Prostate-specific membrane antigen-targeted surgery in prostate cancer: Accurate identification, real-time diagnosis, and precise resection

Radical prostatectomy (RP) combined with pelvic lymph node dissection (PLND) is the first step in multimodal treatment of prostate cancer (PCa) without distant metastases. For a long time, the surgical resection range has been highly dependent on the surgeon's visualization and experience with preoperative imaging. With the rapid development of prostate-specific membrane antigen positron emission tomography and single-photon emission computed tomography (PSMA-PET and PSMA-SPECT), PSMA-targeted surgery has been introduced for a more accurate pathological diagnosis and complete resection of positive surgical margins (PSMs) and micro-lymph node metastases (LNMs). We reviewed PSMA-targeted surgeries, including PSMA-PET-guided prostatic biopsy (PSMA-TB), PSMA-targeted radio-guided surgery (PSMA-RGS), PSMA-targeted fluorescence-guided surgery (PSMA-FGS), and multi-modality/multi-targeted PSMA-targeted surgery. We also discuss the strengths and challenges of PSMA-targeted surgery, and propose that PSMA-targeted surgery could be a great addition to existing surgery protocols, thereby improving the accuracy and convenience of surgery for primary and recurrent PCa in the near future.


Introduction
As the most frequently diagnosed cancer, prostate cancer (PCa) is the second leading cause of death in men worldwide [1].Radical prostatectomy (RP) with pelvic lymph node dissection (PLND) is the optimal choice for patients with intermediate-to high-risk PCa with or without regional lymph node metastases (LNMs) [2,3].Accurate and complete resection of PCa lesions and positive LNMs while preserving healthy tissues as much as possible is essential but difficult, depending on the surgeon's experience in the interpretation of preoperative imaging and anatomical knowledge.Prostate-specific

Ivyspring
International Publisher membrane antigen (PSMA)-targeted surgery has revolutionized traditional surgery and is a new trend in PCa surgery.
Prostate-specific membrane antigen (PSMA), also known as FOLH1 or GCP II, is a folate gamma glutamyl carboxypeptidase that is highly expressed in more than 90% of PCa lesions-including primary lesions, LNMs, and bone metastases-with 100-1000-fold higher expression level than that in normal prostate (NP) tissues [4,5].The significantly higher PSMA expression in PCa makes it an excellent target for PSMA-targeted molecular imaging and therapies [6,7].PSMA-targeted surgery involves using PSMA as a molecular marker to enhance the precision of PCa for an intuitive, fast and reliable way to discriminate PCa tumors from NP tissue, making the outcome of surgery less dependent on the surgeon's own experience and preoperative imaging, visual findings, or frozen sections during surgery.
PSMA-RGS has been clinically applied in hospitals in Europe and Australia, and the first Phase IIa study of PSMA-FGS agents has been completed in 18 PCa patients [15].Although PSMA-FGS began its clinical trials later than PSMA-RGS, it is considered by many to be more convenient than PSMA-TB and PSMA-RGS, due to its radiation-free nature, making it popular among patients and surgeons.
PSMA-targeted surgery has the potential to replace traditional guided surgery methods for PCa.In this review, we aim to provide an overview of the status of PSMA-targeted surgery for PCa, highlighting its efficiency in detecting positive LNMs and positive surgical margins (PSMs).We also focus on patient selection before PSMA-targeted surgery and follow-up after surgery, and the several clinical questions that urgently need to be addressed.

Selecting proper candidates for PSMA-targeted surgery
As shown in Table 1, the inclusion criteria for PSMA-targeted surgeries remain unclear.In most studies on recurrent PCa, PSMA-PET is necessary, whereas the lymph node invasion nomogram is mostly used for patients with primary PCa.In fact, PSMA-PET should be conducted for each patient before prostate biopsy to perform PSMA-TB to improve the diagnostic accuracy of PSMA-PET for clinically significant prostate cancer (csPCa) [17].
Table 1.Inclusion Criteria of patients for PSMA-RGS.

