Vol. 1, 2018 — Featured publications for the IncuCyte® Live-Cell Analysis System

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NEUROSCIENCE

Suppression of MAPK11 or HIPK3 reduces mutant Huntingtin levels in Huntington’s disease models

A novel target discovery approach for neurodegenerative diseases

The root cause of neurodegenerative disorders is often the accumulation of aggregated, misfolded proteins. As a novel approach to identifying druggable targets for neurodegenerative diseases, scientists from Fudan University in Shanghai capitalized on the monogenetic nature of Huntington’s Disease (HD) that is caused by accumulation of mHTT protein produced by the mutant Huntingtin gene as a simple model system for screening for modulators of mHTT protein levels.

Their studies identified MAPK11 and HIPK3 kinases as novel modulators of mHTT levels and confirmed their potential as therapeutic targets for HD both in vitro and in vivo. Their findings show:

• RNAi screens were performed in HD patient fibroblasts and changes in mHTT levels were measured to identify candidate hits. Hits were tested for their ability to reduce HD-relevant neuronal death in hESC-derived neurons. The 11 potential target modulators were enriched in MAPK proteins, suggesting kinase activity was important for modulation of mHTT levels. MAPK11 and HIPK3 targets were validated by siRNA knockdown in the mouse HD striatal cell STHdh^Q7/Q111 and Q47 iPSC-derived neuron cell models and in vivo in Mapk11 and Hipk3 knockout mice.
• Mapk11 and Hipk3 mediated lowering of mHTT and HTT levels in HD models and the modulatory effects are dependent on the presence of mHTT. Mapk11 and Hipk3 kinase activity is important for this regulation, suggesting a druggable target for HD. mHTT may also confer positive effects on MAPK11 and HIPK3 activity, suggesting a positive feedback mechanism where mHTT enhances its own accumulation and, consequently, contributes to disease progression.
• mHTT levels are regulated at the RNA level by Mapk11 through positive regulation of HTT mRNA stability and by HIPK3 through autophagy-mediated HTT protein degradation via the DAXX suppressor.
• IncuCyte software was used to measure caspase-3 activation as a readout for mHTT-mediated toxicity and showed that MAPK11 knockdown significantly lowered mHTT-dependent caspase activation in HD patient iPSC-derived neurons. Heterozygous or homozygous knockout of Mapk11 rescued HD mouse mHTT aggregate, behavioral and gait phenotypes, providing compelling in vivo evidence to target Mapk11 as a potential therapeutic target for HD.

Read the full paper in Cell Res, December 2017.

VIROLOGY

Bunyavirus requirement for endosomal K⁺ reveals new roles of cellular ion channels during infection

Role of cellular K⁺ channels in endosomal transport of viral particles

To infect cells and cause disease, viruses traverse the endocytic network to transport viral particles to their specific cellular destination. Along the way, viruses exploit the endocytic environment and maturation of the endocytic vesicles to trigger the expulsion of its genetic material into the cytosol. This process is regulated by various factors, including pH, proteolysis, and lipid composition.

Scientists from the laboratory of Dr. Jamel Mankouri at the University of Leeds in the United Kingdom had previously identified K⁺ channels as yet another critical factor in trafficking of the prototypic bunyavirus, orthobunyavirus Bunyamwera virus (BUNV) through the endocytic network. Here, the researchers sought to determine why K⁺ channels are critical for BUNV progression through the endocytic network and found:

• Increased endosomal [K⁺] enhances BUMV infection and [K⁺] is related to the BUNV dependence on K⁺ channel activity.
• Using a dual-labelling method for BUNV that discriminates between complete infectious and subviral particles, the K⁺-specific fluorescent probe Asante Potassium Green-4 (AG4) and the IncuCyte system to measure fluorescence, the researchers tracked movement of virions in cells during viral infection and BUNV trafficking through K⁺ containing endosomes. These studies showed that BUNV accumulates in late endosomes with high [K⁺] that is required for initiation of viral infection.
• Blocking of K⁺ channel activity by K⁺ channel inhibitor TEA disrupted normal distribution of K⁺ across the endosomal network, causing a shift in concentration of K⁺ in lysosomes instead of late endosomes, suggesting K⁺ channel modulation influences K⁺ endosomal distribution.
• Experiments using TEA treated cells infected with dual labelled BUNV to track virion trafficking showed that inhibition of K⁺ channels resulted in arrest of BUNV trafficking through the endosomal network and, subsequently, endosomal release of viral particles.

Read the full paper in PloS Pathogens, January 2018.

