ABSTRACT
BACKGROUND: Radiotherapy enhances innate and adaptive anti-tumour immunity. It is unclear whether this effect may be harnessed by combining immunotherapy with radiotherapy fractions used to treat prostate cancer. We investigated tumour immune microenvironment responses of pre-clinical prostate cancer models to radiotherapy. Having defined this landscape, we tested whether radiotherapy-induced tumour growth delay could be enhanced with anti-PD-L1. METHODS: Hypofractionated radiotherapy was delivered to TRAMP-C1 and MyC-CaP flank allografts. Tumour growth delay, tumour immune microenvironment flow-cytometry, and immune gene expression were analysed. TRAMP-C1 allografts were then treated with 3 × 5 Gy ± anti-PD-L1. RESULTS: 3 × 5 Gy caused tumour growth delay in TRAMP-C1 and MyC-CaP. Tumour immune microenvironment changes in TRAMP-C1 at 7 days post-radiotherapy included increased tumour-associated macrophages and dendritic cells and upregulation of PD-1/PD-L1, CD8+ T-cell, dendritic cell, and regulatory T-cell genes. At tumour regrowth post-3 × 5 Gy the tumour immune microenvironment flow-cytometry was similar to control tumours, however CD8+, natural killer and dendritic cell gene transcripts were reduced. PD-L1 inhibition plus 3 × 5 Gy in TRAMP-C1 did not enhance tumour growth delay versus monotherapy. CONCLUSION: 3 × 5 Gy hypofractionated radiotherapy can result in tumour growth delay and immune cell changes in allograft prostate cancer models. Adjuncts beyond immunomodulation may be necessary to improve the radiotherapy-induced anti-tumour response.
Subject(s)
Immune Checkpoint Inhibitors/therapeutic use , Prostatic Neoplasms/therapy , Radiation Dose Hypofractionation , Tumor Microenvironment , Animals , B7-H1 Antigen/analysis , Cell Line, Tumor , Combined Modality Therapy , Disease Models, Animal , Histocompatibility Antigens Class I/analysis , Humans , Male , Mice , Mice, Inbred C57BL , Neoplasm Transplantation , Prostatic Neoplasms/immunology , Prostatic Neoplasms/pathologyABSTRACT
Gemcitabine constitutes one of the backbones for chemotherapy treatment in pancreatic ductal adenocarcinoma (PDAC), but patients often respond poorly to this agent. Molecular markers downstream of gemcitabine treatment in preclinical models may provide an insight into resistance mechanisms. Using cytokine arrays, we identified potential secretory biomarkers of gemcitabine resistance (response) in the transgenic KRasG12D; Trp53R172H; Pdx-1 Cre (KPC) mouse model of PDAC. We verified the oncogenic role of the cytokine tissue inhibitor of matrix metalloproteinases 1 (TIMP1) in primary pancreatic tumors and metastases using both in vitro techniques and animal models. We identified potential pathways affected downstream of TIMP1 using the Illumina Human H12 array. Our findings were validated in both primary and metastatic models of pancreatic cancer. Gemcitabine increased inflammatory cytokines including TIMP1 in the KPC mouse model. TIMP1 was upregulated in patients with pancreatic intraepithelial neoplasias grade 3 and PDAC lesions relative to matched normal pancreatic tissue. In addition, TIMP1 played a role in tumor clonogenic survival and vascular density, while TIMP1 inhibition resensitized tumors to gemcitabine and radiotherapy. We observed a linear relationship between TIMP-1 expression, liver metastatic burden, and infiltration by CD11b+Gr1+ myeloid cells and CD4+CD25+FOXP3+ Tregs, whereas the presence of tumor cells was required for immune cell infiltration. Overall, our results identify TIMP1 upregulation as a resistance mechanism to gemcitabine and provide a rationale for combining chemo/radiotherapy with TIMP1 inhibitors in PDAC. Cancer Res; 77(21); 5952-62. ©2017 AACR.
