Pigment epithelium-derived factor expression and role in follicular development.

RESEARCH QUESTION: What is the involvement of pigment epithelium-derived factor (PEDF), expressed in granulosa cells, in folliculogenesis? DESIGN: mRNA expression of PEDF and other key factors [Cyp19, anti-Müllerian hormone receptor (AMHR) and vascular endothelial growth factor (VEGF)] in mice follicles was examined in order to typify the expression of PEDF in growing follicles and in human primary granulosa cells (hpGC), and to follow the interplay between PEDF and the other main players in folliculogenesis: FSH and AMH. RESULTS: mRNA expression of PEDF increased through folliculogenesis, although the pattern differed from that of the other examined genes, affecting the follicular angiogenic and oxidative balance. In hpGC, prolonged exposure to FSH stimulated the up-regulation of PEDF mRNA. Furthermore, a negative correlation between AMH and PEDF was observed: AMH stimulation reduced the expression of PEDF mRNA and PEDF stimulation reduced the expression of AMHR mRNA. CONCLUSIONS: Folliculogenesis, an intricate process that requires close dialogue between the oocyte and its supporting granulosa cells, is mediated by various endocrine and paracrine factors. The current findings suggest that PEDF, expressed in granulosa cells, is a pro-folliculogenesis player that interacts with FSH and AMH in the process of follicular growth. However, the mechanism of this process is yet to be determined.

Pigment epithelium-derived factor negates oxidative stress in mouse oocytes.

Molecular changes, caused by various environmental factors, affect the quality and developmental potential of oocytes. Oxidative stress (OS) is a major factor involved in various gynecologic disorders and/or in aging. Recent studies suggest that elevated reactive oxygen species (ROS) hamper oocyte quality and future embryonic development. Pigment epithelium-derived factor (PEDF) is a pleiotropic protein, known for its antiangiogenic, anti-inflammatory, and antioxidative properties. Our previous findings demonstrate the antioxidative role of rPEDF in maintaining granulosa cell viability. In the current study, we examined the ability of PEDF to negate the adverse impact of OS on oocytes. Maturation rate of oocytes exposed to OS was significantly lower than that of control oocytes. The number of mtDNA copies in OS-exposed oocytes was significantly higher than in control oocytes (>3 times), whereas ATP concentration was significantly lower. Oocytes exposed to OS demonstrated impaired chromosome arrangement at the metaphase plate. PEDF significantly improved maturation rate of untreated OS-exposed oocytes. Moreover, mtDNA copy number, ATP concentration, and chromosome arrangement at the metaphase plate in rPEDF-treated OS-exposed oocytes were restored to the level of control oocytes. Our findings demonstrate that OS hampers the ability of oocytes to undergo proper in vitro maturation. The energetic balance of OS-exposed oocyte is characterized by excessive mtDNA replication and reduced ATP concentration; it hampers the ability of oocytes to perform high fidelity chromosome segregation. PEDF alleviates this damage, improves the rate of oocyte maturation, and preserves mtDNA level and ATP content, thus enabling oocytes to form proper metaphase plate and improve oocyte competence.

The Role of PEDF in Reproductive Aging of the Ovary.

Reproductive aging is characterized by a decline in ovarian function and in oocytes' quantity and quality. Pigment epithelium-derived factor (PEDF), a pivotal player in ovarian angiogenic and oxidative balance, was evaluated for its involvement in reproductive aging. Our work examines the initial stage of reproductive aging in women and mice, and the involvement of PEDF in the process. Granulosa cells from reproductively-aged (RA) women and mice (36-44 years old and 9-10 months old, respectively) indicated an increase in the level of PEDF mRNA (qPCR), with yet unchanged levels of AMH and FSHR mRNAs. However, the PEDF protein level in individual women showed an intra-cellular decrease (ELISA), along with a decrease in the corresponding follicular fluid, which reflects the secreted fraction of the protein. The in vitro maturation (IVM) rate in the oocytes of RA mice was lower compared with the oocytes of young mice, demonstrated by a reduced polar body extrusion (PBE) rate. The supplementation of PEDF improved the hampered PBE rate, manifested by a higher number of energetically-competent oocytes (ATP concentration and mtDNA copy number of individual oocytes). Our findings propose PEDF as an early marker of reproductive aging, and a possible therapeutic in vitro agent that could enhance the number of good-quality oocytes in older IVF patients.

