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Toxicology and Applied Pharmacology

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Targeting lung cancer stem cells using combination of Tel and Docetaxelliposomes in 3D cultures and tumor xenografts

Peggy Arthura,⁎,1, Nilkumar Patela,⁎,1, Sunil Kumar Surapanenia, Arindam Mondalb,Aragaw Gebeyehua, Arvind Bagdea, Shallu Kutlehriaa, Ebony Nottinghama, Mandip Singha,⁎

a College of Pharmacy and Pharmaceutical Sciences, Florida Agricultural and Mechanical University, Tallahassee, FL 32307, USAb Rutgers University, New Brunswick, NJ, USA

A R T I C L E I N F O

Keywords:Cancer Stem Cells (CSC)Docetaxel Loaded PEGylated Liposomes(DTXPL)Docetaxel (DTX)Telmisartan (Tel)Non-small Cell Lung Cancer (NSCLC)Resistance

A B S T R A C T

Cancer stem cells (CSCs) accounts for recurrence and resistance to chemotherapy in various tumors. Efficacy ofchemotherapeutic drugs is limited by tumor stromal barriers, which hinder their penetration into deep tumorsites. We have earlier shown telmisartan (Tel) pretreatment prior to Docetaxel (DTX) administration enhancesanti-cancer effects in non-small cell lung cancer (NSCLC). Herein, we demonstrated for the first time the efficacyof Docetaxel liposomes (DTXPL) in combination with Tel in 3D cultures of H460 cells by using polysaccharide-based hydrogels (TheWell Biosciences) and also in xenograft model of DTX resistant H460 derived CD133+ lungtumors. DTXPL and Tel combination showed enhanced cytotoxicity in H460 WT 3D cultures by two folds. InH460 3D cultures, Tel pretreatment showed increased liposomal uptake. DTXPL and Tel combination treatedtumors showed reduction in tumor volume (p < .001), increased apoptosis and downregulation of CSC markers(p < .01) in H460 WT and DTX resistant CD133+ xenograft models.

1. Introduction

Despite recent advances in the field of cancer biology for thetherapy of various cancers, clinical progress among lung cancer patientsis not remarkable and lung cancer remains as the prominent cause ofmortality worldwide (Torre et al., 2012; Molina et al., 2008). Althoughdiverse chemotherapeutic drugs have been utilized, they have beenineffective due to their poor uptake and development of drug resistancein tumor cells (Reck et al., n.d.).

In our earlier studies, we have demonstrated improved intratumoraldistribution of docetaxel nanoparticles into the lung tumors by priororal administration of telmisartan (Tel) and also by inhalation admin-istration of Losartan (Godugu et al., n.d.-a). Anti-fibrotic agents like Telhave been used and reported for decreasing interstitial tumor fibrosisby altering TGF-β signaling pathway in lung cancer models and theyalso promote intratumoral distribution of nanoparticles (Sandhiyaet al., 2009; Cabral et al., 2011; Shields et al., n.d.). Role of nanocarriersbehind effective targeted delivery of chemotherapeutics is well de-monstrated in intractable solid tumors (Prud'Homme, 2007; Kano et al.,2007). Recent studies have shown that Tel activates PPAR γ and inhibits

TGF-β induced ECM production in tumors (Benson et al., 2004; Funaoet al., 2009; Lakshmanan et al., n.d.; Geirsson et al., n.d.). Moreover,Ang-II peptides also effect the growth and metastasis of lung cancercells (Rodrigues-Ferreira et al., n.d.). Further, Docetaxel, which is ba-sically a taxane derivative has been widely used as a second line che-motherapeutic agent in lung cancer (He et al., n.d.).

Targeting of cancers using various chemotherapeutic formulationssuch as tumor cell specific nanoparticles, liposomes and specific cellpenetrating peptides is well demonstrated (Sandhiya et al., 2009).These strategies significantly improve the clinical outcome of drugs intumor tissues without disrupting the normal homeostasis of the sur-rounding non-cancerous cells (Sandhiya et al., 2009; Kim et al., n.d.-a;Liu et al., n.d.; Cho et al., 2008; Gobin et al., 2008). Delivery of ther-apeutic payloads to tumor cells is considered as a major clinical chal-lenge due to the presence of dense collagen in solid tumors, whichhinders intratumoral distribution of drugs or delivery systems (Goduguet al., n.d.-a; Cabral et al., n.d.; Shields et al., n.d.; Diop-Frimpong et al.,n.d.; Jain and Stylianopoulos, n.d.). These hindrances cause relapse andpropagation of cancers even after successive treatments and these aredue to the presence of chemoresistant cancer stem cells (CSCs), which

https://doi.org/10.1016/j.taap.2020.115112Received 29 March 2020; Received in revised form 16 May 2020; Accepted 10 June 2020

Abbreviations: CSC, Cancer stem cells; DTXPL, Docetaxel loaded PEGylated liposomes; Tel, Telmisartan; NSCLC, Non-small cell lung cancer; DTX, Docetaxel⁎ Corresponding author.E-mail address: mandip.sachdeva@gmail.com (M. Singh).

1 These two authors contributed equally to this work.

Toxicology and Applied Pharmacology 401 (2020) 115112

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are not effectively targeted by conventional chemotherapeutics and arehaving self-renewal and differentiation ability (Zakaria et al., n.d.).