Subject(s)
Deoxycytidine/analogs & derivatives , Liver Neoplasms/drug therapy , Pancreatic Neoplasms/drug therapy , Tissue Inhibitor of Metalloproteinase-1/genetics , Animals , Antimetabolites, Antineoplastic/pharmacology , Cell Line, Tumor , Deoxycytidine/pharmacology , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/radiation effects , Humans , Liver Neoplasms/radiotherapy , Liver Neoplasms/secondary , Mice, Inbred C57BL , Mice, Knockout , Mice, SCID , Mice, Transgenic , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/radiotherapy , RNA Interference , Tissue Inhibitor of Metalloproteinase-1/metabolism , Tumor Burden/drug effects , Tumor Burden/genetics , Tumor Burden/radiation effects , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , GemcitabineABSTRACT
BACKGROUND AND PURPOSE: Target volume definitions for radiotherapy in pancreatic ductal adenocarcinoma (PDAC) vary substantially. Some groups aim to treat the primary tumor only, whereas others include elective lymph nodes (eLNs). eLNs close to the primary tumor are often included unintentionally within the treatment volume, depending on the respective treatment philosophies. We aimed to measure the percentages of anatomical coverage of eLNs by comparing four different contouring guidelines. PATIENTS AND METHODS: Planning target volumes (PTVs) were contoured using planning computed tomography (CT) scans of 11 patients with PDAC based on the Oxford, RTOG (Radiation Therapy Oncology Group), Michigan, and SCALOP (Selective Chemoradiation in Advanced Localised Pancreatic Cancer trial) guidelines. Clinical target volumes (CTVs) included the peripancreatic, para-aortic, paracaval, celiac trunk, superior mesenteric, and portal vein lymph node areas. Volumetric comparisons of the coverage of all eLN regions were conducted to illustrate the differences between the four contouring strategies. RESULTS: The PTV sizes of the RTOG and Oxford guidelines were comparable. The SCALOP and Michigan PTV sizes were similar to each other and significantly smaller than the RTOG and Oxford PTVs. A large variability of eLN coverage was found for the various subregions according to the respective contouring strategies. CONCLUSION: This is the first study to directly compare the percentage of anatomical coverage of eLNs according to four PTVs in the same patient cohort. Potential practical consequences are discussed in detail.
Subject(s)
Carcinoma, Pancreatic Ductal/diagnosis , Carcinoma, Pancreatic Ductal/radiotherapy , Imaging, Three-Dimensional/standards , Pancreatic Neoplasms/diagnostic imaging , Pancreatic Neoplasms/radiotherapy , Practice Guidelines as Topic , Tomography, X-Ray Computed/standards , Carcinoma, Pancreatic Ductal/secondary , Germany , Humans , Lymphatic Metastasis , Reproducibility of Results , Sensitivity and Specificity , Tumor BurdenABSTRACT
Combined radiochemotherapy is the currently used therapy for locally advanced pancreatic ductal adenocarcinoma (PDAC), but normal tissue toxicity limits its application. Here we test the hypothesis that inhibition of ATR (ATM-Rad3-related) could increase the sensitivity of the cancer cells to radiation or chemotherapy without affecting normal cells. We tested VE-822, an ATR inhibitor, for in vitro and in vivo radiosensitization. Chk1 phosphorylation was used to indicate ATR activity, γH2AX and 53BP1 foci as evidence of DNA damage and Rad51 foci for homologous recombination activity. Sensitivity to radiation (XRT) and gemcitabine was measured with clonogenic assays in vitro and tumor growth delay in vivo. Murine intestinal damage was evaluated after abdominal XRT. VE-822 inhibited ATR in vitro and in vivo. VE-822 decreased maintenance of cell-cycle checkpoints, increased persistent DNA damage and decreased homologous recombination in irradiated cancer cells. VE-822 decreased survival of pancreatic cancer cells but not normal cells in response to XRT or gemcitabine. VE-822 markedly prolonged growth delay of pancreatic cancer xenografts after XRT and gemcitabine-based chemoradiation without augmenting normal cell or tissue toxicity. These findings support ATR inhibition as a promising new approach to improve the therapeutic ration of radiochemotherapy for patients with PDAC.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Isoxazoles/administration & dosage , Pancreatic Neoplasms/radiotherapy , Protein Kinase Inhibitors/administration & dosage , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrazines/administration & dosage , Radiation-Sensitizing Agents/administration & dosage , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/radiation effects , Checkpoint Kinase 1 , DNA Damage/drug effects , DNA Damage/radiation effects , Female , Humans , Mice , Mice, Inbred BALB C , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Phosphorylation/drug effects , Phosphorylation/radiation effects , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Radiation ToleranceABSTRACT
Successful radiosensitization requires that tumor cells become more radiosensitive without causing an equivalent reduction in the survival of cells of the surrounding normal tissues. Since tumor cell radiosensitivity can be influenced by RAS oncogene activation, we have hypothesized that inhibition of oncogenic RAS activity would lead to radiosensitization of tumors with activated RAS. We previously showed in tissue culture that prenyltransferase treatment of cells with activated RAS resulted in radiosensitization, whereas treatment of cells with wild-type RAS had no effect on radiation survival. Here we ask whether the findings obtained in vitro have applicability in vivo. We found that treatment of nude mice bearing T24 tumor cell xenografts with farnesyltransferase inhibitors resulted in a significant and synergistic reduction in tumor cell survival after irradiation. The regrowth of T24 tumors expressing activated RAS was also significantly prolonged by the addition of treatment with farnesyltransferase inhibitors compared to the regrowth after irradiation alone. In contrast, there was no effect on the radiosensitivity of HT-29 tumors expressing wild-type RAS. These results demonstrate that specific radiosensitization of tumors expressing activated RAS oncogenes can be obtained in vivo.
