Sc Tumors Research Articles

Description of the prevalence, histologic characteristics, concomitant abnormalities, and outcomes of mammary gland tumors in companion rats (Rattus norvegicus): 100 cases (1990-2015).

OBJECTIVE To describe the prevalence, histologic characteristics, concomitant abnormalities, and outcomes for various types of mammary gland tumors in companion rats (Rattus norvegicus). DESIGN Retrospective case series. ANIMALS 100 client-owned rats. PROCEDURES Medical records of companion rats that had an SC mass and were examined at a veterinary teaching hospital between 1990 and 2015 were reviewed. Information regarding the signalment, age at mass detection, reproductive sterilization status, histologic diagnosis of the SC mass, location of the initial and all subsequent SC masses, treatments administered, and clinical outcomes was extracted from each record and summarized. RESULTS 105 SC masses were initially detected in 100 rats. The most prevalent SC mass identified was mammary gland fibroadenoma (56/105 [53%]), followed by mammary gland carcinoma (13/105 [12%]). Overall, 26 of 105 (25%) masses were malignant. Sexually intact males were more likely to have nonmammary SC tumors than sexually intact females. In rats receiving no adjunctive treatment after excision of a mammary gland fibroadenoma (n = 16), a second fibroadenoma was detected 1 to 8 months after initial excision, at a median of 4.5 months after surgery. A concomitant pituitary gland tumor was identified in most rats with mammary gland fibroadenoma (21/28 [75%]) and other types of mammary gland tumors (10/17 [59%]). Fourteen of 35 (40%) rats with mammary gland fibroadenoma had concomitant reproductive tract abnormalities. CONCLUSION AND CLINICAL RELEVANCE Results suggested that, like other species, companion rats with SC masses should undergo a thorough diagnostic workup that includes histologic examination of the excised mass.

Abstract 1319: A novel formulation of CX-5461, a small-molecule inhibitor of rRNA synthesis, and its use for treatment of acute myeloid leukemia models

Abstract CX-5461 is a RNA polymerase I inhibitor currently in Phase I clinical trial in Australia for patients with advanced hematologic malignancies. In the pre-clinical setting, CX-5461 is efficacious in a wide range of hematologic and solid tumor models when given orally or intraperitoneally. Currently, the compound is given intravenously (iv) in the first-in-human clinical trial. CX-5461 is solubilized in low pH (3.5) 50 mM sodium phosphate as it is sparingly soluble in water (<1 mg/mL) and at physiological pH. The use of such low pH phosphate buffers affects infusion tolerance. While CX-5461 has nanomolar range activity in vitro, in vivo studies are conducted with either high dosages or frequent dosing. The chemical structure of CX-5461 also suggests potential drug instability under physiological conditions. We have addressed these solubility and stability issues by developing a lipid-based nanoparticulate (LNP) formulation which relies on a previously unrecognized interaction between CX-5461 and copper. Copper (Cu) coordination with CX-5461 was demonstrated using 1H NMR and UV-Vis. Cu-LNPs were prepared with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CHOL) (55:45 molar ratio) using extrusion methods. CX-5461 was loaded into Cu-LNPs at 60°C for 30 minutes and the external buffer was exchanged to Hepes Buffered Saline (pH 7.4). Pharmacokinetic (PK) studies were performed in CD-1 mice given a dose of 30 mg/kg (iv) and plasma concentrations of CX-5461 were determined by HPLC. Efficacy studies were performed in RAG2M mice with established subcutaneous (sc) MV-4-11 tumors and in a MV-4-11 bone marrow engraftment model (30 mg/kg given iv, Q4Dx3). Our 1H NMR data suggests that CX-5461 binds to copper at a 1:1 molar ratio. Copper complexation does not affect the anticancer activity of CX-5461 as determined in several cancer cell lines in vitro. Using copper-complexation as a driving force, CX-5461 was efficiently loaded into LNPs (∼100 nm) prepared to contain copper (300 mM CuSO4). Resulting LNPs have a drug-to-lipid-ratio of 0.2. The LNP formulation has significantly improved the PK profile of CX-5461 and increased the total exposure to drug by an order of magnitude (AUC = 4156 μg-hr/mL for LNP and 319 μg-hr/mL for free CX-5461). The LNP formulation of CX-5461 is more active than the current low-pH formulation of CX-5461 when given iv in leukemia xenograft models. Specifically 37 days after sc tumor cell inoculation the tumor sizes were 720 mm3 and 240 mm3 in control and LNP-CX5461-treated animals respectively. The clinical formulation administered at the same dose exhibited tumor volumes that were comparable to the untreated controls. We have generated the first LNP formulation of CX-5461 using the copper complexation technology developed in our laboratory. LNP-CX-5461 is suitable for iv administration and is more efficacious than the current formulation in our pre-clinical studies. Citation Format: Ada W.Y. Leung, Malathi Anantha, Kathleen E. Prosser, Mohamed Wehbe, Charles J. Walsby, Marcel B. Bally. A novel formulation of CX-5461, a small-molecule inhibitor of rRNA synthesis, and its use for treatment of acute myeloid leukemia models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1319.

