Metabolic changes in glioblastomas in response to choline kinase inhibition: In vivo MRS in rodent models.

Changes in glioblastoma (GBM) metabolism was investigated in response to JAS239, a choline kinase inhibitor, using MRS. In addition to the inhibition of phosphocholine synthesis, we investigated changes in other key metabolic pathways associated with GBM progression and treatment response. Three syngeneic rodent models of GBM were used: F98 (N = 12) and 9L (N = 8) models in rats and GL261 (N = 10) in mice. Rodents were intracranially injected with GBM cells in the right cortex and tumor growth was monitored using T2 -weighted images. Animals were treated once daily with intraperitoneal injections of 4 mg/kg JAS239 (F98 rats, n = 6; 9L rats, n = 6; GL261 mice, n = 5) or saline (control group, F98 rats, n = 6; 9L rats, n = 2; GL261 mice, n = 5) for five consecutive days. Single voxel spectra were acquired on Days 0 (T0, baseline) and 6 (T6, end of treatment) from the tumor as well as the contralateral normal brain using a PRESS sequence. Changes in metabolite ratios (tCho/tCr, tCho/NAA, mI/tCr, Glx/tCr and (Lip + Lac)/Cr) were used to assess metabolic pathway alterations in response to JAS239. Tumor growth arrest was noted in all models in response to JAS239 treatment compared with saline-treated animals, with a significant reduction (p < 0.05) in the F98 model. A reduction in tCho/tCr was observed with JAS239 treatment in all GBM models, indicating reduced phospholipid metabolism, with the highest reduction in 9L followed by GL261 and F98 tumors. A significant reduction (p < 0.05) in the tCho/NAA ratio was observed in the 9L model. A significant reduction in mI/tCr (p < 0.05) was found in JAS239-treated F98 tumors compared with the saline-treated animals. A non-significant trend of reduction in Glx/tCr was observed only in F98 and 9L tumors. JAS239-treated F98 tumors also showed a significant increase in Lip + Lac (p < 0.05), indicating increased cell death. This study demonstrated the utility of MRS in assessing metabolic changes in GBM in response to choline kinase inhibition.

Prospective validation of tumor folate receptor expression density with the association of pafolacianine fluorescence during intraoperative molecular imaging-guided lung cancer resections.

PURPOSE: Pafolacianine, a folate receptor alpha-targeted NIR tracer, has demonstrated clear efficacy in intraoperative molecular imaging-guided (IMI) lung cancer surgery. However, the selection of patients who would benefit from IMI remains challenging given the variability of fluorescence with patient-associated and histopathologic factors. Our goal in this study was to prospectively evaluate whether preoperative FRα/FRß staining can predict pafolacianine-based fluorescence during real-time lung cancer resections. METHODS: This was a prospective study conducted between 2018 and 2022 that reviewed core biopsy and intraoperative data from patients with suspected lung cancer. A total of 196 patients were deemed eligible, of whom core biopsies were taken from 38 patients and assessed for FRα and FRß expression by immunohistochemistry (IHC). All patients underwent infusion of pafolacianine 24 h prior to surgery. Intraoperative fluorescence images were captured with the VisionSense bandpass filter-enabled camera. All histopathologic assessments were performed by a board-certified thoracic pathologist. RESULTS: Of the 38 patients, 5 (13.1%) were found to have benign lesions (necrotizing granulomatous inflammation, lymphoid aggregates) and 1 had metastatic non-lung nodule. Thirty (81.5%) had malignant lesions, with the vast majority (23, 77.4%) being lung adenocarcinoma (7 (22.5%) SCC). None of the benign tumors (0/5, 0%) exhibited in vivo fluorescence (mean TBR of 1.72), while 95% of the malignant tumors fluoresced (mean TBR of 3.11 ± 0.31) compared to squamous cell carcinoma (1.89 ± 0.29) of the lung and sarcomatous lung metastasis (2.32 ± 0.09) (p < 0.01). The TBR was significantly higher in the malignant tumors (p = 0.009). The median FRα and FRß staining intensities were both 1.5 for benign tumors, while the FRα and FRß staining intensities were 3 and 2 for malignant tumors, respectively. Increased FRα expression was significantly associated with the presence of fluorescence (p = 0.01), CONCLUSION: This prospective study sought to determine whether preoperative FRα and FRß expression on core biopsy IHC correlates with intraoperative fluorescence during pafolacianine-guided surgery. These results, although of small sample size, including limited non-adenocarcinoma cohort, suggest that performing FRα IHC on preoperative core biopsies of adenocarcinomas as compared to squamous cell carcinomas could provide low-cost, clinically useful information for optimal patient selection which should be further explored in advanced clinical trials.

