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PDF file - 1.4MB, Fig. S1. IL-1 secreting carcinoma cells are able to induce PGE2 from MSCs; Fig. S2. IL-1 and LoVoCM induce PGE2, IL-6, IL-8 and GRO-� expression in various mesenchymal cells; Fig. S3. MSCs are recruited to tumors in vivo; Fig. S4. LoVo cancer stem cells are ALDH-positive; Fig. S5. The frequency of tumor initiating cells is increased in ALDH1-postive HCC1806 SCCs.; Fig. S6. MSCs increase ALDH high/CD133+ CSC-enriched LoVo cells; Fig. S7. LoVo cells cultured with tdTomato-MSCs have increased numbers of tumor initiating cells; Fig. S8. PGE2-induced tumor initiation in vivo was not further potentiated by IL-6, IL-8, Gro-a and RANTES; Fig. S9. LoVoMSC cocultures secrete PGE2-dependent angiogenic factors; Fig. S10. COX2-PGE2-EP4 signaling is required for the increase of ALDH high LoVo; cells induced by MSCs. Fig. S11. LoVoCM induces fibroblast and myofibroblast markers in MSCs; Fig. S12. ALDH high CSCs are located near COX2-expressing regions in human colorectal cancer tumors.

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Supplementary Methods and Materials, Figure Legends 1-4 from Combined Transductional Untargeting/Retargeting and Transcriptional Restriction Enhances Adenovirus Gene Targeting and Therapy for Hepatic Colorectal Cancer Tumors

Supplementary Figure 1 from Combined Transductional Untargeting/Retargeting and Transcriptional Restriction Enhances Adenovirus Gene Targeting and Therapy for Hepatic Colorectal Cancer Tumors

Supplementary Figure 3 from Combined Transductional Untargeting/Retargeting and Transcriptional Restriction Enhances Adenovirus Gene Targeting and Therapy for Hepatic Colorectal Cancer Tumors

Supplementary Figure 2 from Combined Transductional Untargeting/Retargeting and Transcriptional Restriction Enhances Adenovirus Gene Targeting and Therapy for Hepatic Colorectal Cancer Tumors

Supplementary Figure 4 from Combined Transductional Untargeting/Retargeting and Transcriptional Restriction Enhances Adenovirus Gene Targeting and Therapy for Hepatic Colorectal Cancer Tumors

Mesenchymal stem cells (MSCs) have been used in clinical studies to treat neurological diseases and damage. However, implanted MSCs do not achieve their regenerative effects by differentiating into and replacing neural cells. Instead, MSC secretome components mediate the regenerative effects of MSCs. MSC‐derived extracellular vesicles (EVs)/exosomes carry cargo responsible for rescuing brain damage. We previously showed that EP4 antagonist‐induced MSC EVs/exosomes have enhanced regenerative potential to rescue hippocampal damage, compared with EVs/exosomes from untreated MSCs. Here we show that EP4 antagonist‐induced MSC EVs/exosomes promote neurosphere formation in vitro and increase neurogenesis and neuritogenesis in damaged hippocampi; basal MSC EVs/exosomes do not contribute to these regenerative effects. 2′,3′‐Cyclic nucleotide 3′‐phosphodiesterase (CNP) levels in EP4 antagonist‐induced MSC EVs/exosomes are 20‐fold higher than CNP levels in basal MSC EVs/exosomes. Decreasing elevated exosomal CNP levels in EP4 antagonist‐induced MSC EVs/exosomes reduced the efficacy of these EVs/exosomes in promoting β3‐tubulin polymerization and in converting toxic 2′,3′‐cAMP into neuroprotective adenosine. CNP‐depleted EP4 antagonist‐induced MSC EVs/exosomes lost the ability to promote neurogenesis and neuritogenesis in damaged hippocampi. Systemic administration of EV/exosomes from EP4‐antagonist derived MSC EVs/exosomes repaired cognition, learning, and memory deficiencies in mice caused by hippocampal damage. In contrast, CNP‐depleted EP4 antagonist‐induced MSC EVs/exosomes failed to repair this damage. Exosomal CNP contributes to the ability of EP4 antagonist‐elicited MSC EVs/exosomes to promote neurogenesis and neuritogenesis in damaged hippocampi and recovery of cognition, memory, and learning. This experimental approach should be generally applicable to identifying the role of EV/exosomal components in eliciting a variety of biological responses., EP4 antagonist‐induced mesenchymal stem cell (MSC)‐derived extracellular vesicles (EVs)/exosomes contain elevated cyclic nucleotide 3′‐phosphodiesterase (CNP). Exosomal CNP contributes to the ability of EP4 antagonist‐elicited MSC EVs/exosomes to rescue cognition/learning deficiencies of damaged brain. Decreasing exosomal CNP levels in EP4 antagonist‐induced MSC EVs/exosomes reduced the efficacy of these EVs/exosomes in promoting β3‐tubulin polymerization, in converting toxic 2′,3′‐cAMP into neuroprotective adenosine, and in promoting neurogenesis and neuritogenesis. Scale bars, 50 μm. **P ≤ .005.

