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 found within the tumor microenvironment². Particularly, in the case of glioblastoma, one of the most common and aggressive forms of brain cancer, unraveling key pathways that orchestrate such pro-tumoral scenarios and finding novel compounds that could counteract the depressing response of the immune system might pave the way for future therapies against this incurable disease.

The brain possesses its own immune system cells, and the most relevant cell type are microglia. These cells have been proven to have a rather complex behavior across different central diseases³. In the case of primary brain tumors (e.g., glioblastoma), these cells are shifted toward an anti-inflammatory phenotype that supports tumor cells to colonize the brain parenchyma³. Numerous publications have enhanced the major role of these cells during tumor progression. One of the main reasons for this is that glioma-associated microglia and infiltrated macrophages (GAMs) account for one-third of the total tumor mass, thus suggesting the unequivocal influence of their activation states during brain tumor progression^4,5.

In that vein, fungal β-glucans have been described as potent molecules triggering effective immune responses, including phagocytosis and pro-inflammatory factors production, leading to the elimination of pernicious agents^6,7,8,9,10. Fungal β-glucans have generally been studied using extracts from different mushroom parts. However, the attribution of specific effects requires its purification to avoid ambiguities and to be able to understand the mechanism of action of such molecules as immunomodulatory agents⁸.

In this work, soluble β-glucans are purified from the fruiting body of four different mushrooms, regularly employed as edible (Pleurotus ostreatus and Pleurotus djamor) and as medicinal (Ganoderma lucidum and Hericium erinaceus) mushrooms. In particular, these four mushrooms have great use in the food and pharmaceutical industry and were produced within an environmentally friendly circular economy in a commercial enterprise (see Table of Materials).

In order to lay the foundation for the future use of fungal β-glucans in brain cancer therapies, well-defined purification strategies and preclinical studies delving into their putative interaction with immune system cells are essential to evaluate their potential role as anti-tumor mediators. This work describes the numerous steps of isolation and purification needed to retrieve the soluble β-glucans contained within the fruiting bodies of the selected mushroom. Once successfully purified, microglia cells are activated to enhance their inflammatory phenotype. Mouse glioblastoma cells (GL261) are coated with a different microglia-conditioned medium, previously treated with these extracts, and then its effect on tumor cells' behavior is evaluated. Interestingly, pilot studies from our lab (data not shown) have uncovered how pro-inflammatory microglia may slow tumor cell migration and invasion properties not only in glioblastoma cells but also in other cancer cell lines. This multidisciplinary work may provide a useful tool for oncology researchers to test promising compounds able to boost the immune response in many different types of tumors.

