Supplementary Figure 1-7 on irradiation of CAF; effect of irradiated CAF on cancer cells; glutamine metabolism; proteome analysis of CM CAF; effect of recombinant IGF1 on cancer cells; effect of recombinant IGF1 on metabolism of cancer cells; schematic of in vivo set-up and Figure legends or respective figures

Supplementary Table S1: patient characteristics including age, sex, tumor type, and treatment

Abstract Preoperative radiotherapy (RT) is a mainstay in the management of rectal cancer, a tumor characterized by desmoplastic stroma containing cancer-associated fibroblasts (CAF). Although CAFs are abundantly present, the effects of RT to CAF and its impact on cancer cells are unknown. We evaluated the damage responses of CAF to RT and investigated changes in colorectal cancer cell growth, transcriptome, metabolome, and kinome in response to paracrine signals emerging from irradiated CAF. RT to CAF induced DNA damage, p53 activation, cell-cycle arrest, and secretion of paracrine mediators, including insulin-like growth factor-1 (IGF1). Subsequently, RT-activated CAFs promoted survival of colorectal cancer cells, as well as a metabolic switch favoring glutamine consumption through IGF1 receptor (IGF1R) activation. RT followed by IGF1R neutralization in orthotopic colorectal cancer models reduced the number of mice with organ metastases. Activation of the downstream IGF1R mediator mTOR was significantly higher in matched (intrapatient) samples and in unmatched (interpatient) samples from rectal cancer patients after neoadjuvant chemoradiotherapy. Taken together, our data support the notion that paracrine IGF1/IGF1R signaling initiated by RT-activated CAF worsens colorectal cancer progression, establishing a preclinical rationale to target this activation loop to further improve clinical responses and patient survival. Significance: These findings reveal that paracrine IGF1/IGF1R signaling promotes colorectal cancer progression, establishing a preclinical rationale to target this activation loop. Cancer Res; 78(3); 659–70. ©2017 AACR.

FHL2 is a multifunctional scaffolding protein; its expression is associated with poor prognosis in colorectal cancer. ADAM-17 is a metalloprotease implicated in ectodomain shedding. FHL2 regulates ADAM-17 plasma membrane localisation, and FHL2 deficiency leads to decreased activity of ADAM-17 in mouse macrophages. Presence and relationship of the ADAM-17/FHL2 complex with colorectal cancer progression is unknown. We studied FHL2 and ADAM-17 expression in several colon cancer cell lines by immunocytochemistry and western blot. To highlight the interaction between both molecules, we used the Duolink ® kit for proximity ligation assay on SW480 cells. We also performed proximity ligation assay on biopsies and surgical specimens of colorectal adenocarcinoma and on matched normal mucosa. Furthermore, biopsies of colorectal adenoma with matched normal mucosa were selected. For quantification, pictures of the malignant, adenomatous and normal tissues were taken. Proximity ligation assay signals were quantified. Mean numbers of proximity ligation assay signals and of proximity ligation assay signals/nucleus were calculated. All cell lines showed FHL2 immunoreactivity; strongest positivity was observed in SW480 cells. ADAM-17 was expressed in all cell lines. Proximity ligation assay signals were present in SW480 cells. Quantitative analysis revealed that the interaction between FHL2 and ADAM-17 is more frequent in malignant than in normal tissue (p = 0.005). The mean number of ADAM-17/FHL2 proximity ligation assay signals was higher in colorectal adenocarcinoma than in adenoma with low-grade dysplasia (p = 0.0004). FHL2 interacts with ADAM-17 in normal, dysplastic and malignant colon epithelial cells. Colocalisation of these proteins is more frequent in malignant than in normal and dysplastic cells, suggesting a role for ADAM-17/FHL2 complex in the development of colorectal cancer.

Joke Tommelein , Elly De Vlieghere , Laurine Verset, Elodie Melsens , Justine Leenders, Benedicte Descamps, Annelies Debucquoy, Christian Vanhove, Patrick Pauwels, Christian P. Gespach, Anne Vral, Astrid De Boeck, Karin Haustermans, Pascal de Tullio, Wim Ceelen , Pieter Demetter, Tom Boterberg , Marc Bracke , Olivier De Wever * Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University, Ghent, Belgium Cancer Research Institute Ghent (CRIG), Ghent, Belgium Department of Pathology, Erasme University Hospital, Université Libre de Bruxelles, Brussels, Belgium Department of Surgery, Ghent University Hospital, Ghent, Belgium Center for Interdisciplinary Research on Medicines (CIRM), Université de Liège, Liège, Belgium Department of Electronics and Information System, iMinds-IBiTech-MEDISIP, Ghent University, Ghent, Belgium 7 KU Leuven, University of Leuven, Department of Oncology, Experimental Radiotherapy; University Hospitals Leuven, Radiation Oncology, Leuven, Belgium Center for Oncological Research (CORE), University of Antwerp, Antwerp, Belgium Institut National de la Santé et de la Recherche Médicale, INSERM U938, Molecular and Clinical Oncology, Université Paris VI Pierre et Marie Curie, Paris, France Department of Basic Medical Sciences, Physiology Group, Ghent University, Ghent, Belgium Arnie Charbonneau Cancer Centre, University of Calgary, Calgary, AB, Canada