Preclinical Evaluation of an Activity-Based Probe for Intraoperative Imaging of Esophageal Cancer.

Background: Early detection and complete resection are important prognostic factors for esophageal cancer (EC). Intraoperative molecular imaging (IMI) using tumor-targeted tracers is effective in many cancer types. However, there are no EC-specific IMI tracers. We sought to test a cathepsin activity-based tracer (VGT-309) for EC resection. Methods: Murine (AKR, HNM007) and human (OE19) EC cell lines were screened for cathepsin expression by western blotting. In vitro binding affinity of VGT-309 was evaluated by fluorescence microscopy. Flank tumor models were developed by injecting EC cells into the flanks of BALB/c or athymic nude mice. Mice pretreated with a cathepsin inhibitor (JPM-OEt) were used to confirm on target binding. Animals were injected with 2 mg/kg VGT-309, underwent IMI, and were sacrificed 24 hours after injection. Results: Cathepsins B, L, S, and X were expressed by EC cell lines, and all cell lines were labeled in vitro with VGT-309. Fluorescent signal was eliminated when cells were pretreated with JPM-OEt. On biodistribution analysis, VGT-309 accumulated in the liver, kidneys, and spleen without other organ involvement. VGT-309 selectively accumulated in flank allografts and xenografts, with mean signal-to-background ratio of 5.21 (IQR: 4.18-6.73) for flank allografts and 4.34 (IQR: 3.75-5.02) for flank xenografts. Fluorescence microscopy and histopathological analysis confirmed the selective accumulation of the tracer in tumors compared to background normal tissues. Conclusions: VGT-309 is an effective tracer for IMI of esophageal cancer. There is potential for clinical translation both as an adjunct to endoscopic detection and for complete removal of disease during esophagectomy.

Presence of non-Newtonian fluid in invasive pulmonary mucinous adenocarcinomas impacts fluorescence during intraoperative molecular imaging of lung cancer.

BACKGROUND: Intraoperative molecular imaging (IMI) with folate-targeted NIR tracers has been shown to improve lesion localization in more than 80% of lung adenocarcinomas. However, mucinous adenocarcinomas (MAs) and invasive mucinous adenocarcinomas (IMAs) of the lung, which are variants of adenocarcinoma, appear to have decreased fluorescence despite appropriate folate receptor expression on the tumor surface. We hypothesized that the etiology may be related to light excitation and emission through non-Newtonian fluid (mucin) produced by goblet and columnar cancer cells. METHODS: Intraoperative data for 311 subjects were retrospectively reviewed from a prospectively collected 6-year database. For standardization, all patients underwent infusion of the same targeted molecular optical contrast agent (pafolacianine, folate receptor-targeted NIR fluorochrome) for lung cancer resections. Then, the ratio of the mean fluorescence intensity of the tumors and background tissues (TBR) was calculated. Tumors were examined for mucin, FRa, FRb, and immunofluorescent tracer uptake by a board-certified pathologist. The optical properties of mucin analyzed by imaging software were used to create in vitro gel models to explore the effects on NIR tracer fluorescence intensity. RESULTS: A large proportion (192, 62%) of the patients were female, with an average of 62.8 years and a 34-year mean pack smoking history. There were no severe (Clavien-Dindo > III) complications related to pafolacianine infusion. A total of 195 lesions in the study were adenocarcinomas, of which 19 (6.1%) were of the mucinous subtype. A total of 14/19 of the patients had a smoking history, and more than 74% of the IMA lesions were in the lower lobes. IMA lesions had a lower in situ TBR than nonmucinous adenocarcinomas (2.64 SD 0.23) vs (3.45 SD 0.11), respectively (p < 0.05). Only 9/19 (47%) were localized in situ. Tumor bisection and removal of mucin from IMAs significantly increased pafolacianine fluorescence, with resultant TBR not being significantly different from the control group (4.67 vs 4.89) (p = 0.19). Of the 16 lesions that underwent FR expression analysis, 15/16 had FR presence on cancer cells or tumor-associated macrophages in the tumor microenvironment. There was no statistically significant difference in fluorescence intensity during immunofluorescence analysis (4.99 vs 5.08) (p = 0.16). Physical removal of mucin from IMAs improved the TBR from 3.11 to 4.67 (p < 0.05). In vitro analysis of the impact of synthetic non-Newtonian fluid (agarose 0.5%) on NIR tracer fluorescence showed a decrease in MFI by a factor of 0.25 regardless of the concentration for each 5 mm thickness of mucin. CONCLUSION: The mucinous subtype of lung adenocarcinomas presents a unique challenge in pafolacianine-targeted IMI-guided resections. The presence of non-Newtonian fluids presents a physical barrier that dampens the excitation of the tracer and fluorescence emission detected by the camera. Knowledge of this phenomenon can allow the surgeon to critically analyze lesion fluorescence parameters during IMI-guided lung cancer resections.

