Hans M. Rodermond

Clonogenic assay of cells in vitro.

Clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. The colony is defined to consist of at least 50 cells. The assay essentially tests every cell in the population for its ability to undergo “unlimited” division. Clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. Only a fraction of seeded cells retains the capacity to produce colonies. Before or after treatment, cells are seeded out in appropriate dilutions to form colonies in 1–3 weeks. Colonies are fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v) and counted using a stereomicroscope. A method for the analysis of radiation dose–survival curves is included.

Wnt activity defines colon cancer stem cells and is regulated by the microenvironment

Despite the presence of mutations in APC or β-catenin, which are believed to activate the Wnt signalling cascade constitutively, most colorectal cancers show cellular heterogeneity when β-catenin localization is analysed, indicating a more complex regulation of Wnt signalling. We explored this heterogeneity with a Wnt reporter construct and observed that high Wnt activity functionally designates the colon cancer stem cell (CSC) population. In adenocarcinomas, high activity of the Wnt pathway is observed preferentially in tumour cells located close to stromal myofibroblasts, indicating that Wnt activity and cancer stemness may be regulated by extrinsic cues. In agreement with this notion, myofibroblast-secreted factors, specifically hepatocyte growth factor, activate β-catenin-dependent transcription and subsequently CSC clonogenicity. More significantly, myofibroblast-secreted factors also restore the CSC phenotype in more differentiated tumour cells both in vitro and in vivo. We therefore propose that stemness of colon cancer cells is in part orchestrated by the microenvironment and is a much more dynamic quality than previously expected that can be defined by high Wnt activity.

Poor-prognosis colon cancer is defined by a molecularly distinct subtype and develops from serrated precursor lesions

Colon cancer is a clinically diverse disease. This heterogeneity makes it difficult to determine which patients will benefit most from adjuvant therapy and impedes the development of new targeted agents. More insight into the biological diversity of colon cancers, especially in relation to clinical features, is therefore needed. We demonstrate, using an unsupervised classification strategy involving over 1,100 individuals with colon cancer, that three main molecularly distinct subtypes can be recognized. Two subtypes have been previously identified and are well characterized (chromosomal-instable and microsatellite-instable cancers). The third subtype is largely microsatellite stable and contains relatively more CpG island methylator phenotype–positive carcinomas but cannot be identified on the basis of characteristic mutations. We provide evidence that this subtype relates to sessile-serrated adenomas, which show highly similar gene expression profiles, including upregulation of genes involved in matrix remodeling and epithelial-mesenchymal transition. The identification of this subtype is crucial, as it has a very unfavorable prognosis and, moreover, is refractory to epidermal growth factor receptor–targeted therapy.

Mild hyperthermia inhibits homologous recombination, induces BRCA2 degradation, and sensitizes cancer cells to poly (ADP-ribose) polymerase-1 inhibition

Defective homologous recombination (HR) DNA repair imposed by BRCA1 or BRCA2 deficiency sensitizes cells to poly (ADP-ribose) polymerase (PARP)-1 inhibition and is currently exploited in clinical treatment of HR-deficient tumors. Here we show that mild hyperthermia (41–42.5 °C) induces degradation of BRCA2 and inhibits HR. We demonstrate that hyperthermia can be used to sensitize innately HR-proficient tumor cells to PARP-1 inhibitors and that this effect can be enhanced by heat shock protein inhibition. Our results, obtained from cell lines and in vivo tumor models, enable the design of unique therapeutic strategies involving localized on-demand induction of HR deficiency, an approach that we term induced synthetic lethality.

Methylation of Cancer-Stem-Cell-Associated Wnt Target Genes Predicts Poor Prognosis in Colorectal Cancer Patients

Gene signatures derived from cancer stem cells (CSCs) predict tumor recurrence for many forms of cancer. Here, we derived a gene signature for colorectal CSCs defined by high Wnt signaling activity, which in agreement with previous observations predicts poor prognosis. Surprisingly, however, we found that elevated expression of Wnt targets was actually associated with good prognosis, while patient tumors with low expression of Wnt target genes segregated with immature stem cell signatures. We discovered that several Wnt target genes, including ASCL2 and LGR5, become silenced by CpG island methylation during progression of tumorigenesis, and that their re-expression was associated with reduced tumor growth. Taken together, our data show that promoter methylation of Wnt target genes is a strong predictor for recurrence of colorectal cancer, and suggest that CSC gene signatures, rather than reflecting CSC numbers, may reflect differentiation status of the malignant tissue.

Monoclonal Antibodies Against Lgr5 Identify Human Colorectal Cancer Stem Cells

In colorectal cancer (CRC), a subpopulation of tumor cells, called cancer stem cell (CSC) fraction, is suggested to be responsible for tumor initiation, growth, and metastasis. The search for a reliable marker to identify these CSCs is ongoing as current markers, like CD44 and CD133, are more broadly expressed and therefore are not highly selective and currently also lack function in CSC biology. Here, we analyzed whether the Wnt target Lgr5, which has earlier been identified as a marker for murine intestinal stem cells, could potentially serve as a functional marker for CSCs. Fluorescence‐activated cell sorting‐based detection of Lgr5, using three newly developed antibodies, on primary colorectal tumor cells revealed a clear subpopulation of Epcam+Lgr5+ cells. Similarly, primary CRC‐derived spheroid cultures, known to be enriched for CSCs, contain high levels of Lgr5+ cells, which decrease upon in vitro differentiation of these CSCs. Selection of the Lgr5high CRC cells identified the clonogenic fraction in vitro as well as the tumorigenic population in vivo. Finally, we confirm that Lgr5 expression is dependent on the Wnt pathway and show that Lgr5 overexpression induces clonogenic growth. We thus provide evidence that Lgr5 is, next to a functional intestinal stem cell marker, a selective marker for human colorectal CSCs. STEM CELLS2012;30:2378–2386

Cell survival and radiosensitisation: modulation of the linear and quadratic parameters of the LQ model (Review).

