Massimo Aglietta
e-mail: massimo.aglietta AT ircc.it
website: www.unito.it
affiliation: Università di Torino
research area(s): Experimental Medicine, Cancer Biology
Course:
Biomedical Sciences and Human Oncology
University/Istitution: Università di Torino
University/Istitution: Università di Torino
Present Position:
Professor Medical Oncology, University of Turin
Division Medical Oncology
Istituto per la Ricerca e la Cura del Cancro (I.R.C.C.)
Strada Provinciale 142 - I 10060, Candiolo (Turin), Italy
Phone: (+39) 011 9933628
Fax: (+39) 011 9933299
E-mail: massimo.aglietta@unito.it
BRIEF CRONOLOGY OF EMPLOYMENT
1996 " at present Chief Department Hematology/Oncology, Institute for Cancer Research and Treatment (I.R.C.C), ASO Ordine Mauriziano di Torino, Italy
1990 " at present Full Professor, Internal Medicine (1990-2002); Medical Oncology (from 2002 - present), University of Torino
1990 - 1996: Chief Department Internal Medicine, Ospedale Maggiore, Novara, Italy.
1985 - 1990: Associate Professor, Internal Medicine, University of Torino
1980 - 1985: Assistant Professor, Internal Medicine, University of Torino
1976 - 1978: Scientific cooperator, Radiobiological Institute TNO, Rijswijk, NL
STAGES
1988 (May-Oct):Department of Oncology (Prof. Cavalli), Hospital S. Giovanni, Bellinzona, Switzerland.
1981 (Apr-Jul):Department of Hematology (Prof. N.C. Gorin) Hospital St. Antoine, Paris, France
STUDIES AND SPECIALIZATIONS
1987:Clinical Oncology, cum laude, University of Parma.
1981:Internal Medicine, cum laude, University of Torino.
1976:Degree in Medicine - University of Torino, cum laude.
Experienced in the conduction of clinical trial in compliance with the ICH/GCP procedures (latest training on April 2008)
MD Board Registration n°: 238 - Registered in Biella, Italy
Professor Medical Oncology, University of Turin
Division Medical Oncology
Istituto per la Ricerca e la Cura del Cancro (I.R.C.C.)
Strada Provinciale 142 - I 10060, Candiolo (Turin), Italy
Phone: (+39) 011 9933628
Fax: (+39) 011 9933299
E-mail: massimo.aglietta@unito.it
BRIEF CRONOLOGY OF EMPLOYMENT
1996 " at present Chief Department Hematology/Oncology, Institute for Cancer Research and Treatment (I.R.C.C), ASO Ordine Mauriziano di Torino, Italy
1990 " at present Full Professor, Internal Medicine (1990-2002); Medical Oncology (from 2002 - present), University of Torino
1990 - 1996: Chief Department Internal Medicine, Ospedale Maggiore, Novara, Italy.
1985 - 1990: Associate Professor, Internal Medicine, University of Torino
1980 - 1985: Assistant Professor, Internal Medicine, University of Torino
1976 - 1978: Scientific cooperator, Radiobiological Institute TNO, Rijswijk, NL
STAGES
1988 (May-Oct):Department of Oncology (Prof. Cavalli), Hospital S. Giovanni, Bellinzona, Switzerland.
1981 (Apr-Jul):Department of Hematology (Prof. N.C. Gorin) Hospital St. Antoine, Paris, France
STUDIES AND SPECIALIZATIONS
1987:Clinical Oncology, cum laude, University of Parma.
1981:Internal Medicine, cum laude, University of Torino.
1976:Degree in Medicine - University of Torino, cum laude.
Experienced in the conduction of clinical trial in compliance with the ICH/GCP procedures (latest training on April 2008)
MD Board Registration n°: 238 - Registered in Biella, Italy
The development of new drugs hitting a specific molecular target has been a further step toward a more effective treatment of cancer. The results, however, have been inferior to expectation: in most tumors it has been difficult to find a specific target and , most importantly, the development of resistance is frequent.
