Cancer is a leading cause of mortality throughout the world and new treatments are urgently needed. Recent studies suggest that bone marrow–derived mesenchymal stem cells (MSC) home to and incorporate within tumor tissue. We hypothesized that MSCs engineered to produce and deliver tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a transmembrane protein that causes selective apoptosis of tumor cells, would home to and kill cancer cells in a lung metastatic cancer model. Human MSCs were transduced with TRAIL and the IRES-eGFP reporter gene under the control of a tetracycline promoter using a lentiviral vector. Transduced and activated MSCs caused lung (A549), breast (MDAMB231), squamous (H357), and cervical (Hela) cancer cell apoptosis and death in coculture experiments. Subcutaneous xenograft experiments confirmed that directly delivered TRAIL-expressing MSCs were able to significantly reduce tumor growth [0.12 cm³ (0.04-0.21) versus 0.66 cm³ (0.21-1.11); P < 0.001]. We then found, using a pulmonary metastasis model, systemically delivered MSCs localized to lung metastases and the controlled local delivery of TRAIL completely cleared the metastatic disease in 38% of mice compared with 0% of controls (P < 0.05). This is the first study to show a significant reduction in metastatic tumor burden with frequent eradication of metastases using inducible TRAIL-expressing MSCs. This has a wide potential therapeutic role, which includes the treatment of both primary tumors and their metastases, possibly as an adjuvant therapy in clearing micrometastatic disease following primary tumor resection. [Cancer Res 2009;69(10):4134–42]

Imaging flow cytometry combines the high-throughput capabilities of conventional flow cytometry with single-cell imaging. Here we demonstrate label-free prediction of DNA content and quantification of the mitotic cell cycle phases by applying supervised machine learning to morphological features extracted from brightfield and the typically ignored darkfield images of cells from an imaging flow cytometer. This method facilitates non-destructive monitoring of cells avoiding potentially confounding effects of fluorescent stains while maximizing available fluorescence channels. The method is effective in cell cycle analysis for mammalian cells, both fixed and live, and accurately assesses the impact of a cell cycle mitotic phase blocking agent. As the same method is effective in predicting the DNA content of fission yeast, it is likely to have a broad application to other cell types.

Reports differ as to whether reconstitution of telomerase activity alone is sufficient for immortalization of different types of human somatic cells or whether additional activities encoded by other “immortalizing” genes are also required. Here we show that ectopic expression of either the catalytic subunit of human telomerase (hTERT) or a temperature-sensitive mutant (U19tsA58) of simian virus 40 large-tumor antigen alone was not sufficient for immortalization of freshly isolated normal adult human mammary fibroblasts and endothelial cells. However, a combination of both genes resulted in the efficient generation of immortal cell lines irrespective of the order in which they were introduced or whether they were introduced early or late in the normal proliferative lifespan of the cultures. The order and timing of transduction, however, did influence genomic stability. Karyotype analysis indicated that introduction of both transgenes at early passage, with hTERT first, yielded diploid cell lines. Temperature-shift experiments revealed that maintenance of the immortalized state depended on continued expression of functional U19tsA58 large-tumor antigen, with hTERT alone unable to maintain growth at nonpermissive temperatures for U19tsA58 large-tumor antigen. Such conditional diploid lines may provide a useful resource for both cell engineering and for studies on immortalization and in vitro transformation.

The resolution of recombination intermediates containing Holliday junctions (HJs) is critical for genome maintenance and proper chromosome segregation. Three pathways for HJ processing exist in human cells and involve the following enzymes/complexes: BLM–TopoIIIα–RMI1–RMI2 (BTR complex), SLX1–SLX4–MUS81–EME1 (SLX–MUS complex), and GEN1. Cycling cells preferentially use the BTR complex for the removal of double HJs in S phase, with SLX–MUS and GEN1 acting at temporally distinct phases of the cell cycle. Cells lacking SLX–MUS and GEN1 exhibit chromosome missegregation, micronucleus formation, and elevated levels of 53BP1-positive G1 nuclear bodies, suggesting that defects in chromosome segregation lead to the transmission of extensive DNA damage to daughter cells. In addition, however, we found that the effects of SLX4, MUS81, and GEN1 depletion extend beyond mitosis, since genome instability is observed throughout all phases of the cell cycle. This is exemplified in the form of impaired replication fork movement and S-phase progression, endogenous checkpoint activation, chromosome segmentation, and multinucleation. In contrast to SLX4, SLX1, the nuclease subunit of the SLX1–SLX4 structure-selective nuclease, plays no role in the replication-related phenotypes associated with SLX4/MUS81 and GEN1 depletion. These observations demonstrate that the SLX1–SLX4 nuclease and the SLX4 scaffold play divergent roles in the maintenance of genome integrity in human cells.

Purpose: Previous studies have demonstrated the prognostic importance of the immune microenvironment in follicular lymphoma (FL). To investigate the molecular mechanisms during which tumor-infiltrating T cells (TILs) are altered in the FL microenvironment, we studied highly purified CD4 and CD8 TILs from lymph node biopsies at diagnosis in treatment-naive patients with FL compared with reactive tonsils and the peripheral blood of healthy donors.Patients and Methods: Gene expression profiling of highly purified CD4 and CD8 TILs was performed on the Affymetrix platform. Diagnostic tissue microarrays from an independent patient set (n = 172) were used to verify protein expression and analyze any impact of TIL-expressed genes on outcome. Time-lapse imaging was used to assess T-cell motility.Results: The most upregulated genes in both CD4 and CD8 TILs were PMCH, ETV1, and TNFRSF9. PMCH is not expressed in peripheral blood T cells, but expression is highly induced on culture with FL. Both CD4 and CD8 TILs from patients with FL have significantly impaired motility compared with those of healthy TILs from reactive tonsils and this can be induced on healthy T cells by FL cells. During multivariate analysis, a model incorporating the number and location of T cells expressing PMCH, NAMPT, and ETV1 showed prognostic significance for overall survival and for time to transformation.Conclusion: We showed altered gene expression in TILs in FL and demonstrated that altering the immune microenvironment in FL affects overall survival and time to transformation in this disease.