Year
Country Inclusion Criteria Primary PCa 2015 [13] Germany Primary PCa and suspicion of recurrent LNMs on preoperative PSMA PET/CT 2018 [38], 2021 [49] Germany Suspicion of LNMs on preoperative PSMA PET/CT 2022 [50] Italy Intermediate-or high-risk cN0cM0 PCa at conventional imaging with a risk of LNI of >5% (Briganti nomogram risk) 2022 [51] Australia Primary high-risk PCa (≥cT3a, international society of urological pathology (ISUP) Grade Group ≥3 or PSA of ≥ 15 ng/mL) with potential LNMs (Briganti nomogram risk >10% or on preoperative imaging) Recurrent PCa 2016 [37] Germany Rising PSA level to ≥0.2 ng/mL after primary curative therapy; 68 Ga-PSMA-11 PET-positive soft tissue lesions 2018 [45] Germany Recurrent PCa on preoperative PSMA PET/CT 2022 [42] Netherlands Up to three pelvic PCa recurrences (nodal or local) on preoperative PSMA PET/CT 2022 [54] Germany Recurrent PCa after initial RP with positive LNMs on preoperative PSMA PET/CT 2023 [42] Germany Patients with positive lesion detected on PSMA PET imaging in the pelvis or retroperitoneum suspicious for lymph node metastases (LNMs) or local recurrence.2023 [69] Germany Conforming initial treatment with either radical prostatectomy (RPE) or curatively intended external beam radiation, prostate-specific antigen (PSA) ≥0.5 ng/mL, positive 68 Ga-PSMA I&T PET/CT scan with no more than 2 intense PSMA-positive lymph nodes (maximum uptake at least 6 kBq/mL), and no signs of local recurrence or distant metastases, as well as informed consent to participate in this study.
Compared with the traditional viewpoint of using PSMA-PET as a staging tool after pathologically confirmed PCa, PSMA-TB may further improve the accuracy of biopsy and select suitable patients with higher uptake for PSMA-targeted surgery (Figure 1).Before PSMA-targeted surgery, patients with csPCa must be correctly diagnosed, and the presence of distant metastases (M1 stage) must be excluded.Even in patients with previously negative biopsy results, PSMA-TB can achieve a sensitivity of 100.0%, a specificity of 68.4%, a positive predictive value (PPV) of 66.7%, a negative predictive value (NPV) of 100.0%, and an accuracy of 80.6% for the detection of csPCa [18,19].Furthermore, PSMA-PET and multiparametric magnetic resonance imaging (mpMRI) should both be used as first-line imaging modalities to diagnose and stage primary PCa before prostate biopsy (PB) [6,7].
PSMA-PET clearly illuminates the borders of primary PCa.In comparison between PSMA-PET and robot-assisted radical prostatectomy (RARP) results, the PSMA-TB recommendation correlates better with the final histopathological results of poly(ADP-ribose) polymerase (PARP) tests.PSMA-PET can accurately detect PCa in 93.3% (28/30) and cover the highest Gleason score (GS) in 86.7% (26/30) of patients with intermediate-to-high-risk Pca [20].
For patients with regional LNMs, RP±PLND should be performed.PSMA-PET/CT can be used to exclude patients with distal LNMs (M1a stage), bone metastases (M1b stage), or visceral metastases (M1c stage) from planned curative radical prostatectomy [28].For LNMs with a diameter ≥2.3 mm, PSMA-PET can achieve a detection rate of 50.0%[29].The detection rate goes down significantly for LNMs with a diameter < 2.3 mm; small LNMs can be difficult to discover by preoperative PSMA-PET.
Therefore, PSMA-targeted surgery is needed to illuminate smaller LNMs intraoperatively and provide a chance to completely remove all primary lesions and regional LNMs.Based on the agent, PSMA-targeted surgery can be further divided into PSMA-RGS, PSMA-FGS, and multi-modality PSMA-targeted surgery.

PSMA-RGS
A series of agents have been developed for PSMA-RGS [30].For example, PSMA-I&T can be labeled with 68 Ga for PET imaging, 177 Lu for targeted radionuclide therapy, and 111 In for RGS or single-photon emission computed tomography/ computed tomography (SPECT/CT) [31].Mease et al. first established PSMA-targeted PET agent 18 F-DCFBC and its first in-patient imaging study in five patients with metastatic PCa [32,33].With the advent of 68 Ga-PSMA PET for PCa, PSMA-targeted RGS was introduced to intraoperatively detect PSMA-avid LNMs with atypical localization and inconspicuous morphology.To the best of our knowledge, PSMA-targeted RGS systems, such as PSMA-I&S and PSMA-I&T, are currently the most mature methods for intraoperative navigation in clinical practice (Figure 2A-K).For theranostic purposes, PSMA ligands could be labeled with short half-life imaging isotopes, such as 68 Ga (T 1/2 : 68 minutes), 18 F (T 1/2 : 110 minutes), 111 In (T 1/2 : 2.8 days), 99m Tc (T 1/2 : 6 hours), and for therapeutic isotopes, 177 Lu (T 1/2 : 6.7 days], 225 Ac (T 1/2 : 10.0 days), etc. Documented theranostic isotopes and their characteristics are shown in Table 2. We, aimed to further summarize the recent updates of PSMA-RGS according to agent type.

