Project 2: Cellular topography and function of the breast cancer tissue microenvironment

Abstract/Project Summary
Breast cancer (BC) metastasis is an emergent feature that occurs when the tumor’s ability to recruit metabolic
resources, avoid immune activation, and disseminate to distant sites exceeds the capacity of surrounding normal
tissue to prevent these processes. In line with this, work over the last decade has demonstrated the crucial role
played by the tumor microenvironment (TME) in promoting or deterring BC progression. Tumor cell migration
and immune recruitment have both been shown to be heavily influenced by fibroblasts and the surrounding
extracellular matrix (ECM) they produce. Additionally, protein glycosylation has been shown to modulate these
interactions. To understand how tumor glycosylation and ECM remodeling interact to potentiate BC metastasis,
we will use spatial transcriptomics and two complementary mass spectrometry methods, MIBI-TOF and MALDI,
to identify glycan-dependent, cell-cell, and cell-ECM interactions that shift the TME toward tumor permissive
states. All three analyses will be performed on spatially-coregistered serial sections from the same tissue blocks.
In doing so, comprehensive single-cell maps of each tissue sample constructed by MIBI-TOF can be directly
superimposed with de novo proteomic and transcriptomic data. We will map and enumerate the lineage and
major functional subsets of tumor and stromal cells with respect to relevant therapeutic and molecular
parameters to understand how the BC TME evolves with disease progression. These features will be overlaid
with de novo imaging of tissue glycans to identify potential mechanisms of immune evasion that involve tumor
sialoglycans and macrophage-bound SIGLECs. The frequency and spatial enrichment of these features will be
correlated with spatial transcriptomics data to identify regulatory glycosyltransferases promoting these
interactions. Next, ECM-MALDI and MIBI-TOF data will be used to identify how collagen type, hydroxylation, and
crosslinking shift in coordination with the collagen structure and function of neighboring cell populations. In
particular, we will focus on understanding how the activity of two families of enzymes, prolyl and lysyl
hydroxylases, drive structural changes in the ECM that promote BC metastasis. The clinical significance of these
extracted cellular and molecular definitions of ECM remodeling will be assessed with respect to metastatic risk,
stage, and IC subtype. Taken together, this work will provide an unprecedented view into how TME structure
and cell-cell interactions between tumor and stroma relate to specific facets of the tumor ECM and glycome.

Grant Number: 5U54CA261719-05
NIH Institute/Center: NIH
Principal Investigator: Robert Angelo