InCytes publications Newsletter

Fall 2017 — Featured publications for the IncuCyte® Live-Cell Analysis System


DRUG DISCOVERY

High-throughput functional genetic and compound screens identify targets for senescence induction in cancer

A one-two punch cancer therapy model using senolytic agents

Senescence in cancer provides a fail-safe mechanism against oncogenic transformation and often functions in humans to prevent cancer. Because senescent cells are biologically distinct from other cells, they can be targeted by specific ‘senolytic drugs’ for eradication.

In these studies, researchers at the Netherlands Cancer Institute tested the possibility of utilizing a “one-two punch” approach for cancer therapy where they first induce senescence selectively in cancer cells and follow up with treatment with senolytic drugs to target and kill the senescent cancer cells. Their findings show:

  • A reporter-based CRISPR screen to identify candidate senescence-inducing genes identified SMARCB1, a component of the SWI/SNF chromatin remodeling complex as a top candidate. targeted SMARCB1 gRNAs infected in melanoma cells induced senescence and had an anti-proliferative effect.
  • Targeted SMARCB1 depletion causes downregulation of SOX10 and consequent increased signaling of the MAPK pathway producing a similar state as observed for oncogene-induced senescence. Resistance to vemurafenib was also observed in response to SMARCB1 depletion in melanoma cells.
  • BCL2 family inhibitor ABT263 induced apoptosis of melanoma cells made senescent by SMARCB1 knockout, suggesting the possibility for sequential therapy in which cancer cells are first made senescent and then eradicated by senolytic drugs.
  • A compound screen for senescence induction in RAS mutant lung cancer cells produced 18 aurora kinase inhibitors as top outliers. Aurora kinase inhibitors were shown to induce senescence in lung and various other cancer types as well. Furthermore, treated cells were sensitive to the senolytic agent ABT263.

Read the full paper in Cell Reports, October 2017

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CHEMOTAXIS

Leveraging the IncuCyte technology for higher-throughput and automated chemotaxis assays for target validation and compound characterization

Chemotaxis assays for optimal drug discovery and screening

Chemotaxis of tumor cells and stromal cells in surrounding microenvironment is an essential component of tumor dissemination during progression and metastasis. Chemokines have well-defined roles in directing cell movements necessary for T-cell immune responses and are crucial for the attraction of monocytes and immature dendritic cells (iDCs) to sites of inflammation, and bringing T cells and APCs together. Overall, the need to quantitatively study cell migration has become increasingly important for advancing drug discovery and screening.

Researchers at Bristol-Myers Squibb sought to leverage the IncuCyte® live-cell imaging system for high-throughput and automated chemotaxis/cell migration assays to validate targets and characterize various compounds and their associated behaviors/properties. The authors found:

  • The IncuCyte chemotaxis platform enabled MOA studies of drug targets for their chemotaxis profiling in multiple cell types, including T cells, IDCs and mDCs, and THP-1 and MDA-MB-231 monocytes.
  • Validation data for both adherent and suspension cells characterized migration behaviors and relevant chemokine receptors including MIP-1α/CCR1 (CC chemokine receptor-1), MCP-1/CCR2, and SDF-1α/CXCR4.
  • A dose dependent response is seen for MIP1α and MCP-1, which mediates iDC chemotaxis; SDF-1α/CXCR4 interaction, which regulates MDA-MD-231 migration and can potentially be used as a measurement of breast cancer metastasis; and SDF-1α/CXCR4, which regulates T-cell (Treg and PHA blast) and mDC migration.
  • Chemotaxis assays show high reproducibility, robustness, and precision in comparing the automation assays with manual and analyzing IC50 data for 8 CXCR4 compounds.

Read the full paper in SLAS Discovery, September 2017

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ONCOLOGY

Recurrent ubiquitin B silencing in gynecological cancers establishes dependence on ubiquitin C

A potential novel therapeutic target for gynecological cancers

Bioinformatic approaches that utilize tumor profiling data sets to subcategorize classification of patients into disease subtypes have revolutionized clinical target discovery in oncology, most notably as an alternative approach for identification of targetable loss-of-function or synthetic lethal lesions that lead to cancer, which are not readily druggable.

