Sensitization of drug resistant sarcoma tumors by membrane modulation via short chain sphingolipid-containing nanoparticles

Abstract

Nanoparticles such as liposomes are able to overcome cancer treatment challenges such as Multidrug resistance by increasing the bioavailability of the encapsulated drug, bypassing drug pumps or through targeting resistant cells. Here, we merge enhanced drug delivery by nanotechnology with tumor cell membrane modulation combined in a single formulation. This is achieved through the incorporation of Short Chain Sphingolipids (SCSs) in the liposomal composition, which permeabilizes cell membranes to amphiphilic drugs such as Doxorubicin (Dxr).

To study the mechanism and capability of SCS-containing nanodevices to overcome Dxr resistance, a sensitive uterine sarcoma cell line, MES-SA, and a resistant derived cell line, MES-SA/MX2, were used. The mechanism of resistance was explored by Lipidomics and flow cytometry, revealing significant differences in lipid composition and in P glycoprotein (Pgp) expression.

In vitro assays show that SCS liposomes were able to reverse cell resistance, and importantly, display a higher net effect on resistant than sensitive cells. SCS lipids modulated the cell membrane of MES-SA/MX2 drug resistant cells, while Pgp expression was not affected. Furthermore, SCS-modified liposomes were evaluated in a sarcoma xenograft model on drug accumulation, pharmacokinetics and efficacy. SCS liposomes improved Dxr levels in tumor nuclei of MES-SA/MX2 tumor cells, which was accompanied by a delay in tumor growth of the resistant model. Here we show that Dxr accumulation in tumor cells by SCS-modified liposomes was especially improved in Dxr resistant cells, rendering Dxr as effective as in sensitive cells. Moreover, this phenomenon translated to improved efficacy when Dxr liposomes where modified with SCSs in the drug resistant tumor model, while no benefit was seen in the sensitive tumors.

Introduction

Uterine sarcomas of mesenchymal origin present a very variable prognosis depending on the histologic subtype and tumor grade.^1,2 The incidence of uterine sarcoma is 3–7% of all uterine malignancies and is associated with a poor prognosis when compared to endometrial carcinoma.^1,2 One of the main treatments is Doxorubicin (Dxr)¹, a widely used anthracycline that exert cytotoxicity by intercalation into DNA strands or binding to topoisomerase II.³ Dxr needs to interact with the membrane in order to enter the cancer cell and requires a certain degree of membrane fluidity to be internalized. However, in resistant tumors a lower amount of Dxr is able to enter the cell, limiting the efficacy.⁴

Multidrug resistance (MDR) development is one of the main challenges in cancer treatment. Tumor cells develop several mechanisms to overcome chemotherapy like drug influx decrease or efflux enhancement.⁵ In general, MDR cells are characterized by a higher cell membrane rigidity resulting from a difference in lipid composition between sensitive and resistant cancer cells.⁶ It has been described that resistant cell membranes are enriched in saturated lipids and cholesterol, which is associated with an augmentation of membrane packing. As a consequence of a higher rigidity the permeability is decreased and drugs, such as Dxr, experience more difficulties to enter the cell.⁶ Next to that, drugs that do enter resistant cells are expelled by overexpressed MDR transporters and efflux pumps such as P glycoprotein (Pgp).⁷ The mechanism of action of MDR transporters is often compared with a vacuum cleaner that, by an active pumping process, eliminates drugs from the cytoplasm. Pgp is a member of the ATP-binding cassette superfamily, with several important chemotherapeutics as substrates such as Actinomycin D, Vinca alkaloids, Methotrexate or Dxr.⁷

In order to reduce the important side effects associated to Dxr such as cardiotoxicity and myelotoxicity among others, the drug is administered encapsulated into nanoparticles like liposomes.⁸ Liposomal Dxr formulations present important pharmacokinetic advantages over the free formulation, such as protection of Dxr from undesirable interactions with blood components, increased time of circulation and reduced volume of distribution.⁸ The small size of liposomes, around 100 nm, and the chaotic and immature nature of tumor vessels, allow liposome accumulation in solid tumors, which has been coined Enhanced Permeability and Retention effect (EPR).⁹ Although there is a big controversy around EPR heterogeneity and inter-species differences^10–12, it is still considered the basis of passive tumor accumulation of nanoparticles. Due to the better safety profile, Dxr liposomes (Caelyx®/Doxil®) were approved and are currently used in cancer patients.¹³ However, because these cholesterol containing liposomes are highly stable, they show a slow drug release from the formulation, leading to low levels of bioavailable Dxr and limiting the antitumor effect.^14–16 Therefore, several modifications have been made to improve Doxil-like formulations such as, for instance, triggered release strategies to obtain better bioavailability of the encapsulated drug in the tumor tissue.¹⁷

A novel strategy consists of the use of Short Chain Sphingolipids (SCSs), ceramides with a shorter hydrophobic chain and a sugar moiety in their structure.¹⁸ Once included within liposomal formulations SCSs increase the internalization and antitumor activity of several amphiphilic chemotherapeutics, like Dxr, in cancer cells.^18,19 SCSs mechanism of action result from self-aggregation properties of ceramides and the ability to form transient micro-channels within the cell membrane.^6,20 Thus, once a liposome is in the proximity of cancer cells, SCSs transfer spontaneously from the liposome to incorporate in the cell membrane. As a consequence, the liposome is destabilized and Dxr is released nearby of tumor cells, being able to interact with the formed SCS channels in the tumor cell membrane; therefore the transmembrane drug transport of Dxr is enhanced.²⁰ Previous in vitro studies with SCS-liposomes show a lesser effect of these lipids in membranes of normal (non-tumor) cells, and that resistant tumor cells are more sensitive to SCSs action than ordinary cancer cells, which indicate interesting selective membrane permeabilizing properties.^19,20

The aim of the present work is to investigate whether the improved Dxr accumulation in tumor cells is present also in Dxr resistant tumor cells, and if so whether this translates in a better in vivo efficacy.

