Project: Dissecting phenotypic heterogeneity of human melanoma: building a rationale for active immunotherapies overcoming immunologically-induced dedifferentiation.

Acronym	ITEM (Reference Number: TRS-2015-00000034)
Duration	01/04/2016 - 01/04/2019
Project Topic	Inter- and intralesional tumor heterogeneity is commonly seen in metastatic melanoma, likely playing a major role in resistance to therapy, including immunotherapy. During immunotherapy, many patients experience "mixed response",with some tumor lesions regressing and others progressing. Whereas vaccines targeting a few antigens can "passively" select for antigen-negative tumor cells, even broader spectrum immunotherapies (whole tumor vaccines or immunomodulating antibodies) fail in clearing the tumor in most cases. In this respect, rare tumor cells with stemness properties and distinct expression profiles likely exist in tumor tissue: these cells, being less immunogenic, would be hardly targeted by antitumor immunity. Finally, tumor-specific cytotoxic T lymphocytes can induce dedifferentiation of melanoma cells, possibly underlying tumor relapse after initial response to immunotherapy. HYPOTHESIS Current immunotherapies induce immune response against the "average" antigenic composition of tumor bulk, not being likely able to "see" antigens expressed by rare tumor cells. Moreover, DC vaccines loaded with tumor lysate elicit immunity against the pretherapy tumor tissue,not taking into account the dynamic changes induced by the treatment on tumor cells. Therefore, the identification of antigen sets expressed by distinct melanoma cell subpopulations will allow developing new immunotherapies that concurrently target different subpopulations, including rare or therapyinduced ones. This approach will thereby target the cellular heterogeneity of tumors and minimize the risk of immune-escape of antigen-loss variants. AIMS Primary: to identify nonoverlapping sets of genes expressed by distinct melanoma cell subpopulations and their changes during immunotherapy. Secondary: to validate as antigenic targets the proteins encoded by the genes overespressed by the distinct melanoma cell subpopulations identified in the primary aim. METHODS - Melanoma cell collection: melanoma cells will be obtained by fine needle aspirates (FNA) of metastatic lesions before and after immunotherapy. - Isolation of single cells: CD45- cells obtained by FNA will be isolated with a microchip-based cell isolation system. - Single-cell transcriptomics: SMARTSeq RNA-Seq libraries will be generated for isolated single melanoma cells, tagmented using Nextera XT, and sequenced. - Validation of candidate target antigens: expression of proteins encoded by genes overexpressed in the distinct melanoma cell subpopulations will be tested in metastatic melanoma lesions. Overexpressed proteins will be then tested for their ability to induce CTL cultures in vitro. EXPECTED RESULTS/POTENTIAL IMPACT The identification of nonoverlapping gene sets stably expressed by different melanoma cell subpopulations and their validation as antigenic targets will allow designing new immunotherapies concurrently eliciting immune response against the majority of the patients' tumor cells.
Network	TRANSCAN-2
Call	Joint Transnational Call for Proposals (JTC 2014)

Project partner

Number	Name	Role	Country
1	IRCCS IRST	Coordinator	Italy
2	IGBMC	Partner	France
3	Academic Medical Center, University of Amsterdam	Partner	Netherlands
4	Oslo University Hospital Radiumhospitalet	Partner	Norway