Live Cell Insights Publications Newsletter

Live-Cell Insights Newsletter:
Vol. 2, 2020 — Featured publications for the Incucyte® Live-Cell Analysis System

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2,700 publications and counting!

Incucyte® has reached 2,700 cited publications spanning a wide array of research areas and applications. We’ve experienced over 50% growth in publications in just the last year! Search our publications list to see what exciting research is being published using the Incucyte® Live-Cell Analysis System.

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Therapeutic ACPA inhibits NET formation: a potential therapy for neutrophil-mediated inflammatory diseases

New antibody shows promise for treating issue injury from inflammation

Neutrophil extracellular trap (NET) formation is an antimicrobial activity of neutrophils, designed to protect the host from infection. Neutrophils can form web-like, extra-cellular fiber structures, made of DNA from neutrophils and globular proteins, which have antimicrobial properties and provides a physical barrier to prevent pathogen spread. Excessive NETosis can have harmful effects, however, leading to tissue damage, organ failure, and even death. NETosis plays a role in thrombosis and stroke, sepsis, preeclampsia, and autoimmune conditions such lupus and colitis, among other inflammatory conditions.

The release of NET into extracellular environment during inflammation involves the conversion of arginine to citrulline on histones by peptidyl arginine deiminase type IV (PAD4). Inhibition of PAD4 stops the release of NET, reducing symptoms in mouse models of disease. In this study, Chirivi et al. investigated the effects of engineered therapeutic anti-citrullinated protein antibodies (tACPA) on NET disease pathology in vitro, as well in vivo in four mouse models of disease: collagen antibody-induced arthritis (CAIA), belomycin-induced pulmonary fibrosis (PF), dextran sulfate sodium (DSS)-induced colitis, and lipopolysaccharide (LPS)-induced sepsis.

Key findings include:

  • Specific citrullinated epitopes on histone H2A and H4 were identified as therapeutic targets.

  • tACPA antibody prevented lung fibrosis in PF mice challenged with bleomycin, with significantly less neutrophils found in bronchealveolar lavage fluid than the isotype control, suggesting a reduction in neutrophil inflammation.

  • tACPA prevented inflammation and therefore tissue injury in a mouse model of colitis (DSS).

  • Mice given tACPA, in a LPS-induced model of sepsis, prevented tissue damage and enhanced survival.

  • NET formation was inhibited both in vivo, in a mouse model of peritoneal cell influx, and in vitro.

  • Incucyte® Live-Cell Analysis was used in an immunofluorescence NETosis (NET) assay to capture images of NET release using neutrophils with red nuclear staining treated with green-stained calcium ionophore A23187 (an inducer of NET formation) in the presence of full-length, optimized development candidate antibodies, dc-TACPA or dc-tACPA F (ab’)2 antibody fragments.

  • In vivo, tACPA bound to (pre-) NETS and was then taken up by macrophages.

  • The tACPA antibody shows therapeutic and prophylactic potential for inflammatory disease associated with NET pathology. This was first description on an antibody that interferes with NET expulsion to the extracellular space.

Read the full paper in Cellular & Molecular Immunology, March 2020.



Incucyte® NETosis Assay

An integrated solution to automatically visualize and quantify NETosis, in real time and in 96-well formats — inside your tissue culture incubator.

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Protective autophagy elicited by RAF→MEK→ERK inhibition suggests a treatment strategy for RAS-driven cancers

Combination therapy shows promise for treatment of PDA

Pancreatic ductal adenocarcinoma (PDA) is a particularly deadly cancer associated with KRAS the oncogene and resists treatment. Carcinogenesis of PDA involves the RAF→MEK→ERK pathway, but targeting this pathway has not shown effective clinical benefit.

In this paper by Kinsey et al. from the University of Utah, the authors explored the inhibition of KRAS→RAF→MEK→ERK signaling in PDA. They discovered that inhibition of this pathway promotes autophagy (cellular degradation and recycling of damaged or unnecessary components) providing a protective effect of PDA cells from cytotoxic effects. They explored targeting autophagy as part of a combination treatment strategy for the treatment of RAS-driven cancer.

Key findings include:

  • Inhibition of KRAS→RAF→MEK→ERK pathway promoted autophagy. When MEK1/2 was inhibited, a regulatory pathway of autophagy signaling was activated (LKB1→AMPK→ULK1).