Experimental data

Materials

Hydrogenated soy phosphatidylcholine (HSPC) and DSPE-PEG₂₀₀₀ were purchased from Lipoid (Ludwigshaven, Germany). Short chain sphingolipids 8-Glucosyl-ceramide (C8-GluCer) and Top Fluor Galactosyl Ceramide were from Avanti Polar Lipids (Alabaster, Alabama, USA). PD10 Sephadex columns were obtained from GE Healthcare (Diegem, Belgium). Dxr-HCl (Dxr) was from Pharmachemie (Haarlem, The Netherlands). Cholesterol (Ch), Chloroform, Methanol, HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid), ammonium sulfate, sodium chloride, EDTA, Trizma Hydrochloride, trichloroacetic acid (TCA), sucrose, magnesium sulfate, calcium chloride, Isopropanol, acetic acid, Triton-X-100 and sulforhodamine B (SRB) were from Sigma Aldrich (Zwijndrecht, The Netherlands). McCoy’s 5A medium, PBS, fetal bovine serum (FBS) and Penicillin-streptomycin (P/S) were obtained from GIBCO (Bleiswijk, The Netherlands).

Liposome preparation

Liposomes composed of HSPC:Ch:DSPE-PEG₂₀₀₀ in a molar ratio of 1.85:1:0.15, were developed using the film hydration technique.¹⁹ Briefly, lipids were dissolved in a chloroform:methanol solution [9:1 (v/v)]. Organic solvents were evaporated using a rotary evaporator (Büchi-R144) at 40°C to obtain a lipid film, which was dried under vacuum. SCS liposomes were prepared following the same procedure but adding C8-GluCer (Avanti Polar Lipids, USA) [10% molar ratio] to the lipid composition.¹⁹

The film was hydrated with ammonium sulfate (250 mM, pH 5.5) at 60°C and extruded through 200–50 nm polycarbonate membranes to obtain a homogeneous liposomal population. Active Dxr loading was done using the ammonium sulfate gradient method.²¹ For that, the external buffer was exchanged using a PD10 column (GE Healthcare, Belgium) with Hepes saline buffer (Hepes 10 mM, NaCl 150 mM and EDTA 5 mM, pH 6.7). Afterwards, liposomes were incubated at 60°C with Dxr at 0.1:1 drug:lipid molar ratio for 1 h. Non-encapsulated Dxr was separated by ultracentrifugation at 40,000 rpm in a Beckman ultracentrifuge for 2 h. Liposomes (LP-Dxr or LP-SCS-Dxr) were resuspended in Hepes saline buffer overnight and stored at 4°C until use. Empty liposomes (placebo) were performed following the same procedure without adding Dxr.

The physicochemical characterization of liposomes in terms of particle size, polydispersity index (PDI) and Zeta potential, were determined using the Zetasizer Nano ZS. Formulations were diluted 1:100 (v/v) in deionized water for a convenient scattered intensity on the detector.

In order to characterize Dxr entrapment, lipid concentration was measured by phosphate assay²² and Dxr was measured by fluorimetry (λ_ex475 nm; λ_em 590 nm) using the microplate reader (Victor 1420). Total amount of drug was measured after liposome disruption of an aliquot of liposomes with 1% (v/v) TritonX-100. Encapsulation efficiency was calculated comparing the initial drug:lipid ratio (0.1:1) with the final ratio. Liposome stability was evaluated over 24 h in FBS under shaking.

Cellular assays

All further experiments were done using human uterine sarcoma cell lines MES-SA and MES-SA/MX2, from ATCC. MES-SA/MX2 cells display features of multidrug resistance,²³ being 1000 fold more resistant to mitoxantrone, an anthracenedione antitumor agent, that follows a similar mechanism of action than Dxr.²⁴ Cells were cultured in McCoy’s 5A medium supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were subcultured twice weekly by trypsinization when a confluence of 70% was achieved and maintained in a water saturated atmosphere of 5% CO₂ at 37°C. Cells were tested regularly for mycoplasm using a luminescence assay (LONZA, Basel, Switzerland).

Exploration of Doxorubicin resistance mechanisms

With the aim of determine the mechanism of Dxr resistance and explore whether the influx or the efflux of Dxr are modified in MDR cells, several assays were performed. First, the role of Pgp in the increased efflux of Dxr in resistant cells was evaluated. Hence, the expression of this transporter was determined in both cell lines. Briefly, MES-SA and MES-SA/MX2 cells were seeded in 6 well plates at a concentration of 2×10⁵/well in 3 ml of medium. After 24 h, the medium was removed, cells were washed with 500 μl of PBS twice, and collected using cell scrapers with 200 μl of PBS. Cells were centrifuged at 1,500 rpm for 5 min and transferred to a 96 U bottom plate. Pgp protein was detected by the use of a mouse α-human-Pgp primary antibody (4E3, Abcam) at 1:500 dilution for 30 min at 4°C. After washing, the secondary antibody goat α-mouse AF647 (1:500), was added and incubated in darkness for 15 min at room temperature (RT). Cells were washed and measured using a Becton Dickinson FACScan equipped with Cell Quest software. The analysis of data was done with FlowJo 7.6.1. (Treestar). Regarding the influx mechanism evaluation, the lipid profile of both cell lines was evaluated using Lipidomics to determine whether possible membrane lipid differences may explain Dxr lower uptake in the resistant cell line. In brief, 2×10⁵ cells were cultured in 6 well plates and they were harvested in 200 μl PBS using a scraper when they reached the confluence and frozen at −20°C until analysis. Lipids were extracted using the method of Bligh and Dyer²⁵ and dried lipid extract was redisolved in chloroform:methanol 1:1 (v/v). Chromatography was performed at a flow rate of 1 ml/min on a hydrophilic interaction liquid chromatography (HILIC) column (2.6 μm HILIC 100 Å, 50 × 4.6 mm, Phenomenex, Torrance, CA), by elution with a gradient from Acetonitrile/Acetone (9:1, v/v) to Acetonitrile/H₂O (7:3, v/v) with 10 mM ammonium formate, and both with 0.1% formic acid. The column outlet of the LC was connected to a heated electrospray ionization (hESI) source of a LTQ XL mass spectrometer (ThermoFisher Scientific, Waltham, MA). Full scan spectra were collected in the range from 450–1050 amu in both negative- and positive ionization mode at a scan speed of 3 scans/s.