The linear-quadratic model (LQ model) provides a biologically plausible and experimentally established method to quantitatively describe the dose-response to irradiation in terms of clonogenic survival. In the basic LQ formula, the clonogenic surviving fraction Sd/S₀ following a radiation dose d (Gy) is described by an inverse exponential approximation: Sd/S₀ = e-(αd+βd²), wherein α and β are experimentally derived parameters for the linear and quadratic terms, respectively. Radiation is often combined with other agents to achieve radiosensitisation. In this study, we reviewed radiation enhancement ratios of hyperthermia (HT), halogenated pyrimidines (HPs), various cytostatic drugs and poly(ADP-ribose) polymerase‑1 (PARP1) inhibitors expressed in the parameters α and β derived from cell survival curves of various mammalian cell cultures. A significant change in the α/β ratio is of direct clinical interest for the selection of optimal fractionation schedules in radiation oncology, influencing the dose per fraction, dose fractionation and dose rate in combined treatments. The α/β ratio may increase by a mutually independent increase of α or decrease of β. The results demonstrated that the different agents increased the values of both α and β. However, depending on culture conditions, both parameters can also be separately influenced. Moreover, it appeared that radiosensitisation was more effective in radioresistant cell lines than in radiosensitive cell lines. Furthermore, radiosensitisation is also dependent on the cell cycle stage, such as the plateau or exponentially growing phase, as well as on post-treatment plating conditions. The LQ model provides a useful tool in the quantification of the effects of radiosensitising agents. These insights will help optimize fractionation schedules in multimodality treatments.

Development and characterization of APRIL antagonistic monoclonal antibodies for treatment of B-cell lymphomas

APRIL (A proliferation-inducing ligand) is a TNF family member that binds two TNF receptor family members, TACI and BCMA. It shares these receptors with the closely related TNF family member, B-cell activating factor (BAFF). Contrary to BAFF, APRIL binds heparan sulfate proteoglycans (HSPGs), which regulates cross-linking of APRIL and efficient signaling. APRIL was originally identified as a growth promoter of solid tumors, and more recent evidence defines APRIL also as an important survival factor in several human B-cell malignancies, such as chronic lymphocytic leukemia (CLL). To target APRIL therapeutically, we developed two anti-human APRIL antibodies (hAPRIL.01A and hAPRIL.03A) that block APRIL binding to BCMA and TACI. Their antagonistic properties are unique when compared with a series of commercially available monoclonal anti-human APRIL antibodies as they prevent in vitro proliferation and IgA production of APRIL-reactive B cells. In addition, they effectively impair the CLL-like phenotype of aging APRIL transgenic mice and, more importantly, block APRIL binding to human B-cell lymphomas and prevent the survival effect induced by APRIL. We therefore conclude that these antibodies have potential for further development as therapeutics to target APRIL-dependent survival in B-cell malignancies.

Inhibition of homologous recombination by hyperthermia shunts early double strand break repair to non-homologous end-joining

In S and G2 phase mammalian cells DNA double strand breaks (DSBs) can potentially be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). Results of several studies suggest that these two mechanistically distinct repair pathways can compete for DNA ends. Because HR and NHEJ differ with respect to error susceptibility, generation of chromosome rearrangements, which are potentially carcinogenic products of DSB repair, may depend on the pathway choice. To investigate this hypothesis, the influence of HR and NHEJ inhibition on the frequencies of chromosome aberrations in G2 phase cells was investigated. SW-1573 and RKO cells were treated with mild (41 °C) hyperthermia in order to disable HR and/or NU7441/cisplatin to inactivate NHEJ and frequencies of chromosomal fragments (resulting from unrepaired DSBs) and translocations (products of erroneous DSB rejoining) were studied using premature chromosome condensation (PCC) combined with fluorescence in situ hybridization (FISH). It is shown here that temporary inhibition of HR by hyperthermia results in increased frequency of ionizing-radiation (IR)-induced chromosomal translocations and that this effect is abrogated by NU7441- or cisplatin-mediated inhibition of NHEJ. The results suggest that in the absence of HR, DSB repair is shifted to the error-prone NHEJ pathway resulting in increased frequencies of chromosomal rearrangements. These results might be of consequence for clinical cancer treatment approaches that aim at inhibition of one or more DSB repair pathways.

Targeting colorectal cancer stem cells with inducible caspase-9

Colorectal cancer stem cells (CSCs) drive tumor growth and are suggested to initiate distant metastases. Moreover, colon CSCs are reportedly more resistant to conventional chemotherapy, which is in part due to upregulation of anti-apoptotic Bcl-2 family members. To determine whether we could circumvent this apoptotic blockade, we made use of an inducible active caspase-9 (iCasp9) construct to target CSCs. Dimerization of iCasp9 with AP20187 in HCT116 colorectal cancer cells resulted in massive and rapid induction of apoptosis. In contrast to fluorouracil (5-FU)-induced apoptosis, iCasp9-induced apoptosis was independent of the mitochondrial pathway as evidenced by Bax/Bak double deficient HCT116 cells. Dimerizer treatment of colon CSCs transduced with iCasp9 (CSC-iCasp9) also rapidly induced high levels of apoptosis, while these cells were unresponsive to 5-FU in vitro. More importantly, injection of the dimerizer into mice that developed a colon CSC-iCasp9-induced tumor resulted in a strong decrease in tumor size, an increase in tumor cell apoptosis and a clear loss of CD133+ CSCs. Taken together, our data indicate that dimerization of iCasp9 circumvents the apoptosis block in CSCs, which results in effective tumor regression in vivo.