The importance of cell media in EV cancer treatment

Extracellular vesicles (EVs) provide valuable biomarkers for cancer diagnosis and stable drug vehicles for treatment delivery. To produce effective, non-toxic EV cancer treatments, proper isolation, and appropriate cell culture growth media are essential.

In this article, Promocell details the critical importance of cell media in EV cancer treatment and the challenges that researchers still must overcome.

What are extracellular vesicles?

EVs are produced via the budding of cell membranes. They are then released into the extracellular space to facilitate intercellular communication. EVs are phospholipid bilayer membrane-coated vesicles that transmit specific, bioactive proteins and nucleic acids that modify the behavior of target cells.

They are typically characterized by their biogenesis and size, with a diameter of 50 nm to 1 µm. Three types of EVs exist:¹

• Exosomes
• Microvesicles (ectosomes/shedding vesicles)
• Apoptotic bodies

Exosomes are secreted by various cells, including stroma cells, immune cells, stem cells, and even cancer cells. They are key components of inter-cellular communication and are essential for many pathological and physiological processes.^2,3

Role of EVs in cancer biology

EV cancer treatment research has become incredibly significant within the field of oncology. Approximately ten years ago, research revealed that EVs played sophisticated roles in the initiation, invasion, metastasis, and recurrence of tumorous cells.

Image Credit: PromoCell

EVs enable communication within the tumor microenvironment (TME), transporting bioactive molecules that modify tissue remodeling and the immune response.⁴

Cancer-derived EVs inhibit the immune system’s ability to detect and destroy cancerous cells. They promote nutrient transferal to tumors and, by supporting angiogenesis within the tumor, permit metastasis.

Cancer-derived EVs additionally encourage treatment resistance via the transmission of bioactive molecules (that increase resistance mechanisms and thus survival within the tumor).⁵

Cancer-derived EVs can transform the extracellular matrix and emerging tumor environment (TME) composition, thus promoting tumor growth and invasion.

EVs additionally influence cancer and stromal cell metabolism via the delivery of metabolic enzymes and metabolites that support the energy necessities of the tumor cell.⁵

EV cancer treatment and therapy

Research into EV cancer treatment has gained lots of attention. As they carry molecules that alter target cells, researchers are exploring the potential use of EVs as:

• Diagnostic markers
• Drug delivery vessels
• Therapeutic agents

Extracellular vesicles as biomarkers

The molecules that EVs transport can replicate the molecular and genetic properties of cancerous cells. Thus, EVs have massive potential as biomarkers in cancer diagnosis.

In breast cancer diagnostics, exosomal microRNAs have shown to be particularly powerful biomarkers. They have exhibited various genetic characteristics of different breast cancer types.

Exosomes released by breast cancer cells can additionally inhibit NK cell cytotoxicity and suppress T-cell proliferation, thus impairing the immune response in pre-metastatic tissues.⁵ 

Extracellular vesicles for drug delivery

To specifically target tumor cells, cancer-derived EVs utilize the intrinsic homo-adhesion properties of surface membrane antigens. This is a promising feature for utilizing autologous cancer-derived EVs as vessels for drug delivery.⁶

Unfortunately, the biology and functions of EVs are not completely understood, and EV cancer treatment is still in its early stages of development. Thus, an accurate interpretation of the formation, secretion, and interactions of EVs is crucial to facilitate the translation of EV cancer treatment into clinical practice.

Preparing cancer-derived EVs for therapeutic use

In EV cancer treatment research, four types of cancer-derived EVs have been identified as potential anti-tumor vesicles:

• Irradiated cancer-derived EVs
• Cancer-derived EVs combined with advanced materials, such as nanoparticles
• Cancer-derived EVs loaded with chemotherapeutic drugs
• Genetically engineered cancer-derived EVs

In addition to being manipulated vehicles for anti-tumor drugs, EVs are also acting as immune-active agents. Unfortunately, the preparation, efficacy, and applicability of these techniques have proved to be challenging.¹

Selection and isolation of appropriate extracellular vesicles

To produce EVs with relevant biomolecules, the specific cell type involved in the cancer of interest must first be selected. Post-identification, suitable derived EVs must be isolated and purified using methods like:

• Ultracentrifugation
• Density gradient centrifugation
• Commercial EV isolation kits

Quantification and characterization of extracellular vesicles

The isolated cancer-derived EVs that have preserved their biological cargo and integrity are subsequently quantified and characterized. The type, size distribution, and concentration of EVs, along with their cargo, can be established utilizing:

• Nanoparticle tracking analysis
• Electron microscopy
• Proteomic analysis

Efficacy tests

Manipulation of the TME and tumor progression cell cultures is needed to investigate the biological functions of cancer-derived EVs in intercellular communication.

