Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I–IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab

A combined computational and functional approach identifies IGF2BP2 as a driver of chemoresistance in a wide array of pre-clinical models of colorectal cancer

Aim Chemoresistance is a major cause of treatment failure in colorectal cancer (CRC) therapy. In this study, the impact of the IGF2BP family of RNA-binding proteins on CRC chemoresistance was investigated using in silico, in vitro, and in vivo approaches. Methods Gene expression data from a well-characterized cohort and publicly available cross-linking immunoprecipi‑ tation sequencing (CLIP-Seq) data were collected. Resistance to chemotherapeutics was assessed in patient-derived xenografts (PDXs) and patient-derived organoids (PDOs). Functional studies were performed in 2D and 3D cell culture models, including proliferation, spheroid growth, and mitochondrial respiration analyses. Results We identifed IGF2BP2 as the most abundant IGF2BP in primary and metastastatic CRC, correlating with tumor stage in patient samples and tumor growth in PDXs. IGF2BP2 expression in primary tumor tissue was signif‑ cantly associated with resistance to selumetinib, geftinib, and regorafenib in PDOs and to 5-fuorouracil and oxalipl‑ atin in PDX in vivo. IGF2BP2 knockout (KO) HCT116 cells were more susceptible to regorafenib in 2D and to oxaliplatin, selumitinib, and nintedanib in 3D cell culture. Further, a bioinformatic analysis using CLIP data suggested stabiliza‑ tion of target transcripts in primary and metastatic tumors. Measurement of oxygen consumption rate (OCR) and extracellular acidifcation rate (ECAR) revealed a decreased basal OCR and an increase in glycolytic ATP production rate in IGF2BP2 KO. In addition, real-time reverse transcriptase polymerase chain reaction (qPCR) analysis confrmed decreased expression of genes of the respiratory chain complex I, complex IV, and the outer mitochondrial membrane in IGF2BP2 KO cells. Conclusions IGF2BP2 correlates with CRC tumor growth in vivo and promotes chemoresistance by altering mito‑ chondrial respiratory chain metabolism. As a druggable target, IGF2BP2 could be used in future CRC therapy to overcome CRC chemoresistance

In vitro and in vivo studies of the differentiation potential of human stem cells

