Effect of host immunity on metastatic potential in renal cell carcinoma: the assessment of optimal in vivo models to study metastatic behavior of renal cancer cells.

There has been little information about metastatic behavior of renal cell carcinoma (RCC) cells because human cancers metastasize only rarely in immunodeficient mice. Moreover, it is difficult to know the effect of host immunity on RCC metastasis due to lack of such RCC cells as transplantable in not only xenograft models but also counterparts with intact immunity. Therefore, we scrutinized in vivo metastasis of RCC cells to seek for the optimal preclinical model to study metastatic behavior. The luciferase-expressing three representative human RCC cell lines (Caki-1, A498, and 786-O) and rat ACI-RCC cell which were established in our laboratory were transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice or immunocompetent ACI rats by intracardiac injection as well as orthotopic inoculation. Metastasis was monitored using a bioluminescent imaging technique. Metastasis was rare in the three human RCC cells even when they were directly disseminated into systemic circulation under the condition least susceptible to host immune attack in NOD/SCID mice. In contrast, ACI-RCC cells spontaneously metastasized to pulmonary tissue from orthotopic tumor sites and systemically spread via intracardiac route. Metastases were more extensive when the cells were inoculated into an immunodeficient host, implying suppressive effect of host immunity on colonization of RCC cells. These results suggest that the representative human RCC cells are not adequate resource to study metastasis but that the luciferase-labeled ACI-RCC cell characterized by its luminescent stability, enhanced tumorigenicity, and widespread metastatic potential provides a useful in vivo model for preclinical assessment of cancer progression and potential therapies against RCC.

Establishment and characterization of transplantable, luminescence labeled rat renal cell carcinoma cell lines.

PURPOSE: Since renal cell carcinoma is considered an immunogenic tumor, testing therapeutic strategies has been impeded by the lack of relevant tumor models in immunocompetent animals. Recent advances in bioluminescence imaging permit sensitive in vivo detection and quantification of cells emitting light. Thus, we established bioluminescent rat renal cell carcinoma cell lines for immunocompetent rats. MATERIALS AND METHODS: The rat renal cell carcinoma cell line ACI-RCC stemming from chemically induced renal cell carcinoma in syngeneic ACI rats was stably transfected with a recombinant retroviral vector encoding luciferase genes derived from fireflies (ACI-RCC-ffLuc) or click beetles (ACI-RCC-cbLuc). Cell line growth patterns were characterized by bioluminescence imaging. RESULTS: Linear correlations noted observed between cell number and photon counts in each cell type. ACI-RCC-cbLuc emitted light about 500-fold higher than ACI-RCC-ffLuc. When transplanted subcutaneously, only ACI-RCC-ffLuc grew, possibly because of less antigenicity. ACI-RCC-ffLuc photon emission correlated significantly with subcutaneous tumor size. Orthotopic tumor growth and subsequent metastatic spread were monitored with time by increased photon intensity on bioluminescence imaging. Based on ACI-RCC-cbLuc bioluminescent intensity the in vitro screening test allowed the identification of several anticancer agents, including molecules related to human renal cell carcinoma progression. CONCLUSIONS: The new in vivo rat renal cell carcinoma model with luciferase labeled tumor cells allowed us to monitor tumor growth noninvasively and semiquantitatively by bioluminescence imaging. This model system coupled with in vitro screening permits precise evaluation of tumor behavior in intact animals and determination of the therapeutic efficacy of anticancer agents for renal cell carcinoma.

Validation and Characterization of Platelet-Rich Plasma in the Feline: A Prospective Analysis.

Objective: To quantitate key parameters of the platelet-rich plasma (PRP) product from a commercially available system in healthy, adult felines. Materials and methods: A prospective study was performed from January 2019 to April 2019. 11 adult, healthy cats were used to prospectively analyze a commercially available PRP system. A whole blood sample and a PRP sample that was processed immediately following blood draw according to the manufacturer's protocol were collected from each cat. The mean whole blood and PRP product platelet, RBC, WBC, neutrophil, monocyte, and lymphocyte concentrations were determined. The mean PRP product values were compared to the mean whole blood baseline values using a paired t-test with significance established at p = 0.05. Results: Mean platelet concentration was significantly increased (p = 0.0155). Mean RBC concentration was significantly decreased (p < 0.0001). Mean neutrophil concentration was significantly decreased (p < 0.0001). There was no statistically significant difference in mean WBC, monocyte, and lymphocyte concentrations. Clinical Relevance: The analyzed PRP system increased platelet concentration, while significantly reducing the RBC and neutrophil concentrations. Further study is warranted to determine the clinical applications and efficacy of PRP in felines, and the ideal concentrations of and relationships between platelets, red blood cells, and leukocytes needed for therapeutic effect.

CD4+CD25hiCD127low regulatory T cells are increased in oral squamous cell carcinoma patients.

Regulatory T cells (Tregs), a subset of CD4+ T cells plays a pivotal role in regulating the immune system. An increase in Treg numbers enables cancer progression by dampening the immune system and allowing tumor cells to evade immune detection and destruction. An increase in Treg numbers and expression of inhibitory cytokines including TGF-ß and IL-10 are mechanisms by which Tregs exert their immune suppressive function. However, the presence of Tregs and inhibitory cytokines in oral cancer patients is still unclear. In this study, the presence of circulating Tregs in 39 oral cancer patients and 24 healthy donors was examined by studying the presence of the CD4+CD25hiCD127low cell population in their peripheral blood mononuclear cells using flow cytometry. Serum levels of TGF-ß and IL-10 were measured by ELISA. T cell subsets of OSCC patients were found to differ significantly from healthy donors where a decrease in CD8+ cytotoxic T cells and an increase in Tregs (CD4+CD25hiCD127low) were observed. Further, the ratio of CD8+ T cells/Tregs was also decreased in patients compared to healthy donors. The presence of Tregs was accompanied by a decrease in IL-10 but not TGF-ß secretion in OSCC patients when compared to donors; in addition, the analysis also revealed that an increased presence of Tregs was accompanied by better patient survival. Amongst OSCC patients, smokers had significantly higher levels of TGF-ß. It is apparent that the immune system is compromised in OSCC patients and the characterization of the Treg subpopulation could form a basis for improving our understanding of the perturbations in the immune system that occur during OSCC tumorigenesis.

