Differentially Expressed Genes Correlated with Fibrosis in a Rat Model of Chronic Partial Bladder Outlet Obstruction

Chronic partial bladder outlet obstruction (PBOO) is a prevalent clinical problem that may result from multiple etiologies. PBOO may be a secondary condition to various anatomical and functional abnormalities. Bladder fibrosis is the worst outcome of PBOO. However, gene alterations and the mechanism of fibrosis development after PBOO onset are not clear. Therefore, we aimed to investigate gene expression alterations during chronic PBOO. A rat model of PBOO was established and validated by a significant increase in rat bladder weight. The bladder samples were further analyzed by microarray, and differentially expressed genes (DEGs) that are more related to PBOO compared with the control genes were selected. The data showed that 16 significantly upregulated mRNAs and 3 significantly downregulated mRNAs are involved in fibrosis. Moreover, 13 significantly upregulated mRNAs and 12 significantly downregulated mRNAs are related to TGFB signaling. Twenty-two significantly upregulated mRNAs and nine significantly downregulated mRNAs are related to the extracellular matrix. The genes with differential expressions greater than four-fold included Grem1, Thbs1, Col8a1, Itga5, Tnc, Lox, Timp1, Col4a1, Col4a2, Bhlhe40, Itga1, Tgfb3, and Gadd45b. The gene with a differential expression less than a quarter-fold was Thbs2. These findings show the potential roles of these genes in the physiology of PBOO.

EXTH-29. DUAL TGFB AND PD1 BLOCKADE PROMOTES GERMINAL-CENTER B-CELL IMMUNE RESPONSES AGAINST GLIOBLASTOMA

In contrast to other malignancies such as melanoma and sarcoma, Glioblastoma (GBM) remains difficult to treat with immunotherapies. Recent studies have shown that positive immunotherapeutic responses are mediated by the accumulation of germinal-center-like B cells which are predictive of survival in patients treated with neoadjuvant PD1 blockade. In contrast, GBM-associated B-cells are scarce and the establishment of germinal-center like cells have not been observed. This study seeks to identify how B-cells are driven towards their immunosuppressive phenotypes in GBM and how this prevents immunotherapeutic efficacy. Utilizing single-cell RNA sequencing (scRNA-seq) in a CT2A murine glioma model, TGFb receptors 1 and 3 were identified as the most highly expressed inhibitory receptors on GBM-associated B cells. Furthermore, using scRNA-seq, TGFb1 was identified as the most highly expressed immunosuppressive cytokine in the TME, which was produced principally by tumor-associated myeloid cells (TAMCs). Inhibiting the myeloid compartment using intracranial anti-Gr1 antibody in combination with PD1 blockade resulted in B-cells exhibiting greater proliferation and differentiation into memory B-cells in addition to germinal-center-like B-cells. Further demonstrating B-cell functional reprogramming, autologous T cells isolated from spleens exhibited greater proliferation and robust anti-tumor cytotoxicity when cocultured with tumor-associated B-cells from the dual treatment group. Finally, inhibiting a5b8 integrin, a key complex in releasing active TGFb, increased tumor-infiltrating proliferating B-cells and conferred a long-term survival benefit in the CT2A murine model. Our results demonstrate that the immunosuppressive TME of GBM is influenced by the vital interplay between B-cells and the TME through TGFb signaling. This study highlights the potential therapeutic benefits of targeting the TGFb signaling pathway in conjunction with the current standard of care for GBM.

Cancer environmental immunotherapy: starving tumor cell to death by targeting TGFB on immune cell

The blockage of intersectional communication between tumor and its metabolic and immune microenvironment is now considered a promising solution in treating cancer. Tumors have been identified as a special type of “wounds” that do not heal. Recent studies demonstrate that the lack of the transforming growth factor beta (TGFB) signaling pathway in CD4+ helper T cells induces the remodeling of the intratumoral vascular tissue, like healing “wounds” in damaged tissues caused by tumor overgrowth, which consequently prevents tumor cells from receiving the required nutrients in their microenvironment. TGFB blockade thereby promotes damaged tissue healing, causing tumor cell death as a result of starvation, ultimately obtaining an effective anticancer immunotherapy immune response. Here, we comment on the TGFB-mediated crosstalk between immune system and nutritional supply, highlighting cancer immunotherapeutic strategies targeting environmental immune-metabolism interplay. Cancer environmental immunotherapy targeting TGFB might therefore become one of the most promising treatment strategies for patients with cancer.

