Combined transcriptional, translational and cell surface targeted gene therapy of HER2-positive breast cancer stem cells in three-dimentional cell culture

Purpose Breast Cancer Stem Cells (BCSCs) resist conventional treatments and cause tumor recurrence. Almost 25% of breast cancers overexpress human epidermal growth factor receptor-2 (HER2). Here we developed a novel multi-targeted nanosystem to specically eradicate HER2-positive BCSCs. Plasmids containing CXCR1 promoter, PE38 toxin and 5′UTR of the basic broblast growth factor-2 (bFGF 5'UTR) were constructed. Polyamidoamine (PAMAM) dendrimers functionalized with an anti-HER2 VHH were used for plasmid delivery. Stem proportion of MCF-10A and MDA-MB-231/HER2 + (a cellular model of HER2 overexpression, developed in our lab) were evaluated by mammosphere formation assay. Hanging drop technique was used to produce spheroids. The uptake, gene expression and killing ecacy of the multi-targeted nanosystem were evaluated in both monolayer and spheroid culture. than conventional monolayer culture.


Introduction
BCSCs are a subpopulation of breast cancer cells with selfrenewal and differentiation capacity. Since BCSCs resist conventional treatments and cause tumor recurrence, targeting them may lead to the development of more effective cancer therapies 1 . BCSCs survive and generate mammosphere in low- Page 3/20 adherence serum-free cultures, whereas all the other cell types will undergo anoikis. Each mammosphere represents on average one stem cell of the parental population 2 . Based on this feature, we used mammosphere formation assay to determine the proportion of BCSCs in MDA-MB-231/HER + as well as BCSC-like cells in pseudonormal MCF-10A.
Targeted killer gene therapy is a promising approach for eradicating cancerous cells. Here, we utilized CXCR1 promoter, PE38 toxin and bFGF 5'UTR genetic construct delivery using PAMAM dendrimers functionalized with an anti-HER2 VHH to target HER2-positive BCSCs on transcriptional, translational and cell surface levels simultaneously.
PE38 is a mutated form of Pseudomonas exotoxin A. This highly cytotoxic protein arrests protein synthesis by inactivating eukaryotic elongation factor-2(eEF-2) 3 . To date, many studies have reported that the introduction of PE38 rapidly kills the host cells 4,5 .
In this research we utilized CXCR1 promoter to restrict PE38 transcription to BCSCs and HER2-positive breast cancer cells. CXCR1, a receptor for interleukin-8, is overexpressed in aforementioned cells 6,7 .
With the aim of translational targeting, bFGF 5'UTR was used as an additional discriminatory element to the CXCR1 promoter to improve the stringency and speci city of the treatment. Translation of mRNAs with long and highly structured 5 UTRs is largely dependent on the unwinding activity of eukaryotic translation initiation factor 4E (eIF4E). Eukaryotic IF4E is rare in most cell types 8 . Nonetheless, it is overexpressed in tumorigenic cells and facilitates the translation of GC-rich 5 UTR of the bFGF 9 .
PAMAM dendrimers are attractive vehicles for gene delivery. They effectively condense Nucleic acids and protect them from being degraded by nucleases 10 . In order to avoid off-target effects, dendrimers can be equipped with a variety of tumor-speci c targeting agents such as peptides, aptamers, antibodies and VHHs. VHH is the smallest antigen-binding domain of Camelidae heavy chain antibodies. Their unique characteristics including exquisite a nity and speci city against their targets, high stability and solubility as well as low immunogenicity make them ideal targeting agents for cancer killer gene therapy 11,12 . With the aim of cell surface targeting, we functionalized PAMAM dendrimers with an anti-HER2 VHH.
Almost 25% of breast cancers overexpress HER2 13 . HER2 overexpression confers a higher rate of aggressive metastasis and mortality 14,15 by expanding the BCSC population as well as increasing the tumorigenicity and invasiveness of this population. HER2 is expressed in both bulk tumor and BCSC populations. Some reports suggest that in HER2-nagative breast cancers, HER2 may be selectively expressed in breast cancer stem cells but not bulk cell populations 16,17 . It is assumed that, the considerable therapeutic bene t of HER2-targeting agents (ex. Trastuzumab) might be due to their BCSC targeting capability 18 .
In this study, we exploited the potential of our multi-targeted nanosystem in both monolayer and spheroid culture. Unlike monolayer culture, 3D spheroid culture recaptures many features of the real tumor, such as cell-cell contact, cell-extracellular matrix (ECM) interactions as well as oxygen, nutrient and signal gradients. Hanging drop is an e cient method for cultivating spheroids with uniform size and shape. We utilized the Hanging drop technique to prepare 3D spheroids. One distinct advantage of this technique over other spheroid culture methods, is that this method avoids using uncontrolled mechanical forces and interference from exogenous materials. Indeed, cells accumulate at the bottom of the drop under gravity, secrete their own matrix components and self-assemble into spheroid. These features offer a great potential to study the effect of antitumor treatments 19 . Of note, this study is the rst example of in situ transfection of spheroid in hanging drop culture.
Anti-HER2 VHH production, expression and puri cation The anti-HER2 VHH clone were isolated from a large nanobody library from a one-humped and a twohumped immunized camels, using phage display technique 21 . The nucleotide sequence was sub-cloned into PET28, expressed in E. coli and puri ed using immobilized metal ion a nity chromatography (IMAC) column, as described previously 22 .

