Expanded Circulating Tumor Cells from a Patient with ALK-Positive Lung Cancer Present with EML4-ALK Rearrangement Along with Resistance Mutation and Enable Drug Sensitivity Testing: A Case Study.

The emergence of liquid biopsy using circulating tumor cells (CTCs) as a resource to identify genomic alterations in cancer presents new opportunities for diagnosis, therapy, and surveillance. We identified EML4-ALK gene rearrangement in expanded CTCs from a patient with ALK-positive lung adenocarcinoma. At the time of radiographic progression, CTCs obtained from the patient revealed a drug resistance mutation (i.e., L1196M on the ALK gene). CTCs were expanded ex vivo and drug sensitivity testing was performed using two ALK inhibitors, crizotinib and ceritinib. The half maximal inhibitory concentration of ceritinib was 1664 nM compared with crizotinib (2268 nM), showing that ceritinib was a more potent ALK inhibitor. We show that it is feasible to detect serial genetic alterations in expanded CTCs and perform in vitro drug screening. These findings support the clinical utility of CTCs not only for diagnosis, but also a potential tool for drug sensitivity testing in distinct subsets of lung cancer and for personalized precision medicine.

Oncogenic Potential of CYP24A1 in Lung Adenocarcinoma.

INTRODUCTION: We have previously demonstrated that a subset of lung cancer cells express higher CYP24A1 mRNA, a metabolizing enzyme for 1,25-D3, compared to benign tumors or surrounding normal lung and that high CYP24A1 mRNA expression is associated with poor prognosis in resected lung adenocarcinoma (AC). We hypothesized that CYP24A1 has oncogenic potential and increased CYP24A1 expression may contribute to tumor growth, whereas, CYP24A1 targeting may reduce tumor burden. METHODS: Two low CYP24A1 expressing human lung cancer cell lines (SK-LU-1 and Calu-6) were stably transfected either with an empty lentiviral vector or with the CYP24A1 expressing vector. Over-expression of mRNA and protein levels of CYP24A1 in SK-LU-1 and Calu-6 were confirmed using qRT-PCR and immunoblotting respectively. Next, effects of targeting CYP24A1 were examined in lung cancer cells (A549 and H441), which express higher basal levels of CYP24A1. Finally, we studied the effects of stable knockdown of CYP24A1 in xenograft models. RESULTS: Over-expression of CYP24A1 correlated with accelerated cell growth and invasion compared to control vector-transfected cells. CYP24A1 over-expression also increased RAS protein expression. Knockdown of CYP24A1 using either si- or shRNA reduced CYP24A1 mRNA and protein expression and significantly decreased cell proliferation (30-60%) and reduced mitochondrial DNA content compared to non-targeting (NT) si-/shRNA transfected/transduced cells. Transfection with CYP24A1 siRNA also decreased total RAS protein, thus reducing phosphorylated AKT. Importantly, stable knockdown of CYP24A1 in A549 and H441 lung tumor xenograft models resulted in tumor growth delay and smaller tumor size as evident from tumor bioluminescence and tumor volume measurement studies. Such observations were correlated with decreased tumor cell proliferation as evidenced by reduced Ki67 and Cyclin D staining. CONCLUSIONS: Our data suggest that CYP24A1 has oncogenic properties mediated by increasing RAS signaling, targeting of which may provide an alternate strategy to treat a subset of lung AC.

Expansion of CTCs from early stage lung cancer patients using a microfluidic co-culture model.

