Control of cell penetration enhancer shielding and endosomal escape-kinetics crucial for efficient and biocompatible siRNA delivery.

Although cationic liposomes are efficient carriers for nucleic acid delivery, their toxicity often hampers the clinical translation. Polyethylene glycol (PEG) coating has been largely used to improve their stability and reduce toxicity. Nevertheless, it has been found to decrease the transfection process. In order to exploit the advantages of cationic liposomes and PEG decoration for nucleic acid delivery, liposomes decorated with tetraArg-[G-1]-distearoyl glycerol (Arg4-DAG) dendronic oligo-cationic lipid enhancer (OCE) and PEG-lipid have been investigated. Non decorated or OCE-decorated lipoplexes (OCEfree-LPX and OCE-LPX, respectively) were obtained by lipid film hydration using oligonucleotide (ON) solutions. PEG and OCE/PEG decorated lipoplexes (PEG-OCEfree-LPX and PEG-OCE-LPX, respectively) were obtained by post-insertion of 2 or 5 kDa PEG-DSPE on preformed lipoplexes. The OCE decoration yielded lipoplexes with size of about 240 nm, 84% loading efficiency at 10 N/P ratio, ten times higher than OCEfree-LPX, and prevented the ON release when incubated with physiological heparin concentration or with plasma. The PEG decoration reduced the zeta potential, enhanced the lipoplex stability in serum and decreased both hemolysis and cytotoxicity, while it did not affect the lipoplex size and ON loading. With respect to OCEfree-LPX, the OCE-LPX remarkably associated with cells and were taken up by different cancer cell lines (HeLa and MDA-MB-231). Interestingly, 2 or 5 kDa PEG decoration did not reduce either the cell interaction or the cell up-take of the cationic lipoplexes. With siRNA as a payload, OCE enabled efficient internalization, but endosomal release was hampered. Post-transfection treatment with the lysosomotropic drug chloroquine allowed to identify the optimal time point for endosomal escape. Chloroquine treatment after 12 to 20 h of LPX pre-incubation enabled siRNA mediated target knockdown indicating that this is the time window of endo-lysosomal processing. This indicates that OCE can protect siRNA from lysosomal degradation for up to 20 h, as shown by these rescue experiments.

A distinct stimulatory cDC1 subpopulation amplifies CD8+ T cell responses in tumors for protective anti-cancer immunity.

Type 1 conventional dendritic cells (cDC1) can support T cell responses within tumors but whether this determines protective versus ineffective anti-cancer immunity is poorly understood. Here, we use imaging-based deep learning to identify intratumoral cDC1-CD8+ T cell clustering as a unique feature of protective anti-cancer immunity. These clusters form selectively in stromal tumor regions and constitute niches in which cDC1 activate TCF1+ stem-like CD8+ T cells. We identify a distinct population of immunostimulatory CCR7neg cDC1 that produce CXCL9 to promote cluster formation and cross-present tumor antigens within these niches, which is required for intratumoral CD8+ T cell differentiation and expansion and promotes cancer immune control. Similarly, in human cancers, CCR7neg cDC1 interact with CD8+ T cells in clusters and are associated with patient survival. Our findings reveal an intratumoral phase of the anti-cancer T cell response orchestrated by tumor-residing cDC1 that determines protective versus ineffective immunity and could be exploited for cancer therapy.

Tumor-derived prostaglandin E2 programs cDC1 dysfunction to impair intratumoral orchestration of anti-cancer T cell responses.

Type 1 conventional dendritic cells (cDC1s) are critical for anti-cancer immunity. Protective anti-cancer immunity is thought to require cDC1s to sustain T cell responses within tumors, but it is poorly understood how this function is regulated and whether its subversion contributes to immune evasion. Here, we show that tumor-derived prostaglandin E2 (PGE2) programmed a dysfunctional state in intratumoral cDC1s, disabling their ability to locally orchestrate anti-cancer CD8+ T cell responses. Mechanistically, cAMP signaling downstream of the PGE2-receptors EP2 and EP4 was responsible for the programming of cDC1 dysfunction, which depended on the loss of the transcription factor IRF8. Blockade of the PGE2-EP2/EP4-cDC1 axis prevented cDC1 dysfunction in tumors, locally reinvigorated anti-cancer CD8+ T cell responses, and achieved cancer immune control. In human cDC1s, PGE2-induced dysfunction is conserved and associated with poor cancer patient prognosis. Our findings reveal a cDC1-dependent intratumoral checkpoint for anti-cancer immunity that is targeted by PGE2 for immune evasion.

