Cellular Metrology: Scoping for a Value Proposition in Extra- and Intracellular Measurements

Faruqui, Nilofar; Kummrow, A.; Fu, Boqiang; Divieto, Carla; Rojas, Fabiola; Kisulu, Florence; Cavalcante, Janaína J. V.; Wang, Jing; Campbell, Jonathan J.; Martins, Juliana L.; Choi, Jun-Hyuk; Sassi, Maria Paola; Zucco, Massimo; Vessillier, Sandrine; Zou, Shan; Fujii, Shin-ichiro; Ryadnov, Maxim G.

doi:10.3389/fbioe.2019.00456

Cited by 11 publications

(6 citation statements)

References 45 publications

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“…The effectiveness of non-viral vectors is causally linked to their ability to cross the cellular membrane and release the genetic cargo to allow transgene expression [46,70,155]. Quantitative and correlative measurements are therefore used to assess the safety and the efficacy of the delivery technologies [156][157][158].…”

Section: Evaluation Of Transfection Effectiveness: a Trade-off Betweementioning

confidence: 99%

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

et al. 2020

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Transfection by means of non-viral gene delivery vectors is the cornerstone of modern gene delivery. Despite the resources poured into the development of ever more effective transfectants, improvement is still slow and limited. Of note, the performance of any gene delivery vector in vitro is strictly dependent on several experimental conditions specific to each laboratory. The lack of standard tests has thus largely contributed to the flood of inconsistent data underpinning the reproducibility crisis. A way researchers seek to address this issue is by gauging the effectiveness of newly synthesized gene delivery vectors with respect to benchmarks of seemingly well-known behavior. However, the performance of such reference molecules is also affected by the testing conditions. This survey points to non-standardized transfection settings and limited information on variables deemed relevant in this context as the major cause of such misalignments. This review provides a catalog of conditions optimized for the gold standard and internal reference, 25 kDa polyethyleneimine, that can be profitably replicated across studies for the sake of comparison. Overall, we wish to pave the way for the implementation of standardized protocols in order to make the evaluation of the effectiveness of transfectants as unbiased as possible.

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Section: Evaluation Of Transfection Effectiveness: a Trade-off Betweementioning

confidence: 99%

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

et al. 2020

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“…Leukocyte-based in vitro assays remain one of the gold standards for determining mAb efficacy and predicting adverse responses in patients [ 65 ]. We have previously characterized several assay formats reporting on the impact of FcγRs on mAb mediated immune cell responses in vitro [ 32 , 66 ].…”

Section: Resultsmentioning

confidence: 99%

HIF activation enhances FcγRIIb expression on mononuclear phagocytes impeding tumor targeting antibody immunotherapy

Hussain

Liu

Smith

et al. 2022

J Exp Clin Cancer Res

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Background Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (FcγR) and impaired by the single inhibitory FcγR, FcγRIIb. Methods We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription. Results We report that TAMs are FcγRIIbbright relative to healthy tissue counterparts and under hypoxic conditions, mononuclear phagocytes markedly upregulate FcγRIIb. This enhanced FcγRIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human FcγRIIb+/+ transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of FcγRIIb can partially restore phagocytic function in human monocytes. Conclusion Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of FcγRIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies.

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“…The lack of standardization and harmonization of the results in cell measurements leads to symptomatic irreproducibility of data in laboratory medicine and biotechnology [1], which demands development of metrological support for cellular analysis. Due to the complexity of a cell as an open living system, cell measurable parameters should be considered; a cell should be described using simplified models, and quantitative measurements require the development of adequate reference materials [2].…”

Section: Introductionmentioning

confidence: 99%

Metrology of cellular analysis: problems and solutions

Runov

Шевченко

Goryachaya

et al. 2022

J. Phys.: Conf. Ser.

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Cellular analysis is one of the perspective developing areas of metrology. The complexity of the measuring object requires novel approaches and correct metrological support for the quantitative methods of cellular analysis. Definition of the quantitative parameters and nominal properties of the cell, standardization of methodology – these are main tasks of the Cellular Analysis Working Group of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology. The results of a number of pilot studies carried out by Working Group can be used as a first step establishing metrological traceability of the cell measurements. Development of the stable reference materials for cell quantification is a challenging problem. One of the promising solutions seems to apply synthetic cell-mimicking particles. Currently we are in the process of developing candidate synthetic reference materials for cell measurements.

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Cellular Metrology: Scoping for a Value Proposition in Extra- and Intracellular Measurements

Cited by 11 publications

References 45 publications

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

HIF activation enhances FcγRIIb expression on mononuclear phagocytes impeding tumor targeting antibody immunotherapy

Metrology of cellular analysis: problems and solutions

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