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

Detailed information about Material and Methods used throughout the manuscript including information about cell lines, antibodies, biochemical assays and in vitro and in vivo assays.

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

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.

This work describes the development of an analytical method for quantitative laser ablation tandem ICP-mass spectrometry (LA-ICP-MS/MS) imaging of iron oxide nanoparticles (FeOx NPs) in gelatin microspheres containing CaCO3 crystals (≤30 μm diameter). The strong spectral overlap between the signal of 56Fe+ and those of 40Ar16O+, 38Ar18O+, 40Ar15NH+ and 40Ca16O+ and between those of 40Ca+ and 40Ar+ was overcome by relying on chemical resolution using a mixture of NH3/He (1 : 9) in a mass-shift approach. Product ion scanning allowed Fe(NH3)2+ and CaNH3+ to be identified as the best suited reaction product ions, formed upon reaction of NH3 with Fe+ and Ca+, respectively. The method was validated by using two certified reference materials, i.e. NIST SRM 1577 and 1577a bovine liver, which were solubilized by acidic digestion. Fe-spiked gelatin droplet standards were prepared on a high-purity single-side polished silicon wafer and used as external calibration standards for LA-ICP-MS/MS analysis and a priori, their corresponding Fe concentrations were determined via pneumatic nebulization tandem ICP-MS (PN-ICP-MS/MS) after acidic digestion. High-resolution LA-ICP-MS/MS imaging revealed the mixed structure of the gelatin microspheres containing a high loading of FeOx NPs (2.69 ± 0.91 wt% Fe) in the gelatin phase. The high FeOx NP concentrations are required for magnetic removal after peritoneal injection and are accompanied by numerous CaCO3 crystals that provide an indication about the final porosity of the microspheres.

Here we report that grafting temperature-responsive polymers onto hyaluronic acid allows temperature-induced self-assembly into nanogels with tunable size. These nanogels can be easily loaded with hydrophobic molecules and hold potential for anti-cancer drug delivery towards human ovarian cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in cancer cells without causing damage to normal cells. However, some tumors are resistant to TRAIL monotherapy and clinical studies assessing targeted agents towards the TRAIL receptor have failed to show robust therapeutic activity. Evidence has shown that standard anti-mitotic drugs can induce synergistic apoptosis upon combination with TRAIL via cell cycle arrest. Polo like kinase-1 (PLK1) plays a critical role in different stages of cell cycle progression and mitosis. A number of investigations have demonstrated that PLK1 inhibition causes cell cycle arrest and mitotic catastrophe in non-small cell lung cancer (NSCLC), and we thus postulated that PLK1 inhibition could enhance TRAIL-induced apoptosis. We demonstrate that the combination of a TRAIL receptor agonist and a PLK1 inhibitor synergistically reduces cell viability, and strongly increases apoptosis in NSCLC cellular models. Consistent with our in vitro observations, this drug combination also significantly reduces tumor growth in vivo. Our data additionally reveal that G2/M cell cycle arrest and downregulation of Mcl-1 and signal transducer and activator of transcription 3 (STAT3) activity following PLK1 inhibition may contribute to the sensitization of TRAIL-induced apoptosis in NSCLC. Together, these data support the further exploration of combined TRAIL and PLK1 inhibition in the treatment of NSCLC.

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.

Cancer-associated fibroblasts (CAFs) support cancer growth, invasion, and metastasis. Glucocorticoids (GCs), drugs often administered together with chemotherapy, are steroidal ligands of the glucocorticoid receptor (GR), a transcription factor which upon activation regulates expression of multiple genes involved in suppression of inflammation. We have previously shown that in dexamethasone (Dex)-treated CAFs derived from colon cancer, production and secretion of several factors related to cancer progression, such as tenascin C (TNC) and hepatocyte growth factor (HGF), were strongly suppressed. In this study we show that GCs can neutralize the cancer cell-promoting properties of CAFs. Conditioned medium from solvent-treated CAFs (CMCTRL) stimulates proliferation, motility and stretched morphotype of GRdeficient HCT8/E11 colon cancer cells. Yet, HCT8/E11 proliferation and stretched morphotype are impaired upon treatment with conditioned medium from Dex-treated CAFs (CMDEX), but HCT8/E11 cell migration is slightly increased under these conditions. Moreover, expression and potential activity of MMP-2 is also reduced in CMDEX compared with CMCTRL. These combined in vitro results concur with the results from in vivo chick chorioallantoic membrane assays, where the co-cultures of CAFs with colon cancer cells displayed impaired tumor formation and cancer cell invasion due to Dex administration. Combined, GC treatment influences cancer cell behavior indirectly through effects on CAFs. ispartof: Experimental Cell Research vol:362 issue:2 pages:332-342 ispartof: location:United States status: published