Data were converted to mz(X)ML format and analyzed using XCMS²⁶ version 1.52.0 running under R version 3.4.2. Assignment of lipid classes was done based on chromatographic retention time and subsequent species assignment and carbon de-isotoping was performed in R using an in-silico generated database. Assignment of PC and SM lipid species was based on positive ion mode data, for all other classes, negative ion mode data were used. Even numbered acyl chainlengths were assumed. Lipid class abundance was based on negative ion mode data for all lipid classes. For Principal Component Analysis (PCA), the package pcaMethods was used.²⁷

Short chain sphingolipid mechanism of action

In order to elucidate whether the SCSs acts through the cell membrane or by modifying Pgp expression, MES-SA and MES-SA/MX2 were exposed to different treatments. First, cells were analysed by flow cytometry to determine the effect of SCSs over Pgp. For that, cells were seeded on flat bottom 12 well plates at a final concentration of 5×10⁵ cells/well. When cells reached 80% of confluence, different treatments were added in free serum medium: saline, free SCSs, LP and LP-SCS at a concentration equivalent to 30 μg Dxr/ml.²⁸ After 4 or 24 h of exposure, medium was removed, cells were washed twice and collected using cell scrapers. Cells were transferred to a 96 U bottom plate and stained with α-human-Pgp primary antibody (1:500) for 30 min at 4°C. After washing, the secondary antibody goat α-mouse AF647 (1:500), was added and incubated in darkness for 15 min at room temperature (RT). Cells were washed and measured using a Becton Dickinson FACScan equipped with Cell Quest software. The analysis of data was done with FlowJo 7.6.1.

To determine if SCSs can interact with cell membranes, both cell lines were seeded in 8 well plates (Ibidi, Germany) at a concentration of 8×10⁴ cells per well in 500 μl of medium. Next day, medium was replaced by free serum medium 2 h before starting the treatment. Liposomes were prepared with fluorescent SCSs in their compositionand the following treatments were added to cells: free Dxr, LP-Dxr and LP-SCS-Dxr. In all cases the treatment was 30 μg Dxr/ml.²⁸ Treatments were in contact with cells 4 h and 24 h, the medium refreshed and cells were imaged using confocal microscopy (Zeiss LSM 510 META, Germany). Images were analysed with Zen 2.3. software (Carl Zeiss).

Doxorubicin uptake and cytotoxicity evaluation

Dxr uptake was evaluated by flow cytometry. In brief cells were seeded on flat bottom 12 well plates at a final concentration of 5×10⁵ cells/well. When cells reached 80% of confluence different treatments were added in free serum medium: free Dxr, free SCS/Dxr, LP-Dxr and LP-Dxr-SCS at 30 μg Dxr/ml. Treatments were in contact with cells for 4 h, the medium was refreshed and cells were i) collected, or ii) cultured up to 24 h.

After both different incubation times, cells were washed twice with fresh medium to discard non-incorporated drug and resuspended with cold PBS in FACS tubes. Cellular uptake was measured on a Becton Dickinson FACScan using Cell Quest software and the analysis of data was done with FlowJo 7.6.1.

To evaluate the cytotoxic effect of liposomes compared to free Dxr, MES-SA (2×10⁴) and MES-SA/MX2 (2,5×10⁴) cells were seeded in flat bottom 96 well-plates. Next day cells were exposed to following treatments: Control, Dxr, Free SCS+Dxr, LP-Dxr, LP-Empty, LP-SCS-Dxr and LP-SCS-Empty. Treatments were added in serum free medium in a range from 0 to 100 μM of Dxr for 4 h of exposure. Afterwards, the medium was refreshed, and cells incubated for 24, 48 and 72 h. In the case of empty liposomes (placebo), the amount of added lipid was comparable to the lipid amount of Dxr loaded liposomes. All experiments were performed in triplicate and were repeated at least 3 times with independent liposomal batches.

Cell survival was determined by measuring total cellular protein levels using the SRB assay.²⁹ In short, cells were washed twice with PBS, fixed with 10% trichloroacetic acid (TCA) (1 h at 4°C) and washed with water. Cells were then stained with 0.5% SRB for 15 min and washed 4 times with 1% acetic acid. After drying, protein-bound SRB was dissolved in TRIS buffer (10 mM, pH 9.4) and the absorbance was measured at a wavelength of 540 nm in a plate reader.

The percentage of surviving cells was calculated considering non-treated cells at each time point as reference (100%). Ethanol:water (95:5) used for the administration of SCSs had no effect (data not shown). The IC₅₀ concentration was determined by plotting survival versus the log of the concentration and fitting a non-linear regression curve using GraphPad Prism software v8.01.

