Bright-light Surgery Research Articles

Photoimmunotherapy Inhibits Tumor Recurrence After Surgical Resection on a Pancreatic Cancer Patient-Derived Orthotopic Xenograft (PDOX) Nude Mouse Model.

Photoimmunotherapy (PIT) uses a target-specific photosensitizer based on a near-infrared (NIR) phthalocyanine dye, IR700, to induce tumor necrosis after irradiation with NIR light to kill cancer cells, such as those that remain after surgery. The purpose of the present study was to sterilize the surgical bed after pancreatic cancer resection with PIT in carcinoembryonic antigen (CEA)-expressing, patient-derived, orthotopic xenograft (PDOX) nude mouse models. After confirmation of tumor engraftment, mice were randomized to two groups: bright light surgery (BLS)-only and BLS + PIT. Each treatment arm consisted of seven tumor-bearing mice. BLS was performed under standard bright-field with an MVX10 long-working distance, high-magnification microscope on all mice. For BLS + PIT, anti-CEA antibody conjugated with IR700 (anti-CEA-IR700) (50 µg) was injected intravenously in all mice 24 h before surgery. After the surgery, the resection bed was then irradiated with a red-light-emitting diode at 690 ± 5 nm with a power density of 150 mW/cm(2). Anti-CEA-IR700 labelled and illuminated the pancreatic cancer PDOX. Minimal residual cancer of the PDOX was detected by fluorescence after BLS. The local recurrence rate was 85.7 % for BLS-only and 28.6 % for BLS + PIT-treated mice (p = 0.05). The average recurrent tumor weight was 1149.0 ± 794.6 mg for BLS-only and 210.8 ± 336.9 mg for BLS + PIT-treated mice (p = 0.015). Anti-CEA-IR700 was able to label and illuminate a pancreatic cancer PDOX nude mouse model sufficiently for PIT. PIT reduced recurrence by eliminating remaining residual cancer cells after BLS.

Targeting tumors with a killer-reporter adenovirus for curative fluorescence-guided surgery of soft-tissue sarcoma.

Fluorescence-guided surgery (FGS) of cancer is an area of intense interest. However, FGS of cancer has not yet been shown to be curative due to residual microscopic disease. Human fibrosarcoma HT1080 expressing red fluorescent protein (RFP) was implanted orthotopically in the quadriceps femoris muscle of nude mice. The tumor-bearing mice were injected with high and low-dose telomerase-dependent, green fluorescent protein (GFP)-containing adenovirus OBP-401, which labeled the tumor with GFP. Fluorescence-guided surgery (FGS) or bright light surgery (BLS) was then performed. OBP-401 could label soft-tissue sarcoma (STS) with GFP in situ, concordant with RFP. OBP-401-based FGS resulted in superior resection of STS in the orthotopic model of soft-tissue sarcoma, compared to BLS. High-dose administration of OBP-401 enabled FGS without residual sarcoma cells or local or metastatic recurrence, due to its dual effect of cancer-cell labeling with GFP and killing. High-dose OBP-401 based-FGS improved disease free survival (p = 0.00049) as well as preserved muscle function compared with BLS. High-dose OBP-401-based FGS could cure STS, a presently incurable disease. Since the parent virus of OBP-401, OBP-301, has been previously proven safe in a Phase I clinical trial, it is expected the OBP-401-FGS technology described in the present report should be translatable to the clinic in the near future.

Fluorescence-guided surgery, but not bright-light surgery, prevents local recurrence in a pancreatic cancer patient derived orthotopic xenograft (PDOX) model resistant to neoadjuvant chemotherapy (NAC)

BackgroundThe aim of this study is to determine the efficacy of neoadjuvant chemotherapy (NAC) with gemcitabine (GEM) in combination with fluorescence-guided surgery (FGS) on a pancreatic cancer patient derived orthotopic xenograft (PDOX) model. MethodsA PDOX model was established from a CEA-positive tumor from a patient who had undergone a pancreaticoduodenectomy for pancreatic adenocarcinoma. Mice were randomized to 4 groups: bright light surgery (BLS) only; BLS + NAC; FGS only; and FGS + NAC. An anti-CEA antibody conjugated to DyLight 650 was administered intravenously via the tail vein of mice with a pancreatic cancer PDOX 24 h before surgery. ResultsThe PDOX was clearly labeled with fluorophore-conjugated anti-CEA antibody. Only one out of 8 mice had local recurrence in the FGS only group and zero out of 8 mice had local recurrence in the FGS + NAC which was significantly lower than BLS only or BLS + NAC mice, where local disease recurred in 6 out of 8 mice in each treatment group (p = 0.041 and p = 0.007, respectively). NAC did not significantly reduce recurrence rates when combined with either FGS or BLS. ConclusionThese results indicate that FGS can significantly reduce local recurrence compared to BLS in pancreatic cancer resistant to NAC.