Stem cells are gaining universal attention in various types of can-cers, including lung cancer. A study by Carney et al., demonstrated thatcells (1.5%) taken from adenocarcinoma patients produced colonies in-vitro and transplantation of these into athymic nude mice regeneratedtumors with features characteristic of the original tumors (Carney et al.,1980). Also, Sox2, Oct4, Nanog and CD44 are considered as the char-acteristic cancer stem cell markers which promote tumorigenesis(Hadjimichael et al., n.d.). The transcription factor, NPM1 individuallyforms complexes with Sox2, Oct4 and Nanog in the embryonic stemcells and is involved in tumorigenesis (Johansson and Simonsson, n.d.).Moreover, Sox2 with Oct4 and Nanog activates key tumorigenic factors.CD44, another CSC marker is overexpressed in both the membrane andcytoplasm of the lung cancer cells. CD44 binds to hyaluronan and fa-cilitates epithelial to mesenchymal transition (EMT) in lung cancer cells(Park et al., n.d.). EMT transition mainly helps the lung tumors to evadethe immune system by expressing Programmed Cell Death ReceptorLigand (PDL1) on their surface, which interacts with Programmed CellDeath receptor protein PD1 and thereby blocks the activation of the T-cytotoxic cells against the solid tumors (Gordon et al., n.d.). Twist1along with other transcription factors such as Snail1 and ZEB1 alsoregulates EMT in the tumor cells (Noman et al., n.d.). Cancer cellsgenerate ROS and get sensitized by ROS mediated apoptosis, indicatingthat alterations in the levels of intracellular ROS will determine thesurvival and death of cancer cells (Liou and Storz, n.d.). It is apparentthat killing of the Cancer Stem Cells (CSC) is a serious unmet clinicalneed to curb down the propagation and growth of various cancers in-cluding non-small cell lung cancer.

Several studies have shown the therapeutic potential of gemcita-bine, cisplatin, taxotere, and vinorelbine in decreasing the expression ofcancer stem cell markers. However, development of drug resistance byside populations of cancer stem cells is also well documented(Vinogradov and Wei, n.d.). Hence the need for single punch multi-modal strategy through a suitable drug delivery method, which caneffectively target the cancer stem cells and the different pathwayswhich facilitate the growth of cancer and cancer stem cells is highlyessential.

Liposomes have gained high recognition and attention in the clinicalapplications from over two decades (Goyal et al., 2005). Several lipo-somal formulations are on the market (e.g. Doxil, Daunoxome, Depocyt,Myocet, Marqibo) for treating a variety of cancers (Moosavian et al.,n.d.). Despite so many efforts to improve the effective delivery of cancerchemotherapeutics by using liposomes (Cornelison et al., n.d.), no li-posomal formulation has been approved till now for treating chemo-resistant tumors due to the overexpression of efflux pumps {ATP-binding cassette (ABC) transporters, which include MDR1 (ABCB1),MRP1 (ABCC1) and ABCG2} in tumors. Present on the plasma mem-brane of tumor cells, these transporter proteins function as energydriven pumps, facilitate ATP hydrolysis upon chemotherapeuticsbinding, lower the intracellular drug concentrations (i.e., which inducetoxic effects to tumors), thereby facilitating tumor survival and con-ferring multidrug resistance to tumors (Gottesman et al., 2002).Moreover, dense collagen and fibrin network also play a vital role inhindering the effective intratumoral distribution of the liposomal for-mulations (Cabral et al., n.d.; Shields et al., n.d.; Diop-Frimpong et al.,n.d.; Jain and Stylianopoulos, n.d.; Calcagno and Ambudkar, 2010).

Role of Losartan and Tel in decreasing tumor interstitial fibrosis andpromoting intratumoral distribution of nanoparticles and liposomes iswell documented (Godugu et al., 2013; Patel et al., 2016a; Chen et al.,2018). Tel decreases non-small cell lung cancer (NSCLC) cell pro-liferation by inhibition of PI3K signaling (Zhang and Wang, 2018) andactivation of peroxisome proliferator activated receptor-γ (PPARγ)pathways (Li et al., 2014). Recently, it was reported that Tel improvesthe efficacy of Actinomycin D against lung cancer stem cells (CSCs) byfunctioning as tumor stromal disrupting agent (Green et al., 2019). Tel

treatment increases reactive oxygen species (ROS) production(Rasheduzzaman et al., 2018), which might be also beneficial in tar-geting lung CSCs as ROS regulates Wnt/β-catenin signaling pathway(Hoogeboom and Burgering, 2009; Steinhusen et al., 2000), which ismajorly associated with maintenance, expansion, and epithelial-me-senchymal transition of cancer stem cells (Martin-Orozco et al., 2019).Based on these findings and since CSCs mostly reside in the hypoxicinner core of the tumors, we have selected Tel in our study by assumingthat it significantly decreases collagen production and may facilitateeffective penetration of docetaxel liposomes into the CSCs. In this study,we have evaluated the efficacy of combination therapy of Tel andDTXPL in H460 WT, CD133+ H460 stem cells and DTX resistantCD133+ xenograft models of lung cancer.

2. Methods

2.1. Materials

Tel was purchased from Alpha Aesar (MA 01835). DTX andDulbecco's Modified Eagle Medium (DMEM) media was procured fromSigma Aldrich (St. Louis, MO 63178). Mycoplasma free Non-Small CellLung Cancer (NSCLC) cell line (H-460) was procured from ATCC. Theantibodies were purchased from Cell Signaling Technology, (Danvers,MA 01923). The CD133 stem cell isolation kit, MACS was procuredfrom Miltenyi Biotec (Germany, Catalogue number 130–097-049). Fetalbovine serum (FBS) was obtained from Thomas scientific(Swedesborow, NJ 08085).

2.2. Cell culture

CD133+ stem cells isolated from H-460 cells using MACS separationkit (Miltenyi Biotec, Germany) were cultured in collagen coated T75cm2 tissue culture flasks using DMEM media supplemented with 10%FBS and 0.01% antibiotics (penicillin, streptomycin, neomycin) understandard conditions of 37 °C and 5% CO2 in a controlled humidifiedincubator. Appropriate number of cells were seeded into different well-plates for various experiments once they reach 70–80% confluency.

2.3. Isolation of CD133+ stem cells from H460 cell line

CD133+ stem cells were isolated by using MACS separation kit fromMiltenyi Biotec (Germany). Briefly, the lyophilized MACS magneticmicrobeads were reconstituted by using reconstitution buffer (con-taining stabilizer and 0.05% sodium azide). The cells were then mag-netically labeled using the CD133 MicroBeads as per the MACS se-paration kit protocol and were finally isolated using the MACSseparation column and MACS separator.

