Growth Factor Immobilization to Synthetic Hydrogels: Bioactive bFGF-Functionalized Polyisocyanide Hydrogels.

With its involvement in cell proliferation, migration and differentiation basic fibroblast growth factor (bFGF) has great potential for tissue engineering purposes. So far, however, clinical translation of soluble bFGF-based therapies is unsuccessful, because the required effective doses are often supraphysiological, which may cause adverse effects. An effective solution is growth factor immobilization, whereby bFGF retains its bioactivity at increased efficacy. Studied carriers include films, solid scaffolds, and particles, as well as natural and synthetic hydrogels. However, these synthetic hydrogels poorly resemble the characteristics of the native extracellular matrix (ECM). In this work, bFGF is covalently conjugated to the synthetic, but highly biocompatible, polyisocyanide-based hydrogel (PIC-bFGF), which closely mimics the architecture and mechanical properties of the ECM. The growth factor conjugation protocol is straightforward and readily extrapolated to other growth factors or proteins. The PIC-bFGF hydrogel shows a prolonged bioactivity up to 4 weeks although no clear effects on the ECM metabolism are observed. Beyond the future potential of the PIC-bFGF hydrogel toward various tissue engineering applications, this work underlines that simple biological conjugation procedures are a powerful strategy to induce additional bioactivity in 3D synthetic cell culture matrices.

Synthetic fibrous hydrogels as a platform to decipher cell-matrix mechanical interactions.

Cells continuously sense external forces from their microenvironment, the extracellular matrix (ECM). In turn, they generate contractile forces, which stiffen and remodel this matrix. Although this bidirectional mechanical exchange is crucial for many cell functions, it remains poorly understood. Key challenges are that the majority of available matrices for such studies, either natural or synthetic, are difficult to control or lack biological relevance. Here, we use a synthetic, yet highly biomimetic hydrogel based on polyisocyanide (PIC) polymers to investigate the effects of the fibrous architecture and the nonlinear mechanics on cell-matrix interactions. Live-cell rheology was combined with advanced microscopy-based approaches to understand the mechanisms behind cell-induced matrix stiffening and plastic remodeling. We demonstrate how cell-mediated fiber remodeling and the propagation of fiber displacements are modulated by adjusting the biological and mechanical properties of this material. Moreover, we validate the biological relevance of our results by demonstrating that cellular tractions in PIC gels develop analogously to those in the natural ECM. This study highlights the potential of PIC gels to disentangle complex bidirectional cell-matrix interactions and to improve the design of materials for mechanobiology studies.

Comparison of Osteogenic Capacity and Osteoinduction of Adipose Tissue-Derived Cell Populations.

Stromal vascular fraction (SVF) is the primary isolate obtained after enzymatic digestion of adipose tissue that contains various cell types. Its successful application for cell-based construct preparation in an intra-operative setting for clinical bone augmentation and regeneration has been previously reported. However, the performance of SVF-based constructs compared with traditional ex vivo expanded adipose tissue-derived mesenchymal stromal cells (ATMSCs) remains unclear and direct comparative analyses are scarce. Consequently, we here aimed at comparing the in vitro osteogenic differentiation capacity of donor-matched SVF versus ATMSCs as well as their osteoinductive capacity. Human adipose tissue from nine different donors was used to isolate SVF, which was further purified via plastic-adherence to obtain donor-matched ATMSCs. Both cell populations were immunophenotypically characterized for mesenchymal stromal cell, endothelial, and hematopoietic markers after isolation and immunocytochemical staining was used to identify different cell types during prolonged cell culture. Based on normalization using plastic-adherence fraction determination, SVF and ATMSCs were seeded and cultured in osteogenic differentiation medium for 28 days. Further, SVF and ATMSCs were seeded onto devitalized bovine bone granules and subcutaneously implanted into nude mice. After 42 days of implantation, granules were retrieved, histologically processed, and stained with hematoxylin and eosin (HE) to assess ectopic bone formation. The ATMSCs were shown to be a homogenous cell population during cell culture, whereas SVF cultures consisted of multiple cell types. All donor-matched comparisons showed either accelerated or stronger mineralization for SVF cultures in vitro. However, neither SVF nor ATMSCs loaded on devitalized bone granules induced ectopic bone formation on subcutaneous implantation, as opposed to control granules loaded with bone morphogenetic protein-2 (BMP-2), which triggered ectopic bone formation with 100% incidence. Despite the observed lack of osteoinduction, our findings provide important in vitro evidence on the osteogenic superiority of intra-operatively available SVF as compared with donor-matched ATMSCs. Consequently, further studies should focus on optimizing the efficacy of these cell populations for implementation in orthotopic bone fracture or defect treatment.

