2.1.

Cell Description

Human pancreatic cancer PANC1 cells were purchased from the American Type Culture Collection (ATCC; CRL1469) and grown in Dulbeco’s Modified Eagle Medium (DMEM) with $4.5\text{-}\mathrm{g}/\mathrm{L}$ glucose, l-glutamine, and sodium pyruvate supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. Human pancreatic hTERT-HPNE cells were purchased from ATCC (CRL4023) and grown in DMEM media with $4.5\text{-}\mathrm{g}/\mathrm{L}$ glucose, l-glutamine, and sodium pyruvate supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, and VEGF growth kit (ATCC; PCS100041). Immortalized PANC1 cells between 80 and 100 passages and primary hTERT-HPNE cells between 8 and 15 passages were used in the experiments. The PANC1s usually reached 90% to 100% confluency while the hTERT-HPNEs were about 50% confluent.

2.2.

Microbubbles Description

Because of the overexpression of plectin-1 (Plec1) receptors in pancreatic cancer cells (PANC1), a peptide ligand was developed and added to the microbubble formulation to selectively target the Plec1 receptor.^13,19 To make the targeting ligand, a peptide was covalently attached to the bis-palmitoyl lipid-like moiety via a short polyethyleneglycol spacer (extended span distance 140 Å). The specific peptide sequence was $\mathrm{Lys}-\mathrm{Thr}-\mathrm{Leu}-\mathrm{Leu}-\mathrm{Pro}-\mathrm{Thr}-\mathrm{Pro}-{\mathrm{NH}}_2$. The synthesis of the bioconjugate ligand was performed by solid-phase technology using a Fmoc/tBu protection strategy. The structure and the microbubbles are shown in Fig. 1.

Fig. 1

Targeted microbubbles (top left) were made with the ligand seen below it. Control bubbles (top right) were identical except lacking the targeting ligand. The diameter of the microbubbles generally ranged between 1 and $5\mu \mathrm{m}$. The ligand used was a Plec1 lipid-like bioconjugate. The Plec1 peptide ligand is covalently bound via an amide linkage to a PEGylated spander followed by linkage to diaminobutyrate and two palmitoyl fatty acids. The lipid-like end inserts into the membrane to form a targeted microbubble as demonstrated previously.²⁰

The composition used for the control microbubbles was 2 mol. % DDPE-PEG 2000 and 98 mol. % DPPC dispersed in a propylene glycol, normal saline, glycerol solution ($15:80:5$, $\mathrm{v}:\mathrm{v}:\mathrm{v}$). The composition used for the Plec1 targeted bubbles was 2 mol. % of the targeting ligand and 98 mol. % DPPC similarly dispersed in the propylene glycol, normal saline, and glycerol solution ($15:8:5$, $\mathrm{v}:\mathrm{v}:\mathrm{v}$). In addition, when needed, 0.2% concentration 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, Thermo Fisher, Carlsbad, California) (${\mathrm{Ex}}_{\max }=549\mathrm{nm}$ and ${\mathrm{Em}}_{\max }=565\mathrm{nm}$) was added as a fluorescent label. The total lipid concentration in all formulations was $1\mathrm{mg}/\mathrm{mL}$ and the solution was aliquoted in 1.5-mL increments into 2-mL glass vials, sealed with air-tight septums, and followed by purging with perfluorobutane gas (FluoroMed LP, Round Rock, Texas). The vial was then agitated using a modified dental amalgamator (Lantheus, New York, New York) to form the microbubbles used in imaging.

2.3.

Sample Preparation

Cells were seeded onto poly-d-lysine-coated glass bottomed dishes (MatTek, Ashland, Massachusetts) $\sim 24\mathrm{h}$ prior to imaging in order to ensure adherence. To prepare the cells for imaging, cells were incubated for 5 min with 5-mL media and stained with $5\text{-}\mu \mathrm{L}$ Calcein AM (Thermo Fisher Scientific, Carlsbad, California), followed by additional media and $200\mu \mathrm{L}$ of DiI microbubbles (0.2% concentration). The media cocktail was added to completely fill the dish and parafilm was used to seal it. Due to the buoyancy of the bubbles, the dish was inverted so the bubbles would be in contact with the cells. After incubating with microbubbles for 20 min, the dishes were rinsed twice with Dulbeco’s phosphate-buffered saline to remove any debris or unbound bubbles from the dish. DMEM media was then reintroduced to the dish during imaging. The fluorescent labels, Calcein AM and DiI, were used to confirm microbubbles binding to live cells in the two-photon excitation channel. Coregistration of microbubbles would be confirmed independently by two-photon excitation of the fluorescent labels and the THG signals of the microbubbles. The Calcein AM label, in addition to insuring cell viability, would also provide background contrast in the images to clearly indicate that the microbubbles were binding to the surface of the cells and not to the plate surface.

2.4.

Imaging and Microscope Description

Our multiphoton microscope was designed and built in-house²¹ and is controlled by Labview-based software developed in our research group. A schematic overview of the microscope system is shown in Fig. 2. We used a compact femtosecond fiber laser as the excitation light source. Since MPM is a point imaging technique, a pair of galvometric scan mirrors was used to raster the laser beam in both $X$- and $Y$-dimensions. A collimated laser beam was presented to this raster scanner, after which the beam was expanded to fill the back aperture of the objective. An afocal pair of lenses is used to expand the laser beam by approximately a factor of 4. This telescope setup also served to relay the scan mirror image onto the back aperture of the objective, ensuring that all the scan angles were supported by the imaging system without vignetting.