Abstract IA23: T cell trafficking in lymphoid and non-lymphoid tissues

Abstract Increased representation of CD8 T-lymphocytes in tumors, spontaneously, or after vaccination or adoptive therapy, is essential for immune mediated control of human cancers. T-cell infiltrates are a positive prognostic indicator of patient survival, and it has become clear that the patients who respond clinically to new generation immunotherapies are those in which an immunological infiltrate is already evident prior to treatment. Thus, limited representation of intratumoral T-cells is a major barrier to tumor control, distinct from the barrier created by immunosuppression. Understanding and overcoming this limitation will enable us to extend the effectiveness of many immunotherapies to a broader cross-section of cancer patients. T cell infiltration into lymphoid and non-lymphoid tissues is controlled by homing receptors (HR) that engage corresponding ligands expressed on tissue-associated vasculature. Naïve T-cells enter LN based on expression of L-selectin and CCR7. These HR bind to peripheral node addressin (PNAd) and the chemokines CCL19/CCL21, respectively, which are expressed on specialized lymph node blood vessels called high endothelial venules (HEV). During differentiation, effector T-cells acquire the ability enter peripheral tissues, including tumors, by upregulating other selectins, integrins and chemokine receptors that bind to ligands expressed on inflamed vasculature. However, we lack a full understanding of which HR expressed by CD8 T-cell effectors control their migration into tumors, how this is influenced by expression of the corresponding ligands on tumor vasculature, and whether ligand expression changes as a result of vessel normalization. We have explored the hypothesis that CD8 T-cell effectors activated in different secondary lymphoid organs express distinct HR, which in turn control infiltration into tumors in different anatomical locations. We identified 3 major populations of primary CD8 T-cell effectors based on site of activation and HR expression pattern, and have established which HR enable infiltration of one of these populations into tumors. While it has been assumed that all tumor-infiltrating lymphocytes are effectors that differentiate in tumor-draining LN and subsequently enter the tumor, we have shown that naïve CD8 T-cells also can directly enter tumors. These naïve cells differentiate into functional effectors in the tumor, and can promote its control. Naïve CD8 T-cell infiltration depends on L-selectin and CCR7, as in lymph nodes, and on the development of blood vessels in the tumor that express PNAd and CCL21 and resemble lymph node HEV. Similar lymph node-like vasculature has been reported in human tumors along with ectopic organized lymphoid tissue (tertiary lymphoid organs or TLO), and correlated with longer metastasis-free, disease-free, and overall survival. Development of this vasculature in murine tumors was controlled by a novel mechanism involving effector CD8 T-cells and NK cells that secreted Lymphotoxin-α3 and IFNγ;. This suggests that early infiltration of effectors induces LN-like vasculature, which in turn supports a self-sustaining recruitment of naïve T-cells that differentiate within the tumor. In intraperitoneal but not subcutaneous tumors, this lymph node-like vasculature was also associated with organized aggregates of B-lymphocytes and gp38+ fibroblasts that resembled TLO. The differential expression of these elements in SC and IP tumors reflects differences in immunological tumor microenvironments that remain to be explored further. Nonetheless, these results establish LN-like vasculature as both a consequence of and key contributor to anti-tumor immunity based on its role in promoting T-cell infiltration. Citation Format: Victor H. Engelhard, David Peske, Amber Woods, Nithya Srinivasan, Colin Brinkman, Andrew Ferguson. T cell trafficking in lymphoid and non-lymphoid tissues. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr IA23.