BACKGROUND Based on the clinical investigation in this study, electrocardiography (ECG) devices generated interpretations on atrial fibrillation (AF), premature ventricular contraction (PVC), and premature atrial contraction (PAC) have high ratios of false-positive errors. An ECG interpretation gap exists between ECG devices and cardiologists. OBJECTIVE The aim of this study was to develop an electrocardiogram (ECG) interpreter to improve the performance of AF, PVC, and PAC screening based on an ECG. METHODS In this study, we first adopted a deep learning model to delineate ECG features such as P, QRS, and T waves based on 1160 8–10-s lead I or lead II ECG signals from a clinically-used 12-lead ECG device whose ECG device interpretation is AF as a training dataset. Second, a sliding window with 3-RR intervals in length is applied to the raw ECG to examine the delineated features in the window, and the ECG interpretation is then determined based on experiences of cardiologists. RESULTS The results indicate the following: (1) This delineator achieves a good performance on P-, QRS-, and T- wave delineation with a sensitivity/specificity of 0.94/0.98, 1.00/0.99, and 0.97/0.98, respectively, in 48 10-s test ECGs mixed with true-positive AF and false-positive AF ECGs. (2) As compared to ECG-device generated interpretations, the precision of the detection of AF, PVC, and PAC in this study was increased from 0.77 to 0.86, 0.76 to 0.84, and 0.82 to 0.87 in 188 10-s test ECGs. Finally, (3) the F1 measure, which is a measure of the accuracy of test data but takes false- positives and false-negative into account, on the detection of AF, PVC, and PAC were 0.92, 0.91, and 0.83, respectively. CONCLUSIONS In conclusion, this study improved the precision of ECG devices’ interpretation and bridge the gap between machines’ ECG interpretation and cardiologists and can facilitate clinical applications on one-channel ECG. CLINICALTRIAL

Cells expressing mesenchymal/basal phenotypes in tumors have been associated with stem cell properties. Cancer stem cells (CSCs) are often resistant to conventional chemotherapy. We explored overcoming mesenchymal CSC resistance to chemotherapeutic agents. Our goal was to reduce CSC numbers in vivo, in conjunction with chemotherapy, to reduce tumor burden. Analysis of clinical samples demonstrated that COX-2/PGE2 /EP4 signaling is elevated in basal-like and chemoresistant breast carcinoma and is correlated with survival and relapse of breast cancer. EP4 antagonism elicts a striking shift of breast cancer cells from a mesenchymal/CSC state to a more epithelial non-CSC state. The transition was mediated by EP4 antagonist-induced extracellular vesicles [(EVs)/exosomes] which removed CSC markers, mesenchymal markers, integrins, and drug efflux transporters from the CSCs. In addition, EP4 antagonism-induced CSC EVs/exosomes can convert tumor epithelial/non-CSCs to mesenchymal/CSCs able to give rise to tumors and to promote tumor cell dissemination. Because of its ability to induce a CSC-to-non-CSC transition, EP4 antagonist treatment in vivo reduced the numbers of CSCs within tumors and increased tumor chemosensitivity. EP4 antagonist treatment enhances tumor response to chemotherapy by reducing the numbers of chemotherapy-resistant CSCs available to repopulate the tumor. EP4 antagonism can collaborate with conventional chemotherapy to reduce tumor burden.

