IL-27-engineered CAR.19-NK-92 cells exhibit enhanced therapeutic efficacy.

Chimeric antigen receptor (CAR) engineering of natural killer (NK) cells has shown promising results in early-phase clinical studies. However, advancing CAR-NK cell therapeutic efficacy is imperative. In this study, we investigated the impact of a fourth-generation CD19-targeted CAR (CAR.19) coexpressing IL-27 on NK-92 cells. We observed a significant improvement in NK-92 cell proliferation and cytotoxicity activity against B-cell cancer cell lines, both in vitro and in a xenograft mouse B-cell lymphoma model. Our systematic transcriptome analysis of the activated NK-92 CAR variants further supports the potential of IL-27 in fourth-generation CARs to overcome limitations of NK cell-based targeted tumor therapies by providing essential growth and activation signals. Integrating IL-27 into CAR-NK cells emerges as a promising strategy to enhance their therapeutic potential and elicit robust responses against cancer cells. These findings contribute substantially to the mounting evidence supporting the potential of fourth-generation CAR engineering in advancing NK cell-based immunotherapies.

Evaluation of human adipose-derived mesenchymal stromal cell Toll-like receptor priming and effects on interaction with prostate cancer cells.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are a multipotent cell population of clinical interest because of their ability to migrate to injury and tumor sites, where they may participate in tissue repair and modulation of immune response. Although the processes regulating MSC function are incompletely understood, it has been shown that stimulation of Toll-like receptors (TLRs) can alter MSC activity. More specifically, it has been reported that human bone marrow-derived MSCs can be "polarized" by TLR priming into contrasting immunomodulatory functions, with opposite (supportive or suppressive) roles in tumor progression and inflammation. Adipose-derived MSCs (ASCs) represent a promising alternative MSC subpopulation for therapeutic development because of their relative ease of isolation and higher abundance compared with their bone marrow-derived counterparts; however, the polarization of ASCs remains unreported. METHODS: In this study, we evaluated the phenotypic and functional consequences of short-term, low-level stimulation of ASCs with TLR3 and TLR4 agonists. RESULTS: In these assays, we identified transient gene expression changes resembling the reported pro-inflammatory and anti-inflammatory MSC phenotypes. Furthermore, these priming strategies led to changes in the functional properties of ASCs, affecting their ability to migrate and modulate immune-mediated responses to prostate cancer cells in vitro. CONCLUSIONS: TLR3 stimulation significantly decreased ASC migration, and TLR4 stimulation increased ASC immune-mediated killing potential against prostate cancer cells.

Designing Synthetic Polymers for Nucleic Acid Complexation and Delivery: From Polyplexes to Micelleplexes to Triggered Degradation.

Gene delivery as a therapeutic tool continues to advance toward impacting human health, with several gene therapy products receiving FDA approval over the past 5 years. Despite this important progress, the safety and efficacy of gene therapy methodology requires further improvement to ensure that nucleic acid therapeutics reach the desired targets while minimizing adverse effects. Synthetic polymers offer several enticing features as nucleic acid delivery vectors due to their versatile functionalities and architectures and the ability of synthetic chemists to rapidly build large libraries of polymeric candidates equipped for DNA/RNA complexation and transport. Current synthetic designs are pursuing challenging objectives that seek to improve transfection efficiency and, at the same time, mitigate cytotoxicity. This Perspective will describe recent work in polymer-based gene complexation and delivery vectors in which cationic polyelectrolytes are modified synthetically by introduction of additional components─including hydrophobic, hydrophilic, and fluorinated units─as well as embedding of degradable linkages within the macromolecular structure. As will be seen, recent advances employing these emerging design strategies are promising with respect to their excellent biocompatibility and transfection capability, suggesting continued promise of synthetic polymer gene delivery vectors going forward.

Interleukin-27 Gene Delivery Targeting IL-6Rα-Expressing Cells as a Stress Response Therapy.

