Vol. 1, 2019 — Featured publications for the IncuCyte® Live-Cell Analysis System

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Neuroscience

Increased synapse elimination by microglia in schizophrenia patient-derived models of synaptic pruning

Excessive pruning as a potential target for schizophrenia treatment

The pathophysiology of schizophrenia is not well understood, with over 100 genomic regions associated with increased risk and structural abnormalities. Postmortem brain tissue samples from schizophrenia patients (SZ) indicate a reduction in synapse density, while rodent studies suggest a role for the genetic alleles of compliment component 4 (C4). Excessive synaptic pruning by microglia has been proposed for this reduced density, but the ability to replicate this process in vitro has thus far been limited.

In this study, Sellgren et al. developed a high-throughput, validated synaptosome (SYN) system using human induced microglia-like (iMG) cells, and iPSC-derived neurons to study synaptic pruning. Their data revealed an increased uptake of patient-derived synaptic structures from patient-derived (SZ) samples as compared to matched healthy controls, and this process was complement dependent. Similar results were also found in iMG and iPSC-derived neural cocultures. Though compliment plays a role, and the effects could not be attributed only to the associated human C4 risk factors.

Key findings include:

• IncuCyte® Live-Cell Imaging and Analysis revealed decreased spine density following the co-culture of iMG cells with iPSC-derived neural cultures, coinciding with uptake of post-synaptic density protein (PSD-95) and the presynaptic marker SNAP-25.
• Live cell imaging of pHrodo-labeled SYN uptake, revealed significantly increased phagocytosis in SZ-derived iMG cultures, as compared to matched HC (human control) iMG cultures.
• Combining data from live-cell imaging and confocal microscopy revealed that SZ-derived iMG cells display greater uptake of SYNs than HC-derived iMG cells.
• The antibiotic minocycline reduced synapse uptake in vitro in a dose dependent manner. Interestingly, an accompanying, proof-of-concept review of health records from schizophrenic patients, exposed to minocycline, revealed a slight reduction in the incidence of psychosis.

Read the full paper in Nature Neuroscience, February 2019.

Neuroscience

CD22 blockade restores homeostatic microglial phagocytosis in ageing brains

An important role for CD 22 in age-related brain dysfunction

Microglia in the aged brain have impaired homeostatic function, which affects their ability to appropriately phagocytize CNS perturbations such as myelin debris and protein aggregates. The reasons for this impairment, and the resultant effects, are relatively uncharacterized.

This study by Pluvinage et al., from the Wyss-Coray lab at Stanford University, attempted to further characterize the underlying molecular mechanisms that may be responsible for microglial impairment with age, and attempted to restore microglial phagocytosis and observe the resultant effects on age-related brain dysfunction. CRISPR-Cas9 knockout screening was used for the identification of age-related genetic modifiers of microglial phagocytosis in the BV2 cell line.  This data was combined with RNA sequencing analysis of hippocampal microglia from young and old mice.  The authors then searched for age-upregulated or down-regulated microglial genes that, when subjected to knockout in BV2, resulted in promotion or inhibition of phagocytosis, respectively. 

Key findings include:

• The screens identified CD22, a lectin typically expressed on B cells, as negative regulator of phagocytosis, to be the only gene that fit the established criteria. This gene was upregulated in in aged microglia.  
• CD22 was found to mediate an anti-phagocytic effect of α2,6-linked sialic acid. In addition to flow cytometry, IncuCyte’s real-time, live-cell imaging and analysis was used in vitro to help characterize phagocytosis during this analysis workflow.
• In vivo mice studies were performed to assess the action of CD22 on aged microglia.  Both antibody blockade and genetic ablation resulted in the clearance of myelin debris, α-synuclein fibrils, and Aβ oligomers.
• Long-term blocking of CD-22 in aged mice partially reprogrammed their microglia to a more normal transcriptional sate with improved cognitive function, as assessed through hippocampal-dependent learning and memory performance.
• RNA seq analysis also demonstrated that blocking CD22 reduces the expression neuromodulatory pro-inflammatory molecules, which could also have important implications for other neurodegenerative diseases.

Read the full paper in Nature, April 2019.

Additional commentary on this finding by Wei and Li Frontiers in Immunology, June 2019.

Immunotherapy

Reprogramming human T cell function and specificity with non-viral genome targeting

Pre-clinical exploration of efficient method for T cell engineering

The use of recombinant viral vectors in the pre-clinical development of genetic reprogramming of human T cells has been impeded by manufacturing, testing, and cost constraints. Attempts to use non-viral T cell genome targeting of DNA has been hampered by toxicity, especially with larger dsDNA templates.

In this study by Roth et al. from the Marson lab at the University of California, San Francisco, the authors attempted to reprogram human T-cell function using a CRISPR-Cas9 genomic targeting system for primary human T cells, circumventing the use of viral vectors.  An efficient system was created for inserting large DNA sequences.  This was achieved by performing co-electroporation of human primary T cells with CRISPR-Cas9 RNP complexes as well as long linear dsDNA. The study outlined an efficient method to gene edit human T cells, which remained viable and retained their functionality.

Key findings include:

• This method was efficient, retained cell viability, and is compatible with current manufacturing protocols.  Further, it can accommodate diverse sample types, such as fresh or cryopreserved cells, bulk T cells, sub-populations sorted by FACS, as well as cells from whole blood samples or from leukapheresis procedures.
• To assess the antigen-specific function of the human T cells to targeted tumors, a T-cell in vitro killing assay was performed. The killing of melanoma cell lines that were transduced to express nuclear RFP, in co-culture with modified T cells, was incorporated into this workflow. IncuCyte real-time, live-cell imaging and analysis was to measure cell proliferation and death.
• This reprogramming approach was tested as proof-of-concept for the correction of an IL2RA mutation in autoimmune disease and the replacement of a T-cell receptor locus (TCR) to improve T-cell homing to cancer antigens.

