A comparative study of single or dual treatment of theranostic 188Re-Liposome on microRNA expressive profiles of orthotopic human head and neck tumor model

Background: 188Re-liposome has been used for evaluating the theranostic effi cacy on human head and neck squamous cell carcinoma (HNSCC) at preclinical stages. Here we furthercompared the microRNA expressive profi le in orthtopic HNSCC tumor model exposed to 188Re-liposome. Methods: A single dose or dual doses of 188Re-liposome was intravenously injected into tumor-bearing mice followed by the Cerenkov luminescent imaging (CLI) for monitoring the accumulation of 188Re-liposome in tumors. The microRNA expressive profi le was generated using the TaqMan® OpenArray® Human MicroRNA Panel followed by the DIANA mirPath analysis, KEGG signaling pathways prediction, and Kaplan-Meier survival analysis for predicting the prognostic role of 188Re-liposome aff ected microRNAs. Results: Dual doses of 188Re-liposome exhibited a better tumor suppression than a single dose of 188Re-liposome, including reduced tumor size, Ki-67 proliferative marker, and epithelial-mesenchymal transition (EMT) related factors. The microRNA expressive profi les showed that 22 microRNAs and 19 microRNAs were up-regulated and down-regulated by dual doses of 188Re-liposome, respectively. Concomitantly, these two groups of microRNAs were inversely regulated by a single dose of 188Re-liposome accordingly. These microRNAs infl uenced most downstream genes involved in cancer related signaling pathways. Further, miR-520e and miR-522-3p were down-regulated whereas miR-186-5p and miR-543 were up-regulated by dual doses of 188Re-liposome, and they separately aff ected most of genes involved in their corresponding pathways with high signifi cance. Additionally, high expressions of miR-520e and miR-522-3p were associated with lower survival rate of HNSCC patients. Conclusion: MicroRNA expression could be used to evaluate the therapeutic effi cacy and regarded prognostic factors using diff erent doses of 188Re-liposome. More Information *Address for Correspondence: Yi-Jang Lee, Ph.D., Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, No. 155, Sec. 2, Linong St. Beitou District, 112, Taipei, Taiwan, Tel: +886-2-28267189; Email: yjlee2@ym.edu.tw Submitted: February 20, 2021 Approved: February 24, 2021 Published: February 25, 2021 How to cite this article: Wang SY, Lin LT, Lin BZ, Chang CH, Chang CY, et al. A comparative study of single or dual treatment of theranostic 188Re-Liposome on microRNA expressive profi les of orthotopic human head and neck tumor model. Heighpubs Otolaryngol Rhinol. 2021; 5: 001-012. DOI: 10.29328/journal.hor.1001024 Copyright: © 2021 Wang SY, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Introduction
Radiogenomics is a rising ield of modern applications for radiomics linking to the genetic pro ile of tumor progression to better predict the therapeutic responses from diagnosis to prognosis. A recent review has summarized the applications of radiogenomics in a variety of human cancers [1]. It also directly in luences the gene expressive pro ile of target tissues because of the radiotherapy induced cytotoxicity [2]. Radiopharmaceutical is an ideal candidate for monitoring the ef icacy of drug targeting and therapy using preclinical or clinical imaging modalities. However, radiopharmaceutical is little applied in radiogenomics.
Theranostics is de ined as speci ic targeted diagnosis and therapy using a material with both effects on tumor treatment. Nuclear medicine plays an important role in theranostics as several types of radiopharmaceuticals emit both γ-rays and high energy β particles for diagnosis and therapy, respectively [3]. For instance, rhenium-188 ( 188 Re) emits 85% of 2.12MeV β particles and 15% of 155keV γ-rays during decay, so it belongs to a theranostic radionuclide as well [4]. It is also an attractive and affordable radiopharmaceutical because of its short half-life and on-demand availability using a tungsten-188/ rhenium-188 generator [4]. Moreover, the atomic radius of 188 Re is similar to technetium that has been widely used in clinics [5,6]. The tissue penetration of emitted β particles is about 10 mm, suggesting that it is suitable for the treatment of large-sized or mid-late stage tumors [7]. Accumulated literature have demonstrated that liposome embedded 188 Re ( 188 Re-liposome) is able to target human colorectal cancer, glioblastomas, esophageal cancer, head and neck cancer and lung cancer using the xenograft tumor model [8][9][10][11][12]. Tumor accumulation of 188 Re-liposome is passive on the behalf of the enhanced permeability and retention (EPR) effect, which is dependent on the mal-formation of blood vessels surrounding tumors [13,14]. Previous studies have demonstrated that 188 Re-liposome could in luence the gene expression in human head and neck squamous cell carcinoma (HNSCC), and the let-7 family of microRNA mediated gene expressive pro ile was signi icantly involved in this treatment [12]. Therefore, this inding intrigues us to investigate whether 188 Re-liposome would also in luence the microRNA expressive pro ile.
Although 188 Re-liposome exhibited tumor accumulative property in HNSCC, the therapeutic ef icacy was moderate. Speci ically, regrowth of xenograft tumors were detected when they were treated by a single dose of 188 Re-liposome but not by repeated doses [15]. As chemoresistance is known to be associated with a series of molecular regulation [16], we are interested in investigating the gene expressive pro iles of HNSCC tumors treated with 188 Re-liposome. In this study, we exploited the open arrays of microRNA to analyze over 700 microRNA in orthotopic HNSCC tumor treated with a single dose or repeated doses of 188 Re-liposome. The interested microRNAs were also subjected to survival analysis. The results of 188 Re-liposome modulated expression of microRNA were discussed.