Molecular Landscape of Pelvic Organ Prolapse Provides Insights into Disease Etiology.

Pelvic organ prolapse (POP) represents a major health care burden in women, but its underlying pathophysiological mechanisms have not been elucidated. We first used a case-control design to perform an exome chip study in 526 women with POP and 960 control women to identify single nucleotide variants (SNVs) associated with the disease. We then integrated the functional interactions between the POP candidate proteins derived from the exome chip study and other POP candidate molecules into a molecular landscape. We found significant associations between POP and SNVs in 54 genes. The proteins encoded by 26 of these genes fit into the molecular landscape, together with 43 other POP candidate molecules. The POP landscape is located in and around epithelial cells and fibroblasts of the urogenital tract and harbors four interacting biological processes-epithelial-mesenchymal transition, immune response, modulation of the extracellular matrix, and fibroblast function-that are regulated by sex hormones and TGFB1. Our findings were corroborated by enrichment analyses of differential gene expression data from an independent POP cohort. Lastly, based on the landscape and using vaginal fibroblasts from women with POP, we predicted and showed that metformin alters gene expression in these fibroblasts in a beneficial direction. In conclusion, our integrated molecular landscape of POP provides insights into the biological processes underlying the disease and clues towards novel treatments.

The Effect of Growth Factors on Vaginal Wound Healing: A Systematic Review and Meta-analysis.

Surgical outcomes of pelvic organ prolapse (POP) surgery are poor, resulting in a 20% recurrence risk. Following the hypothesis that impaired wound healing is the main determinant of recurrent POP, growth factors have the potential to promote wound healing and may improve surgical outcomes. In this study, we systematically reviewed the effect of growth factors on vaginal wound healing in both in vitro and animal studies. For each independent comparison, the standardized mean difference and 95% CI were calculated using the Hedges' g correction. Of the 3858 retrieved studies, seven studies were included, of which six were included in meta-analysis (three in vitro studies and four in vivo studies). In vitro, basic fibroblast growth factor (bFGF) promotes proliferation, differentiation, and collagen types I and III production. Epidermal growth factor stimulates proliferation and connective tissue growth factor promotes Tenascin-C expression. These effects, however, are less pronounced in vivo; only bFGF slightly promotes collagen production. The review shows that growth factors, particularly bFGF, are able to promote vaginal wound healing in vitro. The uncertain in vivo findings suggest that preclinical models should be improved. The ultimate goal is to develop effective growth factor-supplemented therapies that improve surgical outcomes for POP.

Intravesical BCG in patients with non-muscle invasive bladder cancer induces trained immunity and decreases respiratory infections.

