Cancer diagnosis, therapy, and monitoring represent fundamental topics of research in medicine and are of utmost importance in healthcare of today’s society. An efficient cancer therapy should possess exceptional abilities not only to ensure a complete removal of the tumor but also to prevent its spreading and invasion to other tissues by metastasis. Current clinical approaches to treat cancer include, and often combine, surgery, chemotherapy, radiation therapy as well as immunotherapy. However, these methods in general still fail to treat highly aggressive metastatic cancers, and present some serious limitations. For instance, irradiation of tumors may damage adjacent healthy tissues, and chemotherapy, which is based on a non-specific systemic distribution regime, requires high drug dosage and promotes severe adverse side effects. For example, the administration of Paclitaxel (PTX), a drug used for the treatment of lung, ovarian, and breast cancers, has been associated with unwanted effects such as hypersensitivity reactions, myelosuppression, and neurotoxicity (1, 2), among others. Doxorubicin (DOX), another drug used in cancer chemotherapy, has also been described to have cardiotoxic side effects (3, 4). Moreover, chemotherapy might turn inefficient due to acquired chemoresistance as exemplified in the case of Gemcitabine – prime therapeutic used to treat pancreatic cancers (5), for DOX (3) and also for PTX (6, 7).

Tumor targeted drug-delivery (Figure 1) represents a promising approach to overcome some of the above mentioned limitations (8). This strategy aims to specifically guide and direct anticancer therapeutics (or imaging agents) to tumor cells without interfering with normal tissues. Such targeted approach relies on the fact that tumor vasculature and tumor cells display a well-differentiated pattern of (over-)expression of specific receptors (i.e., receptors required for tumor angiogenesis), which is consistent with the concept of “Vascular Zip Codes” (9, 10). Targeted drug-delivery methods hence employ small molecules or monoclonal antibodies selective to receptors that are proven to be abnormally expressed on tumors. The conjugation of anticancer drugs to these selective ligands will allow a preferential or selective delivery of the drug to the tumor.

As a result, this technique benefits from several advantages: (i) non-specific interactions with normal tissues are reduced, and thus the adverse side-effects associated to conventional chemotherapy can be minimized. (ii) Site-directed drug release leads to higher local concentrations at the diseased tissue and thus allows dosage reduction. (iii) Acquired chemoresistance can potentially be reduced by co-delivering other therapeutics capable of regulating cancer multi-drug resistance (MDR). To avail these advantages, well accessible cell surface receptors are preferred over intracellular targets where (complex) drug internalization mechanisms need to be taken into consideration. In this regard, one of the most intensely referred class of proteins for targeted therapy is the integrin family (11).

Integrins are heterodimeric transmembrane glycoproteins consisting of an α and a β subunit. In total, 24 different subtypes of integrins that are constituted from 18 α and 8 β subunits have been discovered to date (12). Almost half of them bind to various extra cellular matrix (ECM) proteins such as fibronectin, vitronectin, and collagen through the tripeptide motif Arg-Gly-Asp = RGD [(13), Figure 2], and are vital in the adhesion, signaling, migration, and survival of most cells (14). Integrins have also very important roles in cancer progression and some subtypes have been described to be highly over-expressed on many cancer cells. This is the case of integrins αvβ3, αvβ5, and α5β1, which are crucial mediators of angiogenesis in cancer (8, 15–17). Underlying cause for this is the elevated demand by the enlarging tumor for adequate supply of necessary nutrients and oxygen. In order to meet these demands through blood supply, tumor tissue with a rapidly overgrowing number of cells, signals [via growth factors like vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF)] for increased angiogenesis, a state known as “angiogenic switch.” Sprouting of new blood vessels and overexpression of integrins in tumor tissues and vasculature are thus key features in the pathophysiology of cancer. Other integrins such as αvβ6 and α6β4 are also observed to be expressed on tumor cells (8). Another pivotal function of integrins is the promotion of cell migration by virtue of their binding to ECM components. This phenomenon is responsible for the process of tumor proliferation, migration, invasion, and metastasis (18). These functional aspects together with the high expression levels found on tumor cells have converted integrins into very interesting proteins for targeted cancer diagnosis and therapy studies.

