A pan-cancer agent for screening, resection and wound monitoring via NIR and SWIR imaging

Fluorescence guided surgery (FGS) facilitates real time tumor delineation and is being rapidly established clinically. FGS efficacy is tied to the utilized dye and provided tumor contrast over healthy tissue. Apoptosis, a cancer hallmark, is a desirable target for tumor delineation. Here, we preclinically in vitro and in vivo, validate an apoptosis sensitive commercial carbocyanine dye (CJ215), with absorption and emission spectra suitable for near infrared (NIR, 650–900nm) and shortwave infrared (SWIR, 900–1700nm) fluorescence imaging (NIRFI, SWIRFI). High contrast SWIRFI for solid tumor delineation is demonstrated in multiple murine and human models including breast, prostate, colon, fibrosarcoma and intraperitoneal colorectal metastasis. Organ necropsy and imaging highlighted renal clearance of CJ215. SWIRFI and CJ215 delineated all tumors under ambient lighting with a tumor-to-muscle ratio up to 100 and tumor-to-liver ratio up to 18, from 24 to 168 h post intravenous injection with minimal uptake in healthy organs. Additionally, SWIRFI and CJ215 achieved non-contact quantifiable wound monitoring through commercial bandages. CJ215 provides tumor screening, guided resection, and wound healing assessment compatible with existing and emerging clinical solutions.


Introduction
Surgery is often the rst and sometimes (ideally) the only treatment a cancer patient will undergo.Tumor surgery is evolving but relies on a surgeon's ability to innately determine tumor from healthy tissue under visible light inspection.Fluorescence guided surgery (FGS) improves tumor identi cation and delineation by visualizing uorescent dyes selectively accumulating in tumors.Fluorescence imaging and FGS are heavily used preclinically but not yet widely adopted clinically, with multiple barriers to be overcome by compounds and devices. 1 These limitations include the lack of required expertise in clinical domains and ultimately the need to prove utility to patient outcomes, despite the clinical dominance of optical techniques. 2 Targeted and passive uorophore accumulation imaged via FGS can dramatically improve a surgeon's capability to resect a tumor, aid in nding smaller lesions, identify lesions not detected by conventional methods, and locate lesions which move between pre-op scans and surgery (e.g.bowelassociated). 3,46][7][8][9] Therefore, a novel compound must be developed for each tumor type to be targeted, preclinically tested and then subject to clinical approval, in a time (over a decade) and nancial (~$100-200 million) very costly process. 10,11The "by the minute" charges for surgical room use (avg.USD$46.04/min in 2022) where FGS requires additional time further hinders translation, preventing preclinically validated uorophores from having the highest possible impact. 3,12,13FGS patients normally receive a single dose of the compound, and this (combined with the high costs of bringing such a compound to market) provides yet another barrier to translation. 10,14To overcome this pan-tumor agents are the of some of the highest value in biomedical cancer research, not only for FGS, utilizing e.g., the known lower pH environment of most tumors. 157][18] Cellular damage and apoptosis are highest within the core of a tumor, where hypoxia and reduced blood ow form a non-sustainable environment. 19,20][23][24] Current solutions have neither been clinically translated nor are they produced in larger quantities.There is a clear demand for a scalable solution which should be easy to use, handle, and administer.Additionally, a dye sensitive to apoptosis could potentially have minimal uptake and therefore background in healthy tissue, improving tumor delineation via clearance from non-cancerous (nondamaged) areas.This would inherently provide a pan-cancer targeting approach for numerous clinical applications, and potentially scenarios outside of tumor resection where apoptosis plays a role.Having multiple dye applications could offset current market barriers and improve patient outcomes. 25 this work, we have investigated the potential for delineation of multiple solid tumors and monitoring of wound healing in vivo via a single NIR/SWIR-emitting small molecule dye, CJ215.CJ215 is a commercial, carbocyanine dye, with a molecular weight of ~1330g/mol and is inherently tumor targeting without antibody, peptide or ligand conjugation. 26CJ215 has a strong NIR uorescence band (absorbance from 795-820 and peak emission from 810-840 depending on solution), and like ICG, extends to the SWIR region, potentially useful for SWIRFI in clinical settings.We show cellular localization of CJ215 and its general in vivo tumor speci city.2][33][34][35] CJ215 tumor uptake could be detected with both SWIRFI and NIRFI.SNR and CNR assessment with SWIRFI were performed during screening and resection at >900 nm (sensor response) for all tumor lines and additionally at >1100 and >1300 nm long pass cut offs (via long pass lters) for two tumor lines.Finally, apoptosis is also a known component of wound healing.We therefore evaluated CJ215 for quantitative non-contact and ambient light resistant longitudinal wound assessment through commercial bandages without removing them.7][38] This work establishes CJ215 not only as a pan cancer agent, which can delineate multiple solid tumors with a single systemic intravenous injection but also an agent with applications beyond tumor surgery.We further take advantage of the transparency of plastic compounds in the SWIR region to enable SWIRFI for wound assessment through bandages. 39This work aids the establishment and translation of uorescence for clinical domains, extending beyond FGS and into post-operative care.

Results
Spectral characterization of CJ215: CJ215 is a small molecule carbocyanine dye with a molecular weight of approx.1330g, Supp. 1. First, we pro led the spectral absorption of CJ125 and determined whether it had any SWIR emissive tail, as has been demonstrated for ICG and ICG conjugated cancer targeting probes, Fig. 1 A. 6,40,41 We found that CJ215 underwent a spectral red shifting similar to that seen with ICG when dissolved in fetal bovine serum (FBS), Fig. 1 A. 42 This red shift in FBS was noted at a pH of 4, 6, 7.4, 8 and 10 with CJ215 being highly stable in FBS over 150 mins at all tested pHs, but degrading rapidly in dextrose at a pH of 4, Supp.2. CJ215 underwent further red shifting when dissolved in human serum albumin (HSA), and FBS at a pH of 7.4, followed by a slow blue shift of ~5 nm in HSA and FBS over the course of 96h, Supp.3. Little to no degradation (loss of absorption) of CJ215 was seen in both HSA and FBS.Although the NIR emission of the dye has been shown by the manufacturer, the SWIR emission and SWIRFI capability of the dye was yet unknown.To assess this, we dissolved CJ215 in dextrose, as well as pure or diluted (in PBS) de brinated sheep's blood to simulate human blood, Supp. 4. The peak absorption and emission of CJ215 red shifted by ~25nm in the presence of blood versus dextrose (794 to 818 and 813 to 838 nm, respectively) with SWIR emission extending to ~1550 nm, Fig. 1 B and Supp.4.
