Circulating cell-free DNA methylation patterns as non-invasive biomarkers to monitor colorectal cancer treatment efficacy without referencing primary site mutation profiles.

This study investigates methylation patterns in circulating cell-free DNA (ccfDNA) for their potential role in colorectal cancer (CRC) detection and the monitoring of treatment response. Through methylation microarrays and quantitative PCR assays, we analyzed 440 samples from The Cancer Genome Atlas (TCGA) and an additional 949 CRC samples. We detected partial or extensive methylation in over 85% of cases within three biomarkers: EFEMP1, SFRP2, and UNC5C. A methylation score for at least one of the six candidate regions within these genes' promoters was present in over 95% of CRC cases, suggesting a viable detection method. In evaluating ccfDNA from 97 CRC patients and 62 control subjects, a difference in methylation and recovery signatures was observed. The combined score, integrating both methylation and recovery metrics, showed high diagnostic accuracy, evidenced by an area under the ROC curve of 0.90 (95% CI = 0.86 to 0.94). While correlating with tumor burden, this score gave early insight into disease progression in a small patient cohort. Our results suggest that DNA methylation in ccfDNA could serve as a sensitive biomarker for CRC, offering a less invasive and potentially more cost-effective approach to augment existing cancer detection and monitoring modalities, possibly supporting comprehensive genetic mutation profiling.

Prognostic factors for gastric cancer patients aged ≥ 85 years.

BACKGROUND: As gastric cancer patients aged ≥ 85 years have a short life expectancy and often die from other diseases such as pneumonia, indications for surgery are controversial. In this study, we retrospectively analyzed the prognostic factors of elderly patients with gastric cancer who are candidates for curative gastrectomy. METHODS: Among 114 patients aged ≥ 85 years with gastric cancer at our hospital between 2010 and 2019, prognostic factors were examined using the Cox proportional hazards model in 76 patients excluding those with cStage IVB or endoscopic submucosal dissection. We also analyzed the factors of pneumonia death. RESULTS: cStage was I/IIA/IIB/III/IVA in 37/6/14/14/5 patients, respectively. Treatment included distal gastrectomy in 28 patients, total gastrectomy in 6, local resection in 9, others in 3, and no surgery in 30. In univariate analyses of overall survival, Eastern Cooperative Oncology Group Performance Status, physiological score of Physiological and Operative Severity Score for the enUmeration of Mortality and morbidity (POSSUM), Onodera's prognostic nutritional index, cStage, and treatment were prognostic factors. In a multivariate analysis, POSSUM physiological score, cStage, treatment method {no surgery vs. distal gastrectomy: hazard ratio (HR) 5.78, 95% confidence interval (CI) 2.33-14.3}, (total gastrectomy vs. distal gastrectomy: HR 4.26, 95% CI 1.22-14.9) were independent prognostic factors. In univariate analyses of pneumonia-specific survival, treatment (total gastrectomy vs. distal gastrectomy: HR 6.98, 95% CI 1.18-41.3) was the only prognostic factor. CONCLUSIONS: The prognosis of distal gastrectomy was better than that of non-surgery even in patients aged ≥ 85 years. However, total gastrectomy was considered to be avoidable due to the high rate of postoperative pneumonia death.

Genomically Stable Gastric Cancer Characterized by Hypomethylation in Wnt Signal Cascade.

