SHP2 blockade enhances anti-tumor immunity via tumor cell intrinsic and extrinsic mechanisms.

SHP2 is a ubiquitous tyrosine phosphatase involved in regulating both tumor and immune cell signaling. In this study, we discovered a novel immune modulatory function of SHP2. Targeting this protein with allosteric SHP2 inhibitors promoted anti-tumor immunity, including enhancing T cell cytotoxic function and immune-mediated tumor regression. Knockout of SHP2 using CRISPR/Cas9 gene editing showed that targeting SHP2 in cancer cells contributes to this immune response. Inhibition of SHP2 activity augmented tumor intrinsic IFNγ signaling resulting in enhanced chemoattractant cytokine release and cytotoxic T cell recruitment, as well as increased expression of MHC Class I and PD-L1 on the cancer cell surface. Furthermore, SHP2 inhibition diminished the differentiation and inhibitory function of immune suppressive myeloid cells in the tumor microenvironment. SHP2 inhibition enhanced responses to anti-PD-1 blockade in syngeneic mouse models. Overall, our study reveals novel functions of SHP2 in tumor immunity and proposes that targeting SHP2 is a promising strategy for cancer immunotherapy.

Enabling drug discovery and development through single-cell imaging.

INTRODUCTION: Single-cell imaging-based assays are an area of active and growing investment in drug discovery and development. This approach offers researchers the capability to interrogate rare subpopulations of cells with minimal sample consumption and multiplexed readouts. Recent technological advances in the optical interrogation and manipulation of single cells have substantially increased the throughput and sensitivity of these assays. Areas covered: In this review, the authors focus on three classes of single-cell imaging-based analyses: single-cell microscopy combined with microfluidics, mass spectrometric imaging for subcellular compound localization, and imaging mass cytometry (IMC). They provide an overview of each technology and recent examples of their utility in advancing drug discovery, based on the potential for scalability, multiplexing, and capability to generate definitive data on cellular heterogeneity and target engagement. Expert opinion: Understanding target engagement and heterogeneity at the single-cell level will enable the development of safer and more effective therapies, particularly for new modalities like CAR-T cell therapies and gene editing approaches (AAV, CRISPR). Successful adoption of new single-cell imaging-based approaches in drug discovery will require tandem investment in advanced computational analysis and bioinformatic approaches, due to the complexity and multivariate nature of single-cell imaging data.

Induction of an IFN-Mediated Antiviral Response by a Self-Amplifying RNA Vaccine: Implications for Vaccine Design.

RNA-based vaccines have recently emerged as a promising alternative to the use of DNA-based and viral vector vaccines, in part because of the potential to simplify how vaccines are made and facilitate a rapid response to newly emerging infections. SAM vaccines are based on engineered self-amplifying mRNA (SAM) replicons encoding an Ag, and formulated with a synthetic delivery system, and they induce broad-based immune responses in preclinical animal models. In our study, in vivo imaging shows that after the immunization, SAM Ag expression has an initial gradual increase. Gene expression profiling in injection-site tissues from mice immunized with SAM-based vaccine revealed an early and robust induction of type I IFN and IFN-stimulated responses at the site of injection, concurrent with the preliminary reduced SAM Ag expression. This SAM vaccine-induced type I IFN response has the potential to provide an adjuvant effect on vaccine potency, or, conversely, it might establish a temporary state that limits the initial SAM-encoded Ag expression. To determine the role of the early type I IFN response, SAM vaccines were evaluated in IFN receptor knockout mice. Our data indicate that minimizing the early type I IFN responses may be a useful strategy to increase primary SAM expression and the resulting vaccine potency. RNA sequence modification, delivery optimization, or concurrent use of appropriate compounds might be some of the strategies to finalize this aim.

In vivo imaging with fluorescent smart probes to assess treatment strategies for acute pancreatitis.

BACKGROUND AND AIMS: Endoprotease activation is a key step in acute pancreatitis and early inhibition of these enzymes may protect from organ damage. In vivo models commonly used to evaluate protease inhibitors require animal sacrifice and therefore limit the assessment of dynamic processes. Here, we established a non-invasive fluorescence imaging-based biomarker assay to assess real-time protease inhibition and disease progression in a preclinical model of experimental pancreatitis. METHODS: Edema development and trypsin activation were imaged in a rat caerulein-injection pancreatitis model. A fluorescent "smart" probe, selectively activated by trypsin, was synthesized by labeling with Cy5.5 of a pegylated poly-L-lysine copolymer. Following injection of the probe, trypsin activation was monitored in the presence or absence of inhibitors by in vivo and ex vivo imaging. RESULTS: We established the trypsin-selectivity of the fluorescent probe in vitro using a panel of endopeptidases and specific inhibitor. In vivo, the probe accumulated in the liver and a region attributed to the pancreas by necropsy. A dose dependent decrease of total pancreatic fluorescence signal occurred upon administration of known trypsin inhibitors. The fluorescence-based method was a better predictor of trypsin inhibition than pancreatic to body weight ratio. CONCLUSIONS: We established a fluorescence imaging assay to access trypsin inhibition in real-time in vivo. This method is more sensitive and dynamic than classic tissue sample readouts and could be applied to preclinically optimize trypsin inhibitors towards intrapancreatic target inhibition.