Additionally, the dye underwent an increase in uorescence intensity when present in serum or blood across this spectrum, further enhancing its suitability for in vivo imaging. 43We assessed the ability of SWIRFI to potentially improve tumor delineation through scattering media, by imaging CJ215 through 5 mm of chicken breast, Supp. 5. CJ215 was readily detected at 900 (InGaAs sensor response cutoff), 1000, 1100, 1200 and 1300 nm long pass cut offs (achieved via long pass optical lters) with delineation improving with extended (longer) wavelengths.This improvement is attributed to lower auto uorescence, insensitivity to excitation wavelength (808nm) and reduced stray photon scatter via increased water absorption in this realm of the spectrum, Supp. 5. 44,45 In vitro assessment: Following characterization of the spectrum of CJ215 we assessed the level of CJ215 uptake in vitro in both 4T1 (murine breast cancer) and HT1080 (human brosarcoma) cell lines.We rst assessed the subcellular localization of the dye via NIR point scanning confocal microscopy.After 3hrs of incubation in 4T1 single cells and 3D spheroid structures, CJ215 was found to be localized to mobile vesicles inside both cells and spheroids, likely taken up via endocytosis Fig. 1 C, D. To further assess the uptake of CJ215, 4T1 cells were incubated with the dye for 3 hrs at either 4°C or 37°C.At 4°C endocytosis, as an energy-consuming process is impaired. 46Indeed, CJ215 uptake was reduced by 39.1% in cells at 4°C, indicating that CJ215 is actively transported into cells, Fig. 1 E. We also assessed the effect of inducing apoptosis via staurosporine (Sta) on CJ215 uptake in HT-1080 ( brosarcoma) cells.The addition of staurosporine (Sta), an apoptosis inducer, increased CJ215 cellular uptake by 99.3% compared with untreated control cells, while addition of z-VAD FMK, an inhibitor of Sta induced apoptosis, reduced that uptake by 48.8% (to 51.1%) compared to Sta alone, Fig. 1 F. 47 ICG incubation under comparative conditions did not yield any signi cant change in cellular uptake, Supp.6.Finally, we found that serum-starved 4T1 cells had a 54.3% higher uptake of CJ215 than cells incubated in the presence of serum, as has been shown for other dyes, Supp.7. 43,48 Following xation with PFA, cells retained CJ215 uorescence levels up to 9 days post xation, Supp.8.
In vivo screening: Having determined that induction of apoptosis increased cellular uptake of CJ215 and that the dye was predominantly taken up by cells via active transport, we next assessed CJ215's capability for preclinical tumor screening across four tumor lines in vivo, in mice implanted with a single lesion site.With lower apoptosis levels found in healthy/normal tissues, we hypothesized that tumors would be the main site of CJ125 retention.We utilized a SWIRFI system equipped with an 808 nm laser (300 mW/cm 2 ) to assess CJ215 uptake in male and female mice, across FoxN1 nu , SCID and BALB/c mouse lines under ambient lighting conditions. 41The tested tumor lines comprised xenografted mice bearing breast (4T1, murine, orthotopic), prostate (PC3-PSMA, human, heterotopic), HT1080 ( brosarcoma, human, orthotopic) and CT26 (colon, murine, heterotopic) tumors, Fig. 1 G.All mice were imaged every 24 hr from 1 to 96 hrs and nally at 144 or 168 hrs post injection, Fig. 1 H.The SWIRFI system (>900nm, sensor response) enabled video rate (30 Hz) acquisitions under ambient lighting conditions and readily achieved su cient SNR (>5dB) at all tested timepoints with exposure times as low as 1 ms, Fig. 1 H.In all cases the dye showed highly selective tumor uptake, with su cient SNR achieved within 1 hr post injection.Contrast (CNR), the ability to delineate the tumor from healthy tissue achieved su cient levels (>3dB, Rose criterion) from 24 to 168 hrs, with CNR increasing in all cases with elongated clearance time, Fig. 1 I. 49,50 Images from all mice at all timepoints are also presented, Supp.9-12.All SWIRFI presented throughout this manuscript was performed with the ambient lighting LED on, at no detriment to image delity. 41mor resection: post-euthanasia tumor resection was con rmed in all mice using SWIRFI again under ambient lighting conditions.The assumed primary tumor was resected, and both the excised tumor and remaining tissue (i.e., the entire body) were placed side by side within the SWIRFI system, Fig 2 A. In all cases, the primary site contained enough uorescence to delineate the tumor, while also con rming that the tumor bed was free of residual uorescence.If suspected remnant tumor was left behind, as noted by highly uorescent areas this was then resected.For all tested tumors the primary and potential secondary sites had su cient SNR, Fig 2 B, for detection along with su cient CNR, Fig. 2 C, to be delineated from the tumor bed site.Resection images for all mice and all tumors are also shown, Supp.13-16.
