The tumor microenvironment of colorectal cancer: stromal TLR-4 expression as a potential prognostic marker

<p>Abstract</p> <p>Background</p> <p>Colorectal cancer can be efficiently treated when found at early stages, thus the search for novel markers is of paramount importance. Since inflammation is associated with cancer progression and angiogenesis, we investigated expression of cytokines like IL-6 and other mediators that play a key role in the innate immune system, in particular toll like receptor 4 (TLR4), in the microenvironment of lesions from different stages of colon disease progression, from ulcerative colitis to adenoma and adenocarcinoma to find useful markers.</p> <p>Methods</p> <p>The presence of inflammatory cells and expression of key cytokines involved in the inflammation process were quantified by immunohistochemistry in specific tissue compartments (epithelial, stromal, endothelial) by immunohistochemistry. A murine azoxymethane/dextran sulfate model in which Tir8, a negative regulator of the inflammatory response, was ablated was used to confirm the clinical observations. 116 Archival tissue samples from patients with different stages of colorectal disease: 13 cases of ulcerative colitis (UC), 34 tubular or tubulo-villous adenomas (AD), and 53 infiltrating adenocarcinomas. 16 specimens of healthy mucosa surgically removed with the cancerous tissue were used as a control.</p> <p>Results</p> <p>The differences between healthy tissues and the diverse lesions was characterized by a marked inflammatory-angiogenic reaction, with significantly (P < 0.05) higher numbers of CD68, CD15, and CD31 expressing cells in all diseased tissues that correlated with increasing grade of malignancy. We noted down-regulation of a potential modulator molecule, Hepatocyte Growth Factor, in all diseased tissues (P < 0.05). TLR-4 and IL6 expression in the tumor microenvironment were associated with adenocarcinoma in human samples and in the murine model. We found that adenocarcinoma patients (pT1-4) with higher TLR-4 expression in stromal compartment had a significantly increased risk in disease progression. In those patients with a diagnosis of pT3 (33 cases) colon cancer, those with very high levels of TLR-4 in the tumor stroma relapsed significantly earlier than those with lower expression levels.</p> <p>Conclusions</p> <p>These data suggest that high TLR-4 expression in the tumor microenvironment represents a possible marker of disease progression in colon cancer.</p

Effects of 5-fluorouracil on morphology, cell cycle, proliferation, apoptosis, autophagy and ros production in endothelial cells and cardiomyocytes

Antimetabolites are a class of effective anticancer drugs interfering in essential biochemical processes. 5-Fluorouracil (5-FU) and its prodrug Capecitabine are widely used in the treatment of several solid tumors (gastro-intestinal, gynecological, head and neck, breast carcinomas). Therapy with fluoropyrimidines is associated with a wide range of adverse effects, including diarrhea, dehydration, abdominal pain, nausea, stomatitis, and hand-foot syndrome. Among the 5-FU side effects, increasing attention is given to cardiovascular toxicities induced at different levels and intensities. Since the mechanisms related to 5-FU-induced cardiotoxicity are still unclear, we examined the effects of 5-FU on primary cell cultures of human cardiomyocytes and endothelial cells, which represent two key components of the cardiovascular system. We analyzed at the cellular and molecular level 5-FU effects on cell proliferation, cell cycle, survival and induction of apoptosis, in an experimental cardioncology approach. We observed autophagic features at the ultrastructural and molecular levels, in particular in 5-FU exposed cardiomyocytes. Reactive oxygen species (ROS) elevation characterized the endothelial response. These responses were prevented by a ROS scavenger. We found induction of a senescent phenotype on both cell types treated with 5-FU. In vivo, in a xenograft model of colon cancer, we showed that 5-FU treatment induced ultrastructural changes in the endothelium of various organs. Taken together, our data suggest that 5-FU can affect, both at the cellular and molecular levels, two key cell types of the cardiovascular system, potentially explaining some manifestations of 5-FU-induced cardiovascular toxicity

Cytostatic/cytotoxic effects of 5-FU on cardiomyocytes and endothelial cells.

<p>The cytostatic effect of 5-FU on HCMs (A) was not marked but statistically significant starting at 48 hours and from 10 μM 5-FU. The inhibitory effect of 5-FU was significant in endothelial cells (B) already after 24 hours at the intermediate concentration of 10 μM. The inhibition of cell viability was maintained up to 96 hours. Drug efficacy was also tested on the HT29 and HCT116 colorectal cancer cell lines (C, D). Mean ± S.E.M. of four different experiments for each cell type is shown. (E) MTT data at 72 hours were used to calculate the EC₅₀ values using the non-linear regression function of GraphPad Prism. 5-FU concentrations are reported in μM on a Log₍₁₀₎ scale, DMSO: 0.2% DMSO (vehicle) negative control, 0.1% saponin, positive control.</p

Acidic Vesicular Organelles (AVOs) formation in HCM after 5-FU treatment.