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 fruit bodies at 50 ± 2 °C in a conventional air-drying oven until a constant weight is reached (~24 h).
   3. Ground the dried mushrooms in a blade mill, obtaining about 200 g of powder from each mushroom.
   4. Suspend 100 g of mushroom powder (MP) (P. ostreatus, P. djamor, H. erinaceus, and G. lucidum) in 1,000 mL of H₂O_d. Then, autoclave at 121 °C for 15 min, and finally leave at room temperature for 30 min.
   5. Centrifuge the resulting suspension at 6,000 x g for 10 min at 4 °C.
   6. Dry the precipitate containing insoluble mushroom polysaccharides (IMPs) at 50 ± 2 °C in an air-drying oven for 24 h.
   7. Discard the precipitate and keep the supernatant. Concentrate the supernatant 10 times in a rotary evaporator.
   8. Precipitate the concentrate containing SMPs with ethanol (80% final concentration) at 4 °C overnight.
   9. Centrifuge the ethanol suspension at 6,000 x g for 15 min at 4 °C, retain the pellet (precipitate), and discard the supernatant with a pipette.
   10. Wash the precipitate three times with 80% ethanol before dissolving it in H₂O_d (10% w/v). Adjust the pH to 6.5/7 and the temperature to 37 °C, and treat with 2 U and 4 U of α-amylase and glucoamylase, respectively, to solubilize α-glucans following the manufacturer's instructions (see Table of Materials).
   11. After the treatment with α-amylase/glucoamylase, adjust the pH and temperature to 8.0 and 50 °C, respectively, and treat with alcalase (2.5 U/g of protein) (see Table of Materials) to solubilize the proteins.
       NOTE: This sequential enzymatic treatment removes most α-glucans and proteins that co-precipitate with β-glucans in the ethanol precipitation.
   12. After hydrolysis, centrifuge the hydrolysate at 6,000 x g for 15 min at 4 °C, and the clean supernatant concentrate five times in a rotary evaporator. Precipitate again with 80% ethanol.
   13. Solubilize the resulting precipitate in H₂O_d and dialyze in distilled water for 24 h using cellulose tubing membranes (12,000 Da cut-off membranes; see Table of Materials) to remove low molecular weight molecules. Recover the water-soluble portion and freeze-dry it to produce soluble β-glucans (SβGs).
2. Sugar and protein measurement
   1. Measure the total sugar content of each fraction by the phenol-sulfuric acid method, using glucose as standard⁸.
      NOTE: Quantitation of β-glucan content may also be done by using the β-glucan assay kit (mushroom and yeast; see Table of Materials), based on enzymatic hydrolysis and the activity of oxidoreductases: namely exo-1,3-β-glucanase, glucose oxidase, β-glucosidase, and peroxidase, with the subsequent formation of the quinoneimine. Follow the manufacturer's instructions, with slight modifications.
   2. Use 18 MH₂SO₄ instead of 12 MH₂SO₄.
   3. Evaluate the content of the total glucans and α-glucans separately.
   4. Measure the resulting β-glucan values as the difference between the total glucan and α-glucan (triplicate) values following the Kjeldahl protocol. In certain cases, protein content can be determined by the Lowry method, using albumin to plot the calibration curve^11,12.
3. Ultraviolet absorption spectroscopy analysis
   1. Obtain the SβG ultraviolet (UV) spectra using a UV-visible spectrophotometer (see Table of Materials) by scanning the samples in the 200-400 nm region (Figure 2).
   2. Prepare 1.0 mg/mL of each SβG in H₂O_d, transfer the solution to a quartz cuvette, and scan at room temperature.
4. Molecular weight distribution analysis
   1. Estimate the homoegeneity of SβGs and molecular weight of polymers by size exclusion chromatography (SEC) using a high-performance liquid chromatography (HPLC) system (see Table of Materials) equipped with a refractive index detector and an ultra-hydrogel linear gel-filtration column (300 mm x 7.8 mm; Figure 3).
   2. Perform the assay at 40 °C using deionized water as eluent at a flow rate of 0.5 mL/min^-1 and dextrans (110, 80, and 50 kDa) as standards (see Table of Materials). Collect a 5 mL fraction.
5. Fourier-transform infrared (FTIR) analysis
   1. Record the infrared spectra (Figure 4) on an FTIR spectrometer in the range of 4000-500 cm^-1. The samples should be previously mixed with KBr to form films (standard FTIR procedure; see manufacturer's instructions and Table of Materials).
6. Molecular composition analysis
   1. Estimate the molecular compositions of SβGs by high-performance thin-layer chromatography (HPTLC) as well as gas chromatography coupled to mass spectrometry (GC-MS), following standard procedures¹².