It has become evident that further relevant progresses can derive only from a smart integration of different strategies: a) classic chemotherapy; b) target therapy with monoclonal antibodies or small molecules inhibiting specific targets of the neoplastic cells c) modulation of the tumor environment acting on tumor vessels or on the immune system.
Based on these premises, with a strict interaction between the clinic and the lab, our unit is engaged in projects aiming to overcome the resistance to treatment in specific neoplasias: breast cancer with a particular interest on HER-2 positive subgroups, sarcomas with a specific interest in GIST and osteosarcoma and gastrointestinal tumors.
For all these tumors we conduct GCP protocols in which the study of the tumor is mandatory in order to define markers predictive of response to treatment and the mechanisms underlying the development of tumor resistance.
It has become evident that further relevant progresses can derive only from a smart integration of different strategies: a) classic chemotherapy; b) target therapy with monoclonal antibodies or small molecules inhibiting specific targets of the neoplastic cells c) modulation of the tumor environment acting on tumor vessels or on the immune system.
Based on these premises, with a strict interaction between the clinic and the lab, our unit is engaged in projects aiming to overcome the resistance to treatment in specific neoplasias: breast cancer with a particular interest on HER-2 positive subgroups, sarcomas with a specific interest in GIST and osteosarcoma and gastrointestinal tumors.
For all these tumors we conduct GCP protocols in which the study of the tumor is mandatory in order to define markers predictive of response to treatment and the mechanisms underlying the development of tumor resistance.
1)Joensuu H, De Braud F, Grignagni G, De Pas T, Spitalieri G, Coco P, Spreafico C, Boselli S, Toffalorio F, Bono P, Jalava T, Kappeler C, Aglietta M, Laurent D, Casali PG. Vatalanib for metastatic gastrointestinal stromal tumour (GIST) resistant to imatinib: final results of a phase II study. Br J Cancer. 2011 May24;104(11):1686-90.
2)Grignani G, Palmerini E, Dileo P, Asaftei SD, D'Ambrosio L, Pignochino Y, Mercuri M, Picci P, Fagioli F, Casali PG, Ferrari S, Aglietta M. A phase II trial of sorafenib in relapsed and unresectable high-grade osteosarcoma after failure of standard multimodal therapy: an Italian Sarcoma Group study. Ann Oncol. 2011 Apr 28.
3):Giaccone L, Storer B, Patriarca F, Rotta M, Sorasio R, Allione B, Carnevale-Schianca F, Festuccia M, Brunello L, Omedè P, Bringhen S, Aglietta M, Levis A, Mordini N, Gallamini A, Fanin R, Massaia M, Palumbo A, Ciccone G, Storb R, Gooley TA, Boccadoro M, Bruno B. Long-term follow-up of a comparison of nonmyeloablative allografting with autografting for newly diagnosed myeloma. Blood. 2011 Jun 16;117(24):6721-7.
4)Sangiolo D, Leuci V, Gallo S, Aglietta M, Piacibello W. Gene-modified T lymphocytes in the setting of hematopoietic cell transplantation: potential benefits and possible risks. Expert Opin Biol Ther. 2011 May;11(5):655-66. 6)Peraldo-Neia C, Migliardi G, Mello-Grand M, Montemurro F, Segir R, Pignochino Y, Cavalloni G, Torchio B, Mosso L, Chiorino G, Aglietta M. Epidermal Growth Factor Receptor (EGFR) mutation analysis, gene expression profiling and EGFR protein expression in primary prostate cancer. BMC Cancer. 2011 Jan 25;11:31.