  • MEK1/2 and autophagy was inhibited by combination treatment with trametinib and chloroquine. Incucyte® Live-Cell Imaging and Analysis and Incucyte® Caspase-3/7 Green Reagent was used to perform in vitro apoptosis on three cell types: Mia-PaCa2, BxPC3, and PDX220 (created from a KRAS-mutated PDA PDX). The cells were treated with trametinib, chloroquine, or chloroquine plus trametinib as compared to DMSO controls. Incucyte® Cytotox Red Reagent was used for assessment of cell death. Trametinib and chloroquine were found to act synergistically against the in vitro treated cells.

  • Tumor regression was observed using combination treatment with trametinib plus chloroquine, of patient-derived xenographs mouse models of PDA, NRAS-mutated melanoma, as well as BRAF-mutated colorectal cancers.

  • The authors hypothesized that the inhibitory effects the trametinib and chloroquine combined treatment was the result of tumor cell autonomous induction of autophagy.

  • These findings were translated clinically for the compassionate use of the combined treatment in a PDA patient, as trametinib and hydroxychloroquine are FDA approved drugs. Combined treatment with trametinib and hydroxychloroquine of the PDA patient gave a partial disease response, suggesting a possible new approach for the treatment of RAS-cancers.

Read the full paper in Nature Medicine, March 2019 .



Incucyte® Apoptosis Assays

Detect apoptosis in living cells and in real time using simple mix-an-read protocols.

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Human chimeric antigen receptor macrophages for cancer immunotherapy

CAR macrophages deliver a powerful blow to solid tumors

Macrophages are a key player of innate immunity, performing phagocytosis, cellular cytotoxicity and antigen presentation, and can promote a proinflammatory response when they are outside of the tumor microenvironment (TME). Within the TME however, macrophages can be involved in immunosuppression, and promote invasion and angiogenesis. Human therapeutic trials have been undertaken to administer autologous macrophages to cancer patients as a therapeutic strategy to bolster anti-tumor immunity, but this has not been successful against solid tumors.

In this study by Klinchinsky and colleagues from the University of Pennsylvania, the authors expressed CARs in human macrophages with the hope of directing their phagocytic activities against solid tumors, with the added benefit of stimulating an adaptive immune response. These CAR-M were created by transducing the human monocytic cell line THP-1 with an anti-CD19 CR encoded with the CD3ζ an intracellular domain that has homology to Fc common γ-chain (a signaling molecule for antibody-dependent cellular phagocytosis in macrophages).

Key findings include:

  • Primary human macrophages were successfully genetically engineered using a chimeric adenoviral vector (Ad5f35). Primary human anti-HER2 CAR-Ms were created, and these macrophages displayed a sustained M1 (pro-inflammatory) phenotype.

  • Incucyte® Live-Cell Analysis was performed as part of a bead-based phagocytosis assay of pHrodo beads by UTD or CAR macrophages (CAR-Ms). These CAR-M performed antigen-specific phagocytosis and cleared tumors in vitro.

  • Transfusion of the CAR-M into solid tumor xenograft NOD-SCID and NSGS mouse models reduced tumor burden with extended survival.

  • The CAR-M expressed pro-inflammatory cytokines and chemokines.

  • CAR-M also converted M2 macrophage to M1 (i.e. interferon signaling pattern recognition receptor signaling, TJ1 pathway, and iNOS signaling).

  • Assessment of CARM-M activity demonstrated upregulated antigen presentation, recruitment of cells with antigen presentation, and resistance to immunosuppressive cytokines.

  • The CAR-M also induced a pro-inflammatory TME and bolstered T cell activity in a humanized immune system (HIS) mouse model.

Read the full paper in Nature Biotechnology, March 2020 .



Incucyte® Phagocytosis Assays

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Antibody-mediated delivery of viral epitopes to tumors harnesses CMV-specific T cells for cancer therapy

Alternative treatment approach for cancers resistant to immunotherapy

Immunotherapy efficacy is hampered by specificity problems between T cells and human leukocyte antigen (HLA) epitopes present on the tumor. Responses can be limited by the number of novel tumor epitopes (neoepitopes), due to low tumor mutation burden, which can impair the ability of cytotoxic T Lymphocytes (CTLS) to recognize and respond to the tumor target.

