Cancer-associated fibroblasts are the predominant cell type in the tumor-associated stroma. Their numbers are elevated in tumor stroma compared to stroma found in healthy tissue (34, 35). In many carcinomas, the fraction of CAFs is even greater than the fraction of malignant cells (32). In the tumor microenvironment, tumor and stromal cells upregulate various profibrotic growth factors such as transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF), all of which are main mediators for the transdifferentiation of stromal fibroblasts into CAFs (36, 37). CAFs are phenotypically and functionally distinct from normal stromal fibroblasts (38). CAFs are large spindle-shaped mesenchymal cells that share morphological characteristics of both smooth-muscle cells and fibroblasts (39). Metabolically, CAFs are perpetually activated, proliferate faster, and accumulate greater amounts of ECM constituents than fibroblasts in normal tissues (2, 32, 40). In addition to creating tumor-derived ECM, CAFs have an impact on cancer cell proliferation, invasion, and metastasis through secretion of different growth factors [epidermal growth factor (EGF), FGF, hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1) (2, 3, 31, 34, 41–44)]. CAFs are also involved in the activation of angiogenic programs as well as the recruitment of inflammatory cells (45). Local expression of vascular endothelial growth factor (VEGF) or monocyte chemoattractant protein-1 (MCP-1) by CAFs stimulates angiogenesis and the recruitment of pro-tumor myeloid cells.

As another major component in the tumor stroma, TAMs have emerged as a significant player in the stromal compartment of virtually all types of carcinoma (46). While type M1 macrophages are antigen-presenting cells that incite T cells to mount immune responses, TAMs are M2-type macrophages and tumor promoting. Tumor cells, among other cytokines, produce MCP-1 and colony stimulating factor-1 (CSF-1) which participates in mobilization of TAM-progenitors from the bone marrow and homing to tumor stroma. Homing of TAMs to tumors is also supported by the specific architecture of tumor blood vessels which promote efficient trafficking of blood cells. There is convincing evidence that the extent of MCP-1 expression in human cancers correlated with both TAM infiltration and tumor malignancy (47–53). TAMs contribute to tumor-associated alteration in the ECM by releasing profibrotic growth factors, which then act in an autocrine and/or paracrine manner to differentiate normal stromal fibroblasts into CAFs (33, 37, 46). TAMs also produce growth factors (EGF, HGF, bFGF, and VEGF), cytokines [IL-1, IL-8, and tumor necrosis factor-α (TNFα)], and enzymes [MMP-2, MMP-7, MMP-9, MMP-12, and cyclooxygenase-2 (COX-2)] (46, 54). Additionally, TAMs can suppress anti-tumor immune responses. For example, TAMs secret a distinctive set of cytokines (IL-10 and TGF-β) as well as chemokines [chemokine (C–C motif) ligand (CCL)17, CCL22, and CCL24] favoring recruitment of T_regs and generation of an immune suppressive microenvironment (55–57). As outlined in Section “Epithelial-to-Mesenchymal Transition and Mesenchymal-to-Epithelial Transition in Cancer,” TAMs also promote epithelial-to-mesenchymal transition (EMT) via TGF-β (58) and regulate cancer stem cell (CSC) activities (59) in solid tumors.

Tumor-associated neutrophils comprise another prominent portion of the immune cell infiltrates observed in a wide variety of murine models and human cancers (60–63). Similar to TAMs, products secreted from neutrophils, including reactive oxygen species, cytokine (IL-8), growth factors (VEGF and HGF), and proteinases [arginase (ARG 1), MMP-2, MMP-8, MMP-9, and MMP-13], have defined and specific roles in both regulating tumor cell proliferation, angiogenesis, and metastasis and suppressing the anti-tumor immune response (64).

Once systemically administered drugs reach the tumor sites, they have to exit the tumor vasculature and translocate through the interstitial space in order to reach their target cells. The endothelial cell layer, lining the blood vessels, is thought to present a barrier to macromolecular drugs (20). Transendothelial transport of macromolecular drugs involves a phenomenon known as the enhanced permeability and retention (EPR) effect in solid tumors. The EPR effect is observed for intravenously administered macromolecular anti-cancer drugs that escape renal clearance, due to their large molecular size (10–500 nm). They are mostly unable to pass the tight endothelial junctions of normal blood vessels, but can extravasate and then become trapped in the tumor vicinity (65). Unlike normal tissues that feature an organized vascular network, the blood vessel system in solid tumors is rather chaotic. The endothelial cell layers are poorly aligned (66) and elevated levels of vascular permeability factors generate “leaky” capillaries (65). It is therefore thought that transendothelial transport is not a critical limiting obstacle for large sized drugs.

