Colorectal Tumor Microenvironment Research Articles

PTH-321 Exosomes and microparticles: distinct extracellular compartments which convey genetic information in the colorectal tumour microenvironment

<h3>Introduction</h3> Exosomes and microparticles (MPs) are extracellular vesicles secreted by tumour and stromal cells. Exosomes (40–100 nm) originate in the cytoplasm and are a product of the lysosome-endosome pathway. MPs (100–1000 nm) are larger vesicles which simply bud off from the cell membrane. Exosomes and MPs are known to contain oncogenic (e.g. microRNA-21) and tumour suppressing (e.g. microRNA-200b) microRNAs (miRs). We aim to: (i) screen for cancer-associated microRNAs which are upregulated in exosomes/ MPs from human colorectal cancer fibroblasts; (ii) ascertain the effect of myofibroblast transdifferentiation on cellular and vesicular miR-21 content; (iii) ascertain the effect of epithelial-mesenchymal transition (EMT) on cellular and vesicular miR-200b content; and (iv) compare cellular, exosomal and MP microRNA content for each model above. <h3>Method</h3> Several models were used: (i) primary cancer-associated and normal human colorectal fibroblasts; (ii) HFFF2 fibroblasts – treated with TGF-beta (myofibroblasts) and untreated (fibroblasts); and (iii) SIP-1 induced (EMT positive) and uninduced (EMT negative) DLD-1 colorectal cancer cells. Exosomes and MPs were isolated by selective centrifugation and validated by transmission electron microscopy, western blotting and flow cytometry. A cancer microRNA array (Quantimir) was used to screen for 95 specific microRNAs. Quantitative PCR (TaqMan) was used to measure relative miR-21 and miR-200b concentrations. <h3>Results</h3> Screening revealed distinct microRNA expression profiles in cancer-associated and normal colorectal fibroblasts. MiR-149 and -150 were upregulated in the cellular compartment, miR-92 and -133 in the MP compartment and miR-17–3p and -202 in the exosomal compartment of cancer-associated fibroblasts. Cellular miR-21 concentration was exponentially higher in myofibroblasts than fibroblasts. This was reflected at the exosomal (2.28+/-0.03 vs. 1.00+/-0.04) but not the MP level. SIP induced colorectal cancer cells had 13-fold less miR-200b than uninduced cells. Again, this was reflected at the exosomal (0.24+/-0.01 vs. 1.00+/-0.04) but not the MP level. <h3>Conclusion</h3> MicroRNA screening showed that the cellular, exosomal and MP compartments contain distinct microRNA profiles. In the myofibroblast transdifferentiation and EMT models, exosomal but not MP microRNA concentrations changed significantly and mirrored that of the parent cells. Compared to MPs, exosomes originate deep within the cytoplasm and their proximity to the nuclear machinery may explain this phenomenon. <h3>Disclosure of interest</h3> None Declared.

PTH-320 Exosomes: extracellular vesicles which can immortalise cancer and stromal cells in the colorectal tumour microenvironment

<h3>Introduction</h3> Exosomes are nano-sized (40–100 nm) extracellular vesicles which are exchanged by cancer and stromal cells. They contain proteins and nucleotides (DNA, mRNA, microRNA) and are therefore capable of conveying genetic information. Exosome transfer affects cellular function but this is not well defined in the colorectal cancer (CRC) setting. We aimed to isolate, label and transfer exosomes from colorectal cancer cells to fibroblasts and vice versa. Moreover, we investigated the effect of exosome transfer at the cellular level, focussing on two key pathways: ERK and AKT. <h3>Method</h3> Exosomes were isolated from conditioned media from DLD-1 CRC cells or MRC5 fibroblasts by selective centrifugation and validated by transmission electron microscopy, western blotting and flow cytometry. The resulting exosome pellet was labelled with a lipophilic (green) fluorescent dye and co-cultured with the reciprocal target cell (MRC5 or DLD-1). The cells were then washed thoroughly to remove all extracellular particles and visualised by fluorescence microscopy. Cell lysate was analysed by western blotting for activation of key signalling pathways such as Ras (ERK) and AKT. <h3>Results</h3> Microscopy revealed presence of fluorescent labelled exosomes within both DLD-1 and MRC5 cells following exosome co-culture. There was marked increase in AKT pathway activation in MRC5 fibroblasts after co-culture with DLD-1 CRC exosomes. As a result, significant changes to anti-apoptotic stimuli have been observed. There was a marked increase in ERK pathway activation and moderate increase in AKT activation in DLD-1 CRC cells after co-culture with MRC5 fibroblast exosomes. Again, significant changes to downstream anti-apoptotic signals were observed. <h3>Conclusion</h3> We have shown that CRC exosomes can be transferred to fibroblasts and vice versa. This transfer induces selective phosphorylation of AKT and resistance to apoptosis in recipient fibroblasts and CRC cells. Stromal and tumour-derived exosomes promote resistance to apoptosis and can create an apoptosis resistant niche in the tumour microenvironment. <h3>Disclosure of interest</h3> None Declared.