BACKGROUND: BCG is recommended as intravesical immunotherapy to reduce the risk of tumor recurrence in patients with non-muscle invasive bladder cancer (NMIBC). Currently, it is unknown whether intravesical BCG application induces trained immunity. METHODS: The aim of this research was to determine whether BCG immunotherapy induces trained immunity in NMIBC patients. We conducted a prospective observational cohort study in 17 NMIBC patients scheduled for BCG therapy and measured trained immunity parameters at 9 time points before and during a 1-year BCG maintenance regimen. Ex vivo cytokine production by peripheral blood mononuclear cells, epigenetic modifications, and changes in the monocyte transcriptome were measured. The frequency of respiratory infections was investigated in two larger cohorts of BCG-treated and non-BCG treated NMIBC patients as a surrogate measurement of trained immunity. Gene-based association analysis of genetic variants in candidate trained immunity genes and their association with recurrence-free survival and progression-free survival after BCG therapy was performed to investigate the hypothesized link between trained immunity and clinical response. RESULTS: We found that intravesical BCG does induce trained immunity based on an increased production of TNF and IL-1ß after heterologous ex vivo stimulation of circulating monocytes 6-12 weeks after intravesical BCG treatment; and a 37% decreased risk (OR 0.63 (95% CI 0.40 to 1.01)) for respiratory infections in BCG-treated versus non-BCG-treated NMIBC patients. An epigenomics approach combining chromatin immuno precipitation-sequencing and RNA-sequencing with in vitro trained immunity experiments identified enhanced inflammasome activity in BCG-treated individuals. Finally, germline variation in genes that affect trained immunity was associated with recurrence and progression after BCG therapy in NMIBC. CONCLUSION: We conclude that BCG immunotherapy induces trained immunity in NMIBC patients and this may account for the protective effects against respiratory infections. The data of our gene-based association analysis suggest that a link between trained immunity and oncological outcome may exist. Future studies should further investigate how trained immunity affects the antitumor immune responses in BCG-treated NMIBC patients.

Combination of sunitinib and 177Lu-labeled antibody cG250 targeted radioimmunotherapy: A promising new therapeutic strategy for patients with advanced renal cell cancer.

Sunitinib is an effective treatment for patients with metastatic Renal Cell Carcinoma (mRCC) but ultimately resistance occurs. The aim of this study was to investigate sunitinib resistance in RCCs and to develop therapeutic combination strategies with targeted radioimmunotherapy (RIT). We studied two RCC models, analyzed Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) and AXL/MET expression and performed therapy studies in Balb/cnu/nu mice combining sunitinib and [177Lu]Lu-cG250 RIT (6.5 MBq/10 µg), specifically targeting RCC cells. pAXL and pMET were expressed in sunitinib-resistant SK-RC-52 and absent in sunitinib-sensitive NU12. NGS evaluation showed that expression of VEGFA, VEGFB, VEGFD, PGF and VEGFR1,2,3 was higher and expression of VEGFC and PDGFA was lower in NU12 than in SK-RC-52. Therapy studies combining sunitinib with [177Lu]Lu-cG250 RIT showed that the best response in mice with "resistant" SK-RC-52 tumors was observed with two cycles of Sunitinib and [177Lu]Lu-cG250 RIT, probably due to increased vascular permeability by sunitinib treatment. In the "sensitive" NU12 model, two cycles of [177Lu]Lu-cG250 RIT and two cycles of combination treatment were equally effective. Enhanced therapeutic efficacy was achieved when two agents ([177Lu]Lu-cG250 RIT and sunitinib) that on their own did not induce satisfactory response levels, are combined. Our findings provide a promising new therapeutic strategy for patients with advanced RCC.

Genome-Wide Meta-Analysis Identifies Variants in DSCAM and PDLIM3 That Correlate with Efficacy Outcomes in Metastatic Renal Cell Carcinoma Patients Treated with Sunitinib.

Individual response to sunitinib in metastatic renal cell carcinoma (mRCC) patients is highly variable. Earlier, sunitinib outcome was related to single nucleotide polymorphisms (SNPs) in CYP3A5 and ABCB1. Our aim is to provide novel insights into biological mechanisms underlying sunitinib action. We included mRCC patients from the European EuroTARGET consortium (n = 550) and the RIKEN cohort in Japan (n = 204) which were analysed separately and in a meta-analysis of genome-wide association studies (GWAS). SNPs were tested for association with progression-free survival (PFS) and overall survival (OS) using Cox regression. Summary statistics were combined using a fixed effect meta-analysis. SNP rs28520013 in PDLIM3 and the correlated SNPs rs2205096 and rs111356738 both in DSCAM, showed genome-wide significance (p < 5 × 10−8) with PFS and OS in the meta-analysis. The variant T-allele of rs28520013 associated with an inferior PFS of 5.1 months compared to 12.5 months in non-carriers (p = 4.02 × 10−10, HR = 7.26). T-allele carriers of rs28520013 showed an inferior OS of 6.9 months versus 30.2 months in non-carriers (p = 1.62 × 10−8, HR = 5.96). In this GWAS we identified novel genetic variants in PDLIM3 and DSCAM that impact PFS and OS in mRCC patients receiving sunitinib. The underlying link between the identified genes and the molecular mechanisms of sunitinib action needs to be elucidated.

