Effects of regorafenib on the mononuclear/phagocyte system and how these contribute to the inhibition of colorectal tumors in mice.

BACKGROUND: Regorafenib was previously shown to reduce tumor-associated macrophages and potently inhibit colony-stimulating factor 1 receptor (CSF1R), also known as CD115, in biochemical assays. The CSF1R signaling pathway is essential in the biology of the mononuclear/phagocyte system, which can promote the development of cancer. METHODS: A deeper investigation of regorafenib's effects on CSF1R signaling was performed using preclinical in vitro and in vivo studies with syngeneic CT26 and MC38 mouse models of colorectal cancer. Peripheral blood and tumor tissue were analyzed mechanistically by flow cytometry using antibodies against CD115/CSF1R and F4/80 and by ELISA for chemokine (C-C motif) ligand 2 (CCL2) levels. These read-outs were correlated with drug levels for the detection of pharmacokinetic/pharmacodynamic relationships. RESULTS: Potent inhibition of CSF1R by regorafenib and its metabolites M-2, M-4, and M-5 was confirmed in vitro in RAW264.7 macrophages. The dose-dependent growth inhibition of subcutaneous CT26 tumors by regorafenib was associated with a significant reduction in both the number of CD115hi monocytes in peripheral blood and the number of selective subpopulations of intratumoral F4/80hi tumor-associated macrophages. CCL2 levels were not affected by regorafenib in blood but increased in tumor tissue, which may contribute to drug resistance and prevent complete tumor remission. An inverse relationship between regorafenib concentration and the number of CD115hi monocytes and CCL2 levels was observed in peripheral blood, supporting the mechanistic involvement of regorafenib. CONCLUSIONS: These findings may be clinically useful in optimizing drug dosing using blood-based pharmacodynamic markers and in identifying resistance mechanisms and ways to overcome them by appropriate drug combinations.

LMP1 and EBNA2 constitute a minimal set of EBV genes for transformation of human B cells.

Introduction: Epstein-Barr virus (EBV) infection in humans is associated with a wide range of diseases including malignancies of different origins, most prominently B cells. Several EBV latent genes are thought to act together in B cell immortalization, but a minimal set of EBV genes sufficient for transformation remains to be identified. Methods: Here, we addressed this question by transducing human peripheral B cells from EBV-negative donors with retrovirus expressing the latent EBV genes encoding Latent Membrane Protein (LMP) 1 and 2A and Epstein-Barr Nuclear Antigen (EBNA) 2. Results: LMP1 together with EBNA2, but not LMP1 alone or in combination with LMP2A was able to transform human primary B cells. LMP1/EBNA2-immortalized cell lines shared surface markers with EBV-transformed lymphoblastoid cell lines (LCLs). They showed sustained growth for more than 60 days, albeit at a lower growth rate than EBV-transformed LCLs. LMP1/EBNA2-immortalized cell lines generated tumors when transplanted subcutaneously into severely immunodeficient NOG mice. Conclusion: Our results identify a minimal set of EBV proteins sufficient for B cell transformation.

Rapid testing of candidate oncogenes and tumour suppressor genes in signal transduction and neoplastic transformation.

The COSMIC database (version 94) lists 576 genes in the Cancer Gene Census which have a defined function as drivers of malignancy (oncogenes) or as tumour suppressors (Tier 1). In addition, there are 147 genes with similar functions, but which are less well characterised (Tier 2). Furthermore, next-generation sequencing projects in the context of precision oncology activities are constantly discovering new ones. Since cancer genes differ from their wild-type precursors in numerous molecular and biochemical properties and exert significant differential effects on downstream processes, simple assays that can uncover oncogenic or anti-oncogenic functionality are desirable and may precede more sophisticated analyses. We describe simple functional assays for PTPN11 (protein-tyrosine phosphatase, non-receptor-type 11)/SHP2 mutants, which are typically found in RASopathies and exhibit potential oncogenic activity. We have also designed a functional test for lysyl oxidase (LOX), a prototypical class II tumour suppressor gene whose loss of function may contribute to neoplastic transformation by RAS oncogenes. Moreover, we applied this test to analyse three co-regulated, RAS-responsive genes for transformation-suppressive activity. The integration of these tests into systems biology studies will contribute to a better understanding of cellular networks in cancer.

CD28 Co-Stimulus Achieves Superior CAR T Cell Effector Function against Solid Tumors Than 4-1BB Co-Stimulus.

Spacer or co-stimulatory components in chimeric antigen receptor (CAR) design influence CAR T cell effector function. Few preclinical mouse models optimally support CAR candidate pre-selection for clinical development. Here we use a model in which murine CAR T cells can be exploited with human tumor xenografts. This mouse-in-mouse approach avoids limitations caused by species-specific factors crucial for CAR T cell survival, trafficking and function. We compared trafficking, expansion and tumor control for T cells expressing different CAR construct designs targeting two antigens (L1CAM or HER2), structurally identical except for spacer (long or short) or co-stimulatory (4-1BB or CD28) domains to be evaluated. Using monoclonal, murine-derived L1CAM-specific CAR T cells in Rag-/- mice harboring established xenografted tumors from a human neuroblastoma cell line revealed a clear superiority in CAR T cell trafficking using CD28 co-stimulation. L1CAM-targeting short spacer-CD28/ζ CAR T cells expanded the most at the tumor site and induced initial tumor regression. Treating patient-derived neuroblastoma xenografts with human L1CAM-targeting CAR T cells confirmed the superiority of CD28 co-stimulus. CD28 superiority was also demonstrated with HER2-specific CAR T cells (targeting ovarian carcinoma xenografts). Our findings encourage incorporating CD28 signaling into CAR design for adoptive T cell treatment of solid tumors.