Gene Constructs
The methods of making the gene constructs has been described in detail previously 23 . Brie y, CMV promoter was PCR ampli ed from pGL4.50 (Promega, WI). The PCR product was sub-cloned into the pGL4.14 vector (Promega) to make pGL4.14-CMV (pG-CM).
For cloning of CXCR1 promoter, 1123 bp fragment surrounding the putative promoter region of upstream of CXCR1 gene was ampli ed by PCR from peripheral lymphocytes DNA. The PCR product was inserted into upstream of the luciferase gene in pGL4.14 to make pGL4.14-CXCR1 (pG-CX).
The constructs were puri ed using the endotoxin-free plasmid DNA puri cation kit (MACHEREY-NAGEL), validated by sequencing and then used for cell transfections.
Preparation of VHH-PEG-PAMAM and Trastuzumab-PEG-PAMAM conjugates G5 PAMAM was reacted with NHS-PEG3500-Mal in 2 mL of degassed phosphate buffer (pH 7.5) at a ratio of 1:10 (mol/mol) under N2 atmosphere at room temperature with gentle shaking for 2 h. Removal of unreacted PEG molecules was carried out by ultra ltration using Amicon ultra lters (MWCO 10 kDa; Millipore, Schwalbach, Germany). The number of PEG groups introduced to PAMAM was determined based on TNBSA assay.
For conjugation of the anti-HER2 VHH to maleimide distal end of PEGylated PAMAM dendrimers, the anti-HER2 VHH were rst thiolated using Traut's reagent. Traut's reagent was added to the anti-HER2 VHH in borate buffer (50 mM sodium borate, 0.1M EDTA, pH 8.3), to produce a ratio of 1:10 (mol/mol), and the reaction was performed under N2 atmosphere at room temperature with gentle shaking for 1 h. Removal of unbound Traut molecules and replacing the buffer with phosphate buffer were carried out by ultra ltration using Amicon ultra lters (MWCO 30 kDa).
Thiolated VHH was added to the PEG-PAMAM to produce a ratio of 2:1 (mol/mol), and the reaction was performed under N2 atmosphere at room temperature with gentle shaking for 18 h. The nal conjugates (VHH-PEG-PAMAM) were puri ed using Amicon ultra lters (MWCO 50 kDa), lyophilized and stored at − 80ºC.
All the procedures of preparing Trastuzumab-PEG-PAMAM conjugates were the same as the synthesis of VHH-PEG-PAMAM conjugates except that the molar ratio of Traut reagent to Trastuzumab were (20:1) and removal of unbound Traut molecules were carried out by Amicon ultra lter with a bigger pore size (MWCO 100 kDa).