The present study aimed to investigate the orthotopic growth potential of two generally available esophageal adenocarcinoma cell lines, OE33 and OACM5 1.C, and a third in vivo selected subpopulation, OACM5 1.C SC1. One group of mice was subcutaneously injected in the hind legs. Tumor growth was measured with calipers. Another group was injected orthotopically in the distal esophageal wall through median laparotomy. Tumor development was evaluated macroscopically and confirmed microscopically. A subset of mice was evaluated with magnetic resonance imaging (MRI) to follow tumor progression. Additionally, functional cell line characteristics were evaluated in vitro (clonogenic, collagen invasion and sphere formation assays, and protein analysis of cell-cell adhesion and cytoskeletal proteins) to better understand xenograft behavior. OE33 cells were shown to be epithelial-like, whereas OACM5 1.C and OACM5 1.C SC1 were more mesenchymal-like. The three cell lines were non-invasive into native type I collagen gels. In vivo, OE33 cells led to 63.6 and 100% tumor nodules after orthotopic (n=12) and subcutaneous (n=8) injection, respectively. Adversely, OACM5 1.C cells did not lead to tumor formation after orthotopic injection (n=6) and only 50% of subcutaneous injections led to tumor nodules (n=8). However, the newly established cell line OACM5 1.C SC1 resulted in 33% tumor formation when orthotopically injected (n=6) and in 100% tumors when injected subcutaneously (n=8). The higher xenograft rate of OACM5 1.C SC1 (P

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., SCOPUS: ar.j, info:eu-repo/semantics/published

Background Esophageal cancer is an aggressive disease with poor survival rates. A more patient-tailored approach based on predictive biomarkers could improve outcome. We aimed to predict radiotherapy (RT) response by imaging tumor hypoxia with 18F-FAZA PET/CT in an esophageal adenocarcinoma (EAC) mouse model. Additionally, we investigated the radiosensitizing effect of the hypoxia modifier nimorazole in vitro and in vivo. Methods In vitro MTS cell proliferation assays (OACM5 1.C SC1, human EAC cell line) were performed under normoxic and hypoxic (

Cancer-associated fibroblasts (CAFs) are drivers of tumour progression and are considered as a target and a tool in cancer diagnostic and therapeutic applications. An increased abundance of CAFs or CAF signatures are recognized as a bad prognostic marker in several cancer types. Tumour-environment biomimetics strongly improve our understanding of the communication between CAFs, cancer cells and other host cells. Several experimental drugs targeting CAFs are in clinical trials for multiple tumour entities; alternatively, CAFs can be exploited as a tool to characterize the functionality of circulating tumour cells or to capture them as a tool to prevent metastasis. The continuous interaction between tissue engineers, biomaterial experts and cancer researchers creates the possibility to biomimic the tumour-environment and provides new opportunities in cancer diagnostics and management.

Peritoneal metastasis is life threatening and is the result of an extensive communication between disseminated cancer cells, mesothelial cells and cancer-associated fibroblasts (CAF). CAFs secrete extracellular matrix (ECM) proteins creating a receptive environment for peritoneal implantation. Considering cancer as an ecosystem may provide opportunities to exploit CAFs to create biomimetic traps to deceive and redirect cancer cells. We have designed microparticles (MP) containing a CAF-derived ECM-surface that is intended to compete with natural niches. CAFs were encapsulated in alginate/gelatine beads (500–750 μm in diameter) functionalised with a polyelectrolyte coating (MP[CAF]). The encapsulated CAFs remain viable and metabolically active (≥35 days), when permanently encapsulated. CAF-derived ECM proteins are retained by the non-biodegradable coating. Adhesion experiments mimicking the environment of the peritoneal cavity show the selective capture of floating cancer cells from different tumor origins by MP[CAF] compared to control MP. MP[CAF] are distributed throughout the abdominal cavity without attachment to intestinal organs and without signs of inflammatory reaction. Intraperitoneal delivery of MP[CAF] and sequential removal redirects cancer cell adhesion from the surgical wound to the MP[CAF], delays peritoneal metastasis formation and prolongs animal survival. Our experiments suggest the use of a biomimetic trap based on tumor–environment interactions to delay peritoneal metastasis.