5)Thiel U, Wawer A, Wolf P, Badoglio M, Santucci A, Klingebiel T, Basu O, Borkhardt A, Laws HJ, Kodera Y, Yoshimi A, Peters C, Ladenstein R, Pession A, Prete A, Urban EC, Schwinger W, Bordigoni P, Salmon A, Diaz MA, Afanasyev B, Lisukov I, Morozova E, Toren A, Bielorai B, Korsakas J, Fagioli F, Caselli D, Ehninger G, Gruhn B, Dirksen U, Abdel-Rahman F, Aglietta M, Mastrodicasa E, Torrent M, Corradini P, Demeocq F, Dini G, Dreger P, Eyrich M, Gozdzik J, Guilhot
F, Holler E, Koscielniak E, Messina C, Nachbaur D, Sabbatini R, Oldani E, Ottinger H, Ozsahin H, Schots R, Siena S, Stein J, Sufliarska S, Unal A, Ussowicz M, Schneider P, Woessmann W, Jürgens H, Bregni M, Burdach S; on behalf of the Solid Tumor Working Party (STWP) and the Pediatric Disease Working Party (PDWP) of the European Group for Blood and Marrow Transplantation (EBMT), the Asia Pacific Blood and Marrow Transplantation (APBMT), the Pediatric Registry for Stem. No improvement of survival with reduced- versus high-intensity conditioning for allogeneic stem cell transplants in Ewing tumor patients. Ann Oncol. 2011 Jul;22(7):1614-1621.
6)Valabrega G, Capellero S, Cavalloni G, Zaccarello G, Petrelli A, Migliardi G, Milani A, Peraldo-Neia C, Gammaitoni L, Sapino A, Pecchioni C, Moggio A, Giordano S, Aglietta M, Montemurro F. HER2-positive breast cancer cells resistant to trastuzumab and lapatinib lose reliance upon HER2 and are sensitive to the multitargeted kinase inhibitor sorafenib. Breast Cancer Res Treat. 2010 Dec 9.
7)Cavo M, Tacchetti P, Patriarca F, Petrucci MT, Pantani L, Galli M, Di Raimondo F, Crippa C, Zamagni E, Palumbo A, Offidani M, Corradini P, Narni F, Spadano A, Pescosta N, Deliliers GL, Ledda A, Cellini C, Caravita T, Tosi P, Baccarani M; GIMEMA Italian Myeloma Network. Bortezomib with thalidomide plusdexamethasone compared with thalidomide plus dexamethasone as induction therapy before, and consolidation therapy after, double autologous stem-cell transplantation in newly diagnosed multiple myeloma: a randomised phase 3 study. Lancet. 2010 Dec 18;376(9758):2075-85.
8)Labianca R, Sobrero A, Isa L, Cortesi E, Barni S, Nicolella D, Aglietta M, Lonardi S, Corsi D, Turci D, Beretta GD, Fornarini G, Dapretto E, Floriani I, Zaniboni A; Italian Group for the Study of Gastrointestinal Cancer-GISCAD. Intermittent versus continuous chemotherapy in advanced colorectal cancer: a randomised 'GISCAD' trial. Ann Oncol. 2011 May;22(5):1236-42.
9)Grignani G, Palmerini E, Stacchiotti S, Boglione A, Ferraresi V, Frustaci S, Comandone A, Casali PG, Ferrari S, Aglietta M. A phase 2 trial of imatinib mesylate in patients with recurrent nonresectable chondrosarcomas expressing platelet-derived growth factor receptor-α or -β: An Italian Sarcoma Group study. Cancer. 2011 Feb 15;117(4):826-31. doi: 10.1002/cncr.25632.
10)Tesio M, Golan K, Corso S, Giordano S, Schajnovitz A, Vagima Y, Shivtiel S, Kalinkovich A, Caione L, Gammaitoni L, Laurenti E, Buss EC, Shezen E, Itkin T, Kollet O, Petit I, Trumpp A, Christensen J, Aglietta M, Piacibello W, Lapidot T. Enhanced c-Met activity promotes G-CSF-induced mobilization of hematopoietic progenitor cells via ROS signaling. Blood. 2011 Jan 13;117(2):419-28.
2)Grignani G, Palmerini E, Dileo P, Asaftei SD, D'Ambrosio L, Pignochino Y, Mercuri M, Picci P, Fagioli F, Casali PG, Ferrari S, Aglietta M. A phase II trial of sorafenib in relapsed and unresectable high-grade osteosarcoma after failure of standard multimodal therapy: an Italian Sarcoma Group study. Ann Oncol. 2011 Apr 28.