[COVID-19 vaccination strategy for employees of German hospitals: results of a survey of infection control practitioners within the B-FAST project]. / COVID-19-Impfstrategien für Beschäftigte deutscher Kliniken: Ergebnisse einer Befragung von Leitungen der (Krankenhaus­)Hygiene im Rahmen des B-FAST-Projektes.

BACKGROUND AND AIM: At the beginning of the COVID-19 vaccination campaign in Germany, employees in medical facilities were prioritised for vaccination against SARS-CoV­2 due to the high risk of exposure and contact with vulnerable groups. Hospitals were therefore encouraged to organise and implement the vaccination of their employees as soon as possible. The aim of the study was to record the practice regarding the vaccination strategy for employees in German hospitals. METHODS: In a self-developed cross-sectional study, infection control practitioners of all German university hospitals as well as non-university hospitals in Lower Saxony and Bavaria were surveyed in March 2021. The data were stratified according to the characteristics of university hospitals and non-university hospitals. RESULTS: Of 416 invitations sent out, 100 questionnaires (university hospitals: 33; non-university hospitals: 67) were completed. University hospitals reported greater vaccination capacity than non-university hospitals, but a limiting factor was uncertain vaccine supply. Vaccination information campaigns were planned or had already been conducted in 89% of clinics. About two-thirds of the respondents (70%) said they did not plan to conduct antibody tests on vaccinated employees. A follow-up of vaccinated employees to detect possible SARS-CoV­2 infections by PCR was planned by 41% of the respondents. In case of detection of SARS-CoV­2 infection, 72% of the respondents had planned further diagnostic procedures. DISCUSSION: All hospitals were able to achieve rapid implementation of COVID-19 vaccination of their employees. At the time of the survey, there was also much uncertainty regarding the management of breakthrough infections as well as the need for booster vaccinations.

Electrospun and 3D printed polymeric materials for one-stage critical-size long bone defect regeneration inspired by the Masquelet technique: Recent Advances.

Critical-size long bone defects represent one of the major causes of fracture non-union and remain a significant challenge in orthopaedic surgery. Two-stage procedures such as a Masquelet technique demonstrate high level of success however their main disadvantage is the need for a second surgery, which is required to remove the non-resorbable cement spacer and to place the bone graft into the biological chamber formed by the 'induced membrane'. Recent research efforts have therefore been dedicated towards the design, fabrication and testing of resorbable implants that could mimic the biological functions of the cement spacer and the induced membrane. Amongst the various manufacturing techniques used to fabricate these implants, three-dimensional (3D) printing and electrospinning methods have gained a significant momentum due their high-level controllability, scalable processing and relatively low cost. This review aims to present recent advances in the evaluation of electrospun and 3D printed polymeric materials for critical-size, long bone defect reconstruction, emphasizing both their beneficial properties and current limitations. Furthermore, we present and discuss current state-of-the art techniques required for characterisation of the materials' physical, mechanical and biological characteristics. These represent the essential first steps towards the development of personalised implants for single-surgery, large defect reconstruction in weight-bearing bones.

The BCL-2 family member BOK promotes KRAS-driven lung cancer progression in a p53-dependent manner.

