Murine Pancreatic Adenocarcinoma Dampens SHIP-1 Expression and Alters MDSC Homeostasis and Function

Pancreatic cancer is one of the most aggressive cancers, with tumor-induced myeloid-derived suppressor cells (MDSC) contributing to its pathogenesis and ineffective therapies. In response to cytokine/chemokine receptor activation, src homology 2 domain-containing inositol 5'-phosphatase-1 (SHIP-1) influences phosphatidylinositol-3-kinase (PI3K) signaling events, which regulate immunohomeostasis. We hypothesize that factors from murine pancreatic cancer cells cause the down-regulation of SHIP-1 expression, which may potentially contribute to MDSC expansion, and the suppression of CD8(+) T cell immune responses. Therefore, we sought to determine the role of SHIP-1 in solid tumor progression, such as murine pancreatic cancer.Immunocompetent C57BL/6 mice were inoculated with either murine Panc02 cells (tumor-bearing [TB] mice) or Phosphate Buffer Saline (PBS) (control mice). Cytometric Bead Array (CBA) analysis of supernatants of cultured Panc02 detected pro-inflammatory cytokines such as IL-6, IL-10 and MCP-1. TB mice showed a significant increase in serum levels of pro-inflammatory factors IL-6 and MCP-1 measured by CBA. qRT-PCR and Western blot analyses revealed the in vivo down-regulation of SHIP-1 expression in splenocytes from TB mice. Western blot analyses also detected reduced SHIP-1 activity, increased AKT-1 and BAD hyper-phosphorylation and up-regulation of BCL-2 expression in splenocytes from TB mice. In vitro, qRT-PCR and Western blot analyses detected reduced SHIP-1 mRNA and protein expression in control splenocytes co-cultured with Panc02 cells. Flow cytometry results showed significant expansion of MDSC in peripheral blood and splenocytes from TB mice. AutoMACS sorted TB MDSC exhibited hyper-phosphorylation of AKT-1 and over-expression of BCL-2 detected by western blot analysis. TB MDSC significantly suppressed antigen-specific CD8(+) T cell immune responses in vitro.SHIP-1 may regulate immune development that impacts MDSC expansion and function, contributing to pancreatic tumor progression. Thus, SHIP-1 can be a potential therapeutic target to help restore immunohomeostasis and improve therapeutic responses in patients with pancreatic cancer

Quantified Traveler: Travel Feedback Meets the Cloud to Change Behavior

We describe the design and evaluation of a system named Quantified Traveler (QT). QT is a Computational Travel Feedback System. Travel Feedback is an established programmatic method whereby travelers record travel in diaries, and meet with a counselor who guides her to alternate mode or trip decisions that are more sustainable or otherwise beneficial to society, while still meeting the subject’s mobility needs. QT is a computation surrogate for the counselor. Since counselor costs can limit the size of travel feedback programs, a system such as QT at the low costs of cloud computing, could dramatically increase scale, and thereby sustainable travel. QT uses an app on the phone to collect travel data, a server in the cloud to process it into travel diaries and then a personalized carbon, exercise, time, and cost footprint. The subject is able to see all of this information on the web. We evaluate with 135 subjects to learn if subjects let us use their personal phones and data-plans to build travel diaries, whether they actually use the website to look at their travel information, whether the design creates pro-environmental shifts in psychological variables measured by entry and exit surveys, and finally whether the revealed travel behavior records reduced driving. Before and after statistical analysis and the results from a structural equation model suggest that the results are a qualified success

Quantified Traveler: Travel Feedback Meets the Cloud to Change Behavior

We describe the design and evaluation of a system named Quantified Traveler (QT). QT is a Computational Travel Feedback System. Travel Feedback is an established programmatic method whereby travelers record travel in diaries, and meet with a counselor who guides her to alternate mode or trip decisions that are more sustainable or otherwise beneficial to society, while still meeting the subject’s mobility needs. QT is a computation surrogate for the counselor. Since counselor costs can limit the size of travel feedback programs, a system such as QT at the low costs of cloud computing, could dramatically increase scale, and thereby sustainable travel. QT uses an app on the phone to collect travel data, a server in the cloud to process it into travel diaries and then a personalized carbon, exercise, time, and cost footprint. The subject is able to see all of this information on the web. We evaluate with 135 subjects to learn if subjects let us use their personal phones and data-plans to build travel diaries, whether they actually use the website to look at their travel information, whether the design creates pro-environmental shifts in psychological variables measured by entry and exit surveys, and finally whether the revealed travel behavior records reduced driving. Before and after statistical analysis and the results from a structural equation model suggest that the results are a qualified success

Splenomegaly in TB mice.