The % yield of CD133+ cells from H460 WT cells was 0.9–1% in ourstudy. This mostly depends on how successfully we label CD133+ cellswith CD133 MicroBeads and how efficiently we separate them usingMACS® Column, which was placed in the magnetic field of a MACSSeparator. A growing body of evidences demonstrated % yield ofCD133+ cells ranges from 0.5–1.1% from an initial population of H460WT cells (Yi et al., 2012; Liu et al., 2013; Barr et al., 2013; Wang et al.,2017).

2.4. Preparation of Docetaxel PEGylated liposome

Liposomes were prepared by using modified hydration method asdescribed in our earlier study (Patel et al., 2016a). Briefly, phospholi-pids like DOPC (1, 2-dioleoyl-sn-glycero-3-phosphocholine), choles-terol, PEG 2000 and DTX suspended in chloroform solution were addedto mannitol in a drop wise manner under conditions of constant stirringat 45 °C and then subjected to chloroform evaporation overnight. Re-sultant powder was then dispersed in water and sonicated for 4minusing probe sonicator (Branson Probe Sonicator, USA). Molar ratio of

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DTX to phospholipids, and processing parameters were optimized basedon physical stability. Hydrodynamic diameter of formulation was ana-lyzed by the dynamic light scattering (DLS) method by using NicompPSS particle size analyzer (PSS Systems, USA). Entrapment efficiencywas analyzed through gel permeation chromatography method by usingSephadex G50 column (Sigma Aldrich, USA).

2.5. In-vitro anticancer effects of DTXPL and Tel

Non-small cell lung cancer cell line, H460 WT and CD133+ H460derived stem cells were seeded in 96 well plates (8× 103 cells/well).After 24 h, cells were treated with various concentrations of DTXPL(ranging from 400 to 12.5 nM), Tel (500 to 31.25 μM) and combinationof DTXPL and 50 μM Tel. After 24 h, cells were fixed in 0.25% glutar-aldehyde and were stained with 0.05% crystal violet dye. Percent of cellviability or cell death was calculated by considering DMSO (0.1%)treated cells as 100% viable (zero % cell death). For comparison of 2Dand 3D cytotoxicity data, 2D cultures of H460 WT cells (5× 104 cells/well) were seeded in 24 well plates (same cell number was also used in3D cultures). This cell number was selected from our preliminary op-timization studies for the formation of spheroids in 3D cultures by usingVitrogel 3D RGD. After 18–24 h, cells were treated with various con-centrations of Tel alone, (ranging from to 300 to 37.5 μM), DTX (ran-ging from 25 to 3.125 μM) and the combination of DTX and Tel(keeping the concentration of Tel constant at 50 μM. DMSO (0.1%)treated cells were considered as control. Cell viability was determinedusing crystal violet assay.

2.6. 3D cell culture

VitroGel 3D-RGD solution and dilution solution Type 1 buffer werereceived as a kind gift from TheWell Biosciences (NJ, USA). TheVitroGel 3D-RGD solution was pre-warmed to 37 °C and diluted withdilution buffer in a ratio of 1:2 (hydrogel: dilution solution). The di-luted hydrogel solution was mixed with the cell suspension (i.e.,1.2× 106 cells) and then 250 μL was pipetted into each well of a 24well plate. The plate was then left at room temperature for stabilizationof hydrogel for 15min. 250 μL of organoid media was then added intoeach well to cover the hydrogel. The plate was placed in an incubator at37 °C and media was changed for every 24 h. Spheroid formation wasdetermined by observing under fluorescent microscope (OlympusBX51) after 5 days. After spheroids formation, cells were treated withdifferent concentrations of Tel alone (ranging from 1000 to 125 μM),Docetaxel alone (ranging from 100 to 0.390 μM) and Docetaxel and Telcombination (ranging from 100 to 0.390 μM) for 24 h. The cell vi-abilities of the cells were then determined using MTT assay.

2.7. Coumarin-6 loaded liposomes uptake in H460 WT cells

Drug uptake studies were performed by using Coumarin 6 liposomesin 2D cultures and 3D spheroids of H460 WT cells. In 2D cultures, cells(i.e., 5× 104 cells/well) were seeded in a 24-well plate. After 24 h,wells were pretreated with Tel for 6 h before treatment with Coumarin-6 liposomes for 24 h. Coumarin-6 liposomal uptake in untreated cellswas considered as control. The intensity of green color, which indicatescoumarin-6 uptake in cultures was determined by using.

Image J software. In 3D cultures, H460 WT cells (5× 104 cells/well) were plated using VitroGel 3D RGD and the same procedure wasfollowed as mentioned above.

2.8. Hypoxia assay

Vitrogel 3D LDP2 (based on the polysaccharide, laminin) was usedfor 3D cultures of H460 WT cells seeded at a density of 5× 104 cells/well in a 24 well plate. Hypoxia assay has been performed twice with 4wells/group/study in a 24 well plate. 3D spheroids were maintained in

organoid media for 7 days. 3D spheroids were divided into four groups,each containing 4 wells: DTXPL alone, Tel and DTXPL+Tel group anduntreated (control) cells. The spheroids were subjected to the drugtreatments for 24 h. Image-iT™ Green Hypoxia reagent (ThermoFischer) stock solution (10 μM) was added to the spheroids and thenincubated further at 37 °C for 12 h. 3D spheroid images were capturedusing fluorescent microscope (Olympus BX51) by using standard FITCfilter. This experiment was repeated two times and the intensity ofgreen color as a measurement of hypoxia in 3D cultures was quantifiedby using Image J software.

2.9. DCFDA staining for ROS determination

ROS measurement was performed by using carboxy-H2DCFDA(Sigma Aldrich, USA) dissolved in sterile dimethyl sulfoxide (DMSO).H460 cells (5× 104 cells/well) cultured in 24 well plate by usingVitrogel LDP2 for 5 days were treated with DTXPL, Tel, DTXPL AND Telcombination for 24 h. After treatment, cells were washed with HEPESbuffered salt solution (HBSS) and incubated with 10 μM of carboxy-H2DCFDA at 37 °C for 30min in the dark. The green fluorescent in-tensity was measured using the Infinite 200 PRO multimode platereader (Tecan Group Ltd., Switzerland) at excitation 485 nm andemission 528 nm as described earlier (Sinthupibulyakit et al., n.d.).

