Prolactin receptor-mediated internalization of imaging agents detects epithelial ovarian cancer with enhanced sensitivity and specificity

Abstract

Poor prognosis of ovarian cancer (OvCa), the deadliest of the gynecologic malignancies, reflects major limitations associated with detection and diagnosis. Current methods lack high sensitivity to detect small tumors and high specificity to distinguish malignant from benign tissue, both impeding diagnosis of early and metastatic cancer stages and leading to costly and invasive surgeries. Tissue microarray analysis revealed that >98% of OvCa express the prolactin receptor (PRLR), forming the basis of a new molecular imaging strategy. We fused human placental lactogen (hPL), a specific and tight binding PRLR ligand, to magnetic resonance imaging (gadolinium) and near-infrared fluorescence imaging agents. Both in tissue culture and in mouse models, these imaging bioconjugates underwent selective internalization into OvCa cells via PRLR-mediated endocytosis. Compared to current clinical magnetic resonance imaging techniques, this targeted approach yielded both enhanced signal-to-noise ratio from accumulation of signal via selective internalization and improved specificity conferred by PRLR upregulation in malignant OvCa. These features endow PRLR-targeted imaging with the potential to transform OvCa detection.

Introduction

Advanced ovarian cancer (OvCa) with abdominal spread (stage III & IV) has 5-year survival rates of <30%, while cancers confined to the ovary and the pelvis (stage I and II) have 5-year survival rates of >70% (1). Because the ovaries and the fallopian tube are hidden in the peritoneal cavity, 75% of OvCa remain undiscovered until stage III or IV, after the tumor has metastasized (2). Thus, specific tumor detection poses one of the most important challenges to OvCa research and treatment. Two major goals for current detection methods include (i) detection of tumors when they are still small and curable and (ii) differentiation between benign and malignant ovarian tumors, which would drastically reduce the significant number of unnecessary surgeries (3). Bimanual pelvic examination, transvaginal ultrasound, and serum CA-125 levels have consistently failed to detect early ovarian malignancy (4), and advances in the serum biomarker field have been elusive (5,6). New imaging modalities that have high specificity and sensitivity are therefore urgently needed.

Magnetic resonance imaging (MRI) offers several advantages in the imaging of the pelvis, providing high resolution images of anatomical structures with excellent soft tissue contrast without the use of ionizing radiation (7,8). Upon intravenous (IV) injection of paramagnetic contrast agents (e.g. gadolinium-chelates) (9), solid tumors become contrast-enhanced relative to surrounding tissue due to their altered vascular anatomy causing increased permeability (10). However, the diagnosis of malignant OvCa using gadolinium enhanced MRI still lacks sufficient specificity (11) to distinguish healthy from cancerous tissue, resulting in many unnecessary surgeries for a presumptive malignancy (3) and lacks the sensitivity (12) to detect small, early tumors. One potential strategy to improve sensitivity and specificity involves the development of targeted molecular probes - imaging agents conjugated to ligands that bind specifically to receptors that are over-expressed on cancer cells (13). However, due to lack of viable molecular targets, few targeted imaging probes have emerged for OvCa. Here, we introduce the prolactin receptor (PRLR) as a vehicle for internalization of imaging agents into OvCa cells, engendering a new strategy for targeted molecular imaging of OvCa.

As a member of the cytokine receptor superfamily, PRLR activates kinase mediated signaling networks as a dimer in complex with the pleiotropic protein hormones prolactin, growth hormone, or placental lactogen (14–17). Concomitantly, the heterotrimeric complex formed upon exogenous ligand binding to PRLR induces clathrin-mediated endocytosis (internalization) of the ligand:PRLR complex (18). Based on our own immunohistochemical analysis showing that 98% of all OvCas express the human PRLR, we hypothesized that imaging agents conjugated to a human PRLR ligand will internalize selectively into OvCa cells via the receptor’s natural endocytic mechanism. Herein, we show that imaging conjugates of human placental lactogen (hPL), a specific and high affinity PRLR ligand, internalize efficiently into PRLR positive (PRLR⁺) cancer cells in OvCa mouse models and thereby enable detection OvCa with substantially greater specificity and sensitivity than currently used clinical contrast agents. Molecular PRLR imaging holds the potential to enable a more precise and earlier diagnosis of OvCa and to reduce the number of unnecessary surgeries.

Materials and Methods

Histology

Tissue microarrays were assembled from 28 patients with Fédération Internationale de Gynécologie et d’Obstétrique (FIGO) early stage I/II ovarian cancer and 124 patients with advanced stage III/IV ovarian cancer who had undergone surgery by a gynecologic oncologist at the University of Chicago after obtaining Institutional Review Board (IRB) approval (19). The slides were immunohistochemically stained as previously described (19) using 1:25 dilution of hPRLR antibody (sc-20992, Santa Cruz Biotechnology). Prolactin receptor (PRLR) expression were grouped into levels low, medium, or high. For tissues harvested from mice, immunohistochemical studies on formalin-fixed, deparaffinized sections (5 μM) were performed using hPRLR antibody at 1:250 dilution (19). For frozen section analysis, human and mouse tissues were placed for confocal analysis in OCT fluid and immediately frozen at − 80 °C.

Cell Lines

CaOV3 cells were purchased from ATCC (Manassas, VA) in 2004. HeyA8 and SKOV3ip1 cells were provided by Dr. Gordon Mills (M.D. Anderson Cancer Center, Houston, TX) in 1995. T47D cells were a gift from Charles Clevenger (Northwestern University, Evanston, IL) and acquired between 2003–2006. Cell lines were authenticated using the commercial service, CellCheck (IDEXX Bioresearch). The alleles for 9 short tandem repeat (STR) markers were determined and the results were compared to the profiles from DSMZ, ATCC, JCRB, and RIKEN STR databases. Samples were confirmed to be of human origin and no mammalian inter-species contamination was detected. Samples are tested approximately once per year.

Western blot analysis

Western blot analysis was performed as described (28). The following antibodies were used for our studies: phospho-ERK1/2 and ERK1/2 (Cell-Signaling), actin (Sigma), PRLR (H-300, Santa Cruz Biotechnologies) and hPL (Thermo Fisher Scientific).

