Mitogenic Research Articles

Neuroinflammation as a cause of differential Müller cell regenerative responses to retinal injury.

Unlike mammals, some nonmammalian species recruit Müller glia for retinal regeneration after injury. Identifying the underlying mechanisms may help to foresee regenerative medicine strategies. Using a Xenopus model of retinitis pigmentosa, we found that Müller cells actively proliferate upon photoreceptor degeneration in old tadpoles but not in younger ones. Differences in the inflammatory microenvironment emerged as an explanation for such stage dependency. Functional analyses revealed that enhancing neuroinflammation is sufficient to trigger Müller cell proliferation, not only in young tadpoles but also in mice. In addition, we showed that microglia are absolutely required for the response of mouse Müller cells to mitogenic factors while negatively affecting their neurogenic potential. However, both cell cycle reentry and neurogenic gene expression are allowed when applying sequential pro- and anti-inflammatory treatments. This reveals that inflammation benefits Müller glia proliferation in both regenerative and nonregenerative vertebrates and highlights the importance of sequential inflammatory modulation to create a regenerative permissive microenvironment.

Decoding the Role of Insulin-like Growth Factor 1 and Its Isoforms in Breast Cancer.

Insulin-like Growth Factor-1 (IGF-1) is a crucial mitogenic factor with important functions in the mammary gland, mainly through its interaction with the IGF-1 receptor (IGF-1R). This interaction activates a complex signaling network that promotes cell proliferation, epithelial to mesenchymal transition (EMT) and inhibits apoptosis. Despite extensive research, the precise molecular pathways and intracellular mechanisms activated by IGF-1, in cancer, remain poorly understood. Recent evidence highlights the essential roles of IGF-1 and its isoforms in breast cancer (BC) development, progression, and metastasis. The peptides that define the IGF-1 isoforms-IGF-1Ea, IGF-1Eb, and IGF-1Ec-act as key points of convergence for various signaling pathways that influence the growth, metastasis and survival of BC cells. The aim of this review is to provide a detailed exami-nation of the role of the mature IGF-1 and its isoforms in BC biology and their potential use as possible therapeutical targets.

Assay for interferon gamma release as a novel marker in pediatric patients with systemic lupus erythematosus

BackgroundThe interferon-gamma (IFN-γ) release assay (IGRA) is an important laboratory diagnosis for latent Mycobacterium tuberculosis (TB) infection. The TB-IGRA measures the release of IFN-γ from peripheral blood cells, who are exposed to TB antigen (Ag), mitogen (MT), or negative/nil control (NL) in vitro. While, an exceptional higher TB Ag-NL level will reflect an elevation of peripheral lymphocytes released IFN-γ in a same condition. Therefore, we found that the elevated levels of TB Ag-NL could become a new biomarker for the diagnosis and treatment of pediatric systemic lupus erythematosus (SLE) patients.MethodsWe have analyzed the clinical data of 776 children who are underwent TB-IGRA testing in the Department of Allergy and Rheumatology of Guangzhou Women and Children’s Medical Center from 2018 to 2020. To investigate the association between TB Ag-NL and SLE, we have analyzed the clinical data of 47 SLE patients and TB Ag-NL testing results, and then evaluated the association between TB Ag-NL and SLE disease activity.ResultsThe TB Ag-NL levels were significantly higher in patients with active SLE than those in inactive SLE (p = 0.0002). The TB Ag-NL levels were positively correlated with the SLE disease activity index (SLEDAI) and laboratory diagnosis parameters. The mean value of TB Ag-NL in SLE patients (0.04191 ± 0.07955, IU/mL) were significantly higher than those in patients with juvenile dermatomyositis (JDM) (0.0158 ± 0.0337, IU/mL, p = 0.036), juvenile idiopathic arthritis (JIA) (0.0162 ± 0.0388, IU/mL, p = 0.001), and healthy controls (HC) (0.0001 ± 0.0027, IU/mL, p = 0.0003). Therefore, the elevated TB Ag-NL levels could serve as a potential diagnostic biomarker of SLE, especially for the active SLE.ConclusionThe detection of IFN-γ release levels by the TB-IGRA may be useful to assess SLE disease activity in pediatric patients with active SLE.