Carbonic Anhydrase IX-Targeted α-Radionuclide Therapy with 225Ac Inhibits Tumor Growth in a Renal Cell Carcinoma Model.

In this study, we compared the tumor-targeting properties, therapeutic efficacy, and tolerability of the humanized anti-CAIX antibody (hG250) labeled with either the α-emitter actinium-225 (225Ac) or the ß--emitter lutetium-177 (177Lu) in mice. BALB/c nude mice were grafted with human renal cell carcinoma SK-RC-52 cells and intravenously injected with 30 µg [225Ac] Ac-DOTA-hG250 (225Ac-hG250) or 30 µg [177Lu] Lu-DOTA-hG250 (177Lu-hG250), followed by ex vivo biodistribution studies. Therapeutic efficacy was evaluated in mice receiving 5, 15, and 25 kBq of 225Ac-hG250; 13 MBq of 177Lu-hG250; or no treatment. Tolerability was evaluated in non-tumor-bearing animals. High tumor uptake of both radioimmunoconjugates was observed and increased up to day 7 (212.8 ± 50.2 %IA/g vs. 101.0 ± 18.4 %IA/g for 225Ac-hG250 and 177Lu-hG250, respectively). Survival was significantly prolonged in mice treated with 15 kBq 225Ac-hG250, 25 kBq 225Ac-hG250, and 13 MBq 177Lu-hG250 compared to untreated control (p < 0.05). Non-tumor-bearing mice that received single-dose treatment with 15 or 25 kBq 225Ac-hG250 showed weight loss at the end of the experiment (day 126), and immunohistochemical analysis suggested radiation-induced nephrotoxicity. These results demonstrate the therapeutic potential of CAIX-targeted α-therapy in renal cell carcinoma. Future studies are required to find an optimal balance between therapeutic efficacy and toxicity.

Combining Targeted Radionuclide Therapy and Immune Checkpoint Inhibition for Cancer Treatment.

The development of immunotherapy, in particular immune checkpoint inhibitors (ICI), has revolutionized cancer treatment in the past decades. However, its efficacy is still limited to subgroups of patients with cancer. Therefore, effective treatment combination strategies are needed. Here, radiotherapy is highly promising, as it can induce immunogenic cell death, triggering the release of pro-inflammatory cytokines, thereby creating an immunogenic phenotype and sensitizing tumors to ICI. Recently, targeted radionuclide therapy (TRT) has attained significant interest for cancer treatment. In this approach, a tumor-targeting radiopharmaceutical is used to specifically deliver a therapeutic radiation dose to all tumor cells, including distant metastatic lesions, while limiting radiation exposure to healthy tissue. However, fundamental differences between TRT and conventional radiotherapy make it impossible to directly extrapolate the biological effects from conventional radiotherapy to TRT. In this review, we present a comprehensive overview of studies investigating the immunomodulatory effects of TRT and the efficacy of combined TRT-ICI treatment. Preclinical studies have evaluated a variety of murine cancer models in which α- or ß-emitting radionuclides were directed to a diverse set of targets. In addition, clinical trials are ongoing to assess safety and efficacy of combined TRT-ICI in patients with cancer. Taken together, research indicates that combining TRT and ICI might improve therapeutic response in patients with cancer. Future research has to disclose what the optimal conditions are in terms of dose and treatment schedule to maximize the efficacy of this combined approach.

