*Article* **Magnetic-Guided Axillary UltraSound (MagUS) Sentinel Lymph Node Biopsy and Mapping in Patients with Early Breast Cancer. A Phase 2, Single-Arm Prospective Clinical Trial**

**Allan Jazrawi 1,2, Eirini Pantiora 3,4, Shahin Abdsaleh 3,5, Daniel Vasiliu Bacovia 6, Staffan Eriksson 1,2, Henrik Leonhardt 7, Fredrik Wärnberg 3,4,8 and Andreas Karakatsanis 3,4,\***


**Simple Summary:** Superparamagnetic iron oxide nanoparticles (SPIO) have been shown to identify sentinel lymph nodes (SLNs) in patients with breast cancer. This study investigated whether a minimally invasive approach with MRI-LG after SPIO injection in the breast followed by a magnetic guided axillary ultrasound and core biopsy of the SLN (MagUS) could accurately stage the axilla. The study included not only patients planned for primary surgery but also patients with recurrent cancer after previous surgery, but also patients scheduled for neoadjuvant treatment (NAT). The latter underwent minimally invasive SLNB prior to treatment and had their SLN clipped; surgery in the axilla was performed after NAT. In 79 included patients, MagUS detected all patients with macrometastasis and performed comparably with surgical sentinel lymph node dissection (SLND). It also allowed for marking of the SLN in patients planned for PST and enabled tailored decision making in breast cancer recurrence.

**Abstract:** Lymph Node Dissection (SLND) is standard of care for diagnosing sentinel lymph node (SLN) status in patients with early breast cancer. Study aim was to determine whether the combination of Superparamagnetic iron oxide nanoparticles (SPIO) MRI-lymphography (MRI-LG) and a Magnetic-guided Axillary UltraSound (MagUS) with biopsy can allow for minimally invasive, axillary evaluation to de-escalate surgery. Patients were injected with 2 mL of SPIO and underwent MRI-LG for SN mapping. Thereafter MagUS and core needle biopsy (CNB) were performed. Patients planned for neoadjuvant treatment, the SLN was clipped and SLND was performed after neoadjuvant with the addition of isotope. During surgery, SLNs were controlled for signs of previous biopsy or clip. The primary endpoint was MagUS SLN detection rate, defined as successful SLN detection of at least one SLN of those retrieved in SLND. In 79 patients, 48 underwent upfront surgery, 12 received neoadjuvant and 19 had recurrent cancer. MagUS traced the SLN in all upfront and neoadjuvant cases, detecting all patients with macrometastases (*n* = 10). MagUS missed only one micrometastasis, outperforming baseline axillary ultrasound AUS (AUC: 0.950 vs. 0.508, *p* < 0.001) and showing no discordance to SLND (*p* = 1.000). MagUS provides the niche for minimally invasive axillary mapping that can reduce diagnostic surgery.

**Citation:** Jazrawi, A.; Pantiora, E.; Abdsaleh, S.; Bacovia, D.V.; Eriksson, S.; Leonhardt, H.; Wärnberg, F.; Karakatsanis, A. Magnetic-Guided Axillary UltraSound (MagUS) Sentinel Lymph Node Biopsy and Mapping in Patients with Early Breast Cancer. A Phase 2, Single-Arm Prospective Clinical Trial. *Cancers* **2021**, *13*, 4285. https://doi.org/ 10.3390/cancers13174285

Academic Editors: Moriaki Kusakabe, Akihiro Kuwahata and Katja Pinker-Domenig

Received: 22 June 2021 Accepted: 20 August 2021 Published: 25 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Keywords:** sentinel lymph node biopsy; breast cancer; superparamagnetic iron oxide; magnetic tracer; sentinel lymph node

#### **1. Introduction**

Primary tumor biology and axillary status guide therapeutic decisions in breast cancer treatment [1,2]. Sentinel Lymph Node Dissection (SLND) is considered the standard method of axillary staging, both in upfront surgery as well as after neoadjuvant treatment (NAT) [3–8].

Preoperative identification of patients with a negative SLN, or low-volume axillary disease that does not warrant further surgery, but guides therapeutic decisions, may allow for tailored approaches avoiding upfront SLND [6,9,10]. In patients scheduled for NAT, identifying those with a true negative axilla, but also those with low-volume disease, as de-escalation of axillary surgery after conversion from cN1 to cN0, could be safely attempted. [7,11,12].

At the same time, SLND is not an indolent procedure and is related to complications and considerable short- and long-term morbidity [13–16]. Therefore, non- or minimally invasive modalities have been proposed in order to address this problem. All of them are based on the principle of injecting a contrast interstitially in the breast in the same manner as when SLND is performed. The contrast will then be taken up by the lymphatics and reach the SLNs and will subsequently be visualized by a radiological modality. Previously, several methods such as single-photon emission computed tomography (SPECT), tridimensional computed tomography lymphography (3D-CTLG) or contrast enhanced ultrasound with microbubbles (CEUS) have been evaluated as alternatives to surgery [17–19]. Most of these have shown promising results, but larger studies are missing and, complicated logistics, need for access to nuclear medicine facilities and demanding learning curves are restricting their introduction into clinical practice.

Superparamagnetic iron oxide nanoparticles (SPIO) are used as a SLND tracer with comparable detection to the combination of radioisotope and blue dye, as shown in previous studies [20,21]. Additionally, when SPIO is injected in the breast, it can identify SLNs in axillary magnetic resonance imaging lymphography (MRI-LG) [22]. At the same time, SPIO yields the benefit that it resides in the tissue for a prolonged period of time without migrating to higher lymph node echelons and, thus, allows for the identification for SLNs during a much wider timeframe [23]. In this manner the SLNs that are identified during surgery should be visible in an MRI and, at the same time, transcutaneous signal detected by a magnetic probe, as in surgery, should be able to guide the axillary ultrasound to allow for transcutaneous identification and biopsy of the SLNs. Such a concept would have the perceived advantages of combining and tailoring modalities and at the same time, allowing for preoperative work up in a timeframe wider than the short halftime of Tc99 used for SPECT or that in the case of CEUS [19,24].

The development of an integrated technique bridging non-invasive and minimally invasive procedures for enhancement of the standard, axillary ultrasound-based diagnostic work-up is highly relevant [23–25]. The aim of this study was to determine whether the preoperative work-up with SPIO MRI-LG and Magnetic-guided Axillary UltraSound (MagUS), can accurately localize SLNs and predict SLN status and whether such a technique has the potential of replacing SLN surgery in the future.

#### **2. Methods**

#### *2.1. Patients*

Adult patients with clinically and ultrasound node-negative early breast cancer (cN0) planned for SLND at Uppsala University Hospital, from September 2017 to December 2020, were enrolled in the study after written informed consent. Patients with hypersensitivity to dextran compounds or SPIO, iron overload disease or planned for NAT and monitored

with breast MRI for tumor response, were excluded. If a diagnostic breast MRI was needed, it was performed separately, before SPIO injection and axillary MRI-LG. The study was approved by the Regional Ethics Board in Uppsala (DNR 2016/385).

#### *2.2. MRI-LG*

Patients were injected peritumorally in the breast with 2 mL of SPIO (Magtrace®, Endomag., Cambridge, UK) and underwent MRI-LG one to 14 days after the injection. MRI-LG was performed with the patient in a supine position and adduction of the ipsilateral arm. The examination was performed without iv-contrast and took ca 8 min to complete. In cases of previous breast and axillary surgery or parasternal cancers, the contralateral axilla was also included in the MRI-LG to identify aberrant lymphatic outflow [26]. The MRI images were obtained using a 1,5-T and 3-T system (Philips®, Amsterdam, The Netherlands) with T2W cor, T2\* tra and T2\* cor sequences. Any lymph node with SPIO uptake in a T1 sequence or SPIO related void artifact on T2 sequence was considered a SLN, as previously described imaging was reviewed and the number of identified SLNs was documented [22]. SLN localization was described according to the classification proposed by Clough et al. [27], in relation to the lateral thoracic vein and the second intercostobrachial nerve. SLN metastatic status was assessed according to criteria previously proposed by Motomura et al. [22]; a lymph node was considered non-metastatic if there was a homogenous low intensity signal uptake of SPIO and metastatic if the entire node or a focal area did not show low signal intensity uptake.

