Extracellular lipids of Candida albicans biofilm induce lipid droplet formation and decreased response to a topoisomerase I inhibitor in dysplastic and neoplastic oral cells^*

Abstract

Objective  Some microorganisms, i.e., Candida albicans, have been associated with cancer onset and development, although whether the fungus promotes cancer or whether cancer facilitates the growth of C. albicans is unclear. In this context, microbial-derived molecules can modulate the growth and resistance of cancer cells. This study isolated extracellular lipids (ECL) from a 36-h Candida albicans biofilm incubated with oral dysplastic (DOK) and neoplastic (SCC 25) cells, which were further challenged with the topoisomerase I inhibitor camptothecin (CPT), a lipophilic anti-tumoral molecule.

Methodology  ECL were extracted from a 36-h Candida albicans biofilm with the methanol/chloroform precipitation method and identified with Nuclear Magnetic Resonance (1H-NMR). The MTT tetrazolium assay measured ECL cytotoxicity in DOK and SCC 25 cells, alamarBlue™ assessed cell metabolism, flow cytometry measured cell cycle, and confocal microscopy determined intracellular features.

Results  Three major classes of ECL of C. albicans biofilm were found: phosphatidylinositol (PI), phosphatidylcholine (PC), and phosphatidylglycerol (PG). The ECL of C. albicans biofilm had no cytotoxic effect on neither cell after 24 hours, with a tendency to disturb the SCC 25 cell cycle profile (without statistical significance). The ECL-induced intracellular lipid droplet (LD) formation on both cell lines after 72 hours. In this context, ECL enhanced cell metabolism, decreased the response to CPT, and modified intracellular drug distribution.

Conclusion  The ECL (PI, PC, and PG) of 36-h Candida albicans biofilm directly interacts with dysplastic and neoplastic oral cells, highlighting the relevance of better understanding C. albicans biofilm signaling in the microenvironment of tumor cells.

Candida albicans; Biofilms; Lipids; Oral cancer

Introduction

Many microorganisms, which are critical for oral microenvironment balance, form the oral microbiome. The microbiota residing in the oral cavity prevents the establishment of pathogenic microbes, working as a microbial antagonist.¹ Oral microbiome disruption has affected the course of oral diseases. The microbiome is affected by host immunity, therapies and drugs,² and latrogenic environmental changes, such as orthodontic appliances, dental restorations, implants, and dentures.³

The association between biofilms and cancer has been recently contemplated, particularly biofilms developed by oral plaque microorganisms, such as Fusobacterium nucleatum⁴ and Pseudomonas aeruginosa.⁵ The development of microbial biofilms in either cancer patients or a healthy host correlates to pro-oncogenic features such as E-cadherin loss, increased interleukin-6, and the subsequent activation of the STAT3 transcription factor.⁶ These characteristics highlight the significance of understanding biofilm patterns during cancer onset, development, resistance, and prognosis.

Candida albicans is among the most common opportunistic pathogens that can colonize nearly every human tissue, causing severe invasive infections. The main virulence factor is its ability to produce biofilms. The structure of C. albicans biofilm includes proteins (55%), carbohydrates (25%), lipids (15%), and nucleic acids (5%).⁷

Clinically, C. albicans biofilms are associated with oral candidiasis and Candida leukoplakia.⁸ The literature reports that oral yeast colonization is significantly higher in oral cancer than in non-oral cancer patients.⁹ However, whether the fungus promotes cancer or whether cancer facilitates the growth of C. albicans is unclear; therefore, the cellular signaling between C. albicans and oral cancer cells remains under investigation.¹⁰ In this context, C. albicans can produce carcinogenic compounds, i.e., nitrosamines, which bind to DNA and form adducts that cause genomic instability, activate oncogenes, and initiate cancer development.⁶,¹⁰ Moreover, C. albicans up-regulates oncogenes, potentiates a pre-malignant phenotype, and participates in the early and late stages of malignant promotion and progression of oral cancer.¹⁰,¹¹

The investigation of the interaction of microorganisms and their metabolites with hosts, contributing to tumorigenesis and affecting treatment success or failure, is a currently relevant topic.¹² In a tumor microenvironment, lipid molecules (i.e., phospholipids, fatty acids, triglycerides, sphingolipids, and cholesterol) work as secondary messengers and energy storage, such as lipid droplet (LD) vesicles. LDs are essential organelles found in every cell type, from prokaryotes to eukaryotes, and proposed to be involved as hallmarks of cancer progression and resistance.¹³,¹⁴

Thus, the main goal of this investigation was to verify the presence or absence of interaction between extracellular lipids (ECL) secreted by C. albicans biofilms and dysplastic and neoplastic oral cells, considering the high prevalence of this fungal biofilm in the oral cavity of cancer patients and the relevance of lipids in the tumor cell microenvironment and treatment resistance.

Methodology

Strain and media

This study used the SC 5314 (ATCC™ MYA-2876™) Candida albicans strain. C. albicans cultures were maintained in Sabouraud dextrose agar (SDA; Neogen; Lansing, Michigan, USA) supplemented with chloramphenicol (1 mg/L). For storage, the tubes were maintained at -80°C in a yeast nitrogen base with 20% (v/v) glycerol (YNB; Difco; Sparks, Maryland, USA).

