Periodic Reporting for period 2 - KILL-OR-DIFFERENTIAT (Using cell-cell interactions to unlock new cancer treatments: Forcing neural crest tumors back onto the developmental path)
Periodo di rendicontazione: 2022-04-01 al 2023-09-30
The interactions between tumor and its microenvironment are often critical to uncovering the mechanisms of tumor survival. The tumor can also repress immune response by inducing complex interactions among dozens of immune and stromal cell types that typically make up tumor microenvironment, however those remain largely uncharacterized as we currently lack systematic approaches to uncover relevant cell-cell interactions. The alternative to killing tumor cells, either directly or through immune system, is to force them to differentiate. Such strategy is particularly promising for tumors arising due to failure of progenitor populations to follow proper differentiation cascade. Here as well, the progress has been limited by lack of understanding of the specific intercellular signals that that are disrupted in tumorigenesis. We propose a systematic approach for characterizing cell-cell interactions in complex microenvironments through joint analysis of spatially-resolved and disassociated single-cell transcriptomics. We will apply it to identify inter-cellular signals and pathways that can push tumors of neural crest origin, including as pheochromocytoma (PCC), paraganglioma (PGL) and neuroblastoma (NB), towards terminal differentiation. Building on our expertise with neural crest development, we will use single-cell profiling to map individual tumor cells onto developmental trajectory of neural crest differentiation. We propose to achieve the following objectives:
AIM 1. To profile homeostatic trajectory of neural crest differentiation and identify inter-cellular interactions guiding normal neural crest development using single-cell and spatially-resolved transcriptomics analysis.
AIM 2. Characterize tumor microenvironment composition, cell-cell interactions and intratumoral transcriptional heterogeneity in different pediatric and adult subtypes of NB, PCC and PGL tumors. Map tumor cells onto the homeostatic trajectory to identify corresponding differentiation stage and signaling state of the individual tumor cells. Identify signaling and downstream transcriptional pathways disrupted in tumors.
AIM 3. Predict signalling or transcriptional perturbations that would push tumor cells down homeostatic neural crest differentiation path. Perform pre-clinical validation of such differentiation therapy perturbations in culture and in genetically engineered mouse models and mouse xenografts.
AIM 1. To profile homeostatic trajectory of neural crest differentiation and identify inter-cellular interactions guiding normal neural crest development using single-cell and spatially-resolved transcriptomics analysis.
AIM 2. Characterize tumor microenvironment composition, cell-cell interactions and intratumoral transcriptional heterogeneity in different pediatric and adult subtypes of NB, PCC and PGL tumors. Map tumor cells onto the homeostatic trajectory to identify corresponding differentiation stage and signaling state of the individual tumor cells. Identify signaling and downstream transcriptional pathways disrupted in tumors.
AIM 3. Predict signalling or transcriptional perturbations that would push tumor cells down homeostatic neural crest differentiation path. Perform pre-clinical validation of such differentiation therapy perturbations in culture and in genetically engineered mouse models and mouse xenografts.
From the beginning of the project (during the first reporting period), we largely addressed Aim 1 and Aim2, and to some extent Aim3. Firstly, we significantly improved and extended the single cell map of the entire neural crest lineage, which became a basis for anew hi fi atlas of cell types relevant to the development and origin of a plethora of diseases, such as neuroblastoma (in the first place), but also melanoma, neurofibromatosis and many more. This atlas was already put to work for the analysis of neuroblastoma cell type heterogeneity during the first reporting period. In the parallel, we improved our single cell maps, including spatial maps on the tissue slices, of neuroblastoma cell types directly from human patients, but also from mouse models. Next, we created a principally new method to analyse clonality and cell type heterogeneity in tumors, called NUMBAT. This is a big achievement, as it helps to build the map of transitions between malignant plastic cell populations with different properties. In the following line of experiments, we managed to improve our understanding of unwanted plasticity and transitions from adrenergic into mesenchymal populations of neuroblastoma cells.
Furthermore, generated novel transgenic animals as outlined in aim 2 and observed tumors with 100 % penetrance in these mice. They profiled single-cell transcriptomes these mouse sympatho-adrenal tumors at several stages, from embryonic, pre-neoplastic hyperplasia to pheochromocytoma, neuroblastoma, and composite tumors. They deleted KIF1Bβ and NF1 in the embryonic mouse sympatho-adrenal lineage and observed pheochromocytoma, neuroblastoma, and composite tumors arising in aged mice. Deep single-cell RNA sequencing combined with immunohistochemistry and RNA scope revealed neuroblast-chromaffin cell state transitions at embryonic and postnatal stages driving tumor plasticity. Cancer cells progressively adopt neuroblast lineage identity, computationally predicted to be mediated through a common chromaffin- neuroblast transitional, high-plasticity cell state.
