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Lorem Sit Amet Dolor
Researcher: Lorem Sit Amet

123 abc lane, Townsville, ZZ 00000, USA
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Investigating the role of DIPG-derived exosomes in tumor growth and invasion
Translational
DIPG, Childhood (Brain Cancer)
Lay Summary

Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive and infiltrating brain tumor affecting children for which there is no effective treatment.

Sequencing data have revealed  the existence of well defined subgroups of tumors based on age of onset, specific location in the brain, mutations and clinical outcome. Those tumous are characterized by specific recurrent mutations in histone genes which encodes for H3.3 and H3.1 and another mutation in a bone-related gene called ACVR1 which is largely found mutated with the histone H3.1.

Moreover, the existence of heterogeneity among the DIPG cells composing the tumor mass is increasingly evident. Our recent data suggest the presence of a “network” of distinct subpopulations that taken individually differ in their invasion and migration properties, that interact with each other and with the surrounding microenvironment to promote tumorigenesis and the progression of the tumor.

In this context, a crucial role in the communication between DIPG cells and the microenvironment may be represented by exosomes, cellular microvescicles containing RNA, DNA and proteins.

We hypothesise that the exosomes derived from DIPG cells could mediate the intercellular communication and that they could have a specific "oncogenic signature" that is specific to different molecular subgroup, and therefore could be used as an important diagnostic and prognostic marker.

To define the exosome signature of DIPG, we looked at the proteins enclosed in the exosomes and through which cells could exchange signals.

In particular, we used a panel of patient primary-primary cell lines characterized by the DIPG driver mutations (H3.3 K27M, H3.3 K27M/ACVR1 and H3.1 K27M/ACVR1) cultured in stem cell-like conditions, such as neurospheres (NS) and/or adherent to laminin (L).

Moreover, to better derive specific DIPG oncogenic exosomal signature, we used as a comparison pediatric glioblastoma (pGBM), which arise in different locations but maybe mutated in the same histone genes as DIPG.

The analysis of exosomal protein content showed that DIPG and pGBM carry unique proteins in the exosomes, highlighting the differences between the two tumor types, differences that were clearly shown also for the two DIPG subgroups H3.3 and H3.1

Focus of our study was also another molecule transported in the exosome: the micro-RNAs (miRs). Those are small RNA fragments, that may be exchanged among tumor cells and that we also considered for the identification of exosomal oncogenic signature of DIPG.

In this context, we analyzed the miRNA isolated from different patient primary-derived cell lines including different mutation and location subgroups.  Our data showed that H3.1 K27M/ACVR1 DIPG subgroup was strongly different from H3.3 K27M DIPG and pGBM, concluding that the two main histone H3 gene variants leads to distinct oncogenic programs which could be exploited to develop distinct potential therapeutic strategies.

Moreover, since it has been demonstrated that DIPG and pGBM are heterogeneous tumors characterized by the existence of different cell types that co-operate and communicate one to each other, we also isolated single cells from DIPG and pGBM primary cell lines which were later exploited to give raise to different subpopulations and studied for the exosome.

The single cell derived subpopulations were characterized for their morphology, migration and invasion properties and they were different one from each other underlying the heterogeneous nature of DIPG and pGBM tumor masses. Moreover, we also analyzed the exosome communication between tumor cells, confirming that exomes are an important key of signals exchange.

Having consolidated the role of exosomes in DIPG and pGBM cellular connections and having highlighted some important proteins as well as miRNAs, that could be considered as “oncogenic signature” of DIPG and pGBM, our further aim is to investigate their presence in the exosomes from the peripheral blood of DIPG cancer patients.

This will lead to the identification of new diagnostic markers and prognostic strategies for DIPG and pGBM tumors.

