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Evolution from adherent to suspension: systems biology of HEK293 cell line development.

ABSTRACT: The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.

SUBMITTER: Malm M

PROVIDER: S-EPMC7642379 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Evolution from adherent to suspension: systems biology of HEK293 cell line development.

Malm Magdalena M Saghaleyni Rasool R Lundqvist Magnus M Giudici Marco M Chotteau Veronique V Field Ray R Varley Paul G PG Hatton Diane D Grassi Luigi L Svensson Thomas T Nielsen Jens J Rockberg Johan J

Scientific reports 20201104 1

The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis ...[more]

PMID: 33149219

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Project description:Oncolytic viruses (OVs) represent a novel class of immunotherapeutics under development for the treatment of cancers. OVs that express a cognate or transgenic fusion protein is particularly promising as their enhanced intratumoral spread via syncytia formation can be a potent mechanism for tumor lysis and induction of antitumor immune responses. Rapid and efficient fusion of infected cells results in cell death before high titers are reached. Although this is an attractive safety feature, it also presents unique challenges for large-scale clinical-grade manufacture of OVs. Here we evaluate the use of four different suspension cell lines for the production of a novel fusogenic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV). The candidate cell lines were screened for growth, metabolism, and virus productivity. Permissivity was evaluated based on extracellular infectious virus titers and cell-specific virus yields (CSVYs). For additional process optimizations, virus adaptation and multiplicity of infection (MOI) screenings were performed and confirmed in a 1 L bioreactor. BHK-21 and HEK293SF cells infected at concentrations of 2 × 106 cells/mL were identified as promising candidates for rVSV-NDV production, leading to infectious titers of 3.0 × 108 TCID50/mL and 7.5 × 107 TCID50/mL, and CSVYs of 153 and 9, respectively. Compared to the AGE1.CR.pIX reference produced in adherent cultures, oncolytic potency was not affected by production in suspension cultures and possibly even increased in cultures of HEK293SF and AGE1.CR.pIX. Our study describes promising suspension cell-based processes for efficient large-scale manufacturing of rVSV-NDV. KEY POINTS: • Cell contact-dependent oncolytic virus (OV) replicates in suspension cells. • Oncolytic potency is not encompassed during suspension cultivation. • Media composition, cell line, and MOI are critical process parameters for OV production. • The designed process is scalable and shows great promise for manufacturing clinical-grade material.

Project description:BackgroundAlthough metastasis is the foremost cause of cancer-related death, a specialized mechanism that reprograms anchorage dependency of solid tumor cells into circulating tumor cells (CTCs) during metastatic dissemination remains a critical area of challenge.MethodsWe analyzed blood cell-specific transcripts and selected key Adherent-to-Suspension Transition (AST) factors that are competent to reprogram anchorage dependency of adherent cells into suspension cells in an inducible and reversible manner. The mechanisms of AST were evaluated by a series of in vitro and in vivo assays. Paired samples of primary tumors, CTCs, and metastatic tumors were collected from breast cancer and melanoma mouse xenograft models and patients with de novo metastasis. Analyses of single-cell RNA sequencing (scRNA-seq) and tissue staining were performed to validate the role of AST factors in CTCs. Loss-of-function experiments were performed by shRNA knockdown, gene editing, and pharmacological inhibition to block metastasis and prolong survival.ResultsWe discovered a biological phenomenon referred to as AST that reprograms adherent cells into suspension cells via defined hematopoietic transcriptional regulators, which are hijacked by solid tumor cells to disseminate into CTCs. Induction of AST in adherent cells 1) suppress global integrin/ECM gene expression via Hippo-YAP/TEAD inhibition to evoke spontaneous cell-matrix dissociation and 2) upregulate globin genes that prevent oxidative stress to acquire anoikis resistance, in the absence of lineage differentiation. During dissemination, we uncover the critical roles of AST factors in CTCs derived from patients with de novo metastasis and mouse models. Pharmacological blockade of AST factors via thalidomide derivatives in breast cancer and melanoma cells abrogated CTC formation and suppressed lung metastases without affecting the primary tumor growth.ConclusionWe demonstrate that suspension cells can directly arise from adherent cells by the addition of defined hematopoietic factors that confer metastatic traits. Furthermore, our findings expand the prevailing cancer treatment paradigm toward direct intervention within the metastatic spread of cancer.

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Evolution from adherent to suspension: systems biology of HEK293 cell line development.

Publications

Evolution from adherent to suspension: systems biology of HEK293 cell line development.

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