Our review shortly recapitulates recent developments in integrin targeted cancer therapy, with special focus on targeted delivery of chemotherapy or gene therapy via non-viral vectors like nanoparticles (NPs), micelles, vesicles, or other systems grafted with RGD-based integrin ligands. Considering the vastness of the topic, we have only cited a limited amount of recent works. For previous studies and developments in this field other detailed reviews are available (19–22). Applications based on integrin targeting antibodies and therapies involving the blocking of integrin functions with antagonists and other ligands are not subject of this review.

As previously introduced, the αvβ3 integrin subtype plays a major role in angiogenesis, tumor neovascularization, and tumor metastasis (8). The angiogenic pathways dependent on αvβ3 have been described to be induced by bFGF or tumor necrosis factor α (TNF-α). Its expression is upregulated on angiogenic endothelial cells (36–38) and on various tumor cell lines (39, 40). Antagonistic inhibition of αvβ3 integrin has been shown to suppress angiogenesis (41) and to induce apoptosis (42). The well-established biological roles, high expression on tumor tissues, and the availability of ligands with high affinity, have set αvβ3 the most extensively studied integrin for tumor targeting. The integrin αvβ5 is also involved in angiogenesis but through a distinct pathway stimulated by VEGF or transforming growth factor α (TGF-α) (16). Since most RGD-containing peptidic αvβ3 antagonists also recognize αvβ5, although usually with a lower affinity, these two integrin subtypes are discussed together.

In addition to αvβ3 and αvβ5, an upregulated expression of α5β1 in tumor vasculature and other cancer cells has also been described (36, 53–57). α5β1 primarily recognizes fibronectin through the RGD binding motif. Kim et al. have reported that α5β1 inhibition induces cell apoptosis in endothelial cells (58) and also showed that this integrin mediates the migration of endothelial cells. Noteworthy, it has been shown that α5 might substitute the activity of αv during vasculature remodeling (59). For these reasons, targeting of this integrin has also been approached in cancer therapy.

Kokkoli and co-workers have explored α5β1 integrin for targeting cancer cells by using a fibronectin mimetic α5β1-selective RGD-containing peptide, named PR_b (60) (Figure 2). This group produced DPPC-based liposomal NPs covered by PEG and further decorated with PR_b peptide, and studied their targeting capacity in a CT26.WT mouse colon carcinoma experimental model. The quantities of PEG and peptide were fine-tuned in order to optimize the delivery of the nanovector. By increasing the quantity of conjugated peptide, an enhancement in binding of liposomes to cells was observed, whereas the opposite effect was found when the concentration of PEG was augmented. The cytotoxicity of 5-Fluorouracil carried by these PR_b targeted liposomes was found to be comparable to that of the free drug and better than that of the particles containing only the control GRGDSP sequence, confirming the importance of targeting α5β1 on this cancer model. Similar results were obtained in studies using HCT116 and RKO human colon cancer cells (60). This liposomal system has been further investigated for the delivery and cytotoxicity of DOX to MDA-MB 231 breast cancer cells (61). Confocal microscopy experiments showed that these targeted liposomes were internalized in breast cancer cells via an endocytic pathway, and transferred within the first minutes into early endosomes, and after prolonged times into late endosomes and lysosomes. Particularly at high concentrations, the therapeutic effect of encapsulated DOX in MDA-MB 231 cells was comparable to that of the free DOX.

In a recent approach, PR_b targeted polymersomes have also been explored for siRNA delivery (62). T47D breast cancer cells were studied to check the expression of Orai3. The downregulation of Orai3 levels results in cell apoptosis. The delivery of Orai3 by PR_b-conjugated polymersomes decreased the viability of cancer cells but did not affect non-cancerous MCF10A breast cells. When compared to a commercial transfection agent (Lipofectamine RNAiMAX), the observed therapeutic effect of the polymersome formulation is still moderate. However, this method has not shown any systemic toxicity unlike other transfection reagents.