3):Giaccone L, Storer B, Patriarca F, Rotta M, Sorasio R, Allione B, Carnevale-Schianca F, Festuccia M, Brunello L, Omedè P, Bringhen S, Aglietta M, Levis A, Mordini N, Gallamini A, Fanin R, Massaia M, Palumbo A, Ciccone G, Storb R, Gooley TA, Boccadoro M, Bruno B. Long-term follow-up of a comparison of nonmyeloablative allografting with autografting for newly diagnosed myeloma. Blood. 2011 Jun 16;117(24):6721-7.
4)Sangiolo D, Leuci V, Gallo S, Aglietta M, Piacibello W. Gene-modified T lymphocytes in the setting of hematopoietic cell transplantation: potential benefits and possible risks. Expert Opin Biol Ther. 2011 May;11(5):655-66. 6)Peraldo-Neia C, Migliardi G, Mello-Grand M, Montemurro F, Segir R, Pignochino Y, Cavalloni G, Torchio B, Mosso L, Chiorino G, Aglietta M. Epidermal Growth Factor Receptor (EGFR) mutation analysis, gene expression profiling and EGFR protein expression in primary prostate cancer. BMC Cancer. 2011 Jan 25;11:31.
5)Thiel U, Wawer A, Wolf P, Badoglio M, Santucci A, Klingebiel T, Basu O, Borkhardt A, Laws HJ, Kodera Y, Yoshimi A, Peters C, Ladenstein R, Pession A, Prete A, Urban EC, Schwinger W, Bordigoni P, Salmon A, Diaz MA, Afanasyev B, Lisukov I, Morozova E, Toren A, Bielorai B, Korsakas J, Fagioli F, Caselli D, Ehninger G, Gruhn B, Dirksen U, Abdel-Rahman F, Aglietta M, Mastrodicasa E, Torrent M, Corradini P, Demeocq F, Dini G, Dreger P, Eyrich M, Gozdzik J, Guilhot
F, Holler E, Koscielniak E, Messina C, Nachbaur D, Sabbatini R, Oldani E, Ottinger H, Ozsahin H, Schots R, Siena S, Stein J, Sufliarska S, Unal A, Ussowicz M, Schneider P, Woessmann W, Jürgens H, Bregni M, Burdach S; on behalf of the Solid Tumor Working Party (STWP) and the Pediatric Disease Working Party (PDWP) of the European Group for Blood and Marrow Transplantation (EBMT), the Asia Pacific Blood and Marrow Transplantation (APBMT), the Pediatric Registry for Stem. No improvement of survival with reduced- versus high-intensity conditioning for allogeneic stem cell transplants in Ewing tumor patients. Ann Oncol. 2011 Jul;22(7):1614-1621.
6)Valabrega G, Capellero S, Cavalloni G, Zaccarello G, Petrelli A, Migliardi G, Milani A, Peraldo-Neia C, Gammaitoni L, Sapino A, Pecchioni C, Moggio A, Giordano S, Aglietta M, Montemurro F. HER2-positive breast cancer cells resistant to trastuzumab and lapatinib lose reliance upon HER2 and are sensitive to the multitargeted kinase inhibitor sorafenib. Breast Cancer Res Treat. 2010 Dec 9.
7)Cavo M, Tacchetti P, Patriarca F, Petrucci MT, Pantani L, Galli M, Di Raimondo F, Crippa C, Zamagni E, Palumbo A, Offidani M, Corradini P, Narni F, Spadano A, Pescosta N, Deliliers GL, Ledda A, Cellini C, Caravita T, Tosi P, Baccarani M; GIMEMA Italian Myeloma Network. Bortezomib with thalidomide plusdexamethasone compared with thalidomide plus dexamethasone as induction therapy before, and consolidation therapy after, double autologous stem-cell transplantation in newly diagnosed multiple myeloma: a randomised phase 3 study. Lancet. 2010 Dec 18;376(9758):2075-85.