Biofilm growth and characterization

Candida albicans biofilms were developed in 75-cm² cell culture flasks and grown overnight at 37°C in YNB. Then, cell suspension was standardized to 1 × 10⁷CFU/mL (OD_540nm, 0.845) to form the pre-inoculum. For the inoculum, 1 mL of the pre-inoculum was diluted in 10 mL of fresh YNB medium and grown for eight hours. When the inoculum reached the logarithmic phase (OD_540nm 0.520), it was washed twice in phosphate-buffered saline (PBS; 1.37 M NaCl, 27 mM KCl, 100 mM Na₂HPO₄, 18 mM KH₂PO₄) and centrifuged at 3,200 × g at 4°C. The pellet was resuspended in 20 mL of fresh RPMI-1640 medium (Sigma-Aldrich; Saint Louis, Missouri, USA) and transferred to 75-cm²flasks for the adhesion phase, for 90 minutes at 37°C and 55 rpm. Next, the flasks were washed twice with PBS to remove non-adherent cells. Finally, the biofilms were incubated for 36 hours in 20 mL of fresh RPMI-1640 at 37°C in an orbital shaker (55 rpm). After biofilm growth, the thickness was characterized by double-staining with SYTO9 (green fluorescent nucleic acid stain) and concanavalin-a (cell surface probe) Alexa Fluor 594-conjugated (Invitrogen; Eugene, Oregon, USA) and analyzed with confocal laser scanning microscopy (CLSM). The colonies were counted (CFU) with a serial dilution of 1:1000 of the detached biofilm in sterile PBS. The diluted solution was cultured in an SDA plate and counted after 24 hours. The extracellular components were obtained after 36 hours of biofilm growth. Then, the cultures were gently detached with a cell scraper (Corning Inc., code #3011), preventing fungal cell disruption, combined with the medium (secreted molecules), gently homogenized with a serological pipette, collected, and filtered with a 0.22-µm low protein binding filter (SFCA, Corning; Oneonta, New York, USA). This filtered solution was used for all further assays. The pH was measured with a Qualxtron bench pH meter, model QX 1500 Plus.

Lipid extractions

The extracellular lipids (ECL) in the previously described filtered solution were extracted with the methanol/chloroform precipitation method (2:1, v/v). Cooled methanol (-20°C) was added to the filtered solution. Next, chloroform was added and incubated at room temperature for one hour. After incubation, the lipid-containing phase was collected and dried overnight at room temperature to ensure solvent elimination. Finally, the pellet was suspended in 1 mL of ethanol P.A. and stored at -20°C.

Lipid 1H-NMR analysis

In all experiments, the ECL pool was previously obtained from six (n=6) 36-h-biofilm cultures performed in independent biological replicates. The ECL pool of C. albicans biofilms was measured with the sulfo-phospho-vanillin method.¹⁵ A lyophilized sample from the ECL pool was resuspended in DMSO-d6 for ¹H-NMR analysis. A Bruker Advanced III HD 600 spectrometer was used. The Mestre Nova software analyzed the spectrograms.

Oral squamous cells

DOK cells (Sigma-Aldrich, ECACC™ dysplastic oral keratinocyte) were cultured on 75-cm²cell culture flasks (Corning; Oneonta, New York, USA) in Dulbecco’s modified Eagle’s medium (DMEM; Lonza; Walkersville, Maryland, USA) supplemented with hydrocortisone (5 µg/mL; Sigma-Aldrich; Saint Louis, Missouri, USA) and fetal bovine serum (10% FBS; Gibco; Brazil). SCC 25 cells (ATCC™ primary tongue carcinoma) were cultured in a mixture of DMEM/F-12 medium (1:1) supplemented with HEPES (15 mM), hydrocortisone (400 ng/mL), sodium pyruvate (0.5 mM), and FBS (10%; Gibco; Brazil). The cells were used in the third passage in all the experiments. In a previous study by our research group, 36-h C. albicans biofilms induced the death of normal oral epithelial cells, especially the caspase-3 pathway.¹⁶ Hence, this study did not investigate normal cell phenotypes.

Cell culturing with ECL of C. albicans biofilms

The cells were cultured as described above. DOK and SCC 25 cells were seeded in 24-well plates (Corning; Oneonta, New York, USA) with 40,000 cells/well. After 24 hours, the medium was replaced with keratinocyte basal medium (KBM-Gold; Lonza; Walkersville, Maryland, USA) supplemented with transferrin, insulin, hydrocortisone, epinephrine, gentamicin (Lonza; Walkersville, Maryland, USA), and 0.1% fatty acid-free bovine serum albumin (BSA) (Sigma-Aldrich; Saint Louis, Missouri, USA). Finally, the cells were incubated for 24 and 72 hours at 37°C with 5% CO₂. The controls were a) standard cell culture conditions and b) vehicle control (ethanol v/v) and experimental conditions (ECL v/v). The literature⁷ reports a 15% maximum of lipids in C. albicans biofilms and previous findings of a non-cytotoxic 2% ethanol percentage (data not shown). Therefore, this study used a maximum amount of 2% (v/v) of ECL to stimulate DOK and SCC 25 cells.

MTT assay

Cell viability was measured with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay; Sigma-Aldrich; Saint Louis, Missouri, USA), as previously described.¹⁷ The assay was conducted after 24 hours of cell culturing with ECL of C. albicans biofilms (1% and 2% v/v). A standard control was performed with the cells under normal culture conditions. The vehicle control (1% and 2% ethanol) was also performed. The experiments occurred in four replicates on three independent occasions (n=12).