Peritoneal metastasis is a major cause of death and preclinical models are urgently needed to enhance therapeutic progress. This study reports on a hybrid hydrogel-polylactic acid (PLA) scaffold that mimics the architecture of peritoneal metastases at the qualitative, quantitative and spatial level. Porous PLA scaffolds with controllable pore size, geometry and surface properties are functionalized by type I collagen hydrogel. Co-seeding of cancer-associated fibroblasts (CAF) increases cancer cell adhesion, recovery and exponential growth by in situ heterocellular spheroid formation. Scaffold implantation into the peritoneum allows long-term follow-up (>14 weeks) and results in a time-dependent increase in vascularization, which correlates with cancer cell colonization in vivo. CAF, endothelial cells, macrophages and cancer cells show spatial and quantitative aspects as similarly observed in patient-derived peritoneal metastases. CAF provide long-term secretion of complementary paracrine factors implicated in spheroid formation in vitro as well as in recruitment and organization of host cells in vivo. In conclusion, the multifaceted heterocellular interactions that occur within peritoneal metastases are reproduced in this tissue-engineered implantable scaffold model. (C) 2017 Elsevier Ltd. All rights reserved.

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.

Introduction: Biomimetics of the tumor-environment, an ecosystem, can be applied to create an ecological trap. An ecological trap is an environment of low quality for survival that is preferred by an organism over a better available environment. Carcinoma associated cancer cells (CAFs) produce an extracellular matrix (ECM) that exerts a high attraction to disseminated cancer cells, and is the perfect bait for an ecological trap. Microparticle encapsulated CAFs (MP[CAF]) can redirect adhesion of disseminated cancer cells from the peritoneal wall to the MP[CAF] surface to prevent peritoneal metastasis formation. Materials and Methods: Encapsulation: CAFs cells are encapsulated into microparticles (MP[CAF] 500-700µm) by dripping a mixture of alginate (1.5%), gelatin (0.5%) and CAFs (2.106/ml) in to a CaCl2 bath (1.3%) though a needle (260µm inner diameter) surrounded by an airflow. MP[CAF] where layer-by-layer coated with poly-styrene sulfonate and poly-allyamine (PSS/PAH), with or without the incorporation of iron-oxide nanoparticles between the layers. In vitro confrontation assay: MP[CAF] and MPs without CAFs where confronted with luciferase positive cancer cells (1.105) while shaking. After 48h adhesion the two types of MPs are magnetically separated and the adhesion of SK-OV-3 Luc on the MPs is measured through bioluminescence. In vivo: MP[CAF] (200) are inserted through a small incision in to the abdominal cavity of nude mice. 1.105 ovarian cancer cells (SK-OV-3 Luc IP1) are injected. 24h later MPs with captured cancer cells are removed with a magnet. Sham threated mice underwent the same procedure but now MPs where inserted. Results and Discussion: MP[CAF] are stable and encapsulated CAFs are viable and metabolic active for over 4 weeks. CAF-derived ECM proteins are retained by the PSS/PAH coating. In vitro confrontation show that cancer cells have a higher affinity for MP[CAF] compared to empty MP (fig 1A-B). Small animal MRI imaging shows distribution of MP throughout the abdominal cavity without attachment to intestinal organs and without signs of inflammatory reaction. MP[CAF] trap cancer cells and redirect adhesion away from the wound site. Removal of the MP[CAF] results in a delay peritoneal metastasis formation and a prolonged survival in mice (fig 2A-D).

Ovarian cancer carries a significant mortality. Since symptoms tend to be minimal, the disease is often diagnosed when peritoneal metastases are already present. The standard of care in advanced ovarian cancer consists of platinum-based chemotherapy combined with cytoreductive surgery. Unfortunately, even after optimal cytoreduction and adjuvant chemotherapy, most patients with stage III disease will develop a recurrence. Intraperitoneal administration of chemotherapy is an alternative treatment for patients with localized disease. The pharmacological and physiochemical properties of melflufen, a peptidase potentiated alkylator, raised the hypothesis that this drug could be useful in ovarian cancer and particularily against peritoneal carcinomatosis. In this study the preclinical effects of melflufen were investigated in different ovarian cancer models. Melflufen was active against ovarian cancer cell lines, primary cultures of patient-derived ovarian cancer cells, and inhibited the growth of subcutaneous A2780 ovarian cancer xenografts alone and when combined with gemcitabine or liposomal doxorubicin when administered intravenously. In addition, an intra- and subperitoneal xenograft model showed activity of intraperitoneal administered melflufen for peritoneal carcinomatosis, with minimal side effects and modest systemic exposure. In conclusion, results from this study support further investigations of melflufen for the treatment of peritoneal carcinomatosis from ovarian cancer, both for intravenous and intraperitoneal administration.