Prostaglandin E2 (PGE2)-initiated signaling contributes to stem cell homeostasis and regeneration. However, it is unclear how PGE2 signaling controls cell stemness. This study identifies a previously unknown mechanism by which PGE2/prostaglandin E receptor 4 (EP4) signaling regulates multiple signaling pathways (e.g., PI3K/Akt signaling, TGFβ signaling, Wnt signaling, EGFR signaling) which maintain the basal mammary stem cell phenotype. A shift of basal mammary epithelial stem cells (MaSCs) from a mesenchymal/stem cell state to a non-basal-MaSC state occurs in response to prostaglandin E receptor 4 (EP4) antagonism. EP4 antagonists elicit release of signaling components, by controlling their trafficking into extracellular vesicles/exosomes in a lipid raft/caveolae-dependent manner. Consequently, EP4 antagonism indirectly inactivates, through induced extracellular vesicle/exosome release, pathways required for mammary epithelial stem cell homeostasis, e.g. canonical/noncanonical Wnt, TGFβ and PI3K/Akt pathways. EP4 antagonism causes signaling receptors and signaling components to shift from non-lipid raft fractions to lipid raft fractions, and to then be released in EP4 antagonist-induced extracellular vesicles/exosomes, resulting in the loss of the stem cell state by mammary epithelial stem cells. In contrast, luminal mammary epithelial cells can acquire basal stem cell properties following ingestion of EP4 antagonist-induced stem cell extracellular vesicles/exosomes, and can then form mammary glands. These findings demonstrate that PGE2/EP4 signaling controls homeostasis of mammary epithelial stem cells through regulating extracellular vesicle/exosome release. Reprogramming of mammary epithelial cells can result from EP4-mediated stem cell property transfer by extracellular vesicles/exosomes containing caveolae-associated proteins, between mammary basal and luminal epithelial cells.

Adult brains have limited regenerative capacity. Consequently, both brain damage and neurodegenerative diseases often cause functional impairment for patients. Mesenchymal stem cells (MSCs), one type of adult stem cells, can be isolated from various adult tissues. MSCs have been used in clinical trials to treat human diseases and the therapeutic potentials of the MSC‐derived secretome and extracellular vesicles (EVs) have been under investigation. We found that blocking the prostaglandin E2/prostaglandin E2 receptor 4 (PGE2/EP4) signaling pathway in MSCs with EP4 antagonists increased EV release and promoted the sorting of specific proteins, including anti‐inflammatory cytokines and factors that modify astrocyte function, blood–brain barrier integrity, and microglial migration into the damaged hippocampus, into the EVs. Systemic administration of EP4 antagonist‐elicited MSC EVs repaired deficiencies of cognition, learning and memory, inhibited reactive astrogliosis, attenuated extensive inflammation, reduced microglial infiltration into the damaged hippocampus, and increased blood–brain barrier integrity when administered to mice following hippocampal damage. stem cells translational medicine 2019

Cells expressing mesenchymal/basal phenotypes in tumors have been associated with stem cell properties. Cancer stem cells (CSCs) are often resistant to conventional chemotherapy. We explored overcoming mesenchymal CSC resistance to chemotherapeutic agents. Our goal was to reduce CSC numbers in vivo, in conjunction with chemotherapy, to reduce tumor burden. Analysis of clinical samples demonstrated that COX-2/PGE

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAM(hi)/CD49f(lo) luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAM(lo)/CD49f(hi) basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAM(hi)/CD49f(lo) luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAM(hi)/CD49f(lo) luminal mammary epithelial cells.