Extended spectral emission of CJ215 assessment: The extended spectral emission of carbocyanine dyes into the SWIR spectrum, which only SWIRFI systems can detect, provides improvements in delineation and resolution, especially through scattering media. 44,51We compared the extended spectral emission of CJ215 in HT1080 and CT26 tumor uptake at >900 (sensor response), >1100, and >1300 nm (via long pass optical lters).This was performed to assess increased resolution and penetration via water absorption, which begins to increase >1100 and further increases >1300 nm, Supp. 5. 45 In all cases CJ215 could be detected but as expected, required increasingly longer exposure times at longer wavelength cutoffs (1-2 ms for >900 nm, 100-200 ms for >1100nm and 500-1500ms for >1300nm cutoffs).Tumor SNR was su cient at all timepoints, with CNR permitting tumor delineation for HT1080 tumors at 1 hr but not for the CT26 line, Supp.17.Data combined from both lines, permitted a clearer SNR and CNR assessment and comparison between timepoints.The effect of these cutoffs for resection e cacy was also assessed in both primary sites and potential secondary locations, Fig. 3 A. In all cases, the >1300 nm cutoff provided the lowest SNR and CNR, with >1100 and >900 performing comparably Fig. 3 B,C.a similar trend was identi ed for screening, Supp.17.However, the >1300 nm cutoff did improve delineation of the tumor core for both tested tumor lines, Fig. 3 A, E, F. We found dye localization and core delineation improved both qualitatively and quantitatively at extended wavelengths, most obviously at 1300nm, Fig 3 E, F. Using immunohistochemistry (IHC) for cleaved caspase 3 (CC3, a known apoptosis marker) we con rmed that the core of the tumors had the highest levels of apoptosis, with increased apoptosis observed throughout the tumor, Fig 3 G. 52 CJ215 biodistribution: Whilst SWIRFI is an emerging technique with advantages over established methods, due to the unavailability of imaging systems it remains a restricted resource to many researchers with even fewer clinical options.Additionally, the SWIRFI system employed here had a relatively small FOV, limiting simultaneous imaging of multiple organs.To highlight the suitability of CJ215 for conventional NIRFI, the IVIS Spectrum system (silicon based sensor) was utilized for biodistribution assessment. 53Resected organs were imaged simultaneously with uorescence levels then quanti ed to highlight the various tumor to organ ratios.In all cases, the tumor was always the brightest tissue, achieving tumor:muscle ratios up to 99.63, Fig. 4. For nearly all models, minimal to no remnant uptake was seen in healthy tissues, resulting in e.g., tumor:spleen ratios of up to 47.7 or tumor:liver of 17.86, Fig. 4. Residual uorescence seen in the kidneys highlights the renal clearance of CJ215.Biodistribution from all tumors and all mice are shown along with corresponding H&E staining of select organs, Supp.18-25.During the resection of CT26 tumors, additional uorescence was determined deeper and in proximity to the resected tumor, Fig. 5 A. In two mice (M3, M4) additional areas of increased CJ215 uorescence were identi ed below the resected tumor.These areas were also isolated (Fig. 5 B (ROI-2)) and sent with the primary tumor and select organs sent for histological analysis, Fig. 5  C, D, E. When compared with tumor tissue, these tissues had lower CJ215 uptake (but still elevated over other organs) and were found to be tumor free.These areas were identi ed as small intestine, lymph node, uterine and ovarian tissue with the additional focus in M3 containing a small tumor fragment.Further, IHC staining for cleaved caspase 3 (CC3) Fig. 5 D, an established marker for apoptosis, found increased levels of apoptosis in these tissues.As expected, CT26 primary tumor sites had highly elevated levels of CC3 positive cells, especially within the core of the tumor, Supp.26.The additionally resected tissues which had increased CJ215 uorescence also displayed increased levels of CC3 positive cells, Fig 5 E. 52 Contrast-based SWIRFI for binary tumor delineation: Having determined that simply letting CJ215 clear from healthy tissues improved tumor delineation, we utilized further image processing to improve tumor delineation.This was done with the goal of presenting a surgeon with an e cient method of image display when using CJ215, based upon their selection of a non-tumor background reference point.The entire process was completed using ImageJ with the chosen region recorded, the mean and SD of the ROI calculated, followed by respective framewise and pixelwise correction according to widely established CNR calculations, Eq.1.The resulting image is then displayed as a contrast-based image expressed in CNR (dB), dubbed "contrast mode", Fig 6 A. SWIRFI and CJ215 CNR images can readily provide a surgeon with binary tumor delineation with a 3dB threshold (Rose criterion) found to be effective for all tested tumor lines as seen in Fig 6 B.
Assessment of CJ215 in a metastatic tumor model: The four previously four tumor models are readily and widely used for the assessment of tumor targeting of both uorescent, radioactive, and treatment agents.However, these models represent a single tumor location near the skin surface, where CJ215 could be readily visualized.One signi cantly more challenging location and urgent clinical problem is the surgical removal of peritoneal carcinomatosis. 54,55SW1222 tumors are a human derived colorectal carcinoma tumor line which is highly aggressive and spreads throughout the peritoneal space.This heavily invasive and often palliative surgery requires organ removal during surgery whilst the body cavity is inspected via white light investigation for remnant tumor and would bene t signi cantly from a suitable targeted dye for FGS.This model of metastatic peritoneal spread consisted of SW1222 luciferase expressing patient-derived colorectal cancer cells injected intraperitoneally. 55Once su cient tumor burden was con rmed using luciferase imaging, animals were administered CJ215 as before.