111 In-labeled PSMA-RGS
Although the detection rate of 111 In SPECT/CT is lower than that of 68 Ga-PSMA-11 PET/CT, the longer half-time of 111 In enables 111 In-PSMA-I&T to be appropriate for PSMA-RGS [34].Currently, most PSMA-targeted RGS systems are still highly dependent on γ-detecting probes and γ-emitting radiotracers, such as 111 In and 99m Tc [35]. 111In-PSMA is the most widely used agent for PSMA-RGS, successfully identifying 59% (29/49) PSMA-avid lesions by γ-probe.Among them, 97% of lymph nodes were confirmed as LNMs with a mean size of 7.9 mm (range, 0.8-20.0nm) [36].The effectiveness of 111 In-PSMA RGS is moderate due to interfering tracer accumulation in surrounding organs, leading to a more limited tumor-tobackground ratio (TBR) in vivo compared with ex vivo.This issue might be mitigated by extending the time between the administration of the tracer and the PSMA-RGS procedure.    In-PSMA-I&T 24 h before surgery (Table 3) [13].Although the preliminary case report included only five patients, they successfully proved that PSMA-targeted RGS was feasible and safe [13].Furthermore, Rauscher et al. performed 111 In-PSMA-I&T RGS in 31 patients with recurrent PCa, with a sensitivity of 92.3% and specificity of 93.5% [37] (Table 3).Recently, the DETECT Trial reported that 111 In-PSMA-I&T facilitated the resection of PSMA-avid lesions, of which 97% were confirmed as LNMs in 20 newly diagnosed PCa patients [36].In addition to PSMA-I&T, another theranostic ligand, 111 In-PSMA-617, was used for PET/CT imaging and PSMA-RGS in six primary PCa patients with potential LNMs [38] (Table 3).The sensitivity, specificity, PPV, NPV, and accuracy using cut-off SUVs normalized to lean body mass (SUL) were 92.1%, 98.9%, 94.6%, 98.4%, and 97.7%, respectively [38].In these studies, PSMA-targeted RGS could not discriminate LNMs intraoperatively within the surgery, and a γ-probe was used to confirm LNMs by counts per second (count/s, CPS) after ex vivo removal of the tissue samples [37,38].
Using drop-in γ probes (DIGPs) can overcome the limited maneuverability of traditional laparoscopic gamma probes to facilitate RGS.A DIGP was designed with a 135° grip for optimal performance during robotic-assisted radical prostatectomy (RARP), while a 45° grip DIGP has been shown to be more appropriate for ex vivo clinical setups [39].In one study using a DIGP with nontargeted tracers ( 99m Tc-nanocolloid + ICG), 10 high-risk patients with PCa underwent sentinel pelvic lymph node dissection (PLND) with no side effects [40].In another study, with a median uptake time from injection to surgery of 5.4 h, and ex vivo examinations were also performed with a gamma probe and near-infrared fluorescence (NIRF) imaging [41].All LNMs were identified using DIGP and 91.0% were identified by in vivo NIRF imaging [41].Although sentinel PLND can detect micro-LNMs, its non-targeting nature may limit its further use, especially in patients with recurrent PCa whose lymphatic drainage has been altered by previous PLNDs.Recently, Maurer et al. performed a systematic comparative analysis between robotassisted and open salvage PSMA-RGS, proving the safety and efficiency of robot-assisted PSMA-RGS with a longer operating time and equal estimated blood loss [42].Complete biochemical response (cBR, PSA < 0.2 ng/mL 2-16 weeks after PSMA-RGS) was 57% (34/61) in the open group and 61% (14/24) in the robotic group [42].The same research group further studied the reasons for negative histopathology from PSMA-RGS in 302 patients with recurrent PCa, showing that false positive lesions on PSMA-PET in most cases had negative histopathology and that only a few true-positive lymph nodes were missed by surgery [43].All true-positive lesions were adjacent to the internal iliac arteries or the prostatic fossa [43].

99m Tc-labeled PSMA-RGS
The production and wider applications of PSMA-RGS with 111 In-PSMA-I&T still face some limitations, such as suboptimal nuclear properties, high cost, and limited availability of 111 InCl 3 [12].To overcome the limitations of 111 In-PSMA-I&T in PSMA-RGS, Robu et al. successfully used mercaptoacetyl triserine (MAS 3 ) to replace the DOTAGA chelator in PSMA-I&T for 99m Tc labeling and D-Tyr-D-2-Nal to replace the 3-iodo-D-Tyr-D-Phe sequence to obtain a stronger linker [12].Compared to 111 In, 99m Tc-labeled PSMA-I&T can better meet clinical needs as a more cost-effective and readily available agent.Therefore, 99m Tc-labeled agents were also introduced into the PSMA-RGS.The biodistributions of 111 In labeled and 99m Tc labeled agents are similar, but the absorbed radiation doses can vary greatly; the total body exposure of 111 In is 20-fold higher than that of 99m Tc (0.1 mGy/MBq vs. 0.0047 mGy/MBq) [44].Considering the total body exposure and availability of the two isotopes, technetium is the preferred choice.
In 31 patients with PCa, 99m Tc-PSMA-RGS yielded a sensitivity of 83.6%, specificity of 100.0%, and accuracy of 93.0%.The minimum diameter of detected LNMs was 3 mm [45] (Table 3).99m Tc-PSMA-RGS has also been introduced in robot-assisted PSMA-guided salvage PLND on the robotic da Vinci ® platform [46].After careful measurement of occupational radiation exposure in all personnel, 99m Tc-PSMA-RGS was subsequently introduced in additional clinical trials [47].During the PSMA-targeted surgery with 99m Tc-PSMA-I&S, the measured effective dose ranged from 0 to 5 μSv, including all the personnel during labeling, quality control, syringe preparation, patient administration, patient imaging, and PSMA-targeted surgery.The mean effective doses for anesthesiology technician, scrub nurse, surgical nurse, and surgeon were 0.6 ± 0.5, 3.2 ± 1.3, 0.2 ± 0.4, and 1.0 ± 0.0 μSv, respectively.The total effective dose would be 320 μSv/year if as many as 100 procedures were performed [47]; the maximum effective dose was safe for all personnel.
In vivo and ex vivo gamma probes (Crystal Probe CXS-SG603; Crystal Photonics, Berlin, Germany) were simultaneously used to remove all metastatic lesions depicted on 68 Ga-PSMA-11 PET [45].The cutoff value to discriminate positive specimens from the background was 4 CPS [45] (Table 3).99m Tc-PSMA-I&S was also validated as a safe and effective imaging technique for SPECT/CT in four healthy volunteers and 10 patients with primary PCa for initial staging [48].The injected activities of 562-828 MBq can be translated into effective doses of 3.33-4.42mSv [48].In an ex situ analysis, 99m Tc-PSMA-I&S RGS discriminated LNMs with a sensitivity of 76.6%, specificity of 94.4%, PPV of 89.4%, and NPV of 86.9% [49] (Table 3).
In summary, 99m Tc-PSMA-RGS has been translated into clinical use, facilitating better detection of positive LNMs than conventional ePLND in patients with PCa.The limitations of 99m Tc-PSMA agents include its inability to detect micro-LNMs and lesions with low to intermediate levels of PSMA.Therefore, it is important to develop new strategies to improve the imaging accuracy for better PSMAtargeted surgery.Current strategies include a combination of near-infrared dyes and dual-target methods.

