Circulating Metabolic Markers Identify Patients at Risk for Tumor Recurrence: A Prospective Cohort Study in Colorectal Cancer Surgery.

OBJECTIVE: To investigate the spermidine pathway capability to predict patients at risk for tumor recurrence following colorectal cancer (CRC) surgery. SUMMARY BACKGROUND DATA: Recurrence rates after CRC surgery remain about 20%, despite an optimal technique and adjuvant therapy when necessary. Identification of risk biomarkers of recurrence is an unmet need. The spermidine pathway is indispensable for cell proliferation and differentiation, and is suggested to accelerate tumor spread. METHODS: Prospective cohort study of patients undergoing CRC surgery from 2015 to 2018. Plasma samples were collected before surgery and on postoperative day 4, and the spermidine pathway was assessed through mass spectrometry. Oncological outcomes were registered. RESULTS: 146 patients were included and 24 (16.4%) developed tumor recurrence. Higher levels of preoperative spermidine pathway components (spermidine, spermine, spermidine synthase enzyme, and spermine/arginine balance) were positively associated with recurrence. Surgery promoted a decrease in these pathway elements. The greater the decline was, the lower the risk of recurrence. Preoperative spermidine over the cut-off 0.198 µM displayed a 4.69-fold higher risk of recurrence. The spermine synthase enzyme behaved in the opposite direction. CONCLUSIONS: The spermidine pathway is associated with tumor recurrence following CRC surgery and, after confirmation in larger cohorts, could be translated as a risk biomarker of recurrence into clinical practice.

Circulating metabolic markers after surgery identify patients at risk for severe postoperative complications: a prospective cohort study in colorectal cancer.

BACKGROUND: Early detection of postoperative complications after colorectal cancer (CRC) surgery is associated with improved outcomes. The aim was to investigate early metabolomics signatures capable to detect patients at risk for severe postoperative complications after CRC surgery. MATERIALS AND METHODS: Prospective cohort study of patients undergoing CRC surgery from 2015 to 2018. Plasma samples were collected before and after surgery, and analyzed by mass spectrometry obtaining 188 metabolites and 21 ratios. Postoperative complications were registered with Clavien-Dindo Classification and Comprehensive Complication Index. RESULTS: One hundred forty-six patients were included. Surgery substantially modified metabolome and metabolic changes after surgery were quantitatively associated with the severity of postoperative complications. The strongest positive relationship with both Clavien-Dindo and Comprehensive Complication Index (ß=4.09 and 63.05, P <0.001) corresponded to kynurenine/tryptophan, against an inverse relationship with lysophosphatidylcholines (LPCs) and phosphatidylcholines (PCs). Patients with LPC18:2/PCa36:2 below the cut-off 0.084 µM/µM resulted in a sevenfold higher risk of major complications (OR=7.38, 95% CI: 2.82-21.25, P <0.001), while kynurenine/tryptophan above 0.067 µM/µM a ninefold (OR=9.35, 95% CI: 3.03-32.66, P <0.001). Hexadecanoylcarnitine below 0.093 µM displayed a 12-fold higher risk of anastomotic leakage-related complications (OR=11.99, 95% CI: 2.62-80.79, P =0.004). CONCLUSION: Surgery-induced phospholipids and amino acid dysregulation is associated with the severity of postoperative complications after CRC surgery, including anastomotic leakage-related outcomes. The authors provide quantitative insight on metabolic markers, measuring vulnerability to postoperative morbidity that might help guide early decision-making and improve surgical outcomes.

Modulation of the colon cancer cell phenotype by pro-inflammatory macrophages: A preclinical model of surgery-associated inflammation and tumor recurrence.

Peritoneal infection after colorectal cancer surgery is associated with a higher rate of tumor relapse. We have recently proposed that soluble inflammatory factors released in response to a postoperative infection enhance tumor progression features in residual tumor cells. In an effort to set up models to study the mechanisms of residual tumor cell activation during surgery-associated inflammation, we have analyzed the phenotypic response of colon cancer cell lines to the paracrine effects of THP-1 and U937 differentiated human macrophages, which release an inflammatory medium characteristic of an innate immune response. The exposure of the colon cancer cell lines HT-29 and SW620 to conditioned media isolated from differentiated THP-1 and U937 macrophages induced a mesenchymal-like phenotypic shift, involving the activation of in vitro invasiveness. The inflammatory media activated the ß-catenin/TCF4 transcriptional pathway and induced the expression of several mesenchymal (e.g., FN1 and VIM) and TCF4 target genes (e.g., MMP7, PTGS2, MET, and CCD1). Similarly, differential expression of some transcription factors involved in epithelial-to-mesenchymal transitions (i.e. ZEB1, SNAI1, and SNAI2) was variably observed in the colon cancer cell lines when exposed to the inflammatory media. THP-1 and U937 macrophages, which displayed characteristics of M1 differentiation, overexpressed some cytokines previously shown to be induced in colorectal cancer patients with increased rates of tumor recurrence associated with postoperative peritoneal infections, thus suggesting their pro-tumoral character. Therefore, the environment created by inflammatory M1 macrophages enhances features of epithelial-to-mesenchymal transition, and may be useful as a model to characterize pro-inflammatory cytokines as putative biomarkers of tumor recurrence risk.

