Topical dual-probe staining using quantum dot-labeled antibodies for identifying tumor biomarkers in fresh specimens

Meng, Boyu; Folaron, Margaret R.; Byrd, Brook K.; Samkoe, Kimberley S.; Strawbridge, Rendall S.; Barth, Connor W.; Gibbs, Summer L.; Davis, Scott C.

doi:10.1371/journal.pone.0230267

Cited by 7 publications

(3 citation statements)

References 39 publications

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“…PAI was created for quantitative in vivo imaging, where non-specific accumulation of protein-based, radiolabeled affinity reagents dominated malignant tissue signals but could be corrected for by normalizing the targeted signal to the signal of a co-administered, control antibody labeled with an isotope of different energy [40]. In this reinvigorated technique, spectrally-distinct targeted and untargeted imaging probes are used to correct for non-specific uptake to quantify drug target availability (DTA; also termed "binding potential") [41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57]. We have expanded the PAI technique to measure intracellular targets with the use of spectrally-distinct, fluorescently-labeled targeted and untargeted drug derivatives, such as TKIs [58].…”

Section: Introductionmentioning

confidence: 99%

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

McMahon,

Solanki,

Wang

et al. 2024

Theranostics

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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.

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Section: Introductionmentioning

confidence: 99%

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

McMahon,

Solanki,

Wang

et al. 2024

Theranostics

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“…A robust DDSI protocol with high sensitivity and specificity for differentiation between benign and malignant breast tissues 36 , 37 , 42 – 44 was previously developed and optimized using antibody-based probes on resected specimens to a total staining and imaging time of 8 min for routine intraoperative use with minimal disruption to the clinical workflow. With advancements in FGS technologies and increasing clinical adoption, the overall goal of this study was to expand the DDSI tumor margin assessment methodology to other cancer types, where prostate cancer was evaluated herein.…”

Section: Introductionmentioning

confidence: 99%

Dual probe difference specimen imaging for prostate cancer margin assessment

et al. 2023

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. 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.

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“…Multiple groups have shown that including a spectrally distinct non-targeted isotype probe in the staining solution, acquiring images of both probes in separate channels, and mathematically comparing the two channels helps compensate for imaging system inhomogeneity and produce images of receptor-specific tumor contrast [ 27 , 28 , 35 – 39 ]. This strategy, termed dual-probe difference specimen imaging (DDSI) in our lab, has been applied using antibodies labeled with surface-enhanced Raman scattering (SERS) nanoparticles [ 26 – 28 ], quantum dots [ 40 ] or with spectrally distinct fluorophores [ 35 – 37 ] to target various biomarkers, including ERBB1 (EGFR), ERBB2 (HER2), estrogen receptor, and CD44. Through a series of preclinical studies, we showed that fluorophore-based DDSI of EGFR or HER2 provided high tumor-to-normal diagnostic performance, with an area under the curve (AUC) from receiver operator characteristic (ROC) curves routinely > 0.95 [ 35 – 38 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2023

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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.

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Topical dual-probe staining using quantum dot-labeled antibodies for identifying tumor biomarkers in fresh specimens

Cited by 7 publications

References 39 publications

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

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

Dual probe difference specimen imaging for prostate cancer margin assessment

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

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