Low Serum Concentration in Ovine Embryo Culture Media

Background: Fetal bovine serum (FBS) is rich in nutrients, growth factors, hormones, and other essential compounds necessary for cell growth. However, its use has several disadvantages. Platelets in particular contain the majority of the potent mitogenic factors found in serum. Objectives: The present study aimed to evaluate the effect of platelet lysate (PL) on in vitro embryo production by replacing FBS, to determine if this modified Charles Rosenkrans medium (mCR2aa) can support ovine embryo development compared to the commercially available BO-IVC medium. Methods: In the first experiment, varying concentrations of FBS (0%, 2.5%, 5%, and 10%) and PL (0%, 2.5%, 5%, and 10%) were investigated in the mCR2aa culture medium. In the second experiment, the optimized mCR2aa medium was compared to the BO-IVC medium. Ovine oocytes were matured and fertilized in vitro using BO-IVM Bioscience medium and Brackett and Oliphant medium, respectively. Subsequently, the resulting zygotes were cultured in either mCR2aa medium supplemented with amino acids or BO-IVC medium, according to the experimental design, under a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 38.5ºC. Results: The results indicated that using 5% PL in the mCR2aa culture medium reduced the serum percentage to 2.5%. Conclusions: It was not possible to completely eliminate serum from the mCR2aa medium solely by substituting it with PL. Further studies are needed to explore other serum properties such as chelating agents and antioxidant activities to fully optimize the mCR2aa medium without serum.

Essential growth factor receptors for fibroblast homeostasis and activation: Fibroblast Growth Factor Receptor (FGFR), Platelet Derived Growth Factor Receptor (PDGFR), and Transforming Growth Factor β Receptor (TGFβR).

Fibroblasts are cells of mesenchymal origin that are found throughout the body. While these cells have several functions, their integral roles include maintaining tissue architecture through the production of key extracellular matrix components, and participation in wound healing after injury. Fibroblasts are also key mediators in disease progression during fibrosis, cancer, and other inflammatory diseases. Under these perturbed states, fibroblasts can activate into inflammatory fibroblasts or contractile myofibroblasts. Fibroblasts require various growth factors and mitogenic molecules for survival, proliferation, and differentiation. While the activity of mitogenic growth factors on fibroblasts in vitro was characterized as early as the 1970s, the proliferation and differentiation effects of growth factors on these cells in vivo are unclear. Recent work exploring the heterogeneity of fibroblasts raises questions as to whether all fibroblast cell states exhibit the same growth factor requirements. Here, we will examine and review existing studies on the influence of fibroblast growth factor receptors (FGFRs), platelet-derived growth factor receptors (PDGFRs), and transforming growth factor β receptor (TGFβR) on fibroblast cell states.

A novel method for the establishment of autologous skin cell suspensions: characterisation of cellular sub-populations, epidermal stem cell content and wound response-enhancing biological properties.

Introduction: Autologous cell suspension (ACS)-based therapy represents a highly promising approach for burns and chronic wounds. However, existing technologies have not achieved the desired clinical success due to several limitations. To overcome practical and cost-associated obstacles of existing ACS methods, we have established a novel methodology for rapid, enzymatic disaggregation of human skin cells and their isolation using a procedure that requires no specialist laboratory instrumentation and is performed at room temperature. Methods: Cells were isolated using enzymatic disaggregation of split-thickness human skin followed by several filtration steps for isolation of cell populations, and cell viability was determined. Individual population recovery was confirmed in appropriate culture medium types, and the presence of epidermal stem cells (EpSCs) within keratinocyte sub-populations was defined by flow cytometry via detection of CD49 and CD71. Positive mediators of wound healing secreted by ACS-derived cultures established on a collagen-based wound-bed mimic were detected by proteome arrays and quantified by ELISA, and the role of such mediators was determined by cell proliferation assays. The effect of ACS-derived conditioned-medium on myofibroblasts was investigated using an in-vitro model of myofibroblast differentiation via detection of α-SMA using immunoblotting and immunofluorescence microscopy. Results: Our methodology permitted efficient recovery of keratinocytes, fibroblasts and melanocytes, which remained viable upon long-term culture. ACS-derivatives comprised sub-populations with the CD49-high/CD71-low expression profile known to demarcate EpSCs. Via secretion of mitogenic factors and wound healing-enhancing mediators, the ACS secretome accelerated keratinocyte proliferation and markedly curtailed cytodifferentiation of myofibroblasts, the latter being key mediators of fibrosis and scarring. Discussion: The systematic characterisation of the cell types within our ACS isolates provided evidence for their superior cell viability and the presence of EpSCs that are critical drivers of wound healing. We defined the biological properties of ACS-derived keratinocytes, which include ability to secrete positive mediators of wound healing as well as suppression of myofibroblast cytodifferentiation. Thus, our study provides several lines of evidence that the established ACS isolates comprise highly-viable cell populations which can physically support wound healing and possess biological properties that have the potential to enhance not only the speed but also the quality of wound healing.

