Dendritic Cell Maturation Defines Immunological Responsiveness of Tumors to Radiation Therapy.

Radiation therapy is capable of directing adaptive immune responses against tumors by stimulating the release of endogenous adjuvants and tumor-associated Ags. Within the tumor, conventional type 1 dendritic cells (cDC1s) are uniquely positioned to respond to these signals, uptake exogenous tumor Ags, and migrate to the tumor draining lymph node to initiate cross-priming of tumor-reactive cytotoxic CD8+ T cells. In this study, we report that radiation therapy promotes the activation of intratumoral cDC1s in radioimmunogenic murine tumors, and this process fails to occur in poorly radioimmunogenic murine tumors. In poorly radioimmunogenic tumors, the adjuvant polyinosinic-polycytidylic acid overcomes this failure following radiation and successfully drives intratumoral cDC1 maturation, ultimately resulting in durable tumor cures. Depletion studies revealed that both cDC1 and CD8+ T cells are required for tumor regression following combination therapy. We further demonstrate that treatment with radiation and polyinosinic-polycytidylic acid significantly expands the proportion of proliferating CD8+ T cells in the tumor with enhanced cytolytic potential and requires T cell migration from lymph nodes for therapeutic efficacy. Thus, we conclude that lack of endogenous adjuvant release or active suppression following radiation therapy may limit its efficacy in poorly radioimmunogenic tumors, and coadministration of exogenous adjuvants that promote cDC1 maturation and migration can overcome this limitation to improve tumor control following radiation therapy.

Fluorescent tracking identifies key migratory dendritic cells in the lymph node after radiotherapy.

Radiation therapy generates extensive cancer cell death capable of promoting tumor-specific immunity. Within the tumor, conventional dendritic cells (cDCs) are known to carry tumor-associated antigens to the draining lymph node (TdLN) where they initiate T-cell priming. How radiation influences cDC migration is poorly understood. Here, we show that immunological efficacy of radiation therapy is dependent on cDC migration in radioimmunogenic tumors. Using photoconvertible mice, we demonstrate that radiation impairs cDC migration to the TdLN in poorly radioimmunogenic tumors. Comparative transcriptional analysis revealed that cDCs in radioimmunogenic tumors express genes associated with activation of endogenous adjuvant signaling pathways when compared with poorly radioimmunogenic tumors. Moreover, an exogenous adjuvant combined with radiation increased the number of migrating cDCs in these poorly radioimmunogenic tumors. Taken together, our data demonstrate that cDC migration play a critical role in the response to radiation therapy.

Transient dephosphorylation of p53 serine 376 as an early response to ionizing radiation.

In a previous paper we reported that the cytoplasmic sequestered p53 in cells of the SK-N-SH neuroblastoma cell line could be induced to translocate to the nucleus by exposure to ionizing radiation. We have extended these studies to determine the fate of p53 in HCT116 colorectal carcinoma cells where constitutive p53 protein resides in the nucleus. A continuous increase in the nuclear p53 protein was observed in irradiated cells beginning 1 h after irradiation that persisted for 8 h. Surprisingly, immunofluorescence microscopy revealed a transient, rapid and sensitive increase in a radiation-induced nuclear dephosphorylated p53 using antibody PAb421, which detects p53 when serine 376 is dephosphorylated. The PAb421 epitope was detectable after exposure to radiation doses as low as 0.5 cGy and was 10 to 20 times more sensitive compared to detection of p53 protein levels. The results are consistent with a radiation-induced, sensitive and rapid dephosphorylation of p53 at serine 376. The rapid increase in the nuclear PAb421 epitope was blocked by the protein serine phosphatase inhibitor calyculin A but was not blocked by the protein synthesis inhibitor cycloheximide, suggesting that serine 376 was dephosphorylated by protein serine phosphatase 1 or 2A acting on pre-existing p53 protein. The data suggest that dephosphorylation of serine 376 on constitutive nuclear p53 is a sensitive and early signaling event in the response of cells to DNA damage induced by ionizing radiation.

Differential gene expression in primary human skin keratinocytes and fibroblasts in response to ionizing radiation.

Although skin is usually exposed during human exposures to ionizing radiation, there have been no thorough examinations of the transcriptional response of skin fibroblasts and keratinocytes to radiation. The transcriptional response of quiescent primary fibroblasts and keratinocytes exposed to from 10 cGy to 5 Gy and collected 4 h after treatment was examined. RNA was isolated and examined by microarray analysis for changes in the levels of gene expression. Exposure to ionizing radiation altered the expression of 279 genes across both cell types. Changes in RNA expression could be arranged into three main categories: (1) changes in keratinocytes but not in fibroblasts, (2) changes in fibroblasts but not in keratinocytes, and (3) changes in both. All of these changes were primarily of p53 target genes. Similar radiation-induced changes were induced in immortalized fibroblasts or keratinocytes. In separate experiments, protein was collected and analyzed by Western blotting for expression of proteins observed in microarray experiments to be overexpressed at the mRNA level. Both Q-PCR and Western blot analysis experiments validated these transcription changes. Our results are consistent with changes in the expression of p53 target genes as indicating the magnitude of cell responses to ionizing radiation.

The nuclear matrix shell proteome of human epidermis.

BACKGROUND: Proteomic approaches have identified cancer specific biomarker proteins in the nuclear matrix fraction of cancer cells. We wanted to determine whether a similar approach could be used to investigate melanoma biomarkers. OBJECTIVE: Since it was not clear that a nuclear matrix fraction could be isolated from the intact human epidermis, we first wanted to determine whether a nuclear matrix fraction could be isolated from the intact epidermis of human skin. If this was possible, we secondarily wanted to compare the proteome of cultured melanoma and carcinoma cells to that of the intact epidermis. METHODS: We applied two-dimensional electrophoresis (2DGE) and LC/MS/MS to identify proteins isolated in the nuclear matrix shell protein fraction isolated from the human epidermis and from cultured primary skin and cancer cells. RESULTS: A subcellular fractionation of intact epidermis succeeded in yielding a nuclear matrix shell which made up approximately 40% of total tissue protein. Only 5-10% of total cell protein was fractionated in the nuclear matrix shell of cultured skin cells. The nuclear matrix shell of the intact epidermis was distinguishable from cultured keratinocytes or HaCaT cells by expression of keratin 1. The nuclear matrix of the epidermis was distinguishable from melanocytes and melanoma cells by expression of vimentin in melanocyte-derived cells and by expression of desmoplakin in the intact epidermis. CONCLUSION: The nuclear matrix-intermediate filament system can be isolated from the intact human epidermis. A careful examination of the protein composition of this subcellular fraction from the epidermis and skin cancers may identify useful cancer specific biomarkers.