In vitro and in vivo assessment of a 3D printable gelatin methacrylate hydrogel for bone regeneration applications.

Bone tissue engineering (BTE) has made significant progress in developing and assessing different types of bio-substitutes. However, scaffolds production through standardized methods, as required for good manufacturing process (GMP), and post-transplant in vivo monitoring still limit their translation into the clinic. 3D printed 5% GelMA scaffolds have been prepared through an optimized and reproducible process in this work. Mesenchymal stem cells (MSC) were encapsulated in the 3D printable GelMA ink, and their biological properties were assessed in vitro to evaluate their potential for cell delivery application. Moreover, in vivo implantation of the pristine 3D printed GelMA has been performed in a rat condyle defect model. Whereas optimal tissue integration was observed via histology, no signs of fibrotic encapsulation or inhibited bone formation were attained. A multimodal imaging workflow based on computed tomography (CT) and magnetic resonance imaging (MRI) allowed the simultaneous monitoring of both new bone formation and scaffold degradation. These outcomes point out the direction to undertake in developing 3D printed-based hydrogels for BTE that can allow a faster transition into clinical use.

Molecular Processes in Stress Urinary Incontinence: A Systematic Review of Human and Animal Studies.

Stress urinary incontinence (SUI) is a common and burdensome condition. Because of the large knowledge gap around the molecular processes involved in its pathophysiology, the aim of this review was to provide a systematic overview of genetic variants, gene and protein expression changes related to SUI in human and animal studies. On 5 January 2021, a systematic search was performed in Pubmed, Embase, Web of Science, and the Cochrane library. The screening process and quality assessment were performed in duplicate, using predefined inclusion criteria and different quality assessment tools for human and animal studies respectively. The extracted data were grouped in themes per outcome measure, according to their functions in cellular processes, and synthesized in a narrative review. Finally, 107 studies were included, of which 35 used animal models (rats and mice). Resulting from the most examined processes, the evidence suggests that SUI is associated with altered extracellular matrix metabolism, estrogen receptors, oxidative stress, apoptosis, inflammation, neurodegenerative processes, and muscle cell differentiation and contractility. Due to heterogeneity in the studies (e.g., in examined tissues), the precise contribution of the associated genes and proteins in relation to SUI pathophysiology remained unclear. Future research should focus on possible contributors to these alterations.

Novel Imaging Methods for Renal Mass Characterization: A Collaborative Review.

CONTEXT: The incidental detection of localized renal masses has been rising steadily, but a significant proportion of these tumors are benign or indolent and, in most cases, do not require treatment. At the present time, a majority of patients with an incidentally detected renal tumor undergo treatment for the presumption of cancer, leading to a significant number of unnecessary surgical interventions that can result in complications including loss of renal function. Thus, there exists a clinical need for improved tools to aid in the pretreatment characterization of renal tumors to inform patient management. OBJECTIVE: To systematically review the evidence on noninvasive, imaging-based tools for solid renal mass characterization. EVIDENCE ACQUISITION: The MEDLINE database was systematically searched for relevant studies on novel imaging techniques and interpretative tools for the characterization of solid renal masses, published in the past 10 yr. EVIDENCE SYNTHESIS: Over the past decade, several novel imaging tools have offered promise for the improved characterization of indeterminate renal masses. Technologies of particular note include multiparametric magnetic resonance imaging of the kidney, molecular imaging with targeted radiopharmaceutical agents, and use of radiomics as well as artificial intelligence to enhance the interpretation of imaging studies. Among these, 99mTc-sestamibi single photon emission computed tomography/computed tomography (CT) for the identification of benign renal oncocytomas and hybrid oncocytic chromophobe tumors, and positron emission tomography/CT imaging with radiolabeled girentuximab for the identification of clear cell renal cell carcinoma, are likely to be closest to implementation in clinical practice. CONCLUSIONS: A number of novel imaging tools stand poised to aid in the noninvasive characterization of indeterminate renal masses. In the future, these tools may aid in patient management by providing a comprehensive virtual biopsy, complete with information on tumor histology, underlying molecular abnormalities, and ultimately disease prognosis. PATIENT SUMMARY: Not all renal tumors require treatment, as a significant proportion are either benign or have limited metastatic potential. Several innovative imaging tools have shown promise for their ability to improve the characterization of renal tumors and provide guidance in terms of patient management.