Photoimmunotherapy lowers recurrence after pancreatic cancer surgery in orthotopic nude mouse models

BackgroundPhotoimmunotherapy (PIT) is based on the use of a monoclonal antibody specific to cancer epitopes conjugated to a photosensitizer near-infrared phthalocyanine dye (IR700). In this study, PIT with IR700 conjugated to anti-carcinoembryonic antigen (CEA) was used as an adjunct to surgery in orthotopically-implanted human pancreatic cancer in a nude mouse model to eliminate microscopic disease in the post-surgical tumor bed and prevent local as well as metastatic recurrence. Materials and methodsAthymic nude mice were orthotopically implanted with the human pancreatic cancer cell line BxPC3 expressing green fluorescent protein. After tumor engraftment, the mice were divided into two groups as follows: bright light surgery (BLS) + anti-CEA-IR700 + 690 nm laser (PIT); and BLS only. Anti-CEA-IR700 (100 μg) was administered to the treatment group via tail-vein injection 24 h before therapy. Tumors were resected, and the surgical bed was treated with intraoperative phototherapy at an intensity of 150 mW/cm2 for 30 min. Mice were imaged noninvasively for 8 wk using an OV-100 small animal fluorescence imager. ResultsBLS + PIT reduced local recurrence to 1/7 mice from 7/7 mice with BLS-only (P = 0.001) and metastatic recurrence to 2/7 mice compared with 6/7 mice with BLS-only (P = 0.03). Local tumor growth continued at a rapid rate after BLS-only compared with BLS + PIT where almost no local growth occurred. There was a significant difference in tumor size between mice in the BLS + PIT (2.14 mm2, 95% confidence interval [CI] [−2.06 to 6.34] and BLS-only groups (115.2 mm2, 95% CI [88.8–141.6]) at 6 wk after surgery (P < 0.001). There was also a significant difference in tumor weight between the BLS + PIT group (6.65 mg, 95% CI [−6.35 to 19.65] and BLS-only group (1100 mg, 95% CI [794–1406] at 8 wk after surgery (P < 0.001). ConclusionsPIT holds promise in the treatment of pancreatic cancer and may serve as a useful adjunct to surgery in the eradication of microscopic residual disease that can lead to both local and metastatic recurrence. Further studies are warranted to investigate the potential toxicities of PIT, especially with regard to anastomoses, such as those involved in pancreaticoduodenectomy.

Metastatic recurrence in a pancreatic cancer patient derived orthotopic xenograft (PDOX) nude mouse model is inhibited by neoadjuvant chemotherapy in combination with fluorescence-guided surgery with an anti-CA 19-9-conjugated fluorophore.

The aim of this study is to determine the efficacy of neoadjuvant chemotherapy (NAC) with gemcitabine (GEM) in combination with fluorescence-guided surgery (FGS) on a pancreatic cancer patient derived orthotopic xenograft (PDOX) model. A PDOX model was established from a CA19-9-positive, CEA-negative tumor from a patient who had undergone a pancreaticoduodenectomy for pancreatic adenocarcinoma. Mice were randomized to 4 groups: bright light surgery (BLS) only; BLS+NAC; FGS only; and FGS+NAC. An anti-CA19-9 or anti-CEA antibody conjugated to DyLight 650 was administered intravenously via the tail vein of mice with the pancreatic cancer PDOX 24 hours before surgery. The PDOX was brightly labeled with fluorophore-conjugated anti-CA19-9, but not with a fluorophore-conjugated anti-CEA antibody. FGS was performed using the fluorophore-conjugated anti-CA19-9 antibody. FGS had no benefit over BLS to prevent metastatic recurrence. NAC in combination with BLS did not convey an advantage over BLS to prevent metastatic recurrence. However, FGS+NAC significantly reduced the metastatic recurrence frequency to one of 8 mice, compared to FGS only after which metastasis recurred in 6 out of 8 mice, and BLS+NAC with metastatic recurrence in 7 out of 8 mice (p = 0.041). Thus NAC in combination with FGS can reduce or even eliminate metastatic recurrence of pancreatic cancer sensitive to NAC. The present study further emphasizes the power of the PDOX model which enables metastasis to occur and thereby identify the efficacy of NAC in combination with FGS on metastatic recurrence.

