Colchicine inhibits pressure-induced tumor cell implantation within surgical wounds and enhances tumor-free survival in mice.

Iatrogenic tumor cell implantation within surgical wounds can compromise curative cancer surgery. Adhesion of cancer cells, in particular colon cancer cells, is stimulated by exposure to increased extracellular pressure through a cytoskeleton-dependent signaling mechanism requiring FAK, Src, Akt, and paxillin. Mechanical stimuli during tumor resection may therefore negatively impact patient outcome. We hypothesized that perioperative administration of colchicine, which prevents microtubule polymerization, could disrupt pressure-stimulated tumor cell adhesion to surgical wounds and enhance tumor-free survival. Ex vivo treatment of Co26 and Co51 colon cancer cells with colchicine inhibited pressure-stimulated cell adhesion to murine surgical wounds and blocked pressure-induced FAK and Akt phosphorylation. Surgical wound contamination with pressure-activated Co26 and Co51 cells significantly reduced tumor-free survival compared with contamination with tumor cells under ambient pressure. Mice treated with pressure-activated Co26 and Co51 cells from tumors preoperatively treated with colchicine in vivo displayed reduced surgical site implantation and significantly increased tumor-free survival compared with mice exposed to pressure-activated cells from tumors not pretreated with colchicine. Our data suggest that pressure activation of malignant cells promotes tumor development and impairs tumor-free survival and that perioperative colchicine administration or similar interventions may inhibit this effect.

ERK regulates strain-induced migration and proliferation from different subcellular locations.

Repetitive deformation like that engendered by peristalsis or villous motility stimulates intestinal epithelial proliferation on collagenous substrates and motility across fibronectin, each requiring ERK. We hypothesized that ERK acts differently at different intracellular sites. We stably transfected Caco-2 cells with ERK decoy expression vectors that permit ERK activation but interfere with its downstream signaling. Targeting sequences constrained the decoy inside or outside the nucleus. We assayed proliferation by cell counting and migration by circular wound closure with or without 10% repetitive deformation at 10 cycles/min. Confocal microscopy confirmed localization of the fusion proteins. Inhibition of phosphorylation of cytoplasmic RSK or nuclear Elk confirmed functionality. Both the nuclear-localized and cytosolic-localized ERK decoys prevented deformation-induced proliferation on collagen. Deformation-induced migration on fibronectin was prevented by constraining the decoy in the nucleus but not in the cytosol. Like the nuclear-localized ERK decoy, a Sef-overexpressing adenovirus that sequesters ERK in the cytoplasm also blocked the motogenic and mitogenic effects of strain. Inhibiting RSK or reducing Elk ablated both the mitogenic and motogenic effects of strain. RSK isoform reduction revealed isoform specificity. These results suggest that ERK must translocate to the nucleus to stimulate cell motility while ERK must act in both the cytosol and the nucleus to stimulate proliferation in response to strain. Selectively targeting ERK within different subcellular compartments may modulate or replace physical force effects on the intestinal mucosa to maintain the intestinal mucosal barrier in settings when peristalsis or villous motility are altered and fibronectin is deposited into injured tissue.

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.

The effects of increased extracellular deformation, pressure, and integrin phosphorylation on fibroblast migration.

Wound healing requires fibroblast migration. Increased pressure slows migration and ulcer healing. Pressure also induces beta1 integrin phosphorylation. We hypothesized that beta1 phosphorylation influences cell adhesion and migration. We compared the effects of increased pressure on the adhesion and motility of GD25 beta1-integrin null fibroblasts transfected with wild-type beta1A-integrin, S785A or TT788/9AA (phosphorylation-deficient), or T788D (constitutively phosphomimetic) mutants. GD25 beta1 null cells adhered less than wild type beta1A cells, suggesting adherence by non-integrin mechanisms. Preventing Ser-785 or Thr 788/789 phosphorylation reduced adhesion, suggesting that phosphorylation regulates adhesiveness. Substituting Asp for Thr788 stimulated adhesion on both substrates. Pressure decreased migration in all lines and on all matrixes, the most in wild type beta1A integrin cells and only slightly in beta1A TT788/9AA cells. In comparison, another physical force, repetitive deformation, increased migration in the beta1A integrin T788D, S785A, and wild type cells on fibronectin, and decreased migration on collagen. Deformation did not affect the migration of GD25 beta1-integrin null or TT788/9AA cells. Extracellular signal-regulated kinase (ERK) blockade neither altered basal migration nor prevented pressure inhibition, while the cellular deformation response on fibronectin was altered. beta1-Integrin phosphorylation regulates cellular adhesion and the deformation effects on motility. The pressure-induced motility response is independently regulated.