Effective investigation depends on the use of a suitable cell culture medium that fulfills specific cell culture requirements—this is required to maintain EV viability. It additionally ensures that EVs have no toxic components, contaminants, or immunogenic molecules that could have adverse effects on recipient cells.¹

With a suitable cell culture medium, functional assays can successfully analyze the therapeutic impact of the isolated and adapted EVs on recipient cells. To assess EV cancer treatment efficacy, researchers concentrate on the ability of cancer-derived EVs to:

• Modulate pathways of interest
• Induce apoptosis in targeted cancerous cells
• Affect the overall immune response

Ideal EV isolation and preparation protocols

Protocols for the isolation and efficacy assessment of cancer-derived EVs must be:

• Highly selective
• Easy to follow
• Affordable
• Easily replicated
• Generate a high yield of viable EVs
• Time-efficient and high-through and

To guarantee that EVs are handled and stored correctly, stringent, replicable protocols are also needed. Appropriate cell culture media are crucial for the preservation of EV stability and functionality.

How we can support your EV cancer treatment research

PromoCell offers a cancer media portfolio that provides a practical path for the effective isolation of pure EVs from cancer cell lines or patient-derived cancer cells, such as:

• Cancer Cell Line Medium XF
• Primary Cancer Culture System (PCCS)
• 3D Tumorsphere Medium XF

These media support different cancer cell types, improving their expansion and growth performance. They are, therefore, extremely beneficial for EV production. The media are additionally available for large-scale EV cancer treatment research.

Challenges in isolating cancer-derived EVs

Cell culture-conditioned media offer a more regulated environment for isolated EVs. In EV cancer treatment, the important factors that influence EV yield must be considered.

To begin with, it is imperative to consider whether a culture medium with or without an EV-depleted fetal bovine serum (FBS) would be most appropriate. Abrupt fluctuations in serum-free cultures will likely result in significant cell stress, potentially causing them to modify their EV secretion.⁷

To eradicate FBS-derived EV contaminants for purified EV depletion, EV-depleted FBS media need lengthy ultracentrifugation processes. However, new research revealed that prolonging the ultracentrifugation protocol does not result in an entirely contaminated-free medium. Problematically, any residual bovine small RNA can be erroneously identified as human RNA.⁸

The following are additional factors that must be taken into account in EV cancer therapy protocols:

• Cell culture matrices and plastics
• Exact cell culture medium
• Cell passage, confluency, and viability
• Mycoplasma-status
• Other microbial contaminants⁶

GMP guidelines for EV cancer treatment research

The good manufacturing practices (GMP) grade production method for exosomes must be followed to comply with GMP in EV cancer treatment research. This recommends culture environments, cell types, cultivation systems, and cell culture media. Following EV production, an additional 3-step purification protocol is needed.³

After purification, it is essential to select a method for fully identifying and characterizing exosomes based on their bioactivity and physical properties.

The European Medicines Agency offers scientific recommendations for categorizing advanced therapy medicinal products. This agency promotes scientific progress in cellular biomedicine to enhance disease therapies, providing researchers with recommendations, guidelines, and criteria to advance their medicinal/therapeutic products and enhance directive regulation.                           

References and further reading

1. Lin, W. and Cai, X.D. 2021. Current strategies for cancer cell-derived extracellular vesicles for cancer therapy. Frontiers in Oncology. 11, p.758884. 
2. Pegtel, D.M. and Gould, S.J. 2019. Exosomes. Annual review of biochemistry. 88, pp.487-514.
3. Rezaie, J., Feghhi, M. and Etemadi, T. 2022. A review on exosomes application in clinical trials: Perspective, questions, and challenges. Cell Communication and Signaling, 20(1), pp.1-13.
4. Kosaka, N., Yoshioka, Y., Fujita, Y. and Ochiya, T. 2016. Versatile roles of extracellular vesicles in cancer. The Journal of clinical investigation. 126(4), pp.1163-1172.
5. Kok, V.C. and Yu, C.C. 2020. Cancer-derived exosomes: their role in cancer biology and biomarker development. International Journal of Nanomedicine. pp.8019-8036.
6. Meng, W., He, C., Hao, Y., Wang, L., Li, L. and Zhu, G. 2020. Prospects and challenges of extracellular vesicle-based drug delivery system: Considering cell source. Drug delivery. 27(1), pp.585-598.
7. Allelein, S., Medina-Perez, P., Lopes, A.L.H., Rau, S., Hause, G., Kölsch, A. and Kuhlmeier, D. 2021. Potential and challenges of specifically isolating extracellular vesicles from heterogeneous populations. Scientific Reports. 11(1), p.11585.
8. Mannerström, B., Paananen, R.O., Abu-Shahba, A.G., Moilanen, J., Seppänen-Kaijansinkko, R. and Kaur, S. 2019. Extracellular small non-coding RNA contaminants in fetal bovine serum and serum-free media. Scientific Reports. 9(1), p.5538.

About PromoCell

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