8)Labianca R, Sobrero A, Isa L, Cortesi E, Barni S, Nicolella D, Aglietta M, Lonardi S, Corsi D, Turci D, Beretta GD, Fornarini G, Dapretto E, Floriani I, Zaniboni A; Italian Group for the Study of Gastrointestinal Cancer-GISCAD. Intermittent versus continuous chemotherapy in advanced colorectal cancer: a randomised 'GISCAD' trial. Ann Oncol. 2011 May;22(5):1236-42.
9)Grignani G, Palmerini E, Stacchiotti S, Boglione A, Ferraresi V, Frustaci S, Comandone A, Casali PG, Ferrari S, Aglietta M. A phase 2 trial of imatinib mesylate in patients with recurrent nonresectable chondrosarcomas expressing platelet-derived growth factor receptor-α or -β: An Italian Sarcoma Group study. Cancer. 2011 Feb 15;117(4):826-31. doi: 10.1002/cncr.25632.
10)Tesio M, Golan K, Corso S, Giordano S, Schajnovitz A, Vagima Y, Shivtiel S, Kalinkovich A, Caione L, Gammaitoni L, Laurenti E, Buss EC, Shezen E, Itkin T, Kollet O, Petit I, Trumpp A, Christensen J, Aglietta M, Piacibello W, Lapidot T. Enhanced c-Met activity promotes G-CSF-induced mobilization of hematopoietic progenitor cells via ROS signaling. Blood. 2011 Jan 13;117(2):419-28.
Project Title:
Project Title:
Xenopatient model in rare tumors: a preclinical tool to assay new terapeutic strategies
Mouse cancer models have consistently been used to qualify new anticancer drugs in the development of human clinical trials.
INTRODUCTION
Rodent tumor models currently being used and which include transgenic tumor models, and those generated by planting human tumor cell lines subcutaneously in immunodeficient mice, do not sufficiently represent clinical cancer characteristics, especially with regard to metastasis and drug sensitivity.
The most commonly used cancer drug evaluation model is xenografting, which is generated by injecting cultured human cancer cells (CCL) subcutaneously into immunodeficient mice. Typically, the cancer cells are derived from advanced, highly aggressive or poorly differentiated neoplasms. Nevertheless, the growing tumors lack their original microenvironment, especially tumor-associated stroma, which has recently been shown to be crucial in tumor development and progression. The CCL-based xenograft models in general display limited capability in predicting clinical efficacy of anticancer agents. To address these limitations, efforts have been made to grow histologically intact fresh human tumor tissues in a variety of graft sites in immunodefi cient mice.
The increasingly used patient-derived human tumour tissue (PDTT) xenografts models implanted subcutaneously or in subrenal capsule in immunodeficient mice, such as severe combined immune deficient (SCID) mice, may provide a more accurate reflection of human tumour biological characteristics than tumour cell lines. The ability to passage patients� fresh tumour tissues into large numbers of immunodeficient mice provides possibilities for better preclinical testing of new therapies for the treatment and better outcome for cancer.
These xenografts models retain similar growth and histopathological, immunohistochemical, proteomic, genetic and genomic features as the original cancers and thus should be functional for rapid screening of potential therapeutics.
Main objective of this study is to set up a mouse model of �xenopatients�, i.e. xenografts of patient-derived tumor material, starting from mesenchymal tumors. Metastatic or unresectable relapsing mesenchymal tumors represent an unmet medical need with a dismal prognosis.
PDTT xenograft models, such as bone and soft tissue sarcomas, can offer in vivo experimental tumor models clinically representative of these human cancer types. Moreover it would largely increase the success in identifying new active agents for targeting mesenchymal tumors.
Similarly, individualised models of human cancers would greatly facilitate selecting the best therapy for each individual patient (one patient one mouse).
Specific objectives and Experimental design:
It is believed that PDTT xenograft models possess three general applications.
