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Figure S1 is related to Figure 1 and shows image cytometry gating strategies used in multiplex IHC analysis

File contains supplementary Materials & Methods, supplementary References, and Supplementary Tables S1-S10.

Supplementary Methods and Figure Legends

Supplementary Figure S1. B cells in human PDAC. Supplementary Figure S2. B cells and FcRgamma-positive cells regulate murine PDAC. Supplementary Figure S3. Leukocytes and BTK in human and murine PDAC. Supplementary Figure S4. Macrophage PI3Kgamma activates BTK to promote PDAC progression. Supplementary Figure S5. BTK and PI3Kgamma-inhibition decrease PDAC growth. Supplementary Figure S6. BTK inhibition improves gemcitabine response in late-stage PDAC.

Results of Kinase Inhibitor Screen including compound names, IC50s, and fold change compared to vehicle.

Viability assays, Soft agar, Immunoprecipitation, Kinase inhibitor screen, and Patient outcome data

Results of the Phosphokinase Array with phosphorylation sites, fold change relative to vehicle, and Kmeans clustering.

Supplemental Figures S1-S9. S1: Kinase inhibitor screen and response of OP449 and INK128. S2: CI values for DT1154 combined with INK128 and viability curves. S3: Kmeans clusters and signaling in response to PP2A activators and INK128. S4: Oncoprint and survival curves of patients with alterations in the MYC/mTOR pathway. S5: pT58 MYC expression levels in PDA cells treated with DT1154 and INK128. S6: Signaling with B56a knockdown or T58A MYC overexpression in response to DT1154 and INK128 treatment. S7: Signaling showing decreased expression of MYC increases the efficacy of INK128. S8: Treatment of KPC and KPCM/+ cell lines with BEZ235, Dasatinib, and Sunitinib. S9: Endpoint analysis of long- and short-term in vivo treatment with DT1154 and INK128.

In cancer, kinases are often activated and phosphatases suppressed, leading to aberrant activation of signaling pathways driving cellular proliferation, survival, and therapeutic resistance. Although pancreatic ductal adenocarcinoma (PDA) has historically been refractory to kinase inhibition, therapeutic activation of phosphatases is emerging as a promising strategy to restore balance to these hyperactive signaling cascades. In this study, we hypothesized that phosphatase activation combined with kinase inhibition could deplete oncogenic survival signals to reduce tumor growth. We screened PDA cell lines for kinase inhibitors that could synergize with activation of protein phosphatase 2A (PP2A), a tumor suppressor phosphatase, and determined that activation of PP2A and inhibition of mTOR synergistically increase apoptosis and reduce oncogenic phenotypes in vitro and in vivo. This combination treatment resulted in suppression of AKT/mTOR signaling coupled with reduced expression of c-MYC, an oncoprotein implicated in tumor progression and therapeutic resistance. Forced expression of c-MYC or loss of PP2A B56α, the specific PP2A subunit shown to negatively regulate c-MYC, increased resistance to mTOR inhibition. Conversely, decreased c-MYC expression increased the sensitivity of PDA cells to mTOR inhibition. Together, these studies demonstrate that combined targeting of PP2A and mTOR suppresses proliferative signaling and induces cell death and implicates this combination as a promising therapeutic strategy for patients with PDA.Significance:These findings present a combinatorial strategy targeting serine/threonine protein phosphatase PP2A and mTOR in PDA, a cancer for which there are currently no targeted therapeutic options.