Interleukin-27 (IL-27) has shown promise in halting tumor growth and mediating tumor regression in several models, including prostate cancer. We describe our findings on the effects of IL-27 on the gene expression changes of TC2R prostate adenocarcinoma cells. We utilized RNAseq to assess profile differences between empty vector control, vector delivering IL-27 modified at its C-terminus with a non-specific peptide, and IL-27 modified at the C-terminus with a peptide targeting the IL-6-Rα. The targeted IL-27 had higher bioactivity and activity in vivo in a recent study by our group, but the mechanisms underlying this effect had not been characterized in detail at the gene expression level on tumor cells. In the present work, we sought to examine potential mechanisms for targeted IL-27 enhanced activity directly on tumor cells. The targeted IL-27 appeared to modulate several changes that would be consistent with an anti-tumor effect, including upregulation in the Interferon (IFN) and Interferon regulatory factor (IRF), oxidative phosphorylation, Janus kinase/Signal transducers and activators of transcription (JAK/STAT), and eukaryotic initiation factor 2 (EIF2) signaling. Of these signaling changes predicted by ingenuity pathway analyses (IPA), the novel form also with the highest significance (-log(Benjamini-Hochberg (B-H)) p-value) was the EIF2 signaling upregulation. We validated this predicted change by assaying for eukaryotic initiation factor 2 alpha (eIF2α), or phosphorylated eIF2α (p-eIF2α), and caspase-3 levels. We detected an increase in the phosphorylated form of eIF2α and in the cleaved caspase-3 fraction, indicating that the EIF2 signaling pathway was upregulated in these prostate tumor cells following targeted IL-27 gene delivery. This approach of targeting cytokines to enhance their activity against cancer cells is a novel approach to help augment IL-27's bioactivity and efficacy against prostate tumors and could be extended to other conditions where it could help interfere with the EIF2α pathway and promote caspase-3 activation.

Skeletal muscle signal peptide optimization for enhancing propeptide or cytokine secretion.

We have utilized hidden Markov models using HMMER software to predict and generate putative strong secretory signal peptide sequences for directing efficient secretion of cytokines from skeletal muscle for therapeutic applications. The results show that this approach can analyze signal sequences of a skeletal muscle secretome dataset and classify them, emitting new sequences that are strong candidate skeletal muscle-enriched signal peptides. The emitted signal peptides also were analyzed for their hydropathy and secondary structure profiles as compared to native signal peptides. The emitted signal peptides had a higher degree of hydropathy and helical composition relative to native sequences, which may suggest that these new sequences may hold promize for promoting enhanced secretion of proteins including cytokines or propeptides from skeletal muscle.

Histamine-induced Ca²âº signalling is mediated by TRPM4 channels in human adipose-derived stem cells.

Intracellular Ca2+ oscillations are frequently observed during stem cell differentiation, and there is evidence that it may control adipogenesis. The transient receptor potential melastatin 4 channel (TRPM4) is a key regulator of Ca2+ signals in excitable and non-excitable cells. However, its role in human adipose-derived stem cells (hASCs), in particular during adipogenesis, is unknown. We have investigated TRPM4 in hASCs and examined its impact on histamine-induced Ca2+ signalling and adipogenesis. Using reverse transcription (RT)-PCR, we have identified TRPM4 gene expression in hASCs and human adipose tissue. Electrophysiological recordings revealed currents with the characteristics of those reported for the channel. Furthermore, molecular suppression of TRPM4 with shRNA diminished the Ca2+ signals generated by histamine stimulation, mainly via histamine receptor 1 (H1) receptors. The increases in intracellular Ca2+ were due to influx via voltage-dependent Ca2+ channels (VDCCs) of the L-type (Ca(v)1.2) and release from the endoplasmic reticulum. Inhibition of TRPM4 by shRNA inhibited adipogenesis as indicated by the reduction in lipid droplet accumulation and adipocyte gene expression. These results suggest that TRPM4 is an important regulator of Ca2+ signals generated by histamine in hASCs and is required for adipogenesis.

Transient receptor potential melastatin 4 channel controls calcium signals and dental follicle stem cell differentiation.