Characterization of PG-PAM and VHH-PG-PAM conjugates
Measuring primary amines of PAMAM and PEGylated PAMAM The primary amine group content of PAMAM and PEGylated PAMAM was measured with a spectrophotometer after reaction of the free amine groups with TNBSA, as described by Snyder et al. 24 . The standard curve was generated by the use of glycine serial dilutions (data not shown). The quantity of PEG groups coupled to the PAMAM were calculated by the differences in the amounts of primary amines on PAMAM and PEGylated PAMAM.

Characterization of PG-PAM by FTIR
To verify the PEGylation, 1 mg of unmodi ed G5 PAMAM and PG-PAM conjugates were analyzed by FTIR  Table 1 lists the prepared nanoparticle/gene construct complexes and the corresponding abbreviations.
After that, a volume of 5 µL of the solution were placed on a freshly cleaved untreated mica surface and allowed to stick for 5 min. Then, the excess of sample were removed by careful absorption onto lter paper and the mica surface were further dried under a gentle stream of air at RT. Sample were examined with a JPK AFM (JPK Instruments Co., Germany) using contact mode, HYDRA6V-100N cantilever with pyramidal shape tip, force constant of 0.292 N/m, and resonance frequency of 66 kHz.

Transmission electron microscopy
Transmission electron microscopy (TEM) images were obtained from CEM 902A ZEISS (Jena, Germany) transmission electron microscope with an accelerating voltage of 80 kV, to investigate the size and morphology of the VHH-PG-PAM/pG-CX-bF-PE at N/P ratio of 10.
Flow cytometry analysis to determine Percentage of HER2 expressing cells

Mammosphere cultivation
Mammospheres were generated from 2 × 10 4 single cells seeded in 6-well tissue culture plates coated with 1.5% agarose; containing 2 ml DMEM/F12 (GIBCO) without serum, supplemented with B27 (1:50, Invitrogen), 5 mg/ml insulin (Sigma), 20 ng/ml bFGF (R&D) and 20 ng/ml EGF (R&D). Cultures were incubated in a humidi ed atmosphere at 37°C and 5% CO2 for 3 days without moving or disturbing the plates. After 3 days 400 µl of fresh media was added to each well (without removing the old media). Mammospheres were imaged on day 7. The size and number of mammospheres were quantitated using ImageJ software (NIH, USA). Mammosphere formation e ciency (MFE) was calculated as (the number of mammospheres per well (diameter > 50 µm) divided by the original number of single cells seeded per well) × 100. Experiments were done in duplicates.

Monolayer Cell transfection
For cell transfection, 6 x 10 4 cells/well were seeded and incubated until they reached ∼80% con uency.
Immediately before transfection, the complete medium was removed, cells were washed with PBS, fresh medium without serum and antibiotics was added to each well. The dendriplexes were prepared freshly prior to use and 100 µL of dendriplex solution at a nal transfection DNA concentration of 2 µg /mL − 1 were applied to cultured cells. After 6 h incubation, the transfection solution was removed, cells were washed with PBS, cell-speci c complete medium was added and the culture was further incubated for the time period required for each experiment.
Hanging drop culture MCF-10A and MDA-MB-231/HER + Cell lines were cultured as spheroids in their appropriate media by the hanging drop method. Cells were dissociated from a monolayer cell culture, counted and resuspended in complete medium at a concentration of 3 x 10 4 cells/ml. Thirteen µl aliquots of the suspension (containing 1000 cells each) were deposited on the underside of a 10 cm petri dish lid. The lid was then inverted over the dish lled with 10 ml of PBS (to keep the cells hydrated). The dish was maintained at 37°C in a humidi ed incubator with 5% CO2 for 7 days. The growth media were exchanged every other day by up-righting the lid, taking 10 µL media from each drop and adding 14 µL fresh media into it. Cells were imaged daily and spheroid`s size (average of the major and minor axis length) was measured using the NIH ImageJ software.

