Development and evaluation of a CEACAM6-targeting theranostic nanomedicine for photoacoustic-based diagnosis and chemotherapy of metastatic cancer

Abstract

Metastasis is the leading cause of cancer-related deaths. A number of chemotherapeutic and early diagnosis strategies, including nanomedicine, have been developed to target metastatic tumor cells. However, simultaneous inhibition and imaging of metastasis is yet to be fully achieved. Methods: To overcome this limitation, we have developed human serum albumin-based nanoparticles (tHSA-NPs) with photoacoustic imaging capability, which target carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6). CEACAM6 is highly expressed in metastatic anoikis-resistant tumor cells. Results:In vitro, the CEACAM6-targeting tHSA-NPs efficiently targeted CEACAM6-overexpressing metastatic anoikis-resistant tumor cells. In vivo, CEACAM6-targeting tHSA-NPs administered intravenously to BALB/c nude mice efficiently inhibited lung metastasis in circulating anoikis-resistant tumor cells compared to the controls. In addition, anoikis-resistant tumor cells can be successfully detected by photoacoustic imaging, both in vitro and in vivo, using the intrinsic indocyanine green-binding affinity of albumin. Conclusion: In summary, the CEACAM6-targeting albumin-based nanoparticles allowed the delivery of drugs and photoacoustic imaging to metastatic anoikis-resistant tumor cells in vitro and in vivo. Based on the expression of CEACAM6 in a variety of tumors, CEACAM6-targeting nanomedicine might be used to target various types of metastatic tumor cells.

Keywords: CEACAM6; anoikis resistance; metastasis; nanomedicine; photoacoustic imaging.

Figure 1

Schematic illustration of a CEACAM6-targeting nanomedicine for simultaneous chemotherapy and photoacoustic imaging of metastatic tumor cells. The intravenously administered CEACAM6-targeting albumin-based nanomedicine can target blood-circulating anoikis-resistant (AR) metastatic tumor cells. Encapsulated doxorubicin (DOX) and indocyanine green (ICG) exhibit inhibition and photoacoustic-based diagnosis of tumor metastasis, respectively.

Figure 2

Characteristics of tHSA-NPs with DOX and ICG. (A) Average size and transmission electron micrograph. (B) In vitro release pattern of DOX-loaded tHSA-NPs (D@NPs). (C) Optical absorption spectrum of ICG-conjugated tHSA-NPs (I@NPs) with control groups. (D) Photoacoustic signal intensities of tHSA-NPs and I@NPs with control groups. L7_4 transducer (focal depth: 3 cm) was used to obtain in vitro photoacoustic images.

Figure 3

(A) Illustration of CEACAM6 antibody conjugation onto the surface of tHSA-NPs. (B-C) Size distribution and immunofluorescence patterns with secondary antibody of nanoparticles before and after antibody conjugation.

Figure 4

(A) CEACAM6 overexpression in AR tumor cells (MCF-7, MDA-MB-231 and A549). Immunofluorescence of CEACAM6 protein. (B-C) CEACAM6 mRNA expressions in three cell lines and its quantification from RT-PCR and real-time RT-PCR. (D) CEACAM6 protein expression lasted for 3 days after re-attachment of AR tumor cells in all cell lines.

Figure 5

(A) Cellular targeting of CEACAM6-targeting tHSA-NPs (C6-NPs) to A549 adherent and A549 AR cells during shaking for 30 min. Cell nucleus, F-actin and nanoparticles are shown as blue, green, and red, respectively. The rightmost column depicts merged images of all. (B) Flow cytometry data for quantification of cellular targeting in the same condition.

Figure 6

Cell viability assay with parental A549 adherent and A549 AR cells. Cells were treated at a concentration corresponding to 100 nM DOX for 6 h under (A) static and (B) shaking condition, and further cultured up to 72 h in fresh medium. Bar with dense line and sparse line represent A549 adherent and A549 AR cells, respectively. Asterisk (*) denotes p<0.05 between two groups.

Figure 7

(A-B) In vivo metastasis inhibition of D@C6-NPs and other groups. Right after the intravenous injection of A549 AR cells, the first therapy was conducted. After 3 days, the second therapy was performed. Metastatic nodules in BALB/c nude mouse lungs were photographed and the number of metastatic nodules was quantified. D@C6-NPs group displayed statistical significance (ANOVA, p<0.01). (C) Body weights of mice were recorded at the indicated times. (D) H&E analysis of major organs in control and D@C6-NPs groups.

Figure 8

(A-B) In vitro and in vivo photoacoustic contrast ability of I@C6-NPs. Photoacoustic signals from A549 AR cells after 6 h incubation with I@NPs and I@C6-NPs under shaking conditions and the quantification data. L7_4 transducer (focal depth: 3 cm) was used to obtain ex vivo photoacoustic images. (C) In vivo PA images of metastasized lung clearly showed a PA signal was detected when I@C6-NPs were intravenously injected compared to I@NPs. VisualSonics Vevo LAZR-2100 device equipped with a 40-MHz transducer was used to obtain in vivo photoacoustic images. (D) PA signals inside the lung were quantified (ANOVA, p<0.01). (E) Ex vivo PA imaging of extracted lungs 6 h after the injection of I@NPs and I@C6-NPs. L7_4 transducer was used to obtain ex vivo photoacoustic images.