Intraoperative near infrared functional imaging of rectal cancer using artificial intelligence methods - now and near future state of the art.

Colorectal cancer remains a major cause of cancer death and morbidity worldwide. Surgery is a major treatment modality for primary and, increasingly, secondary curative therapy. However, with more patients being diagnosed with early stage and premalignant disease manifesting as large polyps, greater accuracy in diagnostic and therapeutic precision is needed right from the time of first endoscopic encounter. Rapid advancements in the field of artificial intelligence (AI), coupled with widespread availability of near infrared imaging (currently based around indocyanine green (ICG)) can enable colonoscopic tissue classification and prognostic stratification for significant polyps, in a similar manner to contemporary dynamic radiological perfusion imaging but with the advantage of being able to do so directly within interventional procedural time frames. It can provide an explainable method for immediate digital biopsies that could guide or even replace traditional forceps biopsies and provide guidance re margins (both areas where current practice is only approximately 80% accurate prior to definitive excision). Here, we discuss the concept and practice of AI enhanced ICG perfusion analysis for rectal cancer surgery while highlighting recent and essential near-future advancements. These include breakthrough developments in computer vision and time series analysis that allow for real-time quantification and classification of fluorescent perfusion signals of rectal cancer tissue intraoperatively that accurately distinguish between normal, benign, and malignant tissues in situ endoscopically, which are now undergoing international prospective validation (the Horizon Europe CLASSICA study). Next stage advancements may include detailed digital characterisation of small rectal malignancy based on intraoperative assessment of specific intratumoral fluorescent signal pattern. This could include T staging and intratumoral molecular process profiling (e.g. regarding angiogenesis, differentiation, inflammatory component, and tumour to stroma ratio) with the potential to accurately predict the microscopic local response to nonsurgical treatment enabling personalised therapy via decision support tools. Such advancements are also applicable to the next generation fluorophores and imaging agents currently emerging from clinical trials. In addition, by providing an understandable, applicable method for detailed tissue characterisation visually, such technology paves the way for acceptance of other AI methodology during surgery including, potentially, deep learning methods based on whole screen/video detailing.

Indocyanine green fluorescence visualizes landmark arteries for endoscopic sinus and skull base surgery.

OBJECTIVE: Landmark arteries during endoscopic sinus surgery are currently identified on the basis of anatomy, CT imaging and navigation, and Doppler flowmetry. However, the advantage of intraoperative fluorescence imaging during endoscopic sinus surgery has not been demonstrated. This study aimed to investigate whether Indocyanine Green (ICG) is useful for visualizing landmark arteries during endoscopic sinus and skull base surgery. METHODS: Eight patients who underwent endoscopic sinus and pituitary surgeries and consented to study participation were included. After planned procedures were performed as usual, landmark arteries were examined by ICG endoscope. Recorded video and preoperative CT images were analyzed for identification of five landmark arteries: anterior ethmoidal artery (AEA), posterior ethmoidal artery (PEA), internal carotid artery (ICA), sphenopalatine artery (SPA), and postnasal artery (PNA). Identification of arteries was evaluated three grades: identifiable, locatable, unrecognizable. RESULTS: Eight patients and eleven sides were evaluated. The ICG dose was 2.5 mg/body and a single shot was sufficient for evaluation. 100 % of AEA was identified (9/9 sides), 86 % of PNA (6/7 sides), 56 % of ICA (5/9 sides), and 25 % of PEA and SPA (2/8 sides). CONCLUSION: ICG could visualize landmark arteries, even thin arteries like AEA, during endoscopic sinus and skull base surgeries. Visualization was affected by thickness of bone or soft tissue above arteries, blood clots, sensitivity setting, and angle and distance of near-infrared light irradiation. ICG visualization of landmark arteries may help avoid vascular injuries during endoscopic sinus and skull base surgeries, particularly of AEA, PNA and ICA.

Detection of near-infrared autofluorescence from adrenal neoplasms: An initial experience.