O2-9-2 - Pancreatic Cancer Patient-Derived Orthotopic Xenograft (Pdox) Cured by Fluorescence-Guided Surgery Followed by Uvc

Background: The aim of this study is to determine if ultraviolet light (UVC) irradiation in combination with fluorescence- guided surgery (FGS) can eradicate metastatic human pancreatic cancer in orthotopic nude–mouse models.Methods: Two weeks after orthotopic implantation of human MiaPaCa-2 pancreatic cancer cells, expressing green fluorescent protein (GFP), in nude mice, bright-light surgery (BLS) was performed on all tumor-bearing mice (n = 24). After BLS, mice were randomized into 3 treatment groups; BLS-only (n = 8) or FGS-only (n = 8) or FGS-UVC (n = 8). The residual tumors were resected using a hand-held portable imaging system under fluorescence navigation in mice treated with FGS and FGS-UVC. The surgical resection bed was irradiated with 2700 J/m2 UVC (254 nm) in the mice treated with FGS-UVC.Results: The average residual tumor area after FGS only (n = 16) was significantly smaller than after BLS only (n = 24) (0.135±0.137 mm2 and 3.338±2.929 mm2, respectively; p = 0.007). The BLS-only treated mice had significantly reduced survival compared to FGS- and FGS-UVC treated mice for both relapse-free survival (RFS) (p < 0.001 and p < 0.001, respectively) and overall survival (OS) (p < 0.001 and p < 0.001, respectively). FGS-UVC-treated mice had increased RFS and OS compared to FGS-only treated mice (p = 0.008 and p = 0.025, respectively), with RFS lasting at least 150 days indicating the animals were cured.Conclusion: The results of the present study suggest that UVC irradiation in combination with FGS has clinical potential to increase survival.

Abstract 4375: UVC irradiation in combination with fluorescence-guided surgery cures metastatic human pancreatic cancer in orthotopic mouse models

Abstract The aim of this study was to determine the efficacy of UVC irradiation in combination with fluorescence-guided surgery (FGS) in an orthotopic model of pancreatic cancer. Two weeks after orthotopic implantation of human MiaPaCa-2-GFP pancreatic cancer cells, bright-light surgery (BLS) was performed on all tumor-bearing mice (n=24). Postoperatively, the surgical resection bed was imaged with the OV100 variable magnification imaging system with a magnification of 0.56x to detect residual tumor. The mice which underwent BLS were randomized into 3 groups; BLS only (n=8) or FGS (n=8) or FGS-UVC (n=8). The residual tumors were resected with FGS using a portable, hand-held imaging system. After FGS, the surgical resection bed was imaged with the OV100 with a magnification of 0.89x, to detect minimal residual tumor and was irradiated with 2700 J/m2 UVC (emission peak 254 nm) from the bottom of a chamber using a Benchtop 3UV transilluminator (UVP, LLC, Upland, CA). The average residual tumor area after FGS was significantly smaller than BLS (0.135 ± 0.137 mm2 and 3.338 ± 2.929 mm2, respectively; p=0.007). Relapse-free survival (RFS) and overall survival (OS) were determined in 3 groups. Three-month RFS in the BLS, FGS and FGS-UVC were 0%, 50% and 100%, respectively. Five-month OS in BLS, FGS and FGS-UVC was 10%, 90% and 100%, respectively. FGS and FGS-UVC resulted in significantly increased survival compared to BLS for both RFS and OS. FGS-UVC showed significantly increased survival compared to the FGS for both RFS and OS (p=0.008 and p=0.025, respectively). These results suggests that UVC irradiation in combination with FGS could be a curative strategy for human pancreatic cancer. Citation Format: Yukihiko Hiroshima, Ali Maawy, Sho Sato, Takashi Murakami, Mako Yamamoto, Fuminari Uehara, Shinji Miwa, Shuya Yano, Masashi Momiyama, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Itaru Endo, Robert M. Hoffman. UVC irradiation in combination with fluorescence-guided surgery cures metastatic human pancreatic cancer in orthotopic mouse models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4375. doi:10.1158/1538-7445.AM2014-4375

Abstract 3486: Efficacy of neoadjuvant chemotherapy in combination with fluorescence-guided surgery on a pancreatic cancer patient-derived orthotopic xenograft (PDOX)

