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Small extracellular vesicles promote stiffness-mediated metastasis

Alexandra Sneider, Ying Liu, Bartholomew Starich, Wenxuan Du, Carolyn Marar, Najwa Faqih, Gabrielle E. Ciotti, Joo Ho Kim, Sejal Krishnan, Salma Ibrahim, Muna Igboko, Alexus Locke, Daniel M. Lewis, Hanna Hong, Michelle Karl, Raghav Vij, Gabriella C. Russo, Praful Nair, Estibaliz Gómez-de-Mariscal, Mehran Habibi, Arrate Muñoz-Barrutia, Luo Gu, T.S. Karin Eisinger-Mathason, Denis Wirtz

Posted on: 3 August 2023 , updated on: 29 May 2024

Preprint posted on 5 July 2023

Article now published in Cancer Research Communications at http://dx.doi.org/10.1158/2767-9764.CRC-23-0431

Small but mighty: Sneider and colleagues demonstrate the impact of tissue stiffness on extracellular vesicle-mediated metastasis in breast cancer.

Selected by Jade Chan, yohalie kalukula

Updated 29 May 2024 with a postLight by Jade Chan and Yohalie Kalukula

This highlighted preprint has now been published in Cancer Research Communications, a new journal from the American Association for Cancer Research (AACR). In this study, A. Sneider, Y. Liu, B. Starich and colleagues demonstrate that the function, content, and overall quantity of extracellular vesicles secreted by cancer cells depends on the stiffness of their microenvironment. Importantly, they show that tumour-derived extracellular vesicles can alter the properties of metastatic sites even at great distances from the original tumour.

The preprint article and the published version are largely identical with minor differences in the formatting of the figures, which attests to the scientific rigor of the initial manuscript. Congratulations again to the authors for their contribution to the field!

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

  1. Cox, T. R., & Erler, J. T. (2011). Remodeling and homeostasis of the extracellular matrix: implications for fibrotic diseases and cancer. Dis model mech, 4(2), 165–178.
  2. Colpaert, C., Vermeulen, P., Van Marck, E., and Dirix, L. (2001). The presence of a fibrotic focus is an independent predictor of early metastasis in lymph node-negative breast cancer patients. Am J Surg Pathol 25, 1557–1558.
  3. Hasebe, T., Tsuda, H., Hirohashi, S., Shimosato, Y., Iwai, M., Imoto, S., and Mukai, K. (1996). Fibrotic focus in invasive ductal carcinoma: an indicator of high tumor aggressiveness. Jpn J Cancer Res 87, 385–394.
  4. Hasebe, T., Tsuda, H., Hirohashi, S., Shimosato, Y., Tsubono, Y., Yamamoto, H., and Mukai, K. (1998). Fibrotic focus in infiltrating ductal carcinoma of the breast: a significant histopathological prognostic parameter for predicting the long-term survival of the patients. Breast Cancer Res Treat 49, 195–208.
  5. Hasebe, T., Sasaki, S., Imoto, S., Mukai, K., Yokose, T., and Ochiai, A. (2002). Prognostic Significance of Fibrotic Focus in Invasive Ductal Carcinoma of the Breast: A Prospective Observational Study. Mod Pathol 15, 502–516.
  6. Conklin, M.W., Eickhoff, J.C., Riching, K.M., Pehlke, C.A., Eliceiri, K.W., Provenzano, P.P., Friedl, A., and Keely, P.J. (2011). Aligned Collagen Is a Prognostic Signature for Survival in Human Breast Carcinoma. Am J Pathol 178, 1221–1232.
  7. Miroshnikova, Y. A., Mouw, J. K., Barnes, J. M., Pickup, M. W., Lakins, J. N., Kim, Y., Lobo, K., Persson, A. I., Reis, G. F., McKnight, T. R., Holland, E. C., Phillips, J. J., & Weaver, V. M. (2016). Tissue mechanics promote IDH1-dependent HIF1α-tenascin C feedback to regulate glioblastoma aggression. Nat Cell Biol, 18(12), 1336–1345.
  8. Kalluri, R., and Zeisberg, M. (2006). Fibroblasts in cancer. Nat Rev Cancer 6, 392–401.
  9. van Niel, G., D’Angelo, G., and Raposo, G. (2018). Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol 19, 213.
  10. Marar, C., Starich, B., and Wirtz, D. (2021). Extracellular vesicles in immunomodulation and tumor progression. Nat Immunol, 1–11.
  11. Clayton, A., Mitchell, J.P., Court, J., Linnane, S., Mason, M.D., and Tabi, Z. (2008). Human Tumor-Derived Exosomes Down-Modulate NKG2D Expression. J Immunol 180, 7249–7258.
  12. Hoshino, A., Costa-Silva, B., Shen, T.-L., Rodrigues, G., Hashimoto, A., Tesic Mark, M., Molina, H., Kohsaka, S., Di Giannatale, A., Ceder, S., et al. (2015). Tumour exosome integrins determine organotropic metastasis. Nature 527, 329–335.
  13. Costa-Silva, B., Aiello, N.M., Ocean, A.J., Singh, S., Zhang, H., Thakur, B.K., Becker, A., Hoshino, A., Mark, M.T., Molina, H., et al. (2015). Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat Cell Biol 17, 816–826.
  14. Du, W., Nair, P., Johnston, A., Wu, P.-H., and Wirtz, D. (2022). Cell Trafficking at the Intersection of the Tumor–Immune Compartments. Annu Rev Biomed Eng 24, 275–305.
  15. Hinz, B. (2012). Mechanical Aspects of Lung Fibrosis. Proc Am Thorac Soc 9, 137–147.
  16. Polio, S. R., Kundu, A. N., Dougan, C. E., Birch, N. P., Aurian-Blajeni, D. E., Schiffman, J. D., Crosby, A. J., & Peyton, S. R. (2018). Cross-platform mechanical characterization of lung tissue. PloS one, 13(10), e0204765.
  17. Bhowmick, N.A., Neilson, E.G., and Moses, H.L. (2004). Stromal fibroblasts in cancer initiation and progression. Nature 432, 332–337.
  18. Kaplan, R.N., Riba, R.D., Zacharoulis, S., Bramley, A.H., Vincent, L., Costa, C., MacDonald, D.D., Jin, D.K., Shido, K., Kerns, S.A., et al. (2005). VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature 438, 820–827.

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

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

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