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Projects

Peri-tumoral fibroblasts project
Projects
Peri-tumoral fibroblasts project

// Project

Peri-tumoral fibroblasts

www.taf-research.ro

Period of deployment

2011 – 2016

Project no.

ID 319/2011

Coordinator

"Victor Babeș" University of Medicine and Pharmacy in Timișoara

Project manager

Prof. Dr. Virgil Păunescu

Full name

Peri-tumoral fibroblasts as a new target in anti-tumor therapy – identification of origin, role and characteristic molecular markers

Objectives

General objectives: (1) investigating and validating the origin, role, molecular and functional markers characteristic of TAF, as well as the metabolic pathways activated at the level of tumor stromal cells; (2) identifying the methods of stimulating the immune system against TAF specific antigens.

Specific objectives: (1) Optimizing the isolation and cultivation procedures of peri-tumoral fibroblasts (TAF) derived from solid tumors in order to obtain high purity cell populations; (2) In vitro identification of morphological and functional markers characteristic of peri-tumoral fibroblasts (TAF); (3) Validation of morphological and functional characteristics of TAF in co-culture systems; (4) Comparative evaluation of the genetic characteristics between MSC and TAF; (5) Identification of specific methods for isolation of functionally active human cytotoxic T lymphocytes against TAF specific antigens

Project summary:

The peri-tumoral microenvironment has a key role in the development of malignant processes, so the identification of possible targets at the stromal level is of great interest and could provide complementary strategies to conventional anti-tumor therapies. The inability of immune surveillance to prevent tumor development, as well as the fact that initially effective therapies become ineffective against tumor development over time, suggests that there are mechanisms by which tumors escape immune control. Peri-tumoral fibroblasts (TAFs), which are potentially recruited from tissue stem cells, bone marrow mesenchymal stem cells (MSCs) or result from epithelial-mesenchymal transition, are important for tumor progression, providing the functional and structural supportive environment. The true origin, role and biological characteristics of TAFs are still not well understood. The main objective of this project is the complete characterization of TAFs obtained from solid tumors (breast cancer), to establish their origin and role in the tumor stroma. MSCs will be analyzed in the context of normal/precursor cells, with which TAF will be compared to identify specific markers, which can be used in future studies for anti-tumor therapies. The in vitro cell therapies proposed by this project assume the augmentation of cytotoxic T lymphocyte (CTL) populations, functionally active against TAF specific antigens.

Results:

This project aimed to evaluate the action of modulating the anti-tumor immune response through the action of CD8+ T lymphocytes on the peri-tumoral TAF of the same patient, in vitro, more precisely the targeting of CD8+ T lymphocytes on the FAP-CD26 antigen expressed by TAF isolated from solid tumors. This step was carried out with the help of the technique of using streptamers as a method to isolate CD8+ T lymphocytes active against FAP. Later, the active anti-FAP lymphocytes were put in contact with TAF whose proliferation was evaluated by the xCELLigence method by means of parameters that quantify the number of adhered cells and their degree of dispersion. Several cell populations (TAF) were used in different concentrations, on top of which peripheral blood mononuclear cells, both in the native state, and functionally activated anti-FAP CD8+ T lymphocytes were subsequently added.

The results of these experiments revealed the expression by TAF of some markers that characterize MSC (origin of TAF at the level of MSC) as well as the presence of CD26 at the level of TAF (through the immunocytochemistry method). This aspect made possible the investigation from this experimental stage, which revealed a certain decrease in adhesion and dispersion of TAF in co-culture with active anti-FAP CD8+ lymphocytes, but also with mononuclear cells in their native state, which suggests a potential influence of T lymphocytes on TAF, even in the absence of their activation. Although they were not constant, these results argue for the existence of a lymphocytic cytotoxic effect directed against TAF isolated from solid tumors. This aspect could constitute a basis from which to start for the optimization of TAF isolation and functionally active CD8+ lymphocytes against TAF-specific antigens as well as the extension of these investigations in vivo. 