INTRODUCTION: Gastric cancer is divided into four subtypes by their molecular features linked with genetic alterations, e.g., Epstein-Barr virus (EBV), microsatellite instability-high (MSI-high), chromosomal instability (CIN), and genomically stable (GS), called as TCGA classification. In this study, we tried to clarify the epigenetic features of the four GC subtypes according to aberrant methylation status in 23 loci. METHODS: A total of 98 gastric cancers and their normal gastric mucosa samples were included in this study. We divided gastric cancers into TCGA subtypes which were determined in line with MSI-high, EBV, CIN, to GS by their molecular features. The 13 loci of polymorphic microsatellite sequences were used to determine loss of heterogeneity for the detection of CIN. The MSI status was determined by three mononucleotide repeat markers. Infection of EBV was determined by recovering EBV BNRF1 sequence from genomic DNA collected from gastric cancers. Methylation status of 23 loci was investigated by the combined bisulfite restriction analysis. Status of other findings, e.g., KRAS mutations, HER2 expression status, and infection of helicobacter pylori were confirmed. RESULTS: Gastric cancers were divided into MSI (13%), EBV (7%), CIN (53%), and GS (27%). By histological classification, poorly differentiated adenocarcinoma was more in tumors categorized in MSI-high, and GS and signet-ring cell carcinoma (sig) were more in GS. Among the 23 loci investigated their methylation status, 18 loci were significantly hypermethylated in caner tissues. An unsupervised clustering divided gastric cancers into two clusters and revealed that most GS tumors clustered together in a cluster that exhibited lower methylation levels, distinct from the other subtypes. The inter-variable clustering revealed that a cluster contained the three loci (SFRP2-region 1/2 and APC) belonging to the Wnt signal cascade (Wnt-associated loci). The mean methylation score of Wnt-associated loci was the lowest in GS tumors (MSI-high: 2.7 [95% confidence interval, 2.3-2.9]; EBV: 2.1 [1.2-3.1]; CIN: 2.4 [2.2-2.7]; GS: 1.3 [0.8-0.7]). In contrast, the mean methylation score of the other 15 loci was significantly higher in MSI-high, while that in GS was as same as that in EBV or CIN (MSI-high: 10.4 [8.3-12.4]; EBV: 5.7 [1.7-9.7]; CIN: 4.4 [3.6-5.1]; GS: 3.4 [2.2-4.6]). Additionally, the lower methylation score of Wnt-associated loci was observed only in sig tumors. CONCLUSIONS: GS subtype tumors have the potential to possess distinct signatures in DNA hypomethylation profiles in Wnt signaling pathway, especially in sig.

Regulatory T cells induce a suppressive immune milieu and promote lymph node metastasis in intrahepatic cholangiocarcinoma.

BACKGROUND: Emerging evidence indicates that immunogenicity plays an important role in intrahepatic cholangiocarcinoma (ICC). Herein, we systematically evaluated the clinical relevance of immunogenicity in ICC. METHODS: Highly immunogenic ICCs identified in the public dataset and the Cancer Immunome Atlas (TCIA) were assessed to determine the prognostic impact of immunogenicity in ICC and key components after curative resection. We also investigated the clinical relevance of the immune milieu in ICC. RESULTS: Using the Gene Expression Omnibus dataset 89749 and TCIA, we identified CD8+/forkhead box P3 (FoxP3)+ tumour-infiltrating lymphocytes (TILs), T-cell immunoglobulin and mucin domain 3 (TIM-3) and human leukocyte antigen-A (HLA-A) in highly immunogenic ICCs. Immunohistochemical analysis of the in-house cohort showed that intratumoral FoxP3+ TILs correlated with CD8+ TILs (P = 0.045, Fisher's exact test) and that high FoxP3+/CD8+ ratio (FCR) was an important marker for poor survival (P < 0.001, log-rank test). Furthermore, the FCR was higher in tumour-free lymph nodes in ICCs with lymph node metastases than in those without lymph node metastases (P = 0.003, Mann-Whitney U test). CONCLUSIONS: FCR should be considered an important biomarker that represents the immune environment of ICC based on its potentially important role in tumour progression, especially lymph node metastasis.

Long-term Survival with a Rare Advanced Primary Gastrointestinal Malignant Melanoma Treated with Laparoscopic Surgery/Immune Checkpoint Inhibitor.

Targeted therapies for malignant melanoma have improved patients' prognoses. A primary gastrointestinal malignant melanoma is very rare, with no standard treatment strategy. We treated a 78-year-old Japanese female with advanced primary gastrointestinal melanoma of the descending colon and gallbladder. We administered a multidisciplinary treatment: surgical resection of the descending colon and gallbladder tumors, resection of the metastatic lymph nodes behind the pancreas head, and immune checkpoint antibody-blockade therapy (nivolumab) for ~4 years. PET/CT demonstrated no recurrent lesion for > 3 years. Multidisciplinary therapies (e.g., surgery, chemotherapy, radiotherapy, target therapy, and immune checkpoint antibody-blockade therapy) can successfully treat primary gastrointestinal malignant melanoma.

