SUMMARY Why do opiates make human beings itch ? Spinal opioid-induced itch, a prevalent side effect of pain management, has been considered to occur as a result of pain inhibition. We report that morphine-induced scratching (MIS) is abolished in mice lacking either gastrin-releasing peptide receptor (GRPR) or the μ opioid receptor (MOR). Using exon-specific knockdown, we identified the MOR1D isoform as essential for MIS, whereas MOR1 is important for morphine-induced analgesia (MIA) with no cross activity present. MOR1D and GRPR form constitutive heterodimers in the spinal cord and relay itch information upon morphine activation. Morphine induces internalization of both GRPR and MOR1D, whereas GRP induces that of GRPR but not MOR1D, when co-expressed. Moreover, GRP-induced scratching (GIS) is independent of MOR activation. These results suggest a unidirectional cross-activation of GRPR signaling by MOR1D via heterodimerization, and that opioid-induced itch is an active process concomitant with but independent of opioid analgesia.

Bone metastases occur in ~70% of metastatic breast cancer patients often leading to skeletal injuries. Current treatments are mainly palliative and underscore the unmet clinical need for improved therapies. In this study, we provide preclinical evidence for an antimetastatic therapy based on targeting integrin β3 (β3) which is selectively induced on breast cancer cells in bone by the local bone microenvironment. In a preclinical model of breast cancer, β3 was strongly expressed on bone metastatic cancer cells but not primary mammary tumors or visceral metastases. In tumor tissue from breast cancer patients, β3 was significantly elevated on bone metastases relative to primary tumors from the same patient (n=42). Mechanistic investigations revealed that TGF-β signaling through SMAD2/SMAD3 was necessary for breast cancer induction of β3 within the bone. Using a micelle-based nanoparticle therapy that recognizes integrin αvβ3 (αvβ3-MPs of ~12.5nm), we demonstrated specific localization to breast cancer bone metastases in mice. Using this system for targeted delivery of the chemotherapeutic docetaxel, we showed that bone tumor burden could be reduced significantly with less bone destruction and less hepatotoxicity compared to equimolar doses of free docetaxel. Furthermore, mice treated with αvβ3-MP-docetaxel exhibited a significant decrease in bone-residing tumor cell proliferation compared to free docetaxel. Taken together, our results offer preclinical proof of concept for a method to enhance delivery of chemotherapeutics to breast cancer cells within the bone by exploiting their selective expression of integrin αvβ3 at that metastatic site.

Integrin beta3 is critical for tumor invasion, neoangiogenesis, and inflammation making it a promising cancer target. However, preclinical and clinical data of integrin beta3 antagonists have demonstrated no benefit or worse outcomes. We hypothesized that integrin beta3 could affect tumor immunity and evaluated tumors in mice integrin beta3 could affect tumor immunity and evaluated tumors in mice with deletion of integrin beta3 in macrophage lineage cells (β3KOM). β3KOM mice had increased melanoma and breast cancer growth with increased tumor-promoting M2 macrophages and decreased CD8+ T-cells. Integrin beta3 antagonist, cilengitide, also enhanced tumor growth and increased M2 function. We uncovered a negative feedback loop in M2 myeloid cells wherein integrin beta3 signaling favored STAT1 activation, an M1 polarizing signal, and suppressed M2 polarizing STAT6 activation. Finally, disruption of CD8+ T-cells, macrophages, or macrophage integrin beta3 signaling blocked the tumor-promoting effects of integrin beta3 antagonism. These results suggest that effects of integrin beta3 therapies on immune cells should be considered to improve outcomes.

Testes of sexually mature, as well as newborn and young mice of varying ages were studied by electron microscopy. The seminiferous tubules in the mature mouse possess a single cell layer of extremely flattened cells which form a sheath-like structure around the epithelium of the tubule. These peritubular cells are characterized by cytoplasmic filaments and other features which are typical of smooth muscle cells. A basement lamina is associated with the interstitial or peripheral surface of the cell. Peripherally, there is a n additional cellular layer consisting of connective tissue fibrocytes. In newborn animals, the cells surrounding the tubule epithelium consist of a homogeneous population of fibroblasts, 3-4 layers in thickness. With growth and development of the testes the number of cell layers is reduced and the cells become more attenuated. At 13 days, those cells which are closest to the epithelium show localized aggregates of fine filaments, as well as what appears to be the elaboration of a basement lamina. By 17 days, the cytoplasmic filaments are more numerous and the basement lamina is well defined; by 19 days, the cells closely resemble the peritubular muscle cells of the adult.The probable functional role of these cells is discussed with respect to both sperm transport and the production and maintenance of the surrounding connective tissue stroma.

To further characterize Sertoli cell-germ cell junctional specializations seminiferous tubules from sexually mature Sprague-Dawley rats were dissociated by enzymatic and mechanical methods. Ultrastructural analysis of cell suspensions prepared by incubation in collagenase alone or by mechanical methods revealed that spermatids remained attached to Sertoli cells or Sertoli cell fragments. Such cellular associations were found only between Sertoli cell fragments and spematids in which the developing acrosome had made contact with the plasma membrane (step 8 and subsequent steps of spermiogenesis). Furthermore, the fragments were confined to that region of the plasma membrane over the acrosome. The Sertoli cell half of this adhesive site displayed the typical elements of Sertoli cell junctions, filamentous bundles and associated cisterna of endoplasmic reticulum, in apposition to the spermatids. The spermatids demonstrated no surface specializations at the attachment sites. In contrast, in cell suspensions prepared with trypsin, spermatids were free of attachments to Sertoli cells or their fragments. These results demonstrate that: (1) the junctions act to bind cells together, (2) adhesive type contact is established between Sertoli cells and spermatids at step 8 and subsequent steps of spermiogenesis, (3) contact is restricted to the spermatid plasma membrane over the acrosome, and (4) spermatids can be freed from the junctional specializations by treatment with trypsin.

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Electron microscopic study of the peritubular connective tissue in human testis reveals the presence of "contractile-type" cells rather than of typical fibrocytes. Their cytoplasm has numerous fine filaments and other components. characteristic of smooth muscle cells. The rough-surfaced endoplasmic reticulum, however, is relatively prominent In some instances, the nuclear surface appears scalloped or folded, and the cell surface presents an irregular profile, similar to that of contracted cells.

The role of the stromal compartment in tumor progression is best illustrated in breast cancer bone metastases, where the stromal compartment supports tumor growth, albeit through poorly defined mechanisms. p38MAPKα is frequently expressed in tumor cells and surrounding stromal cells, and its expression levels correlate with poor prognosis. This observation led us to investigate whether inhibition of p38MAPKα could reduce breast cancer metastases in a clinically relevant model. Orally administered, small-molecule inhibitors of p38MAPKα or its downstream kinase MK2 each limited outgrowth of metastatic breast cancer cells in the bone and visceral organs. This effect was primarily mediated by inhibition of the p38MAPKα pathway within the stromal compartment. Beyond effectively limiting metastatic tumor growth, these inhibitors reduced tumor-associated and chemotherapy-induced bone loss, which is a devastating comorbidity that drastically affects quality of life for patients with cancer. These data underscore the vital role played by stromal-derived factors in tumor progression and identify the p38MAPK-MK2 pathway as a promising therapeutic target for metastatic disease and prevention of tumor-induced bone loss. Pharmacologically targeting the stromal p38MAPK-MK2 pathway limits metastatic breast cancer growth, preserves bone quality, and extends survival. .