In North Carolina, rural communities experience high rates of chronic illness due to health inequities exacerbated by the decline of major industries. Community gardens increase access to fresh produce and opportunities for physical activity and may offer additional benefits. These benefits can be difficult to measure as they are often unplanned or unintended. This article describes how we utilized Ripple Effect Mapping (REM), a participatory approach for evaluating complex interventions, to understand the impact of a SNAP-Ed-funded program. We purposively selected six community gardens to participate in 2-hour, facilitated REM sessions. On average, 15 people participated in each session. Participants developed a map of benefits using Appreciative Inquiry, mind mapping, and consensus-building methods. The map organized benefits across three levels: first ripple (individual), second ripple (interpersonal), and third ripple (community). In addition, participants coded benefits using the Community Capitals Framework. After the sessions, the research team extracted identified impacts into a matrix, aligned them with the SNAP-Ed Evaluation Framework, and developed digitized maps. These data corroborated findings from previous evaluations and offered insight into community-identified benefits not previously documented, including other types of capital generated by community gardens in rural communities. In addition, REM was an effective approach to measure and report several SNAP-Ed evaluation indicators, including LT11: Unexpected Benefits. Ultimately, the research team found REM to be an effective community-engaged method for understanding a complex intervention’s benefits while centering participant community voices and transferring ownership of the data to community partners, a key principle in equitable evaluation.

Grocery store intervention trials, including trials testing behavioral economics “nudges,” may change food-purchasing behaviors and improve diet quality. This study aimed to design and evaluate a grocery store healthy checkout lane “nudge” intervention on sales of a targeted healthy item. We conducted a randomized controlled trial based on the behavioral economic concept of cognitive fatigue and the marketing concept of impulse buying. Six grocery stores from one North Carolina-based chain were randomized to the intervention (n = 3) or control (n = 3) condition. Researchers tested a 4-week healthy checkout lane intervention, in which intervention stores moved 6-ounce cans of peanuts to the cash registers. Cashiers were instructed to upsell the peanuts to all shoppers at checkout. While not a component of the intervention, the retailer decreased the price of the peanuts from $1.99 to $1.50 during the first 2 weeks of the intervention. Fidelity to the checkout display was high. Fidelity to the upsell was low. The main outcome measure was aggregated store-level sales of the promoted peanuts for 4 weeks before the intervention and during the 4-week intervention period. On average, sales increased by 10 units/week in intervention stores (5.83 vs. 15.83 units, p = .04) with no significant change in control stores (1.42 vs. 1.17 units, p = .64). The difference (10 vs. −0.25 units, p = .02) was likely due to displaying the peanuts at checkout combined with the price promotion. Larger randomized controlled trials should examine whether healthy checkout lane interventions are effective “nudges” for promoting purchases of healthier foods in grocery stores.

This qualitative study aims to understand how community garden (CG) participants experience the effects of participation on fruit and vegetable (FV) consumption and health behaviors. Semi-structured interviews and focus groups were conducted with a total of 61 CG managers and participants in the fall of 2017, in CGs located in rural low-wealth communities. Five themes were identified: Community gardens influence FV access and consumption, nutritional and agricultural knowledge improve through CG participation, CGs may serve as a kickstart to other improvements to health and wellbeing, CGs have positive impacts on participants’ mental health, and successful gardens contribute to community cohesion.

Tumor necrosis factor may be a mediator of the syndrome of cancer cachexia. Tachyphylaxis or tolerance to the cachectic effects of recombinant tumor necrosis factor (rTNF) has been previously described. In this study, we investigate whether repetitive exposure to rTNF can induce similar tolerance in tumor-bearing (TB) rats and ameliorate cachexia induced by the tumor. In experiment 1, non-tumor-bearing (NTB) and TB rats were randomized to either escalating low doses of rTNF or saline i.p. twice daily for 9 consecutive days. NTB rats treated with rTNF demonstrated a significant decline in food intake and weight change (P less than 0.00001) but soon developed tolerance to the cachectic effects of rTNF; they consumed significantly more food than on the first day of treatment and had weight change similar to NTB rats treated with saline. TB rats treated with rTNF showed a similar significant decline in food intake and weight change (P less than 0.0001) and also demonstrated similar tolerance to the cachectic effects of rTNF with continued treatment. Following treatment, TB rats that had been treated with rTNF ate significantly more and lost less weight than TB rats that had been treated with saline (P less than 0.00001). rTNF treatment of TB rats also demonstrated antineoplastic activity, as estimated tumor weight of tumors from rats treated with rTNF were significantly less than controls (P = 0.003). The anticachexia and antineoplastic effects of rTNF resulted in prolonged survival of TB rats treated with rTNF compared to control TB rats (P = 0.015). Experiment 2 utilized two different rTNF treatment regimens in TB rats: one group received 12 days of escalating doses of rTNF, and another group received 15 days of rTNF treatment. TB rats treated with rTNF again had a significantly greater food intake (P less than 0.00001) and delayed weight loss (P = 0.0001) posttreatment that was further augmented by additional doses of rTNF. Antineoplastic activity of rTNF was less clear, and overall tumor growth curves were not affected by rTNF treatment. Survival of TB rats treated with rTNF was again significantly increased in a dose-dependent manner (P = 0.006). Repeated administration of low doses of rTNF to TB rats induces mild reduction in tumor growth, tolerance to the cachectic effects of rTNF that results in tolerance to the cachectic effects of tumor, and prolongation of survival.