Mesenchymal cells of the tumor-associated stroma are critical determinants of carcinoma cell behavior. We focus here on interactions of carcinoma cells with mesenchymal stem cells (MSC), which are recruited to the tumor stroma and, once present, are able to influence the phenotype of the carcinoma cells. We find that carcinoma cell–derived interleukin-1 (IL-1) induces prostaglandin E2 (PGE2) secretion by MSCs. The resulting PGE2 operates in an autocrine manner, cooperating with ongoing paracrine IL-1 signaling, to induce expression of a group of cytokines by the MSCs. The PGE2 and cytokines then proceed to act in a paracrine fashion on the carcinoma cells to induce activation of β-catenin signaling and formation of cancer stem cells. These observations indicate that MSCs and derived cell types create a cancer stem cell niche to enable tumor progression via release of PGE2 and cytokines. Significance: Although PGE2 has been implicated time and again in fostering tumorigenesis, its effects on carcinoma cells that contribute specifically to tumor formation are poorly understood. Here we show that tumor cells are able to elicit a strong induction of the COX-2/microsomal prostaglandin-E synthase-1 (mPGES-1)/PGE2 axis in MSCs recruited to the tumor-associated stroma by releasing IL-1, which in turn elicits a mesenchymal/stem cell–like phenotype in the carcinoma cells. Cancer Discov; 2(9); 840–55. ©2012 AACR. Read the Commentary on this article by Räsänen and Herlyn, p. 775. This article is highlighted in the In This Issue feature, p. 753.

Adenovirus vectors have a number of advantages for gene therapy. However, because of their lack of tumor tropism and their preference for liver infection following systemic administration, they cannot be used for systemic attack on metastatic disease. Many epithelial tumors (e.g., colon, lung, and breast) express carcinoembryonic antigen (CEA). To block the natural hepatic tropism of adenovirus and to “retarget” the virus to CEA-expressing tumors, we used a bispecific adapter protein (sCAR-MFE), which fuses the ectodomain of the coxsackie/adenovirus receptor (sCAR) with a single-chain anti-CEA antibody (MFE-23). sCAR-MFE untargets adenovirus-directed luciferase transgene expression in the liver by >90% following systemic vector administration. Moreover, sCAR-MFE can “retarget” adenovirus to CEA-positive epithelial tumor cells in cell culture, in s.c. tumor grafts, and in hepatic tumor grafts. The sCAR-MFE bispecific adapter should, therefore, be a powerful agent to retarget adenovirus vectors to epithelial tumor metastases. [Cancer Res 2007;67(11):5354–61]

Steroids are synthesized in adrenal glands and gonads under the control of pituitary peptides. These peptides bind to cell surface receptors to activate the cyclic AMP (cAMP) signaling pathway leading to an increase of steroidogenic gene expression. Exactly how cAMP activates steroidogenic gene expression is not clear, except for the knowledge that transcription factor SF-1 plays a key role. Investigating the factors participating in SF-1 action, we found that c-Jun and homeodomain-interacting protein kinase 3 (HIPK3) were required for basal and cAMP-stimulated expression of one major steroidogenic gene, CYP11A1. HIPK3 enhanced SF-1 activity, and c-Jun was required for the functional interaction of HIPK3 with SF-1. Furthermore, after cAMP stimulation, both c-Jun and Jun N-terminal kinase (JNK) were phosphorylated through HIPK3. These phosphorylations were important for SF-1 activity and CYP11A1 expression. Thus, we have defined HIPK3-mediated JNK activity and c-Jun phosphorylation as important events that increase SF-1 activity for CYP11A1 transcription in response to cAMP. This finding has linked three common factors, HIPK3, JNK, and c-Jun, to the cAMP signaling pathway leading to increased steroidogenic gene expression.

Developing biochemical and cell biological assay for screening biomolecules, evaluating their characteristics in biological processes, and determining their pharmacological effects represents a key technology in biomedical research. A PDMS-based integrated microfluidic platform was fabricated and tested for facilitating the labeling of ligand on the nanogram scale and sequential cell binding analysis in a manner that saves both time and reagents. Within this microfluidic platform, ligand labeling, cell immobolization, and optical analysis are performed in a miniaturized, continuous and semi-automated manner. This microfluidic device for ligand labeling and cell analysis is composed of two functional modules: (i) a circular reaction loop for fluorophore-labeling of the ligand and (ii) four parallel-oriented incubation chambers for immobilization of cells, binding of ligand to different cell populations, and optical evaluation of interactions between the labeled ligand and its cell targets. Epidermal growth factor (EGF) as the ligand and different cell lines with various levels of EGF receptor expression have been utilized to test the feasiblity of this microfluidic platform. When compared to studies with traditional Petri dish handling of cells and tissues, or even microwell analyses, experiments with the microfluidic platform described here are much less time consuming, conserve reagents, and are programmable, which makes these platforms a very promising new tool for biological studies.