A Temperature-Based Easy-Separable (TempEasy) 3D Hydrogel Coculture System.

Interactions between different cell types are crucial for their behavior in tissues, but are rarely considered in 3D in vitro cell culture experiments. One reason is that such coculture experiments are sometimes difficult to perform in 3D or require specialized equipment or know-how. Here, a new 3D cell coculture system is introduced, TempEasy, which is readily applied in any cell culture lab. The matrix material is based on polyisocyanide hydrogels, which closely resemble the mechanical characteristics of the natural extracellular matrix. Gels with different gelation temperatures, seeded with different cells, are placed on top of each other to form an indirect coculture. Cooling reverses gelation, allowing cell harvesting from each layer separately, which benefits downstream analysis. To demonstrate the potential of TempEasy , human adipose stem cells (hADSCs) with vaginal epithelial fibroblasts are cocultured. The analysis of a 7-day coculture shows that hADSCs promote cell-cell interaction of fibroblasts, while fibroblasts promote proliferation and differentiation of hADSCs. TempEasy provides a straightforward operational platform for indirect cocultures of cells of different lineages in well-defined microenvironments.

Mimicking the Biology of Engineered Protein and mRNA Nanoparticle Delivery Using a Versatile Microfluidic Platform.

To investigate the delivery of next-generation macromolecular drugs, such as engineered proteins and mRNA-containing nanoparticles, there is an increasing push towards the use of physiologically relevant disease models that incorporate human cells and do not face ethical dilemmas associated with animal use. Here, we illustrate the versatility and ease of use of a microfluidic platform for studying drug delivery using high-resolution microscopy in 3D. Using this microfluidic platform, we successfully demonstrate the specific targeting of carbonic anhydrase IX (CAIX) on cells overexpressing the protein in a tumor-mimicking chip system using affibodies, with CAIX-negative cells and non-binding affibodies as controls. Furthermore, we demonstrate this system's feasibility for testing mRNA-containing biomaterials designed to regenerate bone defects. To this end, peptide- and lipid-based mRNA formulations were successfully mixed with colloidal gelatin in microfluidic devices, while translational activity was studied by the expression of a green fluorescent protein. This microfluidic platform enables the testing of mRNA delivery from colloidal biomaterials of relatively high densities, which represents a first important step towards a bone-on-a-chip platform. Collectively, by illustrating the ease of adaptation of our microfluidic platform towards use in distinct applications, we show that our microfluidic chip represents a powerful and flexible way to investigate drug delivery in 3D disease-mimicking culture systems that recapitulate key parameters associated with in vivo drug application.

Isolation of multipotent progenitor cells from pleura and pericardium for tracheal tissue engineering purposes.

Tissue engineering (TE) of long tracheal segments is conceptually appealing for patients with inoperable tracheal pathology. In tracheal TE, stem cells isolated from bone marrow or adipose tissue have been employed, but the ideal cell source has yet to be determined. When considering the origin of stem cells, cells isolated from a source embryonically related to the trachea may be more similar. In this study, we investigated the feasibility of isolating progenitor cells from pleura and pericard as an alternative cells source for tracheal tissue engineering. Porcine progenitor cells were isolated from pleura, pericard, trachea and adipose tissue and expanded in culture. Isolated cells were characterized by PCR, RNA sequencing, differentiation assays and cell survival assays and were compared to trachea and adipose-derived progenitor cells. Progenitor-like cells were successfully isolated and expanded from pericard and pleura as indicated by gene expression and functional analyses. Gene expression analysis and RNA sequencing showed a stem cell signature indicating multipotency, albeit that subtle differences between different cell sources were visible. Functional analysis revealed that these cells were able to differentiate towards chondrogenic, osteogenic and adipogenic lineages. Isolation of progenitor cells from pericard and pleura with stem cell features is feasible. Although functional differences with adipose-derived stem cells were limited, based on their gene expression, pericard- and pleura-derived stem cells may represent a superior autologous cell source for cell seeding in tracheal tissue engineering.

