mTOR Regulation of N-Myc Downstream Regulated 1 (NDRG1) Phosphorylation in Clear Cell Renal Cell Carcinoma.

The mechanistic target of rapamycin (mTOR) kinase is a component of two signaling complexes that are known as mTOR complex 1 (mTORC1) and mTORC2. We sought to identify mTOR-phosphorylated proteins that are differently expressed in clinically resected clear cell renal cell carcinoma (ccRCC) relative to pair-matched normal renal tissue. Using a proteomic array, we found N-Myc Downstream Regulated 1 (NDRG1) showed the greatest increase (3.3-fold) in phosphorylation (on Thr346) in ccRCC. This was associated with an increase in total NDRG1. RICTOR is a required subunit in mTORC2, and its knockdown decreased total and phospho-NDRG1 (Thr346) but not NDRG1 mRNA. The dual mTORC1/2 inhibitor, Torin 2, significantly reduced (by ~100%) phospho-NDRG1 (Thr346). Rapamycin is a selective mTORC1 inhibitor that had no effect on the levels of total NDRG1 or phospho-NDRG1 (Thr346). The reduction in phospho-NDRG1 (Thr346) due to the inhibition of mTORC2 corresponded with a decrease in the percentage of live cells, which was correlated with an increase in apoptosis. Rapamycin had no effect on ccRCC cell viability. Collectively, these data show that mTORC2 mediates the phosphorylation of NDRG1 (Thr346) in ccRCC. We hypothesize that RICTOR and mTORC2-mediated phosphorylation of NDRG1 (Thr346) promotes the viability of ccRCC cells.

Osteoregenerative Potential of 3D-Printed Poly ε-Caprolactone Tissue Scaffolds In Vitro Using Minimally Manipulative Expansion of Primary Human Bone Marrow Stem Cells.

The repair of orthopedic and maxillofacial defects in modern medicine currently relies heavily on the use of autograft, allograft, void fillers, or other structural material composites. This study examines the in vitro osteo regenerative potential of polycaprolactone (PCL) tissue scaffolding, fabricated via a three-dimensional (3D) additive manufacturing technology, i.e., a pneumatic micro extrusion (PME) process. The objectives of this study were: (i) To examine the innate osteoinductive and osteoconductive potential of 3D-printed PCL tissue scaffolding and (ii) To perform a direct in vitro comparison of 3D-printed PCL scaffolding with allograft Allowash® cancellous bone cubes with regards to cell-scaffold interactions and biocompatibility with three primary human bone marrow (hBM) stem cell lines. This study specifically examined cell survival, cell integration, intra-scaffold cell proliferation, and differentiation of progenitor cells to investigate the potential of 3D-printed PCL scaffolds as an alternative to allograft bone material for the repair of orthopedic injuries. We found that mechanically robust PCL bone scaffolds can be fabricated via the PME process and the resulting material did not elicit detectable cytotoxicity. When the widely used osteogenic model SAOS-2 was cultured in PCL extract medium, no detectable effect was observed on cell viability or proliferation with multiple test groups showing viability ranges of 92.2% to 100% relative to a control group with a standard deviation of ±10%. In addition, we found that the honeycomb infill pattern of the 3D-printed PCL scaffold allowed for superior mesenchymal stem-cell integration, proliferation, and biomass increase. When healthy and active primary hBM cell lines, having documented in vitro growth rates with doubling times of 23.9, 24.67, and 30.94 h, were cultured directly into 3D-printed PCL scaffolds, impressive biomass increase values were observed. It was found that the PCL scaffolding material allowed for biomass increase values of 17.17%, 17.14%, and 18.18%, compared to values of 4.29% for allograph material cultured under identical parameters. It was also found that the honeycomb scaffold infill pattern was superior to the cubic and rectangular matrix structures, and provided a superior microenvironment for osteogenic and hematopoietic progenitor cell activity and auto-differentiation of primary hBM stem cells. Histological and immunohistochemical studies performed in this work confirmed the regenerative potential of PCL matrices in the orthopedic setting by displaying the integration, self-organization, and auto-differentiation of hBM progenitor cells within the matrix. Differentiation products including mineralization, self-organizing "proto-osteon" structures, and in vitro erythropoiesis were observed in conjunction with the documented expression of expected bone marrow differentiative markers including CD-99 (>70%), CD-71 (>60%), and CD-61 (>5%). All of the studies were conducted without the addition of any exogenous chemical or hormonal stimulation and exclusively utilized the abiotic and inert material polycaprolactone; setting this work apart from the vast majority of contemporary investigations into synthetic bone scaffold fabrication In summary, this study demonstrates the unique clinical potential of 3D-printed PCL scaffolds for stem cell expansion and incorporation into advanced microstructures created via PME manufacturing to generate a physiologically inert temporary bony defect graft with significant autograft features for enhanced end-stage healing.