Indocyanine green fluorescence-guided surgery in head and neck cancer: A systematic review.

OBJECTIVE: To assess the feasibility and effectiveness of indocyanine green (ICG) for image-guided resection of head and neck cancer (HNC). DATA SOURCES: PubMed, Embase, and Scopus databases. REVIEW METHODS: Searches were conducted from database inception to February 2022. Patient and study characteristics, imaging parameters, and imaging efficacy data were extracted from each study. RESULTS: Nine studies met inclusion criteria, representing 103 head and neck tumors. Weighted mean ICG dose and imaging time were 1.27 mg/kg and 11.77 h, respectively. Among the five studies that provided quantitative metrics of imaging efficacy, average ICG tumor-to-background ratio (TBR) was 1.56 and weighted mean ONM-100 TBR was 3.64. Pooled sensitivity and specificity across the five studies were 91.7 % and 71.9 %, respectively. CONCLUSION: FGS with ICG may facilitate real-time tumor-margin delineation to improve margin clearance rates and progression-free survival. Future studies with validated, quantitative metrics of imaging success are necessary to further evaluate the prognostic benefit of these techniques.

Combined fluorescence-guided surgery and photodynamic therapy for glioblastoma multiforme using cyanine and chlorin nanocluster.

INTRODUCTION: Glioblastoma multiforme (GBM) is the most common primary intracranial malignancy; survival can be improved by maximizing the extent-of-resection. METHODS: A near-infrared fluorophore (Indocyanine-Green, ICG) was combined with a photosensitizer (Chlorin-e6, Ce6) on the surface of superparamagnetic-iron-oxide-nanoparticles (SPIONs), all FDA-approved for clinical use, yielding a nanocluster (ICS) using a microemulsion. The physical-chemical properties of the ICS were systematically evaluated. Efficacy of photodynamic therapy (PDT) was evaluated in vitro with GL261 cells and in vivo in a subtotal resection trial using a syngeneic flank tumor model. NIR imaging properties of ICS were evaluated in both a flank and an intracranial GBM model. RESULTS: ICS demonstrated high ICG and Ce6 encapsulation efficiency, high payload capacity, and chemical stability in physiologic conditions. In vitro cell studies demonstrated significant PDT-induced cytotoxicity using ICS. Preclinical animal studies demonstrated that the nanoclusters can be detected through NIR imaging in both flank and intracranial GBM tumors (ex: 745 nm, em: 800 nm; mean signal-to-background 8.5 ± 0.6). In the flank residual tumor PDT trial, subjects treated with PDT demonstrated significantly enhanced local control of recurrent neoplasm starting on postoperative day 8 (23.1 mm3 vs 150.5 mm3, p = 0.045), and the treatment effect amplified to final mean volumes of 220.4 mm3 vs 806.1 mm3 on day 23 (p = 0.0055). CONCLUSION: A multimodal theragnostic agent comprised solely of FDA-approved components was developed to couple optical imaging and PDT. The findings demonstrated evidence for the potential theragnostic benefit of ICS in surgical oncology that is conducive to clinical integration.

Imaging of cancer lipid metabolism in response to therapy.

Lipids represent a diverse array of molecules essential to the cell's structure, defense, energy, and communication. Lipid metabolism can often become dysregulated during tumor development. During cancer therapy, targeted inhibition of cell proliferation can likewise cause widespread and drastic changes in lipid composition. Molecular imaging techniques have been developed to monitor altered lipid profiles as a biomarker for cancer diagnosis and treatment response. For decades, MRS has been the dominant non-invasive technique for studying lipid metabolite levels. Recent insights into the oncogenic transformations driving changes in lipid metabolism have revealed new mechanisms and signaling molecules that can be exploited using optical imaging, mass spectrometry imaging, and positron emission tomography. These novel imaging modalities have provided researchers with a diverse toolbox to examine changes in lipids in response to a wide array of anticancer strategies including chemotherapy, radiation therapy, signal transduction inhibitors, gene therapy, immunotherapy, or a combination of these strategies. The understanding of lipid metabolism in response to cancer therapy continues to evolve as each therapeutic method emerges, and this review seeks to summarize the current field and areas of unmet needs.