Real-Time Fluorescence Image-Guided Oncolytic Virotherapy for Precise Cancer Treatment.

Oncolytic virotherapy is one of the most promising, emerging cancer therapeutics. We generated three types of telomerase-specific replication-competent oncolytic adenovirus: OBP-301; a green fluorescent protein (GFP)-expressing adenovirus, OBP-401; and Killer-Red-armed OBP-301. These oncolytic adenoviruses are driven by the human telomerase reverse transcriptase (hTERT) promoter; therefore, they conditionally replicate preferentially in cancer cells. Fluorescence imaging enables visualization of invasion and metastasis in vivo at the subcellular level; including molecular dynamics of cancer cells, resulting in greater precision therapy. In the present review, we focused on fluorescence imaging applications to develop precision targeting for oncolytic virotherapy. Cell-cycle imaging with the fluorescence ubiquitination cell cycle indicator (FUCCI) demonstrated that combination therapy of an oncolytic adenovirus and a cytotoxic agent could precisely target quiescent, chemoresistant cancer stem cells (CSCs) based on decoying the cancer cells to cycle to S-phase by viral treatment, thereby rendering them chemosensitive. Non-invasive fluorescence imaging demonstrated that complete tumor resection with a precise margin, preservation of function, and prevention of distant metastasis, was achieved with fluorescence-guided surgery (FGS) with a GFP-reporter adenovirus. A combination of fluorescence imaging and laser ablation using a KillerRed-protein reporter adenovirus resulted in effective photodynamic cancer therapy (PDT). Thus, imaging technology and the designer oncolytic adenoviruses may have clinical potential for precise cancer targeting by indicating the optimal time for administering therapeutic agents; accurate surgical guidance for complete resection of tumors; and precise targeted cancer-specific photosensitization.

Enhanced Metastatic Recurrence Via Lymphatic Trafficking of a High-Metastatic Variant of Human Triple-Negative Breast Cancer After Surgical Resection in Orthotopic Nude Mouse Models.

We previously developed and characterized a highly invasive and metastatic triple-negative breast cancer (TNBC) variant by serial orthotopic implantation of MDA-MB-231 human breast cancer cells in nude mice. Eventually, a highly invasive and metastatic variant of human TNBC was isolated after lymph node metastases was harvested and orthotopically re-implanted into the mammary gland of nude mice for two cycles. The variant thereby isolated is highly invasive in the mammary gland and metastasized to lymph nodes in 10 of 12 mice compared to 2 of 12 of the parental cell line. In the present report, we observed that high-metastatic MDA-MB-231H-RFP cells produced significantly larger subcutaneous tumors compared with parental MDA-MB-231 cells in nude mice. Extensive lymphatic trafficking by high-metastatic MDA-MB-231 cells was also observed. High-metastatic MDA-MB-231 developed larger recurrent tumors 2 weeks after tumor resection compared with tumors that were not resected in orthotopic models. Surgical resection of the MDA-MB-231 high-metastatic variant primary tumor in orthotopic models also resulted in rapid and enhanced lymphatic trafficking of residual cancer cells and extensive lymph node and lung metastasis that did not occur in the non-surgical mice. These results suggest that surgical resection of high metastatic TNBC can greatly increase the malignancy of residual cancer. J. Cell. Biochem. 118: 559-569, 2017. © 2016 Wiley Periodicals, Inc.

Therapeutic Cell-Cycle-Decoy Efficacy of a Telomerase-Dependent Adenovirus in an Orthotopic Model of Chemotherapy-Resistant Human Stomach Carcinomatosis Peritonitis Visualized With FUCCI Imaging.