Abstract The aim of this study is to determine the efficacy of neoadjuvant chemotherapy (NAC) in combination with fluorescence-guided surgery (FGS) on pancreatic cancer patient derived orthotopic xenograft (PDOX). We established the PDOX from the patient suffering from metastatic CA19-9-positive pancreatic cancer. Forty nude mice were implanted with tumor using surgical orthotopic implantation (SOI) on the nude mouse pancreas. Four weeks after implantation, the mice with tumor were randomized into 4 treatment groups: (1) bright-light surgery (BLS) only; (2) FGS; (3) NAC-BLS; (4) NAC-FGS. Gemcitabine (80 mg/kg, ip, weekly, 3 weeks) was used for NAC. Seven weeks after implantation, BLS was performed on all tumor-bearing mice. A monoclonal anti-human CA19-9 antibody conjugated with Dylight 650 was delivered to tumor-bearing mice in the FGS groups as a single intravenous dose 24 hours before BLS. Postoperatively, the surgical resection bed of FGS mice were imaged with the OV100 fluorescence imaging system to detect fluorescent residual tumors. The fluorescent residual tumors of FGS groups were then resected under fluorescence navigation. The average resected tumor weight of each group was as follows: (1) BLS, 188.5 ± 50.0 mg; (2) FGS, 278.0 ± 111.4 mg; (3) NAC-BLS, 84.5 ± 48.7 mg; (4) NAC-FGS, 141.8 ± 48.9 mg. The average resected tumor weight of NAC groups was significantly less than the non-NAC groups (113.1 ± 57.0 mg and 235.6 ± 94.3 mg, respectively; p&amp;gt;0.001). The average resected tumor weight of the FGS groups was significantly larger than the BLS groups (213.4 ± 107.1 mg and 136.5 ± 73.8 mg, respectively; p=0.016). Eight weeks after resection, we performed laparotomy to examine recurrence. The recurrence rate of each group was as follows: (1) BLS, 9 / 9 (100%); (2) FGS, 4 / 8 (50%); (3) NAC-BLS, 6 / 9 (66.7%); (4) NAC-FGS, 2 / 8 (25%). The recurrence rate of FGS groups was significantly lower than BLS groups (6 /16; 37.5% and 15 / 18; 83.3%, respectively; p=0.012), whereas there was no significant difference between FGS and NAC-FGS groups (p=0.608). There was no significant difference in the total recurrence rate between NAC and non-NAC groups (p=0.157). However NAC significantly reduced peritoneal recurrence (29.4 % and 0%, respectively; p=0.044). The results indicate that NAC in combination with FGS might reduce or even eliminate the peritoneal recurrence of pancreatic cancer. Citation Format: Yukihiko Hiroshima, Ali Maawy, Yong Zhang, Sho Sato, Takashi Murakami, Mako Yamamoto, Fuminari Uehara, Shinji Miwa, Shuya Yano, Masashi Momiyama, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Itaru Endo, Robert M. Hoffman. Efficacy of neoadjuvant chemotherapy in combination with fluorescence-guided surgery on a pancreatic cancer patient-derived orthotopic xenograft (PDOX). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3486. doi:10.1158/1538-7445.AM2014-3486

Abstract 4313: Efficacy of fluorescence-guided surgery on primary human osteosarcoma

Abstract We investigated the effectiveness of fluorescence-guided surgery (FGS) to improve outcomes in an orthotopic mouse model of human osteosarcoma. 143B-RFP human osteosarcoma cells were injected into the intramedullary cavity of the tibia in 32 nude mice. Two weeks after the implantation, 16 mice underwent FGS, and the other 16 mice underwent bright-light surgery (BLS). Half of the BLS and FGS mice (8 mice in each group) underwent adjuvant chemotherapy with cisplatin. Weekly imaging of the mice was performed and RFP-expressing areas were recorded. The fluorescent area of residual tumor after BLS and FGS were 10.2 ± 2.4 mm2 and 0.1 ± 0.1 mm2, respectively (P &amp;lt; 0.001). The residual tumor grew after BLS and BLS+CDDP. In contrast, the FGS mice and the FGS+CDDP mice had almost no tumor growth. Disease-free survival (DFS) in the BLS-, the BLS+CDDP-, the FGS-, and the FGS+CDDP-treated mice was 12.5%, 37.5%, 75.0%, and 87.5%, respectively. The FGS-treated mice had significantly higher DFS rate than the BLS-treated mice (P = 0.021), and the FGS+CDDP-treated mice had significantly higher DFS rate than the BLS+CDDP-treated mice (P = 0.043). Although chemotherapy significantly reduced multiple metastases (P = 0.033), there was no significant correlation between FGS and lung metastasis. Our study showed that FGS significantly reduced the recurrence of the tumor, but did not reduce lung metastasis. To reduce tumor recurrence and multiple metastases, the combination of FGS and chemotherapy may be necessary in the clinic. Citation Format: Shinji Miwa, Yukihiko Hiroshima, Shuya Yano, Yasunori Matsumoto, Fuminari Uehara, Mako Yamamoto, Hiroaki Kimura, Katsuhiro Hayashi, Hiroyuki Tsuchiya, Robert M. Hoffman. Efficacy of fluorescence-guided surgery on primary human osteosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4313. doi:10.1158/1538-7445.AM2014-4313