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.

FAK association with multiple signal proteins mediates pressure-induced colon cancer cell adhesion via a Src-dependent PI3K/Akt pathway.

Cancer cell adhesion is traditionally viewed as random, occurring if the cell's receptors match the substrate. Cancer cells are subjected to pressure and shear during growth against a constraining stroma, surgical manipulation, and passage through the venous and lymphatic system. Cells shed into a cavity such as the abdomen postoperatively also experience increased pressure from postoperative edema. Increased extracellular pressure stimulates integrin-mediated cancer cell adhesion via FAK and Src. PI 3-kinase (PI3K) inhibitors (LY294002 or wortmannin), Akt inhibitors, or Akt1 siRNA blocked adhesion stimulated by 15 mmHg pressure in SW620 or primary human malignant colonocytes. Pressure activated PI3K, tyrosine-phosphorylated and membrane-translocated the p85 subunit, and phosphorylated Akt. PI3K inhibitor (LY294002) prevented pressure-stimulated Akt Ser473 and FAK Tyr397, but not FAK576 or Src416 phosphorylation. PP2 inhibited PI3K activity and Akt phosphorylation. FAK siRNA did not affect pressure-induced PI3K activation but blocked Akt phosphorylation. Pressure also stimulated FAK or FAKY397F mutant translocation to the membrane. Akt inhibitor IV blocked pressure-induced Akt and FAK translocation. Pressure activated Src- and PI3K-dependently induced p85 interaction with FAK, and FAK with beta1 integrin. These results delineate a novel force-activated inside-out Src/PI3K/FAK/Akt pathway by which cancer cells regulate their own adhesion. These signals may be potential targets for inhibition of metastatic adhesion.

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.

Molecular Programming of Tumor-Infiltrating CD8+ T Cells and IL15 Resistance.

Despite clinical potential and recent advances, durable immunotherapeutic ablation of solid tumors is not routinely achieved. IL15 expands natural killer cell (NK), natural killer T cell (NKT) and CD8(+) T-cell numbers and engages the cytotoxic program, and thus is under evaluation for potentiation of cancer immunotherapy. We found that short-term therapy with IL15 bound to soluble IL15 receptor α-Fc (IL15cx; a form of IL15 with increased half-life and activity) was ineffective in the treatment of autochthonous PyMT murine mammary tumors, despite abundant CD8(+) T-cell infiltration. Probing of this poor responsiveness revealed that IL15cx only weakly activated intratumoral CD8(+) T cells, even though cells in the lung and spleen were activated and dramatically expanded. Tumor-infiltrating CD8(+) T cells exhibited cell-extrinsic and cell-intrinsic resistance to IL15. Our data showed that in the case of persistent viral or tumor antigen, single-agent systemic IL15cx treatment primarily expanded antigen-irrelevant or extratumoral CD8(+) T cells. We identified exhaustion, tissue-resident memory, and tumor-specific molecules expressed in tumor-infiltrating CD8(+) T cells, which may allow therapeutic targeting or programming of specific subsets to evade loss of function and cytokine resistance, and, in turn, increase the efficacy of IL2/15 adjuvant cytokine therapy. Cancer Immunol Res; 4(9); 799-811. ©2016 AACR.

The Diagnostic Significance of a Mucocele of the Appendix.