Identification of immunogenic MAGED4B peptides for vaccine development in oral cancer immunotherapy.

The ever-increasing number of tumor-associated antigens has provided a major stimulus for the development of therapeutic peptides vaccines. Tumor-associated peptides can induce high immune response rates and have been developed as vaccines for several types of solid tumors, and many are at various stages of clinical testing. MAGED4B, a melanoma antigen, is overexpressed in oral squamous cell carcinoma (OSCC) and this expression promotes proliferation and cell migration. In this study, we have identified 9 short peptides derived from MAGED4B protein that are restricted in binding to the HLA subtypes common in the Asian population (HLA-A2, A11, and A24). The peptides had good binding affinity with the MHC-Class I molecules and stimulated ex-vivo IFN-gamma and Granzyme-B production in blood samples from OSCC patients, suggesting that they are immunogenic. Further, T cells stimulated with peptide-pulsed dendritic cells showed enhanced T-cell cytotoxic activity against MAGED4B-overexpressing OSCC cell lines. In summary, we have identified MAGED4B peptides that induce anti-tumor immune responses advocating that they could be further developed as vaccine candidates for the treatment of OSCC.

CXCL17 expression by tumor cells recruits CD11b+Gr1 high F4/80- cells and promotes tumor progression.

BACKGROUND: Chemokines are involved in multiple aspects of pathogenesis and cellular trafficking in tumorigenesis. In this study, we report that the latest member of the C-X-C-type chemokines, CXCL17 (DMC/VCC-1), recruits immature myeloid-derived cells and enhances early tumor progression. METHODOLOGY/PRINCIPAL FINDINGS: CXCL17 was preferentially expressed in some aggressive types of gastrointestinal, breast, and lung cancer cells. CXCL17 expression did not impart NIH3T3 cells with oncogenic potential in vitro, but CXCL17-expressing NIH3T3 cells could form vasculature-rich tumors in immunodeficient mice. Our data showed that CXCL17-expressing tumor cells increased immature CD11b(+)Gr1(+) myeloid-derived cells at tumor sites in mice and promoted CD31(+) tumor angiogenesis. Extensive chemotactic assays proved that CXCL17-responding cells were CD11b(+)Gr1(high)F4/80(-) cells (≈ 90%) with a neutrophil-like morphology in vitro. Although CXCL17 expression could not increase the number of CD11b(+)Gr1(+) cells in tumor-burdened SCID mice or promote metastases of low metastatic colon cancer cells, the existence of CXCL17-responding myeloid-derived cells caused a striking enhancement of xenograft tumor formation. CONCLUSIONS/SIGNIFICANCE: These results suggest that aberrant expression of CXCL17 in tumor cells recruits immature myeloid-derived cells and promotes tumor progression through angiogenesis.

Transcriptional modulation using HDACi depsipeptide promotes immune cell-mediated tumor destruction of murine B16 melanoma.

With melanoma, as with many other malignancies, aberrant transcriptional repression is a hallmark of refractory cancer. To restore gene expression, use of a histone deacetylase inhibitor (HDACi) is expected to be effective. Our recent DNA micro-array analysis showed that the HDACi depsipeptide (FK228) significantly enhances gp100 antigen expression. Herein, we demonstrate that depsipeptide promotes tumor-specific T-cell-mediated killing of B16/F10 murine melanoma cells. First, by a quantitative assay of caspase-3/7 activity, a sublethal dose of depsipeptide was determined (ED50: 5 nM), in which p21(Waf1/Cip1) and Fas were sufficiently evoked concomitantly with histone H3 acetylation. Second, the sublethal dose of depsipeptide treatment with either a recombinant Fas ligand or tumor-specific T cells synergistically enhanced apoptotic cell death in B16/F10 cells in vitro. Furthermore, we found that depsipeptide increased levels of perforin in T cells. Finally, in vivo metastatic growth of B16/F10 in the lung was significantly inhibited by a combination of depsipeptide treatment and immune cell adoptive transfer from immunized mice using irradiated B16 cells and gp100-specific (Pmel-1) TCR transgenic mice (P<0.05, vs cell transfer alone). Consequently, employment of a transcriptional modulation strategy using HDACis might prove to be a useful pretreatment for human melanoma immunotherapy.

Toriello-Carey syndrome: delineation and review.

Toriello and Carey [1988: Am J Med Genet 31:17-23] first described a syndrome with component manifestations of corpus callosum agenesis, unusual facial appearance, Robin sequence, and other anomalies. This was termed the Toriello-Carey syndrome by Lacombe et al. [1992: Am J Med Genet 42:374-376]. Since then, 11 reports describing 16 additional children have been published; in addition, we have had the opportunity to review over 30 unpublished cases. However, for various reasons, only 25 of the unpublished patients were included in this review. Based on this total, we can begin to better delineate this syndrome, as well as provide some information on natural history.