In situ single-cell therapeutic response imaging facilitated by the TRIPODD fluorescence imaging platform.

Purpose: Small molecule drugs such as tyrosine kinase inhibitors (TKIs) targeting tumoral molecular dependencies have become standard of care for numerous cancer types. Notably, epidermal growth factor receptor (EGFR) TKIs (e.g., erlotinib, afatinib, osimertinib) are the current first-line treatment for non-small cell lung cancer (NSCLC) due to their improved therapeutic outcomes for EGFR mutated and overexpressing disease over traditional platinum-based chemotherapy. However, many NSCLC tumors develop resistance to EGFR TKI therapy causing disease progression. Currently, the relationship between in situ drug target availability (DTA), local protein expression and therapeutic response cannot be accurately assessed using existing analytical tools despite being crucial to understanding the mechanism of therapeutic efficacy. Procedure: We have previously reported development of our fluorescence imaging platform termed TRIPODD (Therapeutic Response Imaging through Proteomic and Optical Drug Distribution) that is capable of simultaneous quantification of single-cell DTA and protein expression with preserved spatial context within a tumor. TRIPODD combines two complementary fluorescence imaging techniques: intracellular paired agent imaging (iPAI) to measure DTA and cyclic immunofluorescence (cyCIF), which utilizes oligonucleotide conjugated antibodies (Ab-oligos) for spatial proteomic expression profiling on tissue samples. Herein, TRIPODD was modified and optimized to provide a downstream analysis of therapeutic response through single-cell DTA and proteomic response imaging. Results: We successfully performed sequential imaging of iPAI and cyCIF resulting in high dimensional imaging and biomarker assessment to quantify single-cell DTA and local protein expression on erlotinib treated NSCLC models. Pharmacodynamic and pharmacokinetic studies of the erlotinib iPAI probes revealed that administration of 2.5 mg/kg each of the targeted and untargeted probe 4 h prior to tumor collection enabled calculation of DTA values with high Pearson correlation to EGFR, the erlotinib molecular target, expression in the tumors. Analysis of single-cell biomarker expression revealed that a single erlotinib dose was insufficient to enact a measurable decrease in the EGFR signaling cascade protein expression, where only the DTA metric detected the presence of bound erlotinib. Conclusion: We demonstrated the capability of TRIPODD to evaluate therapeutic response imaging to erlotinib treatment as it relates to signaling inhibition, DTA, proliferation, and apoptosis with preserved spatial context.

Ultra high content analyses of circulating and tumor associated hybrid cells reveal phenotypic heterogeneity.

Persistently high, worldwide mortality from cancer highlights the unresolved challenges of disease surveillance and detection that impact survival. Development of a non-invasive, blood-based biomarker would transform survival from cancer. We demonstrate the functionality of ultra-high content analyses of a newly identified population of tumor cells that are hybrids between neoplastic and immune cells in patient matched tumor and peripheral blood specimens. Using oligonucleotide conjugated antibodies (Ab-oligo) permitting cyclic immunofluorescence (cyCIF), we present analyses of phenotypes among tumor and peripheral blood hybrid cells. Interestingly, the majority of circulating hybrid cell (CHC) subpopulations were not identified in tumor-associated hybrids. These results highlight the efficacy of ultra-high content phenotypic analyses using Ab-oligo based cyCIF applied to both tumor and peripheral blood specimens. The combination of a multiplex phenotypic profiling platform that is gentle enough to analyze blood to detect and evaluate disseminated tumor cells represents a novel approach to exploring novel tumor biology and potential utility for developing the population as a blood-based biomarker in cancer.

Nerve Visualization using Phenoxazine-Based Near-Infrared Fluorophores to Guide Prostatectomy.

