Multimodal Treatment Eliminates Cancer Stem Cells and Leads to Long-Term Survival in Primary Human Pancreatic Cancer Tissue Xenografts.

Copyright: 2013 Hermann et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.PURPOSE: In spite of intense research efforts, pancreatic ductal adenocarcinoma remains one of the most deadly malignancies in the world. We and others have previously identified a subpopulation of pancreatic cancer stem cells within the tumor as a critical therapeutic target and additionally shown that the tumor stroma represents not only a restrictive barrier for successful drug delivery, but also serves as a paracrine niche for cancer stem cells. Therefore, we embarked on a large-scale investigation on the effects of combining chemotherapy, hedgehog pathway inhibition, and mTOR inhibition in a preclinical mouse model of pancreatic cancer. EXPERIMENTAL DESIGN: Prospective and randomized testing in a set of almost 200 subcutaneous and orthotopic implanted whole-tissue primary human tumor xenografts. RESULTS: The combined targeting of highly chemoresistant cancer stem cells as well as their more differentiated progenies, together with abrogation of the tumor microenvironment by targeting the stroma and enhancing tissue penetration of the chemotherapeutic agent translated into significantly prolonged survival in preclinical models of human pancreatic cancer. Most pronounced therapeutic effects were observed in gemcitabine-resistant patient-derived tumors. Intriguingly, the proposed triple therapy approach could be further enhanced by using a PEGylated formulation of gemcitabine, which significantly increased its bioavailability and tissue penetration, resulting in a further improved overall outcome. CONCLUSIONS: This multimodal therapeutic strategy should be further explored in the clinical setting as its success may eventually improve the poor prognosis of patients with pancreatic ductal adenocarcinoma

Serum‐Derived Small Extracellular Vesicles From Diabetic Mice Impair Angiogenic Property of Microvascular Endothelial Cells: Role of EZH2

Background Impaired angiogenic abilities of the microvascular endothelial cell (MVEC) play a crucial role in diabetes mellitus–impaired ischemic tissue repair. However, the underlying mechanisms of diabetes mellitus–impaired MVEC function remain unclear. We studied the role of serum‐derived small extracellular vesicles (ssEVs) in diabetes mellitus–impaired MVEC function. Methods and Results ssEVs were isolated from 8‐week‐old male db/db and db/+ mice by ultracentrifugation and size/number were determined by the Nano‐sight tracking system. Diabetic ssEVs significantly impaired tube formation and migration abilities of human MVECs. Furthermore, local transplantation of diabetic ssEVs strikingly reduced blood perfusion and capillary/arteriole density in ischemic hind limb of wildtype C57BL/6J mice. Diabetic ssEVs decreased secretion/expression of several pro‐angiogenic factors in human MVECs. Mechanistically, expression of enhancer of zest homolog 2 (EZH2), the major methyltransferase responsible for catalyzing H3K27me3 (a transcription repressive maker), and H3K27me3 was increased in MVECs from db/db mice. Diabetic ssEVs increased EZH2 and H3K27me3 expression/activity in human MVECs. Expression of EZH2 mRNA was increased in diabetic ssEVs. EZH2‐specific inhibitor significantly reversed diabetic ssEVs‐enhanced expression of EZH2 and H3K27me3, impaired expression of angiogenic factors, and improved blood perfusion and vessel density in ischemic hind limb of C57BL/6J mice. Finally, EZH2 inactivation repressed diabetic ssEVs‐induced H3K27me3 expression at promoter of pro‐angiogenic genes. Conclusions Diabetic ssEVs impair the angiogenic property of MVECs via, at least partially, transferring EZH2 mRNA to MVECs, thus inducing the epigenetic mechanism involving EZH2‐enhanced expression of H3K27me3 and consequent silencing of pro‐angiogenic genes. Our findings unravel the cellular mechanism and expand the scope of bloodborne substances that impair MVEC function in diabetes mellitus

Assessment of in vivo biocompatibility/safety.

<p>(<b>A</b>) Body weights were recorded for all mice throughout the first 100 days of the experiment. (<b>B</b>) White blood cell counts of all mice were assessed at the end of the administration period of the triple combination.</p

Comparison of the <i>in vitro</i> and <i>in vivo</i> effects of Pegylated Gemcitabine.

<p>(<b>A</b>) <i>In vitro</i> effects of Gem and PEG-Gem on apoptosis and cell death as well as CD133 expression (cumulative results of cells obtained from different xenografts). (<b>B</b>) Kaplan-Meier Curve depicting cumulative survival time of all mice pooled by treatment group. For illustrative purposes, selected survival curves of Fig. 2D are depicted again. (<b>C</b>) Tumor growth curves for primary whole-tissue xenografts implanted subcutaneously and orthotopically, respectively. Continuous line depicts Gem+vehicle, dashed line depicts Gem+ SIBI, dotted line depicts Gem+SIBI+Rapa. (<b>D</b>) Kaplan-Meier Curve depicting cumulative survival time of all mice pooled by treatment group. For illustrative purposes, selected survival curves of Fig. 2D are depicted again.</p

Combination therapy in a representative set of pancreatic ductal adenocarcinoma.

<p>(<b>A–F</b>) Tumor growth curves for primary whole-tissue xenografts PDAC-265, PDAC-185, JH051, 247, Pax22, and 354 implanted subcutaneously and orthotopically. Continuous line depicts Gem+vehicle, dashed line depicts Gem+SIBI, dotted line depicts Gem+SIBI+Rapa (n≥6 per group). (<b>G</b>) Kaplan-Meier Curve depicting cumulative survival time of all mice pooled by treatment group.</p

Effect of combination therapy on cancer stem cell content.

<p>(<b>A</b>) Representative flow cytometry plots and (<b>B</b>) quantification of cancer stem cell (EpCAM⁺CD133⁺CD44⁺) content of tumors in the respective treatment group (cumulative results of cells obtained from different xenografts). (<b>C</b>) Representative images and quantification of secondary sphere formation of treated PDAC-Pax22 tumors explanted at the end of the experiment (d200).</p

Effect of combination therapy on tumor composition.

<p>(<b>A</b>) Representative histological pictures showing stroma content in the respective treatment groups in gemcitabine resistant orthotopic tumors (<b>PDAC-185, upper panel</b>), (<b>Pax22, lower panel</b>). (<b>B</b>) Quantification of stroma content throughout the different treated xenografts.</p

Targeting of sonic hedgehog and mTOR in pancreatic ductal adenocarcinoma.

<p>(<b>A</b>) Fold increase mRNA expression levels of SHH, GLI-1, and GLI-2 of sphere-derived vs. adherent cells. (<b>B</b>) Western blot analysis of mTOR pathway activity via the assessment of S6 kinase expression (upper panel) and phosphorylation (lower panel) in adherent primary cells versus stem cell-enriched sphere-derived cells. (<b>C</b>) Illustration of experimental setup. Duration of triple therapy is marked by a dark grey box (day 21 to 48), Gem monotherapy with a light grey box (day 48 to 81).</p