Rapid tumor cell proliferation and weakly developed lymphatics cause high IFP (67, 68) and blood vessel remodeling by intussusception (69) or compression (70). Additionally, the increased hydraulic conductivity of “leaky” capillaries can further increase the IFP in tumors (71). Together, this leads to an imbalance in blood flow and nutrient supply within the tumor microenvironment. The uniformly high IFP in the center of solid tumors drops toward the periphery (72), which could negatively affect drug extravasations in the high-pressure regions. Cells that are distant to blood vessels (100–200 μm) and located in high-pressure regions subsequently constitute large areas of hypoxic, necrotic, or semi-necrotic tissue. This exacerbates the tendency of tumor cells to overproduce and release lactic acids within these regions, which results in acidosis (73). Moreover, the vascular surface area per unit tissue weight is decreasing with tumor growth, which further limits transvascular exchange for large tumors when compared to small tumors (74, 75). In contrast, cells situated in the invasive front benefit from the enhanced vascular permeability that supplies adequate amounts of macromolecules for rapid tumor growth (76). Furthermore, the blood flow rates in non-necrotic regions can be substantially higher than in the surrounding normal tissue (77). It is therefore expected that the uptake of drugs in solid tumors is heterogeneous and the general distribution might decrease with increasing tumor weight. It is thought that induction of massive cell death by chemo- and radio-therapy can lower the IFP in tumors (78). The application of chemotherapy to lower the IFP is also used in approaches to “normalize” the tumor vasculature. Anti-angiogenic drugs are thought to compensate for the pro-angiogenic factors that are extensively produced in the tumor in order to eliminate “leaky” blood vessels. Ideally, this would lead to a more organized blood vessel system that features more functional and more uniformly perfused capillaries within solid tumors. On the other hand, this would also inhibit the extravasation of large drugs.

Two major consequences of abnormal microcirculation in solid tumors are hypoxia and low extracellular pH. Hypoxia or oxygen deprivation is a key factor in tumor progression and resistance to therapy. The most important regulatory factor of the hypoxia-signaling pathway activity in cells is hypoxia-inducible transcription factor 1 (HIF-1α). Under hypoxic condition, tumors produce a number of chemokines that attract and differentiate CAFs, TAMs, and TANs. Approaches that reduced intratumoral hypoxia therefore block pro-tumoral functions of these cells, including the production of stroma proteins. It is therefore thought that hypoxia targeting strategies improve the intratumoral penetration of drugs (79).

Tight junctions play a key role in the formation of epithelial sheets. Strictly linked to tight junctions is a barrier function within a sheet of cells that restricts ions and small molecules to pass through the paracellular space between two adjacent epithelial cells (80). Additionally, tight junctions function as a “fence” that separates the apical and basal membrane compartments in an individual cell (81). Importantly, the tight junction strands on one cell are associated laterally to tight junction strands of opposing membranes on neighboring cells (82). The permselective barrier function is based on occludins and claudins, two types of transmembrane proteins that have been identified among more than 40 proteins within tight junctions (81, 83). Other tight junction transmembrane proteins comprise the singlespan junctional adhesion molecules (JAMs) or coxsackie and adenovirus receptor (CAR) (83–86) and the lately identified tetraspan tricellulin, which is enriched in areas where three cells meet (86).

The major transmembrane proteins of adherens junctions are classical cadherins, such as epithelial cadherin (E-cadherin). Members of this protein superfamily promote homophilic intercellular adhesion in a Ca²⁺-dependent manner. Their cytoplasmic domain binds cytosolic catenins that link the cadherin/catenin complex to the actin cytoskeleton. The formation of adherens junctions consequently leads to the assembly of tight junctions, but E-cadherin is dispensable for tight junction maintenance (87). A central role in maintenance and initiation of an epithelial phenotype is attributed to E-cadherin, the core protein of adherens junctions. In addition to its homophilic intercellular adhesive features, the extracellular E-cadherin domain functions as a direct repressor of receptor tyrosine kinase (RTK) signaling via blockage of FGF or EGF ligand binding stimulation (88, 89). The cytoplasmic part of E-cadherin connects to the actin cytoskeleton and also influences a number of signaling pathways via direct binding to p120- and β-catenin. When adjunct to E-cadherin at the membrane, β-catenin inhibits cell growth (90), whereas its translocation to the nucleus activates canonical Wnt signaling (91). Similarly, p120-catenin stabilizes E-cadherin at the membrane, while blocking NF-κB and Ras-MAPK signaling (92, 93).