Identification of a Unique Inhibitor-Binding Site on Choline Kinase α.

Choline kinase α (ChoKα) is an enzyme that is upregulated in many types of cancer and has been shown to be tumorigenic. As such, it makes a promising target for inhibiting tumor growth. Though there have been several inhibitors synthesized for ChoKα, not all of them demonstrate the same efficacy in vivo, though the reasons behind this difference in potency are not clear. One particular inhibitor, designated TCD-717, has recently completed phase I clinical trials. Cell culture and in vitro studies support the powerful inhibitory effect TCD-717 has on ChoKα, but an examination of the inhibitor's interaction with the ChoKα enzyme has been missing prior to this work. Here we detail the 2.35 Å structure of ChoKα in complex with TCD-717. Examination of this structure in conjunction with kinetic assays reveals that TCD-717 does not bind directly in the choline pocket as do previously characterized ChoKα inhibitors, but rather in a proximal but novel location near the surface of the enzyme. The unique binding site identified for TCD-717 lends insight for the future design of more potent in vivo inhibitors for ChoKα.

Calcium-axonemal microtubuli interactions underlie mechanism(s) of primary cilia morphological changes.

We have used cell culture of astrocytes aligned within microchannels to investigate calcium effects on primary cilia morphology. In the absence of calcium and in the presence of flow of media (10 µL.s-1) the majority (90%) of primary cilia showed reversible bending with an average curvature of 2.1 ± 0.9 × 10-4 nm-1. When 1.0 mM calcium was present, 90% of cilia underwent bending. Forty percent of these cilia demonstrated strong irreversible bending, resulting in a final average curvature of 3.9 ± 1 × 10-4 nm-1, while 50% of cilia underwent bending similar to that observed during calcium-free flow. The average length of cilia was shifted toward shorter values (3.67 ± 0.34 µm) when exposed to excess calcium (1.0 mM), compared to media devoid of calcium (3.96 ± 0.26 µm). The number of primary cilia that became curved after calcium application was reduced when the cell culture was pre-incubated with 15 µM of the microtubule stabilizer, taxol, for 60 min prior to calcium application. Calcium caused single microtubules to curve at a concentration ≈1.0 mM in vitro, but at higher concentration (≈1.5 mM) multiple microtubule curving occurred. Additionally, calcium causes microtubule-associated protein-2 conformational changes and its dislocation from the microtubule wall at the location of microtubule curvature. A very small amount of calcium, that is 1.45 × 1011 times lower than the maximal capacity of TRPPs calcium channels, may cause gross morphological changes (curving) of primary cilia, while global cytosol calcium levels are expected to remain unchanged. These findings reflect the non-linear manner in which primary cilia may respond to calcium signaling, which in turn may influence the course of development of ciliopathies and cancer.

Intraoperative Molecular Imaging Combined With Positron Emission Tomography Improves Surgical Management of Peripheral Malignant Pulmonary Nodules.

OBJECTIVE: To determine if intraoperative molecular imaging (IMI) can improve detection of malignant pulmonary nodules. BACKGROUND: 18-Fluorodeoxyglucose positron emission tomography (PET) is commonly utilized in preoperative assessment of patients with solid malignancies; however, false negatives and false positives remain major limitations. Using patients with pulmonary nodules as a study model, we hypothesized that IMI with a folate receptor targeted near-infrared contrast agent (OTL38) can improve malignant pulmonary nodule identification when combined with PET. METHODS: Fifty patients with pulmonary nodules with imaging features suspicious for malignancy underwent preoperative PET. Patients then received OTL38 before pulmonary resection. During resection, IMI was utilized to evaluate known pulmonary nodules and identify synchronous lesions. Tumor size, PET standardized uptake value, and IMI tumor-to-background ratios were compared for known and synchronous nodules via paired and unpaired t tests, when appropriate. Test characteristics of PET and IMI with OTL38 were compared. RESULTS: IMI identified 56 of 59 (94.9%) malignant pulmonary nodules identified by preoperative imaging. IMI located an additional 9 malignant lesions not identified preoperatively. Nodules only detected by IMI were smaller than nodules detected preoperatively (0.5 vs 2.4 cm; P < 0.01), but displayed similar fluorescence (tumor-to-background ratio 3.3 and 3.1; P = 0.50). Sensitivity of IMI and PET were 95.6% and 73.5% (P = 0.001), respectively; and positive predictive values were 94.2% and 89.3%, respectively (P > 0.05). Additionally, utilization of IMI clinically upstaged 6 (12%) subjects and improved management of 15 (30%) subjects. CONCLUSIONS: These data suggest that combining IMI with PET may provide superior oncologic outcomes for patients with resectable lung cancer.