Glioma-associated microglia/macrophages augment tumorigenicity in canine astrocytoma, a naturally occurring model of human glioma

Background Glioma-associated microglia/macrophage (GAM) markedly influence glioma progression. Under the influence of transforming growth factor beta (TGFB), GAM are polarized toward a tumor-supportive phenotype. However, neither therapeutic targeting of GAM recruitment, nor TGF Beta (TGFB) signaling demonstrated efficacy in glioma patients despite efficacy in preclinical models, underscoring the need for a comprehensive understanding of the TGFB/GAM axis. Spontaneously occurring canine gliomas share many features with human glioma and provide a complementary translational animal model for further study. Given the importance of GAM and TGFB in human glioma, the aims of this study were to further define the GAM-associated molecular profile and the relevance of TGFB signaling in canine glioma that may serve as the basis for future translational studies. Methods GAM morphometry, levels of GAM-associated molecules, and the canonical TGFB signaling axis were compared in archived samples of canine astrocytomas versus normal canine brain. Further, the effect of TGFB on the malignant phenotype of canine astrocytoma cells was evaluated. Results GAMs diffusely infiltrated canine astrocytomas. GAM density was increased in high-grade tumors that correlated with a pro-tumorigenic molecular signature and up-regulation of the canonical TGFB signaling axis. Moreover, TGFB1 enhanced migration of canine astrocytoma cells in vitro. Conclusions Canine astrocytomas share a similar GAM-associated immune landscape with human adult glioma. Our data also support a contributing role for TGFB1 signaling in the malignant phenotype of canine astrocytoma. These data further support naturally occurring canine glioma as a valid model for investigation of GAM-associated therapeutic strategies for human malignant glioma.

Elucidating the Cellular Mechanism for E2-induced Dermal Fibrosis

Background: Both TGFb and estradiol (E2), a form of estrogen, are pro-fibrotic in the skin. In the connective tissue disease, systemic sclerosis (SSc), both TGFb and E2 are likely pathogenic. Yet, the regulation of TGFb in E2-induced dermal fibrosis remains ill-defined. Elucidating those regulatory mechanisms will improve the understanding of fibrotic disease pathogenesis and set the stage for developing potential therapeutics. Using E2-stimulated primary human dermal fibroblasts in vitro and human skin tissue ex vivo, we identified the important regulatory proteins for TGFb and investigated the extracellular matrix (ECM) components that are directly stimulated by E2-induced TGFb signaling.Methods: We used primary human dermal fibroblasts in vitro and human skin tissue ex vivo stimulated with E2 or vehicle (ethanol) to measure TGFb1, TGFb2 levels using quantitative PCR (qPCR). To identify the necessary cell signaling proteins in E2-induced TGFb1 and TGFb2 transcription, human dermal fibroblasts were pre-treated with an inhibitor of the extracellular signal-regulated kinase/ mitogen-activated protein kinase (ERK/MAPK) pathway, U0126. Finally, human skin tissue ex vivo was pre-treated with SB-431542, a TGFb receptor inhibitor, to establish the effects of TGFb signaling on E2-induced collagen 22A1 (Col22A1) transcription. Statistical tests used included parametric and non-parametric ANOVA, student’s T-test and Wilcoxon matched-pairs signed rank test. Significance was defined as p-value ≤ 0.05.Results: We found that TGFb1, TGFb2, and collagen 22A1 (Col22A1), a TGFb-responsive gene, are induced in response to E2 stimulation. Mechanistically, Col22A1 induction is blocked by the TGFb receptor inhibitor SB-431542, despite E2 stimulation. Additionally, inhibiting E2-induced ERK/MAPK activation and early growth response 1 (EGR1) transcription prevents E2-induced increase in TGFb1 and TGFb2 transcription and translation. Conclusions: We conclude that E2-induced dermal fibrosis occurs in part through induction of TGFb1, 2 and Col22A1, which is regulated through EGR1 and the MAPK pathway. Thus, blocking estrogen signaling and/or production may be a novel therapeutic option in pro-fibrotic diseases.

