Besides conventional therapies for cancer, such as surgery, radiation, chemotherapy and molecularly targeted therapy, recent exciting progress in immunotherapy has ushered in a new era of cancer treatment [1]. Immunotherapy usually targets the immune system to provoke a systemic response against cancer cells and has shown unprecedented and durable efficacy in advanced cancer patients [2]. Increasing evidence suggests that tumors mainly use immune checkpoints as an important mechanism to escape antitumor immune responses [3]. Among them, programmed cell death protein 1 (PD-1) and its ligand, PD-1 ligand (PD-L1 or B7H1), form the most exciting pathway, as far as cancer therapy methods are concerned [4]. A variety of cancers show increased expression of PD-L1 and currently PD-L1 status is the only approved diagnostic biomarker for initiating immunotherapy [5]. To date, PD-1/PD-L1 antibodies have been translated into survival benefits and are broadly used in practice for treatment of several cancers [6]. However, the response rates in many cancers are not impressive with a large proportion of non-responders, accompanied by immune-related adverse events in some patients receiving PD-1/PD-L1 − targeted therapeutics [7, 8]. Thus, there is a need to stratify patients who might benefit from immune checkpoint inhibitors.

Currently, immunohistochemistry (IHC) is an existing companion diagnostic to guide patient selection, therapeutic monitoring, and improve overall response rates [9, 10]. However, tissue-based methods are invasive and prone to sampling errors. Moreover, Biopsy-based IHC often fails to reveal intra-tumor and inter-tumor heterogeneity [11, 12]. Although PD-L1 testing is now established as a logical and commonly examined biomarker for predicting the responses, challenges still remain. 1) Heterogeneity is the major obstacle to achieve consistency in PD-L1 testing. 2) PD-L1 is expressed transiently on various cell types and rarely only on cancer cells. 3) PD-L1 expression can change spontaneously and evolve when tumor cells encounter immune effector cells and may be influenced by previous treatments such as surgical resection, chemotherapy, or targeted drug therapy. 4) It is not established which scoring system (e.g., TPS and CPS scoring method) is optimal in clinical settings. 5) Differential expression of PD-L1 between primary and metastatic sites indicate a single PD-L1 test is not sufficient [13, 14]. Overall, there remains a great need to develop a unified standard for defining PD-L1 positivity and internationally guiding better clinical practice in larger patient populations.

Molecular imaging, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), and fluorescence molecular imaging (FMI), but especially the former two modalities, can overcome the inherent limitations of IHC and achieve non-invasive visualization and dynamic quantification across the body [15, 16]. Tremendous progress has been made in molecular imaging of the PD-1/PD-L1 axis, and in addition, tracers derived from full-length antibodies and antibody mimetics have been developed, including but not limited to, atezolizumab-based tracers [15, 17], 18F-BMS-986192 [18], and 18F-anti-PD-L1 Adnectin [19], etc.) [20]. However, optical imaging with FMI has limitations related to autofluorescence and imaging depth limitation [21]. Imaging with PET or SPECT may also present certain limitations associated with ionization radiation and administered dosage [22]. Consequently, alternative methods for sensitive and quantitative assessment of PD-L1 are urgent to guide therapy decisions. Magnetic particle imaging (MPI) is an emerging real-time imaging modality that directly detects superparamagnetic iron oxide nanoparticle (SPION)-based tracers in biological tissue with superb contrast, high sensitivity with linear quantitation, no ionizing radiation, and no depth attenuation. [23, 24]. SPIONs are a family of contrast agents in magnetic resonance imaging (MRI) as they provide an excellent nanoplatform for imaging various tumors, and they are currently being investigated for clinical use based on MPI [25, 26]. Targeted SPIONs conjugated with specific ligands, including antibodies, peptides, and nucleotides, are considered ideal imaging probes with high biocompatibility and low toxicity [27, 28]. Among them, Ferucarbotran/Resovist is still the standard of reference in MPI and one of the most ideal SPIONs commercially available [29]. Although still in its nascent stage, MPI has already gained attention and shown clinical translational potential in imaging cancer and guiding therapy [30]. Moreover, simulation studies on the design of a functional MPI brain imager proved promising for human scale systems [31]. However, no prior study has explored the possibility of MPI in monitoring PD-L1.

