Effects of Cetuximab and Erlotinib on the behaviour of cancer stem cells in head and neck squamous cell carcinoma

Abstract

The therapeutic responses of many solid tumours to chemo- and radio-therapies are far from fully effective but therapies targeting malignancy-related cellular changes show promise for further control. In head and neck squamous cell carcinoma, the epidermal growth factor receptor (EGFR) is commonly overexpressed and investigation of agents that block this receptor indicate a limited response when used alone but an ability to enhance the actions of other drugs. The hierarchical stem cell patterns present in tumours generate cellular heterogeneity and this is further complicated by cancer stem cells (CSC) shifting between epithelial (Epi-CSC) and mesenchymal (EMT-CSC) states. To clarify how such heterogeneity influences responses to EGFR blocking, we examined the effects of Cetuximab and Erlotinib on the cell sub-populations in HNSCC cell lines. These agents reduced cell proliferation for all subpopulations but induced little cell death. They did however induce large shifts of cells between the EMT-CSC, Epi-CSC and differentiating cell compartments. Loss of EMT-CSCs reduced cell motility and is expected to reduce invasion and metastasis. EGFR blocking also induced shifts of Epi-CSCs into the differentiating cell compartment which typically has greater sensitivity to chemo/radiation, an effect expected to enhance the overall response of tumour cell populations to adjunctive therapies.

Keywords: Cetuximab; EMT; HNSCC; cancer stem cells; differentiation.

Figure 1. Effects of Cetuximab and Erlotinib on cell proliferation and apoptosis in HNSCC cell lines

Percentage of total number of CA1 (A) and Luc4 (B) cells compared to controls after 3 days of treatment with Cetuximab and Erlotinib at various concentrations. (C) Cell morphology of CA1 and Luc4 after treatment with Cetuximab (100 µg/ml) and Erlotinib (500 ng/ml) for 3 days (Scale = 50μm). (D) Total number of CA1 and Luc4 after each day of treatment. (E) Analysis of Annexin V positive cells after 3 days treatment with either Cetuximab or Erlotinib. (F) Representative images of IdU positive cells (green) in CA1 and Luc4 after treatment with Cetuximab and Erlotinib (Scale = 25μm). (G) Quantification of the number of IdU positive cells. (H) FACS analysis of expression of CD44 and ESA on CA1 and Luc4 cells after 3 days of treatment with Cetuximab or Erlotinib showing decrease of cells with CD44^high/ESA^high expression and increase the CD44^high/ESA^low fraction. (I) Quantification of data depicted in H, showing the percentage of CD44^high/ESA^low, CD44^high/ESA^high and CD44^low fractions. For this and all subsequent figures; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 2. Cetuximab and Erlotinib decrease clonogenicity, proliferation rates and EGFR expression

(A) Effects of treatment on colony forming ability. (B) Quantification of the number of colonies formed in CA1 and Luc4 cell lines. (C) Number of spheres formed after 3 days of treatment. (D) FACS plots of changes in the number of cells expressing cell-surface EGFR. (E, F) Comparisons of percentages of EGFR expressing cells in the two cell lines after treatment. (G) Altered patterns of EGFR staining after treatment. (H) Western blots showing protein levels for EGFR, pEGFR, and pERK. Altered patterns of cell proliferation (I–K), accumulation (L) and apoptosis (M) of cell sub-fractions following treatment. (N) Levels of Cyclin D1 were reduced in all sub-fractions.

Figure 3. Cetuximab and Erlotinib induce cellular differentiation

(A) Mean cell size of CD44^high/ESA^low, CD44^high/ESA^high and CD44^low fractions in CA1 and Luc4 cell lines after treatment. Representative graphs showing (B) increased size and (C) increased cytoplasmic/nuclear ratio of CD44^high/ESA^low cells. In control cultures, expression of the differentiation markers Calgranulin B and Involucrin is normally lowest in CD44^high/ESA^low sub-population (D) but is increased in all fractions (E) after treatment. (F) qPCR indicated reduced Vimentin and increased E-cadherin expression of EMT fractions after treatment. (G) Control cultures show some cohesive colonies staining for E-cadherin (green) surrounded by scattered DAPI-positive E-cadherin negative cells. After treatment with Cetuximab or Erlotinib scattered cells were lost and nearly all cells were E-cadherin positive. (H) Control cultures show numerous scattered vimentin-positive (green) cells which, after treatment are reduced in number and tend to cluster into and around epithelial-like colonies (Scale for G and H = 25 μm) Inserts show colonies at higher magnification.

Figure 4. Changes in cell transitions, motility, and response to cisplatin induced by treatment

(A) Isolated populations of CD44^high/ESA^low cells retain EMT characteristics 3 days after plating but treated with Cetuximab or Erlotinib begin to form clustered epithelial colonies (^*). Three days after plating a mixture of EGFP⁺ EMT cells with unlabeled parental cells, EGFP⁺ cells retain their EMT-like morphology but treatment with Cetuximab or Erlotinib induces transition into an epithelial phenotype (Scale = 25μm). (B–D). Images of scratches made in control and treated cultures showed reduced closure of scratches after treatment. (E) Cell counts after treatment with Cisplatin alone or combined with EGFR inhibition indicate more effective action of the combined drugs. (F) Cytometry assessing CD44 and ESA expression identifies 3 cell sub-populations in HNSCC cell lines. The majority cell population is ESA^high and contains a CD44^high/ ESA^high epithelial stem cell population (A), and a CD44^low differentiating non-stem cell population (B) (see Figure 2A). A third population (C) is CD44^high/ESA^low and corresponds to cells that have undergone EMT. Three transitions occur – EMT, MET and transition into differentiation. Blocking the EGFR markedly enhanced the transitions indicated by red arrows.