Fluorescence microscopy

We grew cells to ~70% confluency on 12 mm glass coverslips placed in 60 mm plates over three days and then starved with serum-free media overnight. The cells were then incubated with 500 nM imaging agent for 6 hours at 37 °C in serum-free media at indicated zinc concentration, washed thrice with PBS, fixed in 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS, and mounted in Prolong Gold with DAPI (Invitrogen). For immunofluorescence studies, cells were permeabilized with 0.1% Triton X-100 in PBS, followed by incubation with 1:250 dilution of anti-hPRLR antibody (sc-20992) overnight, and briefly with a 1:1000 dilution of Alexa 488 labeled mouse anti-rabbit antibody (Invitrogen) before mounting.

Frozen sections of human tissue specimen for hPL*-Cy5 binding were probed with 500 nM hPL*-Cy5 in the presence of 20 μm zinc overnight, washed thoroughly with dPBS, and mounted using the immnunofluorescence protocol. Data was analyzed with an Olympus FV1000 (Olympus America). After processing of frozen xenograft mouse tumors by the HTRC, we used mounted tissue protocol described above for confocal analysis. These samples were analyzed using a DSU Spinning Disk Confocal (Olympus America). All data were processed using ImageJ software (v1.42q, NIH).

Tumor xenografts

For subcutaneous (SQ) tumor imaging of ovarian cancer, we injected female, athymic nude mice (5–6 weeks old) near the thigh with 1 × 10⁶ normal or transfected HeyA8 cells, 2 × 10⁶ SKOV3ip1 cells, or 5 × 10⁶ CaOV3 cells with growth-factor reduced matrigel (BD Biosciences) at a 2:1 ratio. The xenograft model for metastases was previously described (19,20). Briefly, 1 × 10⁶ GFP-Luc cells were intraperitoneally (i.p.) injected into 5–6 week old, female, athymic nude mice. The Institutional Animal Care and Use Committee (IACUC) at the University of Chicago approved all protocols presented in these studies.

Near-infrared fluorescent imaging

Mice (22.9 ± 0.7 g) with SQ HeyA8 tumors (normal and GFP-Luc) that weighed the indicated size were imaged. For specificity experiments, shScram (l. leg) and shPRLR (r. leg) HeyA8 cells were subcutaneously implanted on contralateral legs and allowed tumors to grow for seven to ten days. For all experiments, we made IV injections of indicated imaging agents (0.21 μmol/kg Cy5.5) and analyzed animals using fluorescence molecular tomography instrument (FMT1, VisEn Imaging) at the 680 nm channel. Total Cy5.5 (pmols) were calculated by drawing a 3D region of interest (ROI) around indicated tumor areas and plotted values as a function of tumor mass – acquired as a wet mass (g) after euthanasia and dissection.

Ex vivo tissue imaging

Mice were euthanized mice at indicated times after hPL*-Cy5.5 injection, organs immediately dissected, and organs analyzed in an OV100 Small Animal Imaging System (Olympus America) using the indicated filter. For IP metastases imaging, hPL*-Cy5.5 (0.21 μmol/kg Cy5.5) in mice fourteen days after cell implantation, euthanized 8 HPI, dissected organs, and analyzed using the OV100 system.

Bioluminescence imaging

Ten minutes prior to imaging and 24 HPI of indicated imaging agent, 200 μL of 15 mg/mL D-luciferin (Gold Biotechnology) was injected as an IP bolus in animals, and imaged SQ tumor margins on the Xenogen IVIS 200 (Calipar) at indicated intensities. Tumor margin with representative BLI were correlated with 2D MR images for approximation of 2D slice acquisition.

Animal MR imaging

MR images were obtained from imaged mice (20.7 ± 0.7 g) with SQ tumors (62.1 ± 13.5 mg) seven to ten days after implantation in a 30-cm horizontal bore Bruker BioSpec 9.4 Tesla Small Animal MR System (Bruker-Biospin). A homebuilt half-open birdcage coil (21) was used to acquire T₁-weighted low resolution 2D gradient echo images with the following parameters: fat suppression turned-on; flip angle, 30°; repetition time 250 ms; echo time 5 ms; field of view, 2.56 × 2.56 cm; matrix size, 128 × 128, two averages. High-resolution images (matrix size, 256 × 256 with two averages) were then acquired. We serially imaged the mice at time 0 HPI, immediately after IV injection (low dose, 0.36 μmol/kg gadolinium; high dose, 100 μmol/kg gadolinium), 4 HPI, and 24 HPI, with the best images acquired at 24 HPI. We acquired R₁ values using RAREVTR pulse-sequence. We analyzed R₁ values of tumors in slices with high T₁-weighted signal increases (regions of interest, ROI) by normalizing to surrounding muscle tissue. We then subtracted this value from the R₁ tumor to muscle ratio (same ROI) at 0 HPI. We harvested the organs 24 HPI for ICP-MS analysis.

Relaxivity (r₁)

The T₁ values of contrast agents were obtained in PBS using a 5.6 cm vertical bore, 11.7 Tesla vertical magnet (Oxford Instruments) using a Bruker DRX-500 MHZ Avance Spectrometer (Bruker Instruments) at the University of Illinois (Chicago, IL) A Rapid Acquisition with Relaxation Enhancement with Variable Repetition Time (RAREVTR) pulse sequence was used to obtain T₁ values. Relaxivity (r₁) was calculated using linear regression analysis of 1/T1 as function of gadolinium or protein concentrations.

ICP-MS

Synthesized MRI contrast agents were quantified for gadolinium in a trace-metal free 3% HNO₃ (Fluka) matrix solution by X series II (Thermo Electron) ICP-MS at Northwestern University (Evanston, IL). For cell analysis, we washed cells thrice with dPBS, gently removed them from plate surface using a cell lifter, dried at 80 °C, digested with 100% HNO₃ and then diluted to 3% HNO₃. Likewise, animal tissues were harvested at 24 hours post-injection (HPI) of gadolinium contrast agents given as an intravenous (IV) bolus, weighed, digested with 100% HNO₃ and then diluted to 3% HNO₃ for gadolinium analysis.

