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  • br Discussion br Despite several decades of investigation in

    2020-08-18

    
    4. Discussion
    Despite several decades of investigation into biology and treatment of PC, there is still a lack of deep understanding of the causes and pa-thogenesis of PC and more effective therapeutics, making PC one of the most lethal malignancies. Recent findings on the association of dysre-gulation of miRNAs with pathogenesis and progression of PC offer clue to developing miRNA-based therapies [12–16,28]. After revealing a significant downregulation of miR-1291 in human PC tissues and a tumor suppressive action of miR-1291 [17], we demonstrated in the present study that miR-1291 prodrug monotherapy (10–20 μg/mouse or 0.5–1 mg/kg, i.v.) was as effective as Gem-nP (300-40 μg/mouse; 7.5/1 ratio; i.v.) for the control of PC growth in PANC-1 xenograft and PDX mouse models, while combination therapy offered the greatest degrees of suppression. The optimal outcome of combination treatment with miR-1291 and Gem-nP was associated with an increased level of apoptosis.
    Research and development of new miRNA therapeutics are limited to the use of miRNA mimics made in test tubes by chemical synthesis, as well as the access to large quantities of miRNA agents required for animal and human studies [23]. Distinguished from the conventional synthetic miRNA agents, the present study investigated the efficacy of a bioengineered miR-1291 prodrug that was produced in living cells, and purified by a fast protein liquid chromatography (FPLC) method to high degree of homogeneity on large scale [22,29]. Biologic miR-1291 pro-drug was selectively processed to mature miR-1291 in human PC Puromycin and xenograft tumor tissues, which consequently modulated target gene expression and improved the efficacy of Gem-nP.
    PANC-1 cell line is well known for its resistance to chemotherapy which is at least partly due to the overexpression of efflux transporter ABCC1/MRP1, and our previous studies have demonstrated the sup-pression of ABCC1 by miR-1291 in PANC-1 cells [19]. Most im-portantly, PANC-1 is a cell line directly derived from human pancreas/ ducts with epithelioid carcinoma [30], whereas the AsPC-1 cell line is derived from mouse xenografts established with ascites cells of a patient with pancreatic cancer [31]. Therefore, the PANC-1 cell line should be closer and more relevant to human PC and was chosen to establish
    Fig. 4. Combination therapy with miR-1291 prodrug and Gem-nP is the most effective in suppressing PANC-1 xenograft tumor growth in mice and all therapies are well tolerated. (A) Timeline of the establishment of PANC-1 xenograft mouse model and drug treatment. (B) PANC-1 xenograft tumor growth was reduced to the greatest degree by combination treatment with miR-1291 prodrug and Gem-nP. *P < 0.05, ***P < 0.001 (2-way ANOVA with Bonferroni post-tests). (C) Visual comparison of dissected tumors from mice with different treatments. (D) Weights of the dissected xenograft tumors. *P < 0.05 (1-way ANOVA). (E) Body weights were not altered by drug treatment. (F) Blood biochemistry profiles including alanine transaminase (ALT), aspartate transaminase (AST), creatinine, blood urea nitrogen (BUN), and total bilirubin showed no significant difference by different treatments. Values are mean ± SD (N = 6 per group, except N = 3 for blood chemistry profiles). The ranges of individual markers (derived from BALB/c mice; Comparative Pathology Laboratory at UC-Davis) were marked as references.
    xenograft mouse models for therapy studies. While both miR-1291 and Gem-nP monotherapy showed an overall effectiveness in controlling PANC-1 xenograft tumor growth, intra-individual variation was ob-vious. Even with a small sample size of six mice per group, three sub-jects were sensitive to miR-1291 and Gem-nP monotherapy, whereas the other three showed relatively poor responses. In contrast, combi-nation therapy with miR-1291 and Gem-nP, while well tolerated in mice, was able to ubiquitously suppress tumor growth and to a greater extent than either Gem-nP or miR-1291 alone, demonstrating the ad-vantage of combination treatment than monotherapy.
    To better recapitulate the properties of original patient tumors and reflect the efficiency of new therapies in patients, an increasing number of PDX models have been used for studying cancer biology and asses-sing new drugs [25,32–35]. In current study, PDX models from three different PC patients were established and utilized to evaluate miR-1291 monotherapy and combination treatment with Gem-nP. Con-sistent with findings from PANC-1 xenograft mouse models, miR-1291 prodrug was effective to reduce PDX tumor growth and improve the efficacy of Gem-nP, while histopathology analysis indicated that PDX tumor indeed better preserved the histological features of clinical PC than PANC-1 xenograft tumors (data no shown). As manifested by the increased c-caspase-3 levels, reduction of PDX progression by combi-nation therapy was attributable to the induction of apoptosis, which is also in accordance with in vitro data. Moreover, different PDX tumor