Spheroid transfection
On the seventh day following spheroid generation, the complete medium was replaced with PBS and subsequently with fresh medium without serum and antibiotics, using sequential pipetting method. The dendriplexes were prepared freshly prior to use and were carefully introduced to the individual spheroids by taking 14 µL media from each drop and adding 14 µL of dendriplex solution to them. After 6 h incubation, the medium was replaced with the complete medium, using the sequential pipetting method.
The hanging drop culture was further incubated for the time period required for each experiment. Transfected spheroids were then collected and dissociated to single cells by pipetting through a 200 µl pipet tip. The obtained single cells were used for cellular uptake, PE38 mRNA Expression or cytotoxicity assays as described in subsequent Methods sections. For each assay, three identical experiments were performed and 20 spheroids were analyzed per condition. Real-time PCR were performed by Rotorgene 3000 series PCR machine (Corbett Research, San Francisco, USA) using RealQ Plus 2x Master Mix Green (Amplicon, Denmark). All mRNA quanti cation data were normalized to β-actin. The primers for amplifying PE38 and beta-actin were as follows: PE38 for: 5 AGGACCTCGACGCGATCTG, PE38 Rev: 5 TCAGGCTGGTGCGGTAGAAG, β-actin for: 5 TCC CTGGAGAAGAGCTACG and β-actin Rev: 5 GTAGTTTCGTGGATGCCACA. Relative PE38 mRNA expression in different samples were calculated by Pfa method. The assay was repeated three times.

Cytotoxicity study
To evaluate PE38 cytotoxicity, cell viability of transfected cells vs. non-transfected cells were determined by MTT assay. Brie y, cells were seeded and transfected as described for monolayer or spheroid culture, 6 h after transfection with different dendriplexes, transfection solution was removed, cells were washed with PBS and complete medium was added to each well. After 48 hours incubation, the complete medium was replaced by the fresh medium without serum and antibiotics, then 1 mg/mL MTT reagent (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma) was added to each well and followed as the manufacturer recommended. The assay was repeated three times.

Statistical analysis
Page 10/20 The statistical analysis was performed using GraphPad PRISM v.6.01 (GraphPad Software Inc, CA, USA). All data subjected to statistical analysis were obtained from at least three parallel experiments. Unpaired Student's t-test was used to determine signi cant differences between each group. Two-way ANOVA with Tukey's multiple comparison post hoc test was used to determine signi cant differences between different groups. A p-value ≤ 0.05 was considered statistically signi cant.

Results And Discussion
Characterization of PG-PAM and VHH-PG-PAM conjugates Measuring primary amines of PAMAM and PEGylated PAMAM TNBSA assay con rmed that PEGylation of dendrimers with molar ratios of 10 yielded conjugates containing an average of 19 PEG chains per PAMAM molecule.

Characterization of PG-PAM by FTIR
The formation of PG-PAM conjugates was further con rmed by FTIR spectra. In FTIR spectra (Fig. 1a), the peak at 3300 cm − 1 due to -NH-groups of PAMAM stretching vibration is evident. The characteristic peaks of methyl C = O stretching of carbonyl groups of PAMAM internal amides or PG-PAM amide bonds is found at 1650 cm − 1 . Besides, the peak at 1110 cm − 1 due to stretching vibration of -CH2-O-CH2etheric bonds of PEG molecules, indicating that PEG chains are successfully attached to PAMAM dendrimers.