#### *2.3. Magnetic Guided Axillary UltraSound (MagUS) and Core Needle Biopsy (CNB)*

After reviewing of MRI-LG, the radiologist performed a second look axillary ultrasound in another session. The examination was focused to the area where the SLNs were identified on MRI (Figure 1). After a primary assessment for lymph nodes, a handheld magnetometer (Sentimag®, Endomag, Cambridge, UK) was used to identify the "pre-incision hotspot" which is the area with the highest magnetic uptake on the skin, and concordance with the MRI localization was registered.

**Figure 1.** (**a**,**b**). Visualization of SLN with MRI before and after SPIO. In an enhancement of the SLN is visualized after injection of SPIO. The red circle visualizes the enhanced SLN after the injection of SPIO.

Subsequently, the identified lymph node(s) were assessed, and the percutaneous CNB of the SLN was performed with ultrasound guidance under local anesthesia (Figure 2). The CNB was evaluated for the presence of brown staining and magnetic uptake with the SentiMag probe (Figure 3). If more than one pathological lymph nodes were identified at this stage, the protocol stated that multiple efforts could be performed only after patient

consent; otherwise, if the bioptic material obtained was considered representative and adequate, only the most prominent node was biopsied. Standard histopathologic analyses to assess metastasis was also performed, including verification of SPIO presence in the SLN. In patients undergoing NAT, the SLN was clipped simultaneously after the CNB, at the same session. When CNB was completed, the area was scanned for bleeding.

**Figure 2.** (**a**,**b**). MagUS with the SLN visualized in the red circle (**left**). Magnetic probe localizes the magnetic "hotspot" and after that CNB is performed (**right**). Monitor width 3.9 cm.

**Figure 3.** MagUS SLN-biopsy specimen (size 1 cm).

The study protocol ruled that the first five patients would undergo axillary MRI-LG before and after SPIO administration, and that MagUS and CNB was performed in the operation theatre, after the induction of anesthesia and right before surgery. In cases of recurrent breast cancer with aberrant SLN localization on MRI-LG and MagUS, a decision to attempt SLND was made at the multidisciplinary conference and after discussion with the patient. In patients undergoing NAT, a new axillary MRI-LG was performed after NAT, with no subsequent SPIO injection to see whether SPIO uptake in the SLNs was still visible. The number and localization of SLNs on MRI images was documented and axillary transcutaneous SentiMag signal was recorded. During subsequent SLND, concomitant radioisotope injection was administered and during surgery we registered which SLNs were magnetic, radioactive or both as well as the signal of the clipped node with both tracers.

#### *2.4. Surgery and Specimen Pathology*

During surgery, SLND was performed and the retrieved SLNDs were controlled macroscopically and microscopically for signs of previous biopsy, hematoma or the presence of clip, if placed. Standard pathology of the SLN specimen served as a reference to the microscopical examination of the CNB.

The entire MagUs flowchart is summarized in Figure 4.


**Figure 4.** Flowchart showing the MagUS process.

#### *2.5. Trial Design and Study Endpoints*

To assess whether the MagUS concept has the niche to replace surgical axillary evaluation (SLND), it was necessary to ensure concordance and agreement across the different modalities. With other words, it was necessary to verify that the SLNs identified and retrieved during surgery, were the same lymph nodes visualized on the MRI and the same that were detected by the magnetic probe, identified by the ultrasound and subsequently biopsied with a core needle. The common denominator was the presence of SPIO in the node and how this is demonstrated throughout the different modalities (MRI, MagUS, Surgery). Therefore, the outcome of interest was a minimum agreement in the assessment obtained by the MRI/MagUS with the standard of care, that is surgery. For this, it was clinically relevant to assess if the technique at hand is feasible, before venturing on a large clinical trial. Subsequently, the MagUS trial was conceived as a single stage phase 2 trial following the A'Hern's design [28]. For a one-sided test a type one error a = 0.025 and 80% power, a sample size of 75 or more was required between a maximum futility proportion of 95% (corresponding to the proportion of successful detection above which the method can be further considered) and a minimum efficacy of proportion of 85% (corresponding to the proportion of successful detection under which, the method should not warrant further investigation).

The primary endpoint was determination of the MagUS SLN detection rate, defined as successful SLN detection of at least one SLN of those retrieved in the following SLND. Secondary endpoints were false-negative rate (FNR) of the MagUS technique, defined as no diagnosis of SLN metastasis (index test = negative) but presence of metastases by histopathology in any of the retrieved SLNs (reference test = positive) and overall accuracy, sensitivity, specificity and positive and negative predictive value (PPV, NPV).

Another aim of the study was to determine whether the MagUS technique could improve preoperative workup accuracy. For this, discordance in axillary evaluation from baseline clinical and ultrasonographical assessment was assessed.

Subgroup analyses were carried out to review the role of each component of the MagUS technique (MRI-LG. MagUS and MagUS core biopsy) and their potential role in tailored axillary mapping and inform on a future phase 3 trial.

The manuscript was prepared according to the Standards for the Reporting of Diagnostic Accuracy Studies (STARD) statement [29]. Descriptive statistics were performed by means of median (range) for continuous variables. Subsequently, non-parametric tests were used for comparisons. The McNemar's test was used for the assessment of discordance in paired observations. For diagnostic accuracy statistics, Receiver Operating Characteristics (ROC) curves were constructed and the area under the curve (AUC) is provided. Effect sizes are provided with 95% confidence intervals (95% CI). Data analyses were performed using SPSS (V 26.0. IBM Corp, Armonk, NY, USA) and Stata®, version 16 (StataCorp LP, College Station, TX, USA).

#### **3. Results**

The study is summarized in (Figure 5) and patient characteristics are presented in Table 1. In a total of 79 patients, 48 had early breast cancer and underwent upfront surgery, 12 underwent NAT and 19 had recurrent breast cancer after previous breast and axillary surgery.

**Figure 5.** STARD flow diagram. \* MRI\_LG: Magnetic resonance imaging Lymphography. SLND: Sentinel Lymph Node Dissection.

**Table 1.** Patient characteristics.



**Table 1.** *Cont.*

MRI-LG was performed a median of 3 days after SPIO injection (range 1–12) and the MagUS with transcutaneous SLNB ± SLN clipping a median of 3 days (range 1–5) after MRI-LG. In all 73 patients where MagUS SLNB was performed, transcutaneous detection was successful and the SLN was located. Minimally invasive SLNB (MagUS CNB) retrieved lymphatic tissue with magnetic signal on the SentiMag® probe, and the presence of SPIO was confirmed on post-operative histopathology. At surgery, the node with signs of previous biopsy and/or clip was always retrieved. In one case, the lymph node that was biopsied was a non-sentinel node (i.e., ex vivo signal less than 10% of the signal of the SLN with the maximal signal), but the true SLN was just behind it and recovered during SLND.

Metastases on specimen pathology was found in 11 patients (11/73, 15.1%, 95% confidence intervals: 7.8; 25.4). MagUS identified all patients with SLN macrometastases (*n* = 10) and missed only one SLN with a micrometastasis, resulting in a FNR of 8.3% and an overall accuracy of 98.6% (Tables 2 and 3). In terms of diagnostic performance, when compared to the results of surgical pathology, MagUS performed very accurately (AUC: 0.955; 0.865, 1.000, *p* < 0.001) whereas AUS was not predictive at all (AUC: 0.505; 0.410, 0.601, *p* = 0.916).