Cell line
Human FaDu head and neck squamous cell carcinoma cells (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI-1640 (Life Technologies Inc., Carlsbad, CA, USA) medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamate, 50 unit/ml penicillin and 50 μg/ml streptomycin (Invitrogen, Carlsbad, CA). The pH of medium was adjusted by sodium bicarbonate. Cells were incubated at 37 o C in a humidi ied incubator with 5% CO 2 , and passaged every two days.

Preparation of Rhenium-188 liposomal drug
The procedures of 188 Re-liposome preparation of validation were followed as described previously [17]. The dose of each injection is 640 μCi corresponding to 80% maximum tolerated dose (MTD) [9].

HNSCC orthotopic tumor model
Four-week old male BALB/c nude mice were used to establish the orthotopic tumor model (National Laboratory Animal Center of Taiwan, Tainan, Taiwan). FaDu cells (1 x 10 6 ) were resuspended in 100 μl of OPTI-MEM and injected into the buccal positions of mice (N = 5) using a 27G insulin needle. Tumors could form about 3 weeks after tumor implantation. The study has been approved by the Institutional Animal Care and Utilization Committee (IACUC) of National Yang-Ming University (case no. 1061010).

Cerenkov luminescent imaging (CLI) and tumor resection
CLI was performed at 24 hours after the administration of 188 Re-liposome. The signals were acquired by the in vivo Imaging System (IVIS 50, Perkin Elmer Inc., Waltham, MA, USA). Regions of interest (ROIs) were delineated on the tumor around the mouth. For evaluation of tumor response to 188 Reliposome, resection of tumors with various treatment were performed and compared by size.

MicroRNA expressive profi ling analysis
TaqMan ® OpenArray ® Human MicroRNA Panel (Thermo Fisher Scienti ic Inc., Waltham, MA, USA) was used to detect the microRNA expression in orthotopic HNSCC tumors treated with different regimens of 188 Re-liposome and compared to untreated control. The operation was performed in the Sequencing Core Facility of National Yang Ming University Genome Center (YMGC). For analysis, the expression levels of the two experimental groups (dual doses and single dose of 188 Re-liposome) were de ined by the Mean Relative Quanti ication (RQ) normalized to the control group. The expression level changes between the groups were demonstrated by log2 ratio. All the identi ied assay id of each miRNA probes was converted to miRBase ID (v22) referring to the manufacturer's handbook. The lists were input to DIANA mirPath v.3 (http://snf-515788.vm.okeanos.grnet.gr/) for Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, referencing the DIANA-MicroT prediction [20]. The overall involvement and the co-regulated gene prediction were concluded with p -value threshold of 0.05 and MicroT threshold of 0.8 by genes union and genes intersection function, respectively.

Survival analysis for microRNA
The association of microRNA of interest with public human HNSCC microRNA database was determined by an online Kaplan-Meier plotter [21]. In the database, the miRNA subsystems include 11k samples from 20 different cancer types, which include 523 human HNSCC cases. The signi icance of microRNA associated survival rate was determined by a log-rank test.

Statistics
Data were represented as the mean ± S.D. from triplicate independent results, and the statistical analysis was performed using t-test. The statistical signi icance was set at p < 0.05.

Eff ects of single dose and dual doses of 188 Re-liposome on the growth of orthotopic HNSCC tumor model
The regimen of 188 Re-liposome treatment and the timeline of imaging evaluation and tumor resection on HNSCC tumorbearing mice were schemed ( Figure 1A). Because 188 Reliposome emits high energy β-particles, the drug accumulation at tumor site can be detected by the Cerenkov luminescent imaging (CLI) in vivo. Compared to the untreated control, both strategies of 188 Re-liposome treatment showed CLI signals in tumor-bearing mice ( Figure 1B). Although dual doses did not exhibit increased accumulation of 188 Re-liposome at tumor lesion, the tumor size was apparently smaller than that treated with a single dose of 188 Re-liposome ( Figure 1C). These results implied that dual doses of 188 Re-liposome would be more effective than a single dose of 188 Re-liposome on suppression of tumor growth in vivo.

