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Supplementary tables S1-6. Supplementary table S1 Primers used for RT-qPCR analyses. Supplementary table S2 Clinical variables associated with differences in gene expression among 119 resected liver metastases. Supplementary table S3 613 genes whose expression was significantly upregulated and 481 genes whose expression was significantly (p

Purpose: Chemotherapy treatment of metastatic colon cancer ultimately fails due to development of drug resistance. Identification of chemotherapy-induced changes in tumor biology may provide insight into drug resistance mechanisms.Experimental Design: We studied gene expression differences between groups of liver metastases that were exposed to preoperative chemotherapy or not. Multiple patient-derived colonosphere cultures were used to assess how chemotherapy alters energy metabolism by measuring mitochondrial biomass, oxygen consumption, and lactate production. Genetically manipulated colonosphere-initiated tumors were used to assess how altered energy metabolism affects chemotherapy efficacy.Results: Gene ontology and pathway enrichment analysis revealed significant upregulation of genes involved in oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis in metastases that were exposed to chemotherapy. This suggested chemotherapy induces a shift in tumor metabolism from glycolysis towards OXPHOS. Indeed, chemotreatment of patient-derived colonosphere cultures resulted in an increase of mitochondrial biomass, increased expression of respiratory chain enzymes, and higher rates of oxygen consumption. This was mediated by the histone deacetylase sirtuin-1 (SIRT1) and its substrate, the transcriptional coactivator PGC1α. Knockdown of SIRT1 or PGC1α prevented chemotherapy-induced OXPHOS and significantly sensitized patient-derived colonospheres as well as tumor xenografts to chemotherapy.Conclusions: Chemotherapy of colorectal tumors induces a SIRT1/PGC1α-dependent increase in OXPHOS that promotes tumor survival during treatment. This phenomenon is also observed in chemotherapy-exposed resected liver metastases, strongly suggesting that chemotherapy induces long-lasting changes in tumor metabolism that potentially interfere with drug efficacy. In conclusion, we propose a novel mechanism of chemotherapy resistance that may be clinically relevant and therapeutically exploitable. Clin Cancer Res; 21(12); 2870–9. ©2015 AACR.

Supplementary Figures S1-3. Supplementary figure S1 Cytochrome c oxidase (COX IV) is highly expressed in chemotherapy-treated liver metastases Supplementary figure S2 Expression changes of glycolytic and OXPHOS genes in response to chemotherapy. Supplementary figure S3 Oxygen consumption rates of colonospheres treated with chemotherapy +/- nicotinamide

Supplementary Table S5. Primer pairs used for RT-qPCR analysis.

Supplementary Figure S2. Loss of GPx2 impairs differentiation of CSCs.

Supplementary Figure Legends. Legends for Supplementary Figures S1-S6.

Supplementary Materials and Methods. Description of additional methods and procedures used in the study.

Colorectal tumorigenesis is accompanied by the generation of oxidative stress, but how this controls tumor development is poorly understood. Here, we studied how the H2O2-reducing enzyme glutathione peroxidase 2 (GPx2) regulates H2O2 stress and differentiation in patient-derived “colonosphere” cultures. GPx2 silencing caused accumulation of radical oxygen species, sensitization to H2O2-induced apoptosis, and strongly reduced clone- and metastasis-forming capacity. Neutralization of radical oxygen species restored clonogenic capacity. Surprisingly, GPx2-suppressed cells also lacked differentiation potential and formed slow-growing undifferentiated tumors. GPx2 overexpression stimulated multilineage differentiation, proliferation, and tumor growth without reducing the tumor-initiating capacity. Finally, GPx2 expression was inversely correlated with H2O2-stress signatures in human colon tumor cohorts, but positively correlated with differentiation and proliferation. Moreover, high GPx2 expression was associated with early tumor recurrence, particularly in the recently identified aggressive subtype of human colon cancer. We conclude that H2O2 neutralization by GPx2 is essential for maintaining clonogenic and metastatic capacity, but also for the generation of differentiated proliferating tumor mass. The results reveal an unexpected redox-controlled link between tumor mass formation and metastatic capacity. Cancer Res; 74(22); 6717–30. ©2014 AACR.