Finally, to compare both cell lines and evaluate in depth the SCSs effect, two calculations were done. First the Loss of Resistance Factor (LRF), which represents the variation provided by SCSs, under the combination with Dxr. It was calculated with the following formula:

$$LRF=\frac{DxrI{C}_{50}}{SCS+Dxr\hspace{0.17em}I{C}_{50}}$$

Secondly, the Resistance Ratio (RR) was calculated following the formula written below, were IC₅₀ MES-SA/MX2 is the IC₅₀ value of the resistant cell line and IC₅₀ MES-SA is the IC₅₀ value of the sensitive cell line for each condition tested:

$$RR=\frac{I{C}_{50}MESSA/MX2}{I{C}_{50}MESSA}$$

Thus, there are 3 possible situations:

RR>1 indicates resistance

RR=1 means both cell lines showed similar cytotoxic effect

RR<1 means sensitization

When IC₅₀ is >50 (not reached at 50 μM), a value of 50 was fixed to do the calculations.

In vivo assays

All in vivo experiments were done with male athymic nude mice, due to the human origin of the utero sarcoma cell lines, weighing 25–30 g and purchased from Envigo (Horst, The Netherlands). Animals were housed in microisolator cages under positive-pressure ventilation and maintained in closed-shelf laminar-flow racks, kept under standard laboratory conditions. Sterilized food and water were available ad libitum. The protocols of the study were approved by the Animal Experimentation Committee of the Erasmus MC and in accordance with the applicable European guidelines (2322 105-11-50 and 2419 105-11-55).

To induce a tumor, 15×10⁶ MES-SA or MES-SA/MX2 cells in 100 μl PBS were subcutaneously injected into the right flank of mice. Tumour growth and body weight were recorded every other day. When tumor volume was about 1 cm in diameter, animals were sacrificed and tumor divided into 3×3 mm pieces and use as a donor tumor. Tumor pieces were implanted subcutaneously in right flanks of mice used for pharmacokinetics, biodistribution and efficiency assays as indicated below.

Pharmacokinetics in tumor bearing mice

Tumor pieces from the donor tumor were implanted subcutaneously and tumor volume and body weight were recorded every two days, starting one week after tumor implantation. When tumor size was about 9×9 mm, animals were randomly divided in three groups (n = 9/group) to receive intravenously 4 mg/kg of free Dxr, LP-Dxr or LP-SCS-Dxr.

Blood samples (150 μL) were collected from the tail in heparinised capillary tubes at chosen times from 5 min to 24 h after treatment administration and immediately cooled on ice. Plasma was obtained from centrifugation at 3,500 g for 15 min at 4°C, and stored at −20°C until analysis. Total Dxr concentrations were determined in a fixed volume of 50 μl of plasma mixed with 50 μl of TritonX-100 (10%) to disrupt liposomes. In that way total Dxr was measured by fluorimetry (λ_ex475 nm; λ_em 590 nm) using a Wallac Victor 1420 microplate reader.

Dxr fluorescent signal at each time point (F_n) was compared to the 5 min signal (F₅), which was considered as 100% of administered treatment using the following formula:

$$F\left(\%\right)=\frac{F_n}{F_5}x100$$

Biodistribution of Doxorubicin liposomes

Tumor pieces of MES-SA or MES-SA/MX2 were implanted subcutaneously in 36 mice per cell line. Tumor volume and body weight were recorded every two days. When tumor reached at about 9×9 mm in size, animals were randomly divided in four groups (n = 9/group): 4 mg/kg of free Dxr, LP-Dxr, LP-SCS-Dxr or saline, intravenously. At 4, 12 or 24 h after treatment, a blood sample was collected from 3 mice/group followed by a perfusion with 20 ml of PBS trough the right ventricle of the heart to eliminate the main blood from the body. Afterwards, different organs and tumors were harvested.

To quantify total Dxr, 200 μl of the homogenate were mixed with 10% TritonX-100, 200 μL of water and 1.5 ml of acidified isopropanol. Mixture was stirred and Dxr was extracted overnight at −20°C. Tubes were centrifuged at 15,000 g for 20 min and supernatant was use to quantify total Dxr fluorescence (λ_ex475 nm; λ_em 590 nm). In all cases, to correct non-specific background fluorescence, control signal of each tissue was subtracted from sample signal. Besides, fluorescence was divided by the correspondent organ weight to normalize the signal.

In order to quantify nuclear Dxr, 200 μl of homogenate were mixed with 100 μl of sucrose (1.8 M) to preserve and separate nuclei from cell debris. After 10 min of centrifugation at 1,000 g and 4°C, supernatants were obtained and mixed with 10% TritonX-100, 200 μL of water and 1.5 ml of acidified isopropanol to extract Dxr. Following overnight incubation, tubes were centrifuged at 15,000 g for 20 min and Dxr supernatant was quantified by fluorimetry. Each sample was corrected for background (non treated samples). The percentage of nuclear Dxr was referred to total Dxr amount to calculate the percentages. Nuclei fluorescence was divided by the organ weight to normalize the value. In both cases data are expressed as micro equivalents of Dxr/g of tissue, as this assay does not discriminate between Dxr and Dxr metabolites.

Efficacy study

Tumor pieces from the donor tumor were implanted subcutaneously and tumor volume and body weight were recorded every two days, starting one week after tumor implantation. When tumors were around 6×6 mm, animals were randomly divided into 6 groups (n = 6/group) to receive intravenously the following treatments: Saline, free Dxr, LP-Dxr, LP-SCS-Dxr, LP-Empty and LP-SCS-Empty. Dxr dose was in all cases 4 mg/kg and the empty liposome dose was equivalent to Dxr liposomes. Treatments were administered every 5 days with a total of 3 doses. Tumor volume (V) was determined by measuring in two directions with a caliper and calculated using the formula:

$$V=\frac{A{B}^2}{2}$$

where A is the largest diameter and B the smallest diameter. Tumor and weight were followed after treatment, and animals were sacrificed by cervical dislocation when required.