Eff ects of single dose and dual doses of 188 Re-liposome on the expression of Ki67 biomarker
To determine if different effects of tumor suppression by single dose and dual doses of 188 Re-liposome was associated with tumor proliferation, the Ki-67 proliferative marker was examined in sections of resected tumors. Compared to the untreated control and the single dose, tumors treated with dual doses of 188 Re-liposome expressed a very low level of Ki-67 using IHC staining (Figure 2A). The result was also quanti ied by IHC scoring ( Figure 2B). Therefore, dual doses of 188 Re-liposome could better suppress the proliferation of tumor cells in vivo.

Eff ects of single dose and dual doses of 188 Re-liposome on the expression of EMT related biomarkers
In addition to the Ki-67 proliferative marker, we also compared the expression of biomarkers associated with EMT mechanism in HNSCC tumors treated with a single dose and dual doses of 188 Re-liposome. E-cadherin, vimentin, Snail, Slug, and ZEB-1 were examined, and the results showed that dual doses of 188 Re-liposome exhibited stronger effects on suppression of EMT by inducing E-cadherin, and inhibiting vimentin, Snail and Slug, respectively ( Figure 3). ZEB-1 was the only EMT promoting molecule suppressed equally by both single dose and dual doses of 188 Re-liposome ( Figure 3). According to the expression of these molecules, it suggests that dual doses of 188 Re-liposome enhanced the therapeutic ef icacy by suppressing tumor proliferation and metastasis concomitantly.

Demonstration of potent microRNA regulated by single dose and dual doses of 188 Re-liposome
According to the KEGG pathway analysis by the microRNA openarray dataset, the intracellular signaling pathways of HNSCC tumors in luenced by dual doses of 188 Re-liposome were ranked by p -value. Thirty-nine and 62 pathways were signi icantly affected by the microRNAs down-regulated and up-regulated by dual doses of 188 Re-liposome, respectively (p < 0.05). According to the rank of p -value, the top 10 signaling pathways affected by miRNAs that were downregulated or up-regulated by dual doses of 188 Re-liposome were different and were shown in table 3 and table 4. Compared to other microRNAs down-regulated by dual doses of 188 Re-liposome, miR-520e and miR-522-3p affected most of the genes involved in that top 10 pathways (Table 3). On the other hand, miR-186-5p and miR-543 were involved in   (Table 4). We next compared the downstream genes regulated by miR-520e and miR-522-3p and found that SMAD2, FZD3, PIK3CA, and JAK1 genes were involved in these two microRNAs ( Figure 5A). For miR-186-5p and miR-543, 13 genes including FGF12, SMAD2, CBL, PTCH1, TPR, CDK6, FZD3, HDAC2. PIAS2, PIK3CA, PTEN, CREBBP, and XIAP were co-regulated ( Figure 5B). Surprisingly, SMAD2, FZD3, and PIK3CA genes were commonly regulated by these four miRNAs, even though miR520e and miR-522-3p were down-regulated, but miR-186-5p and miR-543 were up-regulated by dual doses of 188 Re-liposome. The full list of downstream genes regulated by these four microRNAs was provided (Supplementary data 2).

