Lung cancer is one of the leading causes of cancer-related deaths in the United States. A major hurdle for improved therapies is immune suppression mediated by the tumor and its microenvironment. The lung tumor microenvironment (TME) contains large numbers of tumor-associated macrophages (TAMs), which suppress the adaptive immune response, increase neo-vascularization of the tumor, and provide pro-tumor factors to promote tumor growth. CD11b is highly expressed on myeloid cells, including TAMs, where it forms a heterodimeric integrin receptor with CD18 (known as CD11b/CD18, Mac-1, CR3, and αMβ2), and plays an important role in recruitment and biological functions of these cells, and is a validated therapeutic target. Here, we describe our pre-clinical studies targeting CD11b in the context of lung cancer, using pharmacologic and genetic approaches that work via positive allosteric modulation of CD11b function. GB1275 is a novel small molecule modulator of CD11b that is currently in Phase 1/2 clinical development. We assess GB1275 treatment effects on tumor growth and immune infiltrates in the murine Lewis Lung Carcinoma (LLC) syngeneic tumor model. Additionally, as an orthogonal approach to determine mechanisms of action, we utilize our recently developed novel CD11b knock-in (KI) mouse that constitutively expresses CD11b containing an activating isoleucine to glycine substitution at residue 332 in the ligand binding CD11b A-domain (I332G) that acts as a positive allosteric modulator of CD11b activity. We report that pharmacologic modulation of CD11b with GB1275 significantly reduces LLC tumor growth. CD11b KI mice similarly show significant reduction in both the size and rate of LLC tumor growth, as compared to WT mice, mimicking our observed treatment effects with GB1275. Tumor profiling revealed a significant reduction in TAM infiltration in GB1275-treated and in CD11b KI mice, increase in the ratio of M1/M2-like TAMs, and concomitant increase in cytotoxic T cells. The profiling also showed a significant decrease in CCL2 levels and a concomitant reduction in Ly6C hi monocytes in circulation in both groups. These findings suggest that positive allosteric modulation of CD11b reduces TAM density and reprograms them to enhance the adaptive immune response and is a novel therapeutic strategy against lung cancer.

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SUMMARY Spatial organization of molecules and cells in complex tissue microenvironments provides essential organizational cues in health and disease. A significant need exists for improved visualization of these spatial relationships. Here, we describe a multiplex immunofluorescence imaging method, termed SeqStain, that uses fluorescent-DNA-labeled antibodies for immunofluorescent staining and nuclease treatment for de-staining that allows selective enzymatic removal of the fluorescent signal. SeqStain can be used with primary antibodies, secondary antibodies, and antibody fragments to efficiently analyze complex cells and tissues. Additionally, incorporation of specific endonuclease restriction sites in antibody labels allows for selective removal of fluorescent signals while retaining other signals that can serve as marks for subsequent analyses. The application of SeqStain on human kidney tissue provided a spatialomic profile of the organization of >25 markers in the kidney, highlighting it as a versatile, easy-to-use, and gentle new technique for spatialomic analyses of complex microenvironments.

Background and purposeRecent reports from our laboratory demonstrated the post-ischaemic expression profile of various matrix metalloproteinases (MMPs) in rats and the detrimental role of MMP-12 in post-stroke brain damage. We hypothesise that the post-stroke dysregulation of MMPs is similar across species and that genetic deletion of MMP-12 would not affect the post-stroke expression of other MMPs. We tested our hypothesis by determining the pre-ischaemic and post-ischaemic expression profile of MMPs in wild-type and MMP-12 knockout mice.MethodsFocal cerebral ischaemia was induced in wild-type and MMP-12 knockout mice by middle cerebral artery occlusion procedure by insertion of a monofilament suture. One hour after ischaemia, reperfusion was initiated by removing the monofilament. One day after reperfusion, ischaemic brain tissues from various groups of mice were collected, and total RNA was isolated and subjected to cDNA synthesis followed by PCR analysis.ResultsAlthough the post-stroke expression profile of MMPs in the ischaemic brain of mice is different from rats, there is a clear species similarity in the expression of MMP-12, which was found to be predominantly upregulated in both species. Further, the post-stroke induction or inhibition of various MMPs in MMP-12 knockout mice is different from their respective expression profile in wild-type mice. Moreover, the brain mRNA expression profile of various MMPs in MMP-12 knockout mice under normal conditions is also different to their expression in wild-type mice.ConclusionsIn the ischaemic brain, MMP-12 upregulates several fold higher than any other MMP. Mice derived with the genetic deletion of MMP-12 are constitutive and have altered MMP expression profile both under normal and ischaemic conditions.