<p>AVOs formation is associated with the establishment of an autophagic process. Prolonged treatment with 5-FU significantly increased AVOs accumulation (*P<0.05; **P<0.01; ***P<0.005; ****P<0.0001) in HCM (A) as compared to control. Both 48 as well as 96 hours treatment of HUVEC (B) did not significantly influence the amount of acidic compartment staining over time. Mean ± S.E.M. of four different experiments per cell types are shown.</p

LC-3 expression as autophagic marker induced by 5-FU treatment.

<p>Staining with anti-LC3 showed punctae formation associated with the induction of the autophagic process. HCM (A) at all concentrations evaluated showed presence of LC-3 positive vesicles, while in HUVEC (B) the presence of these structures were not detectable unless using the highest 5-FU concentration. Cloroquine used as a positive control (Ctrl+) clearly induced vesicle formation. (C) Western blot analyses confirmed LC3 expression and increase on HCMs at the higher concentration of the drug.</p

<i>In vivo</i> effect of 5-FU.

<p>The CT26 murine colon adenocarcinoma cell line was used to evaluate effect of 5-FU <i>in vivo</i> on BALB/c mice. Sensitivity to 5-FU was demonstrated both <i>in vitro</i> in MTT (A, inset) and <i>in vivo</i> (A, B). Both treatment schedules (1 mg/kg every day, 10 mg/kg every other day) significantly reduced tumor growth (A) and weight upon sacrifice (B) as compared to control (PBS). Hematoxylin/eosin staining of renal and cardiac tissues from PBS and 5-FU treated animals (C). Transmission electron microscopy of renal tissues showed alterations (cytoplasmic vacuolization, membrane breakage) of endothelial cells (insets) in both treated groups with respect to controls (D). Cardiac tissues showed intact cardiomyocytes (nuclei indicated by thick white arrows) and occasional minor alterations of endothelial nuclei (thin black arrows) at the 10 mg/kg dose.</p

Effects of 5-FU on HCMs and HUVECs integrity and cycling ability.

<p>Membrane damage was calculated as the ratio between released LDH and total LDH. The effect of 5-FU on LDH release was statistically significant at 5-FU concentrations higher that 10 μM, less marked on cardiomyocytes (*P<0.05; **P<0.001) (A) with respect to HUVECs (****P<0.0001) (B). Mean ± S.E.M. of three different experiments for each cell type is shown. After 96 hours of 5-FU treatment cells were collected and stained with Annexin-V/7AAD to determine induction of apoptosis. Cumulative graphs of three independent experiments showing Annexin V⁺/7AAD^± cells are shown. Higher concentrations induced statistically significant amount of apoptotic cells as compared to negative control both in HCM (* P<0.05) (C) and HUVEC (**P<0.01; ***P<0.005) (D). Impairment of proliferative capacity was assessed using BrdU incorporation after 84 hours of treatment with 5-FU. BrdU-unlabeled cells (BrdU^-) represent cells in G1 and G2/M phases, BrdU-labeled cells (BrdU⁺) represent replicating cells in S phase. Staining of HCMs (E) revealed a certain degree of replicating capability in the negative control and at the lower concentrations of 5-FU, with complete absence of cells in the BrdU⁺ fraction in doses of 10 μM or more. Endothelial cells (F) display a dose dependent accumulation of BrdU^- cells in G0 in the presence of 5-FU. Mean ± S.E.M. of three different experiments for each cell type is shown. Negative controls (Ctrl-) consisted of cells treated with vehicle alone (0.2% DMSO), positive controls (Ctrl+) were cells treated with 0.1% saponin.</p

Effects of a ROS scavenger on ROS and AVOs formation.

<p>HCM and HUVE cells treated with 5-FU at the indicated concentrations together with the ROS scavenger NAC (10 mM) resulted in a reduction of ROS production. Both relative ROS as percentage of negative control (A, B) and representative histograms of flow cytometric data (C, D) are shown; light grey histograms 10 μM 5-FU, dark grey histograms 100 μM 5-FU, black histograms 1000 μM 5-FU; grey shaded histograms = negative control (Ctrl-); solid lines = 5-FU; dotted lines = 5-FU+NAC. NAC also reduced formation of AVOs in HCM (E), suggesting that ROS is also involved in the autophagic response in these cells. Since endothelial cells did not show induction of AVOs, NAC has little effect on AVO production in these cells (F). *P<0.05; **P<0.01. Mean ± S.E.M. of three different experiments for each cell type is shown.</p