Participation in scientific events:

  1. Bojin F, Ordodi V, Gavriliuc O, Cristea M, Anghel S, Gruia A, Mic FA, Cean A, Lazarovicz R, Toma M, Marusciac L, Tanasie G, Tatu C, Panaitescu C, Paunescu V. In vitro models of adipogenesis for understanding molecular mechanisms underlying the occurrence of obesity. Conference and Advanced Translational Research Workshop: Chronic Diseases – From Pathogenesis to Therapy, 25-27 October 2012, Timisoara. Abstract book pg. 37, ISBN 978-606-8054-93-3.
  2. Cernescu L, Toma M, Bojin F, Gavriliuc O, Anghel S, Cristea M, Gruia A, Tatu C, Tanasie G, Panaitescu C, Paunescu V. Proliferative potential of double positive CD4+/CD8+ T cells in co-culture system with tumor cells. The 24th Annual Immunology Conference, 27-29 September 2012, Bucharest. Abstract Book pg. 102, ISBN 978-606-8003-24-5.
  3. Bojin F, Ordodi V, Anghel S, Cristea M, Gavriliuc O, Marusciac L, Toma M, Lazarovicz R, Panaitescu C, Paunescu V. Na+/K+ – ATP-ase blockers inhibit functionality of bone marrow-derived mesenchymal stem cells. Conference and Advanced Research Workshop – Sudden Cardiac Death & Cardioprotection, 6-9 September 2012, Timisoara. Abstract Book pg. 97, ISBN 978-606-8054-80-3; 978-963-306-162-6.
  4. Bojin F, Gavriliuc OI, Cristea MI, Gruia AT, Anghel S, Tatu CS, Tanasie G, Panaitescu C, Tatu CA, Paunescu V. Ouabain inhibits in vitro proliferation of tumor-associated fibroblasts and cancer cell lines. European Journal of Cancer 48, Suppl. 5 (2012): S52; Proceedings Book, 22nd Biennial Congress of the European Association of Cancer Research – From basic research to personalized cancer treatment, 7-10 July 2012, Barcelona, Spain.
  5. Bojin F, Ordodi V, Cristea M, Anghel S, Gruia A, Tanasie G, Tatu C, Panaitescu C, Paunescu V. Introspection in molecular expression pattern of human mesenchymal stem cells-differentiated adipocytes. Academician Nicolae Cajal Symposium, Bucharest, March 28-31 2012, Abstract book pg. 10.
  6. Paunescu V, Bojin F, Ordodi V, Gavriliuc O, Cristea M, Anghel S, Gruia A, Mic FA, Tanasie G, Tatu C, Panaitescu C. Molecular expression pattern of human mesenchymal stem cells-differentiated adipocytes. Physiology-Physiology, 2012; supplement: 22 – The 11th National Congress of the Romanian Society of Physiological Sciences, 10-12 May, Timisoara, Romania.
  7. Anghel S, Bojin F, Tatu CA, Ordodi V, Gruia A, Rosca A, Tanasie G, Tatu C, Panaitescu C, Paunescu V. Differentiation mechanisms and pathways of dog periosteum-derived stem cells. Physiology-Physiology, 2012; supplement: 30 – The 11th National Congress of the Romanian Society of Physiological Sciences, 10-12 May, Timisoara, Romania.
  8. Bojin F, Ordodi V, Gavriliuc O, Cristea M, Anghel S, Gruia A, Nistor D, Crisnic D, Boleman A, Tanasie G, Tatu C, Panaitescu C, Paunescu V. Na/K pump inhibitors decrease proliferation of normal and tumor cells. Physiology-Physiology, 2012; supplement: 34 – The 11th National Congress of the Romanian Society of Physiological Sciences, 10-12 May, Timisoara, Romania.
  9. Cristea M, Bojin F, Gai E, Rosca A, Tatu C, Paunescu V. Anti-tumoral effects of Aristolochia clematitis aqueous extract on tumor cell lines. Physiology-Physiology, 2012; supplement: 39 – The 11th National Congress of the Romanian Society of Physiological Sciences, 10-12 May, Timisoara, Romania.
  10. Deica M, Sima R, Cernat L, Cristea M, Bojin F, Paunescu V. Effects of chemotherapeutic drugs and adjuvant therapy mixtures on normal and tumor cells – in vitro studies. Physiology-Physiology, 2012; supplement: 40 – The 11th National Congress of the Romanian Society of Physiological Sciences, 10-12 May, Timisoara, Romania.
  11. Gai E, Gruia A, Boldeanu F, Cristea M, Bojin F, Serban M, Arghirescu S, Jinca C, Balint Gib L, Ordodi V, Dumitrascu V, Tatu C, Lungeanu D, Paunescu V. Frequency of HLA-A allele correlated with high risk of hematological diseases. Physiology-Physiology, 2012; supplement: 43 – The 11th National Congress of the Romanian Society of Physiological Sciences, 10-12 May, Timisoara, Romania.
