The In vivo CAM assays platform provides scientific expertise and services using of the chick embryo model, more specifically, assays based on the chorioallantoic membrane – the CAM.

Created in 2012, the platform offers researchers additional or alternative in vivo tools (complying with the 3Rs policy) that are reliable and cost and time efficient.

Due to the CAM structure and its easy access, CAM assays constitute attractive preclinical in vivo tools for drug screening and/or vascular growth studies. Associated with the chick natural immunoincompetence, CAM assays can also be used to study complex cancer features and the effect of potential therapeutic molecules.

The platform ensures:

• Protocol design
• Experimental execution
• Analysis of the results
• Data interpretation.

 Assays are available for all scientific community to analyze functional features such as:

• Angiogenesis
• Tumorigenesis
• Cell Invasion
• Metastisation
• Vascular Permeability

The available collection of functional assays can be applied to a diversity of test conditions:

• Cells previously grown in culture
• Biomaterials assembled with or without cells
• Drugs, tested directly on the CAM or on CAM xenograft tumours
• Conditioned mediums
• Supernatants enriched in exosomes or other vesicles or components
• Extracts from plants or microorganisms.

The platform is exclusively dedicated to CAM assays and is fully equipped to address new challenges and widen the model applications in a range of research topics. The In vivo CAM assays platform’s work is validated by our publication track record, and by the national and international network of collaborators and clients, both from academia and industry.

ROOM

The in vivo CAM assays platform is located at i3s East wing, Room 107

CONTACTS

Marta Teixeira Pinto

mtpinto@i3s.up.pt

Ext.: 2070

SCHEDULING

The scheduling and design of the assays is personalized. Thus, it is necessary to schedule an initial meeting with head of the Platform to initiate the process.

In vivo analysis of functional features:

• Angiogenesis (e.g.)
• Tumourigenesis (e.g.)
• Cell Invasion (e.g.)
• Metastisation 
• Vascular Permeability (e.g.)

Multiple test conditions:

• cells previously grown in culture (normal or tumour)
• biomaterials (assembled with or without cells)
• drugs, tested directly on the CAM (anti- or pro- angiogenic activity)
• drugs, tested on CAM xenograft tumours (anti- or pro- tumourigenic activity)
• conditioned mediums (from cultured cells)
• supernatants enriched in components of interest (exosomes, etc)
• extracts from plants or microorganisms or derived proteins
• etc

THE CHICK EMBRYO MODEL

The chick embryo has long been used as a model organism in a number of areas including oncobiology and biomaterial research. The embryo is surrounded by the chorioallantoic membrane (CAM), a highly vascularised extra-embryonic membrane that can be used to graft human cells. When grafted on CAM, tumour cells are capable of stimulating the formation of new blood vessels, benefiting from blood supply. This allows them to develop in a similar manner as in their native microenvironment i.e. to proliferate, invade and metastasise to the chick embryonic organs.

The in vivo chick embryo model presents several important advantages:

• It fulfils the 3Rs policy.  Both the Directive 2010/63/EU of the European Parliament, and the Portuguese law DL 113/2013,  on the protection of animals used for scientific purposes, do not contain any kind of restriction to the use of non-mammal embryos
• The embryo is naturally immunoincompetent, thus easily allowing mammal cell xenografts
• It requires short experimental times

ROOM: The in vivo CAM assays platform is located at i3s East wing, Room 107

CONTACTS: Marta Teixeira Pinto mtpinto@i3s.up.pt; Ext.: 2070

SCHEDULING: The scheduling and design of the assays is personalized. Thus, it is necessary to schedule an initial meeting with head of the Platform to initiate the process.

Pinto MT, Ribeiro AS, Paredes J: The Chick CAM as an In Vivo System to Study Stem Cell Activity. In: Methods in Molecular Biology. vol. 2572; 2023: 155-166.