Cell cycle assay by flow cytometry

DOK and SCC 25 cells (2.5 × 10⁵ cells/well) were seeded into six-well plates (Corning™, NY, USA) in triplicate (n=3) and cultured for 24 hours with ECL of C. albicans biofilms (2% v/v), as previously described. For the control, the cells were grown under standard conditions. The monolayer was dissociated and detached with Accutase (Sigma-Aldrich™, St. Louis, MO, USA) for 10 minutes at 37°C. Next, cells were centrifuged at 400× g for five minutes at 4°C. The pellet was resuspended in 100 μL of PBS and received 900 μL of chilled 70% ethanol. The cells were incubated overnight at 4°C. Then, the suspension was centrifuged at 700× g for five minutes at 4°C. The supernatant was discarded and the pellet was resuspended in 100 μL of a cell cycle solution (3.4 mM of tris HCl, pH 7.4; 0.1% Triton X-100; 700 U/L of RNAse (DNAse free)), 10 mM of NaCl (Sigma-Aldrich™, St. Louis, MO, USA), and 30 µg/mL of propidium iodide (Invitrogen^TM, Thermo Fischer Scientific Inc., MA, USA). The tubes were incubated for 20 minutes on ice and protected from light. The suspension was transferred to a cytometry tube and subjected to flow cytometry analysis (BD FACSAria™ III cell sorter). A side-scatter (SSC) versus forward-scatter (FSC) located the homogeneous cell population with the BD FACS Diva software. This population originated from an SSC-A x SSC-W dotplot, followed by an FSC-A x FSC-W graphic, to exclude debris and doublets. The PE-A × PE-W dotplot graph detected 10,000 events. The Watson Pragmatic model of FlowJo™ Software - version 10 analyzed the distribution of cell cycle phases.

Confocal laser scanning microscopy (CLSM)

Nile Red (2 mg/mL; Sigma-Aldrich; Saint Louis, Missouri, USA) staining identified lipids in C. albicans biofilms and lipid droplet (LD) vesicles in oral cell cultures.¹⁸,¹⁹ C. albicans biofilm was grown in six-well plates (Falcon; Durham, North Carolina, USA). After 36 hours, the Nile Red solution was added and incubated at 37°C for 10 minutes. CLSM detection used a Carl Zeiss LSM 800 confocal microscope. The parameters for detecting neutral lipids were 488 nm excitation and up to 540 nm emission (yellow to green fluorescence), and for polar lipids, 561 nm excitation and above 650 nm emission (red fluorescence). All experiments occurred in triplicate on two different occasions (n=6). For oral cells, the Nile Red stock solution was diluted in acetone P.A. (2 mg/mL, use 1:1000). LD formation was evaluated only after 72 hours of oral cell culturing with ECL of C. albicans biofilms, according to the literature.²⁰ After 72 hours, the oral cells were washed with PBS. Subsequently, each well received 1 mL of fresh culture medium and 1 µL of Nile Red stock solution. Cells were incubated for 10 minutes. Then, CLSM detected LD formation in oral cells.¹⁹

Camptothecin challenge assay

Camptothecin (CPT) is a lipophilic molecule effective against many types of tumors. The molecular target of CPT is human DNA topoisomerase I (topo I). CPT forms a (topo)-I-CPT-DNA covalent complex, mediating DNA damage and leading cells to apoptosis.²¹ Hence, CPT became an anti-tumoral lipophilic drug for challenging oral cells. DOK and SCC 25 cells were seeded at 40,000 cells/well (24-well plates) in KBM supplemented with transferrin, insulin, hydrocortisone, epinephrine, gentamicin (Lonza; Walkersville, Maryland, USA), and 0.1% (v/v) fatty acid-free BSA (Sigma-Aldrich; Saint Louis, Missouri, USA). C. albicans lipids (2% v/v) were added to the experimental samples for 72 hours. Then, the wells were washed with PBS and received fresh KBM without BSA. The topoisomerase I inhibitor CPT (10 µg/mL) (Sigma-Aldrich; Saint Louis, Missouri, USA) was used for the challenge assay (four hours of incubation with CPT). The controls were a) standard cell culture conditions b) vehicle control (ethanol 2% v/v), c) ECL of C. albicans biofilms 2% v/v, d) ECL of C. albicans biofilms 2% v/v + 10 µg/mL of CPT, and e) 10 µg/mL of CPT. All experiments occurred in four replicates on three independent occasions (n=12).

AlamarBlue™ assay

The alamarBlue™ assay quantified cellular metabolism. After the CPT challenge assay, 100 µL of alamarBlue™ (Invitrogen; Eugene, Oregon, USA) were added to each well and incubated for four hours at 37°C with 5% CO₂. A Fluoroskan FL (Thermo Fischer Scientific Inc., MA, USA) measured fluorescence at 545 nm excitation and 590 nm emission.

Camptothecin uptake and co-localization assay

Confocal laser scanning microscopy (CLSM) identified the intracellular co-localization of the CPT drug in LD vesicles. After the CPT challenge assay, DOK and SCC 25 cells were washed with PBS once, and each well received fresh cell culture media. Only cells treated with CPT, previously incubated with ECL of C. albicans or not, were evaluated in this assay. Images were obtained with a CLSM Carl Zeiss LSM 800. The CLSM parameters were brightfield and 405 nm laser range up to 470 nm for CPT uptake detection. The LD double staining co-localizing protocol used 488 nm and 561 nm laser sources. All experiments occurred in triplicate on two different occasions (n=6).

Statistical analysis

GraphPad Prism vs 8.0 analyzed all the data in this study. All tests considered a 5% significance level. The Shapiro-Wilk normality test analyzed the data, which presented a normal distribution. The data were subjected to one-way ANOVA with Tukey’s post-test to compare the groups.

Results

The 3D biofilm architecture of Candida albicans was shown with confocal laser scanning microscopy (CLSM) after 36 hours of development. The biofilm was stained with SYTO9 and concanavalin-a at 594 nm, reaching a thickness of around 121.88 µm (Figure 1.A). The biofilms grew to 8.11±0.24 Log₁₀ (corresponding to 3.72×10⁸ CFU/mL), and the average pH was 6.68±0.12 (SD). These results came from six independent biological replicates (n=6).