Statistics

Statistical analysis was performed using t of student and Kruskall Wallis or ANOVA test followed by U of Mann Whitney comparison. P-values lower than 0.05 were considered as statistically significant. All parametric values are expressed as mean ± standard deviation (SD). Calculations were performed using Graphpad v8.01. In vivo efficacy assay statistics were done using Rstudio (TumGrowth package from Kroemerlab).

Results and Discussion

While chemotherapy is one of the most important pillars of cancer therapy, an unacceptable percentage of patients do not benefit from the treatment. The main reasons for that are undesired dose-limiting side effects and low drug levels at the target site, resulting from a rapid clearance and low tumor tissue penetration.¹³ Nanotechnology has been a very useful tool to overcome these issues, mainly improving pharmacokinetic properties of the encapsulated drug, with enhancement of tumor accumulation, accompanied by reduction of side effects. An increasing number of formulations are currently being evaluated in clinical trials or are already commercialized.³⁰ However, no formulation has been approved yet for MDR tumors in spite of some promising examples of tumors sensitization using nanoparticles.^31,32 In the present work we use an SCS-liposomal formulation to try to overcome the Dxr resistance of cancer cells. For that, Dxr resistant (Dxr IC₅₀ 41.72 μM) and sensitive (Dxr IC₅₀ 0.478 μM) cell lines were used (Supplementary figure S1).

Resistance mechanism evaluation

It is necessary to take into account that Dxr uptake is higher in MES-SA cells (Supplementary figure S1A). With the aim to explore the reasons behind Dxr resistance, the influx and efflux mechanisms of Dxr were evaluated. First, we evaluated the efflux of Dxr, related with the expression of Pgp in both cell lines. As shown in Figure 1, the resistant cell line has clearly higher Pgp surface expression compared to the sensitive cell line. Interestingly, a second peak was observed in MES-SA/MX2 cells that may correspond to a Pgp negative or lower expression cells, indicating possibly loss of resistance during culture or a smaller heterogeneous population (Figure 1A). On the contrary, MES-SA cells are almost 100% negative for Pgp expression. In fact, there is no significant difference with non-stained cells, as shown in Figure 1B.

Figure 1.

Evaluation of Pgp expression on MES-SA and MES-SA/MX2 human uterine sarcoma cell lines by flow cytometry. A) Representative histograms showing distribution of positive and negative (non stained) populations. B) Quantification of Pgp expression. Red color shows non-stained population (negative control) and α-Pgp stained results are depicted in blue. ** p<0.01.

Next, to evaluate the possible influx differences, lipidomics was performed to explore differences in lipid composition between sensitive and resistant cell lines. A total of 228 phospholipid species belonging to seven different phospholipid classes were identified upon lipidomic profiling of MES-SA and MES-SA/MX2 cells (Figure 2). Principal component analysis (PCA) was used, based on linear combinations of individual lipid species levels, in such a way that these new principal components best preserve the original variance in the data set.³³ The analysis reveals that both cell lines have a significantly different lipid composition as can be concluded from the clear separation of both cell lines on principal component 1 (p = 0.002), which accounts for 52% of all variance in the original dataset, while principal component 2 explains the 26% (Figure 2A). A deeper analysis to determine which lipid species contribute most to this difference is shown in panel 2B, where the contribution of each lipid species to PC-1 and PC-2 is plotted. The difference in lipidome composition results from changes in many lipid species, but taking into account Figure 2A, it can be concluded that MES-SA/MX2 is enriched in lipids species such as PI 38:5, PI 36:2 or PG 34:1, whereas MES-SA cell line is enriched in PI 38:4 or PI 38:3.

Figure 2.

Principal component analysis (PCA) of phospholipids found in the MES-SA and MES-SA/MX2 cell lines. A) The PCA score plot shows a clear difference in lipid profiles between the two cell lines. B) The PCA loadings plot demonstrates that many species contribute to the observed difference in panel A. Lipid species are colored by their lipid class and the size of the dot reflects the abundance of the species. C) Lipid class abundance as detected by LC-MS analysis of both cell lines. Plotted are the contributions of the lipid classes ± standard deviation to the total MS signal when separating lipid extracts of MES-SA and MES-SA/MX2 cell lines. D) Contribution of lipids based on the number of carbon atoms (y axis) in the fatty radyl groups of glycerophospholipids in the extracts; E) Degree of saturation of glycerophospholipid species in the lipid extracts. Bars represent the abundance of lipid species with unsaturated bonds (y axis) * p < 0.05; ** p < 0.01. PG: Phosphatidylglycerol; BMP: Bis-monoacylglycerophosphate; PI: Phosphatidylinositol; PE: Phosphatidylethanolamine; PS: Phosphatidylserine; PC: Phosphatidylcholine; SM: Sphyngomyeline.

PCA analysis is powerful in showing differences at the species level when there is no clear change at the class level as shown in Figure 2C, where the only significant difference (p<0.05) is found at PG. We then looked for changes in acyl chain length as represented by the total number of carbon atoms in the fatty acyl chains as it has been related before with thickness and diffusion rate of the membrane³⁴. Figure 2D shows that MES-SA/MX2 cells have more 30 and 32 carbon atoms lipid species (p<0.01) and less 40 carbon atoms lipids (p<0.05). The average number of carbon atoms in acyl chains was 36.65 in the MES-SA/MX2 cell line versus 36.86 in MES-SA. Although this difference is statistically significant (p<0.05), it is not likely that this 0.6% difference represents a physiologically relevant effect on membrane thickness. The distribution of lipid unsaturation in lipid species shows that the resistant MES-SA/MX2 cell line had more than two-fold unsaturated species at the expense of four-fold unsaturated species (Figure E).