Considerations regarding PSMA-RGS
Regarding PSMA-RGS, several key areas need further investigation to optimize patient care and outcomes.The follow-up of PCa patients receiving PSMA-RGS needs to be standardized and include an evaluation of whether positive LNMs may be missed in PSMA-RGS and their impact on prognosis.Requirements and patient selection criteria need to be established for PSMA-targeted RGS candidates, to be verified before the injection of radiotracers.Lastly, it is also critical to assess the prescribed imaging strategy, including the first preoperative imaging ( 68 Ga-PSMA-PET/CT) and the subsequent second preoperative imaging (PSMA SPECT/CT) after administering the radiotracer for PSMA-RGS.
To investigate the oncological outcomes of patients with salvage PLND via PSMA-RGS, Knipper et al. showed that, in 94% (343/364) of patients receiving PSMA-RGS, 48.1% (165/343) can reach a PSA level of <0.2 ng/mL at 2-16 weeks after PSMA-RGS, with a 2-year biochemical recurrence-free survival of 32.0% and therapy-free survival of 58.0% [54] (Table 3).Adjuvant systemic therapy should be added to further improve the overall survival in patients with recurrent PCa undergoing PSMA-RGS [55].For PSMA-targeted RGS, both robot-assisted and open surgeries are appropriate for patients with recurrent PCa.Ambrosini et al. compared 24 patients (28%) undergoing robot-assisted RGS with 61 patients (72%) receiving open PSMA-RGS and found that they had comparable oncological outcomes with no safety concerns [42].
PSMA-PET imaging and SPECT/CT imaging should both be performed preoperatively with different purposes.PSMA-PET imaging before or after biopsy provides accurate staging, while SPECT/CT confirms the presence of PSMA-avid lesions and tracer uptake.PSMA-PET/CT can detect nearly double the number of LNMs compared with 99m Tc-PSMA-I&S SPECT/CT during PSMA RGS preoperative imaging [45,56] (Figure 1).As 99m Tc-PSMA-I&S SPECT/CT clearly has an inferior resolution, it should only be considered for preoperative imaging when PSMA PET is unavailable [57].
As shown in Figure 1, patients with suspected PCa are recommended to undergo serum PSA testing and PSMA-PET before biopsy for PSMA-TB and accurate staging.PSMA SPECT/CT is performed after radiotracer injection to confirm specific uptake by PCa lesions.After 3 months, postoperative PSMA-PET is performed to evaluate the surgical outcome.The performance of PSMA-targeted surgery can be improved using similar approaches to those applied to PSMA-targeted theranostic agents, such as 177 Lu-PSMA-617.Both PSMA-targeted surgery and radioligand therapy require a high tumor uptake and TBR, as well as a relatively long circulation half-life.For this purpose, albumin binders have been conjugated with PSMA-targeted radioligands to prolong blood retention time with minimal adverse effects [58].Albumin, as one of most abundant proteins in human serum, exerts an important role as a carrier protein of hormones or fatty acids [58].Its strengths, including nontoxicity, prolonged half-time, biodegradability and absence of immunogenicity enabled it as a promising candidate for theranostic agents [58].The albumin binding Evans blue (EB) moiety has proven to be a safe and effective way to improve the pharmacokinetics of PSMA-617.Compared with 177 Lu-PSMA-617, 177 Lu-EB-PSMA-617 accumulates significantly more in PSMA + PCa tumors, reduces the needed dose of radioactivity [59,60], is safe, and shows improved pharmacokinetics and therapeutic efficacy in 76 patients with progressive metastatic castration-resistant prostate cancer (mCRPC) [60][61][62][63].Another EB modified PSMA radioligand 177 Lu-LNC1003 can further improve therapeutic efficacy by greatly enhancing uptake and retention, even in PCa with moderate level of PSMA expression [64,65].Another series of dual PSMA and albumin binding ligands were also reported, such as PSMA+albumin (RPS-025, RPS-063, RPS-067, RPS-072) [66][67][68].For example, the dual-targeted dye RPS-027 showed higher tumor uptake and increased tumor-to-tissue distribution than MIP-1095 in PSMA-targeted α-therapy [66].Then, a series of ligands (RPS-061, RPS-063, RPS-066, RPS-067, RPS-068, and RPS-069) based RPS-027 was developed for higher tumor uptake and lower kidney uptake, but just in its preclinical models [67].Among all albumin-binding agents, LNC1003 has just finished its Phase 1 trial and showed best potential in clinical translation.In addition to 111 In and 99m Tc, 67 Ga with three low gamma emission energy levels at 93, 185, and 300 keV has also been introduced to synthesize 67 Ga-PSMA I&T, with the aim of overcoming the potential disadvantages of currently used radionuclides such as 111 In, 99m Tc, and 68 Ga [69] (Table 3).With a longer half-life, 67 Ga-PSMA RGS can be performed 1 day after injection of 67 Ga-PSMA I&T, with the option of a further delay of 1 or 2 more days [69].
PSMA PET/CT has also been used for intraoperative imaging [79].Intraoperative PET/CT imaging (AURA10; XEOS Medical, Gent, Belgium) was first used to detect specimens from RP and lymphadenectomy ex vivo in a prospective two-center feasibility study [80,81].This technique can provide a quicker method to detect PSMs in resected specimens than frozen section analysis; however, this type of imaging also has obvious shortcomings.The ex vivo specimen PET/CT imaging cannot determine whether all positive lesions (prostatic lesions and LNMs) have been totally removed by surgery because no postoperative PSMA PET/CT is performed, whether the estimated dose per procedure is safe for personnel, and whether the ex vivo imaging can correctly match the final pathological results of the whole prostate.Therefore, we believe that ex vivo PET/CT imaging of specimens should not be the preferred method for PSMA-RGS.

