1.

Introduction

One of the main reasons for locoregional treatment failure in oncology is the inability to fully excise the (primary) tumor during surgery. For example, complete resection of cancer is particularly difficult when the tumor is nonpalpable. In such instances, surgical guidance techniques that help optimize the excision are expected to have a positive influence on the clinical outcome.

Initial detection of nonpalpable lesions at pre-operative imaging [Fig. 1 ] is often followed by image-guided markation of the lesion [Fig. 1]. These procedures typically aim to mark the center of the lesion using guide-wire localization (WL), radio-guided occult lesion localization (ROLL), or radioactive seed localization (RSL). In WL, a hook is placed in the center of the tumor and attached to a guide-wire^{1, 2} along which the surgeon can excise the tissue. In ROLL, a nonspecific radiotracer ( ${}^{99\mathrm{m}}\mathrm{Tc}$ -NanoColl or sulfur-colloid) is injected into the tumor prior to surgery.³ The radioactive signal is then used to acoustically localize the lesion intra-operatively using a gamma probe. Radioactive seed localization (RSL) provides an alternative radio guidance method to ROLL using titanium marker seeds filled with a small amount of radioactive iodine $\left({}^{125}\mathrm{I}\right)$ .^{4, 5, 6, 7}

Fig. 1

Schematic representation of the combined pre-, intra-, and post-operative imaging paradigm that can be achieved with multimodal marker seeds. (a) Tumor diagnosing imaging procedure. (b) Image-guided placement of bracketing tumor marker seeds. (c) Pre-operative validation of the marker placement accuracy. (d) Optical intra-operative detection of the tumor markers. (e) Geographical (re)orientation of the excised tissue section at pathology.

Image-guided localization is typically carried out using x-ray imaging [such as (stereotactic) mammography (XM)], ultrasonography (US), or magnetic resonance imaging (MRI).^{4, 6, 7, 8} Although WL, ROLL, and RSL all aid in the localization of cancer during surgery, accurate marker placement remains crucial. Hence, accurate verification of the marker relative to the tumor location in one or multiple preoperative imaging modalities may be useful. This strategy requires the localization markers to be visible in the same modality that was used to visualize the tumor. Clearly, the latter may vary for different tumor types and sites.

Intra-operative fluorescence imaging is emerging as an optical detection method in surgical oncology. Fluorescent dyes such as fluorescein and indocyanine green (ICG) are used in several different applications in patients, mostly for superficial imaging of blood flow or lymph node detection.^{9, 10, 11, 12} Surprisingly, the potential of this optical modality in tumor-localization applications is currently unexplored. Nevertheless, it can be used to overcome an important shortcoming of the current acoustic radioguidance methods—namely, by providing real-time optical guidance.

To eliminate the need for margin re-excision, it is vital that surgical resection margins do not contain any residual tumor tissue. For this reason, an additional small tumor-free margin around the tumor, the so-called surgical safety margin, is often excised.^{13, 14} Different fluorescent wavelengths have different degrees of tissue penetration. While visual dyes (emission $400\text{to}600\mathrm{nm}$ ) can be detected only superficially, far-red and near-infrared dyes (emissions $⩾650\mathrm{nm}$ ) give a much higher degree of tissue penetration, but can be detected only with a light-sensitive camera system.¹⁵ This difference in tissue penetration can be used to provide a rough estimate of the depth of the marker in the tissue. Multispectral fluorescence imaging thereby enables differentiation between simultaneous emissions at different wavelengths.^{16, 17} Specifically for this purpose, we have previously developed dual-emissive (inorganic) dyes that have a visible green emission and a far-red emission that can be accurately detected without autofluorescence.¹⁸

Improved macroscopic localization methods may also be beneficial post-operatively at pathology. To report the presence of cancer on the resection edges back to the surgeon for potential re-excision, the pathologist typically indicates the direction in which the positive edge was observed. Commonly, India ink and sutures are used to mark the original orientation of the excised tissue in the patient.¹⁹ Three-dimensional bracketing of a lesion with differently “colored” marker seeds may provide an opportunity to record the relative location and orientation of the tumor in individual patients.

The technical challenge we have set out to solve is the development of marker seeds that (1) can be accurately placed at different positions surrounding the lesion and thereby implementing a safety margin [Fig. 1]; (2) can be pre-operatively detected using the same imaging technique employed to diagnose the lesion rather than only with the modality used for bracketing [Fig. 1]; (3) can be optically located during surgical procedures [Fig. 1]; and (4) will provide information about specimen orientation for pathologists [Fig. 1] after excision of the lesion. To incorporate all these properties into a single marker, we have generated multimodal marker seeds that are visible with fluorescence imaging, XM, US, MRI, computed tomography (CT), and single-photon emission computed tomography (SPECT). We have studied their potential in providing surgical guidance in pre-, intra-, and post-operative phantom experiments and in experiments in dead surplus mice.