  12. Bojin F, Cernescu L, Cean A, Tanasie G, Panaitescu C, Paunescu V. Proliferative potential of double positive CD4+/CD8+ T lymphocytes in coculture with tumor cells. Abstract Book of 15th International Congress of Immunology, Milan, Italy, August 22-27, 2013, pg. 835, Frontiers – on line.
  13. Lazarovicz R, Toma M, Gavriliuc O, Marusciac L, Gruia A, Tatu C, Tanasie G, Panaitescu C, Bojin F, Paunescu V. Repurposing Na+/K+ pump blockers for in vitro suppression of tumor development. Abstract Book of 15th International Congress of Immunology, Milan, Italy, August 22-27, 2013, pg. 843, Frontiers – on line.
  14. Toma M, Lazarovicz R, Gavriliuc O, Tatu CA, Ordodi V, Rosca A, Cristea M, Gruia A, Anghel S, Tatu C, Tanasie G, Panaitescu C, Bojin F, Paunescu V. Tumor associated fibroblasts are endowed with increased ability to support tumor development. Abstract Book of 15th International Congress of Immunology, Milan, Italy, August 22-27, 2013, pg. 963, Frontiers – on line.
  15. Bojin F, Gavriliuc O, Tanasie G, Tatu C, Panaitescu C, Cernescu L, Marusciac L, Toma M, Lazarovicz R, Paunescu V. Targeted methods for isolation of human cytotoxic T lymphocytes against tumor microenvironment. Abstract Book of Physiology Conference – New facet of function and dysfunction. Current progress in biomedical sciences and medical education. 09-10 May 2013, Iasi, Romania
  16. Panaitescu C, Marusciac L, Ordodi V, Tanasie G, Tatu C, Toma M, Lazarovicz R, Gavriliuc O, Bojin F, Paunescu V. Effects of CD29 siRNA specific inhibition on mesenchymal stem cells migration capacity in inflammatory lesions. "Academician Nicolae Cajal" Symposium. Virology and Molecular Medicine, Genomics, Molecular and Cellular Biology, 27-29 March 2013, Bucharest. Abstract Book pg. 8.
  17. Paunescu V, Gavriliuc O, Tanasie G, Tatu C, Panaitescu C, Cernescu L, Marusciac L, Toma M, Lazarovicz R, Bojin F. New approaches in isolation of human cytotoxic T lymphocytes functionally active against TAF-derived specific antigens. "Academician Nicolae Cajal" Symposium. Virology and Molecular Medicine, Genomics, Molecular and Cellular Biology, 27-29 March 2013, Bucharest. Abstract Book pg. 9.
  18. Bojin F, Paunescu V. Tissue resident stem cells in normal and neoplastic conditions. IANR VI & 10th GCNN Conference, 4-7 April 2013, Bucharest, Romania, Abstract Book.
  19. Bojin F, Gavriliuc OI, Tatu CS, Tanasie G, Panaitescu C, Tatu CA, Paunescu V. Cytotoxic T lymphocytes functionally active against tumor microenvironment-derived specific antigen. 23rd Biennial Congress of the European Association of Cancer Research (EACR-23) – From basic research to personalized treatment in cancer, Munich, Germany 5 – 8 July 2014, Proceedings Book, European Journal of Cancer 2014; 50(suppl. 5) pp.891, ISSN 0959-8049.
  20. Gavriliuc O, Bojin F, Paunescu V. Are tumor associated fibroblasts activated mesenchymal stem cells? Stem cells and epigenetics in cancer. Hong Kong, China 16-18 October 2014, Abstract Book, pp 42.
  21. Paunescu V, Bojin F, Tatu C, Gavriliuc O, Tanasie G, Tatu CS, Panaitescu C. Adult mesenchymal stem cells and tumor-associated fibroblasts: insights on morphological and functional characteristics. International Society for Regenerative Medicine and Surgery First Congress. New Frontiers in Regenerative Medicine and Surgery. Interdisciplinarity, Research and Clinical Applications. May 14-16, 2015, Bucharest, Romania.
  22. Paunescu V, Bojin F, Tatu C, Gavriliuc O, Panaitescu C, Ordodi V, Tanasie G. Healing of skin lesions in animal model induced by adult mesenchymal stem cells and derivatives. International Society for Regenerative Medicine and Surgery First Congress. New Frontiers in Regenerative Medicine and Surgery. Interdisciplinarity, Research and Clinical Applications. May 14-16, 2015, Bucharest, Romania.