In a search for next generation treatment options for ovarian cancers, researchers at the Novartis Institutes for Biomedical Research used The Cancer Genome Atlas (TCGA) data set to identify a recurrent repression of the ubiquitin B (UBB) gene in high-grade serous ovarian cancer (HGSOC) and other gynecological cancers. Using bioinformatic analysis and cell lines in which UBB is strongly suppressed, the researchers demonstrated the therapeutic value of the observed UBB silencing event. Their studies showed:

  • UBB silencing is correlated with strong UBB DNA methylation and the proliferative subtype of ovarian cancer. UBB silencing is most prevalent in 3 gynecological cancers: HGSOC, UCEC and UCS and is associated with poor survival. Similar parallels were observed in cell lines profiled as having strongly decreased UBB expression (UBBLO) by the Cancer Cell Line Encyclopedia (CCLE), further validating a link between UBB silencing and gynecological cancers.
  • Morphology, caspase 3/7 and colony formation assays using real-time microscopy showed synthetic lethality results from siRNA knockdown of UBC in UBBLO cells. UBC depletion in UBBLO but not UBBWT cells led to cell death and long-term reduction in colony growth rates, demonstrating dependence UBBLO cells on UBC for viability.
  • While knockdown of UBB or UBC in UBBWT cells was tolerated, silencing of both resulted in synthetic lethality. Furthermore, overexpression of exogenous UBB in UBBLO cells suppresses UBC dependency, suggesting that synthetic lethality in UBBLO cells is due to a decrease in the cellular ubiquitin pool that establishes a dependence on UBC. Free ubiquitin and protein-ubiquitin conjugates levels, as well as protein turnover, decreased with knock down of UBC in UBBLO cells, suggesting that ubiquitin pool depletion is affecting diverse cell systems by preventing efficient protein turnover.
  • UBBLO cells with inducible UBC were orthotopically transplanted to produce xenograft models of ovarian cancer. Inducible expression of a UBC-targeting shRNA in these mice led to tumor regression and substantial long-term survival benefit, validating the therapeutic potential of targeting UBB silencing for clinically aggressive HGSOC subtypes in a more physiologically relevant model.

Read the full paper in The Journal of Clinical Investigation, November 2017

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EPIGENETICS

Repression of stress-induced LINE-1 expression protects cancer cell subpopulations from lethal drug exposure

Overcoming drug resistance by disruption of epigenetic state

Drug-resistant cell populations within heterogeneous tumors can serve as the founders of disease relapse and remains a barrier for successful cancer treatment. Epigenetic repression of highly repetitive genome elements contributes to the survival of these drug tolerant subpopulations of cancer cells and has been shown to counterbalance drug-induced expression. This balance can promote genomic stability and selective fitness in drug resistant cancer subpopulations where conditions would normally be lethal.

Therefore, it is increasingly important to disrupt the drug-tolerant state and counteract acquired resistance these subpopulations have to anti-cancer drugs to effectively treat cancers in the clinic. Guler et al. investigated the genomes of a cancer cell subpopulation and its survival to treatment with otherwise lethal drugs, known as drug-tolerant persisters (DTPs). The authors found the following:

  • Drug tolerant cancer cell subpopulations harbor a repressed chromatin state, and disruption of this repressed chromatin state is lethal to these cells, which can be partially rescued by reducing LINE-1 expression.
  • Drug exposure induces the expression of repeat elements and interferon stimulated genes.
  • The survival of drug tolerant cells required an increase in H3K9me3 over LINE-1 elements. Therefore, H3K9 methyltransferases are required for establishment of a drug tolerant state.
  • HDAC and other epigenetic inhibitors, such as G9A and EZH2 inhibitors, as measured by IncuCyte® live-cell imaging platform, prevent the establishment of a heterochromatin state in the dormant DTP population, which results in reduced DTP numbers.
  • DNA demethylating agents, in combination with HDAC inhibitors, show promise in early clinical studies in relapsed cancer patients.

Read the full paper in Cancer Cell, August 2017

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ONCOLOGY

Dual “mAb” HER family blockade in head and neck cancer human cell lines combined with photon therapy

Triple Therapy to improve head and neck cancer treatment and prognosis

Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide by incidence, with an estimated 600,000 new cases reported each year worldwide. HNSCC are highly resistant to treatment and have a low survival rate and high rate of tumor recurrence. Epidermal growth factor (EGFR) is overexpressed in more than 90% of HNSCC and is a predictor of poor prognosis. Where EGFR is overexpressed in HNSCC, HER2 and HER3 are also expressed.

Little data exists on a HER2-HER3 blockade and invasion/migration in HNSCC. The aim of this study was to explore the effect of a pan-HER blockade on proliferation, migration and invasion of human HNSCC (and corresponding head and neck cancer stem cells, CSCs population) via combined treatment of cetuximab and pertuzumab, in the presence or absence of photo irradiation. The authors found the following:

  • Cetuximab strongly inhibited a HNSCC chemo- and radioresistant human cell line (SQ20B) and a hypopharyngeal carcinoma cell line (FaDu) cell proliferation, migration and invasion. However, cetuximab had little effect on SQ20B-CSC.
  • Cetuximab-pertuzumab drug treatment combined with radiation was a significant and effective treatment for inhibiting SQ20, FaDu, and SQ20B-CSC as measured by proliferation, migration and invasion assays on the IncuCyte Zoom® live cell imaging system.
  • Furthermore, cetuximab-pertuzumab with 10 Gy photo irradiation was shown to switch off phosphor-AKT and phosphor-MEK1/2 expression in these three cell populations.
  • By affecting and blocking the downstream AKT-mTOR and Ras-MAPK signaling pathways, this dual “mAB” HER family blockade along with photon therapy (triple therapy) shows promise in treating HNSCC.