BACKGROUND AND OBJECTIVES: Fluorescence from adrenal tumors can be detected with near-infrared imaging after injection with indocyanine green. However, it is unknown if adrenal tumors exhibit autofluorescence. The aim of this study was to determine whether adrenal tumors emit near-infrared autofluorescence (NIRAF). METHODS: This was a prospective study of patients who underwent minimally invasive adrenalectomy at a tertiary center. Intraoperative images were analyzed to detect NIRAF with a 750 nm camera. Descriptive and comparative statistical analyses were performed. RESULTS: Twenty-five adrenalectomies were examined. Only 11 tumors (44%), that originated from the cortex exhibited autofluorescence. A contrast distinction between the tumor and retroperitoneum was observed in 23 patients, whereas a contrast distinction between the tumor and normal adrenocortical tissue was seen in 12 patients. The overall fluorescence intensity of adrenal tumors was found to be variable and ranging between 0.3 and 5.6 times that of the background tissue. Pheochromocytoma, malignancy and adrenal cyst did not demonstrate NIRAF. CONCLUSION: This is the first study to show that adrenocortical tissue can demonstrate NIRAF. The pattern of fluorescence was similar to that observed after indocyanine green injection in our historical experience. NIRAF has a potential to be used as an intraoperative optical adjunct during adrenalectomy.

5-Aminolevulinic acid fluorescence guided surgery for recurrent high-grade gliomas.

INTRODUCTION: Fluorescence guided surgery (FGS) with five-aminolevulinic acid (5-ALA) is expected to revolutionize neurosurgical care of patients with high-grade gliomas (HGG). After the recent landmark FDA approval, this optical agent is now available to neurosurgeons in the United States. METHODS: This review is designed to highlight the evidence for the use of 5-ALA in recurrent HGG surgery for the neurosurgical community. The manuscript was prepared in accordance with the PRISMA guidelines. RESULTS: Intra-operatively, a strong fluorescent signal is highly correlated with the presence of cellular tumor in recurrent HGG, giving it a high positive predictive value (PPV). Similar to what is observed in primary HGG surgery, false-negative results can occur if tumor cells do not emit fluorescence. In addition, false-positive fluorescence signals in tissues devoid of tumor cells can be observed more frequently in recurrent HGG compared to the primary setting. However, these areas overwhelmingly contain reactive/regressive tissue, resection of which is unlikely to cause functional deficits. The safety profile of 5-ALA is similarly favorable in primary and recurrent HGG. CONCLUSIONS: 5-ALA FGS is a powerful adjunct in the resection of recurrent HGG with a high PPV and favorable safety profile. It is therefore the authors' opinion to routinely employ this fluorescent agent as a standard of care.

Advantages of patient-derived orthotopic mouse models and genetic reporters for developing fluorescence-guided surgery.

Fluorescence-guided surgery can enhance the surgeon's ability to achieve a complete oncologic resection. There are a number of tumor-specific probes being developed with many preclinical mouse models to evaluate their efficacy. The current review discusses the different preclinical mouse models in the setting of probe evaluation and highlights the advantages of patient-derived orthotopic xenografts (PDOX) mouse models and genetic reporters to develop fluorescence-guided surgery.

Preclinical evaluation of near-infrared (NIR) fluorescently labeled cetuximab as a potential tool for fluorescence-guided surgery.

The high rate of recurrence in patients with pancreatic ductal adenocarcinoma (PDAC) could be reduced by supporting the surgeons in discriminating healthy from diseased tissues with intraoperative fluorescence-guidance. Here, we studied the suitability of Cetuximab, a therapeutic monoclonal antibody targeting the human epidermal growth factor receptor (EGFR), near-infrared (NIR) fluorescently labeled as a new tool for fluorescence-guided surgery. Distribution and binding of systemically injected Cetuximab Alexa Fluor 647 conjugate (Cetux-Alexa-647) and the co-injected control human IgG Alexa Fluor 750 conjugate (hIgG-Alexa-750) was studied over 48 h by NIR fluorescence imaging in mice bearing human orthotopic AsPC-1 and MIA PaCa-2 PDAC tumors. Cetux-Alexa-647, but not the control hIgG-Alexa-750 fluorescence, was specifically detected in vivo in both primary pancreatic tumors with maximum fluorescence intensities at 24 h, and in metastases of AsPC-1 tumors as small as 1 mm. Lifetime analysis and NIR fluorescence microscopy of tumor sections confirmed the binding specificity of Cetux-Alexa-647 to PDAC cells. Comparable results were obtained with Cetuximab conjugated to Alexa Fluor 750 dye (Cetux-Alexa-750). Fluorescence-guided dissection, performed 24 h after injection of Cetuximab conjugated to IRDye 800CW (Cetux-800CW), enabled a real-time delineation of AsPC-1 tumor margins, and small metastases. Odyssey scans revealed that only the vital part of the tumor, but not the necrotic part was stained with Cetux-800CW. NIR fluorescently labeled Cetuximab may be a promising tool that can be applied for fluorescence-guided surgery to visualize tumor margins and metastatic sites in order to allow a precise surgical resection.