**Table 2.** Comparison between MagUS and final pathology.


Mc Nemar's test, *p* = 1.000.


**Table 3.** Diagnostic performance of the MagUS technique.

The number of SLNs identified on MRI-LG (median 4, range 1–6) did not differ from the number of SLNs retrieved (median 3, range 1–6) (Wilcoxon signed rank test, *p* = 0.331) with high correlation (Cronbach's Alpha = 0.719; 0.481, 0.848, *p* < 0.001). Additionally, topographic concordance between MRI-LG, MagUS and SLND was 100%. In 63 patients (86%), the nodes were located medial to the lateral thoracic vein and caudal to the intercostobrachial nerve.

In patients receiving NAT, the MagUS allowed for accurate axillary mapping, identification and clipping of the true SLN prior to the initiation of NAT. After the completion of NAT, a median of 130 days (range 86–140) after SPIO injection, the SLNs were still visualized in MRI-LG and were detectable during surgery in all patients. There was excellent correlation between the number of SLNs identified on MRI (median 4, range 2–6) and the magnetic SLNs retrieved (median 3.5, range 1–6) with Cronbach's Alpha = 0.919; 0.699, 0.978, *p* < 0.001.

In patients with local recurrence after previous breast and axillary surgery (*n* = 19), MagUS showed either aberrant lymphatic outflow or no outflow in 9 patients (47.3%), preventing unnecessary ipsilateral axillary exploration. In the remaining 10 patients, both MagUS SLNB and subsequent surgery were successful.

#### **4. Discussion**

In this phase 2 trial, the MagUS technique (MRI-LG and MagUS) provided comparable results in accuracy and FNR with the standard of SLND. It was more accurate than the standard b-mode AUS in preoperatively detecting low-volume axillary disease. In this trial, it was demonstrated that accurate minimally invasive axillary staging can be achieved with a multimodal platform that can be modified to meet tailored patient needs.

SLND is not an indolent procedure and is related to short- and long-term morbidity such as postoperative pain, restricted shoulder range of motion, axillary web syndrome and lymphedema, as suggested in recent meta-analysis [13,14,30]. These findings indicate the need of establishing techniques for less invasive axillary staging that might result in less surgery, less subsequent postoperative complications and a reduction of costs and resources related with surgery [31,32]. Additionally, this MagUS workup can be performed in a wide timeframe and in an outpatient basis, as SPIO resides in the tissue a long period of time.

Recently, the necessity of surgical axillary mapping has been challenged in particular clinical scenarios. Observational data suggest that SLND may be safely omitted in older patients with primary tumors with small size and favorable biology [33–35]. The SOUND randomized trial examines whether a negative AUS can allow for the omission of SLND in patients with unifocal tumors < 2 cm planned for breast conservation and radiotherapy [36]. However, this approach does not take in consideration recent data that suggest that, in women with small tumors that are SLN negative, radiotherapy may be safely omitted nor that diagnosis of low-volume axillary disease, may allow for tailoring of radiotherapy or systemic treatment [6,9,37–39]. The results of the MagUS trial suggest that this technique may be used instead of SLND in selected cases.

It has been shown that 25% of patients considered as cN0 by AUS+/−FNAC will have a positive SLN in surgery. MagUS has the potential to correctly identify this lowvolume axillary disease group, so that further treatment decisions may be tailored but without further axillary surgery, as it has been shown in landmark trials such as AMAROS, ACOSOG Z0011 or, more recently, the RxPonder trial [6,9,40]. Reversely, in women with one positive lymph node on standard AUS, MagUS could assess the volume of axillary disease in a more accurate manner. This is a group that often harbors a higher nodal disease burden [41]. However, other studies show that this is explained by the fact that the sensitivity of AUS + FNAC increases significantly in patients with higher risk for nodal metastasis [42]. At the same time, up to 43.2% of this patient group, will be found to have two or less metastatic nodes, meaning that ALND will have been overtreatment [10]. If MagUS shows that there is only low-volume axillary disease, then the patient may have the possibility to avoid overtreatment and tailor treatment decisions may be made after discussion in the multidisciplinary meeting [43].

Subsequently, MagUS may also address issues regarding axillary staging in the setting of NAT, as it yields the potential of differentiating patients that are clinically node negative from those who are also SLN negative prior to NAT. In this manner, therapeutic decisions regarding the axilla, such as axillary radiotherapy may be better tailored, while its definitive role in this setting remains still to be elucidated [44,45]. At the same time, it may answer whether, in cN positive patients, the metastatic node is a sentinel or if, at presentation, there are non-sentinel metastases, which is suggestive of a higher axillary nodal burden. In this manner, it becomes safer to identify more appropriate potential candidates for axillary conservation post-NAT as recently suggested in the Lucerne toolbox [12]. Moreover, MRI-LG before and after NAT allows for an estimate of the number of SLNs in the axilla. This may address the problem of FNR after NAT, that has been discussed in landmark trials, such as Sentina and ACOSOG Z1071 [46–50]. In these trials FNR was shown to decrease with the removal of ≥3 nodes, including clipped nodes, if such, whereas double tracer was shown to increase detection rate [7,46–50]. In the present study, post-NAT MRI-LG showed uptake in the same SLNs, suggesting that SPIO did not migrate in higher nodal echelons during NAT. Intraoperatively, there was transcutaneous magnetic signal and SLNs were detected in all cases. It may be so that, a MagUS could be repeated after NAT to allow for more focused axillary evaluation, as standard AUS has not shown promising results in this setting [51]. As omission of axillary surgery post neoadjuvant is discussed in several breast cancer subtypes, provided that there is pathologic complete response (PCR) in the breast, MagUS could provide a safer manner to discuss omission of surgery, rather than, in case of non-PCR, performing SLND that will be subject to the risk of false negatives post NAT and after a previous excision in the breast [52,53]. A given restriction is that SPIO injection in the breast impairs the diagnostic accuracy of the MRI, suggesting that the tumor response should be performed with other modalities. Reassuringly, modalities, such as ultrasound and PET-CT have shown comparable accuracy in this setting, without the known risk of false positive findings from the MRI [54–57].

Evaluating nodal status for breast cancer after previous breast and axillary surgery is a challenge. SLN detection rate is lower and aberrant, extra-axillary lymphatic drainage is not unusual [26,58,59]. For this reason, the use of preoperative mapping by means of scintigraphy is recommended in this setting. However, whilst accurate, scintigraphy complicates logistics and this is why it recent data suggest that it is no longer necessary for patients without previous breast or axillary surgery undergoing upfront SLND [60]. MagUS has, in this setting, allowed for tailored patient treatment with flexibility, as the MRI-LG performed preoperatively, allowed in good time to know whether SLND would be attempted on the day of surgery. In this manner, logistics were facilitated, and treatment decisions could be tailored with more precision and accuracy.

The strictly controlled study design allowed for safe results, despite the absence of a control arm. However, this is a phase 2 trial and these results need to be refined and reproduced in a larger scale. Consequently, a phase 3 randomized controlled trial is needed prior to standardization and routine adaptation of the technique instead of surgical SLND. The results suggest that MagUS has the potential to provide a substantial niche to avoid axillary surgery. The cost of surgery is the most substantial, especially if one takes the expenses related with leave of absence, morbidity and complication risks into consideration. Moreover, it is currently unclear whether the technique will always be implemented with the combination of an MRI and MagUS, something which might complicate and prolong the preoperative assessment of the patient. Finally if clinical MRI of the breast is intended, it should be performed first, to be followed by MagUs in another, different session. However, study results suggest that in women without risk factors for decreased ultrasound accuracy and transcutaneous magnetic probe detection (obesity, previous axillary surgery, etc.), MagUS and CNB were sufficient to accurately stage the axilla, suggesting that MRI is probably necessary in a small subgroup of patients (obesity, previous axillary surgery, etc.). This means that tailoring the technique to the specific patient will result in different routines and probably costs. Another substantial benefit is that this can be performed during the period between diagnosis a breast surgery, so that axillary mapping can be performed preoperatively and on an outpatient basis.