Figure 1
Candida albicans biofilm characterization. (Upper panel) A. CLSM of a 3D architecture of the 36-h C. albicans biofilm stained with SYTO9 and concanavalin Alexa-Fluor 594 nm. The biofilm presented a green-yellow-red fluorescence and thickness of 121.88 µm. (Down panel) B. CLSM images of the 36-h C. albicans biofilm with Nile Red staining. (B.a) Neutral lipids stained in yellow. (B.b) Polar lipids stained in red. (B.c) Lipid storage (Gold-yellow intracellular spots – white arrow). (B.d). Detailed lipid storage. (n=6).

A Nile Red stain identified lipids in fresh 36-h biofilm cultures (Figure 1.B). Neutral (Figure 1.B.a) and polar (Figure 1.B.b) lipids were detected in the 36-h biofilm. Image merging allowed identifying lipid storages in the biofilm (Figure 1.B.c) and detailing them (Figure 1.B.d). Then, the extracellular lipid (ECL) pool produced by the 36-h C. albicans biofilm was quantified, reaching a concentration of 3.5 mg/mL. All further assays were performed with this ECL pool.

The ¹H-NMR spectroscopy analyzed the ECL pool to identify its main composition. Figure 2 shows the peaks corresponding to the hydrogen resonance of lipids. The hydrogen from the CH₃-free aliphatic terminal, common to all lipids, corresponds to the weak peak at 0.8 ppm (Figure 2, a). The hydrogen from methylene protons of aliphatic chains (-CH₂) n, β-methylene protons of carbonyl –OCO−CH₂−CH₂−, and methylene protons in the α-position of –CH₂−CH=CH− double bond peaks appear at 1.2-2.50 ppm (Figure 2, b). The CH₂CO residues peak at 2.53 ppm (Figure 2, c). The methylene protons of the heteroatom connected to phosphorus (CH₂−O−P), typical of phospholipids, appear at 4.43 ppm (Figure 2, d), as well –C1H₂ in the glycerol backbone in phospholipids (Figure 2, c).²² The methyl protons of charged nitrogen N-(CH₃)³, typical of choline, appear at 3.36 ppm (Figure 2, e).²²,²³ The C₆HOH of inositol and CH₂O of glycerol, typical of glycolipids, appear at 3.42 ppm (Figure 2, f). Double bond protons with cis conformation in the aliphatic chain (−CH=HC) appear at 5.47 ppm (Figure 2, g).²³ The ¹H-NMR strongly suggested that phosphatidylinositol (PI), phosphatidylcholine (PC), and phosphatidylglycerol (PG) lipids prevailed in the extracts.

Figure 2
Spectrogram of lipids (ECL) extracted from C. albicans biofilm. Resonance was performed from a lyophilized sample of C. albicans lipids previously extracted from six biofilms (n=6). The sample was resuspended in DMSO for 1H-NMR analysis. (a) CH3 residues. (b) CH2 residues from methylene protons of aliphatic chains –(CH2) n, β-methylene protons of carbonyl –OCO−CH2−CH2−, and methylene protons in the α-position of –CH2−CH=CH− double bonds. (c) CH2CO residues. (d) Methyl protons of charged nitrogen N-(CH3)+3, C-OH (C1, C4) of phosphatidylinositol, and glycerol and CH3 of methyl glycerol. The identified lipids were phosphatidylinositol (PI); phosphatidylcholine (PC), and phosphatidylglycerol (PG).

The ECL pool helped explain the presence or absence of direct interaction between pre-malignant (DOK cells) and malignant (SCC 25) oral cells ECL of C. albicans biofilms. A cytotoxicity curve was made for both cells using the vehicle control (ethanol), achieving the optimal amount (2% v/v) of solvent (ethanol) for the next steps (data not shown). DOK and SCC 25 cells were incubated for 24 hours with ECL of C. albicans biofilms (1% and 2% v/v). A statistical difference (**p<0.01) in the cell viability (MTT assay, n=12) of DOK cells when exposed to ECL 2% (v/v) was found, but not to ECL 1% (v/v) (Figure 3. A). Even when verifying differences in the cell viability assay, the cell cycle profile (n=3) of DOK cells did not change (Figure 3.a blue arrows). The SCC 25 cells showed a difference between ECL (1% and 2% v/v) and the control in the MTT cell viability assay (n=12), *p<0.05 (Figure 3. B). At a higher ECL concentration (2% v/v), SCC 25 cells showed a different cell cycle profile, with a G1 phase enlargement and a G2/M phase flattening, without statistical significance (n=3) (Figure 3.b, red arrows compared to blue arrows).

Figure 3
Cell viability and cell cycle profile. The MTT assay assessed cell viability (n=12). (A) Cell viability assay for DOK cells. A statistical difference between the group exposed to ECL 2% (v/v) and the control (**p<0.01) was found. (B) Cell viability assay for SCC 25 cells. A statistical difference between both groups exposed to ECL and the control (*p<0.05) was found. The cell cycle assay of DOK cells (n=3) (a) showed no tendency to change the cell cycle profile between CT and ECL (blue arrows). SCC 25 cells (n=3) (b) showed a tendency to disturb the cell cycle profile, with a G1 phase enlargement and a G2/M phase flattening, without statistical significance (n=3), comparing the CT (blue arrow) and ECL (red arrow) groups.

As for intracellular changes, a CLSM Nile Red assay was performed for 72 hours after the incubation of DOK and SCC 25 cells with ECL 2% (v/v) of C. albicans biofilms (n=6). First, control cells (Figure 4 column A) and vehicle control cells (Figure 4 column B) did not show significant changes in the amount and size of intracellular lipid droplets (LD). The ECL group induced an extensive intracellular LD formation in DOK and SCC 25 cells compared to controls (Figure 4 column C, showing bright yellow spots – white arrows).

Figure 4
Confocal laser screening microscopy (CLSM) for DOK and SCC 25 cells cultured with ECL of C. albicans biofilm. CLSM of LD in DOK and SCC 25 cells after culturing for 72 hours with ECL (2% v/v) of C. albicans. (A) DOK and SCC 25 cells (control), (B) DOK and SCC 25 cells (vehicle), and (C) DOK and SCC 25 cells (ECL 2% v/v), LD vesicles (arrows), n=6.