Executive Summary

Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor which affects children and for which there is no effective treatment at the moment.  We and others have recently shown that DIPG is characterized by a remarkable degree of intratumoral heterogeneity and consists of distinct genetically and phenotypically heterogeneous subclonal populations. It is well recognized that exosomes mediate the cross-talk among the tumor cells and between the tumor and its microenvironment. We hypothesize that the DIPG subclonal network is sustained through paracrine signaling and that exosomes are involved in this intratumor cross-talk promoting tumorigenesis and tumor progression. To understand the role that exosomes play in DIPG tumorigenesis and determine the mechanistic basis of the exosome-mediated interclonal communication, we aim to: i. determine the specific DIPG-exosome signature within the distinct mutational subgroups and within distinct sub-clonal populations; ii. define the exosome-mediated mechanisms of crosstalk between distinct DIPG subclones and determine the functional consequences of such uptake. To address these aims we  specifically isolated and characterized exosomes derived from different DIPG patient primary-derived cells. We found that exosomes derived from DIPG displays a variable cargo of total protein with a trend for DIPG lines which is higher compared to the exosomes derived from GBM. Electron microscopy showed that the population we isolated is compatible with exosomes with a size ranging between 50-80 nm. Using proteomic analysis we identified first a common signature for the DIPG derived exosomes compared to the GBM exososomes and interestingly a peculiar signature related to the different DIPG molecular subgroups. The miRNAs analysis of DIPG-derived exosomes suggested that their miRNAs profile is driven by the two main histone H3 variants leading to two distinct oncogenic programmes with distinct potential specific therapeutic targets  In our project we aim also to investigate  the specific profile of exosomes derived from DIPG subclonal populations and analyze the functional consequences (effect on growth and invasion) of exosomal uptake in co-culture experiments of DIPG subclones. We have successfully transduced one DIPG and one GBM primary derived cell line using 6 different lentiviral vectors expressing each a different fluorescent protein in order to perform long term co-culture experiment and be able to distinguish the dinamics of direct cell-cell interaction via exosomes. So far we demonstrated despite cell transduction, heterogeneity is preserved in the DIPG and GBM cell in terms of morphology, cell growth, migration and invasion. Moreover we have show that labeled exosomes derived from the bulky population are acrively uptaken from a derived subclone. This confirm our hypothesis that exosomes represent a potential shuttle for exchange of oncogenic signals between glioma subpopulations. Our goal is indeed to better elucidate the mechanisms of cell-cell communication that mediate the DIPG growth and invasion. Our future plan in the second year of this project will be i) to validate exosomal miRNAs on additional DIPG cell lines and in patient tissue samples; ii) to validate, by modulation of their expression specific miRNAs in DIPG primary derived cell lines; iii) to evaluate the exosomal miRNAs in plasma samples from patients affected by DIPG; iv) to validate specific exosomal proteins in tissue patients.We have already collected plasma samples from 8 DIPG and GBM patients and we estimate we will collect 6-8 more during this year.

We believe that this study will lead to the identification of potential new targets and the development of new diagnostic/prognostic tools for patients affected by DIPG.

Our preliminary data demonstrate that tumor-secreted exosomes can be efficiently used to track the communication among subclonal population in DIPG tumors and the activation of specific oncogenic pathways. Dr. Mara Vinci and collaborators have already demonstrated that DIPG is a heterogeneous tumor and that it is characterised by a complex subclonal architecture where distinct subclones co-operate to display the more aggressive phenotype, in particular in terms of migration and invasion (Vinci et al., Nature Medicine 2018, accepted). Her expertise with the collaboration of Dr. Angela Di Giannatale, who is a pediatric oncologist who works with her team on understanding the role of tumor-secreted exosomes in the tumor cross-talk, will ensure that the study will be carried out according to plan. Furthermore the collaboration with other experts (Dr.Putignani and Dr. Levimortera for the proteomic, Dr. Masotti for the miRNA analysis and Prof. Novelli for the FISH study) will enable a complementary synergy fundamental for the success of this project.

Description of Research Proposal

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Sed hendrerit vitae purus et tempor. Aenean vitae varius velit. Nullam aliquet ipsum elit. Mauris vestibulum purus et metus imperdiet, quis gravida eros pretium. Curabitur rutrum nunc vitae tincidunt condimentum. Aenean sit amet augue velit. Nunc tristique quis lorem id pharetra. Pellentesque sollicitudin, eros sed egestas rutrum, nulla nisl sodales elit, ut imperdiet nunc lectus sit amet nulla. Ut malesuada finibus libero. Curabitur mi dolor, sollicitudin quis bibendum quis, dictum sed enim.