Statistical analysis

For PRLR analysis in TMA studies (Table 1), the p-values were calculated using the Kruskal-Wallis tests except for age at diagnosis, stage, and grading where the p-value is from Spearman rank correlation analysis. For studies in presented in Supplementary Table 1, McNemar’s test was performed to compare the proportion of ovarian tumors with a score of 3 to the proportion of omentum samples with a score of 3 (and likewise for the comparison of ovarian versus peritoneal samples). Linear regression analysis was used in the comparison of %ID to tumor mass and calculating R₁ values for contrast agents. In the case of comparing gadolinium uptake in HeyA8 cells, statistical comparisons were performed by analysis of variance. For all other data, Student’s t tests were used assuming equal variances to compare data pairs. Values of p ≤ 0.05 were considered significant and reported data as mean values ± s.e.m.

Table 1. Clinical data of PRLR expression in patients with FIGO stage I–IV epithelial ovarian/fallopian/peritoneal cancer, n = 152.

FIGO (Fédération Internationale de Gynécologie et d’Obstétrique). The p-values were calculated using the Kruskal-Wallis tests except for age at diagnosis, stage, and grading where the p-value is from Spearman rank correlation analysis.

	Total # (%)	Prolactin Receptor Score
		Low	Moderate	High	p-value
OvCa/Fallopian tube/peritoneal cancers	152 (100)	2 (1.3)	24 (15.8)	126 (82.9)
Age at diagnosis (years) [n=152]					0.08
Median [min-max]	58 [36–88]	51.5 [48–55]	54.5 [39–77]	59 [36–88]
FIGO Stage					0.02
I	20 (13.2)	2 (10.0)	8 (40.0)	10 (50.0)
II	8 (5.3)	0	1 (12.5)	7 (87.5)
III	86 (56.6)	0	9 (10.5)	77 (89.5)
IV	38 (25.0)	0	6 (15.8)	32 (84.2)
Unknown	0	0	0	0
Histology					<0.01
Serous-papillary	107 (70.4)	0	10 (9.4)	97 (90.7)
Endometrioid	21 (13.8)	0	5 (23.8)	16 (76.2)
Clear cell	13 (8.6)	1 (1.7)	3 (23.1)	9 (69.2)
Mucinous	11 (7.2)	1 (9.1)	6 (54.6)	4 (36.4)
Grading					0.04
G1	12 (7.9)	0	4 (33.3)	8 (66.7)
G2	34 (22.4)	1 (2.9)	7 (20.6)	26 (76.5)
G3	106 (69.7)	1 (0.9)	13 (12.3)	92 (86.8)
Largest residual tumor at the end of surgery					0.17a
≤ 1 cm	93 (61.2)	2 (2.2)	17 (18.3)	74 (79.6)
> 1 cm	58 (38.2)	0	7 (12.1)	51 (87.9)
Unknown	1 (0.7)	0	0	1 (100)
Chemotherapy Type					0.12
Neo-adjuvant	17 (11.2)	0	1 (5.9)	16 (94.1)
Primary	124 (81.6)	2 (1.6)	19 (15.3)	103 (83.1)
None	11 (7.2)	0	4 (36.4)	7 (63.6)
Chemotherapy					0.26
Taxane/Platinumb	129 (84.9)	2 (1.6)	19 (14.7)	108 (83.7)
Platinum containingc	6 (3.9)	0	1 (16.7)	5 (83.3)
Otherd	6 (3.9)	0	0	6 (100)
No chemotherapy	11 (7.2)	0	4 (36.4)	7 (63.6)

^aAnalysis excluded one patient with missing data.

^bOne patient received only Paclitaxel

^cThree patients had platinum single agent chemotherapy and three patients had platinum with another chemotherapy drug.

^dFive patients had Gemcitabine and one patient was part of a phase I study.

Results

The prolactin receptor is over-expressed on human malignant epithelial ovarian cancers

We conducted immunohistochemical analysis on tissue microarrays containing FIGO stage I-IV human OvCa (n = 152) and normal ovarian tissue (n=10) with categorization of PRLR expression into three groups: low, moderate, and high (Fig. 1a, Table 1 and Supplementary Fig. 1). Moderate to high PRLR expression was observed in 98% of samples and localized to the epithelial cancer cell compartment but not the tumor stroma such as fibroblasts and mesothelial cells (Supplementary Fig. 2a). Moderate to high PRLR expression was present regardless of histological type, grade, and stage (Table 1). In contrast, analysis of normal ovarian tissue (n=10) showed very little receptor expression (Fig. 1a, top left, and Supplementary Fig. 2b), consistent with a previous report (22). Furthermore, we observed no significant difference in PRLR expression between primary ovarian tumors and the corresponding tumor metastases in the peritoneum, suggesting that molecular imaging of PRLR should detect OvCa independent of anatomical location (Supplementary Table 1). Collectively, these data establish PRLR as an up-regulated cell-surface receptor in epithelial OvCa and implicate it as a potential imaging target both for detecting OvCa and for differentiating malignant ovarian tissue (PRLR⁺) from benign tissue (PRLR⁻).

Figure 1. Imaging agents conjugated to human placental lactogen bind to prolactin receptor positive ovarian cancer cells and tissue and stimulate ERK phosphorylation.

(a) Top panel: Immunohistochemical sections of a normal ovary, a serous OvCa, and the corresponding omental metastasis from the serous OvCa stained with PRLR antibody. Images were taken at 25x magnification. Bottom panel: Cryosection of corresponding ovarian tissue. hPL*-Cy5 was added in the presence of ZnCl₂ to the tissue, subsequently stained for PRLR (green), and counterstained with DAPI (blue) to detect the nuclei. Cy5 (red). White bar (50 μm).

(b) Left: Interaction of ovine placental lactogen (yellow) with rat extracellular domain of PRLR (green). Locations of Zn²⁺ (blue) and cysteine mutation (orange) are modeled into diagram from PDB 1F6F using PyMol software. Right: Schematic diagrams of imaging conjugates hPL*-Cy5.5 and hPL*-GdDTPA.

(c) Left: Analysis of hPL*-gadolinium conjugates by isoelectric focusing. Isoelectric gel shows purified products after each step of synthesis. S (isoelectric focusing standard), hPL (WT hPL), and pI (isoelectric point). Right: Analysis of imaging conjugates by SDS-PAGE followed by coomassie stain. Gel shows final purified products before cell and animal studies. S (standard), hPL (WT hPL).

