Jörg A. Krüger

Targeting tumor-associated fibroblasts improves cancer chemotherapy by increasing intratumoral drug uptake

Tumor-associated fibroblasts are key regulators of tumorigenesis. In contrast to tumor cells, which are genetically unstable and mutate frequently, the presence of genetically more stable fibroblasts in the tumor-stromal compartment makes them an optimal target for cancer immunotherapy. These cells are also the primary source of collagen type I, which contributes to decreased chemotherapeutic drug uptake in tumors and plays a significant role in regulating tumor sensitivity to a variety of chemotherapies. To specifically kill tumor-associated fibroblasts, we constructed an oral DNA vaccine targeting fibroblast activation protein (FAP), which is specifically overexpressed by fibroblasts in the tumor stroma. Through CD8+ T cell-mediated killing of tumor-associated fibroblasts, our vaccine successfully suppressed primary tumor cell growth and metastasis of multidrug-resistant murine colon and breast carcinoma. Furthermore, tumor tissue of FAP-vaccinated mice revealed markedly decreased collagen type I expression and up to 70% greater uptake of chemotherapeutic drugs. Most importantly, pFap-vaccinated mice treated with chemotherapy showed a 3-fold prolongation in lifespan and marked suppression of tumor growth, with 50% of the animals completely rejecting a tumor cell challenge. This strategy opens a new venue for the combination of immuno- and chemotherapies.

Immunostimulatory Effects of Low-Dose Cyclophosphamide Are Controlled by Inducible Nitric Oxide Synthase

Cyclophosphamide is a widely used chemotherapeutic drug that was recently applied as either an antiangiogenic/antivasculogenic or an immunostimulatory agent in combination with cancer immunotherapies. It has been previously shown that cyclophosphamide augments the efficacy of antitumor immune responses by depleting CD4+ CD25+ T regulatory cells and increasing both T-lymphocyte proliferation and T memory cells. Furthermore, cyclophosphamide was shown to mediate killing of circulating endothelial progenitors. However, the molecular basis for these observations has not yet been elucidated. We show here that the cyclophosphamide-mediated inhibition of inducible nitric oxide synthase is directly linked to its immunostimulatory but not to its antivasculogenic effects. Moreover, combined application of cyclophosphamide with a novel, oral DNA vaccine targeting platelet-derived growth factor B (PDGF-B), overexpressed by proliferating endothelial cells in the tumor vasculature, not only completely inhibited the growth of different tumor types but also led to tumor rejections in mice. These findings provide a new rationale at the molecular level for the combination of chemotherapy and immunotherapy in cancer treatment.

Quantitative Mass Spectrometry Identifies Drug Targets in Cancer Stem Cell-Containing Side Population

A multifaceted approach is presented as a general strategy to identify new drug targets in a breast cancer stem cell‐containing side population. The approach we have utilized combines side population cell sorting and stable isotope labeling by amino acids in cell culture with mass spectrometry to compare and identify proteins with differential expression profiles between side population cells, know to be enriched in cancer stem cells, and nonside population cells, which are depleted in cancer stem cells, for two breast cancer cell lines, MCF7 and MDA‐MB231. Almost 900 proteins were quantified, and several important proteins in cell cycle control and differentiation were found to be upregulated in the cancer stem cell‐containing side population. Most interestingly, a splice isoform of pyruvate kinase M2 as well as peroxiredoxin 6 were found to be downregulated. The differential levels of three of these proteins, thymosin β4 (TB4), proliferation‐associated protein 2G4, and SIAH‐interacting protein, were validated using Western blot. Furthermore, functional validation provided clear evidence that elevated TB4 expression contributes to drug resistance in the stem cell population. Small interfering RNA silencing of TB4 led to a loss of chemoresistance in two separate breast cancer populations. These proteins likely contribute to resistance in the cancer stem cell‐containing side population, and their altered expression in a tumor causes clinical resistance to chemotherapy. The ability to perform quantitative mass spectrometry has enabled the identification of a series of proteins that could serve as future therapeutic targets.