Structural Dissection of Crotalicidin, a Rattlesnake Venom Cathelicidin, Retrieves a Fragment with Antimicrobial and Antitumor Activity.

In silico dissection of crotalicidin (Ctn), a cathelicidin from a South American pit viper, yielded fragments Ctn[1-14] and Ctn[15-34], which were tested to ascertain to what extent they reproduced the structure and activity of the parent peptide. NMR data showing Ctn to be α-helical at the N-terminus and unstructured at the C-terminus were matched by similar data from the fragments. The peptides were tested against Gram-positive and -negative bacteria and for toxicity against both tumor and healthy cells. Despite its amphipathic α-helical structure, Ctn[1-14] was totally inert toward bacteria or eukaryotic cells. In contrast, unstructured Ctn[15-34] replicated the activity of parent Ctn against Gram-negative bacteria and tumor cells while being significantly less toxic toward eukaryotic cells. This selectivity for bacteria and tumor cells, plus a stability to serum well above that of Ctn, portrays Ctn[15-34] as an appealing candidate for further development as an anti-infective or antitumor lead.

Postoperative peritoneal infection enhances migration and invasion capacities of tumor cells in vitro: an insight into the association between anastomotic leak and recurrence after surgery for colorectal cancer.

OBJECTIVE: The aim of this study was to investigate the effect of postoperative peritoneal infection on proliferation, migration, and invasion capacities of cancer cells lines in vitro after surgery for colorectal cancer. BACKGROUND: Anastomotic leakage is associated with higher rates of recurrence after surgery for colorectal cancer. However, the mechanisms responsible are unknown. We hypothesized that the infection-induced inflammatory response may enhance tumor progression features of residual cancer cells. METHODS: Prospective matched cohort study. Patients undergoing surgery for colorectal cancer with curative intent (January 2008-March 2012) were included. Patients who had an anastomotic leak or intra-abdominal abscess were included in the infection group (n=47). For each case patient, another patient with an uncomplicated postoperative course was selected for the control group (n=47).In vitro treatments on cancer cell lines (MDA-MB-231 and SW620) were performed using baseline and postoperative serum and peritoneal fluid samples to determine cell proliferation and cell migration/invasion activities. RESULTS: Postoperative peritoneal fluid from infected patients enhanced both cell migration (infection: 140±85 vs control: 94±30; P=0.016) and cell invasion (infection: 117±31 vs control: 103±16; P=0.024) capacities of cancer cell lines. With serum samples, these effects were only observed in cell migration assays (infection: 98±28 vs control: 87±17; P=0.005). Some minor activation of cell proliferation was observed by treatment with serum from infection group. Two-year cumulative disease-free survival was significantly lower in patients with postoperative peritoneal infection (infection: 77.6% vs control: 90.6%; P=0.032). CONCLUSIONS: Our results suggest that postoperative peritoneal infection enhances the invasive capacity of residual tumor cells after surgery, thus facilitating their growth to recurrent tumors.

The Polycomb group protein RING1B is overexpressed in ductal breast carcinoma and is required to sustain FAK steady state levels in breast cancer epithelial cells.

In early stages of metastasis malignant cells must acquire phenotypic changes to enhance their migratory behavior and their ability to breach the matrix surrounding tumors and blood vessel walls. Epigenetic regulation of gene expression allows the acquisition of these features that, once tumoral cells have escape from the primary tumor, can be reverted. Here we report that the expression of the Polycomb epigenetic repressor Ring1B is enhanced in tumoral cells that invade the stroma in human ductal breast carcinoma and its expression is coincident with that of Fak in these tumors. Ring1B knockdown in breast cancer cell lines revealed that Ring1B is required to sustain Fak expression in basal conditions as well as in Tgfß-treated cells. Functionally, endogenous Ring1B is required for cell migration and invasion in vitro and for in vivo invasion of the mammary fat pad by tumoral cells. Finally we identify p63 as a target of Ring1B to regulate Fak expression: Ring1B depletion results in enhanced p63 expression, which in turns represses Fak expression. Importantly, Fak downregulation upon Ring1B depletion is dependent on p63 expression. Our findings provide new insights in the biology of the breast carcinoma and open new avenues for breast cancer prognosis and therapy.

