Chanae R. Hardamon

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

BACKGROUND 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. STUDY DESIGN 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. RESULTS 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). CONCLUSIONS Surgical outcomes were improved in pancreatic cancer using fluorescence-guidance. This novel approach has significant potential to improve surgical treatment of cancer.

Macrophage PI3Kγ Drives Pancreatic Ductal Adenocarcinoma Progression

UNLABELLED Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a low 5-year survival rate, yet new immunotherapeutic modalities may offer hope for this and other intractable cancers. Here, we report that inhibitory targeting of PI3Kγ, a key macrophage lipid kinase, stimulates antitumor immune responses, leading to improved survival and responsiveness to standard-of-care chemotherapy in animal models of PDAC. PI3Kγ selectively drives immunosuppressive transcriptional programming in macrophages that inhibits adaptive immune responses and promotes tumor cell invasion and desmoplasia in PDAC. Blockade of PI3Kγ in PDAC-bearing mice reprograms tumor-associated macrophages to stimulate CD8(+) T-cell-mediated tumor suppression and to inhibit tumor cell invasion, metastasis, and desmoplasia. These data indicate the central role that macrophage PI3Kγ plays in PDAC progression and demonstrate that pharmacologic inhibition of PI3Kγ represents a new therapeutic modality for this devastating tumor type. SIGNIFICANCE We report here that PI3Kγ regulates macrophage transcriptional programming, leading to T-cell suppression, desmoplasia, and metastasis in pancreas adenocarcinoma. Genetic or pharmacologic inhibition of PI3Kγ restores antitumor immune responses and improves responsiveness to standard-of-care chemotherapy. PI3Kγ represents a new therapeutic immune target for pancreas cancer. Cancer Discov; 6(8); 870-85. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 803.

An LED light source and novel fluorophore combinations improve fluorescence laparoscopic detection of metastatic pancreatic cancer in orthotopic mouse models.

BACKGROUND The aim of this study was to improve fluorescence laparoscopy of pancreatic cancer in an orthotopic mouse model with the use of a light-emitting diode (LED) light source and optimal fluorophore combinations. STUDY DESIGN Human pancreatic cancer models were established with fluorescent FG-RFP, MiaPaca2-GFP, BxPC-3-RFP, and BxPC-3 cancer cells implanted in 6-week-old female athymic mice. Two weeks postimplantation, diagnostic laparoscopy was performed with a Stryker L9000 LED light source or a Stryker X8000 xenon light source 24 hours after tail-vein injection of CEA antibodies conjugated with Alexa 488 or Alexa 555. Cancer lesions were detected and localized under each light mode. Intravital images were also obtained with the OV-100 Olympus and Maestro CRI Small Animal Imaging Systems, serving as a positive control. Tumors were collected for histologic analysis. RESULTS Fluorescence laparoscopy with a 495-nm emission filter and an LED light source enabled real-time visualization of the fluorescence-labeled tumor deposits in the peritoneal cavity. The simultaneous use of different fluorophores (Alexa 488 and Alexa 555), conjugated to antibodies, brightened the fluorescence signal, enhancing detection of submillimeter lesions without compromising background illumination. Adjustments to the LED light source permitted simultaneous detection of tumor lesions of different fluorescent colors and surrounding structures with minimal autofluorescence. CONCLUSIONS Using an LED light source with adjustments to the red, blue, and green wavelengths, it is possible to simultaneously identify tumor metastases expressing fluorescent proteins of different wavelengths, which greatly enhanced the signal without compromising background illumination. Development of this fluorescence laparoscopy technology for clinical use can improve staging and resection of pancreatic cancer.

The nuclear factor-κB pathway down-regulates expression of the NKG2D ligand H60a in vitro: implications for use of nuclear factor-κB inhibitors in cancer therapy

NKG2D ligands are cell surface proteins that activate NKG2D, a receptor used by natural killer (NK) cells to detect virus‐infected and transformed cells. When tumour cells express high levels of NKG2D ligands, they are rejected by the immune system. Hence, reagents that increase NKG2D ligand expression on tumour cells can be important for tumour immunotherapy. To identify genes that regulate the NKG2D ligand H60a, we performed a microarray analysis of 3′‐methylcholanthrene‐induced sarcoma cell lines expressing high versus low H60a levels. A20, an inhibitor of nuclear factor‐κB (NF‐κB) activation, was differentially expressed in H60a‐hi sarcoma cells. Correspondingly, treatment of tumour cells with inhibitors of NF‐κB activation, such as sulfasalazine (slz), BAY‐11‐7085, or a non‐phosphorylatable IκB, led to increased levels of H60a protein, whereas transduction of cells with an active form of IκB kinase‐β (IKKβ) led to decreased levels of H60a. The regulation probably occurred at the transcriptional level, because NF‐κB pathway inhibition led to increased H60a transcripts and promoter activity. Moreover, treatment of tumour cells with slz enhanced their killing by NK cells in vitro, suggesting that NF‐κB inhibition can lead to tumour cell rejection. Indeed, when we blocked the NF‐κB pathway specifically in tumour cells, there was decreased tumour growth in wild‐type but not immune‐deficient mice. Our results suggest that reagents that can block NF‐κB activity specifically in the tumour and not the host immune cells would be efficacious for tumour therapy.

