HaCaT-ras A5 cell

HaCaT-ras A5 cells are a spontaneously immortalized, non-tumorigenic human skin keratinocyte cell line, instrumental in the study of tumour microenvironment interactions and the progression of skin carcinoma. Originating from a 62-year-old Caucasian male, these cells have undergone clonal selection and mutagenesis, which, coupled with autocrine growth factor regulation, enable the formation of slow-growing, highly differentiated benign cystic tumours in Balb/c-nu/nu mice. This makes them a valuable model for investigating the cellular dynamics and molecular mechanisms of tumour progression in vivo. The HaCaT-ras A5 cells are particularly useful for elucidating the complex interactions between tumour cells and surrounding stromal cells, including fibroblasts, immune cells, and endothelial cells. These interactions are mediated by the secretion of various signalling molecules such as growth factors, cytokines, and proteases, among which interleukin-6 (IL-6) plays a pivotal role. IL-6 is known to become dysregulated in many cancer types, primarily through overexpression or persistent activation of the STAT3 transcription factor. Research has shown that IL-6 stimulation of HaCaT-ras A5 cells significantly increases their proliferation via the JAK/STAT signalling pathway, while fibroblasts remain unaffected due to a more potent inhibition by SOCS3, a negative regulator of this pathway. This differential response has been captured in a mathematical model describing the dynamics of STAT3 and SOCS3, providing a deeper understanding of cell-specific signalling cascades. Furthermore, IL-6 not only directly affects HaCaT-ras A5 cell proliferation but also indirectly influences the cellular environment through the activation of a network of growth factors such as HGF, KGF, VEGF, and IL-8. Gene expression analysis involving over 16,000 genes revealed that IL-6 stimulation upregulates 19 genes related to the interferon signal pathway in both HaCaT-ras A5 cells and fibroblasts, which correlates with the observed growth inhibition in fibroblasts. The discovery of the crucial role of SerpinB4 in the proliferation of HaCaT-ras A5 cells, confirmed through siRNA knockdown experiments, underscores the intricate regulation by IL-6 in both tumour and stromal cells. This comprehensive understanding of IL-6’s roles enhances the potential for developing targeted therapeutic strategies aimed at modulating IL-6 signalling pathways in the tumour microenvironment. Overall, HaCaT-ras A5 cells offer a robust model for exploring the complex interplay within the tumour microenvironment, paving the way for novel approaches in cancer research and therapy development.

SKU:BHC11100894

• SDS (86D22C7F91E842DC9AE6A777B4D21EFA)
• Product Sheet (86D22C7F91E842DC9AE6A777B4D21EFA)

• Protein expression: P53 (+), CEA (+),
• Tumorigenic: Formation of benign tumors in Balb/c-nu/nu mice.
• Karyotype: Aneuploid (hypotetraploid)

• cultureMedium: DMEM, w: 4.5 g/L Glucose, w: 4 mM L-Glutamine, w: 3.7 g/L NaHCO3, w: 1.0 mM Sodium pyruvate (Cytion article number 820300a)
• supplements: Supplement the medium with 10% FBS
• dissociationReagent: The 1:1 mixture of EDTA (stock. 0.05%) and trypsin (stock: 0.1%) must be prepared each time ahead of detaching the cells using PBS without Ca2+ and Mg2+ to provide a physiologic osmolarity. Ready-to-use mixtures of trypsin/EDTA are not recommended, as this may result in cell clumps. As an alternative, TrypLETM Express (Life Technologies) instead of trypsin/EDTA can be used. The protocol of the manufacturer should be followed.
• subculturing:
  • Discard Old Medium: Remove the old medium from the flasks.
  • Wash Cells: Add 3-5 ml of PBS (without calcium and magnesium) to T25 flasks, or 5-10 ml to T75 flasks, to wash the adherent cells.
  • Add EDTA Solution: Cover the cell layer completely with a freshly prepared 0.05% EDTA solution-use 1-2 ml for T25 flasks and 2.5 ml for T75 flasks.
  • Incubation: Incubate the flasks at 37 degrees Celsius for 10 minutes.
  • Add Trypsin/EDTA Solution: Following the incubation, add a freshly prepared trypsin/EDTA solution (0.05% trypsin, 0.025% EDTA) to the flasks, ensuring the cells are fully covered-use 1 ml for T25 flasks and 2.5 ml for T75 flasks.
  • Monitor Detachment: Observe the cells, which should detach within 1-2 minutes.
  • Neutralize Trypsin: Add FBS-containing cell culture medium to stop the trypsin activity.
  • Transfer Cells: Dispense the cell suspension into new flasks pre-filled with fresh culture medium.
• seedingDensity: 1 x 104 cells/cm2
• fluidRenewal: 2 times per week
• freezeMedium: As a cryopreservation medium, use complete growth medium (including FBS) + 10% DMSO for adequate post-thaw viability, or CM-1 (Cytion catalog number 800100), which includes optimized osmoprotectants and metabolic stabilizers to enhance recovery and reduce cryo-induced stress.