Abstract 4311: Fluorescence-guided surgery of pancreatic cancer using activatable cell penetrating peptides (ACPPs) in orthotopic mouse models

Abstract Surgical resection with complete removal of tumor remains one of the most effective modalities in the treatment of cancer. The aim of this study was to evaluate the efficacy of using activatable cell penetrating peptides (ACPPs) conjugated to Cy5 fluorophores to accurately label primary pancreatic tumor for fluorescence-guided surgery (FGS) in orthotopic mouse models. Orthotopic mouse models of human pancreatic cancer using surgical implantation of tumor fragments of the MiaPaca-2-GFP pancreatic cancer cell line were established. Two weeks after tumor implantation, the tumors were resected with bright light surgery (BLS) or FGS. FGS was performed with a florescence-dissecting microscope (MVX10, Olympus) 2 hours after periorbital injection of ACPPs. Completeness of resection was assessed from postoperative images obtained with the Maestro CRI Small Imaging System. Postoperatively, we performed whole body imaging of the mice to assess for recurrence. At eight weeks, mice were sacrificed to evaluate pancreatic tumor burden, as well as local and distant recurrence. Cy-5-conjugated ACPPs, cleavable by MMP-2 and MMP-9 permitted clear visualization of tumor margins for fluorescence-guided resection. With FGS, no residual tumor was detected in the postoperative surgical site in all 16 mice. One mouse out of 16 undergoing BLS had evidence of residual tumor postoperatively. At 8 weeks, mice in the BLS group demonstrated larger primary tumors compared to mice in the FGS group (1.72 g ± SE 0.58 vs 0.25 g ± SE 0.14, p=0.026). Mean disease-free survival (DFS) was significantly lengthened from 5.33 weeks in the BLS group to 7.38 weeks in the FGS group (p=0.02). Furthermore, recurrence rates were lower in the FGS group (38% vs 73%, p=0.049). This translated into a lower local and distant recurrence rate for FGS (31% vs 67% and 25% vs 60%, respectively). ACPPs can provide an alternative method to accurately and effectively label cancer cells for improved resection of tumors that do not express specific tumor antigens and result in better outcomes post-resection. Citation Format: Cristina A. Metildi, Sharmeela Kaushal, Csilla N. Felsen, Quyen T. Nguyen, Robert M. Hoffman, Roger Y. Tsien, Michael Bouvet. Fluorescence-guided surgery of pancreatic cancer using activatable cell penetrating peptides (ACPPs) in orthotopic mouse models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4311. doi:10.1158/1538-7445.AM2014-4311

Fluorescence-guided surgery improves outcome in an orthotopic osteosarcoma nude-mouse model.

In order to develop a model for fluorescence-guided surgery (FGS), 143B human osteosarcoma cells expressing red fluorescent protein (RFP) were injected into the intramedullary cavity of the tibia in nude mice. The fluorescent areas of residual tumors after bright-light surgery (BLS) and FGS were 10.2 ± 2.4 mm(2) and 0.1 ± 0.1 mm(2) , respectively (p<0.001). The BLS-treated mice and BLS+cisplatinum (CDDP)-treated mice had significant recurrence. In contrast, the FGS mice and FGS+CDDP mice had very little recurring tumor growth. Disease-free survival (DFS) in the BLS-, BLS+CDDP-, FGS-, and FGS+CDDP-treated mice was 12.5%, 37.5%, 75.0%, and 87.5%, respectively. The FGS-treated mice had a significantly higher DFS rate than the BLS-treated mice (p=0.021). The FGS+CDDP-treated mice had significantly higher DFS rate than the BLS+CDDP-treated mice (p=0.043). Although chemotherapy significantly reduced multiple metastases (p=0.033), there was no significant correlation between FGS and lung metastasis. FGS significantly reduced the recurrence of the primary tumor but did not reduce lung metastasis. The combination of FGS and adjuvant CDDP reduced tumor recurrence and prevented multiple metastases. FGS and adjuvant chemotherapy should be performed as early as possible in the disease to prevent both recurrence and metastatic development.

Fluorescence-guided surgery in combination with UVC irradiation cures metastatic human pancreatic cancer in orthotopic mouse models.