PROFILING OF Y1 CELLS TREATED WITH FGF-2 REVEALS PARALLELS WITH ONCOGENE-INDUCED SENESCENCE

ABSTRACTParadoxically, oncogenes that drive cell cycle progression may also trigger pathways leading to senescence, thereby inhibiting the growth of tumorigenic cells. Along these lines, Y1 cells, which carry an amplification of Ras, become senescent after treatment with the mitogen FGF-2. To understand how FGF-2 promotes senescence, we profiled the epigenome, transcriptome, proteome, and phospho-proteome of Y1 cells stimulated with FGF-2. FGF-2 caused delayed acetylation of histone H4 and higher levels of H3K27me3. Sequencing analysis revealed decreased expression of cell cycle-related genes with concomitant loss of H3K27ac. In contrast, FGF-2 promoted the expression of p21, various cytokines, and MAPK-related genes. Nuclear envelope proteins, particularly lamin B1, displayed increased phosphorylation in response to FGF-2. Proteome analysis suggested alterations in cellular metabolism, as evident by modulated expression of enzymes involved in purine biosynthesis, tRNA aminoacylation, and the TCA cycle. Altogether, the response of Y1 cells to FGF-2 is consistent with oncogene-induced senescence. We propose that Y1 cells enter senescence due to deficient cyclin expression and high levels of p21, which may stem from DNA damage or TGFb signaling.

Multiple Roles of Transforming Growth Factor Beta in Amyotrophic Lateral Sclerosis

Transforming growth factor beta (TGFB) is a pleiotropic cytokine known to be dysregulated in many neurodegenerative disorders and particularly in amyotrophic lateral sclerosis (ALS). This motor neuronal disease is non-cell autonomous, as it affects not only motor neurons but also the surrounding glial cells, and the target skeletal muscle fibers. Here, we analyze the multiple roles of TGFB in these cell types, and how TGFB signaling is altered in ALS tissues. Data reported support a crucial involvement of TGFB in the etiology and progression of ALS, leading us to hypothesize that an imbalance of TGFB signaling, diminished at the pre-symptomatic stage and then increased with time, could be linked to ALS progression. A reduced stimulation of the TGFB pathway at the beginning of disease blocks its neuroprotective effects and promotes glutamate excitotoxicity. At later disease stages, the persistent activation of the TGFB pathway promotes an excessive microglial activation and strengthens muscular dysfunction. The therapeutic potential of TGFB is discussed, in order to foster new approaches to treat ALS.

Tgfb3 collaborates with PP2A and notch signaling pathways to inhibit retina regeneration

Neuronal degeneration in the zebrafish retina stimulates Müller glia (MG) to proliferate and generate multipotent progenitors for retinal repair. Controlling this proliferation is critical to successful regeneration. Previous studies reported that retinal injury stimulates pSmad3 signaling in injury-responsive MG. Contrary to these findings, we report pSmad3 expression is restricted to quiescent MG and suppressed in injury-responsive MG. Our data indicates that Tgfb3 is the ligand responsible for regulating pSmad3 expression. Remarkably, although overexpression of either Tgfb1b or Tgfb3 can stimulate pSmad3 expression in the injured retina, only Tgfb3 inhibits injury-dependent MG proliferation; suggesting the involvement of a non-canonical Tgfb signaling pathway. Furthermore, inhibition of Alk5, PP2A or Notch signaling rescues MG proliferation in Tgfb3 overexpressing zebrafish. Finally, we report that this Tgfb3 signaling pathway is active in zebrafish MG, but not those in mice, which may contribute to the different regenerative capabilities of MG from fish and mammals.