Small extracellular vesicles promote stiffness-mediated metastasis

Introduction

The diverse functions carried out by the different tissues and organs in our bodies are not only influenced by biochemical signalling, but by physical cues like stiffness as well. Different tissues exhibit different average stiffnesses, ranging from 100 Pa in the brain to over 10⁶ Pa in bone (1). Tissue stiffness is largely influenced by the extracellular matrix (ECM), a mesh-like network composed of proteins such as collagens. Changes to the mechanical properties of the ECM during tumorigenesis have been reported in several solid tumour types (2-6). For example, tissue stiffness is correlated with tumour aggression and grade in glioma (7). Tissue stiffness can also influence cells within the tumour microenvironment. For example, cancer-associated fibroblasts (CAFs) increase collagen deposition in proximity to tumour cells, which can promote metastatic behaviours (8).

One way in which cancer cells can communicate with their environment is through the secretion of extracellular vesicles (EVs), which are 30-150 nm wide and contain proteins and nucleic acids (9). Due to their tiny size, tumour-derived EVs can travel long distances from their cells of origin and influence processes such as immune evasion and metastatic niche priming (10-13).

In almost all prior studies on EVs, cancer cells were cultured on plastic, which does not accurately reflect stiffness levels experienced by cancer cells in vivo and may affect the protein cargo carried by EVs. In this study, the authors used a combination of cell culture, mouse models, and zebrafish models of metastasis to determine the impact of physiologically relevant tissue stiffness on EV-mediated metastatic dissemination.

Key Findings

Matrix stiffness impacts EV quantity and protein cargo

The authors measured the stiffness of breast tumour tissues from patients and found a wide range of stiffnesses, from ~3 kPa to over ~80 kPa. Breast cancer is known to commonly metastasize to the lung, which has a stiffness range of 0.5-5 kPa. Using fresh tumour samples for compression analysis, the authors found that stiff tumour regions (>10 kPA) released more extracellular vesicles (EVs) than soft regions (<10 kPA).

The authors also wondered whether stiffness affected the protein cargo within EVs. Thus, they developed “soft” (0.5 kPa) and “stiff” (25 kPa) matrices to model physiologically relevant stiffness levels. When the authors isolated EVs from highly invasive, triple-negative breast cancer cells cultured on soft or stiff matrices, they found striking differences in the protein content between these conditions through mass spectrometry. Proteins enriched in stiff EVs compared to soft EVs were primarily involved in tumorigenesis, cell adhesion, and cell migration, prompting the authors to investigate whether matrix stiffness could affect the ability of EVs to spread to different organs.

Stiff EVs show enhanced biodistribution in vivo

To see whether matrix stiffness affected the ability of EVs to spread, the authors injected immunocompromised mice with fluorescent EVs via the tail vein and tracked them throughout the body. Using near-infrared imaging, they observed that stiff EVs had a greater signal intensity in the lungs, liver, and spleen compared to soft EVs. Importantly, this observation in their mouse model mimics human breast cancer, which mainly metastasizes to the lungs.

But how do stiff EVs distribute more readily than soft EVs? The authors hypothesized that stiff versus soft EVs differed in their ability to bind to ECM proteins. Using an ECM binding assay, they found that stiff EVs attached more strongly to collagen IV than soft EVs. This enhanced binding could potentially be due to the enrichment of adhesion proteins like CD44 in stiff versus soft EVs.

Stiff EVs promote cancer cell dissemination and survival in vivo

Based on the accumulation of stiff EVs within common tumour secondary sites, the authors wondered whether stiff EVs could affect circulating cancer cells during metastasis (14). Indeed, the escape of cancer cells from the primary tumour is often choreographed by chemotaxis, the biased migration of cells under a chemical gradient. Therefore, they tested the chemotactic properties of stiff versus soft EVs using a transwell assay. In this experiment, breast cancer cells were separated from EV concentrated media by a semipermeable membrane. After 16 hours, cell migration towards stiff EVs was threefold higher than towards soft EVs.