We have established an orthotopic nude-mouse model of gastric cancer carcinomatosis peritonitis, a recalcitrant disease in human patients. Human MKN45 poorly-differentiated human gastric cancer cells developed carcinomatosis peritonitis upon orthotopic transplantation in nude mice. The MKN45 cells expressed the fluorescent ubiquitination-based cell cycle indicator (FUCCI) that color codes the phases of the cell cycle. The intra-peritoneal tumors and ascites contained mostly quiescent G1 /Go cancer cells visualized as red by FUCCI imaging. Cisplatinum (CDDP) treatment did not reduce bloody ascites, and larger tumors formed in the peritoneal cavity after CDDP treatment in an early-stage carcinomatosis peritonitis orthotopic mouse model. Paclitaxel-treated mice had reduced ascites, but also had large tumor masses in the peritonium after treatment with cancer cells mostly in G0 /G1 , visualized by FUCCI red. In contrast, OBP-301 telomerase-dependent adenovirus-treated mice had no ascites and only small tumor nodules consisting of cancer cells mostly in S/G2 phases in the early-stage carcinomatosis peritonitis model, visualized by FUCCI green. Furthermore, OBP-301 significantly reduced the size of tumors (P < 0.01) and ascites even in a late-stage carcinomatosis peritonitis model. These results suggest that quiescent peritoneally-disseminated gastric cancer cells are resistant to conventional chemotherapy, but OBP-301 significantly reduced the weight of the tumors and increased survival, suggesting clinical potential. J. Cell. Biochem. 118: 3635-3642, 2017. © 2016 Wiley Periodicals, Inc.

Use of αv Integrin Linked to GFP to Image Molecular Dynamics in Trafficking Cancer-Cell Emboli.

Human osteosarcoma cells with an αv integrin green fluorescent protein (GFP) fusion gene were previously established and imaged in vitro and in vivo. In the present study, we imaged the interaction of αv integrin-GFP in osteosarcoma cells and collagen fibers in vascular-trafficking osteosarcoma emboli in nude mice. Human 143B osteosarcoma cells, expressing αv integrin-GFP, were injected by a vascular route in an abdominal skin flap in nude mice. Osteosarcoma cells were fluorescently imaged in the epigastric cranialis vein in the abdominal skin flap by confocal microscopy. Collagen fibers were imaged in reflectance mode. At early stages of tumor embolus-formation, cancer cells adhered firmly to each other, diffusely expressing αv integrin-GFP. Two weeks after injection, collagen fiber scaffolds were visualized at the margins of tumor emboli or within them. Four weeks after injection, cancer cells invading from emboli were strongly expressing αv integrin-GFP, and were aligned along collagen fibers. The results suggest αv integrin and collagen fiber scaffolds are important for tumor embolus formation, which are potential seeds of metastasis. J. Cell. Biochem. 118: 26-30, 2017. © 2016 Wiley Periodicals, Inc.

Real-Time In Vivo Confocal Fluorescence Imaging of Prostate Cancer Bone-Marrow Micrometastasis Development at the Cellular Level in Nude Mice.

In the present report, we demonstrate in vivo fluorescence imaging of bone-marrow micrometastasis of prostate cancer at the cellular level in nude mice. PC-3 human prostate cancer cells labeled with green fluorescent protein (GFP) or red fluorescent protein (RFP) were injected into the left ventricle or intratibial bone marrow of nude mice. PC-3-GFP, as well as selected high metastatic variants of PC-3-GFP, PC-3-GFP-BM6 or PC-3-RFP were visualized by real-time fluorescence imaging, to traffic and grow in the bone marrow. Formation of bone marrow micrometastasis could be imaged at the single-cell level in live mice, using confocal microscopy. The ability to track bone marrow micrometastasis in real time at the cellular level provides a visual target for evaluating new therapeutics for this recalcitrant disease. J. Cell. Biochem. 117: 2533-2537, 2016. © 2016 Wiley Periodicals, Inc.

Experimental Curative Fluorescence-guided Surgery of Highly Invasive Glioblastoma Multiforme Selectively Labeled With a Killer-reporter Adenovirus.

Fluorescence-guided surgery (FGS) of cancer is an area of intense current interest. However, although benefits have been demonstrated with FGS, curative strategies need to be developed. Glioblastoma multiforme (GBM) is one of the most invasive of cancers and is not totally resectable using standard bright-light surgery (BLS) or current FGS strategies. We report here a curative strategy for FGS of GBM. In this study, telomerase-dependent adenovirus OBP-401 infection brightly and selectively labeled GBM with green fluorescent protein (GFP) for FGS in orthotopic nude mouse models. OBP-401-based FGS enabled curative resection of GBM without recurrence for at least 150 days, compared to less than 30 days with BLS.

Real-time fluorescence imaging of the DNA damage repair response during mitosis.