Radiofrequency is a secure and effective method for pancreatic transection in laparoscopic distal pancreatectomy: results of a randomized, controlled trial in an experimental model.

BACKGROUND: Postoperative pancreatic fistula (PPF) is the most frequent and serious complication after laparoscopic distal pancreatectomy (LDP). Our goal was to compare the performance, in terms of PPF prevention, and safety of a radiofrequency (RF)-assisted transection device versus a stapler device in a porcine LDP model. METHODS: Thirty-two animals were randomly divided into two groups to perform LDP using a RF-assisted device (RF group; n = 16) and stapler device (ST group; n = 16) and necropsied 4 weeks after surgery. The primary endpoint was the incidence of PPF. Secondary endpoints were surgery/transection time, intra/postoperative complications/deaths, postoperative plasmatic amylase and glucose concentration, peritoneal liquid amylase and interleukin 6 (IL-6) concentrations, weight variations, and histopathological changes. RESULTS: Two clinical and one biochemical PPF were observed in the ST and RF groups respectively. Peritoneal amylase concentration was significantly higher in the RF group 4 days after surgery, but this difference was no longer present at necropsy. Both groups presented a significant decrease in peritoneal IL-6 concentration during the postoperative follow-up, with no differences between the groups. RF group animals showed a higher postoperative weight gain. In the histopathological exam, all RF group animals showed a common pattern of central coagulative necrosis of the parenchymal surface, surrounded by a thick fibrosis, which sealed main and secondary pancreatic ducts and was not found in ST group. CONCLUSIONS: The fibrosis caused by an RF-assisted device can be at least as safe and effective as stapler compression to achieve pancreatic parenchyma sealing in a porcine LDP model.

Preoperative administration of erythropoietin stimulates tumor recurrence after surgical excision of colon cancer in mice by a vascular endothelial growth factor-independent mechanism.

BACKGROUND: It has been suggested that preoperative administration of erythropoietin (Epo) in patients with gastrointestinal cancer reduces transfusional needs and is also associated with lower morbidity. On the other hand, experimental and clinical studies show that Epo might enhance tumor growth and angiogenesis. Our aim was to ascertain whether preoperative administration of Epo has any effect on tumor recurrence after curative surgery using an experimental model of colon cancer. MATERIALS AND METHODS: We induced tumors by injecting B51LiM colon cancer cells into the cecal wall of Balb/c mice. We randomized the animals into three groups of treatment with (1) recombinant human Epo, (2) recombinant mouse Epo, or (3) vehicle alone, for 12 d until cecectomy. On postoperative day 12, we killed mice and analyzed tumor recurrence. We measured serum levels of vascular endothelial growth factor and determined vascular endothelial growth factor expression and tumor microvessel density by immunohistochemistry. We also investigated the in vitro effect of Epo on B51LiM cell line proliferation. RESULTS: All three groups displayed tumor recurrence, but the final tumor load score and total tumoral weight were higher in the two groups that included Epo. The differences were statistically significant when we compared the recombinant mouse Epo group with the control group. We found no evidence of increased angiogenesis or enhanced cell proliferation as possible mechanisms of Epo-induced recurrence. CONCLUSIONS: Preoperative administration of Epo stimulates tumor recurrence in an animal model of colon cancer. Our results point to the need for further research on the mechanisms of tumor growth enhancement by Epo, to better understand the benefits or disadvantages of Epo treatment.

Postoperative intra-abdominal infection increases angiogenesis and tumor recurrence after surgical excision of colon cancer in mice.

