Tumor secretome-mediated pro-tumoral microenvironment in diffuse intrinsic pontine glioma

Abstract

[eng] The development of immunotherapy for DIPG is justified by its dismal prognosis and the need for radically new therapeutic approaches for this disease. Recent success in treating melanoma, renal cell carcinoma, non-small lung cancer or prostate cancer with immune checkpoint inhibitors, or treating leukemia, carcinomas, and even glioblastoma multiforme with CAR-T cells has triggered interest in this approach. To optimize immunotherapy for DIPG, we must understand the immune escape mechanisms exerted by these tumors, likely through the generation of a pro-tumoral microenvironment, characterized by the presence of anti-inflammatory cytokines, immunosuppressive cells, or neovascularization. Previous work found that the microenvironment of DIPG is non-inflammatory, with a low or absent expression of immunomodulatory molecules, including immune checkpoints PD-L1 and CTLA-4. While such findings are clear regarding the absence of inflammation, it remains unknown which molecules or mechanisms are responsible for the acquisition of immune deprivation, as no classical chemokines have been identified to date as overexpressed in this cancer. A precise and in-depth analysis of the immune infiltrate of DIPG and the study of its secretome may unveil immunological mechanisms by which the tumor is able to escape and grow. Such knowledge would contribute to identify potential immune-related druggable targets, necessary for increasing the lifespan and survival of the patients. In this thesis, I will approach (i) the characterization of DIPG immunoenvironment,

(ii) the identification of potential cancer-secreted proteins responsible for the microenvironmental phenotype in the tumor, and (iii) the testing of a novel therapy targeting one of the proposed mechanisms.

Our general hypotheses are that (i) immunohistochemical markers in DIPG tissues will be consistent with immune suppression, including low counts of TILs and high counts of TAMs, consistent with a cold tumor microenvironment; (ii) primary DIPG cells in culture secrete specific cytokines, which are shared among different patients, constituting a common “DIPG secretome”; (iii) the secretome of DIPG modifies different cellular components of the microenvironment, such as mesenchymal stem cells (MSCs), endothelial cells and macrophages, favoring the establishment of a pro-tumorigenic niche; and (iv) blocking cytokine-activated pathways leads to anticancer activity in DIPG and pHGG models, in vitro and in vivo. To address our hypotheses, we set three main objectives: I. To characterize the immune cell infiltrate in DIPG and the immune escape mechanisms. We will quantify TIL and TAM populations in a large set of DIPG tissue samples from biopsies and necropsies of patients, and we will compare them to the counts obtained from non- tumoral brainstems. We will study the expression of the immune checkpoints PD-L1, CTLA-4 and B7-H3 by immunohistochemistry (IHC) in the same samples. We will also study the expression of these proteins in vitro, in primary cell cultures. II. To characterize the DIPG secretome by identifying specific cytokines secreted by primary DIPG cells in culture, which are common among DIPG patients. We will analyze the expression of soluble and secreted proteins in tissue samples, in primary DIPG cultures, and in CSF and serum from patients. Then, we will expose cells of the tumor microenvironment, such as MSCs, endothelial cells and peripheral blood mononuclear cell (PBMC)-derived macrophages, to the DIPG

secretomes and to the recombinant versions of the identified cytokines, to address phenotypic changes in the cells. III. To evaluate a new therapy for DIPG blocking the pathway activated by osteopontin and CHI3L1, two cytokines overexpressed in the secretome of the tumor. We will study whether sorafenib, an FDA- approved drug targeting one of the pathways activated by osteopontin and CHI3L1, presents anticancer activity against DIPG models. We will also assess changes on microenvironmental components after treatment.