The potential utility of circulating tumor cells (CTCs) to guide clinical care in oncology patients has gained momentum with emerging micro- and nanotechnologies. Establishing the role of CTCs in tumor progression and metastasis depends both on enumeration and on obtaining sufficient numbers of CTCs for downstream assays. The numbers of CTCs are few in early stages of cancer, limiting detailed molecular characterization. Recent attempts in the literature to culture CTCs isolated from metastatic patients using monoculture have had limited success rates of less than 20%. Herein, we have developed a novel in-situ capture and culture methodology for ex-vivo expansion of CTCs using a three dimensional co-culture model, simulating a tumor microenvironment to support tumor development. We have successfully expanded CTCs isolated from 14 of 19 early stage lung cancer patients. Expanded lung CTCs carried mutations of the TP53 gene identical to those observed in the matched primary tumors. Next-generation sequencing further revealed additional matched mutations between primary tumor and CTCs of cancer-related genes. This strategy sets the stage to further characterize the biology of CTCs derived from patients with early lung cancers, thereby leading to a better understanding of these putative drivers of metastasis.

KRAS-G12C mutation is associated with poor outcome in surgically resected lung adenocarcinoma.

INTRODUCTION: The aim of this study was to examine the effects of KRAS mutant subtypes on the outcome of patients with resected lung adenocarcinoma (AC). METHODS: Using clinical and sequencing data, we identified 179 patients with resected lung AC for whom KRAS mutational status was determined. A multivariate Cox model was used to identify factors associated with disease-free survival (DFS) and overall survival (OS). Publicly available mutation and gene-expression data from lung cancer cell lines and lung AC were used to assess whether distinct KRAS mutant variants have a different profile. RESULTS: Patients with KRAS mutation had a significantly shorter DFS compared with those with KRAS wild-type (p = 0.009). Patients with KRAS-G12C mutant tumors had significantly shorter DFS compared with other KRAS mutants and KRAS wild-type tumors (p < 0.001). In the multivariate Cox model, KRAS-G12C remained as an independent prognostic marker for DFS (Hazard ratio = 2.46, 95% confidence interval 1.51-4.00, p < 0.001) and for OS (Hazard ratio = 2.35, 95% confidence interval 1.35-4.10, p = 0.003). No genes were statistically significant when comparing the mutational or transcriptional profile of lung cancer cell lines and lung AC harboring KRAS-G12C with other KRAS mutant subtypes. Gene set enrichment analysis revealed that KRAS-G12C mutants overexpressed epithelial to mesenchymal transition genes and expressed lower levels of genes predicting KRAS dependency. CONCLUSIONS: KRAS-G12C mutation is associated with worse DFS and OS in resected lung AC. Gene-expression profiles in lung cancer cell lines and surgically resected lung AC revealed that KRAS-G12C mutants had an epithelial to mesenchymal transition and a KRAS-independent phenotype.

Propofol's effects on phagocytosis, proliferation, nitrate production, and cytokine secretion in pressure-stimulated microglial cells.

BACKGROUND: Intracranial hypertension complicates severe traumatic brain injury frequently and might be associated with poor outcomes. Traumatic brain injury induces a neuroinflammatory response by microglial activation and upregulation of proinflammatory cytokines, such as interleukin-1ß, tumor necrosis factor alpha, and interleukin-6. To elucidate the effect of increased intracranial pressure on microglial function, we studied the effects of increased extracellular pressure on primary human microglial cell phagocytosis, proliferation, cytokine secretion, and total nitrate production. In addition, because many patients receive propofol during anesthesia or intensive care unit sedation, we evaluated whether propofol alters the effects of pressure. METHODS: Human microglial cells were pretreated with (2.5-20 µg/mL) propofol or Intralipid as a vehicle control were incubated at ambient atmospheric pressure or at 15 or 30 mm Hg increased pressure for 2 h for phagocytosis assays or 24 h for proliferation, cytokine secretion, and total nitrate production studies. Phagocytosis was determined by incorporation of intracellular fluorescent latex beads. Tumor necrosis factor alpha, interleukin-1ß, and interleukin-6 were assayed by sandwich enzyme-linked immunosorbent assay and total nitrate by Greiss reagent. RESULTS: Increased extracellular pressure stimulated phagocytosis versus untreated microglial cells or cells treated with an Intralipid vehicle control. Propofol also stimulated microglial phagocytosis at ambient pressure. Increased pressure, however, decreased phagocytosis in the presence of propofol. Pressure also increased microglial tumor necrosis factor-α and interleukin-1ß secretion and propofol pretreatment blocked the pressure-stimulated effect. Interleukin-6 production was not altered either by pressure or by propofol. Pressure also induced total nitrate secretion, and propofol pretreatment decreased basal as well as pressure-induced microglial nitrate production. CONCLUSION: Extracellular pressures consistent with increased intracranial pressure after a head injury activate inflammatory signals in human primary microglial cells in vitro, stimulating phagocytosis, proliferation, tumor necrosis factor-α, interleukin-1ß, and total nitrate secretion but not affecting interleukin-6. Such inflammatory events may contribute to the worsened prognosis of traumatic brain injury after increased intracranial pressure. Because propofol alleviated these potentially proinflammatory effects, these results suggest that the inflammatory cascade activated by intracranial pressure might be targeted by propofol in patients with increased intracranial pressure after traumatic brain injury.