IMMUNO-ONCOLOGY

Macrophage-dependent cytoplasmic transfer during melanoma invasion in vivo

A new mode of contact-dependent cell-cell communication in the tumor microenvironment

To gain a better understanding of cell-cell interactions in the tumor microenvironment and how they influence tumor motility and dissemination, a collaborative research effort led by Dr. Cecilia Moens at Fred Hutchinson Cancer Research Center sought to capture the dynamics that occur in interactions between tumor cells and tumor-associated macrophages using high-resolution imaging of in vivo model systems.

Tumor-associated macrophages undergo a phenotypic switch through signaling in the tumor microenvironment, leading them to promote tumorigenesis and be associated with poor prognosis. Researchers sought to improve current end-point and in vitro methods to study macrophage interaction with tumor cells by utilizing in vivo zebrafish xenotransplant and mouse models to study the melanoma tumor microenvironment. These studies uncovered:

• Using an adaptation of human-in-fish xenotransplantation amenable to high-resolution imaging where human melanoma cells were transplanted to the zebrafish larval hindbrain ventricle that has many cell types present in the tumor microenvironment, they found that tumor cells disseminated from the hindbrain ventricle to distant sites in the larval fish. Metastatic cells disseminated at a higher frequency than primary tumors.
• They observed close and dynamic interactions between host macrophages and transplanted melanoma cells. Using morpholino knockdown of macrophages, they found that macrophage-tumor interactions were necessary for melanoma tumor dissemination. Furthermore, using a TNF-α reporter, they showed that M2 macrophages predominantly populate the progressive tumor microenvironment suggesting that M2, rather than M1, macrophages are responsible for promoting metastasis.
• Using a Cre/LoxP system as a readout of cytoplasmic transfer, they showed that macrophages transfer cytoplasmic molecules to tumor cells and that this cytoplasmic transfer is correlated with tumor cell dissemination both in in vivo zebrafish and in mammalian syngeneic melanoma models. Cytoplasmic transfer is a contact-mediated process, requiring recruitment of macrophages to tumor cells.
• Increased macrophage-tumor cell contact and cytoplasmic transfer correlates with tumor cell dissemination in vivo. Movies of co-cultures of MC38 colorectal tumor cells and primary macrophages were generated using the IncuCyte system to track tumor cell motility. These movies show that cytoplasmic transfer alters tumor cell migration in vitro.

Read the full paper in Developmental Cell, December 2017.

Cell Biology

Cell-derived matrices for studying cell proliferation and directional migration in a complex 3D microenvironment

Recapitulating the cellular microenvironment in vitro

Conventional monolayer cell cultures on non-biological rigid surfaces lack multiple aspects of the in vivo cellular environment including regulatory cues from extracellular matrix (ECM) components. 3D scaffold culture models incorporating purified ECM components offer a more physiological growth environment than 2D models but do not fully recapitulate the protein complexity of the in vivo ECM. Cell-derived matrices (CDM) consist of naturally secreted ECM components and closely reproduce the physiological properties of the cellular microenvironment.

Researchers at the University of Turku, Finland, report a reproducible method for CDM production from fibroblasts and illustrate applications of CDMs as follows:

• A 17-step protocol incorporates culture of fibroblasts in confluent monolayers on gelatinized coverslips for 7 to 21 days followed by cell denudation and removal of nucleic acids, resulting in thin (17 μm) cell-free CDMs. During culture, matrix deposition can be manipulated by additives such as the addition of ascorbic acid to increase collagen content. Effective extraction of cells from the fibrillar CDMs is critical and relies on an alkaline buffer combined with a nonionic detergent.
• The CDM provides a structured matrix that induces spontaneous cell elongation, polarization, and migration, and is amenable to immunofluorescence staining and high-magnification live-cell imaging and analysis using the IncuCyte® Live-Cell Analysis System. Organization, mechanical properties, and composition of CDMs produced by different stromal cell types can be assessed using protocols for immunofluorescence, electron microscopy, or mass spectrometry.
• Real-time fully automated proliferation assays of cells cultured in CDM using the IncuCyte allow for the assessment of cell-stroma interactions, with the potential for drug screening. Observations include cancer-cell-growth-inhibitory properties of CDM produced by normal fibroblasts from cancer patients compared to CDM produced by the same cells following immortalization using telomerase.
• Experiments of cell migration in CDM can be imaged using the IncuCyte and showed that migration persistence but not speed depends on ECM properties, with directional migration following CDM topography.
• Protocols can be adapted to produce CDM from non-fibroblastic cell types.

Read the full paper in Nature Protocols, November 2017.

ONCOLOGY

Specific targeting of melanotic cells with peptide ligated photosensitizers for photodynamic therapy

A novel approach to targeted and personalized treatments of benign and malign pigmented lesions

Photodynamic therapy (PDT) is a non-systemic cancer treatment utilizing photosensitizing drugs in combination with specific light exposure. PDT with proto-porphyrin IX precursors, which are preferentially metabolized by hyperactive cells, has applications in urology, gastroenterology and dermatology to kill precancerous and malignant cells, but is ineffective against melanomas and more advanced cancers.