Repetitive sublethal doses of tumor necrosis factor (TNF) can induce tolerance or tachyphylaxis to the toxic effects of TNF. Because tumor-bearing (TB) mice are more sensitive to the toxic effects of TNF, this study investigates whether similar tolerance occurs in TB mice and whether it affects the antitumor response of TNF. Nontumor-bearing C3H/Hen mice were treated with twice daily i.p. sublethal escalating doses of human recombinant TNF (2, 2, 3, 3, 4, and 4 micrograms i.p. every 12 h for 6 days) and were challenged 2 days later with a lethal i.v. dose (40 micrograms) of TNF. TNF-pretreated mice had 100% survival as compared to 0% survival in control mice previously treated with saline (P less than 0.01). Tumor-bearing C57BL/6 mice bearing an MCA-106 or MCA-102 sarcoma were treated with an identical TNF-tolerizing regimen (2, 2, 3, 3, 4, and 4 micrograms i.p. every 12 h for 6 days) beginning 3 days following tumor inoculation and were similarly more resistant to a subsequent 100% lethal i.v. treatment dose of TNF than control TB mice. A significantly greater percentage of TNF-pretreated mice bearing the MCA-106 sarcoma survived treatment doses of 8, 12, and 16 micrograms of TNF i.v. than control TB mice. Similarly, a significantly greater percentage of TNF-pretreated mice bearing the MCA-102 sarcoma survived treatment doses of 6 and 9 micrograms of TNF i.v. than control TB mice. However, the ability to administer higher doses of TNF i.v. to TNF-pretreated TB mice did not improve therapeutic efficacy. In mice bearing the MCA-106 tumor the most efficacious treatment responses were seen in animals that were previously naive to TNF, and treatment toxicity (lethality) correlated directly with antitumor efficacy such that larger treatment doses of TNF in tolerant mice resulted in similar antitumor effects as smaller treatment doses in control TB mice. In mice bearing the MCA-102 tumor, equitoxic treatment doses of TNF produced similar antitumor effects in both control and tolerant TB mice. There were no differences in cure rate for TNF-tolerant or control TB mice bearing either tumor. The results suggest that TNF tolerance occurs in TB mice and reduces the toxicity as well as the therapeutic efficacy of TNF.

As part of a study to manage islet cell tumors in patients with multiple endocrine neoplasia (MEN), patients with MEN I and Zollinger-Ellison syndrome (ZES) underwent surgery if a pancreatic islet cell tumor was identified on imaging studies. Patients with MEN I and either insulinoma or vasoactive intestinal polypeptide tumor (VIPoma) underwent surgery whether or not a tumor was identified. Each patient underwent preoperative portal venous sampling (PVS). Nine patients with MEN I and one with MEN II underwent surgery; seven had ZES, one had insulinoma, one had VIPoma, and one had both insulinoma and ZES. Eight of the nine patients with MEN I had an identifiable hormone gradient on PVS. Islet cell tumors were removed from the pancreas of each patient; two patients also had duodenal wall tumors, and three patients had malignant islet cell tumors. No patient with ZES and MEN I was cured of ZES despite the fact that islet cell tumor was removed from the region of the gastrin gradient in five of six patients. The single patient with MEN II and ZES and the three additional patients with MEN I and either insulinoma or VIPoma were cured by islet cell tumor resection. The results indicate that islet cell tumors in patients with MEN I can be both extrapancreatic and malignant. In patients with MEN I and ZES, ZES cannot be cured by tumor resection, and PVS cannot be used to select patients for curative surgery. It appears that gastrinoma in patients with MEN II, as well as either insulinoma or VIPoma in patients with MEN I, can be cured by islet cell tumor resection.

