Strategies to increase drug penetration in solid tumors

Abstract

Despite significant improvement in modalities for treatment of cancer that led to a longer survival period, the death rate of patients with solid tumors has not changed during the last decades. Emerging studies have identified several physical barriers that limit the therapeutic efficacy of cancer therapeutic agents such as monoclonal antibodies, chemotherapeutic agents, anti-tumor immune cells, and gene therapeutics. Most solid tumors are of epithelial origin and, although malignant cells are de-differentiated, they maintain intercellular junctions, a key feature of epithelial cells, both in the primary tumor as well as in metastatic lesions. Furthermore, nests of malignant epithelial tumor cells are shielded by layers of extracellular matrix (ECM) proteins (e.g., collagen, elastin, fibronectin, laminin) whereby tumor vasculature rarely penetrates into the tumor nests. In this chapter, we will review potential strategies to modulate the ECM and epithelial junctions to enhance the intratumoral diffusion and/or to remove physical masking of target receptors on malignant cells. We will focus on peptides that bind to the junction protein desmoglein 2 and trigger intracellular signaling, resulting in the transient opening of intercellular junctions. Intravenous injection of these junction openers increased the efficacy and safety of therapies with monoclonal antibodies, chemotherapeutics, and T cells in mouse tumor models and was safe in non-human primates. Furthermore, we will summarize approaches to transiently degrade ECM proteins or downregulate their expression. Among these approaches is the intratumoral expression of relaxin or decorin after adenovirus- or stem cell-mediated gene transfer. We will provide examples that relaxin-based approaches increase the anti-tumor efficacy of oncolytic viruses, monoclonal antibodies, and T cells.

Keywords: epithelial junctions; extracellular matrix; junction opener; relaxin; tumor stroma; tumor-associated macrophages.

Figure 1

Tumor stroma. (A) Schematic representation of tumor stroma components. The tumor stroma is composed of stromal cells (fibroblasts, macrophages, neutrophils, and mesenchymal stem cells) as well as extracellular matrix. (B) Sections from breast cancer patient biopsies (stage III and IV). DSG2 staining appears in brown. Malignant cells are DSG2-positive and form nests that are surrounded by tumor stroma containing DSG2-negative stroma cells such as fibroblasts. (C) Immunofluorescence analysis for Her2/neu (green) and the stroma protein laminin (red) on a breast cancer section. The scale bar is 20 μm.

Figure 2

Architecture of epithelial cells. (A) Adjacent epithelial cells maintain several intercellular junctions and an apical-basal polarity. Tight junctions seal the paracellular space close to the apical side. Initial cell contact is initiated by cadherins in the adherens junction complex that is situated underneath tight junctions. Adherens junction complexes encircle cells as an adherens belt, which connects to the F-actin cytoskeleton. Desmosomes are spot-like adhesions randomly arranged on lateral sides of plasma membranes. A key desmosomal junction protein is desmoglein 2 (DSG2). (B) Target receptors for cancer therapy are often trapped in epithelial junctions. Shown are breast cancer cells stained for Her2/neu (the target receptor for trastuzumab/Herceptin™) and the junction protein claudin 7. The merged image shows colocalization of both proteins in junctions between cells.

Figure 3

Therapeutic effect of relaxin-expressing oncolytic adenovirus. (A) ECM acts as a physical barrier in solid tumors, so that interstitial viral penetration and cell-to-cell spread of conventional oncolytic adenoviruses is restricted to the site of administration, leading to limited therapeutic efficacy. (B) Relaxin-expressing oncolytic adenovirus decreases ECM components within a tumor mass and increases its tumor penetration and dispersion, thereby eliciting improved antitumor efficacy.

Figure 4

Junction opener 1 (JO-1). (A) Schematic structure of JO-1 The Ad serotype 3 fiber knob domain and one fiber shaft motif was fused through a flexible linker to a homodimerizing K-coil domain (182). The protein is self-dimerizing and can be purified by His-Ni-NTA affinity chromatography. (B) Mode of action. JO-1 binds with picomolar avidity to DSG2. In epithelial cancer cells, DSG2 is overexpressed and exposed on the cell surface with preferential localization to desmosomes. JO-1 binding to DSG2 triggers cleavage of DSG2 dimers between neighboring cells and the transient activation of EMT pathways. This triggers junction opening and relocalization of target receptors that are often trapped in epithelial junctions. Junction opening allows for access of drugs (for example mAbs) to their target receptors. (C) Transmission electron microscopy of junctional areas of T84 cells. Cells were either treated with PBS (upper panel) or JO-1 (lower panel) for 1 h on ice, washed, and then incubated for 15 min at 37°C. At this time, the electron-dense dye ruthenium red (Ru) (1) was added together with the fixative. If tight junctions (above the desmosomes) are closed, the dye only stains the apical membrane (black line). If tight junctions are open, the dye penetrates between the cells and stains the baso-lateral membrane. JO-1 also mediates the partial dissociation of desmosomes (D). The scale bar is 0.5 μm.

Figure 5

Junction opener 1 increases tumor penetration and efficacy of PEGylated liposomal doxorubicin (PLD)/Doxil™. Studies were performed in mice with mammary fat pad tumors derived from ovc316 cells (135, 206). Ovc316 cells are Her2/neu positive epithelial tumor cells derived from an ovarian cancer biopsy. (A) Scheme of experiment on tumor penetration of PLD. Mice were intravenously injected with PBS or JO-1 (2 mg/kg) followed by PLD or PBS 1 h later. Two hours after PBS or PLD injection, mice were sacrificed and tumors harvested. (B) Immunofluorescence analysis for PLD on tumor sections with anti-PEG antibodies. PLT appears in red. The scale bar is 20 μm. Notably, free PEG is poorly detected by ELISA or immunohistochemistry (184). (C) PLD concentrations in tumors measured by ELISA (N = 3). (D) Therapy study in mice with ovc316 tumors. Treatment was started when tumors reached a volume of 100 mm³ (D0). Mice were injected intravenously with 2 mg/kg JO-1 or PBS, followed by an intravenous injection of PLD (1 mg/kg) or PBS 1 h later. Treatment was repeated weekly (N = 5).