In this study by Millar et al., the authors describe an approach using endogenous, bystander T cells directed against non-cancer antigen targets. Using CD8+ T cells targeted against cytomegalovirus, which are often found in healthy individuals, the authors redirect them using antibody-peptide epitope conjugates (APECS). The APECS contain CMV-derived epitopes conjugated to antibodies directed against the tumors. This alternative retargeting approach may be especially well-suited for tumors with low tumor mutation burden (TMB) and limited antigenicity.

Key findings include:

  • A high frequency of circulating CMV cytotoxic T lymphocytes (CTLS) were found in cancer patients. Most of these cells were effector memory T cells, suggesting functional immunity.

  • APECs were created containing CMV-derived epitopes that were also conjugated to tumor-targeting antibodies via metalloprotease-sensitive linkers and were able to activate CMV-CTLs.

  • The antibody specificity of the APECS for their targets was tested on antigen-expressing and non-expressing cell lines. The APEC effectively reprogrammed target antigen-expressing tumors.

  • The authors tested the ability of APEC to activate T cells in a limited subset of CTL to avoid problems such as cytokine release syndrome and Treg activation, which can be problematic with other approaches. Using an MMP14-activated cAPEC, they discovered that activation was restricted to epitope-specific CTL.

  • Epitope specific CTL from PBMC isolates were also tested. Incucyte® Live -Cell Imaging and Analysis was used to assess T-cell cytotoxicity of CMV-CTL expanded in vitro against APEC-treated MDA-MB-231 tumor cells, and time-lapse videos of T-cell cytotoxicity were prepared.

  • In vivo studies with patient derived xenograft (PDX) models for breast, liver, and lung cancer, using a broadly reactive MMP14-APEC, showed efficacy. APEC could penetrate to the center of tumors, becoming activated and displaying cytotoxic capability.

  • This study demonstrates that the APECS were able to perform redirection of non-viral-specific T-cell response against tumors.

Read the full paper in Nature Biotechnology, April 2020 .



Incucyte® Immune Cell Killing Assays

An integrated solution for real-time visualization and automated analysis of immune cell-mediated killing of tumor cells – all within your tissue culture incubator.

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Analysis of a-synuclein species enriched from cerebral cortex of humans with sporadic dementia with Lewy bodies

New insights from the first characterization of αSyn-specific biochemical differences in DLB

Dementia with Lewy bodies (DLB) is characterized by intraneuronal aggregates of the protein αsynuclein (αSyn) that may lead to cellular disorders. The underlying mechanisms of synucleinopathies and neurodegeneration is not known, though some clues have been found through investigations of insoluble, filamentous forms of in Lewy bodies and syn aggregates, as well as soluble, diffusible αSyn species.

In this international, multi-institutional study, Sanderson and colleagues performed the first characterization of αSyn-specific biochemical differences in DLB. Their analysis of sequential protein extracts from a cohort of post-mortem DLB patients and controls revealed new insights into the pathological mechanisms underlying this debilitating disease.

Key findings include:

  • Fractionation of post-mortem cortical tissue extracts from 22 patients with DLB was performed along with 18 non-synucleinopathy controls to identify brain pieces with a high pathology burden. Analysis revealed that there was a shift of both cytosolic and membrane-bound αSyn to aggregated forms in DLB.

  • Aqueous, cytosolic extracts from the cortex were prepared to isolate soluble species that might be associated with toxicity and pathogenesis. This aqueous fraction and the insoluble fraction with Lewy aggregates were assessed for bioactivity and cytotoxicity using reporter assays.

  • As it was previously shown that αSyn can permeabilize membranes, Incucyte® Live-Cell Imaging and Analysis was used in an A53T-α-synuclein-yellow fluorescent protein inclusion formation assay, testing the ability of recombinant monomer or pre-formed fibrils to permeabilize lipid vesicle mixtures. The cytosolic fractions were found to be responsible for increased permeability of membrane phospholipids.

  • High molecular weight, soluble αSyn fractions were tested against primary rat hippocampal neurons. Incucyte® was also used to monitor neurite length and branch points in real time for 72 hrs, with no alterations in neurite length or branch point number.