Indocyanine green (ICG) fluorescence-guided laparoscopic adrenalectomy.

OBJECTIVE: Laparoscopic adrenalectomy has become the standard of care for many adrenal tumors. However, the success of the operation hinges on identifying the adrenal vein and complete tumor resection. We demonstrate the use of a commercially available near infrared fluorescent imaging system to clearly delineate the vascular anatomy of adrenal neoplasms and enhance the border between tumor and normal tissue. We hypothesize that this will increase the safety of laparoscopic adrenalectomy. MATERIALS AND METHODS: We performed laparoscopic adrenalectomy utilizing indocyanine green (ICG) and a specialized laparoscopic fluorescence imaging system on four consecutive patients undergoing laparoscopic adrenalectomy over a 4-month period. RESULTS: The adrenal arteries and vein were vividly enhanced with ICG fluorescence guidance, and the border between tumor and adjacent tissue was clearly demarcated. The operations were performed safely with minimal blood loss and short operative times. There were no complications. CONCLUSIONS: Adrenal neoplasms can be resected laparoscopically under ICG fluorescence guidance and can be used to clearly identify vascular structures and enhance the borders of the tumor. This technique allows for clear identification of the adrenal vein and has the potential to improve the safety of laparoscopic adrenalectomy.

Improved disease-free survival and overall survival after fluorescence-guided surgery of liver metastasis in an orthotopic nude mouse model.

BACKGROUND: In the present study, we sought to determine if fluorescence-guided surgery (FGS) would improve survival compared to standard bright light surgery (BLS) in an experimental colorectal liver metastasis nude mouse model. METHODS: Orthotopic nude-mouse models of human HT-29-GFP colon cancer liver metastasis were established in the left lobe of the liver of mice. Fourteen mice with a single liver metastasis were randomized into FGS or BLS groups of seven each. FGS of liver metastasis was performed using a hand-held portable fluorescence imaging system (Dino-Lite) to visualize the GFP fluorescence of the metastasis. The BLS- and FGS-treated mice were followed by weekly fluorescence imaging in order to detect recurrence. RESULTS: The bright fluorescence of GFP provided sufficient illumination to accurately distinguish the margins of the metastasis within the liver. Recurrence occurred in multiple sites including the liver, lung, and other organs in the BLS-treated mice but was significantly reduced in FGS-treated mice. The FGS-treated mice had significantly prolonged disease-free survival (P = 0.001) and overall survival (P = 0.027) compared to BLS-treated mice. CONCLUSION: The results of the present report demonstrate the feasibility and efficacy of FGS for liver metastasis and suggest its important clinical potential.

In Vivo Labeling and Detection of Circulating Tumor Cells in Mice Using OTL38.

PURPOSE: We recently developed an optical instrument to non-invasively detect fluorescently labeled circulating tumor cells (CTCs) in mice called 'Diffuse in vivo Flow Cytometry' (DiFC). OTL38 is a folate receptor (FR) targeted near-infrared (NIR) contrast agent that is FDA approved for use in fluorescence guided surgery of ovarian and lung cancer. In this work, we investigated the use OTL38 for in vivo labeling and detection of FR + CTCs with DiFC. PROCEDURES: We tested OTL38 labeling of FR + cancer cell lines (IGROV-1 and L1210A) as well as FR- MM.1S cells in suspensions of Human Peripheral Blood Mononuclear cells (PBMCs) in vitro. We also tested OTL38 labeling and NIR-DIFC detection of FR + L1210A cells in blood circulation in nude mice in vivo. RESULTS: 62% of IGROV-1 and 83% of L1210A were labeled above non-specific background levels in suspensions of PBMCs in vitro compared to only 2% of FR- MM.1S cells. L1210A cells could be labeled with OTL38 directly in circulation in vivo and externally detected using NIR-DiFC in mice with low false positive detection rates. CONCLUSIONS: This work shows the feasibility of labeling CTCs in vivo with OTL38 and detection with DiFC. Although further refinement of the DiFC instrument and signal processing algorithms and testing with other animal models is needed, this work may eventually pave the way for human use of DiFC.