Metabolic implications of dietary trans-fatty acids.

Dietary trans-fatty acids are associated with increased risk of cardiovascular disease and have been implicated in the incidence of obesity and type 2 diabetes mellitus (T2DM). It is established that high-fat saturated diets, relative to low-fat diets, induce adiposity and whole-body insulin resistance. Here, we test the hypothesis that markers of an obese, prediabetic state (fatty liver, visceral fat accumulation, insulin resistance) are also worsened with provision of a low-fat diet containing elaidic acid (18:1t), the predominant trans-fatty acid isomer found in the human food supply. Male 8-week-old Sprague-Dawley rats were fed a 10% trans-fatty acid enriched (LF-trans) diet for 8 weeks. At baseline, 3 and 6 weeks, in vivo magnetic resonance spectroscopy (1H-MR) assessed intramyocellular lipid (IMCL) and intrahepatic lipid (IHL) content. Euglycemic-hyperinsulinemic clamps (week 8) determined whole-body and tissue-specific insulin sensitivity followed by high-resolution ex vivo 1H-NMR to assess tissue biochemistry. Rats fed the LF-trans diet were in positive energy balance, largely explained by increased energy intake, and showed significantly increased visceral fat and liver lipid accumulation relative to the low-fat control diet. Net glycogen synthesis was also increased in the LF-trans group. A reduction in glucose disposal, independent of IMCL accumulation was observed in rats fed the LF-trans diet, whereas in rats fed a 45% saturated fat (HF-sat) diet, impaired glucose disposal corresponded to increased IMCLTA. Neither diet induced an increase in IMCLsoleus. These findings imply that trans-fatty acids may alter nutrient handling in liver, adipose tissue, and skeletal muscle and that the mechanism by which trans-fatty acids induce insulin resistance differs from diets enriched with saturated fats.

Acute impairment of rat renal function by L -NAME as measured using dynamic MRI.

OBJECTIVE: To assess the feasibility of utilizing dynamic contrast enhanced (DCE)-MRI for depicting the effects of N (G)-nitro-L -arginine methylester (L -NAME), a nitric oxide synthase (NOS) inhibitor, on glomerular filtration rate (GFR) in rats. Since Gd-DTPA is mainly cleared through the kidneys, a first-order kinetic model was used to estimate GFR based on a clearance index (k ( cl )) that describes the tracer transport rate from the renal cortex to the outer medulla. MATERIALS AND METHODS: Normotensive Sprague-Dawley rats were infused with either vehicle (0.9% NaCl) or one of three doses of L -NAME (1, 3 or 10 mg/kg) for 30 min prior to imaging. In a separate set of animals, systolic blood pressure (SBP) was measured for all treatment groups. RESULTS: L -NAME caused a significant increase in SBP at all doses when compared to pre-dose values and at the two highest doses, post-infusion, when compared to vehicle. Administration of L -NAME also led to dose-dependent changes in the rate of Gd-DTPA uptake and tracer concentrations reached in selected regions of the kidney. The k ( cl ) measurements indicated a significant impairment of GFR following NOS blockade at the highest dose of L -NAME. CONCLUSION: DCE-MRI method detected changes in GFR in response to NO inhibition with L -NAME. This non-invasive technique could be used in longitudinal studies in preclinical and clinical settings offering a rapid assessment of single-kidney function.

Targeting of the antivascular drug combretastatin to irradiated tumors results in tumor growth delay.

PURPOSE: The aim of the study is to evaluate the effects of targeting the antivascular drug combretastatin to irradiated mouse melanomas. METHODS: Combretastatin was incorporated into liposomes with surfaces modified by the addition of cyclo(Arg-Gly-Asp-D-Phe-Cys) (RGD) to create an immunoliposome (IL). This addition of RGD allows the liposome to be preferentially targeted to alphavbeta3, an integrin up-regulated in the vasculature of irradiated tumors. C57BL mice bearing a transplanted B16-F10 melanoma were randomly assigned to one of the following treatment groups: untreated, a single dose of 5-Gy radiation (IR), IL (14.5 mg/kg of combretastatin), 5-Gy radiation plus IL, and a systemic administration of free drug (81.0 mg/kg of combretastatin). RESULTS: In this transplanted tumor model, there was no significant increase in the volume of the IL + IR (5 Gy) treated tumors during the initial 6 days posttreatment; all other treatment groups exhibited exponential growth curves after day 3. The IL + IR (5 Gy) treatment resulted in a 5.1-day tumor growth delay compared to untreated controls. CONCLUSIONS: These findings indicate that preferential targeting of antivascular drugs to irradiated tumors results in significant tumor growth delay.