SWIRFI was performed at >900, >1100 and >1300 nm cut offs to assess tumor screening.Tumor visualization of the SW1222 model without prior knowledge of lesion location was initially di cult to distinguish over endogenous colon signal, Fig. 7 A, B, C Supp. 27.As before, necropsy biodistribution was performed with NIRFI at 168 hrs post injection, Fig. 7 D, Supp.28.We found that tumors were widely dispersed throughout the peritoneum were highly necrotic (essentially dead tissue), with patchy uorescence seen across identi ed tumors, Fig. 7 B, C. In all mice, highly uorescent lesions were seen adjacent to the spleen often engul ng the pancreas (Splenic Tum.), Fig. 7 D and were con rmed to be tumorous via H&E staining, Fig. 7 E Wound monitoring: During hair depilation in the CT26 model slight skin surface damage was inadvertently caused which then proceeded to form scabs and heal over time.We incidentally noticed high levels of CJ215 uptake in these areas, along with tumor uptake, Supp.30.Over the 168hr imaging period, the wound uorescence decreased with progressing wound healing but remained unchanged in the tumor.Spurred on by this nding, we assessed the ability of SWIRFI to image CJ215 through a variety of commercially available bandages, Supp.31.SWIRFI readily detected CJ215 through all tested bandages, the majority of which were opaque in visible light.Encouraged by this observation, we next implemented a controlled wound monitoring experiment where mice underwent a skin incision with a scalpel, followed by absorbable stitch closing, and were intravenously administered CJ215.Hydrogel burn bandages were placed over the wound during imaging as they provided a non-stick barrier at the wound site, are highly scattering, hindering visible light wound inspection, and provided suitable intensity losses and SNR changes in phantom testing, Supp.31.Mice were imaged at >900, >1100 and >1300 nm with and without a bandage from 2 to 240 hrs (10 days) post-surgery and CJ215 injection, Fig. 8 A, B and Supp.32-35.The bandage was only placed on mice during imaging and was removed before they awoke to prevent discomfort to mice, inadvertent bandage consumption, or bandage degradation.We found that after a single injection of CJ215 the intensity of dye uptake corresponded with wound healing progression, using CNR as a metric for wound assessment, with >1300 nm providing the best wound CNR, along with the wound being readily delineated through the bandage during the main healing phase, Fig. 8 C. The reduction in signal by the bandage prevented reliable wound delineation at 168 hrs and onwards.Wound CNR was detectible from 1-2 hours post injection, highlighting the quick uptake and targeting to wounds of CJ215.CNR peaked 48 hrs post injection then dropped over time until the wound was no longer detectible i.e., completely healed, with longer exposure times utilized between 72 & 240 hrs post injection to ensure su cient signal collection.CNR was found to provide a quantitative metric for wound healing assessment in line with visible light inspection of wound healing but, importantly, also useable when removal of wound dressing is detrimental to the patient.To con rm lack of wound uptake once healed, mice then received a second injection of CJ215 and were imaged every 24 hrs from 1 to 48 hrs post injection.As opposed to right after the initial surgery, su cient contrast could not be detected within the now healed wound region in all mice, with necropsy based biodistribution showing no signi cant difference in uorescence intensity in the wound area versus normal skin, Supp.36.H&E further con rmed the normal physiology of the wound area, Supp.37.

Discussion
In this work, we have validated the use of the commercial carbocyanine dye, CJ215, for tumor delineation in preclinical cancer models as well as monitoring of naïve and dressed wounds.CJ215 ful lls many requirements for clinical translation and importantly, is optically like ICG and therefore compatible with the wide array of already existing clinical FGS systems, Fig 1 A. CJ215 can take advantage of the reduced optical tissue scatter and absorption in the NIR, which aside from FDA approval, attributes the dominance of ICG.Furthermore, CJ215 has a strong SWIR emission tail, Fig 1 B, which provides advantages over NIR and visible emission ranges. 44We set out to elucidate the potential tumor targeting capability of CJ215 by assessing its uptake in vitro.CJ215 appeared to be endocytosed and spread across multiple mobile vesicles within 4T1 single cells and spheroids, Fig 1 C, D, Supp.Videos 1, 2. CJ215 uptake is increased in cells which are actively metabolic, as shown by the incubation at 4° vs 37°C , Fig 1 E. We show that induction of apoptosis (cell death) with staurosporine doubled cellular uptake of CJ215 in HT1080 cells, while apoptosis inhibition tempered this uptake, indicating that the dye is more readily taken up by apoptotic cells Fig 1 F. 47 Notably, in the presence of FBS (serum) cellular uptake of CJ215 was reduced by over 50%, Supp.7, with CJ215 found to be stable (still uorescent) in formaldehyde over the course of days, Supp.8. CJ215 showed excellent uptake in a variety of tumor models in vivo, representing some of the most prevalent cancers including breast (4T1), prostate (PC3-PSMA), brosarcoma (HT1080) and colon (CT26), Fig 1 G. High uptake was seen in all models across multiple mouse lines, both in male and female mice, with human and murine derived tumors in various orthotopic and heterotopic locations, Fig 1 G.Our previously established ambient light resistant SWIRFI method and CJ215 could delineate tumors within 1 hr post injection, with highly conclusive delineation at 24 to 168 hrs, Fig. 1 H, I. 41 Tumor contrast steadily increased over time, in line with previous investigations with other carbocyanine based probes, 41 Tumor resection performed on euthanized mice found that SWIRFI with CJ215 enabled con rmation of tumor resection, identi cation of remnant tumor, and highlighted other suspect regions with the tumor site always having the brightest uorescence, Fig 2 .SWIRFI enabled investigation of the extended spectral emission of CJ215 which current silicon based sensors cannot detect, Supp 5. 6, 40,51 We assessed the reduction in photon scatter of CJ215 by increased water absorption at >1100 and >1300 nm long pass cutoffs to improve tumor CNR, Fig 3 .For both screening and resection these wavelengths ultimately did not provide a major advantage in tumor delineation, due to the high tumor uptake of CJ215 and at the cost of non-video rate imaging, Fig 3 B-D.
However, the >1300nm cutoff provided both qualitative and quantitative non-destructive insight to CJ215 distribution within the tumor core, highlighting tumor heterogeneity, Fig 3 E, F. This core uptake was re ected in CC3 (apoptosis) staining via IHC, con rming the observations made via SWIRFI, Fig 3 E, G. SWIRFI has optical bene ts but remains a novel and often inaccessible tool due to limited access to suitable imaging equipment.The utilized SWIRFI system has a reduced FOV (a single mouse), making multiple organ comparisons cumbersome.We performed NIRFI necropsy biodistribution post resection on an IVIS system with a larger FOV (up to 5 mice), further con rming both CJ215 tumor localization and suitability for NIRFI, The exact cause of cellular damage and apoptosis in these regions is unclear, however they were predominantly near the primary tumor site and may have been compressed as it proliferated, causing cell stress and apoptosis.