Desmosomes are molecular complexes of cell adhesion proteins and linking proteins that attach the cell surface adhesion proteins to intracellular keratin cytoskeletal filaments. The cell adhesion proteins of the desmosome, desmoglein, and desmocollin, are members of the cadherin family of cell adhesion molecules. They are transmembrane proteins that bridge the space between adjacent epithelial cells by way of homophilic binding of their extracellular domains to other desmosomal cadherins on the adjacent cell. Both have five extracellular domains, and have calcium-binding motifs. One of these junction proteins, DSG2, is upregulated in malignant cells (94, 95). DSG2 contains four extracellular cadherin domains (ECDs; ECD1–ECD4), which link neighboring cells to each other through homodimers. ECDs are linked via an extracellular anchor and membrane-spanning domain to the intracellular anchor and intracellular cadherin-typical sequence (ICS) motifs. The role of the conserved ICS is not known, although consensus sites for protein kinase C phosphorylation and a caspase-3 cleavage site have been identified and could contribute to signaling (96). Desmosomes probably do not directly regulate paracellular permeability, but they seem to do this indirectly by altering the structure and the stability of tight junctions (97).

During progression toward metastatic disease carcinoma cells engage EMT. The EMT program can be activated by a multitude of factors secreted by tumor stroma cells, which triggers a complex signaling network including TGF-β, Wnt, HGF, EGF, and PDGF pathways (115). The morphological changes that occur during EMT are a consequence of diverse molecular mechanisms that contribute to the acquisition of mesenchymal features. A central event during EMT is the functional loss of E-cadherin. Subsequent breakdown of intercellular epithelial junctions plays a major role in cancer progression, where E-cadherin therefore acts as a repressor of invasion (116). Accordingly, the reduced expression of this major regulator of the epithelial phenotype is associated with poor prognosis in several cancers (117). The loss of E-cadherin and several other epithelial genes including multiple members of the claudin family as well as occludin is mainly regulated via transcriptional repression by EMT inducers that include transcription factors Snail, ZEB, Twist, FOXC2, and E47 (118–126). Notably, these EMT inducers also act as positive regulators of gene expression for several mesenchymal genes (127, 128). A consequent event in EMT is the change from E-cadherin to N-cadherin (129). The importance of this cadherin switch is highlighted by the fact that homophilic intercellular junctions formed by N-cadherin are less resistant to rupture under physiological stress conditions, when compared with E-cadherin (130). Additionally, a shift from several keratins (-8, -9, and -18) to vimentin occurs, resulting in a more flexible cytoskeleton (131, 132). Concomitantly with the acquisition of such mesenchymal features, the expression of several ECM proteins is induced. Fibronectin, collagen precursors, laminin, and vitronectin are all reported to be elevated in mesenchymal cells (133). These and other proteins, including Src kinase, integrin-linked kinase, integrin β-5, and MMPs, are upregulated during EMT, have an impact on cytoskeletal remodeling, and promote cell motility (104).

Successful EMT induction ultimately enables cancer cells to leave the primary tumor, enter the bloodstream, and attach to distant organ sites in order to build metastases. The endpoint of this process however, involves the reversed process (MET), where cells that underwent EMT regain epithelial properties and form tumors that histopathologically resemble the primary cancer (110, 134). Although the underlying mechanisms are currently unknown, it is likely that MET events are initiated due to the lack of EMT-inducing signals at attachment sites of metastatic tumor cells. In support, numerous examples of advanced carcinomas exist, showing that mesenchymal cells can regain characteristics of epithelial cells or undergo MET (104). It is now generally accepted that the reverting to an epithelial phenotype through MET represents a protective mechanism against host-immune attacks and creates resistance to anti-cancer drugs. The transdifferentiation into an epithelial phenotype and the formation of tight junctions between malignant cells that prevent penetration of host anti-tumor immune cells, host anti-tumor antibodies, and therapeutics represents one of the most basic cancer resistance mechanisms.