The response to DNA damage during mitosis was visualized using real-time fluorescence imaging of focus formation by the DNA-damage repair (DDR) response protein 53BP1 linked to green fluorescent protein (GFP) (53BP1-GFP) in the MiaPaCa-2(Tet-On) pancreatic cancer cell line. To observe 53BP1-GFP foci during mitosis, MiaPaCa-2(Tet-On) 53BP1-GFP cells were imaged every 30 min by confocal microscopy. Time-lapse imaging demonstrated that 11.4 ± 2.1% of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells had increased focus formation over time. Non-mitotic cells did not have an increase in 53BP1-GFP focus formation over time. Some of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells with focus formation became apoptotic. The results of the present report suggest that DNA strand breaks occur during mitosis and undergo repair, which may cause some of the mitotic cells to enter apoptosis in a phenomenon possibly related to mitotic catastrophe.

Osteosarcoma cells enhance angiogenesis visualized by color-coded imaging in the in vivo Gelfoam® assay.

We previously described a color-coded imaging model that can quantify the length of nascent blood vessels using Gelfoam® implanted in nestin-driven green fluorescent protein (ND-GFP) nude mice. In ND-GFP mice, nascent blood vessels are labeled with GFP. We report here that osteosarcoma cells promote angiogenesis in the Gelfoam® angiogenesis assay in ND-GFP mice. Gelfoam® was initially transplanted subcutaneously in the flank of transgenic ND-GFP nude mice. Seven days after transplantation of Gelfoam®, skin flaps were made and human 143B osteosarcoma cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in cytoplasm were injected into the transplanted Gelfoam®. The control-group mice had only implanted Gelfoam®. Skin flaps were made at days 14, 21, and 28 after transplantation of the Gelfoam® to allow imaging of vascularization in the Gelfoam® using a variable-magnification small animal imaging system and confocal fluorescence microscopy. ND-GFP expressing nascent blood vessels penetrated and spread into the Gelfoam® in a time-dependent manner in both control and osteosarcoma-implanted mice. ND-GFP expressing blood vessels in the Gelfoam® of the osteosarcoma-implanted mice were associated with the cancer cells and larger and longer than in the Gelfoam®-only implanted mice (P < 0.01). The results presented in this report demonstrate strong angiogenesis induction by osteosarcoma cells and suggest this process is a potential therapeutic target for this disease.

3-dimensional tissue is formed from cancer cells in vitro on Gelfoam®, but not on Matrigel™.

Cell and tissue culture can be performed on different substrates such as on plastic, in Matrigel™, and on Gelfoam(®), a sponge matrix. Each of these substrates consists of a very different surface, ranging from hard and inflexible, a gel, and a sponge-matrix, respectively. Folkman and Moscona found that cell shape was tightly coupled to DNA synthesis and cell growth. Therefore, the flexibility of a substrate is important for cells to maintain their optimal shape. Human osteosarcoma cells, stably expressing a fusion protein of α(v) integrin and green fluorescent protein (GFP), grew as a simple monolayer without any structure formation on the surface of a plastic dish. When the osteosarcoma cells were cultured within Matrigel™, the cancer cells formed colonies but no other structures. When the cancer cells were seeded on Gelfoam(®), the cells formed three-dimensional tissue-like structures. The behavior of 143B osteosarcoma cells on Gelfoam(®) in culture is remarkably different from those of these cells in monolayer culture or in Matrigel™. Tissue-like structures were observed only in Gelfoam(®) culture. The data in this report suggest a flexible structural substrate such as Gelfoam(®) provides a more in vivo-like culture condition than monolayer culture or Matrigel(TM) and that Matrigel(TM) does not result in actual three-dimensional culture.

Efficacy of tumor-targeting Salmonella typhimurium A1-R on nude mouse models of metastatic and disseminated human ovarian cancer.