Con rmation of the high tumor uptake, seen during SWIRFI screening, spurred us to present images in a way that would be most useful for FGS.Image presentation during surgery is an integral component of FGS when assessing novel systems and probes, and ultimately is where impact and utility of the approach are determined. 60 With the success and high tumor localization of CJ215 in single site tumor models, we then assessed its potential in a more challenging way.The SW1222 cell line is a human derived, highly aggressive, and metastatic tumor model which spreads throughout the peritoneal space.Current clinical treatment includes surgical removal, where white light visual inspection is utilized in a highly invasive surgery.This procedure comprises organ repositioning with peritoneal inspection and often fails to detect and remove all lesions.CJ215 was assessed in one such model where mice received SW1222 cells injected intraperitoneally, Fig 7 A. 55 Tumor growth was con rmed with luciferase, but SWIRFI and CJ215 (even at extended wavelengths e.g., >1300nm) did not conclusively delineate tumors through the peritoneum, Fig 62 This is further highlighted by the high presence of disrupted blood vessels and necrotic tissue morphology in H&E staining of main tumors, versus the more viable and less necrotic splenic/pancreatic tumors which presented higher tumor:muscle ratios.
Hair depilation during CT26 tumor imaging in icted minor wounds.These wounds showed high CJ215 uptake, with CJ215 clearing from them over time as they healed but remaining in the adjacent tumor, Supp.Fig 30.This feature of CJ215 uptake is reminiscent of previous descriptions of tumors being wounds which do not heal. 63SWIR imaging is commonly used in industrial settings to detect e.g., liquids through substances which are opaque in the visible spectrum and therefore also opaque to the human eye. 39We combined this aspect of SWIR sensors with SWIRFI to detect CJ215 through a variety of commercial bandages in phantoms, Supp.Throughout this work we have shown that CJ215 is highly tumor selective with excellent clearance from healthy organs.This clearance further aids tumor delineation and even enabled the detection of tumors on or close to non-affected organs.CJ215 displayed notable spectral changes when dissolved in FBS, HSA or blood compared to dextrose eluding to that in vivo, CJ215 is binding to serum and other components of blood.This is evidenced by the shift in absorption peak of CJ215 in dextrose, HSA, FBS and blood (795, 805, 810 and 818nm, respectively Supp.Fig 2-4), with each solution presenting an increasingly convoluted environment and more components for CJ215 to bind to, more akin to in vivo settings.Importantly following an initial red spectral shift, CJ215 underwent a blue shift over the course of days (~5-10nm) potentially forming a covalent adduct with albumin and blood components, as shown for other carbocyanine dyes. 43,48In comparison to previous work, CJ215 contains a methoxy group on the cyclohexenyl ring at the center of the heptamethine bridge as opposed to a chloride group along with some other subtle chemical differences. 43This methoxy group and likely CJ215 as a whole is less reactive than a carbocyanine dye with a chloride group at the same location, explaining the slow blue shift (days) in the presence of blood components.Based on the biodistribution with renal clearance and in vitro experimentation, CJ215 is likely binding to blood components in vivo which are then preferentially taken up by apoptotic and stressed cells including tumors and wound healing areas. 48This is further con rmed by the serum free in vitro experiments and biodistribution similarity to radiolabeled albumin, with protein catabolism by tumors and wound healing being a predominant driver of uptake. 43,67e limitation of CJ125 is that based on our experimentation, CJ215 will likely not be of use in highly advanced and necrotic (dead) tumor tissue with poor vasculature as shown in the SW1222 model.However, surgery would not be performed at this level of tumor burden.This is unlike the tested single site tumor models, where CJ215 is readily delivered via the blood stream and vasculature, dispersed throughout the tumor itself and then trapped within highly apoptotic cores.Nevertheless, the SW1222 model did validate the ability of CJ215 to delineate peritoneal metastasis in multiple dispersed lesions.
Due to the ability of CJ215 to highlight and be retained in apoptotic tissue, CJ215 may not be suitable for FGS in patients which have already received e.g., adjuvant chemotherapeutics which are known to cause off target cell death. 68This would likely increase non-tumorous background uptake, potentially reducing tumor CNR or leading to false positives as seen in the CT26 model, however, uptake in the tumor could be increased as well.Conversely, due to the low uptake in healthy tissue and established biodistribution seen here, CJ215 could be used to study these affects and the organ protection provided by co-administered compounds.Fortunately, surgery is often the rst treatment for most solid tumors, highlighting the suitability of CJ215 for tumor FGS within current surgical guidelines.Whilst toxicology has not yet been con rmed, no adverse events were reported in any of the mice used herein with all mice surviving the injections and main organs appearing histologically normal.Repeated injections also caused no issue and as CJ215 is renally cleared, it may reduce potential liver toxicity.
In summary, CJ215 provides a unique dye for tumor and wound monitoring through binding to mostly apoptotic cells, enabling delineation of various tumors in murine models (all tested models have been included and we have yet to nd a solid model which CJ215 does not delineate) and quantitative wound monitoring.These aspects position CJ215 as a pan cancer agent, with high tumor selectivity for multiple applications reducing the often-limiting factor of FDA approval pipeline costs and bridging the "valley" between preclinical and clinical translation.Finally, CJ215 is a commercial product facilitating rapid replication of these results and further investigation by any researcher with a suitable device e.g., IVIS.
This is in stark comparison to the burden shouldered by academic labs where mass production of custom compounds is often unachievable and not a focus.
In vitro assessment: 4T1 (ATCC® CRL-2539) cells were cultured in RPMI media (supplemented with Pen-Strep and FBS) until con uent in 6 well plastic poly-d-lysine coated plates with an embedded glass cover slip (P06G-1.5-10-F,MatTek, USA).In these conditions 4T1 cells formed a mix of single cells and spheroid aggregations.Cells were washed once with fresh medium, then incubated (incubator, 37°C, 5% CO 2 ) with CJ215 (suspended in cell culture medium) for 3 hrs at a concentration of 0.645 mM. 1 µl of stock DAPI solution (62248, Thermo Fisher Scienti c, USA) was added to each well at 2hrs 40 mins into CJ215 incubation.At 3hrs well media was aspirated, cells washed once with fresh medium and then 1 ml of medium was added to each well.Point scanning confocal microscopy was then performed (STELLARIS 8, Leica, USA) within an hour, recording various images (stacks, videos) with appropriate DAPI, CJ215 and polarized white light settings.Images were imported into ImageJ, split by color with gaussian blur (sigma = 1) applied to DAPI and CJ215 channels followed by contrast adjustment for easier visualization prior to remerging the channels.