PSMA-FGS in clinical stage
Compared to PSMA-RGS, PSMA-FGS is a more promising and attractive strategy for intraoperative navigation because it is more intuitive.It is dynamically fitted for the da Vinci surgical system, equipped with a fluorescent imaging system.Currently, OTL78 and IS-002 are two agents for PSMA-FGS that have entered clinical trials [14][15][16] (Table 4).The median size of correctly detected LNMs by PSMA-FGS-using OLT 78 was 1.9 mm (IQR 1.8-2.8),much smaller than that of PSMA-RGS.The exact resolution of IS-002 is yet to be determined.The sensitivity of ex vivo imaging with OLT78 was higher than its in vivo imaging, because the limited range of motion of a laparoscope and limited range of fluorescence may cause difficulties in the in vivo visualization of the apex of the prostate, which is located in the deep region of pelvis [15].Compared with the laparoscope, robotic surgery systems may greatly facilitate deep pelvic visualization and can be better integrated into a robot-assisted fluorescenceguided surgery workflow.
In a single-arm, phase 2a, feasibility trial, Stibbe et al. showed that OTL78 was well tolerated and had the potential to achieve complete oncological resection of PSMs and positive LNMs, with the optimal dose of 30 µg/kg at 24 h preoperatively (Figures 4-5) [15].Similarly, Nguyen et al. conducted a phase 1, single-center, dose-escalation study to evaluate another near-infrared agent, IS-002, in 24 men with high-risk PCa by RP+ePLND [16].The PSMA-FGS with IS-002 achieved a PPV of 97% and NPV of 45% for LNMs, and PPV of 100% and NPV of 80% for locoregional/residual disease detection with an injection dose of 25 µg/kg (Figures 4-5) [16].These studies using OTL78 or IS-002 demonstrate the excellent performance of PSMA-FGS in the detection of PCa lesions and LNMs, as clinicians prefer real-time imaging and radiation-free agents.

PSMA FGS in the preclinical stage
In addition to OTL78 and IS-002, other agents for PSMA FGS are still in their respective preclinical stages (Figure 2L-W).Based on the binding motif, PSMA-targeted agents in FGS can be divided into antibody-conjugated NIRF dyes, small-moleculeconjugated NIRF dyes, and activable NIRF dyes.

Antibody-conjugated NIRF agents
In 2011, a humanized anti-PSMA antibody, huJ591, was conjugated with indocyanine green (ICG); however, huJ591-ICG was quenched until uptake by PSMA + PCa tumors [82] (Table 5).The quenching ability of huJ591-ICG was mainly due to the unique property of ICG, which loses its fluorescence when bound to the antibody, but regains fluorescence when it is separated from the antibody after internalization and degradation of the ICG conjugate in the lysosome [82].Despite the advantages of ICG, such as documented safety and deep tissue penetration, the fluorescence quenching could hinder the visibility and identification of target areas.To maintain fluorescence and bypass the quenching ability of ICG conjugates, a series of IRDyes were developed and conjugated with huJ591.The IRDye800CW is a hydrophilic dye that can be easily synthesized and maintained in vitro and in vivo.
IRDye800CW was introduced to replace ICG to bind huJ591 as ProsaFluor (huJ591-ICG-IRDye800CW) [83] (Table 5).Unlike IRDye800CW, IRDye700DX is another dye that could provide simultaneous NIRF imaging and NIR-infrared photoimmunotherapy, where IRDye700DX, upon activation by NIR light of a wavelength that correlates to the absorption spectrum of the agent, produces reactive oxygen species (ROS) and thus induces localized ROS-mediated cell death in targeted cells.IRDye700DX is a hydrophilic dye that differentiates it from previous hydrophobic dyes, with associated advantages including easier distribution throughout the body without the need for complex solubilizing agents, no aggregation in aqueous solutions leading to less non-specific binding to various tissues, facilitated conjugation with targeting molecules, and therefore overall better biocompatibility.
Both IRDye800CW and IRDye700DX have been approved by the US Food and Drug Administration (FDA) to start clinical trials, indicating their safety for use in FGS.
Antibodies or antibody fragment conjugates can be used for the optical imaging of PSMA-positive tumors.The advantages of antibody conjugates include a long circulation half-life, providing a long time window for FGS, and reliable safety because most of the antibodies have been approved by the FDA for treatment in clinical trials.However, due to the nonspecific uptake of antibodies by the liver and spleen, only a small portion of the conjugates are available to bind the PSMA-positive tumor.Therefore, for PSMA-FGS, a focus has instead been placed on small-molecule conjugated NIRF agents.
To evaluate the performance of the PSMAtargeted agents, PSMA-negative PC3 cells were transfected with a PSMA-expressing lentivirus to obtain PC3-PSMA + cells, which were used to evaluate the efficacy of PSMA-targeted NIRF dyes.Similarly, IRDye700DX was also conjugated to PSMA-617 as IRDye700DX-PSMA for preliminary validation in PC3-PSMA + tumor mice [101].Derks et al. modified the PSMA peptide linker from glutamic acid to a lysine residue and synthesized three dualmodality DOTA(GA)-IRDye700DX-PSMA ligands (PSMA0N01, PSMA-N02, and PSMA-N03) based on the existing PSMA backbone structures (PSMA-1007/PSMA-617) [102] (Table 5).Dell'Oglio et al. developed and validated the performance of intraoperative PSMA-mediated FGS using EuK-(SO3)Cy5mas 3 in a porcine model, demonstrating its clinical translational potential [103] (Table 5).However, while the porcine model enables the study of robot-assisted radical RP within a large animal model, unlike humans or mice, it does not have PSMA-positive tumors; therefore, targeted agents still need further evaluation in mouse models and clinical trials.