2. In vitro study of β-glucan-induced microglia stimulation

1. Cell culture of mouse glioblastoma and microglia cells in 8-well chamber slides
   NOTE: This protocol is specific for GL261 (glioblastoma) and BV2 (microglia) cell lines. However, with slight modifications, these steps could potentially be used to study other cancer and immune cell lines.
   1. Prepare Dulbecco's modified Eagle's medium (DMEM) complete medium modified with L-glutamin, 4.5 g/L D-glucose, and without pyruvate. Add 10% of fetal bovine serum (FBS) and 1% penicillin/streptomycin (see Table of Materials). Pre-warm the material in a water bath at 37 °C for 15 min.
   2. Thaw frozen BV2 and GL261 aliquots into a water bath (37 °C) for 2 min, and just before they completely thaw, carry them into a laminar flow hood and plate the cells into two different sterile T25 flasks (one for each cell line).
   3. Incubate the T25 flasks at 37 °C, 5% CO₂ until the culture is confluent.
      NOTE: Depending on the freezing conditions and the time under cryopreservation, the time until confluence may vary. These cell lines usually require between 3 to 5 days to reach confluency in a T75 flask.
   4. After the BV2 cell culture becomes confluent, transfer it into 8-well chamber slides 0.6 x 10⁶ cells/well. Keep the 8-well chamber slides in the incubator for 24 h.
   5. Once the microglia cells are plated into the 8-well chamber slides, repeat the same protocol with the GL261 cells.
2. Activation of microglia with β-glucans
   1. Coat the BV2 cells with four different β-glucans (P. ostreatus, P. djamor, G. lucidum, and H. erinaceus) at a 0.2 mg/mL concentration for 72 h. One experimental condition must remain untreated (normal medium), acting as the control group.
   2. Collect the supernatant with a pipette after 72 h and pass the remaining volume through a 0.20 µm syringe filter. Then, freeze the supernatant at -80 °C for at least 24 h.
3. Treatment of GL261 with pre-activated microglia-conditioned medium
   1. Once the GL261 is 80% confluent within the 8-well chamber slides, add β-glucan-treated microglial medium (step 2.2.2) at a final volume concentration of 25% for 72 h (total volume: 250 µl/well).
   2. Remove the medium after 72 h incubation and discard it.
   3. Wash the cells with phosphate buffer saline (PBS; pH 7.4, 0.1 M) three times for 5 minutes.
   4. Fix the cells by adding 200 µL of 4% paraformaldehyde (PFA) at 4 °C for 15 min.
      NOTE: Depending on the different antibodies that might be used for immunofluorescence, fixation methods may differ from typical 4% PFA. Alcohol-based fixatives may be more efficient in preserving certain epitopes.
4. Immunofluorescence study
   1. Wash the samples with PBS with triton X (PBST; 0.01%) for 10 min three times.
   2. Remove the PBST and add bovine serum albumin (BSA) blocking buffer 10% in PBST (Table 1) for 30 min at room temperature.
   3. Remove the blocking buffer and add 200 µL per well of PBS containing the primary antibodies mixture (1:500 rat Ki-67 monoclonal antibody and 1:500 rabbit cleaved caspase-3 antibody; see Table of Materials). Incubate overnight at 4 °C.
   4. After 24 h of incubation at 4 °C, leave the samples at room temperature for 30 min.
   5. Wash the wells three times for 10 min with PBS on a shaker (low speed).
   6. Remove the PBS and replace with 200 µL per well of PBS containing the mixture of secondary antibodies (1:200 donkey anti-rat IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 and 1:200 donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 647; see Table of Materials) for 45 min at room temperature in the dark.
   7. Wash the samples with PBS for 10 min on a multipurpose shaker.
   8. Remove the PBS and add 200 µL per well of 4′,6-diamidino-2-phenylindole (DAPI) diluted in PBS (1:5,000) for 1 min.
   9. Remove DAPI (see Table of materials) and wash the cells for 5 min in PBS.
   10. Remove the well frame and add 50 µL of PBS:glycerol (1:1) on each well and cover with a coverslip.
   11. Seal the slides with nail polish.
   12. Acquire images at 20x using a confocal microscope system (see Table of materials).

3. Quantification and analysis of tumor cell proliferation and apoptosis

NOTE: In order to measure the potential effect of the different β-glucans on tumor cell proliferation and apoptosis, an in-house script was used in ImageJ software to quantify the number of positive pixels of Ki67 (proliferation) and cCasp3 (apoptosis)¹³.

1. Open ImageJ. Click on the Plugins button. Click on Coloc2, a plugin previously installed in the plugin folder, and finally select the image to analyze¹¹.
   NOTE: This plugin was available following previous contact with Dr. Vasiliki Economopoulos (veconom@uwo.ca). Instead of the script, both ImageJ and Fiji software have different tools for colocalization analysis (see Table of materials), with similar properties.
2. Set thresholds according to the control (untreated, DMEM only) conditions. Click on the OK button.
   NOTE: In order to avoid background and intensity discrepancies, all images must be taken under the same conditions. Preferably, imaging sessions should be performed on the same day, and microscope parameters unaltered across images.
3. Ensure that the resulting images of the colocalized pixels and a summary window providing the percentage or raw number of pixels above the threshold pop up. Normalize the results with respect to the control (untreated) conditions.
   NOTE: All data are given as mean ± SEM. Statistical analysis was performed using graphing and analysis software (see Table of Materials). A one-way ANOVA with Tukey's multiple comparison test was used. Errors are represented as standard error of the mean (s.e.m.) (*p < 0.05, **p < 0.01).

Representative 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 large amount of protein content in SMPs. However, the enzymatic treatment with α-amylase/glucoamylase and protease reduced the amount of protein and increased the β-glucan concentration.