Peixoto J, Príncipe C, Pestana A, Osório H, Pinto MT, Prazeres H, Soares P, Lima RT: Using a Dual CRISPR/Cas9 Approach to Gain Insight into the Role of LRP1B in Glioblastoma. In: International Journal of Molecular Sciences. vol. 24; 2023.

Padrão T, Dias J, Carvalho Â, Pinto MT, Monteiro FJ, Sousa SR: Vancomycin-loaded bone substitute as a ready-to-use drug delivery system to treat osteomyelitis. Ceram Int 2023, 49(15):24771-24782.

Moura D, Pereira AT, Ferreira HP, Barrias CC, Magalhaes FD, Bergmeister H, Goncalves IC: Poly(2-hydroxyethyl methacrylate) hydrogels containing graphene-based materials for blood-contacting applications: From soft inert to strong degradable material. Acta Biomater 2023, 164:253-268.

Mota R, Lima RT, Flores C, Silva JF, Cruz B, Alves B, Pinto MT, Adessi A, Pereira SB, De Philippis R et al: Assessing the Antitumor Potential of Variants of the Extracellular Carbohydrate Polymer from Synechocystis DeltasigF Mutant. Polymers (Basel) 2023, 15(6).

Malespin-Bendana W, Ferreira RM, Pinto MT, Figueiredo C, Alpizar-Alpizar W, Une C, Figueroa-Protti L, Ramirez V: Helicobacter pylori infection induces abnormal expression of pro-angiogenic gene ANGPT2 and miR-203a in AGS gastric cell line. Braz J Microbiol 2023, 54(2):791-801.

Fischer D, Fluegen G, Garcia P, Ghaffari-Tabrizi-Wizsy N, Gribaldo L, Huang RY, Rasche V, Ribatti D, Rousset X, Pinto MT et al: The CAM Model-Q&A with Experts. Cancers (Basel) 2022, 15(1).

Fiordalisi MF, Ferreira JR, Pinto ML, Ribeiro-Machado C, Teixeira Pinto M, Oliveira MJ, Barbosa MA, Madeira Gonçalves R, Caldeira J: The impact of matrix age on intervertebral disc regeneration. Biomaterials Advances 2022, 143:213192.

Baptista-Silva S, Bernardes BG, Borges S, Rodrigues I, Fernandes R, Gomes-Guerreiro S, Pinto MT, Pintado M, Soares R, Costa R, Oliveira AL: Exploring Silk Sericin for Diabetic Wounds: An In Situ-Forming Hydrogel to Protect against Oxidative Stress and Improve Tissue Healing and Regeneration. Biomolecules 2022, 12(6).

Tokarchuk I, Janser FA, Schläfli AM, Pinto MT, Humbert M, Niklaus NJ, Berezowska S, Langer R, Tschan MP: Increased LAMP2A levels correlate with a shorter disease-free survival of HER2 negative breast cancer patients and increased breast cancer cell viability. Biochemical and Biophysical Research Communications 2021, 569:47-53.

Silva BR, Rebelo R, Rodrigues JM, Xavier CPR, Vasconcelos MH, Queiroz MRP: Synthesis of Novel Methyl 3-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates and Antitumor Activity Evaluation: Studies In Vitro and In Ovo Grafts of Chick Chorioallantoic Membrane (CAM) with a Triple Negative Breast Cancer Cell Line. Molecules 2021, 26(6).

Pinto MT, Ribeiro AS, Conde I, Carvalho R, Paredes J: The chick chorioallantoic membrane model: A new in vivo tool to evaluate breast cancer stem cell activity. International Journal of Molecular Sciences 2021, 22(1):1-15.

Pinto F, Santos-Ferreira L, Pinto MT, Gomes C, Reis CA: The Extracellular Small Leucine-Rich Proteoglycan Biglycan Is a Key Player in Gastric Cancer Aggressiveness. Cancers (Basel) 2021, 13(6).