Abstract 1002: Norrin controls colon cancer angiogenesis

Abstract The purposes of the study: Colorectal (CRC) tumors depend on mediators in the tumor microenvironment, such as vascular endothelial growth factor, to promote angiogenesis. Norrin is a ligand that binds to the cell surface Frizzled-4 receptor, stimulating canonical Wnt signaling, and is known to mediate blood vessel growth in the eye and ear. We investigate here the role of Norrin secreted by colon cancer cells in endothelial cell growth and motility as well as the expression of the Norrin signaling pathway components in the CRC tumor microenvironment. Experimental procedures: Primary human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein and a HUVEC-derived endothelial cell line, EAhy926 were utilized for in vitro angiogenesis assays measuring cell motility and in vitro angiogenesis. Experiments were performed with and without the addition of Norrin-containing conditioned medium from a transfected CRC cell line CaCO2 and with and without blocking anti-Norrin antibodies. Tissue specimens from human CRC patients were evaluated with a double signal amplification system to define the expression of Norrin, Frizzled-4, LRP5 and TSPAN12 in tumor blood vessels. Summary of data: Blocking generated by the endothelial cells themselves Norrin reduced endothelial cell motility, branch point number during in vitro angiogenesis (1/sq. mm), and the network length (mm/sq. mm) during in vitro angiogenesis. Norrin conditioned medium produced by CRC cell line CaCO2 transfected with Norrin expression construct increased endothelial cell motility. Colorectal tumors express Norrin protein. Endothelial cells in the colorectal tumor blood vessels contain all of the components of the Norrin signaling pathway needed for the endothelial cells to respond to Norrin in the tumor microenvironment. Statement of the conclusions: This study presents both in vitro and in vivo data that colorectal tumors use Norrin protein as an alternative mechanism that promotes angiogenesis in the CRC tumor microenvironment. Citation Format: Kestutis Planutis, Marina Planoutene, Randall F. Holcombe. Norrin controls colon cancer angiogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1002. doi:10.1158/1538-7445.AM2014-1002

Dendritic Cell Cancer Vaccines for Treatment of Colon Cancer

Despite advances in treatment modalities, colorectal cancer (CRC) still accounts for about half a million deaths yearly worldwide. The majority of metastatic CRC are unresectable, and of those who undergo resection, few are cured. Advances in chemotherapy and targeted therapy have improved survival, but options remain limited, making the case for incorporating novel modalities in CRC treatment paradigm. Our improved understanding of the tumor immune microenvironment had led to the development of novel immunotherapeutic agents that have been tested in clinical trials such as cancer vaccines. Cancer vaccines are designed to activate the effector immune cells in order to generate an immune response against tumors. Dendritic cell vaccines offer potential benefits for CRC patients but requires further evaluation. We reviewed the role of dendritic cells in the colorectal tumor microenvironment and dendritic cancer vaccine studies in colorectal cancer.

A novel signaling pathway regulates colon cancer angiogenesis through Norrin.

Norrin binds to the frizzled-4 receptor, stimulating canonical Wnt signaling. We investigate here the role of colorectal cancer (CRC) produced Norrin in endothelial cell growth, motility, and blood vessel formation, as well as the expression of the Norrin signaling pathway components in the CRC tumor microenvironment. Norrin conditioned medium produced by CRC cell line CaCO2 transfected with Norrin expression construct increased endothelial cell motility. Blocking Norrin signaling reduced endothelial cell motility, branch point number (1/mm2), and the network length (mm/mm2) during in vitro angiogenesis. Colorectal tumors express Norrin protein. Endothelial cells in the colorectal tumor microenvironment contain all of the components of the Norrin signaling pathway needed to respond to Norrin protein. This study presents data that Norrin may play a role in the regulation of angiogenesis in the colorectal cancer tumor microenvironment.