Activatable NIRF dyes
PSMA also has glutamate carboxypeptidase (CP) activity.Activatable dyes, namely "turn on/turn off" agents, can highlight tumors with a lower background (Figure 3A-B).Kawatani et al. developed 5GluAF-2MeTG as the first in-class activatable NIRF dye for FGS patients with PCa [104] (Table 5).5GluAF-2MeTG was designed based on previous findings that aryl glutamate conjugates with an azoformyl linker can be recognized by PSMA and have a sufficiently low lowest unoccupied molecular orbital energy level to quench the dye via photoinduced electron transfer [104].After incubation with recombinant human PSMA, 5GluAF-Fl showed significantly increased (>200 fold) fluorescence, which was mainly converted to highly fluorescent fluorescein [104].Xing et al. developed a self-quenching NIRF dye, Cy-KUE-OA, with dual PCa-membrane affinity.The targeted dye was anchored to the phospholipids of the cell membrane of PCa cells, dequenched, and showed strong Cy7 fluorescent light.In addition to in vitro and in vivo imaging in PSMA + PCa tumor-bearing mice, the study further confirmed the performance of Cy-KUE-OA on surgically resected specimens of PCa tissues, LNMs, and healthy tissues from patients [105] (Table 5).Compared to previous preclinical studies, the validation of resected PCa specimens in this study was solid for clinical translation, providing a new method to validate the performance of dyes.The resected specimen validation in this study is an excellent method to validate the performance of dyes, bridging the gap between preclinical studies and clinical trials.