Development of silica-encapsulated silver nanoparticles as contrast agents intended for dual-energy mammography.

OBJECTIVE: Dual-energy (DE) mammography has recently entered the clinic. Previous theoretical and phantom studies demonstrated that silver provides greater contrast than iodine for this technique. Our objective was to characterize and evaluate in vivo a prototype silver contrast agent ultimately intended for DE mammography. METHODS: The prototype silver contrast agent was synthesized using a three-step process: synthesis of a silver core, silica encapsulation and PEG coating. The nanoparticles were then injected into mice to determine their accumulation in various organs, blood half-life and dual-energy contrast. All animal procedures were approved by the institutional animal care and use committee. RESULTS: The final diameter of the nanoparticles was measured to be 102 (±9) nm. The particles were removed from the vascular circulation with a half-life of 15 min, and accumulated in macrophage-rich organs such as the liver, spleen and lymph nodes. Dual-energy subtraction techniques increased the signal difference-to-noise ratio of the particles by as much as a factor of 15.2 compared to the single-energy images. These nanoparticles produced no adverse effects in mice. CONCLUSION: Silver nanoparticles are an effective contrast agent for dual-energy x-ray imaging. With further design improvements, silver nanoparticles may prove valuable in breast cancer screening and diagnosis. KEY POINTS: • Silver has potential as a contrast agent for DE mammography. • Silica-coated silver nanoparticles are biocompatible and suited for in vivo use. • Silver nanoparticles produce strong contrast in vivo using DE mammography imaging systems.

Synthesis and Evaluation of Cytosolic Phospholipase A(2) Activatable Fluorophores for Cancer Imaging.

Activatable fluorophores selective to cytosolic phospholipase A2 (cPLA2) were synthesized and evaluated for their ability to image triple negative breast cancer cells. The activatable constructs were synthesized by esterification of a small molecule fluorophore with a fatty acid resulting in ablated fluorescence. Selectivity for cPLA2 was generated through the choice of fluorophore and fatty acid. Esterification with arachidonic acid was sufficient to impart specificity to cPLA2 when compared to esterification with palmitic acid. In vitro analysis of probes incorporated into phosphatidylcholine liposomes demonstrated that a nonselective phospholipase (sPLA2 group IB) was able to hydrolyze both arachidonate and palmitate coupled fluorophores resulting in the generation of fluorescence. Of the four fluorophores tested, DDAO (7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)) was observed to perform optimally in vitro and was analyzed further in 4175-Luc+ cells, a metastatic triple negative human breast cancer cell line expressing high levels of cPLA2. In contrast to the in vitro analysis, DDAO arachidonate was shown to activate selectively in 4175-Luc+ cells compared to the control DDAO palmitate as measured by fluorescence microscopy and quantitated with fluorescence spectroscopy. The addition of two agents known to activate cPLA2 enhanced DDAO arachidonate fluorescence without inducing any change to DDAO palmitate. Inhibition of cPLA2 resulted in reduced fluorescence of DDAO arachidonate but not DDAO palmitate. Together, we report the synthesis of a cPLA2 selective activatable fluorophore capable of detecting cPLA2 in triple negative breast cancer cells.

Implementation of two-dimensional L-COSY at 7 Tesla: an investigation of reproducibility in human brain.