The Apoptotic Effect of HIF-1α Inhibition Combined with Glucose plus Insulin Treatment on Gastric Cancer under Hypoxic Conditions.

Gastric cancer grows under a hypoxic environment. HIF-1α is known to play an important role in controlling the production of reactive oxygen species (ROS) in the mitochondria under hypoxic conditions. We previously established HIF-1α knockdown (KD) cells and control (SC) cells in the 58As9 gastric cancer cell line. In this study, we revealed that KD cells, but not SC cells, induced apoptosis under conditions of hypoxia (1% O2) due to excessive production of ROS. A quantitative RT-PCR analysis demonstrated that the expressions of ten genes, which are involved in the control mechanisms of ROS (including the Warburg effect, mitophagy, electron transport chain [ETC] modification and ROS scavenging), were regulated by HIF-1α. Moreover, the promotion of glucose uptake by glucose plus insulin (GI) treatment enhanced the apoptotic effect, which was accompanied by further ROS production in hypoxic KD cells. A Western blot analysis showed that the membranous expression of GLUT1 in KD cells was elevated by glucose and/or insulin treatments, indicating that the GI-induced glucose uptake is mediated by the increased translocation of GLUT1 on the cell membrane. Finally, the anti-tumor effect of HIF-1α knockdown (KD) plus GI was evaluated using a tumor xenograft model, where a hypoxic environment naturally exists. As a result, the GI treatment strongly inhibited the growth of the KD tumors whereby cell apoptosis was highly induced in comparison to the control treatment. In contrast, the growth of the SC tumors expressing HIF-1α was not affected by the GI treatment. Taken together, the results suggest that HIF-1α inhibition plus GI may be an ideal therapy, because the apoptosis due to the destruction of ROS homeostasis is specifically induced in gastric cancer that grows under a hypoxic environment, but not in the normal tissue under the aerobic conditions.

Effector TCD8 use different homing receptor/ligand interactions to enter subcutaneous and intraperitoneal B16 melanoma tumors (TUM7P.1022)

Abstract Immunotherapies have the potential to control cancer, but are currently only effective in patients with a preexisting TCD8 infiltrate. TCD8 infiltration into tissues relies on interactions between homing receptors on TCD8 and ligands on the vasculature. However, those required for entry into tumors are incompletely characterized. Using B16 melanoma expressing the strong antigen ovalbumin, the homing receptor ligands VCAM-1 and E-selectin were more highly expressed on SC tumor vasculature, while MadCAM-1 was expressed more highly on IP tumor vasculature. B16 parental tumors and tumors grown in Rag1KO animals expressed negligible levels of VCAM-1 and MadCAM-1, suggesting that activated adaptive immune cells that previously infiltrated the tumor drove their expression. Our previous work showed that anatomical site of TCD8 priming results in differential expression of α4β1, α4β7, and E-Selectin Ligand (ESL) on effectors. We showed that α4β1+ effectors enter SC and IP tumors via interaction with VCAM-1, while ESL+ effectors preferentially enter SC tumors via interaction with E-Selectin. Unexpectedly, α4β7 and MadCAM-1 did not mediate entry into IP tumors, apparently because MadCAM-1 was not expressed on the luminal surface. Our results suggest that the commonly used SC vaccination route generates effectors that may not home efficiently to visceral metastases. Vaccination targeting non-skin draining LN may generate effectors that infiltrate visceral metastases more efficiently.

Establishment and characterization of novel human primary and metastatic anaplastic thyroid cancer cell lines and their genomic evolution over a year as a primagraft.