Firstly, they can be used as an in vivo screening tool to test novel drugs with therapeutic potential in cancer treatment.
Secondly, they can be used to evaluate key markers of response and resistance to drugs.
Finally, they can be applied to achieve individualized chemotherapy regimens by preclinically assessing the chemosensitivity of tumors to registered anticancer agents in vivo.
1) Xenopatient generation: a) engraftment assay: Tumor surgical biopsies diced into 2 � 2 � 3-mm3 pieces and placed in medium supplemented with 20% foetal bovine serum and 0.05% penicillin/streptomycin will be implanted into 5- to 6-week-old female severe combined immune deficient (SCID) mice under anesthesia with isoflurane, by a small incision and subcutaneous pocket made in each side of the lower back in which one tumor piece will be deposited in each pocket. Additional tissue samples will be immediately snap frozen and stored at -80 °C for genetic, genomic and protein analyses. For each tumour type, 2 mice will be used. Growth of established tumor xenografts will monitored weekly by vernier calliper measurement of tumor length and width.
b) Expansion assay: At a size of about 1�2 cm3, tumors will be removed for serial transplantation. Tumor-bearing animals will be anesthetized with diethyl ether and sacrificed by cervical dislocation. Tumors will be minced under sterile conditions and implanted in successive SCID mice, as described above. From the 2 mice used to engraft each tumour type, 4 mice will be generated.
2) Treatment assay: following transplantation to expand the cohort of mice, tumors will be allowed to grow to 200�500 mm3 before initiation of treatment for chemosensitivity testing.
3) Cryopreservation and tissue bank generation: Numerous samples from early passages should be stored in the tissue bank, cryopreserved in liquid nitrogen and used for further experiments. Additional tissue samples were immediately snap frozen and stored at -80 °C for genetic, genomic and protein analyses.
INTRODUCTION
Rodent tumor models currently being used and which include transgenic tumor models, and those generated by planting human tumor cell lines subcutaneously in immunodeficient mice, do not sufficiently represent clinical cancer characteristics, especially with regard to metastasis and drug sensitivity.
The most commonly used cancer drug evaluation model is xenografting, which is generated by injecting cultured human cancer cells (CCL) subcutaneously into immunodeficient mice. Typically, the cancer cells are derived from advanced, highly aggressive or poorly differentiated neoplasms. Nevertheless, the growing tumors lack their original microenvironment, especially tumor-associated stroma, which has recently been shown to be crucial in tumor development and progression. The CCL-based xenograft models in general display limited capability in predicting clinical efficacy of anticancer agents. To address these limitations, efforts have been made to grow histologically intact fresh human tumor tissues in a variety of graft sites in immunodefi cient mice.
The increasingly used patient-derived human tumour tissue (PDTT) xenografts models implanted subcutaneously or in subrenal capsule in immunodeficient mice, such as severe combined immune deficient (SCID) mice, may provide a more accurate reflection of human tumour biological characteristics than tumour cell lines. The ability to passage patients� fresh tumour tissues into large numbers of immunodeficient mice provides possibilities for better preclinical testing of new therapies for the treatment and better outcome for cancer.
These xenografts models retain similar growth and histopathological, immunohistochemical, proteomic, genetic and genomic features as the original cancers and thus should be functional for rapid screening of potential therapeutics.
Main objective of this study is to set up a mouse model of �xenopatients�, i.e. xenografts of patient-derived tumor material, starting from mesenchymal tumors. Metastatic or unresectable relapsing mesenchymal tumors represent an unmet medical need with a dismal prognosis.
PDTT xenograft models, such as bone and soft tissue sarcomas, can offer in vivo experimental tumor models clinically representative of these human cancer types. Moreover it would largely increase the success in identifying new active agents for targeting mesenchymal tumors.
Similarly, individualised models of human cancers would greatly facilitate selecting the best therapy for each individual patient (one patient one mouse).
Specific objectives and Experimental design:
It is believed that PDTT xenograft models possess three general applications.