A variety of cancer entities are driven by KRAS mutations, which remain difficult to target clinically. Survival pathways, such as resistance to cell death, may represent a promising treatment approach in KRAS mutated cancers. Based on the frequently observed genomic deletions of BCL-2-related ovarian killer (BOK) in cancer patients, we explored the function of BOK in a mutant KrasG12D-driven murine model of lung cancer. Using KrasG12D/+ Bok-/- mice, we observed an overall tumor-promoting function of BOK in vivo. Specifically, loss of BOK reduced proliferation both in cell lines in vitro as well as in KrasG12D-driven tumor lesions in vivo. During tumor development in vivo, loss of BOK resulted in a lower tumor burden, with fewer, smaller, and less advanced tumors. Using KrasG12D/+ Tp53Δ/Δ Bok-/- mice, we identified that this phenotype was entirely dependent on the presence of functional p53. Furthermore, analysis of a human dataset of untreated early-stage lung tumors did not identify any common deletion of the BOK locus, independently of the TP53 status or the histopathological classification. Taken together our data indicate that BOK supports tumor progression in Kras-driven lung cancer.

Detection and differentiation of murine leukemia virus (MLV) and murine stem cell virus (MSCV) and therefrom derived nucleic acids.

Murine leukemia virus (MLV) and murine stem cell virus (MSCV) and derived retroviral vectors are widely used to study retrovirus biology and as tools for gene delivery. The method described here represents a quantitative real time PCR (qPCR) with hydrolysis probe that can be applied within classical qPCR as well as in digital droplet PCR (ddPCR). The method targets a 60 bp long fragment located within the U5 region of the MLV/MSCV genome sequence. For the here described method a LOD95% of 25 copies per PCR reaction (DNA) and 80 copies per PCR reaction (RNA) was determined, and PCR efficiencies of 92.5 % and 98.5 %, respectively, were observed. This method enables the fast and simple titration of viral genomic RNA present in retroviral vector stocks for accurate and consistent transduction experiments. Furthermore, it enables the detection of proviral and transfer plasmid derived DNA sequences and can be modified to differentiate between retroviral RNA and DNA.

XIAP restrains TNF-driven intestinal inflammation and dysbiosis by promoting innate immune responses of Paneth and dendritic cells.

Deficiency in X-linked inhibitor of apoptosis protein (XIAP) is the cause for X-linked lymphoproliferative syndrome 2 (XLP2). About one-third of these patients suffer from severe and therapy-refractory inflammatory bowel disease (IBD), but the exact cause of this pathogenesis remains undefined. Here, we used XIAP-deficient mice to characterize the mechanisms underlying intestinal inflammation. In Xiap−/− mice, we observed spontaneous terminal ileitis and microbial dysbiosis characterized by a reduction of Clostridia species. We showed that in inflamed mice, both TNF receptor 1 and 2 (TNFR1/2) cooperated in promoting ileitis by targeting TLR5-expressing Paneth cells (PCs) or dendritic cells (DCs). Using intestinal organoids and in vivo modeling, we demonstrated that TLR5 signaling triggered TNF production, which induced PC dysfunction mediated by TNFR1. TNFR2 acted upon lamina propria immune cells. scRNA-seq identified a DC population expressing TLR5, in which Tnfr2 expression was also elevated. Thus, the combined activity of TLR5 and TNFR2 signaling may be responsible for DC loss in lamina propria of Xiap−/− mice. Consequently, both Tnfr1−/−Xiap−/− and Tnfr2−/−Xiap−/− mice were rescued from dysbiosis and intestinal inflammation. Furthermore, RNA-seq of ileal crypts revealed that in inflamed Xiap−/− mice, TLR5 signaling was abrogated, linking aberrant TNF responses with the development of a dysbiosis. Evidence for TNFR2 signaling driving intestinal inflammation was detected in XLP2 patient samples. Together, these data point toward a key role of XIAP in mediating resilience of TLR5-expressing PCs and intestinal DCs, allowing them to maintain tissue integrity and microbiota homeostasis.

CD47-targeted cancer immunogene therapy: Secreted SIRPα-Fc fusion protein eradicates tumors by macrophage and NK cell activation.