Fluorescence-guided surgery (FGS) is poised to revolutionize surgical medicine through near-infrared (NIR) fluorophores for tissue- and disease-specific contrast. Clinical open and laparoscopic FGS vision systems operate nearly exclusively at NIR wavelengths. However, tissue-specific NIR contrast agents compatible with clinically available imaging systems are lacking, leaving nerve tissue identification during prostatectomy a persistent challenge. Here, it is shown that combining drug-like molecular design concepts and fluorophore chemistry enabled the production of a library of NIR phenoxazine-based fluorophores for intraoperative nerve-specific imaging. The lead candidate readily delineated prostatic nerves in the canine and iliac plexus in the swine using the clinical da Vinci Surgical System that has been popularized for minimally invasive prostatectomy procedures. These results demonstrate the feasibility of molecular engineering of NIR nerve-binding fluorophores for ready integration into the existing surgical workflow, paving the path for clinical translation to reduce morbidity from nerve injury for prostate cancer patients.

Detection of neoplastic-immune hybrid cells with metastatic properties in uveal melanoma.

Background: Uveal melanoma is the most common non-cutaneous melanoma and is an intraocular malignancy affecting nearly 7,000 individuals per year worldwide. Of these, approximately 50% will progress to metastatic disease for which there are currently no effective therapies. Despite advances in molecular profiling and metastatic stratification of uveal melanoma tumors, little is known regarding their underlying biology of metastasis. Our group has identified a disseminated neoplastic cell population characterized by co-expression of immune and melanoma proteins, circulating hybrid cells (hybrids), in patients with uveal melanoma. Compared to circulating tumor cells, which lack expression of immune proteins, hybrids are detected at an increased prevalence in peripheral blood and can be used as a non-invasive biomarker to predict metastatic progression. Methods: To ascertain mechanisms underlying enhanced hybrid cell dissemination we identified hybrid cells within primary uveal melanoma tumors using single cell RNA sequencing and evaluated their gene expression and predicted ligand-receptor interactions in relation to other melanoma and immune cells within the primary tumor. We then verified expression of upregulated hybrid pathways within patient-matched tumor and peripheral blood hybrids using cyclic immunofluorescence and quantified their protein expression relative to other non-hybrid tumor and disseminated tumor cells. Results: Among the top upregulated genes and pathways in hybrid cells were those involved in enhanced cell motility and cytoskeletal rearrangement, immune evasion, and altered cellular metabolism. In patient-matched tumor and peripheral blood, we verified gene expression by examining concordant protein expression for each pathway category: TMSB10 (cell motility), CD74 (immune evasion) and GPX1 (metabolism). Both TMSB10 and GPX1 were expressed on significantly higher numbers of disseminated hybrid cells compared to circulating tumor cells, and CD74 and GPX1 were expressed on more disseminated hybrids than tumor-resident hybrids. Lastly, we identified that hybrid cells express ligand-receptor signaling pathways implicated in promoting metastasis including GAS6-AXL, CXCL12-CXCR4, LGALS9-P4HB and IGF1-IGFR1. Conclusion: These findings highlight the importance of TMSB10, GPX1 and CD74 for successful hybrid cell dissemination and survival in circulation. Our results contribute to the understanding of uveal melanoma tumor progression and interactions between tumor cells and immune cells in the tumor microenvironment that may promote metastasis.

Analysis of uveal melanoma scRNA sequencing data identifies neoplastic-immune hybrid cells that exhibit metastatic potential.