Peritoneal metastases cause cancer mortality and preclinical models are urgently needed to boost therapeutic progress. This study reports on a hybrid hydrogel-polylactic acid (PLA) scaffold that mimics the architecture of peritoneal metastases at the qualitative, quantitative and spatial level. Porous PLA scaffolds with controllable pore size, shape, and surface properties are functionalized by type I collagen hydrogel and co-seeding of cancer-associated fibroblast (CAF) increases cancer cell adhesion, recovery, and exponential growth by in situ heterocellular spheroid formation. Scaffold implantation at the peritoneal wall allows long-term follow-up (>14 weeks) and results in a time-dependent increase in vascularization, which correlates with cancer cell colonization in vivo. CAF, endothelial cells, macrophages and cancer cells show spatial and quantitative aspects as similarly observed in patient-derived peritoneal metastases. CAF provide long-term secretion of complementary paracrine factors implicated in spheroid formation in vitro as well as in recruitment and organization of host cells in vivo. In conclusion, the multifaceted heterocellular interactions that occur within peritoneal metastases are reproduced in this tissue-engineered implantable scaffold model. Citation Format: Olivier De Wever, Emiel Dejaeghere, Elly De Vlieghere. Heterocellular 3D scaffolds as biomimetic to recapitulate the tumor microenvironment of peritoneal metastases in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5027.

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.

For the development of effective anti-cancer vaccines, tumor associated antigens need to be internalized by antigen presenting cells alongside specific co-stimulatory signals. Interestingly, relative to soluble antigens, nano-and micro-particulate antigens are much better presented to CD8 T cells, a crucial step in the induction of cytotoxic T cells that can eliminate malignant cells. In this regard, a generic strategy to encapsulate cancer cell derived proteins into a particulate delivery system would be of high interest. Here we present a versatile approach to incorporate cancer cell proteins into polymeric capsules using the cells themselves as templates for layer-by-layer assembly of complimentary interacting species. After coating, the cells are killed by hypo-osmotic treatment leading to bio-hybrid capsules loaded with cell lysate. Particular focus is devoted in this work on choosing the optimal coating components and conditions to maximize cell membrane integrity during the coating process, minimize pre-mature protein release and achieve optimal encapsulation of cell lysate upon lysis of the cells. To further underline the generic nature of our approach, we demonstrate that heat shock proteins, important immune-activators, can be induced and encapsulated into the bio-hybrid capsules.

Cancer progression is characterized by a complex reciprocity between neoplastic epithelium and adjacent stromal cells. In ductal carcinoma in situ (DCIS) of the breast, both reduced stromal decorin expression and myxoid stroma are correlated with increased recurrence risk. In this study, we aimed to investigate paracrine regulation of expression of decorin and related extracellular matrix (ECM) proteins in cancer-associated fibroblasts (CAFs). Transforming growth factor-β1 (TGF-β1) was identified as a competent ECM modulator, as it reduced decorin and strongly enhanced versican, biglycan and type I collagen expression. Similar but less pronounced effects were observed when fibroblasts were treated with basic fibroblast growth factor (bFGF). Despite this concerted ECM modulation, TGF-β1 and bFGF differentially regulated alpha-smooth muscle actin (α-SMA) expression, which is often proposed as a CAF-marker. Cancer cell-derived secretomes induced versican and biglycan expression in fibroblasts. Immunohistochemistry on twenty DCIS specimens showed a trend toward periductal versican overexpression in DCIS with myxoid stroma. Cancer cell adhesion was inhibited by decorin, but not by CAF-derived matrices. Cancer cells presented significantly enhanced spreading when seeded on matrices derived from TGF-β1-treated CAF. Altogether these data indicate that preinvasive cancerous lesions might modulate the composition of surrounding stroma through TGF-β1 release to obtain an invasion-permissive microenvironment.