(d) Ligand stimulated phosphorylation of ERK-MAPK. T47D cells were stimulated with the indicated concentrations of hPL, hPL*-GdDTPA, and TM hPL*-GdDTPA for 0.5 h. Total ERK (ERK) and phosphorylated ERK (pERK) were detected by immunoblotting. Blots were stripped and re-probed with antibody to ERK.

Conjugates of hPL bind to PRLR⁺ tissues and cells

To allow for attachment of imaging agents to hPL, we generated an isoleucine to cysteine mutation at residue 138 located in a flexible loop region (I138C, referred to hereafter as hPL*), expressed recombinant hPL* in E. coli, and purified it using a C-terminal His₆-tag. According to the crystal structure of ovine placental lactogen (an hPL analogue) in complex with rat PRLR extracellular domain (a human PRLR analogue), the flexible loop resides outside the binding interface, suggesting that mutation within this region will not interfere with hormone binding to the receptor (Fig. 1b) (17). Upon incubation with excess maleimide-Cy5 dye under mild reducing conditions, hPL* becomes quantitatively labeled (1:1 stoichiometric ratio of dye:protein after purification) but not when pre-treated with excess iodoacetamide (Supplementary Fig. 3), consistent with labeling at C138. As a control we constructed a variant of hPL* containing three alanine mutations (TM hPL*) known to reduce binding to the receptor (23). These mutations reside in the site 1 binding region, which includes two residues (E174, H18) in the ligand:receptor interface that interact with a zinc ion, a required cofactor for hPL binding to PRLR (Supplementary Fig. 4) (23). The TM variant also conjugated to maleimide dyes at 1:1 stoichiometric ratios. Purified products indicated single species (Fig. 1c).

We incubated hPL*-Cy5 with frozen sections of normal and malignant ovarian tissue in the presence of zinc (ZnCl₂). After washing the incubated tissue, the malignant cells but not the normal cells retained Cy5 fluorescence. This signal corresponded to PRLR expression levels and depended upon the presence of zinc consistent with a signal resulting from hPL*-Cy5 binding directly to PRLR (Fig. 1a, bottom row). These results suggest that hPL*-conjugates may offer a strategy to differentiate between benign (PRLR⁻) tissue and malignant (PRLR⁺) tissue.

To test the efficacy of our concept for clinical MRI, we conjugated hPL* and TM hPL* to a gadolinium contrast agent, GdDTPA (Fig. 1b–c). We linked the DTPA moiety to the proteins using the bifunctional crosslinking agent SPDP, which allowed conjugation of the chelating agent by disulfide exchange. Under the same conditions, wild-type hPL, which lacks a free cysteine, did not react (Supplementary Fig. 6a). To load gadolinium ion into hPL*-DTPA and TM hPL*-DTPA, conjugates were treated with excess gadolinium chloride and dialyzed to remove free gadolinium ion. Isoelectric focusing showed a clear shift in pI after DTPA conjugation and gadolinium loading (Fig. 1c). Analysis by inductively coupled plasma mass spectrometry (ICP-MS) revealed ~1:1 ratio of gadolinium to protein in each case (Supplementary Fig. 6b).

We first tested whether the hPL I138C mutation and subsequent conjugations affected ligand-induced PRLR-mediated signaling using both T47D cells, a breast cancer cell line over-expressing the long isoform of PRLR, and HeyA8 cells, an OvCa cell line over-expressing a shorter PRLR isoform (Supplementary Fig. 6). Incubation of these cells with hPL, hPL*-GdDTPA, or hPL*-Cy5 in the presence of zinc induced ERK phosphorylation, a downstream PRLR signaling effect (Fig. 1d) (24). In contrast, cells incubated with TM hPL ^*-GdDTPA, hPL*-GdDTPA without zinc, or hPL*-GdDTPA in the presence of the PRLR-mediated signaling inhibitor, cyclosporine A (CsA) (25), showed attenuated ERK phosphorylation (Fig. 1d and Supplementary Fig. 7). Together, the results indicate that the observed signaling hinges on the integrity of the binding interface between receptor and hormone. To address concerns for possible side effects caused by hPL* activation of the signaling pathway, we assessed the tumorigenic potential of hPL*-conjugate exposure, given as a single dose, by counting the colonies formed on soft agar plates of PRLR⁺ OvCa cells. Our results indicate that a short-term exposure (6 hours) – a situation encountered for diagnostic purposes - does not increase tumorogenic activity, whereas long term exposure (> 15 days) appears to increase activity (Supplementary Fig. 8). Overall, our data show that the site-specific modification of the flexible loop region in hPL allows for construction of hPL* conjugates that retain the ability to induce receptor-mediated signaling.

PRLR⁺ OvCa cells, but not PRLR⁻ cells, internalize hPL* imaging conjugates

Having demonstrated downstream signaling activity, we proceeded to test whether hPL* and associated conjugates internalize into PRLR⁺ cancer cells, which would allow for the accumulation and retention of imaging agents inside PRLR⁺ cancer cells compared to PRLR⁻ cells. We analyzed cell lysates after incubation of HeyA8 cells with hPL* at an endocytosis permissive temperature (37 °C) and at an endocytosis restrictive temperature (4 °C). Analysis of untreated cell lysates with an anti-hPL antibody confirmed that HeyA8 cells had very little, if any, endogenous hPL. However, lysate analysis after incubation of HeyA8 cells with hPL* showed significant intracellular levels of hPL* at 37 °C compared to incubation at 4 °C, suggesting active, rather than passive, internalization and accumulation (Fig. 2a, top panel). These findings are consistent with results for PRLR-mediated internalization of prolactin (26,27).

Figure 2. Imaging agents conjugated to human placental lactogen are internalized into prolactin receptor positive ovarian cancer cells by receptor-mediated endocytosis.

(a) Immunoblot analysis of internalization of hPL*, hPL*-conjugates, and triple mutant (TM) hPL*-conjugates. Top panel: Detection of hPL* in HeyA8 cell lysates before incubation (time 0 h) and after incubation at 4 °C and 37 °C. Middle panel: Detection of hPL*- or TM hPL*-conjugates in solution. Bottom panel: Upper, detection of hPL* and TM hPL* conjugates in cell lysates of HeyA8 cells. Lower, hPL* conjugates in HeyA8 cells transfected with short-hairpin RNA-expressing plasmids containing a scrambled (shScram) or PRLR (shPRLR) sequence. All blots were stripped and re-probed with actin antibody.

