Xiaoyin Xu

Selective inhibition of JNK with a peptide inhibitor attenuates pain hypersensitivity and tumor growth in a mouse skin cancer pain model

Cancer pain significantly affects the quality of cancer patients, and current treatments for this pain are limited. C-Jun N-terminal kinase (JNK) has been implicated in tumor growth and neuropathic pain sensitization. We investigated the role of JNK in cancer pain and tumor growth in a skin cancer pain model. Injection of luciferase-transfected B16-Fluc melanoma cells into a hindpaw of mouse induced robust tumor growth, as indicated by increase in paw volume and fluorescence intensity. Pain hypersensitivity in this model developed rapidly (<5 days) and reached a peak in 2 weeks, and was characterized by mechanical allodynia and heat hyperalgesia. Tumor growth was associated with JNK activation in tumor mass, dorsal root ganglion (DRG), and spinal cord and a peripheral neuropathy, such as loss of nerve fibers in the hindpaw skin and induction of ATF-3 expression in DRG neurons. Repeated systemic injections of D-JNKI-1 (6 mg/kg, i.p.), a selective and cell-permeable peptide inhibitor of JNK, produced an accumulative inhibition of mechanical allodynia and heat hyperalgesia. A bolus spinal injection of D-JNKI-1 also inhibited mechanical allodynia. Further, JNK inhibition suppressed tumor growth in vivo and melanoma cell proliferation in vitro. In contrast, repeated injections of morphine (5 mg/kg), a commonly used analgesic for terminal cancer, produced analgesic tolerance after 1 day and did not inhibit tumor growth. Our data reveal a marked peripheral neuropathy in this skin cancer model and important roles of the JNK pathway in cancer pain development and tumor growth. JNK inhibitors such as D-JNKI-1 may be used to treat cancer pain.

Prevention of nodal metastases in breast cancer following the lymphatic migration of paclitaxel-loaded expansile nanoparticles.

Although breast cancer patients with localized disease exhibit an excellent long-term prognosis, up to 40% of patients treated with local resection alone may harbor occult nodal metastatic disease leading to increased locoregional recurrence and decreased survival. Given the potential for targeted drug delivery to result in more efficacious locoregional control with less morbidity, the current study assessed the ability of drug-loaded polymeric expansile nanoparticles (eNP) to migrate from the site of tumor to regional lymph nodes, locally deliver a chemotherapeutic payload, and prevent primary tumor growth as well as lymph node metastases. Expansile nanoparticles entered tumor cells and paclitaxel-loaded eNP (Pax-eNP) exhibited dose-dependent cytotoxicity in vitro and significantly decreased tumor doubling time in vivo against human triple negative breast cancer in both microscopic and established murine breast cancer models. Furthermore, migration of Pax-eNP to axillary lymph nodes resulted in higher intranodal paclitaxel concentrations and a significantly lower incidence of lymph node metastases. These findings demonstrate that lymphatic migration of drug-loaded eNP provides regionally targeted delivery of chemotherapy to both decrease local tumor growth and strategically prevent the development of nodal metastases within the regional tumor-draining lymph node basin.

Treating triple-negative breast cancer by a combination of rapamycin and cyclophosphamide: An in vivo bioluminescence imaging study

Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, has been shown to inhibit the growth of oestrogen positive breast cancer. However, triple-negative (TN) breast cancer is resistant to rapamycin treatment in vitro. We set to test a combination treatment of rapamycin with DNA-damage agent, cyclophosphamide, in a TN breast cancer model. By binding to and disrupting cellular DNA, cyclophosphamide kills cells via interfering with their normal functions. We assessed the responses of nude mice bearing tumour xenografts of TN MDA-MB-231 cells to the combination of rapamycin and cyclophosphamide in both orthotopic mammary and lung-metastasis models. We tracked tumour growth and metastasis by bioluminescent imaging and examined the expression of Ki67, CD34 and HIF-1alpha in tumour tissues by immunohistochemistry and apoptosis index with TUNEL assay, and found that MDA-MB-231 cells are sensitive to rapamycin therapy in orthotopic mammary, but not in lung with metastasis. Rapamycin when combined with cyclophosphamide is found to have a more significant effect in reducing tumour volume and metastasis with a much improved survival rate. Our data also show that the sensitivity of TN tumours to rapamycin is associated with the microenvironment of the tumour cells. The data indicate that in a relatively hypoxic environment HIF-1alpha may play a role in mediating the anti-cancer effect of rapamycin and cyclophosphamide may prevent the feedback activation of Akt by rapamycin. Overall our results show that rapamycin plus cyclophosphamide can achieve an improved efficacy in suppressing tumour growth and metastasis, suggesting that the combination therapy can be a promising treatment option for TN cancer.