Importantly, changes between EMT and MET occur gradually, which leads to a wide range of intermediate cell stages that consequently possess an E/M hybrid phenotype. The E/M hybrid phenotype is especially prominent in the invasive front of several carcinomas where it has also initially been linked to cells with a stem cell-like phenotype (112, 113). We have recently shown in cancer cells derived from ovarian cancer biopsies that CSCs generate mesenchymal cells via EMT in vitro and undergo MET to form tumors containing epithelial cells when injected into immunodeficient mice (135). A marker combination widely used to identify CSCs in multiple cancers including prostate, pancreatic, and colon is EpCAM and CD44 (136, 137). Interestingly, the expression of these proteins can be accredited to epithelial and mesenchymal cells, respectively, suggesting a more general pattern of an E/M hybrid phenotype for tumor-initiating cells (TICs) (25). Very recently, work by Yu et al. demonstrated that cells, which leave the primary tumor, possess an epithelial or E/M hybrid phenotype. In the bloodstream these circulating tumor cells are bound by platelets, which trigger EMT via TGF-β signaling (138). However, cells undergoing EMT that leave the primary tumor experience a proliferation arrest, which is mediated by EMT inducers, like Twist1. In order to reenter a proliferative stage that allows for colonization and macrometastasis, the downregulation of Twist1 and concomitant MET is critically needed, while ongoing EMT signaling leads to dormancy and micrometastases at sites of reattachment (139). Additionally, it was recently reported that two distinct types of EMT exist in carcinomas, depending on the presence or absence of EMT inducer paired-related homeobox transcription factor 1 (Prrx1). When Prrx1 is expressed in cells undergoing Twist1-induced EMT, a CSC pattern is suppressed and cells fail to colonize. After Prrx1 is downregulated and other EMT inducers, such as Twist1 or ZEB1 have vanished, MET occurs and metastatic growth can be initiated (140). Notably, MET has also been reported in non-epithelial cancers, e.g., sarcoma (141, 142).

Wang et al. suggested that at least two mechanisms are involved in JO-1-mediated opening of tight junctions: DSG2 cleavage/internalization and EMT-like intracellular signaling. Epithelial cells are linked to each other by homodimers of DSG2. Studies in vitro and in xenograft models have shown that the DSG2 ECD is cleaved upon binding of JO-1, which results in DSG2 internalization (109). On the other hand, a series of data indicate that Ad3 binding triggers EMT-like signaling, which most likely involves the intracellular domain (ICD) of DSG2. Using mRNA expression arrays and qRT-PCR, Wang et al. found 12 h after incubation of polarized breast cancer epithelial cells with a JO-1-like ligand that 430 genes were upregulated and 352 genes were downregulated compared to incubation with a control protein (181). mRNA expression profiling revealed the activation of pathways involved in EMT (MAPK/ERK, adherens junctions, focal adhesion, and regulation of actin cytoskeleton signaling). Further studies showed an increase in PI3K and MAPK/ERK1/2 phosphorylation within 1 h after incubation with Ad type 3 pento-dodecahedral particles or JO-1 (109, 181). PI3K and MAPK/ERK1/2 activation was significantly decreased in cells in which DSG2 expression was suppressed by siRNA. Beyer et al. also found that subsequently to MAPK and PI3K activation, the protein levels of E-cadherin, a key junction protein, decreased in epithelial cells, indicating a down-regulation of gene expression of junction proteins (109).

Beyer et al. have also shown in mouse xenograft tumor models that the i.v. administration of JO-1-mediated junction opening in epithelial tumors (183). The changes triggered by JO-1 were detectable within 1 h after its i.v. injection. This, subsequently, enabled the increased intratumoral penetration of the anti-Her2/neu mAb trastuzumab (109). These biological effects of JO-1 translated into an increased therapeutic efficacy of several mAbs, including trastuzumab and cetuximab, in xenograft tumor models, e.g., models of colon, breast, gastric, lung, and ovarian cancer (109). JO-1 co-administration also enhanced the therapeutic efficacy of several chemotherapy drugs, including PEGylated liposomal doxorubicin (PLD or Doxil^®), paclitaxel (Taxol^®), nanoparticle albumin bound paclitaxel (Abraxane^®), and irinotecan (Camptosar^®) in tumor xenograft models of breast, lung, and prostate cancer (183). Furthermore, chemotherapy doses could be decreased without compromising the anti-tumor effects due to JO-1 co-therapy. This also provided protective effects to normal tissues (183). For example, we showed that the ability of JO-1 to open intercellular junctions in tumors increased the uptake and amount of chemotherapeutics in the tumor environment (Figures 5A–C). This then resulted in reduced drug levels in normal tissues, thereby providing a larger therapeutic window. Immunofluorescence analysis of tissue sections also revealed higher levels of PLD in tumors of JO-1 + PLD treated mice compared to mice treated with PLD alone. In these animals, PLD is found to be more broadly distributed over a greater distance from blood vessels, suggesting better intratumoral penetration and absorption by tumor tissue (Figure 5B). Using an ELISA to measure PEGylated compounds in tissues (184), we found more PLD in tumors and less in normal tissues of mice that received JO-1 prior to i.v. PLD injection (Figure 5C). Better intratumoral penetration and accumulation of PLD after JO-1-mediated junction opening, resulted in enhanced therapeutic efficacy of PLD chemotherapy. This was shown in a model with mammary fat pad tumors derived from primary ovarian cancer cells (obtained from a patient biopsy) (135). This cell line was nearly resistant to PLD injected intravenously at a dose that corresponds to PLD doses used in patients. Importantly, JO-1 pre-treatment significantly improved PLD therapy (Figure 5D). JO-1 also relieved adverse side effects from PLD treatment, e.g., liver enzymes (AST, ALT, and alkaline phosphatase) were significantly decreased in animals treated with JO-1 and PLD compared to mice treated with PLD alone. Mice that received JO-1 injections also had less severe tissue damage in the bone marrow and intestine caused by PLD treatment (183).

