Metastasis is a complex multi-step process by which cancer cells disseminate from a primary site to distant organs. The transcription factor Twist has been shown to play a key role in metastasis in a mouse mammary tumor metastasis model. It has also been implicated in invasive human cancers and correlated with increased incidence of metastasis and poor disease progression in vivo. One cellular function of Twist that contributes to metastasis is to promote Epithelial-Mesenchymal-Transition, allowing cancer cells to lose cell to cell adhesion and gain motility. However, the role of Twist in other steps of the metastatic cascade has not yet been examined. My project focuses on the role of Twist in promoting tumor invasion and the formation of invasive cellular structures called invadopodia. Fluorescent gelatin degradation assays as well as immunofluorescence assays demonstrate that Twist is necessary and sufficient for local matrix degradation and invadopodia formation. It was unclear the mechanism by which Twist induced invadopodia formation. I hypothesize that Twist may regulate proteases that are associated with invadopodia, playing a key role in invadopodia formation and matrix degradation. My study showed that Twist expression specifically upregulated the expression of three proteases associated with invadopodia: ADAM12, FAPa, and DPPIV. I further investigated the role of ADAM12 studying matrix degradation and invadopodia formation. We found that expression of ADAM12 is necessary for proper matrix degradation and functional invadopodia formation. Together, these results indicate that induction of ADAM12 plays a critical role in Twist-induced matrix degradation and local invasion

Fluorescence-guided surgery can enhance the surgeon's ability to achieve a complete oncologic resection. There are a number of tumor-specific probes being developed with many preclinical mouse models to evaluate their efficacy. The current review discusses the different preclinical mouse models in the setting of probe evaluation and highlights the advantages of patient-derived orthotopic xenografts (PDOX) mouse models and genetic reporters to develop fluorescence-guided surgery.

BACKGROUND: Surgical orthotopic implantation of human colon cancer tissue to the ceca of mice has been used to mimic behavior of cancer in human patients for the development of precision cancer medicine. However, with the current method of serosal surface implantation (SSI) of pieces of human colon cancer tissue, cancer cells are exposed to the peritoneum, which can artificially increase the rate of peritoneal carcinomatosis (PC) during the disease course. The objective of the present study was to introduce a tumor-sealing method (TSM) and compare it with SSI for the ability to produce clinically-relevant metastases without artificial PC. MATERIALS AND METHODS: HCT116 colon cancer cells transfected with green fluorescence protein (GFP) were cultured and then injected into the subcutaneous layer of athymic nude mice. Subcutaneous tumors were allowed to grow sufficiently to supply adequate tumor for orthotopic implantation. For SSI, a 1 mm3-sized tumor fragment was sutured to partially torn serosa of the cecum. For TSM, the blind end of the cecum was folded over the tumor fragment and sealed with sutures. At 20 days after implantation, all mice were opened to visualize PC by intravital fluorescence imaging. At necropsy, distant metastasis was investigated using frozen section of whole blocks of organs. RESULTS: At 20 days after implantation, PC rates in the SSI group and the TSM group were 80% (12/15) and 20% (3/15), respectively (p<0.001). The liver metastasis rate was 41.7% (5/12) in the SSI group and 50% (5/10) in the TSM group (p=0.696). The lung metastasis rate was 0% (0/12) in the SSI group and 10% (1/10) in the TSM group (p=0.201). The mean survival of mice without PC on the 20th day was significantly longer than that of mice with PC on the 20th day (69.1±14.7 vs. 44.5±12.4 days, p=0.001). CONCLUSION: These results suggest that TSM might be a more patient-like and useful method as a model of metastatic colon cancer than SSI.

Mucins (MUC1-MUC24) are a family of glycoproteins involved in cell signaling and barrier protection. They have been implicated in the progression of numerous malignancies including gastric, pancreatic, ovarian, breast, and lung cancer. Mucins have also been extensively studied with respect to colorectal cancer. They have been found to have diverse expression profiles amongst the normal colon, benign hyperplastic polyps, pre-malignant polyps, and colon cancers. Those expressed in the normal colon include MUC2, MUC3, MUC4, MUC11, MUC12, MUC13, MUC15 (at low levels), and MUC21. Whereas MUC5, MUC6, MUC16, and MUC20 are absent from the normal colon and are expressed in colorectal cancers. MUC1, MUC2, MUC4, MUC5AC, and MUC6 are currently the most widely covered in the literature regarding their role in the progression from normal colonic tissue to cancer.

Colorectal cancer (CRC) is a common cause of cancer and cancer-related death. Surgery is the only curative modality. Fluorescence-enhanced visualization of CRC with targeted fluorescent probes that can delineate boundaries and target tumor-specific biomarkers can increase rates of curative resection. Approaches to enhancing visualization of the tumor-to-normal tissue interface are active areas of investigation. Nonspecific dyes are the most-used approach, but tumor-specific targeting agents are progressing in clinical trials. The present narrative review describes the principles of fluorescence targeting of CRC for diagnosis and fluorescence-guided surgery with molecular biomarkers for preclinical or clinical evaluation.

Tumor-specific targeting with fluorescent probes can enhance contrast for identification of cancer during surgical resection and visualize otherwise invisible tumor margins. Nanobodies are the smallest naturally-occurring antigen-binding molecules with rapid pharmacokinetics. The present work demonstrates the efficacy of a fluorescent anti-CEA nanobody conjugated to an IR800 dye to target and label patient derived pancreatic cancer xenografts. After intravenous administration, the probe rapidly localized to the pancreatic cancer tumors within an hour and had a tumor-to-background ratio of 2.0 by 3 h. The fluorescence signal was durable over a prolonged period of time. With the rapid kinetics afforded by fluorescent nanobodies, both targeting and imaging can be performed on the same day as surgery.