beta(1)-integrin mediates pressure-stimulated phagocytosis.

BACKGROUND: Extracellular pressure alterations in infection, inflammation, or positive pressure ventilation may influence macrophage phagocytosis. We hypothesized that pressure modulates beta1-integrins to stimulate phagocytosis. METHODS: We assayed fibroblast phagocytosis of fluorescent latex beads at ambient or 20 mm Hg increased pressure, and macrophage integrin phosphorylation by Western blot. RESULTS: Pressure did not alter phagocytosis in beta(1)-integrin null GD25 fibroblasts, but stimulated phagocytosis in fibroblasts expressing wild-type beta(1)-integrin. In phorbol myristate acetate-differentiated THP-1 macrophages, pressure stimulated beta(1)-integrin T788/789 phosphorylation, but not S785 phosphorylation. Furthermore, pressure stimulated phagocytosis in cells expressing an inactivating S785A point mutation or a T788D substitution to mimic a constitutively phosphorylated threonine, but not in cells expressing an inactivating TT788/9AA mutation. CONCLUSIONS: The effects of pressure on phagocytosis are not limited to macrophages but generalize to other phagocytic cells. These results suggest that pressure stimulates phagocytosis via increasing beta(1)-integrin T789 phosphorylation. Interventions that target beta(1)-integrin threonine 789 phosphorylation may modulate phagocytic function.

Irrigant divalent cation concentrations influence bacterial adhesion.

BACKGROUND: Surgical wounds are frequently contaminated by microbes, but rarely become infected if the bacterial burden is low, and irrigation is used to reduce contamination. Wound fluids are low in calcium and high in magnesium. We hypothesized that manipulating irrigant divalent cation concentrations might influence bacterial adhesion. METHODS: Staphylococcus aureus, E. coli, and Pseudomonas aeruginosa were stained with fluorescent calcein AM before plating onto fibroblast monolayers, collagen I, or uncoated bacteriologic plastic. After 1 h, wells were washed with HEPES-buffered pH-balanced sterile water without or with 5 mM CaCl(2), 5 mM MgCl(2), or 1 mM EDTA+EGTA, and the remaining adherent bacteria were assayed fluorometrically. RESULTS: Supplementing the irrigation with magnesium or chelators increased but calcium-supplemented irrigation reduced bacterial adhesion to collagen or fibroblasts. Nonspecific electrostatic bacterial adhesion to uncoated plastic was unaffected by calcium. CONCLUSION: Bacterial adhesion to mammalian cells and matrix proteins is influenced by divalent cations, and pathogenic bacteria may be adapted to adhere under the low calcium high magnesium conditions in wounds. Although these results await confirmation for other bacteria, and in vivo validation and safety-testing, they suggest that supplementing wound irrigation with 5 mM CaCl(2) may reduce bacterial adhesion and subsequent wound infection.

Increased extracellular pressure provides a novel adjuvant stimulus for enhancement of conventional dendritic cell maturation strategies.