The potential of indocyanine green fluorescence detection in surgical cut margin of breast conserving surgery.

Background: Fluorescence-guided surgery (FGS) is a cutting-edge technology that uses near-infrared (NIR) fluorescence imaging to guide surgeons in surgery. Indocyanine green (ICG) is a fluorescent dye, which can be used for in vivo imaging of tumor cells. We aimed to explore the use of ICG fluorescence-guided technology as a rapid intraoperative margin assessment method for breast cancer surgery. In addition, we also compared the dose selection of ICG. Methods: This was a non-randomized prospective cohort study. Data were collected between August 2021 and October 2022 in the Division of Breast Surgery, Department of General Surgery, Nanjing Drum Tower Hospital, the Affiliated Hospital of Medical School, Nanjing University. Upon specimen removal, tumor margins were immediately analyzed by ICG fluorescence detection and then sent to the pathology department for intraoperative frozen section analysis and subsequent routine pathological examination. Abnormal margin rates were calculated and compared using intraoperative frozen section analysis and under the guidance of ICG fluorescence. Results: The study included 69 cases of breast cancer patients who underwent tumor resection assisted by ICG fluorescence-guided technology, including 18 patients with a 0.5 mg/kg dose and 51 patients with a 1.0 mg/kg dose. According to the study findings, the ICG test achieved a sensitivity of 81.82% and a specificity of 75.82%. At a dose of 0.5 mg/kg, the sensitivity was 66.67% whereas the specificity was 93.33%. At the dose of 1 mg/kg, the sensitivity was 87.5%, and the specificity was 74.42%. Similarly, for intraoperative frozen section analysis, the sensitivity was 81.82%, but the specificity was enhanced to 94.83%. Positive surgical cut margin was not identified in 2/69 by ICG fluorescence and frozen section analysis respectively. Conclusions: The sensitivity of ICG fluorescence detection is comparable to that of frozen section analysis, but the specificity is poor. The sensitivity increased and the specificity decreased at 1 mg/kg compared to the 0.5 mg/kg dose. ICG fluorescence can be used as a supplementary tool for frozen section analysis. These findings support further development and clinical performance assessment of ICG fluorescence.

Intraoperative microscopic autofluorescence detection and characterization in brain tumors using stimulated Raman histology and two-photon fluorescence.

Introduction: The intrinsic autofluorescence of biological tissues interferes with the detection of fluorophores administered for fluorescence guidance, an emerging auxiliary technique in oncological surgery. Yet, autofluorescence of the human brain and its neoplasia is sparsely examined. This study aims to assess autofluorescence of the brain and its neoplasia on a microscopic level by stimulated Raman histology (SRH) combined with two-photon fluorescence. Methods: With this experimentally established label-free microscopy technique unprocessed tissue can be imaged and analyzed within minutes and the process is easily incorporated in the surgical workflow. In a prospective observational study, we analyzed 397 SRH and corresponding autofluorescence images of 162 samples from 81 consecutive patients that underwent brain tumor surgery. Small tissue samples were squashed on a slide for imaging. SRH and fluorescence images were acquired with a dual wavelength laser (790 nm and 1020 nm) for excitation. In these images tumor and non-tumor regions were identified by a convolutional neural network that reliably differentiates between tumor, healthy brain tissue and low quality SRH images. The identified areas were used to define regions.of- interests (ROIs) and the mean fluorescence intensity was measured. Results: In healthy brain tissue, we found an increased mean autofluorescence signal in the gray (11.86, SD 2.61, n=29) compared to the white matter (5.99, SD 5.14, n=11, p<0.01) and in the cerebrum (11.83, SD 3.29, n=33) versus the cerebellum (2.82, SD 0.93, n=7, p<0.001), respectively. The signal of carcinoma metastases, meningiomas, gliomas and pituitary adenomas was significantly lower (each p<0.05) compared to the autofluorescence in the cerebrum and dura, and significantly higher (each p<0.05) compared to the cerebellum. Melanoma metastases were found to have a higher fluorescent signal (p<0.01) compared to cerebrum and cerebellum. Discussion: In conclusion we found that autofluorescence in the brain varies depending on the tissue type and localization and differs significantly among various brain tumors. This needs to be considered for interpreting photon signal during fluorescence-guided brain tumor surgery.