Ziel der Untersuchung war eine vergleichende Analyse des Differenzierungspotenzials von humanen adulten und embryonalen Stammzellen. Dabei wurden humane embryonale Stammzellen (hESZ) ungerichtet und spontan in vitro differenziert, während humane adulte Stammzellen (CD34+) erstmals in vitro mittels Ko-Kultur auf murinen AML12 Zellen gerichtet differenziert wurden. In vivo wurde die Kapazität zur Bildung von Teratomen von spontan vordifferenzierten hESZ in immundefizienten Mäusen umfassend geprüft. Auch wurden adulte CD34+ - Zellen direkt in die Leber von neugeborenen und adulten immundefizienten Mäusen appliziert. Die hESZ wurden auf unterschiedlichen Matrizen, wie murinen und humanen Zellen kultiviert und zeigten ein vergleichbares Expressionsprofil. Die Analyse der Markerexpression der undifferenzierten und spontan differenzierten Zellen erfolgte auf RNA-Ebene. Die spontane in vitro Differenzierung der hESZ zeigte zum frühen Zeitpunkt (14 Tage) einen leichten Anstieg der frühen endodermalen Marker und des hepatischen Differenzierungsmarkers Albumin. Zum späteren Zeitpunkt (82 Tage) wurde auf der Ebene der endodermalen Differenzierung eine höhere Expression der hepatischen Transkriptionsfaktoren und des endodermalen Markers verzeichnet, der ektodermale Marker war dagegen herunterreguliert. Die Pluripotenzmarker zeigten zu beiden Zeitpunkten eine gleich bleibend hohe Expression. Die mittels Ko-Kultur hepatisch induzierten CD34+ Zellen zeigten eine schwach ausgeprägte endodermale Differenzierung, nachgewiesen durch die Expression von Connexinen und Zytokeratinen. Wesentliche Änderungen der Zellmorphologie waren nicht nachweisbar. Die undifferenzierten hESZ exprimierten auf hohem Niveau Transkripte für Pluripotenz und die drei Keimblätter, zudem führten sie in vivo zur Teratombildung. Die spontane Differenzierung der hESZ in vitro über die Zeit (25, 60 und 89 Tage) führte zu einer niedrigeren Wachstumsrate und zu einem einheitlicheren histopathologischen Phänotyp in vivo. Weiterhin wurden in Teratomen von 25 und 60 Tagen vordifferenzierten Zellen frühe endodermale und hepatische Marker gefunden, jedoch keine in Teratomen von 89 Tage vordifferenzierten Zellen. Die intrahepatisch transplantierten CD34+ Zellen in neugeborenen Mäusen führte zu einem Anwachsen in unterschiedlichen Organen, wie Knochenmark, Milz und Thymus, sowie zu einer exklusiven Expression vom B-Zell Marker CD19. Nach Applikation in die Lebern von adulten Mäusen wurde dagegen kein Anwachsen in den Organen detektiert. Zusammenfassend wurde festgestellt, dass die humanen embryonalen, als auch adulten Stammzellen durch Anwendung unterschiedlicher in vitro und in vivo Methoden in Zellen mit endodermal-hepatischen, als auch hämatopoetischen Merkmalen, differenzieren.In this study we analyzed the spontaneous differentiation of human embryonic stem cells (hESC) in vitro and in vivo and we investigate the influence of in vitro differentiation on in vivo teratoma formation in immunodeficient mice. Further, the hematopoetic stem cells (CD34+) were comparatively examined with respect to their normal and induced hepatic differentiation potential. In addition, we proved the engraftment of CD34+ cells in vivo after intrahepatical transplantation in immunodeficient mice. The SA002 human embryonic stem cells were cultured on irradiated mouse embryonic fibroblasts, on irradiated human foreskin fibroblasts, or feeder-free using matrigel. The CD34+ stem cells were cultured according to in-house established in vitro protocols. The marker expression of undifferentiated and differentiated stem cells was monitored by real time RT-PCR. Further microarray profiling, electron microscopy, FACS-analysis and dye transfer methods, as well as immunohistochemistry were used to determine the differentiation status of stem cell preparations. Undifferentiated SA002 stem cells were cultured under different conditions. These morphologically undifferentiated cells had a comparable marker expression profile, with high expression levels of pluripotency markers and low-to-moderate expression levels of germ layer markers. The undifferentiated CD34+ cells showed a high expression for CD34 transcript whereas endodermal markers were expressed on low level. The in vitro spontaneously differentiated SA002 stem cells demonstrated at early time points a slight upregulation of endodermal marker expression and of the liver marker albumin. At later time points, the expression of transcripts for hepatic and endodermal markers was upregulated, whereas expression of the ectodermal marker transcripts was downregulated. Expression of pluripotency markers remained high, and hematopoetic markers were not expressed. The induced hepatic CD34 cells showed a low endodermal differentiation potential with expression of transcripts for connexins and cytokeratins. Further, significant changes in cell morphology were not detectable. SA002 cells that showed spontaneous partial differentiation in vitro had a low teratoma formation capacity in vivo. Cells that were partially differentiated led to slower growing teratomas with more uniform histology. The intrahepatically transplanted CD34+ cells into newborn mice led to an engraftment in various organs like bone marrow, spleen and thymus as well as to an exclusive expression of B-cell markers. In summary, we determined that human embryonic as well as adult stem cells differentiate in cells with endodermal-hepatic properties using different methods. The results presented in this study can contribute to experimental research, especially the understanding of processes of organ differentiation, as well as the application of cell therapy in the context of regeneration

Heterogeneous pathway activation and drug response modelled in colorectal-tumor-derived 3D cultures.

Organoid cultures derived from colorectal cancer (CRC) samples are increasingly used as preclinical models for studying tumor biology and the effects of targeted therapies under conditions capturing in vitro the genetic make-up of heterogeneous and even individual neoplasms. While 3D cultures are initiated from surgical specimens comprising multiple cell populations, the impact of tumor heterogeneity on drug effects in organoid cultures has not been addressed systematically. Here we have used a cohort of well-characterized CRC organoids to study the influence of tumor heterogeneity on the activity of the KRAS/MAPK-signaling pathway and the consequences of treatment by inhibitors targeting EGFR and downstream effectors. MAPK signaling, analyzed by targeted proteomics, shows unexpected heterogeneity irrespective of RAS mutations and is associated with variable responses to EGFR inhibition. In addition, we obtained evidence for intratumoral heterogeneity in drug response among parallel "sibling" 3D cultures established from a single KRAS-mutant CRC. Our results imply that separate testing of drug effects in multiple subpopulations may help to elucidate molecular correlates of tumor heterogeneity and to improve therapy response prediction in patients