Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin. In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Contralateral suppression of aldosterone at adrenal venous sampling predicts hyperkalemia following adrenalectomy for primary aldosteronism.

BACKGROUND: We aimed to determine whether a greater degree of contralateral suppression of aldosterone secretion at adrenal venous sampling predicted the development of postoperative hyperkalemia after unilateral adrenalectomy for primary aldosteronism. METHODS: A retrospective analysis of patients undergoing unilateral adrenalectomy for primary aldosteronism between 2004-2015 was performed. Clinical and biochemical parameters of patients who developed hyperkalemia (≥5.2 mmol/L) after unilateral adreanlectomy were compared with those who remained normokalemic. The contralateral suppression index was defined as the aldosterone-to-cortisol ratio from the nondominant adrenal vein divided by the aldosterone-to-cortisol ratio from the external iliac vein. RESULTS: Of 192 patients who met criteria for inclusion, 12 (6.3%) developed hyperkalemia (median serum potassium 5.5 mmol/L, range 5.2-6.2 mmol/L), with a median time to onset of 13.5 days (range 7-55 days). Five patients had transiently increased serum potassium concentrations that normalized spontaneously. Four patients received mineralocorticoid replacement therapy with fludrocortisone. On univariate analysis, hyperkalemic patients had slightly greater preoperative serum creatinine levels (1.2 vs 1.0 mg/dL, P = .01), higher postoperative creatinine (1.3 vs 1.0 mg/dL, P = .02), lesser median contralateral suppression index (0.14 vs 0.27, P = .03), and larger adenomas (1.9 vs 1.4 cm, P = .02). On multivariable logistic regression, the contralateral suppression index remained the only significant predictor of postoperative hyperkalemia (P = .04) with an optimal cut-off of <0.47. CONCLUSION: Hyperkalemia after unilateral adrenalectomy for primary aldosteronism is uncommon and usually transient, but may require mineralocorticoid supplementation. Patients with a contralateral suppression index of <0.47 require meticulous follow-up and monitoring of serum potassium concentrations after unilateral adrenalectomy.

Scrambler Therapy for the management of chronic pain.

PURPOSE: Chronic pain is a widespread and debilitating condition, encountered by physicians in a variety of practice settings. Although many pharmacologic and behavioral strategies exist for the management of this condition, treatment is often unsatisfactory. Scrambler Therapy is a novel, non-invasive pain modifying technique that utilizes trans-cutaneous electrical stimulation of pain fibers with the intent of re-organizing maladaptive signaling pathways. This review was conducted to further evaluate what is known regarding the mechanisms and mechanics of Scrambler Therapy and to investigate the preliminary data pertaining to the efficacy of this treatment modality. METHODS: The PubMed/Medline, SCOPUS, EMBASE, and Google Scholar databases were searched for all articles published on Scrambler Therapy prior to November 2015. All case studies and clinical trials were evaluated and reported in a descriptive manner. RESULTS: To date, 20 reports, of varying scientific quality, have been published regarding this device; all but one small study, published only as an abstract, provided results that appear positive. CONCLUSION: The positive findings from preliminary studies with Scrambler Therapy support that this device provides benefit for patients with refractory pain syndromes. Larger, randomized studies are required to further evaluate the efficacy of this approach.

Linked deficiencies in extracellular PP(i) and osteopontin mediate pathologic calcification associated with defective PC-1 and ANK expression.

Osteopontin and PP(i) both suppress hydroxyapatite deposition. Extracellular PP(i) deficiency causes spontaneous hypercalcification, yet unchallenged osteopontin knockout mice have only subtle mineralization abnormalities. We report that extracellular PP(i) deficiency promotes osteopontin deficiency and correction of osteopontin deficiency prevents hypercalcification, suggesting synergistic inhibition of hydroxyapatite deposition. Nucleotide pyrophosphatase phosphodiesterase (NPP) isozymes including PC-1 (NPP1) function partly to generate PP(i), a physiologic calcification inhibitor. PP(i) transport is modulated by the membrane channel protein ANK. Spontaneous articular cartilage calcification, increased vertebral cortical bone formation, and peripheral joint and intervertebral ossific ankylosis are associated with both PC-1 deficiency and expression of truncated ANK in ank/ank mice. To assess how PC-1, ANK, and PP(i) regulate both calcification and cell differentiation, we studied cultured PC-1 -/- and ank/ank mouse calvarial osteoblasts. PC-1 -/- osteoblasts demonstrated approximately 50% depressed NPP activity and markedly lowered extracellular PP(i) associated with hypercalcification. These abnormalities were rescued by transfection of PC-1 but not of the NPP isozyme B10/NPP3. PC-1 -/- and ank/ank cultured osteoblasts demonstrated not only comparable extracellular PP(i) depression and hypercalcification but also marked reduction in expression of osteopontin (OPN), another direct calcification inhibitor. Soluble PC-1 (which corrected extracellular PP(i) and OPN), and OPN itself (> or = 15 pg/ml), corrected hypercalcification by PC-1 -/- and ank/ank osteoblasts. Thus, linked regulatory effects on extracellular PP(i) and OPN expression mediate the ability of PC-1 and ANK to regulate calcification.