(b) Confocal microscopy analysis of hPL*-Cy5 internalization. Top row: Addition of hPL*-Cy5 and TM hPL*-Cy5 to PRLR⁺ HeyA8 cells. Cells were stained for PRLR (green). Bottom row: Addition of hPL*-Cy5 to PRLR⁺ shScram cells or to PRLR⁻ shPRLR cells. Cells with plasmids express GFP (green).

All experiments were performed in the presence of 20 μM ZnCl₂ (unless otherwise indicated). Images were taken after 6 h of incubation. All cells were counterstained with DAPI (blue) to detect the nuclei. Cy5 signal (red). White bar (30 μm).

(c) Inductively coupled mass spectrometry (ICP-MS) of gadolinium internalization. Left panel: Detection of gadolinium in PRLR⁺ HeyA8 cell lysates after incubation with Magnevist^™, hPL*-GdDTPA, or TM hPL*-GdDTPA (n=4). Results were normalized to uptake of GdDTPA alone. p < 0.005 ANOVA analysis. Right panel: Detection of gadolinium in PRLR⁺ shScram and PRLR⁻ shPRLR cell lysates after incubation with hPL*-GdDTPA. Results were normalized to uptake by shScram cells and represent at least n=5 samples. Asterisk, p < 0.05 Student’s t test.

All experiments were performed in the presence of 20 μM ZnCl₂ (unless otherwise indicated). Samples were analyzed by inductively-coupled plasma mass spectrometry (ICP-MS) after 6 h of incubation.

We next tested the imaging conjugates hPL*-Cy5 and hPL*-GdDTPA for internalization. Immunoblot analysis demonstrated zinc dependent internalization of hPL*-Cy5 and hPL*-GdDTPA into PRLR⁺ HeyA8 cells. In contrast, TM hPL*-Cy5 and TM hPL*-GdDTPA, which have reduced affinity for the receptor, showed severely attenuated levels of uptake (Fig. 2a, bottom panel). These differences did not arise from biases in anti-hPL antibody for detection of hPL*-conjugates compared to TM hPL*-conjugates (Fig. 2a, middle panel). We further confirmed internalization of hPL*-Cy5 into HeyA8 cells by confocal microscopy (Fig. 2b, top panel) and internalization of hPL*-GdDTPA by measurement of gadolinium content of cell lysates using ICP-MS (Fig. 2c). Unconjugated GdDTPA (Magnevist^™), hPL*-GdDTPA in the absence of zinc, and TM hPL*-GdDTPA each showed a ~4-fold reduction of internalization compared to hPL*-GdDTPA (Fig. 2c). We obtained similar results using T47D cells (Supplementary Fig. 9). These observations and the requirement for zinc strongly suggest active internalization of hPL*-conjugates via PRLR-mediated endocytosis.

To directly establish the dependence of internalization upon the presence of the PRLR, we stably transfected HeyA8 cells lines with plasmids that express green fluorescent protein (GFP) and contain either a short hairpin (sh) RNA with a scrambled sequence (shScram) or a human PRLR shRNA (shPRLR) cassette. Analysis by Western blot and immunoblotting showed that shPRLR cells expressed less PRLR than untransfected, shScram, or the GFP-luciferase expressing HeyA8 cells (GFP-Luc) (28) (Supplementary Fig. 10 and compare with Supplementary Fig. 6a). Cells transfected with the shPRLR plasmid exhibited reduced hPL*-Cy5 and hPL*-GdDTPA internalization compared to cells transfected with the shScram plasmid (Fig. 2a–c). Together these results show that hPL*-conjugates internalize specifically into PRLR⁺ cancer cells through PRLR-mediated endocytosis.

hPL*-Cy5.5 localizes to xenograft PRLR⁺ tumors in mice through PRLR-mediated endocytosis

Unmodified HeyA8, GFP-Luc HeyA8, and shScram HeyA8 cells formed solid, PRLR⁺ serous-papillary tumors when implanted subcutaneously (SQ) in athymic nude mice, whereas solid, serous-papillary tumors formed from shPRLR HeyA8 cells had reduced PRLR expression (Fig. 3a and Supplementary Fig. 11). As expected, muscle tissue surrounding tumors showed undetectable PRLR expression in all implanted mice (Fig. 3a). We also observed PRLR expression in murine ovarian tissue (29) in contrast to normal human ovarian tissue, which has undetectable to low levels of PRLR (30) (Fig. 1a, top panel).

Figure 3. hPL*-Cy5 localizes to PRLR expressing tumors in vivo.

(a) Comparison of immunohistochemical (IHC) sections of HeyA8 xenograft tumor (human), mouse muscle tissue, and mouse ovarian tissues stained with anti-PRLR antibody. Additionally, xenograft tumors of HeyA8 cells transfected with plasmids expressing scrambled short hairpin RNA (shScram) or PRLR short hairpin RNA (shPRLR) are shown. Serial sections were stained with hematoxylin and eosin (H&E). Black bar (100 μm).

(b) Imaging of tumors by bioluminescence (BLI) and by near infrared fluorescence (NIRF) imaging using fluorescence molecular tomography (FMT). PRLR⁺ HeyA8 tumors expressing GFP and luciferase (GFP-Luc) were grown subcutaneously in nude mice for 8 days. hPL*-Cy5.5 and TM hPL*-Cy5.5 were injected at equivalent dosages and imaged by NIRF imaging using FMT at 24 HPI. Immediately aftewards, mice were injected with D-luciferin and imaged by BLI. Images from FMT depict volume renderings of tumor areas taken at the same color gating. BLI exposure times of hPL*-Cy5.5 and TM hPL*-Cy5.5 treated mice were 5 s and 1 s, respectively.

(c) Correlation of tumor mass and Cy5.5 uptake. Linear regression analysis of tumor mass versus Cy5.5 uptake (percent injected dose – ID) using hPL*-Cy5.5 (diamonds) and TM hPL*-Cy5.5 (squares) at 24 HPI.

(d) Ex vivo fluorescence images of organs. Organs were harvested from hPL*-Cy5.5 injected mice (left) compared to TM hPL*-Cy5.5 injected mice (right) at 24 HPI. White bar (10 mm).