The lipid species PC 36:4 and PI 36:2 contribute most to this difference (see Supplementary Figure S2 for boxplots of these species). Therefore, the MES-SA/MX2 cell line is enriched in lipids with saturated acyl chains that are more tightly packed and ordered than the unsaturated counterparts³⁴.

Several authors reported differences in saturation levels comparing resistant and non-resistant cancer tissues, although in general it strongly depends on the cancer type and the stage.^35–37 In our case, the lipidomic profile shows a significant difference in lipid composition: MES-SA/MX2 cell line has enrichment in shorter glycerophospholipids and a different unsaturation pattern. We hypothesize that these differences may be involved in a change on membrane assemblage and cell permeability that contributes to the resistant profile. In fact, the increase of unsaturated lipids in the membrane of cells has been related with an enhancement on lipid package and a permeability reduction to molecules like chemotherapeutics.³⁷

The cell entrance mechanism of Dxr involves a flip-flop cell membrane interaction and therefore any change in the lipid composition can affect the entrance of the chemotherapeutic.^20,38 Thus, this difference in the unsaturation lipids among MES-SA and MES-SA/MX2 cell lines could lead to a decrease of Dxr uptake in the resistant cell line.

Thus, there is a combination of resistance factors. Firstly, the increase of membrane rigidity that decreases the Dxr entrance in the resistant cell line. Secondly the overexpression of Pgp which pumps out the already low amounts of Dxr that enter the cell. We therefore conclude that there is not a unique explanation for the establishment of MDR mechanism.

SCSs effect over cells

Once these two mechanisms of resistance were stablished, we analyzed whether the SCS treatment is able to modify them. It was proposed that SCSs transfers spontaneously from the liposome to the cell membrane, where SCSs cluster and form transient channels which allows Dxr entrance into the cell.^20,39 Hence, the Pgp expression under treatment and the incorporation of SCSs to the membrane were evaluated.

As expected, SCS lipids were able to sensitize MES-SA/MX2 cells administered in free form and in the absence of FBS (see supplementary data S3 for more details). However, these lipids do not form micelles and it is not possible to administer them in free form. Therefore they were included into liposomes that allow us the co-delivery of both therapeutics.

Doxil-like liposomes were prepared by adding 10% of SCSs to the composition³⁹, which did not modify the physicochemical characteristics of the liposomes as can be seen in supplementary material S4 and Cryo-TEM images confirm intact while elongated Dxr-containing liposomes.

Cells were exposed for 4 and 24 h to free SCS, SCS liposomes and conventional liposomes and the Pgp expression was evaluated by flow cytometry. Figure 3 shows that MES-SA cells remained negative for Pgp whereas MES-SA/MX2 cells were still positive after all treatments. Both cell lines showed no significant differences after SCSs treatment, indicating that SCS lipids are not interfering at all with Pgp expression in the cell membrane.

Figure 3.

Pgp expression in MES-SA and MES-SA/MX2 after 4 or 24 h of exposure to different treatments. Data correspond to 2 independent experiments carried out in triplicate.

Regarding SCSs incorporation in the cell membrane, a confocal assay was performed by preparing SCS liposomes using fluorescent SCS. Figure 4 shows the incorporation of fluorescent SCS in cells after 4 h of exposure. We observed a higher degree of interaction of SCSs with MESA-SA/MX2 cell compared to MESA-SA cells. Hence, there is a weaker SCS signal, in green, in MES-SA cells in comparison with MES-SA/MX2 cell line, where the SCSs incorporation to cells is clearly higher.

Figure 4.

Confocal images (20X) of sensitive and resistant cell lines after incubation with LP-SCS-Dxr prepared with fluorescent SCS lipids for 4 h and 24 h. The white bar represents 100 μm of length.

It is remarkable that in the presence of LP-SCS-Dxr, the fluorescent signal pattern of Dxr changes, being more homogeneous and affecting almost all cells (Figure 4). This may be explained by the effect of SCSs on all cells and as such equalizing uptake among the cells. For more detailed images of Dxr uptake by confocal microscopy see Supplementary Figure S5.

Dxr uptake and cytotoxicity assays

These results underline the statement made above that SCSs have a more profound effect on resistant cells. In consequence, and in line with the results obtained with confocal microscopy, there is a more efficient uptake of Dxr by resistant cells.

As shown in Figure 5, Dxr uptake analyzed by flow cytometry showed that resistant cells had low Dxr uptake after 4 and 24 h of exposure than MES-SA cells. Again, the resistant cell line consisted of two populations as two peaks appeared; one with low Dxr uptake and a smaller population with more drug uptake, comparable to the sensitive cell line and probably related with the lower Pgp expression cells. However, the combination of SCSs with free or encapsulated drug improves Dxr uptake significantly, especially by MES-SA/MX2 (Figure 5B and C), where Dxr levels triplicate the one of non SCS treated cells.

Figure 5.

Dxr uptake in MES-SA and MES-SA/MX2 uterine sarcoma cell lines. A) Representative histograms obtained after 4 or 24 h of exposure to different treatments; B) MES-SA cell line analysis of FACS results; C) MES-SA/MX2 analysis of the cytometry assay. The experiment was repeated 3 times in triplicate.

Already at 4 h of exposure an obvious and significant effect of SCSs on Dxr uptake was observed, which continued to increase after that showing a time dependent mechanism. The SCS-effect administered in free form was more important compared with SCSs on liposomes, that becomes statistically significant after 24 h in MES-SA/MX2 cells (p<0.05). Thus, it seems that in the SCS liposomes the time dependence is more relevant.