Active transport was assessed also using 4T1 cells incubated in pre-coated plastic 6 well plates (no glass coverslip).Cells were washed once and then placed into either a fridge or incubator for 10 mins prior to incubation with CJ215 as performed for confocal imaging.CJ215 uptake was assessed using the 700 and 800 channels on an Odyssey CLx, with image analysis performed on .tiffimages consisting of determining the mean uorescence within each well (Fiji/ImageJ). 69HT1080 (ATCC® CCL-121™) cells (75,000 cells/well) were seeded on a 24 well plate with 500 µL of cell medium (DMEM, with Pen-Strep and 10% FBS).After 24 hours, the cells had attached to the plate surface and Staurosporine was added to the wells with a nal concentration of 2 µM Staurosporine.The cells were incubated with Staurosporine for 18 hours.5 µL CJ215 stock solution (0.5 mg/mL) was mixed with 1.2 mL media and 50 µL dye-media mix was added to each well.Dye was incubated with the cells for an additional 6 hours, then cells were washed with PBS once.The plate was imaged and quanti ed as before (Odyssey CLx and Fiji/ImageJ).Serum free and serum uptake of CJ215 was assessed with 4T1 cells plated on 6 well plates, as before.
Cells were starved in serum free conditions (cell medium without FBS) for 3 hours prior to addition of and incubation with CJ215, also as before.Cells were washed once with respective media and then xed and imaged in 1 ml of formaldehyde solution (formaldehyde solution 4%, microscopy grade, 1.00496.5000,Sigma-Aldrich, USA) and imaged as before (Odyssey CLx).Formaldehyde stability of CJ215 was assessed with the same cells stored at 4°C for up to 9 days post xation, again imaged as before.
In vivo experimentation: Single tumor model sites were generated with multiple cells, mouse lines and sexes.In brief, 3.0x10 5 4T1 cells suspended in 30µL of Matrigel were injected into the mammary pad of female FoxN1 nu mice.5x10 6 PC3 cells expressing human prostate speci c membrane antigen (PC3-PSMA) suspended in 50µL of PBS were subcutaneously injected into the ank of male NOD.Cg-Prkdcscid/J mice.5x10 6 HT1080 cells suspended in 100µL of 50:50 Matrigel and PBS were subcutaneously injected into the ank, at the top of the leg in female FoxN1 nu mice.2x10 6 CT26 cells suspended in PBS were subcutaneously injected into the ank of female BALB/cAnN mice.Mice were utilized at various ages, with tumors allowed to proliferate until tumors at various sizes could be visually determined, approx.>50 mm 3 .5.0x10 6 SW1222 cells expressing luciferase suspended in 100 µl of cell medium were intraperitoneally injected into female FoxN1 nu mice, sporadically forming tumor lesions, and assessed using luciferin.Female FoxN1 nu mice (bearing no tumors) were also used for wound healing experiments.
In all cases mice received a 2mg/kg intravenous injection of CJ215 suspended in clinical grade dextrose.
Upon receipt of CJ215 it was suspended in dextrose, aliquoted into glass dram vials and lyophilized for storage at -20°C.Injection solutions were prepared within 1 hr prior to administration, with solutions appearing dark green.The dye did not form any noticeable clumps, could be readily resuspended in dextrose simply using a pipette and animal injections went smoothly.CJ215 was not handled in any overly restrictive or particular manner and no adverse event was noticed post dye injection.All mouse handling, experimentation, imaging, and housing was performed according to NIH guidelines and via approved IACUC protocols at MSKCC.Mice received food and water ad libitum, under a 12 hr on/off light cycle, 5 mice per cage with food.FoxN1 nu mice used in tumor experiments were fed either a solid amoxicillin or Sulfatrim based diet to counteract skin infections.SW1222 and wound healing mice were placed on a low auto uorescence diet to counteract observed intestinal signals, with wound healing mice receiving Sulfatrim water to counteract skin infections.Surgery was performed on mice in line with preapproved protocols with mice having anesthesia induced at 3% v/v followed by maintenance at 1-2% v/v in oxygen and placement on a heating pad.Prior to surgery the mice received subcutaneously received buprenorphine (0.5 mg/kg) and meloxicam (2mg/kg) with bupivacaine (0.1cc) administered at the incision site for numbing.The surgical eld was cleaned for surgery using sets of alternating scrubs, (alternating either povidine-iodine (Betadine), chlorhexidine and 70% isopropyl alcohol).All surgical instruments were sterilized by steam sterilization and using a glass bead sterilizer between animals.A scalpel was used to create a ~1cm incision with wound sealing performed with mono lament absorbable suture (monocryl).Mice were monitored post recovery every 24hrs and received meloxicam (2mg/kg) subcutaneously for up to 72hrs post-surgery.
Tumor resection was only performed on mice which had been euthanized via CO 2 inhalation, in line with approved protocols.Following this the tumor was resected under white light visualization with resection con rmed using SWIRFI.Necropsy biodistribution was assessed by isolating the following organs: tumor or wound site, liver, kidneys, lungs, brain, spleen, pancreas, stomach, small and large intestines, skin, bone, muscle, and heart.

SWIRFI and NIRFI:
Ambient light resistant SWIRFI was performed as previously described. 41In brief, a hyperspectral SWIR system (IR-VIVO, Photon etc., Canada) equipped with an 808 nm laser (~300 mW/cm 2 ) for dye excitation, a 940 nm LED for SWIR white light (re ectance imaging) and an RGB LED was used for SWIRFI.The ambient lighting LED was on for all SWIRFI images presented throughout this manuscript.Extended emission imaging was achieved using suitable long pass optical lters (Thorlabs, NJ, USA) mounted in front of the lens of the system in a custom mount.Exposure times ranged from 1-1500 ms with data stored in a .h5format.Images were converted to tiff les with corrections then performed in Fiji/ImageJ as before including dark noise subtraction, outlier removal, and cropping. 41De ned ROIs were used to assess brightness, SNR and CNR, also as previously described. 41NIRFI for biodistribution was performed using an IVIS Spectrum (PerkinElmer, USA) with ICG based lter sets (745 excitation and 840 nm emission), at exposure times of 5-20s, high lamp, f# of 1 and small binning.Organ quanti cation was performed in ImageJ/Fiji with ROIs drawn across each organ in the raw luminescent tiff le exported from the IVIS.This system was also used for luciferase imaging of SW1222 mice in bioluminescence mode.For all live animal imaging, mice were anaesthetized using iso urane inhalation at an induction of 3% v/v and maintenance of 1-2% v/v in O 2 and were placed on a heating pad during imaging.
