The combination of a low-dose chemotherapeutic agent, 5-fluorouracil, and an adenoviral tumor vaccine has a synergistic benefit on survival in a tumor model system.

Standard cancer therapies, particularly those involving chemotherapy, are in need of modifications that both reduce short-term and long-term side effects as well as improve the overall survival of cancer patients. Here we show that combining low-dose chemotherapy with a therapeutic vaccination using an adenovirus encoding a model tumor-associated antigen, ovalbumin (Ad5-OVA), had a synergistic impact on survival in tumor-challenged mice. Mice that received the combinatorial treatment of Ad5-OVA plus low-dose 5-fluorouracil (5-FU) had a 95% survival rate compared to 7% and 30% survival rates for Ad5-OVA alone and 5-FU alone respectively. The presence of 5-FU enhanced the levels of OVA-specific CD8(+) T lymphocytes in the spleens and draining lymph nodes of Ad5-OVA-treated mice, a phenomenon that was dependent on the mice having been tumor-challenged. Thus 5-FU may have enhanced survival of Ad5-OVA-treated mice by enhancing the tumor-specific immune response combined with eliminating tumor bulk. We also investigated the possibility that the observed therapeutic benefit may have been derived from the capacity of 5-FU to deplete MDSC populations. The findings presented here promote the concept of combining adenoviral cancer vaccines with low-dose chemotherapy

Effect of depleting MDSCs from variously vaccinated tumor-bearing mice on survival and tumor-specific T lymphocyte responses.

<p><i>A</i>. Tumor-bearing mice were treated with multiple i.p. doses of RB6-8C5 (as described in materials and methods section) and draining lymph node and spleen cells were isolated after 6 days of depletion (day 12 post-tumor challenge) and stained for the presence of gMDSCs (CD11b⁺Ly6C^+lowLy6G⁺) and mMDSCs (CD11b⁺Ly6C^+hiLy6G⁻). Data is presented as a percentage of total lymph node or spleen populations. Student T-test was used to determine statistical significance. <i>B.</i> Survival analysis: C57BL/6 mice were challenged with E.G7 and then, 7 days post tumor challenge, were treated as indicated. Survival curve represents pooled data from 2 independent experiments where a total of n = 8 mice/treatment group were used. Statistical analysis (Log-rank (Mantel-Cox) test) of survival data revealed that only the Ad5-OVA plus 5-FU treatment to be significantly different from all other treatments (*** P<0.001). <i>C.</i> Two weeks post-treatment the levels of OVA-specific CD8⁺ T cells, as a percentage of total CD8⁺CD3⁺ cells, was determined using a fluorescently-tagged OVA tetramer (see materials and methods for further details), in PBLs. Results displayed represent pooled data from 2 independent experiments and were performed in conjunction with the survival studies (see above (<i>B</i>.)). Statistical significance was determined using an ANOVA one way analysis of variance (with Tukey post-test) (*<i>P</i><0.05). All error bars represent standard error of the mean.</p

Anti-tumor effect and survival in mice treated with Ad5-OVA and/or 5-FU.

<p>C57BL/6 mice were challenged with E.G7 and then, 7 days post tumor challenge, given: no treatment (naïve); Ad5-OVA; Ad5-OVA +5-FU; or 5-FU. <i>A–D.</i> The tumor volumes for each mouse from one representative experiment are shown. <i>E.</i> Survival graph representing four pooled experiments. Total number of mice for each treatment was: n = 15 for naïve group, n = 14 for Ad5-OVA alone group, n = 21 for Ad5-OVA +5-FU group, n = 10 for Ad5-LacZ +5-FU group, and n = 15 for the 5-FU alone group. Statistical analysis (Log-rank (Mantel-Cox) test) of survival data revealed that mice survived significantly longer, compared to untreated mice, when treated with Ad5-OVA alone (p<0.001 (***⁽¹⁾)), 5-FU alone (p<0.001(***⁽²⁾)) or Ad5-LacZ +5-FU (p<0.001(***⁽³⁾), and that mice treated with Ad5-OVA +5-FU in combination survived significantly longer than untreated mice (p<0.001 (***⁽⁴⁾) and mice treated with Ad5-OVA alone (p<0.001 (***⁽⁵⁾)), 5-FU alone (p<0.001(***⁽⁶⁾)) and Ad5-LacZ +5-FU (p = 0.001 (***⁽⁷⁾).</p

Treg levels in spleen and draining lymph node.

