20 research outputs found
Restriction landmark genomic scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations.
BackgroundRestriction landmark genomic scanning (RLGS) is one of the most successfully applied methods for the identification of aberrant CpG island hypermethylation in cancer, as well as the identification of tissue specific methylation of CpG islands. However, a limitation to the utility of this method has been the ability to assign specific genomic sequences to RLGS spots, a process commonly referred to as "RLGS spot cloning."ResultsWe report the development of a virtual RLGS method (vRLGS) that allows for RLGS spot identification in any sequenced genome and with any enzyme combination. We report significant improvements in predicting DNA fragment migration patterns by incorporating sequence information into the migration models, and demonstrate a median Euclidian distance between actual and predicted spot migration of 0.18 centimeters for the most complex human RLGS pattern. We report the confirmed identification of 795 human and 530 mouse RLGS spots for the most commonly used enzyme combinations. We also developed a method to filter the virtual spots to reduce the number of extra spots seen on a virtual profile for both the mouse and human genomes. We demonstrate use of this filter to simplify spot cloning and to assist in the identification of spots exhibiting tissue-specific methylation.ConclusionThe new vRLGS system reported here is highly robust for the identification of novel RLGS spots. The migration models developed are not specific to the genome being studied or the enzyme combination being used, making this tool broadly applicable. The identification of hundreds of mouse and human RLGS spot loci confirms the strong bias of RLGS studies to focus on CpG islands and provides a valuable resource to rapidly study their methylation
Downregulation and/or Release of NKG2D Ligands as Immune Evasion Strategy of Human Neuroblastoma
Neuroblastoma (NB) is a pediatric extracranial tumor characterized by downregulation of human leukocyte antigen class I and defects of the antigen processing machinery, two features that make it an appropriate target for natural killer (NK)-mediated lysis. NKG2D is an activating immunoreceptor expressed by cytotoxic T lymphocytes and NK cells. The ligands for NKG2D are the major histocompatibility complex class I-related chain (MIC)A and MICB glycoproteins, and the UL-16-binding proteins (ULBPs). Here, the expression of NKG2D ligands was investigated in human primary NB tumors and cell lines because scanty information is available on this issue. MICA, MICB, and ULBP transcripts were found in most tumors and cell lines. MICA protein was detected in some NB cell lines but not in primary tumors. A soluble form of MICA (sMICA) was identified in most patient sera and in some cell line supernatants. sMICA downregulated surface NKG2D in normal peripheral blood CD8+ cells and decreased NK-mediated killing of MICA+ NB cells. MICB was detected exclusively in the cytosol of primary tumors and cell lines. Approximately 50% of primary tumors expressed ULBP-2, but not ULBP-1 or -3. ULBP-3 was expressed in 5 of 9 cell lines, ULBP-2 in 2 of 9, whereas ULBP-1 was never detected. These studies delineate novel potential pathways of tumor escape and immunodeficiency in NB
Expression and Functional Analysis of Human Leukocyte Antigen Class I Antigen-Processing Machinery in Medulloblastoma
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Immune Adjuvant Activity of Pre-Resectional Radiofrequency Ablation Protects against Local and Systemic Recurrence in Aggressive Murine Colorectal Cancer
Purpose While surgical resection is a cornerstone of cancer treatment, local and distant recurrences continue to adversely affect outcome in a significant proportion of patients. Evidence that an alternative debulking strategy involving radiofrequency ablation (RFA) induces antitumor immunity prompted the current investigation of the efficacy of performing RFA prior to surgical resection (pre-resectional RFA) in a preclinical mouse model. Experimental Design Therapeutic efficacy and systemic immune responses were assessed following pre-resectional RFA treatment of murine CT26 colon adenocarcinoma. Results: Treatment with pre-resectional RFA significantly delayed tumor growth and improved overall survival compared to sham surgery, RFA, or resection alone. Mice in the pre-resectional RFA group that achieved a complete response demonstrated durable antitumor immunity upon tumor re-challenge. Failure to achieve a therapeutic benefit in immunodeficient mice confirmed that tumor control by pre-resectional RFA depends on an intact adaptive immune response rather than changes in physical parameters that make ablated tumors more amenable to a complete surgical excision. RFA causes a marked increase in intratumoral CD8+ T lymphocyte infiltration, thus substantially enhancing the ratio of CD8+ effector T cells: FoxP3+ regulatory T cells. Importantly, pre-resectional RFA significantly increases the number of antigen-specific CD8+ T cells within the tumor microenvironment and tumor-draining lymph node but had no impact on infiltration by myeloid-derived suppressor cells, M1 macrophages or M2 macrophages at tumor sites or in peripheral lymphoid organs (i.e., spleen). Finally, pre-resectional RFA of primary tumors delayed growth of distant tumors through a mechanism that depends on systemic CD8+ T cell-mediated antitumor immunity. Conclusion: Improved survival and antitumor systemic immunity elicited by pre-resectional RFA support the translational potential of this neoadjuvant treatment for cancer patients with high-risk of local and systemic recurrence
Pre-resectional RFA improves local CT26 tumor control and survival.