2.

Materials and methods

2.1.

Dual-Emissive Dyes

Lipid-coated $\mathrm{In}\mathrm{P}∕\mathrm{Zn}\mathrm{S}$ particles were synthesized and irradiated as described previously.¹⁸ These particles have two separate emissions—namely, an exciton emission at ${\lambda }_{\mathit{em}}=520\mathrm{nm}$ , $600\mathrm{nm}$ , or $660\mathrm{nm}$ (short lifetime) and a defect emission at ${\lambda }_{\mathit{max}}=660\mathrm{nm}$ [long lifetime $>60\min$ ; see Figs. 2 and 2 ]. The latter emission can be preilluminated $({\lambda }_{\mathit{em}}=366\mathrm{nm})$ and detected without real-time excitation.

Fig. 2

Dual-emissive $\mathrm{In}\mathrm{P}∕\mathrm{Zn}\mathrm{Sn}$ particles and generation of multimodal marker seeds. (a) Schematic representation of dual-emissive $\mathrm{In}\mathrm{P}∕\mathrm{Zn}\mathrm{S}$ particles. (b) Normalized fluorescent emission spectra with ${\lambda }_{\mathit{em}}=520\mathrm{nm}$ (exciton green) and ${\lambda }_{\mathit{em}}=660\mathrm{nm}$ (defect; red). (c) Schematic generation of multimodal marker seeds that incorporate dual-emissive dyes and ${}^{99\mathrm{m}}\mathrm{Tc}$ lipids. (Color online only.)

For safety margin estimation, green particles $({\lambda }_{\mathit{em}}=520\mathrm{nm})$ were used. For geographic tissue orientation, green, orange $({\lambda }_{\mathit{em}}=600\mathrm{nm})$ , and red $({\lambda }_{\mathit{em}}=660\mathrm{nm})$ particles were used. Herein, the colors were used for visual detection.

3.

Results

To combine the ability to pre-operatively set and validate preferred resection margins with optical detection and safety margin estimation, multimodal and dual-emissive marker seeds were generated. The potential of these marker seeds for pre-operative tumor bracketing, intra-operative surgical guidance, and post-operative geographic reorientation was studied.

To illustrate the multimodal seed concept and the potential of these seeds in vivo, seeds were first implanted into the prostate of a dead surplus mouse. After implantation with a hollow needle into the prostate, a SPECT/CT image was made, clearly visualizing the location of the radioactive seed with respect to the anatomy of the mouse (Fig. 3 ; SPECT/CT). Following the pre-operative SPECT/CT, intra-operative multispectral fluorescence imaging enabled detection of the seed (Fig. 3). Here, the defect emission allows detection without autofluorescence.¹⁸ In addition, the visual exciton emission of the $\mathrm{In}\mathrm{P}∕\mathrm{Zn}\mathrm{S}$ -particles in the seed could also be detected by eye after excitation with a UV light source [flashlight; Fig. 3]. Due to the small size of the animal and relatively large size of the marker seed, these experiments unfortunately did not enable a form of surgical safety margin estimation.

Fig. 3

Imaging of multimodal marker seeds: (a) Schematic representation of the placement of the seed in a mouse prostate. (b) 3-D SPECT/CT image visualizes the seed localization (color-coding: fire) with respect to the mouse anatomy, followed by (c) $660\mathrm{nm}$ emission (color-coding: glow), (d) $520\mathrm{nm}$ emission (color-coding: rainbow) and (e) a visual fluorescent (orange) image of the exposed marker seed. (Color online only.)

The potential use of multimodal marker seeds in surgical safety margin estimation was further investigated in a tissue phantom using a surrogate tumor. After placement under US guidance [Figs. 4 and 4(bI)], the distinctive contrast-generating properties of the seeds could be exploited to differentiate the seeds from their environment. T2-weighted MR imaging provided a clear distinction between the glass seed itself (black) and the lipid content of the seed [white; see Fig. 4(bII)]. With x ray, the difference in density provides a clear distinction between the tissue, the silicone rubber, the titanium shell of the ${}^{125}\mathrm{I}$ -seed, and the glass of the multimodal marker seeds [Fig. 4(bIII)]. Dual-isotope SPECT/CT allowed differentiation between the ${}^{125}\mathrm{I}$ -seed and the ${}^{99\mathrm{m}}\mathrm{Tc}$ -containing multimodal marker seeds [see Fig. 4(bIV)].