Elevations in the intracellular Ca(2+) concentration are a phenomena commonly observed during stem cell differentiation but cease after the process is complete. The transient receptor potential melastatin 4 (TRPM4) is an ion channel that controls Ca(2+) signals in excitable and nonexcitable cells. However, its role in stem cells remains unknown. The aim of this study was to characterize TRPM4 in rat dental follicle stem cells (DFSCs) and to determine its impact on Ca(2+) signaling and the differentiation process. We identified TRPM4 gene expression in DFSCs, but not TRPM5, a closely related channel with similar function. Perfusion of cells with increasing buffered Ca(2+) resulted in a concentration-dependent activation of currents typical for TRPM4, which were also voltage-dependent and had Na(+) conductivity. Molecular suppression with shRNA decreased channel activity and cell proliferation during osteogenesis but not adipogenesis. As a result, enhanced mineralization and phosphatase enzyme activity were observed during osteoblast formation, although DFSCs failed to differentiate into adipocytes. Furthermore, the normal agonist-induced first and secondary phases of Ca(2+) signals were transformed into a gradual and sustained increase which confirmed the channels' ability to control Ca(2+) signaling. Using whole genome microarray analysis, we identified several genes impacted by TRPM4 during DFSC differentiation. These findings suggest an inhibitory role for TRPM4 on osteogenesis while it appears to be required for adipogenesis. The data also provide a potential link between the Ca(2+) signaling pattern and gene expression during stem cell differentiation.

Human adipose-derived mesenchymal stromal cell pigment epithelium-derived factor cytotherapy modifies genetic and epigenetic profiles of prostate cancer cells.

BACKGROUND AIMS: Adipose-derived mesenchymal stromal cells (ASCs) are promising tools for delivery of cytotherapy against cancer. However, ASCs can exert profound effects on biological behavior of tumor cells. Our study aimed to examine the influence of ASCs on gene expression and epigenetic methylation profiles of prostate cancer cells as well as the impact of expressing a therapeutic gene on modifying the interaction between ASCs and prostate cancer cells. METHODS: ASCs were modified by lentiviral transduction to express either green fluorescent protein as a control or pigment epithelium-derived factor (PEDF) as a therapeutic molecule. PC3 prostate cancer cells were cultured in the presence of ASC culture-conditioned media (CCM), and effects on PC3 or DU145. Ras cells were examined by means of real-time quantitative polymerase chain reaction, EpiTect methyl prostate cancer-focused real-time quantitative polymerase chain reaction arrays, and luciferase reporter assays. RESULTS: ASCs transduced with lentiviral vectors were able to mediate expression of several tumor-inhibitory genes, some of which correlated with epigenetic methylation changes on cocultured PC3 prostate cancer cells. When PC3 cells were cultured with ASC-PEDF CCM, we observed a shift in the balance of gene expression toward tumor inhibition, which suggests that PEDF reduces the potential tumor-promoting activity of unmodified ASCs. CONCLUSIONS: These results suggest that ASC-PEDF CCM can promote reprogramming of tumor cells in a paracrine manner. An improved understanding of genetic and epigenetic events in prostate cancer growth in response to PEDF paracrine therapy would enable a more effective use of ASC-PEDF, with the goal of achieving safer yet more potent anti-tumor effects.

Polymer-peptide delivery platforms: effect of oligopeptide orientation on polymer-based DNA delivery.

The success of nonviral transfection using polymers hinges on efficient nuclear uptake of nucleic acid cargo and overcoming intra- and extracellular barriers. By incorporating PKKKRKV heptapeptide pendent groups as nuclear localization signals (NLS) on a polymer backbone, we demonstrate protein expression levels higher than those obtained from JetPEI and Lipofectamine 2000, the latter being notorious for coupling high transfection efficiency with cytotoxicity. The orientation of the NLS peptide grafts markedly affected transfection performance. Polymers with the sequence attached to the backbone from the valine residue achieved a level of nuclear translocation higher than the levels of those having the NLS groups attached in the opposite orientation. The differences in nuclear localization and DNA complexation strength between the two orientations correlated with a striking difference in protein expression, both in cell culture and in vivo. Polyplexes formed from these comb polymer structures exhibited transfection efficiencies superior to those of Lipofectamine 2000 but with greatly reduced toxicity. Moreover, these novel polymers, when administered by intramuscular ultrasound-mediated delivery, allowed a high level of reporter gene expression in mice, demonstrating their therapeutic promise in vivo.

Transcriptomic analysis of mouse TRAMP cell lines and tumors provide insights into shared pathways and therapeutic targets.