2.10. Animals

BALB/c mice (female 6- weeks old, athymic Nude-Foxn1nu) werepurchased from Envigo. The mice were housed and maintained inspecific pathogen-free conditions in the facility approved by theAmerican Association for Accreditation of Laboratory Animal Care.Food and water were provided to the animals in standard housed cages.Animals were maintained under standard conditions of 37 °C and 60%relative humidity. All experiments were done in accordance with theguidelines of the Institutional Animal Care and Use Committee (IACUC)at Florida A&M University. Animals were acclimatized for 1 week priorto the tumor studies.

2.11. Inoculation of H460 WT cells in mice and antitumor study (1stround)

H460 WT cells were cultured in RPMI 1640 media supplementedwith 10% FBS under standard conditions of 5% CO2 and 37 °C in acontrolled humidified incubator. H460 WT cells (2.5× 106 cells/an-imal) were mixed with Matrigel (1:1) and were then subcutaneouslyinjected into the right flank of each mice. Two weeks post tumor cellsimplantation, DTXPL and Tel treatment was started. Mice were ran-domly divided into 4 groups (Untreated, DTXPL alone, Tel and DTXPL+ Tel) with 4 animals per each group. Tel oral treatment was started onthe 15th day after inoculation of cells, followed by DTXPL IV admin-istration on the 17th day. Tel (10mg/kg) was administered every daywhereas DTXPL (5mg/kg) was administered twice a week. Tumor vo-lume was measured on every 4th day during 2 weeks of treatment byusing a digital Vernier caliper.

2.12. Inoculation of CD133+ stems cells derived from H460 WT tumors inmice and antitumor study (2nd round)

CD133+ stem cells were isolated from H460 WT cells obtained fromthe DTXPL treated H460 tumor tissue. CD133+ stem cells were grownin RPMI 1640 media containing 10% FBS and standard antibiotic mix at5% CO2 and 37 °C. The animals were randomized into 4 groups (4animals per each group). The CD133+ cells (2.5× 106 cells/animal)were mixed with Matrigel (1:1) and were subcutaneously injected intothe right flank of mice. The animals were subjected to the same treat-ments and tumor measurements were carried out as followed during thefirst round.

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2.13. Inoculation of Docetaxel-resistant CD133+ stem cells from docetaxeltreated tumors in mice and antitumor study (3rd round)

Docetaxel-resistant CD133+ stem cells were isolated from DTXPL-treated CD133+ tumor tissue from the second round of animal ex-periment. The CD133+ stem cells were grown in RPMI 1640 mediacontaining 10% FBS and standard antibiotic mix at 5% CO2 and 37 °C.These resistant cells (2.5× 106 cells/animal) were mixed with Matrigel(1:1) and were subcutaneously injected into the right flank of the mice.There were three treatment groups (Untreated, DTXPL, and DTXPL+Tel) with four mice per group in this study.

2.14. Western blot analysis

Proteins were extracted from tumor tissues collected from control(untreated) and treated groups by using RIPA lysis buffer (50mM Tris-HCL, pH 8.0, with 150mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxychlorate, and 0.1% sodium dodecyl sulfate)with protease inhibitor and 500mM phenyl methyl sulfonyl fluoride(Godugu et al., n.d.-b). Protein estimation was carried out by using BCAProtein Assay Reagent Kit (PIERCE, Rockford, IL). Equal amounts ofsupernatant protein (50 μg) from the control and treatment groups weredenatured by boiling for 5min in SDS sample buffer, and were sepa-rated by 10% SDS-PAGE, transferred to nitrocellulose membranes forimmunoblotting. Membranes were blocked with 5% skim milk in Tris-buffered saline buffer containing Tween 20 [10mM Tris-HCL (pH 7.6),150Mm NaCl, and 0.5% Tween 20] and were probed with Sox2, CD44s{CD44 (156-3C11) Mouse mAb #3570}, which recognizes the epitopecentered around Pro210 of human CD44 (UniProt ID P16070), MMP9,Survivin, Caspase 3, Cyclin D1, p-STAT 3, p-STAT 3, ABCG2 and ABCC1antibodies (Cell Signaling and Technology) in 1:1000 dilutions.Horseradish peroxidase-conjugated secondary antibodies (Cell Sig-naling Technology) were used. Proteins were visualized by using en-hanced chemiluminescent solution (Biorad) and were exposed to Che-midoc Instrument (Biorad).

2.15. Statistical analysis

Tumor volume was measured by using digital Vernier caliper in-strument and the tumor volumes were calculated by the formulaTV=½ ab2, where ‘a’ and ‘b’ represent the length and width of thetumors and TV is tumor volume. All data were presented as themean ± standard deviation (SD). The significance of difference amongthe treatment groups was determined by using either Student's t-test orone-way ANOVA through using GraphPad prism version 5.0 (CA, USA),where p value less than 0.05 between the groups was considered asstatistically significant.

3. Results

3.1. Formulation characterization

Docetaxel pegylated liposome formulation (DTXPL) was preparedaccording to the previously established modified hydration method asdescribed (Patel et al., 2016a). Briefly, liposomes prepared with un-saturated phospholipid (DOPC) showed five-fold higher stability incomparison to those prepared using saturated lipid (DPPC). The amountof cholesterol used also plays a crucial role for determining liposomalstability and retention of DTX in the formulation. It was observed thatbatch prepared with DOPC and 3mol% DTX with 15mol% of choles-terol showed maximum stability at room temperature for 3–4 days,while cholesterol content above 40% precipitates DTX rapidly within2–3 h. Moreover, liposomes prepared at hydration temperature of 45 °Cshowed higher physical stability for 4 days in comparison to thoseprepared at 10 and 25 °C, which showed almost similar stability for upto 2–3 days at room temperature. Finally, the mole percent of DTX was

optimized based on the stability of liposomes. Increasing DTX molepercent from 3 to 5 profoundly reduced the stability of liposomes. So,the batch prepared using 1mg/mL of DTX concentration with 3mol%of DTX was evaluated in our study. The particle size of optimizedDTXPL was observed to be 133.2 ± 11.7 nm with a polydispersityindex value of 0.207 ± 0.0113. The entrapment efficiency of DTX inliposomes was found to be 96.4 ± 2.45%. Optimized DTXPL for-mulation prepared using DTX:DOPC:Cholesterol:DSPE-PEG (2000) in3:50:30:15 weight ratio was found to be physically and chemicallystable for 2months at 2–8 °C.

