To define the mechanisms regulating cell survival and growth of HNSCC, HSC–3 cells were subjected to cell culture in 3 dimensions (3D culture) as multicellular aggregates (MCA) also called spheroids. ErbB3 expression was significantly increased following MCA formation compared with monolayer (ML) culture at 24 h (not shown here).To determine the importance of elevated ErbB3 in downstream signalling, MCA were treated with the ErbB3 ligand, HRG–β1 or with EGF as a reference control. HRG–β1 induced strong p–ErbB3 compared with the non–treated control (not shown). In addition, HRG–β1 treatment also increased the p–ErbB2 level and led to significant induction of p–AKT. Like HRG–β1, EGF treatment also induced p–ErbB3, albeit at lower levels. As expected, EGF treatment led to increased induction of p–ErbB2, p–EGFR and p–AKT levels.The authors next tested if elevated ErbB3 level may modulate cell survival and growth.
Figure 1ABHRG:–+Figure 1: (A) HSC–3 cells cultured as MCA for 24 h were treated with or without HRG–β1 (100 ng ml−1).Phase–contrast images of MCA were taken after an additional 48 h (scale bar, 100 μm). (B)HSC–3 cells cultured as in A) were treated with HRG–β1 (100 ng ml−1) and processed for immunoblotting with indicated antibodies.The authors further demonstrated that elevated ErbB3 gene expression triggered by the 3D culture is regulated transcriptionally through specific activation of the ErbB3 promoter (data not shown here).
Figure 2Figure 2: (A) HSC–3 cells were either cultured as ML or as MCA with increasing cell density (a=3.3 × 104;b=6.6 × 104;c=5 × 105; and d=1 × 106cells per ml). After 24 h, cell lysates was processed for immunoblotting. (B)Phase–contrast photomicrographs of the cell density–dependent MCA formation described in (A) (scale bar, 100 μm). (C)ML cultures of HNSCC cells were incubated under normoxia (N, 20% O2) conditions or under hypoxia (H, 1% O2) conditions in a Hypoxystation, for 16 h at 37°C. Cell extracts were processed for immunoblotting as indicated. (D)HSC–3 cells exposed to hypoxia (H) or normoxia (N) conditions as in C, and processed for immunostaining with antibodies to ErbB3 and HIF–1α (scale bar, 20 μm). (E)HSC–3 cells cultured as MCA were labelled with pimonidazole (PIM, marker of hypoxia) followed by immunostaining with antibodies to PIM, HIF–1α and ErbB3 (A, C and E) and phase–contrast microscopy (B, D and F; scale bar, 20 μm). (F)Representative H&E staining ofHNSCC xenograft tumour cryosection confirming presence of tumour islands/nests (arrows). Magnification × 10. Lower panel indicates a magnified image of the area outlined by dashed box. (G)HNSCC xenograft tumour sections immunostained with ErbB3 or HIF–1α(scale bar, 50 μm). What is shown is a representative experiment that was performed at least three independent times. The images show the general trend of ErbB3/HIF expression in MCA/tumor sections.
Answer ALL Questions (80 marks)
1. The authors mentionspheroid as being a good model for cancer research. Do you agree with them? Provide a well–argued answer (10 marks)
2. Based on Figure 1 A, B, describe the effects of HRG–β1 treatment on the MCA. What can you conclude on the effects of the high levels of erbB3 found in resistant tumours? (10 marks)
3. Figure 2 analysis and interpretation, answer the following questions to guide you (20 marks): In Figure 2A,and using knowledge from themodule,what does the HIF–1α staining tell you? What information do you get from Figure 2C, D? Is Figure 2E surprising, why?
4. In Figure 2F,G, the authors mention tumour xenograft. What do you think of this model?Based on what you learned in the module, do you think they could have usedotherpreclinical rodent approach, how? What would have been the advantages and drawbacks?(20 marks)
5. Based on results shown in Figure 2, which strategy would you use to decrease erbB3 levels? (5 marks)
6. How does this study exemplify the role of the micro–environment on cancer cells?Based on what was covered in the module, canyou briefly describe another example? (15 marks