BACKGROUND: Recent reports have suggested that anastomotic leakage is associated with greater rates of tumor recurrence and cancer-specific mortality after surgery for colorectal cancer. The impact of postoperative intra-abdominal infection on long-term oncologic results, however, is still controversial, and no direct causal relationship has been found between both processes. Our aim was to investigate the influence of postoperative intraabdominal infection on angiogenesis and tumor growth in an animal model of colon cancer. METHODS: Balb/c mice were randomized immediately after injection of 5x10(6) B51LiM cells into the cecal wall into 2 groups: cecal resection without postoperative infection (group 1), and cecal resection with postoperative intra-abdominal infection (group 2). A total of 18 days after cell injection, cecectomy was performed, and infection was induced in group 2 by intraperitoneal injection of 3x10(8) colony-forming units of Bacteroides fragilis. On postoperative day 12, the mice were killed. RESULTS: Comparing group 1 with group 2, tumor recurrence was more frequent in animals with intraabdominal infection (65% vs 100%, respectively; P=02). VEGF serum levels were greater at the time of sacrifice in the group with infection (11+/-10 vs 30+/-23 pg/mL; P<.05). Tumor angiogenesis was also increased in the postoperative infection group. The mean (+/-standard deviation) microvessel density was 16+/-7 versus 28+/-11 vessels per high-power field (P<.05). CONCLUSION: We concluded that postoperative intra-abdominal infection increases angiogenesis and tumor recurrence after operative excision of a colon cancer in mice.

Cyclin D1 negatively regulates the expression of differentiation genes in HT-29 M6 mucus-secreting colon cancer cells.

HT-29 M6 colon cancer cells differentiate to a mucus-secreting phenotype in culture. We found that the pattern of cyclin D1 expression in HT-29 M6 cells did not correlate with instances of cell proliferation but was specifically induced during a dedifferentiation process following disaggregation of epithelial cell layers, even under conditions that did not allow cell cycle reentrance. Interestingly, ectopic expression of cyclin D1 in differentiated cells led to the inhibition of the transcriptional activity of differentiation gene promoters, such as the mucin MUC1. We thus propose that the overexpression of cyclin D1 found in colon cancer favours tumour dedifferentiation as one mechanism of tumour progression.

A phase II trial of fixed-dosed rate gemcitabine in platinum-resistant ovarian cancer: a GEICO (Grupo Español de Investigación en Cáncer de Ovario) Trial.

OBJECTIVES: Gemcitabine has well-recognized activity in the treatment of ovarian cancer. Fixed-dose rate (FDR) delivery has been proposed as a more rationale way to administer gemcitabine, to avoid saturation of the enzyme that catalyzes its intracellular transformation into the active metabolites, difluorodeoxycitidine biphosphate, and triphosphate. Our aim was to assess clinical activity of gemcitabine delivered by FDR infusion in patients with platinum resistant ovarian cancer. MATERIALS AND METHODS: Patients with platinum-resistant ovarian cancer received gemcitabine 1000 mg/m(2) over 120 minutes on days 1 and 8 of each cycle. Cycles were repeated every 3 weeks, and up to 6 cycles were delivered. RESULTS: Forty-eight patients were included in the study. Among 41 patients evaluable for response, 9 clinical responses (1 complete response and 8 partial responses) were observed, achieving a global response rate of 22%. Grade 3 to 4 hematological toxicity consisted of anemia (15% of patients), neutropenia (24%), and thrombopenia (10%). One patient died due to septic shock. The main grade 3 to 4 nonhematological toxicity was asthenia (7 patients, 17%). CONCLUSION: Activity of gemcitabine administered by FDR infusion in patients with platinum-resistant ovarian cancer seems similar to that achieved using 30-minute infusions, with higher toxicity.

In vitro differentiation of HT-29 M6 mucus-secreting colon cancer cells involves a trychostatin A and p27(KIP1)-inducible transcriptional program of gene expression.

Tumor cell dedifferentiation-such as the loss of cell-to-cell adhesion in epithelial tumors-is associated with tumor progression. To better understand the mechanisms that maintain carcinoma cells in a differentiated state, we have dissected in vitro differentiation pathways in the mucus-secretor HT-29 M6 colon cancer cell line, which spontaneously differentiates in postconfluent cultures. By lowering the extracellular calcium concentration to levels that prevent intercellular adhesion and epithelial polarization, our results reveal that differentiation is calcium-dependent and involves: (i) a process of cell cycle exit to G(0) and (ii) the induction of a transcriptional program of differentiation gene expression (i.e., mucins MUC1 and MUC5AC, and the apical membrane peptidase DPPIV). In calcium-deprived, non-differentiated postconfluent cultures, differentiation gene promoters are repressed by a trichostatin A (TSA)-sensitive mechanism, indicating that loss of gene expression by dedifferentiation is driven by histone deacetylases (HDAC). Since TSA treatment or extracellular calcium restoration allow gene promoter activation to similar levels, we suggest that induction of differentiation is one mechanism of HDAC inhibitor antitumor action. Moreover, transcriptional de-repression can also be induced in non-differentiating culture conditions by overexpressing the cyclin-dependent kinase inhibitor p27(KIP1), which is normally induced during spontaneous differentiation. Since p27(KIP1) downregulation in colon cancer is associated with poor prognosis independently of tumor cell division rates, we propose that p27 (KIP1) may prevent tumor progression by, at least in part, enhancing the expression of some differentiation genes. Therefore, the HT-29 M6 model allows the identification of some basic mechanisms of cancer cell differentiation control, so far revealing HDAC and p27(KIP1) as key regulatory factors of differentiation gene expression.