Full articles:

  1. Anastasiu MD, Cean A, Bojin MF, Panaitescu C, Tanasie G Paunescu V. Explants-isolated Human Placenta and Umbilical Cord Cells Share Characteristics of Both Epithelial and Mesenchymal Stem Cells. Rom J Morphol Embryol 2016; 57(2): 383-390.
  2. Hurmuz M, Bojin F, Ionac M, Tatu F, Puscasiu D, Tatu C. Plastic Adherence Method for Isolation of Stem Cells Derived from Infrapatellar Fat Pad. Plastic materials 2016; 53(3): 553-556.
  3. Bojin F, Gruia A, Ordodi V, Mic F, Marusciac L, Gavriliuc O, Tatu CA, Paunescu V. Features of lipid metabolism along differentiation pathway of human mesenchymal stem cells towards mature adipocytes. Submitted for publication – PLOS One, 2012.
  4. Cean A, Istratoaie B, Cristea M, Ivan A, Anghel S, Bojin F, Tanasie G, Tatu C, Panaitescu C, Paunescu V. The morphology of colonies from breast cancer SK-BR-3 cells in semisolid media depends on the media composition. Physiology – Physiology, 2012; 22(75): 9-14.
  5. Marusciac L, Panaitescu C, Paunescu V. Interactions between mesenchymal stem cells and cellular effectors of the immune system. Physiology – Physiology, 2012; 22(73): 4-10.
  6. Paunescu V, Bojin FM, Gavriliuc OI, Taculescu EA, Ianos R, Ordodi VL, Iman VF, Tatu CA. Enucleation: a possible mechanism of cancer cell death. J Cell Mol Med. 2014; 18(6): 962-5.
  7. Tatu CS, Bojin FM, Gruia AT, Ordodi VI, Mic FA, Iman V, Cean A, Gavriliuc OI, Paunescu V. Features of Lipid Metabolism along Differentiation Pathway of Human Mesenchymal Stem Cells towards Mature Adipocytes. Romanian Biotechnological Letters, 2014; 19(2): 9257-71.
  8. Tatu RF, Anusca D, Groza S, Marusciac L, Bojin F, Tatu CS, Hurmuz M, Paunescu V. Morphological and Functional Characterization of Femoral Head Drilling-Derived Mesenchymal Stem Cells. Rom J Morphol Embryol, 2014; 55.
  9. Deica M, Sima R, Cernat L, Bojin F, Tatu C, Tanasie G, Panaitescu C, Paunescu V. In Vitro Toxicity Studies on Breast Cancer Cell Line SK-BR3 and Tumor-Associated Fibroblasts (TAF). Physiology – Physiology, 2014; 24(81): 17-21.
  10. Deica M, Sima R, Cernat L, Bojin F, Tatu C, Tanasie G, Panaitescu C, Paunescu V. Changes Induced by Addition of Metformin in Culture Media of Tumor-Associated Fibroblasts (TAF). Physiology – Physiology, 2014; 24(81): 44-48.
  11. Ivanescu R, Lazarovicz R, Bojin F, Panaitescu C, Paunescu V, Tanasie G. Epithelial markers expressed by human mesenchymal stem cells upon in vitro induction. Physiology – Physiology, 2015; 25(85): 31-34.
  12. Anastasiu MD, Cean A, Bojin F, Gavriliuc O, Gluhovschi A, Anastasiu D, Craina M, Tatu C, Tanasie G, Panaitescu C, Paunescu V. Placental Stem Cells Modulate Tumor Formation in Mouse Model of Uterine Sarcoma. Physiology – Physiology, 2015; 25(85): 26-30.
  13. Anastasiu MD, Cernat L, Bojin F, Gavriliuc O, Gluhovschi A, Anastasiu D, Craina M, Crisnic D, Tatu C, Tanasie G, Panaitescu C, Paunescu V. In Vitro Assessment of Tumor-Associated Fibroblasts' Proliferation Ability and Viability. Physiology – Physiology, 2015; 25(87): 10-15.
  14. Ivanescu R, Toma M, Lazarovicz R, Bojin F, Nistor D, Panaitescu C, Paunescu V, Tanasie G. In Vitro Morphological Characterization of Endothelial Cells Derived from Bovine Cornea. Physiology – Physiology, 2015; 25(87): 36-40.

Fig. 1. Cell types used in project experiments

Fig. 2. Immunostaining of MSC and TAF cell populations

Fig. 3. Immunocytochemical and RT-PCR evaluation of alpha-SMA expression level in MSC and TAF cell populations (left panel) and evolution over time of FAP (fibroblast activation protein) expression evaluated by immunophenotypic (right panel)