Uveal melanoma (UM) is the most common non-cutaneous melanoma and is an intraocular malignancy that affects nearly 7,000 individuals per year worldwide. Of these, nearly 50% will progress to metastatic disease for which there are currently no effective therapies. Despite advances in the molecular profiling and metastatic stratification of class 1 and 2 UM tumors, little is known regarding the underlying biology of UM metastasis. Our group has identified a disseminated tumor cell population characterized by co-expression of immune and melanoma proteins, (circulating hybrid cells (CHCs), in patients with UM. Compared to circulating tumor cells, CHCs are detected at an increased prevalence in peripheral blood and can be used as a non-invasive biomarker to predict metastatic progression. To identify mechanisms underlying enhanced hybrid cell dissemination we sought to identify hybrid cells within a primary UM single cell RNA-seq dataset. Using rigorous doublet discrimination approaches, we identified UM hybrids and evaluated their gene expression, predicted ligand-receptor status, and cell-cell communication state in relation to other melanoma and immune cells within the primary tumor. We identified several genes and pathways upregulated in hybrid cells, including those involved in enhancing cell motility and cytoskeleton rearrangement, evading immune detection, and altering cellular metabolism. In addition, we identified that hybrid cells express ligand-receptor signaling pathways implicated in promoting cancer metastasis including IGF1-IGFR1, GAS6-AXL, LGALS9-P4HB, APP-CD74 and CXCL12-CXCR4. These results contribute to our understanding of tumor progression and interactions between tumor cells and immune cells in the UM microenvironment that may promote metastasis.

Nerve-Sparing Gynecologic Surgery Enabled by A Near-Infrared Nerve-Specific Fluorophore Using Existing Clinical Fluorescence Imaging Systems.

Patients undergoing gynecological procedures suffer from lasting side effects due to intraoperative nerve damage. Small, delicate nerves with complex and nonuniform branching patterns in the female pelvic neuroanatomy make nerve-sparing efforts during standard gynecological procedures such as hysterectomy, cystectomy, and colorectal cancer resection difficult, and thus many patients are left with incontinence and sexual dysfunction. Herein, a near-infrared (NIR) fluorescent nerve-specific contrast agent, LGW08-35, that is spectrally compatible with clinical fluorescence guided surgery (FGS) systems is formulated and characterized for rapid implementation for nerve-sparing gynecologic surgeries. The toxicology, pharmacokinetics (PK), and pharmacodynamics (PD) of micelle formulated LGW08-35 are examined, enabling the determination of the optimal imaging doses and time points, blood and tissue uptake parameters, and maximum tolerated dose (MTD). Application of the formulated fluorophore to imaging of female rat and swine pelvic neuroanatomy validates the continued clinical translation and use for real-time identification of important nerves such as the femoral, sciatic, lumbar, iliac, and hypogastric nerves. Further development of LGW08-35 for clinical use will unlock a valuable tool for surgeons in direct visualization of important nerves and contribute to the ongoing characterization of the female pelvic neuroanatomy to eliminate the debilitating side effects of nerve damage during gynecological procedures.

Improving precision surgery: A review of current intraoperative nerve tissue fluorescence imaging.

Iatrogenic nerve injury represents one of the most feared surgical complications and remains a major morbidity across many surgical specialties. Currently, no clinically approved technique can directly enhance intraoperative nerve visualization, where intraoperative nerve identification continues to challenge even experienced surgeons. Fluorescence-guided surgery (FGS) has been successfully integrated into clinical medicine to improve safety and efficacy in the surgical arena. A number of tissue- and disease-specific contrast agents are in the clinical translation pipeline for future FGS integration. Within this context, a diverse repertoire of fluorescent tracers have been developed to improve surgeons' intraoperative vision. This review aims to convey the recent developments for nerve-specific FGS and its potential for clinical translation.

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.

Use of Freshly Amputated Human Limbs for Pre-Clinical Evaluation of Molecular-Targeted Fluorescent Probes.

We have co-developed a first-in-kind model of fluorophore testing in freshly amputated human limbs. Ex vivo human tissue provides a unique opportunity for the testing of pre-clinical fluorescent agents, collection of imaging data, and histopathologic examination in human tissue prior to performing in vivo experiments. Existing pre-clinical fluorescent agent studies rely primarily on animal models, which do not directly predict fluorophore performance in humans and can result in wasted resources and time if an agent proves ineffective in early human trials. Because fluorophores have no desired therapeutic effect, their clinical utility is based solely on their safety and ability to highlight tissues of interest. Advancing to human trials even via the FDA's phase 0/microdose pathway still requires substantial resources, single-species pharmacokinetic testing, and toxicity testing. In a recently concluded study using amputated human lower limbs, we were able to test successfully a nerve-specific fluorophore in pre-clinical development. This study used systemic administration via vascular cannulization and a cardiac perfusion pump. We envision that this model may assist with early lead agent testing selection for fluorophores with various targets and mechanisms.