Reverting the molecular fingerprint of tumor dormancy as a therapeutic strategy for glioblastoma.

Glioblastoma is an aggressive and invasive brain malignancy with high mortality rates despite current treatment modalities. In this study, we show that a 7-gene signature, previously found to govern the switch of glioblastomas from dormancy to aggressive tumor growth, correlates with improved overall survival of patients with glioblastoma. Using glioblastoma dormancy models, we validated the role of 2 genes from the signature, thrombospondin-1 ( TSP-1) and epidermal growth factor receptor ( EGFR), as regulators of glioblastoma dormancy and explored their therapeutic potential. EGFR up-regulation was reversed using EGFR small interfering RNA polyplex, antibody, or small-molecule inhibitor. The diminished function of TSP-1 was augmented via a peptidomimetic. The combination of EGFR inhibition and TSP-1 restoration led to enhanced therapeutic efficacy in vitro, in 3-dimensional patient-derived spheroids, and in a subcutaneous human glioblastoma model in vivo. Systemic administration of the combination therapy to mice bearing intracranial murine glioblastoma resulted in marginal therapeutic outcomes, probably due to brain delivery challenges, p53 mutation status, and the aggressive nature of the selected cell line. Nevertheless, this study provides a proof of concept for exploiting regulators of tumor dormancy for glioblastoma therapy. This therapeutic strategy can be exploited for future investigations using a variety of therapeutic entities that manipulate the expression of dormancy-associated genes in glioblastoma as well as in other cancer types.-Tiram, G., Ferber, S., Ofek, P., Eldar-Boock, A., Ben-Shushan, D., Yeini, E., Krivitsky, A., Blatt, R., Almog, N., Henkin, J., Amsalem, O., Yavin, E., Cohen, G., Lazarovici, P., Lee, J. S., Ruppin, E., Milyavsky, M., Grossman, R., Ram, Z., Calderón, M., Haag, R., Satchi-Fainaro, R. Reverting the molecular fingerprint of tumor dormancy as a therapeutic strategy for glioblastoma.

ortho-Chlorination of phenoxy 1,2-dioxetane yields superior chemiluminescent probes for in vitro and in vivo imaging.

A recent methodology, developed by our group, has enabled a dramatic improvement in the emissive nature of the excited species, formed during the chemiexcitation of dioxetanes under physiological conditions. This approach has resulted in the discovery of distinct phenoxy-dioxetane luminophores that produce a chemiluminescence signal via a direct-mode of emission. Here, we show a significant pKa effect of our new phenoxy-dioxetanes on their chemiexcitation and on their ability to serve as chemiluminescent turn-ON probes for biological applications. Using an appropriate phenoxy-dioxetane probe with a direct-mode of emission, we were able to image ß-galactosidase activity, in cancer cells and in tumor-bearing mice. To the best of our knowledge, this is the first example to demonstrate in vitro and in vivo endogenous enzymatic chemiluminescence images obtained by a single-component phenoxy-dioxetane probe. We anticipate that our strategy, for the design and synthesis of such distinct luminophores, will assist in providing new effective turn-ON probes for non-invasive intravital chemiluminescence imaging techniques.

PEGylated dendritic polyglycerol conjugate targeting NCAM-expressing neuroblastoma: Limitations and challenges.

Neural cell adhesion molecule (NCAM) is found to be a stem-cell marker in several tumor types and its overexpression is known to correlate with increased metastatic capacity. To combine extravasation- and ligand-dependent targeting to NCAM overexpressing-cells in the tumor microenvironment, we developed a PEGylated NCAM-targeted dendritic polyglycerol (PG) conjugate. Here, we describe the synthesis, physico-chemical characterization and biological evaluation of a PG conjugate bearing the mitotic inhibitor paclitaxel (PTX) and an NCAM-targeting peptide (NTP). PG-NTP-PTX-PEG was evaluated for its ability to inhibit neuroblastoma progression in vitro and in vivo as compared to non-targeted derivatives and free drug. NCAM-targeted conjugate inhibited the migration of proliferating endothelial cells, suggesting it would be able to inhibit tumor angiogenesis. The targeting conjugate provided an improved binding and uptake on IMR-32 cells compared to non-targeted control. However, these results did not translate to our in vivo model on orthotopic neuroblastoma bearing mice.