(e) Confocal microscopy of frozen sections of xenograft GFP-Luc tumors in mice injected with hPL*-Cy5.5 or TM hPL*-Cy5.5. Cells were counterstained with DAPI (blue) to detect the nuclei. Cy5.5 (red), GFP (green), co-localization of GFP and Cy5.5 (yellow & white arrows), and white bar (20 μm).

(f) Imaging of PRLR⁺ shScram tumors compared to PRLR⁻ shPRLR tumors. Tumors were grown subcutaneously on contralateral legs in same mouse for 9 days. Left panel: NIRF image by FMT from hPL*-Cy5.5 injected mice at 8 HPI. Right panel: Quantification of Cy5.5 signal from FMT at 8 HPI normalized to mass of tumors (n=4). Asterisk, p < 0.01 Student’s t test.

We then proceeded to test whether the near-infrared fluorescence (NIRF) imaging probe hPL*-Cy5.5 internalizes specifically and accumulates in PRLR⁺ OvCa xenografts in vivo. Intravenous injection of hPL*-Cy5.5 into mice bearing > 2 mm-sized HeyA8 xenografts followed by imaging using fluorescence molecular tomography (FMT) at 24 hours post-injection (HPI) showed that hPL*-Cy5.5 localizes to PRLR⁺ GFP-Luc tumors (Fig 3b). In contrast, TM hPL*-Cy5.5 showed reduced localization. Localization of NIRF signal from hPL*-Cy5.5 and TM hPL*-Cy5.5 correlated with tumor borders defined by bioluminescence imaging (BLI) (Fig. 3b). Quantification of Cy5.5 uptake as a function of tumor weight confirmed the increased uptake of hPL*-Cy5.5 relative to TM hPL*-Cy5.5 (Fig. 3c). hPL*-Cy5.5 accumulation in PRLR⁺ HeyA8 tumors occurred in a time-dependent manner (Supplementary Figure 12a), with maximal Cy5.5 signal at 72 HPI and clearance from the tumor by 192 HPI (Supplementary Figure 12b). We observe exponential decay pharmacokinetics of hPL*-Cy5 in the blood, which is similar to other reported protein-imaging conjugates (Supplementary Figure 12c) (31–33). Ex vivo imaging of organs from mice at 24 HPI (Fig. 3d) indicated that beyond the expected localization to PRLR⁺ murine ovarian tissue, as well as liver and kidneys (due to contrast agent metabolism and clearance), hPL*-Cy5.5 preferentially localized to the PRLR⁺ tumor. In contrast, TM hPL*-Cy5.5 exhibited decreased localization to the tumor relative to hPL*-Cy5.5. Notably, hPL*-Cy5.5 does not localize to the PRLR⁻ muscle tissue (Fig. 3d and Supplementary Figure 12d). Confocal imaging of GFP-Luc HeyA8 xenografts confirmed that hPL*-Cy5.5 localizes to and internalizes into PRLR⁺ tumors (Fig. 3e). As OvCa is a heterogeneous disease with many different subtypes (20), we also tested CaOV3 cells, which form an adenocarcinoma SQ xenograft tumor (34) with high expression of PRLR (Supplementary Fig. 10). We analyzed the CaOV3 xenograft for its capacity to internalize hPL*-Cy5.5 and confirmed tissue-specific uptake by NIRF and ex vivo imaging, consistent with our findings of hPL*-Cy5.5 uptake in PRLR⁺ HeyA8 xenografts (Supplementary Fig. 13). Together, the data strongly suggest that the PRLR mediates hPL*-Cy5.5 uptake in vivo.

To test the potential for hPL*-guided imaging to directly differentiate PRLR⁻ (benign) tissue from PRLR⁺ (malignant) tissue in vivo, we used hPL*-Cy5.5 to image mice implanted with both SQ shPRLR and shScram tumors on contra-lateral hind-limbs with NIRF. As revealed by NIRF using FMT technology, shScram tumors accumulated significantly more hPL*-Cy5.5 at 8 HPI than did shPRLR tumors (Fig. 3f). Additional ex vivo images of excised tumors using Cy5.5 and GFP filters confirmed that the Cy5.5 signal in shPRLR tumors localized to non-GFP expressing tumor tissue (PRLR⁺) with very little co-localization to PRLR⁻ GFP expressing tissue. In contrast, Cy5.5 and GFP signals co-localized in shScram tumors (Supplementary Fig. 14). Thus, hPL*-Cy5.5 can distinguish between high and low PRLR expressing tumors within the same mouse and also can differentiate regions of high and low PRLR expression within the same tumor. Distribution of fluorescence signal in various organs corresponded to results obtained by hPL*-Cy5.5 imaging of live animals bearing GFP-Luc and normal HeyA8 tumors. Overall, these data demonstrate that solid, PRLR⁺ ovarian tumor xenografts internalize hPL*-Cy5.5 in a PRLR-dependent manner and suggest that molecular PRLR imaging can distinguish benign tissue from malignant OvCa.

hPL*-Cy5.5 detects small, PRLR⁺ peritoneal OvCa – a model for early stage OvCa imaging

HeyA8 OvCa cells and GFP-Luc HeyA8 cells form small, solid, PRLR⁺ disseminated peritoneal tumors after intraperitoneal (IP) implantation, mimicking the distribution of human OvCa (Supplementary Fig. 15a) (34). To test the sensitivity of molecular PRLR imaging, we targeted small, PRLR⁺ tumors formed from GFP-Luc HeyA8 cells in this intraperitoneal model of OvCa. Ex vivo confocal imaging of peritoneal area 8 HPI of hPL*-Cy5.5 showed tumor nodules within abdominal fat with GFP and Cy5.5 signal co-localization (Supplementary Fig. 15b). Confocal microscopy of frozen tissue confirmed Cy5.5 signal in tumor tissue (Supplementary Fig. 15c) compared to tumor tissue from a non-injected animal. These data demonstrate sensitivity of hPL*-conjugates for detecting small tumors in both an IP and SQ OvCa model. Additionally, we detected no differences in PRLR expression between IP and SQ implanted tumors (Supplementary Fig. 15a and Fig. 3a). We also note that PRLR expression was not biased by location in human OvCa either (Supplementary Table 1).

hPL*-GdDTPA enhances T₁-weighted MRI signals from PRLR⁺ tumors

Having demonstrated the efficacy of molecular PRLR imaging for detecting malignant tumors using NIRF, we proceeded to test PRLR directed imaging using MRI. In solution at 11.7 T, the two contrast agents, hPL*-GDTPA and TM hPL*-GdDTPA, showed small increases in relaxivities (r₁) compared to Magnevist^™ (Figure 4a and Supplementary Fig. 16a). The small increase in r₁ after conjugation of GdDTPA to hPL* or TM hPL* is consistent with the small differences observed by other high molecular weight gadolinium chelates at high magnetic field strengths (35,36). Given recent concerns of gadolinium stability and toxicity (37), we verified that hPL*-GdDTPA is stable at 37 °C for 24 hours compared to Omniscan^™, a gadolinium chelate with low stability (Supplementary Fig. 16b).