BACKGROUND: Our aim was to evaluate the ease and utility of using indocyanine green fluorescence angiography for intraoperative localization of the parathyroid glands. METHODS: Indocyanine green fluorescence angiography was performed during 60 parathyroidectomies for primary hyperparathyroidism during a 22-month period. Indocyanine green was administered intravenously to guide operative navigation using a commercially available fluorescence imaging system. Video files were graded by 3 independent surgeons for strength of enhancement using an adapted numeric scoring system. RESULTS: There were 46 (77%) female patients and 14 (23%) male patients whose ages ranged from 17 to 87 (average 60) years old. Of the 60 patients, 43 (71.6%) showed strong enhancement, 13 (21.7%) demonstrated mild to moderate vascular enhancement, and 4 (6.7%) exhibited little or no vascular enhancement. Of the 54 patients who had a preoperative sestamibi scan, a parathyroid adenoma was identified in 36, while 18 failed to localize. Of the 18 patients who failed to localize, all 18 patients (100%) had an adenoma that fluoresced on indocyanine green imaging. The operations were performed safely with minimal blood loss and short operative times. CONCLUSION: Indocyanine green angiography has the potential to assist surgeons in identifying parathyroid glands rapidly with minimal risk.

Background/purpose

Development of a humanized fluorophore-conjugated antibody that can improve contrast for fluorescence-guided oncologic surgeries.

Methods

BxPC-3-GFP pancreatic cancer cells were injected into flanks of nude mice. Fragments of subcutaneous tumors were grafted onto the pancreatic tail of recipient mice to create orthotopic xenograft models of pancreatic cancer. After tumors developed for 4 weeks, a humanized anti-carcinoembryonic antigen antibody conjugated to an 800 nm near-infrared fluorescent dye (hM5A-IR800) was injected intravenously. Mice were imaged at 6, 12, 24, 48, and 72 h after injection.

Results

Fluorescence imaging showed that hM5A-IR800 specifically localized to BxPC-3 human pancreatic cancer cells. The fluorescent probe localized to cell surfaces in vitro and specifically co-localized with green fluorescent protein-labeled tumors in an orthotopic pancreatic xenograft model in vivo. Serial imaging at specific time points showed peak signal intensity of the orthotopic pancreatic tumor at 48 h; this time point corresponded with a maximal tumor-to-background ratio (TBR) of 16.6 at 48 h.

Discussion

hM5A-IR800 was successfully able to specifically label orthotopic pancreatic tumors in situ. The longer wavelength allowed deeper tissue penetration, particularly in tumor areas covered by normal pancreatic parenchyma. The probe had expected kinetics for an antibody-fluorophore conjugate, with the peak signal intensity reached at 48 h. A clear tumor signal was observed with a TBR > 5 at all time points, with high contrast (TBR of 16.6) at 48 h.

Conclusion

hM5A-IR800 demonstrated excellent tumor localization and a very bright signal. It is a promising agent for future clinical fluorescence-guided surgery applications.

Introduction

Claudins are tight-junction proteins, which maintain an epithelial barrier in normal colon cells. Overexpression of Claudin-1 has been implicated for development of colon cancer. We postulated that Claudin-1 may be a useful target in near-infrared imaging and fluorescence-guided surgery.

Methods

We conjugated Claudin-1 antibody to LI-COR IR800DyeCW (Claudin-1-IRDye800CW). Western blotting of 9 human colon cancer cell lysates was performed. Animal imaging was performed with the LI-COR Pearl Trilogy Fluorescence Imaging System. A dose-response study was carried out with subcutaneous LS174T colon cancer cell line models. Increasing doses of Claudin-1-IRDye800CW via tail vein injection were administered to three groups of mice. Two groups of mice were used as controls (antibody alone, and dye alone). In vivo imaging was performed at 24, 48, and 72 h after administration of the conjugated dye. Orthotopic implantation of patient-derived tumors and cell lines was performed and peritoneal carcinomatosis models were created. After tumor growth, mice were administered Claudin-1-IRDye800CW and imaged in vivo 48 h later. The mice were euthanized and laparotomy was performed to assess internal organs and toxicity.

Results

Western blotting revealed that all colon cancer cell lysates expressed varying amounts of Claudin-1. All tumors demonstrated strong and specific fluorescence labeling at 800 nm, even with the lowest dose of 12.5 μg of Claudin-1-IRDye800CW.

Conclusions

Claudin-1 is a useful target for near-infrared antibody-based imaging for visualization of colorectal tumors for future use in fluorescence-guided surgery.

A major barrier to the diagnosis and effective treatment of solid-tumor cancers is the difficulty in detection and visualization of tumor margins in primary and metastatic disease. The use of fluorescence can augment the surgeon's ability to detect cancer and aid in its resection. Several cancer types express carcinoembryonic antigen (CEA) including colorectal, pancreatic and gastric cancer. Antibodies to CEA have been developed and tagged with near-infrared fluorescent dyes. This review article surveyed the use of CEA antibodies conjugated to fluorescent probes for in vivo studies since 1990. PubMed and Google Scholar databases were queried, and 900 titles and abstracts were screened. Fifty-nine entries were identified as possibly meeting inclusion/exclusion criteria and were reviewed in full. Forty articles were included in the review and their citations were screened for additional entries. A total of 44 articles were included in the final review. The use of fluorescent anti-CEA antibodies has been shown to improve detection and resection of tumors in both murine models and clinically. The cumulative results indicate that fluorescent-conjugated anti-CEA antibodies have important potential to improve cancer diagnosis and surgery. In an emerging technology, anti-CEA fluorescent antibodies have also been successfully used for photoimmunotherapy treatment for cancer.