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Immune monitoring of blood and tumor microenvironment

Immune responses can lead to treatment success in cancer patients. Tumor antigen-specific T cells play leading roles, and a large number of additional biological mechanisms can influence the clinical outcome. Monitoring immune responses in cancer patients provides information on disease-induced immune properties (baseline), and on treatment efficacy. In this chapter we outline the current status of immune monitoring assays. They must be critically evaluated and optimized, with the aim to selectively focus on those approaches that provide in vivo relevant results, paving the way to selectively apply meaningful assays and to identify prognostic and predictive biomarkers that support clinical care.

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Acridine orange exhibits photodamage in human bladder cancer cells under blue light exposure

Human bladder cancer (BC) cells exhibit a high basal level of autophagic activity with accumulation of acridine-orange(AO)-stained acidic vesicular organelles. The rapid AO relocalization was observed in treated BC cells under blue-light emission. To investigate the cytotoxic effects of AO on human BC cell lines under blue-light exposure, human immortalized uroepithelial (SV-Huc-1) and BC cell lines (5637 and T24) were treated with indicated concentrations of AO or blue-light exposure alone and in combination. The cell viability was then determined using WST-1, time-lapse imaging with a Cytosmart System and continuous quantification with a multi-mode image-based reader. Treatment of AO or blue-light exposure alone did not cause a significant loss of viability in BC cells. However, AO exhibited a dose-dependent increment of cytotoxicity toward BC cells under blue-light exposure. Furthermore, the tumor formation of BC cells with treatment was significantly reduced when evaluated in a mouse xenograft model. The photodamage caused by AO was nearly neglected in SV-Huc-1 cells, suggesting a differential effect of this treatment between cancer and normal cells. In summary, AO, as a photosensitizer, disrupts acidic organelles and induces cancer cell death in BC cells under blue-light irradiation. Our findings may serve as a novel therapeutic strategy against human BC.

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Time-lapse microscopic observation of non-dividing cells in cultured human osteosarcoma MG-63 cell line

Cancer stem cells resemble normal tissue-specific stem cells in many aspects, such as self-renewal and plasticity. Like their non-malignant counterparts, cancer stem cells are suggested to exhibit a relative quiescence. The established cancer cell lines reportedly harbor slow-proliferating cells that are positive for some cancer stem cells markers. However, the fate of these cells and their progeny remains unknown. We used time-lapse microscopy and the contrast-based segmentation algorithm to identify and monitor actively dividing and non-dividing cells in human osteosarcoma MG-63 cell line. Within the monitored field of view the non-dividing cells were represented by three cells that never divided, and one cell that attempted to divide, but failed cytokinesis, and later, after significantly prolonged division, produced the progeny with enlarged segmented nuclei, thus pointing to a possible mitotic catastrophe. Together, these cells initially constituted about 6.2% of the total number of seeded cells, yet only 0.02% of all cells at the end of the observation period when cells became confluent. Non-dividing cells were characterized by rounded shape, dark nuclei, random cytoplasmic streaming and subtle oscillatory movement, however, they did not migrate and rarely formed cell-cell contacts as compared to actively dividing cells. Our data indicate that the observed non-dividing MG-63 cells do not have a growth advantage over other cells and, therefore, they do not contribute to the cancer stem cells pool.

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Tenascin-C and Integrin α9 mediate interactions of prostate cancer with the bone microenvironment

Deposition of the extracellular matrix protein tenascin-C is part of the reactive stroma response, which has a critical role in prostate cancer progression. Here, we report that tenascin C is expressed in the bone endosteum and is associated with formation of prostate bone metastases. Metastatic cells cultured on osteo-mimetic surfaces coated with tenascin C exhibited enhanced adhesion and colony formation as mediated by integrin α9β1. In addition, metastatic cells preferentially migrated and colonized tenascin-C-coated trabecular bone xenografts in a novel system that employed chorioallantoic membranes of fertilized chicken eggs as host. Overall, our studies deepen knowledge about reactive stroma responses in the bone endosteum that accompany prostate cancer metastasis to trabecular bone, with potential implications to therapeutically target this process in patients.

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Medium supplement to increase the efficiency of oocyte maturation and embryo culture in vitro

The present invention provides a novel oocyte maturation medium or/and embryo culture medium with a chemically defined supplement to produce matured oocytes at high efficiency. The inventive medium or supplement comprises three growth factors, namely, fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF-1) in a synergistic combination. Methods for oocyte and embryo culture are also provided.

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Quadrupling efficiency in production of genetically modified pigs through improved oocyte maturation

Assisted reproductive technologies in all mammals are critically dependent on the quality of the oocytes used to produce embryos. For reasons not fully clear, oocytes matured in vitro tend to be much less competent to become fertilized, advance to the blastocyst stage, and give rise to live young than their in vivo-produced counterparts, particularly if they are derived from immature females. Here we show that a chemically defined maturation medium supplemented with three cytokines (FGF2, LIF, and IGF1) in combination, so-called “FLI medium,” improves nuclear maturation of oocytes in cumulus–oocyte complexes derived from immature pig ovaries and provides a twofold increase in the efficiency of blastocyst production after in vitro fertilization. Transfer of such blastocysts to recipient females doubles mean litter size to about nine piglets per litter. Maturation of oocytes in FLI medium, therefore, effectively provides a fourfold increase in piglets born per oocyte collected. As they progress in culture, the FLI-matured cumulus–oocyte complexes display distinctly different kinetics of MAPK activation in the cumulus cells, much increased cumulus cell expansion, and an accelerated severance of cytoplasmic projections between the cumulus cells outside the zona pellucida and the oocyte within. These events likely underpin the improvement in oocyte quality achieved by using the FLI medium.