Near-Infrared Dioxetane Luminophores with Direct Chemiluminescence Emission Mode.

Chemiluminescent luminophores are considered as one of the most sensitive families of probes for detection and imaging applications. Due to their high signal-to-noise ratios, luminophores with near-infrared (NIR) emission are particularly important for in vivo use. In addition, light with such long wavelength has significantly greater capability for penetration through organic tissue. So far, only a few reports have described the use of chemiluminescence systems for in vivo imaging. Such systems are always based on an energy-transfer process from a chemiluminescent precursor to a nearby emissive fluorescent dye. Here, we describe the development of the first chemiluminescent luminophores with a direct mode of NIR light emission that are suitable for use under physiological conditions. Our strategy is based on incorporation of a substituent with an extended π-electron system on the excited species obtained during the chemiexcitation pathway of Schaap's adamantylidene-dioxetane probe. In this manner, we designed and synthesized two new luminophores with direct light emission wavelength in the NIR region. Masking of the luminophores with analyte-responsive groups has resulted in turn-ON probes for detection and imaging of ß-galactosidase and hydrogen peroxide. The probes' ability to image their corresponding analyte/enzyme was effectively demonstrated in vitro for ß-galactosidase activity and in vivo in a mouse model of inflammation. We anticipate that our strategy for obtaining NIR luminophores will open new doors for further exploration of complex biomolecular systems using non-invasive intravital chemiluminescence imaging techniques.

Remarkable Enhancement of Chemiluminescent Signal by Dioxetane-Fluorophore Conjugates: Turn-ON Chemiluminescence Probes with Color Modulation for Sensing and Imaging.

Chemiluminescence is among the most sensitive methods for achieving a high signal-to-noise ratio in various chemical and biological applications. We have developed a modular practical synthetic route for preparation of turn-ON fluorophore-tethered dioxetane chemiluminescent probes. The chemiluminescent emission of the probes was significantly amplified through an energy-transfer mechanism under physiological conditions. Two probes were composed with green and near-infrared (NIR) fluorescent dyes tethered to Schaap's dioxetane. While both probes were able to provide chemiluminescence in vivo images following subcutaneous injection, only the NIR probe could provide a chemiluminescence image following intraperitoneal injection. These are the first in vivo images produced by Schaap's dioxetane chemiluminescence probes with no need of an enhancer. Previously, chemiluminescence cell images could only be obtained with a luciferin-based probe. Our NIR probe was able to image cells transfected with ß-galactosidase gene by chemiluminescence microscopy. We also report, for the first time, the instability of dioxetane-fluorophore conjugates to ambient light. Our synthetic route effectively overcomes this limitation through a late-stage functionalization of the dioxetane intermediate. We anticipate that our practical synthetic methodology will be useful for preparation of various chemiluminescent probes for numerous applications.

Novel pullulan bioconjugate for selective breast cancer bone metastases treatment.

A novel polysaccharide bioconjugate was designed to selectively target breast cancer bone metastases using a bisphosphonate moiety (alendronate, ALN). Paclitaxel (PTX) was first covalently conjugated to pullulan (Pull) through a Cathepsin K-sensitive tetrapeptide spacer followed by a self-immolative aminobenzyl alcohol spacer to obtain Pull-(GGPNle-φ-PTX). ALN was then conjugated to the polymeric backbone of Pull-(GGPNle-φ-PTX) via a PEG spacer. The final bioconjugate Pull-(GGPNle-φ-PTX)-(PEG-ALN) was found to assemble into colloidal spherical structures, which were physically and chemically stable under physiological conditions. In vitro studies showed that Pull-(GGPNle-φ-PTX)-(PEG-ALN) had strong affinity for hydroxyapatite, which simulates the bone tissue. Paclitaxel was rapidly released from the bioconjugate by Cathepsin K cleavage under pathological conditions. All studies performed using human MDA-MB-231-BM (bone metastases-originated clone), murine 4T1 breast cancer cells, murine K7M2, and human SAOS-2 osteosarcoma cells showed that the bioconjugate exerted an enhanced antiproliferative activity compared to the conjugate without the ALN. Furthermore, the nanoconjugate inhibited the migration of cancer cells and further displayed potent anti-angiogenic activity. In conclusion, the results showed that this conjugate has an excellent potential for selective treatment of bone neoplasms such as breast cancer bone metastases and osteosarcoma.

