A study led by researchers at the Olivia Newton-John Cancer Research Institute (ONJCRI), La Trobe University (LTU), The Walter and Eliza Hall Institute, and the University of Melbourne has used a novel optical color-coding barcoded cancer cell tracking strategy to investigate metastatic heterogeneity in breast cancer.

The study's findings suggest this strategy not only enables comparison of metastatic seeding dynamics between different organs using high-resolution imaging, but also has identified new site-driven gene expression signatures associated with lung and liver metastases.

"This strategy allowed us to identify the fitness of up to 31 different clones in visceral organs most commonly affected by breast cancer metastases," said study co-corresponding author Delphine Merino, laboratory head at ONJCRI and LTU's School of Cancer Medicine.

"Understanding how cancer clones interact, survive and grow in distant organs, will help us to design better therapies to prevent [tumorigenesis] or stop tumor progression," said Merino.

Breast cancer is a highly heterogeneous disease, with widely variable clinical outcomes within and across molecular subtypes, depending on the propensity of individual cancer cells to metastasize and to escape standard therapies.

Single-cell sequencing studies have shown that patients' tumors can be highly heterogenous. "This genomic heterogeneity is thought to be associated with poor outcomes, as heterogenous tumors are more likely to contain aggressive and drug-resistant clones," said Merino.

Among the different breast cancer subtypes, triple-negative breast cancer (TNBC) is a highly heterogenous disease associated with a particularly poor prognosis and a higher 5-year post-diagnostic recurrence risk than other breast cancer subtypes.

"Not only is TNBC a more aggressive subtype compared to others, but also there is a lack of targeted therapies for this subtype," Merino told BioWorld Science.

Furthermore, evidence increasingly suggests that clonal heterogeneity plays an important role in metastatic growth in TNBC, with high rates of metastasis being seen to visceral organs including lungs and liver.

However, the nature of the clonal interactions involved in this highly selective process remains unclear.

Reported in the July 7, 2021, edition of Science Advances, the new study investigated metastatic heterogeneity in TNBC using an optical barcoding approach based on a panel of multicolor Lentiviral Gene Ontology (LeGO) vectors.


Recently developed to enable better spatiotemporal tracking of individual clones within heterogeneous tumor cell types, this approach can image, quantify and sort labeled cell populations by microscopy and flow cytometry at the single-cell level.

However, while it allows long-term in vitro tracking of labeled cells and their progeny, the number of colors that can be used to track cancer cells in vivo is limited, and increasing the number of color combinations that can be detected and quantified in vivo is challenging.

To investigate how TNBC cells interact with and adapt to visceral organs, the researchers optimized LeGO technology to visualize and quantify in vivo 31 cancer cell subclones barcoded with specific fluorescent tags.

This mapping of the clonal composition of thousands of metastases in the lungs and liver revealed that metastases were highly polyclonal in lungs but not in the liver, while subclonal transcriptomes were shown to vary according to their metastatic niche.

"Tumor and metastasis heterogeneity is thought to play an important role in tumor progression and drug resistance, while polyclonal metastases may be more likely to contain cells that can escape treatment," said Merino.

"Furthermore, cancer cells expressing different genes in different microenvironments may have responded differently to cancer treatments," she added.

The research group further identified a reversible niche-driven transcriptomic signature that was shown to be conserved in autopsy-sampled lung and liver metastases.

"Cells adopt different gene expression profiles to adapt to their microenvironment and some of these genes may be pharmacologically inhibited to kill metastases," said Merino.

Within this genetic signature, the tumor necrosis factor-alpha (TNF-alpha) pathway was found to be differentially upregulated in lung versus liver metastases.

"The upregulation of this pathway in lung metastases was observed in several patient samples," Merino said.

"Although TNF-alpha inhibition with etanercept affected metastasis heterogeneity in the lungs, it did not have a significant impact on the survival of the metastatic clones. However, inhibition downstream of TNF-alpha using the inhibitor of apoptosis proteins (IAP) birinapant strongly impacted clone survival," she said.

"These results indicate that these preclinical models can be extremely valuable in studying the impact of pharmacological inhibitors on the interaction and survival of metastatic clones in multiple organs," said Merino, noting that collectively the study's findings may have future breast cancer management implications.

Management implications

"The long-term objective is to identify, in these signatures, genes or pathways that can be efficiently targeted with existing drugs, for treating patients with advanced cancer," she said.

Looking forward, "it would be interesting to investigate whether the TNF-alpha pathway is directly responsible for the high degree of metastatic heterogeneity observed in the lungs.

"From a translational viewpoint, it would be interesting to test the effect of these drugs, in combination with other agents, in a larger number of preclinical models, which will help us to design new strategies to kill metastases and improve the outcomes of patients with TNBC."