Dendritic cell (DC)-based vaccine strategies have gained increasing popularity in recent years. Methods for ex vivo generation of immunocompetent mature DCs still require optimization. DCs have been shown to phenotypically mature under elevated pressure. We compared the effects of pressure on DC maturation with LPS- and cytokine-stimulation. Human monocyte-derived immature or LPS- and cytokine-matured DCs were exposed to ambient or 40 mmHg increased pressure for 12h, then assessed for expression of CD80, CD86, CD40, MHC-I/II, and inflammatory cytokine production. DCs were also evaluated for capacity to stimulate T-cell proliferation by co-culture with allogeneic lymphocytes. Pressure significantly increased cytokine production and expression of all surface molecules on immature DC other than MHC-I and CD40. Pressure/LPS-treated DCs displayed further upregulation of MHC-I, CD40, and IL-12p70. Cytokine-matured DCs appeared less responsive to pressure. T-cell proliferation correlated with MHC expression. Results suggest mechanical stimulation of DCs may provide a useful adjuvant to TLR-agonist maturation strategies.

Propofol inhibits pressure-stimulated macrophage phagocytosis via the GABAA receptor and dysregulation of p130cas phosphorylation.

Surgical stress and anesthesia result in systemic immunosuppression. Propofol, a commonly used anesthetic agent, alters immune cell functions. Previously, we demonstrated that extracellular pressure increases macrophage phagocytosis. We hypothesized that propofol might influence pressure-induced macrophage phagocytosis in monocytes from patients undergoing surgery. Pressure (20 mmHg above ambient pressure) augmented phagocytosis in monocytes from non-propofol-anesthetized patients but reduced phagocytosis in monocytes from propofol-anesthetized patients. In vitro, propofol stimulated phagocytosis but reversed pressure-induced phagocytosis in THP-1 macrophages and monocytes from healthy volunteers. The GABA(A) receptor antagonists picrotoxin and SR-95531 did not affect basal THP-1 phagocytosis or prevent pressure-stimulated phagocytosis. However, picrotoxin and SR-95531 negated the inhibitory effect of pressure in propofol-treated cells without altering propofol-induced phagocytosis. Phosphorylation of the adaptor protein p130cas was inversely related to phagocytosis: it was inhibited by pressure or propofol but increased by pressure + propofol compared with propofol alone. Reduction of p130cas by small interfering RNA in THP-1 macrophages increased basal phagocytosis and prevented pressure and propofol effects. In conclusion, propofol may alter macrophage responses to pressure via the GABA(A) receptor and p130cas, whereas pressure also acts via p130cas but independently of GABA(A) receptors. p130cas may be an important target for modulation of macrophage function in anesthetized patients.

Extracellular-regulated kinase activation regulates replication of Mycobacterium avium intracellularly in primary human monocytes.

Mycobacterium avium-intracellulare (MAI) is a ubiquitous environmental pathogen that causes disseminated infection in immunocompromised patients, such as those with human immunodeficiency virus, interleukin-12 deficiency, or interferon-gamma receptor mutation. Colony morphotypes are associated with MAI pathogenicity. Our previous studies have reported that smooth-transparent (SmT) morphotypes are more virulent and induce less cytokine (interleukin-1beta and tumor necrosis factor-alpha) production by human monocytes than the smooth-domed (SmD) morphotypes. Mitogen-activated protein (MAP) kinases such as extracellular-regulated kinase (ERK) are activated by the phagocytosis of particle antigens in macrophages, and this ERK activation subsequently influences cytokine expression and the control of intracellular pathogen growth. The influence of MAP kinase activation on MAI replication in human monocytes was examined. Peripheral blood monocytes isolated from healthy subjects by Ficoll-Hypaque sedimentation were infected with virulent SmT or avirulent SmD MAI without or with MAP kinase inhibitors. MAP kinase activities were determined by in vitro kinase assay, intracellular MAI growth by CFU assay, and cytokines by enzyme-linked immunosorbent assay. MAI infection induced ERK and p38 activation. Inhibition of ERK by PD98059, but not p38, significantly increased intracellular MAI growth. Tumor necrosis factor-alpha release and interleukin-1beta production in response to MAI were reduced by MAP kinase inhibition. p38 inhibition tended to reduce cytokine production more substantially. These data suggest that ERK activation limits intra-monocytic MAI replication and enhances monocytic cytokine release, whereas p38 activation influences only cytokine release. The effect of MAP kinases on MAI growth might thus be mediated by the modulation of cytokine production.