Unusual Presentation of Primary Pulmonary Sarcomatous Cancer With Brain Metastasis: A Case Report.

Pulmonary sarcomatous carcinoma is a rare subtype of non-small cell lung cancer (NSCLC). This cancer has very low survival rates primarily due to its aggressive nature and propensity for early spread to abdominal organs and the skeletal system. Remarkably, brain metastasis is observed at later stages of the disease, likely attributing to the high fatality rate after the disease progresses to the brain tissue. In our case, a 79-year-old female with a 45-pack-year smoking history sought medical attention at a primary care clinic due to a 3-month history of recurrent right-sided chest pain. Notably, she denied cough, sputum production, palpitations, or syncope. CT chest revealed a 6.8 x 3.5 cm mass in the right upper lobe (RUL) of the lung, with evidence of obstruction and infiltration of the adjacent chest wall. A PET scan indicated increased uptake in the mass and the presence of smaller pulmonary nodules in both lungs, and multiple nodules in the upper left arm, abdomen, right inguinal region, left thigh, and cecum. Importantly, no intracranial lesions were detected. A subsequent colonoscopy yielded normal findings. Histopathologic examination of the lung mass and cell markers was consistent with a diagnosis of sarcomatous carcinoma of the lung. Only three days after the initial clinic visit, the patient presented with numbness and tingling in her lower extremities. Brain MRI revealed multiple bilateral brain metastases accompanied by significant vasogenic edema, prompting treatment with steroid therapy and brain radiation therapy. Subsequent chemotherapy/immunotherapy with Nab-paclitaxel /carboplatin/atezolizumab was initiated but led to significant treatment-related toxicities. Consequently, the treatment plan was adjusted to a single dose of single-agent immunotherapy using pembrolizumab. Unfortunately, the patient chose to discontinue treatment and eventually passed away after 13 days of palliative care. Compared to other lung cancer subtypes, brain metastasis in sarcomatous lung cancer is infrequent due to its lower prevalence among all lung cancer cases. Furthermore, sarcomatous lung cancer has a reduced propensity for developing brain metastasis when compared to other forms of non-small cell lung cancer (NSCLC). Regrettably, the prognosis for sarcomatous lung cancer with brain metastasis remains generally unfavorable, signaling an advanced stage of the disease with limited treatment options.

A Rare Presentation of Small Bowel Perforation Secondary to Microscopic Metastasis of Non-small Cell Lung Cancer (NSCLC): A Case Report.

Gastrointestinal tract perforation (GITP) due to metastatic lung cancer is an exceptionally rare occurrence. Symptoms can range from mild abdominal discomfort to severe and life-threatening bowel perforation. In this case presentation, we describe an unusual instance involving squamous non-small cell lung cancer (NSCLC), where microscopic metastases in the small bowel led to bowel perforation. Our patient, a 71-year-old male with a history of stage IIIa squamous cell carcinoma in the right lung and smoking history, completed chemoradiation therapy and is currently undergoing treatment with durvalumab. He presented to the ED with complaints of abdominal pain, nausea, and abdominal distention. His review of systems revealed no other significant issues, and his vital signs were stable. However, the abdominal examination revealed noticeable distention with tenderness upon palpation and guarding. Laboratory results were significant for leukocytosis with a left shift of neutrophils and mildly elevated kidney function. A CT scan of the abdomen and pelvis revealed widespread pneumoperitoneum, indicating a bowel perforation. Consequently, the patient underwent an urgent exploratory laparotomy, during which a small bowel perforation measuring 0.6 cm x 0.3 cm in the jejunum was identified, necessitating the resection of the affected bowel segment. Intraoperative esophagogastroduodenoscopy (EGD) showed normal findings. The histopathological examination of the resected bowel revealed clusters of squamous cell carcinoma with a desmoplastic reaction, affecting the submucosal and muscular layers at the site of the defect, with surgical margins free of tumor or inflammation. This finding indicated metastatic disease originating from the known lung squamous cell carcinoma. After the operation, the patient was admitted to the ICU due to septic shock caused by E. coli and Klebsiella peritonitis, requiring intubation and circulatory support with pressors. Ultimately, he was discharged following treatment. This case underscores the rarity of symptomatic bowel perforation from micro-metastasis in squamous NSCLC and emphasizes the need for rigorous assessment and timely surgical intervention. However, it is important to recognize the significant risk of complications and a high mortality rate, leading to a challenging prognosis. As such, individuals with a known history of lung carcinoma who present with abdominal symptoms should undergo comprehensive evaluation to prevent life-threatening complications through early intervention.