Histological Assessment: Para n embedded organs (tumor or wound, liver, kidney, spleen, and muscle) were prepared as previously described to generate either on 5 µm thick unstained or H&E stained (Leica Autostainer, ST5010) slides. 41H&E slides were imaged using an automated slide scanner at 20x magni cation (0.8 NA, 3DHistech, Budapest, Hungary) with representative images shown at 20x (SlideViewer, Version 2.5, 3DHistech).Immunohistochemistry for CC3 was performed on para n sections using a Leica Bond RX automated stainer.After heat-induced epitope retrieval in a pH 6.0 buffer, anti-CC3 antibody (Cell Signaling 9661) was applied at a concentration of 1:250, followed by a polymer detection reagent kit according to the manufacturer's instructions (DS9800, Novocastra Bond Polymer Re ne Detection, Leica Biosystems).The chromogen indicating positive immunoreactivity was 3,3 diaminobenzidine tetrachloride (DAB), and sections were counterstained with hematoxylin.CC3 IHC images were acquired an Olympus VS200 slides scanner and VS200 ASW 3.
Necropsy and histological analysis of additional regions of interest during tumor resection.A) During resection and necropsy small areas (labelled as 3.1, 3.2, were identi ed and found to be highly uorescent (NIRFI, IVIS Spectrum, 745nm excitation, 840nm emission) over background areas and muscle tissue.B) Quanti cation of ROIs with ROI to muscle ratios shown (italics).Left, H&E staining of resected tissues from M3 identi ed (clockwise) as a lymph node surrounded by adipose tissue (3.1), the small intestine with pancreas and a small neoplastic tumor area not bound to any identi able tissue (3.2) and nally the reproductive organs including the uterus, oviduct, ovaries (3.3).Right, H&E staining of resected tissues from M4 identi ed (clockwise) as the skin and subcutis (3.1), a lymph node surrounded by adipose tissue (3.2) and nally the small intestine also with adipose tissue (3.3)  Presented images are single frames from post-processed (corrected) video rate acquisitions, the entire videos are available in Supplemental Video 3. Non-speci c uptake is present at the tail of the HT1080 mouse, but the tumor is delineated over surrounding tissue.
, Supp.29.Highly necrotic tumors still achieved a tumor:muscle ratio of 7.89, Fig.7 D. Splenic tumors had an improved tumor:muscle ratio of 10.38 and 8.80 over the spleen itself Fig.7 D, appearing less necrotic (H&E staining, Fig.7 E), highlighting the ability of CJ215 to delineate organ bound tumor masses and uptake in viable tumors.

Fig 4 .
Tumors had the brightest uorescence in all experiments with non-tumor bearing organs having minimal residual dye uptake.This biodistribution enabled un precedented tumor:muscle ratios of up to 100 where anything above 2 or even 1.5 is considered successful, Fig 4.56 Furthermore, CJ215 cleared from vital organs such as the liver, spleen and pancreas enabling tumor:organ ratios in the mid 40s,Fig 4.Although not present in these single site models, this highlights that CJ215 has the potential to delineate various organ metastases with high contrast for improved delineation.Importantly, these ratios were achieved by simply allowing CJ215 to clear from healthy tissue after a single systemic intravenous injection.CJ215 cleared renally as highlighted by the retained kidney uorescence, in stark comparison to other dyes or nanoparticle-based approaches where liver clearance can be a predominant factor, Fig 4.15,[57][58][59] Select organs from these mice were assessed via histopathology (H&E staining), con rming tumor tissue from healthy organs, and assessing additionally resected regions.All suspected tumor regions were con rmed to be tumorous via H&E staining.During CT26 resection and necropsy, additional areas aside from the primary tumor demonstrated elevated organ:muscle ratios, Fig 5 A, B. These regions were con rmed to be lymph node, uterine, ovarian, colon as well as a single tumor site, Fig 5 C. IHC for CC3 (apoptosis marker) was performed on these tissues, highlighting their increased apoptotic levels, Fig 5 D. This also con rmed the highly apoptotic core seen at the center of tumors, where CJ215 uptake had been brightest, Fig 3 G, Supp.Fig 26.

7 A
, Supp.Fig 22.At 168 hrs post injection, SWIRFI resection was attempted with tumors being identi ed as solid, black necrotic tissue (likely dead), widely dispersed throughout the peritoneum, and bound to the intestines and spleen, Fig 7 B. NIRFI con rmed lower tumor uptake in the most necrotic tissues but did highlight large lesions spread around the spleen and pancreas, which could be readily delineated, Fig 7 B, Supp.Fig 23.Even though less successful than single site models, CJ215 achieved tumor:muscle ratios of up to 10 within these tumors with an uneven distribution across highly necrotic areas, Fig 7 C. Due to the rapid growth of these tumors and their widespread dispersion, they lacked viable vascular networks within necrotic tissue at the injection timepoint.The non-existent metabolic activity, and poor blood delivery in dead and pervasively necrotic tissue hinders CJ215 delivery and therefore uptake in such lesions, Fig 7 B, Supp.Fig 24.

Fig 31 .