One of two chondrocyte-expressed isoforms of cartilage intermediate-layer protein functions as an insulin-like growth factor 1 antagonist.

OBJECTIVE: Aging and osteoarthritic (OA) cartilage commonly demonstrate enhanced expression of the large, transforming growth factor beta (TGFbeta)-inducible glycoprotein cartilage intermediate-layer protein (CILP) as well as enhanced extracellular inorganic pyrophosphate (PPi) that promotes the deposition of calcium pyrophosphate dihydrate crystals. In normal chondrocytes, TGFbeta induces elevated chondrocyte extracellular PPi. Insulin-like growth factor 1 (IGF-1) normally blocks this response and reduces extracellular PPi. However, chondrocyte resistance to IGF-1 is observed in OA and aging. Because CILP was reported to chromatographically fractionate with PPi-generating nucleotide pyrophosphatase phosphodiesterase (NPP) activity, it has been broadly assumed that CILP itself has NPP activity. Our objective was to directly define CILP functions and their relationship to IGF-1 in chondrocytes. METHODS: Using primary cultures of articular chondrocytes from the knee, we defined the function of the previously described CILP (CILP-1) and of a recently described 50.6% identical protein that we designated the CILP-2 isoform. RESULTS: Both CILP isoforms were constitutively expressed by primary cultured articular chondrocytes, but only CILP-1 expression was detectable in cultured knee meniscal cartilage cells. Neither CILP isoform had intrinsic NPP activity. But CILP-1 blocked the ability of IGF-1 to decrease extracellular PPi, an activity specific for the CILP-1 N-terminal domain. The CILP-1 N-terminal domain also suppressed IGF-1-induced (but not TGFbeta-induced) proliferation and sulfated proteoglycan synthesis, and it inhibited ligand-induced IGF-1 receptor autophosphorylation. CONCLUSION: Two CILP isoforms are differentially expressed by chondrocytes. Neither CILP isoform exhibits PPi-generating NPP activity. But, increased expression of CILP-1, via N-terminal domain-mediated inhibitory effects of CILP-1 on chondrocyte IGF-1 responsiveness, could impair chondrocyte growth and matrix repair and indirectly promote PPi supersaturation in aging and OA cartilage.

The predictive value of laboratory findings in patients with primary hyperparathyroidism.

BACKGROUND: Despite extensive preoperative imaging and intraoperative "gadgetry" to facilitate localization of abnormal parathyroid glands, the onus of identification and resection remains with the surgeon in the operating room. We pondered the relative usefulness of routine laboratory studies to the surgeon as predictive guides to the intraoperative findings in patients with primary hyperparathyroidism (HPT). STUDY DESIGN: Pre- and postoperative laboratory data were analyzed in 166 consecutive patients (1998 to 1999) undergoing successful cervical exploration for sporadic primary HPT and were correlated with the pathologic findings. Patients with secondary HPT, multiple endocrine neoplasia, familial HPT, and parathyroid carcinoma were not included in this study. RESULTS: One hundred eighteen women and 48 men (mean age = 63 years) with parathyroid adenoma (n = 155) and sporadic hyperplasia (n = 11) were evaluated. Mean parathormone (PTH) levels varied little with the pathology: adenoma = 9.6 pmol/L and hyperplasia = 10.2 pmol/L (p > 0.05). In patients with parathyroid adenoma, analysis of preoperative measures showed a positive correlation (r = 0.48, p < 0.0001) with PTH and gland weight. The correlation appeared to be the strongest in the tails of the distribution; in 22 of 23 (96%) cases with PTH levels lower than 6 pmol/L, the offending lesion or lesions were less than 400 mg; in all six cases with PTH levels higher than 18.0 pmol/L, the abnormal gland or glands weighed more than 800 mg. PTH levels between 6 and 18 pmol/L revealed mean adenoma weight of 757 mg (median = 420 mg; range = 50 to 5,500 mg). CONCLUSIONS: Extreme values of PTH in patients with single-gland parathyroid disease alert the surgeon to the likelihood of small or large parathyroid adenomas. Laboratory studies do not differentiate adenoma from hyperplasia, nor do they pinpoint the size of abnormal glands with moderate-range PTH values.