<p>C57BL/6 mice were challenged with E.G7 and then, 7 days post tumor challenge, were treated as indicated. On day 5 post treatment the levels of Tregs (Foxp3⁺), as a percentage of total CD4⁺CD3⁺ cells, was determined using direct immunofluorescence (see materials and methods for further details) on the samples from the spleen (<i>A</i>) and the lymph node <i>(B</i>). Results displayed are from one representative experiment (n = 4 mice per group). These results were reproducible. Using an ANOVA one way analysis of variance (with Tukey post-test) no statistically significant differences were observed in independent experiments nor after pooling of the data. Error bars represent standard error of the mean.</p

Levels of gMDSCs and mMDSCs in the spleen and draining lymph node.

<p>C57BL/6 mice were challenged with E.G7 cells and then, 7 days post tumor challenge, were treated as indicated and then 5 days later, spleen and draining lymph nodes were harvested and stained using direct immunofluorescence for detection mMDSCs and gMDSCs and then analyzed using flow cytometry (see materials and methods for further details). <i>A</i>. Example of FloJo-generated dot-plots showing how mMDSCs and gMDSCs were delineated in samples obtained from the spleen. Total splenocytes were gated using a forward scatter (FSC) versus side scatter (SSC) dot-plot (<i>top row</i>). These cells were further gated to select for CD11b^+hi cells using a SSC versus CD11b dot-plot (<i>middle row</i>). Then these CD11b+hi cells were further defined using a Ly6G versus Ly6C dot-plot (<i>bottom row</i>). mMDSCs were defined as CD11b⁺Ly6G⁻Ly6C^+hi (<i>circled (left side)</i>) and gMDSCs were defined as CD11b⁺Ly6G⁺Ly6C^+low (<i>circled (right side)</i>). A similar method of analysis was used for cells obtained from the draining lymph node. <i>B–E</i>. On day 5 post treatment, the levels of mMDSCs (<i>B</i> and <i>D</i>) and gMDSCs (<i>C</i> and <i>E</i>), as a percentage of total cells, were determined, in both the spleen (<i>B</i> and <i>C</i>) and the draining lymph node (<i>D</i> and <i>E</i>). Mean percentage values (and range) for the naïve groups prior to normalization were as follows: mMDSCs in the lymph node = 0.24% (range: 0.02–0.84%); mMDSCs in the spleen = 0.26% (range: 0.02–0.63%); gMDSCs in the lymph node = 0.03% (range: 0.01–0.08%); gMDSCs in the spleen = 1.35% (0.87–4.1%). Results displayed are derived from pooled data from four separate experiments, where the number of mice/treatment group in each experiment was n = 1, n = 2, n = 2, n = 4 and therefore a total of n = 9 mice from each pooled treatment group were analyzed. Statistical significance was determined using an ANOVA one way analysis of variance (with Tukey post-test) (*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001)). Error bars represent standard error of the mean.</p

OVA-specific CD8⁺ T cell responses in tumor challenged and non-tumor-challenged mice.

<p>C57BL/6 mice were challenged with E.G7 and then, 7 days post tumor challenge, were treated as indicated. <i>A</i> and <i>B</i>. On day 9 post treatment the levels of OVA-specific CD8+ T cells, as a percentage of total CD8+CD3+ cells, were determined using a fluorescently-tagged OVA tetramer (see materials and methods for further details) in samples from the spleen (<i>A</i>) and the lymph node (<i>B</i>). The percentages were normalized against the mean values obtained for the naïve groups. Mean percentage values for the naïve group prior to normalization were 0.26% (range 0.16–0.39%) for the lymph node and 0.68% (range 0.54–0.89%) for the spleen. Data represents pooled data from four separate experiments, where the number of mice/treatment group in each experiment was n = 1, n = 2, n = 2 and n = 4 and therefore a total of n = 9 mice from each pooled treatment group were analyzed. Statistical significance was determined using an ANOVA one way analysis of variance (with Tukey post-test) (**<i>P</i><0.01, ***<i>P</i><0.001). <i>C</i><b>.</b> Non-tumor-challenged mice (n = 4 per group) were vaccinated with indicated treatments and then the levels of OVA-specific CD8⁺ T cells in the peripheral blood were measured on days 7, 15 and 21. These results were shown to be reproducible. Error bars represent standard error of the mean.</p