To evaluate the impact of SCSs and Dxr uptake in the selected cell lines, a cytotoxicity assay was performed. Cells were exposed to increasing amounts of several treatments for 4 h and the survival was determined at 72 h.

Comparable to free SCSs, the incorporation of SCSs in Dxr liposomes augments cytotoxicity towards MES-SA/MX2 in a higher extent than to MES-SA cells (Figure 6). In fact, it reaches an effect comparable to free Dxr plus SCSs. Regarding IC₅₀ values, the addition of free SCSs decreased the RR 4-fold (Table 1A).

Figure 6.

Cytotoxicity profiles of A) MES-SA and B) MES-SA/MX2 cells at 72 h after 4 h of exposure to Dxr, free or encapsulated, combined with SCSs in free form or incorporated in liposomes. Cell survival was calculated as percentage of non-treated cells. Average of 3 experiments in triplicates +/− SD is depicted.

Table 1.

IC₅₀ values obtained after 4 h of treatment exposure (average and standard deviation of 3 independent experiments) for sensitive and resistant cell lines. A) IC₅₀ values obtained at 24, 48 and 72 h after treatment. B) These values were used to calculate the RR, dividing the IC₅₀ of MES-SA/MX2 cell line by IC₅₀ values of MES-SA cell line. 1: p<0.05 vs Free Dxr; 2: p<0.05 vs Dxr + SCSs; 3:p<0.05 vs LP-Dxr.

A)	MES-SA
	Dxr	Dxr + SCSs	LP-Dxr	LP-SCS-Dxr
24 h	2.30 ± 0.69	1.42 ± 0.32	> 501.2	> 501.2
48 h	2.09 ± 0.80	1.22 ± 0.44	> 501.2	30.44 ± 6.551.2.3
72 h	1.99 ± 1.27	1.59 ± 1.66	> 501.2	29.05 ± 8.391.2.3
	MES-SA/MX2
	Dxr	Dxr + SCSs	LP-Dxr	LP-SCS-Dxr
24 h	> 50	25.24 ± 3.741	> 502	53.92 ± 8.042
48 h	36.16 ± 7.17	10.24 ± 0.041	> 501.2	26.59 ± 8.202.3
72 h	28.49 ± 10.18	6.70 ± 3.341	> 501.2	23.66 ± 4.262.3
B)	Loss of Resistance Factor
	MES-SA		MES-SA/MX2
	Dxr/Dxr + SCSs	LP-Dxr / LP-SCS-Dxrd	Dxr/Dxr + SCSs	LP-Dxr / LP-SCS-Dxr
24 h	1.69 ± 0.55	1 ± 0	1.99 ± 0.21	0.93 ± 0.09
48 h	1.76 ± 0.55	1.81 ± 0.35	3.53 ± 0.48	1.94 ± 0.43
72 h	1.702 ± 1.52	1.83 ± 0.61	5.15 ± 3.76	2.13 ± 0.27
C)	Resistance ratio
	Dxr	Dxr + SCSs	LP-Dxr	LP-SCS-Dxr
24 h	23.33 ± 8.08	17.85 ± 0.99	1.00	1.07 ± 0.11
48 h	14.48 ± 2.99	9.12 ± 3.24	1.00	0.96 ± 0.30
72 h	17.35 ± 6.83	4.36 ± 2.94	1.00	0.72 ± 0.11

When SCSs are incorporated in liposomes a RR below 1 is reached, indicating that the resistant cells were sensitized to Dxr, becoming as sensitive to Dxr as the sensitive cell line. In fact, the LRF shows no change in MES-SA cell line over time, regardless of treatments, whereas there is a clear time dependence in MES-SA/MX2 cells, as LRF increases in both treatments in time (Table 1B).

With these in vitro experiments we confirm the proposed hypothesis, where SCSs transfers spontaneously from liposomes when encountering a resistant tumor cell and modulates the cell membrane permeability, which improves drug influx.²⁰ Moreover, this transference of SCSs not only results in the permeabilization of resistant cells, but at the same time liposomes are being destabilized, favoring Dxr release in the proximity of tumor cells.⁶ This is not a minor effect, as the rigidness and stability of Doxil-like liposomes is one of the main reasons for their low efficacy.^40,41

Biodistribution and Pharmacokinetics

Based on the in vitro results, the behavior of SCS formulations was evaluated in vivo. For that, mice bearing MES-SA or MES-SA/MX2 tumors of around 100 mm³ were treated intravenously with free Dxr, LP-Dxr or LP-SCS-Dxr, and Dxr. Pharmacokinetics and tissue distribution was evaluated. We observed that blood residence of Dxr was independent of type of tumor (data not shown) and therefore these results were pooled (Figure 7).

Figure 7.

Pharmacokinetic profile of different treatments in tumor bearing mice. Dots represent the average of 6 mice and bars, the standard deviation.* p<0.05.

In agreement with the literature, free Dxr was rapidly eliminated from blood circulation while encapsulated Dxr circulated for 16–18 h.^42–47 For LP-SCS-Dxr, in line with similar formulations⁴⁸, circulation time was shorter, around 8 h. Apparently, the presence of SCSs in liposomes either promotes clearance of the liposome or destabilizes Dxr encapsulation, probably due to serum proteins interacting with SCSs. We found the same issue in in vitro assays. A deeper research needs to be done to tackle the serum and SCSs interaction question. Nevertheless, around 20% of the administered dose was still circulating at the 24 h time point.

