We monitored real-time in vivo levels of serotonin release in the digestive system of intact zebrafish embryos during early development (5 dpf) using differential pulse voltammetry with implanted carbon fiber microelectrodes modified with carbon nanotubes dispersed in nafion. A detection limit of 1 nM, a linear range between 5 to 200 nM and a sensitivity of 83.65 nA·μM −1 were recorded. The microelectrodes were implanted at various locations in the intestine of zebrafish embryos. Serotonin levels of up to 29.9(±1.13) nM were measured in vivo in normal physiological conditions. Measurements were performed in intact live embryos without additional perturbation beyond electrode insertion. The sensor was able to quantify pharmacological alterations in serotonin release and provide the longitudinal distribution of this neurotransmitter along the intestine with high spatial resolution. In the presence of fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), concentrations of 54.1(±1.05) nM were recorded while in the presence of p-chloro-phenylalanine (PCPA), a tryptophan hydroxylase inhibitor, the serotonin levels decreased to 7.2(±0.45) nM. The variation of serotonin levels was correlated with immunohistochemical analysis. We have demonstrated the first use of electrochemical microsensors for in vivo monitoring of intestinal serotonin levels in intact zebrafish embryos.

Fewer than half of patients with systemic sclerosis demonstrate modified Rodnan skin score improvement during mycophenolate mofetil (MMF) treatment. To understand the molecular basis for this observation, we extended our prior studies and characterized molecular and cellular changes in skin biopsies from subjects with systemic sclerosis treated with MMF. Eleven subjects completed ≥24 months of MMF therapy. Two distinct skin gene expression trajectories were observed across six of these subjects. Three of the six subjects showed attenuation of the inflammatory signature by 24 months, paralleling reductions in CCL2 mRNA expression in skin and reduced numbers of macrophages and myeloid dendritic cells in skin biopsies. MMF cessation at 24 months resulted in an increased inflammatory score, increased CCL2 mRNA and protein levels, modified Rodnan skin score rebound, and increased numbers of skin myeloid cells in these subjects. In contrast, three other subjects remained on MMF >24 months and showed a persistent decrease in inflammatory score, decreasing or stable modified Rodnan skin score, CCL2 mRNA reductions, sera CCL2 protein levels trending downward, reduction in monocyte migration, and no increase in skin myeloid cell numbers. These data summarize molecular changes during MMF therapy that suggest reduction of innate immune cell numbers, possibly by attenuating expression of chemokines, including CCL2.

ONCR-177 is an engineered recombinant oncolytic herpes simplex virus (HSV) with complementary safety mechanisms, including tissue-specific miRNA attenuation and mutant UL37 to inhibit replication, neuropathic activity, and latency in normal cells. ONCR-177 is armed with five transgenes for IL12, FLT3LG (extracellular domain), CCL4, and antagonists to immune checkpoints PD-1 and CTLA-4. In vitro assays demonstrated that targeted miRNAs could efficiently suppress ONCR-177 replication and transgene expression, as could the HSV-1 standard-of-care therapy acyclovir. Although ONCR-177 was oncolytic across a panel of human cancer cell lines, including in the presence of type I IFN, replication was suppressed in human pluripotent stem cell–derived neurons, cardiomyocytes, and hepatocytes. Dendritic cells activated with ONCR-177 tumor lysates efficiently stimulated tumor antigen–specific CD8+ T-cell responses. In vivo, biodistribution analyses suggested that viral copy number and transgene expression peaked approximately 24 to 72 hours after injection and remained primarily within the injected tumor. Intratumoral administration of ONCR-177 mouse surrogate virus, mONCR-171, was efficacious across a panel of syngeneic bilateral mouse tumor models, resulting in partial or complete tumor regressions that translated into significant survival benefits and to the elicitation of a protective memory response. Antitumor effects correlated with local and distant intratumoral infiltration of several immune effector cell types, consistent with the proposed functions of the transgenes. The addition of systemic anti–PD-1 augmented the efficacy of mONCR-171, particularly for abscopal tumors. Based in part upon these preclinical results, ONCR-177 is being evaluated in patients with metastatic cancer (ONCR-177-101, NCT04348916).