Phytosulfokine alpha enhances regeneration of transformed and untransformed protoplasts of Brassica oleracea.

Phytosulfokine-α (PSK-α) is a disulfated pentapeptide (YIYTQ) acting as an intercellular signal peptide and growth factor. It was originally isolated from conditioned medium of asparagus mesophyll cell cultures in 1996 and later characterized as a hormone-like signal molecule with important roles in numerous processes of in vivo plant growth and development. It is currently becoming a valuable mitogenic factor in plant breeding and biotechnology due to its stimulatory effect on in vitro cell elongation, proliferation and differentiation. The focus of our work was to review current knowledge about the roles of PSK-α in plant biotechnology and to evaluate its influence on the regeneration of protoplasts of four Brassica oleracea cultivars (two cauliflower and two cabbage) cultured under two distinctive protocols and with different protoplast densities. Protoplast regeneration was studied due to its high value for plant genome editing, which is generally limited by the inefficient regeneration of treated protoplasts of numerous important plant genotypes. Our hypothesis was that the stress related to PEG-mediated protoplast transformation and the following decrease in viable protoplast density in culture could be alleviated by the addition of PSK-α to the culture medium. We therefore tested whether PSK-α could increase cell division at the early stages of culture (5 and 15 days after protoplast isolation) and stimulate the formation of microcallus colonies up to the 30st day of culture and to evaluate its influence on callus organogenesis leading to shoot regeneration. The PSK-α showed a strong stimulatory effect on untransformed protoplast regeneration already during the first days of culture, accelerating cell division up to 5.3-fold and the formation of multicellular microcallus colonies up to 37.0-fold. The beneficial influence was retained at later stages of regeneration, when PSK improved shoot organogenesis even if it was present only during the first 10 days of culture. The highest numbers of shoots, however, were regenerated when PSK was present during the first days of culture and later in solid shoot regeneration medium. Finally, the addition of PSK-α to PEG-transformed protoplasts significantly enhanced their division rate and the formation of microcallus colonies in selection media, up to 44.0-fold.

Essential growth factor receptors for fibroblast homeostasis and activation

Fibroblasts are cells of mesenchymal origin that are found throughout the body. While these cells have several functions, their integral roles include maintaining tissue architecture through the production of key extracellular matrix components, and participation in wound healing after injury. Fibroblasts are also key mediators in disease progression during fibrosis, cancer, and other inflammatory diseases. Under these perturbed states, fibroblasts can activate into inflammatory fibroblasts or contractile myofibroblasts. Fibroblasts require various growth factors and mitogenic molecules for survival, proliferation, and differentiation. While the activity of mitogenic growth factors on fibroblasts in vitro was characterized as early as the 1970s, the proliferation and differentiation effects of growth factors on these cells in vivo are unclear. Moreover, recent work exploring the heterogeneity of fibroblasts raises questions as to whether all fibroblast cell states exhibit the same growth factor requirements. Here, we will examine and review existing growth factors known to influence fibroblast homeostasis to begin unpacking the potential growth factors that may influence in vivo fibroblast cell states.

The effect of phytosulfokine alpha on haploid embryogenesis and gene expression of Brassica napus microspore cultures.

