Pparg, a nuclear receptor, is downregulated in basal subtype bladder cancers that tend to be muscle invasive and amplified in luminal subtype bladder cancers that tend to be non-muscle invasive. ...Bladder cancers derive from the urothelium, one of the most quiescent epithelia in the body, which is composed of basal, intermediate, and superficial cells. We find that expression of an activated form of Pparg (VP16;Pparg) in basal progenitors induces formation of superficial cells in situ, that exit the cell cycle, and do not form tumors. Expression in basal progenitors that have been activated by mild injury however, results in luminal tumor formation. We find that these tumors are immune deserted, which may be linked to down-regulation of Nf-kb, a Pparg target. Interestingly, some luminal tumors begin to shift to basal subtype tumors with time, down-regulating Pparg and other luminal markers. Our findings have important implications for treatment and diagnosis of bladder cancer.
The binding site barrier (BSB) was originally proposed to describe the binding behavior of antibodies to cells peripheral to blood vessels, preventing their further penetration into the tumors. Yet, ...it is revisited herein to describe the intratumoral cellular disposition of nanoparticles (NPs). Specifically, the BSB limits NP diffusion and results in unintended internalization of NPs by stroma cells localized near blood vessels. This not only limits the therapeutic outcome but also promotes adverse off-target effects. In the current study, it was shown that tumor-associated fibroblast cells (TAFs) are the major component of the BSB, particularly in tumors with a stroma-vessel architecture where the location of TAFs aligns with blood vessels. Specifically, TAF distance to blood vessels, expression of receptor proteins, and binding affinity affect the intensity of the BSB. The physical barrier elicited by extracellular matrix also prolongs the retention of NPs in the stroma, potentially contributing to the BSB. The influence of particle size on the BSB was also investigated. The strongest BSB effect was found with small (∼18 nm) NPs targeted with the anisamide ligand. The uptake of these NPs by TAFs was about 7-fold higher than that of the other cells 16 h post-intravenous injection. This was because TAFs also expressed the sigma receptor under the influence of TGF-β secreted by the tumor cells. Overall, the current study underscores the importance of BSBs in the delivery of nanotherapeutics and provides a rationale for exploiting BSBs to target TAFs.
Abstract Background An early report on the molecular subtyping of muscle-invasive bladder cancer (MIBC) by gene expression suggested that response to neoadjuvant chemotherapy (NAC) varies by subtype. ...Objective To investigate the ability of molecular subtypes to predict pathological downstaging and survival after NAC. Design, setting, and participants Whole transcriptome profiling was performed on pre-NAC transurethral resection specimens from 343 patients with MIBC. Samples were classified according to four published molecular subtyping methods. We developed a single-sample genomic subtyping classifier (GSC) to predict consensus subtypes (claudin-low, basal, luminal-infiltrated and luminal) with highest clinical impact in the context of NAC. Overall survival (OS) according to subtype was analyzed and compared with OS in 476 non-NAC cases (published datasets). Intervention Gene expression analysis was used to assign subtypes. Outcome measurements and statistical analysis Receiver-operating characteristics were used to determine the accuracy of GSC. The effect of GSC on survival was estimated by Cox proportional hazard regression models. Results and limitations The models generated subtype calls in expected ratios with high concordance across subtyping methods. GSC was able to predict four consensus molecular subtypes with high accuracy (73%), and clinical significance of the predicted consensus subtypes could be validated in independent NAC and non-NAC datasets. Luminal tumors had the best OS with and without NAC. Claudin-low tumors were associated with poor OS irrespective of treatment regimen. Basal tumors showed the most improvement in OS with NAC compared with surgery alone. The main limitations of our study are its retrospective design and comparison across datasets. Conclusions Molecular subtyping may have an impact on patient benefit to NAC. If validated in additional studies, our results suggest that patients with basal tumors should be prioritized for NAC. We discovered the first single-sample classifier to subtype MIBC, which may be suitable for integration into routine clinical practice. Patient summary Different molecular subtypes can be identified in muscle-invasive bladder cancer. Although cisplatin-based neoadjuvant chemotherapy improves patient outcomes, we identified that the benefit is highest in patients with basal tumors. Our newly discovered classifier can identify these molecular subtypes in a single patient and could be integrated into routine clinical practice after further validation.
The tumor microenvironment (TME) serves as a multidrug resistant center for tumors under the assault of chemotherapy and a physiological barrier against the penetration of therapeutic nanoparticles ...(NPs). Previous studies have indicated the ability for therapeutic NP to distribute into, and deplete tumor-associated fibroblasts (TAFs) for improved therapeutic outcomes. However, a drug resistant phenotype gradually arises after repeated doses of chemotherapeutic NP. Herein, the acquisition of drug resistant phenotypes in the TME after repeated cisplatin NP treatment was examined. Particularly, this study was aimed at investigating the effects of NP damaged TAFs on neighboring cells and alteration of stromal structure after cisplatin treatment. Findings suggested that while off-targeted NP damaged TAFs and inhibited tumor growth after an initial dose, chronic exposure to cisplatin NP led to elevated secretion of Wnt16 in a paracrine manner in TAFs. Wnt16 upregulation was then attributed to heightened tumor cell resistance and stroma reconstruction. Results attest to the efficacy of Wnt16 knockdown in damaged TAFs as a promising combinatory strategy to improve efficacy of cisplatin NP in a stroma-rich bladder cancer model.