Multi-modality/multi-targeted PSMA-targeted surgery
With regard to the future development of PSMA-guided surgery, fluorescent and radioactive tracers are complementary and used in combination with multi-modality-guided surgery [106].Because PSMA-RGS and PSMA-FGS have their own strengths and shortcomings, multi-modality PSMA-targeted dyes with radiotracers and fluorescent dyes in a hybrid tracer were simultaneously developed for PSMA-RGS and PSMA-FGS (Figure 3 C-F).Integrating radioactive and fluorescent signatures into a single dye can enable surgeons to improve their intraoperative performance.The PSMA-11-based multi-modality agent 68 Ga-Glu-urea-Lys-HBED-CC-IRDye800CW ( 68 Ga-PSMA-11-IRDye800CW) also achieved accurate intraoperative detection of PSMA + PCa tumors in tumor-bearing mice and healthy pigs [107].To overcome the nonspecific accumulation of PSMA-11 in non-malignant tissues, a histidine-(H) and glutamic acid (E)-containing linker ((HE) 3 -linker) was introduced between the chelator and the PSMA-binding motif [108].Compared to 68 Ga-PSMA-11-IRDye800CW, 68 Ga-Glu-urea-Lys-(HE)3-HBED-CC-IRDye800CW ( 68 Ga-PSMA-914) can be used for preoperative PET imaging and PSMA-guided PARP with faster excretion and a lower background signal [109].
Another agent, NYM016, was designed using the NIRF dye Cy7 and NOTA chelator for 68 Ga labeling. 68Ga-NYM016 was evaluated in a patient with recurrent PCa with metastatic lesions, showing an SUV max of 18.93 in recurrent lesions [110].Furthermore, Aras et al. synthesized a multimodality agent, 18 F-BF3-Cy3-ACUPA, and determined its safety and feasibility in 10 PCa patients by preoperative PET and intraoperative FGS [111].Harmatys et al. designed 64 Cu-LC-Pyro, a low molecular weight PSMAtargeted agent with long plasma circulation time and high tumor-to-background ratio (10:1) for PET/CT, NIRF imaging and NIR-PIT at the same time [112].
Cordonnier et al. designed an oleic acid-coated spherical NaYF4:Yb,Tm@NaYF4 core/shell upconversion nanoparticle (UCNP) labeled with 125 I for PSMA-targeted FGS/RGS in LNCaP-Luc-bearing mice [113].Cheng et al. developed a theranostic nanotexaphyrin for PSMA targeted SPECT/CT imaging and PIT through the chelation of metal isotopes ( 111 In/ 175 Lu), achieving a TBR of 2.7 [114].Although the multi-modality method combines the strengths of PSMA-RGS and PSMA-FGS, challenges have also been exemplified.For instance, the diagnostic standard for PCa lesions and LNMs in multi-modality PSMA-targeted surgery, decisions regarding contradictory results of PSMA-RGS and PSMA-FGS, and the hybrid modality weakening the performance of each imaging modality remain to be elucidated.Radiation and contradictory results from multi-modality imaging may limit its further development and clinical translation.[16].(E) White light and sensitive Firefly images of an area where residual disease was identified in the anterior apical prostate resection bed near the urethra.Sensitive Firefly pinpointed (Fig. 2E) highly specific IS-002 fluorescent signal in the resection bed not deemed suspicious in white light endoscopy that was subsequently biopsied [16].(F) White light and sensitive Firefly images after biopsy.Insert in Figure 1F shows a strong fluorescent signal in the biopsied tissue, which was confirmed tumor positive following frozen sectioning.Further inspection of the prostate surface revealed (Fig. 2E) a fluorescent signal corresponding to a transected focal tumor extension.Gold arrows indicate biopsy location; gold stars mark transected tumor extension on prostate surface in Figures 2D and 2F.Dotted white lines (Fig. 2E) indicate instrument and prostate boundaries [16].The figures(A-C) were adapted with permission from [15,52], copyright 2022 Lancet Oncology © Elsevier, Inc.The figures (D-F) were adapted with permission from [16], copyright 2023 Eur Urol Oncol © Elsevier, Inc.It is well known that PSMA expression is heterogeneous and can vary between patients, lesions in the same patient, or even within the same lesion.To better recognize all LNMs during PSMA RGS, multi-targeting methods have also been attempted (Figure 3G-K).Integrin αvβ3 is a reasonable co-target with PSMA for neo-vasculature which incorporates the Arg-Gly-Asp (RGD) binding motif.EUKL-cRGDfK-IRDye800 could be used to illuminate either the PSMA + or integrin αvβ3 + xenografts in mice [115].Dual-targeting agents with PSMA-targeting ability and complementary molecules have also been introduced [116].PSMA and gastrin-releasing peptide receptor (GRPR) are complementarily expressed in PCa tumors, as has been validated in prostatectomy samples and patients [117][118][119].For targeting GRPR, RM-26 (GRPR antagonist) and its dual-targeting radiotracers (RGD-RM26-03, denoted as LNC1015) have showed favorable pharmacokinetics and high clinical feasibility for PET/CT imaging of cancer [120][121][122][123][124]. Similarly, Schollhammer et al. also retrospectively studied 20 frozen samples with various GS group using 111 In-PSMA-617 and GRPR antagonist 111 In-RM2 [117].They found that 111 In-PSMA-617 had a higher uptake in the higher GS group, whereas 111 In-RM2 had higher binding in the lower GS group [117].The dual-tracer method was also validated in recent clinical studies.Qiu et al. also showed that dual-tracer (NOTA-P2-RM26 and PSMA-617) PET/CT-targeted biopsy (TB) achieved a higher PCa detection rate (69.77%) than systematic biopsy SB (29.29%) or mpMRI-TB (36.14%) [125].GRPR/PSMA TB is better than GRPR TB or PSMA TB alone: the csPCa omission diagnostic rate of dual-tracer PET/CT-TB (15.38%) was significantly lower than 68 Ga-GRPR PET/CT-TB (48.72%) or 68 Ga-PSMA PET/CT-TB (28.21%) [125].Recently, a PSMA/GRPR-targeting radioligand named 68 Ga-BQ7812 was also developed but just in preclinical stage [126].This compound holds promise for increasing the chance of specific tumor binding, thereby improving detection accuracy, especially in patients with heterogeneous expression of either target.Fibroblast activation protein alpha (FAP) is a transmembrane protease highly expressed in the tumor microenvironment of PCa [127].Boinapally et al. developed small-molecule PSMA-and FAPtargeting moieties with 64 Cu, named 64 Cu-FP-L1 and 64 Cu-FP-L2, respectively [116], with the potential to be used for PSMA-FAP dual-targeted guided PCa surgery.These PSMA/FAP dual-targeted radioligands enable imaging of lesions expressing FAP, PSMA, or both on the tumor cell surface or within the tumor microenvironment.These multi-targeted dyes were developed for patients with varying levels of PSMA expression in PCa tumors, including those with high, low, or no PSMA expression.While as nearly all treatment-naïve patients receiving PSMA-RGS had PSMA-positive PCa tumors expressing 10-100-fold more PSMA than surrounding benign prostatic tissues [5] and loss of PSMA expression is commonly observed in patients with CRPC who do not usually undergo further surgery, a well-performing PSMA single-targeted dye may suffice for most clinical needs in PSMA-RGS or PSMA-FGS, multi-targeted conjugates offer promise for patients with PCa tumors displaying low or reduced PSMA expression.These conjugates provide additional advantages for managing heterogeneous tumors.
Thus, we believe that establishment and clinical validation of a well-performing PSMA-targeted dye (such as LNC1003) with reliable safety and a long operation time window can meet the greatest clinical need for PSMA-targeted surgery.For a better understanding of the development of PSMA-targeted surgery, a chronological timeline highlighting major events and significant agents for PSMA-targeted guided surgery was shown in Figure 7.