MagUS seems to be a method that can allow for alternatives to surgical axillary mapping. It comes to add to the armamentarium of other minimally invasive techniques that have previously been proposed [17,19,22,61] allowing for tailored axillary mapping in breast cancer. Its presumed advantages are the combination of different imaging modalities, together with that SPIO remains in the node a longer period, so as to allow for delayed SLND. Technique refinement and larger studies will allow for elucidation of the possibilities and its role in breast cancer diagnosis and treatment.

#### **5. Conclusions**

MagUS provides the niche for minimally invasive axillary mapping that can meet tailored patient needs and reduce diagnostic surgery. A phase 3 RCT is planned to further evaluate the technique.

**Author Contributions:** Conceptualization, A.K.; methodology, A.K.; software, A.K.; validation, A.K.; formal analysis, A.K.; investigation, A.J., E.P., S.A., D.V.B., F.W., S.E. and A.K.; resources, A.J., E.P., S.A. and D.V.B.; data curation, F.W. and A.K.; writing—original draft preparation, A.J.; writing—review and editing, A.J., E.P., D.V.B., S.A., F.W., H.L., S.E. and A.K.; visualization, A.J.; supervision, F.W., S.E. and A.K.; project administration, F.W. and A.K.; funding acquisition, A.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** Institutional grants by Uppsala University and the Swedish Association for Breast Cancer ("Bröstcancerförbundet", www.brostcancerforbundet.se, ref. KDB1621/17) are acknowledged. Sponsors and funding bodies had no role in study design, data collection, analysis, or interpretation.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Helsinki Declaration of ethical principles involving human subjects and was approved by Uppsala University regional ethical committee (decision number 2017/063).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to ethical considerations and data regulations.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Review* **Application of Magnetic Nanoparticles for Rapid Detection and In Situ Diagnosis in Clinical Oncology**

**Tatsuya Onishi 1,†, Kisyo Mihara 2,†, Sachiko Matsuda 3,\*, Satoshi Sakamoto 4, Akihiro Kuwahata 5, Masaki Sekino 6, Moriaki Kusakabe 7,8, Hiroshi Handa <sup>9</sup> and Yuko Kitagawa <sup>3</sup>**


**Simple Summary:** Screening, monitoring, and diagnostic methods in oncology are a critical part of treatment. The currently used clinical methods have limitations, most notably the time, cost, and special facilities required for radioisotope-based techniques. The use of magnetic nanoparticles is an alternative approach that offers faster analyses with safer materials over a wide range of oncological applications, such as the detection of cancer biomarkers and immunostaining. Furthermore, magnetic nanoparticles, such as superparamagnetic iron oxide nanoparticles, can detect sentinel lymph nodes for breast cancer in a clinical setting, as well as those for gallbladder cancer in animal models within a timeframe that would enable them to be used during surgery with a magnetic probe.

**Abstract:** Screening, monitoring, and diagnosis are critical in oncology treatment. However, there are limitations with the current clinical methods, notably the time, cost, and special facilities required for radioisotope-based methods. An alternative approach, which uses magnetic beads, offers faster analyses with safer materials over a wide range of oncological applications. Magnetic beads have been used to detect extracellular vesicles (EVs) in the serum of pancreatic cancer patients with statistically different EV levels in preoperative, postoperative, and negative control samples. By incorporating fluorescence, magnetic beads have been used to quantitatively measure prostate-specific antigen (PSA), a prostate cancer biomarker, which is sensitive enough even at levels found in healthy patients. Immunostaining has also been incorporated with magnetic beads and compared with conventional immunohistochemical methods to detect lesions; the results suggest that immunostained magnetic beads could be used for pathological diagnosis during surgery. Furthermore, magnetic nanoparticles, such as superparamagnetic iron oxide nanoparticles (SPIONs), can detect sentinel lymph nodes in breast cancer in a clinical setting, as well as those in gallbladder cancer in animal models, in a surgery-applicable timeframe. Ultimately, recent research into the applications of magnetic beads in oncology suggests that the screening, monitoring, and diagnosis of cancers could be improved and made more accessible through the adoption of this technology.

**Citation:** Onishi, T.; Mihara, K.; Matsuda, S.; Sakamoto, S.; Kuwahata, A.; Sekino, M.; Kusakabe, M.; Handa, H.; Kitagawa, Y. Application of Magnetic Nanoparticles for Rapid Detection and In Situ Diagnosis in Clinical Oncology. *Cancers* **2022**, *14*, 364. https://doi.org/10.3390/ cancers14020364

Academic Editor: Manfred Ogris

Received: 14 December 2021 Accepted: 10 January 2022 Published: 12 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Keywords:** magnetic nanoparticles; in situ diagnosis; rapid detection; extracellular vesical quantification; presurgical screening; pathological diagnosis; sentinel node mapping

#### **1. Introduction**

Magnetic nanoparticles (MNPs) have recently been applied to life sciences as well as clinical settings. MNPs comprise aggregates of iron oxide (FeO, Fe2O3, and Fe3O4) or ferrite particles (which contain iron oxide as the main component) in the nanometer order, which are dispersed or embedded in polymers, such as polysaccharide, polystyrene, silica, and agarose [1]. Their application to life science research stems from the ability to separate, guide, and detect MNPs using magnetic fields. Additionally, MNPs can be processed to furnish their surface with a variety of functions. Recognition sites, such as functional groups and biomolecules, are immobilized on the surface of the beads and are used to recognize targets for separation or detection [1]. The physical size and magnetization strength of the beads are roughly proportional to the number of iron oxide particles in the polymer. Protein purification and cell separation applications require strong magnetic particles, whereby micro-sized magnetic particles are used with a magnetic field [2]. For stem cell differentiation experiments and gene transfer applications, small magnetic particles (<100 nm) are generally used [3]. Furthermore, some nanosized magnetic particles, such as superparamagnetic iron oxide nanoparticles (SPIONs), are biocompatible and can be used internally in magnetic resonance imaging (MRI) contrast media for the liver [4].

We focused on the applications of MNPs in oncology from a surgeon's perspective when monitoring biomarkers before and after surgery, and for intraoperative diagnosis during surgery (Figure 1). In this review, we provide an overview of the application of MNPs in oncology.

**Figure 1.** The concept of this review. The usage of magnetic nanoparticles (MNPs) was divided into two objectives: monitoring of biomarkers (before/after surgery) and intraoperative diagnosis (during surgery). The types of MNPs and examples of their use are indicated. FG, ferrite and glycidyl methacrylate; FF, fluorescent FG; SPIONs, superparamagnetic iron oxide nanoparticles; EVs, extracellular vesicles; PSA, prostate-specific antigen; SLN, sentinel lymph node; MRIF, magnetically promoted rapid immunofluorescence.

#### **2. Monitoring Biomarkers before and after Surgery**

In oncological clinical settings, early detection and accurate diagnosis are important for cancer treatment, both before and after surgery. Enzyme-linked immuno-sorbent assay (ELISA) [5–7], which uses antigen–antibody reactions as its detection mechanism, is widely used to detect cancer biomarkers in serum for screening or monitoring before surgery, but the enzymatic method is time consuming. However, the MNP method accelerates the antigen–antibody reaction. This is a different mechanism to magnetic separation, in which antibody-immobilized MNPs can be attracted to immobilized antigen via a magnetic field. In this section, we describe two examples of the MNP method for biomarker detection: pancreas cancer-specific extracellular vesicles (EVs) using ferrite and glycidyl methacrylate (FG) beads and prostate-specific antigen (PSA) using fluorescent FG (FF) beads.