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To investigate how cells sense stiffness in settings structurally similar to native extracellular matrices, we designed a synthetic fibrous material with tunable mechanics and user-defined ...architecture. In contrast to flat hydrogel surfaces, these fibrous materials recapitulated cell-matrix interactions observed with collagen matrices including stellate cell morphologies, cell-mediated realignment of fibres, and bulk contraction of the material. Increasing the stiffness of flat hydrogel surfaces induced mesenchymal stem cell spreading and proliferation; however, increasing fibre stiffness instead suppressed spreading and proliferation for certain network architectures. Lower fibre stiffness permitted active cellular forces to recruit nearby fibres, dynamically increasing ligand density at the cell surface and promoting the formation of focal adhesions and related signalling. These studies demonstrate a departure from the well-described relationship between material stiffness and spreading established with hydrogel surfaces, and introduce fibre recruitment as a previously undescribed mechanism by which cells probe and respond to mechanics in fibrillar matrices.
Biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) is linked to the development of hereditary (VHL-associated) and sporadic clear-cell renal carcinomas as well as other ...abnormalities. The VHL gene product, pVHL, is part of an E3 ubiquitin ligase complex that targets the alpha subunits of the heterodimeric transcription factor HIF (hypoxia-inducible factor) for degradation in the presence of oxygen. Here we report that a HIF2alpha variant lacking both of its two prolyl hydroxylation/pVHL-binding sites prevents tumor inhibition by pVHL in a DNA-binding dependent manner. Conversely, downregulation of HIF2alpha with short hairpin RNAs is sufficient to suppress tumor formation by pVHL-defective renal carcinoma cells. These results establish that tumor suppression by pVHL is linked to regulation of HIF target genes.
Tumors grown in a stroma-rich mouse model resembling clinically advanced bladder carcinoma with UMUC3 and NIH 3T3 cells have high levels of fibroblasts and an accelerated tumor growth rate. We used ...this model to investigate the synergistic effect of combined gemcitabine monophosphate (GMP) nanoparticles and Cisplatin nanoparticles (Combo NP) on tumor-associated fibroblasts (TAFs). A single injection of Combo NP had synergistic anti-tumor effects while the same molar ratio of combined GMP and Cisplatin delivered as free drug (Combo Free) fell outside of the synergistic range. Combo NP nearly halted tumor growth with little evidence of general toxicity while Combo Free had only a modest inhibitory effect at 16mg/kg GMP and 1.6mg/kg Cisplatin. Combo NP increased levels of apoptosis within the tumor by approximately 1.3 folds (TUNEL analysis) and decreased α-SMA-positive fibroblast recruitment by more than 87% (immunofluorescence) after multiple injections compared with Combo Free, GMP NP or Cisplatin NP alone. The TAF-targeting capability of Combo NP was evaluated by double staining for TUNEL and α-SMA at various time points after a single injection. On day one after injection, 57% of the TUNEL-positive cells were identified as α-SMA-positive fibroblasts. By day four, tumor stroma was 85% depleted and 87% of the remaining TAFs were TUNEL-positive. Combo NP-treated tumors became 2.75 folds more permeable than those treated with Combo Free as measured by Evans Blue. We conclude that the antineoplastic effect of Combo NP works by first targeting TAFs and is more effective as an anti-tumor therapy than Combo Free, GMP NP or Cisplatin NP alone.
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The Cancer/Testes (CT) Antigen HORMAD1 is germ cell-restricted and plays developmental roles in generation and processing of meiotic DNA Double Strand Breaks (DSB). Many tumors aberrantly overexpress ...HORMAD1 yet the potential impact of this CT antigen on cancer biology is unclear. We tested a potential role of HORMAD1 in genome maintenance in lung adenocarcinoma cells. We show that HORMAD1 re-distributes to nuclear foci and co-localizes with the DSB marker γH2AX in response to ionizing radiation (IR) and chemotherapeutic agents. The HORMA domain and C-term disordered oligomerization motif are necessary for localization of HORMAD1 to IR-induced foci (IRIF). HORMAD1-depleted cells are sensitive to IR and camptothecin. In reporter assays, Homologous Recombination (HR)-mediated repair of targeted ISce1-induced DSBs is attenuated in HORMAD1-depleted cells. In Non-Homologous End Joining (NHEJ) reporter assays, HORMAD1-depletion does not affect repair of ISce1-induced DSB. Early DSB signaling events (including ATM phosphorylation and formation of γH2AX, 53BP1 and NBS1 foci) are intact in HORMAD1-depleted cells. However, generation of RPA-ssDNA foci and redistribution of RAD51 to DSB are compromised in HORMAD1-depleted cells, suggesting that HORMAD1 promotes DSB resection. HORMAD1-mediated HR is a neomorphic activity that is independent of its meiotic partners (including HORMAD2 and CCDC36. Bioinformatic analysis of TCGA data show that similar to known HR pathway genes HORMAD1 is overexpressed in lung adenocarcinomas. Overexpression of HR genes is associated with specific mutational profiles (including copy number variation). Taken together, we identify HORMAD1-dependent DSB repair as a new mechanism of radioresistance and a probable determinant of mutability in lung adenocarcinoma.
High-grade urothelial cancer contains intrinsic molecular subtypes that exhibit differences in underlying tumor biology and can be divided into luminal-like and basal-like subtypes. We describe here ...the first subtype-specific murine models of bladder cancer and show that Upk3a-Cre
; Trp53
; Pten
; Rosa26
(UPPL, luminal-like) and BBN (basal-like) tumors are more faithful to human bladder cancer than the widely used MB49 cells. Following engraftment into immunocompetent C57BL/6 mice, BBN tumors were more responsive to PD-1 inhibition than UPPL tumors. Responding tumors within the BBN model showed differences in immune microenvironment composition, including increased ratios of CD8
:CD4
and memory:regulatory T cells. Finally, we predicted and confirmed immunogenicity of tumor neoantigens in each model. These UPPL and BBN models will be a valuable resource for future studies examining bladder cancer biology and immunotherapy.
This work establishes human-relevant mouse models of bladder cancer.
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