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.

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.

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.

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.

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.

Correction: Sun et al. Generation of the Chondroprotective Proteomes by Activating PI3K and TNFα Signaling. Cancers 2022, 14, 3039.

In the original article [...].

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.

Enhancing Prednisone-Based Arthritis Therapy with Targeted IL-27 Gene Delivery.

Rheumatoid arthritis (RA) is a chronic autoimmune disease which is characterized primarily by synovial hyperplasia and accumulation of several types of immune infiltrates that promote progressive destruction of the articular structure. Glucocorticoids are often prescribed to treat RA because of their strong anti-inflammatory and immunosuppressive effects. However, their application must be limited to the short-term due to a risk of adverse events. In the present study, we examined the potential combination of low-dose prednisone with gene delivery of an agent of promising and complementary effectiveness in RA, interleukin (IL)-27. IL-27 has been shown to have anti-inflammatory potential, while also acting as an effective bone-normalization agent in prior reports. The present report examined a version of IL-27 targeted at the C-terminus with a short 'peptide L' (pepL, LSLITRL) that binds the interleukin 6 receptor α (IL-6Rα) upregulated during inflammation. By focusing on this targeted form, IL-27pepL or 27pL, we examined whether the anti-inflammatory potential of prednisone (at a relatively low dose and short duration) could be further enhanced in the presence of 27pL as a therapy adjuvant. Our results indicate that 27pL represents a novel tool for use as an adjuvant with current therapeutics, such as prednisone, against inflammatory conditions.

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.

Reengineering Tumor Microenvironment with Sequential Interleukin Delivery.

Some cytokines can reengineer anti-tumor immunity to modify the tumor micro-environment. Interleukin-27 (IL-27) can partially reduce tumor growth in several animal models, including prostate cancer. We hypothesized that addition of IL-18, which can induce the proliferation of several immune effector cells through inducing IFNγ could synergize with IL-27 to enhance tumor growth control. We describe our findings on the effects of IL-27 gene delivery on prostate cancer cells and how sequential therapy with IL-18 enhanced the efficacy of IL-27. The combination of IL-27 followed by IL-18 (27→18) successfully reduced cancer cell viability, with significant effects in cell culture and in an immunocompetent mouse model. We also examined a novel chimeric cytokine, comprising an IL-27 targeted at the C-terminus with a short peptide, LSLITRL (27pepL). This novel cytokine targets a receptor upregulated in tumor cells (IL-6Rα) via the pepL ligand. Interestingly, when we compared the 27→18 combination with the single 27pepL therapy, we observed a similar efficacy for both. This efficacy was further enhanced when 27pepL was sequenced with IL-18 (27pepL→18). The observed reduction in tumor growth and significantly enriched canonical pathways and upstream regulators, as well as specific immune effector signatures (as determined by bioinformatics analyses in the tumor microenvironment) supported the therapeutic design, whereby IL-27 or 27pepL can be more effective when delivered with IL-18. This cytokine sequencing approach allows flexible incorporation of both gene delivery and recombinant cytokines as tools to augment IL-27's bioactivity and reengineer efficacy against prostate tumors and may prove applicable in other therapeutic settings.

Sonodelivery in Skeletal Muscle: Current Approaches and Future Potential.

There are currently multiple approaches to facilitate gene therapy via intramuscular gene delivery, such as electroporation, viral delivery, or direct DNA injection with or without polymeric carriers. Each of these methods has benefits, but each method also has shortcomings preventing it from being established as the ideal technique. A promising method, ultrasound-mediated gene delivery (or sonodelivery) is inexpensive, widely available, reusable, minimally invasive, and safe. Hurdles to utilizing sonodelivery include choosing from a large variety of conditions, which are often dependent on the equipment and/or research group, and moderate transfection efficiencies when compared to some other gene delivery methods. In this review, we provide a comprehensive look at the breadth of sonodelivery techniques for intramuscular gene delivery and suggest future directions for this continuously evolving field.

Ligand-Mediated Targeting of Cytokine Interleukin-27 Enhances Its Bioactivity In Vivo.