The aim of this study is to determine if ultraviolet light (UVC) irradiation in combination with fluorescence-guided surgery (FGS) can eradicate metastatic human pancreatic cancer in orthotopic nude–mouse models. Two weeks after orthotopic implantation of human MiaPaCa-2 pancreatic cancer cells, expressing green fluorescent protein (GFP), in nude mice, bright-light surgery (BLS) was performed on all tumor-bearing mice (n = 24). After BLS, mice were randomized into 3 treatment groups; BLS-only (n = 8) or FGS (n = 8) or FGS-UVC (n = 8). The residual tumors were resected using a hand-held portable imaging system under fluorescence navigation in mice treated with FGS and FGS-UVC. The surgical resection bed was irradiated with 2700 J/m2 UVC (254 nm) in the mice treated with FGS-UVC. The average residual tumor area after FGS (n = 16) was significantly smaller than after BLS only (n = 24) (0.135±0.137 mm2 and 3.338±2.929 mm2, respectively; p = 0.007). The BLS treated mice had significantly reduced survival compared to FGS- and FGS-UVC-treated mice for both relapse-free survival (RFS) (p<0.001 and p<0.001, respectively) and overall survival (OS) (p<0.001 and p<0.001, respectively). FGS-UVC-treated mice had increased RFS and OS compared to FGS-only treated mice (p = 0.008 and p = 0.025, respectively); with RFS lasting at least 150 days indicating the animals were cured. The results of the present study suggest that UVC irradiation in combination with FGS has clinical potential to increase survival.

Fluorescence-guided surgery of prostate cancer bone metastasis

BackgroundThe aim of this study is to investigate the effectiveness of fluorescence-guided surgery (FGS) of prostate cancer experimental skeletal metastasis. Materials and methodsGreen 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. ResultsThe 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. ConclusionsFGS 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.

Fluorescence-guided Surgery with a Fluorophore-conjugated Antibody to Carcinoembryonic Antigen (CEA), that Highlights the Tumor, Improves Surgical Resection and Increases Survival in Orthotopic Mouse Models of Human Pancreatic Cancer

We have developed a method of distinguishing normal tissue from pancreatic cancer in vivo using fluorophore-conjugated antibody to carcinoembryonic antigen (CEA). The objective of this study was to evaluate whether fluorescence-guided surgery (FGS) with a fluorophore-conjugated antibody to CEA, to highlight the tumor, can improve surgical resection and increase disease-free survival (DFS) and overall survival (OS) in orthotopic mouse models of human pancreatic cancer. We established nude-mouse models of human pancreatic cancer with surgical orthotopic implantation of the human BxPC-3 pancreatic cancer. Orthotopic tumors were allowed to develop for 2 weeks. Mice then underwent bright-light surgery (BLS) or FGS 24 h after intravenous injection of anti-CEA-Alexa Fluor 488. Completeness of resection was assessed from postoperative imaging. Mice were followed postoperatively until premorbid to determine DFS and OS. Complete resection was achieved in 92 % of mice in the FGS group compared to 45.5 % in the BLS group (p = 0.001). FGS resulted in a smaller postoperative tumor burden (p = 0.01). Cure rates with FGS compared to BLS improved from 4.5 to 40 %, respectively (p = 0.01), and 1-year postoperative survival rates increased from 0 % with BLS to 28 % with FGS (p = 0.01). Median DFS increased from 5 weeks with BLS to 11 weeks with FGS (p = 0.0003). Median OS increased from 13.5 weeks with BLS to 22 weeks with FGS (p = 0.001). FGS resulted in greater cure rates and longer DFS and OS using a fluorophore-conjugated anti-CEA antibody. FGS has potential to improve the surgical treatment of pancreatic cancer.

Successful Fluorescence-Guided Surgery on Human Colon Cancer Patient-Derived Orthotopic Xenograft Mouse Models Using a Fluorophore-Conjugated Anti-CEA Antibody and a Portable Imaging System

Fluorescence-guided surgery (FGS) can enable successful cancer surgery where bright-light surgery often cannot. There are three important issues for FGS going forward toward the clinic: (a) proper tumor labeling, (b) a simple portable imaging system for the operating room, and (c) patient-like mouse models in which to develop the technology. The present report addresses all three. Patient colon tumors were initially established subcutaneously in nonobese diabetic (NOD)/severe combined immune deficiency (SCID) mice immediately after surgery. The tumors were then harvested from NOD/SCID mice and passed orthotopically in nude mice to make patient-derived orthotopic xenograft (PDOX) models. Eight weeks after orthotopic implantation, a monoclonal anti-carcinoembryonic antigen (CEA) antibody conjugated with AlexaFluor 488 (Molecular Probes Inc., Eugene, OR) was delivered to the PDOX models as a single intravenous dose 24 hours before laparotomy. A hand-held portable fluorescence imaging device was used. The primary tumor was clearly visible at laparotomy with the portable fluorescence imaging system. Frozen section microscopy of the resected specimen demonstrated that the anti-CEA antibody selectively labeled cancer cells in the colon cancer PDOX. The tumor was completely resected under fluorescence navigation. Histologic evaluation of the resected specimen demonstrated that cancer cells were not present in the margins, indicating successful tumor resection. The FGS animals remained tumor free for over 6 months. The results of the present report indicate that FGS using a fluorophore-conjugated anti-CEA antibody and portable imaging system improves efficacy of resection for CEA-positive colorectal cancer. These data provide the basis for clinical trials.