CD47 protects healthy cells from macrophage attack by binding to signal regulatory protein α (SIRPα), while its upregulation in cancer prevents immune clearance. Systemic treatment with CD47 antibodies requires a weakened Fc-mediated effector function or lower CD47-binding affinity to prevent side effects. Our approach combines "the best of both worlds," i.e., maximized CD47 binding and full Fc-mediated immune activity, by exploiting gene therapy for paracrine release. We developed a plasmid vector encoding for the secreted fusion protein sCV1-hIgG1, comprising highly efficient CD47-blocking moiety CV1 and Fc domain of human immunoglobulin G1 (IgG1) with maximized immune activation. sCV1-hIgG1 exhibited a potent bystander effect, blocking CD47 on all cells via fusion protein secreted from only a fraction of cells or when transferring transfection supernatant to untransfected cells. The CpG-free plasmid ensured sustained secretion of sCV1-hIgG1. In orthotopic human triple-negative breast cancer in CB17-severe combined immunodeficiency (SCID) mice, ex vivo transfection significantly delayed tumor growth and eradicated one-third of tumors. In intratumoral transfection experiments, CD47 blockage and increased migration of macrophages into the tumor were observed within 17 h of a single injection. Natural killer (NK) cell-mediated lysis of sCV1-hIgG1-expressing cells was demonstrated in vitro. Taken together, this approach also opens the opportunity to block, in principle, any immune checkpoints.

Auto-aggressive CXCR6+ CD8 T cells cause liver immune pathology in NASH.

Nonalcoholic steatohepatitis (NASH) is a manifestation of systemic metabolic disease related to obesity, and causes liver disease and cancer1,2. The accumulation of metabolites leads to cell stress and inflammation in the liver3, but mechanistic understandings of liver damage in NASH are incomplete. Here, using a preclinical mouse model that displays key features of human NASH (hereafter, NASH mice), we found an indispensable role for T cells in liver immunopathology. We detected the hepatic accumulation of CD8 T cells with phenotypes that combined tissue residency (CXCR6) with effector (granzyme) and exhaustion (PD1) characteristics. Liver CXCR6+ CD8 T cells were characterized by low activity of the FOXO1 transcription factor, and were abundant in NASH mice and in patients with NASH. Mechanistically, IL-15 induced FOXO1 downregulation and CXCR6 upregulation, which together rendered liver-resident CXCR6+ CD8 T cells susceptible to metabolic stimuli (including acetate and extracellular ATP) and collectively triggered auto-aggression. CXCR6+ CD8 T cells from the livers of NASH mice or of patients with NASH had similar transcriptional signatures, and showed auto-aggressive killing of cells in an MHC-class-I-independent fashion after signalling through P2X7 purinergic receptors. This killing by auto-aggressive CD8 T cells fundamentally differed from that by antigen-specific cells, which mechanistically distinguishes auto-aggressive and protective T cell immunity.

MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically.

Evasion of programmed cell death represents a critical form of oncogene addiction in cancer cells. Understanding the molecular mechanisms underpinning cancer cell survival despite the oncogenic stress could provide a molecular basis for potential therapeutic interventions. Here we explore the role of pro-survival genes in cancer cell integrity during clonal evolution in non-small cell lung cancer (NSCLC). We identify gains of MCL-1 at high frequency in multiple independent NSCLC cohorts, occurring both clonally and subclonally. Clonal loss of functional TP53 is significantly associated with subclonal gains of MCL-1. In mice, tumour progression is delayed upon pharmacologic or genetic inhibition of MCL-1. These findings reveal that MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically.

Combined Chemisorption and Complexation Generate siRNA Nanocarriers with Biophysics Optimized for Efficient Gene Knockdown and Air-Blood Barrier Crossing.

