The role of vascular endothelial growth factor and matrix metalloproteinases in canine lymphoma: in vivo and in vitro study

Background: Canine lymphoma represents the most frequent haematopoietic cancer and it shares some similarities with human non-Hodgkin lymphoma. Matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) play a coordinated role during invasion and proliferation of malignant cells; however, little is known about their role in canine haematologic malignancies. The aim of this study was to investigate the mRNA and protein expression of VEGF and the most relevant MMPs in canine lymphoma. Lymph node aspirates from 26 B-cell and 21 T-cell lymphomas were collected. The protein expression levels of MMP-9, MMP-2 and VEGF-A were evaluated by immunocytochemistry, and the mRNA levels of MMP-2, MMP-9, MT1-MMP, TIMP-1, TIMP-2, RECK, VEGF-A and VEGF-164 were measured using quantitative RT-PCR.Results: MT1-MMP, TIMP-1 and RECK mRNA levels were significantly higher in T-cell lymphomas than in B-cell lymphomas. Higher mRNA and protein levels of MMP-9 and VEGF-A were observed in T-cell lymphomas than in B-cell lymphomas and healthy control lymph nodes. A positive correlation was found between MMP-9 and VEGF-A in T-cell lymphomas. Moreover, MMP-9, MT1-MMP, TIMP-1 and VEGF-A were expressed at the highest levels in high-grade T-cell lymphomas.Conclusions: This study provides new information on the expression of different MMPs and VEGF in canine lymphoma, suggesting a possible correlation between different MMPs and VEGF, immunophenotype and prognosis

Breed-associated risks for developing canine lymphoma differ among countries: an European canine lymphoma network study

Canine breeds may be considered good animal models for the study of genetic predisposition to cancer, as they represent genetic clusters. From epidemiologic and case collection studies it emerges that some breeds are more likely to develop lymphoma or specific subtypes of lymphoma but available data are variable and geographically inconsistent. This study was born in the context of the European Canine Lymphoma Network with the aim of investigating the breed prevalence of canine lymphoma in different European countries and of investigating possible breed risk of lymphoma overall and/or different lymphoma subtypes

Rabbit derived VL single-domains as promising scaffolds to generate antibody–drug conjugates

© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.Antibody-drug conjugates (ADCs) are among the fastest-growing classes of therapeutics in oncology. Although ADCs are in the spotlight, they still present significant engineering challenges. Therefore, there is an urgent need to develop more stable and effective ADCs. Most rabbit light chains have an extra disulfide bridge, that links the variable and constant domains, between Cys80 and Cys171, which is not found in the human or mouse. Thus, to develop a new generation of ADCs, we explored the potential of rabbit-derived VL-single-domain antibody scaffolds (sdAbs) to selectively conjugate a payload to Cys80. Hence, a rabbit sdAb library directed towards canine non-Hodgkin lymphoma (cNHL) was subjected to in vitro and in vivo phage display. This allowed the identification of several highly specific VL-sdAbs, including C5, which specifically target cNHL cells in vitro and present promising in vivo tumor uptake. C5 was selected for SN-38 site-selective payload conjugation through its exposed free Cys80 to generate a stable and homogenous C5-DAB-SN-38. C5-DAB-SN-38 exhibited potent cytotoxicity activity against cNHL cells while inhibiting DNA-TopoI activity. Overall, our strategy validates a platform to develop a novel class of ADCs that combines the benefits of rabbit VL-sdAb scaffolds and the canine lymphoma model as a powerful framework for clinically translation of novel therapeutics for cancer.This work was supported by the Portuguese Funding Agency, Fundação para a Ciência e Tecnologia, FCT IP (SAICT/2017/32085, PTDC/QUI-OUT/3989/2021 and Ph.D. fellowship SFRH/BD/131468/2017 to ASA and SFRH/BD/90514/2012 to JD). CIISA has provided support through Project UIDB/00276/2020, funded by FCT and LA/P/0059/2020-AL4AnimalS. Research Institute for Medicines (iMed.ULisboa) acknowledges the financial support of Fundação para a Ciência e Tecnologia (Projects: PTDC/QUI-OUT/3989/2021; UIDB/04138/2020 and UIDP/04138/2020). The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project Nº 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC).info:eu-repo/semantics/publishedVersio

NVX-412, a new oncology drug candidate, induces S-phase arrest and DNA damage in cancer cells in a p53-independent manner.