Background Physical activity is associated with a lower risk of colorectal cancer (CRC) and CRC‐specific mortality. However, evidence for a causal relationship between physical activity and disease progression is lacking. Here, we have used CRC organoids to create a novel mouse model for spontaneous metastasis formation to multiple organs. We have used this model to assess the influence of voluntary exercise on disease progression. Methods Collagen‐embedded murine colorectal tumour organoids were transplanted into the livers of immunocompetent C57Bl/6 mice using microsurgery. Voluntary exercise in tumour‐bearing mice was modelled by offering running wheels continuously (n = 12) or 3 h/day (n = 12) versus no wheel access (n = 12). Running wheel revolutions were cumulatively measured every 30 min and physical activity was continuously monitored by infrared cameras. Food intake was monitored throughout the experiment and body composition was assessed with echoMRI. Animals were sacrificed 14 weeks after tumour initiation. Tumour load was quantified by EpCAM immunohistochemistry staining. Systemic inflammation parameters were assessed in blood plasma by a multiplex immunoassay. Results Tumour growth was initiated by implantation of CRC organoids into the livers of immunocompetent mice. The resulting tumours spontaneously formed distant metastases to non‐implanted liver lobes and to the lungs. Mice with access to the running wheels for 3 h/day ran relatively short distances (2.3 ± 0.3 km/night; 221 ± 29 km total distance) with relatively high intensity (wheel revolutions/h). Mice with continuous access to the running wheels ran significantly longer distances (6.6 ± 3.0 km/night; 600 ± 290 km total distance) with a significantly lower intensity. Both exercise groups showed increased lean body mass, and decreased fat mass and body weight compared with tumour‐bearing control mice. Food intake was unaffected by exercise or tumour growth. Primary tumour growth was not significantly affected by exercise. However, mice with continuous wheel access (long distance‐lower intensity group) displayed increased lung metastasis and decreased liver metastasis formation, when compared with the sedentary control group. Short distance‐higher intensity exercise did not affect metastasis formation. Analysis of blood cytokine levels revealed that mice with continuous wheel access displayed signs of systemic inflammation. Conclusions These results suggest that exercise has the potential to influence the patterns and extent of metastasis in CRC, and that the degree and intensity of exercise are likely to be important variables. Confirmation of these results in additional preclinical models with or without systemic treatment is warranted.

BackgroundThe immunogenic nature of metastatic colorectal cancer (CRC) with high microsatellite instability (MSI-H) underlies their responsiveness to immune checkpoint blockade (ICB). However, resistance to ICB is commonly observed, and is associated with the presence of peritoneal-metastases and ascites formation. The mechanisms underlying this site-specific benefit of ICB are unknown.MethodsWe created a novel model for spontaneous multiorgan metastasis in MSI-H CRC tumors by transplanting patient-derived organoids (PDO) into the cecum of humanized mice. Anti-programmed cell death protein-1 (PD-1) and anti-cytotoxic T-lymphocytes-associated protein 4 (CTLA-4) ICB treatment effects were analyzed in relation to the immune context of primary tumors, liver metastases, and peritoneal metastases. Immune profiling was performed by immunohistochemistry, flow cytometry and single-cell RNA sequencing. The role of B cells was assessed by antibody-mediated depletion. Immunosuppressive cytokine levels (interleukin (IL)-10, transforming growth factor (TGF)b1, TGFb2, TGFb3) were determined in ascites and serum samples by ELISA.ResultsPDO-initiated primary tumors spontaneously metastasized to the liver and the peritoneum. Peritoneal-metastasis formation was accompanied by the accumulation of ascites. ICB completely cleared liver metastases and reduced primary tumor mass but had no effect on peritoneal metastases. This mimics clinical observations. After therapy discontinuation, primary tumor masses progressively decreased, but peritoneal metastases displayed unabated growth. Therapy efficacy correlated with the formation of tertiary lymphoid structures (TLS)—containing B cells and juxtaposed T cells—and with expression of an interferon-γ signature together with the B cell chemoattractant CXCL13. B cell depletion prevented liver-metastasis clearance by anti-CTLA-4 treatment. Peritoneal metastases were devoid of B cells and TLS, while the T cells in these lesions displayed a dysfunctional phenotype. Ascites samples from patients with cancer with peritoneal metastases and from the mouse model contained significantly higher levels of IL-10, TGFb1, TGFb2 and TGFb3 than serum samples.ConclusionsBy combining organoid and humanized mouse technologies, we present a novel model for spontaneous multiorgan metastasis by MSI-H CRC, in which the clinically observed organ site-dependent benefit of ICB is recapitulated. Moreover, we provide empirical evidence for a critical role for B cells in the generation of site-dependent antitumor immunity following anti-CTLA-4 treatment. High levels of immunosuppressive cytokines in ascites may underlie the observed resistance of peritoneal metastases to ICB.