The direct effect of SARS-CoV-2 virus vaccination on human ovarian granulosa cells explains menstrual irregularities.

Following administration of the SARS-CoV-2 vaccine, many women worldwide reported short-term menstrual irregularities. Although menstrual bleeding, "the fifth vital sign", is experienced by more than 300 million people on any given day worldwide, these changes were only partially studied. Irregular periods are important well beyond fertility and the discomfort they impose; they are associated with the risk of cardiovascular morbidity, chronic diseases, and premature mortality. Pre-clinical examination of the vaccine polymeric envelope indicates its accumulation in the ovaries. The somatic endocrine cells of the ovarian follicle - the granulosa cells (GCs)-participate in the strict hypothalamic-pituitary-ovarian (HPO) feedback loop that governs the menstrual cycle via endocrine and paracrine regulators, as AMH and Inhibins. We aimed to unravel the direct effect of the COVID-19 vaccine on GCs and link their post-vaccine activity to changes in menstrual patterns. Human primary GCs exposed in-vitro to the Pfizer COVID-19 vaccine BNT162b2, demonstrated no change in their viability but altered mRNA transcripts, specifically of the regulatory key factors: InhibinB was upregulated, whereas AMH was downregulated. We further examined pre- and post-vaccination blood samples from individual women and found a 2-3 folds change in the post-vaccination FSH/InhibinB protein level ratio, compared to their pre-vaccination values. This altered expression of InhibinB could significantly impact the HPO axis in vaccinated women and may ultimately influence the endometrium cyclicity, manifested clinically by the commonly reported changes in menstrual bleeding patterns.

Combining TIGIT Blockade with MDSC Inhibition Hinders Breast Cancer Bone Metastasis by Activating Antitumor Immunity.

Bone is the most common site of breast cancer metastasis. Bone metastasis is incurable and is associated with severe morbidity. Utilizing an immunocompetent mouse model of spontaneous breast cancer bone metastasis, we profiled the immune transcriptome of bone metastatic lesions and peripheral bone marrow at distinct metastatic stages, revealing dynamic changes during the metastatic process. We show that cross-talk between granulocytes and T cells is central to shaping an immunosuppressive microenvironment. Specifically, we identified the PD-1 and TIGIT signaling axes and the proinflammatory cytokine IL1ß as central players in the interactions between granulocytes and T cells. Targeting these pathways in vivo resulted in attenuated bone metastasis and improved survival, by reactivating antitumor immunity. Analysis of patient samples revealed that TIGIT and IL1ß are prominent in human bone metastasis. Our findings suggest that cotargeting immunosuppressive granulocytes and dysfunctional T cells may be a promising novel therapeutic strategy to inhibit bone metastasis. Significance: Temporal transcriptome profiling of the immune microenvironment in breast cancer bone metastasis revealed key communication pathways between dysfunctional T cells and myeloid derived suppressor cells. Cotargeting of TIGIT and IL1ß inhibited bone metastasis and improved survival. Validation in patient data implicated these targets as a novel promising approach to treat human bone metastasis.

Hormonal regulation of pigment epithelium-derived factor (PEDF) in granulosa cells.

Angiogenesis is critical for the development of ovarian follicles. Blood vessels are abrogated from the follicle until ovulation, when they invade it to support the developing corpus luteum. Granulosa cells are known to secrete anti-angiogenic factors that shield against premature vascularization; however, their molecular identity is yet to be defined. In this study we address the physiological role of pigment epithelium-derived factor (PEDF), a well-known angiogenic inhibitor, in granulosa cells. We have shown that human and mouse primary granulosa cells express and secrete PEDF, and characterized its hormonal regulation. Stimulation of granulosa cells with increasing doses of estrogen caused a gradual decrease in the PEDF secretion, while stimulation with progesterone caused an abrupt decrease in its secretion. Moreover, We have shown, by time- and dose-response experiments, that the secreted PEDF and vascular endothelial growth factor (VEGF) were inversely regulated by hCG; namely, PEDF level was nearly undetectable under high doses of hCG, while VEGF level was significantly elevated. The anti-angiogenic nature of the PEDF secreted from granulosa cells was examined by migration, proliferation and tube formation assays in cultures of human umbilical vein endothelial cells. Depleting PEDF from primary granulosa cells conditioned media accelerated endothelial cells proliferation, migration and tube formation. Collectively, the dynamic expression of PEDF that inversely portrays VEGF expression may imply its putative role as a physiological negative regulator of follicular angiogenesis.