Microspore embryogenesis (ME) is the most powerful tool for creating homozygous lines in plant breeding and molecular biology research. It is still based mainly on the reprogramming of microspores by temperature, osmotic and/or nutrient stress. New compounds are being sought that could increase the efficiency of microspore embryogenesis or even induce the formation of haploid embryos from recalcitrant genotypes. Among these, the mitogenic factor phytosulfokine alpha (PSK-α) is promising due to its broad spectrum of activity in vivo and in vitro. The aim of our study was to investigate the effect of PSK-α on haploid embryogenesis from microspores of oilseed rape (Brassica napus L., DH4079), one of the most important oil crops and a model plant for studying the molecular mechanisms controlling embryo formation. We tested different concentrations (0, 0.01, 0.1 and 1 µM) of the peptide and evaluated its effect on microspore viability and embryo regeneration after four weeks of culture. Our results showed a positive correlation between addition of PSK-α and cultured microspore viability and a positive effect also on the number of developed embryos. The analysis of transcriptomes across three time points (day 0, 2 and 4) with or without PSK-α supplementation (15 RNA libraries in total) unveiled differentially expressed genes pivotal in cell division, microspore embryogenesis, and subsequent regeneration. PCA grouped transcriptomes by RNA sampling time, with the first two principal components explaining 56.8% variability. On day 2 with PSK, 45 genes (15 up- and 30 down-regulated) were differentially expressed when PSK-α was added and their number increased to 304 by day 4 (30 up- and 274 down-regulated). PSK, PSKR, and PSI gene expression analysis revealed dynamic patterns, with PSK2 displaying the highest increase and overall expression during microspore culture at days 2 and 4. Despite some variations, only PSK1 showed significant differential expression upon PSK-α addition. Of 16 ME-related molecular markers, 3 and 15 exhibited significant differential expression in PSK-supplemented cultures at days 2 and 4, respectively. Embryo-specific markers predominantly expressed after 4 days of culture, with higher expression in medium without PSK, while on day 0, numerous sporophyte-specific markers were highly expressed.

FGF9, a Potent Mitogen, Is a New Ligand for Integrin αvβ3, and the FGF9 Mutant Defective in Integrin Binding Acts as an Antagonist.

FGF9 is a potent mitogen and survival factor, but FGF9 protein levels are generally low and restricted to a few adult organs. Aberrant expression of FGF9 usually results in cancer. However, the mechanism of FGF9 action has not been fully established. Previous studies showed that FGF1 and FGF2 directly bind to integrin αvβ3, and this interaction is critical for signaling functions (FGF-integrin crosstalk). FGF1 and FGF2 mutants defective in integrin binding were defective in signaling, whereas the mutants still bound to FGFR suppressed angiogenesis and tumor growth, indicating that they act as antagonists. We hypothesize that FGF9 requires direct integrin binding for signaling. Here, we show that docking simulation of the interaction between FGF9 and αvβ3 predicted that FGF9 binds to the classical ligand-binding site of αvβ3. We show that FGF9 bound to integrin αvβ3 and generated FGF9 mutants in the predicted integrin-binding interface. An FGF9 mutant (R108E) was defective in integrin binding, activating FRS2α and ERK1/2, inducing DNA synthesis, cancer cell migration, and invasion in vitro. R108E suppressed DNA synthesis and activation of FRS2α and ERK1/2 induced by WT FGF9 (dominant-negative effect). These findings indicate that FGF9 requires direct integrin binding for signaling and that R108E has potential as an antagonist to FGF9 signaling.

Overexpression and Purification of Mitogenic and Metabolic Fibroblast Growth Factors.