The new molecular entity quinoxalinhydrazide derivative NVX-412 was identified as a promising drug candidate for the treatment of various cancer types due to its strong cytotoxic activity and relative specificity. Here, we provide first data about the mechanisms of action of NVX-412. We show that NVX-412 exerts its anti-neoplastic activity in a p53-independent manner and induces S-phase arrest and DNA damage as assessed by γH2AX staining. We suggest a bi-modal (dose-dependent) mode of action of NVX-412, being primarily cytostatic at lower and predominantly cytotoxic at higher concentrations. Based on the broad and consistent anti-neoplastic activity observed, NVX-412 holds promise as an effective drug candidate for the treatment of various cancer types, especially for hematological malignancies with highly unmet medical need

NVX-412 induces significant increase in cell sizes at IC₅₀ and higher concentrations in HCT116, HeLa and HT-29 cells.

<p>HCT116, HeLa and HT-29 cells were treated for up to 72 hours as indicated. A DMSO control was performed but did not show any differences compared to UTC. After 24, 48 and 72 hours pictures were taken (<b>A</b>, <b>C</b>, <b>E</b>). Panels <b>B</b>, <b>D</b> and <b>F</b> show quantification of cell sizes after 48 hours treatment with indicated concentrations of NVX-412. Box plots represent data of cells counted within 5 different fields of view. Statistical analysis was performed with GraphPad Prism 5.0. **** indicates p value <0.0001 (Mann-Whitney U Test). <b>A</b>, <b>B</b>: HCT116. <b>C</b>, <b>D</b>: HeLa. <b>E</b>, <b>F</b>: HT-29. UTC, Untreated Control; CPT, Camptothecin.</p

NCI-60 DTP human tumor cell line screen shows strong anti-cancer activity in various cancer types.

<p>A panel of 59 human tumor cell lines of diverse tumor entities was included in a NCI screen. This table shows the mean IC₅₀ values in nM after 48 hours incubation for the various cancer types for 57 of them. Results for two cell lines were identified as outliers and were not included in the calculation of the mean IC₅₀. Numbers in brackets indicate the number of cell lines tested within a certain cancer type. NCI, National Cancer Institute; DTP, Developmental Therapeutics Program.</p

Chemical structure of NVX-412.

<p><b>A:</b> Pyrazine-2-carboxylic acid N’-(7-fluoro-pyrrolo[1,2-α]quinoxalin-4-yl)-hydrazide-oxalic acid co-crystal; Molecular Formula: C₁₈H₁₃FN₆O₅, Molecular Weight: 412 g/mol <b>B:</b> Solvent accessible mesh model. White: carbon; yellow: fluorine; blue: nitrogen; red: oxygen. Both structures were generated with ChemBio 3D Ultra 12.0 (CambridgeSoft, MA, USA).</p

NVX-412 induces S-phase arrest and DNA damage and reduces DNA replication rate.

<p><b>A:</b> Cell cycle analyses by flow cytometry for HT-29, HeLa and HCT116 cells treated for 24 hours as indicated with NVX-412 or CPT. Percentages of cells in G1, S and G2/M phase of the cell cycle are shown. Cells were analyzed using a BD FACScan. <b>B:</b> NVX-412 reduces DNA replication in a reversible manner in HeLa and HCT116 cells. HeLa and HCT116 cells were treated for 24 hours as indicated. Additionally, after 24 hours of treatment cells were allowed to recover for 24 hours in normal growth medium without NVX-412 or CPT. DNA replication rate was analyzed by BrdU incorporation. <b>C:</b> Western Blot for pChk1(Ser296) in HCT116 cells after 0–48 hours incubation with 150 nM NVX-412. <b>D:</b> NVX-412 induces γH2AX in HeLa cells in a reversible manner. Cells were treated for 3 and 24 hours as indicated. Additionally, after 24 hours of treatment cells were allowed to recover for 24 hours in normal growth medium without NVX-412 or CPT. Data represent fold change in average γH2AX fluorescence intensity per nucleus (± SD) as quantified from immunofluorescence stainings. UTC, Untreated Control; CPT, Camptothecin. Mean values of at least 2 independent experiments are shown. Statistical analysis was performed with GraphPad Prism 5.0. **** indicates p value <0.0001 (Two-way ANOVA).</p

IC₅₀ values for 21 cell lines of various tumor entities.

<p>The table shows IC₅₀ values after 72 hours incubation for 21 cell lines originating from diverse tumor entities of two species. IC₅₀ values were calculated with GraphPad Prism 5.0 based on at least two independent experiments performed in duplicates.</p

Breed-associated risks for developing canine lymphoma differ among countries : an European canine lymphoma network study

Canine breeds may be considered good animal models for the study of genetic predisposition to cancer, as they represent genetic clusters. From epidemiologic and case collection studies it emerges that some breeds are more likely to develop lymphoma or specific subtypes of lymphoma but available data are variable and geographically inconsistent. This study was born in the context of the European Canine Lymphoma Network with the aim of investigating the breed prevalence of canine lymphoma in different European countries and of investigating possible breed risk of lymphoma overall and/or different lymphoma subtypes