Diabetic glomerular injury is a major complication of diabetes mellitus and is the leading cause of end stage renal disease (ESRD). Healthy podocytes are essential for glomerular function and health. Injury or loss of these cells results in increased proteinuria and kidney dysfunction and is a common finding in various glomerulopathies. Thus, mechanistic understanding of pathways that protect podocytes from damage are essential for development of future therapeutics. MicroRNA-146a (miR-146a) is a negative regulator of inflammation and is highly expressed in myeloid cells and podocytes. We previously reported that miR-146a levels are significantly reduced in the glomeruli of patients with diabetic nephropathy (DN). Here we report generation of mice with selective deletion of miR-146a in podocytes and use of these mice in models of glomerular injury. Induction of glomerular injury in C57BL/6 wildtype mice (WT) and podocyte-specific miR-146a knockout (Pod-miR146a–/–) animals via administration of low-dose lipopolysaccharide (LPS) or nephrotoxic serum (NTS) resulted in increased proteinuria in the knockout mice, suggesting that podocyte-expressed miR-146a protects these cells, and thus glomeruli, from damage. Furthermore, induction of hyperglycemia using streptozotocin (STZ) also resulted in an accelerated development of glomerulopathy and a rapid increase in proteinuria in the knockout animals, as compared to the WT animals, further confirming the protective role of podocyte-expressed miR-146a. We also confirmed that the direct miR-146a target, ErbB4, was significantly upregulated in the diseased glomeruli and erlotinib, an ErbB4 and EGFR inhibitor, reducedits upregulation and the proteinuria in treated animals. Primary miR146–/– podocytes from these animals also showed a basally upregulated TGFβ-Smad3 signaling in vitro. Taken together, this study shows that podocyte-specific miR-146a is imperative for protecting podocytes from glomerular damage, via modulation of ErbB4/EGFR, TGFβ, and linked downstream signaling.

Introduction: Multiple pieces of evidence suggest that the elevated endogenous tPA (endo-tPA) levels in the brain after ischemic stroke are neurotoxic and contribute to the ongoing brain damage. The neurotoxicity of endo-tPA could be due to activation of apoptotic cell signaling processes, extracellular matrix degradation, or increase in permeability of the neurovascular unit. The purpose of this study is to investigate the effect of specific endo-tPA suppression on post-stroke brain injury and neurological recovery. Methods: Male Sprague-Dawley rats were subjected to a suture model middle cerebral artery occlusion (MCAO) procedure followed by reperfusion. To achieve the specific knockdown of endo-tPA, plasmids expressing shRNAs specific to rat tPA (tPAsh) formulated as nanoparticles were administered immediately after reperfusion to rats intravenously via tail vein at a dose of 1 mg/kg body weight. Untreated, and vehicle/tPAsh-treated stroke-induced rats along with sham-operated rats were sacrificed at various post-reperfusion time points. We performed various techniques such as TTC staining, real-time PCR, and immunoblot analysis to determine both the efficiency of tPAsh plasmids and efficacy of tPAsh treatment on post-stroke brain injury. To assess the effect of tPAsh treatment on post-stroke neurological recovery, we performed modified neurological severity scores, modified adhesive removal, and beam walking tests at regular intervals until 14 days reperfusion. Results: Endo-tPA protein expression in the ischemic brain of rats was increased until 5 days reperfusion. Treatment with tPAsh formulation reduced the protein expression of tPA in the ischemic brain of rats and thereby demonstrated the in vivo efficiency of plasmids expressing tPA shRNAs. The MCAO procedure-induced mortality is comparable across the cohorts. Specific knockdown of endo-tPA reduced the infarct volume and facilitated the post-stroke neurological recovery. Conclusions: Preventing the induction of post-stroke endo-tPA levels in rat brain attenuates ischemic brain damage and improves post-stroke neurological recovery.

We recently reported for the first time the detrimental role of matrix metalloproteinase (MMP)‐12 on post‐stroke brain damage. We hypothesize that the post‐stroke MMPs dysregulation is similar across species and the genetic deletion of MMP‐12 does not affect the post‐stroke expression of other MMPs. We tested our hypothesis by determining the pre‐ and post‐ischemic expression profile of MMPs in MMP‐12 knockout and respective wild type mice. Focal cerebral ischemia was induced in MMP‐12 knockout and wild type mice by middle cerebral artery occlusion procedure by insertion of a monofilament suture. One hour after ischemia, reperfusion was initiated by removing the monofilament. One‐day after reperfusion, ischemic brain tissues from various groups of mice were collected for real‐time PCR analysis and PCR analysis followed by agarose gel electrophoresis. Although the post‐stroke regulation of several MMPs was altered in wild type mice, huge upregulation of MMP‐12 (~763 fold over sham) was noticed in mice similar to rats as reported earlier by our group. Unlike in rats, MMP‐8 was prominently upregulated (~280 fold over sham) in ischemic mice brains. As expected, MMP‐12 expression is absent in the ischemic brains of MMP‐12 knockout mice. Except the expression of MMP‐9, the expression of several other MMPs such as MMP‐1a, −2, −8, −10, −15, −16, −17, −21 and −28 was altered in the ischemic brain of MMP‐12 knockout mice as compared to wild type mice under similar experimental conditions. In addition, the expression of various MMPs in MMP‐12 knockout mice under non‐ischemic conditions was altered as compared to the wild type mice that did not subject to ischemia. Based on our results, we conclude that the mice with genetic deletion of MMP‐12 are constitutive in nature that warrants the attention of their use in elucidating the role of MMP‐12 in post‐stroke brain damage.Support or Funding InformationThis work was supported in part by the OSF HealthCare Foundation and OSF Illinois Neurological Institute, Peoria, IL.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.