MiR-21 and its target gene CCL20 are both highly overexpressed in the microenvironment of colorectal tumors: Significance of their regulation

Recently, we reported a functional interaction between miR-21 and its identified chemokine target CCL20 in colorectal cancer (CRC) cell lines. Here, we investigated whether such functional interactions are permitted at the cellular level which would require an inverse correlation of expression and also co-expression of miR-21 and CCL20 in the same cell. Expression profiling was performed using qPCR, and ELISA, in situ hybridization and immunohistochemistry were applied for the presentation of their cellular localization. We demonstrated that miR-21 as well as CCL20 were both significantly upregulated in CRC tissues; thus, showing no antidromic expression pattern. This provided an initial clue that miR-21 and CCL20 may not be expressed in the same cell. In addition, we located miR-21 expression at the cellular level predominantly in stromal cells such as tumor-associated fibroblasts and to a minor degree in immune cells such as macrophages and lymphocytes. Likewise, CCL20 expression was primarily detected in tumor-infiltrating immune cells. Thus, investigating the cellular localization of miR-21 and its target CCL20 revealed that both molecules are expressed predominantly in the microenvironment of CRC tumors.

Mesenchymal stem cells in the colorectal tumor microenvironment: Recent progress and implications

Mesenchymal stem cells (MSCs) are nonhematopoietic multipotent adult stem cells. They have been shown to have a natural tropism for many tumors types, including colorectal, and are capable of escaping host immune surveillance. MSCs are known to engraft at tumors and integrate into their architecture, potentially as carcinoma-associated fibroblasts. In contrast with other malignancies, our understanding of the interactions between colorectal cancer cells and MSCs remains limited. Considering the established importance of inflammation in the colorectal cancer primary tumor microenvironment and the role of stromal cells in this process, there is a potential wealth of information to be gleaned from further investigation of interactions between these cell populations. Epithelial-mesenchymal transition is central to colorectal cancer progression and MSCs have also been implicated in this process. This review explores the current knowledge (both in vitro and in vivo) of interactions between colorectal cancer cells and MSCs. It highlights potential effects of cell source, number and ratio on outcome of in vivo studies and explores strategies to more accurately explore their role in the primary tumor microenvironment. As our understanding of the underlying molecular processes in colorectal cancer develops, elucidation of these interactions will be central to development of novel therapeutic strategies for this prevalent disease.

Abstract B12: The effects of Irinophore C™ on the microenvironment and vasculature of a primary orthotopic model of colorectal cancer.

Abstract Introduction: Colorectal cancer is a common cancer that accounts for ∼10% of cancer cell deaths in North America. There are numerous in vivo xenograft models for colorectal cancer available, but in general, these are derived from immortalized cell lines and fail to capture the complexities and heterogeneity of tumors seen in the clinic. Our group has developed a series of orthotopic, primary tumors from samples of human colorectal cancer tissue obtained during surgical resection that maintain the characteristics of the original sample. The effects of Irinophore C™, (a liposomal formulation of irinotecan that is more efficacious and less toxic than the parent drug) on the microenvironment and vascular function of these primary colorectal tumors were examined in the present study. Materials and Methods: Primary tumor tissues, obtained from colorectal cancer patients, were validated by a reference pathologist and implanted subcutaneously in SCID mice. Small pieces of subcutaneous tumors that grew successfully were then passaged orthotopically on the ascending colon of new mice. When these tumors reached ∼200mm3, groups of mice were treated with vehicle control (0.9% saline), irinotecan (50mg/kg), or Irinophore C™ (25mg/kg) once a week for 6 weeks. Separate groups of tumors were harvested on days 3 and 42 after treatment started. Immunofluorescence staining was performed on tumor cryosections followed by computerized image analysis to determine levels of cell proliferation, apoptosis, hypoxia, and vessel density. Results: 4 of 14 samples were successfully propagated orthotopically and were characterized by a reference pathologist. The tumors maintain their original morphology, and one is a highly mucinous adenocarcinoma whereas the others are typical colorectal adenocarcinomas. Treatment with irinotecan and Irinophore C™ reduced tumor volumes by 54% and 92%, respectively, compared to the vehicle control. No toxic effects were seen with Irinophore C™. Immunostaining data showed that the distance between blood vessels remained the same for Irinotecan when compared to control, but increased with Irinophore C™ treatment (+30%; P&amp;lt;0.01). However, perfusion was not significantly different between the three treatment groups. Furthermore, compared to the vehicle treatment group, tumors treated with irinotecan are 42% (P&amp;lt;0.05) more necrotic and tumors treated with Irinophore C™ are 97% less hypoxic. Conclusion: An orthotopic model of colorectal cancer was successfully developed using patient tumor materials that retained the morphology of the primary tumor. Irinophore C™ was more effective in controlling tumor growth than irinotecan despite being delivered at a lower dose. In addition, treatment with Irinophore C™ appeared to improve overall vascular function in the tumor. Based on these data, it is clear that Irinophore C™ has different mechanisms of action and is more efficacious than irinotecan against primary models of colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B12.