Expression of poly-ADP-ribose polymerase (PARP) in endometrial adenocarcinoma: Prognostic potential.

BACKGROUND: In the United States endometrial carcinoma is the most common female gynecologic malignancy. An average of more than 60,000 new cases of endometrial carcinomas have been diagnosed yearly over the past 5 years, with a higher incidence occurring in the central Appalachian states of Ohio and West Virginia. In the U.S., the national average of newly diagnosed endometrial carcinomas is 26.8 in every 100,000 women, while in the states of Ohio and West Virginia the average is 30.5 and 31.1 in every 100,000 women, respectively. This notable increase in the incidence of endometrial carcinomas may be due a variety of elevated risk factors including but not limited to: tobacco use, obesity, and genetic predisposition of the predominant demographic. The American Cancer Society estimates that approximately 55,000 new cases of endometrial carcinoma will be diagnosed in 2020 yet, this disease is widely considered understudied and under-represented in mainstream cancer research circles. METHODS: The aim of this study was to quantitate the co-expression of two DNA repair proteins poly-ADP-ribose polymerase 1 and 2 (Parp-1 and Parp-2) by enzyme- linked immuno-sorbent assay (ELISA) in 60 endometrioid endometrial tumor samples and compare their expression to matched non-malignant endometrial tissue from the same corresponding donors from central Appalachia. RESULTS: We found that Parp-1 was significantly overexpressed in endometrial carcinoma relative to corresponding normal tissue. This overexpression implicates Parp inhibition therapy as a possible treatment for the disease. Our results also found a protective effect of native Parp-2 expression in non-malignant endometrial tissue with each 1 ng/mL increase in PARP-2 concentration in normal tissue was associated with a 10 % reduction in the hazard of tumor progression (HR = 0.90; p = 0.039) and a 21 % reduction in the hazard of death (HR = 0.79; p = 0.044). CONCLUSIONS: This study demonstrated the over-expression of the druggable target Parp-1 in endometrial adenocarcinoma and observed a strong negative correlation of native Parp-2 expression and disease progression via the quantification of the Parp proteins using enzyme- linked immuno-sorbent assay (ELISA) assays.

Minimally Manipulative Method for the Expansion of Human Bone Marrow Mesenchymal Stem Cells to Treat Osseous Defects.

Lack of standardization of clinically compliant culture protocols of mesenchymal stem cells for re-implantation in humans have hindered clinical progress in the field of tissue regeneration to repair maxillofacial and orthopedic defects. The goal of this study was to establish a clinically relevant osteogenic protocol for collection and expansion of autologous stem cells to be used at Marshall University for re-implantation and repair of maxillofacial and orthopedic conditions. Human bone marrow (hBM) samples were collected from patients undergoing intramedullary nail fixation for closed femoral fractures. hBM mesenchymal cells were expanded by growing them first in Petri dishes for two weeks, followed by a week of culture using Perfecta 3D Hanging Drop Plates®. Various scaffold materials were tested and analyzed for cellular integration, vitality, and differentiation capacity of harvested hBM-MSCs including: 60/40 blend of hydroxyapatite biomatrix; Acellular bone composite discs; Allowash®, cancellous bone cubes; PLGA (poly lactic-co-glycolic acid); and Woven chitin derived fiber. We found that the 3D spheroid culture allowed production of hBM mesenchymal cells that retained osteoblast differentiation capacity over a monolayer culture of hBM-MSCs without the need to use chemical or hormonal modulation. We also observed that hydroxyapatite and Allowash cancellous bone scaffolds allowed better cell integration and viability properties as compared to other materials tested in this study. In conclusion, the multimodal culture methodology we developed creates actively differentiating stem-cell spheroids that can then be readily utilized in clinical practices to improve the regeneration of tissues of the head and the body.

Environments Associated with Moderate-to-Vigorous Physical Activity and Sedentary Behavior of Colorectal Cancer Survivors.

