Prostate cancer (PCa) is the second leading cause of cancer deaths among American men. Unfortunately, there is no cure once the tumor is established within the bone niche. Although osteocytes are master regulators of bone homeostasis and remodeling, their role in supporting PCa metastases remains poorly defined. This is largely due to a lack of suitable ex vivo models capable of recapitulating the physiological behavior of primary osteocytes. To address this need, we integrated an engineered bone tissue model formed by 3D-networked primary human osteocytes, with conditionally reprogrammed (CR) primary human PCa cells. CR PCa cells induced a significant increase in the expression of fibroblast growth factor 23 (FGF23) by osteocytes. The expression of the Wnt inhibitors sclerostin and dickkopf-1 (Dkk-1), exhibited contrasting trends, where sclerostin decreased while Dkk-1 increased. Furthermore, alkaline phosphatase (ALP) was induced with a concomitant increase in mineralization, consistent with the predominantly osteoblastic PCa-bone metastasis niche seen in patients. Lastly, we confirmed that traditional 2D culture failed to reproduce these key responses, making the use of our ex vivo engineered human 3D bone tissue an ideal platform for modeling PCa-bone interactions.

In the current study we evaluated the effects of immunoproteasome inhibition using ONX 0914 (formerly PR-957) to ameliorate graft-versus-host disease (GVHD). ONX 0914, an LMP7-selective epoxyketone inhibitor of the immunoproteasome, has been shown to reduce cytokine production in activated monocytes and T cells and attenuate disease progression in mouse models of rheumatoid arthritis, colitis, systemic lupus erythematosus, and, more recently, encephalomyelitis. Inhibition of LMP7 with ONX 0914 in the B10.BR→CBA MHC-matched/minor histocompatibility antigen (miHA)-disparate murine blood and marrow transplant (BMT) model caused a modest but significant improvement in the survival of mice experiencing GVHD. Concomitant with these results, in vitro mixed lymphocyte cultures revealed that stimulator splenocytes, but not responder T cells, treated with ONX 0914 resulted in decreased IFN-γ production by allogeneic T cells in both MHC-disparate (B10.BR anti-B6) and miHA-mismatched (B10.BR anti-CBA) settings. In addition, a reduction in the expression of the MHC class I-restricted SIINFEKL peptide was observed in splenocytes from transgenic C57BL/6-Tg(CAG-OVA)916Jen/J mice exposed to ONX 0914. Taken together, these data support that LMP7 inhibition in the context of BMT modulates allogeneic responses by decreasing endogenous miHA presentation and that the consequential reduction in allogeneic stimulation and cytokine production reduces GVHD development.

De novo cancer‐targeting immunostimulatory peptides have been designed and developed as synthetic antibody mimics. A series of bifunctional peptides incorporating NKp30‐binding and NK‐cell‐activating domains were synthesized as linear dimers and then extended into branching trimeric peptides by the incorporation of GRP78‐targeting and tumor‐cell‐binding sequences. A selected trimeric peptide from this small set of peptides displayed binding capabilities on GRP78+ HepG2 and A549 target cells. Cell binding diminished in the presence of an anti‐GRP78 peptide blocker, thus suggesting GRP78‐binding dependence. Similarly, the selected trimeric peptide was also found to exhibit NK cell binding in an NKp30‐dependent manner, which translated into NK cell activation as indicated by cytokine secretion. In co‐culture, fluorescence microscopy revealed that the target GFP‐expressing A549 cells were visibly associated with the effector NK cells when pre‐activated with lead trimeric peptide. Accordingly, A549 cells were found to be compromised, as evidenced by the loss of GFP signal and notable detection of early‐/late‐stage apoptosis. Investigation of the immunological markers related to toxicity revealed detectable secretion of pro‐inflammatory cytokines and chemokines, including IFN‐γ, TNF‐α, and IL‐8. Furthermore, administration of peptide‐activated NK cells into A549‐tumor‐bearing mice resulted in a consistent decrease in tumor growth when compared to the untreated control group. Taken together, the identification of a lead trimeric peptide capable of targeting and activating NK cells’ immunotoxicity directly towards GRP78+/B7H6‐ tumors provides a novel proof‐of‐concept for the development of cancer‐targeting immunostimulatory peptide ligands that mimic antibody‐targeting and ‐activating functions related to cancer immunotherapy applications.