  • Incucyte® Live-Cell Imaging and Analysis, along with the Incucyte® Neurotrack Analysis Software Module were used to assess average neurite length and cell body cluster area in neurons treated with recombinant αSyn monomer or Syn6 DLB. Incucyte® capture of the morphological changes revealed that neurites treated with recombinant aSyn monomer were largely intact, whereas neurons treated with Syn6 DLB brain (shown to have the active species of αSyn) displayed neurite retraction.

  • The insoluble, Lewy-associated fractions caused morphological changes of human stem cell derived neurons.

  • LB associated α-synuclein therefore drives neurotoxicity while α-synuclein soluble brain aggregates drive pathogenesis 

Read the full paper in Brain Communications, 2020.



Incucyte® Neurite Analysis Assays

Perform automated, continuous, non-invasive imaging and measurement of neurite outgrowth and generate meaningful, kinetic data with visual confirmation of biological changes.

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Exosomal communication by metastatic osteosarcoma cells modulates alveolar macrophages to an M2 tumor-promoting phenotype and inhibits tumoricidal functions

Important insights on tumor cell microenvironment immunosuppression

Poor patient survival and metastasis in osteosarcoma is associated with tumor-promoting, immunosuppressive M2 macrophages. The presence of the M1 antitumorogenic macrophages, however, is associated with an anti-metastatic influence, augmentation of the adaptive immune response, and longer survival. Exosomes are vesicles for intracellular communication, have been demonstrated to influence macrophage phenotype and activation, and can be released by cancer cells.

In this comprehensive study by Wolf-Dennen, Gordon and Kleinerman from MD Anderson Cancer Center, the authors attempt to explore M2 dominance and poor patient survival in osteosarcoma. They examined the influence of exosomes from mon-metastatic and metastatic osteosarcoma cell lines on macrophage phenotype and function, which revealed new important insights on cellular signaling of tumor-associated macrophages in osteosarcoma.

Key findings include:

  • MHS mouse alveolar macrophages, administered exosomes from K7M3 and DLMA metastatic sublines, induced phenotypic expression of M2 Macrophage markers (IL-10, TGFB2, and CCL2 mRNA), along with decreased functional activity of phagocytosis, efferocytosis, and macrophage-mediated killing of tumor cells.

  • Conversely, the administration of exosomes from the non-metastatic Dunn and K7 cells did not induce expression of M2 markers, nor impact the functional activity of phagocytosis, efferocytosis, or cell killing.

  • The Incucyte® Live-Cell Analysis System was used to capture the uptake of fluorescently labeled exosomes from osteosarcoma cells and fibroblasts by mouse-derived alveolar macrophages.

  • The Incucyte® and non-perturbing reagents were used to study how metastatic osteosarcoma cell exosomes cause inhibition of alveolar macrophage phagocytosis. Incucyte® pHrodo Red Labeling Reagent was used to label osteosarcoma cells. The labeled cells were then added to murine macrophage (MHS) cells. Phagocytosis was captured and analyzed phagocytosis in real time, revealing inhibition of this functional output for K7M3 and DLM8.

  • To assess efferocytosis (phagocytic clearance of dying cells), osteosarcoma cells and fibroblasts, treated with gemcitabin, were labeled with Incucyte® pHrodo for 24 h, then cultured with MHS cell culture. Incucyte® integrated software was used to determine efferocytosis, with significant inhibition.

  • The Live-Cell Analysis System and the Incucyte® Caspase 3/7 Green Reagent were used to assess macrophage-mediated tumor cell killing. Metastatic osteosarcoma cell exosomes decreased macrophage-mediated tumor cell killing.

  • Of note, the metastatic osteosarcoma cell exosomes increased TGFB2 secretion, and this signaling pathway is associated with tumor suppression. Inhibition of TGB2 stopped the suppression from alveolar macrophages on metastatic osteosarcoma cell exosomes.

  • Taken together, the authors suggest that exosomes from the metastatic osteosarcoma cells have the capacity to modulate cellular signaling of tumor-associated macrophages, promoting the development of an M2 phenotype, enhancing immunosuppression in the tumor microenvironment via TGFB2 production.

Read the full paper in OncoImmunology, March 2020 .



Incucyte® pHrodo® Red Cell Labeling Kit for Phagocytosis

The Incucyte® pHrodo® Red Cell Labeling Kit enables the labeling of your choice of target cells with a pH-sensitive fluorophore for use in Incucyte® Phagocytosis Assays.

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