UV spectra of the different SβGs showed no UV absorption peaks at 260 and 280 nm (Figure 2A), indicating that SβGs lacked nucleic acids (260 nm) and proteins (280 nm). Furthermore, the homogeneity of SβGs was tested following UV-visible absorption spectroelectrochemistry (SEC). The chromatograms (Figure 3) showed good homogeneity, with a main sharp and single peak at 8.20, 10.5, 10,9, and 11.3 min for H. erinaceus, G. lucidum, P. ostreatus, and P. djamor, respectively. These data suggest that the fraction is consistent with homopolymers. Moreover, the weight-averaged Mw was calculated as around 120.8, 92.8, 80.7, and 75.9 kDa for H. erinaceus, G. lucidum, P. ostreatus, and P. djamor, respectively, according to the calibration curve equation (y = -0.0655x + 2.6194; R² = 0.9951).

The FTIR spectra measured molecular vibrations that corresponded to covalent polysaccharide bonds (Figure 4). The spectra exhibited a broad and intense hydroxyl group at around 3,435 cm^-1. It also showed a weak C-H-stretching peak at around 2,922 cm^-1, corresponding to polysaccharides¹⁴. Furthermore, the absorbance at around 1,641 cm^-1 could be assigned to amide I¹⁵, related to the elongation vibrations of the C=O and CN groups. The signal at around 1,154 cm^-1 might be due to C-O-C asymmetric stretching of the glycosidic linkage¹⁶. Finally, the band at around 1,072 cm^-1 indicated C-O stretching of β-glucans¹⁶. The weak absorption near 893 cm^-1 might be due to the asymmetric refractive vibration of β-pyranose, showing the β-configuration of sugar units¹⁷. Overall, SβGs were found to mainly consist of carbohydrate conjugated with a minimal amount of protein.

The monosaccharide profile of SβGs was further studied by HPTLC and GC-MS. The presence of a large amount of D-glucose with smaller amounts of D-galactose and D-mannose and a trace of D-xylose, D-rhamnose, D-fucose, and L-arabinose was confirmed. Table 3 summarises the results obtained for SβGs of H. erinaceus, G. lucidum, P. ostreatus, and P. djamor.

Microglia-conditioned medium, pre-activated with β-glucan, induced apoptosis in cancer cells
Once β-glucans from the selected mushrooms were successfully isolated and fully characterized, they were added to the microglia cell culture (BV2). At 72 h after the addition of microglia-conditioned medium to the GL261 cells (Figure 5), the expression of two key markers for proliferation (Ki67) and apoptosis (cleaved caspase 3 [cCasp3]) were measured by immunofluorescence. Using an in-house script in ImageJ software, the number of positive pixels for each fluorescent channel was quantified, and thus the way in which the potential effect of β-glucan-induced microglial activation may affect tumor cells behavior was analyzed. Using control samples as a threshold for the intensity of each fluorophore, the script provided the number of pixels and, thus, indicated the expression for each marker after the different experimental conditions (Figure 6).

Interestingly, GL261 did not suffer any significant difference regarding tumor proliferation once exposed to the different microglia-conditioned media (Figure 7). However, P. djamor (B) and H. erinaceus (C) showed a strong induction (approximately sixfold and ninefold increase, respectively) of cCasp3.

Figure 1: Isolation protocol. Schematic of the protocol to isolate and purify SβG preparation from P. streatus, P. djamor, H. erinaceus, and G. lucidum. Please click here to view a larger version of this figure.

Figure 2: UV spectra of β-glucans. UV spectra in the 200-400 nm region of (A) P. ostreatus, (B) G. lucidum, (C) P. djamor, and (D) H. erinaceus. Please click here to view a larger version of this figure.

Figure 3: Size exclusion chromatograms. Size exclusion chromatograms of (A) P. ostreatus, (B) G. lucidum, (C) P. djamor, and (D) H. erinaceus. Please click here to view a larger version of this figure.

Figure 4: FTIR spectra. Fourier-transform infrared (FTIR) spectra of (A) P. ostreatus, (B) G. lucidum, (C) P. djamor, and (D) H. erinaceus. Please click here to view a larger version of this figure.