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Tumor-associated macrophages can account for up to 50% of the tumor mass in breast cancer patients and high TAM density is associated with poor clinical prognosis. Because TAMs enhance tumor growth, development, and metastatic potential, redirection of TAM activation may have significant therapeutic benefit. Our studies in primary human macrophages and murine breast TAMs suggest that the synthetic oleanane triterpenoid CDDO-methyl ester (CDDO-Me) reprograms the activation profile of TAMs from tumor-promoting to tumor-inhibiting. We show that CDDO-Me treatment inhibits expression of IL-10 and VEGF in stimulated human M2 macrophages and TAMs but increases expression of TNF-α and IL-6. Surface expression of CD206 and CD163, which are characteristic of M2 activation, is significantly attenuated by CDDO-Me. In contrast, CDDO-Me up-regulates surface expression of HLA-DR and CD80, which are markers of M1 activation, and importantly potentiates macrophage activation of autologous T cells but inhibits endothelial cell vascularization. These results show for the first time that CDDO-Me redirects activation of M2 macrophages and TAMs from immune-suppressive to immune-stimulatory, and implicate a role for CDDO-Me as an immunotherapeutic in the treatment of breast and potentially other types of cancer.

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the tumor microenvironment (tMe) is an essential contributor to the development and progression of malignancy. Within the tMe, tumor associated macrophages (tAMs) mediate angiogenesis, metastasis, and immunosuppression, which inhibits infiltration of tumor-specific cytotoxic CD8+ t cells. in previous work, we demonstrated that the synthetic triterpenoid cDDo-methyl ester (cDDo-Me) converts breast tAMs from a tumor-promoting to a tumor-inhibiting activation state in vitro. We show now that CDDO-Me remodels the breast TME, redirecting TAM activation and T cell tumor infiltration in vivo. We demonstrate that CDDO-Me significantly attenuates IL-10 and VEGF expression but stimulates TNF production, and reduces surface expression of CD206 and CD115, markers of immunosuppressive TAMs. CDDO-Me treatment redirects the TAM transcriptional profile, inducing signaling pathways associated with immune stimulation, and inhibits TAM tumor infiltration, consistent with decreased expression of CCL2. In CDDO-Me-treated mice, both the absolute number and proportion of splenic CD4 + t cells were reduced, while the proportion of CD8 + T cells was significantly increased in both tumors and spleen. Moreover, mice fed CDDO-Me demonstrated significant reductions in numbers of CD4 + Foxp3 + regulatory T cells within tumors. These results demonstrate for the first time that CDDO-Me relieves immunosuppression in the breast tMe and unleashes host adaptive anti-tumor immunity.The tumor microenvironment (TME) provides the interface for communication between malignant and immune cells. As cells accumulate genetic and epigenetic aberrations, they recruit immune and other cellular mediators that collectively establish chronic inflammation in the TME. Mounting evidence demonstrates that interaction between tumor cells and immune cells in the TME facilitates tumor immune evasion by redirecting immune cell activation from a state of immune stimulation to immune suppression.The activation state and effector function of tumor-associated lymphoid and myeloid cells are profoundly influenced by the local TME, and these cells are likely an essential component of most tumors, regardless of tumorigenic trigger 1,2 . Tumor associated macrophages (TAMs) are key orchestrators of tumor cell survival and metastasis and shape adaptive immune responses via interaction with CD4 + and CD8 + T cell populations, among others 3 . TAMs recruit monocytes to the TME by secreting chemokines including CCL2, and promote angiogenesis through production of vascular endothelial growth factor (VEGF) 4,5 , transforming growth factor β (TGF-β) and matrix metalloproteinases (MMPs) 6 . Immunosuppression in the TME is mediated by TAMs through production of TGF-β, IL-10, and arginase 1 7 , which inhibit T cell activation and survival.TAMs can account for up to 50% of the tumor mass in breast cancer and genetic depletion of TAMs results in delayed tumor progression 8 , indicating that these cells play a non-redundant role. High TAM volumes are associated with poor clinical outcome for s...

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