Peritoneal metastases in colorectal cancer (CRC) belong to Consensus Molecular Subtype 4 (CMS4) and are associated with poor prognosis. Conventional imaging modalities, such as Computed Tomography (CT) and Fluorodeoxyglucose-Positron Emission Tomography (FDG-PET), perform very poorly in the detection of peritoneal metastases. However, the stroma-rich nature of these lesions provides a basis for developing molecular imaging strategies. In this study, conducted from 2019 to 2021, we aimed to generate a Platelet-Derived Growth Factor Receptor beta (PDGFRB)-binding molecular imaging tracer for the detection of CMS4 CRC, including peritoneal metastases. The expression of PDGFRB mRNA discriminated CMS4 from CMS1-3 (AUROC = 0.86 (95% CI 0.85–0.88)) and was associated with poor relapse-free survival. PDGFRB mRNA and protein levels were very high in all human peritoneal metastases examined (n = 66). Therefore, we generated a PDGFRB-targeting llama nanobody (VHH1E12). Biotin-labelled VHH1E12 bound to immobilized human and mouse PDGFRB with high affinity (EC50 human PDGFRB = 7 nM; EC50 murine PDGFRB = 0.8 nM), and to PDGFRB-expressing HEK293 cells grown in vitro. A pharmacokinetic analysis of IRDye-800CW-conjugated VHH1E12 in mice showed that the plasma half-life was 6 min. IRDye-800CW-conjugated VHH1E12 specifically accumulated in experimentally induced colorectal cancer peritoneal metastases in mice. A tissue analysis subsequently demonstrated co-localization of the nanobody with PDGFRB expression in the tumour stroma. Our results demonstrate the potential value of PDGFRB-targeted molecular imaging as a novel strategy for the non-invasive detection of CMS4 CRC, in particular, peritoneal metastases.

