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.

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.

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.

Oligonucleotide conjugated antibody strategies for cyclic immunostaining.

A number of highly multiplexed immunostaining and imaging methods have advanced spatial proteomics of cancer for improved treatment strategies. While a variety of methods have been developed, the most widely used methods are limited by harmful signal removal techniques, difficulties with reagent production and antigen sensitivity. Multiplexed immunostaining employing oligonucleotide (oligos)-barcoded antibodies is an alternative approach that is growing in popularity. However, challenges remain in consistent conjugation of oligos to antibodies with maintained antigenicity as well as non-destructive, robust and cost-effective signal removal methods. Herein, a variety of oligo conjugation and signal removal methods were evaluated in the development of a robust oligo conjugated antibody cyclic immunofluorescence (Ab-oligo cyCIF) methodology. Both non- and site-specific conjugation strategies were assessed to label antibodies, where site-specific conjugation resulted in higher retained binding affinity and antigen-specific staining. A variety of fluorescence signal removal methods were also evaluated, where incorporation of a photocleavable link (PCL) resulted in full fluorescence signal removal with minimal tissue disruption. In summary, this work resulted in an optimized Ab-oligo cyCIF platform capable of generating high dimensional images to characterize the spatial proteomics of the hallmarks of cancer.

TRIPODD: a Novel Fluorescence Imaging Platform for In Situ Quantification of Drug Distribution and Therapeutic Response.

PURPOSE: Personalized medicine has largely failed to produce curative therapies in advanced cancer patients. Evaluation of in situ drug target availability (DTA) concomitant with local protein expression is critical to an accurate assessment of therapeutic efficacy, but tools capable of both are currently lacking. PROCEDURE: We developed and optimized a fluorescence imaging platform termed TRIPODD (Therapeutic Response Imaging through Proteomic and Optical Drug Distribution), resulting in the only methodology capable of simultaneous quantification of single-cell DTA and protein expression with preserved spatial context within a tumor. Using TRIPODD, we demonstrate the feasibility of combining two complementary fluorescence imaging techniques, intracellular paired agent imaging (iPAI) and cyclic immunofluorescence (cyCIF), conducted with oligonucleotide-conjugated antibodies (Ab-oligos) on tissue samples. RESULTS: We successfully performed sequential imaging on a single tissue section of iPAI to capture single-cell DTA and local protein expression heterogeneity using Ab-oligo cyCIF. Fluorescence imaging data acquisition was followed by spatial registration resulting in high dimensional data correlating DTA to protein expression at the single-cell level where uptake of a targeted probe alone was not well correlated to protein expression. CONCLUSION: Herein, we demonstrated the utility of TRIPODD as a powerful imaging platform capable of interpreting tumor heterogeneity for a mechanistic understanding of therapeutic response and resistance through quantification of drug target availability and proteomic response with preserved spatial context at single-cell resolution.

Oligonucleotide conjugated antibodies permit highly multiplexed immunofluorescence for future use in clinical histopathology.

SIGNIFICANCE: Advanced genetic characterization has informed cancer heterogeneity and the challenge it poses to effective therapy; however, current methods lack spatial context, which is vital to successful cancer therapy. Conventional immunolabeling, commonplace in the clinic, can provide spatial context to protein expression. However, these techniques are spectrally limited, resulting in inadequate capacity to resolve the heterogenous cell subpopulations within a tumor. AIM: We developed and optimized oligonucleotide conjugated antibodies (Ab-oligo) to facilitate cyclic immunofluorescence (cyCIF), resulting in high-dimensional immunostaining. APPROACH: We employed a site-specific conjugation strategy to label antibodies with unique oligonucleotide sequences, which were hybridized in situ with their complementary oligonucleotide sequence tagged with a conventional fluorophore. Antibody concentration, imaging strand concentration, and configuration as well as signal removal strategies were optimized to generate maximal staining intensity using our Ab-oligo cyCIF strategy. RESULTS: We successfully generated 14 Ab-oligo conjugates and validated their antigen specificity, which was maintained in single color staining studies. With the validated antibodies, we generated up to 14-color imaging data sets of human breast cancer tissues. CONCLUSIONS: Herein, we demonstrated the utility of Ab-oligo cyCIF as a platform for highly multiplexed imaging, its utility to measure tumor heterogeneity, and its potential for future use in clinical histopathology.

Cardiac ErbB-1/ErbB-2 mutant expression in young adult mice leads to cardiac dysfunction.

Multiple factors lead to the development and maintenance of chronic heart failure. Blockade of ErbB-2 or ErbB-4 tyrosine kinase receptor signaling leads to dilated cardiomyopathy. ErbB-1 may protect the heart against stress-induced injury and its ligand; epidermal growth factor (EGF) increases myocardial contractility, whereas heparin-binding EGF is essential for normal cardiac function. However, the role of ErbB-1 in control of cardiac function is not clear. We hypothesized that ErbB-1 is essential for maintaining adult cardiac function. Using the ecdysone-inducible gene expression system, we expressed humanized cardiomyocyte-specific dominant-negative ErbB-1 mutant receptors (hErbB-1-mut) in young adult mice that block endogenous cardiac ErbB-1 signaling. Molecular, morphological, and physiological tests (under anesthesia) were performed. As a result, hErbB-1-mut was expressed selectively in cardiomyocytes leading to the blockade of endogenous ErbB-1 phosphorylation and ErbB-2 transphosphorylation. An increase in left ventricular mass, atrial natriuretic factor expression, and histological changes were indicative of cardiac hypertrophy. Cardiac dilation, numerous cardiac lesions, and the loss of the clear boundary between cardiac fibrils were noted histologically. Early and long-term hErbB-1-mut induction led to a significant decrease in fractional shortening and to significant increases in left ventricular end-systolic diameter and volume. The treatment of adenylyl cyclase activator (forskolin analog) normalized the depressed cardiac function. Resting cardiac function returned to normal after reversing mutant expression. A 4-day survival rate of transverse-aortic constricted hErbB-1-mut mice was only 20% compared with 100% in controls. In conclusion, these observations indicate that the blockade of cardiac ErbB-1 signaling leads to the blockade of ErbB-2 signaling and that together they result in cardiac dysfunction.