C Terminus of Clostridium perfringens Enterotoxin Downregulates CLDN4 and Sensitizes Ovarian Cancer Cells to Taxol and Carboplatin

Purpose: We have previously shown that CLDN4 (encoding claudin-4), a cell tight junction (TJ) protein, is highly expressed in human epithelial ovarian carcinomas (EOC) but undetectable in normal ovaries. CLDN4 has been identified as a specific receptor for C terminus of Clostridium perfringens enterotoxin (C-CPE), a nontoxic molecule that may disrupt TJ barrier function and enhance cellular absorption. The purpose of this study was to determine the potential clinical applications of C-CPE and its effects on CLDN4 expression in EOC. Experimental Design: Using a 3-dimensional culture model and monolayer culture of EOC cells, we examined the effects of C-CPE on CLDN4 expression by quantitative real-time PCR, immunofluorescence, and Western blot. The synergistic effect of C-CPE to clinically relevant chemotherapies (Taxol and Carboplatin) was observed in EOC culture and xenograft mice. Furthermore, we determined through oligonucleotide microarray analysis that the transcript profile alterations dysregulated as a consequence of C-CPE treatment. Results: C-CPE treatment decreased protein expression and relocated CLDN4 from cell–cell contact regions to the cytoplasm. Particularly, C-CPE sensitized EOC cells to chemotherapeutic administration at low dosages and significantly inhibited tumor growth in a nontoxic manner. Furthermore, we provided genome-wide molecular evidence that C-CPE treatment is involved in the stimulation of the ubiquitin–proteasome pathway and the inhibition of cell metabolism in EOC cells. Conclusions: The addition of C-CPE can enhance the effectiveness of Taxol or Carboplatin and significantly inhibited EOC cell growth in a CLDN4-dependent manner, suggesting that C-CPE may have promising therapeutic potential for EOC. Clin Cancer Res; 17(5); 1065–74. ©2010 AACR.

In vivo Fluorescence Imaging of Muscle Cell Regeneration by Transplanted EGFP-labeled Myoblasts

In vivo fluorescence imaging (FLI) enables monitoring fluorescent protein (FP)-labeled cells and proteins in living organisms noninvasively. Here, we examined whether this modality could reach a sufficient sensitivity to allow evaluation of the regeneration process of enhanced green fluorescent protein (eGFP)-labeled muscle precursors (myoblasts). Using a basic FLI station, we were able to detect clear fluorescence signals generated by 40,000 labeled cells injected into a tibialis anterior (TA) muscle of mouse. We observed that the signal declined to approximately 25% on the 48 hours of cell injection followed by a recovery starting at the second day and reached a peak of approximately 45% of the original signal by the 7th day, suggesting that the survived population underwent a limited run of proliferation before differentiation. To assess whether transplanted myoblasts could form satellite cells, we injured the transplanted muscles repeatedly with cardiotoxin. We observed a recovery of fluorescence signal following a disappearance of the signal after each cardiotoxin injection. Histology results showed donor-derived cells located underneath basal membrane and expressing Pax7, confirming that the regeneration observed by imaging was indeed mediated by donor-derived satellite cells. Our results show that FLI is a powerful tool that can extend our ability to unveil complicated biological processes such as stem cell-mediated regeneration.

A method based on rank-ordered filter to detect edges in cellular image

To extract morphological features about nuclei from microscopy cellular image, it is usually required to find the edges of nuclei at first. Standard edge detection methods may not produce satisfactory results due to the varying brightness and background in cellular image. It is important to extract close, smooth, and correct edges in order to compute features like compactness, convexity, roundness, and etc. We present a new method to detect edges of nuclei in microscopy images. The method is based on using median filtering to compute the total variation with respect to the central pixel in a filter window. This step exploits one important feature of median filter, i.e., within the filter window, the total variation (TV) with respect to the median is always less than or equal to the TV with respect to the original center pixel. In other words, median filtering looks for the output that minimizes the total variation within the filtering window. The resulting image has enhanced contrast along the boundary of nuclei. As the final step, we use popular edge detection methods such as Canny detector and Laplacian of Gaussian to find edges of nuclei in the image. Examples from processing real cellular image obtained by light microscope show that the method obtains better edges in terms of connectivity, smoothness, and closely following the boundary of nuclei.