Antiangiogenic antitumor activity of HPMA copolymer-paclitaxel-alendronate conjugate on breast cancer bone metastasis mouse model.

Polymer therapeutics have shown promise as tumor-targeted drug delivery systems in mice. The multivalency of polymers allows the attachment of different functional agents to a polymeric backbone, including chemotherapeutic and antiangiogenic drugs, as well as targeting moieties, such as the bone-targeting agent alendronate (ALN). We previously reported the conjugation of ALN and the chemotherapeutic drug paclitaxel (PTX) with N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer. The in vitro physicochemical properties, cancer cytotoxicity and antiangiogenic activity of HPMA copolymer-PTX-ALN conjugate were extensively characterized. The reported results warranted in vivo evaluations of the conjugate. In this manuscript, we evaluated the in vivo anticancer and antiangiogenic activity of HPMA copolymer-PTX-ALN conjugate. The conjugate exhibited an antiangiogenic effect by decreasing microvessel density (MVD), and inducing apoptotic circulating endothelial cells (CEC) following treatment of the mice. Using intravital imaging system and mCherry-labeled breast cancer cell lines, we were able to monitor noninvasively the progression of orthotopic metastatic tumors injected into the tibia of the mice. HPMA copolymer-PTX-ALN conjugate showed the greatest antitumor efficacy on mCherry-labeled 4T1 mammary adenocarcinoma inoculated into the tibia, as compared with PTX alone or in combination with ALN. Treatment with the bone-targeted polymeric conjugate demonstrated improved efficacy, was better tolerated, and was more easily administered intravenously than the clinically used PTX formulated in Cremophor/ethanol.

Microengineered perfusable 3D-bioprinted glioblastoma model for in vivo mimicry of tumor microenvironment.

Many drugs show promising results in laboratory research but eventually fail clinical trials. We hypothesize that one main reason for this translational gap is that current cancer models are inadequate. Most models lack the tumor-stroma interactions, which are essential for proper representation of cancer complexed biology. Therefore, we recapitulated the tumor heterogenic microenvironment by creating fibrin glioblastoma bioink consisting of patient-derived glioblastoma cells, astrocytes, and microglia. In addition, perfusable blood vessels were created using a sacrificial bioink coated with brain pericytes and endothelial cells. We observed similar growth curves, drug response, and genetic signature of glioblastoma cells grown in our 3D-bioink platform and in orthotopic cancer mouse models as opposed to 2D culture on rigid plastic plates. Our 3D-bioprinted model could be the basis for potentially replacing cell cultures and animal models as a powerful platform for rapid, reproducible, and robust target discovery; personalized therapy screening; and drug development.

Bone metastasis is associated with acquisition of mesenchymal phenotype and immune suppression in a model of spontaneous breast cancer metastasis.

The most common site of breast cancer metastasis is the bone, occurring in approximately 70% of patients with advanced disease. Bone metastasis is associated with severe morbidities and high mortality. Therefore, deeper understanding of the mechanisms that enable bone-metastatic relapse are urgently needed. We report the establishment and characterization of a bone-seeking variant of breast cancer cells that spontaneously forms aggressive bone metastases following surgical resection of primary tumor. We characterized the modifications in the immune milieu during early and late stages of metastatic relapse and found that the formation of bone metastases is associated with systemic changes, as well as modifications of the bone microenvironment towards an immune suppressive milieu. Furthermore, we characterized the intrinsic changes in breast cancer cells that facilitate bone-tropism and found that they acquire mesenchymal and osteomimetic features. This model provides a clinically relevant platform to study the functional interactions between breast cancer cells and the bone microenvironment, in an effort to identify novel targets for intervention.

Rational Design of Polyglutamic Acid Delivering an Optimized Combination of Drugs Targeting Mutated BRAF and MEK in Melanoma.