3.2. In-vitro anticancer studies

In order to evaluate the efficacy of the combination therapy in non-small cell lung cancer, we used H460 wild type and CD133+ stem cellsderived from H460 cells. The experimental design began by evaluatingthe cytotoxic potential of the combination therapy, DTXPL formulationas well as the free drug through crystal violet-based cytotoxicity assay.For combination studies with DTXPL, 50 μM of Tel was used for allexperiments because Tel till 50 μM did not show any significant cyto-toxicity in 2D cultures. As summarized in Table 1, IC50 values of DTXPL,DTXPL in combination with 50 μM Tel, and Tel alone in H460 WT cellswere observed to be 151.23 ± 39.198 nM, 86.989 ± 13.547 nM and117.2 ± 4.212 μM respectively. In CD133+ H460 derived.

stem cells, the IC50 values of DTXPL, DTXPL in combination with50 μM Tel and Tel alone were observed to be 130.875 ± 9.949 nM,113.857 ± 13.057 nM and 106.125 ± 4.429 μM. The effect of DTXPLin combination with 50 μM Tel was further evaluated by performingcytotoxicity assay in 3D cultures using Vitrogel RGD hydrogel (TheWellBioscience). The IC50 values of the treatments in 2D and 3D cultureswith the same number of cells seeded were compared to determine theeffect of the presence of extracellular matrix (i.e., in 3D cultures) inaltering the cytotoxic effects of treatments. We observed increased IC50

values with all the treatments in 3D cultures. IC50 values of DTXPL,DTXPL in combination with 50 μM Tel and Tel alone in 3D cultures ofH460 WT cells were observed to be 811.35 ± 199.19 μM,95.78 ± 0.371 μM and 832.085 ± 5.913 μM respectively as summar-ized in Table 3. Cytotoxicity assay revealed an eight-fold decrease in theIC50 value with combination group of DTXPL and Tel when comparedto DTXPL in 3D cultures. However, we observed 2-fold decrease in theIC50 value with the combination group of DTX and Tel treatment incomparison to DTX in 3D cultures (Table 2).

Table 1Cytotoxicity assays of DTXPL, Tel and DTXPL and Tel combination in 2D cul-tures of H460 WT and CD133+ stem cells derived from H460 WT cells. Datashown was representative of 3 independent experiments and expressed asMean ± SD.

Treatments H460 WT (2D) CD133+ stem cells (2D)

DTXPL IC50 (nM) 151.23 ± 39.198 130.875 ± 9.949DTXPL IC50 (nM)+ 50 μM Tel 86.989 ± 13.547 113.8571 ± 13.057Tel IC50 (μM) 117.2 ± 4.212 106.125 ± 4.429

Table 2Comparison of the IC50 values of DTX, Tel and DTX and Tel combination in 2Dand 3D cultures of H460 WT with equal number of cells seeded (5×104 cells/well). Data shown was representative of 3 independent experiments and ex-pressed as Mean ± SD.

Treatment 2D (IC50 values) 3D (IC50 values)

DTX+ Tel (μM) 12.19 ± 0.12 34.093 ± 1.173DTX (μM) 19.73 ± 1.146 70.050 ± 11.819Tel (μM) 170.377 ± 26.34 832.085 ± 5.913

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3.3. Liposomal uptake studies

Fluorescent images in Fig. 1B and C demonstrated that Coumarin-6loaded liposomal uptake showed similar fluorescence intensity in bothcontrol (untreated) and Tel pre-treated H460 cells in 2D cultures.However, there was a significant increase in the uptake of Coumarin-6liposomes (p < .05) in the Tel pretreated cells when compared to un-treated cells in 3D cultures as shown in Fig. 1C. In 3D cultures of H460WT cells, Tel pretreatment increased the uptake of Coumarin-6 lipo-somes by 3.7-fold when compared to untreated cells. However, in 2Dcultures, the liposomal uptake was 1.2-fold higher in Tel pre-treatedcells.

3.4. Hypoxia assay

3D cultures of H460 WT cells were treated with DTXPL, Tel andDTXPL+ Tel in order to determine their effects on hypoxic environ-ment of the spheroids. DTXPL did not show any significant reduction inthe hypoxia of the spheroids as measured by the fluorescent intensities.In our study, we observed that DTXPL and Tel combination significantlydecreased hypoxia (p < .001) in 3D spheroids (Fig. 2), which closelyresemble in-vivo tumor environment. Tel alone also decreased hypoxiato a certain extent in 3D spheroids. This suggests that duringTel+DTXPL combination treatment, Tel disrupts tumor stroma andfacilitates DTXPL more inside the 3D spheroids, thereby producingsignificant anti-cancer effects by decreasing the hypoxic conditions oftumor microenvironment along with decreasing docetaxel resistance.

3.5. DCFDA staining for ROS determination

Reactive oxygen species (ROS) are toxic to cells and can also induceoxidative damage to lipids, proteins, and DNA. We assessed the levels ofintracellular ROS generation upon treatment with DTXPL, Tel andcombination group of DTXPL and Tel by performing DCFDA assay toascertain the role of oxidative stress in cellular damage induced by Teland DTXPL. We observed significant increase in the generation of ROSlevels with DTXPL and Tel combination treatment group (p < .05)when compared to the untreated cells (Fig. 3). This indicates that ROS

Table 3Cytotoxicity assays of DTXPL, Tel and DTXPL and Tel combination in3D cultures of H460 WT cells (5×104 cells/well). Data shown wasrepresentative of 3 independent experiments and expressed asMean ± SD.