#### *2.1. Measuring a Biomarker in Serum Using FG Beads* 2.1.1. FG Beads

Handa's group initially developed affinity latex beads, styrene–GMA (SG) beads, which have a polystyrene core and glycidyl methacrylate (GMA) on their surface, known as poly GMA beads (Figure 2A left) [8]. Poly GMA beads have epoxy groups that can immobilize proteins, nucleic acids, and low-molecular-weight compounds. Additionally, the group found that carboxyl and thiol groups bind to the ferrite surface [9,10]. On the basis of these findings, 35–40 nm ferrite was coupled with the adaptor molecule and then coated with a copolymer of styrene and GMA, followed by coating with GMA [10] to generate the FG beads (Figure 2A, middle). FG beads have a 200 nm diameter with several encapsulated ferrite nanoparticles. Similar to the SG beads, specific ligands can be bound to the GMA surface to enable it to bind target molecules (Figure 2A, right). Because of the ferrite core, it can then be attracted or separated using magnetic forces.

**Figure 2.** (**A**): Construction of SG and ferrite and glycidyl methacrylate (FG) beads. SG beads are composed of styrene and glycidyl methacrylate (GMA) (left). FG beads are prepared with surfacemodified ferrite particles, styrene, and GMA (middle). Transmission electron microscopy image is shown (middle). Antibodies can be immobilized on the surface of FG beads (right). Modified from Inomata et al. and Nishino et al. (**B**): Schematic image of the quantification of extracellular vesicles (EVs). Candidate lectins were coated on the optical disc of the ExoCounter system. Lectin-binding EVs in the sera of pancreatic cancer patient or cell lines were captured on the disc and labeled with anti-CD9 Ab-conjugated nanoparticles. The absolute numbers of labeled EVs were quantified using the optical disc drive of the ExoCounter. Modified from Yokose et al.

#### 2.1.2. Screening or Monitoring of EVs with FG Beads

EVs are granular substances with a diameter of 50–150 nm, and they are secreted by cells [11,12]. Lipids and proteins derived from cell membranes are contained on the surface of EVs, and inside the EVs are intracellular substances, such as nucleic acids [13] (including microRNA, messenger RNA, and DNA) and proteins [14]. Recently, it has been suggested that EVs are involved in cancer development. EVs released from cancer cells are known to function in ways that favor cancer cells, such as cell survival, malignant transformation, and metastasis.

The surface proteins on EVs reflect parental cells, such as CD147 from colorectal cancer cells [15,16], human epidermal growth factor receptor 2 (HER2) from breast cancer cells [17], and CD91 from lung cancer cells [18]. Therefore, measuring specific EVs released from cancer cells has potential in cancer screening and monitoring. The methods used to count EVs are mainly conventional particle-counting methods, such as nanoparticle tracking analysis [19,20] and tunable resistive pulse sensing [21,22], or labeling-detection methods, such as ELISA [18,23] and flow cytometry [14,24].

The ExoCounter system is a unique assay system that uses FG beads to count the absolute number of EVs and analyze surface proteins simultaneously. The system uses an optical disc with periodic grooves that are 160 nm wide at the bottom and 260 nm wide at the top. Individual EVs can be bound at the bottom of the groove and FG beads at the top (Figure 2B). The basic reaction mechanism is a magnetically prompted rapid sandwich immunoassay. Using an optical head based on Blu-ray disc technology, EVs modified with nanoparticles are detected one by one. The immunoassay uses antibodycoated detection FG beads and samples placed on a capture antibody- or ligand-coated optical disc (Figure 2B). A magnet is attached under the disc for 1–2 min to concentrate the FG beads onto the immobilized capture antibody or ligand, and then unbound FG beads are washed out. The captured FG beads are counted by an optical pickup composed of a laser diode and a photodetector.

The ExoCounter system has been used to analyze pancreatic cancer patient serum, in which EVs with glycoprotein are bound to Agaricus bisporus agglutinin (ABA) or Amaranthus caudatus agglutinin (ACA) using CD9 antibody-coated FG beads to detect EVs on an ABA- or ACA-coated disc [25]. Using this method, EVs that have a carbohydrate chain that binds to ABA or ACA can be detected. EV quantification was performed on 90 samples from pancreatic cancer patients (68 preoperative and 22 postoperative samples) and 77 negative control serum samples [25]. The ABA-binding and ACA-binding EVs were significantly higher in the preoperative pancreatic cancer patients than in the negative controls (*p* < 0.001 and *p* < 0.001, respectively) (Figure 3) [25]. Furthermore, the number of labeled EVs was significantly reduced in the post-pancreatectomy sera, almost to the same level as that of the negative controls (*p* < 0.001 and *p* < 0.001, respectively) (Figure 3) [25]. The measurement that captures the characteristics of EVs is quite unique.

**Figure 3.** Quantification of ABA- and ACA-positive EVs from the sera of preoperative and postoperative pancreatic cancer patients and negative controls. Patient sera were analyzed using ABA- or ACA-coated discs and anti-CD9 Ab-conjugated beads with ExoCounter. Adapted from Yokose et al. \*\* *p* < 0.01.

#### *2.2. Measuring a Biomarker in Serum Using FF Beads* 2.2.1. FF Beads

The next generation of FG beads is fluorescent FG beads (FF beads). Generally, fluorescent substances are immobilized on the polymer surface by covalency or affinity. However, a unique feature of FF beads is that fluorescent substances, such as europium complexes (Eu (TTA)3 (TOPO)2), can be encapsulated. Europium complexes emit fluorescence at 618 nm under light excitation at 340 nm. FG beads are tolerant to several organic solvents and expand or shrink depending on the type of solvent. When acetone is used, the surface polymer of FF beads swells along with the encapsulated fluorescent substance, and then returns to its original configuration in water (Figure 4). The fluorescence can be directly observed with a fluorescence detector or microscope. In addition to their magnetic attraction function, signal amplification is not necessary, which enables fast and highly sensitive disease diagnosis [26–28].

**Figure 4.** Scheme of FF beads. FF beads were prepared by encapsulating fluorescent materials in FG beads. Antibodies were immobilized on FF beads. FF beads emit fluorescence upon exposure to UV excitation. Modified from Kabe et al.

#### 2.2.2. Screening or Monitoring of Cancer Biomarkers with FF Beads

FF beads were used to measure PSA, a widely used biomarker in patients with prostate cancer, using a magnetically prompted rapid sandwich immunoassay [26]. Detection was undertaken by measuring the fluorescence intensity. The detected antibody-coated FF beads and samples were placed on an antibody-coated capture microplate, and a magnet was attached under the plate for 1–2 min to concentrate the FF beads onto the immobilized antibody. The unbound FF beads were washed out, and the fluorescence of the remaining FF beads was held on the plate through the antigen–antibody reaction, which was then measured directly. When the limit of quantification (LOQ) was defined as the lowest concentration measurable intraassay (CV < 20%) in the sandwich immunoassay with FF beads, then the LOQ of this method was estimated to be 0.02 ng/mL for PSA in serum [26].

Clinical examination of prostate cancer requires the detection of PSA in serum over a range of 0.1 to 10 ng/mL [29,30]. Magnetically prompted rapid sandwich immunoassay is therefore sufficient to analyze a healthy donor who would generally have low concentrations of PSA (<0.1 ng/mL) and patients with prostate cancer who would have concentrations >4.0 ng/mL [26].

#### **3. Intraoperative Diagnosis during Surgery**

Cancerous areas are surgically removed and diagnosed pathologically during surgery, often with lymph nodes. The powerful application of MNPs in intraoperative situations includes sentinel lymph node (SLN) mapping and the rapid diagnosis of metastasis in SLNs. Currently, radioisotope (RI) tracers and blue dye are used as the gold standard for SLN mapping during surgery [31,32]; however, the RI method risks radiation exposure to both patients and medical personnel. Furthermore, the locations at which it can be used are limited because RI methods require nuclear medicine facilities. Using biocompatible MNPs, such as SPIONs, SLN detection can be performed without a special RI facility. Moreover, this MRI contrast media can drain into SLNs faster than RI and can be detected using a magnetometer.