The present study focused on comparing the gene expression profiles of different mouse models of prostate cancer, focusing on the TRAMP transgenic model and its derived cell lines and extending the comparisons to relevant genetically engineered mouse models and human prostate cancer datasets. Employing RNA sequencing, we examined different levels of prostate cancer aggressiveness from the original TRAMP cells to the TRAMP-C2 (TC2) derived cell line and extending to the aggressive TC2-Ras (TC2R) cells and tumors. TC2R acquire the ability to grow in bone tissue upon implantation, unlike the parental TC2 cells. Analysis identified upregulated genes in cell cycle regulation, immune response, and mitotic processes in TRAMP compared to wild-type tissues. TC2 cells exhibited unique gene profiles enriched in ECM organization and tissue development pathways, while TC2R cells showed increased cytokine signaling and motility genes, with decreased ECM and immune response pathways. In vivo TC2R models demonstrated enhanced ECM organization and receptor tyrosine kinase signaling in tumors, notably enriching immune processes and collagen degradation pathways in intratibial tumors. Comparative analysis among mouse and human datasets showed overlaps, particularly in pathways relating to mitotic cycle regulation, ECM organization, and immune interactions. A gene signature identified in TC2R tumors correlated with aggressive tumor behavior and poor survival in human datasets. Further immune cell landscape analysis of TC2R tumors revealed altered T cell subsets and macrophages, confirmed in single-cell RNA-seq from human samples. TC2R models thus hold significant promise in helping advance preclinical therapeutics, potentially contributing to improved prostate cancer patient outcomes.

Granular Hydrogels Improve Myogenic Invasion and Repair after Volumetric Muscle Loss.

Skeletal muscle injuries including volumetric muscle loss (VML) lead to excessive tissue scarring and permanent functional disability. Despite its high prevalence, there is currently no effective treatment for VML. Bioengineering interventions such as biomaterials that fill the VML defect to support cell and tissue growth are a promising therapeutic strategy. However, traditional biomaterials developed for this purpose lack the pore features needed to support cell infiltration. The present study investigates for the first time, the impact of granular hydrogels on muscle repair - hypothesizing that their flowability will permit conformable filling of the defect site and their inherent porosity will support the invasion of native myogenic cells, leading to effective muscle repair. Small and large microparticle fragments are prepared from photocurable hyaluronic acid polymer via extrusion fragmentation and facile size sorting. In assembled granular hydrogels, particle size and degree of packing significantly influence pore features, rheological behavior, and injectability. Using a mouse model of VML, it is demonstrated that, in contrast to bulk hydrogels, granular hydrogels support early-stage (satellite cell invasion) and late-stage (myofiber regeneration) muscle repair processes. Together, these results highlight the promising potential of injectable and porous granular hydrogels in supporting endogenous repair after severe muscle injury.

Interleukin-27 expression modifies prostate cancer cell crosstalk with bone and immune cells in vitro.

Prostate cancer is frequently associated with bone metastases, where the crosstalk between tumor cells and key cells of the bone microenvironment (osteoblasts, osteoclasts, immune cells) amplifies tumor growth. We have explored the potential of a novel cytokine, interleukin-27 (IL-27), for inhibiting this malignant crosstalk, and have examined the effect of autocrine IL-27 on prostate cancer cell gene expression, as well as the effect of paracrine IL-27 on gene expression in bone and T cells. In prostate tumor cells, IL-27 upregulated genes related to its signaling pathway while downregulating malignancy-related receptors and cytokine genes involved in gp130 signaling, as well as several protease genes. In both undifferentiated and differentiated osteoblasts, IL-27 modulated upregulation of genes related to its own signaling pathway as well as pro-osteogenic genes. In osteoclasts, IL-27 downregulated several genes typically involved in malignancy and also downregulated osteoclastogenesis-related genes. Furthermore, an osteogenesis-focused real-time PCR array revealed a more extensive profile of pro-osteogenic gene changes in both osteoblasts and osteoclasts. In T-lymphocyte cells, IL-27 upregulated several activation-related genes and also genes related to the IL-27 signaling pathway and downregulated several genes that could modulate osteoclastogenesis. Overall, our results suggest that IL-27 may be able to modify interactions between prostate tumor and bone microenvironment cells and thus could be used as a multifunctional therapeutic for restoring bone homeostasis while treating metastatic prostate tumors.