Treatment IC50 values

DTXPL (μM) 811.35 ± 199.19DTXPL (μM)+50 μM Tel 95.78 ± 0.371Tel (μM) 832.085 ± 5.913

Fig. 1. Distribution of coumarin-6 liposomes in 2D and 3D cultures of H460 WT cells. (A) Microscopic image of 3D spheroid formed by using Vitrogel RGD (After5 days). (B) Fluorescent images in 2D cultures of control and Tel pretreated cells. (C) Fluorescent images in 3D cultures of control and Tel pretreated cells. Greenfluorescence intensity in 2D and 3D culture was calculated and expressed as mean ± SD. p-values were calculated using t-test and *p < .05 was considered to besignificant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Hypoxia assay in 3D cultures of H460 WT cells. (A) Fluorescence images of hypoxia staining in 3D cultures of untreated, DTXPL, Tel, and DTXPL + Telcombination treated H460 cells. (B) Mean fluorescence intensity in 3D cultures was expressed as mean ± SD (*** p < .001).

Fig. 3. ROS determination in 3D culture of H460 WT cells. (A) Fluorescence images showing ROS generation in 3D cultures of untreated, DTXPL, Tel and DTXPL +Tel combination treated cells (B) Mean fluorescence intensity in 3D cultures was expressed as mean ± SD (* p < .05).

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generation induced by combination of Tel and DTXPL group was re-sponsible for increased cytotoxicity in H460 WT cells when comparedto untreated cells.

3.6. Antitumor study

The antitumor studies in animals were performed in multiple roundsby inoculation of tumor derived cells from previous experiments. Theobjective here was to create a drug resistant cancer stem cell model todetermine the efficacy of the treatments in all resistant tumors and stemcell enriched tumors as seen in many cancer cases. Tel oral adminis-tration followed by IV administration of DTXPL significantly decreasedtumor volume in H460 WT (Fig. 4A), CD133+ H460 stem cells (Fig. 4B)and DTX resistant CD133+ (Fig. 4c) xenograft models of lung cancer.The reason behind the observed increased anti-cancer efficacy with Teland DTXPL combination in all these xenograft models was due to dis-ruption of tumor stroma by Tel, which thereby facilitates DTXPL ef-fectively into the targeted tumors to show its anti-cancer effects. Tumorvolume was significantly lower in the combination group (p < .001)and DTXPL treated group (p < .01). For the third round of animal

studies, we removed Tel alone group because during previous rounds ofantitumor studies, we did not observe any significant difference be-tween the control and Tel treatment. The stem cells isolated from H460cells were characterized by western blotting for the presence of CD133.As shown in Fig. 4D & 4E, we observed significant expression of CD133in the H460 derived stem cells (p < .01) before and after the animalstudies when compared to H460 WT cells, indicating that stem cellpopulation was maintained in our animal experiments.

3.7. Western blot analysis

Western blotting of the tumor lysates showed that DTXPL + Teldecreased the protein expression of anti-apoptotic marker, survivin(p < .001) and caspase 3 (p < .01) respectively in comparison tocontrol (untreated) group (Fig. 5A). Significant down regulation of theextracellular matrix (ECM) marker, MMP9 (p < .05) was also observedin DTXPL+Tel treated tumors (Fig. 5A). DTX treated group did notshow any effect in the deregulation of stem cell markers in DTX re-sistant CD133+ stem cell xenograft model (Fig. 5B). However, DTXPL+ Tel decreased the protein expression of lung cancer stem cell markers

Fig. 4. Tel oral administration followed by DTXPL significantly decreased the tumor volume in (A) H460 xenograft model of lung cancer, (B) CD133+ stem cellsderived from H460 WT cells xenograft model of lung cancer, (C) DTX resistant CD133+ lung tumor xenografts. Tumor volume was measured for every 4 days byusing digital Vernier caliper. The results were described as mean ± SD (n=4) (**P < .01, ***p < .001). (D) Characterization of CD133 expression in both H460WT and CD133+ H460 derived stem cells before injection of cells into mice (**p < .01). (E) Characterization of CD133 expression in both H460 WT and CD133+

H460 derived stem cells after animal experiment (** P < .01).

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such as SOX 2 (p < .05) and CD44 (p < .001) respectively in DTXresistant CD133+ stem cell xenograft model, demonstrating the efficacyof combination treatment of DTXPL+Tel when compared to DTX alonein DTX resistant CD133+ stem cell xenograft model.

4. Discussion

Docetaxel in liposomal formulation (DTXPL) was prepared by themodified hydration method as described (Patel et al., 2016a), which iswell established in our laboratory. Preliminary physical characteriza-tion studies revealed that DTXPL liposomes of 133.2 ± 11.7 nm dia-meter were found to be stable at 2–8 °C for 2months with an entrap-ment efficiency of 96.4 ± 2.45%. Liposomes are preferred to be storedat 2–8 °C for longer stability as it helps them to maintain their structure.Freezing or long term storage at room temperature will disrupt thestructure of liposomes, which leads to precipitation of drugs (Bulbakeet al., 2017).

We have earlier reported that Tel pretreatment significantly im-proved the delivery of DTX liposomes into A549 lung tumors by acting

as an anti-fibrotic agent, which disrupts tumor stromal barriers andhelps in effective permeation of DTX liposomes into the deeper layers oftumors (Patel et al., 2016a). However, tumor regression and resistanceto chemotherapeutics is still a major clinical concern due to the pre-sence of cancer stem cells (Abdullah and EK-H, 2013; Phi et al., 2018).Cancer stem cells (CSCs) play an important role in drug resistance,relapse and metastasis of various tumors (Vinogradov and Wei, n.d.;Prieto-Vila et al., n.d.). This is the first report demonstrating the com-binatorial anti-cancer effects of Tel and Docetaxel liposomes (DTXPL) in3D cultures of H460 wild type (H460-WT) cells as well as in xenografttumor models of lung tumors developed by inoculation of CD133+ stemcells derived from H460 WT tumors and DTX resistant CD133+ stemcells derived from DTX treated tumors.

This study also aims to further demonstrate the therapeutic benefitof using 3D cultures over 2D culture methods for in-vitro evaluation ofanti-cancer effect of the combination therapy as well as investigating itsefficacy in cancer stem cell animal model.