We have examined the role of a novel targeted cytokine, interleukin-27 (IL-27), modified at the C terminus with a dual targeting and therapeutic heptapeptide, in treating prostate cancer. IL-27 has shown promise in halting tumor growth and mediating tumor regression in several cancer models, including prostate cancer. We describe our findings on the effects of targeted IL-27 gene delivery on prostate cancer cells in vitro and in vivo and how the targeting enhances bioactivity of the IL-27 cytokine. We applied the IL-27 gene delivery protocol utilizing sonoporation (sonodelivery) with the goal of reducing prostate tumor growth in an immunocompetent TC2R C57/BL6 model. The reduction in tumor growth and effector cellular profiles implicate targeted IL-27 as more effective than an untargeted version of IL-27 in promoting bioactivity, as assessed by STAT1 and IFN-γ reporter genes. Moreover, enhanced antitumor effects and significantly higher accumulation of natural killer T (NKT) and CD8 effector cells in the tumors were observed. These results support a novel IL-27-based targeting strategy that is promising since it shows improved therapeutic efficacy while utilizing simple and effective sonodelivery methods.

Polymer-mediated gene therapy: Recent advances and merging of delivery techniques.

The ability to safely and precisely deliver genetic materials to target sites in complex biological environments is vital to the success of gene therapy. Numerous viral and nonviral vectors have been developed and evaluated for their safety and efficacy. This study will feature progress in synthetic polymers as nonviral vectors, which benefit from their chemical versatility, biocompatibility, and ability to carry both therapeutic cargo and targeting moieties. The combination of synthetic gene carrying constructs with advanced delivery techniques promises new therapeutic options for treating and curing genetic disorders. This article is characterized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies.

Lysines residing in putative Small Ubiquitin-like MOdifier (SUMO) motifs regulate fate and function of 37 KDa laminin receptor.

There is a putative precursor to mature receptor relationship between 37 Laminin Receptor (LR) and 67 LR. As such, the pair are frequently referred to as a single entity, the 37/67 kDa Laminin Receptor (37/67 LR) and 67 LR was identified as a laminin binding entity. 37/67 LR has been of clinical interest for many years, as 37/67 LR is a prognostic indicator for many cancers including breast, lung, colon, and prostate. However, the genesis of 67 LR is controversial, and confounded by its stability under SDS-PAGE conditions, a lack of splice variants, and the existence of post-translational modifications that cannot account for the mass discrepancy between 37 and 67 LR. In the present work, we mutated potential SUMO motif sites (Lysine residues) in 37 LR and generated a series of 37 LR-expressing plasmids with a C-terminal histidine tag. We report an inability to detect 67 LR formation, suggesting that SUMOylation does not appear to directly occur at the lysine residues proposed. However, the work revealed that these lysine mutations still appear to be important and can impact the fate and function of 37 LR, by impairing half-life and steady state pre-mRNA levels. These results suggest that the Lys residues within putative SUMO motifs of 37 LR are important for 37 LR function.

A dock derived compound against laminin receptor (37 LR) exhibits anti-cancer properties in a prostate cancer cell line model.

Laminin receptor (67 LR) is a 67 kDa protein derived from a 37 kDa precursor (37 LR). 37/67 LR is a strong clinical correlate for progression, aggression, and chemotherapeutic relapse of several cancers including breast, prostate, and colon. The ability of 37/67 LR to promote cancer cell aggressiveness is further increased by its ability to transduce physiochemical and mechanosensing signals in endothelial cells and modulate angiogenesis. Recently, it was demonstrated that 37/67 LR modulates the anti-angiogenic potential of the secreted glycoprotein pigment epithelium-derived factor (PEDF). Restoration of PEDF balance is a desirable therapeutic outcome, and we sought to identify a small molecule that could recapitulate known signaling properties of PEDF but without the additional complications of peptide formulation or gene delivery safety validation. We used an in silico drug discovery approach to target the interaction interface between PEDF and 37 LR. Following cell based counter screening and binding validation, we characterized a hit compound's anti-viability, activation of PEDF signaling-related genes, anti-wound healing, and anti-cancer signaling properties. This hit compound has potential for future development as a lead compound for treating tumor growth and inhibiting angiogenesis.

Strategies for reducing inflammation and promoting bone repair in arthritis.

Chronic forms of arthritis encompass many joint inflammatory disorders, including rheumatoid arthritis (RA), an autoimmune inflammatory disease, and osteoarthritis (OA), typically a 'wear and tear' condition that is now known to also have an inflammatory etiology. The impact of inflammation in the disease prognosis and joint degradation due to impaired repair mechanisms has long been recognized for RA, and now also for OA. Both forms of arthritis are prevalent chronic health conditions, and despite recent advances, their treatment still represents an unmet medical need because of safety and efficacy concerns with currently prescribed drugs. There is an urgent need to develop and test new drugs that selectively target inflamed joints and to control articular inflammation while preventing healthy tissue damage. The therapeutics developed for RA might be useful for OA, since studies in humans and animal models demonstrate a key role for chronic, low-grade inflammation in the pathogenesis of OA. In this review, we discuss current and emerging new therapies for management of inflammation and promotion of cartilage and/or bone repair in RA and OA.