PURPOSE: Physical activity (PA) is an effective intervention for improving the quality of life of colorectal cancer survivors (CRC) and may reduce the risk of cancer recurrence and cancer specific and all-cause mortality. However, most CRC survivors are not sufficiently active to receive these benefits. Sedentary behavior (SB) has also been linked to morbidity and mortality risk independent of activity level, thereby presenting an additional opportunity to improve health outcomes of CRC survivors. The built environment is known to influence PA and SB; however, little is known about where CRC survivors engage in PA and SB. The objective of this exploratory study was to objectively identify locations where CRC survivors engage in PA and SB in order to inform health promoting interventions. METHOD: Activity and location of CRC survivors (n = 31) was monitored for 1 week between January 2014 and April 2015 in Nova Scotia, Canada. Bouts of PA and SB were time-matched with GPS data to attribute bouts to specific geographic locations. RESULTS: Participants' home environment was the main location for both time spent in PA bouts (73.7 %) and time spent in SB bouts (90.5 %). CONCLUSION: This study is the first to objectively identify environments where CRC survivors are active and sedentary. These findings highlight the importance of considering the home environment when developing intervention strategies to increase PA and reduce SB in CRC survivors.

A new complementary procedure for patients affected by head and neck cancer: Chemo-predictive assay.

INTRODUCTION: Administration of ineffective anticancer therapy is associated with unnecessary toxicity and development of resistant clones. Cancer stem cells (CSCs) resist chemotherapy, thereby causing relapse of the disease. Thus, development of a test that identifies the most effective chemotherapy management offers great promise for individualized anticancer treatments. We have developed an ex vivo chemotherapy drug response assay (ChemoID(®)), which measures the sensitivity of CSCs as well as the bulk of tumor cells to a variety of chemotherapy agents to assist an oncologist in making treatment decisions. METHODS: Three patients affected by oral cancer were referred. RESULTS: Biopsy showed a well-differentiated squamous cell carcinoma (G1) in case 1, a G2 adenocarcinoma in case 2 and a G3 squamous cell carcinoma in case 3. In all of the three cases, after clinical inspection and suspicion of a diagnosis of cancer, a double biopsy was performed. One specimen was sent to the ChemoID laboratory for chemosensitivity assay and the other for histological analysis. Chemotherapy dose response curves were generated, and grouped in 3 categories: 1. No response (less than 30% cell kill), Intermediate (30-60% cell kill), and 3. Sensitive (60% cell kill or above). CONCLUSIONS: This procedure may be useful in helping physicians choose an effective chemotherapy regimen for head and neck cancer patients and lower treatment costs by eliminating ineffective chemotherapies and unnecessary toxicity particularly in elderly patients.

Chemo-predictive assay for targeting cancer stem-like cells in patients affected by brain tumors.

Administration of ineffective anticancer therapy is associated with unnecessary toxicity and development of resistant clones. Cancer stem-like cells (CSLCs) resist chemotherapy, thereby causing relapse of the disease. Thus, development of a test that identifies the most effective chemotherapy management offers great promise for individualized anticancer treatments. We have developed an ex vivo chemotherapy sensitivity assay (ChemoID), which measures the sensitivity of CSLCs as well as the bulk of tumor cells to a variety of chemotherapy agents. Two patients, a 21-year old male (patient 1) and a 5-month female (patient 2), affected by anaplastic WHO grade-III ependymoma were screened using the ChemoID assay. Patient 1 was found sensitive to the combination of irinotecan and bevacizumab, which resulted in a prolonged disease progression free period of 18 months. Following recurrence, the combination of various chemotherapy drugs was tested again with the ChemoID assay. We found that benzyl isothiocyanate (BITC) greatly increased the chemosensitivity of the ependymoma cells to the combination of irinotecan and bevacizumab. After patient 1 was treated for two months with irinotecan, bevacizumab and supplements of cruciferous vegetable extracts containing BITC, we observed over 50% tumoral regression in comparison with pre-ChemoID scan as evidenced by MRI. Patient 2 was found resistant to all treatments tested and following 6 cycles of vincristine, carboplatin, cyclophosphamide, etoposide, and cisplatin in various combinations, the tumor of this patient rapidly progressed and proton beam therapy was recommended. As expected animal studies conducted with patient derived xenografts treated with ChemoID screened drugs recapitulated the clinical observation. This assay demonstrates that patients with the same histological stage and grade of cancer may vary considerably in their clinical response, suggesting that ChemoID testing which measures the sensitivity of CSLCs as well as the bulk of tumor cells to a variety of chemotherapy agents could lead to more effective and personalized anticancer treatments in the future.