Read the full paper in International Journal of Molecular Medicine. February 2017

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DRUG DISCOVERY

Inhibition of the sarco/endoplasmic reticulum (ER) Ca2+-ATPase by thapsigargin analogs induces cell death via ER Ca2+ depletion and the unfolded protein response

Mechanisms of Tg-induced apoptosis in prostate and breast cancer cells

Thapsigargin (Tg), a sesquiterpene lactone, blocks the sarco/endoplasmic reticulum (ER) Ca2+ATPase (SERCA), disrupting normal Ca2+ homeostasis in cells and eventually leading to apoptosis. Due to this apoptotic effect, analogs of Tg are being utilized as cancer therapeutics, but their cytotoxicity has warranted production of prodrugs that can be activated to target specific cancer types.

To optimize Tg prodrug efficacy, Sehgal et al. sought to understand how Tg and Tg analogs interact with and affect SERCA function to induce cell death in prostate and breast cancer cells. To this end, the researchers used a set of O(8)-sidechain-substituted Tg analogs that can be conjugated and utilized for prodrug synthesis, a Boc-protected derivative of 8ADT (Boc-8ADT) analog with a strongly apoptotic effect similar to that of Tg, and EpoTg, a Tg derivative with a high affinity for SERCA but poor ability to induce apoptosis, and demonstrated:

  • Despite differences in the reaction kinetics of Ca2+ release from SERCA, and variations in their effect on cells, all of the above analogs, whether or not suitable for prodrug synthesis, have strong inhibitory effects on SERCA enzyme activity.
  • Live-cell imaging of cell proliferation, apoptosis and morphology showed variations among the Tg analogs, but all caused depletion of ER Ca2+ stores followed by reduced cell proliferation and morphological changes. All analogs induced cell death in a concentration-dependent manner, with similar cytotoxic efficiency as observed for maximal Tg levels in all cell lines. However, concentrations for maximal cell death varied substantially between analogs.
  • Tg and the O(8)-substituted analogs displayed conventional SERCA inhibitor behavior in efficiently inhibiting Ca2+ binding and transport and ATP hydrolysis. Boc-8ADT showed slower kinetics and no tangible effects on cytosolic Ca2+ levels, despite displaying the highest level of toxicity, demonstrating that high cytosolic Ca2+ levels observed for Tg is not necessary for its apoptotic effect. EpoTg shows a high affinity for SERCA, but retained ATPase activity even at high concentrations with an appreciable degree of Ca2+ accumulation observed in SR vesicles.
  • Knockdown of store-operated Ca2+ entry (SOCE) components Orai1 and STIM1 did not reduce toxicity, indicating that SOCE and high cytosolic Ca2+ levels are not critical for Tg-induced apoptosis. However, Tg ER Ca2+ depletion correlated with Tg-induced cell death, as did an unfolded protein response (UPR), suggesting they are crucial for initiation of apoptosis at low concentrations of these compounds.

Read the full paper in The Journal of Biological Chemistry, September 2017

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STEM CELLS

Chemically-induced photoreceptor degeneration and protection in mouse iPSC-derived three-dimensional retinal organoids

3D organoid disease modeling for drug effect monitoring and screening

Self-organizing 3D tissue engineering allows researchers to obtain tissue and cells that mimic in vivo development. Utilizing iPSC-derived 3D tissues has broad applicability, including the reproduction and modeling of photoreceptor degeneration.

Ito et al. investigated whether induced photoreceptor cell death in mouse iPSC-derived 3D retinal organoids (3D-retinas) by 4-OHT would induce photoreceptor degeneration in mouse retinal implants. A fluorescent live-cell imaging system allowed for the measurement and quantification of degeneration-related properties. The authors reported the following:

  • Successful induction of in vitro photoreceptor degeneration in 3D retinas differentiated from mouse iPSCs was seen via treatment of chemicals used in ex vivo experiments, such as 4-OHT and DES.
  • The protective effects of commonly used ophthalmic supplements such as vitamin E, which target 4-OHT induced degeneration, was quantified by live-cell imaging of rod-photoreceptor-derived GFP fluorescence. 400 uM of Vitamin E showed the strongest protective effect.
  • By microarray analysis, Vitamin E was shown to suppress genes related to oxidative stress and ER stress, while lutein was observed, in part, to suppress genes related to oxidative stress.
  • The investigated in vitro photoreceptor degeneration model proves useful for studying the protective effect of drugs against some diseases of the retina.

Read the full paper in Stem Cell Research, October 2017

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