Fig. 2

Schematic of lab-built multiphoton microscope, configured for this project.

For this study, we imaged with a 1040-nm femtosecond fiber laser as the illumination source. The Zeiss Achroplan $40\times$ 0.75 numerical aperture (NA) water-immersion microscope objective was used to image the samples. The signal light returning from the sample was split into two channels by a 538-nm longpass dichroic mirror (Semrock), detected by Hamamatsu H-10721 Photomultiplier Tubes, and their signal amplified with Stanford Research SR570 preamplifiers. The transmitted channel had a 750-nm shortpass filter to remove any remaining 1040-nm light from reaching the detector, allowing the detection of two-photon-excited fluorescence (2PEF). The reflected channel had a 340-nm bandpass filter with a 22-nm full width at half maximum pass window to isolate the THG signal.

2.5.

Blinded Study Methodology

Four sets of experiments (one experimental and three controls) were conducted using the following in order to investigate the selectivity of the targeted microbubbles: (1) PANC1 incubated with targeted microbubbles (experimental), (2) PANC1 incubated with nontargeted microbubbles (control), (3) noncancerous ductal epithelial pancreatic cells (hTERT) incubated with targeted microbubbles (control), and (4) noncancerous pancreatic cells incubated with nontargeted microbubbles (control). Each set of experiments was performed at least 10 times to confirm the accuracy of the results. When imaging the cells, the researcher who performed the multiphoton imaging task was blinded as to which microbubbles were used in the sample being imaged in order to eliminate bias when observing and analyzing the images.

2.6.

Statistical Approach

In order to create a more quantitative assessment of the number of bubbles in each cell dish, we define a bubble index

4.1.

False Positives

It is noted that the targeting ligand for the microbubbles is highly selective; however, there is always the possibility of nonspecific binding to noncancerous cells. The possibility for bubbles to be trapped in tightly packed cells also leads to the chance for control bubbles to be seen in the cancerous cells. During our experiments, we occasionally saw microbubbles in small numbers binding in one of the three control experiments. We also discovered that it was possible for the bubbles to adhere to the bottom of the tray of the hTERT cells due to the poly-d-lysine coating at the bottom of the tray, leading to a few false positives. However, these bubbles were isolated in pockets of cells or adherent to the bottom of the cell culture plate. These effects were reduced by identifying only those microbubbles bound over the calcein background or directly on the fringe of the calcein fluorescence (i.e., a cell edge).

Proof of these results is shown in Fig. 8, where Fig. 8(a) shows hTERT cells with targeted bubbles, Fig. 8(b) shows PANC1 cells with targeted bubbles, and Fig. 8(c) shows a region in a targeted dish of hTERT cells with no cells in the field of view.

Fig. 8

(a) hTERT cells and targeted bubbles. (b) PANC1 cells and targeted bubbles. The bubbles bind directly to the cells in the PANC1 cells, as they appear next to the membrane. The bubbles in the hTERT dish were almost $10\mu \mathrm{m}$ away from the cell membrane. (c) Bubbles seen sticking to the poly-d-lysine coating with no cells present.

The difference could be seen in the distance between the bubbles between the two groups. In the inset for each cell set, it could be seen how the cells bound directly to the cells in the PANC1 set, as they appeared adjacent to the membrane. By contrast, the bubbles in the hTERT appeared $10\mu \mathrm{m}$ or more from the cells themselves, indicating they could not be binding to the cells, and must be adhering to the poly-d-lysine coating. As shown in Fig. 8(c), bubbles could even be occasionally found sticking to the poly-d-lysine coating with no cells present. Because this condition only exists in this experimental setting, this would probably not be an issue in a clinical setting. These bubbles were not counted for computing the bubble index for that set of images, because they were not binding to the cell itself.

4.2.

Discussion

The long-term focus of this project is to design a point-of-care methodology that can determine a cancer-free resection using targeted microbubbles. Because it is possible to miniaturize a multiphoton into a probe form factor,²⁸ these bubbles could be imaged in a surgical setting, allowing the physician to rapidly determine whether the resected margins are indeed cancer free. In addition, we have previously demonstrated the use of second-harmonic generation and THG as a method of label-free cancer determination in Barrett’s esophagus and ovarian cancer.^16,17 We plan to combine these two methods in the future to gather information both about the surface of a sample with the bubbles and potentially up to 1-mm deep through tissue to examine beneath the surgical margin. By adding the capability for a physician to look deeper through tissue, we believe that we can further increase confidence that the cancerous tissue has been removed, thereby increasing the probability of long-term survival of the patient. As an intermediate step, we will evaluate this technique in identifying cancer in samples of pancreatic tissue.

4.3.

Conclusion

In this study, we demonstrated a method of identifying cancerous cell lines using peptide ligands, lipid microbubbles, and MPM. We believe that this could lead to a fast and accurate method for examining the entire cut surface during surgery. This is in sharp contrast to the current state of the art, where long surgery times and small sampling area make survival less likely. We found on average that the bubbles appeared over 100 times more often in the cancerous cells compared to the healthy cells. Throughout the study, the bubbles bound to the cancer cells in higher frequency across the entire sample surface, clearly separating it from the noncancerous cell lines, which only occasionally showed bubbles in a few locations.