Appendiceal mucoceles are rare lesions with a variable clinical presentation often identified incidentally on imaging or at laparotomy/laparoscopy for an unrelated diagnosis. Mucocele of the appendix may be a benign or malignant process, making early recognition based on symptoms and key radiographic characteristics of the utmost importance for optimal patient management. Here we present the case of a patient presenting with non-specific abdominal complaints suffering from appendiceal mucocele perforation due to low-grade mucinous adenocarcinoma.

Ex vivo interleukin-12-priming during CD8(+) T cell activation dramatically improves adoptive T cell transfer antitumor efficacy in a lymphodepleted host.

BACKGROUND: Clinical application of adoptive T cell therapy has been hindered by an inability to generate adequate numbers of nontolerized, functionally active, tumor-specific T cells, which can persist in vivo. In order to address this, we evaluated the impact of interleukin (IL)-12 signaling during tumor-specific CD8(+) T cell priming in terms of persistence and antitumor efficacy using an established B16 melanoma tumor adoptive therapy model. STUDY DESIGN: B6 mice were injected subcutaneously with B16 melanoma tumor cells. On day 12 of tumor growth, mice were preconditioned with cyclophosphamide (4mg dose, intraperitoneally), and 1 day later were treated by adoptive transfer of tumor-specific pmel-1 CD8(+) T cells primed ex vivo 3 days earlier with both IL-12 and antigen (hGP100(25-33) peptide) or antigen only. Tumors were measured biweekly, and infused donor T cells were analyzed for persistence, localization to the tumor, phenotype, and effector function. RESULTS: Adoptive transfer of tumor-specific CD8(+) T cells primed with IL-12 was significantly more effective in reducing tumor burden in mice preconditioned with cyclophosphamide compared with transfer of T cells primed without IL-12. This enhanced antitumor response was associated with increased frequencies of infused T cells in the periphery and tumor as well as elevated expression of effector molecules including granzyme B and interferon-γ (IFNγ). CONCLUSIONS: Our findings demonstrate that ex vivo priming of tumor-specific CD8(+) T cells with IL-12 dramatically improves their in vivo persistence and therapeutic ability on transfer to tumor-bearing mice. These findings can be directly applied as novel clinical trial strategies.

Isoform-specific modulation of pressure-stimulated cancer cell proliferation and adhesion by α-actinin.

BACKGROUND: Intratumoral pressure may stimulate cancer proliferation whereas intravascular pressure promotes metastatic adhesion. α-Actinin proteins facilitate focal adhesion formation and link focal adhesion complexes to the cytoskeleton. We hypothesized that α-actinin is the mechanotransducer that mediates the effects of pressure on cancer cell proliferation and adhesion. METHODS: We treated SW620 colon cancer cells with specific short interfering RNA to reduce α-actinin-1 and/or α-actinin-4, the 2 key epithelial isoforms. Proliferation was measured in adherent cells by microculture tetrazolium (MTT) assay after 24 hours at ambient or 40 mm Hg increased pressure. For comparison, we evaluated the effects of 30 minutes of ambient or 15-mm Hg increased pressure on adhesion of suspended SW620 cells. Because the transcription factor nuclear factor-κB (NF-κB) influences proliferation, we used co-immunoprecipitation to evaluate NF-κB-α-actinin association and a lentiviral reporter assay for NF-κB activity. RESULTS: A total of 40 mm Hg increased pressure increased SW620 proliferation 41% ± 6% (n = 10; P < .05) versus ambient pressure controls. Reducing α-actinin-1 and α-actinin-4 together or α-actinin-4 alone blocked this effect, but reducing α-actinin-1 alone did not (n = 6; P < .05). We observed a 72% ± 11% increase in NF-κB activity (n = 6; P < .05), and increased association between NF-κB and α-actinin-4 in adherent cells under pressure. NF-κB and α-actinin-1 did not co-immunoprecipitate. However, reducing α-actinin-4 did not prevent pressure-induced NF-κB activation (n = 8). CONCLUSIONS: α-actinin-4 may mediate pressure stimulation of proliferation within large rapidly growing tumors, perhaps by binding transcription factors such as NF-κB. α-actinins may be important targets to inhibit cancer proliferation and metastasis.