First demonstration of a novel nerve-targeting fluorophore in a cohort of ex vivo human tissues.

Iatrogenic nerve injury is a common complication across all surgical specialties. Better nerve visualization and identification during surgery will improve outcomes and reduce nerve injuries. The Gibbs Laboratory at Oregon Health and Science University has developed a library of near-infrared, nerve-specific fluorophores to highlight nerves intraoperatively and aid surgeons in nerve identification and visualization; the current lead agent is LGW16-03. Prior to this study, testing of LGW16-03 was restricted to animal models; therefore, it was unknown how LGW16-03 performs in human tissue. To advance LGW16-03 to clinic, we sought to test this current lead agent in ex vivo human tissues from a cohort of patients and determine if the route of administration affects LGW16-03 fluorescence contrast between nerves and adjacent background tissues (muscle and adipose). LGW16-03 was applied to ex vivo human tissue from lower limb amputations via two strategies: (1) systemic administration of the fluorophore using our first-in-kind model for fluorophore testing, and (2) topical application of the fluorophore. Results showed no statistical difference between topical and systemic administration. However, in vivo human validation of these findings is required.

Dual probe difference specimen imaging for prostate cancer margin assessment.

Significance: Positive margin status due to incomplete removal of tumor tissue during radical prostatectomy for high-risk localized prostate cancer requires reoperation or adjuvant therapy, which increases morbidity and mortality. Adverse effects of prostate cancer treatments commonly include erectile dysfunction, urinary incontinence, and bowel dysfunction, making successful initial curative prostatectomy imperative. Aim: Current intraoperative tumor margin assessment is largely limited to frozen section analysis, which is a lengthy, labor-intensive process that is obtrusive to the clinical workflow within the operating room (OR). Therefore, a rapid method for prostate cancer margin assessment in the OR could improve outcomes for patients. Approach: Dual probe difference specimen imaging (DDSI), which uses paired antibody-based probes that are labeled with spectrally distinct fluorophores, was shown herein for prostate cancer margin assessment. The paired antibody-based probes consisted of a targeted probe to prostate-specific membrane antigen (PSMA) and an untargeted probe, which were used as a cocktail to stain resected murine tissue specimens including prostate tumor, adipose, muscle, and normal prostate. Ratiometric images (i.e., DDSI) of the difference between targeted and untargeted probe uptake were calculated and evaluated for accuracy using receiver operator characteristic curve analysis with area under the curve values used to evaluate the utility of the DDSI method to detect PSMA positive prostate cancer. Results: Targeted and untargeted probe uptake was similar between the high and low PSMA expressing tumor due to nonspecific probe uptake after topical administration. The ratiometric DDSI approach showed substantial contrast difference between the PSMA positive tumors and their respective normal tissues (prostate, adipose, muscle). Furthermore, DDSI showed substantial contrast difference between the high PSMA expressing tumors and the minimally PSMA expressing tumors due to the ratiometric correction for the nonspecific uptake patterns in resected tissues. Conclusions: Previous work has shown that ratiometic imaging has strong predictive value for breast cancer margin status using topical administration. Translation of the ratiometric DDSI methodology herein from breast to prostate cancers demonstrates it as a robust, ratiometric technique that provides a molecularly specific imaging modality for intraoperative margin detection. Using the validated DDSI protocol on resected prostate cancers permitted rapid and accurate assessment of PSMA status as a surrogate for prostate cancer margin status. Future studies will further evaluate the utility of this technology to quantitatively characterize prostate margin status using PSMA as a biomarker.

OregonFluor enables quantitative intracellular paired agent imaging to assess drug target availability in live cells and tissues.