Evaluating Receptor-Specific Fresh Specimen Staining for Tumor Margin Detection in Clinical Breast Specimens.

PURPOSE: Reliable and rapid identification of tumor in the margins of breast specimens during breast-conserving surgery to reduce repeat surgery rates is an active area of investigation. Dual-stain difference imaging (DDSI) is one of many approaches under evaluation for this application. This technique aims to topically apply fluorescent stain pairs (one targeted to a receptor-of-interest and the other a spectrally distinct isotype), image both stains, and compute a normalized difference image between the two channels. Prior evaluation and optimization in a variety of preclinical models produced encouraging diagnostic performance. Herein, we report on a pilot clinical study which evaluated HER2-targeted DDSI on 11 human breast specimens. PROCEDURES: Gross sections from 11 freshly excised mastectomy specimens were processed using a HER2-receptor-targeted DDSI protocol shortly after resection. After staining with the dual-probe protocol, specimens were imaged on a fluorescence scanner, followed by tissue fixation for hematoxylin and eosin and anti-HER2 immunohistochemical staining. Receiver operator characteristic curves and area under the curve (AUC) analysis were used to assess diagnostic performance of the resulting images. Performance values were also compared to expression level determined from IHC staining. RESULTS: Eight of the 11 specimens presented with distinguishable invasive ductal carcinoma and/or were not affected by an imaging artifact. In these specimens, the DDSI technique provided an AUC = 0.90 ± 0.07 for tumor-to-adipose tissue and 0.81 ± 0.15 for tumor-to-glandular tissue, which was significantly higher than AUC values recovered from images of the targeted probe alone. DDSI values and diagnostic performance did not correlate with HER2 expression level, and tumors with low HER2 expression often produced high AUC, suggesting that even the low expression levels were enough to help distinguish tumor. CONCLUSIONS: The results from this preliminary study of rapid receptor-specific staining in human specimens were consistent with prior preclinical results and demonstrated promising diagnostic potential.

NIRDye 812: A molecular platform tailored for multimodal bioimaging applications of targeted fluorescence- and photoacoustic-guided surgery.

The primary treatment for malignant tumors remains to be surgical removal of the diseased tissue. The presence or absence of residual diseased tissue at the tumor margin is the strongest predictor of postoperative prognosis and recurrence. Accordingly, reliance on the ability of surgeons to visually distinguish diseased tissue from healthy tissue unambiguously in real time is crucial. Near infrared-I (NIRI) fluorescence-emitting targeting biomolecular constructs such as anticancer antibody-fluorophore conjugates, namely cetuximab-IRDye® 800CW (CTB-IRDye® 800CW), are FDA-approved for clinical trial usage in the fluorescence-guided resection of diseased tissue due to affording improved direct visualization of tumor tissue when compared to the use of either the unaided eye under standard white light illumination (WLI) surgical techniques or non-targeting fluorophores. Unfortunately, though helpful, CTB-IRDye® 800CW affords limited (i) identification of diseased tissue and (ii) tumor margin delineation, because the immunoconjugate generates suboptimal tumor-to-background ratios (TBRs) as a result of its spectral/photophysical profiles poorly aligning with the fixed optical windows of pre-/clinical setups. As such, CTB-IRDye® 800CW is more prone to affording incomplete resection compared to if TBRs were higher due to otherwise. To aid in accurately identifying deep-seated diseased tissue, photoacoustic (PA) tomography has been implemented alongside CTB-IRDye® 800CW to achieve PA signals that could result in higher TBRs. However, in clinical trial practice, using IRDye® 800CW for PA imaging also yields subpar TBRs due to it affording low PA signals. To overcome such limitations, we developed NIRDye 812, a structurally-modified topological equivalent of IRDye® 800CW, to confer it the capability to yield both higher TBRs and superior PA signal than that of the equivalent CTB-conjugate and fluorophore IRDye® 800CW itself, respectively. To do so, we substituted the oxygen atom at its meso-position with a sulfur atom. CTB-NIRDye 812 demonstrated a red-shifted absorption wavelength at 796 nm and a peak NIR-I fluorescence emission wavelength at 820 nm, which better dovetails with the fixed windows of preinstalled fixed emission filters within commercial pre-/clinical NIR-I fluorescence imaging instruments. Overall, CTB-NIRDye 812 provided a âˆ¼ 2-fold increase in TBRs compared to those of CTB-IRDye® 800CW in vivo. Also, NIRDye 812 displayed an ∼60% higher PA signal than that of IRDye® 800CW. Collectively, we achieved our goal of improving upon the spectral/photophysical and PA properties of IRDye® 800CW via introducing a subtle modification to its electronic core such that its CTB immunoconjugate could potentially allow for fast track or breakthrough designation by the FDA due to its near-identical structure displaying considerably improved efficacy.