DOK and SCC 25 cells were challenged with the lipophilic topoisomerase I inhibitor camptothecin (CPT), attempting to understand whether the ECL of C. albicans biofilms could change cellular susceptibility to anti-tumoral drugs. DOK cells showed a difference between the control and ECL 2% (****p<0.0001) and the control and CPT (***p<0.001). There was a difference between ECL 2% and CPT (****p<0.0001) and ECL 2% and ECL 2% + CPT (***p<0.001), demonstrating mitigation of the CPT effect. SCC 25 cells showed a difference between the control and ECL 2% (***p<0.001) and ECL 2% + CPT (****p<0.0001) and CPT (*p<0.05). Both treated ECL 2% cells (ECL 2% and ECL 2% + CPT) showed a difference compared to CPT (for both groups, ****p<0.0001). However, no differences between ECL 2% and ECL 2% + CPT were found, demonstrating resistance to CPT when previously culturing cells with ECL 2%.

CPT uptake was studied with CLSM (n=6). Figures 5.a (DOK cells) and 5.b (SCC 25 cells) showed that the ECL of C. albicans biofilms did not change the amount of CPT internalization. However, a high-resolution CLSM tool with a Nile Red double staining (n=6) detected a different pattern of CPT intracellular distribution (Figure 6). In the absence of ECL incubation, DOK cells had a higher perinuclear CPT retention (Figure 6. upper panel A - red arrows) than the cytoplasmic distribution of the ECL group (Figure 6. upper panel E - white arrows). A positive co-localization with the formed LD and the CPT (Figure 6. upper panels F, G, and H – yellow to white spots, respectively) was found, with a positive accumulation of CPT in LD. This behavior was poorly detected without ECL (Figure 6. upper panels C, D, and E). For SCC 25 cells, cytoplasmic aggregates were more pronounced in the group previously cultured with ECL of C. albicans biofilms (Figure 6. down panel A compared to E – white arrows). These aggregates showed a positive CPT-LD co-localization (Figure 6. down panels B, C, D, F, G, H – white arrows) with a large amount and size of yellow to white spots in the group previously cultured with ECL of C. albicans (Figure 6. down panels F, G, H – white arrows). Red arrows show no co-localization between CPT and LD. White arrows show a positive co-localization between CPT and LD.

Figure 5
AlamarBlue and CPT uptake of DOK and SCC 25 cells cultured with ECL of C. albicans biofilm. DOK and SCC 25 cells were cultured with ECL of C. albicans for 72 hours. Then, the cells were challenged with the topoisomerase I inhibitor CPT for another four hours, followed by the alamarBlueTM assay (n=12). DOK cells (A) showed statistical differences between all groups. SCC 25 cells (B) showed statistical differences between all groups, except for the cells exposed to ECL 2% v/v and ECL 2% v/v + CPT (*p<0.05; ***p<0.001; ****p<0.0001). CPT uptake with brightfield and 405 nm fluorescence emission (arrows) for (a) DOK (n = 6) and (b) SCC 25 (n=6) cells.

Figure 6
Cell double staining of CPT and 2% ECL of 36-h C. albicans biofilms with Nile Red and high-resolution CLSM. DOK cells (upper panel). CPT detection (A - blue panel white arrows). Nile Red neutral lipids (B - green panel). Nile Red polar lipids (C - red panel). Co-localization of CPT and LD (D – merged panel, white arrows) (n=6). SCC 25 cells (down panel). CPT detection (A – blue panel, white arrows). Nile Red neutral lipids (B - green panel). Nile Red polar lipids (C - red panel). Co-localization of CPT and LD (D - merged panel, white arrows) (n=6). Red arrows show no co-localization between CPT and LD. White arrows show a positive co-localization between CPT and LD.

Discussion

The opportunistic fungus C. albicans has received much attention over the last decade, mainly due to its ability to produce biofilms. Some fingerprints, i.e., lipid molecules, from the extracellular matrix of C. albicans biofilms play a critical role in infection and fungal resistance.⁷ Most of these molecules consist of neutral classes of free fatty acids, triacylglycerols, glycerolipids, sphingolipids, and phospholipids (i.e., phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol). The SC 5314 strain showed significant differences in the distribution of lipid classes between biofilm and planktonic growth, i.e., biofilms with higher lipid levels than planktonic cells.²⁴

It is worth noting that C. albicans infects hosts not as free-living planktonic organisms, but as a multicellular biofilm, developing chronic infections mainly in the oral cavity. Chronic inflammation has been linked to many carcinogenesis stages,²⁵,²⁶ and biofilm formation has been attributed to a potential etiological role in the development of some cancers.²⁷-²⁹ Recent clinical studies have linked fungi (e.g., C. albicans) and cancer, claiming the need for more in vitro and in vivo studies in this area.³⁰,³¹

In a tumor microenvironment, the resident microbiota and its metabolites trigger metabolic signaling pathways, which further promote or suppress the malignant behavior of host cells.³² In this study, the entire fresh 36-h C. albicans biofilm produced lipid molecules, as Radulovic, et al described.³³ (2013). Phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylinositol (PI) molecules were the three major identified lipid classes, named extracellular lipids (ECL), and these findings correlate to previous studies.²⁴,³⁴

The ECL in this study induced lipid droplet (LD) formation in DOK and SCC 25 cells. LD vesicles represent energy storage readily available to support anoikic resistance and cancer cell invasiveness.²⁰ LD is involved in lipid and protein storage, transportation, and degradation, contributing to many diseases, including cancer. Regarding C. albicans infections, unknown mechanisms described LD biogenesis in macrophages and hepatocytes in a rat model,³⁵ and we found no descriptions for oral dysplastic or neoplastic cells.