Henriques TB, Dos Santos DZ, Dos Santos Guimaraes I, Tessarollo NG, Lyra-Junior PCM, Mesquita P, Padua D, Amaral AL, Cavadas B, Pereira L et al: Inhibition of CXCR2 plays a pivotal role in re-sensitizing ovarian cancer to cisplatin treatment. Aging (Albany NY) 2021, 13(10):13405-13420.

Giao T, Saavedra J, Vieira JR, Pinto MT, Arsequell G, Cardoso I: Neuroprotection in early stages of Alzheimer's disease is promoted by transthyretin angiogenic properties. Alzheimers Res Ther 2021, 13(1):143.

Sousa AR, Martins-Cruz C, Oliveira MB, Mano JF: One-Step Rapid Fabrication of Cell-Only Living Fibers. Adv Mater 2020, 32(2):e1906305.

Silva AS, Santos LF, Mendes MC, Mano JF: Multi-layer pre-vascularized magnetic cell sheets for bone regeneration. Biomaterials 2020, 231:119664.

Loureiro J, Torres AL, Neto T, Aguiar P, Barrias CC, Pinto MT, Amaral IF: Corrigendum to "Conjugation of the T1 sequence from CCN1 to fibrin hydrogels for therapeutic vascularization" [Mater. Sci. & Eng. C. 104 (2019) 109847]. Mater Sci Eng C Mater Biol Appl 2020, 108:110514.

Leite M, Marques MS, Melo J, Pinto MT, Cavadas B, Aroso M, Gomez-Lazaro M, Seruca R, Figueiredo C: Helicobacter Pylori Targets the EPHA2 Receptor Tyrosine Kinase in Gastric Cells Modulating Key Cellular Functions. Cells 2020, 9(2).

Dionisio MR, Vieira AF, Carvalho R, Conde I, Oliveira M, Gomes M, Pinto MT, Pereira P, Pimentel J, Souza C et al: BR-BCSC Signature: The Cancer Stem Cell Profile Enriched in Brain Metastases that Predicts a Worse Prognosis in Lymph Node-Positive Breast Cancer. Cells 2020, 9(11).

Coelho CC, Padrao T, Costa L, Pinto MT, Costa PC, Domingues VF, Quadros PA, Monteiro FJ, Sousa SR: The antibacterial and angiogenic effect of magnesium oxide in a hydroxyapatite bone substitute. Sci Rep 2020, 10(1):19098.

Alves MM, Mil-Homens D, Pinto S, Santos CF, Montemor MF: Antagonist biocompatibilities of Zn-based materials functionalized with physiological active metal oxides. Colloids Surf B Biointerfaces 2020, 191:110990.

Marques FG, Poli E, Rino J, Pinto MT, Diegues I, Pina F, Rosa Santos SC: Low Doses of Ionizing Radiation Enhance the Angiogenic Potential of Adipocyte Conditioned Medium. Radiat Res 2019, 192(5):517-526.

Loureiro J, Torres AL, Neto T, Aguiar P, Barrias CC, Pinto MT, Amaral IF: Conjugation of the T1 sequence from CCN1 to fibrin hydrogels for therapeutic vascularization. Mater Sci Eng C Mater Biol Appl 2019, 104:109847.

Janser FA, Ney P, Pinto MT, Langer R, Tschan MP: The Chick Chorioallantoic Membrane (CAM) Assay as a Three-dimensional Model to Study Autophagy in Cancer Cells. Bio Protoc 2019, 9(13):e3290.

Ghazaryan N, Movsisyan N, Macedo JC, Vaz S, Ayvazyan N, Pardo L, Logarinho E: The antitumor efficacy of monomeric disintegrin obtustatin in S-180 sarcoma mouse model. Invest New Drugs 2019, 37(5):1044-1051.

Freitas D, Campos D, Gomes J, Pinto F, Macedo JA, Matos R, Mereiter S, Pinto MT, Polonia A, Gartner F et al: O-glycans truncation modulates gastric cancer cell signaling and transcription leading to a more aggressive phenotype. EBioMedicine 2019, 40:349-362.