The integrin subtype αvβ6 is expressed at low or undetectable levels in most adult epithelia, but may be upregulated during inflammation and wound healing (8). αvβ6 preferentially binds to TGF-β1 latency associated peptide (LAP) (63), but can also recognize the ECM proteins tenascin and fibronectin (64). In this regard, αvβ6 is biologically important for the activation of TGF-β1 and has been shown to control TGF-β activity or signaling in fibrosis and to play a crucial role in TGF-β-integrin crosstalk in carcinomas (65). Furthermore, αvβ6 was found to be significantly upregulated in tumor tissues (8) and in certain cancer types including colon (66), ovarian carcinoma (67), and in early stage of non-small cell lung cancer (NSCLC), which is associated with poor patient survival (68, 69). Other studies have shown that αvβ6 expression is correlated with the development of metastasis in gastric cancer and the enhanced survival and invasive potential of carcinoma cells (70, 71). This pathological relevance has turned αvβ6 into a promising target for tumor diagnostics and antitumor therapy.

To date, several linear and cyclic peptides as well as peptidomimetics have been developed to target specifically the αvβ6 integrin subtype (68, 70, 72–74). For instance, the high affinity αvβ6-specific 20-mer peptide H2009.1 (75) was conjugated as a tetramer to a poly-glutamic acid polymer carrying DOX, and was shown to specifically target αvβ6-expressing cells in vitro (76). In another work, the selectivity of this peptide toward αvβ6 was exploited to guide fluorescent quantum dots to lung adenocarcinoma cell line H2009 in vitro (68). Recently, this peptide has also been conjugated to a water soluble PTX conjugate resulting in selective cytotoxicity for the αvβ6-expressing NSCLC cell line (77). The conjugate was able to reduce the rate of tumor growth in vivo, however without an increased benefit over the use of free PTX. Furthermore, the same peptide was used to investigate the multimeric effect on functionalized liposomes (78). In this study, liposomes displaying tetramers of the H2009.1 peptide demonstrated higher drug delivery and toxicity toward αvβ6-expressing cells than liposomes displaying single copies of H2009.1, even if the total number of peptides bound to each liposome was identical. In another approach, H2009.1 was used to functionalize the surface of multifunctional micelles encapsulated with SPIO and DOX for MRI and drug-delivery applications, respectively (79). The functionalized micelles significantly increased cell targeting and uptake in αvβ6-expressing H2009 cells, as verified by MRI and confocal imaging.

A20FMDV2 (80, 81) is another αvβ6-selecitve 20-mer peptide (Figure 2) that can be used for targeted therapies. As an example, this peptide was radiolabeled on solid phase using 4-[¹⁸F]fluorobenzoic acid and the conjugate was selectively uptaken by αvβ6-positive tumors but not by αvβ6-negative tumors, as monitored in mice by PET (70). In a similar approach, A20FMDV2 was conjugated to 5-[¹⁸F]fluoro-1-pentyne via an azide-based 1,3-dipolar cycloaddition (click chemistry). However, no difference in tumor targeting in vivo was observed for such strategy compared to the previous labeling method (82). ¹⁸F-labeled derivatives of the same peptide were described to improve tumor uptake capacity in BxPC-3 (pancreatic cancer) xenograft-bearing mice over [¹⁸F]-FDG (83). Recently, A20FMDV2 was conjugated to an ¹⁸F-based tracer by copper-free, strain promoted click chemistry. However, the resulting derivative did not show a remarkable in vivo tumor uptake by mouse with mouse model DX3puroβ6-tumor (84). Furthermore, A20FMDV2 was conjugated to DTPA and labeled with ¹¹¹In for SPECT imaging. In this study, the conjugate showed specific localization in αvβ6-tissues, and displayed increased uptake in an αvβ6-positive tumor and in a mouse xenograft model bearing breast tumors that express αvβ6 endogenously (85). Additionally, A20FMDV2 was incorporated into a recombinant adenovirus type 5 (Ad5) leading to increased cytotoxicity on a panel of αvβ6-positive human carcinoma cell lines in vitro and enhancement in tumor uptake and improved tumor transduction in an αvβ6-positive xenograft model in vivo over the Ad5 wild type (86).

In another approach pursued by the Gambhir research group, cystine knot peptides showing high affinity for αvβ6 but none for the related subtypes αvβ3, αvβ5, and α5β1 were developed and conjugated to ⁶⁴Cu-DOTA for PET-based tumor imaging (87). Injection of these conjugates into mice bearing either αvβ6-positive BxPC-3 xenografts or αvβ6-negative tumors, and monitoring by PET imaging, showed αvβ6-selective targeting for the tumors expressing αvβ6. In a recent study (88), two cystine knot peptides were labeled with ¹⁸F-fluorobenzoate and their capacity to be uptaken by tumor cells assessed in vivo. PET imaging revealed for both peptides specific targeting of αvβ6-positive BxPC-3 xenografted tumors over αvβ6-negative HEK 293 tumors. These results illustrate the potential of the described strategies to be clinically used in PET imaging of αvβ6-over-expressing tumors.