Fibroblast growth factors (FGFs) are proteins with a vast array of biological activity, such as cell development and repair, glucose and bile acid metabolisms, and wound healing. Due to their critical and diverse physiological functions, FGFs are believed to possess potential as therapeutic agents for many diseases and conditions that warrant further investigations. Thus, a simple, cost-efficient method to purify these biologically active signaling proteins is desirable. Herein, we introduce such techniques to purify FGFs that possess either high heparin-binding affinity or low to no heparin-binding affinity. This method takes advantage of the high affinity toward heparin sulfate from paracrine FGF1 to isolate the targeted protein. It also accounts for FGF members that have low heparin affinity, such as the metabolic FGFs, by introducing poly-histidine tags in the recombinant protein in combination with the immobilized metal affinity chromatography. Subsequently, the purified FGF products are separated from the other small protein by high-speed centrifugation. Products are then subjected to other biophysical experiments like SDS-PAGE, mass spectrometry, circular dichroism, intrinsic fluorescence, isothermal titration calorimetry, differential scanning calorimetry, and biological cell activity assay to confirm that the target proteins are purified with intact native conformation and no significant change in the intrinsic characteristics and biological activities.

MiRNAs from Inflamed Gingiva Link Gene Signaling to Increased MET Expression

Several array-based microRNA (miRNA) expression studies independently showed increased expression of miRNAs hsa-miR-130a-3p, -142-3p, -144-3p, -144-5p, -223-3p, -17-5p, and -30e-5p in gingiva affected by periodontal inflammation. We aimed to determine direct target genes and signaling pathways regulated by these miRNAs to identify processes relevant to gingival inflammatory responses and tissue homeostasis. We transfected miRNA mimics (mirVana) for each of the 7 miRNAs separately into human primary gingival fibroblasts cultured from 3 different donors. Following RNA sequencing, differential gene expression and second-generation gene set enrichment analyses were performed. miRNA inhibition and upregulation was validated at the transcript and protein levels using quantitative reverse transcriptase polymerase chain reaction, Western blotting, and reporter gene assays. All 7 miRNAs significantly increased expression of the gene MET proto-oncogene, receptor tyrosine kinase (MET). Expression of known periodontitis risk genes CPEB1, ABCA1, and ATP6V1C1 was significantly repressed by hsa-miR-130a-3p, -144-3p, and -144-5p, respectively. The genes WASL, ENPP5, ARL6IP1, and IDH1 showed the most significant and strongest downregulation after hsa-miR-142-3p, -17-5p, -223-3p, and -30e-5p transfection, respectively. The most significantly regulated gene set of each miRNA related to cell cycle (hsa-miRNA-144-3p and -5p [Padj = 4 × 10−40 and Padj = 4 × 10−6], -miR-17-5p [Padj = 9.5 × 10−23], -miR-30e-5p [Padj = 8.2 × 10−18], -miR-130a-3p [Padj = 5 × 10−15]), integrin cell surface interaction (-miR-223-3p [Padj = 2.4 × 10−7]), and interferon signaling (-miR-142-3p [Padj = 5 × 10−11]). At the end of acute inflammation, gingival miRNAs bring together complex regulatory networks that lead to increased expression of the gene MET. This underscores the importance of mesenchymal cell migration and invasion during gingival tissue remodeling and proliferation in restoring periodontal tissue homeostasis after active inflammation. MET, a receptor of the mitogenic hepatocyte growth factor fibroblast secreted, is a core gene of this process.

Sodium glucose co-transporter 2 inhibition increases epidermal growth factor expression and improves outcomes in patients with type 2 diabetes

Underlying molecular mechanisms of the kidney protective effects of sodium glucose co-transporter 2 (SGLT2) inhibitors are not fully elucidated. Therefore, we studied the association between urinary epidermal growth factor (uEGF), a mitogenic factor involved in kidney repair, and kidney outcomes in patients with type 2 diabetes (T2D). The underlying molecular mechanisms of the SGLT2 inhibitor canagliflozin on EGF using single-cell RNA sequencing from kidney tissue were examined. Urinary EGF-to-creatinine ratio (uEGF/Cr) was measured in 3521 CANagliflozin cardioVascular Assessment Study (CANVAS) participants at baseline and week 52. Associations of uEGF/Cr with kidney outcome were assessed using multivariable-adjusted Cox regression models. Single-cell RNA sequencing was performed using protocol kidney biopsy tissue from ten young patients with T2D on SGLT2i, six patients with T2D on standard care only, and six healthy controls (HCs). In CANVAS, each doubling in baseline uEGF/Cr was associated with a 12% (95% confidence interval 1-22) decreased risk of kidney outcome. uEGF/Cr decreased after 52 weeks with placebo and remained stable with canagliflozin (between-group difference +7.3% (2.0-12.8). In young persons with T2D, EGF mRNA was primarily expressed in the thick ascending loop of Henle. Expression in biopsies from T2D without SGLT2i was significantly lower compared to HCs, whereas treatment with SGLT2i increased EGF levels closer to the healthy state. In young persons with T2D without SGLT2i, endothelin-1 emerged as a key regulator of the EGF co-expression network. SGLT2i treatment was associated with a shift towards normal EGF expression. Thus, decreased uEGF represents increased risk of kidney disease progression in patients with T2D. Canagliflozin increased kidney tissue expression of EGF and was associated with a downstream signaling cascade linked to tubular repair and reversal of tubular injury.

