# Modeling Metastasis and Acquired Drug Resistance Using Circulating Tumor Cells

> **NIH NIH R01** · MASSACHUSETTS GENERAL HOSPITAL · 2024 · $399,000

## Abstract

PROJECT SUMMARY
Advanced hormone receptor positive (HR+) breast cancers, the most common subtype, are
initially responsive to multiple endocrine interventions, but they ultimately develop drug resistance.
Circulating tumor cells (CTCs) underlie the blood-borne metastatic spread of cancer, and they
also provide a noninvasive source to sample tumor cells during the course of therapy and acquired
resistance. Using a microfluidic enrichment technology that preserves CTC viability, we successfully
established a cohort of 15 CTC-derived longterm cultures from women with refractory HR+ breast
cancer. A subset of these shows the expected acquired ESR1 and PIK3CA mutations, confirmed in
matched metastatic tumor biopsies. However, the most common, and least well understood, correlate of
endocrine resistance is loss of estrogen receptor-α (ER) expression by cancer cells. In these cases,
cultured CTCs recapitulate ER silencing, and some are biphenotypic, with coexisting ER+ and ER-
subpopulations derived from the same patient. Single cell-derived colonies show that ER+ CTCs can
produce ER- progeny, pointing to likely epigenetic mechanisms and underlying cell plasticity. We
propose to investigate the epigenetic regulation of ER expression loss and its potential restoration,
using genomic analyses combined with CRISPR functional screens. In Aim 1, we will compare
chromatin landscapes of isogenic ER+ and ER- CTC subpopulations, define their distinct functional
properties and identify factors that modulate their interconversion in vitro. In Aim 2, we will use undertake
CRISPR screens to identify genes capable of restoring ER expression to CTC lines from HR+ breast
cancers that have lost endogenous ER expression following endocrine therapy. Preliminary data
indicate the feasibility of this approach, and we will explore mechanisms underlying restoration of
ER expression, and whether this is accompanied by a return to ER-dependent proliferation. Together,
these Aims address the plasticity of ER expression in patient-derived cultured CTCs that
recapitulate clinical treatment exposures and tumor adaptation mechanisms. Loss of ER expression in HR
+ breast cancer may also lead to dependence on alternative oncogenic drivers, that may be constitute
drug targets in refractory HR+ breast cancer. In Aim 3, we apply a chemical proteomic strategy in CTC
lines, combining cysteine cross-linking with mass spectrometry to identify all ligandable cysteine residues
within cellular proteins. Preliminary data show multiple cysteine-targetable proteins in CTCs that are not
present in untreated breast cancer lines, and we will use CRISPR screens to identify those required for
proliferation, followed by identification of tool compounds from a specialized library of cysteine-reactive
covalent inhibitors for functional analyses. Together, these complementary approaches address the loss
of ER expression in advanced HR+ breast cancers, with the goal of overcoming acquired resistance to
endo...

## Key facts

- **NIH application ID:** 10783084
- **Project number:** 5R01CA129933-17
- **Recipient organization:** MASSACHUSETTS GENERAL HOSPITAL
- **Principal Investigator:** Daniel A. Haber
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $399,000
- **Award type:** 5
- **Project period:** 2008-04-01 → 2028-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10783084

## Citation

> US National Institutes of Health, RePORTER application 10783084, Modeling Metastasis and Acquired Drug Resistance Using Circulating Tumor Cells (5R01CA129933-17). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10783084. Licensed CC0.

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