DPPG2-based thermosensitive liposomes as drug delivery system for effective muscle-invasive bladder cancer treatment in vivo.

PURPOSE: Recommended treatments for muscle-invasive bladder cancer (MIBC) come with considerable morbidity. Hyperthermia (HT) triggered drug release from phosphatidylglycerol-based thermosensitive liposomes (DPPG2-TSL) might prevent surgical bladder removal and toxicity from systemic chemotherapy. We aimed to assess the efficacy of DPPG2-TSL with HT in a syngeneic orthotopic rat urothelial carcinoma model. METHODS: A total of 191 female Fischer F344 rats were used. Bladder tumors were initiated by inoculation of AY-27 cells and tumor-bearing rats were selected with cystoscopy and semi-randomized over treatment groups. On days 5 and 8, animals were treated with DOX in different treatment modalities: intravenous (iv) DPPG2-TSL-DOX with HT, iv free DOX without HT, intravesical DOX without HT, intravesical DOX with HT or no treatment (control group), respectively. Animals were euthanized on day 14 and complete tumor response was assessed by histopathological evaluation. RESULTS: Iv DPPG2-TSL-DOX + HT resulted in a favorable rate of animals with complete tumor response (70%), compared to iv free DOX (18%, p = .02), no treatment (0%, p = .001), and intravesical DOX with (43%, p = .35) or without HT (50%, p = .41). All rats receiving intravesical DOX with HT and 24% of rats treated with DPPG2-TSL-DOX containing the same DOX dose with HT had to be euthanized before day 14 because of substantial bodyweight loss, which was associated with dilated ureters urine retention in a few rats. CONCLUSION: Treatment with DPPG2-TSL-DOX combined with intravesical HT outperformed systemic and intravesical DOX in vivo. There might be a role for DPPG2-TSL encapsulating chemotherapeutics in the treatment of MIBC in the future.

Simultaneous Recording of the Uptake and Conversion of Glucose and Choline in Tumors by Deuterium Metabolic Imaging.

Increased glucose and choline uptake are hallmarks of cancer. We investigated whether the uptake and conversion of [2H9]choline alone and together with that of [6,6'-2H2]glucose can be assessed in tumors via deuterium metabolic imaging (DMI) after administering these compounds. Therefore, tumors with human renal carcinoma cells were grown subcutaneously in mice. Isoflurane anesthetized mice were IV infused in the MR magnet for ~20 s with ~0.2 mL solutions containing either [2H9]choline (0.05 g/kg) alone or together with [6,6'-2H2]glucose (1.3 g/kg). 2H MR was performed on a 11.7T MR system with a home-built 2H/1H coil using a 90° excitation pulse and 400 ms repetition time. 3D DMI was recorded at high resolution (2 × 2 × 2 mm) in 37 min or at low resolution (3.7 × 3.7 × 3.7 mm) in 2:24 min. Absolute tissue concentrations were calculated assuming natural deuterated water [HOD] = 13.7 mM. Within 5 min after [2H9]choline infusion, its signal appeared in tumor spectra representing a concentration increase to 0.3-1.2 mM, which then slowly decreased or remained constant over 100 min. In plasma, [2H9]choline disappeared within 15 min post-infusion, implying that its signal arises from tumor tissue and not from blood. After infusing a mixture of [2H9]choline and [6,6'-2H2]glucose, their signals were observed separately in tumor 2H spectra. Over time, the [2H9]choline signal broadened, possibly due to conversion to other choline compounds, [[6,6'-2H2]glucose] declined, [HOD] increased and a lactate signal appeared, reflecting glycolysis. Metabolic maps of 2H compounds, reconstructed from high resolution DMIs, showed their spatial tumor accumulation. As choline infusion and glucose DMI is feasible in patients, their simultaneous detection has clinical potential for tumor characterization.