Self-assembly of gelatin and collagen in the polyvinyl alcohol substrate and its influence on cell adhesion, proliferation, shape, spreading and differentiation.

Culture substrates display profound influence on biological and developmental characteristic of cells cultured in vitro. This study investigates the influence of polyvinyl alcohol (PVA) substrates blended with different concentration of collagen or/and gelatin on the cell adhesion, proliferation, shape, spreading, and differentiation of stem cells. The collagen/gelatin blended PVA substrates were prepared by air drying. During drying, blended collagen or/and gelatin can self-assemble into macro-scale nucleated particles or branched fibrils in the PVA substrates that can be observed under the optical microscope. These collagen/gelatin blended substrates revealed different surface topography, z-average, roughness, surface adhesion and Young's modulus as examined by the atomic force microscope (AFM). The results of Fourier transform infrared spectroscopy (FTIR) analysis indicated that the absorption of amide I (1,600-1,700cm-1) and amide II (1,500-1,600cm-1) groups increased with increasing collagen and gelatin concentration blended and the potential of fibril formation. These collagen or/and gelatin blended PVA substrates showed enhanced NIH-3T3 fibroblast adhesion as comparing with the pure PVA, control tissue culture polystyrene, conventional collagen-coated and gelatin-coated wells. These highly adhesive PVA substrates also exhibit inhibited cell spreading and proliferation. It is also found that the shape of NIH-3T3 fibroblasts can be switched between oval, spindle and flattened shapes depending on the concentration of collagen or/and gelatin blended. For inductive differentiation of stem cells, it is found that number and ration of neural differentiation of rat cerebral cortical neural stem cells increase with the decreasing collagen concentration in the collagen-blended PVA substrates. Moreover, the PVA substrates blended with collagen or collagen and gelatin can efficiently support and conduct human pluripotent stem cells to differentiate into Oil-Red-O- and UCP-1-positive brown-adipocyte-like cells via ectodermal lineage without the addition of mitogenic factors. These results provide a useful and alternative platform for controlling cell behavior in vitro and may be helpful for future application in the field of regenerative medicine and tissue engineering.

How to initiate tissue regeneration by generating mutually exclusive cell states.

A healing wound is made of cells that paused proliferation and secrete mitogenic factors and cells that proliferate to replace those lost to cell death. A new study in PLOS Biology describes how 2 conserved signaling pathways could repress each other to create these mutually exclusive cell states.

Influence of Media Composition on the Level of Bovine Satellite Cell Proliferation.

It is predicted that already in 2040, 35% of requirements for meat will be provided by in vitro production. Recreating the course of myogenesis in vitro, and thus resembling a structure of muscle tissue, is the basis for research focusing on obtaining cultured meat and requires providing relevant factors supporting the proliferation of satellite cells-being precursors of skeletal muscles. The present work aimed to develop the composition of the medium that would most effectively stimulate the proliferation of bovine satellite cells (BSCs). The modeling and optimization methods included the measurements of the synergistic, co-stimulatory effect of three medium components: the amount of glucose, the type of serum (bovine or horse), and the amount of mitogenic factor-bFGF. Additionally, the qPCR analyses determined the expression of genes involved in myogenesis, such as Pax7 and Myogenic Regulatory Factors, depending on the level of the tested factor. The results showed significant positive effects of serum type (bovine serum) and mitogenic factor (addition of 10 ng/mL bFGF) on the proliferation rate. In turn, qPCR analysis displayed no significant differences in the relative expression level of Pax7 genes and MRF factors for both factors. However, a statistically higher Pax7 and Myf5 gene expression level was revealed when a low glucose medium was used (p < 0.05). In conclusion, the components of the medium, such as bovine serum and the addition of a mitogenic factor at the level of 10 ng/mL, ensure a higher proliferation rate of BSCs and lower glucose content ensured the expression of crucial genes in the self-renewal of the satellite cell population.