Figure 4. hPL*-GdDTPA enhances signals from PRLR⁺ tumors by MRI.

(a) Relaxivity (r₁) of synthesized or commercially available gadolinium contrast agents at 11.7 T at room temperature. r₁ values represent the slope of 1/T₁ as function of gadolinium concentration. The error is reported as a 95% confidence interval using linear regression analysis on four data points.

(b) T₁-weighted axial images of mice before and 24 HPI with equivalent dosages of hPL*-GdDTPA, TM hPL*-GdDTPA, or Magnevist^™. Bioluminescence images (BLI) were taken immediately after acquiring 24 HPI images and indicate approximate location of axial images shown by MRI (white ovals). BLI of hPL*-GdDTPA, TM hPL*-GdDTPA, and Magnevist^™ were taken with 8 s, 20 s, and 45 s exposure time, respectively. Mice were anesthetized and revived between scans. Tumor tissue (dotted white line), muscle (m).

(c) Change in tumor/muscle relaxation rate ratio 24 HPI of contrast agents using MRI. The change in relaxation rate (ΔR₁) was calculated as the relaxation rate of the tumor tissue normalized to relaxation rate of surrounding muscle at 24 HPI subtracted from the same value calculated before injection of contrast agents. Data points are the mean ± s.e.m. from n = 3 animals. Asterisk, p < 0.05 Student’s t test.

(d) Biodistribution of gadolinium in PRLR⁺ HeyA8 xenografts and mouse organs at 24 HPI of equivalent dosages of contrast agents. Tissues were digested with acid and quantified for gadolinium content using inductively coupled mass spectrometry (ICP-MS). Values were normalized to injected dose (ID) and tissue mass. Results were further normalized to data from Magnevist^™ injection. FT – Fallopian Tube. Data points are the mean ± s.e.m. at least n = 3 animals. Single * and double **, p < 0.05 Student’s t test.

We then tested the ability of hPL*-GdDTPA to detect cancer cells by MRI. Using an in vitro MRI assay similar to that described for T47D cells (38) and previously used for imaging pancreatic islet cells by one of our laboratories (39), we incubated T47D and HeyA8 cells with hPL*-GdDTPA and compared the results to analogous incubations with GdDTPA and PBS. Results showed T₁-weighted signal enhancements and reduction of T₁ relaxation times for T47D and HeyA8 cells treated with hPL*-GdDTPA compared to controls (Supplementary Fig. 17).

We then tested the ability of hPL*-GdDTPA to detect solid, PRLR⁺ tumors formed by HeyA8 cells. In initial experiments, we imaged mice continuously under anesthesia after hPL*-GdDTPA injection. Subtraction of T₁-weighted images acquired at 2 and 4 HPI from pre-contrast images confirmed that T₁-weighted enhancement in the tumor were occurring as a consequence of hPL*-GdDTPA injection (Supplementary Fig. 18a). By comparison, attempts to image solid, PRLR⁺ GFP-Luc and PRLR⁺ SKOV3ip1 tumors (an ovarian tumor derived from SQ implanted SKOV3ip1 cells) immediately after IV Magnevist^TM injection at clinical doses (100 μmol/kg) showed poor signal enhancement compared to surrounding muscle tissue (Supplementary Fig. 18b, c). We observed T₁-weighted enhancement of PRLR⁺ tumors by hPL*-GdDTPA during serial imaging (Supplementary Fig. 19a).

We then compared hPL*-GdDTPA to TM hPL*-GdDTPA and Magnevist^™. In mice bearing PRLR⁺, GFP-Luc tumors, IV injection of hPL*-GdDTPA at 0.36 μmol/kg resulted in increased T₁-weighted image contrast at 4 and 24 HPI compared to equivalent dosage of Magnevist^™ or TM hPL*-GdDPTA (Fig. 4b). BLI imaging showed that the contrast enhancement corresponded to locations of maximal luciferin signal and included the interior regions of the tumor tissue (Fig. 4b). Measured relaxivities (calculated as a ratio of tumor relaxivity to muscle relaxivity at 24 HPI subtracted from the ratio calculated before injection) confirmed T₁ contrast enhancement at 24 HPI (Fig. 4c and Supplementary Fig. 19b). Tissue biodistribution of gadolinium qualitatively and quantitatively matched hPL*-Cy5.5 distribution (Fig. 3d and Supplementary Fig. 19c) and confirmed that tumors isolated from mice treated with hPL*-GdDTPA contained more gadolinium than did tumors isolated from mice treated with TM hPL*-GdDTPA or Magnevist^™ (Fig. 4d and Supplementary Fig. 19d). These data strongly suggest that hPL*-GdDTPA localizes and accumulates to PRLR⁺ tumors in a manner similar to hPL*-Cy5.5 and enables detection by currently used clinical imaging.

Additionally, we tested hPL*-GdDTPA for its ability to image a different solid, PRLR⁺ ovarian tumor with moderate to high PRLR expression derived from SQ implanted SKOV3ip1 cells (Fig. 5a, b). IV Injection of hPL*-GdDTPA increased signal intensity of tumors in T₁-weighted MR images compared to TM hPL*-GdDTPA (Fig. 5b) indicating that contrast enhancement provided by hPL*-GdDTPA was not unique to HeyA8 tumors. Lastly, we attempted MR imaging of a SQ tumor implanted near the abdomen and successfully detected an OvCa tumor after IV injection of hPL*-GdDTPA (Supplementary Fig. 20). The results indicated signal enhancement in tumors compared to small colon, large colon, and peritoneum. We did not find PRLR in omental tissue and detected minimal localization of hPL*-GdDTPA to omentum (Fig. 4d).