The DNA damage checkpoint is activated during residual tumour cell survival to methotrexate treatment as an initial step of acquired drug resistance.

In the process of acquired drug resistance, the absence of tumour cell subpopulations already resistant before treatment implies an initial adaptive stage of cell growth following drug exposure that, under the selective pressure of the drug, allows the emergence of stably resistant cell variants. Here, we show that p53-defective HT-29 colon cancer cells overcome methotrexate-induced cell death owing to DNA damage checkpoint-mediated cell survival at the adaptive stage that precedes stable resistance acquisition. HT-29 cell cycle progression was dramatically delayed in the presence of a lethal dose of methotrexate, leading to DNA damage during S-phase transition and to cell death as treated cells progressed to G2 and M phases. As a result, the DNA damage checkpoint was induced as indicated by the presence of activated phosphorylated forms of checkpoint proteins Chk1 and Rad9. As we recently described, in-vitro resistance to methotrexate occurs without cell subpopulations already resistant before treatment, hence resistance is acquired through a multistep process that includes an early stage of transient cell survival. Our present results showed that this acute cell survival stage was due to a minor percentage of cells that could complete the first division cycle after drug exposure. Cell survival was enhanced by drug withdrawal during S-phase transition and suppressed if drug withdrawal was followed by treatment with the checkpoint-inhibitor drug caffeine. These results thus point to checkpoint-mediated transient adaptation as a target to prevent the emergence of acquired resistance to methotrexate.

Methotrexate resistance in vitro is achieved by a dynamic selectionprocess of tumor cell variants emerging during treatment.

Genetic instability leads to tumor heterogeneity, which in turn provides a source of cell variants responsible for drug resistance. However, the source of resistant cells during the process of acquired resistance is poorly understood. Our aim has been to characterize the mechanism by which acquired resistance to methotrexate emerges during the course of cancer cell treatment in vitro. We recently demonstrated that, in vitro, HT-29 colon cancer cells become transiently sensitive to methotrexate by depleting the extracellular milieu of survival factors; on the other hand, the cell population under treatment can reversibly adapt to grow below a critical cell density in the presence of the drug. Here, we show that this adapted cell population gives rise to permanent resistant populations through repeated cycles of cell death and growth. This increased cell turnover, but not merely cell proliferation, is required for the appearance of increasing degrees of stable resistance that are progressively selected by drug pressure. Such a process, taking place in multiple steps, is here designated "dynamic selection." The analysis of sensitive and resistant HT-29 cell populations revealed that methotrexate induces genomic instability--characterized by centrosome amplification and aberrant chromosome recombination--leading to a low-level amplification of the 5q chromosome arm as one of the earliest genetic events selected during treatment. Therefore, this model provides a mechanism by which a tumor cell population lacking resistant subpopulations before treatment is able to acquire the genetic changes required for stable drug resistance.

Low tumor cell density environment yields survival advantage of tumor cells exposed to MTX in vitro.

Stable resistance to methotrexate has been well characterized after prolonged treatment of the HT-29 colon cancer cell line, but the mechanism of cell survival at the early stages of the drug resistance process still remains unclear. Here, we demonstrate that human cancer cells in vitro are sensitive to methotrexate only above a critical cell culture density, which specifically coincides with their ability to deplete the extracellular nucleosides from a fully supplemented culture medium. At lower cell densities, extracellular nucleosides remain intact and allow salvage nucleotide synthesis that renders cells insensitive to the drug. Consistently, medium conditioned by cells seeded at standard cell densities sensitizes low cell density cultures. Extracellular nucleosides are the determinants of sensitivity because the latter effect can be mimicked with the use of inhibitors of nucleoside cellular import and reversed by supplying exogenous thymidine and hypoxanthine. Interestingly, treatment at a sensitizing cell density does not preclude the survival of less than 1% of the cells--which have no intrinsic resistance--owing to the inability of the dying cell population to condition the culture medium; this population thus survives indefinitely to continuous treatment by keeping adapted to a low cell number. This cell density-dependent adaptive process accounts for the initial steps of in vitro resistance to methotrexate (MTX) and provides a novel mechanistic insight into the cell population dynamics of cell survival and cell death during drug treatment.