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 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.

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.

The induction of macrophage-deactivating (interleukin-10 [IL-10] and transforming growth factor beta [TGF-␤]) and macrophage-activating (IL-1, IL-6, and tumor necrosis factor alpha [TNF-␣]) cytokines by lipoarabinomannan (LAM) from pathogenic Mycobacterium tuberculosis Erdman and H37Rv strains (ManLAM) and nonpathogenic mycobacteria (AraLAM) in human blood monocytes was examined. ManLAM was significantly less potent in induction of TNF-␣, IL-1, IL-6, and IL-10 protein and mRNA, whereas its ability to induce TGF-␤ was similar to that of AraLAM. Differences in induction of TNF-␣ mRNA by the two LAM preparations only became apparent at late time points of culture (24 h). The induction of TNF-␣ and IL-1 by purified protein derivative of M. tuberculosis was significantly stronger than that by ManLAM. Pretreatment of monocytes with ManLAM did not, however, interfere with cytokine induction by lipopolysaccharide or AraLAM. The extensive mannosyl capping of arabinose termini of ManLAM may underlie the lack of ability to induce some cytokines (IL-1, TNF-␣, and IL-10) and the retained ability to induce TGF-␤. The latter may have a role in shifting the cytokine milieu in favor of survival of M. tuberculosis.

We have previously demonstrated that constant 20 mmHg extracellular pressure increases serumopsonized 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 MAPK␣ 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 Ϯ 20.5% in DMSO-treated THP-1 macrophages and by 20.9 Ϯ 9% in primary monocytes (P Ͻ 0.05 each). SB-203580 (20 M) reduced basal p38 MAPK phosphorylation by 34.7 Ϯ 2.1% in THP-1 macrophages and prevented pressure activation of p38. p38 MAPK␣ 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 MAPK␣ siRNA reduced total FAK protein by 40 -50%, and total ERK by 10 -15%, but increased phosphorylated ERK 1.4 Ϯ 0.1-fold. p38 MAPK␣ 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 FAKdependent inverse effect on p38 MAPK␣ that might subsequently downregulate ERK.force; inflammation; infection; leukocyte; mechanotransduction; signal transduction MONOCYTES AND MACROPHAGES are recruited to sites of inflammation and play critical roles in innate host defense mechanisms. Tissue pressure is often altered in association with inflammation or infection. Mechanical stimuli such as pressure are known to modulate cellular morphology and function in other cell types (7,38,39,73,79). We have previously reported that constant low extracellular pressure increases phagocytosis by phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages (67). Although focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) are activated by physical force caused by pressure in some other cell types (2,4,44,75,76,80), the effect of pressure on macrophage phagocytosis appears partially mediated by inhibition of a FAK-ERK signal pathway (67). Like ERK, the mitogen-activated protein kinase (MAPK) p38 is activated by various stress stimuli, including lipopolysaccharide (LPS) stimulation in macrophages and physical force...