Read the full paper in Nature, July 2018.

Drug Development

Prioritization of cancer therapeutic targets using CRISPR-Cas9 screens

A framework—and a step forward—for the discovery of more specific drug targets

Efforts toward targeted cancer therapy continue to expand, yet the actual efficacy of this approach has been less than optimal.  This is partially due to limited identification of new therapeutic targets. An approach is need to advance and prioritize target identification.

In this paper by Behan, Iorio, and Picco, et al., the authors performed genome-scale CRISPR-Cas9 fitness screens in over 300 cancer cell lines representing 30 types of cancer (over 18,000 genes) in order to prioritize the unbiased discovery of drug candidates.  This study presents a valuable framework for early drug discovery that may be used for more specific target identification with the goal of improving cancer therapeutic efficacy.

Key findings include:

• Genes responsible for cancer cell fitness (required for growth and viability) were analyzed in the CRISPR-Cas9 screens of cancer cell lines.  This approach was able to classify essential and non-essential genes with high sensitivity, specificity and precision. A set of pan-core fitness genes was determined, which also 132 additional, previously unidentified genes.
• Information on cell fitness effects, genomic biomarkers, and target tractability for drug development were combined to prioritize genes for the identification of new targets.
• This strategy was verified in an investigation of WRN helicase (Werner syndrome ATP-dependent helicase) as a target for MSI cancers. In vivo validation of MSI cells to WRN knockout was performed using a mouse xenograft model with a doxycycline-inducible WRN sgRNA system with HCT116 cells.
• IncuCyte phase contrast imaging used to study growth rate of cloned inducible WRN sgRNA-expressing HCT116 cells after conditional WRN knockout.  Cells were then inoculated into NOD/SCID mice, tumors established, and the mice were treated with doxycycline, resulting in significant growth suppression of these tumors and a reduction of proliferating cells.

Read the full paper in Nature, April 2019.

Immunotherapy

CAR-T cells secreting BiTEs circumvent antigen escape without detectable toxicity

Overcoming barriers to immune therapy for glioblastoma

Glioblastoma is especially difficult to treat using CAR-T cells directed against a single target. The heterogeneous nature of the tumor antigens, and outgrowth of tumors lacking the target allows for tumor escape from therapy.

In this communication, Choi et al. of the Maus laboratory at Harvard describe their efforts to combine CARs with BiTES, in a complementary manner, into one single T-cell product, CART-EGFRvIII.BiTE-EGFR, for administration as a more effective immune therapy for solid tumors. This construct was developed for the generation of CAR specific for glioblastoma-specific tumor antigen (EGFRvIII), and a bispecific T-Cell engager (BiTE) that is directed against wild-type EGFR (amplified in glioblastoma, not in normal brain) in an attempt to overcome antigen escape, immune suppression, and the exhaustion of T cells. This construct was developed to achieve targeting of multiple antigens and recruitment and activation of bystander T cells.

Key findings include:

• The construct, CART-EGFRvIII.BiTE-EGFR, could elicit both potent and specific antitumor activity in heterogeneous tumors, which was augmented by local BiTE secretion by CAR-T recruitment of bystander effectors.
• Redirection of T-cells through both CARS and BiTES enabled T cells to maintain their ability to lyse tumor cells using BiTE-mediated cytotoxicity and was effective against heterogeneous tumors.
• The combination of CARs and BiTES for co-stimulation enabled a reversal of T-cell exhaustion.
• The antitumor activity of the CART-EGFRvIII.BiTE-EGFR construct against BT74 (established glioblastoma PDX neurosphere) was assessed using IncuCyte live-cell analysis, and evaluation of their morphology revealed selective antitumor activity in wells with CAR-T cells that secreted BiTE-EGFR, as compared to control wells (BiTE-CD19 or UTD T-cell controls).

Read the full paper in Nature Biotechnology, July 2019.

Oncology

BACH1 Stabilization by Antioxidants Stimulates Lung Cancer Metastasis

Antioxidants: friend or foe? It depends on the context.

The production of reactive oxygen species (ROS) by cancers had previously prompted consumption of antioxidant supplements for cancer prevention.  However, recent data indicates that the value of administering antioxidants depends on the cancer and the context. Indeed, ROS have also been found to have anti-tumorigenic roles, preventing tumor progression, which may can be circumvented by antioxidants.  In the case of lung cancer, it was previously known that dietary antioxidant supplementation and activation of endogenous antioxidants may actually cause the progression of early lung tumors.  However, the potential influence of antioxidants on metastasis of lung cancer was uncharacterized.

In this study by Wiel et al., the authors combined genomic, metabolic, and data mining, with mouse models to study the role of ROS and antioxidants in the development of lung cancer metastasis, focusing on the transcription factor BACH1, which is responsive to oxidative stress.

Key findings include:

• The long-term supplementation with the anti-oxidants N-acetylcysteine and vitamin E were found to promote KRAS lung cancer metastasis in mice through reduction of free heme levels and the stabilization of BACH1 transcription.
• BACH 1 activation of Hk2 and GAPDH transcription was found to promote glycolysis-induced metastasis. This can be stimulated with antioxidants and inhibited during oxidative stress— a previously unknown mechanism. Glycolysis was found to drive the migration and metastasis, which was both antioxidant- and BACH 1-dependent. IncuCyte live cell imaging and analysis was used at several points in the assessment of migration by scratch wound closure, along with proliferation measures.
• BACH1 and associated upstream/downstream proteins may be potential drug targets for inhibiting of lung cancer metastasis, especially in patients with NRF2/KEAP1 mutations.

Read the full paper in Cell, July 2019.