The resected lymph nodes can be examined pathologically during surgery. Rapid diagnosis of cancer or metastasis in SLNs is necessary for surgical decision making. To visualize cancer or metastasis, immunostaining can increase the accuracy of diagnosis, but it is usually time consuming.

In this section, magnetic methods for SLN detection and rapid immunostaining are described.

#### *3.1. Detecting Sentinel Node during Surgery Using SPIONs*

Lymph nodes are responsible for trapping foreign substances, such as pathogens, before they can spread throughout the body, and eliminating them through an immune response [33]. Metastasis to regional lymph nodes is the most important prognostic indicator of outcome in patients with solid tumors. Tumor cells that have invaded the stroma can reach regional lymph nodes through the lymphatic capillaries and trunks around the tumor, forming lymph node metastases [33]. In melanoma [34] and breast cancer [35], the SLN theory has been established, whereby tumor cells that invade the lymphatic vessels first metastasize to specific lymph nodes, the so-called SLNs [36], and then to regional lymph nodes and organs throughout the body.

Pathologic examination of SLNs during surgery could provide information about the staging of regional lymph nodes. If the SLN is demonstrated to be cancer negative, then radical lymph node dissection would not be necessary. Recently, the applications of SLN theory were reported to be beneficial for many cancers, such as skin [36], breast [37], gastrointestinal [38], and gynecological cancers [39]. There could even be benefits during laparoscopic surgery [40].

The standard approach for the detection of SLNs is the dual-tracer method using an RI tracer (radiolabeled tin colloid) and blue dye [41]. However, the use of RIs requires a nuclear medicine facility. Furthermore, the RI tracer must be injected 2–24 h prior to surgery for accurate SLN detection [32]. These issues indicate the need for non-radioactive, rapid-assessment tracers with an ability to reliably detect SLNs. The RI method could therefore be replaced by a magnetic method.

#### 3.1.1. SPIONs

SPIONs can be categorized as MNPs. SPIONs, such as Sienna+ and Resovist, are hydrophilic colloidal solutions of γ-Fe2O3 coated with carboxydextran. The diameter of the iron oxide particles is 4–10 nm, and the total size of SPIONs is approximately 60 nm. SPIONs are biocompatible and are specifically taken up by reticuloendothelial tissues (Kupffer cells), mainly in the liver. MRI is a diagnostic approach that uses a receiving coil to acquire the radio waves generated when a high-frequency magnetic field is applied to hydrogen atoms in a living body, causing a resonance phenomenon, and creates an image on the basis of the signal data. SPIONs are used as a negative contrast agent because they have a strong transverse relaxation time (T2) shortening effect and decrease the MR signal. After administration to the human body, SPIONs are rapidly taken up by Kupffer cells in the liver. Kupffer cells are not present in cancerous tissues and, thus, exert a contrast effect in MRI [42].

SPIONs have also been used as tracers for SLN biopsy. Following injection around the tumor, SPIONs are taken up by the SLNs and detected by a dedicated probe [43]. In this section, we focus on SLN detection by SPIONs.

#### 3.1.2. Magnetic Probes

Magnetic Probes for Breast Cancer

Magnetic field detectors are necessary to detect SPIONs in SLNs for SLN mapping. A number of magnetic probes have been developed. For example, Sentimag is based

on the mechanism of an AC pickup coil that is commercially available and is one of the most widely used in clinical settings [43–49]. Other magnetic probes that are based on the fundamental mode of orthogonal fluxgate (FM-OFG) [50–53], such as a magnetic tunneling junction (MTJ) sensor [54] and negatively charged nitrogen-vacancy centers in diamonds, have been developed. DiffMag is based on a pickup coil with AC and DC differential magnetometry [55,56]. These magnetic probes have demonstrated the ability to detect between 280 ng and 500 μg SPIONs from a distance of 1 mm to 2.5 cm.

Sekino et al. [57] showed that the amount of iron uptake in SLNs in breast cancer patients was approximately 140 ± 80 μg [57], which was 0.3% of the injection dose (1.6 mL of Resovist) that contained 44.6 mg of iron. Therefore, the magnetic probe is required to have a detection ability in the order of 100 μg at a typical distance of 2–3 cm for breast cancer to be applicable in the clinic.

A magnetic probe developed by Sekino and Kusakabe's group employed a permanent magnet and a Hall-effect magnetic sensor with a code-less handheld shape [57]. This probe is also commercially available as a medical device (Matrix Cell Research Institute Inc., Ibaraki, Japan, CE mark 93/42EEC; NB:0344, EC certificate No.4201663CE01). The major feature of this probe is that it allows precise positioning of the sensor with respect to the magnetic null point (where the magnetic flux density is zero) to remove environmental effects, such as any ambient magnetic fields and temperature effects. Other features of this probe are its easy handling for surgeons during operations because of its compact shape and low weight (108 g), and its code-less appearance. This probe can detect 56, 140, 280, and 560 μg SPIONs at a distance of 7, 9, 11, and 15 mm, respectively.

#### Magnetic Probe for Laparoscopic Study

Laparoscopic surgery is a less intensive method of surgery in which an endoscope and forceps are manipulated in four to five small incisions with ports (trocars) [58]. Usually, two sizes of ports are used, and the inner diameter of the larger port is 12 mm. Therefore, there is the need for a magnetic probe of a suitable shape for laparoscopic surgery. The differences between magnetic probes used for breast cancer and laparoscopic surgery are shown in Table 1 [57,59].


**Table 1.** Comparison of the probes for breast cancer and laparoscopic surgery.

The benefit of using magnetic nanoparticles, such as SPIONs, for SLN mapping in laparoscopic surgery is not just to avoid RI exposure, but because of the speed at which SPIONs can drain to SLNs from the injection site. SPIONs drain quicker than RI tracers [60], meaning that SPIONs could be used as an SLN detection tracer during surgery. Another benefit is the detection distance, which is shorter than that of RI. Furthermore, the strength of the RI tracer signal means that signals from the injection site can interfere with the detection signal from SLNs [41,61,62]. This so-called shine-through effect is especially pronounced in the narrow intraperitoneal space and is not an issue with magnetic nanoparticles.

Kuwahata et al. [63] developed an AC/DC probe magnetic sensor for laparoscopic surgery. This probe employs a nonlinear response from the magnetic nanoparticles magnetized by an alternating magnetic field with a static magnetic field to achieve sensitive detection. The probe showed a longitudinal detection length of 10 mm for 140 μg iron; the detection limit is approximately 280 ng from a 1 mm distance. The suitability of the probe was demonstrated using a porcine model.

#### 3.1.3. SLN Detection during Surgery

#### Breast Cancer

Magnetic tracers are taken up by macrophages in the lymph nodes and detected by a handheld magnetometer [43]. In a previous study, it was shown that SPIONs reach the axillary lymph nodes within minutes after injection into the breast [60]. To detect SPIONs, several magnetometers have been developed [43,56,64].

In the EU, Sienna+ (a suspension of SPIONs) and Sentimag (a specialized probe) are used for SLN biopsy of breast cancer. Sienna+ is injected into the tumor periphery to reach the SLNs and can be identified by Sentimag. Sienna+ is a suspension of dark grains and can be recognized as a dye. A meta-analysis of clinical trials of SLN biopsies using magnetic detection systems showed that the identification rate of SLNs was not inferior to that of simultaneous administration of radiocolloid ± dye (conventional method vs. magnetic method: 96.8% vs. 97.1%).