Firstly, they can be used as an in vivo screening tool to test novel drugs with therapeutic potential in cancer treatment.
Secondly, they can be used to evaluate key markers of response and resistance to drugs.
Finally, they can be applied to achieve individualized chemotherapy regimens by preclinically assessing the chemosensitivity of tumors to registered anticancer agents in vivo.
1) Xenopatient generation: a) engraftment assay: Tumor surgical biopsies diced into 2 � 2 � 3-mm3 pieces and placed in medium supplemented with 20% foetal bovine serum and 0.05% penicillin/streptomycin will be implanted into 5- to 6-week-old female severe combined immune deficient (SCID) mice under anesthesia with isoflurane, by a small incision and subcutaneous pocket made in each side of the lower back in which one tumor piece will be deposited in each pocket. Additional tissue samples will be immediately snap frozen and stored at -80 °C for genetic, genomic and protein analyses. For each tumour type, 2 mice will be used. Growth of established tumor xenografts will monitored weekly by vernier calliper measurement of tumor length and width.
b) Expansion assay: At a size of about 1�2 cm3, tumors will be removed for serial transplantation. Tumor-bearing animals will be anesthetized with diethyl ether and sacrificed by cervical dislocation. Tumors will be minced under sterile conditions and implanted in successive SCID mice, as described above. From the 2 mice used to engraft each tumour type, 4 mice will be generated.
2) Treatment assay: following transplantation to expand the cohort of mice, tumors will be allowed to grow to 200�500 mm3 before initiation of treatment for chemosensitivity testing.
3) Cryopreservation and tissue bank generation: Numerous samples from early passages should be stored in the tissue bank, cryopreserved in liquid nitrogen and used for further experiments. Additional tissue samples were immediately snap frozen and stored at -80 °C for genetic, genomic and protein analyses.
Project Title:
Preclinical model of cancer adoptive immunotherapy with Cytokine Induced Killer Cells
General aim of our research activity is to investigate and optimize at a preclinical level a new immunotherapy strategy for the treatment of metastatic solid tumors refractory to standard treatments. The project is designed with a clinical perspective with the aim of providing reliable biological basis for subsequent clinical protocols. We will focus on a new form of adoptive immunotherapy, based on Cytokine-Induced Killer (CIK) cells, our main settings will be centered on mesenchymal tumors (bone and soft tissue sarcomas) and melanoma but the final result will be a �transversal� model that might result effective against several tumor types.
MAIN OBJECTIVE will be to define and optimize a preclinical model of adoptive immunotherapy with CIK cells for the treatment of solid tumors. As previously said we will primarily focus on mesenchymal tumors (bone and soft tissue sarcomas) and melanoma but our model has the potential to be extended to many other tumor types.
SPECIFIC OBJECTIVES AND EXPERIMENTAL DESIGN:
a) Generation of CIK cells. CIK cells will be expanded from patients with metastatic mesenchymal tumors or melanoma as well as from control healthy donors. The expansion of CIK cells will occur over 3-4 weeks according to standard procedures with the timed addition of IFN-γ, Ab anti-CD3 and IL2 (Lu and Negrin, 1994). Ex-vivo cultures will start from fresh or cryopreserved peripheral blood mononuclear cells (PBMC). We will weekly assess the expansion rate and phenotypic profile by flow cytometry (CD3; CD4: CD56; CD8; NKG2D) to provide quantitative and qualitative information about patients derived CIK cells to be compared with results obtained from healthy controls. At the end of the ex-vivo expansion a small amount of CIK cells (5-10 x10^6) will be used fresh, to test their tumor-killing ability in-vitro, the rest will be cryopreserved for subsequent infusions into a preclinical in-vivo murine model.
