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Osteosarcoma (OSA) is a heterogeneous and aggressive solid tumor of the bone. We recently identified the colony stimulating factor 1 receptor (Csf1r) gene as a novel driver of osteosarcomagenesis in mice using the Sleeping Beauty (SB) transposon mutagenesis system. Here, we report that a CSF1R-CSF1 autocrine/paracrine signaling mechanism is constitutively activated in a subset of human OSA cases and is critical for promoting tumor growth and contributes to metastasis. We examined CSF1R and CSF1 expression in OSAs. We utilized gain-of-function and loss-of-function studies (GOF/LOF) to evaluate properties of cellular transformation, downstream signaling, and mechanisms of CSF1R-CSF1 action. Genetic perturbation of CSF1R in immortalized osteoblasts and human OSA cell lines significantly altered oncogenic properties, which were dependent on the CSF1R-CSF1 autocrine/paracrine signaling. These functional alterations were associated with changes in the known CSF1R downstream ERK effector pathway and mitotic cell cycle arrest. We evaluated the recently FDA-approved CSF1R inhibitor Pexidartinib (PLX3397) in OSA cell lines in vitro and in vivo in cell line and patient-derived xenografts. Pharmacological inhibition of CSF1R signaling recapitulated the in vitro genetic alterations. Moreover, in orthotopic OSA cell line and subcutaneous patient-derived xenograft (PDX)-injected mouse models, PLX3397 treatment significantly inhibited local OSA tumor growth and lessened metastatic burden. In summary, CSF1R is utilized by OSA cells to promote tumorigenesis and may represent a new molecular target for therapy., Competing Interests: Declaration of Competing Interest Dr. Largaespada is the co-founder and co-owner of several biotechnology companies including NeoClone Biotechnologies, Inc., Discovery Genomics, Inc. (recently acquired by Immunsoft, Inc.), and B-MoGen Biotechnologies, Inc. (recently acquired by bio-techne corporation). He consults for Genentech, Inc., which is funding some of his research. Dr. Largaespada holds equity in and serves as the Chief Scientific Officer of Surrogen, a subsidiary of Recombinetics, a genome-editing company. The business of all these companies is unrelated to the contents of this manuscript. Other authors have no conflicts of interest to disclose., (Copyright © 2020. Published by Elsevier Inc.)

Aromatase is a key enzyme responsible for the biosynthesis of estrogen from testosterone. Although recent evidence indicates that spinal cord aromatase participates in nociceptive processing, the mechanisms underlying its regulation and its involvement in nociception remain unclear. The present study focuses on the potential role of astrocyte aromatase in formalin-induced acute pain and begins to uncover one mechanism by which spinal aromatase activation is controlled. Following intraplantar formalin injection, nociceptive responses were quantified and immunohistochemistry/co-immunoprecipitation assays were used to investigate the changes in spinal Fos expression and the phospho-serine levels of spinal aromatase. Intrathecal (i.t.) injection of letrozole (an aromatase inhibitor) mitigated both the late phase formalin-induced nociceptive responses and formalin-induced spinal Fos expression. Furthermore, formalin-injected mice showed significantly reduced phospho-serine levels of aromatase, which is associated with the rapid activation of this enzyme. However, sigma-1 receptor inhibition with i.t. BD1047 blocked the dephosphorylation of aromatase and potentiated the pharmacological effect of letrozole on formalin-induced nociceptive responses. In addition, i.t. administration of a sub-effective dose of BD1047 potentiated the pharmacological effect of cyclosporin A (a calcineurin inhibitor) on both the formalin-induced reduction in phospho-serine levels of aromatase and nociceptive behavior. These results suggest that dephosphorylation is an important regulatory mechanism involved in the rapid activation of aromatase and that spinal sigma-1 receptors mediate this dephosphorylation of aromatase through an intrinsic calcineurin pathway., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Most acute and chronic animal models of pain rely heavily on reflexive assays for evaluating levels of nociception, which involves removing the animal from its normal social environment. Here, we examine and characterize the influence of social interactions on inflammatory pain-evoked changes in movement in two different mouse strains. To produce inflammatory nociception, we injected CFA bilaterally into the hind paws of Balb/c and C3H mice and then recorded exploratory locomotor activity using an automated detector system to first evaluate the effects of social behavior on nociception. Secondly, we determined if carprofen administration altered the effects of social behavior on nociceptive-evoked movement. This methodology was expanded to create a novel thermal activity assay to objectively measure the effect of heat and cold on CFA-evoked animal movement in paired animals. Paired Balb/c and C3H mice exhibited significant hyper-locomotion that lasted for 3h post-injection in Balb/c, but only 1h post-injection in C3H. Single Balb/c mice only showed increased activity for 1h post-injection, while single C3H mice showed no increase. This CFA-induced increase in activity in paired animals was highly inversely correlated with mechanical allodynia as measured using standard Von Frey filaments. Carprofen administration completely blocked this CFA-induced hyperlocomotor activity. Both heat and cold induced a significant increase in locomotor activity in paired mice injected with CFA, while having no effect on activity in control mice injected with saline. The results presented here indicate that social interactions greatly influence inflammatory pain-induced changes in locomotor activity and indicate that the use of movement-based assays to evaluate nociception in paired mice may provide an alternative and more sensitive method to quantify nociception and characterize novel analgesic effects over time in the context of social interactions in rodent models of pain., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The therapeutic management of chronic pain associated with many cancers is problematic due to the development of tolerance and other adverse effects during the disease progression. Recently we reported on a bivalent ligand (MMG22) containing both mu agonist and mGluR5 antagonist pharmacophores that produced potent antinociception in mice with LPS-induced acute inflammatory pain via a putative MOR-mGluR5 heteromer. In the present study we have investigated the antinociception of MMG22 in a mouse model of bone cancer pain to determine its effectiveness in reducing this type of chronic nociception. There was a 572-fold increase in the potency of MMG22 over a period of 3-21 days that correlated with the progressive increase in hyperalgesia induced by bone tumor growth following implantation of fibrosarcoma cells in mice. The enhancement of antinociception with the progression of the cancer is possibly due to inhibition of NMDA receptor-mediated hyperalgesia via antagonism of mGluR5 and concomitant activation of MOR by the MMG22-occupied heteromer. Notably, MMG22 was 3.6-million-fold more potent than morphine at PID 21. Since MMG22 exhibited a 250,000-times greater potency than that of a mixture of the mu opioid (M19) agonist and mGluR5 antagonist (MG20) monovalent ligands, the data suggest that targeting the putative MOR-mGluR5 heteromer is far superior to univalent interaction with receptors in reducing tumor-induced nociception. In view of the high potency, long duration (>24h) of action and minimal side effects, MMG22 has the potential to be a superior pharmacological agent than morphine and other opiates in the treatment of chronic cancer pain and to serve as a novel pharmacologic tool., (Copyright © 2014 Elsevier B.V. All rights reserved.)