Clinical tests using SPIONs and blue dye tracers in patients with breast cancer have shown that handheld magnetic probes are useful for detecting SLNs containing magnetic nanoparticles [65]. A multicenter study of breast cancer SLN biopsies using TAKUMI and Resovist (ferucarbotran) as a tracer showed that the identification rate of SLNs was not inferior to that of the RI method (RI method vs. magnetic method: 98.1% vs. 94.8%) [66].

#### Gallbladder Cancer

SLN mapping is challenging for cancers of difficult-to-access visceral organs, such as the gallbladder. This is because the standard method of RI use requires preoperative tracer injection. Indocyanine green (ICG) fluorescence imaging is a promising tool for SLN detection in patients with breast, gastric [67], and colorectal cancers [68]. Lymph flow and SLNs are detected soon after injection with a fluorescence imaging system, even in dense adipose tissue. However, because the ICG tracer is small, it passes through downstream lymph nodes, making it difficult to quantitatively analyze SLNs [69]. Magnetic methods to detect intra-abdominal SLNs can be used to overcome these challenges and have been effectively applied.

In a gallbladder cancer feasibility study using an animal model, the TAKUMI probe, which includes a Hall sensor, was modified for laparoscopic use [59]. Its feasibility for detecting SLNs of the gallbladder was evaluated using a laparoscopic dual-tracer method by injecting ICG and SPIONs into five wild-type pigs without cancer and one immunodeficient (RAG2-knockout) cancer-bearing pig. The laparoscopic probe identified the SPIONs in the lymph nodes of four out of the five wild-type pigs during surgery (Figure 5). The magnetic field counts were 2.5–15.9 μT, and fluorescence was detected in SLNs in all five pigs.

ICG shows a visual lymph-flow map, and SPIONs more accurately identify each SLN with a measurable magnetic field, which is similar to the RI method. It was confirmed using a RAG2-knockout porcine gallbladder cancer model with lymph node metastases that SLN mapping is effective under tumor-burden circumstances. We identified an SLN in the laparoscopic investigation, and the magnetic field count was 3.5 μT. The SLN was histologically determined to be one of two metastatic lymph nodes [59]. This result suggested the possibility of identifying SLNs in the intra-abdominal cavity organs.

#### *3.2. Magnetically Promoted Rapid Immunofluorescence (MRIF) Staining Using FF Beads*

Resected SLNs are examined pathologically. Here, we describe the rapid immunostaining of SLNs with positive images observed by fluorescence microscopy.

**Figure 5.** Laparoscopic sentinel lymph node (SLN) detection with a mixed tracer in porcine surgery. (**A**): Injection of the mixed indocyanine green (ICG) dye and magnetic tracer into the gallbladder wall. (**B**): ICG fluorescence signals detected by near-infrared laparoscopy. (**C**): Magnetic field evaluation of lymph nodes with the laparoscopic magnetic probe (yellow arrow). (**D**): Fluorescence signal-oriented identification of SLNs by the magnetic method. (**E**): Resection of the detected SLNs. (**F**): Brown pigmentation with the magnetic tracer in one resected regional lymph node among five. Modified from Mihara et al.

#### 3.2.1. Europium Single Staining

Accurate identification of the extent of a lesion allows the surgeon to minimize removal during minimally invasive surgery of solid tumors. Thus, there is a need for the rapid diagnosis of lesion characteristics and progression during surgery [70,71]. Generally, snap-frozen sections are prepared during surgery and stained with hematoxylin–eosin (HE) for examination by a pathologist. Although HE staining can provide rapid diagnosis, diagnosis can be difficult, such as in cases with small lesions. Immunostaining is one approach to increase the diagnostic accuracy. The avidin–biotin complex method is a commonly used immunostaining system that involves four sequential steps: (1) primary antibody staining; (2) biotin-labeled secondary antibody staining; (3) avidin–biotin–peroxidase complex formation; and (4) development by diaminobenzidine (DAB) staining. Antigen–antibody reaction steps by primary and secondary antibodies are particularly time consuming, and the method is not suitable for rapid intraoperative diagnosis. Thus, there have been attempts to shorten the time of the procedure using ultrasound [72] and microwaves [72,73] that accelerate the antigen–antibody reaction with a stirring effect in addition to Brownian motion. Alternatively, Onishi et al. used FF beads to develop MRIF staining, which shortens reaction and washing times using a magnet [26,74]. MRIF can be performed in two steps without secondary antibody, signal amplification, or DAB staining: (1) incubation with antibody-coated FF beads and (2) washing, because the antigen–antibody complex can be directly observed using a fluorescence microscope to observe the fluorescent material encapsulated in the FF beads (Figure 6). This procedure reduces the time to a 1 min reaction and 1 min wash step with a magnet when applied to frozen sections of xenografted samples of A431 human epidermoid cancer cells that express high levels of epidermal growth factor receptor (EGFR) and anti-EGFR antibody-europium encapsulated FF beads (Figure 7A) [74].

**Figure 6.** Scheme of magnetically promoted rapid immunofluorescence. 1: Diluted FF beads are dripped onto tumor cells, and the slide is vigorously agitated on the magnet; 2: the slide is inverted into a plastic container and washed with a magnet; 3, 4: FF beads bound to tumor cells can be observed directly by fluorescence microscopy. Modified from Onishi et al.

**Figure 7.** (**A**): Staining of A431 cells by hematoxylin–eosin (HE) (**left**), conventional immunostaining (**middle**), and MRIF (**right**). Images of an A431 (human epidermoid cancer cells with high expression of epidermal growth factor receptor (EGFR)) xenograft in pigs. (**B**): Staining image of a human breast cancer metastatic lymph node by HE (**left**), conventional immunostaining (**middle**), and MRIF (**right**) incubated with anti-pan-cytokeratin antibody-coated FF beads. Image of a paraffin-embedded tissue array of a stained human breast cancer metastatic lymph node. Scale bar = 250 μm and 25 μm for high magnification. Adapted from Onishi et al.

The strength of the magnetic force is critical for obtaining maximum results; therefore, a jig was prepared, and the relationship between the magnetic force and the distance from a 10 mm diameter and 24 mm length cylindrical magnet was examined. The magnetic force (F) acting on an FF bead was calculated as F = −∇(−*mb*·*B*), where *mb* is the magnetic moment of the FF beads, and B is the magnetic field strength of the magnet [75]. The distribution of the magnetic force was stronger at the margins than at the center of the magnet; therefore, we decided to agitate the magnet to obtain uniform staining [74]. The optimal distance between the A431 xenograft samples and the magnet using anti-EGFR antibody-coated FF beads for a 1 min incubation was within 2–5 mm, whereby the magnetic force =7.79 × <sup>10</sup>−<sup>15</sup> N to 3.35 × <sup>10</sup>−<sup>15</sup> N. A distance shorter than 2.0 mm showed unwanted background staining, and a distance greater than 5 mm showed insufficient staining. We also examined the optimal distance for washing. A distance from the samples to the magnet of 11 mm with a magnetic force of 4.78 × <sup>10</sup>−<sup>16</sup> N showed the best result for anti-EGFR antibody-coated FF beads. A distance >11 mm showed unwanted background staining. The staining efficiency was confirmed by the staining of breast cancer clinical samples for cytokeratin (CK), which is present in all epithelial cells, even in tumorigenesis, and is a widely used epithelial marker. Anti-pan-cytokeratin antibody (AE1/AE3) was used in this study. Figure 7B shows similar patterns of staining by conventional immunostaining and MRIF, which is consistent with the cancer region observed in the HE-stained section. The positive rates of conventional immunostaining were compared with MRIF staining using anti-pancytokeratin antibody-coated FF beads and clinical tissue array samples. The positive rate of conventional immunostaining was 96.5% (276/286) and that of MRIF was 92.7% (265/286). The coincidence rate was 94.8% (271/286) [74]. Normal tissue (i.e., breast tissue, tonsil, and lymph nodes) was analyzed. The positive rate of conventional immunostaining was 26.3% (25/95) and that of MRIF was 32.6% (31/95) [74]. The coincidence rate was 91.6% (87/95) (Table 2). Under optimal conditions, this ultrarapid immunostaining approach may be an ancillary method for pathological diagnosis during surgery.