Increased pressure stimulates aberrant dendritic cell maturation.

Patients with malignancy typically exhibit abnormal dendritic cell profiles. Interstitial tumor pressure is increased 20-50 mmHg over that in normal tissue. We hypothesized that elevated pressure in the tumor microenvironment may influence dendritic cell (DC) phenotype and function. Monocyte-derived immature and mature DC isolated from healthy human donors were exposed to either ambient or 40 mmHg increased pressure at 37 degrees C for 12 hours, then assessed for expression of CD80, CD86, CD83, CD40, MHC-I and MHC-II. IL-12 production and phagocytosis of CFSE-labeled tumor lysate were assessed in parallel. Elevated pressure significantly increased expression of all co-stimulatory and MHC molecules on mature DC. Immature DC significantly increased expression of CD80, CD86, CD83 and MHC-II, but not MHC-I and CD40, versus ambient pressure controls. Pressure-treated immature DC phenotypically resembled mature DC controls, but produced low IL-12. Phenotypic maturation correlated with decreased phagocytic capacity. These results suggest increased extracellular pressure may cause aberrant DC maturation and impair tumor immunosurveillance.

Akt2, but not Akt1 or Akt3 mediates pressure-stimulated serum-opsonized latex bead phagocytosis through activating mTOR and p70 S6 kinase.

Monocytes and macrophages play critical roles in innate host defense and are sensitive to mechanical stimuli. Tissue pressure is often altered in association with inflammation or infection. Low pressure (20 mmHg), equivalent to normal tissue pressure, increases phagocytosis by primary monocytes and PMA-differentiated THP-1 macrophages, in part by FAK and ERK inhibition and p38 activation. PI-3K is required for macrophage phagocytosis, but whether PI-3K mediates pressure-stimulated phagocytosis is not known. Furthermore, little is known about the role played by the PI-3K downstream Kinases, Akt, and p70 S6 kinase (p70S6K) in modulating macrophage phagocytosis. Thus, we studied the contribution of PI-3K, Akt, and p70S6K to pressure-increased serum-opsonized bead phagocytosis. Pressure-induced p85 PI-3K translocation from cytosolic to membrane fractions and increased Akt activation by 36.1 +/- 12.0% in THP-1 macrophages. LY294002 or Akt inhibitor IV abrogated pressure-stimulated but not basal phagocytosis. Basal Akt activation was inhibited 90% by LY294002 and 70% by Akt inhibitor IV. Each inhibitor prevented Akt activation by pressure. SiRNA targeted to Akt1, Akt2, or Akt3 reduced Akt1, Akt2, and Akt3 expression by 50%, 45%, and 40%, respectively. However, only Akt2SiRNA abrogated the pressure-stimulated phagocytosis without affecting basal. Pressure also activated mTOR and p70S6K. mTORSiRNA and p70S6K inhibition by rapamycin or p70S6KSiRNA blocked pressure-induced, but not basal, phagocytosis. Changes in tissue pressure during inflammation may regulate macrophage phagocytosis by activation of PI-3K, which activates Akt2, mTOR, and p70S6K.

Activation of p38 MAPKalpha by extracellular pressure mediates the stimulation of macrophage phagocytosis by pressure.

