Specific Targeting of Mesothelin-Expressing Malignant Cells Using Nanobody-Functionalized Magneto-Fluorescent Nanoassemblies

Our plain-English summary of Specific Targeting of Mesothelin-Expressing Malignant Cells Using Nanobody-Functionalized Magneto-Fluorescent Nanoassemblies, by T. Briolay, J. Fresquet, D. Meyer, et al (International Journal of Nanomedicine, 2024).

The study

• Authors: T. Briolay, J. Fresquet, D. Meyer, B. Kerfelec, P. Chames, E. Ishow, C. Blanquart
• Journal: International Journal of Nanomedicine
• Published: 2024;19:633-650
• Cited by: 10 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Introduction: Most current anti-cancer therapies are associated with major side effects due to a lack of tumor specificity. Appropriate vectorization of drugs using engineered nanovectors is known to increase local concentration of therapeutic molecules in tumors while minimizing their side effects. Mesothelin (MSLN) is a well-known tumor associated antigen overexpressed in many malignancies, in particular in malignant pleural mesothelioma (MPM), and various MSLN-targeting anticancer therapies are currently evaluated in preclinical and clinical assays. In this study, we described, for the first time, the functionalization of fluorescent organic nanoassemblies (NA) with a nanobody (Nb) targeting MSLN for the specific targeting of MSLN expressing MPM cancer cells.

Methods: Cell lines from different cancer origin expressing or not MSLN were used. An Nb directed against MSLN was coupled to fluorescent NA using click chemistry. A panel of endocytosis inhibitors was used to study targeted NA internalization by cells. Cancer cells were grown in 2D or 3D and under a flow to evaluate the specificity of the targeted NA. Binding and internalization of the targeted NA were studied using flow cytometry, confocal microscopy and transmission electron microscopy.

Results: We show that the targeted NA specifically bind to MSLN-expressing tumor cells. Moreover, such functionalized NA appear to be internalized more rapidly and in significantly larger proportions compared to naked ones in MSLN+ MPM cells, thereby demonstrating both the functionality and interest of the active targeting strategy. We demonstrated that targeted NA are mainly internalized through a clathrin-independent/dynamin-dependent endocytosis pathway and are directed to lysosomes for degradation. A 3D cell culture model based on MSLN-expressing multicellular tumor spheroids reveals NA penetration in the first superficial layers.

Conclusion: Altogether, these results open the path to novel anticancer strategies based on MSLN-activated internalization of NA incorporating drugs to promote specific accumulation of active treatments in tumors.

© 2024 Briolay et al.

In plain English

This was a laboratory study of cancer cells in dishes. No experiments were done in people or animals, though some cell lines came from patients' samples; asbestos exposure was not examined. The authors, in France, attached a nanobody (a small antibody fragment) that recognizes mesothelin, a protein on many malignant pleural mesothelioma cells, to tiny fluorescent, magnetic particles. They compared these targeted particles with untargeted ones. Test cells included a mesothelioma line with very low mesothelin (Meso34), the same line engineered to overexpress it (Meso34-MSLN), and other cancer lines. Targeted particles did not change Meso34 or Meso34-MSLN viability over 72 hours, even at high amounts. About 65% of Meso34-MSLN cells were labeled by targeted particles, versus under 10% by untargeted ones. Targeted particles entered Meso34-MSLN cells faster and in larger amounts. Entry was energy-dependent and partly blocked by dynasore; most particles ended up in lysosomes, the cells' degradation compartments. In 3D cell clusters, particles reached only the outer layers. Under flow, they bound almost only to mesothelin-expressing cells. The authors concluded that the approach opens prospects for delivering drugs specifically to mesothelin-expressing cancer cells.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) experiment on cultured cancer cell lines, including flat (2D) cultures, 3D multicellular spheroids and cultures under a flow of medium
• Who was studied: Human cancer cell lines in Nantes, France. The pleural mesothelioma line Meso34 (very low mesothelin) and Meso34-MSLN, the same line engineered to overexpress mesothelin. Also the mesothelioma line Meso13, the ovarian carcinoma line A1847 and the lung adenocarcinoma line ADCA153. Some cell lines were established from patients' samples; the mesothelioma and adenocarcinoma lines came from patients' pleural effusions.
• Exposure: No asbestos or fiber exposure was studied. Cells were exposed to magneto-fluorescent organic nanoassemblies either without a targeting agent (FONmagc) or carrying an anti-mesothelin nanobody (FONmagc-Nb).
• Main results:
  • The viability of Meso34 and Meso34-MSLN cells (the two lines tested for toxicity) did not change after 72 hours with high amounts of the targeted particles.
  • In Meso34-MSLN cells, about 65% of cells were labeled with the targeted particles, compared with less than 10% with the untargeted particles.
  • In Meso34 cells (low mesothelin), internalization did not differ between targeted and untargeted particles. In Meso34-MSLN cells, internalization of the targeted particles was faster and stronger.
  • In cell lines with natural (endogenous) mesothelin, the text reports a higher signal with targeted than with untargeted particles. The signal was not fully in line with mesothelin levels: ADCA153 had less mesothelin than A1847 but took up more targeted particles.
  • In Meso34-MSLN cells, incubation at 4°C reduced uptake of targeted particles by about 80%, and dynasore reduced the signal by about 40%.
  • In Meso34-MSLN cells, about 5% of the particle signal overlapped with acidic compartments (probably lysosomes) at 6 hours, and around 60% at 24 hours.
  • In 3D spheroids of Meso34-MSLN cells, particles entered the first cell layers but not the innermost layers.
  • Under a flow of medium over a mixed culture, the targeted particles bound almost only to mesothelin-expressing cells.
• The authors' conclusion: The authors concluded that the targeted particles can specifically bind mesothelin-expressing cancer cells in coculture, 3D and flow models. They concluded that active targeting sped up internalization and specific accumulation in these cells, opening prospects for local, specific drug delivery.
• Limitations the authors state:
  • The innermost layers of the spheroids appeared inaccessible to the particles.
  • The authors stated that the way particles enter cells might differ in other mesothelin-expressing cell types or other mesothelioma cell lines.
  • Electron microscopy sizing could not be done on the final nanobody-coupled particles because of the presence of salts.

Topics

Medical terms in this study, explained in our Lexicon: Mesothelin, Pleural Mesothelioma, Transmission Electron Microscopy (TEM).

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