Current nucleic acid (NA) nanotherapeutic approaches face challenges because of shortcomings such as limited control on loading efficiency, complex formulation procedure involving purification steps, low load of NA cargo per nanoparticle, endosomal trapping, and hampered release inside the cell. When combined, these factors significantly limit the amount of biologically active NA delivered per cell in vitro, delivered dosages in vivo for a prolonged biological effect, and the upscalability potential, thereby warranting early consideration in the design and developmental phase. Here, we report a versatile nanotherapeutic platform, termed auropolyplexes, for improved and efficient delivery of small interfering RNA (siRNA). Semitelechelic, thiolated linear polyethylenimine (PEI) was chemisorbed onto gold nanoparticles to endow them with positive charge. A simple two-step complexation method offers tunable loading of siRNA at concentrations relevant for in vivo studies and the flexibility for inclusion of multiple functionalities without any purification steps. SiRNA was electrostatically complexed with these cationic gold nanoparticles and further condensed with polycation or polyethyleneglycol-polycation conjugates. The resulting auropolyplexes ensured complete complexation of siRNA into nanoparticles with a high load of ∼15,500 siRNA molecules/nanoparticle. After efficient internalization into the tumor cell, an 80% knockdown of the luciferase reporter gene was achieved. Auropolyplexes were applied intratracheally in Balb/c mice for pulmonary delivery, and their biodistribution were studied spatio-temporally and quantitatively by optical tomography. Auropolyplexes were well tolerated with ∼25% of the siRNA dose remaining in the lungs after 24 h. Importantly, siRNA was released from auropolyplexes in vivo and a fraction also crossed the air-blood barrier, which was then excreted via kidneys, whereas >97% of gold nanoparticles were retained in the lung. Linear PEI-based auropolyplexes offer a combination of successful endosomal escape and better biocompatibility profile in vivo. Taken together, combined chemisorption and complexation endow auropolyplexes with crucial biophysical attributes, enabling a versatile and upscalable nanogold-based platform for siRNA delivery in vitro and in vivo.

Regulatory myeloid cells paralyze T cells through cell-cell transfer of the metabolite methylglyoxal.

Regulatory myeloid immune cells, such as myeloid-derived suppressor cells (MDSCs), populate inflamed or cancerous tissue and block immune cell effector functions. The lack of mechanistic insight into MDSC suppressive activity and a marker for their identification has hampered attempts to overcome T cell inhibition and unleash anti-cancer immunity. Here, we report that human MDSCs were characterized by strongly reduced metabolism and conferred this compromised metabolic state to CD8+ T cells, thereby paralyzing their effector functions. We identified accumulation of the dicarbonyl radical methylglyoxal, generated by semicarbazide-sensitive amine oxidase, to cause the metabolic phenotype of MDSCs and MDSC-mediated paralysis of CD8+ T cells. In a murine cancer model, neutralization of dicarbonyl activity overcame MDSC-mediated T cell suppression and, together with checkpoint inhibition, improved the efficacy of cancer immune therapy. Our results identify the dicarbonyl methylglyoxal as a marker metabolite for MDSCs that mediates T cell paralysis and can serve as a target to improve cancer immune therapy.

Bifunctional Labeling of Rabbit Mesenchymal Stem Cells for MR Imaging and Fluorescence Microscopy.

PURPOSE: Longitudinal imaging studies are important in the translational process of stem cell-based therapies. Small animal imaging models are widely available and practical but insufficiently depict important morphologic detail. In contrary, large animal models are logistically challenging and costly but offer greater imaging quality. In order to combine the advantages of both, we developed an intermediate-sized rabbit animal model for cartilage imaging studies. PROCEDURES: Rabbit mesenchymal stem cells (rMSC) were isolated as primary cultures from the bone marrow of New Zealand white rabbits. rMSC were subsequentially transduced lentivirally with eGFP and magnetically labeled with the iron oxide ferucarbotran. eGFP expression was evaluated by flow cytometry and iron uptake was analyzed by isotope dilution mass spectrometry and Prussian blue staining. Fluorescence microscopy of eGFP-transduced rMSC was performed. Viability and induction of apoptosis were assessed by XTT and caspase-3/-7 measurements. The chondrogenic potential of labeled cells was quantified by glycosaminoglycan contents in TGF-ß3 induced pellet cultures. Labeled and unlabeled cells underwent magnetic resonance imaging (MRI) at 1.5 T before and after differentiation using T1-, T2-, and T2*-weighted pulse sequences. Relaxation rates were calculated. rMSCs were implanted in fibrin clots in osteochondral defects of cadaveric rabbit knees and imaged by 7 T MRI. T2* maps were calculated. Statistical analyses were performed using multiple regression models. RESULTS: Efficiency of lentiviral transduction was greater than 90 %. Fluorescence signal was dose dependent. Cellular iron uptake was significant for all concentrations (p < 0.05) and dose dependent (3.3-56.5 pg Fe/cell). Labeled rMSC showed a strong, dose-dependent contrast on all MR pulse sequences and a significant decrease in T2 and T2* relaxation rates. Compared with non-transduced or unlabeled controls, there were no adverse effects on cell viability, rate of apoptosis, or chondrogenic differentiation. MRI of labeled rMSCs in osteochondral defects showed a significant signal of the transplant with additional high-resolution anatomical information. CONCLUSIONS: This intermediate-sized rabbit model and its bifunctional labeling technique allow for improved depiction of anatomic detail for noninvasive in vivo rMSC tracking with MRI and for immunohistological correlation by fluorescence microscopy.