<p><b>(A)</b> Time schedule outlining the different treatments used. BALB/c mice (10–12 mice/group) were injected s.c. with 10<sup>6</sup> CT26 cells into the left flank on day 0. Treatments were performed 10 days after tumor innoculation. Primary tumors were treated as indicated with sham surgery, resection (Rxn), or RFA on days 10 or 17. <b>(B)</b> Schematic showing sequence of administration of RFA (90°C for 1 minute) 7 days prior to surgical resection. Tumor growth curves <b>(C)</b> and survival curves <b>(D)</b> of mice bearing CT26 tumors in different treatment groups. In <b>(C)</b>, the number of long-term survivors without tumor recurrence at 150 days is indicated for each experimental group; for <b>(D)</b> *<i>P</i> < 0.001 for pre-resectional RFA group compared to all other groups. <b>(E)</b> Surviving mice (n = 9) in the pre-resectional RFA group were rechallenged with 10<sup>6</sup> CT26 cells into the contralateral (right) flank on day 150. Tumor growth curves are depicted in which <i>T</i> = 0 corresponds to the time of injection of secondary tumors. As a control, tumor growth was monitored following inoculation of the same tumor cell dose into non-tumor (NT)-experienced naive BALB/c mice (n = 10); *<i>P</i> < 0.005 as determined by Kaplan-Meier analysis. One representative experiment of ≥ three independent experiments is shown.</p
Survival benefit of pre-resectional RFA depends on adaptive immunity.
<p><b>(A)</b> Time schedule outlining treatment groups. SCID mice (5 mice/group) were implanted s.c. with 10<sup>6</sup> CT26 cells into the left flank on day 0. Treatment groups incuded sham surgery, resection (Rxn), or RFA on days 10 or 17. <b>(B)</b> Survival curves are representative of two independent experiments; n.s., not significant as determined by Kaplan-Meier analysis.</p
Pre-resectional RFA elicits systemic antitumor effects in distant tumors.
<p><b>(A)</b> Time schedule outlining different treatments used in the experiements. BALB/c mice (5 mice/group) were injected s.c. with 10<sup>6</sup> CT26 cells into the left flank on day 0 to establish primary tumor, followed by s.c. injection of 10<sup>6</sup> CT26 cells in the right flank on day 7 to establish secondary tumor. Primary tumors or skin (at region outside of primary tumor; upper back) were treated as indicated with sham surgery, resection (Rxn), or RFA on days 10 or 17. <b>(B)</b> Growth curves of secondary CT26 tumors implanted s.c. on contralateral flank. *<i>P</i> <0.05. <b>(C)</b> Representative photomicrographs and quantification of immunostained endogenous CD8<sup>+</sup> T cells in distant secondary s.c. CT26 tumors. *<i>P</i> < 0.001; scale bar, 100 μm.</p