Metastasis of cancer is the process by which the primary tumor spreads from the original site to other parts of the body. Palliative care is the most common treatment option available for patients with metastatic cancer, but most patients eventually succumb to the disease due to the lack of therapeutics that can specifically target disseminated tumors [1]. Currently, diagnosis of metastasis primarily relies on approaches such as computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), X-ray, blood tests for metastasis markers, ultrasound, and biopsy analyses depending on the cancer type. Even when metastasized tumors are resected, there is a high chance of relapse because of incomplete removal of lesions. Patients with brain metastasis have particularly poor survivability partly due to the blood-brain barrier restricting therapy delivery [2]. Developing novel strategies to help in early detection and real-time guidance in resection of metastatic tumors would provide better chance of survival for patients with advanced cancer [3]. Treating cancer metastasis is also immensely challenging due to tumor heterogeneity and the diversity of cancer subtypes, which limit opportunities for developing universally applicable approaches. Current treatment options available for patients with metastasis include surgery, proton therapy, immunotherapy, chemotherapy, radiation, or immunotherapy. For many cancers, available treatment approaches are not adequate to completely eradicate metastatic cells, which have a tendency to undergo transformation to progressively aggressive variants. There is much effort put into designing improved targeted imaging probes and therapeutic modalities to efficiently diagnose and treat cancer metastasis [4,5]. Examples of ongoing efforts in the development of targeted imaging and therapeutic strategies are discussed below.

In a recent study, an orthotopic mouse model of triple-negative breast cancer (TNBC) 4T1 cells grafted into the mammary fat pad of immunocompetent mice was used to screen a phage display library for cyclic combinatorial peptides that home to spontaneous pulmonary metastasis [6]. After intravenous injection of the peptides displayed on phage, metastatic cells were sorted by fluorescence-activated cell sorting (FACS) from the lung cell suspension and enriched in repeated selection rounds in mice with metastasis. This led to recovery of a number of peptide sequences that were enriched. Two lead Breast Lung Metastasis-homing Peptides (BLMP) peptides, CLRHSSKIC (BLMP5) and CRAGVGRGC (BLMP6), were tested and validated. Both peptides were confirmed to bind murine and human cells derived from breast carcinoma in culture. Homing of peptides was validated in vivo by injecting peptide-phage clones into mice and measuring their tissue localization. Metastasis homing of BLMP6 conjugated with a Cy3 fluorophore was also validated. Tissue analysis of primary tumors revealed that these peptides have the affinity for receptors expressed in metastasis and some primary tumor cells, Colocalization with a mesenchymal N-cadherin−expressing cells indicated that both peptides are selective for cancer cells that have undergone the epithelial-to-mesenchymal transition (EMT), a process linked with metastatic dissemination. To assess their potential for in vivo imaging, these peptides were first labeled with gallium-67 (⁶⁷Ga) to quantitatively measure tissue distribution. Ex vivo tissue analysis with ⁶⁷Ga-BLMP6 showed selective uptake in experimental lung metastases formed by intravenously injected B16F10 melanoma cells. In contrast, ⁶⁷Ga-BLMP5 displayed relatively more uptake in primary tumors than ⁶⁷Ga-BLMP6. Importantly, elevated BLMP6 signal was seen in kidneys that had renal metastases, indicating that the peptide binds to a receptor expressed on metastatic cells irrespective of the secondary organ. Additionally, PET imaging with the ⁶⁸Ga-labeled analogs showed that BLMP6 also homes to lung metastases formed by B16F10 melanoma cells. This finding indicated that the peptide binds to a receptor expressed on metastatic cells irrespective of tumor origin.

To test the utility of the BLMP peptides as delivery systems for therapeutic payloads, they were synthesized as conjugates containing an apoptosis-inducing moiety [6]. To measure uptake by cancer cells, these hunter-killer (HK) peptides were biotinylated and labeled with Cy3-strepavidin. The analysis confirmed the HK-BLMP6 had specificity for cancer cells. Cytotoxicity assays demonstrated that HK-BLMP6 peptide has a higher efficiency in killing cancer cells than HK-BLMP5, providing further evidence that the two peptides bind to different receptors. Upon treatment of mice grafted with 4T1 cells, it was observed that HK-BLMP6 reduces metastatic burden, while having little effect on primary tumors [6]. Further validation of BLMP peptide derivatives will be needed to assess their clinical potential. Identification of receptors recognized by these peptides may pave the way for improved targeted imaging probes and antibody-based therapeutics.

In addition to peptides, other platforms have shown a potential in metastasis imaging. For example, NP-Q-NO₂ was reported as an activable nanoprobe that is used for multispectral optoacoustic tomography (MSOT) that activates and produces near-infrared (NIR) fluorescence and optoacoustic signals by aggregation-induced emission in the presence of nitroreductase [7].

