Combination chemotherapy and radiation of human squamous cell carcinoma of the head and neck augments CTL-mediated lysis - PubMed (original) (raw)

Combination chemotherapy and radiation of human squamous cell carcinoma of the head and neck augments CTL-mediated lysis

Alexander Gelbard et al. Clin Cancer Res. 2006.

Abstract

Purpose: The combination of systemic multiagent chemotherapy (5-fluorouracil + cisplatin) and tumor irradiation is standard of care for head and neck squamous cell carcinoma (HNSCC). Furthermore, it has been shown that sublethal doses of radiation or chemotherapeutic drugs in diverse cancer types may alter the phenotype or biology of neoplastic cells, making them more susceptible to CTL-mediated cytotoxicity. However, little is known about the potential synergistic effect of drug plus radiation on CTL killing. Here, we examined whether the combination of two chemotherapeutics and ionizing radiation enhanced CTL-mediated destruction of HNSCC more so than either modality separately, as well as the basis for the enhanced tumor cell lysis.

Experimental design: Several HNSCC cell lines with distinct biological features were treated with sublethal doses of cisplatin and 5-fluorouracil for 24 hours and with 10-Gy irradiation. Seventy-two hours postirradiation, tumor cells were exposed to an antigen-specific CD8+ CTL directed against carcinoembryonic antigen or MUC-1.

Results: In three of three tumor cell lines tested, enhanced CTL activity was observed when the two modalities (chemotherapy and radiation) were combined as compared with target cells exposed to either modality separately. CTL-mediated lysis was MHC restricted and antigen specific and occurred almost entirely via the perforin pathway. Moreover, the combination treatment regimen led to a 50% reduction in Bcl-2 expression whereas single modality treatment had little bearing on the expression of this antiapoptotic gene.

Conclusions: Overall, these results reveal that (a) CTL killing can be enhanced by combining multiagent chemotherapy and radiation and (b) combination treatment enhanced or sensitized HNSCC to the perforin pathway, perhaps by down-regulating Bcl-2 expression. These studies thus form the rational basis for clinical trials of immunotherapy concomitant with the current standard of care of HNSCC.

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Figures

Fig. 1. Combination therapy with multiagent chemotherapy (CDDP and 5-FU) and irradiation (10 Gy) is sublethal

Serial cell counts of a representative cell line (HN 12) were obtained to confirm that the combination of chemotherapy and radiation was sublethal. Tumor cells received no treatment (open squares), or the combination of chemotherapy and 10Gy external beam radiation (closed squares). Following treatment, cells were recultured and serial cell counts were obtained at 1, 3, 5 and 7 days post-therapy.

Fig. 2. HNSCC cells treated with chemotherapy and radiation demonstrate significantly increased sensitivity to Ag-specific cytotoxic T-cell killing

HNSCC tumor cells received no treatment (control), chemotherapy (CDDP and 5-FU), irradiation (10 Gy), or the combination of chemotherapy and irradiation. Following therapy, cells were recultured for 72 hours. Cells were then labeled with 111In and co-incubated with HLA-A2 restricted CEA-specific CTL for 18 hours at an E:T ratio of 30:1. Panels A–C, Cell lines shown were both CEA- and HLA-A2 positive. Panel D, Treated cells were co-incubated with HLA-A2 restricted MUC-1-specific CTL for 18 hours at an E:T ratio of 30:1. Panel E, Blocking of CEA-specific cytotoxicity of HN-4 cells with anti-HLA-A2 mAb. Panel F, HN 30 is a CEA positive, HLA-A2 negative cell line and is shown as a negative control.

Fig. 3. Treatment of HNSCC tumor cells with chemotherapy and irradiation modulates tumor phenotype

HNSCC tumor cells received no therapy (control), chemotherapy (CDDP and 5-FU, irradiation (10 Gy), or the combination of chemotherapy and irradiation. Panel A, Cells were recultured for 72 hours and then analyzed by flow cytometry. Cells were analyzed for Fas, MUC-1, ICAM-1 and MHC class I surface expression after each treatment. Intracellular levels of CEA expression were also measured. Numbers indicate the percentage of positive cells. Numbers in parentheses denote the mean fluorescent intensity. Isotype control antibody staining was less than 5% in all samples. Bold indicates marked upregulation (increased cell surface levels of 10% or more or a >1.5-fold increase of mean fluorescent intensity not observed in isotype control). Panels B–E: Cells were analyzed for intracellular CEA levels. Dashed lines depict staining with isotype control antibody, solid lines depict CEA expression of HNSCC tumor cells that received no therapy (control), while solid histograms depict CEA expression of HNSCC tumor cells that received the combination of chemotherapy and irradiation.

Fig. 4. The enhanced CTL killing of HNSCC after combination therapy is perforin dependent

Panel A, The Fas signaling pathway of tumor cell line HN-4 was determined to be defective. HN-4 cells were incubated with Fas cross-linking antibody CH11, or IgM isotype control antibody (shaded area) for 3 hours. Cells were then fixed and intracellular staining of caspase 3 (solid thick line) was preformed with FITC-labeled fluorescent antibody. Caspase expression was quantified via flow cytometry. Panels B and C, Functional abrogation of perforin pathway resulted in the loss of enhanced sensitivity of treated tumor cells to CTL killing. Tumor cells received no treatment (control), or the combination of chemotherapy and 10Gy external beam radiation. Following treatment, cells were recultured for 72 hours. Cytotoxic T lymphocytes were then incubated with either PBS (Panel B) or Concanamycin A (100nm) (Panel C) for 3 hours at 37°C. Following incubation, tumor cells were labeled with 111In and co-incubated with the CTL for 18 hours at an E:T ratio of 30:1.

Fig. 5. The combination of chemotherapy and radiation reduces tumor cell expression of anti-apoptotic gene BCL-2

HN 4 tumor cells received no treatment (control), chemotherapy, 10 Gy external beam irradiation, or the combination of chemotherapy and radiation. Following treatment, cells were recultured for 24 hours. Cellular mRNA was then harvested and real-time polymerase chain reactions (PCR) were performed. Pro and anti-apoptotic genes expression levels were normalized to housekeeping gene GAPDH mRNA levels for each sample. Panel F: Persistence of BCL-2 downregulation. HN 4 cells received no treatment (open squares) or the combination of chemotherapy and radiation (closed squares )). At the indicated time points, mRNA was harvested and real-time PCR were performed for BCL-2.

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Combination chemotherapy and radiation of human squamous cell carcinoma of the head and neck augments CTL-mediated lysis - PubMed (original) (raw)