We report here the efficacy of tumor-targeting Salmonella typhimurium A1-R (A1-R) on mouse models of disseminated and metastatic ovarian cancer. The proliferation-inhibitory efficacy of A1-R on human ovarian cancer cell lines (SKOV-3-GFP, OVCAR-3-RFP) was initially demonstrated in vitro. Orthotopic and dissemination mouse models of ovarian cancer were made with the human ovarian cancer cell line SKOV-3-GFP. After tumor implantation, the mice were treated with A1-R (5 × 10(7) colony-forming units [CFU], i.v.), and there were no severe adverse events observed. In the orthotopic model, tumor volume after treatment was 276 ± 60.8 mm(3), compared to 930 ± 342 mm(3) in the untreated control group (P = 0.022). There was also a significant difference in survival between treated mice and untreated mice in a peritoneal dissemination model (P = 0.005). The results of this report demonstrate that A1-R is effective for highly aggressive human ovarian cancer in metastatic and dissemination mouse models and suggest its clinical potential for this highly treatment-resistant disease.

Comparison of UVB and UVC effects on the DNA damage-response protein 53BP1 in human pancreatic cancer.

We have previously demonstrated that ultraviolet (UV) light is effective against a variety of cancer cells expressing fluorescent proteins in vivo as well as in vitro. In the present report, we compared the DNA damage repair (DDR) response of pancreatic cancer cells after UVB or UVC irradiation. The UV-induced DNA damage repair was imaged with green fluorescent protein (GFP) fused to the DDR-related chromatin-binding protein 53BP1 in MiaPaCa-2 human pancreatic cancer cells growing in 3D Gelfoam® histoculture and in superficial tumors grown in nude mice. 53BP1-GFP forms foci during DNA damage repair. A clonogenic assay in 2D monolayer culture initially showed that UVC and UVB inhibited MiaPaCa-2 cell proliferation in a dose-dependent manner, with UVC having more efficacy. Three-dimensional Gelfoam® histocultures and confocal imaging enabled 53BP1-GFP foci to be observed within 1 h after UV irradiation, indicating the onset of DDR response. UVB-induced 53BP1-GFP focus formation was observed up to a depth of 120 µm in MiaPaCa-2 cells on Gelfoam® compared to 80 µm for UVC. UVB-induced 53BP1-GFP focus formation was observed up to a depth of 80 µm in MiaPaCa-2 cells, implanted within skin flaps in mice, at a significantly greater extent than UVC. MiaPaCa-2 cells irradiated by UVB or UVC in the skin-flap mouse model had a significant decrease in tumor growth compared to untreated controls with UVB having more efficacy than UVC. Our results demonstrate that UVB has greater tissue penetration than UVC because of its longer wavelength and has clinical potential for eradicating superficial cancer.

Efficacy of Salmonella typhimurium A1-R versus chemotherapy on a pancreatic cancer patient-derived orthotopic xenograft (PDOX).

The aim of this study is to determine the efficacy of tumor-targeting Salmonella typhimurium A1-R (A1-R) on pancreatic cancer patient-derived orthotopic xenografts (PDOX). The PDOX model was originally established from a pancreatic cancer patient in SCID-NOD mice. The pancreatic cancer PDOX was subsequently transplanted by surgical orthotopic implantation (SOI) in transgenic nude red fluorescent protein (RFP) mice in order that the PDOX stably acquired red fluorescent protein (RFP)-expressing stroma for the purpose of imaging the tumor after passage to non-transgenic nude mice in order to visualize tumor growth and drug efficacy. The nude mice with human pancreatic PDOX were treated with A1-R or standard chemotherapy, including gemcitabine (GEM), which is first-line therapy for pancreatic cancer, for comparison of efficacy. A1-R treatment significantly reduced tumor weight, as well as tumor fluorescence area, compared to untreated control (P = 0.011), with comparable efficacy of GEM, CDDP, and 5-FU. Histopathological response to treatment was defined according to Evans's criteria and A1-R had increased efficacy compared to standard chemotherapy. The present report is the first to show that A1-R is effective against a very low-passage patient tumor, in this case, pancreatic cancer. The data of the present report suggest A1-1 will have clinical activity in pancreatic cancer, a highly lethal and treatment-resistant disease and may be most effectively used in combination with other agents.

Fluorescence-guided surgery of prostate cancer bone metastasis.