Fig. 4

“Pre-operative” multimodal seed bracketing and placement delineation in a tumor phantom. (a) 3-D tumor bracketing with multimodal marker seeds. Phantom contains a surrogate tumor in a fresh piece of tenderloin. (b) Pre-operative assessment of the marker seed localization using (I) ultrasound—the marker seeds deflect the ultrasound signal enabling accurate detection; (II) T2-weighted MRI—the marker seeds can be accurately discriminated from the surrounding by the lack of signal from the seed and the white signal from its contents; (III) a planar x-ray image—the high attenuation of the seed material is easily detected; and (IV) SPECT/CT—dual-isotope SPECT enables differentiation between ${}^{99\mathrm{m}}\mathrm{Tc}$ -containing multimodal marker seeds (color-coding: green) and the ${}^{125}\mathrm{I}$ containing iodine seed (color-coding: fire). (Color online only.)

To mimic intra-operative surgical safety margin estimation, the phantom was imaged in a multispectral manner at different stages during the resection. For a representation of the tissue penetration at the different emissions, see Fig. 5 . Signal-to-background ratios (SBRs) were used to present the ability to detect the different emissions at a different distance from the seed to the surface. The preilluminated defect emission could already be detected while the seed was situated at a distance of $12\mathrm{mm}$ $(\mathrm{SBR}=2.2)$ , followed by a ten-fold increased SBR at a distance of $5\mathrm{mm}$ [Fig. 5]. In contrast, the exciton signal could be detected only starting at a distance to the surface of $5\mathrm{mm}$ $(\mathrm{SBR}=2.5)$ .

Fig. 5

Margin estimation based on multispectral imaging of dual-emissive seeds. (a) Schematic representation of the tissue penetration depth of the different optical emissions from the marker seeds; fluorescence excitation ( ${\lambda }_{\mathit{ex}}=366\mathrm{nm}$ ; blue) and the emissions ${\lambda }_{\mathit{max}}=520\mathrm{nm}$ (green) and ${\lambda }_{\mathit{max}}=660\mathrm{nm}$ (red). (b) Detection of $660\mathrm{nm}$ (color-coding: glow) and $520\mathrm{nm}$ (color-coding; rainbow) emissions during the layer-by-layer exposure of the marker seeds. Fluorescent signal intensities are depicted as signal-to-background ratio (SBR). (Color online only.)

To exploit the value of the fluorescence detection in geographical pathologic guidance, seeds filled with differently colored $\mathrm{In}\mathrm{P}∕\mathrm{Zn}\mathrm{S}$ particles were used. These particles emit light at different wavelengths and thus have different visual colors: green: $520\mathrm{nm}$ ; orange: $600\mathrm{nm}$ ; and red: $660\mathrm{nm}$ [see Fig. 6 ]. To prove the concept of using 3-D bracketing with marker seeds in geographic tissue (re)orientation, the differently colored seeds were placed on the different sides of a dice. Seeds filled with red particles were placed at position one and six, seeds filled with orange particles were placed at the two and five position, and seeds filled with green particles were placed at position three and four of the dice [Figs. 6 and 6]. When placed under a UV light source, it was possible to visually discriminate between the different orientations. The same differentiation is of course possible using different settings on a (multispectral) fluorescence camera.

Fig. 6

3-D tumor bracketing for (re)orientation at pathology: (a) Differences in emission peaks of the different $\mathrm{In}\mathrm{P}∕\mathrm{Zn}\mathrm{S}$ particles. (b) Schematic representation of 3-D tumor bracketing with differently colored seeds (green/orange/red). (c) Proof of concept on a die allowing accurate visual identification and differentiation of the marker seeds. (Color online only.)

4.

Discussion

In the clinic, tumor-lesions are often marked before surgical removal. Multimodal markers may extend the use of current tumor marking approaches, as they are applicable during the entire pre-, intra-, and post-operative trajectory. The different properties of our experimental multimodal seeds can be used for (1) accurate seed placement, (2) verification of the placement accuracy, (3) fluorescence guided excision with surgical safety-margins, and (4) ex vivo verification of original specimen orientation.

5.

Conclusions

We have provided a proof of concept for using multimodal marker seeds in tumor bracketing and surgical guidance. Using the unique multimodal properties of the seeds, placement accuracy can be validated and resection margins can be predetermined. Moreover, multispectral imaging of the dual-emissive fluorescent content provides the opportunity for estimation of surgical safety margins. Last, the differently colored fluorescent particles provide geographical information to the pathologist regarding the original orientation of the lesion in the patient.