Op initiatief van de gemeente Amsterdam is in 2020 een pilot transferbegeleiding van start gegaan. Hierbinnen krijgen jongeren coaching tijdens overstapmomenten in hun schoolloopbaan. De pilot is gevolgd door onderzoek en daarvan wordt in dit rapport verslag gedaan. Daarnaast wordt er onderzocht hoe jongeren uit PRO/VSO beter begeleid kunnen worden in hun overstap naar vervolgonderwijs en/of van school naar werk. De eerste stap binnen de pilot betrof het informeren van zorgcoördinatoren en mentoren over de pilot. Als tweede stap zijn als transferbegeleiders coaches van MEE geselecteerd en nader geïnformeerd over de werkwijze. Gedurende het traject zijn ook coaches van Jongeren Die het Kunnen (JDK) bij het traject betrokken. De coaches hadden geen specifieke ‘transferbegeleidingswerkwijze’ die zij gebruikten en de coaching kon daarom niet op basis hiervan worden geëvalueerd. Het onderzoek kreeg daarmee een meer volgend en ondersteunend karakter. In de matching van jongeren en coaches stond de klik tussen jongeren en coaches centraal, daarom zijn er filmpjes gemaakt van de coaches waar leerlingen een keuze uit konden maken. Vervolgens is een speeddate met de coaches georganiseerd waar alle leerlingen een coach hebben gekozen. De pilot is gestart met 8 leerlingen. In de pilot zijn alle sleutelfiguren binnen het coaching traject geïnterviewd (coaches, leerlingen, betrokken docenten en ouders/verzorgers). De vragen waar jongeren mee worstelden waren zeer divers en liepen uiteen van heel praktische vragen tot zeer diepgaande vragen. Coaches hielpen door een jongere te begeleiden bij een praktisch vraagstuk zoals een bijbaantje regelen of bij minder ‘hands on’ problemen door een luisterend oor te zijn. Deze veelheid aan hulpvragen in combinatie met specifieke uitdagingen maakte dat er maatwerk nodig was. De coaches vonden sommige casussen daardoor zwaar en gaven aan behoefte te hebben aan meer input voor de coaching en zelf ook aan meer begeleiding en sturing. Uit de literatuur en gehouden interviews blijkt relatieopbouw van belang. De relatie heeft grote invloed op de slagingskans van een hulptraject. Om een relatie op te bouwen moet een professional onder andere menselijkheid, lef en empathie tonen en de jongere als een gelijke beschouwen. De coaches gaven aan dat het veel tijd kost – meer ook dan aanvankelijk werd gedacht - om elkaar te leren kennen en vertrouwen te winnen. De meeste tijd ging dan ook uit naar deze eerste fase van het traject. Coaches en jongeren voerden hierbij diverse activiteiten uit, zoals samen sporten, wandelen of samen eten. De jongeren stellen zich vaak niet makkelijk open. Ook blijkt open en betrokken bejegening te werken. Dit houdt onder andere in dat de professional betrouwbaar en betrokken is, naast de client staat, respect toont, voor goed contact zorgt en positieve feedback geeft. Motiverende gespreksvoering kan hierbij ingezet worden. Jongeren willen zich gezien en gehoord voelen. Dit betekent dat de professional interesse toont, inlevingsvermogen heeft, meedenkt en de jongeren betrekt in de samenwerking en bovendien een intrinsieke motivatie heeft om jongeren te helpen. Het feit dat de jongeren een coach kregen uit hun zelf opgestelde top 3 heeft volgens zowel coaches als jongeren bijgedragen aan de motivatie voor het coaching traject. Tevens helpt het als de professional 1) echt is, dus iets van zichzelf laat zien in het contact met de jongere en een rolmodel is en 2) betrouwbaar is, dus doen wat je zegt en zeggen wat je doet, beschikbaar zijn en de tijd nemen. Daarbij moet niet het behandelplan, maar het perspectief van de jongere zelf leidend zijn in de werkrelatie. Wanneer het netwerk van de jongere karig is, is de rol van de transfercoach extra belangrijk. De veilige omgeving behouden of (opnieuw) op zetten en nog meer proberen in te spelen op de leefwereld van de jongere is essentieel. Al met al kan worden geconcludeerd dat het hebben van transferbegeleiding goed is voor een jongere. Of het daadwerkelijk helpt bij de overstap is vooralsnog lastig te beoordelen, echter lijkt het wel een stap in de goede richting te zijn.

Professionals werkzaam in de jeugdzorg passen doorgaans veel verschillende methoden toe in hun werk met kinderen, jongeren en hun ouders. Het vergt oefening en ervaring om deze methoden kwalitatief goed toe te passen en toe te blijven passen. Reflectie op en feedback over de toepassing van de specifieke competenties (kennis, vaardigheden, houding) in de dagelijkse praktijk zijn daarbij essentieel. Op dit moment zijn er geen goed ontworpen tools ter ondersteuning van effectieve reflectie tijdens de training of daarna, in het dagelijkse werk met kinderen, jongeren en hun ouders. De bestaande instrumenten op papier zijn nuttig, maar zijn arbeidsintensief en missen directe feedback en personalisatie.

The presence of BRAF