b)CIKs� tumor-killing ability. We will assess the tumoricidal activity of CIK cells against commercially available and autologous tumor cells, both in vitro and in xenograft murine models. Patient-derived tumor cell lines will be established, whenever possible, starting from fresh tumor biopsies. Non-tumoral targets like human fibroblasts or allogeneic irradiated PBMC will be used as control and to confirm the safety of such approach. The essays in-vitro will be performed with a non-radioactive-essay staining target cells with CFSE and analyzing their immune-mediated killing by flow cytometry (Jedema et al., 2004; Introna M. 2010). We will consider as significant a specific tumor lysis > 25% with a 40:1 effector/target ratio. Cytotoxic curves will be compared with controls. It will be important to correlate the observed tumor-killing capacity with the spontaneous expression of CIKs� target molecules on tumor cells (MIC A/B; ULBPs proteins). In-vivo tumor killing. The test in-vivo will be performed on immunocompromised mice bearing patients� tumor xenografts. We will wait for the tumor xenograft to become measurable, then we will infuse CIK cells (5-10x10^6 per infusion) intravenously, and repeat the infusions weekly for 3 weeks or longer, according to the total number of CIKs available. Mice will be sacrificed when tumor volume reaches a maximum of 2 cm or unacceptable toxicity occurs. We will compare tumor growth and survival rates of treated mice with untreated controls. Removed tumor samples will be evaluated analyzed for lymphocytes infiltration. We expect to treat 6 mice per patient, an equal number of untreated mice will be used as controls.
MAIN OBJECTIVE will be to define and optimize a preclinical model of adoptive immunotherapy with CIK cells for the treatment of solid tumors. As previously said we will primarily focus on mesenchymal tumors (bone and soft tissue sarcomas) and melanoma but our model has the potential to be extended to many other tumor types.
SPECIFIC OBJECTIVES AND EXPERIMENTAL DESIGN:
a) Generation of CIK cells. CIK cells will be expanded from patients with metastatic mesenchymal tumors or melanoma as well as from control healthy donors. The expansion of CIK cells will occur over 3-4 weeks according to standard procedures with the timed addition of IFN-γ, Ab anti-CD3 and IL2 (Lu and Negrin, 1994). Ex-vivo cultures will start from fresh or cryopreserved peripheral blood mononuclear cells (PBMC). We will weekly assess the expansion rate and phenotypic profile by flow cytometry (CD3; CD4: CD56; CD8; NKG2D) to provide quantitative and qualitative information about patients derived CIK cells to be compared with results obtained from healthy controls. At the end of the ex-vivo expansion a small amount of CIK cells (5-10 x10^6) will be used fresh, to test their tumor-killing ability in-vitro, the rest will be cryopreserved for subsequent infusions into a preclinical in-vivo murine model.
b)CIKs� tumor-killing ability. We will assess the tumoricidal activity of CIK cells against commercially available and autologous tumor cells, both in vitro and in xenograft murine models. Patient-derived tumor cell lines will be established, whenever possible, starting from fresh tumor biopsies. Non-tumoral targets like human fibroblasts or allogeneic irradiated PBMC will be used as control and to confirm the safety of such approach. The essays in-vitro will be performed with a non-radioactive-essay staining target cells with CFSE and analyzing their immune-mediated killing by flow cytometry (Jedema et al., 2004; Introna M. 2010). We will consider as significant a specific tumor lysis > 25% with a 40:1 effector/target ratio. Cytotoxic curves will be compared with controls. It will be important to correlate the observed tumor-killing capacity with the spontaneous expression of CIKs� target molecules on tumor cells (MIC A/B; ULBPs proteins). In-vivo tumor killing. The test in-vivo will be performed on immunocompromised mice bearing patients� tumor xenografts. We will wait for the tumor xenograft to become measurable, then we will infuse CIK cells (5-10x10^6 per infusion) intravenously, and repeat the infusions weekly for 3 weeks or longer, according to the total number of CIKs available. Mice will be sacrificed when tumor volume reaches a maximum of 2 cm or unacceptable toxicity occurs. We will compare tumor growth and survival rates of treated mice with untreated controls. Removed tumor samples will be evaluated analyzed for lymphocytes infiltration. We expect to treat 6 mice per patient, an equal number of untreated mice will be used as controls.