Fluorescently labeled chimeric anti‐CEA antibody improves detection and resection of human colon cancer in a patient‐derived orthotopic xenograft (PDOX) nude mouse model

The aim of this study was to evaluate a new fluorescently labeled chimeric anti-CEA antibody for improved detection and resection of colon cancer. Frozen tumor and normal human tissue samples were stained with chimeric and mouse antibody-fluorophore conjugates for comparison. Mice with patient-derived orthotopic xenografts (PDOX) of colon cancer underwent fluorescence-guided surgery (FGS) or bright-light surgery (BLS) 24 hr after tail vein injection of fluorophore-conjugated chimeric anti-CEA antibody. Resection completeness was assessed using postoperative images. Mice were followed for 6 months for recurrence. The fluorophore conjugation efficiency (dye/mole ratio) improved from 3-4 to >5.5 with the chimeric CEA antibody compared to mouse anti-CEA antibody. CEA-expressing tumors labeled with chimeric CEA antibody provided a brighter fluorescence signal on frozen human tumor tissues (P = 0.046) and demonstrated consistently lower fluorescence signals in normal human tissues compared to mouse antibody. Chimeric CEA antibody accurately labeled PDOX colon cancer in nude mice, enabling improved detection of tumor margins for more effective FGS. The R0 resection rate increased from 86% to 96% with FGS compared to BLS. Improved conjugating efficiency and labeling with chimeric fluorophore-conjugated antibody resulted in better detection and resection of human colon cancer in an orthotopic mouse model.

Fluorescence-guided surgery of human colon cancer increases complete resection resulting in cures in an orthotopic nude mouse model

BackgroundWe inquired if fluorescence-guided surgery (FGS) could improve surgical outcomes in fluorescent orthotopic nude mouse models of human colon cancer. MethodsWe established fluorescent orthotopic mouse models of human colon cancer expressing a fluorescent protein. Tumors were resected under bright light surgery (BLS) or FGS. Pre- and post-operative images with the OV-100 Small Animal Imaging System (Olympus Corp, Tokyo Japan) were obtained to assess the extent of surgical resection. ResultsAll mice with primary tumor that had undergone FGS had complete resection compared with 58% of mice in the BLS group (P = 0.001). FGS resulted in decreased recurrence compared with BLS (33% versus 62%, P = 0.049) and lengthened disease-free median survival from 9 to >36 wk. The median overall survival increased from 16 wk in the BLS group to 31 weeks in the FGS group. FGS resulted in a cure in 67% of mice (alive without evidence of tumor at >6 mo after surgery) compared with only 37% of mice that underwent BLS (P = 0.049). ConclusionsSurgical outcomes in orthotopic nude mouse models of human colon cancer were significantly improved with FGS. The present study can be translated to the clinic by various effective methods of fluorescently labeling tumors.

Fluorescence-Guided Surgery Allows for More Complete Resection of Pancreatic Cancer, Resulting in Longer Disease-Free Survival Compared with Standard Surgery in Orthotopic Mouse Models

Negative surgical margins are vital to achieve cure and prolong survival in patients with pancreatic cancer. We inquired if fluorescence-guided surgery (FGS) could improve surgical outcomes and reduce recurrence rates in orthotopic mouse models of human pancreatic cancer. A randomized active-control preclinical trial comparing bright light surgery (BLS) to FGS was used. Orthotopic mouse models of human pancreatic cancer were established using the BxPC-3 pancreatic cancer cell line expressing red fluorescent protein (RFP). Two weeks after orthotopic implantation, tumors were resected with BLS or FGS. Pre- and postoperative images were obtained with the OV-100 Small Animal Imaging System to assess completeness of surgical resection in real time. Postoperatively, noninvasive whole body imaging was done to assess recurrence and follow tumor progression. Six weeks postoperatively, mice were sacrificed to evaluate primary pancreatic and metastatic tumor burden at autopsy. A more complete resection of pancreatic cancer was achieved using FGS compared with BLS: 98.9% vs 77.1%, p = 0.005. The majority of mice undergoing BLS (63.2%) had evidence of gross disease with no complete resections; 20% of mice undergoing FGS had complete resection and an additional 75% had only minimal residual disease (p = 0.0001). The mean postoperative tumor burden was significantly less with FGS compared with BLS: 0.08 ± 0.06 mm(2) vs 2.64 ± 0.63 mm(2), p = 0.001. The primary tumor burden at termination was significantly less with FGS compared with BLS: 19.3 ± 5.3 mm(2) vs 6.2 ± 3.6 mm(2), p = 0.048. FGS resulted in significantly longer disease-free survival than BLS (p = 0.02, hazard ratio = 0.39, 95% CI 0.17, 0.88). Surgical outcomes were improved in pancreatic cancer using fluorescence-guidance. This novel approach has significant potential to improve surgical treatment of cancer.