Correia CR, Bjorge IM, Zeng J, Matsusaki M, Mano JF: Liquefied Microcapsules as Dual-Microcarriers for 3D+3D Bottom-Up Tissue Engineering. Adv Healthc Mater 2019, 8(22):e1901221.

Torres AL, Bidarra SJ, Pinto MT, Aguiar PC, Silva EA, Barrias CC: Guiding morphogenesis in cell-instructive microgels for therapeutic angiogenesis. Biomaterials 2018, 154:34-47.

Santos M, Pereira PM, Varanda AS, Carvalho J, Azevedo M, Mateus DD, Mendes N, Oliveira P, Trindade F, Pinto MT et al: Codon misreading tRNAs promote tumor growth in mice. RNA Biology 2018, 15(6):773-786.

Bauman E, Feijao T, Carvalho DTO, Granja PL, Barrias CC: Xeno-free pre-vascularized spheroids for therapeutic applications. Sci Rep 2018, 8(1):230.

Ribeiro-Rodrigues TM, Laundos TL, Pereira-Carvalho R, Batista-Almeida D, Pereira R, Coelho-Santos V, Silva AP, Fernandes R, Zuzarte M, Enguita FJ et al: Exosomes secreted by cardiomyocytes subjected to ischaemia promote cardiac angiogenesis. Cardiovascular Research 2017, 113(11):1338-1350.

Prazeres H, Torres J, Rodrigues F, Pinto MT, Pastoriza MC, Gomes D, Cameselle-Teijeiro J, Vidal A, Martins TC, Sobrinho-Simões M, Soares P: Corrigendum: Chromosomal, epigenetic and microRNA-mediated inactivation of LRP1B, a modulator of the extracellular environment of thyroid cancer cells (Oncogene (2010) 30 (1302-1317) DOI: 10.1038/onc.2010.512). Oncogene 2017, 36(1):146.

Pópulo H, Nunes B, Sampaio C, Batista R, Pinto MT, Gaspar TB, Miranda-Alves L, Cai RZ, Zhang XY, Schally AV et al: Inhibitory Effects of Antagonists of Growth Hormone-Releasing Hormone (GHRH) in Thyroid Cancer. Hormones and Cancer 2017, 8(5-6):314-324.

Olivera-Severo D, Uberti AF, Marques MS, Pinto MT, Gomez-Lazaro M, Figueiredo C, Leite M, Carlini CR: A new role for Helicobacter pylori urease: Contributions to angiogenesis. Frontiers in Microbiology 2017, 8(SEP).

Teresa Pinto A, Laranjeiro Pinto M, Patrícia Cardoso A, Monteiro C, Teixeira Pinto M, Filipe Maia A, Castro P, Figueira R, Monteiro A, Marques M et al: Ionizing radiation modulates human macrophages towards a pro-inflammatory phenotype preserving their pro-invasive and pro-angiogenic capacities. Scientific Reports 2016, 6.

Resende C, Regalo G, Durães C, Pinto MT, Wen X, Figueiredo C, Carneiro F, Machado JC: Interleukin-1B signalling leads to increased survival of gastric carcinoma cells through a CREB-C/EBPβ-associated mechanism. Gastric Cancer 2016, 19(1):74-84.

Pinto AT, Pinto ML, Velho S, Pinto MT, Cardoso AP, Figueira R, Monteiro A, Marques M, Seruca R, Barbosa MA et al: Intricate macrophage-colorectal cancer cell communication in response to radiation. PLoS ONE 2016, 11(8).

Ferreira LB, Tavares C, Pestana A, Pereira CL, Eloy C, Pinto MT, Castro P, Batista R, Rios E, Sobrinho-Simões M et al: Osteopontin-a splice variant is overexpressed in papillary thyroid carcinoma and modulates invasive behavior. Oncotarget 2016, 7(32):52003-52016.