Effect of lithium salts on lactate dehydrogenase, adenylate kinase, and 1-phosphofructokinase activities

Inhibitions of 30 nM rabbit muscle 1-phosphofructokinase (PFK-1) by lithium, potassium, and sodium salts showed inhibition or not depending upon the anion present. Generally, potassium salts were more potent inhibitors than sodium salts; the extent of inhibition by lithium salts also varied with the anion. Li2CO3 was a relatively potent inhibitor of PFK-1 but LiCl and lithium acetate were not. Our results suggest that extents of inhibition by monovalent salts were due to both cations and anions, and the latter needs to be considered before inhibition can be credited to the cation. An explanation for monovalent salt inhibitions is proffered involving interactions of both cations and anions at negative and positive sites of PFK-1 that affect enzyme activity. Our studies suggest that lithium cations per se are not inhibitors: the inhibitors are the lithium salts, and we suggest that in vitro studies involving the effects of monovalent salts on enzymes should involve more than one anion.

Effect of ammonium, sodium, and potassium ions on rabbit muscle phosphofructokinase-1 and adenylate kinase activities

This report shows that 30 nM PFK-1 and 30 nM AK were both affected by the presence of NH4+, Na+, and K+ salts but with opposite consequences. Low concentrations of PFK-1 lose about half of its activity as a result of dilution and become susceptible to further activity losses owing to the presence of monovalent salts. On the other hand low concentrations of AK lose about 75 percent of its activity but regains activity losses owing to the presence of monovalent salts. It was determined that regain of AK activity did not appear to be a reflection of a major effect on the Km value of either AMP or ATP. Dilution to 30 nM AK resulted in no increase Km values compared to Km values at 140 nM AK. Dilution caused major decreases in the maximum velocities, Vmax, when ATP or fructose 6-phosphate was the variable substrate. It was shown in earlier reports that these same low concentrations of PFK-1 and AK were susceptible inhibitions by ascorbate. These attributes are discussed as they may relate to the role of ascorbate facilitation glycogen synthesis in resting muscle and the role that the cytoskeleton infrastructure scaffold may play is also discussed.

Abstract IA22: Innate immune cell PI3K gamma as a target for suppression of pancreatic ductal adenocarcinoma

Ductal adenocarcinoma of the pancreas (PDAC) is a devastating disease that will afflict greater than 40,000 Americans. Less than 5% of these new pancreatic cancer patients will survive 5 years following diagnosis. PDAC is rarely detected in the early stages, with more than 80% of patients presenting with locally unresectable or metastatic disease at the time of diagnosis. As this form of cancer is also resistant to current cytotoxic therapies and ionizing radiation, novel therapeutic and diagnostic approaches are desperately needed to improve patient outcome. A characteristic feature of PDAC is the presence of an abundant, inflammatory infiltrate. In response to tumor-derived chemoattractants, we found that myeloid cells invade tumors where they promote immunosuppression, resulting in tumor growth and metastasis. We discovered that myeloid cell PI3-kinase gamma (PI3Kgamma) controls tumor immunosuppression, as PI3Kgamma signaling inhibits pro-inflammatory gene expression responses in macrophages, monocytes and granulocytes and promotes anti-inflammatory responses. Suppression of PI3Kgamma in mutant mice and in mice treated with pharmacologic inhibitors of PI3Kgamma promotes CD8+ T cell anti-tumor immune responses, leading to a 50% reduction in tumor growth and metastasis in orthotopic mouse models of pancreatic carcinoma and significant extension of survival in GEM models of PDAC (PDX1-cre; LSL-KrasG12D/+; LSL-Trp53 R172H/+). Furthermore, PI3Kgamma inhibition blocks macrophage stimulation of collagen deposition by fibroblasts in PDAC. We are exploring the combination of PI3kinase gamma inhibition with tumor cell and T cell targeted therapeutics could significantly reduce tumor growth and metastasis of PDAC. These approaches could improve patient outcomes by reducing tumor progression and preserving organ function by inhibiting desmoplasia. Citation Format: Megan Kaneda, Chanae Hardamon, Michael C. Schmid, Michael Bouvet, Franco Novelli, Emilio Hirsch, Andrew Lowy, Judith A. Varner. Innate immune cell PI3K gamma as a target for suppression of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr IA22.