BACKGROUND: The aim of this study is to investigate the effectiveness of fluorescence-guided surgery (FGS) of prostate cancer experimental skeletal metastasis. MATERIALS AND METHODS: Green fluorescent protein-expressing PC-3 human prostate cancer cells (PC-3-green fluorescent protein) were injected into the intramedullary cavity of the tibia in 32 nude mice. After 2 wk, 16 of the mice underwent FGS; the other 16 mice underwent bright-light surgery (BLS). Half of BLS and FGS mice (8 mice in each group) received zoledronic acid (ZOL). Weekly fluorescence imaging of the mice was performed. Six weeks after surgery, metastases to lung and inguinal lymph node were evaluated by fluorescence imaging. RESULTS: The percentage of residual tumor after BLS and FGS was 9.9 ± 2.2% and 0.9 ± 0.3%, respectively (P < 0.001). FGS reduced recurrent cancer growth compared with BLS (P < 0.005). Although FGS alone had no significant effect on inguinal lymph node metastases, lung metastasis or disease-free survival (DFS), ZOL in combination with FGS significantly increased DFS (P = 0.01) in comparison with the combination of BLS and ZOL. ZOL reduced lymph node metastases (P = 0.033) but not lung metastasis. CONCLUSIONS: FGS significantly reduced recurrence of experimental prostate cancer bone metastasis compared with BLS. The combination of FGS and ZOL increased DFS over BLS and ZOL. ZOL inhibited lymph node metastasis but not lung metastasis.

Hand-held high-resolution fluorescence imaging system for fluorescence-guided surgery of patient and cell-line pancreatic tumors growing orthotopically in nude mice.

BACKGROUND: In this study, we investigated the advantages of fluorescence-guided surgery (FGS) in mice of a portable hand-sized imaging system compared with a large fluorescence imaging system or a long-working-distance fluorescence microscope. METHODS: Mouse models of human pancreatic cancer for FGS included the following: (1) MiaPaCa-2-expressing green fluorescent protein, (2) BxPC3 labeled with Alexa Fluor 488-conjucated anti-carcinoembryonic antigen (CEA) antibody, and (3) patient-derived orthotopic xenograft (PDOX) labeled with Alexa Fluor 488-conjugated anti-carbohydrate antigen 19-9 antibody. RESULTS: Each device could clearly detect the primary MiaPaCa-2-green fluorescent protein tumor and any residual tumor after FGS. In the BxPC3 model labeled with Alexa Fluor 488-conjugated anti-CEA, each device could detect the primary tumor, but the MVX10 could not clearly detect the residual tumor remaining after FGS whereas the other devices could. In the PDOX model labeled with Alexa Fluor 488-conjugated anti-carbohydrate antigen 19-9, only the portable hand-held device could distinguish the residual tumor from the background, and complete resection of the residual tumor was achieved under fluorescence navigation. CONCLUSIONS: The results described in the present report suggest that the hand-held mobile imaging system can be applied to the clinic for FGS because of its convenient size and high sensitivity which should help make FGS widely used.

Imaging UVC-induced DNA damage response in models of minimal cancer.

We have previously demonstrated that the ultraviolet (UV) light is effective against a variety of cancer cells in vivo as well as in vitro. In the present report, we imaged the DNA damage repair response of minimal cancer after UVC irradiation. DNA-damage repair response to UV irradiation was imaged on tumors growing in 3D culture and in superficial tumors grown in vivo. UV-induced DNA damage repair was imaged with GFP fused to the DNA damage response (DDR)-related chromatin-binding protein 53BP1 in MiaPaCa-2 human pancreatic cancer cells. Three-dimensional Gelfoam® histocultures and confocal imaging enabled 53BP1-GFP nuclear foci to be observed within 1 h after UVC irradiation, indicating the onset of DNA damage repair response. A clonogenic assay showed that UVC inhibited MiaPaCa-2 cell proliferation in a dose-dependent manner, while UVA and UVB showed little effect on cell proliferation. Induction of UV-induced 53BP1-GFP focus formation was limited up to a depth of 40 µm in 3D-culture of MiaPaCa-2 cells. The MiaPaCa-2 cells irradiated by UVC light in a skin-flap mouse model had a significant decrease of tumor growth compared to untreated controls. Our results also demonstrate that 53BP1-GFP is an imageable marker of UV-induced DNA damage repair response of minimal cancer and that UVC is a useful tool for the treatment of residual cancer since UVC can kill superficial cancer cells without damage to deep tissue.