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  • br All treatments were well tolerated in mice as

    2020-08-07


    All treatments were well tolerated in mice as animal body weights showed no significant differences among different groups (Fig. 4E). To further examine the safety of drug treatments, blood Z VAD FMK profiles were determined (Fig. 4F). All markers of liver and kidney functions including alanine aminotransferase (ALT), aspartate amino-transferase (AST), total bilirubin, blood urea nitrogen (BUN) and creatinine were within the normal ranges, except ALT levels in two mice (one from miR-1291 monotherapy and one from Gem-nP mono-treatment) slightly exceeded the normal range. However, there was no significant difference in each blood biomarker between any treatment groups, suggesting that therapies did not cause any hepatic or renal toxicity. Together, the results indicate that systemic administration of therapeutic doses of miR-1291 prodrug or Gem-nP alone, or in com-bination are well tolerated in PANC-1 xenograft mouse models.
    3.6. Efficacy of miR-1291 prodrug treatment alone and in combination with Gem-nP chemotherapy in three different PDX mouse models
    Compared to xenograft models derived from cancer cell lines, PDX tumor models may better preserve the heterogeneity and histological characteristics of the original tumors [25–27]. Therefore, we estab-lished three PDX models from clinical PC samples and employed them to further assess miR-1291 prodrug therapies. The first PDX model (PA-0387, Fig. 5) was subjected to the same dose regimens as those used in PANC-1 xenograft mouse models. Similarly, RT-qPCR analyses con-firmed high levels of miR-1291 in PDX tissues at 24 h after systemic administration of a single dose of miR-1291 prodrug (Supplementary Fig. S5B). Therapy data showed that treatment with miR-1291 prodrug or Gem-nP alone significantly reduced PDX PA-0387 tumor growth, as compared to buffer or MSA control; and combination treatment showed the highest degree of inhibition (Fig. 5A). Likewise, visual inspection of dissected tumors (Fig. 5B) and examination of final tumor weights (Fig. 5C) supported the effectiveness of miR-1291 prodrug alone, Gem-nP alone, and their combination in the control of PDX PA-0387, while there was no statistical difference between mono- and combination therapy. H&E staining demonstrated that PDXs indeed showed the histologic phenotypic characteristics close to clinical pancreatic ade-nocarcinomas (Supplementary Fig. S6). Furthermore, im-munohistochemistry studies showed that there was no difference in cell proliferation (Ki-67 staining) between different treatment groups, while tumors from combination group showed the highest levels of apoptosis
    Fig. 3. Bioengineered miR-1291 prodrug sensitizes human pancreatic cancer cells to Gem-nP. Compared to the MSA control, a low dose of MSA/miR-1291 had minimal effects on AsPC-1 (A) and PANC-1 (C) cell proliferation, whereas it significantly (P < 0.001; 2-way ANOVA with Bonferroni posttests) improved the sensitivity of AsPC-1 (B) and PANC-1 (D) cells to Gem-nP, which was also indicated by the estimated pharmacodynamic parameters (E). AsPC-1 and PANC-1 cells were treated with MSA/mir-1291 or MSA control alone (A, C) or in combination with various concentrations of Gem-nP (B, D; shown are total concentrations of Gem-nP at a fixed ratio of 8: 1) for 48 h, and cell viability was determined by CellTiter-Glo assay. Values are mean ± SD (N = 3). *P < 0.05; **P < 0.01, compared to corresponding MSA control treatment (Student's t-test).
    (c-caspase-3) (Fig. 5D), supporting the induction of apoptosis as a major mechanism behind their antitumor activities. In addition, none of the animals showed any signs of stress, and there was no significant dif-ference in body weights (Fig. 5E) and blood biochemistry profiles (Fig. 5F) among different treatments, suggesting that all drug treat-ments were safe to PDX-bearing mice.
    Another PDX model, PA-0375, was utilized to critically assess the efficacy of miR-1291 prodrug monotherapy and combination therapy with Gem-nP, by following the same dosing regimens for PANC-1 xe-nograft models (Fig. 4A). Our data showed that treatment with either miR-1291 prodrug or Gem-nP, alone or in combination, was able to significantly suppress PA-0375 PDX tumor growth in mice (Fig. 6A–C). While it was not statistically different between mono- and combination therapy, combination therapy obviously produced the greatest extent of inhibition.
    Because the third PDX model, PA-0327, was more aggressive than the other two PDX models, we refined the dosing regimens by in-creasing miR-1291 prodrug dose to 20 μg/mouse for both mono- and combination therapy, while using the same dose of Gem-nP. Optimal outcomes were surprisingly observed (Fig. 6D–F). Compared to buffer and MSA treatment, monotherapy with miR-1291 prodrug or Gem-nP significantly reduced PA-0327 PDX to a similar level (∼50%), which was indicated by tumor growth over time (Fig. 6D), visual inspection of dissected tumors (Fig. 6E) and quantitative measurement of tumor weights (Fig. 6F) at the end of the study. Most importantly, co-ad-ministration of miR-1291 prodrug and Gem-nP chemotherapeutics could suppress PDX progression to the greatest degree (> 80%) that was also significantly different from monotherapy (Fig. 6D). The strongest antitumor effects of combination therapy were also demon-strated by visual inspection (Fig. 6E) and weighting (Fig. 6F) of the dissected tumors. In addition, body weight of PDX-bearing animals did not show any significant difference among different treatment groups (Supplementary Fig. S7), indicating that all treatments were well tol-erated in mice.