Pellentesque nibh erat, egestas sit amet sagittis malesuada, rhoncus at neque. Mauris maximus commodo tortor, non egestas magna finibus vitae. Sed hendrerit nulla vel venenatis tempor. Suspendisse tristique tincidunt libero et placerat. Nam tincidunt condimentum lorem, vel pharetra est iaculis sed. Nullam ac tincidunt orci. Quisque pharetra ut sem sit amet aliquam. Phasellus risus libero, varius in condimentum vel, commodo id ipsum. Aliquam in metus cursus, mattis diam ut, aliquam magna. Suspendisse facilisis dui et orci varius, suscipit facilisis augue dapibus. In eget nibh ipsum. Suspendisse eget pharetra est, quis condimentum felis. Fusce scelerisque congue libero, sed aliquam mi elementum a. Etiam scelerisque ante non auctor porta. Nam eu nunc id ex finibus dictum. Praesent dui ex, dictum ac massa eget, rutrum gravida nisi.

Sed egestas arcu in dui euismod, eget faucibus massa iaculis. Etiam efficitur lectus et purus lobortis, ac blandit eros rutrum. Proin bibendum consectetur leo vel gravida. Etiam et ultricies sapien. Nam lacinia tellus erat, id facilisis est consequat et. Morbi quis risus in neque iaculis pharetra ut consectetur libero. Aenean feugiat tempor mi eu posuere.

Budget

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer gravida non felis non euismod. Fusce finibus aliquet consequat. Nam ac metus bibendum, iaculis purus sed, suscipit ligula. Proin et nisi libero. Mauris non urna urna. Nullam augue eros, fringilla sed mauris vitae, porta tincidunt risus. Aliquam sed tincidunt sem. Quisque lacinia quam tortor, imperdiet efficitur odio iaculis in. Sed ultricies condimentum volutpat. Vivamus dignissim faucibus porta.

Curabitur ut ipsum non odio malesuada vulputate. Morbi maximus, est eu lobortis molestie, tortor sapien hendrerit nisi, in cursus odio diam ut odio. Fusce pulvinar volutpat velit. Aliquam erat volutpat. Integer rhoncus mollis suscipit. Praesent non ipsum mollis, finibus nunc a, scelerisque nibh. In feugiat iaculis velit, eu semper lacus dignissim nec. Praesent vitae nisi leo. Cras venenatis dictum magna ut semper. Sed eget eros nibh. Sed vitae quam sed dolor faucibus elementum. Curabitur interdum porttitor finibus. Nullam tincidunt odio lectus, sit amet rhoncus libero dapibus sed. Sed mollis egestas enim, vel porta tortor volutpat eget.

Morbi orci urna, ornare non pretium eget, pulvinar eget magna. Ut consectetur efficitur varius. Fusce ac aliquet mauris, at mattis ligula. Quisque est libero, interdum id orci et, ornare luctus diam. Proin commodo lectus id accumsan blandit. Nulla eu turpis interdum, luctus ante ac, imperdiet tellus. In semper enim eu tristique aliquam.

Collaborations and Conflicts of Interest

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer gravida non felis non euismod. Fusce finibus aliquet consequat. Nam ac metus bibendum, iaculis purus sed, suscipit ligula. Proin et nisi libero. Mauris non urna urna. Nullam augue eros, fringilla sed mauris vitae, porta tincidunt risus. Aliquam sed tincidunt sem. Quisque lacinia quam tortor, imperdiet efficitur odio iaculis in. Sed ultricies condimentum volutpat. Vivamus dignissim faucibus porta.

Curabitur ut ipsum non odio malesuada vulputate. Morbi maximus, est eu lobortis molestie, tortor sapien hendrerit nisi, in cursus odio diam ut odio. Fusce pulvinar volutpat velit. Aliquam erat volutpat. Integer rhoncus mollis suscipit. Praesent non ipsum mollis, finibus nunc a, scelerisque nibh. In feugiat iaculis velit, eu semper lacus dignissim nec. Praesent vitae nisi leo. Cras venenatis dictum magna ut semper. Sed eget eros nibh. Sed vitae quam sed dolor faucibus elementum. Curabitur interdum porttitor finibus. Nullam tincidunt odio lectus, sit amet rhoncus libero dapibus sed. Sed mollis egestas enim, vel porta tortor volutpat eget.