Estrada MF, Rebelo SP, Davies EJ, Pinto MT, Pereira H, Santo VE, Smalley MJ, Barry ST, Gualda EJ, Alves PM et al: Modelling the tumour microenvironment in long-term microencapsulated 3D co-cultures recapitulates phenotypic features of disease progression. Biomaterials 2016, 78:50-61.

Almeida MI, Silva AM, Vasconcelos DM, Almeida CR, Caires H, Pinto MT, Calin GA, Santos SG, Barbosa MA: miR-195 in human primary mesenchymal stromal/stem cells regulates proliferation, osteogenesis and paracrine effect on angiogenesis. Oncotarget 2016, 7(1):7-22.

Cardoso AP, Pinto ML, Pinto AT, Pinto MT, Monteiro C, Oliveira MI, Santos SG, Relvas JB, Seruca R, Mantovani A et al: Matrix metalloproteases as maestros for the dual role of LPS- and IL-10-stimulated macrophages in cancer cell behaviour. BMC Cancer 2015, 15(1).

Caldeira J, Figueiredo J, Brás-Pereira C, Carneiro P, Moreira AM, Pinto MT, Relvas JB, Carneiro F, Barbosa M, Casares F et al: E-cadherin-defective gastric cancer cells depend on Laminin to survive and invade. Human Molecular Genetics 2015, 24(20):5891-5900.

Simões-correia J, Silva DI, Melo S, Figueiredo J, Caldeira J, Pinto MT, Girão H, Pereira P, Seruca R: DNAJB4 molecular chaperone distinguishes WT from mutant E-cadherin, determining their fate in vitro and in vivo. Human Molecular Genetics 2014, 23(8):2094-2105.

Cardoso AP, Pinto ML, Pinto AT, Oliveira MI, Pinto MT, Gonçalves R, Relvas JB, Figueiredo C, Seruca R, Mantovani A et al: Macrophages stimulate gastric and colorectal cancer invasion through EGFR Y 1086, c-Src, Erk1/2 and Akt phosphorylation and smallGTPase activity. Oncogene 2014, 33(16):2123-2133.

Gomes C, Osório H, Pinto MT, Campos D, Oliveira MJ, Reis CA: Expression of ST3GAL4 Leads to SLex Expression and Induces c-Met Activation and an Invasive Phenotype in Gastric Carcinoma Cells. PLoS ONE 2013, 8(6).

Pinheiro H, Carvalho J, Oliveira P, Ferreira D, Pinto MT, Osoquot, rio H, Licastro D, Bordeira c, o R et al: Transcription initiation arising from E-cadherin/CDH1 intron2: A novel protein isoform that increases gastric cancer cell invasion and angiogenesis. Human Molecular Genetics 2012, 21(19):4253-4269.

Moutinho RP, Coelho L, Silva A, Lobo Pereira JA, Pinto M, Baptista IP: Validation of a dental image-analyzer tool to measure the radiographic defect angle of the intrabony defect in periodontitis patients. Journal of Periodontal Research 2012, 47(6):695-700.

Caldeira J, Simões-Correia J, Paredes J, Pinto MT, Sousa S, Corso G, Marrelli D, Roviello F, Pereira PS, Weil D et al: CPEB1, a novel gene silenced in gastric cancer: A Drosophila approach. Gut 2012, 61(8):1115-1123.

Prazeres H, Torres J, Rodrigues F, Pinto M, Pastoriza MC, Gomes D, Cameselle-Teijeiro J, Vidal A, Martins TC, Sobrinho-Simões M, Soares P: Chromosomal, epigenetic and microRNA-mediated inactivation of LRP1B, a modulator of the extracellular environment of thyroid cancer cells. Oncogene 2011, 30(11):1302-1317.

Pinto M, Soares P, Ribatti D: Thyroid hormone as a regulator of tumor induced angiogenesis. Cancer Letters 2011, 301(2):119-126.

DIRECTIVE 2010/63/EU OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 22 September 2010 on the protection of animals used for scientific purposes

Decreto-Lei n.º 113/2013

i3S Scientific Platforms Acknowledgements