Dendriplex characterisation
Gel retardation assay Agarose electrophoretic mobility retardation assay was performed using PAM/pG-CX-bF-PE, PG-PAM /pG-CX-bF-PE and VHH-PG-PAM/pG-CX-bF-PE, at N/P ratio of 10. The puri ed pG-CX-bF-PE plasmid moved toward anode (Fig. 2a, lane 1). In contrast, Fig. 2a, lanes 2 shows that, not only PAMAM reversed the plasmid electrophoresis pattern, but also the negatively charged Bromophenol Blue dye were also electrophoresed to the opposite direction, cathode. This result suggest that the overall charge of PAM/pG-CX-bF-PE were comparatively positive. Figure 2a, lanes 3 and 4 show that, PG-PAM and VHH-PG-PAM interacted su ciently with the pG-CX-bF-PE and neutralized its negative charge so the plasmid were retarded in the well.

Dynamic light scattering and zeta potential measurements
The size and zeta potential of the dendriplexes at N/P ratio of 10 were measured by zetasizer Nano ZS. According to results presented in Table 2, all the dendriplexes showed the appropriate size and charge density. Low polydispersity indexes indicate the formation of homogeneous and aggregate free dendriplexes.
It seems that by conjugating PEG and VHH to the PAMAM dendrimers, the size of dendriplexes increased, while the zeta potential were decreased. These ndings imply that both PEG and VHH were successfully attached to the dendrimers and shielded the positive charges of the free amines on the PAMAM surface. However, the size of dendriplexes ranged between 105.6 and 154.7 nm and the zeta potential ranged between 26.8 and 5.2 mV which are considered to be appropriate for endocytosis.

Atomic force microscopy
The morphology of VHH-PG-PAM/pG-CX-bF-PE at an N/P ratio of 10, were observed by AFM (Fig. 2b). The result obtained revealed that the dendriplexes formed spherical particles with an average diameter of 121 ± 2 nm and a narrow size distribution, falling within the optimum size requirements (100-200 nm) for e cient cellular endocytosis.

