Isolation and Purification of Fungal β-Glucan as an Immunotherapy Strategy for Glioblastoma

Summary

The present protocol describes the purification steps and subsequent studies of four different fungal β-glucans as potential immunomodulatory molecules that enhance the anti-tumoral properties of microglia against glioblastoma cells.

Abstract

One of the biggest challenges in developing effective therapies against glioblastoma is overcoming the strong immune suppression within the tumor microenvironment. Immunotherapy has emerged as an effective strategy to turn the immune system response against tumor cells. Glioma-associated macrophages and microglia (GAMs) are major drivers of such anti-inflammatory scenarios. Therefore, enhancing the anti-cancerous response in GAMs may represent a potential co-adjuvant therapy to treat glioblastoma patients. In that vein, fungal β-glucan molecules have long been known as potent immune modulators. Their ability to stimulate the innate immune activity and improve treatment response has been described. Those modulating features are partly attributed to their ability to bind to pattern recognition receptors, which, interestingly, are greatly expressed in GAMs. Thus, this work is focused on the isolation, purification, and subsequent use of fungal β-glucans to enhance the tumoricidal response of microglia against glioblastoma cells. The mouse glioblastoma (GL261) and microglia (BV-2) cell lines are used to test the immunomodulatory properties of four different fungal β-glucans extracted from mushrooms heavily used in the current biopharmaceutical industry: Pleurotus ostreatus, Pleurotus djamor, Hericium erinaceus, and Ganoderma lucidum. To test these compounds, co-stimulation assays were performed to measure the effect of a pre-activated microglia-conditioned medium on the proliferation and apoptosis activation in glioblastoma cells.

Introduction

Despite the advent of novel achievements in the field of neuro-oncology, the life expectancy of glioblastoma patients remains meager. Gold-standard therapies against brain tumors are based on the amalgamation of surgery, radiotherapy, and chemotherapy. However, in the last decade, immunotherapy has emerged as a powerful strategy to treat different types of cancer¹. Thus, the possibility of harnessing the body's immune response against tumor cells has recently become the fourth pillar of oncology.

It has long been known that one of the biggest challenges in the field is to overcome the strong immunosuppression fou....

Protocol

The four different mushroom variants described in this protocol were obtained from a commercial source (see Table of Materials).

1. Isolation of fungal β-glucans

1. Extraction and isolation of soluble mushroom polysaccharides
   NOTE: Soluble mushroom polysaccharides (SMPs) were obtained according to the procedure schematically shown in Figure 1.
   1. Gently rinse fresh P. ostreatus, P. djamor, H. erinaceus, and G. lucidum fruiting bodies (about 2,000 g/mushroom) in distilled water five times.
   2. Dry the ....

Results

Successful purification of β-glucans
The mass of MP, SMPs, and SβGs obtained from fruiting bodies of P. ostreatus, P. djamor, G. lucidum, and H. erinaceus following the extraction and purification process is summarised in Table 1. The basic composition (total carbohydrates, β-glucans, and protein) of MP, SMPs, and SβGs obtained from the fungi is depicted in Table 2. These results show how the protocol allowed the retrieval of a lar.......

Discussion

This work describes the use of well-established techniques to successfully isolate, purify, and characterize the content of SβGs from four different fungi. The results showed how after hot water extraction of SMPs, obtained from P. ostreatus, P. djamor, G. lucidum, and H. erinaceus, followed by hydrolytic treatment with α-amylase, glucosidase, and protease, the content of α-glucan and protein were reduced, thus significantly enriching the amount of pure SβGs.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
8-well chamber slides	Thermo Fisher, USA	171080
Air-drying oven	J.P. Selecta S.A., Spain	2000210
Albumin	Sigma-Aldrich, St. Louis	A7030
Alcalase	Novozymes, Denmark	protease
Alexa Fluor 488	Thermofisher, USA	A32731
Alexa Fluor 647	Thermofisher, USA	A32728
Blade mill	Retsch, Germany	SM100
Bovine Serum Albumin	MERK, Germany	A9418
Cellulose tubing membrane	Sigma-Aldrich, St. Louis	D9402
Centrifuge	MERK, Germany	Eppendorf, 5810R
Colocalisation pluggins	ImageJ		(https://imagej.net/imaging/colocalization-analysis )
DAPI	MERK, Germany	28718-90-3
Dextrans	Pharmacosmos, Holbalk, Denmark	Dextran 410, 80, 50
Dulbecco´s modified Eagle´s medium, Gluta MAXTM	Gibco, Life Technologies, Carlsbad, CA, USA	10564011
Extenda (α- Amylase/Glucoamylase)	Novozymes, Denmark
Fetal bovine serum	Gibco, Life Technologies, Carlsbad, CA, USA	A4736301
FT-IR spectromete	Bruker-Vertex, Switzerland	VERTEX 70v
Graphing and analysis software	GraphPad Prism (GraphPad Software, Inc.)
H2SO4
HPLC system	Waters Corp, Milford, MA, USA	Waters 2695 HPLC
Incubator	Eppedorf	Galaxy 170S
Mass Spectometer	Q Exactive GC, Thermo Scientific	725500
Paraformaldehyde	MERK, Germany	P6148
Penicillin/streptomycin	Sigma-Aldrich, St. Louis	P4458
pH meter	Crison, Barcelona, Spain	Basic 20
Phosphate-buffered saline	Gibco, Life Technologies, Carlsbad, CA, USA	1010-015
Rabbit Cleaved Caspase-3 (Asp175) Antibody	Abcam, UK	ab243998
Rat Ki-67 Monoclonal	Thermofisher, USA	MA5-14520
Rotary evaporator	Büchi Ibérica S.L.U., Spain	El Rotavapor R-100
Ultra-hydrogel linear gel-filtration column (300 mm x 7.8 mm)	Waters Corp, Milford, MA, USA	WAT011545
UV-Visible spectrophotometer	Amersham Bioscience, UK	Ultrospec 2100 pro
VectaMount	Vector Laboratories, C.A, USA	H-5000-60
Water bath	J.P. Selecta S.A., Spain
Zeiss LSM 7 DUO Confocal Microscope System.	Zeiss, Germany
β-glucan Assay Kit	Megazyme, Bray, Co. Wicklow, Ireland	K-BGLU
β-glucans	Setas y Hongos del Sur, S.L.		Supplied the four variants of mushrooms