3′-Chloro-5,7-dimethoxyisoflavone inhibits TNFα-induced CXCL10 gene transcription by suppressing the NF-κB pathway in HCT116 human colon cancer cells

Tumor necrosis factor α (TNFα) is a major inflammatory cytokine that plays important roles in progression of tumorigenesis in the tumor microenvironment. CXC chemokine ligand 10 (CXCL10), expression of which is stimulated by TNFα, is involved in tumor migration, invasion, and metastasis. 3′-Chloro-5,7-dimethoxyisoflavone (CDMF) is a synthetic isoflavone derivative. Here, we found that CDMF inhibits TNFα-induced invasive motility of human colon cancer cells. We tested whether CDMF would inhibit TNFα-induced CXCL10 expression using reverse transcription-PCR, quantitative real-time PCR, and enzyme-linked immunosorbent assay in HCT116 cells. CXCL10 expression, stimulated by TNFα, was suppressed by CDMF. The transcription factor nuclear factor-κB (NF-κB) is involved in TNFα-induced transcriptional activation of the CXCL10 gene promoter. Point mutation of the NF-κB binding site abolished TNFα-induced CXCL10 promoter activity. We next examined the effect of CDMF on TNFα-induced NF-κB activity. CDMF strongly inhibited both TNFα-induced IκB phosphorylation on Ser-32 and p65/RelA phosphorylation on Ser-536. Additionally, CDMF almost blocked TNFα-induced NF-κB-dependent transcriptional activity, as demonstrated by a NF-κB cis-acting reporter assay. Overall, our results indicate that CDMF suppresses production of CXCL10, by TNFα, through inhibition of NF-κB in HCT116 cells. We propose that CDMF may have beneficial effects in reducing TNFα-induced inflammatory responses, which are essential for tumor development in the colorectal tumor microenvironment.

The Colorectal Tumor Microenvironment: The Next Decade

Colorectal cancer cells establish a crosstalk with the tumor microenvironment, such that implantation and development of the tumor is generally favoured. CRC progression depends on mutations in the tumor's oncogenic pathways as well as metastasis suppressor genes, but is also influenced by the inflammatory components in the microenvironment. Inflammation results from the dietary intakes and is either compounded or counterbalanced by our lifestyles. Whether driven by intrinsic pathways or infection, inflammation produces a massive influx of cytokines and chemokines. Currently, in colorectal cancer, the best approach to counter this inflammatory wave in the microenvironment appears to be CCL2 cytokine targeting. A fairly new avenue of discovery has identified microRNAs regulating colorectal cancer-mediated inflammation, and in particular the IL-6 pro-inflammatory pathway that induces pro-apoptotic genes and HIF1α-elicited VEGF secretion. miRNAs also play a significant role in controlling metabolic genes such as the upregulation of the fatty acid synthase gene with the concomitant down-regulation of the carnitine palmitoyl transferase 1 gene. Within the metastatic environment, the Discoidin domain receptor-2 (DDR2) gene encodes a tyrosine kinase receptor for fibrillar collagen that contributes to colorectal cancer metastasis by increasing myofibroblasts, neoangiogenic vessels and proliferating cancer cells. Ongoing identification of gene signatures differentiating between primary tumor cells and their metastatic counterparts promises a wealth of new targets to be exploited for further therapeutic use within the next decade.

Matrix Metalloproteinase-14 (MT1-MMP)–Mediated Endoglin Shedding Inhibits Tumor Angiogenesis