A wide variety of carrier systems have been described to achieve tumor-specific therapeutic effects via integrin targeting. The principal success of this strategy is evidenced by two main observations – the dosage of drug has been usually reduced and an enhanced (and often selective) activity against tumors is achieved. The data obtained from independent studies using different carrier systems are promising and there is therefore hope to bring the targeted delivery methods into practice. However, a number of aspects related to the use of these drug-delivery systems in cancer therapy should be carefully considered.

In the first place, comparative studies between distinct carrier systems are missing. Such studies could provide useful insights on their relative advantages and disadvantages, and help in their further development and optimization. Detailed studies concerning the systemic toxicity and long-term side effects of the drug-delivery vectors in physiological systems are also essential. Another important aspect to optimize the concentration of drugs in cancer therapy would be to evaluate the efficiency of drug uptake with regard to the overall administered dose, but most studies have only rated the efficiency of the targeted systems in comparison to untargeted systems, without mentioning about the concentrations of the drug used. The investigation of the metabolic stability of these systems in gut and liver as well as their bioavailability profile would also be crucial to improve the efficacy of the therapy. Further optimization of such drug formulations could be directed toward new routes of administration, including, though certainly difficult, orally available conjugates.

It should be mentioned that most studies in this field rank the antitumor potency of the targeted systems based on the reduction in tumor volume and size, parameters that will however not entirely assure the success of the therapy. More satisfactory would be to carry out longer experiments to ensure the complete removal of tumors and arrest of resurrections. In this regard, recent findings have suggested that antiangiogenic therapeutics that aim at treating cancer primarily through reduction and control of tumor growth, may, in some cases, indirectly promote cancer invasiveness and metastasis (89, 90). This ultimately alarms development of targeted therapies which can inhibit multiple cellular functions and affecting not only cell survival in situ but also mechanisms involved in the promotion and progression of metastasis. Further investigations on this matter should include the study of targeted therapy on early stage and late stage tumors, and the effect (if any) of these strategies in the development of drug resistance mechanisms by some tumors. Additionally, treatment of cancer often necessitates a combination therapy (combination of different therapeutics or therapies). In this respect, it is demanding to study the usage of targeted approaches for delivering multiple drugs or therapies either by a single carrier system or multiple carrier systems. These studies are further pending in literature. Most of the studies on targeted gene delivery have used luciferase model system. Though it is a good analogous system for understanding gene delivery, proper experimental gene therapy studies aimed to treat cancers are to be extensively studied.

The choice of an optimal integrin ligand is another aspect of paramount importance in the design of integrin-based targeted therapies in cancer. This will depend on the differential pattern of integrin expression in cancer cell types and the biological activity and selectivity profiles of the targeting ligands. Many applications have used linear or cyclic RGD peptides to deliver drugs or nucleotides to tumors. Most of these peptides are active for αvβ3; however, it is often ignored that these ligands may target other integrin subtypes as well. This might not be relevant as long as simplified cellular or experimental animal models are investigated. However, it may raise safety concerns if clinical applications in humans are to be envisaged. E.g., the habitually used peptide – c(RGDfX), developed in our group long ago (25, 30), has about 1 nM affinity for αvβ3 and is certainly selective against αIIbβ3 (low affinity for the platelet receptor). Nonetheless, the compound also has affinity in the low nanomolar range for αvβ5 (7.6 nM) and α5β1 (15 nM) (73). Thus, the use of c(RGDfX) might not always provide enough selectivity to distinguish between distinct cell types. In this regard, our group has recently developed (91, 92) and functionalized (93, 94) peptidomimetics which can clearly discriminate between αvβ3 and α5β1. Application of such single integrin subtype selective ligands will enable a selective and controlled delivery of drugs to tumors, taking advantage of the distinct patterns of integrin expression found for each cancer type.

It is on the basis of these considerations that targeted therapy with integrin ligands be translated into clinical studies, and be demonstrated whether such strategy will result in a clear benefit for cancer patients.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.