Transmission electron microscopy
Transmission electron microscopy was performed to further investigate the size and morphology of VHH-PG-PAM/pG-CX-bF-PE dendriplexes. TEM result was in accordance with the AFM data, showing spherical structures with an average particle size of 118 ± 7 nm (Fig. 2c). However, the particle size visualized by TEM and AFM were smaller than those determined by DLS. The most probable explanation would be that TEM and AFM determine the dry particle size, whereas DLS re ects the hydrodynamic size.
There is discernible differences in BCSC pools between MCF-10A and MDA-MB-231/HER2 + Mammosphere assay was used to evaluate the stem property of the cell lines. According to Fig. 4a Mammosphere formation assay identi es BCSCs based on their resistance to anoikis in nonadherent/serum-free conditions. This technique is considered to be more solid than conventional BCSC speci c marker identi cation since cell surface markers vary in nitely from one breast cancer cell to another 25 .
Morphology of spheroids in hanging drops.
We utilized the hanging drop technique to generate spheroids with uniform shape and size. MCF-10A grown in hanging drop culture formed thoroughly loose aggregates exposing free areas between the cells.
Based on these results, MCF-10A aggregates were excluded from further investigation and MDA-MB-231/HER + spheroids were selected for subsequent studies.
MDA-MB-231/HER + spheroids were maintained in hanging drop culture for 7 days and imaged daily. As illustrated in Fig. 5, spheroids of different drops were consistent in both size and shape. On the rst day spheroids measured approximately 203 µm. From rst to sixth day the spheroids diameter increased slightly. On the sixth day their average size reached to 484 µm. However, by day 7 it decreased to 439 µm, indicating spheroids started to become denser. Several researchers suggest that spheroids of diameter ranging between 300 and 500 µm accurately mimic real tumors [26][27][28] .
As it can be seen in Fig. 5, with increasing length of hanging drop culture, spheroids became a little darker in color, which is an indicative of gradual compactness 29 .
On the seventh day following hanging drop culture, spheroids were transfected with the previously mentioned dendriplexes. It is noticeable that, spheroids were washed and transfected while they were in the drops, which to the best of our knowledge has not been previously accomplished.
Next, the transfected spheroids were used to evaluate the discrimination of our treatment e cacy in MDA-MB-231/HER2 + monolayer and spheroid culture.
Anti-HER2 VHH functionalization improved cellular uptake in the HER2 overexpressing cell line.
Fluorescence microscopy was employed to compare the uptake e ciency of dendrimers and evaluate targeting e ciency of the anti-HER2 VHH. As it can be seen in Fig. 6 Comparison of MDA-MB-231/HER2 + grown as monolayer vs spheroid revealed that internalization of all the dendriplexes were signi cantly lower in the spheroids.
A possible reason for the lower uptake rate in spheroids might be the penetration resistance inherent in their structure. Spheroid is a three-dimensional (3D) model that closely resembles small avascular tumors and micrometastases in that they contain a proliferative outer shell, a relatively large zone of hypoxic quiescent cells and a relatively small necrotic area at the center. This 3D structure exhibit cell-to-cell and cell-to-matrix interactions as well as nutrient, pH and oxygen gradients. These characteristics make them proportional to penetration so that they are not e ciently transfected. This kind of resistance might not be observed when cells are cultured as monolayer 32,33 .
In First, we will discuss the cytotoxic effect of the different dendriplexes in the three cell lines (cultured as monolayer) together, then MDA-MB-231/HER2 + grown as monolayer and spheroid will be compared.
PAM/pG (empty vector) was used to determine the toxicity of the transfection process and as shown in These results also demonstrate that, combination of the cell surface and transcriptional targeting, exerted a synergistic targeting effect in HER2 + BCSCs.
When transfected with VHH-PG-PAM/pG-CX-bF-PE (targeted on cell surface, transcriptional and translational levels), MCF-10A and MDA-MB-231 exhibited 61% and 38% viability, respectively. But MDA-MB-231/HER2 + showed 15% recovery of the cells. Distinct viability variation between the cell lines, indicates the synergistic effect of these three targeting elements when combined together. It is noticeable that, viability of VHH-PG-PAM/pG-CX-bF-PE treated groups had a tendency to increase as compared to that of VHH-PG-PAM/pG-CX-PE treated groups, probably because the highly structured, GC rich 5' UTR of the bFGF hinders e cient translation. Translation of mRNAs with highly structured bFGF 5′UTR is particularly dependent on the unwinding activity of eIF4E. Higher levels of eIF4E in breast cancer cell lines, relative to non-malignant MCF10A cells have been reported previously [34][35][36]  Comparison of MDA-MB-231/HER2 + grown as monolayer vs spheroid revealed that MDA-MB-231/HER2 + spheroid followed the same viability pattern as its monolayer, upon all the treatments, except the viability were signi cantly higher upon all the treatments (p < 0.01). These results further validated those obtained by cellular uptake and real-time PCR experiments. Indeed, previous studies have demonstrated a differential response to compounds when cells grown as spheroid compared to monolayer 38,39 . A good illustration would be the study of Carver et al. 40 . They demonstrated that the delivery of oligonucleotides with Lipofectamine lipoplex and PEI polyplex was signi cantly attenuated in spheroid models compared to monolayer cultures. Further, their results showed that only cells located at the periphery of the spheroid received the oligonucleotides.

Conclusion
In summary, we successfully developed a welldesigned multi-targeted nanosystem using anti-HER2 VHH functionalized PAMAM, CXCR1 promoter, PE38 toxin and bFGF 5′UTR. This nanosystem was selectively internalized, speci cally transcribed, e ciently translated and caused a selective cytotoxicity in HER2positive BCSCs. Our data suggest that this novel multi-targeted nanosystem would be a potent strategy for selective cancer killer gene therapy.
We further demonstrated that the e cacy of our targeted gene therapy was lower in spheroid models compared to monolayer cultures, indicating that anticancer therapy assessments using spheroid might be more predictive of clinical e cacy than conventional monolayer cell culture.

Declarations Data Availability
All data generated or analyzed during this study are included in this published article