Obesity/Type II diabetes alters macrophage polarization resulting in a fibrotic tendon healing response

<div><p>Type II Diabetes (T2DM) dramatically impairs the tendon healing response, resulting in decreased collagen organization and mechanics relative to non-diabetic tendons. Despite this burden, there remains a paucity of information regarding the mechanisms that govern impaired healing of diabetic tendons. Mice were placed on either a high fat diet (T2DM) or low fat diet (lean) and underwent flexor tendon transection and repair surgery. Healing was assessed via mechanical testing, histology and changes in gene expression associated with collagen synthesis, matrix remodeling, and macrophage polarization. Obese/diabetic tendons healed with increased scar formation and impaired mechanical properties. Consistent with this, prolonged and excess expression of extracellular matrix (ECM) components were observed in obese/T2DM tendons. Macrophages are involved in both inflammatory and matrix deposition processes during healing. Obese/T2DM tendons healed with increased expression of markers of pro-inflammatory M1 macrophages, and elevated and prolonged expression of M2 macrophages markers that are involved in ECM deposition. Here we demonstrate that tendons from obese/diabetic mice heal with increased scar formation and increased M2 polarization, identifying excess M2 macrophage activity and matrix synthesis as a potential mechanism of the fibrotic healing phenotype observed in T2DM tendons, and as such a potential target to improve tendon healing in T2DM.</p></div

Systemic EP4 Inhibition Increases Adhesion Formation in a Murine Model of Flexor Tendon Repair.

Flexor tendon injuries are a common clinical problem, and repairs are frequently complicated by post-operative adhesions forming between the tendon and surrounding soft tissue. Prostaglandin E2 and the EP4 receptor have been implicated in this process following tendon injury; thus, we hypothesized that inhibiting EP4 after tendon injury would attenuate adhesion formation. A model of flexor tendon laceration and repair was utilized in C57BL/6J female mice to evaluate the effects of EP4 inhibition on adhesion formation and matrix deposition during flexor tendon repair. Systemic EP4 antagonist or vehicle control was given by intraperitoneal injection during the late proliferative phase of healing, and outcomes were analyzed for range of motion, biomechanics, histology, and genetic changes. Repairs treated with an EP4 antagonist demonstrated significant decreases in range of motion with increased resistance to gliding within the first three weeks after injury, suggesting greater adhesion formation. Histologic analysis of the repair site revealed a more robust granulation zone in the EP4 antagonist treated repairs, with early polarization for type III collagen by picrosirius red staining, findings consistent with functional outcomes. RT-PCR analysis demonstrated accelerated peaks in F4/80 and type III collagen (Col3a1) expression in the antagonist group, along with decreases in type I collagen (Col1a1). Mmp9 expression was significantly increased after discontinuing the antagonist, consistent with its role in mediating adhesion formation. Mmp2, which contributes to repair site remodeling, increases steadily between 10 and 28 days post-repair in the EP4 antagonist group, consistent with the increased matrix and granulation zones requiring remodeling in these repairs. These findings suggest that systemic EP4 antagonism leads to increased adhesion formation and matrix deposition during flexor tendon healing. Counter to our hypothesis that EP4 antagonism would improve the healing phenotype, these results highlight the complex role of EP4 signaling during tendon repair

Relative expression of collagen and matrix metalloproteinase genes are altered in HFD.

<p>Relative mRNA expression of (A) type III (<i>Col3a1</i>) and (B) type I collagen (<i>Col1a1</i>), (C) <i>Mmp9</i>, and (D) <i>Mmp2</i> were determined by RT-PCR between 3 and 28 days post-repair. Data were normalized to the internal control <i>β-actin</i>. Fold changes are reported relative to LFD day 3 expression. White bars = LFD; black bars = HFD, (*) indicates p<0.05, (**) indicates p<0.001, (***) indicates p<0.0001 between HFD and LFD.</p