PURPOSE: To evaluate the utility of two-dimensional (2D) Localized Correlated Spectroscopy (L-COSY) in metabolic profiling of the human brain at 7 Tesla (T). MATERIALS AND METHODS: The 2D L-COSY sequence was implemented at 7 T and its reliability was assessed by test-retest studies of a metabolite phantom and a healthy volunteer. L-COSY data were acquired from the occipital lobe of healthy subjects (n = 6; all male; age, 30-72 years) to assess intersubject variability. Additionally, two subjects underwent scans from the parieto-occipital region, basal ganglia, frontal lobe or dorsolateral prefrontal cortex to test the versatility of L-COSY in studying differing anatomy. Integral/volume measurements of L-COSY spectra were used to estimate normalized metabolite-to-creatine concentrations. RESULTS: Phantom test-retest studies revealed coefficients of variation (CVs) of 3-20% for most metabolites. Human 2D L-COSY spectra permitted detection of several metabolite resonances from multiple locations and inter-subject variation studies demonstrated CVs of 4-26%. Cross-peaks from gamma-aminobutyric acid (GABA), isoleucine (Ile), lysine (Lys) and Ethanolamine (Eth) were quantified, which are not readily resolvable with conventional one-dimensional (1D) MR spectroscopy. CONCLUSION: 2D L-COSY at 7 T demonstrated improved sensitivity in detecting additional metabolites with reliability comparable to established techniques at lower fields, which may aid in the metabolic assessment of diseased states.

Inhibition of glioblastoma cell proliferation and invasion by the choline-kinase inhibitor JAS239 varies with cell type and hypoxia.

Background: Elevated choline kinase alpha (ChoK) is observed in most solid tumours including glioblastomas (GBM), yet until recently, inhibitors of ChoK have demonstrated limited efficacy in GBM models. Given that hypoxia is associated with GBM therapy resistance, we hypothesised that tumour hypoxia could be responsible for such limitations. We therefore evaluated in GBM cells, the effect of hypoxia on the function of JAS239, a potent ChoK inhibitor. Methods: Rodent (F98 and 9L) and human (U-87 MG and U-251 MG) GBM cell lines were subjected to 72 hours of hypoxia conditioning and treated with JAS239 for 24 hours. NMR metabolomic measurements and analyses were performed to evaluate the signalling pathways involved. In addition, cell proliferation, cell cycle progression and cell invasion were measured in cell monolayers and 3D spheroids, with or without JAS239 treatment in normoxic or hypoxic cells to assess how hypoxia affects JAS239 function. Results: Hypoxia and JAS239 treatment led to significant changes in the cellular metabolic pathways, specifically the phospholipid and glycolytic pathways associated with a reduction in cell proliferation via induced cell cycle arrest. Interestingly, JAS239 also impaired GBM invasion. However, JAS239 effects were variable depending on the cell line, reflecting the inherent heterogeneity observed in GBMs. Conclusion: Our findings indicate that JAS239 and hypoxia can deregulate cellular metabolism, inhibit proliferation and alter cell invasion. These results may be useful for the design of new therapeutic strategies based on ChoK inhibition that can act on multiple pro-tumorigenic features.

A Phase 2 Multicenter Clinical Trial of Intraoperative Molecular Imaging of Lung Cancer with a pH-Activatable Nanoprobe.

PURPOSE: Intraoperative molecular imaging (IMI) uses tumor-targeted optical contrast agents to improve identification and clearance of cancer. Recently, a probe has been developed that only fluoresces when activated in an acidic pH, which is common to many malignancies. We report the first multicenter Phase 2 trial of a pH-activatable nanoprobe (pegsitacianine, ONM-100) for IMI of lung cancer. METHODS: Patients with suspected or biopsy-confirmed lung cancer scheduled for sublobar resection were administered a single intravenous infusion of pegsitacianine (1 mg/kg) one to three days prior to surgery. Intraoperatively, the patients underwent a white light thoracoscopic evaluation, and then were imaged with an NIR thoracoscope to detect tumor fluorescence. The primary study endpoint was the proportion of patients with a clinically significant event (CSE) which was defined as an intraoperative discovery during IMI that led to a change in the surgical procedure. Possible CSEs included (i) localizing the index lung nodule that could not be located by white light, (ii) identifying a synchronous malignant lesion, or (iii) recognizing a close surgical margin (< = 10 mm). Secondary endpoints were sensitivity, specificity, NPV, and PPV of pegsitacianine in detecting tumor-containing tissue. The safety evaluation was based on adverse event reporting, clinical laboratory parameters, and physical examinations. RESULTS: Twenty patients were confirmed as eligible and administered pegsitacianine. Most of the patients were female (n = 12 [60%]), middle-aged (mean age 63.4 years), and former smokers (n = 13 [65%], 28.6 mean pack years). Mean lesion size was 1.9 cm, and most lesions (n = 17 [85%]) were malignant. The most common histologic subtype was adenocarcinoma (n = 9). By utilizing IMI with pegsitacianine, one patient had a CSE in the detection of a close margin and another had localization of a tumor not detectable by traditional surgical means. Six of 19 (31.6%) malignant lesions fluoresced with mean tumor-to-background ratio (TBR) of 3.00, as compared to TBR of 1.20 for benign lesions (n = 3). Sensitivity and specificity of pegsitacianine-based IMI for detecting malignant tissue was 31.6% and 33.3%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) of pegsitacianine-based IMI was 75% and 7.1%, respectively. Pegsitacianine-based imaging was not effective in differentiating benign and malignant lymph nodes. From a safety perspective, no drug-related serious adverse events occurred. Four patients experienced mild pegsitacianine-related infusion reactions which required discontinuing the study drug with complete resolution of symptoms. CONCLUSIONS: Pegsitacianine-based IMI, though well tolerated from a safety perspective, does not consistently label lung tumors during resection and does not provide significant clinical benefit over existing standards of surgical care. The biology of lung tumors may not be as acidic as other solid tumors in the body thereby not activating the probe as predicted.