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

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

Abstract 514: A novel dual color mouse model for studying the tumor microenvironment of pancreatic adenocarcinoma

We have developed a novel, dual color, immunocompetent mouse model for studying the pancreatic tumor microenvironment using fluorescence imaging. To develop this model, we used mice engineered to constitutively express a conditional tdTomato fluorescent transgene that converts to the expression of EGFP following Cre recombinase-mediated intramolecular rearrangement of the transgene. The reporter transgene is driven by a strong, ubiquitous promoter from a well-characterized genomic locus. Both tdTomato and EGFP fluorescent proteins are membrane-targeted, allowing distinction of single cells and cell processes within the microenvironmental setting. For our studies of the pancreatic microenvironment, we used PdxCre (Pdx-1-Cre) transgenic mice. PdxCre mice express Cre recombinase within the developing pancreas during embryogenesis. To generate mice in which the pancreatic parenchyma, expressing membrane-targeted green fluorescence (mG), is highlighted against the red fluorescent (mT) backlight of stromal and non-pancreatic tissues, we crossed PdxCre transgenic mice with conditional fluorescent (mTmG) indicator mice. Both lines are on a mixed C57Bl/6 × Sv129 background, backcrossed several generations onto C57Bl/6. We used PDA4964 pancreatic adenocarcinoma cells, derived from genetically engineered triple transgenic (LSL-KrasG12D/+;LSL-Trp53R172H/+; PdxCre) mutant mice, to generate orthotopic tumors in the pancreata of PdxCre+, mTmG+ dual transgenic, dual color mice. Using a dosage of 1 × 106 tumor cells per mouse, half of the mice developed tumors within 4-6 weeks. Ex vivo imaging of orthotopic tumor-bearing mice at necropsy revealed large pancreatic tumors comprised of non-fluorescent tumor cells supported and nourished by red fluorescent host stromal and vascular cells. The expanding tumor masses distorted and displaced the adjacent green fluorescent host pancreatic parenchyma. Upon microscopic examination of the tumor-host interface, cell borders of individual host pancreatic parenchymal and stromal cells were clearly outlined. Our preliminary findings indicate that this novel, dual color, immunocompetent mouse model will be valuable for studying the pancreatic tumor microenvironment using fluorescence imaging. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 514. doi:10.1158/1538-7445.AM2011-514

Abstract 5323: Staging of metastatic pancreatic cancer is facilitated by fluorescence laparoscopy

The aim of this report was to improve the real-time imaging of fluorescence laparoscopy with the introduction of an LED light source. We also attempted to determine the optimal fluorophore to improve accurate identification and localization of tumor deposits without the loss of background illumination. Human pancreatic cancer orthotopic models were established with intraperitoneal injections of RFP-expressing FG or BxPC-3, or non-fluorescent BxPC-3 human pancreatic cancer cells into 6-week-old female athymic mice. One to 2 weeks post implantation, diagnostic laparoscopy was performed with a Stryker L9000 LED light source or a Stryker X8000 xenon light source 24 hours after tail vein injection of Alexa 488-conjugated anti-CEA. An attempt was made to detect and localize all cancer lesions under each light mode. After laparoscopy, the animals were sacrificed and abdominal cavities exposed. Images of the lesions were obtained with the Maestro CRI Small Animal Imaging System serving as a positive control. Tumors were collected and processed for histologic review. Fluorescence laparoscopy with the use of a 495-nm emission filter and an LED light source enabled real-time visualization of the fluorescence-labeled tumor metastases in the peritoneal cavity. With adjustments to the LED light source, we could simultaneously detect tumor lesions of different fluorescent colors and surrounding structures with minimal autofluorescence. Overall, fluorescence laparoscopy using the LED light source afforded accurate detection of more lesions compared to standard xenon bright field laparoscopy. In addition, several tumor deposits of less than 1mm were identified and localized under FL. Such lesions were not detected under bright field laparoscopy. All identified lesions, when possible, were verified histologically. In this study, we have thus demonstrated that using an LED light source with adjustments to the red, blue and green wavelengths, we can simultaneously identify fluorescent tumor metastases of different wavelengths without compromising background illumination. The further development of this technology can serve as a novel tool in the staging and treatment of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5323. doi:10.1158/1538-7445.AM2011-5323