In order to evaluate the efficacy of the combination therapy, weused both wild type H-460 cells as well as CD133+ stem cells derived

Fig. 5. Western blot analysis of lung tumor lysate. (A) Western blot images of different protein expressions in H460 tumor lysates. Quantitative analysis of Survivin,Caspase 3, Cyclin D1, P Stat −3, and MMP9 expression. Data were representative of three different experiments and were presented as mean, and error bars refer toSD. (*p < .05, **p < .01, ***p < .001 was considered significant when compared to control). (B) Western blot analysis of stem cell and drug resistant proteinmarkers in DTX resistant CD133+ H-460 stem cells tumor lysate. Data were representative of three different experiments and were presented as mean, and error barsrefer to SD. (*p < .05, **p < .01, ***p < .001).

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from H-460 cells. DTXPL in combination with 50 μM of Tel showed aslight decrease in the IC50 value when compared to the single treatmentof DTXPL in both H460 and CD133+ H460 derived stem cells for 24 h.However, the efficacy of DTXPL and Tel combination was more pro-minent in 3D cultures when compared to 2D cultures due the presenceof extracellular matrix in the 3D culture system. 3D culture systemshave been proven to be more reflective of in-vivo cellular responses ascompared to 2D cultures due to the presence of cellular communicationand the development of extracellular matrix (Ravi et al., n.d.). 3D ex-tracellular matrix (ECM) environment developed by using matrigels orsynthetic matrices can provide effective quantitative analysis of cellmigration, differentiation, survival, and growth (Griffith and Swartz,2006). Cells cultured in 3D systems provide more contact space formechanical inputs and cell adhesion, which play an important role forintegrin ligation, cell contraction and even intracellular signaling(Suuronen et al., 2005; Louekari, 2004).

Coumarin-6 liposomes are widely used for evaluating uptake invarious cancer cells (Wei et al., n.d.; Luo et al., n.d.). Coumarin-6 li-posomes uptake studies showed similar fluorescence intensity in boththe control and Tel pre-treated 2D cultures of H460 cells. However, in3D cultures, we observed a significant increase in the uptake of Cou-marin-6 (3.7-fold) in the Tel pretreated cells when compared to theuntreated cells (control). In 3D cultures unlike 2D cultures, there is thepresence of collagen and cellular network, which gets disrupted by pre-treatment with Tel and thereby facilitates increased uptake of Cou-marin-6. This data signifies the importance of Tel pretreatment (i.e.,orally) before the intravenous administration of DTXPL in lung tumorxenografts. This data also supports our previous findings, which haveshown increased uptake of Coumarin-6 in Tel pre-treated A549 tumorxenografts when comparted to control (Patel et al., 2016a). Herein,both control and Tel treated groups showed similar uptake of Cou-marin-6 in normal lung tissue. However, Tel pre-treated A549 tumorsshowed significant increase in the uptake of Coumarin-6 when com-pared to untreated tumors, demonstrating the role of Tel as a tumordisrupting agent, which helps in the increased uptake of Coumarin-6 in-vivo.

Hypoxia is the decrease in the regular level of tissue oxygen tension,that occurs usually in solid tumors as well as in NSCLC. Cancer cells gothrough a sequence of genetic and metabolic modifications that en-hance their survival and proliferation. However, hypoxia during somecases is unsuitable for cell growth (Kunz and Ibrahim, 2003; Harris,2002). Hypoxic conditions in tumors have been proven to be predictivecause of poorly clinical outcome for cancer patients, which leads to anincreased risk of tumor invasion and metastasis (Postovit et al., 2002;Krishnamachary et al., 2003), as well as resistance to some anticanceragents (Frederiksen et al., 2003). There was a significant decrease(p < .001) in hypoxia when cells are treated with combination therapyof DTXPL and Tel when compared to untreated 3D cultures, suggestingthe role of combination therapy in overcoming resistance and invasion.

ROS levels were determined after 24 h of treatment with DTXPL, Teland combination of DTXPL and Tel using DCFDA assay in 3D cultures.This was to assess the association between ROS generation and apop-tosis induced by the combination of DTXPL and Tel treatment. ROSactivates various signaling pathways in multiple cancers, which areinvolved in glucose metabolism, cell proliferation and differentiation(Storz, 2005). However, cancer cell cycle arrest and apoptosis can alsobe caused by increased ROS levels as a result of treatment with antic-ancer agents. Apoptosis has been associated with increased mitochon-drial oxidative stress, which leads to the release of cytochrome C andeventually caspase activation and cell death (Simon et al., 2000;Cadenas, 2004). A significant increase in ROS was observed with thecombination treatment, which explains the induction of apoptosis inH460 cells. The efficacy of combination treatment of Tel and DTXPLwas also evaluated in various xenograft models of lung tumors devel-oped by subcutaneous administration of H460 WT cells, and CD133+

stem cells derived from H460 WT tumors and DTX resistant CD133+

stem cells in athymic nude mice. This study aims to develop a drugresistant cancer stem cell model to determine the efficacy of thetreatments in both resistant and stem cell regenerated tumors, whichare seen in many patients. Earlier reports have shown the efficacy of Tel(i.e., at a dose of 5mg/kg body weight) in improving the efficacy ofdocetaxel liposomes (i.e., at a dose of 5mg/kg body weight) in xeno-graft models of lung cancer (Patel et al., 2016a; Patel et al., 2016b). Wealso have used the same dose of Tel in all our xenograft models but wehave increased the dose of docetaxel liposomes to 10mg/kg bodyweight in our xenograft models as we are evaluating their effect onCD133+ and DTX resistant CD133+ H460 xenograft models also, whichgrow very aggressively and are more resistant to DTXPL in comparisonto H460 WT xenograft model. Tel oral administration followed by in-travenous administration of docetaxel liposomes significantly inhibitedthe tumor growth in H460 WT, CD133+ H460 stem cells, and DTXresistant CD133+ H460 stem cell models of lung tumors in athymicnude mice when compared to control and Tel treatment. We observedsimilar trend in tumor volume during all three rounds of our animalstudies, demonstrating the effect of the combination therapy in com-parison to the control and DTXPL treatment.