Hematoporphyrin-derived photosensitizers such as 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide (HPPH) may be effective for PDT of invasive cancers, but effective targeted delivery approaches preventing collateral damage to healthy tissues are lacking. Investigators at the Singapore Agency for Science, Technology and Research now report the development of a peptide-ligated photosensitizer with specificity for melanocytes and melanoma that spares surrounding keratinocytes. Their report shows:

• Synthetic constructs were designed to incorporate an α-melanocyte-stimulating hormone (α-MSH) octapeptide conjugated to the photosensitizers HPPH (NAP-HPPH) or methylene blue (NAP-MB), which have absorption maxima at near-infrared wavelengths characterized by deep tissue penetration. The octapeptide component binds to the melanocortin-1 receptor (MCR1), which is highly expressed on melanocytes and melanoma cells, and is taken up by receptor-mediated internalization.
• High-throughput PDT screening of skin cell monolayer cultures was accomplished using a patented-LED device capable of delivering light at specific wavelengths and integrated into the IncuCyte, enabling real-time live imaging and analysis of cell morphology, cell density, and cell proliferation over time. Screening results demonstrated selective MC-1 dependent uptake of peptide conjugates by melanocytes and melanoma cells but not keratinocytes, and a physiologic time course of melatonin production in response to the peptide derivatives.
• NAP-HPPH and NAP-MB peptide conjugates were non-toxic to skin cells under dark conditions. In combination with subsequent application of sequential LED light dosage at near-IR wavelengths (660 nm), both NAP-HPPH and NAP-MB were highly cytotoxic to mouse B16-F10 melanoma cells, whereas NAP-MB treated N/TERT-1 keratinocytes remained unaffected and exhibited normal proliferation and morphology, as demonstrated with IncuCyte data.
• IncuCyte data also demonstrated that treatment with NAP-MB followed by 660 nm light exposure was cytotoxic to human primary melanocytes and human FM55 melanoma cells but not N/TERT-1 keratinocytes, indicating specific targeting of melanoma cells with minimal damage to surrounding keratinocytes.

Read the full paper in Scientific Reports, November 2017.

ONCOLOGY

KRAS dimerization impacts MEK inhibitor sensitivity and oncogenic activity of mutant KRAS

Targeting oncogenic KRAS dimerization as an exploitable strategy for the treatment of cancer

KRAS is one of the most frequently mutated oncogenes detected in cancers, including lung adenocarcinoma (LUAD), and can result in constitutively activated MAPK survival signaling in tumor cells. KRAS mutant cancers are typically resistant to targeted therapies such as RAF/MEK/ERK inhibition and don’t respond well to standard of care chemotherapy so the need for effective treatment is urgent. This study looks to understand the critical role of wild-type KRAS and mutant KRAS dimerization in tumor fitness and resistance to MEK inhibitors, which are used to disrupt the MAPK signaling pathway that promotes tumor cell proliferation, angiogenesis, and resistance to apoptosis.

A collaboration between researchers from Dana Farber Cancer Institute and other institutions and working with a KRAS loss of heterozygosity (LOH) model that is genetically inducible, and a dimerization-deficient (charge-reversal) D154Q mutant KRAS, at the alpha4-alpha5 KRAS dimer interface, interrogated the role of KRAS dimerization, both in vitro and in vivo, in KRAS-mutant cancers, uncovering new insights that reveal a potential therapeutic strategy for further exploration. These studies show:

• Elimination of wild-type KRAS markedly increased both cell proliferation and levels of mutant KRAS-GTP in RAS-less MEFs (LOH model) transduced with different human HA-tagged KRAS mutants.
• Dimerization is required to sustain the oncogenic function of mutant KRAS. Oncogenic KRAS in a GTP-bound monomeric state failed to support cell proliferation in vitro, by growth assessment assays using the IncuCyte Live-Cell Analysis System, and in vivo possibly as a consequence of diminished activation of downstream MAPK signaling.
• In addition, disruption of KRAS dimerization increased apoptosis in vitro per Caspase-3/7 activity test with the resulting increase in fluorescence over time measured by the IncuCyte.
• When dimerization was impaired with charge-reversal D154Q KRAS mutant, sensitivity to MEK inhibitors increased in vitro as determined by growth rate analysis performed in the IncuCyte of transduced LUAD cancer cell lines H23 or H358 cells. These data demonstrate that D154Q abrogates the dimerization of both wild-type and mutant KRAS but without altering other fundamental biochemical properties of KRAS such as intrinsic GTPase activity, GEF or GAP sensitivity, or CRAF binding.

Read the full paper in Cell, February 2018.