Therefore, the significance of LD has provided a compelling insight into cancer treatment. LD is present in some breast and colon adenocarcinomas resistant to chemotherapeutical drugs.¹⁴,³⁶ Many mechanisms related to the environment explain the resistance mediated by lipids in cancer cells. For example, resistance to tyrosine kinase inhibitors is associated with new lipid biosynthesis.³⁷ Drug-resistant cancer cells have shown increased LD numbers. These organelles were postulated to sequester hydrophobic agents, reducing drug effectiveness and activating the ERK/Akt/mTOR survival pathway.³⁷,³⁸ In the presence of ECL of C. albicans biofilm, the anti-tumoral effect of the camptothecin (CPT) lipophilic drug was mitigated and abolished in DOK and SCC 25 cells, respectively. This cellular behavior occurred with different patterns of intracellular drug uptake, mainly inside the LD vesicles induced by C. albicans lipids. Also, LD was already described as a positive marker of malignant phenotype progression.³⁹ Thus, a positive correlation between lipids (PC, PG, and PI) of C. albicans biofilms and the in vitro LD formation in oral dysplastic and neoplastic cells addresses the relevance of better understanding this pathogen-host interaction in a tumor environment.

Overall, our findings and those from recent clinical studies³⁰,³¹ require further investigation. We should note that the tumoral microenvironment is a complex network that includes many tissues, immune cells, and the microbiome formed by many bacteria, fungi, and viruses living in the same environment, thus contributing to cancer.

Conclusion

This study described for the first time the direct interaction between lipids of Candida albicans biofilms and oral dysplastic and neoplastic cells. Phosphatidylcholine, phosphatidylglycerol, and phosphatidylinositol were identified and enhanced cellular metabolism, lipid droplet formation, and anti-tumoral drug resistance. Thus, this study addressed the relevance of better understanding the signaling of C. albicans biofilms in the oral tumor microenvironment.

Acknowledgment

School of Dentistry (FOAr/UNESP), CAPES, CNPq, Chemical Institute (IQ/UNESP), Bioscience and Biotechnology Applied to Pharmacy Program (BBAF/UNESP).