Anaplastic thyroid cancer (ATC) has no effective treatment, resulting in a high rate of mortality. We established cell lines from a primary ATC and its lymph node metastasis, and investigated the molecular factors and genomic changes associated with tumor growth. The aim of the study was to understand the molecular and genomic changes of highly aggressive ATC and its clonal evolution to develop rational therapies. We established unique cell lines from primary (OGK-P) and metastatic (OGK-M) ATC specimen, as well as primagraft from the metastatic ATC, which was serially xeno-transplanted for more than 1 year in NOD scid gamma mice were established. These cell lines and primagraft were used as tools to examine gene expression, copy number changes, and somatic mutations using RNA array, SNP Chip, and whole exome sequencing. Mice carrying sc (OGK-P and OGK-M) tumors developed splenomegaly and neutrophilia with high expression of cytokines including CSF1, CSF2, CSF3, IL-1β, and IL-6. Levels of HIF-1α and its targeted genes were also elevated in these tumors. The treatment of tumor carrying mice with Bevacizumab effectively decreased tumor growth, macrophage infiltration, and peripheral WBCs. SNP chip analysis showed homozygous deletion of exons 3-22 of the PARD3 gene in the cells. Forced expression of PARD3 decreased cell proliferation, motility, and invasiveness, restores cell-cell contacts and enhanced cell adhesion. Next generation exome sequencing identified the somatic changes present in the primary, metastatic, and primagraft tumors demonstrating evolution of the mutational signature over the year of passage in vivo. To our knowledge, we established the first paired human primary and metastatic ATC cell lines offering unique possibilities for comparative functional investigations in vitro and in vivo. Our exome sequencing also identified novel mutations, as well as clonal evolution in both the metastasis and primagraft.

399 Rho-GTPase, RAC1 and Cdc42 mediates Wnt–beta-catenin signals for metastasis associated phenotypes in TNBC: A proof of concept study

Material and Methods: For in vitro experiments, cells were exposed to paclitaxel and/or TAS-119. Cell viability was determined by measuring cellular ATP concentration. For in vivo experiments, antitumor efficacy was assessed in subcutaneous HCC-1806-luc (triple negative breast cancer) and NCI-H460-luc (non-small cell lung cancer) xenograft models. The potential of taxane therapy with TAS-119 was evaluated in non-small cell lung cancer orthotopic models of H460-luc tumor in nude mice. To monitor tumor burden, in vivo bioluminescence imaging in individual mice was monitored weekly. Results: TAS-119 selectively inhibited phosphorylation of Aurora A without affecting the activity of Aurora B and Aurora C in NCI-H460 cells. TAS119 showed synergistic cell growth inhibition in combination with paclitaxel in NCI-H460 and HCC1806 cells. TAS-119 clearly enhanced antitumor efficacy of docetaxel in NCI-H460-luc and paclitaxel in HCC-1806-luc nude mice with sc tumors. In the lung orthotopic models, TAS-119 with docetaxel improved median survival, with significant benefit of the combination (155% ILS) over docetaxel alone (67% ILS). Conclusions: TAS-119 has a potential as an enhancer of taxane therapy in NSCLC and TNBC cells both in vitro and in vivo.

400 Optimization of novel pyrido[2,3-b]pyrazine based small molecule fibroblast growth factor receptor 1, 2, 3 & 4 (FGFR) inhibitors into a potential clinical candidate

Material and Methods: For in vitro experiments, cells were exposed to paclitaxel and/or TAS-119. Cell viability was determined by measuring cellular ATP concentration. For in vivo experiments, antitumor efficacy was assessed in subcutaneous HCC-1806-luc (triple negative breast cancer) and NCI-H460-luc (non-small cell lung cancer) xenograft models. The potential of taxane therapy with TAS-119 was evaluated in non-small cell lung cancer orthotopic models of H460-luc tumor in nude mice. To monitor tumor burden, in vivo bioluminescence imaging in individual mice was monitored weekly. Results: TAS-119 selectively inhibited phosphorylation of Aurora A without affecting the activity of Aurora B and Aurora C in NCI-H460 cells. TAS119 showed synergistic cell growth inhibition in combination with paclitaxel in NCI-H460 and HCC1806 cells. TAS-119 clearly enhanced antitumor efficacy of docetaxel in NCI-H460-luc and paclitaxel in HCC-1806-luc nude mice with sc tumors. In the lung orthotopic models, TAS-119 with docetaxel improved median survival, with significant benefit of the combination (155% ILS) over docetaxel alone (67% ILS). Conclusions: TAS-119 has a potential as an enhancer of taxane therapy in NSCLC and TNBC cells both in vitro and in vivo.