Figure 5: Schematic of the co-stimulation assays. Co-stimulation assay where mouse microglia (BV2) cells were coated for 72 h with β-glucans. After being cryopreserved and filtered, the supernatant was collected and transferred to GL261 cell culture (25%) for 72 h. Please click here to view a larger version of this figure.

Figure 6: Proliferation and apoptosis images. Immunofluorescence images showing a triple colocalization of GL261 with DAPI (blue), Ki67 (green), and cCasp3 (red). The 'Prob Coloc' script (bottom images) allowed quantifying the number of positive pixels from each marker and colocalization amongst them. Scale bar: 10 µm. Please click here to view a larger version of this figure.

Figure 7: Quantitation of proliferation rates and apoptosis. Normalized values with respect to the control conditions (DMEM) of Ki67 (left) and cCasp3 (right) expression in GL261 cells after the microglia-conditioned medium exposition. (A) Pleurotus ostreatus, (B) Pleurotus djamor, (C) Hericium erinaceus y, (D) Ganoderma lucidum. Errors are represented as s.e.m. (*p < 0.05, **p < 0.01). Please click here to view a larger version of this figure.

	MP (g)	SMPs (g)	SβGs (g)
P. ostreatus	201.3 ± 2.2	14.4 ± 0.9 (7.1%)	5.3 ± 0.2 (2.6%)
P. djamor	200.8 ± 1.9	13.5 ± 0.6 (6.7%)	4.9 ± 0.3 (2.4%)
G. lucidum	201.8 ± 1.6	14.7 ± 1.2 (7.3%)	5.5 ± 0.2 (2.7%)
H. erinaceus	204.2 ± 1.2	15.4 ± 0.8 (7.5%)	5.7 ± 0.2 (2.8%)

Table 1: Table of glucan content. Mass balance for obtaining MP, SMPs, and SβGs from fruiting bodies of P. ostreatus, P. djamor, G. lucidum, and H. erinaceus.

		MP	SMPs	SβGs
	CHt (%)	67.3 ± 1.9	53.8 ± 2.3	90.1 ± 1.2
P. ostreatus	β-Glucan (%)	22.7 ± 1.4	31.3 ± 2.4	89.4 ± 2.3
	Protein (%)	21.5±0.9	19.6 ± 0.8*	0.4 ± 0.1*
	CHt (%)	68.3 ± 2.1	61.4 ± 3.1	93.4 ± 1.1
P. djamor	β-Glucan (%)	24.3 ± 2.8	30.8 ± 3.5	91.3 ± 3.4
	Protein (%)	19.9 ± 1.0	22.3 ± 1.1*	0.9 ± 0.2*
G. lucidum	CHt (%)	66.4 ± 1.8	56.8 ± 2.9	93.7 ± 0.9
	β-Glucan (%)	22.5 ± 1.9	24.9 ± 3.1	92.0 ± 2.6
	Protein (%)	18.9 ± 0.8	21.4 ± 0.6*	1.5 ± 0.4*
H. erinaceus	CHt (%)	67.4 ± 1.2	58.9 ± 1.9	93.8 ± 1.4
	β-Glucan (%)	23.9 ± 1.6	35.9 ± 2.1	91.8 ± 2.8
	Protein (%)	17.6 ± 1.3	22.7 ± 1.8*	1.3 ± 0.2*

Table 2: Total carbohydrates. Dry weight (g) content of total carbohydrates (CH_t), β-glucan, and protein of MP, SMPs, and SβGs. Protein content (MP) quantified by the Kjeldah method¹². (*, proteins quantified by the Lowry method⁹).

	D-Gluc (%)	D-Mann (%)	D-Gala (%)	D-Fuco (%)	D-Xylo (%)	D-Rham (%)	L-Arab (%)
H. erinaceus	91.6 ± 0.6	3.8 ± 0.1	2.1 ± 0.2	0.5 ± 0.2	0.8 ± 0.2	0.3 ± 0.1	n.d
G. lucidum	94.3 ± 0.8	2.6 ± 0.2	1.9 ± 0.2	0.3 ± 0.1	0.9 ± 0.2	n.d.	0.4 ± 0.1
P. ostreatus	93.8 ± 0.5	4.4 ± 0.1	0.8 ± 0.1	0.4 ± 0.1	n.d.	n.d.	n.d.
P. djamor	95.2 ± 0.7	1.7 ± 0.2	1.1 ± 0.1	0.3 ± 0.1	n.d.	n.d.	n.d.