Endoglin is a transforming growth factor-beta coreceptor with a crucial role in angiogenesis. A soluble form of endoglin is present in the circulation, but the role of soluble endoglin (sEndoglin) is poorly understood. In addition, the endoglin shedding mechanism is not known. Therefore, we examined the role of sEndoglin in tumor angiogenesis and the mechanism by which the extracellular domain of endoglin is released from the membrane.In colorectal cancer specimens, we observed high endothelial endoglin protein expression, accompanied with slightly lower sEndoglin levels in the circulation, compared with healthy controls. In vitro analysis using endothelial sprouting assays revealed that sEndoglin reduced spontaneous and vascular endothelial growth factor-induced endothelial sprouting. Human umbilical vascular endothelial cells were found to secrete high levels of sEndoglin. Endoglin shedding was inhibited by matrix metalloproteinase (MMP) inhibitors and MMP-14 short hairpin RNA, indicating MMP-14 as the major endoglin shedding protease. Coexpression of endoglin and membrane-bound MMP-14 led to a strong increase in sEndoglin levels. Endoglin shedding required a direct interaction between endoglin and membrane-localized MMP-14. Using cleavage site mutants, we determined that MMP-14 cleaved endoglin at a site in close proximity to the transmembrane domain. Taken together, this study shows that MMP-14 mediates endoglin shedding, which may regulate the angiogenic potential of endothelial cells in the (colorectal) tumor microenvironment.

Coordination of Intratumoral Immune Reaction and Human Colorectal Cancer Recurrence

A role for the immune system in controlling the progression of solid tumors has been established in several mouse models. However, the effect of immune responses and tumor escape on patient prognosis in the context of human cancer is poorly understood. Here, we investigate the cellular and molecular parameters that could describe in situ immune responses in human colorectal cancer according to clinical parameters of metastatic lymph node or distant organ invasion (META- or META+ patients). Primary tumor samples of colorectal carcinoma were analyzed by integrating large-scale phenotypic (flow cytometry, 39 patients) and gene expression (real time reverse transcription-PCR, 103 patients) data sets related to immune and protumoral processes. In META- colorectal cancer primary tumors with high densities of T cells, we observed significant positive correlations between markers of innate immune cells [tumor-associated macrophages, dendritic cells, natural killer (NK) cells, and NKT cells] and markers of early-activated T cells. Significant correlations were also observed between markers of cytotoxic and effector memory T-cell subpopulations. These correlation profiles were absent in tumors with low T-cell infiltrates and were altered in META+ tumors with high T-cell infiltrates. We show that the coexpression of genes mediating cytotoxicity (GNLY) and Th1 adaptive immune responses (IRF1) accurately predicted patient survival independently of the metastatic status. High intratumoral mRNA expression of the proangiogenic mediator vascular endothelial growth factor was associated with significantly reduced survival rates in patients expressing high mRNA levels of GNLY. Investigation of the colorectal cancer primary tumor microenvironment allowed us to uncover the association of favorable outcomes with efficient coordination of the intratumoral immune response.

15-Deoxy-Δ 12,14-prostaglandin J 2 down-regulates CXCR4 on carcinoma cells through PPARγ- and NFκB-mediated pathways

The chemokine receptor CXCR4 plays a key role in the metastasis of colorectal cancer and its growth at metastatic sites. Here, we have investigated the mechanisms by which CXCR4 on cancer cells might be regulated by eicosanoids present within the colorectal tumor microenvironment. We show that prostaglandins PGE 2, PGA 2, PGD 2, PGJ 2 and 15dPGJ 2 each down-regulates CXCR4 receptor expression on human colorectal carcinoma cells to differing degrees. The most potent of these were PGD 2 and its metabolites PGJ 2 and 15dPGJ 2. Down-regulation was most rapid with the end-product 15dPGJ 2 and was accompanied by a marked reduction in CXCR4 mRNA. 15dPGJ 2 is known to be a ligand for the nuclear receptor PPARγ. Down-regulation of CXCR4 was also observed with the PPARγ agonist rosiglitazone, while 15dPGJ 2-induced CXCR4 down-regulation was substantially diminished by the PPARγ antagonists GW9662 and T0070907. These data support the involvement of PPARγ. However, the 15dPGJ 2 analogue CAY10410, which can act on PPARγ but which lacks the intrinsic cyclopentenone structure found in 15dPGJ 2, down-regulated CXCR4 substantially less potently than 15dPGJ 2. The cyclopentenone grouping is known to inhibit the activity of NFκB. Consistent with an additional role for NFκB, we found that the cyclopentenone prostaglandin PGA 2 and cyclopentenone itself could also down-regulate CXCR4. Immunolocalization studies showed that the cellular context was sufficient to trigger a focal nuclear pattern of NFκB p50 and that 15dPGJ 2 interfered with this p50 nuclear localization. These data suggest that 15dPGJ 2 can down-regulate CXCR4 on cancer cells through both PPARγ and NFκB. 15dPGJ 2, present within the tumor microenvironment, may act to down-regulate CXCR4 and impact upon the overall process of tumor expansion.