Furthermore, generated novel transgenic animals as outlined in aim 2 and observed tumors with 100 % penetrance in these mice. They profiled single-cell transcriptomes these mouse sympatho-adrenal tumors at several stages, from embryonic, pre-neoplastic hyperplasia to pheochromocytoma, neuroblastoma, and composite tumors. They deleted KIF1Bβ and NF1 in the embryonic mouse sympatho-adrenal lineage and observed pheochromocytoma, neuroblastoma, and composite tumors arising in aged mice. Deep single-cell RNA sequencing combined with immunohistochemistry and RNA scope revealed neuroblast-chromaffin cell state transitions at embryonic and postnatal stages driving tumor plasticity. Cancer cells progressively adopt neuroblast lineage identity, computationally predicted to be mediated through a common chromaffin- neuroblast transitional, high-plasticity cell state.
The major progress beyond the state of the art includes the two main points:
1. We developed a principally new method for calling the tumor-related mutations in single cell data for building clonal trees of clonal development within tumors (this helps to understand the repertoire of malignant cell types in the tumor and their relations with each other including unwanted plasticity). This method is just published in highly prestigious Nature Biotechnology journal and is called NUMBAT:
https://github.com/kharchenkolab/numbat
Here we provide a more detailed description why this is important: distinguishing malignant cells in neuroblastoma samples has become a critical challenge for understanding neuroblastoma origins. To meet that challenge, we have developed a powerful computational technique for detecting genomic aberrations characteristic of tumor cells from single-cell RNA-seq data, called Numbat (Gao et al., Nature Biotech’22). It takes advantage of the recent progress in population genetics, which has enabled us to predict maternal and paternal alleles of individuals from haplotype data with high accuracy. Numbat uses this population-based phasing to significantly increase the sensitivity and specificity in detecting copy-number alternations and loss-of-heterozygosity events. Using this new method we have been able to confirm the surprising presence of an early developmental precursor population in some of the neuroblastoma patients.
2. Another major breakthrough is the discovery of human-specific aspects of sympatho-adrenal and neural crest development published in Nature Genetics paper, which is focused on characterizing human embryological aspects of chromaffin cell formation within healthy adrenal gland. This knowledge if of high relevance to neuroblastoma research, as it helps to understand why mouse models of neuroblastoma poorly recapitulate the original human disease. This study is showing how human development differs from mouse quire radically in terms of aspects related to sympatho-adrenal lineage progression with branching transitions between sympathetic, chromaffin, bridge and schwann cell precursor cell types giving rise to neuroblastoma of different subtypes. The new human-specific embryological finds will definitely provide a better ground for classifying tumor cell type heterogeneity and monitoring transitions between malignant cell types by comparing them to healthy human transitions.
1. We developed a principally new method for calling the tumor-related mutations in single cell data for building clonal trees of clonal development within tumors (this helps to understand the repertoire of malignant cell types in the tumor and their relations with each other including unwanted plasticity). This method is just published in highly prestigious Nature Biotechnology journal and is called NUMBAT:
https://github.com/kharchenkolab/numbat
Here we provide a more detailed description why this is important: distinguishing malignant cells in neuroblastoma samples has become a critical challenge for understanding neuroblastoma origins. To meet that challenge, we have developed a powerful computational technique for detecting genomic aberrations characteristic of tumor cells from single-cell RNA-seq data, called Numbat (Gao et al., Nature Biotech’22). It takes advantage of the recent progress in population genetics, which has enabled us to predict maternal and paternal alleles of individuals from haplotype data with high accuracy. Numbat uses this population-based phasing to significantly increase the sensitivity and specificity in detecting copy-number alternations and loss-of-heterozygosity events. Using this new method we have been able to confirm the surprising presence of an early developmental precursor population in some of the neuroblastoma patients.
2. Another major breakthrough is the discovery of human-specific aspects of sympatho-adrenal and neural crest development published in Nature Genetics paper, which is focused on characterizing human embryological aspects of chromaffin cell formation within healthy adrenal gland. This knowledge if of high relevance to neuroblastoma research, as it helps to understand why mouse models of neuroblastoma poorly recapitulate the original human disease. This study is showing how human development differs from mouse quire radically in terms of aspects related to sympatho-adrenal lineage progression with branching transitions between sympathetic, chromaffin, bridge and schwann cell precursor cell types giving rise to neuroblastoma of different subtypes. The new human-specific embryological finds will definitely provide a better ground for classifying tumor cell type heterogeneity and monitoring transitions between malignant cell types by comparing them to healthy human transitions.