Cabozantinib and IL-27 combinatorial therapy for bone-metastatic prostate cancer.

Introduction: Prostate cancer is the second leading cause of cancer-related death among American men. Prostate tumor cells exhibit significant tropism for the bone and once metastasis occurs, survival rates fall significantly. Current treatment options are not curative and focus on symptom management. Immunotherapies are rapidly emerging as a possible therapeutic option for a variety of cancers including prostate cancer, however, variable patient response remains a concern. Chemotherapies, like cabozantinib, can have immune-priming effects which sensitize tumors to immunotherapies. Additionally, lower doses of chemotherapy can be used in this context which can reduce patient side effects. We hypothesized that a combination of chemotherapy (cabozantinib) and immunotherapy [Interleukin-27 (IL-27)] could be used to treat bone-metastatic prostate cancer and exert pro-osteogenic effects. IL-27 is a multi-functional cytokine, which promotes immune cell recruitment to tumors, while also promoting bone repair. Methods: To test this hypothesis, in vivo experiments were performed where syngeneic C57BL/6J mice were implanted intratibially with TRAMP-C2ras-Luc cells that are able to form tumors in bone. Immunotherapy was administered in the form of intramuscular gene therapy, delivering plasmid DNA encoding a reporter gene (Lucia), and/or a therapeutic gene (IL-27). Sonoporation was used to aid gene delivery. Following immunotherapy, the animals received either cabozantinib or a vehicle control by oral gavage. Bioluminescence imaging was used to monitor tumor size over time. Results: Combinatorial therapy inhibited tumor growth and improved survival. Further, RNA sequencing was used to investigate the mechanisms involved. Microcomputed tomography and differentiation assays indicated that the combination therapy improved bone quality by enhancing osteoblast differentiation and inhibiting osteoclast differentiation. Discussion: Our conclusion is that a chemo-immunotherapy approach such as the one examined in this work has potential to emerge as a novel therapeutic strategy for treating bone-metastatic prostate cancer. This approach will enable a significant reduction in chemotherapy-associated toxicity, enhance sensitivity to immunotherapy, and improve bone quality.

Poly I:C-priming of adipose-derived mesenchymal stromal cells promotes a pro-tumorigenic phenotype in an immunocompetent mouse model of prostate cancer.

Introduction: Mesenchymal stromal cells (MSC) are envisioned as a potential cellular vehicle for targeted cancer therapies due to their tumor tropism and immune permissiveness. An obstacle in their use is the duality in their interactions within tumors, rendering them pro-tumorigenic or anti-tumorigenic, in a context dependent manner. MSC preconditioning, or priming, has been proposed as a strategy for directing the effector properties of MSC at tumor sites. Methods: We primed human MSC derived from adipose tissues (ASC), a clinically advantageous MSC source, utilizing toll-like receptor agonists. Subsequently, we explored the consequences in tumor progression and transcriptome upon the interaction of tumor cells with primed or unprimed ASC in an in vivo model of prostate cancer, the second most common cancer and second leading cause of cancer related death in men in the USA. Results and discussion: In the studied model, poly I:C-primed ASC were found to significantly accelerate tumor growth progression. And while unprimed and LPS-primed ASC did not exert a significant effect on tumor growth at the macroscopic level, gene expression analyses suggested that all treatments promoted distinct modulatory effects in the tumor microenvironment, including altered modulation of angiogenesis, and immune response processes. However, the effects resulting from the collective interaction across these processes must be sufficiently skewed in a pro-tumorigenic or anti-tumorigenic direction for evidence of tumor progression modulation to be detectable at the macroscopic level. Our study highlights potential MSC-tumor microenvironment interactions that may be leveraged and should be considered in the development of cancer therapeutics utilizing MSC.

Is Osteopontin a Good Marker for Bone Metastasis in Canine Mammary Gland Tumor and Prostate Cancer?