Abstract 3120: RUNX2 rescues the inhibitory effect of FGFR-2 silencing and increases the metastatic potential of IBH-6 human breast cancer xenografts

Abstract Carcinoma associated fibroblasts participate in the regulation of breast cancer growth. We have shown that FGF2 is one of the growth factors involved in tumor progression that activates FGFR-2 and progesterone receptors (PR) increasing the proliferation of breast cancer cells (Cerliani et al, Cancer Res 2011). In addition, we have also shown an increased expression of RUNX2 in tumor cells with acquired hormone independence. It has been suggested that RUNX2 regulates the expression of FGFR2, and on the other hand that FGF2 increases RUNX2 expression. The aim of this study was to evaluate the interplay between FGFR-2 and RUNX2 using a human breast cancer xenograft model. IBH-6 cells express ERα, PR, FGFR 1 to 4 and RUNX2 and grow in vivo in immunocompromised mice without hormone supply giving rise to invasive carcinomas. IBH-6 cells were stably transfected with a specific plasmid designed to knock down the expression of FGFR-2 (shFGFR-2) or with a control plasmid (Scrambled, SC). The decrease of FGFR-2 was confirmed by Western Blot and immunohistochemistry. No differences were found between both cell lines in the proliferation assays performed in vitro. For the in vivo experiments, 3.106 cells were s.c. inoculated into the flank of nude mice (nu/nu). The shFGFR-2 tumors were significantly smaller than control tumors (p<0.001), showed a less aggressive phenotype, a lower proliferation index (Ki67, p<0.01), diminished ratio of pErk/Erk and pAkt/Akt (p<0.05) and a decrease in CCND1 and RUNX2 expression as compared with control SC tumor samples. In order to evaluate if RUNX2 was able to bypass the inhibitory effect of shFGFR-2, cells were cotransfected with the expression plasmid for RUNX2 and were then inoculated in mice as previously described. An increase in tumor size and in aggressiveness associated with higher levels of CCND1 was observed in shFGFR-2/RUNX2 as compared with control (p<0.05) or shFGFR-2 (p<0.01) tumors. Interestingly, a high number of lung and liver metastases were observed in all the shFGFR-2/RUNX2 animals. Our results indicate that FGFR-2 is a key protein involved in mammary tumor growth. The fact that no changes were registered between shFGFR-2 and control cells growing in plastic lends support to our hypothesis postulating a paracrine effect between stromal and parenchymal tissue involving FGFR-2. Since RUNX2 bypasses the inhibitory effect induced by shFGFR-2 we propose that the signaling pathways involved are FGFR-2-independent supporting the hypothesis that FGF2 activates FGFR-2 and in turn RUNX2. Currently we are studying the signaling pathways involved in RUNX2 mediated tumor growth. The results reported here emphasize the use of FGFR-2 inhibitors in combination with standard therapy in breast cancer. Citation Format: Cecilia Pérez Piñero, María May, Isabel A. Lüthy, Claudia Lanari. RUNX2 rescues the inhibitory effect of FGFR-2 silencing and increases the metastatic potential of IBH-6 human breast cancer xenografts. [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 3120. doi:10.1158/1538-7445.AM2014-3120

Treatment of human hepatocellular carcinoma by the oncolytic herpes simplex virus G47delta.

