Invadopodia: a new therapeutic target to block cancer metastasis

Abstract

Cancer cells become dangerous when they acquire the ability to invade through physical barriers in the body and disseminate to distant sites. Recent evidence has demonstrated that cancer cells utilize specialized structures called invadopodia, unique protrusions that concentrate proteases such as matrix metalloproteinases (MMPs), to escape blood vessels during the process of extravasation. Perhaps most exciting is the fact that inhibition of invadopodia through genetic or pharmacological means reduces the ability of cancer cells to extravasate and effectively blocks metastasis. This opens the door for the development of novel therapies targeting invadopodia and cancer metastasis.

Keywords:

• cancer
• cell migration
• intravital imaging
• invadopodia
• metastasis

Metastasis is responsible for the majority of human cancer deaths; yet, no therapies exist that target this clinically relevant process. At a basic level, metastasis comprises a cascade of sequential events that include local invasion, intravasation, survival in the circulation, extravasation and colonization. Of these, extravasation has been somewhat a neglected child of the metastatic cascade. This is due, in part, to difficulties in modeling it effectively. Circulating cancer cells lodge in small capillaries and extravasate at random, deep within tissues, which precludes their direct visualization and study [1]. For this reason, our knowledge of cancer cell extravasation has been lacking, and is limited by the use of overly simplistic in vitro models or the analysis of fixed tissue samples. Recently, we have shown for the first time that extravasation is an active process that requires the coordination of invadopodia – highly specialized structures unique to invasive cancer cells [2]. To study extravasation in a relevant in vivo environment, we developed a novel animal model based on ex ovo cultured avian embryos that allows simultaneous quantitative imaging of both extravasating tumor cells and adjacent vasculature at high resolution [3–6]. The ex ovo chorioallantoic membrane model is ideal for intravital imaging of cancer due to its accessibility, transparency and high level of vascularization [3–6]. Furthermore, multi-color high-resolution imaging can be performed for long periods of time (>36 h), as the model does not require surgical intervention, host immunosuppression or demanding culture conditions [7,8]. Our recently published work, which builds upon previous studies using mouse and zebrafish models, reveals the active and mechanistically complex nature of extravasation and highlights its potential as a therapeutic target [2].

Our intravital imaging experiments revealed that circulating cancer cells lodge within the capillaries and begin to probe their environment using rounded, short-lived protrusions while migrating within the vasculature using a morphologically amoeboid mechanism. In contrast to immune cells, we found that cancer cells do not roll, but exclusively employ active migration, reinforcing the idea that cancer and immune cells use distinct extravasation mechanisms [1,2,9]. Within 4–6 h after arriving in the capillaries, cancer cells initiate the formation of a single dominant protrusion that crosses the endothelial layer into the extravascular stroma. We confirmed that these dominant protrusions are indeed invadopodia, evidenced by the localization of invadopodia-specific Tks4 and Tks5 adaptor proteins, the actin cytoskeleton branching protein cortactin and the extracellular matrix-degrading MT1-matrix metalloproteinases (MMP) protease. Inhibition of Tks4, Tks5 or cortactin using siRNA abrogates the formation of invadopodia and effectively blocks cancer cell extravasation. Furthermore, pharmacological blockade of Src, a major tyrosine kinase regulating invadopodia formation and tumor cell invasion, also blocks the formation of invadopodia in intravascular cancer cells and prevents their extravasation [10]. The utilization of invadopodia by extravasating cancer cells appears to be a general feature of cancer, as we observed them in breast (MDA-MB-231), fibrosarcoma (HT1080) and squamous carcinoma (HEp3) cells.

The identification of invadopodia as key components of the extravasation machinery represents a promising new avenue to target metastasis therapeutically. Invadopodia biogenesis relies on well-characterized cell signaling pathways that can be successfully targeted by existing therapeutics. Specific inhibitors targeting key invadopodia signaling hubs (Src, Arg/c-Abl/MMPs) currently exist [10–12]. Indeed, we showed that relatively low sub-toxic doses of the Src inhibitor Saracatinib effectively inhibit invadopodia formation in cancer cells, preventing them from escaping the lung vasculature and establishing distant metastases. Also, the unique structural components of invadopodia present another appealing therapeutic target. Invadopodia assembly consists of: precisely coordinated vesicle transport (controlled by kinesin motors); actin polymerization and branching (controlled by Arp2/3/cofilin/cortactin/Tks5); membrane cell adhesion (controlled by integrins); and localized extracellular matrix proteolysis (controlled by Tks4/MMPs/ADAM10). As we have shown, inhibition of several of these components (cortactin, Tks4, Tks5) results in a twofold reduction in cancer cell metastasis [2]. While the targeting of invadopodia-specific vesicle transport machinery or components of cell adhesion is yet to be investigated, it is likely that their function is required for successful cancer cell extravasation and metastasis. Targeting multiple components of invadopodia structure and function may allow for still greater efficacy.

The location of extravasating cancer cells is ideal from a drug delivery standpoint. While the delivery of therapeutic antibodies and biologics into solid tumors at sufficient doses can be a significant problem [13], extravasating cancer cells are not protected by physical barriers such as the vascular wall or tissue stroma and, therefore, can be readily targeted. This presents an opportunity to preferentially target cancer cells in the vascular compartment, as lower doses should achieve a desirable therapeutic effect in the bloodstream. Targeting MMPs, for example, on intravascular cancer cells to block cancer extravasation should require a lower dose than those in solid tumors, which should reduce associated side effects [10,13].

The development of a highly accessible in vivo model for extravasation opens the door for further interrogation of the basic biology underlying metastasis. Answering several pressing questions should further promote design of metastasis-targeting therapies. How different is structural organization of invadopodia in vivo from that previously described in vitro? Does invadopodia precursor formation in vivo require integrin-mediated adhesion or it relays on the stiffness of the local vascular wall? What are the similarities and differences between cancer cell invadopodia that are assembled during extravasation, intravasation and invasion? Are the mechanisms that control immune cell extravasation distinct from those that control extravasation of cancer cells? Our work suggests that there may be a significant difference, which opens an exciting possibility of selectively targeting the extravasation of cancer cells while preserving the immune system of cancer patients.

It is important to consider the potential therapeutic window of extravasation-targeting approaches. First, these types of therapies should benefit those with aggressive metastatic cancers, positive lymph nodes and/or already documented metastasis by preventing further dissemination. Second, these approaches may be of benefit to those undergoing removal of the primary tumor or tumor biopsy due to the induced entry of tumor cells into the circulation.

Taken together, this work opens up unique possibilities for further discovery in basic, translational and clinical research. The confirmation that key in vitro invasion machinery is indeed responsible for one of the major metastatic steps in vivo will drive the development of new anti-metastatic therapeutics. Furthermore, the establishment and validation of a new in vivo model that allows for robust, quantitative imaging of cancer cell metastasis paves the way for new genetic and pharmacological approaches that target in this clinically important process.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.