Osteopontin (OPN) is a protein synthesized by a large number of cells, and its overexpression has been associated with the development and prognosis of cancer. OPN overexpression has been claimed to be a marker for the development of bone metastasis in human cancers, but no prior research has investigated the association between OPN expression and the metastasis of canine mammary gland tumors (MGTs) and prostate cancer (PC). Therefore, we investigated OPN expression in MGTs and PC samples from 50 canine patients with or without metastasis (bone vs. other sites). Higher OPN expression was detected in primary tumor samples from animals with bone metastasis than in those without bone involvement (p = 0.0321). In MGT samples, a significantly lower survival rate was observed in patients with higher OPN expression (p = 0.0171). In animals with PC, there was a strong trend toward lower survival in animals with positive OPN expression; however, this trend was not statistically significant (p = 0.0779). From these findings, it can be concluded that OPN may be a promising target for future MGTs and PC studies because of its role in enhancing cell invasion and metastasis.

Engineering NK-CAR.19 cells with the IL-15/IL-15Rα complex improved proliferation and anti-tumor effect in vivo.

Introduction: Natural killer 92 (NK-92) cells are an attractive therapeutic approach as alternative chimeric antigen receptor (CAR) carriers, different from T cells, once they can be used in the allogeneic setting. The modest in vivo outcomes observed with NK-92 cells continue to present hurdles in successfully translating NK-92 cell therapies into clinical applications. Adoptive transfer of CAR-NK-92 cells holds out the promise of therapeutic benefit at a lower rate of adverse events due to the absence of GvHD and cytokine release syndrome. However, it has not achieved breakthrough clinical results yet, and further improvement of CAR-NK-92 cells is necessary. Methods: In this study, we conducted a comparative analysis between CD19-targeted CAR (CAR.19) co-expressing IL-15 (CAR.19-IL15) with IL-15/IL-15Rα (CAR.19-IL15/IL15Rα) to promote NK cell proliferation, activation, and cytotoxic activity against B-cell leukemia. CAR constructs were cloned into lentiviral vector and transduced into NK-92 cell line. Potency of CAR-NK cells were assessed against CD19-expressing cell lines NALM-6 or Raji in vitro and in vivo in a murine model. Tumor burden was measured by bioluminescence. Results: We demonstrated that a fourth- generation CD19-targeted CAR (CAR.19) co-expressing IL-15 linked to its receptor IL-15/IL-15Rα (CAR.19-IL-15/IL-15Rα) significantly enhanced NK-92 cell proliferation, proinflammatory cytokine secretion, and cytotoxic activity against B-cell cancer cell lines in vitro and in a xenograft mouse model. Conclusion: Together with the results of the systematic analysis of the transcriptome of activated NK-92 CAR variants, this supports the notion that IL-15/IL-15Rα comprising fourth-generation CARs may overcome the limitations of NK-92 cell-based targeted tumor therapies in vivo by providing the necessary growth and activation signals.

Mutant Hras(G12V) and Kras(G12D) have overlapping, but non-identical effects on hepatocyte growth and transformation frequency in transgenic mice.

BACKGROUND: Mouse hepatocarcinogenesis is associated with mutations in Hras, but infrequently in Kras. The effect on carcinogenesis of developmental age at the time of ras mutation remains unknown. AIM: We sought to compare quantitatively the effects of expressing mutant H- or Kras genes in fetal vs. adult mouse liver. METHODS: We established an inducible system of gene expression in mouse liver to define disease pathogenesis associated with activation of oncogene expression. RESULTS: Diffuse expression of either oncogene in fetal or adult hepatocytes caused hepatomegaly. For mutant Hras(G12V), this phenotype was almost fully reversible and accompanied by apoptosis, indicating that maintenance of hepatomegaly requires continuous Hras(G12V) expression. We also examined the effect of ras expression on growth of transplanted hepatocytes in an in vivo system that allows us to quantify hepatocyte growth effects in both permissive and restrictive hepatic growth environments. Mutant Kras(G12D) had no effect on hepatocyte growth in this system. In contrast, Hras(G12V) induced increased hepatocyte focus growth in quiescent liver, the hallmark of a cell autonomous growth stimulus. Hras(G12V) also increased the fraction of donor hepatocyte foci that displayed extreme growth, a characteristic of preneoplastic lesions. CONCLUSIONS: The primary effect of diffuse, whole-liver expression of either mutant ras gene in fetal or adult mouse liver is diffuse and progressive hepatic growth. Hras(G12V) mutation influences hepatocarcinogenesis by conferring cell autonomous growth potential upon foci of expressing cells and by increasing the risk of neoplastic progression. Kras(G12D) does not share these latter carcinogenic effects in mouse liver.

