Table 1.
Primer sequences used in RT- PCR assays.
Fig 1.
Loss of epithelial morphology.
The inflammatory medium conditioned by macrophages induced a morphological shift in the epithelial colon cancer cell lines HT-29 and SW620 that was reminiscent of an epithelial-to-mesenchymal transition. Phase contrast microscopy images were obtained after treatment of colon cancer cell cultures for 2 days with the indicated conditioned media. CON, standard medium; MONO-CM, conditioned medium from non-activated monocytes; MACRO-CM, conditioned medium from differentiated macrophages.
Fig 2.
Cell line-dependent cell proliferation and toxicity.
The inflammatory medium conditioned by macrophages provoked a slowdown of cell proliferation in the HT-29 and SW620 cell lines, whereas it induced cell death in Caco2 cells. A, cell proliferation assays on HT-29 and SW620 during continuous treatment with the indicated conditioned media. Cell cultures were seeded at 3x104/cm2 and 2x104/cm2, respectively, and viable cells were calculated by trypan blue exclussion during the course of continuous treatment. The graph shows a representative experiment made in duplicates. B, cell countings were performed on the sixth day of the experiment shown in A and are expressed as the mean ± SD from 4 independent experiments (*, p = 0.068 with respect to CON or MONO-CM according to the Wilcoxon sign-rank test). C, cell viability was analyzed by clonogenicity assays as described in Materials and Methods, revealing that HT-29 cells preserved almost full viability after treatment with MACRO-CM for 48 hours while Caco2 cells underwent massive cell death. Results are shown as the percentage of visible colonies respect to 100% viability in cultures that had been treated with standard medium (CON). Percentage values are the mean ± SD from 6 independent experiments (*, p<0.050 with respect to CON or MONO-CM according to the Wilcoxon sign-rank test). CON, standard medium; MONO-CM, conditioned medium from non-activated monocytes; MACRO-CM, conditioned medium from differentiated macrophages.
Fig 3.
In vitro cell migration and invasion.
The inflammatory medium conditioned by macrophages induced in vitro migration and invasion activities in colon cancer cells. A, wound healing assays using the HT-29 cell line. Confluent monolayers of HT-29 cell cultures were scrapped with a rubber tip (0 hours) and thereafter treated with the indicated conditioned media for 48 hours. Cell scattering and increased healing migration were apparent after 48 hours of treatment with MACRO-CM. B, images of crossed wounds were taken after 24 and 48 hours of continuous treatment in the experiment shown in A, and the closing distances near the cross corners were measured. The graph shows the mean (mm) ± error of two independent experiments, each one integrating an average of the healing distances of several measures in each cell monolayer. C, in vitro migration and invasion assays were performed using HT-29 or SW620 cells cultured on transwells for 24 hours in the presence of the indicated conditioned media. Results are the mean ± SD of cells moving through the transwell relative to the basal migration or invasion using standard medium as a control (CON) from at least 6 independent experiments in each culture condition. (*, p<0.03 with respect to CON or MONO-CM according to the Wilcoxon sign-rank test). CON, standard medium; MONO-CM, conditioned medium from non-activated monocytes; MACRO-CM, conditioned medium from differentiated macrophages.
Fig 4.
PMA-differentiated THP-1 and U937 macrophages displayed features of a M1 type phenotype.
PMA-differentiated THP-1 and U937 macrophages (MACRO) were harvested in parallel with non-treated cultures (MONO), and total RNA isolated. Some cultures were further incubated in the presence of medroxyprogesterone (MPA) for 24 hours or left untreated (MPA-CON), and similarly harvested and total RNA isolated. RNA samples were reverse transcribed to cDNA, and Real Time PCR analysis was performed using oligonucleotide primers specific for the indicated genes (see Material and Methods). Markers of the M1 phenotype (IL6, IL1B, CXCL10) displayed strong induction during PMA-differentiation but were abruptly suppressed with further incubation with the M2 inducer MPA. In contrast, induction of the M2 markers CD163 and IL10 was modest, and in the case of CD163 potently enhanced by the M2 inducer MPA. Results are shown as the relative levels of each mRNA in the differentiated cell lines (MACRO, MPA-CON, MPA) respect to the non-treated monocytic cell lines (MONO) and expressed as the mean ± error of at least 2 independent experiments.
Fig 5.
β-catenin/TCF4 transcriptional activity.