**Table 2.** Coincidence ratio between conventional IHC and MRIF staining.

MRIF, magnetically promoted rapid immunofluorescence.

#### 3.2.2. Multi-Colored Staining

Using several hydrophobic fluorophores that can be embedded into the polymer layers of the beads, the construction of multi-colored FF beads becomes possible. We applied a series of compounds, such as 3-dimesityl boryl-2,2 -bithiophene and 5,5 -dimesityl-3 dimesityl boryl-2,2 -bithiophene, which contain boron, to create multi-colored FF beads (patent: JP 6409173). Through the fluorescent labeling of target markers, multi-MRIF would be achieved. Figure 8 shows HE staining, conventional IHC staining, europium single staining, and europium double staining of human lymph nodes with metastasis by multi-MRIF. We designed antibody-coated FF beads to emit fluorescence independently. FF beads were coated with antibodies against CK19, which is expressed in epithelial cells, and tenascin C (TNC), which is a glycoprotein that is expressed in the extracellular matrix around cancer cells. Because some triple-negative breast cancers do not express CK19, tenascin C is a good candidate to compensate for CK19 to increase the detection rate of triple-negative breast cancer. Anti-CK19 antibody-coated FF beads show green fluorescence, and anti-TNC antibody-coated FF beads show red fluorescence. Conventional immunostaining with pan-CK was well correlated with single MRIF staining with pan-CK antibody-coated FF beads, which showed magenta fluorescence derived from europium

complexes. The blue color was nuclear with DAPI staining. For CK19 and TNC double staining, both sets of FF beads were equally mixed and stained under the same magnetic conditions as EGFR for a 1 min reaction and a 1 min wash. CK19 (Figure 8D) and tenascin C (Figure 8E) were mostly stained in cancerous regions. Figure 8G shows merged images from D, E, and F. There is still a need to optimize the conditions because the antibody affinity is varied; however, this result demonstrates the possibility of double staining in one step. Furthermore, when frozen sections of six human metastatic lymph node samples from breast cancer were stained with IHC and MRIF, all lymph nodes were positive with a 100% concordance rate. In short, we successfully performed fluorescence multiplex staining of human breast cancer metastatic lymph nodes by binding antibodies against CK19 and TNC to FF beads containing different fluorophores. Because the system is applicable to frozen sections, it enables rapid diagnosis and meets clinical needs.

**Figure 8.** Staining of a frozen tissue section of a human breast cancer metastatic lymph node. (**A**): Hematoxylin and eosin (HE) staining. (**B**): Conventional immunostaining (diaminobenzidine). (**C**): Single staining of magnetically promoted rapid immunofluorescence (MRIF) with anti-pancytokeratin antibody-coated FF beads that emitted magenta fluorescence. Multi-colored MRIF using anti-CK19 antibody-coated F beads that emitted green fluorescence (**D**), anti-TNC antibodycoated FF beads that emitted red fluorescence (**E**), DAPI staining (**F**), and merged images (**G**). Scale bar = 1000 μm Adapted from Onishi et al. and new data.

#### **4. Discussion and Future Perspectives**

In this review, we described the applications of MNPs in oncology from a surgeon's perspective of monitoring biomarkers before and after surgery, and for intraoperative diagnosis during surgery. Pancreatic cancer-specific EVs and a cancer-specific antigen, PSA, were measured by the magnetic method, which could be used for monitoring cancer development before and after surgery. SLN detection can be performed during surgery by the magnetic method, and immunostaining can even be completed during surgery. Laboratory techniques related to surgical procedures can be undertaken magnetically.

Notably, EV detection and immunostaining are quite unique. Most EV methods require an extraction step of EVs from serum or plasma, but this magnetic method uses serum directly and involves counting the absolute number of EVs that express characteristic cell surface proteins. Pancreatic cancer is one of the most aggressive cancer types because it is difficult to identify in the early stages [76]. This method has potential as an early detection tool. Immunostaining is a powerful tool to increase the accuracy of diagnosis, but to contribute to decisions on surgical procedure, staining must be completed within 20 min [77]. MRIF requires only a 1 min reaction and a 1 min wash, and, thus, this method has the potential for practical application in the clinic. Moreover, because it is easy to define MRIF as positive and negative, it can be automated, reducing the requirement for a pathologist.

The magnetic SLN method is a promising alternative to the RI method. Moreover, it has the potential for clinical application to the laparoscopic method for detecting SLN metastasis from cancers of visceral organs, which are difficult to examine via the surface of the body or by endoscopy. These procedures can enable the identification of SLNs for almost all intra-abdominal organs that are laparoscopically accessible. Moreover, the long shelf life and easy handling of SPIONs and their detector permit the accurate diagnosis of metastatic cancers in mid- to small-scale medical facilities and developing countries.

Because europium is toxic, FG beads also have the potential for magnetic sensing with magnetic probes. Magnetic sensing activities strongly depend on magnetic characteristics, such as the magnetic moment. Compared with the magnetic moment of Resovist (approximately 50 emu/g) [78], the magnetic moment of the beads (20 emu/g) [10] is relatively small. Considering the detectable distance of Resovist of 9 mm with a magnetic probe as demonstrated by Sekino et al. [57], the detectable distance of the FF beads could be several millimeters. This expected magnetic sensing activity potentially enables the intra-abdominal detection of cancer and lymph nodes at a proximal distance.

The problems relating to the rate of false positives and false negatives that this type of methodology generates in each of its applications should be addressed, for example, which test confirms that the biological matrix has correctly come into contact with the analytical system in the presence of a negative result. However, regarding EV measurement, a lectin array [25] could be used to confirm the result; however, the results of lectin arrays are relative and are not quantitative. PSA measurements should be confirmed by conventional methods, such as ELISA, but the authors did not examine the associated rate of false positives and false negatives. The sentinel node is defined as the first lymph node that cancer cells reach, and the number of nodes may vary depending on the detection method. There are usually one or two for the RI method and more for the dye method. It is therefore difficult to discuss false positives and false negatives. In this review, we described the ICG dye method and the SPION method. Regarding MRIF staining, Onishi et al. [74] used conventional immunostaining to confirm that the antibody had correctly come into contact with the antigen and described the concordance rate because tissue array samples are not always serial sections.

#### **5. Conclusions**

Screening, monitoring, and diagnosis are critical in oncology treatment. However, current clinical methods are time consuming. The use of magnetic nanoparticles is an alternative approach that offers faster analyses over a wide range of oncological applications, such as the detection of cancer biomarkers and immunostaining. Radioisotope tracers are used for SLN mapping during cancer surgery; however, the RI method risks radiation exposure to both patients and medical personnel and requires nuclear medicine facilities. Using biocompatible MNPs, such as SPIONs, SLN detection can be performed safely without a special RI facility. The magnetic method is an interesting approach and its use is expected in more applications. It is hoped that large-scale clinical trials will be undertaken to demonstrate its usefulness and to validate it for clinical diagnosis.

**Author Contributions:** T.O., K.M. and S.M. designed and drafted the manuscript. S.S., A.K., M.S., M.K., H.H. and Y.K. drafted a part of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This review received no external funding.

**Acknowledgments:** The authors thank Y. Nakamura and S. Matsuda for their continued support. We thank Shaun Galbraith and H. Nikki March for editing a draft of this manuscript and for helping to draft the abstract.

**Conflicts of Interest:** M.K. is a member of Matrix Cell Research Institute Inc., which is TAKUMI's manufacturer. The other authors have no competing interests for this review.

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