Hydrogel bandages, used in burn wound healing, were chosen to track wound healing in mice post a controlled incision, Fig 8 A, B, Supp.Video 4. 64,65 Similar to tumor delineation, CNR could be inversely used to elucidate wound healing progression completely noninvasively after a single systemic injection of CJ215, Fig 8 C. CNR increased up to 48hrs post injection and then decreased as wounds healed in line with visible inspection (without bandages) with the >1300nm cutoff providing the highest CNR, albeit not video rate, Fig 8 C, Supp.Fig 32-35.Having completely healed and following a second injection of CJ215, the wound area showed only minimal uptake, found to be in line with non-injured skin, Supp.Fig 36.These mice also provided further biodistribution data of CJ215 in non-tumor bearing conditions, con rming the renal clearance as seen at later timepoints and again the lack of uptake in other organs, Supp.Fig 37.This aspect of CJ215 and SWIRFI provide an objective, rapid (seconds), and quantitative solution in chronic wound monitoring with potential clinical applications in e.g., diabetic and burn wound victims.66This will be especially useful where bandage removal may cause pain to victims or pose a risk of infection.64Based on these experiments a patient (even those not undergoing tumor resection) could receive a single injection of CJ215 upon surgery completion with wound healing tracked for days during post-operative care.SWIRFI and CJ215 can readily visualize bandage covered wounds under ambient lighting conditions providing an additional application of clinical uorescence imaging, not limited to only tumor resection.

Figure 3 Extended
Figure 3

Figure 8
Figure 8 Su cient SNR was always achieved with SWIRFI, the main tumor delineation factor was concluded to be contrast (CNR), as expected, Fig 2 H, I. Here, we present video rate images in a contrast mode where the user selects a non-tumorous background reference point, Fig 6 A. A custom script in ImageJ facilitated this, across SWIRFI videos at >900 nm and achieved pixelwise image correction at a rate of ~40 ms per image, Fig 6 B, Supp.Video 3. The 3dB threshold was found to be successful in highlighting tumor areas for all lines, ful lling the Rose criterion. 49,50Additionally, the conversion of images from a linear to a logarithmic scale counteracted the high tumor core uptake of CJ215 easing entire tumor delineation, Fig 6 B CT26 model.This method enables binary tumor delineation, on the y for FGS and can be readily implemented by other researchers and for other probes.
Graphics processing unit (GPU) implementation (outside the scope of this work, but possible with ImageJ) could decrease image processing time to enable real-time visualization (assuming image handoff from memory to GPU is e cient) during FGS as opposed to in post processing with few ms acquisition times.61 MSKCC and Ileana Miranda of the Laboratory of Comparative Pathology at MSKCC.We would like to thank Eric Chan, Eric Rosiek & Yevgeniy Romin for of the Molecular Cytology core at MSKCC for microscopy assistance.Disclosures: D.A.H. is a co-founder and o cer with equity interest in Lime Therapeutics, Inc., and cofounder with equity interest in Selectin Therapeutics Inc., and Resident Diagnostics, Inc., and a member of the scienti c advisory board of Concarlo Therapeutics, Inc., Nanorobotics Inc., and Mediphage Bioceuticals, Inc.The remaining authors declare no competing interests.CJ215 was a gift from Proimaging who did not sponsor this research aside from providing CJ215.Data Availability: All data and code required used throughout this manuscript is available from the corresponding authors upon request.Note, all code to process images was peformed in ImageJ via already available plug-ins.
The script calculated the mean and SD of this ROI for all frames in a video image and uses the CNR formula commonly utilized to present images in "contrast mode".CJ215 spectral, in vitro, and in vivo assessment.A) The absorption of CJ215 dissolved in either fetal bovine serum (solid line) or dextrose (dotted line) was assessed at concentrations from 20.30 µM to 0.203 µM.The entire absorption spectrum red shifted in serum 12nm from a peak of 798 to 810 nm, like that of ICG.B) The SWIR emission tail of the dye was characterized from 950 to 1550 nm with notable emission past 1100 nm and a 4x increase in intensity when dissolved in serum vs dextrose.C) Representative single cell NIR confocal microscopy localization uptake of CJ215 (green, 3hr incubation period) in 4T1 cells, stained with DAPI (nucleus) with polarized white light images overlayed (grayscale).
). B) SNR and CNR quanti cation of the HT1080 tumor line, with >1100 presenting the highest contrast but not signi cantly improved over >900 nm.C) SNR and CNR quanti cation of the CT26 tumor line, with no difference found in CNR between tested cutoffs.D) Combined assessment of HT1080 and CT26 with >1300 nm found to provide the lowest SNR compared to other cutoffs with no signi cant difference in CNR found when combining both groups.In all cases the mean, SD and replicates are shown (n=4 per tumor line, n=8 for combined) with statistical analysis shown based on a non-paired or matched Welch and Brown-Forsythe ANOVA with Dunnet T3 multiple comparisons test.Thresholds are shown as before (5dB and 3dB for SNR and CNR, respectively).E) Zoomed in view of the CT26 tumor at 900, 1100 and 1300 nm.F) Vertical and horizontal full width at half maximum (FWHM) assessment (dotted grey lines, E) of the tumor at all wavelengths.The qualitative improvement in tumor core delineation seen at >1100 and best at >1300 nm, is also observed quantitatively.G) Corresponding cleaved caspase 3 (CC3) immunohistochemistry staining of the CT26 tumor shown in A and E, showing highest levels of apoptosis at the tumor core with positively stained cells present throughout the sample (i-iv).Black arrows show the approximate slice location with corresponding white arrows in E.
. D) Cleaved caspase 3 positive (CC3+, for apoptosis i.e., damaged cells) IHC staining on a consecutive slice for each mouse sample.Labelled boxes highlight ROIs with increased levels of CC3 speci c staining.Non-speci c CC3 staining widely seen in the small intestine of M3 should be ignored.E) Zoomed in areas as highlighted at various magni cation levels: 3.1 Small to moderate number of CC3+ cells were seen in the lymph node cortex and paracortex.3.2 CC3+ cells were found in the tumor region.3.3a CC3+ cells are highlighted in the granulosa of ovarian follicles.3.3b Small number of CC3+ cells are seen within the endometrial epithelium.4.1 No CC3+ cells were seen in the skin.4.2 Moderate number of CC3+ cells are seen within the crypts with a small number in the enterocytes of villi.4.3a & 4.3b to moderated number of CC3+ cells were seen throughout lymph node cortex and paracortex.