A Biodegradable "One-For-All" Nanoparticle for Multimodality Imaging and Enhanced Photothermal Treatment of Breast Cancer.

Silver sulfide nanoparticles (Ag2S-NP) hold promise for various optical-based biomedical applications, such as near-infrared fluorescence (NIRF) imaging, photoacoustics (PA), and photothermal therapy (PTT). However, their NIR absorbance is relatively low, and previous formulations are synthesized using toxic precursors under harsh conditions and are not effectively cleared due to their large size. Herein, sub-5 nm Ag2S-NP are synthesized and encapsulated in biodegradable, polymeric nanoparticles (AgPCPP). All syntheses are conducted using biocompatible, aqueous reagents under ambient conditions. The encapsulation of Ag2S-NP in polymeric nanospheres greatly increases their NIR absorbance, resulting in enhanced optical imaging and PTT effects. AgPCPP nanoparticles exhibit potent contrast properties suitable for PA and NIRF imaging, as well as for computed tomography (CT). Furthermore, AgPCPP nanoparticles readily improve the conspicuity of breast tumors in vivo. Under NIR laser irradiation, AgPCPP nanoparticles significantly reduce breast tumor growth, leading to prolonged survival compared to free Ag2S-NP. Over time, AgPCPP retention in tissues gradually decreases, without any signs of acute toxicity, providing strong evidence of their safety and biodegradability. Therefore, AgPCPP may serve as a "one-for-all" theranostic agent that degrades into small components for excretion after fulfilling diagnostic and therapeutic tasks, offering good prospects for clinical translation.

Decreased lactate concentration and glycolytic enzyme expression reflect inhibition of mTOR signal transduction pathway in B-cell lymphoma.

The application of kinase inhibitors in cancer treatment is growing rapidly. However, methods for monitoring the effectiveness of the inhibitors are still poorly developed and currently rely mainly on the tracking of changes in the tumor volume, a rather late and relatively insensitive marker of the therapeutic response. In contrast, MRS can detect changes in cell metabolism and has the potential to provide early and patient-specific markers of drug activity. Using human B-cell lymphoma models and MRS, we have demonstrated that the inhibition of the mTOR signaling pathway can be detected in malignant cells in vitro and noninvasively in vivo by the measurement of lactate levels. An mTOR inhibitor, rapamycin, suppressed lactic acid production in lymphoma cell line cultures and also diminished steady-state lactate levels in xenotransplants. The inhibition was time dependent and was first detectable 8 h after drug administration in cell cultures. In xenotransplants, 2 days of rapamycin treatment produced significant changes in lactic acid concentration in the tumor measured in vivo, which were followed by tumor growth arrest and tumor volume regression. The rapamycin-induced changes in lactate production were strongly correlated with the inhibition of expression of hexokinase II, the key enzyme in the glycolytic pathway. These studies suggest that MRS or (18) F-fluorodeoxyglucose positron emission tomography (FDG PET) detection of changes in glucose metabolism may represent effective noninvasive methods for the monitoring of mTOR targeting therapy in lymphomas and other malignancies. Furthermore, the measurement of glucose metabolic inhibition by MRS or FDG PET imaging may also prove to be effective in monitoring the efficacy of other kinase inhibitors given that the rapamycin-sensitive mTOR lies downstream of many oncogenic signaling pathways.

Three-Dimensional Near-Infrared Specimen Mapping Can Identify the Distance from the Tumor to the Surgical Margin During Resection of Pulmonary Ground Glass Opacities.