Luminescent and fluorescent triple reporter plasmid constructs for Wnt, Hedgehog and Notch pathway.

Tracking the activity of signalling pathways is a fundamental method for basic science, as well as in cancer- and pharmaceutical research. The developmental pathways Wnt, Hedgehog and Notch are frequently deregulated in cancers and represent a valuable target for the discovery of novel anticancer compounds. Here we present reporter systems for tracking activity of these pathways by using specific promoter elements driving the expression of either sensitive luciferases or fluorescent proteins. A high level of sensitivity was obtained using the luciferase reporter genes for firefly (FLuc), secreted Gaussia (GLuc) and synthetic NanoLuc (NLuc). As fluorescent reporter proteins, mTurqouise2, tdTomato and iRFP720 were chosen. Specificity of pathway activity was validated by co-transfection with pathway activating genes, showing significant response to induction. In addition, multi-gene plasmids were cloned, allowing the detection of all three pathways by one vector. By using the multi-gene vector 3P-Luc (wnt-NLuc, hedgehog-FLuc, Notch-GLuc), we could unambiguously demonstrate the crosstalk between pathways, while excluding cross reactivity of luciferase substrates. First studies with synthetic compounds confirmed the applicability of the system for future drug screening approaches.

Age-Related Gliosis Promotes Central Nervous System Lymphoma through CCL19-Mediated Tumor Cell Retention.

How lymphoma cells (LCs) invade the brain during the development of central nervous system lymphoma (CNSL) is unclear. We found that NF-κB-induced gliosis promotes CNSL in immunocompetent mice. Gliosis elevated cell-adhesion molecules, which increased LCs in the brain but was insufficient to induce CNSL. Astrocyte-derived CCL19 was required for gliosis-induced CNSL. Deleting CCL19 in mice or CCR7 from LCs abrogated CNSL development. Two-photon microscopy revealed LCs transiently entering normal brain parenchyma. Astrocytic CCL19 enhanced parenchymal CNS retention of LCs, thereby promoting CNSL formation. Aged, gliotic wild-type mice were more susceptible to forming CNSL than young wild-type mice, and astrocytic CCL19 was observed in both human gliosis and CNSL. Therefore, CCL19-CCR7 interactions may underlie an increased age-related risk for CNSL.

Gene editing enables T-cell engineering to redirect antigen specificity for potent tumor rejection.

Adoptive transfer of TCR transgenic T cells holds great promise for treating various cancers. So far, mainly semi-randomly integrating vectors have been used to genetically modify T cells. These carry the risk of insertional mutagenesis, and the sole addition of an exogenous TCR potentially results in the mispairing of TCR chains with endogenous ones. Established approaches using nonviral vectors, such as transposons, already reduce the risk of insertional mutagenesis but have not accomplished site-specific integration. Here, we used CRISPR-Cas9 RNPs and adeno-associated virus 6 for gene targeting to deliver an engineered TCR gene specifically to the TCR alpha constant locus, thus placing it under endogenous transcriptional control. Our data demonstrate that this approach replaces the endogenous TCR, functionally redirects the edited T cells' specificity in vitro, and facilitates potent tumor rejection in an in vivo xenograft model.