References

• ¹- Irfan M, Delgado RZ, Frias-Lopez J. The Oral microbiome and cancer. Front Immunol. 2020;11:591088. doi: 10.3389/fimmu.2020.591088
  » https://doi.org/10.3389/fimmu.2020.591088
• ²- Börnigen D, Ren B, Pickard R, Li J, Ozer E, Hartmann EM, et al. Alterations in oral bacterial communities are associated with risk factors for oral and oropharyngeal cancer. Sci Rep. 2017;7(1):17686. doi: 10.1038/s41598-017-17795-z
  » https://doi.org/10.1038/s41598-017-17795-z
• ³- Pokrowiecki R, Mielczarek A, Zaręba T, Tyski S. Oral microbiome and peri-implant diseases: where are we now? Ther Clin Risk Manag. 2017;13:1529-42. doi: 10.2147/TCRM.S139795
  » https://doi.org/10.2147/TCRM.S139795
• ⁴- Brennan CA, Garrett WS. Fusobacterium nucleatum: symbiont, opportunist and oncobacterium. Nat Rev Microbiol. 2019;17(3):156-66. doi: 10.1038/s41579-018-0129-6
  » https://doi.org/10.1038/s41579-018-0129-6
• ⁵- Al-hebshi NN, Nasher AT, Maryoud MY, Homeida HE, Chen T, Idris AM, et al. Inflammatory bacteriome featuring Fusobacterium nucleatum and Pseudomonas aeruginosa identified in association with oral squamous cell carcinoma. Sci Rep. 2017;7(1):1834. doi: 10.1038/s41598-017-02079-3
  » https://doi.org/10.1038/s41598-017-02079-3
• ⁶- Johnson CH, Dejea CM, Edler D, Hoang LT, Santidrian AF, Felding BH, et al. Metabolism links bacterial biofilms and colon carcinogenesis. Cell Metab. 2015;21(6):891-7. doi: 10.1016/j.cmet.2015.04.011
  » https://doi.org/10.1016/j.cmet.2015.04.011
• ⁷- Zarnowski R, Westler WM, Lacmbouh GA, Marita JM, Bothe JR, Bernhardt J, et al. Novel entries in a fungal biofilm matrix encyclopedia. mBio. 2014;5(4):e01333-14. doi: 10.1128/mBio.01333-14
  » https://doi.org/10.1128/mBio.01333-14
• ⁸- Sokol H, Leducq V, Aschard H, Pham H-P, Jegou S, Landman C, et al. Fungal microbiota dysbiosis in IBD. Gut. 2017;66(6):1039-48. doi: 10.1136/gutjnl-2015-310746
  » https://doi.org/10.1136/gutjnl-2015-310746
• ⁹- Alnuaimi AD, Wiesenfeld D, O’Brien-Simpson NM, Reynolds EC, McCullough MJ. Oral Candida colonization in oral cancer patients and its relationship with traditional risk factors of oral cancer: a matched case-control study. Oral Oncol. 2015;51(2):139-45. doi: 10.1016/j.oraloncology.2014.11.008
  » https://doi.org/10.1016/j.oraloncology.2014.11.008
• ¹⁰- Vadovics M, Ho J, Igaz N, Alföldi R, Rakk D, Veres E, et al. Candida albicans enhances the progression of oral squamous cell carcinoma in vitro and in vivo. mBio. 2022;13(1):e03144-21. doi: 10.1128/mBio.03144-21
  » https://doi.org/10.1128/mBio.03144-21
• ¹¹- Amaya Arbeláez MI, Silva AC, Navegante G, Valente V, Barbugli PA, Vergani CE. Proto-oncogenes and cell cycle gene expression in normal and neoplastic oral epithelial cells stimulated with soluble factors from single and dual biofilms of Candida albicans and Staphylococcus aureus. Front Cell Infect Microbiol. 2021;11:627043. doi: 10.3389/fcimb.2021.627043
  » https://doi.org/10.3389/fcimb.2021.627043
• ¹²- Bullman S, Pedamallu CS, Sicinska E, Clancy TE, Zhang X, Cai D, et al. Analysis of Fusobacterium persistence and antibiotic response in colorectal cancer. Science. 2017;358(6369):1443-8. doi: 10.1126/science.aal5240
  » https://doi.org/10.1126/science.aal5240
• ¹³- Buechler C, Aslanidis C. Role of lipids in pathophysiology, diagnosis and therapy of hepatocellular carcinoma. Biochim Biophys Acta Mol Cell Biol Lipids. 2020;1865(5):158658. doi: 10.1016/j.bbalip.2020.158658
  » https://doi.org/10.1016/j.bbalip.2020.158658
• ¹⁴- Montopoli M, Bellanda M, Lonardoni F, Ragazzi E, Dorigo P, Froldi G, et al. “Metabolic Reprogramming” in ovarian cancer cells resistant to cisplatin. Curr Cancer Drug Targets. 2011;11(2):226-35. doi: 10.2174/156800911794328501
  » https://doi.org/10.2174/156800911794328501
• ¹⁵- Nunes TS, Rosa LM, Vega-Chacón Y, Mima EG. Fungistatic action of N-Acetylcysteine on Candida albicans biofilms and its interaction with antifungal agents. Microorganisms. 2020;8(7):980. doi: 10.3390/microorganisms8070980
  » https://doi.org/10.3390/microorganisms8070980
• ¹⁶- Dias KC, Barbugli PA, Vergani CE. Influence of different buffers (HEPES/MOPS) on keratinocyte cell viability and microbial growth. J Microbiol Methods. 2016;125:40-2. doi: 10.1016/j.mimet.2016.03.018
  » https://doi.org/10.1016/j.mimet.2016.03.018
• ¹⁷- Dias KC, Sousa DL, Barbugli PA, Cerri PS, Salih VM, Vergani CE. Development and characterization of a 3D oral mucosa model as a tool for host-pathogen interactions. J Microbiol Methods. 2018;152:52-60. doi: 10.1016/j.mimet.2018.07.004
  » https://doi.org/10.1016/j.mimet.2018.07.004
• ¹⁸- Larsen P, Olesen BH, Nielsen PH, Nielsen JL. Quantification of lipids and protein in thin biofilms by fluorescence staining. Biofouling. 2008;24(4):241-50. doi: 10.1080/08927010802040255
  » https://doi.org/10.1080/08927010802040255
• ¹⁹- Antunes P, Cruz A, Barbosa J, Bonifácio VD, Pinto SN. Lipid droplets in cancer: from composition and role to imaging and therapeutics. Molecules. 2022;27(3):991. doi: 10.3390/molecules27030991
  » https://doi.org/10.3390/molecules27030991
• ²⁰- Corbet C, Bastien E, Jesus JP, Dierge E, Martherus R, Vander Linden C, et al. TGFβ2-induced formation of lipid droplets supports acidosis-driven EMT and the metastatic spreading of cancer cells. Nat Commun. 2020;11(1):454. doi: 10.1038/s41467-019-14262-3
  » https://doi.org/10.1038/s41467-019-14262-3
• ²¹- Liu LF, Desai SD, Li TK, Mao Y, Sun M, Sim SP. Mechanism of action of camptothecin. Ann N Y Acad Sci. 2000;922:1-10. doi: 10.1111/j.1749-6632.2000.tb07020.x
  » https://doi.org/10.1111/j.1749-6632.2000.tb07020.x
• ²²- Mika A, Kaczynski Z, Stepnowski P, Kaczor M, Proczko-Stepaniak M, Kaska L, et al. Potential application of 1H NMR for routine serum lipidome analysis: evaluation of effects of bariatric surgery. Sci Rep. 2017;7(1):15530. doi: 10.1038/s41598-017-15346-0