But what would happen in a more complex and challenging microenvironment? To model this scenario, the authors created a zebrafish xenograft model. In this experiment, stiff or soft EVs were injected into the yolk sac of zebrafish embryos 2 days post-fertilization, followed by injection of cancer cells. Cell dissemination and the leakage of cancer cells into perivascular locations (extravasation) were assessed 24h and 72h post injection, respectively. In the absence of EVs, only 4.2% of cancer cells were able to disseminate. Strikingly, 33.8% of embryos injected with stiff EVs showed disseminated cells in contrast to only 10.7% of embryos injected with soft EVs. Similarly, 20% of embryos treated with stiff EVs had extravasated cells, whereas none of the embryos injected with soft EVs exhibited extravasation.

Soft EVs transform fibroblasts into CAF-like cells

The authors investigated whether stiff versus soft EVs from breast cancer cells had different effects on tumour formation at secondary sites by transforming resident fibroblasts. Indeed, the nesting of future sites of metastasis is a process orchestrated by the crosstalk between the microenvironment and transformed resident stromal cells, such as cancer associated fibroblasts (CAF). Since EVs showed greater retention in the lungs in vivo, the authors assessed EV-mediated changes in the phenotype of normal lung fibroblasts.

Lung tissue is relatively soft (0.5-5 kPa), similar to normal breast tissue (15,16). Lung fibroblasts treated with soft EVs upregulated CAF-associated genes such as smooth muscle actin, collagen I, and VEGFA (8,17) compared to fibroblasts treated with stiff EVs. Next, the authors investigated the expression of inflammatory S100 proteins since they were found to be critical for successful pre-metastatic niche formation (18). Lung fibroblasts exposed to soft EVs upregulated the expression of four S100 genes, whereas treatment with stiff EVs resulted in downregulation.

Altogether, the authors found that in contrast to stiff EVs, soft EVs induce a potent CAF-like phenotype in lung fibroblasts, highlighting the different roles played by soft versus stiff EVs during the metastatic cascade.

Above: A summary of the different effects of stiff versus soft EVs during metastasis. Tumour tissue stiffens over the course of tumour development, resulting in a change in the protein content of EVs. Stiff EVs upregulate adhesion molecules and distribute more readily into distant sites. Tumour derived EVs with a ‘soft’ gene expression profile promote a CAF identity in neighboring fibroblasts. Schematic created using BioRender.

Why we chose this preprint

Yohalie: This preprint holds particular significance given the growing interest in extracellular vesicles (EV) as new biomarkers in cancer diagnosis. The authors demonstrate how EVs can exhibit distinct roles in the metastatic cascade based on their production conditions. They highlight the crucial role of soft EVs in preparing distant organs during late-stage metastasis, while stiff EVs are crucial for cell dissemination during early-stage metastasis.

Jade: I found this preprint particularly interesting due to the finding that the stiffness of the primary tumour could affect the success of metastatic colonization even at great distances from the original site. Solid tumours exhibit a high degree of intra- and intertumoral heterogeneity in terms of stiffness, and I believe this preprint nicely highlights how different steps in the metastatic process (from escaping the primary tumour to remodelling resident cells of the metastatic niche) are influenced by the mechanical properties of the original tumour.

 Questions for the authors

1. What upstream signalling events occur in tumour cells that determine the protein cargo of stiff versus soft EVs?
2. Are there in vitro assays to assess matrix remodelling by lung derived fibroblasts upon treatment with soft EVs? Would there be differences in fiber alignment, or changes to the architecture of the collagen matrix?
3. Can stiff EVs impact fibroblasts at the primary tumour site to help cells escape the tumour?
4. In vivo, most matrices are viscoelastic. Do you think the content of EVs could be impacted by matrix viscosity?
5. Which molecules contained by stiff versus soft EVs could be responsible for inducing a CAF-like phenotype?

 References

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Tags: breast cancer, extracellular matrix, mechanobiology, metastasis, vesicle

doi: https://doi.org/10.1242/prelights.35186

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