Impact of c-MYC expression on proliferation, differentiation, and risk of neoplastic transformation of human mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cells isolated from bone marrow (MSC) represent an attractive source of adult stem cells for regenerative medicine. However, thorough research is required into their clinical application safety issues concerning a risk of potential neoplastic degeneration in a process of MSC propagation in cell culture for therapeutic applications. Expansion protocols could preselect MSC with elevated levels of growth-promoting transcription factors with oncogenic potential, such as c-MYC. We addressed the question whether c-MYC expression affects the growth and differentiation potential of human MSC upon extensive passaging in cell culture and assessed a risk of tumorigenic transformation caused by MSC overexpressing c-MYC in vivo. METHODS: MSC were subjected to retroviral transduction to induce expression of c-MYC, or GFP, as a control. Cells were expanded, and effects of c-MYC overexpression on osteogenesis, adipogenesis, and chondrogenesis were monitored. Ectopic bone formation properties were tested in SCID mice. A potential risk of tumorigenesis imposed by MSC with c-MYC overexpression was evaluated. RESULTS: C-MYC levels accumulated during ex vivo passaging, and overexpression enabled the transformed MSC to significantly overgrow competing control cells in culture. C-MYC-MSC acquired enhanced biological functions of c-MYC: its increased DNA-binding activity, elevated expression of the c-MYC-binding partner MAX, and induction of antagonists P19ARF/P16INK4A. Overexpression of c-MYC stimulated MSC proliferation and reduced osteogenic, adipogenic, and chondrogenic differentiation. Surprisingly, c-MYC overexpression also caused an increased COL10A1/COL2A1 expression ratio upon chondrogenesis, suggesting a role in hypertrophic degeneration. However, the in vivo ectopic bone formation ability of c-MYC-transduced MSC remained comparable to control GFP-MSC. There was no indication of tumor growth in any tissue after transplantation of c-MYC-MSC in mice. CONCLUSIONS: C-MYC expression promoted high proliferation rates of MSC, attenuated but not abrogated their differentiation capacity, and did not immediately lead to tumor formation in the tested in vivo mouse model. However, upregulation of MYC antagonists P19ARF/P16INK4A promoting apoptosis and senescence, as well as an observed shift towards a hypertrophic collagen phenotype and cartilage degeneration, point to lack of safety for clinical application of MSC that were manipulated to overexpress c-MYC for their better expansion.

Loss of endogenous RNF43 function enhances proliferation and tumour growth of intestinal and gastric cells.

Ring finger protein 43 (RNF43) is an E3 ubiquitin ligase that has been described to be frequently mutated in gastrointestinal cancers. RNF43 downregulation was associated with distant metastasis, TNM stage and poorer survival in patients with gastric and colorectal cancers. Functional analysis has shown that overexpressed RNF43 negatively regulates Wnt signalling by ubiquitinating Frizzled receptors and targeting them for degradation and by sequestering T-cell factor 4 (TCF4) to the nuclear membrane, thereby inhibiting Wnt-mediated transcription. In the stomach, RNF43 overexpression was shown to impair stem-like properties and to be negatively correlated with expression of Wnt-target genes. In this study, we show that RNF43 knockdown enhances the tumourigenic potential of gastric and colorectal cancer cell lines in vitro and in vivo. Thus, loss of RNF43 leads to increased proliferation and anchorage-independent growth as well as increased invasive capacity. In a xenograft model, RNF43 depletion enhanced tumour growth. Furthermore, we established two mouse models in which mutations in the RING domain of RNF43 were introduced. In the intestine and colon, loss of Rnf43 did not induce changes in epithelial architecture or proliferation. In contrast, in the stomach, thickening of the mucosa, hyperplasia and cellular atypia were observed in these mice. Notably, this was independent of elevated Wnt signalling. Together, our results show that RNF43 plays a tumour suppressive role in gastric and colorectal cancer cells and that the loss of its function alters gastric tissue homeostasis in vivo.