Fluorescence Guidance and Intraoperative Adjuvants to Maximize Extent of Resection.

Safely maximizing extent of resection has become the central goal in glioma surgery. Especially in eloquent cortex, the goal of maximal resection is balanced with neurological risk. As new technologies emerge in the field of neurosurgery, the standards for maximal safe resection have been elevated. Fluorescence-guided surgery, intraoperative magnetic resonance imaging, and microscopic imaging methods are among the most well-validated tools available to enhance the level of accuracy and safety in glioma surgery. Each technology uses a different characteristic of glioma tissue to identify and differentiate tumor tissue from normal brain and is most effective in the context of anatomic, connectomic, and neurophysiologic context. While each tool is able to enhance resection, multiple modalities are often used in conjunction to achieve maximal safe resection. This paper reviews the mechanism and utility of the major adjuncts available for use in glioma surgery, especially in tumors within eloquent areas, and puts forth the foundation for a unified approach to how leverage currently available technology to ensure maximal safe resection.

Quicker, deeper and stronger imaging: A review of tumor-targeted, near-infrared fluorescent dyes for fluorescence guided surgery in the preclinical and clinical stages.

Cancer is a public health problem and the main cause of human mortality and morbidity worldwide. Complete removal of tumors and metastatic lymph nodes in surgery is significantly beneficial for the prognosis of patients. Tumor-targeted, near-infrared fluorescent (NIRF) imaging is an emerging field of real-time intraoperative cancer imaging based on tumor-targeted NIRF dyes. Targeted NIRF dyes contain NIRF fluorophores and specific binding ligands such as antibodies, peptides and small molecules. The present article reviews recently updated tumor-targeted NIRF dyes for the molecular imaging of malignant tumors in the preclinical stage and clinical trials. The strengths and challenges of NIRF agents with tumor-targeting ability are also summarized. Smaller ligands, near infrared II dyes, dual-modality dyes and activatable dyes may contribute to quicker, deeper, stronger imaging in the nearest future. In this review, we highlighted tumor-targeted NIRF dyes for fluorescence-guided surgery and their potential clinical translation.

Fluorescence-guided hepatobiliary surgery with long and short wavelength fluorophores.

IMPORTANCE: Fluorescence-guided surgery (FGS) is a potentially powerful tool for hepatobiliary (HPB) surgery. The high sensitivity of fluorescence navigation is especially useful in settings where tactile feedback is limited. OBJECTIVE: The present narrative review evaluates literature on the use of FDA-approved fluorophores such as methylene blue (MB), 5-aminolevulinic acid (5-ALA), and indocyanine green (ICG) for clinical intra-operative image-guidance during HPB surgery. EVIDENCE REVIEW: Approaches such as dosing, timing, imaging devices and comparative endpoints are summarized. The feasibility and safety of fluorophores in visualizing the biliary tree, identify biliary leaks, outline anatomic hepatic segments, identify tumors, and evaluate perfusion and graft function in liver transplants are discussed. FINDINGS: Tumor-specific probes are a promising advancement in FGS with a greater degree of specificity. The current status of tumor-specific probes being evaluated in clinical trials are summarized. CONCLUSIONS AND RELEVANCE FOR REVIEWS: Relevant discussion of promising tumor-specific probes in pre-clinical development are discussed. Fluorescence-guidance in HPB surgery is relatively new, but current literature shows that the dyes are reliably able to outline desired structures with a variety of dosing, timing, and imaging devices to provide real-time intra-operative anatomic information to surgeons. Development of tumor-specific probes will further advance the field of HPB surgery especially during oncologic resections.

Enhancement of Cancer-Specific Protoporphyrin IX Fluorescence by Targeting Oncogenic Ras/MEK Pathway.

