Bioluminescence imaging in live cells and animals

Cho

et al. 2019

Preprint

Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. We previously reported SPARK (Kim et al., 2017), a transcription factor that is activated by the coincidence of blue light and a PPI. Here, we report an improved, second-generation SPARK2 that incorporates a luciferase moiety to control the light-sensitive LOV domain. SPARK2 can be temporally gated by either external light or addition of a small-molecule luciferin, which causes luciferase to open LOV via proximity-dependent BRET. Furthermore, the nested "AND" gate design of SPARK2-in which both protease recruitment to the membrane-anchored transcription factor and LOV domain opening are regulated by the PPI of interest-yields a lowerbackground system and improved PPI specificity. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.

Section: Selection Of Nanoluc As the Luciferase Donor For Lovmentioning

confidence: 99%

Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

Cho

et al. 2019

Preprint

“…2A and 2B). As bioluminescence detection generally has a negligible background [17,18], we hypothesized that luciferase-expressing tumor-cell variants may have survived in a dormant state in the caecum of tumor-rejecting mice [19]. Adaptive immune cells, in particular CD4 + and CD8 + T-lymphocytes, are essential for the establishment and maintenance of tumor dormancy [20].…”

Section: In Rejecting Mice Tumor Cells Do Not Survive In a Dormant Stmentioning

confidence: 99%

Colon-specific immune microenvironment regulates cancer progression versus rejection

Trimaglio

Tilkin–Mariamé

Féliu

et al. 2020

Preprint

Background:Immunotherapies have achieved clinical benefit in many types of cancer but remain limited to a subset of patients in colorectal cancer (CRC). Resistance to immunotherapy can be attributed in part to tissue-specific factors constraining antitumor immunity. Thus, a better understanding of how the colon microenvironment shapes the immune response to CRC is needed to identify mechanisms of resistance to immunotherapies and guide the development of novel therapeutics. Methods:In an orthotopic mouse model of MC38-CRC, tumor progression was monitored by bioluminescence imaging and the immune signatures were assessed at a transcriptional level using NanoString and at a cellular level by flow cytometry.Results: Despite initial tumor growth in all mice, only 25 to 35% of mice developed a progressive lethal CRC while the remaining animals spontaneously rejected their solid tumor.No tumor rejection was observed in the absence of adaptive immunity, nor when MC38 cells were injected in non-orthotopic locations, subcutaneously or into the liver. We observed that progressive CRC tumors exhibited a protumor immune response, characterized by a regulatory T-lymphocyte pattern, discernible shortly post-tumor implantation, as well as suppressive myeloid cells. In contrast, tumor-rejecting mice presented an early inflammatory response and an antitumor microenvironment enriched in CD8 + T cells. Conclusions:Taken together, our data demonstrate the role of the colon microenvironment in regulating the balance between anti or protumor immune responses. While emphasizing the relevance of the CRC orthotopic model, they set the basis for exploring the impact of the identified signatures in colon cancer response to immunotherapy.

“…This interpretation is in conflict with our previous observation that direct injection of DTZ into blank mice caused negligible signals, in addition to a plethora of publications which successfully used CTZ or CTZ analogs for in vivo BLI. 17,[22][23][24][25] In this manuscript, we re-investigated the performance of Akaluc and Antares2 bioluminescent reporters for tracking i.v.-injected cells in mice, and by using hydrodynamic transfection and xenograft tumor mouse models. We identified several factors, such as ATP dependency, serum stability, and molecular size, which significantly affected BLI results.…”

Section: Figure 2 Chemical Structures and Bioluminescence Emission Wmentioning

confidence: 99%

Identification of Factors Complicating Bioluminescence Imaging

Yeh

Chen

et al. 2019

Preprint

In vivo bioluminescence imaging (BLI) has become a standard, non-invasive imaging modality for following gene expression or the fate of proteins and cells in living animals. Currently, bioluminescent reporters used in laboratories are mostly derivatives of two major luciferase families: ATP-dependent insect luciferases and ATP-independent marine luciferases. Inconsistent results have been reported for experiments using different bioluminescent reporters and users are often confused when trying to choose an optimal bioluminescent reporter for a given research purpose. Herein, we re-examined inconsistency in several experimental settings and identified factors, such as ATP dependency, serum stability, and molecular size, which significantly affected BLI results. We expect this study will make the research community aware of these factors and facilitate more accurate interpretation of BLI data by considering the nature of each bioluminescent reporter. Corresponding Author