Sensitizing thermochemotherapy with a PARP1-inhibitor

Cis-diamminedichloroplatinum(II) (cisplatin, cDDP) is an effective chemotherapeutic agent that induces DNA double strand breaks (DSBs), primarily in replicating cells. Generally, such DSBs can be repaired by the classical or backup non-homologous end joining (c-NHEJ/b-NHEJ) or homologous recombination (HR). Therefore, inhibiting these pathways in cancer cells should enhance the efficiency of cDDP treatments. Indeed, inhibition of HR by hyperthermia (HT) sensitizes cancer cells to cDDP and in the Netherlands this combination is a standard treatment option for recurrent cervical cancer after previous radiotherapy. Additionally, cDDP has been demonstrated to disrupt c-NHEJ, which likely further increases the treatment efficacy. However, if one of these pathways is blocked, DSB repair functions can be sustained by the Poly-(ADP-ribose)-polymerase1 (PARP1)-dependent b-NHEJ. Therefore, disabling b-NHEJ should, in principle, further inhibit the repair of cDDP-induced DNA lesions and enhance the toxicity of thermochemotherapy. To explore this hypothesis, we treated a panel of cancer cell lines with HT, cDDP and a PARP1-i and measured various end-point relevant in cancer treatment. Our results demonstrate that PARP1-i does not considerably increase the efficacy of HT combined with standard, commonly used cDDP concentrations. However, in the presence of a PARP1-i, ten-fold lower concentration of cDDP can be used to induce similar cytotoxic effects. PARP1 inhibition may thus permit a substantial lowering of cDDP concentrations without diminishing treatment efficacy, potentially reducing systemic side effects

Enhancing synthetic lethality of PARP-inhibitor and cisplatin in BRCA-proficient tumour cells with hyperthermia

Background: Poly-(ADP-ribose)-polymerase1 (PARP1) is involved in repair of DNA single strand breaks. PARP1-inhibitors (PARP1-i) cause an accumulation of DNA double strand breaks, which are generally repaired by homologous recombination (HR). Therefore, cancer cells harboring HR deficiencies are exceptionally sensitive to PARP1-i. For patients with HR-proficient tumors, HR can be temporarily inhibited by hyperthermia, thereby inducing synthetic lethal conditions in every tumor type. Since cisplatin is successfully used combined with hyperthermia (thermochemotherapy), we investigated the effectiveness of combining PARP1-i with thermochemotherapy. Results: The in vitro data demonstrate a decreased in cell survival after addition of PARP1-i to thermochemotherapy, which can be explained by increased DNA damage induction and less DSB repair. These in vitro findings are in line with in vivo model, in which a decreased tumor growth is observed upon addition of PARP1-i. Materials and Methods: Survival of three HR-proficient cell lines after cisplatin, hyperthermia and/or PARP1-i was studied. Cell cycle analyses, quantification of γ-H2AX foci and apoptotic assays were performed to understand these survival data. The effects of treatments were further evaluated by monitoring tumor responses in an in vivo rat model. Conclusions: Our results in HR-proficient cell lines suggest that PARP1-i combined with thermochemotherapy can be a promising clinical approach for all tumors independent of HR status

Elevated temperatures and longer durations improve the efficacy of oxaliplatin- and mitomycin C-based hyperthermic intraperitoneal chemotherapy in a confirmed rat model for peritoneal metastasis of colorectal cancer origin

Introduction: In patients with limited peritoneal metastasis (PM) originating from colorectal cancer, cytoreductive surgery (CRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC) is a potentially curative treatment option. This combined treatment modality using HIPEC with mitomycin C (MMC) for 90 minutes proved to be superior to systemic chemotherapy alone, but no benefit of adding HIPEC to CRS alone was shown using oxaliplatin-based HIPEC during 30 minutes. We investigated the impact of treatment temperature and duration as relevant HIPEC parameters for these two chemotherapeutic agents in representative preclinical models. The temperature- and duration- dependent efficacy for both oxaliplatin and MMC was evaluated in an in vitro setting and in a representative animal model. Methods: In 130 WAG/Rij rats, PM were established through i.p. injections of rat CC-531 colon carcinoma cells with a signature similar to the dominant treatment-resistant CMS4 type human colorectal PM. Tumor growth was monitored twice per week using ultrasound, and HIPEC was applied when most tumors were 4-6 mm. A semi-open four-inflow HIPEC setup was used to circulate oxaliplatin or MMC through the peritoneum for 30, 60 or 90 minutes with inflow temperatures of 38°C or 42°C to achieve temperatures in the peritoneum of 37°C or 41°C. Tumors, healthy tissue and blood were collected directly or 48 hours after treatment to assess the platinum uptake, level of apoptosis and proliferation and to determine the healthy tissue toxicity. Results: In vitro results show a temperature- and duration- dependent efficacy for both oxaliplatin and MMC in both CC-531 cells and organoids. Temperature distribution throughout the peritoneum of the rats was stable with normothermic and hyperthermic average temperatures in the peritoneum ranging from 36.95-37.63°C and 40.51-41.37°C, respectively. Treatments resulted in minimal body weight decrease (&lt;10%) and only 7/130 rats did not reach the endpoint of 48 hours after treatment. Conclusions: Both elevated temperatures and longer treatment duration resulted in a higher platinum uptake, significantly increased apoptosis and lower proliferation in PM tumor lesions, without enhanced normal tissue toxicity. Our results demonstrated that oxaliplatin- and MMC-based HIPEC procedures are both temperature- and duration-dependent in an in vivo tumor model.</p

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

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;30:2378238

Residual Late Radiation Damage in Mouse Stromal Tissue Assessed by the Tumor Bed Effect

Irradiation of murine subcutaneous stroma before implantation of tumor cells leads to retarded tumor growth. This effect is called Tumor Bed Effect (TBE) and can be used to assess the sensitivity of stromal tissue to radiation. We tested the ability of stromal tissue to recover from X-ray-induced damage as a function of the time interval between X-irradiation and implantation of tumor cells over a period of 195 days. We also assessed the effects of a second test treatment of X-irradiation before implantation to assess residual damage by the first radiation treatment. The tumor bed effect in C57Bl10xDBA2 mice observed after X-ray treatment and implantation of M8013 cells (from a transplantable mouse mammary carcinoma) declines with the time that elapses between X-rays and implantation. Implantation of tumor cells 195 days after initial irradiation of 10 or 20 Gy resulted in a considerably smaller TBE. The half-time of the decay is estimated as about 50 days. The extent of the recovery was then tested in two-fraction experiments, with radiation fractions separated by intervals of 30 or 180 days. In the experiment with re-irradiation at an interval of 30 days after the first radiation dose of 20 Gy hardly any recovery was observed, whereas at an interval of 180 days a considerable recovery was observed. We presume that the recovery in TBE that was observed a long time after the irradiation results from a proliferative stimulus to endothelial cells which takes place during the post-irradiation period. The proliferative response leads to cell death of the X-ray damaged endothelial cells and thereafter these are replaced by healthy cell

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 include