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Platforms for manufacturing allogenic, autologous and iPSC cell therapy produts: An industry perspective

As cell therapy processes mature from benchtop research protocols to industrial processes capable of manufacturing market-relevant numbers of doses, new cell manufacturing platforms are required. Here we give an overview of the platforms and technologies currently available to manufacture allogeneic cell products, such as mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), and technologies for mass production of autologous cell therapies via scale-out. These technologies include bioreactors, microcarriers, cell separation and cryopreservation equipment, molecular biology tools for iPSC generation, and single-use controlled-environment systems for autologous cell production. These platforms address the challenges of manufacturing cell products in greater numbers while maintaining process robustness and product quality.

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Preclinical evaluation of the imipridone family, analogs of clinical stage anti-cancer small molecule ONC201, reveals potent anti-cancer effects of ONC212

Anti-cancer small molecule ONC201 upregulates the integrated stress response (ISR) and acts as a dual inactivator of Akt/ERK, leading to TRAIL gene activation. ONC201 is under investigation in multiple clinical trials to treat patients with cancer. Given the unique imipridone core chemical structure of ONC201, we synthesized a series of analogs to identify additional compounds with distinct therapeutic properties. Several imipridones with a broad range of in vitro potencies were identified in an exploration of chemical derivatives. Based on in vitro potency in human cancer cell lines and lack of toxicity to normal human fibroblasts, imipridones ONC206 and ONC212 were prioritized for further study. Both analogs inhibited colony formation, and induced apoptosis and downstream signaling that involves the integrated stress response and Akt/ERK, similar to ONC201. Compared to ONC201, ONC206 demonstrated improved inhibition of cell migration while ONC212 exhibited rapid kinetics of activity. ONC212 was further tested in >1000 human cancer cell lines in vitro and evaluated for safety and anti-tumor efficacy in vivo. ONC212 exhibited broad-spectrum efficacy at nanomolar concentrations across solid tumors and hematological malignancies. Skin cancer emerged as a tumor type with improved efficacy relative to ONC201. Orally administered ONC212 displayed potent anti-tumor effects in vivo, a broad therapeutic window and a favorable PK profile. ONC212 was efficacious in vivo in BRAF V600E melanoma models that are less sensitive to ONC201. Based on these findings, ONC212 warrants further development as a drug candidate. It is clear that therapeutic utility extends beyond ONC201 to include additional imipridones.

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Simple and Easy Monitoring of Tube Formation and Migration Assays with the CytoSMART™ Live Cell Imaging System

Movement of cells plays a critical role in the development of cancer. Analyzing the motility of cells in appropriate cell culture models is therefore an important tool for cancer researchers. Live cell imaging is particularly well suited to capture dynamic processes in cell culture. The CytoSMART™ Device is an easy-to-use, small and affordable live cell monitoring system suitable for the label-free analysis of cell motility within a standard cell culture incubator. Label-free approaches offer the benefit of cell analysis without potential cytotoxic or other side-effects of the used markers or dyes on the cells. This presentation shows the suitability of the CytoSMART™ Device for the analysis of different cancer-relevant assays. The formation of new blood vessels is required to ensure sufficient nutrient and oxygen supply and to allow solid tumors to grow beyond a certain size. This process can be mimicked in cell culture models in so-called tube formation assays. In this study, Human Umbilical Vein Endothelial Cells (HUVEC) were seeded on Engelbreth-Holm Swarm Sarcoma derived Basement Membrane Extract (BME). The resulting formation of endothelial tubes was monitored with the CytoSMART™ Device. Subsequently, the average length of the formed tubes as well as the number of closed tube circles was quantitatively determined. In addition, the impact of Suramin on tube formation was evaluated as an example for a tube formation inhibiting compound. The migration of cancer cells is also required for the growth and in particular the metastasis of tumors. In a first example, the closure of a so-called wound or scratch in a confluent monolayer of cancer cells was monitored with the CytoSMART™ Device. Determining the migration speed of the cells by measuring the speed of wound closure is a simple assay to determine the migration potential of cancer cells. Modifying the cancer cells, e.g. by knocking down specific genes with siRNA, can help to identify genes that play a role in cell migration. Compounds that are expected to reduce cell motility and therefore reduce the metastasis potential of cancer cells can be easily tested. In a second example, the invasion of cancer cells into a three-dimensional (3D) matrix was analyzed. Tumor cells were embedded in a cancer-relevant matrix and their invasion into the 3D matrix was documented with the CytoSMART™ Device. While this type of model is slightly more difficult in set-up and analysis compared to simple scratch assays, it may reflect better the in vivo situation where solid tumors develop within three-dimensional tissues. The images captured with the CytoSMART™ System were quantitatively analyzed using appropriate software. Overall, the CytoSMART™ System is an easy-to-use, small and affordable live cell imaging system suitable for the label-free analysis of different cancer-relevant assays.

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Novel Technologies for the Precise Monitoring of Chemotaxis: Investigating Aberrant Chemotactic Behavior in Fibroblasts from Patients with Lung Disease

Chemotaxis is a mechanism that provides directional cellular movement in response to alterations in the chemical composition of the immediate environment. The mechanism, in its simplest form, governs a wide range of biological processes: from targeting the movement of neutrophils toward a site of infection to providing cancer cells with a means of entering circulation during metastasis. It is also well known that chemotactic cell migration is a significant factor in the coordination of many physiological processes, such as wound healing and embryonic development. Due to the far-reaching implications of chemotactic activity in clinical research, it will come as no surprise that the underlying mechanisms that govern the process have long been investigated. There are several well-established techniques, but recent technological advances enable scientists to now view chemotaxis in much finer detail.

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