Biological impact of mechanical stimuli on tumor metastasis.

Increasing evidence suggests tumor cell exposure to mechanical stimuli during the perioperative period as well as throughout the normal disease process may have a discernable impact on tumor metastasis and patient outcome. In vitro studies have demonstrated that transient exposure to increased extracellular pressure and shear forces modulates integrin binding affinity and stimulates cancer cell adhesion through a cytoskeleton- and focal adhesion complex-dependent signaling mechanism. More prolonged exposure to elevated pressures stimulates tumor cell proliferation by a distinct signaling pathway. Whether pressure effects on cell adhesion and proliferation pose biological ramifications in vivo remained unknown. We recently reported that pressure activation of malignant cells does indeed have a biological impact on surgical wound implantation, tumor development and tumor-free survival in a murine colon tumor model. Moreover, this effect can be disrupted by preoperative administration of colchicine. Taken together with previous work from our laboratory and others, these findings suggest that further elucidation of the mechanical signaling pathways governing pressure-stimulated tumor cell adhesion and proliferation may identify novel therapeutic targets for the treatment and prevention of tumor metastasis.

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.

Increased extracellular pressure enhances cancer cell integrin-binding affinity through phosphorylation of beta1-integrin at threonine 788/789.

Increased extracellular pressure stimulates beta1-integrin-dependent cancer cell adhesion. We asked whether pressure-induced adhesion is mediated by changes in beta1-integrin binding affinity or avidity and whether these changes are phosphorylation dependent. We evaluated integrin affinity and clustering in human SW620 colon cancer cells by measuring differences in binding between soluble Arg-Gly-Asp (RGD)-Fc ligands and RGD-Fc-F(ab')2 multimeric complexes under ambient and 15-mmHg increased pressures. Phosphorylation of beta1-integrin S785 and T788/9 residues in SW620 and primary malignant colonocytes was assessed in parallel. We further used GD25-beta1-integrin-null murine fibroblasts stably transfected with either wild-type beta1A-integrin, S785A, TT788/9AA, or T788D mutants to investigate the role of beta1-integrin site-specific phosphorylation. SW620 binding of RGD-Fc-F(ab')2 multimeric complexes, but not soluble RGD-Fc ligands, was sensitive to integrin clustering. RGD-Fc ligand binding was significantly increased under elevated pressure, suggesting that pressure modulates beta1-integrin affinity. Pressure stimulated both beta1-integrin S785 and T788/9 phosphorylation. GD25-beta1A-integrin wild-type and S785A cells displayed an increase in adhesion to fibronectin under elevated pressure, an effect absent in beta1-integrin-null and TT788/9AA cells. T788D substitution significantly elevated basal cell adhesion but displayed no further increase under pressure. These results suggest pressure-induced cell adhesion is mediated by beta1-integrin T788/9 phosphorylation-dependent changes in integrin binding affinity.

Strain-induced proliferation requires the phosphatidylinositol 3-kinase/AKT/glycogen synthase kinase pathway.