The Immunomodulatory Effects of Mesenchymal Stem Cell Polarization within the Tumor Microenvironment Niche.

Mesenchymal stem cells (MSCs) represent a promising tool for cell therapy, particularly for their antitumor effects. This cell population can be isolated from multiple tissue sources and also display an innate ability to home to areas of inflammation, such as tumors. Upon entry into the tumor microenvironment niche, MSCs promote or inhibit tumor progression by various mechanisms, largely through the release of soluble factors. These factors can be immunomodulatory by activating or inhibiting both the adaptive and innate immune responses. The mechanisms by which MSCs modulate the immune response are not well understood. Because of this, the relationship between MSCs and immune cells within the tumor microenvironment niche continues to be an active area of research in order to help explain the apparent contradictory findings currently available in the literature. The ongoing research aims to enhance the potential of MSCs in future therapeutic applications.

Combination of Interleukin-27 and MicroRNA for Enhancing Expression of Anti-Inflammatory and Proosteogenic Genes.

Remission of inflammation has become an achievable goal in inflammatory or rheumatoid arthritis (RA); however, bone erosion continues in many patients. Interleukin- (IL-) 27 regulates immune and bone cell balance and also suppresses activities of several inflammatory cell types in RA. Despite its promise, challenges to clinical translation of IL-27 have been its partial effects in vivo. Due to their ability to modulate plasticity of bone and immune cell differentiation, we examined the potential for several microRNA (miR) candidates in enhancing the effects of IL-27. Using differentiation, luciferase, and real time quantitative PCR assays, we show that IL-27 promotes osteoblast differentiation, reduces expression of osteoblast inhibitory genes, and reduces osteoclast differentiation, and results suggest a potential coordination with TGFß/BMP/SMAD and JAK/STAT pathways. We selected miRNA regulators of these and related pathways to examine whether the effects of IL-27 could be augmented for therapeutic applications. miR-29b and miR-21 augmented IL-27 proosteogenic while downregulating osteoclastogenic signals and also worked to reduce inflammatory signaling in activated macrophages, while miR-21 and miR-20b worked with IL-27 to reduce inflammatory gene expression in fibroblasts and T cells. It appears that several miRNAs can be utilized to enhance IL-27's impact on modulating osteogenesis and reducing proinflammatory signaling.

In Vivo Exposure to Inorganic Arsenic Alters Differentiation-Specific Gene Expression of Adipose-Derived Mesenchymal Stem/Stromal Cells in C57BL/6J Mouse Model.

The number of mesenchymal stem cell (MSC) therapeutic modalities has grown in recent years. Adipose-derived mesenchymal stem/stromal cells (ASCs) can be isolated and expanded relatively easily as compared with their bone-marrow counterparts, making them a particularly promising source of MSCs. And although the biological mechanisms surrounding ASCs are actively being investigated, little is known about the effects that in vivo environmental exposures might have on their ability to properly differentiate. Therefore, we hypothesized that ASCs isolated from mice exposed to inorganic arsenic (iAs) would have an altered response towards adipogenic, osteogenic, and/or chondrogenic differentiation. To test this hypothesis, C57BL/6J male mice were provided drinking water containing 0, 300, or 1000 ppb iAs. ASCs were then isolated and differentiated, which was assessed by immunocytochemistry and real-time quantitative PCR (RT-qPCR). Our results showed that total urinary arsenic equilibrated within 1 week of exposure to iAs and was maintained throughout the study. ASCs isolated from each exposure group maintained differentiation capabilities for each lineage. The magnitude of differentiation-specific gene expression, however, appeared to be concentration dependent. For osteogenesis and chondrogenesis, differentiation-specific gene expression decreased, whereas adipogenesis showed a biphasic response with an initial decrease followed by an increase in adipogenic-related gene expression following iAs exposure. These results suggest that the level in which differentiation-specific genes are induced within these stromal cells might be sensitive to environmental contaminants. These findings highlight the need to take into account potential environmental exposures prior to selecting stromal cell donors, so ASCs can achieve optimal efficiency in regenerative therapy applications.