High-dose per Fraction Radiotherapy Induces Both Antitumor Immunity and Immunosuppressive Responses in Prostate Tumors.

PURPOSE: The use of high-dose per fraction radiotherapy delivered as stereotactic body radiotherapy is a standard of care for prostate cancer. It is hypothesized that high-dose radiotherapy may enhance or suppress tumor-reactive immunity. The objective of this study was to assess both antitumor and immunosuppressive effects induced by high-dose radiotherapy in prostate cancer coclinical models, and ultimately, to test whether a combination of radiotherapy with targeted immunotherapy can enhance antitumor immunity. EXPERIMENTAL DESIGN: We studied the effects of high-dose per fraction radiotherapy with and without anti-Gr-1 using syngeneic murine allograft prostate cancer models. The dynamic change of immune populations, including tumor-infiltrating lymphocytes (TIL), T regulatory cells (Treg), and myeloid-derived suppressive cells (MDSC), was evaluated using flow cytometry and IHC. RESULTS: Coclinical prostate cancer models demonstrated that high-dose per fraction radiotherapy induced a rapid increase of tumor-infiltrating MDSCs and a subsequent rise of CD8 TILs and circulating CD8 T effector memory cells. These radiation-induced CD8 TILs were more functionally potent than those from nonirradiated controls. While systemic depletion of MDSCs by anti-Gr-1 effectively prevented MDSC tumor infiltration, it did not enhance radiotherapy-induced antitumor immunity due to a compensatory expansion of Treg-mediated immune suppression. CONCLUSIONS: In allograft prostate cancer models, high-dose radiotherapy induced an early rise of MDSCs, followed by a transient increase of functionally active CD8 TILs. However, systemic depletion of MDSC did not augment the antitumor efficacy of high-dose radiotherapy due to a compensatory Treg response, indicating blocking both MDSCs and Tregs might be necessary to enhance radiotherapy-induced antitumor immunity.

Near-Infrared Dye-Labeled Anti-Prostate Stem Cell Antigen Minibody Enables Real-Time Fluorescence Imaging and Targeted Surgery in Translational Mouse Models.

PURPOSE: The inability to intraoperatively distinguish primary tumor, as well as lymphatic spread, increases the probability of positive surgical margins, tumor recurrence, and surgical toxicity. The goal of this study was to develop a tumor-specific optical probe for real-time fluorescence-guided surgery. EXPERIMENTAL DESIGN: A humanized antibody fragment against PSCA (A11 minibody, A11 Mb) was conjugated with a near-infrared fluorophore, IRDye800CW. The integrity and binding of the probe to PSCA were confirmed by gel electrophoresis, size-exclusion chromatography, and flow cytometry, respectively. The ability of the probe to detect tumor-infiltrated lymph nodes and metastatic lesions was evaluated in 2 xenograft models, as well as in transgenic mice expressing human PSCA (hPSCA). An invasive intramuscular model was utilized to evaluate the efficacy of the A11 Mb-IRDye800CW-guided surgery. RESULTS: A11 Mb was successfully conjugated with IRDye800CW and retained specific binding to PSCA. In vivo imaging showed maximal signal-to-background ratios at 48 hours. The A11 Mb-IRDye800CW specifically detected PSCA-positive primary tumors, tumor-infiltrated lymph nodes, and distant metastases with high contrast. Fluorescence guidance facilitated more complete tumor resection, reduced tumor recurrence, and improved overall survival, compared with conventional white light surgery. The probe successfully identified primary orthotopic tumors and metastatic lesions in hPSCA transgenic mice. CONCLUSIONS: Real-time fluorescence image-guided surgery with A11 Mb-IRDye800CW enabled detection of lymph node metastases and positive surgical margins, facilitated more complete tumor removal, and improved survival, compared with white light surgery. These results may be translatable into clinical practice to improve surgical and patient outcomes.

Target genes of the largest human SWI/SNF complex subunit control cell growth.

The largest subunit of the mammalian SWI/SNF-A or BAF (BRG1-associated factor) chromatin-remodelling complex is encoded by two related cDNAs hOsa1/BAF250a and hOsa2/BAF250b that are unique to the BAF complex and absent in the related PBAF (Polybromo BAF). hOsa/BAF250 has been shown to interact with transcriptional activators and bind to DNA suggesting that it acts to target the remodelling complex to chromatin. To better understand the functions of hOsa2, we established inducible stable HeLa cell lines over-expressing FLAG-hOsa2 or a derivative lacking the ARID (AT-rich interactive domain) DNA-binding domain. Immunopurification of complexes containing hOsa2 that was followed by mass spectrometry and immunoblotting demonstrated the presence of BRG1 and known BAFs, but not hOsa1 or hBRM. Deletion of the ARID did not compromise the integrity of the complex. Induction of hOsa2 expression caused impaired cell growth and accumulation of cells in the G0/G1 cell cycle phase. Elevated levels of the p53 and p21 proteins were detected in these cells while c-Myc mRNA and protein levels were found to decrease. Chromatin immunoprecipitation and reporter assays suggested that hOsa2 had a direct effect on c-myc and p21 promoter activity. Thus hOsa2 plays an important role in controlling genes regulating the cell cycle.