The intestinal epithelium is repetitively deformed by shear, peristalsis, and villous motility. Such repetitive deformation stimulates the proliferation of intestinal epithelial cells on collagen or laminin substrates via ERK, but the upstream mediators of this effect are poorly understood. We hypothesized that the phosphatidylinositol 3-kinase (PI3K)/AKT cascade mediates this mitogenic effect. PI3K, AKT, and glycogen synthase kinase-3beta (GSK-3beta) were phosphorylated by 10 cycles/min strain at an average 10% deformation, and pharmacologic blockade of these molecules or reduction by small interfering RNA (siRNA) prevented the mitogenic effect of strain in Caco-2 or IEC-6 intestinal epithelial cells. Strain MAPK activation required PI3K but not AKT. AKT isoform-specific siRNA transfection demonstrated that AKT2 but not AKT1 is required for GSK-3beta phosphorylation and the strain mitogenic effect. Furthermore, overexpression of AKT1 or an AKT chimera including the PH domain and hinge region of AKT2 and the catalytic domain and C-tail of AKT1 prevented strain activation of GSK-3beta, but overexpression of AKT2 or a chimera including the PH domain and hinge region of AKT1 and the catalytic domain and C-tail of AKT2 did not. These data delineate a role for PI3K, AKT2, and GSK-3beta in the mitogenic effect of strain. PI3K is required for both ERK and AKT2 activation, whereas AKT2 is sequentially required for GSK-3beta. Furthermore, AKT2 specificity requires its catalytic domain and tail region. Manipulating this pathway may prevent mucosal atrophy and maintain the mucosal barrier in conditions such as ileus, sepsis, and prolonged fasting when peristalsis and villous motility are decreased and the mucosal barrier fails.

SiRNA-mediated reduction of alpha-actinin-1 inhibits pressure-induced murine tumor cell wound implantation and enhances tumor-free survival.

Viable cancer cells can commonly be recovered from surgical sites and venous blood during tumor resection. The adhesion of these cells to surrounding tissues may impact patient outcomes. Iatrogenic exposure to increased extracellular pressure modulates integrin binding affinity and stimulates colon cancer cell adhesion in vitro through an alpha-actinin-1-dependent signaling pathway. We hypothesized that preoperative small interfering RNA-mediated silencing of alpha-actinin-1 in tumor tissue could disrupt pressure-stimulated cancer cell adhesion to murine surgical wounds and thereby enhance subsequent tumor-free survival. Reducing alpha-actinin-1 in CT26 murine adenocarcinoma cells blocked cell adhesion to collagen in vitro and similarly inhibited pressure-induced CT26 implantation in murine surgical wounds in vivo. Surgical wound contamination with pressure-activated CT26 cells significantly reduced tumor-free survival compared to contamination with tumor cells maintained under ambient pressure. However, mice treated with pressure-activated CT26 cells preoperatively transfected with alpha-actinin-1-specific small interfering RNA displayed reduced surgical site implantation and increased tumor-free survival compared to mice exposed to pressure-activated cells expressing normal levels of alpha-actinin-1 protein. These results suggest that pressure activation of malignant cells promotes tumor development and impairs tumor-free survival. alpha-Actinin-1 may be an effective therapeutic target to inhibit perioperative pressure-stimulated tumor cell implantation.

Cytoskeletal signaling by way of alpha-actinin-1 mediates ERK1/2 activation by repetitive deformation in human Caco2 intestinal epithelial cells.

BACKGROUND: Repetitive deformation stimulates proliferation in human Caco2 intestinal epithelial cells by way of an ERK1/2-dependent pathway. We examined the effects of cytoskeletal perturbation on deformation-induced signaling in Caco2 cells. METHODS: The Caco2 cell cytoskeleton was disrupted with either cytochalasin D, phalloidin, colchicine, or paclitaxel. Levels of alpha-actinin-1 and -4 and paxillin were reduced by specific small interfering RNA. Cells on collagen I-precoated membranes were subjected to 10% repetitive deformation at 10 cycles/min. After 1 hour, cells were lysed for Western blot analysis. RESULTS: Strain-activated ERK1/2, focal adhesion kinase, and Src phosphorylation in dimethyl sulfoxide- and/or nontargeting small interfering RNA-treated control cell populations. Cytochalasin D and paclitaxel, but not phalloidin and colchicine, blocked ERK1/2 phosphorylation. A decrease in alpha-actinin-1, but not in alpha-actinin-4 or paxillin, inhibited ERK1/2 and focal adhesion kinase phosphorylation, whereas Src activation appears to be independent of these effects. CONCLUSIONS: The intestinal epithelial cell cytoskeleton may transduce mechanical signals by way of alpha-actinin-1 into the focal adhesion complex, culminating in ERK1/2 activation and proliferation.