A Prostate-Specific Membrane Antigen-Targeted Near-Infrared Conjugate for Identifying Pulmonary Squamous Cell Carcinoma during Resection

Abstract

Pulmonary squamous cell carcinoma is the second most common lung cancer subtype and has a low 5-year survival rate at 17.6%. Complete resection with negative margins can be curative, but a high number of patients suffer early postoperative recurrence due to inadequate disease clearance at the index operation. Intraoperative molecular imaging (IMI) with tumor-targeted optical contrast agents is effective in improving resection completeness for other tumor types, but there are no IMI tracers targeted to pulmonary squamous cell carcinoma. In this report, we describe the use of a novel prostate-specific membrane antigen (PSMA)-targeted near-infrared conjugate (OTL78) to identify pulmonary squamous cell carcinoma. We identified PSMA as a viable target by examining its expression in human lung tumor specimens from a surgical cohort. Ninety-four percent of tumors expressed PSMA in either the pulmonary squamous cells or the tumor neovasculature. Using in vitro and in vivo models, we found that OTL78 reliably localized pulmonary squamous cell carcinoma in a PSMA-dependent manner. Finally, we found that IMI with OTL78 markedly improved surgeons' ability to identify residual disease after surgery in a preclinical model. Ultimately, this novel optical tracer may aid surgical resection of pulmonary squamous cell carcinoma and potentially improve long-term outcomes.

Figure 1.

Immunohistochemical analysis of PSMA expression in a cohort of patients with surgically resectable pulmonary squamous cell carcinoma. A. Representative staining intensities of pulmonary squamous cell carcinoma specimens scored as 0, no staining; 1+, weak staining; 2+, moderate staining; and 3+, strong staining. B. Representative image of PSMA-positive tumor neovascular endothelial (NEC) staining. C. Percent of specimens with PSMA expression in tumor cells or tumor NECs, stratified by final pathologic stage. PSMA was robustly expressed at all stages of disease and there were no significant differences by stage.

Figure 2.

Optical properties of OTL78 and in vitro labeling of human pulmonary squamous cell carcinoma cell lines. A. Chemical structure of OTL78 consisting of a high-affinity PSMA ligand coupled to the fluorophore S0456 (molecular weight: 1793.37 a.m.u.). Absorption and emission spectra at right show peak excitation at a wavelength of 774–776 nm and peak emission at 794–796 nm. B. In vitro OTL78 binding potential was evaluated for several human pulmonary squamous cell carcinoma lines. 22rv1 (a known high-PSMA-expressing prostate cancer cell line) was used as a positive control, while KB (a cervical carcinoma cell line with low PSMA expression) was used as a negative control. At left are representative flow cytometry tracings of cells after exposure to OTL78-spiked media (1 μM) for 4 hours. Mean fluorescence intensity (MFI) of OTL78-exposed cells corresponds to the blue histogram; unstained cells were used as a baseline (red histogram). Cells co-cultured with OTL78 were examined by fluorescence microscopy (green pseudocoloration) (right). Cells were counterstained with an anti-EPCAM antibody conjugated to a FITC fluorophore (red pseudocoloration). C. OTL78 labeling of human pulmonary squamous cell carcinoma cells (H513) in a concentration dependent manner. Cells were imaged 2 hours after OTL78 administration. OTL78 is shown in green overlaid upon DAPI staining. Inset images are at 100x. Kd by mean fluorescence intensity was calculated to be 58.2 nM. D. OTL78 labeling of human pulmonary squamous cell carcinoma cells (H513) in a time-dependent manner. OTL78 was administered at a concentration of 20 nM. OTL78 is shown in green overlaid upon DAPI staining. Inset images are at 100x.

Figure 3.

Dosing, timing, and biodistribution of OTL78 in mice bearing flank xenografts. Mice bearing 22rv1 and KB flank xenografts were administered OTL78 at increasing dosing levels then imaged with the Pearl Trilogy in vivo Imaging System. A. Representative images of mice at various times after intravenous drug delivery. B. Signal-to background ratios (SBRs) were obtained for each dosing level and plotted over time from drug delivery. C. Four hours after delivery of OTL78 at 2 mg/kg, mice bearing 22rv1 and KB xenografts were euthanized to determine drug biodistribution. Fluorescence of organs and tumors were obtained using the Pearl Trilogy. D. Bar graph demonstrating fluorescence of flank tumors as compared to other organs.

Figure 4.

OTL78 accumulates in pulmonary squamous cell carcinoma xenografts. Mice (n=3/group) bearing 22rv1, KB, H1264, and H513 flank xenografts were administered 2 mg/kg OTL78 and imaged with a NIR compatible imaging system four hours after injection. A. Representative images of mice from each group. The left column shows white light imaging with red arrow denoting the location of the flank xenograft. The center column shows near-infrared images, and the rightmost column shows NIR overlaid on white light image. B. SBRs from the flank xenografts. Each point represents a single tumor with means and error bars displayed for each group. C. Mean fluorescence intensity of tumor and background tissue. Lines represent paired tumor and background measurements from the same mouse. Mean bars with standard error are shown. ***: p<0.0001.n.s.: not significant.

Figure 5.

Correlative ex vivo histopathology of flank xenografts and association of fluorescence with density of PSMA expression. All mice were administered 2 mg/kg OTL78 and mice were sacrificed 4 hours after OTL78 administration. A. Representative images from 22rv1, H513, H1264, and KB xenografts showing macroscopic tumor fluorescence, hematoxylin and eosin staining, immunohistochemical staining for PSMA, and immunofluorescence by fluorescence microscopy. B. Mean tumor MFI for each xenograft group plotted adjacently to PSMA density (% PSMA-positive cells/HPF) with error bars for the respective groups. C. Scatter plot of mean fluorescence intensity vs. PSMA density showing strong correlation between the two (R²=0.8508). Each point represents a single tumor.

Figure 6.

IMI with OTL78 improves identification of residual disease after resection of pulmonary squamous cell carcinoma xenografts. A. Schematic of study design. Mice bearing H1264 and H513 flank xenografts (n=10/cell line) were randomized into resection with residual disease or complete (R0) resection. All mice were administered 2 mg/kg OTL78 and mice were sacrificed 4 hours after OTL78 administration. Two blinded surgeons were then asked to grade margins as positive or negative, without knowledge of the number of positive or negative margins. One investigator could use only traditional methods (visual inspection and finger palpation) and the other could use traditional methods aided by intraoperative imaging with OTL78. B. Summary of the accuracy of margins assessed by traditional methods alone compared to those assessed with the aid of OTL78 IMI. C. Representative images of positive and negative margins as seen by the surgeon using traditional methods (left column) and the one aided by IMI with OTL78 (rightmost three columns).