Abstract A053: Epigenomic analyses of circulating tumor DNA in patients with metastatic castration resistant prostate cancer (mCRPC) treated with immune checkpoint blockade and PARP inhibition

Abstract Background: Molecular characterization of mCRPC has revealed disruption of DNA damage repair genes that may improve sensitivity to PARP inhibitors such as olaparib (O), which in turn may further complement the activity of the PD-L1 immune checkpoint inhibitor, durvalumab (D), through mechanisms such as activation of the STING pathway. We hypothesized that (epi)genomic analyses of circulating tumor DNA (ctDNA) would identify mechanisms of treatment response in patients with mCRPC treated with O plus D (NCT02484404). Methods: Eligible patients for this study had mCRPC with prior treatment by abiraterone and/or enzalutamide. Study participants were administered 300 mg of O twice daily and 1500 mg of D every 4 weeks. We obtained blood samples in Streck tubes from 38 (out of 60) study participants. Plasma samples were acquired at baseline and at progression. Buffy coat was isolated from each sample. Genomic DNA and cell-free DNA (cfDNA) were separately isolated and assembled into sequencing libraries. Low-pass whole-genome sequencing (3-4 × coverage) was performed for somatic copy number and fragmentomic analyses. In a subset of samples with the greatest cfDNA content, chromatin immunoprecipitation sequencing (ChIP-seq) with antibodies against H3K4me2 and H3K4me3 was performed directly in additional aliquots of plasma. Results: Amongst the 38 patients with at least one cfDNA sample, 5 patients had a radiographic partial response (PR) per RECIST v1.1, 28 patients had stable disease (SD), and 6 patients had progressive disease (PD) during the first two months of treatment. At baseline, ctDNA content in plasma was significantly lower in patients who went on to have PR or SD versus PD (p=0.02). However, upon progression, ctDNA content in plasma was not significantly different between groups. Losses to PTEN and NKX3-1 were significantly enriched in ctDNA from patients with PD (p=0.041 and p=0.047, respectively). By contrast, biallelic combined losses to BRCA2, RB1, BRCA1 and TP53 were observed in all PRs at baseline but not in any patient with PD (p=0.048). In patients with SD, longitudinal fragmentomic analysis inferred that the activity of the mitogenic KLF4 transcription factor (TF) was the most increased upon progression, while activity of the protective HIF2A TF was the most decreased. However, the TF binding motifs predicted by H3K4me2 and H3K4me3 cfDNA ChIP-seq did not significantly change in patients with PD. Conclusions: Distinctive genomic and epigenomic profiles were observed in plasma from patients with responsive vs. progressive disease, and the profiles may predict responses to combination D+O therapy. Deeper whole-genome sequencing of these samples currently in progress will reveal the contribution of germline and somatic point mutations to clinical response. Ongoing efforts to estimate the phylogenetic architecture of patients’ tumors from these plasma samples will enable the inference of drug response phenotypes as a function of evolutionary potential and potentially identify new opportunities to target treatment-resistant disease. Citation Format: Anna Baj, Clara C. Y. Seo, Nicholas T. Terrigino, John R. Bright, S. Thomas Hennigan, Isaiah M. King, Shana Y. Trostel, William D. Figg, William L. Dahut, Jung-Min Lee, David Y. Takeda, Fatima Karzai, Adam G. Sowalsky. Epigenomic analyses of circulating tumor DNA in patients with metastatic castration resistant prostate cancer (mCRPC) treated with immune checkpoint blockade and PARP inhibition [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A053.

Therapeutic potential of adipose tissue-derivatives in modern dermatology.