Table 3: Characterization of monosaccharides. Monosaccharide profile of SβGs of P. ostreatus, P. djamor, G. lucidum, and H. erinaceus (n.d., some monosaccharides were undetectable).

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.

Despite this, we observed that most of the fungal β-glucans were water-insoluble during the purification process. The main interest of the study was the SβGs, owing to their medical/pharmaceutical properties^10,18. Furthermore, thanks to hydrolytic processing, ethanol precipitation, and dialysis, soluble low-molecular-weight carbohydrates, peptides, oligopeptides, and amino acids were successfully removed from SMPs, showing a similar efficiency to other previous works using a different type of mushroom but with similar processes^19,20.

To test the purity of SβGs, the UV spectra of the different SβGs were investigated by UV spectrophotometry, scanning the samples in the 200-400 nm region. No UV absorption peaks were observed at 260 and 280 nm, indicating that SβGs had neither nucleic acids (260 nm) nor proteins (280 nm), thus showing again that β-glucans mainly constituted the SβGs. Although UV spectra in the region of 200-400 nm showed the absence of any defined/sharp pick at 280 nm, a small peak could still be observed. This could be explained by the presence of a small amount of polysaccharide-bound proteins, agreeing with the results shown in Table 2. However, it is interesting to consider that such a negligible amount of polysaccharides could also be explained by the delayed access to the remaining proteins, which may be shielded by glucans or by steric effects that prevent the complete degradation of glucan-bound proteins. Importantly, the homogeneity of SβGs was further tested by SEC, a powerful analytical technique used to purify dissolved molecules by size, which confirmed the protocol results.

Additionally, FTIR spectra was used to measure molecular vibrations corresponding to covalent polysaccharide bonds. As previously described, similar spectra were obtained for all four fungi. The spectra feature exhibited the presence of polysaccharides¹⁴, amide I¹⁵, and β-glucans¹⁶. The weak absorption near 893 cm^-1 suggested the β-configuration of sugar units¹⁷. Overall, SβGs were found to be mainly formed by carbohydrates conjugated with minimal protein. Regarding the interest in using SβGs as immunomodulatory molecules, it is worth highlighting that polysaccharide-protein complexes are often noted for their immunomodulatory benefits²¹.

Finally, the monosaccharide profile of SβGs was studied by HPTLC and GC-MS. The presence of a large amount of D-glucose with smaller amounts of D-galactose and D-mannose and traces of D-xylose, D-rhamnose, and D-fucose strictly implies that the predominant component in SβGs is β-glucan. However, it is important to clarify that our purification system results from optimizing different classical procedures. We are currently working on improving several steps, mainly focused on chromatography techniques (size exclusion and ion exchange chromatography).

Regarding the main aim of this work, which was to test the impact of β-glucans on immune cells, once the four different types of β-glucans were successfully purified, their potential effects on the activation of microglia were tested. Immunofluorescence was used against two gold-standard markers for proliferation and apoptosis^22,23.

Despite no statistical differences in tumor proliferation rates, P. ostreatus (A) and H. erinaceus (C) were able to drop Ki67 expression by up to 50%. The lack of significance is likely due to the high variance in the study regarding G. lucidum. Furthermore, the induction of apoptosis in cancer cells is a rather interesting therapeutic approach, and P. djamor (B) and H. erinaceus (C) showed a significant induction of cCasp3 levels, suggesting the activation of the cell death program. All these results are in accordance with previous studies that showed the anti-tumoral effect of these fungi in other types of tumors^24,25. The experiments were performed in duplicate, maintaining the same conditions and led by the same investigators. Software-based analysis of the results from the immunofluorescence studies supports an unbiased approach and enhances the potential reach of this study.

In general, these studies have a main challenge regarding the use of β-glucans, which is the purity of the compounds. It is mandatory to pursue the highest standards in order to confirm that the observed effects, after their use in immunologic studies, are exclusively provoked by the carbohydrates, and not owing to proteins or other structures that may remain attached to them if the purification is not adequately performed. One extra step that may be considered in future studies is the use of chromatography techniques (e.g., ion exchange or size exclusion).

The overall conclusion after the studies places H. erinaceus as the top candidate as a potential immunotherapeutic option for the treatment of glioblastoma, owing to its ability to drop tumor proliferation (~50%) and induce strong activation of the cell death program in glioblastoma cells.

Materials

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