Paclitaxel-Loaded Expansile Nanoparticles in a Multimodal Treatment Model of Malignant Mesothelioma

BACKGROUND Malignant mesothelioma has a poor prognosis even when treated aggressively with multimodal therapy. Traditional murine tumor models can be used to evaluate drug efficacy and toxicity in malignant mesothelioma, but not to assess the effect of a multimodal approach that includes the surgical resection of tumor. We therefore developed a murine model of multimodal therapy in which we evaluated paclitaxel-loaded expansile nanoparticles (Pax-eNP) for delivering intracavitary chemotherapy in malignant mesothelioma. METHODS Paclitaxel-loaded expansile nanoparticles (Pax-eNP) of 100 nm, designed to release drug at an endosomal pH below 5, were synthesized. Xenografts of human malignant mesothelioma were established intraperitoneally in nude mice, followed by cytoreductive surgery (CRS) via laparotomy, and with omentectomy and resection of abdominal fat pads done 14 days later. At fascial closure, 10 mg/kg paclitaxel was delivered as traditional paclitaxel/paclitaxel Cremophor-EL (Pax-CE) or Pax-eNP. Morbidity and survival were assessed over a period of 90 days. RESULTS Cytoreductive surgery in mice was feasible and reproducible, and incurred less than 5% operative mortality. By itself, CRS did not significantly prolong survival; however, the addition of intraoperative Pax-CE or Pax-eNP significantly increased survival as compared with that of mice with untreated disease. In the case of Pax-eNP, the increase in survival was also statistically significant as compared with that following resection alone. CONCLUSIONS A murine model of CRS for malignant mesothelioma allows the in vivo assessment of multimodal therapy, including nanoparticle delivery. Combination therapy was superior to no treatment or CRS alone in prolonging survival. Treatment with Pax-eNP improved overall survival in the setting of CRS, suggesting that Pax-eNP merits further evaluation for intracavitary drug delivery following the surgical resection of malignant mesothelioma.

Mononuclear Cells from Dedifferentiation of Mouse Myotubes Display Remarkable Regenerative Capability

Certain lower organisms achieve organ regeneration by reverting differentiated cells into tissue‐specific progenitors that re‐enter embryonic programs. During muscle regeneration in the urodele amphibian, postmitotic multinucleated skeletal myofibers transform into mononucleated proliferating cells upon injury, and a transcription factor‐msx1 plays a role in their reprograming. Whether this powerful regeneration strategy can be leveraged in mammals remains unknown, as it has not been demonstrated that the dedifferentiated progenitor cells arising from muscle cells overexpressing Msx1 are lineage‐specific and possess the same potent regenerative capability as their amphibian counterparts. Here, we show that ectopic expression of Msx1 reprograms postmitotic, multinucleated, primary mouse myotubes to become proliferating mononuclear cells. These dedifferentiated cells reactivate genes expressed by embryonic muscle progenitor cells and generate only muscle tissue in vivo both in an ectopic location and inside existing muscle. More importantly, distinct from adult muscle satellite cells, these cells appear both to fuse with existing fibers and to regenerate myofibers in a robust and time‐dependent manner. Upon transplantation into a degenerating muscle, these dedifferentiated cells generated a large number of myofibers that increased over time and replenished almost half of the cross‐sectional area of the muscle in only 12 weeks. Our study demonstrates that mammals can harness a muscle regeneration strategy used by lower organisms when the same molecular pathway is activated. Stem Cells 2014;32:2492–2501

An image processing pipeline to detect and segment nuclei in muscle fiber microscopic images.

Muscle fiber images play an important role in the medical diagnosis and treatment of many muscular diseases. The number of nuclei in skeletal muscle fiber images is a key bio‐marker of the diagnosis of muscular dystrophy. In nuclei segmentation one primary challenge is to correctly separate the clustered nuclei. In this article, we developed an image processing pipeline to automatically detect, segment, and analyze nuclei in microscopic image of muscle fibers. The pipeline consists of image pre‐processing, identification of isolated nuclei, identification and segmentation of clustered nuclei, and quantitative analysis. Nuclei are initially extracted from background by using local Otsus threshold. Based on analysis of morphological features of the isolated nuclei, including their areas, compactness, and major axis lengths, a Bayesian network is trained and applied to identify isolated nuclei from clustered nuclei and artifacts in all the images. Then a two‐step refined watershed algorithm is applied to segment clustered nuclei. After segmentation, the nuclei can be quantified for statistical analysis. Comparing the segmented results with those of manual analysis and an existing technique, we find that our proposed image processing pipeline achieves good performance with high accuracy and precision. The presented image processing pipeline can therefore help biologists increase their throughput and objectivity in analyzing large numbers of nuclei in muscle fiber images. Microsc. Res. Tech. 77:547–559, 2014.

An image processing approach to analyze morphological features of microscopic images of muscle fibers.

We present an image processing approach to automatically analyze duo-channel microscopic images of muscular fiber nuclei and cytoplasm. Nuclei and cytoplasm play a critical role in determining the health and functioning of muscular fibers as changes of nuclei and cytoplasm manifest in many diseases such as muscular dystrophy and hypertrophy. Quantitative evaluation of muscle fiber nuclei and cytoplasm thus is of great importance to researchers in musculoskeletal studies. The proposed computational approach consists of steps of image processing to segment and delineate cytoplasm and identify nuclei in two-channel images. Morphological operations like skeletonization is applied to extract the length of cytoplasm for quantification. We tested the approach on real images and found that it can achieve high accuracy, objectivity, and robustness.