Identification of a Folate Receptor-Targeted Near-Infrared Molecular Contrast Agent to Localize Pulmonary Adenocarcinomas

Abstract

Non-small cell lung cancer (NSCLC) is the number one cancer killer in the United States. Despite attempted curative surgical resection, nearly 40% of patients succumb to recurrent disease. High recurrence rates may be partially explained by data suggesting that 20% of NSCLC patients harbor synchronous disease that is missed during resection. In this report, we describe the use of a novel folate receptor-targeted near-infrared contrast agent (OTL38) to improve the intraoperative localization of NSCLC during pulmonary resection. Using optical phantoms, fluorescent imaging with OTL38 was associated with less autofluorescence and greater depth of detection compared to traditional optical contrast agents. Next, in in vitro and in vivo NSCLC models, OTL38 reliably localized NSCLC models in a folate receptor-dependent manner. Before testing intraoperative molecular imaging with OTL38 in humans, folate receptor-alpha expression was confirmed to be present in 86% of pulmonary adenocarcinomas upon histopathologic review of 100 human pulmonary resection specimens. Lastly, in a human feasibility study, intraoperative molecular imaging with OTL38 accurately identified 100% of pulmonary adenocarcinomas and allowed for identification of additional subcentimeter neoplastic processes in 30% of subjects. This technology may enhance the surgeon's ability to identify NSCLC during oncologic resection and potentially improve long-term outcomes.

Keywords: folate receptor alpha; intraoperative imaging; pulmonary adenocarcinoma; surgery.

Graphical abstract

Figure 1

Optical Properties of OTL38 Are Superior to Those of FITC, PPIX and ICG Contrast agents (1 × 10⁻⁵M) were suspended in a 1.7-mL Eppendorf tube. Tubes were then inserted into pneumonectomy specimen at variable depths (0 mm, 1 mm, 5 mm, and 10 mm). Representative data obtained using a specimen from a deceased 72-year-old male with no smoking history. (A) Fluorescent overlay images were obtained using the FloCam imaging system. (B) Autofluorescence of pulmonary parenchyma was quantified under lighting conditions specific to each optical agent. (C) Tumor-to-background ratios (TBRs) were calculated using ROI software for each imaging agent with respect to depth from the pleural surface. Results expressed as mean (SD). ns, not significant (p > 0.05); *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2

OTL38 Binds Human NSCLC Models In Vitro and Is Proportional to FRα Expression In vitro FRα expression and OTL38 binding potential was evaluated for several human NSCLC lines. KB (a known high-FRα-expressing nasopharyngeal carcinoma model) was used as positive control, while NCIN87 (a gastric carcinoma with low FRα expression) was used as a negative control. Representative flow cytometry tracings of cells after exposure to OTL38-spiked media (1 μM) for 4 hr (left). Mean fluorescence intensity (MFI) of OTL38-exposed cells corresponds to the blue histogram; unstained cells were used as a baseline (red histogram). Cells co-cultured with OTL38 were examined by fluorescence microscopy (green pseudocoloration) (middle). Cells were counterstained with an anti-EPCAM antibody conjugated to a FITC fluorophore (red pseudocoloration). Representative images of cell lines that were immunostained for FRα using the anti-FRα monoclonal antibody (mAb) 26B3.F2 (right).

Figure 3

OTL38 Accumulates in FRα-Expressing NSCLC Xenografts Mice bearing A549 flank xenografts were administered OTL38 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) Tumor-to background ratios (TBRs) were obtained for each dosing level and plotted over time from drug delivery. (C) 24 hr after delivery of OTL38 at 0.025 mg/kg, mice bearing A549 and NCIN87 were euthanized to determine drug biodistribution. Fluorescence of organs and tumors were obtained using the Pearl Trilogy. (D) Bar graph demonstrating fluorescence of flank A549, NCIN87, and kidneys are provided. (E) Mice with established orthotopic pulmonary A549 xenografts (12 days) were imaged 24 hr after receiving OTL38 at 0.025 mg/kg. Results expressed as mean (SD). **p < 0.01.

Figure 4

Staining Patterns and Intensities for FRα Expression in Pulmonary Adenocarcinoma (A–D) Representative staining of pulmonary adenocarcinoma specimen scored as 0, no staining (A); 1+, weak staining (B), 2+, moderate staining (C); and 3+, strong staining (D).

Figure 5

OTL38 Results in Fluorescence in Human FRα Expressing Pulmonary Adenocarcinomas Representative data from the first 5 subjects enrolled in a pilot study involving IMI with OTL38 (0.025 mg/kg). Approximately 4 hr after intravenous delivery, subjects underwent minimally invasive pulmonary resection (VATS). Preoperative CT (column 1) and PET (column 2) scans are provided. Intraoperative bright-field (column 3) and fluorescent overlay views (column 4) during VATS resection. H&E (column 5) and FRα immunohistochemistry (column 6) of resected tumors.

Figure 6

OTL38 Accumulates within Pulmonary Adenocarcinomas in Areas of FRα Expression Representative histopathologic analysis (subject 3) of resected pulmonary adenocarcinoma. Whole-section images were obtained and evaluated using H&E staining, FRα immunohistochemistry, and NIR microscopic scanning (top row). The tumor (outlined in dash marks) demonstrated strong fluorescence, particularly in areas of FRα expression. We noted increased fluorescence in areas of strong FRα (*, second row) and moderate levels in those areas with less intense expression (**, third row). Normal pulmonary parenchyma displayed negligible fluorescence (***, bottom row). *, area of tumor with strong FRα expression; **, area of tumor with moderate FRα expression; ***, normal lung parenchyma.