CRISPR-Cas9-Mediated ELANE Mutation Correction in Hematopoietic Stem and Progenitor Cells to Treat Severe Congenital Neutropenia.

Severe congenital neutropenia (SCN) is a monogenic disorder. SCN patients are prone to recurrent life-threatening infections. The main causes of SCN are autosomal dominant mutations in the ELANE gene that lead to a block in neutrophil differentiation. In this study, we use CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)6 as a donor template delivery system to repair the ELANEL172P mutation in SCN patient-derived hematopoietic stem and progenitor cells (HSPCs). We used a single guide RNA (sgRNA) specifically targeting the mutant allele, and an sgRNA targeting exon 4 of ELANE. Using the latter sgRNA, ∼34% of the known ELANE mutations can in principle be repaired. We achieved gene correction efficiencies of up to 40% (with sgELANE-ex4) and 56% (with sgELANE-L172P) in the SCN patient-derived HSPCs. Gene repair restored neutrophil differentiation in vitro and in vivo upon HSPC transplantation into humanized mice. Mature edited neutrophils expressed normal elastase levels and behaved normally in functional assays. Thus, we provide a proof of principle for using CRISPR-Cas9 to correct ELANE mutations in patient-derived HSPCs, which may translate into gene therapy for SCN.

Periodic harvesting of embryonic stem cells from a hollow-fiber membrane based four-compartment bioreactor.

Different types of stem cells have been investigated for applications in drug screening and toxicity testing. In order to provide sufficient numbers of cells for such in vitro applications a scale-up of stem cell culture is necessary. Bioreactors for dynamic three-dimensional (3D) culture of growing cells offer the option for culturing large amounts of stem cells at high densities in a closed system. We describe a method for periodic harvesting of pluripotent stem cells (PSC) during expansion in a perfused 3D hollow-fiber membrane bioreactor, using mouse embryonic stem cells (mESC) as a model cell line. A number of 100 × 10(6) mESC were seeded in bioreactors in the presence of mouse embryonic fibroblasts (MEF) as feeder cells. Over a cultivation interval of nine days cells were harvested by trypsin perfusion and mechanical agitation every second to third culture day. A mean of 380 × 10(6) mESC could be removed with every harvest. Subsequent to harvesting, cells continued growing in the bioreactor, as determined by increasing glucose consumption and lactate production. Immunocytochemical staining and mRNA expression analysis of markers for pluripotency and the three germ layers showed a similar expression of most markers in the harvested cells and in mESC control cultures. In conclusion, successful expansion and harvesting of viable mESC from bioreactor cultures with preservation of sterility was shown. The present study is the first one showing the feasibility of periodic harvesting of adherent cells from a continuously perfused four-compartment bioreactor including further cultivation of remaining cells.

Cell differentiation mediated by co-culture of human umbilical cord blood stem cells with murine hepatic cells.

In the present study, purified human cord blood stem cells were co-cultivated with murine hepatic alpha mouse liver 12 (AML12) cells to compare the effect on endodermal stem cell differentiation by either direct cell-cell interaction or by soluble factors in conditioned hepatic cell medium. With that approach, we want to mimic in vitro the situation of preclinical transplantation experiments using human cells in mice. Cord blood stem cells, cultivated with hepatic conditioned medium, showed a low endodermal differentiation but an increased connexin 32 (Cx32) and Cx43, and cytokeratin 8 (CK8) and CK19 expression was monitored by reverse transcription polymerase chain reaction (RT-PCR). Microarray profiling indicated that in cultivated cord blood cells, 604 genes were upregulated 2-fold, with the highest expression for epithelial CK19 and epithelial cadherin (E-cadherin). On ultrastructural level, there were no major changes in the cellular morphology, except a higher presence of phago(ly)some-like structures observed. Direct co-culture of AML12 cells with cord blood cells led to less incisive differentiation with increased sex-determining region Y-box 17 (SOX17), Cx32 and Cx43, as well as epithelial CK8 and CK19 expressions. On ultrastructural level, tight cell contacts along the plasma membranes were revealed. FACS analysis in co-cultivated cells quantified dye exchange on low level, as also proved by time relapse video-imaging of labelled cells. Modulators of gap junction formation influenced dye transfer between the co-cultured cells, whereby retinoic acid increased and 3-heptanol reduced the dye transfer. The study indicated that the cell-co-cultured model of human umbilical cord blood cells and murine AML12 cells may be a suitable approach to study some aspects of endodermal/hepatic cell differentiation induction.