In order to evaluate whether the faster clearance of SCS formulations is reflected by the Dxr organ biodistribution, uptake in tumor and other tissues were studied. Therefore, Dxr was quantified in mice bearing sensitive and resistant tumors, treated with SCSs and non-SCS Dxr liposomes. At different time points after treatment (4, 12 and 24 h), organs were harvested and Dxr content analyzed by fluorescence. The signal is expressed after normalization per tissue weight to decrease inter-individual differences.

Dxr changes in tissue distribution between MESA-SA and MESA-SA/MX2-bearing mice were not observed, except for the accumulation in the spleen at 24 h (Supplementary Figure S5). Also, between LP-Dxr and LP-SCS-Dxr non-obvious differences were seen.

Dxr accumulation in tumor tissue is shown in more detail in Figure 8. Dxr levels are similar comparing both tumor types. However, the accumulation of Dxr in tumors is dependent on the formulation.

Figure 8.

Dxr quantification in Tumor tissue in detail at 4, 12 and 24 h after treatment administration. Bars correspond to 3 mice values +/− SD.* p< 0.05; ** p<0.01.

Free Dxr showed no differences over time, being lower in the resistant tumor. The encapsulation of Dxr enhanced tumor accumulation from 12 h onwards. SCS liposomes were able to increase the drug amount in tumor tissue significantly only in the MES-SA/MX2 model, which is in line with the results shown above (Figure 8).

It has been claimed that total accumulation of Dxr in tumors may not give a good indication of activity, as it does not discriminate between bioavailable and entrapped Dxr and does not necessarily reflect presence of Dxr at the target, the nucleus.¹⁵ Therefore, we further analyzed the accumulation of Dxr in nuclei in the same samples as above.

As expected, free Dxr accumulation was less in resistant cells nuclei, compared to sensitive cells (Figure 9A), while administration of LP-Dxr resulted in comparable nuclear Dxr levels (Figure 9B). Importantly, administration of LP-SCS-Dxr resulted in an augmented accumulation of Dxr in nuclei of resistant tumor cells (Figure 9C).

Figure 9.

Percentages of nuclear Dxr in tumor tissue at 4, 12 and 24 h. A) Free Dxr treated mice; B) LP-Dxr treated group; C) LP-SCS-Dxr treated mice. Columns represent percentages of 3 samples, and bars the SD.* p < 0.05.

Efficacy evaluation of SCS liposomes

As we observed an improved accumulation of Dxr when SCSs were included in the liposomes, an efficacy assay was performed to evaluate the antitumoral effect of LP-SCS-Dxr (Figure 10). It is necessary to take into account the different growth profile of both tumor models. Thus, the doubling time of MES-SA tumor was 4 days compared to MES-SA/MX2 that corresponded to 6 days^49,50. Dxr acts as an intercalating agent and therefore it is more effective in fast growing tumors, apart from the resistance itself.

Figure 10.

Tumor growth profiles after 60 days of A) MES-SA and B) MES-SA/MX2 tumor-bearing mice. Mice were treated with PBS (control), free Dxr, LP-Dxr or LP-SCS-Dxr for three times at 3, 7 and 11 days when tumor reached the proper size. Lines represent the average of 6 mice +/− SD. All groups showed a statistically significant difference compared to control.

While the administration of LP-Dxr delayed tumor growth in all cases regarding free Dxr treatment, SCS-containing liposomes did not further improve the tumor growth control given by Dxr liposomes in mice bearing sensitive tumors. This result was expected as all our data consistently shows that SCSs do not affect the sensitive MES-SA cell line.

However, administration of LP-SCS-Dxr had a significant inhibitory effect in MESA-SA/MX2-bearing mice compared to LP-Dxr, although all mice showed eventually outgrowth of the tumor. At this point it is necessary to analyze all in vivo data as a whole. An unfavorable pharmacokinetic profile of SCS liposomes compared to non modified Doxil-like liposomes was observed, probably as a result of serum components interaction with SCSs. Nevertheless, Dxr encapsulated in liposomes is able to accumulate in tumor tissue 2–3 times more than Dxr administered in free form. And, more importantly, the nuclei accumulation of Dxr delivered by SCS liposomes is improved in MES-SA/MX2 tumor cells compared to sensitive tumor cells. Together, incorporation of SCSs, while no changing the biodistribution pattern, improved the effect on the resistant tumor. This result is comparable with previous publications, that show an increase in nuclear drug accumulation with other formulations while maintaining comparable biodistribution⁵¹. Although stability and pharmacokinetics need to be further improved, we conclude that SCS-modified Dxr liposomes exert a better antitumor effect, showing the important possibilities of this formulation.

Conclusion

The present work shows that Dxr resistant cancer cells exhibit differences in the composition of the cell membrane and Pgp expression when compared to sensitive cells. We demonstrate that SCSs improves the therapeutic potential of Dxr liposomes by selectively enhancing the membrane permeability of resistant tumor cells, while having no effect on sensitive cells. Besides, the effect SCSs inflicts does not result from alteration in the Pgp expression. Thus, the reason behind this selectivity should be likely related to the cell membrane composition, which is enriched in unsaturated and shorter lipids in the case of MES-SA/MX2.

We confirmed the resistant cell membrane modulation in several in vitro assays that showed the sensitization capacity of these SCS lipids. Besides, in vivo assays show that in spite of a shorter blood residence time of LP-SCS-Dxr, these liposomes were able to reach the tumor and overcome, at least partially, the resistance of MES-SA/MX2. Importantly, we show that the effect SCSs have on tumor growth correlates with augmented drug accumulation in vitro and in vivo specifically in resistant tumor cells

We hypothesize that improvement in pharmacokinetics will achieve a further enhancement of the antitumor effect. Finally, we propose a combined therapy with a Pgp inhibit or to obtain a better antitumor efficacy and to achieve more complete sensitization of resistant tumors.⁵²