Association of miRNAs aff ected by 188 Re-liposome and patients survival
We next analyzed the association of miR-520e, miR-522-3p, miR-186-5p, and miR-543 with patients survival using the public on-line Kaplan-Meier Plotter tool (see Materials and Methods). High expression of miR-520e and miR-522-3p was associated with a reduced survival rate, while that of miR-186-5p was marginally associated with an increased survival rate of HNSCC cancer patients ( Figure 6). On the other hand, higher expression of miR-543 was also associated with lower survival rate (data not shown). As mentioned above, miR-520e and miR-522-3p were suppressed by dual doses of 188 Re-liposome treatment, but miR-186-5p was up-regulated accordingly. Hence, it may suggest that these microRNAs could be used as prognostic factors for HNSCC patients treated with 188 Re-liposome.      Re-liposome has been demonstrated to be effective on suppression a variety of human cancers. Most of the studies focused on the biodistribution, tumor targeting, pharmaceutic kinetics and dosimetry previously [9,10,17,[22][23][24]. A phase 0 clinical trial has also shown that 188 Re-liposome is a potent theranostic agent for cancer treatment [25]. In this study, we irst demonstrated that dual doses of 188 Re-liposome exhibited stronger effects than a single dose of 188 Re-liposome on suppression of orthotopic HNSCC tumor growth and EMT phenomenon. We have recently showed that repeated treatment of 188 Re-liposome on tumor-bearing mice do not cause acute toxicity but reduce blood cell counts [15]. The time interval between the two administrations of 188 Re-liposome was over 10 half-lives of this isotope, yet the responses of HNSCC tumors were still enhanced. It is speculated that tumor insulted by irst dose of 188 Re-liposome has not been fully repaired before the second doses of 188 Re-liposome. Notably, the dose of dual treatments were equal and both were at 80% MTD. Thus a sublethal damage repair should not be raised to compromise the ef icacy of 188 Re-liposome at second treatment.
Most of microRNAs down-regulated by dual doses of 188 Re-liposome but up-regulated by a single dose of 188 Reliposome, or vice versa, were associated with genes involved in pathways in cancer according to the KEGG pathway analysis. Although the p -value of this category was not smallest in affected pathways (Table 1 and 2), it is convinced that dual doses of 188 Re-liposome would vigorously in luence microRNA related genes in tumor ablation. Interestingly, the pathway of proteoglycan in cancer accounted for the smallest p -value in both conditions of microRNA regulated by dual doses of 188 Re-liposome. The signaling of hyaluronan, heparan sulfate proteoglycans, chondroitin sulfate/dermatan sulfate proteoglycan, and keratan sulfate proteoglycan are involved in proteoglycan in cancer pathway, and they are essential for cell adhesion, migration and angiogenesis in KEGG pathway. Indeed, the role of proteoglycan in tumor microenvironment and angiogenesis has been reported [26][27][28]. Thus, this bioinformatics analysis for microRNA regulation was consistent with the tumor suppressive effects caused by dual doses of 188 Re-liposome. The p -value of Hippo signaling pathway was the same with proteoglycan in cancer pathway only in microRNAs down-regulated by dual doses of 188 Re-liposome. This pathway was even not ranked as primary pathway affected by 188 Re-liposome up-regulated microRNAs ( Table 2). As Hippo signaling is a critical tumor suppressor pathway, it would be interesting to further investigate how 188 Re-liposome regulate this signaling pathway.
According to the data analysis of TaqMan ® Openarray Human MicroRNA Panel, miR-520e and miR-522-3p were down-regulated by dual doses of 188 Re-liposome but they were also induced by a single dose of 188 Re-liposome. On the contrary, miR-186-5p and miR-543 were regulated oppositely by the same regime. Although these microRNAs did not rank as top change by dual doses over a single dose of 188 Re-liposome, they in luenced most genes in top 10 pathways affected by dual doses of 188 Re-liposome. The Venn diagram showed that miR-520e and miR-522-3p co-regulated 4 downstream genes, and miR-186-5p and miR-543 co-regulated 13 downstream genes. Surprisingly, these two groups of microRNAs responded oppositely to dual doses of 188 Re-liposome in luenced 3 common genes, that is, SMAD2, FZD3, and PIK3CA. SMAD2 mediates transforming growth factor-β (TGF-β) relayed signaling pathway, and it works differentially with other SMAD isoform [29]. TGF-β signaling pathway contains both tumor suppressor and oncogene actions mediated by SMAD3 [30]. However, several lines of evidence showed that SMAD2 belongs to tumor suppressor gene of TGF-β signaling pathway [31,32]. Little is known about the function of FZD3 (Frizzled 3 receptor) gene, although a recent report demonstrated that down-regulation of FZD3 gene suppresses human melanoma tumorigenesis independent of WNT signaling [33]. PIK3CA (Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha) gene is also regarded an oncogene, and is usually mutated in cancer cells [34]. It is believed that other genes independently regulated by these two groups of microRNAs are also associated with tumorigenesis. Although we focused on these four microRNAs up-regulated or downregulated by dual doses of 188 Re-liposome, other microRNAs are not excluded and further investigation should be required.
For clinical relevant, we applied the Kaplan-Meier survival analysis to examine if the 188 Re-liposome regulated microRNA would be associated with the survival rate of HNSCC patients. For dual doses of 188 Re-liposome down-regulated miR-520e and miR-522-3p, both of them exhibit reduced survival rates at high expression. Notably, these two microRNAs were upregulated by a single dose of 188 Re-liposome. Dual doses of 188 Re-liposome up-regulated miR-186-5p was associated with enhanced survival rate at high expression, but the signi icance is margin. MiR-181c-5p was ranked as highest D/S ratio, and this microRNA exhibited signi icant increase of survival rate at high expression (Supplementary data 3). However, miR-872 had the lowest D/S ratio but the association of this microRNA with survival rate was unavailable using the online KM plot tool. Although current study could not determine the biological signi icance of these microRNA in modulating the therapeutic ef icacy of 188 Re-liposome, they might be considered as prognostic factors for different regime of 188 Reliposome treatment.

Conclusion
Current data demonstrated that dual doses of 188 Reliposome exhibited better tumor ablation than a single dose of 188 Re-liposome on the orthotopic HNSCC tumor model. We further found that several microRNAs were inversely regulated by these two regimes of 188 Re-liposome treatment.