Figure 5. hPL*-GdDTPA enhances signals from PRLR⁺ SKOV3ip1 tumors by MRI.

(a) Immunohistochemical (IHC) stain with anti-PRLR antibody in HeyA8 and SKOV3ip1 xenografts. Black bar (100 μm).

(b) T₁-weighted axial images of tumors through lower abdominal quadrants before injection and at 24 HPI of indicated contrast agent. Tumor tissue (dotted white line), muscle (m).

Discussion

Ovarian cancer remains the deadliest of gynecological malignancies because current methods for detection have low specificity, confounding efforts to identify disease onset or disease recurrence after surgery (7). Moreover, the current practice of MR imaging of the pelvis following intravenous injection of the gadolinium contrast agent, Magnevist^TM often poorly distinguishes whether an observed mass contains benign or malignant tissue (11), further confounding efforts to diagnose and treat the disease (3). Here we have demonstrated proof-of-concept for a new, targeted molecular imaging approach for OvCa based upon upregulation of the PRLR and its capacity to mediate ligand-induced endocytosis. Levine et al previously showed that >80% of OvCas express weak to high levels of PRLR regardless of stage, grade, and histology (22). Our own analysis confirmed and extended these observations, revealing that a majority (>98%) of OvCas express moderate to high levels of the PRLR. Additionally, we compared metastases to primary tumors and found little difference in PRLR expression between these two subgroups.

To exploit the receptor’s capacity to mediate endocytosis, we established chemistry for conjugation of imaging agents to a flexible loop within hPL located outside the receptor interface without affecting the capacity for PRLR mediated endocytosis. hPL has several advantages as a vehicle for imaging: it binds specifically to PRLR with high affinity (23), promotes its own internalization via the PRLR (18), and allows informed design of bioconjugates because the binding epitope has been defined structurally (17). Moreover, hPL is expressed in the placenta and circulates in the serum of pregnant woman at high levels for several weeks (40). This suggests little competition of hPL*-conjugates with endogenous hormone, since pregnant patients are unlikely to be in the OvCa patient population, and suggests that high levels of hormone are tolerated by patients without detrimental effects.

Despite these advantages, the capacity of hPL*-conjugates to trigger PRLR signaling raises concerns about possible tumor induction. Given the half-life of our conjugate and clearance by 6 HPI, injection of small amounts of contrast agent required for diagnostic imaging is not expected to have significant effects on tumor growth. Our experiments suggest that only prolonged exposures to hPL*-conjugates induced tumor growth. Before proceeding to clinical imaging of PRLR using hPL*-conjugates, further validation and toxicity studies in non-murine animal and non-human primate models must be completed.

Cell lines and tumors carrying PRLR efficiently internalized imaging agents fused to hPL, whereas those lacking the receptor or expressing low levels of the receptor due to RNAi knockdown did not, suggesting a specific mechanism of internalization via PRLR. Less efficient internalization of the weaker binding TM hPL*-conjugates provides further support for a specific mechanism. In mouse models, the imaging agents accumulated into multiple OvCa tumors expressing moderate to high levels of PRLR relative to surrounding tissue. The NIRF probe, hPL*-Cy5.5, readily detected tumors in mice imaged by FMT, while the contrast agent, hPL*-GdDTPA, readily detected tumors by MRI. We systematically show that a single gadolinium-chelate conjugated to hPL* allowed detection of PRLR⁺ OvCa tumors, likely due to a combination of high affinity of hPL to PRLR and high-expression levels of PRLR in OvCa. Although not directly tested in our work, we hypothesize that signal enhancement on T₁-weigted imaging may be augmented by the increased relaxivity of our agent in the intracellular environment, which is analogous to the liver-specific gadolinium chelates that are used clinically (41). Additionally, we speculate that hPL*-GdDTPA may have a higher relaxivity at 1.5 T or 3.0 T, magnetic field strengths used clinically, owing to the slower rotational correlation times of high molecular weight gadolinium chelates (42).

Our imaging platform achieved sufficient sensitivity to detect small tumors (10 mg) and metastatic OvCa, conferring nearly a 100-fold improvement in the OvCa detection threshold compared to GdDTPA, a contrast agent used in the clinic currently. Coupled with the expression of PRLR in early stage tumors (Table 1), our ability to image multiple human OvCa xenografts that express moderate to high levels of PRLR supports the potential for clinical translatability and possible improvement of the MRI detection sensitivity of human OvCa in patients.

As we proceed towards translating molecular PRLR imaging clinically, further optimization of hPL*-conjugates may be necessary. Testing the distribution and metabolism of hPL*-conjugates in non-murine and primate animal models may indicate necessary alterations of the protein, chelating moiety, or conjugation chemistry. Other design considerations include using antibody fragments that bind specifically to PRLR as designed by one of our laboratories (43) or increasing the gadolinium payload by coupling hPL* to particles carrying multiple gadolinium ions (unpublished data).

Taken together the PRLR targeted imaging platform may endow clinicians with a potential method to distinguish malignant from nonmalignant tissue with high specificity, engendering both a pre-operative diagnostic tool and a post-operative surveillance tool for tumor detection and recurrence that could reduce the large number of unnecessary surgeries. Internalization of folate bioconjugates via folate receptor mediated endocytosis has enabled detection of metastatic OvCas in humans undergoing reductive surgery (44), progressing beyond detection of tumors in animal models. Considering that PRLR upregulation occurs with greater frequency in OvCa than does folate receptor upregulation (>98% versus 72%, c.f. this work and reference 39), analogous approaches using PRLR mediated internalization of hPL bioconjugates could potentially be beneficial to a greater fraction of OvCa patients.

In summary, upregulation of PRLR in OvCa and its use through targeted molecular imaging sets the stage for advances in the non-invasive diagnosis and treatment of OvCa. In developing this technology further, it will be necessary to more fully define PRLR expression ranges with respect to OvCa and other gynecological pathologies. We envision adapting our approach to other imaging modalities such as positron emission tomography (PET) or combined approaches such as MRI/PET. We speculate that our PRLR imaging concept would confer similar advantages to diagnosis of breast cancers, which have strongly up-regulated PRLR expression compared to normal breast tissue (46). Beyond imaging, the capacity of PRLR targeting to internalize cargo in a cell-type specific manner may hold promise as a therapeutic delivery system.