We have previously demonstrated that constant 20 mmHg extracellular pressure increases serum-opsonized latex bead phagocytosis by phorbol 12-myristate 13-acetate (PMA)- differentiated THP-1 macrophages in part by inhibiting focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Because p38 MAPK is activated by physical forces in other cells, we hypothesized that modulation of p38 MAPK might also contribute to the stimulation of macrophage phagocytosis by pressure. We studied phagocytosis in PMA-differentiated THP-1 macrophages, primary human monocytes, and human monocyte-derived macrophages (MDM). p38 MAPK activation was inhibited using SB-203580 or by p38 MAPKalpha small interfering RNA (siRNA). Pressure increased phagocytosis in primary monocytes and MDM as in THP-1 cells. Increased extracellular pressure for 30 min increased phosphorylated p38 MAPK by 46.4 +/- 20.5% in DMSO-treated THP-1 macrophages and by 20.9 +/- 9% in primary monocytes (P < 0.05 each). SB-203580 (20 microM) reduced basal p38 MAPK phosphorylation by 34.7 +/- 2.1% in THP-1 macrophages and prevented pressure activation of p38. p38 MAPKalpha siRNA reduced total p38 MAPK protein by 50-60%. Neither SB-203580 in THP-1 cells and peripheral monocytes nor p38 MAPK siRNA in THP-1 cells affected basal phagocytosis, but each abolished pressure-stimulated phagocytosis. SB-203580 did not affect basal or pressure-reduced FAK activation in THP-1 macrophages, but significantly attenuated the reduction in ERK phosphorylation associated with pressure. p38 MAPKalpha siRNA reduced total FAK protein by 40-50%, and total ERK by 10-15%, but increased phosphorylated ERK 1.4 +/- 0.1-fold. p38 MAPKalpha siRNA transfection did not affect the inhibition of FAK-Y397 phosphorylation by pressure but prevented inhibition of ERK phosphorylation. Changes in extracellular pressure during infection or inflammation regulate macrophage phagocytosis by a FAK-dependent inverse effect on p38 MAPKalpha that might subsequently downregulate ERK.

Extracellular pressure stimulates macrophage phagocytosis by inhibiting a pathway involving FAK and ERK.

We hypothesized that changes in extracellular pressure during inflammation or infection regulate macrophage phagocytosis through modulating the focal adhesion kinase (FAK)-ERK pathway. Undifferentiated (monocyte-like) or PMA-differentiated (macrophage-like) THP-1 cells were incubated at 37 degrees C with serum-opsonized latex beads under ambient or 20-mmHg increased pressure. Pressure did not affect monocyte phagocytosis but significantly increased macrophage phagocytosis (29.9 +/- 1.8 vs. 42.0 +/- 1.6%, n = 9, P < 0.001). THP-1 macrophages constitutively expressed activated FAK, ERK, and Src. Exposure of macrophages to pressure decreased ERK and FAK-Y397 phosphorylation (77.6 +/- 7.9%, n = 7, P < 0.05) but did not alter FAK-Y576 or Src phosphorylation. FAK small interfering RNA (SiRNA) reduced FAK expression by >75% and the basal amount of phosphorylated FAK by 25% and significantly increased basal macrophage phagocytosis (P < 0.05). Pressure inhibited FAK-Y397 phosphorylation in mock-transfected or scrambled SiRNA-transfected macrophages, but phosphorylated FAK was not significantly reduced further by pressure in cells transfected with FAK SiRNA. Pressure increased phagocytosis in all three groups. However, FAK-SiRNA-transfected cells exhibited only 40% of the pressure effect on phagocytosis observed in scrambled SiRNA-transfected cells so that phagocytosis inversely paralleled FAK activation. PD-98059 (50 microM), an ERK activation inhibitor, increased basal phagocytosis (26.9 +/- 1.8 vs. 31.7 +/- 1.1%, n = 15, P < 0.05), but pressure did not further increase phagocytosis in PD-98059-treated cells. Pressure also inhibited ERK activation after mock transfection or transfection with scrambled SiRNA, but transfection of FAK SiRNA abolished ERK inhibition by pressure. Pressure did not increase phagocytosis in MonoMac-1 cells that do not express FAK. Increased extracellular pressure during infection or inflammation enhances macrophage phagocytosis by inhibiting FAK and, consequently, decreasing ERK activation.