Abstract 372: Disease-free survival and overall survival from colon cancer is improved with fluorescence-guided surgery in metastatic mouse models

Abstract The aim of this study was to improve disease-free survival and overall survival in orthotopic nude mouse models of human colon cancer with fluorescence-guided surgery (FGS). Fluorescent orthotopic or carcinomatosis models were established in nude mice using human colon cancer cell lines HCT-116 and HT-29 expressing either green fluorescent protein (GFP) or red fluorescent protein (RFP). The tumors were later resected by bright light surgery (BLS) or FGS. In orthotopic and carcinomatosis models, pre- and post-operative images were obtained with the OV-100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan) to assess the extent of surgical resection. In the orthotopic model, whole body imaging of the mice was performed in the postoperative period to assess cancer recurrence and to follow subsequent tumor progression. The mice were sacrificed when they became premorbid and their abdomens were exposed for intravital and ex vivo imaging. Tumor burden was measured in mm2 using ImageJ v1.440. A greater extent of tumor resection in mice with carcinomatosis was achieved using FGS compared to BLS (99.9% vs. 76.9%, p = 0.006). Furthermore, all mice with localized disease had a complete surgical resection with FGS. In contrast, complete resection was achieved in only 56% of the mice undergoing BLS. (p=0.001) Fewer mice in the FGS group had evidence of tumor recurrence (33%) compared to mice in the BLS group (58%), lengthening disease-free survival from 9 weeks in the BLS group to 27 weeks in the FGS group. Overall survival of the mice also increased from 17 weeks in the BLS group to 29 weeks in the FGS group. The results of the present study demonstrate that improved surgical outcomes in the treatment of colon cancer can be achieved with FGS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 372. doi:1538-7445.AM2012-372

Abstract 375: Fluorescence-guided surgery leads to improved resection of primary pancreatic cancer and prolonged survival

Abstract The aim of this study was to determine if fluorescence-guided surgery (FGS) could improve surgical outcomes, reduce recurrence rates and improve overall survival in orthotopic mouse models of human pancreatic cancer. Orthotopic mouse models of human pancreatic cancer were established using the BxPC-3 RFP-expressing pancreatic cancer cell line in nude mice. Two weeks after implantation, the mice were randomized to undergo bright light surgery (BLS) or fluorescence-guided surgery (FGS). Pre- and postoperative images were obtained with the Olympus OV-100 Small Animal Imaging System to assess completeness of surgical resection. Whole body imaging of the mice was performed weekly in the postoperative period to assess for recurrence and follow tumor progression. Additionally, half of the mice were randomly selected to undergo 4 weeks of postoperative gemcitabine treatment. At six weeks postoperatively, or when premorbid, the mice were sacrificed and primary pancreatic tumor burden was measured using ImageJ v1.440. A more complete resection of pancreatic cancer was achieved using FGS compared to BLS: 98.9% vs 77.1%, p=0.005. Sixty-three percent of the mice undergoing BLS had evidence of gross residual disease, whereas 20% of mice undergoing FGS received a complete resection and an additional 75% of the mice were left with minimal residual disease (p=0.0001). At six weeks, the primary pancreatic tumor burden was significantly less with FGS compared to BLS: 19.341 ± 5.26 mm2 vs. 6.194 ± 3.61 mm2, p=0.048. In a pilot survival study, FGS alone was associated with a decreased risk of tumor recurrence (HR=0.093, 95% CI 0.013-0.645), and mean overall survival was lengthened from 18.5 weeks to 28.2 weeks. The addition of adjuvant gemcitabine (GEM) to FGS further lengthened mean overall survival to 42.75 weeks (p=0.019). Three of the four mice in the FGS with adjuvant gemcitabine group were alive at 12 months postoperatively and all of the mice (n=4) were free of tumor at time of sacrifice. In this study, we achieved improved surgical outcomes in primary pancreatic cancer under fluorescence-guidance. FGS afforded a more complete resection of primary tumor and improved disease-free survival and overall survival. The combination of FGS and GEM lengthened disease-free survival and improved overall survival. This novel approach has potential to improve outcomes in the surgical treatment of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 375. doi:1538-7445.AM2012-375