Non-destructive fluorophore diffusion across cell membranes to provide an unbiased fluorescence intensity readout is critical for quantitative imaging applications in live cells and tissues. Commercially available small-molecule fluorophores have been engineered for biological compatibility, imparting high water solubility by modifying rhodamine and cyanine dye scaffolds with multiple sulfonate groups. The resulting net negative charge, however, often renders these fluorophores cell-membrane-impermeant. Here we report the design and development of our biologically compatible, water-soluble and cell-membrane-permeable fluorophores, termed OregonFluor (ORFluor). By adapting previously established ratiometric imaging methodology using bio-affinity agents, it is now possible to use small-molecule ORFluor-labelled therapeutic inhibitors to quantitatively visualize their intracellular distribution and protein target-specific binding, providing a chemical toolkit for quantifying drug target availability in live cells and tissues.

Robust biomarker discovery through multiplatform multiplex image analysis of breast cancer clinical cohorts.

Spatial profiling of tissues promises to elucidate tumor-microenvironment interactions and enable development of spatial biomarkers to predict patient response to immunotherapy and other therapeutics. However, spatial biomarker discovery is often carried out on a single patient cohort or imaging technology, limiting statistical power and increasing the likelihood of technical artifacts. In order to analyze multiple patient cohorts profiled on different platforms, we developed methods for comparative data analysis from three disparate multiplex imaging technologies: 1) cyclic immunofluorescence data we generated from 102 breast cancer patients with clinical follow-up, in addition to publicly available 2) imaging mass cytometry and 3) multiplex ion-beam imaging data. We demonstrate similar single-cell phenotyping results across breast cancer patient cohorts imaged with these three technologies and identify cellular abundance and proximity-based biomarkers with prognostic value across platforms. In multiple platforms, we identified lymphocyte infiltration as independently associated with longer survival in triple negative and high-proliferation breast tumors. Then, a comparison of nine spatial analysis methods revealed robust spatial biomarkers. In estrogen receptor-positive disease, quiescent stromal cells close to tumor were more abundant in good prognosis tumors while tumor neighborhoods of mixed fibroblast phenotypes were enriched in poor prognosis tumors. In triple-negative breast cancer (TNBC), macrophage proximity to tumor and B cell proximity to T cells were greater in good prognosis tumors, while tumor neighborhoods of vimentin-positive fibroblasts were enriched in poor prognosis tumors. We also tested previously published spatial biomarkers in our ensemble cohort, reproducing the positive prognostic value of isolated lymphocytes and lymphocyte occupancy and failing to reproduce the prognostic value of tumor-immune mixing score in TNBC. In conclusion, we demonstrate assembly of larger clinical cohorts from diverse platforms to aid in prognostic spatial biomarker identification and validation.

Flexible Cyclic Immunofluorescence (cyCIF) Using Oligonucleotide Barcoded Antibodies.

Advances in our understanding of the complex, multifaceted interactions between tumor epithelia, immune infiltrate, and tumor microenvironmental cells have been driven by highly multiplexed imaging technologies. These techniques are capable of labeling many more biomarkers than conventional immunostaining methods. However, multiplexed imaging techniques suffer from low detection sensitivity, cell loss-particularly in fragile samples-, and challenges with antibody labeling. Herein, we developed and optimized an oligonucleotide antibody barcoding strategy for cyclic immunofluorescence (cyCIF) that can be amplified to increase the detection efficiency of low-abundance antigens. Stained fluorescence signals can be readily removed using ultraviolet light treatment, preserving tissue and fragile cell sample integrity. We also extended the oligonucleotide barcoding strategy to secondary antibodies to enable the inclusion of difficult-to-label primary antibodies in a cyCIF panel. Using both the amplification oligonucleotides to label DNA barcoded antibodies and in situ hybridization of multiple fluorescently labeled oligonucleotides resulted in signal amplification and increased signal-to-background ratios. This procedure was optimized through the examination of staining parameters including staining oligonucleotide concentration, staining temperature, and oligonucleotide sequence design, resulting in a robust amplification technique. As a proof-of-concept, we demonstrate the flexibility of our cyCIF strategy by simultaneously imaging with the original oligonucleotide conjugated antibody (Ab-oligo) cyCIF strategy, the novel Ab-oligo cyCIF amplification strategy, as well as direct and indirect immunofluorescence to generate highly multiplexed images.