Generation of the Chondroprotective Proteomes by Activating PI3K and TNFα Signaling.

PURPOSE: To develop a novel treatment option for Chondrosarcoma (CS) and inflammatory arthritis, we evaluated a counterintuitive approach of activating tumorigenic and inflammatory signaling for generating joint-protective proteomes. METHODS: We employed mesenchymal stem cells and chondrocytes to generate chondroprotective proteomes by activating PI3K signaling and the administration of TNFα. The efficacy of the proteomes was examined using human and mouse cell lines as well as a mouse model of CS. The regulatory mechanism was analyzed using mass spectrometry-based whole-genome proteomics. RESULTS: While tumor progression and inflammatory responses were promoted by activating PI3K signaling and the administration of TNFα to CS cells and chondrocytes, those cells paradoxically generated a chondroprotective conditioned medium (CM). The application of CM downregulated tumorigenic genes in CS cells and TNFα and MMP13 in chondrocytes. Mechanistically, Hsp90ab1 was enriched in the chondroprotective CM, and it immunoprecipitated GAPDH. Extracellular GAPDH interacted with L1CAM and inhibited tumorigenic behaviors, whereas intracellular GAPDH downregulated p38 and exerted anti-inflammatory effects. CONCLUSIONS: We demonstrated that the unconventional approach of activating oncogenic and inflammatory signaling can generate chondroprotective proteomes. The role of Hsp90ab1 and GAPDH differed in their locations and they acted as the uncommon protectors of the joint tissue from tumor and inflammatory responses.

Immunotherapy by mesenchymal stromal cell delivery of oncolytic viruses for treating metastatic tumors.

Oncolytic viruses (OVs) have emerged as a very promising anti-cancer therapeutic strategy in the past decades. However, despite their pre-clinical promise, many OV clinical evaluations for cancer therapy have highlighted the continued need for their improved delivery and targeting. Mesenchymal stromal cells (MSCs) have emerged as excellent candidate vehicles for the delivery of OVs due to their tumor-homing properties and low immunogenicity. MSCs can enhance OV delivery by protecting viruses from rapid clearance following administration and also by more efficiently targeting tumor sites, consequently augmenting the therapeutic potential of OVs. MSCs can function as "biological factories," enabling OV amplification within these cells to promote tumor lysis following MSC-OV arrival at the tumor site. MSC-OVs can promote enhanced safety profiles and therapeutic effects relative to OVs alone. In this review we explore the general characteristics of MSCs as delivery tools for cancer therapeutic agents. Furthermore, we discuss the potential of OVs as immune therapeutics and highlight some of the promising applications stemming from combining MSCs to achieve enhanced delivery and anti-tumor effectiveness of OVs at different pre-clinical and clinical stages. We further provide potential pitfalls of the MSC-OV platform and the strategies under development for enhancing the efficacy of these emerging therapeutics.

A Second-Generation Nanoluc-IL27 Fusion Cytokine for Targeted-Gene-Therapy Applications.

An emerging approach in treating skeletal malignancies utilizes osteoimmunology to investigate new multifunctional immune-stimulatory agents that can simultaneously combat tumor growth and promote bone repair. We have hypothesized that cytokine Interleukin-27 (IL-27) is an excellent candidate biologic to help rebalance the prostate tumor cells and bone cell environment. In this work, we examined the proof of principle for a short, secreted luciferase (Nanoluc or Nluc) fusion with IL-27 to produce a novel cytokine-based biologic (Nluc-27), whereby we examined its efficacy in vitro in reducing prostate tumor growth and rebalancing bone cell proliferation and differentiation. This work demonstrates the targeting and anti-tumor efficacy of the Nluc-27 fusion cytokine in cancer and bone cell models. The fusion cytokine is detectable in conditioned media, and bioactive in different cell systems. This novel Nluc-27 cytokine will allow flexible incorporation of other targeting domains and may serve as flexible tool to augment IL-27's bioactivity and reengineer its efficacy against prostate tumor or bone cells, and may prove applicable to several other cell types for targeted gene therapy applications.