Roland C. M. Vulders

A Doxorubicin Prodrug Activated by the Staudinger Reaction

Much effort has been devoted to prodrug systems that effect drug release at the tumor through enzymatic action. To widen the scope of prodrug therapy, the use of the selective Staudinger reaction as prodrug activator, instead of relying on enzymatic action, was investigated. Doxorubicin was conjugated to a p-azidobenzyl trigger that is cleaved after reacting with the chemical activator, triphenylphosphine. The prodrug activation was confirmed in water, cell growth medium, and serum, using HPLC and LCMS. Next, this approach was tested in a cell proliferation assay with A431 human vulvar skin squamous carcinoma cells. The doxorubicin prodrug was shown to exhibit a 176-fold higher IC50 of 15.1 microM vs 0.086 microM for the parent drug, doxorubicin. Addition of triphenylphosphine (5 x 60 microM in 72 h) to the prodrug in cell culture effected the complete recovery of the activity of the parent drug as evidenced by an IC50 value of 0.074 microM. Furthermore, high levels of triphenylphosphine were tolerated well by the cells. The demonstrated usefulness of the Staudinger reaction in cell culture and its in vivo potential opens up new avenues for prodrug therapy.

Complete sequence-based pathway analysis by differential on-chip DNA and RNA extraction from a single cell

We demonstrate on-chip, differential DNA and RNA extraction from a single cell using a microfluidic chip and a two-stage lysis protocol. This method enables direct use of the whole extract, without additional washing steps, reducing sample loss. Using this method, the tumor driving pathway in individual cells from a colorectal cancer cell line was determined by applying a Bayesian computational pathway model to sequences obtained from the RNA fraction of a single cell and, the mutations driving the pathway were determined by analyzing sequences obtained from the DNA fraction of the same single cell. This combined functional and mutational pathway assessment of a single cell could be of significant value for dissecting cellular heterogeneity in tumors and analyzing single circulating tumor cells.

Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device.

Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cancer derived cell lines (LS174T, LS180 and RKO) and fresh colorectal tumors have been individually trapped, their genomes extracted and prepared for sequencing using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06%) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as the lysis is in sub-nanoliter volumes. Our data thus demonstrates that high quality whole genome sequencing of single cells can be achieved using a relatively simple, inexpensive and scalable device. Detection of genetic heterogeneity at the single cell level, as we have demonstrated for freshly obtained single cancer cells, could soon become available as a clinical tool to precisely match treatment with the properties of a patients own tumor.

Sequencing Metrics of Human Genomes Extracted from Single Cancer Cells Individually Isolated in a Valveless Microfluidic Device

Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cell (LS174T, LS180 and RKO) lines and fresh colorectal cancers are individually trapped, their genomes extracted and prepared for sequencing, using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06 %) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as extraction is in sub-nanoliter volumes. Our data also demonstrates that high quality single cell sequencing can be achieved using a relatively simple, inexpensive and scalable device.

Abstract 4552: Pretargeted radioimmunoimaging and -therapy in tumor-bearing mice using a bioorthogonal reaction

A major challenge of radioimmunotherapy (RIT) of cancer is to enhance the nuclear radiation dose delivered to the tumor while minimizing the dose in healthy tissues. Monoclonal antibodies (mAb) circulate for a long time and only slowly accumulate in the tumor, which, when used for RIT, leads to dose-limiting side effects in healthy organs. This efficacy-limiting factor can be circumvented by pretargeting, which involves tumor targeting of a mAb followed by binding of a small radiolabeled probe to the tumor-bound mAb. The superior image contrast and the ability to administer higher (therapeutic) radiation doses compared to directly labeled mAbs is offset by the drawbacks of the current biological pretargeting systems, involving either immunogenicity issues or extensive re-engineering of the parent mAb. To address this, we designed a novel pretargeting approach based on the bio-orthogonal chemical inverse-electron-demand Diels Alder reaction, employing a trans-cyclooctene-conjugated mAb and a radiolabeled tetrazine derivative, and we evaluated the in vitro stability and reactivity, and tumor targeting in mice bearing colorectal xenografts. The in vitro stability and reactivity of 111 In-labeled tetrazine and anti-TAG72 mAb CC49 functionalized with trans-cyclooctene (TCO) moieties through lysine residue conjugation were monitored in PBS, serum and blood. In vivo pretargeting was performed in LS174T-tumored mice using 125 I-labeled CC49-TCO and 111 In-labeled tetrazine and assessed by dual isotope biodistribution and SPECT imaging with a nanoSPECT/CT. When 111 In-tetrazine was administered to mice 1 day after CC49-TCO, the chemically-tagged tumors reacted rapidly with 111 In-tetrazine, resulting in pronounced radioactivity localization throughout the tumor and good tumor contrast, as demonstrated by SPECT/CT imaging of live mice 3 h post injection: 4.18%ID/g, tumor-to-muscle ratio (T/M)=13.1. In mice treated with unmodified CC49, the tumor could not be discriminated from the surrounding tissue (0.28 %ID/g, T/M=0.5). Mice treated with TCO-modified rituximab, which lacks specificity for TAG72, showed the expected retention of 111 In-tetrazine in blood and non-target organs, and a much reduced tumor accumulation (1.02 %ID/g, T/M=2.1). Corresponding biodistribution experiments revealed a remarkable 52-57% reaction yield between TCO and tetrazine moieties present in tumor and blood. We have demonstrated the first use of a chemical reaction between two exogenous moieties in living animals for the non-invasive imaging of low-abundance targets in clinically relevant conditions. The inverse-electron-demand Diels Alder reaction has the potential to improve the state of the art of pretargeted RIT and can be applied to a range of antibodies due to its universal and straightforward conjugation chemistry. The validation for pre-targeted RIT in LS174T-tumored mice is underway. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4552.