The timely diagnosis and therapeutic monitoring of human renal cell carcinoma (RCC) is limited by the lack of specific biomarkers. To identify candidate RCC biomarkers, we used 2-DE gel electrophoresis with mass spectrometry and 2-DE spot intensity-based ROC analysis to analyze 18 sets of paired normal and RCC tumor tissue including conventional, papillary, and chromophobe subtypes. Validation was performed with RCC patient plasma samples and confirmed by clustergram, shRNA, and immunohistochemistry assays. Cardinal candidates were evaluated by ELISA. The leading candidate biomarker that was upregulated in RCC samples according to the clustergram and validation analysis was nicotinamide N-methyltransferase (NNMT) (13/15, P < 0.0001). Other upregulated candidate biomarkers that were identified by this method include ferritin, hNSE, NM23, secretagogin, and L-plastin. The upregulation of NNMT in RCC was confirmed by immunoblotting and immunohistochemistry. Analysis of fractionated membrane-associated proteins identified CAP-G, mitofillin, tubulin alpha, RBBP7, and HSP27. Of these, RBBP7 and HSP27 were highly expressed in the chromophobe subtype of RCC (3/3) but were absent from conventional RCC (0/3). The triple combination of the NNMT, FTL, and hNSE biomarkers had the highest predictive capacity of 0.993, while NNMT was the single, most powerful candidate diagnostic biomarker for all types of RCC.

IntroductionAlthough development of anoikis-resistant myofibroblasts during tissue remodeling is known to be associated with tumor invasion, the mechanism by which myofibroblasts become resistant to anoikis is unknown. We previously demonstrated laminin-332 upregulation in the fibrosis around invasive ductal carcinoma (IDC). Because laminin-332 promotes cell survival through binding to integrins, we hypothesized that invasive breast cancer cells confer an anoikis-resistant phenotype on myofibroblasts by upregulating laminin-332 expression during tissue remodeling. Here, we demonstrate that invasive breast cancer cells induce laminin-332 upregulation and integrin β4 neoexpression in myofibroblasts to confer an anoikis-resistant phenotype.MethodsThree types of fibroblasts were isolated from the tumor burden, the fibrosis, and normal tissue of patients with early stage IDC (less than 10 mm diameter), designated cancer-associated fibroblasts (CAFs), interface fibroblasts (InFs), and normal breast fibroblasts (NBFs), respectively. To investigate direct and indirect crosstalk with tumor cells, fibroblasts were co-cultured with invasive MDA-MB-231 or noninvasive MCF7 cells or in conditioned medium. Anoikis resistance of fibroblasts was measured by cell viability and caspase-3 activity after incubation on poly-HEMA coated plates for 72 hours. Involvement of laminin-332/integrin α3β1 or α6β4 signaling in anoikis resistance was confirmed by treatment with purified laminin-332 or blocking antibodies against laminin-332, integrin β1, or integrin β4.ResultsMDA-MB-231 cells induced laminin-332 upregulation and integrin β4 neoexpression in fibroblasts, leading to anoikis resistance. InFs showed a higher endogenous level of laminin-332 than did CAFs and NBFs. After stimulation with MDA-MB-231-conditioned medium, laminin-332 expression of InFs was dramatically increased and maintained under anoikis conditions. Laminin-332 upregulation was also observed in CAFs and NBFs, but at a lower level than in InFs. Laminin-332 induced Akt (Ser473) phosphorylation by binding to integrin α3β1. Integrin β4 neoexpression induced laminin-332-independent Rac1 activation and promoted anoikis resistance in fibroblasts approximately twofold more effectively than did laminin-332, regardless of the type of fibroblast. In addition, integrin β4 expression suppressed fibroblast aggregation in conditions of anoikis.ConclusionInvasive breast cancer cells confer an anoikis-resistant phenotype on myofibroblasts during tissue remodeling by inducing laminin-332 upregulation and integrin β4 neoexpression. Interface fibroblasts appear to be the primary myofibroblasts that interact with invasive tumor cells during tissue remodeling.

Cochlea macrophages regulate cochlea inflammation and may harbors the potentials to protect hearing function from injury, including acoustic overstimulation. Cochlea macrophage numbers increase at 3–7 days after acoustic stimulation. However, the exact timing of macrophage infiltration and maturation from inflammatory monocytes is unclear. Furthermore, neutrophils may also be involved in this process. Therefore, in this study, we investigated time-dependent immune cell infiltration, macrophage transformation, and neutrophil involvement following acoustic stimulation. Flow cytometry and immunofluorescence were conducted in C-X3-C motif chemokine receptor 1 (CX3CR1)+/GFP mice after acoustic overstimulation (at baseline and at 1, 2, 3, and 5 days after exposure to 120 dB for 1 h) to identify inflammatory monocytes in the cochlea. RNA-sequencing and quantitative polymerase chain reaction were performed to identify differentially expressed genes. Inflammatory monocytes infiltrated into the lower portion of the lateral wall within 2 days after acoustic overstimulation (dpn), followed by transformation into macrophages at 3–5 dpn via CX3CR1 upregulation and Ly6C downregulation. In addition, inflammatory monocytes were aggregated inside the collecting venule only at 1 dpn. Neutrophils were not a major type of phagocyte during this response. The gene encoding C-C motif chemokine ligand 2 gene was significantly upregulated as early as 3 h after acoustic overstimulation. Given these results, treatment to control immune response after a noise-induced hearing loss should be applied as soon as possible.

Radioresistance is one of the main determinants of treatment outcome in oral squamous cell carcinoma (OSCC), and treatment of radioresistant OSCC is difficult due to cross resistance to other conventional treatments. We aimed to identify whether genetically modified oncolytic adenovirus expressing relaxin (RLX), which affects collagen metabolism, can effectively inhibit growth of the radioresistant OSCC. Therapeutic effect of oncolytic adenovirus was compared between radiosensitive and radioresistant OSCC cell lines in vitro and in vivo, and spread of adenovirus throughout the tumor mass was verified by immunohistochemistry (IHC). Oncolytic adenovirus effectively killed cancer cells and there was no significant difference in the cytotoxic effect between radiosensitive and radioresistant OSCC cell lines. In animal experiments, the adenovirus significantly reduced the size of tumor, and there was no significant difference between radiosensitive and radioresistant OSCC. In IHC, RLX expressing adenovirus showed better proliferation and eliminated collagens more effectively compared to RLX nonexpressing adenovirus. These findings suggested that genetically modified oncolytic adenovirus can effectively inhibit growth of the radioresistant OSCC and might be a new therapeutic option in radioresistant OSCC.

No abstract