In breast cancer xenograft sections and in cultured breast cancer cells, Beyer et al. found co-staining of Her2/neu and the adherens junction protein claudin 7 (183). Confocal microscopy of breast cancer BT474 cells confirmed the trapping of Her2/neu in lateral junctions. Incubation of the Her2/neu positive breast cancer cell lines BT474 or HCC1954 with JO-1 changed the composition of the lateral epithelial junctions within 1 h. As a result of this, Her2/neu staining at the cells surface became more intense, while it faded in areas distal of the cell surface. This suggests that JO-1-mediated junction opening triggered a translocation of Her2/neu from lateral membranes to the cell surface.

Junction opener 1 does not efficiently bind to mouse (185), hamster, or dog DSG2 (André Lieber, unpublished data). To perform efficacy and safety studies in a small animal model, we generated human DSG2-transgenic mice that expressed human DSG2 at a level and in a pattern similar to humans (185). Using the hDSG2-transgenic mouse model with syngeneic hDSG2^high tumors, we demonstrated that JO-1 predominantly accumulates in tumors (183). This could be explained by either one of the following factors: (i) the overexpression of DSG2 by tumor cells, (ii) better accessibility of DSG2 on tumor cells due to a lack of strict cell polarization compared to DSG2-expressing normal epithelial cells, or (iii) a high degree of vascularization and vascular permeability in tumors.

The i.v. injection of JO-1 at a dose of 2 mg/kg into hDSG2-transgenic mice had no observed adverse side effects, except for mild, transient diarrhea. There were also no abnormalities found in laboratory parameters as well as histopathological studies of tissues. We speculate that this is due to the fact that DSG2 in tissues, other than the tumor and a subset of epithelial cells in the intestine/colon, is not accessible to i.v. injected JO-1. The hDSG2-transgenic mouse model was also used to obtain biodistribution and pharmacokinetics data for JO-1 (183).

Recently, we started safety studies with JO-1 in macaques. So far, we injected two animals intravenously with JO-1 at a dose of 0.6 mg/kg and performed a full necropsy 3 days later. Behavior and health was normal in both animals. In blood and tissue analyses, we did not find hematological or histological abnormalities, except for a mild inflammation in the small intestine. JO-1 binding to DSG2 on tumor cells triggers pathways involved in EMT, a process which, as mentioned above, has been associated with tumor metastasis. Over 20 in vivo studies conducted with JO-1 combined with a range of cancer therapeutics in various different cancers with long-term follow-up, have not provided any evidence of metastases (183). Transient activation of the EMT pathway is only one of many steps required for tumor metastasis. Detachment of tumor cells from epithelial cancers and their subsequent migration is only possible after long-term crosstalk between malignant cells and the tumor microenvironment, resulting in changes in the tumor stroma and phenotypic reprograming of epithelial cells into mesenchymal cells (186).

Junction opener 1 is a protein derived form Ad3 and therefore potentially immunogenic. This might not be a critical issue if JO-1 is used in combination with chemotherapy, which suppresses immune responses (187–189). In addition, Beyer et al. have shown that JO-1 remains active in vitro and in vivo, even in the presence of anti-JO-1 antibodies generated by the JO-1 vaccination of mice (183). This may be due to the fact that JO-1 binds to DSG2 with a very high avidity, thus potentially disrupting the complexes between JO-1 and antibodies against JO-1. Notably, JO-1 is a dimer of a trimeric fiber knob, which contributes to the picomolar avidity to DSG2 (182). Wang et al. performed repeated injections of JO-1 in an immunocompetent hDSG2 mouse tumor model to test the effect of anti-JO-1 antibodies on the therapeutic efficacy of JO-1 (185). Importantly JO-1 had an enhancing effect on PLD therapy after repeated JO-1 pre-treatment, demonstrating that JO-1 continues to be effective after multiple treatment cycles, even in the presence of detectable antibodies.