Abstract Preoperative radiotherapy is a mainstay in current management of colorectal cancer. Several tumor types, including colorectal cancer, show an abundant desmoplastic stroma, characterized by cancer-associated fibroblasts (CAFs). Nevertheless the effects of radiation to the CAFs in the stroma and its reciprocal impact on the response of CRC cells to irradiation have not been studied. We investigated the functional (cell number, morphotype) and biochemical (transcriptomics, metabolomics and kinomics) phenotype of CRC cells in response to paracrine signals from irradiated CAFs. Using neutralizing antibodies and small molecule inhibitors, we analysed the role of tyrosine-kinase receptor activation in CRC cells in response to supernatants from irradiated CAFs. Furthermore, the impact of tyrosine-kinase receptor neutralization in combination with RT was evaluated in an orthotopic CRC model. The level of mTOR activation, a downstream tyrosine-kinase receptor intermediate, was analysed in response to neoadjuvant treatment in human CRC specimens. Further work will reveal which soluble factor(s) are implicated in the communication between irradiated CAFs and colorectal cancer cells. Citation Format: Olivier De Wever, Joke Tommelein, Marc Bracke, Tom Boterberg, Pieter Demetter, Laurin Verset. Radiotherapy-induced damage to cancer-associated fibroblasts and its reciprocal influence on cancer progression: Impact of soluble factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5907. doi:10.1158/1538-7445.AM2017-5907

In patients with rectal prolapse is the prevalence of colorectal cancer increased, suggesting that a colorectal tumor may induce rectal prolapse. Establishment of tumor xenografts in immunodeficient mice after orthotopic inoculations of human colorectal cancer cells into the caecal wall is a widely used approach for the study of human colorectal cancer progression and preclinical evaluation of therapeutics. Remarkably, 70% of young mice carrying a COLO320DM caecal tumor showed symptoms of intussusception of the large bowel associated with intestinal lumen obstruction and rectal prolapse. The quantity of the COLO320DM bioluminescent signal of the first three weeks post-inoculation predicts prolapse in young mice. Rectal prolapse was not observed in adult mice carrying a COLO320DM caecal tumor or young mice carrying a HT29 caecal tumor. In contrast to HT29 tumors, which showed local invasion and metastasis, COLO320DM tumors demonstrated a non-invasive tumor with pushing borders without presence of metastasis. In conclusion, rectal prolapse can be linked to a non-invasive, space-occupying COLO320DM tumor in the gastrointestinal tract of young immunodeficient mice. These data reveal a model that can clarify the association of patients showing rectal prolapse with colorectal cancer.

Colorectal cancer (CRC) progression and eventually metastasis is directed in many aspects by a circuitous ecosystem consisting of an extracellular matrix scaffold populated by cancer-associated fibroblasts (CAFs), endothelial cells, and diverse immune cells. CAFs are recruited from local tissue-resident fibroblasts or pericryptal fibroblasts and distant fibroblast precursors. CAFs are highly abundant in CRC. In this review, we apply the metastasis-promoting communication of colorectal CAFs to 10 cancer hallmarks described by Hanahan and Weinberg. CAFs influence innate and adaptive tumor immune responses. Using datasets from previously published work, we re-explore the potential messages implicated in this process. Fibroblasts present in metastasis (metastasis-associated fibroblasts) from CRC may have other characteristics and functional roles than CAFs in the primary tumor. Since CAFs connect metastasis-promoting communication, CAF markers are potential prognostic biomarkers. CAFs and their products are possible targets for novel therapeutic strategies.

BACKGROUND AND PURPOSE:Cancer-associated fibroblasts (CAFs) are increasingly recognised as promoters of tumour progression. It is poorly investigated whether cancer management protocols, such as neoadjuvant radio(chemo)therapy, have an impact on CAFs and, by consequence, on tumour progression. This prompted us to study the impact of neoadjuvant radio(chemo)therapy on the α-SMA/epithelial area ratio in rectal cancer, and the impact of this ratio on recurrence-free survival.MATERIAL AND METHODS:Immunohistochemistry for the CAF marker α-SMA and the proliferation marker Ki67 was performed on sections from 98 rectal cancers of which 62 had undergone neoadjuvant radio(chemo)therapy.RESULTS:Computer-assisted quantitative analysis showed that the α-SMA/neoplastic epithelial area ratio was higher after neoadjuvant therapy, and that rectal cancers with high α-SMA/epithelial area ratio had low proliferation rates. Interestingly, the α-SMA/epithelial area ratio was an adverse prognostic factor with regard to recurrence-free survival in univariate analysis. In addition, multivariate analysis showed that an α-SMA/epithelial area ratio above 1 provides an independent prognostic value associated with a poor recurrence-free survival.CONCLUSION:These results suggest that neoadjuvant treatment has an impact on CAFs in rectal cancer. The correlation of CAFs with decreased recurrence-free survival and abundant experimental data in the literature suggest that under certain circumstances, not yet very well understood, CAFs may favour tumour progression.

401 Background: Four-and-a-half LIM domains protein 2 (FHL2) is a component of the focal adhesion structures and has been suggested to play a role in cancer progression. It has been shown to be overexpressed in colorectal cancer (CRC). Here we examined a possible prognostic value of FHL2 in CRC. Methods: Immunohistochemistry for FHL2 was performed on 296 CRCs without distant metastases at time of surgery. Staining in the epithelial compartment was quantitatively evaluated using image analysis, and results were related to clinical variables. Antibody specificity was tested using siRNA transfection in hTERT-immortalised myofibroblasts. FHL2 expression was investigated also by Western blotting in sporadic (HT29) and HNPCC-derived (HCT116 and HCT8/E11) colon cancer cell lines. Results: Varying degrees of cytoplasmic FHL2 expression by neoplastic epithelial cells were detectable in all cases. Higher FHL2 expression in the epithelial compartment was an independent adverse prognostic factor. Expression in the tumour invasion front (p < 0.001) as well as in the centre of the tumour (p < 0.001) was associated with metachronous metastases. No relation between FHL2 expression and microsatellite instability could be demonstrated. Conclusions: Higher FHL2 expression is involved in CRC progression and correlates with the development of metachronous metastases, suggesting that FHL2 is an independent adverse prognostic indicator for CRC.