability of patients impaired the development of effective therapies and consequently, the survival rate is dramatically poor. Dogs spontaneously develop histologically similar neoplasia, hemangiosarcoma (HSA), that is relatively common representing 5-7% of all canine malignant tumors with an incidence that is significantly higher in breeds such as German shepherd and boxer. Both AS and HSA are considered to originate from hematopoietic endothelial progenitor cells. Despite the fact that AS is predominantly cutaneous in humans and HSA is visceral in the vast majority of dogs, metastasis are mostly localized in lungs and liver in both models and comparative genomic analyses confirmed strong similarities between AS and HSA as well as the high but convergent heterogeneity within and between patients of both species. At protein level, immune histochemical analyses of canine samples indicated the overexpression of CD31, VEGFR1, VEGFR2, VEGFR3, PDGFRA/B, MUC18, c-Kit and FGFR1 in at least part of the evaluated specimens and consequently such receptors could serve as HSA membrane, accessible markers. Data relative to AS cell surface targetable biomarkers are more limited in number but suggest overexpression of VEGF receptors as well as of c-Kit. It would be particularly useful to identify further surface biomarkers shared by AS and HSA cells to develop targeting strategies suitable for both species. The HSA prevalence and the prior knowledge justifies the interest for exploring HSA as a tractable model for studying human AS, developing drug- and immunotherapies as well as for testing experimental therapies in clinical trials.

This project general aim is to isolate a set of immunoreagents suitable for the characterization, diagnostics and, potentially, therapy of (splenic) HSA in dogs. They should target biomarkers exposed at the cell surface, allowing the selective targeting of the corresponding cells in tissues. Furthermore, they could serve as reagents for implementing similar diagnostic protocols in human AS. However, information and reagent transfer between HSA and AS will only be suitable and reliable if the similarity between the two biological systems is sufficiently high. Consequently, the conservation of epitopes between HSA and AS cells needs to be first be evaluated and, for accomplishing this task, we’ll exploit the large pool of anti-HSA reagents that will be recovered by panning ligand libraries against the tumor biomarkers to obtain a multidimensional profile of both HSA and AS exposed proteomes. The achievement of these objectives will be obtained by means of a multidisciplinary, collaborative team involving the complementary expertise of UNG (nanobody technology) and of two strategic partners, Hilde de Rooster at Ghent University (veterinary oncology surgery) and Antonio Cosma at the Luxembourg Institute of Health (flow and mass cytometry).

Project implementation:

After having fixed the legal issues and hired dedicated young researchers, the lab activities moved ahead and we have isolated nanobodies against several antigens that have been reported being biomarkers of hemangiosarcoma/angiosarcoma in the literature. More problematic resulted the panning on whole cells because there are no available human cell lines and we could use only the canine ones. But the relevance of comparative oncology is perfectly evidenced by this condition: the research of rare human tumor diseases can advance because we can exploit biological samples recovered from corresponding canine patients and gain information suitable for both species.

WP2, Binder isolation and production. Task 2.1, Panning on known biomarkers: sets of binders have been selected. Task 2.2, Panning on whole cells: this has been the most problematic task because nobody of the groups we contacted for obtaining human AS cells replied. Our initial aim was to perform a double panning (canine + human cells) to identify cross-reacting binders, but this was not feasible because of the lack of human material. Therefore, we decided to proceed with a panning on canine cells and look for cross-reacting binders during the characterization step. Task 2.3, Binder production and characterization: indeed, the production will be performed for the whole project period, but we got enough material for the immediate tests. The results from Task 3.1 required the reorganization of the workflow. Task 2.4, Binder structural determination and in silico optimization: we advanced faster than expected, specifically in the in silico optimization.

WP3, Displayed proteome characterization. Task 3.1, Reagent preparation optimization: a comprehensive set of options was compared and clear conclusions were obtained. These results will impact Task 2.3 because impose a reorganization of the binder production pipeline. Tasks 3.2 and 3.3, Flow and mass-cytometry on cell lines: these tasks proceed in parallel because of the common issues related to the reagent preparation.

WP5, Reagent validation on tumors. Task 5.1, New cell line preparation: The objective was achieved and the protocol was set for additional isolation of cell cultures, if necessary for the widening of research activities. Tasks 5.2/5.3, IHC and CIS on tumors: CIS has not completed yet, but IHC was extended to a larger number of biomarkers with respect to the initial plan.

Publications:

Waheed Y, Mojumdar A, Shafiq M, de Marco A, De March M (2024) The fork remodeler Helicase-like Transcription Factor in cancer development: all at once. BBA - Molecular Basis of Disease. BBA Mol Basis Dis 1870:167280 COBISS.SI-ID 198569219

Štrancar A, D’Ercole C, Nakić M, Cikatricisova L, De March M, de Marco A (2024) A practical guide for the quality evaluation of fluobodies/chromobodies. Biomolecules 14:587 COBISS.SI-ID 195579651

de Marco A (2025) Recent advances in recombinant production of soluble proteins in E. coli. Microb Cell Factories 24:21 COBISS.SI-ID 222590211

LUONG, Huyen Thuc Tran, REVETS, Dominique, DE MARCH, Matteo, VERCAMMEN, Sofie, DE ROOSTER, Hilde, DE MARCO, Ario, COSMA, Antonio. Common targetable biomarkers in canine hemangiosarcoma and human angiosarcoma. V: Atelier de l'Inserm 280 : exploiter le potentiel des NANOBODIES®: d’outils de recherche à agents thérapeutiques. COBISS.SI-ID 229432323