  » https://doi.org/10.1038/s41598-017-15346-0
• ²³- Mor NC, Correia BS, Valc AL, Tasic L. A protocol for fish lipid analysis using Nuclear Magnetic Resonance Spectroscopy. J Braz Chem Soc. 2020;31(4):662-72. doi: 10.21577/0103-5053.20190230
  » https://doi.org/10.21577/0103-5053.20190230
• ²⁴- Lattif AA, Mukherjee PK, Chandra J, Roth MR, Welti R, Rouabhia M, et al. Lipidomics of Candida albicans biofilms reveals phase-dependent production of phospholipid molecular classes and role for lipid rafts in biofilm formation. Microbiology (Reading). 2011;157(Pt 11):3232-42. doi: 10.1099/mic.0.051086-0
  » https://doi.org/10.1099/mic.0.051086-0
• ²⁵- Armstrong MJ, Mullins CD, Gronseth GS, Gagliardi AR. Impact of patient involvement on clinical practice guideline development: a parallel group study. Implement Sci. 2018;13(1):55. doi: 10.1186/s13012-018-0745-6
  » https://doi.org/10.1186/s13012-018-0745-6
• ²⁶- Plummer M, de Martel C, Vignat J, Ferlay J, Bray F, Franceschi S. Global burden of cancers attributable to infections in 2012: a synthetic analysis. Lancet Glob Health. 2016;4(9):e609-16. doi: 10.1016/S2214-109X(16)30143-7
  » https://doi.org/10.1016/S2214-109X(16)30143-7
• ²⁷- Dejea CM, Fathi P, Craig JM, Boleij A, Taddese R, Geis AL, et al. Patients with familial adenomatous polyposis harbor colonic biofilms containing tumorigenic bacteria. Science. 2018;359(6375):592-7. doi: 10.1126/science.aah3648
  » https://doi.org/10.1126/science.aah3648
• ²⁸- Dejea CM, Wick EC, Hechenbleikner EM, White JR, Welch JLM, Rossetti BJ, et al. Microbiota organization is a distinct feature of proximal colorectal cancers. Proc Natl Acad Sci U S A. 2014;111(51):18321-6. doi: 10.1073/pnas.1406199111
  » https://doi.org/10.1073/pnas.1406199111
• ²⁹- Deng Y, Liu SY, Chua SL, Khoo BL. The effects of biofilms on tumor progression in a 3D cancer-biofilm microfluidic model. Biosens Bioelectron. 2021;180:113113. doi: 10.1016/j.bios.2021.113113
  » https://doi.org/10.1016/j.bios.2021.113113
• ³⁰- Narunsky-Haziza L, Sepich-Poore GD, Livyatan I, Asraf O, Martino C, Nejman D, et al. Pan-cancer analyses reveal cancer-type-specific fungal ecologies and bacteriome interactions. Cell. 2022;185(20):3789-806.e17. doi: 10.1016/j.cell.2022.09.005
  » https://doi.org/10.1016/j.cell.2022.09.005
• ³¹- Dohlman AB, Klug J, Mesko M, Gao IH, Lipkin SM, Shen X, et al. A pan-cancer mycobiome analysis reveals fungal involvement in gastrointestinal and lung tumors. Cell. 2022;185(20):3807-22.e12. doi: 10.1016/j.cell.2022.09.015
  » https://doi.org/10.1016/j.cell.2022.09.015
• ³²- Liu NN, Ma Q, Ge Y, Yi CX, Wei LQ, Tan JC, et al. Microbiome dysbiosis in lung cancer: from composition to therapy. NPJ Precis Oncol. 2020;4(1):33. doi: 10.1038/s41698-020-00138-z
  » https://doi.org/10.1038/s41698-020-00138-z
• ³³- Radulovic M, Knittelfelder O, Cristobal-Sarramian A, Kolb D, Wolinski H, Kohlwein SD. The emergence of lipid droplets in yeast: current status and experimental approaches. Curr Genet. 2013;59(4):231-42. doi: 10.1007/s00294-013-0407-9
  » https://doi.org/10.1007/s00294-013-0407-9
• ³⁴- Zarnowski R, Sanchez H, Covelli AS, Dominguez E, Jaromin A, Bernhardt J, et al. Candida albicans biofilm-induced vesicles confer drug resistance through matrix biogenesis. PLoS Biol. 2018;16(10):e2006872. doi: 10.1371/journal.pbio.2006872
  » https://doi.org/10.1371/journal.pbio.2006872
• ³⁵- Paraje MG, Correa SG, Renna MS, Theumer M, Sotomayor CE. Candida albicans-secreted lipase induces injury and steatosis in immune and parenchymal cells. Can J Microbiol. 2008;54(8):647-59. doi: 10.1139/w08-048
  » https://doi.org/10.1139/w08-048
• ³⁶- Santini MT, Romano R, Rainaldi G, Filippini P, Bravo E, Porcu L, et al. The relationship between 1H-NMR mobile lipid intensity and cholesterol in two human tumor multidrug resistant cell lines (MCF-7 and LoVo). Biochim Biophys Acta. 2001;1531(1-2):111-31. doi: 10.1016/s1388-1981(01)00093-2
  » https://doi.org/10.1016/s1388-1981(01)00093-2
• ³⁷- Butler LM, Perone Y, Dehairs J, Lupien LE, de Laat V, Talebi A, et al. Lipids and cancer: emerging roles in pathogenesis, diagnosis and therapeutic intervention. Adv Drug Deliv Rev. 2020;159:245-93. doi: 10.1016/j.addr.2020.07.013
  » https://doi.org/10.1016/j.addr.2020.07.013
• ³⁸- Verbrugge SE, Al M, Assaraf YG, Kammerer S, Chandrupatla DM, Honeywell R, et al. Multifactorial resistance to aminopeptidase inhibitor prodrug CHR2863 in myeloid leukemia cells: down-regulation of carboxylesterase 1, drug sequestration in lipid droplets and pro-survival activation ERK/Akt/mTOR. Oncotarget. 2016;7(5):5240-57. doi: 10.18632/oncotarget.6169
  » https://doi.org/10.18632/oncotarget.6169
• ³⁹- Cruz AL, Barreto EA, Fazolini NP, Viola JP, Bozza PT. Lipid droplets: platforms with multiple functions in cancer hallmarks. Cell Death Dis. 2020;11(2):105. doi: 10.1038/s41419-020-2297-3
  » https://doi.org/10.1038/s41419-020-2297-3

• Data availability statement
• The datasets generated during the current study are available from the corresponding author on reasonable request.
• *
  The manuscript is derived from a master’s dissertation of the program Biociências e Biotecnologia Aplicadas a Farmácia da Faculdade de Ciências Farmacêuticas, UNESP-AQA. This is the respective access address: https://repositorio.unesp.br/handle/11449/213767
• Funding: This work was supported by the Coordination of Higher Education and Graduate Training – Brazil (CAPES) under grant 001, the National Council for Scientific and Technological Development (CNPq) Foundation of Brazil under grant 431895/2016-3, the São Paulo State Research Support Foundation (FAPESP) under grant 2019/07574-9.

Publication Dates

• Publication in this collection
  03 Feb 2023
• Date of issue
  2022

History

• Received
  18 Aug 2022
• Reviewed
  20 Oct 2022
• Accepted
  24 Oct 2022