IFN-γ Rα Is a Key Determinant of CD8+ T Cell-Mediated Tumor Elimination or Tumor Escape and Relapse in FVB Mouse

<div><p>During the past decade, the dual function of the immune system in tumor inhibition and tumor progression has become appreciated. We have previously reported that neu-specific T cells can induce rejection of neu positive mouse mammary carcinoma (MMC) and also facilitate tumor relapse by inducing neu antigen loss and epithelial to mesenchymal transition (EMT). Here, we sought to determine the mechanism by which CD8+ T cells either eliminate the tumor, or maintain tumor cells in a dormant state and eventually facilitate tumor relapse. We show that tumor cells that express high levels of IFN-γ Rα are eliminated by CD8+ T cells. In contrast, tumor cells that express low levels of IFN-γ Rα do not die but remain dormant and quiescent in the presence of IFN-γ producing CD8+ T cells until they hide themselves from the adaptive immune system by losing the tumor antigen, neu. Relapsed tumor cells show CD44+CD24- phenotype with higher rates of tumorigenesis, <i>in vivo</i>. Acquisition of CD44+CD24- phenotype in relapsed tumors was not solely due to Darwinian selection. Our data suggest that tumor cells control the outcome of tumor immune surveillance through modulation of the expression of    IFN-γ Rα.</p> </div

The CD44+CD24- stem-like population and CD44+CD24+ population of WT MMC respond similarly to IFN-γ.

<p>WT MMC tumor cells were cultured with or without IFN-γ (50 ng/ml/10⁶ cells) for 3 days and CD44+CD24- as CD44+CD24- populations were analyzed for viability (Annexin V-/PI-) and proliferation (BrdU) by flow cytometry (A), these two population where sorted from WT MMC cells using a BD FACSAria III cell sorter and cultured for 60 days, after that analysis of CD24, CD44, Sca1 and neu was done (B) by flow cytometry. Also analysis of relapsed ANV tumor is shown. Data represent two independent experiments.</p

CD44+CD24- stem-like tumor cells show greater tumorigenicity compared with CD44+CD24+ population of WT MMC.

<p>WT MMC tumor cells were sorted into CD44+CD24- and CD44+CD24- populations and on day twenty one, 50,000 of the sorted cells were injected into mice (n=2) and tumor growth was observed. Sorted tumor cells were also cultured for 10 days (in triplicates) and counted using trypan blue exclusion. Fold of expansions are shown (B).</p

Status of IFN-γ Rα expression in the tumor cells determines the type of response to IFN-γ.

<p>WT MMC tumor cells were cultured with IFN-γ (50 ng/ml/10⁶ cells) for two weeks and were analyzed at different time points for viability (Annexin V-/PI-) by flow cytometry (A), cell proliferation by trypan blue exclusion (B), neu expression (C) and evaluation of CD44+CD24- population (D) by flow cytometry. E) dnIFN-γ Rα MMC or IFN-γ Rα++ MMC were also culture in the absence or presence of IFN-γ (50 ng/ml/10⁶ cells) for 3 days and percent viable cells were counted by trypan blue exclusion. Data represent 2-3 independent experiments.</p

Comparison of Cervical Spine Anatomy in Calves, Pigs and Humans

<div><p>Background Context</p><p>Animals are commonly used to model the human spine for <i>in vitro</i> and <i>in vivo</i> experiments. Many studies have investigated similarities and differences between animals and humans in the lumbar and thoracic vertebrae. However, a quantitative anatomic comparison of calf, pig, and human cervical spines has not been reported.</p><p>Purpose</p><p>To compare fundamental structural similarities and differences in vertebral bodies from the cervical spines of commonly used experimental animal models and humans.</p><p>Study Design</p><p>Anatomical morphometric analysis was performed on cervical vertebra specimens harvested from humans and two common large animals (i.e., calves and pigs).</p><p>Methods</p><p>Multiple morphometric parameters were directly measured from cervical spine specimens of twelve pigs, twelve calves and twelve human adult cadavers. The following anatomical parameters were measured: vertebral body width (VBW), vertebral body depth (VBD), vertebral body height (VBH), spinal canal width (SCW), spinal canal depth (SCD), pedicle width (PW), pedicle depth (PD), pedicle inclination (PI), dens width (DW), dens depth (DD), total vertebral width (TVW), and total vertebral depth (TVD).</p><p>Results</p><p>The atlantoaxial (C1–2) joint in pigs is similar to that in humans and could serve as a human substitute. The pig cervical spine is highly similar to the human cervical spine, except for two large transverse processes in the anterior regions ofC4–C6. The width and depth of the calf odontoid process were larger than those in humans. VBW and VBD of calf cervical vertebrae were larger than those in humans, but the spinal canal was smaller. Calf C7 was relatively similar to human C7, thus, it may be a good substitute.</p><p>Conclusion</p><p>Pig cervical vertebrae were more suitable human substitutions than calf cervical vertebrae, especially with respect to C1, C2, and C7. The biomechanical properties of nerve vascular anatomy and various segment functions in pig and calf cervical vertebrae must be considered when selecting an animal model for research on the spine.</p></div

Presentation_1_SRA inhibition improves antitumor potency of antigen-targeted chaperone vaccine.pptx

We have previously demonstrated that scavenger receptor A (SRA) acts as an immunosuppressive regulator of dendritic cell (DC) function in activating antitumor T cells. Here we investigate the potential of inhibiting SRA activity to enhance DC-targeted chaperone vaccines including one that was recently evaluated in melanoma patients. We show that short hairpin RNA-mediated SRA silencing significantly enhances the immunogenicity of DCs that have captured chaperone vaccines designed to target melanoma (i.e., hsp110-gp100) and breast cancer (i.e., hsp110-HER/Neu-ICD). SRA downregulation results in heightened activation of antigen-specific T cells and increased CD8+ T cell-dependent tumor inhibition. Additionally, small interfering RNA (siRNA) complexed with the biodegradable, biocompatible chitosan as a carrier can efficiently reduce SRA expression on CD11c+ DCs in vitro and in vivo. Our proof-of-concept study shows that direct administration of the chitosan-siRNA complex to mice promotes chaperone vaccine-elicited cytotoxic T lymphocyte (CTL) response, culminating in improved eradication of experimental melanoma metastases. Targeting SRA with this chitosan-siRNA regimen combined with the chaperone vaccine also leads to reprogramming of the tumor environment, indicated by elevation of the cytokine genes (i.e., ifng, il12) known to skew Th1-like cellular immunity and increased tumor infiltration by IFN-γ+CD8+ CTLs as well as IL-12+CD11c+ DCs. Given the promising antitumor activity and safety profile of chaperone vaccine in cancer patients, further optimization of the chitosan-siRNA formulation to potentially broaden the immunotherapeutic benefits of chaperone vaccine is warranted.</p

Anatomical Parameters and Abbreviations.

<p>Anatomical Parameters and Abbreviations.</p

Comparison of pedicle properties (mean ± stand deviation).

<p>Comparison of pedicle properties (mean ± stand deviation).</p

Anatomical parameters.

<p>(VBW) vertebral body width, (VBD) vertebral body depth, (VBH) vertebral body height, (SCW) spinal canal width, (SCD) spinal canal depth, (PW) pedicle width, (PH) pedicle height,(PI) pedicle inclination, (DD) dens depth, (DW) dens width, (TVD) total vertebral depth, and (TVW) total vertebral width.</p

Comparison of vertebral body width (mean ± stand deviation).

<p>Comparison of vertebral body width (mean ± stand deviation).</p