Protoporphyrin IX (PpIX) is an endogenous fluorescent molecule that selectively accumulates in cancer cells treated with the heme precursor 5-aminolevulinic acid (5-ALA). This cancer-specific accumulation of PpIX is used to distinguish tumor from normal tissues in fluorescence-guided surgery (FGS) and to destroy cancer cells by photodynamic therapy (PDT). In this study, we demonstrate that oncogenic Ras/mitogen-activated protein kinase kinase (MEK) pathway can modulate PpIX accumulation in cancer cells. Methods: To identify Ras downstream elements involved in PpIX accumulation, chemical inhibitors were used. To demonstrate the increase of PpIX accumulation by MEK inhibition, different human normal and cancer cell lines, BALB/c mice bearing mammary 4T1 tumors and athymic nude mice bearing human tumors were used. To identify the mechanisms of PpIX regulation by MEK, biochemical and molecular biological experiments were conducted. Results: Inhibition of one of the Ras downstream elements, MEK, promoted PpIX accumulation in cancer cells treated with 5-ALA, while inhibitors against other Ras downstream elements did not. Increased PpIX accumulation with MEK inhibition was observed in different types of human cancer cell lines, but not in normal cell lines. We identified two independent cellular mechanisms that underlie this effect in cancer cells. MEK inhibition reduced PpIX efflux from cancer cells by decreasing the expression level of ATP binding cassette subfamily B member 1 (ABCB1) transporter. In addition, the activity of ferrochelatase (FECH), the enzyme responsible for converting PpIX to heme, was reduced by MEK inhibition. Finally, we found that in vivo treatment with MEK inhibitors increased PpIX accumulation (2.2- to 2.4-fold) within mammary 4T1 tumors in BALB/c mice injected with 5-ALA without any change in normal organs. Similar results were also observed in a human tumor xenograft model. Conclusion: Our study demonstrates that inhibition of oncogenic Ras/MEK significantly enhances PpIX accumulation in vitro and in vivo in a cancer-specific manner. Thus, suppressing the Ras/MEK pathway may be a viable strategy to selectively intensify PpIX fluorescence in cancer cells and improve its clinical applications in FGS.

Current status and future perspectives of fluorescence-guided surgery for cancer.

Curative cancer surgery is dependent on the removal of all primary tumor and metastatic cancer cells. Preoperative imaging, intraoperative inspection and palpation, as well as pathological margin confirmation aid the surgeon, but these methods are lacking in sensitivity and can be highly subjective. Techniques in fluorescence-guided surgery (FGS) are emerging that selectively illuminate cancer cells, enhancing the distinction between tumors and surrounding tissues with the potential for single-cell sensitivity. FGS enhances tumor detection, surgical navigation, margin confirmation, and in some cases can be combined with therapeutic techniques to eliminate microscopic disease. In this review, we describe the preclinical developments and currently-used techniques for FGS.

Fluorescence-guided surgery of a highly-metastatic variant of human triple-negative breast cancer targeted with a cancer-specific GFP adenovirus prevents recurrence.

We have previously developed a genetically-engineered GFP-expressing telomerase-dependent adenovirus, OBP-401, which can selectively illuminate cancer cells. In the present report, we demonstrate that targeting a triple-negative high-invasive human breast cancer, orthotopically-growing in nude mice, with OBP-401 enables curative fluorescence-guided surgery (FGS). OBP-401 enabled complete resection and prevented local recurrence and greatly inhibited lymph-node metastasis due to the ability of the virus to selectively label and subsequently kill cancer cells. In contrast, residual breast cancer cells become more aggressive after bright (white)-light surgery (BLS). OBP-401-based FGS also improved the overall survival compared with conventional BLS. Thus, metastasis from a highly-aggressive triple-negative breast cancer can be prevented by FGS in a clinically-relevant mouse model.

Adenoviral targeting of malignant melanoma for fluorescence-guided surgery prevents recurrence in orthotopic nude-mouse models.

Malignant melanoma requires precise resection in order to avoid metastatic recurrence. We report here that the telomerase-dependent, green fluorescent protein (GFP)-containing adenovirus OBP-401 could label malignant melanoma with GFP in situ in orthotopic mouse models. OBP-401-based fluorescence-guided surgery (FGS) resulted in the complete resection of malignant melanoma in the orthotopic models, where conventional bright-light surgery (BLS) could not. High-dose administration of OBP-401 enabled FGS without residual cancer cells or recurrence, due to its dual effect of cancer-cell labeling with GFP and killing.