Conclusions
PSMA-targeted surgery can lead to significant changes in traditional surgical procedures for PCa because of its high sensitivity in the detection of PSMs and micro-LNMs compared to any preoperative imaging or visualization.PSMA-targeted surgery may benefit PCa patients with PSMA-TB because of a more accurate pathological diagnosis by PSMA-RGS or PSMA-FGS for a more accurate resection of all prostatic and metastatic lesions.PSMA-TB, PSMA-RGS, and PSMA-FGS should be used as overall strategies for patients with primary csPCa or recurrent PCa, thereby greatly facilitating clinical diagnosis and surgical treatment.Among the above strategies, PSMA-RGS has entered clinical use in many trials, but we believe that PSMA-FGS will be a developing trend in the near future, as it is radiation-free and intuitively used in real-time within surgery.The multi-targeted method may be more suitable for CRPC radiotherapy than PSMA-targeted surgery.
Multi-modality methods have strengths, but the shortcomings of both agents and contradictory results may limit their use.Importantly, the close correlation among preoperative imaging, PSMA-TB, PSMA-FGS, and follow-up evaluations after surgery requires further investigation.However, a well-performing clinical standard to select the most suitable candidates for PSMA-RGS and PSMA-FGS is still lacking.More attention should be paid to potential PSMA-FGS dyes, and their efficacy should be validated in more clinical trials in the near future.With the rapid development of PSMA-targeted dyes, PSMA-targeted surgery, and its related standard flow would completely change conventional surgical methods and improve the level of precision surgery for all patients with PCa.

Figure 1 .
Figure 1.The procedures of PSMA-targeted RGS/FGS.The SPECT/CT on a day before PSMA-RGS can ensure that the radiotracers for PSMA-RGS have been circulated to the PCa lesions and LNMs.

Figure 4 .
Figure 4. Robot-assisted 99m Tc-PSMA radio-guided surgery procedure.Preoperative (A) CT, (B) PSMA PET/CT, and (C) 99m Tc-PSMA-I&S SPECT/CT imaging demonstrate a prevesical lesion (indicated with an arrow) in a patient with biochemical recurrence after primary treatment for prostate cancer.(D) During the surgery, the DROP-IN probe is inserted in the abdomen through a trocar (E) and is autonomously maneuvered by the surgeon using a Da Vinci surgical console.(F, G) In vivo radioactivity measurement of a suspected prostate cancer recurrence.(H) Ex vivo radioactivity measurement of the resected specimen.Histological analysis revealed a lymph node metastasis on (I) hematoxylin and eosin staining and (J) PSMA immunohistochemistry. CT = computed tomography; PET = positron emission tomography; PSMA = prostate-specific membrane antigen; SPECT = single-photon emission CT.The figures were adapted with permission from [52], copyright 2022 European Urology © Elsevier, Inc.

Figure 5 .
Figure 5. Fluorescence workflow for surgical margin in apical prostate resection of OTL78(A-C) and IS-002 (D-F).(A)The entire prostate is imaged directly after resection ex vivo on the backtable.Increased fluorescence signal is visible at the apex[15].(B) Subsequent assessment of the resection bed shows residual fluorescence signal (indicated with an arrow) where the apex used to be[15].(C) A fluorescent, PSMA-expressing positive surgical margin is seen on ex vivo gross macroscopy[15].(D) White light and sensitive Firefly image of the excised prostate 24 h after IS-002 injection (100 lg/kg) showing a strong, focal fluorescent signal in the primary tumor[16].(E) White light and sensitive Firefly images of an area where residual disease was identified in the anterior apical prostate resection bed near the urethra.Sensitive Firefly pinpointed (Fig.2E) highly specific IS-002 fluorescent signal in the resection bed not deemed suspicious in white light endoscopy that was subsequently biopsied[16].(F) White light and sensitive Firefly images after biopsy.Insert in Figure1Fshows a strong fluorescent signal in the biopsied tissue, which was confirmed tumor positive following frozen sectioning.Further inspection of the prostate surface revealed (Fig.2E) a fluorescent signal corresponding to a transected focal tumor extension.Gold arrows indicate biopsy location; gold stars mark transected tumor extension on prostate surface in Figures2D and 2F.Dotted white lines (Fig.2E) indicate instrument and prostate boundaries[16].The figures(A-C) were adapted with permission from[15,52], copyright 2022 Lancet Oncology © Elsevier, Inc.The figures (D-F) were adapted with permission from[16], copyright 2023 Eur Urol Oncol © Elsevier, Inc.

Figure 7 .
Figure 7. Chronological timeline highlighting major events and significant agents for PSMA-targeted guided surgery.

Table 2 .
Isotopes and relevant characteristics for PSMA-targeted radionuclides.

Country Retro/ pros Patient number Patient type Preoperative staging Gamma probe Agents for PSMA-RGS Age tPSA (ng/ml) GS sensitivit y specificit y PPV NPV Accuracy
In 2015, Maurer et al. first traced LNMs intraoperatively with a gamma probe in five patients receiving injections of

Table 5 .
Potential agents for PSMA-FGS in the preclinical stage