CD133 is often used as a marker for separation of CSC populationfrom various tumors, predominantly gliomas and carcinomas (Kimet al., n.d.-b). In our study, CD133+ stem cells isolated from H460 WTcells showed excellent purity, which was confirmed by performingwestern blotting. We observed increased CD133 expression, almostaround four-fold significantly higher (**P < .01) in isolated CD133+

stem cells when compared to H460 WT cells. Various reports haveearlier demonstrated the purity of CD133+ stem cells as 75–85%, whenisolated from human fetal brain, melanoma, non-small cell lung andcolon cancer cells followed by characterization through flow-cyto-metry, immunocytochemistry and western blotting (Liu et al., 2013;Vincent et al., 2014; Yu et al., 2004; Zhang et al., 2015; Welte et al.,2013).

Western blotting of the cell lysates revealed that DTXPL + Telgroup showed significant down regulation of lung cancer stem cellsmarkers such as SOX 2 and CD44. This implies that there has been asignificant inhibition of the chemo-resistant populations of cancer cells(CSCs), which possesses the self-renewal and differentiation abilities.Cluster of differentiation 44 (CD44), a multifunctional class I trans-membrane glycoprotein, which acts as a specific receptor for hyaluronicacid (HA), is expressed at low levels on the surface of several normalcells and is highly expressed in almost every cancer cell in its standardor variant form (Ponta et al., 2003; Naor et al., 2009; Arpicco et al.,2013). High levels of CD44 are associated with poor prognosis, me-tastasis, progression, cancer stem cells (CSCs) survival, self-renewal anddrug resistance in various tumors (Visvader and Lindeman, 2008; Leunget al., 2010; Weber et al., 2002; East and Hart, 1993; Hu et al., 2018).CD44 standard (CD44s), the most prolific isoform of CD44 is highlyexpressed in solid tumors (Sneath and Mangham, 1998; Zöller, 1995).Specific CD44 variant (CD44v) and CD44 standard (CD44s) regulatemetastasis and epithelial to mesenchymal transition (EMT) respectivelyby activating different signaling pathways, which all contribute to poorsurvival of distinct cancer patients (Brown et al., 2011; Dang et al.,2015; Okabe et al., 2014; Ye et al., 2019; Zhang et al., 2018). In ourstudy, we have observed decreased expression of CD44s with Tel andDTXPL combination, which demonstrates the efficacy of combination indecreasing the stemness in DTX resistant CD133+ H460 stem cell xe-nograft model. According to studies by Ota et al. (2009), resistance tochemotherapeutic agents could be caused by ABCG2, which serves as aprognostic marker of survival in patients with advanced non-small celllung cancer (Ota et al., 2008). The side population cell properties oflung cancer cells which possess stem-like characteristics are mainly dueto ABCG2 (92, 93). ABCG2 partly contributes to the multi-drug re-sistance to anticancer agents and its overexpression is also associatedwith adverse prognosis in some cancers (Kunz and Ibrahim, 2003). Inthe DTX resistant CD133+ H460 derived stem cell model, we observed

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an increased expression of ABCG2 and ABCC1 in DTXPL alone treatedtumors and decreased expression ABCG2 and ABCC1 in DTXPL and Telcombination treated tumors. ABCC1, however is more highly expressedin the DTXPL treated tumors when compared to the control group. Thisshows that the DTX resistant tumors express higher drug resistantmarkers upon treatment with the DTXPL. MMPs are crucial for the re-lease of cytokines and growth factors from ECM as well as in the recycleof ECM. High levels of MMP2, produced by tumor stromal cells areresponsible for invasion and metastasis. DTXPL and Tel combinationsignificantly reduced MMP9 expression demonstrating the effect of Telon the disruption of the ECM. Since we observed decreased hypoxia(i.e., in 3D spheroids of H460 WT cells), CSCs and drug resistancemarkers with DTXPL and Tel combination treatment for 2 weeks in DTXresistant CD133+ H460 xenograft model, we conclude that Tel dis-ruption of tumor stroma effectively facilitates DTXPL into the CSCs andthereby contributes to increased anti-cancer effects in DTX resistantCD133+ H460 stem cell xenograft model. Moreover, we think thatthese effects might sustain longer even after 2 weeks but as we observedthat tumor was growing very fast in control animals (i.e., increasedtumor volume as measured by Vernier caliper for a total period of4 weeks) and in order to maintain the same time point for evaluatingmolecular changes, we have sacrificed all the animals after only2 weeks' treatment of DTXPL and Tel combination in CD133+ and DTXresistant CD133+ H460 xenograft models. The effectiveness of DTXPLand Tel combination will not be diminished after 2 weeks in xenograftmodels as Patel et al., 2016 (work conducted in our laboratory) hasshown that this combination therapy is effective even after 6 weeks inA549 xenograft model and animals showed 80% survival with thiscombination treatment (Patel et al., 2016a). In summary, it appears thatTel in this combination treatment works by multiple pathways in-cluding, apoptosis, anti-angiogenesis, anti-fibrotic and downregulatingdrug resistant genes. Currently we are performing RNA seq analysis tofurther look for downstream targets which contribute to stemness andresistance in lung tumors.

5. Conclusion

Tel pretreatment enhances the penetration of DTXPL and therebycontributes to increased anticancer efficacy when compared to DTX andTel alone treated tumors. Pretreatment of tumors via oral administra-tion of an antifibrotic agent such as Tel has great potential and could bebeneficial in improving the outcome of various anticancer nano for-mulations clinically.

Author contributions

P.A, N·P, S·K·S, A.M, A.G, A.B, S.K and E.N designed and conductedthe research and experiments. P.A, S. K·S and M.S. wrote the manu-script. The study was supervised by M.S. All authors read and approvedthe final manuscript.

Declaration of Competing Interest

All authors declare no conflicts of interests.

Acknowledgements

This work is supported by the National Institute on Minority Healthand Health Disparities of National Institutes of Health (RCMI5U54MD007582-35) and NSF-CREST center for Complex MaterialDesign for Multidimensional Additive processing (CoManD) award #1735968.

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