The inflammatory media conditioned by THP-1 or U937 macrophages induced the activity of the transcription factor β-catenin/TCF4 in SW620 colon cancer cells. Cultures of HT-29 or SW620 cells were transfected with the Firefly luciferase reporter plasmid Top, which contains an artificial gene promoter with TCF4-responsive DNA binding sites. As controls, the vector containing the TCF4 DNA binding sites mutated (Fop) and the empty vector (vector) were transfected in parallel. After 4 hours of transfection, cell cultures were treated with the indicated conditioned media for 24 hours and finally harvested. Results are shown relative to the TOP activity of cells treated with standard medium (CON) and expressed as the mean ± SD of at least 3 independent experiments (*, p = 0.068 with respect to CON or MONO-CM according to the Wilcoxon sign-rank test). CON, standard medium; MONO-CM, conditioned medium from non-activated monocytes; MACRO-CM, conditioned medium from differentiated macrophages.
Fig 6.
Gene expression analysis of EMT-related markers.
The inflammatory medium conditioned by THP-1 or U937 macrophages induced the expression of several genes related to the EMT process. Total RNA was isolated from HT-29 or SW620 cell cultures that had been treated with the indicated conditioned media for 48 hours. RNA samples were reverse transcribed to cDNA, and Real Time PCR analysis was performed using oligonucleotide primers specific for the indicated genes (see Material and Methods). Genes analyzed were: FN1 (fibronectin 1), VIM (vimentin), ITGB6 (integrin subunit beta 6), and S100A4 (S100 calcium binding protein A4). Results are shown as the relative levels of each mRNA respect to the sample treated with standard medium (CON) and expressed as the mean ± SD of at least 3 independent experiments. CON, standard medium; MONO-CM, conditioned medium from non-activated monocytes; MACRO-CM, conditioned medium from differentiated macrophages.
Fig 7.
Gene expression analysis of β-catenin/TCF4 transcriptional target genes.
The inflammatory medium conditioned by THP-1 or U937 macrophages induced the expression of some targets of the β-catenin/TCF4 transcriptional pathway. Total RNA was isolated from HT-29 or SW620 cell cultures that had been treated with the indicated conditioned media for 48 hours. RNA samples were reverse transcribed to cDNA, and Real Time PCR analysis was performed using oligonucleotide primers specific for the indicated genes (see Material and Methods). Genes analyzed were: MMP7 (matrix metallopeptidase 7), PTGS2 (prostaglandin-endoperoxide synthase 2, or COX-2), MET (MET proto-oncogene, receptor tyrosine kinase), and CCND1 (cyclin D1). Results are shown as the relative levels of each mRNA respect to the sample treated with standard medium (CON) and expressed as the mean ± SD of at least 3 independent experiments. CON, standard medium; MONO-CM, conditioned medium from non-activated monocytes; MACRO-CM, conditioned medium from differentiated macrophages.
Fig 8.
Gene expression analysis of EMT-related transcription factors.
The inflammatory medium conditioned by THP-1 or U937 macrophages induced the expression of some transcription factor genes known as mediators of the EMT process. Total RNA was isolated from HT-29 or SW620 cell cultures that had been treated with the indicated conditioned media for 48 hours. RNA samples were reverse transcribed to cDNA, and Real Time PCR analysis was performed using oligonucleotide primers specific for the indicated genes (see Material and Methods). Genes analyzed were: ZEB1 (zinc finger E-box binding homeobox 1), SNAI1 (snail family transcriptional repressor 1, or Snail), and SNAI2 (snail family transcriptional repressor 2, or Slug). Results are shown as the relative levels of each mRNA respect to the sample treated with standard medium (CON) and expressed as the mean ± SD of at least 3 independent experiments. N.D., not detectable; CON, standard medium; MONO-CM, conditioned medium from non-activated monocytes; MACRO-CM, conditioned medium from differentiated macrophages.
Fig 9.
Cytokine genes induced by PMA-differentiated THP-1 and U937 macrophages suggested common mechanisms with the tumor recurrence in colorectal cancer patients undergoing postoperative peritoneal infections.
PMA-differentiated THP-1 and U937 macrophages (MACRO) were harvested in parellel with non-treated cultures (MONO), and total RNA isolated. RNA samples were reverse transcribed to cDNA, and Real Time PCR analysis was performed using oligonucleotide primers specific for the indicated genes (see Material and Methods). Genes analyzed corresponded to a gene panel differentially expressed in peripheral blood leukocytes of colorectal cancer patients undergoing postoperative peritoneal infetions (see reference 14): MMP9 (matrix metallopeptidase 9), IL1R2 (interleukin 1 receptor type 2), PLSCR1 (phospholipid scramblase 1), TRPS1 (transcriptional repressor GATA binding 1), ORM1 (orosomucoid 1), HP (haptoglobin), ADAM9 (ADAM metallopeptidase domain 9), and PDGFC (platelet derived growth factor C). Results are shown as the relative levels of each mRNA in the PMA treated cell lines (MACRO) respect to the non-treated monocytic cell lines (MONO) and expressed as the mean ± SD of at least 3 independent experiments. N.D., not detectable.