molecular heterogeneity

Personalized Spatial Regulatory Networks to Decode Breast Cancer Microenvironments

PROJECT SUMMARY Triple-negative breast cancer (TNBC) is an aggressive subtype with early recurrence, high metastatic burden, and limited treatment options. While genomic alterations contribute to its progression, epigenetic plasticity and spatial organization within the tumor microenvironment (TME) play critical roles in intra-tumor heterogeneity, immune evasion, and therapy resistance, yet remain poorly understood. To address this, we will develop a cost- effective and scalable methodology that integrates spatial ATAC-seq, spatial in situ transcriptomics (Xenium), and single-nucleus (sn) Epi Multiome sequencing (snRNA-seq + snATAC-seq) from core-needle biopsies, enabling high-resolution mapping of gene regulatory networks within the intact TME. Our preliminary data from six TNBC biopsies demonstrate that spatial in situ transcriptomics and spatial ATAC-seq provide critical insights into tissue architecture but suffer from data sparsity, necessitating the integration of single-nucleus Epi Multiome data to enhance cell-type annotation and impute missing genomic features. In Aim 1, we will establish a multi- modal workflow that maximizes molecular insights from limited biopsy material by optimizing tissue-preserving and multiplexed sequencing approaches. This includes leveraging patient-specific genetic variation to deconvolute nuclei-derived data and linking it to spatial transcriptomic and spatial chromatin accessibility profiles. In Aim 2, we will develop a computational framework to integrate these multi-layered datasets, enabling spatially resolved epigenomic-transcriptomic analysis that identifies key regulatory chromatin elements and transcriptional programs associated with TNBC progression, immune infiltration, and therapy resistance. This project will generate the first comprehensive, patient-specific spatial regulatory atlas of TNBC, providing fundamental insights into how chromatin accessibility and gene expression interact within the TME. Ultimately, this work will pave the way for novel precision oncology strategies, biomarker discovery, and the development of targeted therapies that address TNBC’s spatial and molecular heterogeneity.

Organization of Midbrain Serotonin System

The serotonin system is the most frequently targeted neural system pharmacologically for treating psychiatric disorders, including depression and anxiety. Serotonin neurons of the dorsal and median raphe nuclei (DR, MR) collectively innervate the entire forebrain and midbrain, modulating diverse physiology and behaviour. By using viral-genetic methods, we found that DR serotonin system contains parallel sub-systems that differ in input and output connectivity, physiological response properties, and behavioural functions. To gain a fundamental understanding of the molecular heterogeneity of DR and MR, we used single-cell RNA - sequencing (scRNA-seq) to generate a comprehensive dataset comprising eleven transcriptomically distinct serotonin neuron clusters. We generated novel intersectional viral-genetic tools to access specific subpopulations. Whole-brain axonal projection mapping revealed that the molecular features of these distinct serotonin groups reflect their anatomical organization and provide tools for future exploration of the full projection map of molecularly defined serotonin groups. The molecular architecture of serotonin system lays the foundation for integrating anatomical, neurochemical, physiological, and behavioural functions.

Functional and Molecular Heterogeneity in the Developing Vestibulo-Ocular Reflex Circuit

Molecular Heterogeneity of Cajal-Retzius neurons in the Developing Cortex Underlies their Circuit Connectivity and Sensory Driven responses