Targeted therapies against cancer can relieve symptoms and induce remission, however, they often present limited duration of disease control, cause side effects and often induce acquired resistance. Therefore, there is a great motivation to develop a unique delivery system, targeted to the tumor, in which we can combine several active entities, increase the therapeutic index by reducing systemic exposure, and enhance their synergistic activity. To meet these goals, we chose the biocompatible and biodegradable poly(α,L-glutamic acid) (PGA) as a nanocarrier that facilitates extravasation-dependent tumor targeting delivery. The RAS/RAF/MEK/ERK pathway when aberrantly activated in melanoma, can lead to uncontrolled cell proliferation, induced invasion, and reduced apoptosis. Here, we selected two drugs targeting this pathway; a MEK1/2 inhibitor (selumetinib; SLM) and a modified BRAF inhibitor (modified dabrafenib; mDBF), that exhibited synergism in vitro. We synthesized and characterized our nanomedicine of PGA conjugated to SLM and mDBF (PGA-SLM-mDBF). PGA-SLM-mDBF inhibited the proliferation of melanoma cells and decreased their migratory and sprouting abilities without inducing a hemolytic effect. Moreover, the polymer-2-drugs conjugate exhibited superior anti-tumor activity in comparison with the two separate polymer-drug conjugates in vitro and with free drugs in a mouse model of primary melanoma and prolonged survival at a lower dose.

Light emission enhancement by supramolecular complexation of chemiluminescence probes designed for bioimaging.

Chemiluminescence offers advantages over fluorescence for bioimaging, since an external light source is unnecessary with chemiluminescent agents. This report demonstrates the first encapsulation of chemiluminescence phenoxy-adamantyl-1,2-dioxetane probes with trimethyl ß-cyclodextrin. Clear proof for the formation of a 1 : 1 host-guest complex between the adamantyl-1,2-dioxetane probe and trimethyl ß-cyclodextrin was provided by mass spectroscopy and NMR experiments. The calculated association constant of this host-guest system, 253 M-1, indicates the formation of a stable inclusion complex. The inclusion complex significantly amplified the light emission intensity relative to the noncomplexed probe under physiological conditions. Complexation of adamantyl-dioxetane with fluorogenic dye-tethered cyclodextrin resulted in light emission through energy transfer to a wavelength that corresponds to the fluorescent emission of the conjugated dye. Remarkably, the light emission intensity of this inclusion complex was approximately 1500-fold higher than that of the non-complexed adamantyl-dioxetane guest. We present the first demonstration of microscopic cell images obtained using a chemiluminescence supramolecular dioxetane probe and demonstrate the utility of these supramolecular complexes by imaging of enzymatic activity and bio-analytes in vitro and in vivo. We anticipate that the described chemiluminescence supramolecular dioxetane probes will find use in various biological applications.

Image-guided surgery using near-infrared Turn-ON fluorescent nanoprobes for precise detection of tumor margins.

Complete tumor removal during surgery has a great impact on patient survival. To that end, the surgeon should detect the tumor, remove it and validate that there are no residual cancer cells left behind. Residual cells at the incision margin of the tissue removed during surgery are associated with tumor recurrence and poor prognosis for the patient. In order to remove the tumor tissue completely with minimal collateral damage to healthy tissue, there is a need for diagnostic tools that will differentiate between the tumor and its normal surroundings. Methods: We designed, synthesized and characterized three novel polymeric Turn-ON probes that will be activated at the tumor site by cysteine cathepsins that are highly expressed in multiple tumor types. Utilizing orthotopic breast cancer and melanoma models, which spontaneously metastasize to the brain, we studied the kinetics of our polymeric Turn-ON nano-probes. Results: To date, numerous low molecular weight cathepsin-sensitive substrates have been reported, however, most of them suffer from rapid clearance and reduced signal shortly after administration. Here, we show an improved tumor-to-background ratio upon activation of our Turn-ON probes by cathepsins. The signal obtained from the tumor was stable and delineated the tumor boundaries during the whole surgical procedure, enabling accurate resection. Conclusions: Our findings show that the control groups of tumor-bearing mice, which underwent either standard surgery under white light only or under the fluorescence guidance of the commercially-available imaging agents ProSense® 680 or 5-aminolevulinic acid (5-ALA), survived for less time and suffered from tumor recurrence earlier than the group that underwent image-guided surgery (IGS) using our Turn-ON probes. Our "smart" polymeric probes can potentially assist surgeons' decision in real-time during surgery regarding the tumor margins needed to be removed, leading to improved patient outcome.