Tumor necrosis factor (TNF) is a macrophage product released in response to endotoxin that has been implicated as a cause of the toxicity and lethality seen in septic shock. Previous work suggests that tolerance to nutritional and lethal effects of TNF occur after repeated exposure to recombinant tumor necrosis factor (rTNF). In this study pretreatment of rats with a single low intravenous dose of rTNF prevented subsequent death when a lethal dose of rTNF was administered 24 hours later (tolerance or tachyphylaxis). Pretreatment with rTNF also afforded protection against the lethal effects of either endotoxin or cecal ligation and puncture when rats were challenged 24 hours later. Recombinant TNF injected 6 hours after cecal ligation and puncture initially resulted in a significant survival advantage for treated animals. When this experiment was repeated with a different lot of rTNF, however, the therapeutic benefit of rTNF was not obtained until the dose was decreased by a factor of 10. Protection against the lethal effects of cecal ligation and puncture did not occur when rTNF was given 24 hours after the insult. A single low dose of rTNF can result in tolerance or tachyphylaxis to the lethal effects of TNF. The results suggest that the early administration of low-dose rTNF may be useful in the prevention and treatment of the lethality of sepsis.

This study evaluated the in vitro renin release, tissue cyclic AMP content (TcAMPc), and tissue renin content (TRC) changes with time, in response to administration of dopamine (DOP) and of the dopamine-receptor blocking agent pimozide (PIM) to renal cortical slices from sodium deficient (SD) rats. Addition of 10(-3)M DOP to the slice preparation resulted in a gradual stimulation of RR with time, which was significantly different from that seen in control samples after 60 min of incubation. In contrast, TcAMPc of the DOP-treated samples was significantly greater than that of controls after 5 min of incubation. At 60 min, mean TRC of DOP-treated samples was greater than that of controls but not significantly. Two PIM doses (10(-8)M and 10(-6)M, whether added alone or together with 10(-3)M DOP to the cortical slice system, significantly increased RR in each instance while simultaneously depressing TcAMP content markedly below that of unstimulated controls at all incubation times examined. Mean TRC of pimozide-treated samples was also lower than that of controls by 60 min. These in vitro data in the SD rat suggest that: 1) stimulation of renin release by DOP is time-dependent and is mediated by a TcAMP-generating mechanism, and 2) the increase in renin release by PIM administration appears to involve pharmacological inactivation of TcAMP-generating pathways and disruption of membrane permeability, leading to uncontrolled RR.

Inguinal and femoral hernias are the most common conditions for which primary care physicians refer patients for surgical management. Hernias usually present as swelling accompanied by pain or a dragging sensation in the groin. Most hernias can be diagnosed based on the history and clinical examination, but ultrasonography may be useful in differentiating a hernia from other causes of groin swelling. Surgical repair is usually advised because of the danger of incarceration and strangulation, particularly with femoral hernias. Three major types of open repair are currently used, and laparoscopic techniques are also employed. The choice of technique depends on several factors, including the type of hernia, anesthetic considerations, cost, period of postoperative disability and the surgeon's expertise. Following initial herniorrhaphy, complication and recurrence rates are generally low. Laparoscopic techniques make it possible for patients to return to normal activities more quickly, but they are more costly than open procedures. In addition, they require general anesthesia, and the long-term hernia recurrence rate with these procedures is unknown.