Preclinical evaluation of molecularly targeted fluorescent probes in perfused amputated human limbs.

Significance: This first-in-kind, perfused, and amputated human limb model allows for the collection of human data in preclinical selection of lead fluorescent agents. The model facilitates more accurate selection and testing of fluorophores with human-specific physiology, such as differential uptake and signal in fat between animal and human models with zero risk to human patients. Preclinical testing using this approach may also allow for the determination of tissue toxicity, clearance time of fluorophores, and the production of harmful metabolites. Aim: This study was conducted to determine the fluorescence intensity values and tissue specificity of a preclinical, nerve tissue targeted fluorophore, as well as the capacity of this first-in-kind model to be used for lead fluorescent agent selection in the future. Approach: Freshly amputated human limbs were perfused for 30 min prior to in situ and ex vivo imaging of nerves with both open-field and closed-field commercial fluorescence imaging systems. Results: In situ, open-field imaging demonstrated a signal-to-background ratio (SBR) of 4.7 when comparing the nerve with adjacent muscle tissue. Closed-field imaging demonstrated an SBR of 3.8 when the nerve was compared with adipose tissue and 4.8 when the nerve was compared with muscle. Conclusions: This model demonstrates an opportunity for preclinical testing, evaluation, and selection of fluorophores for use in clinical trials as well as an opportunity to study peripheral pathologies in a controlled environment.

Current and Future Applications of Fluorescence Guidance in Orthopaedic Surgery.

Fluorescence-guided surgery (FGS) is an evolving field that seeks to identify important anatomic structures or physiologic phenomena with helpful relevance to the execution of surgical procedures. Fluorescence labeling occurs generally via the administration of fluorescent reporters that may be molecularly targeted, enzyme-activated, or untargeted, vascular probes. Fluorescence guidance has substantially changed care strategies in numerous surgical fields; however, investigation and adoption in orthopaedic surgery have lagged. FGS shows the potential for improving patient care in orthopaedics via several applications including disease diagnosis, perfusion-based tissue healing capacity assessment, infection/tumor eradication, and anatomic structure identification. This review highlights current and future applications of fluorescence guidance in orthopaedics and identifies key challenges to translation and potential solutions.

Circulating Neoplastic-Immune Hybrid Cells Predict Metastatic Progression in Uveal Melanoma.

Background: Uveal melanoma is an aggressive cancer with high metastatic risk. Recently, we identified a circulating cancer cell population that co-expresses neoplastic and leukocyte antigens, termed circulating hybrid cells (CHCs). In other cancers, CHCs are more numerous and better predict oncologic outcomes compared to circulating tumor cells (CTCs). We sought to investigate the potential of CHCs as a prognostic biomarker in uveal melanoma. Methods: We isolated peripheral blood monocular cells from uveal melanoma patients at the time of primary treatment and used antibodies against leukocyte and melanoma markers to identify and enumerate CHCs and CTCs by immunocytochemistry. Results: Using a multi-marker approach to capture the heterogeneous disseminated tumor cell population, detection of CHCs was highly sensitive in uveal melanoma patients regardless of disease stage. CHCs were detected in 100% of stage I-III uveal melanoma patients (entire cohort, n = 68), whereas CTCs were detected in 58.8% of patients. CHCs were detected at levels statically higher than CTCs across all stages (p = 0.05). Moreover, CHC levels, but not CTCs, predicted 3 year progression-free survival (p < 0.03) and overall survival (p < 0.04). Conclusion: CHCs are a novel and promising prognostic biomarker in uveal melanoma.