<p>(<b>A</b>) Spleens from TB and control. mice. (<b>B</b>) Weights of spleens from TB and control mice. Represented is the mean ± S.E.M. of control (n = 4) compared to TB (n = 4) mice. ***<i>p</i><0.001; *<i>p</i><0.05 (by two-tailed Student's <i>t</i> test).</p

Increase in Pro-inflammatory factors in murine Panc02 cells and TB mice.

<p>(<b>A</b>) Pro-inflammatory cytokine production by Panc02 cells as determined by CBA and flow cytometry analysis of culture supernatant. (<b>B</b>) Growth curve of TB mice inoculated with murine Panc02 cells. (<b>C</b>) Cytokine profiles of TB and control mice as measured by CBA assay. Represented is the mean ± S.E.M. of control (n = 4) compared to TB (n = 4) mice. ***<i>p</i><0.001 (by two-tailed Student's <i>t</i> test).</p

Activation of pro-survival pathways in splenocytes TB mice.

<p>(<b>A</b>) Western blot analysis of phospho-SHIP-1 (Tyr1020) expression in whole splenocytes from control and TB mice. (<b>B</b>) Western blot analysis of phospho-AKT-1 (Ser473) and AKT 1/2/3 protein expression in whole splenocytes from control and TB mice. (<b>C</b>) Western blot analysis of phospho-BAD (Ser112) and BAD protein expression in whole splenocytes from control and TB mice, (<b>D</b>) Western blot analysis of BCL-2 protein expression in whole splenocytes from control and TB mice. To control for equal protein loading the blot was re-probed with an antibody specific for β-actin. Control (n = 3) compared to TB (n = 3) mice.</p

Expansion of MDSC in peripheral blood and splenocytes from TB mice.

<p>(<b>A</b>) MDSC percentages in peripheral blood (PB) from TB and control mice. Shown are representative flow cytometry results of Mac-1⁺Gr-1⁺ MDSC. (<b>B</b>) Represented is the Mean ± S.E.M of the relative percentages of MDSC in TB and control mice. (<b>C</b>) MDSC percentages in splenocytes (SP) from TB and control mice. Shown are representative flow cytometry results of Mac-1⁺Gr-1⁺ MDSC. (<b>D</b>) Represented is the Mean ± S.E.M of the relative percentages of MDSC in the splenocytes of TB and control mice. Control (n = 3) compared to TB (n = 3) mice **<i>p</i><0.01; ***<i>p</i><0.001 (by two-tailed Student's <i>t</i> test).</p

Down-regulation of SHIP-1 in splenocytes from TB mice.

<p>(<b>A</b>) Western blot analysis of SHIP-1 protein expression in whole splenocytes from control and TB mice. *(<b>B</b>) qRT-PCR analysis of SHIP-1 mRNA expression in whole splenocytes from control and TB mice. (<b>C</b>) Western blot analysis of SHIP-2 protein expression in whole splenocytes from control and TB mice. (<b>D</b>) PTEN protein expression in whole splenocytes from control and TB mice. To control for equal protein loading the blot was re-probed with an antibody specific for β-actin. *Represented is the mean ± S.E.M. of control (n = 3) compared to TB (n = 3) mice. <i>p</i><0.05 (by two-tailed Student's <i>t</i> test).</p

Loss of homeostasis and function of MDSC in splenocytes from TB mice.

<p>(<b>A</b>) Western blot analysis of phospho-AKT-1 (Ser473) and BCL-2 protein expression in Gr-1⁺ enriched MDSC splenocytes from TB and control mice. To control for equal protein loading the blot was re-probed with an antibody specific for β-actin. (<b>B</b>) TB Gr1⁺ MDSC significantly suppress antigen-specific T cell responses (OT-I specific (CD8⁺) T cells primed with OVA-peptide pulsed DC (DC-OVA)) compared to control Gr1⁺ MDSC <i>in vitro</i>. Controls for the assay included T cells incubated alone in complete medium (CM) or DC-OVA. Control (n = 3) compared to TB (n = 3) mice. **<i>p</i><0.01; ***<i>p</i><0.001 (by two-tailed Student's <i>t</i> test).</p