Phase I study to assess safety, biodistribution and radiation dosimetry for 89Zr-girentuximab in patients with renal cell carcinoma.

PURPOSE: In this phase I study, we evaluated the safety, biodistribution and dosimetry of [89Zr]Zr-DFO-girentuximab (89Zr-girentuximab) PET/CT imaging in patients with suspicion of clear cell renal cell carcinoma (ccRCC). METHODS: Ten eligible patients received an intravenous administration of 37 MBq (± 10%) of 89Zr-girentuximab at mass doses of 5 mg or 10 mg. Safety was evaluated according to the NCI CTCAE (version 4.03). Biodistribution and normal organ dosimetry was performed based on PET/CT images acquired at 0.5, 4, 24, 72 and 168 h post-administration. Additionally, tumour dosimetry was performed in patients with confirmed ccRCC and visible tumour uptake on PET/CT imaging. RESULTS: 89Zr-girentuximab was administered in ten patients as per protocol. No treatment-related adverse events ≥ grade 3 were reported. 89Zr-girentuximab imaging allowed successful differentiation between ccRCC and non-ccRCC lesions in all patients, as confirmed with histological data. Dosimetry analysis using OLINDA/EXM 2.1 showed that the organs receiving the highest doses (mean ± SD) were the liver (1.86 ± 0.40 mGy/MBq), the kidneys (1.50 ± 0.22 mGy/MBq) and the heart wall (1.45 ± 0.19 mGy/MBq), with a mean whole body effective dose of 0.57 ± 0.08 mSv/MBq. Tumour dosimetry was performed in the 6 patients with histologically confirmed ccRCC resulting in a median tumour-absorbed dose of 4.03 mGy/MBq (range 1.90-11.6 mGy/MBq). CONCLUSIONS: This study demonstrates that 89Zr-girentuximab is safe and well tolerated for the administered activities and mass doses and allows quantitative assessment of 89Zr-girentuximab PET/CT imaging in patients with suspicion of ccRCC. TRIAL REGISTRATION: NCT03556046-14th of June, 2018.

Lysine methyltransferase G9a is an important modulator of trained immunity.

OBJECTIVES: Histone methyltransferase G9a, also known as Euchromatic Histone Lysine Methyltransferase 2 (EHMT2), mediates H3K9 methylation which is associated with transcriptional repression. It possesses immunomodulatory effects and is overexpressed in multiple types of cancer. In this study, we investigated the role of G9a in the induction of trained immunity, a de facto innate immune memory, and its effects in non-muscle-invasive bladder cancer (NMIBC) patients treated with intravesical Bacillus Calmette-Guérin (BCG). METHODS: EHMT2 expression was assessed upon induction of trained immunity by RNA sequencing and Western blotting. G9a inhibitor BIX-01294 was used to investigate the effect on trained immunity responses in vitro. Subsequent cytokine production was measured by ELISA, epigenetic modifications were measured by ChIP-qPCR, Seahorse technology was used to measure metabolic changes, and a luminescence assay was used to measure ROS release. RNA sequencing was performed on BIX-01294-treated monocytes ex vivo. RESULTS: The expression of EHMT2 mRNA and protein decreased in monocytes during induction of trained immunity. G9a inhibition by BIX-01294 induced trained immunity and amplified trained immunity responses evoked by various microbial ligands in vitro. This was accompanied by decreased H3K9me2 at the promoters of pro-inflammatory genes. G9a inhibition was also associated with amplified ex vivo trained immunity responses in circulating monocytes of NMIBC patients. Additionally, altered RNA expression of inflammatory genes in monocytes of NMIBC patients was observed upon ex vivo G9a inhibition. Furthermore, intravesical BCG therapy decreased H3K9me2 at the promoter of pro-inflammatory genes. CONCLUSION: Inhibition of G9a is important in the induction of trained immunity, and G9a may represent a novel therapeutic target in NMIBC patients.