In this issue of European Journal of Nuclear Medicine and Molecular Imaging, Peng et al. reported a new imaging probe (MFNPs-aPDL1) with MPI/FMI dual-modality to dynamically visualize and quantify whole-body PD-L1 in different tumor xenografts with varying PD-L1 expression levels [32]. The fluorescence confocal microscopy imaging data showed the low-toxic MFNPs-aPDL1 nanoprobe can specifically bind to the PD-L1 on CT26 tumor cells and PD-L1 constitutive expressing CHO-PDL1 tumor cells. Moreover, targeted binding of MFNPs-aPDL1 is correlated with PD-L1 expression level. MFNPs-aPDL1 successfully detected CT26 tumors exhibit higher MPI signal and longer retention (especially from 12 − 48 h), confirmed by ex vivo MPI of tumors. FMI with MFNPs-aPDL1 showed specific and targeted signal in CT26 tumor 24 h post-injection, mirroring MPI observation. However, it was not a whole-body detection because of the limited penetration depth of fluorescence. These results suggest that MPI is suitable for in vivo quantitative imaging and characterization of molecular events. To further examine the sensitivity and ability of MFNPs-aPDL1 MPI in quantitatively detecting in vivo PD-L1 expression, the authors constructed tumor-bearing mice with CHO-PDL1 and control CHO lesions. The data showed that MPI signal was significantly higher in the CHO-PDL1 xenografts than in the CHO xenografts, which was also validated by ex vivo MPI. qPCR and western blot data demonstrated specific PD-L1 expression in CHO-PDL1 but not in CHO. Furthermore, the MPI-guided strategy performed in CT26-tumor-bearing mice showed that aPD-L1 + IFN-γ treatment can more effectively inhibit tumor growth, which may provide better therapeutic efficacy and facilitate the clinical optimization of cancer immunotherapy.

This work is of importance since this is the first combination of MPI and a newly developed imaging probe (MFNPs-aPDL1), which indeed exhibited targeted and specific PD-L1 binding. The conjugation of aPD-L1 with magnetic fluorescent hybrid nanoparticles facilitated targeted binding and detection of PD-L1 in the tumor microenvironment. Moreover, to achieve reliable and standardized MPI images, a local maximum point-based threshold expansion image processing algorithm was developed to enable the elimination of the artifacts and optimization of the MPI images, leading to precise delineation of MPI signals for more accurate quantification of the varying PD-L1 levels. In addition, FMI was adopted as a complementary imaging modality, which can validate the in vivo MPI observation. MPI possesses high sensitivity and no image depth limitation and FMI has high spatial resolution. Hence, the combination of MPI with FMI can provide more detailed in vivo characterization of PD-L1 expression. Elevated PD-L1 expression levels in MPI induced by the IFN-γ treatment accompanied with enhanced immunotherapeutic efficacy further demonstrate the feasibility of the imaging approach. In general, the new imaging method is a potential approach for the in vivo characterization of immune checkpoint molecule expression.

Meanwhile, considerable effort has been made in recent years to detect biomarkers in vivo through two complementary FMI and MPI techniques. For example, a dual-modality molecular imaging probe (SPIONs@A-T NPs) manifested the feasibility of diagnostic evaluation to distinguish lymph node metastases from normal lymph nodes in a preclinical breast cancer model [33]. In this work, the unique ATP-responsive fluorescence design allows FMI to specifically light up the lymphatic metastasis, and MPI can obviate the limitation of imaging depth of FMI and ensure sensitive imaging of metastatic tumor cells. Another study described a plectin-1 peptide (PTP)-targeted conjugation (PTP-Fe3O4-IRDye800CW) which was applied to both the subcutaneous and orthotopic pancreatic ductal adenocarcinoma (PDAC) mice models for FMI/MPI/MRI multimodality imaging [28]. The authors found that this multimodality imaging probe showed more precise detection of PDAC with outstanding targeting ability and exhibited good safety. In another example, a multimodal imaging agent (5HFeC NPs) was designed and used for active myeloperoxidase (MPO) targeting [34]. The in vivo-specific targeting ability of 5HFeC NPs was evaluated in an established MPO-implanted mouse model using FMI and MPI, which showed 5HFeC NPs possessed high contrast enhancement in plaques of the abdominal aorta. These studies have demonstrated the great importance of MPI in precise detection of biomarkers. However, MPI shows low spatial resolution and no anatomical information. Thus, we expect to see follow-up studies that develop multimodal imaging, which can integrate the respective complementary advantages of different imaging modalities (MPI, FMI, and MRI, etc.) and provide more accurate and deeper anatomical details for precise imaging.

Now that this pioneering study has been carried out, a few aspects of MPI-guided immunotherapy may deserve to be further investigated in the future. First, compared with traditional in vivo studies of cancer therapeutics, the study of checkpoint blockers requires a focus on host–tumor interactions, specifically the immune–tumor interface. As there are inherent differences between human and murine immune systems [35], further investigation of MPI-guided immunotherapy in human or humanized xenograft models will be warranted and studies investigating predictive algorithms built on tumor-host interaction data could better stratify patients for immunotherapy. In addition, the anti-tumor effects of MPI in various other solid tumor types also need to be validated. Second, the selection of potential MPI tracers is currently limited, which heavily depends on advances in nanoparticle fabrication and theoretical models of nanoscale magnetization [36]. Also, there is not yet a comprehensive understanding of the underlying physics of MPI tracer response. As such, future concerted theoretical and experimental work is crucial to gain an in-depth understanding of magnetic relaxation processes that govern MPI tracers and the development of optimized MPI tracers.