Stem cell-mediated therapies in combination with biomaterial and growth factor-based approaches in regenerative medicine are rapidly evolving with increasing application beyond the dermatologic field. Adipose-derived stem cells (ADSCs) are the more frequently used adult stem cells due to their abundance and easy access. In the case of volumetric defects, adipose tissue can take the shape of defects, restoring the volume and enhancing the regeneration of receiving tissue. When regenerative purposes prevail on volume restoration, the stromal vascular fraction (SVF) rich in staminal cells, purified mesenchymal stem cells (MSCs) or their cell-free derivatives grafting are favoured. The therapeutic efficacy of acellular approaches is explained by the fact that a significant part of the natural propensity of stem cells to repair damaged tissue is ascribable to their secretory activity that combines mitogenic factors, cytokines, chemokines and extracellular matrix components. Therefore, the secretome's ability to modulate multiple targets simultaneously demonstrated preclinical and clinical efficacy in reversing pathological mechanisms of complex conditions such atopic dermatitis (AD), vitiligo, psoriasis, acne and Lichen sclerosus (LS), non-resolving wounds and alopecia. This review analysing bothin vivoandin vitromodels gives an overview of the clinical relevance of adipose tissue-derivatives such as autologous fat graft, stromal vascular fraction, purified stem cells and secretome for skin disorders application. Finally, we highlighted the major disease-specific limitations and the future perspective in this field.

Insulin-like Growth Factor-2 (IGF-2) in Fibrosis.

The insulin family consists of insulin, insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), their receptors (IR, IGF-1R and IGF-2R), and their binding proteins. All three ligands are involved in cell proliferation, apoptosis, protein synthesis and metabolism due to their homologous sequences and structural similarities. Insulin-like growth factor 2, a member of the insulin family, plays an important role in embryonic development, metabolic disorders, and tumorigenesis by combining with three receptors with different degrees of affinity. The main pathological feature of various fibrotic diseases is the excessive deposition of extracellular matrix (ECM) after tissue and organ damage, which eventually results in organic dysfunction because scar formation replaces tissue parenchyma. As a mitogenic factor, IGF-2 is overexpressed in many fibrotic diseases. It can promote the proliferation of fibroblasts significantly, as well as the production of ECM in a time- and dose-dependent manner. This review aims to describe the expression changes and fibrosis-promoting effects of IGF-2 in the skin, oral cavity, heart, lung, liver, and kidney fibrotic tissues.

Free-standing multilayer films as growth factor reservoirs for future wound dressing applications

Chronic skin wounds place a high burden on patients and health care systems. The use of angiogenic and mitogenic growth factors can facilitate the healing but growth factors are quickly inactivated by the wound environment if added exogenously. Here, free-standing multilayer films (FSF) are fabricated from chitosan and alginate as opposing polyelectrolytes in an alternating manner using layer-by-layer technique. One hundred bilayers form an about 450 μm thick, detachable free-standing film that is subsequently crosslinked by either ethyl (dimethylaminopropyl) carbodiimide combined with N-hydroxysuccinimide (E-FSF) or genipin (G-FSF). The characterization of swelling, oxygen permeability and crosslinking density shows reduced swelling and oxygen permeability for both crosslinked films compared to non-crosslinked films (N-FSF). Loading of fibroblast growth factor 2 (FGF2) into the films results in a sustained release from crosslinked FSF in comparison to non-crosslinked FSF. Biocompatibility studies in vitro with human dermal fibroblasts cultured underneath the films demonstrate increased cell growth and cell migration for all films with and without FGF2. Especially G-FSF loaded with FGF2 greatly increases cell proliferation and migration. In vivo biocompatibility studies by subcutaneous implantation in mice show that E-FSF causes an inflammatory tissue response that is absent in the case of G-FSF. N-FSF also represents a biocompatible film but shows early degradation. All FSF possess antibacterial properties against gram+ and gram- bacteria demonstrated by an agar diffusion disc assay. In summary, FSF made of alginate and chitosan crosslinked with genipin can act as a reservoir for the sustained release of FGF2, possessing high biocompatibility in vitro and in vivo. Moreover, G-FSF promotes growth and migration of human dermal fibroblasts and has antibacterial properties, which makes it an interesting candidate for bioactive wound.