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IMR90 ER:RAS model

IMR90 ER:RAS is the cell culture model of oncogene-induced senescence (OIS). This system is based on non-immortalized, primary IMR90 human fetal lung fibroblasts that were made to express conditional oncogenic construct ER:RAS. The oncogene is inactive until triggered by the researcher upon the start of the experiment.

Principle of the experiment: addition of 4OHT to the culture medium triggers senescence in model cells. From McHugh et al., 2023.

Setup

The baseline IMR90 cells are transduced with a chimeric fusion oncogene construct called ER:RAS. It consists of the HRASG12V oncogene fused to the ligand-binding domain of a mutant estrogen receptor (ER).

There are 2 states in this model:

  1. Inducer absent, oncogene inactive: The ER:RAS protein is normally bound by heat shock proteins (HSPs) and kept inactive in the cytoplasm.
  2. Inducer present, oncogene active: Adding the inducer 4-hydroxy-tamoxifen (4-OHT) to the culture medium triggers a conformational change in the estrogen receptor, displacing HSPs and activating robust RAS signaling.

Left: control IMR90 cells. Right: IMR90 cells displaying oncogene induced senescence. From McHugh et al., 2023.

Once 4-OHT is added, the transduced IMR90 cells demonstrate classic senescence markers:

  • Irreversible G1 Growth Arrest: Mediated through the synchronous activation of the p53-p21 and p16INK4a-RB tumor suppressor pathways.
  • Morphological Changes: Cells flatten, become enlarged, and exhibit a distinct, vacuolated cytoplasm.
  • Metabolic Changes: Increased lysosomal activity detectable via positive Senescence-Associated β-Galactosidase (SA-β-gal) staining at pH 6.0.
  • SASP Activation: Robust production of the Senescence-Associated Secretory Phenotype, including pro-inflammatory cytokines like IL-6 and IL-8.
  • Nuclear Changes: Formation of Senescence-Associated Heterochromatin Foci (SAHF) and persistent DNA damage response (DDR) activation.

Rationale

Primary human fibroblasts do not undergo spontaneous transformation easily and have an intact tumor-suppressor barrier. The system provides:

  • Synchronicity: Unlike replicative senescence (which takes months and happens asynchronously), the IMR90 ER:RAS system forces an entire cell population into a coordinated senescent state within 6 to 7 days.
  • Experimental Control: Uninduced cells (treated with vehicle control) serve as an identical, actively proliferating baseline, minimizing genetic background discrepancies.