In another nanoparticle-based approach, CdSe/ZnS quantum dots (QD) linked with alpha-fetoprotein (AFP) monoclonal antibody (mAb) were used. The AFP mAB linked to the quantum dots (QD-AFP-AB), were shown to bind to human hepatocellular carcinoma (HCC) cells. These QD probes were shown to be tolerable in vivo and enable targeting of both primary tumor and metastases in hepatocellular carcinoma [8].

Lapatinib, a tyrosine kinase inhibitor binding to epithelial growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2), conjugated to a fluorescent dye S0456, was also used for metastasis detection [9]. In that study, S0456-HPPA-NHS conjugated with Lapatinib (LP-S) was validated by using oral squamous cell carcinoma cell (OSCC) lines with varying degree of EGFR expression in vitro. An OSCC cell line CAL27 was used for in vivo comparison of LP-S with indocyanine green (ICG), which is clinically approved for NIR imaging. LP-S was shown to be a better probe for real time NIR imaging, and subsequent evaluation, demonstrated the effectiveness of LP-S for targeting tumor metastases in lymph nodes and its potential for fluorescence-guided surgery [9].

Tumor metastasis targeting peptide 1 (TMTP1) is another peptide-based agent that has been preclinically evaluated in an HCC model of SMMC-7721 cells grafted subcutaneously into immunodeficient mice [10]. TMTP1 is a peptide with 5 amino acid (NVVRQ) chain, which was selected from a FliTrx bacterial peptide library and was found to bind highly metastatic cancer cell lines. The peptide was radiolabeled with lutetium (¹⁷⁷Lu), dodecane tetraacetic acid (DOTA) and Evan’s Blue (EB) to design [¹⁷⁷Lu]Lu-DOTA-EB-TMTP1, a tumor-targeting peptide that can specifically deliver cell-killing radiation to metastatic cancer cells. When evaluated in vivo, this theranostic compound exhibited high retention in HCC tumors, which led to potent antitumor activity and significantly higher survival in treated mice [10].

Antibodies, which tend to have higher affinity and specificity than small peptides, have also been tested for therapy delivery to metastases. After screening against proteins of the cadherin family for targets which are responsible for maintaining epithelial structures and adhesive properties in cells, 23C6 was selected as an antibody with dual affinity against epithelial E-cadherin (CDH1) and mesenchymal OB-cadherin (CDH11). In vivo experiments were performed using 4T1 cells in an immunocompetent mouse model, as well as TNBC MDA-MB-231 cells and pancreatic ductal adenocarcinoma cells (PDAC9) for xenograft models in immunodeficient mice. In models used, 23C6 was shown to suppress metastatic burden without affecting the progression of primary tumors. Importantly, this antibody showed no toxicity in healthy organs [11].

Various other approaches have been attempted for therapeutic metastasis targeting. One study used emtansine, a highly potent microtubule inhibitor that becomes active once it is internalized by a cancer cell, encapsulated in pH-sensitive liposomes [12]. These emtansine liposomes were coated with macrophage membranes with high expression of α4 and β1 integrins, which were extracted from the murine macrophage cell line RAW 264.7. In vivo evaluation of the membrane-decorated emtansine liposome (MEL) showed higher therapeutic efficacy when tested against normal emtansine encapsulated liposomes and the efficacy of the MEL was lower when the α4β1 integrin markers were blocked [12].

Another strategy used a mechanoresponsive cell system (MRCS) to selectively detect and treat cancer metastasis [13]. Mesenchymal stem cells (MSC) were used to develop an MRCS that use Yes-associated proteins (YAP)/ transcriptional co-activator with PDZ binding motif (TAZ) as an on/off switch to validate the response to matrix stiffness in a breast cancer cell line. The reporters were replaced with cytosine deaminase (CD) creating MRCS-CD, which locally activates the prodrug 5-fluorocytosine (5-FC) in response to increased stiffness and, hence, enables tumor cell killing. Homing of the MRCs was validated in vivo by inducing lung metastasis of MDA-MB-231 cells in immunocompromised mice, and treatment with the MRCS-CD confirmed specific targeting of metastatic cells, which were induced to undergo apoptosis [13].

While notable progress is being made, the heterogeneity of metastases continues to pose formidable challenges. The nuances of specific metastatic sites and unique features of metastases derived from distinct types of cancer at different stages of their growth will be important to consider in evaluating newly developed agents. Identification of biomarkers and cell surface receptors specific for metastatic cells will be instrumental for the development of targeted therapeutics and imaging probes. Targets of non-cancer cells marking metastases could also be considered. Future efforts can utilize established platform technologies that have proven to be clinically effective in targeting primary tumors [14]. The demonstrated ability to target metastatic cells with a variety of delivery systems, ranging from low molecular peptides to biologics, particle-based agents, and gene delivery vectors, (viral and non-viral), outlines promising opportunities for interdisciplinary approaches and translational research.

This work was supported by grants W81XWH-22-1-1002 and W81XWH-22-1-1004 from the US Department of Defense and in part by Bovay Foundation and Levy-Longenbaugh Fund.

The authors declare no conflict of interest.