BACKGROUND: Lung cancers can recur locally due to inadequate resection margins. Achieving adequate margin distances is challenging in pulmonary ground glass opacities (GGOs) because they are not easily palpable. To improve margin assessment during resection of GGOs, we propose a novel technique, three-dimensional near-infrared specimen mapping (3D-NSM). METHODS: Twenty patients with a cT1 GGO were enrolled and received a fluorescent tracer preoperatively. After resection, specimens underwent 3D-NSM in the operating room. Margins were graded as positive or negative based upon fluorescence at the staple line. Images were analyzed using ImageJ to quantify the distance from the tumor edge to the nearest staple line. This margin distance calculated by 3D-NSM was compared to the margin distance reported on final pathology several days postoperatively. RESULTS: 3D-NSM identified 20/20 GGOs with no false positive or false negative diagnoses. Mean fluorescence intensity for lesions was 110.92 arbitrary units (A.U.) (IQR: 77.77-122.03 A.U.) compared to 23.68 A.U. (IQR: 19.60-27.06 A.U.) for background lung parenchyma (p < 0.0001). There were 4 tumor-positive or close margins in the study cohort, and all 4 (100%) were identified by 3D-NSM. 3D-NSM margin distances were nearly identical to margin distances reported on final pathology (R2 = 0.9362). 3D-NSM slightly under-predicted margin distance, and the median difference in margins was 1.9 mm (IQR 0.5-4.3 mm). CONCLUSIONS: 3D-NSM rapidly localizes GGOs by fluorescence and detects tumor-positive or close surgical margins. 3D-NSM can accurately quantify the resection margin distance as compared to formal pathology, which allows surgeons to rapidly determine whether sublobar resection margin distances are adequate.

A pH-Activatable Nanoprobe Labels Diverse Histologic Subtypes of Human Lung Cancer During Resection.

BACKGROUND: Intraoperative molecular imaging (IMI) uses tumor-targeted optical contrast agents to improve identification and clearance of cancer during surgery. Recently, pH-activatable contrast agents have been developed but none has been tested in lung cancer. Here, we report the successful clinical translation of pegsitacianine (ONM-100), a pH-activatable nanoprobe, for fluorescence-guided lung cancer resection. METHODS: We first characterized the pH setpoint for pegsitacianine fluorescence activation in vitro. We then optimized the specificity, dosing, and timing of pegsitacianine in murine flank xenograft models of lung adenocarcinoma and squamous cell carcinoma. Finally, we tested pegsitacianine in humans undergoing lung cancer surgery as part of an ongoing phase 2 trial. RESULTS: We found that the fluorescence activation of pegsitacianine occurred below physiologic pH in vitro. Using preclinical models of lung cancer, we found that the probe selectively labeled both adenocarcinoma and squamous cell carcinoma xenografts (mean tumor-to-background ratio [TBR] > 2.0 for all cell lines). In the human pilot study, we report cases in which pegsitacianine localized pulmonary adenocarcinoma and pulmonary squamous cell carcinoma (TBRs= 2.7 and 2.4) in real time to illustrate its successful clinical translation and potential to improve surgical management. CONCLUSIONS: This translational study demonstrates the feasibility of pegsitacianine as an IMI probe to label the two most common histologic subtypes of human lung cancer.

Intraoperative molecular imaging: 3rd biennial clinical trials update.

Significance: This third biennial intraoperative molecular imaging (IMI) conference shows how optical contrast agents have been applied to develop clinically significant endpoints that improve precision cancer surgery. Aim: National and international experts on IMI presented ongoing clinical trials in cancer surgery and preclinical work. Previously known dyes (with broader applications), new dyes, novel nonfluorescence-based imaging techniques, pediatric dyes, and normal tissue dyes were discussed. Approach: Principal investigators presenting at the Perelman School of Medicine Abramson Cancer Center's third clinical trials update on IMI were selected to discuss their clinical trials and endpoints. Results: Dyes that are FDA-approved or currently under clinical investigation in phase 1, 2, and 3 trials were discussed. Sections on how to move benchwork research to the bedside were also included. There was also a dedicated section for pediatric dyes and nonfluorescence-based dyes that have been newly developed. Conclusions: IMI is a valuable adjunct in precision cancer surgery and has broad applications in multiple subspecialties. It has been reliably used to alter the surgical course of patients and in clinical decision making. There remain gaps in the utilization of IMI in certain subspecialties and potential for developing newer and improved dyes and imaging techniques.