BackgroundOncolytic herpes simplex virus (HSV) can replicate in and kill cancer cells while sparing the adjacent normal tissue. Hepatocellular carcinoma (HCC) is amongst the most common and lethal cancers, especially in Third World countries. In this study, the cytotoxicity of a third-generation oncolytic HSV, G47Δ, was investigated in different human HCC cell lines and in an immortalized human hepatic cell line. Additionally, subcutaneous models of HCC were established to evaluate the in vivo anti-tumor efficacy of G47Δ.MethodsThe HepG2, HepB, SMMC-7721, BEL-7404, and BEL-7405 human HCC cell lines and the HL-7702 human hepatic immortalized cell lines were infected with G47Δ at different multiplicities of infection (MOIs). The viability of infected cells was determined, and the G47Δ replication was identified by X-gal staining for LacZ expression. Two subcutaneous (s.c.) HCC tumor models of HCC were also established in Balb/c nude mice, which were intratumorally(i.t.) treated with either G47Δ or mock virus. Tumor volume and mouse survival times were documented.ResultsMore than 95% of the HepG2, Hep3B,and SMMC-7721 HCC cells were killed on by day 5 after infection with a MOI’s of 0.01. For the HL-7702 human hepatic immortalized cells, 100% of the cells were killed on by day 5 after infection with a MOI’s of 0.01. The BEL-7404 HCC cell line was less susceptible with about 70% cells were killed by day 5 after infection with a MOI’s of 0.01. Whereas the BEL-7405 HCC cells were the least susceptible, with only 30% of the cells were killed. Both the SMMC-7721 and BEL-7404 cells form aggressive sc tumor models. G47Δ replicates in the tumors, such that most of the tumors regressed after the G47Δ-treatment, and treated tumor-bearing mice survived much longer than the control animals.ConclusionsG47Δ effectively kills human HCC cells and an immortalized hepatic cell line at low MOI. Intra-tumor injection of G47Δ can induce a therapeutic effect and prolong the survival of treated mice bearing SMMC-7721 and BEL-7404 subcutaneously (s.c.) tumors. Thus, G47Δ may be useful as a novel therapeutic agent for HCC.

AB0811 Total Antioxidant Status and Oxidative Stress in Patients with Osteopenia and Osteoporosis

BackgroundOsteoporosis (OP) is the most common metabolic bone disorders. OP results from an imbalance between bone formation and resorption in favor of the later1. Increased oxidative stress (OS) in bone...

Combination of 13-Cis retinoic acid and lovastatin: marked antitumor potential in vivo in a pheochromocytoma allograft model in female athymic nude mice.

Currently, there are no reliably effective therapeutic options for metastatic pheochromocytoma (PCC) and paraganglioma. Moreover, there are no therapies that may prevent the onset or progression of tumors in patients with succinate dehydrogenase type B mutations, which are associated with very aggressive tumors. Therefore, we tested the approved and well-tolerated drugs lovastatin and 13-cis-retinoic acid (13cRA) in vitro in an aggressive PCC mouse cell line, mouse tumor tissue-derived (MTT) cells, and in vivo in a PCC allograft nude mouse model, in therapeutically relevant doses. Treatment was started 24 hours before sc tumor cell injection and continued for 30 more days. Tumor sizes were measured from outside by caliper and sizes of viable tumor mass by bioluminescence imaging. Lovastatin showed antiproliferative effects in vitro and led to significantly smaller tumor sizes in vivo compared with vehicle treatment. 13cRA promoted tumor cell growth in vitro and led to significantly larger viable tumor mass and significantly faster increase of viable tumor mass in vivo over time compared with vehicle, lovastatin, and combination treatment. However, when combined with lovastatin, 13cRA enhanced the antiproliferative effect of lovastatin in vivo. The combination-treated mice showed slowest tumor growth of all groups with significantly slower tumor growth compared with the vehicle-treated mice and significantly smaller tumor sizes. Moreover, the combination-treated group displayed the smallest size of viable tumor mass and the slowest increase in viable tumor mass over time of all groups, with a significant difference compared with the vehicle- and 13cRA-treated group. The combination-treated tumors showed highest extent of necrosis, lowest median microvessel density and highest expression of α-smooth muscle actin. The combination of high microvessel density and low α-smooth muscle actin is a predictor of poor prognosis in other tumor entities. Therefore, this drug combination may be a well-tolerated novel therapeutic or preventive option for malignant PCC.

T-cell therapy of pancreatic cancer

The integrin avb6 is expressed on the tumour cell surface in >90% of pancreatic ductal carcinomas (PDACs) and high levels of expression of the b6 gene correlates with reduced overall survival from PDAC (HR1⁄41.74, Logrank p1⁄46x10-4). Using a panel of human PDAC cell lines and avb6blocking antibodies we find that in PDAC cells avb6 mediates cell migration, invasion, proliferation and survival. Antibody blockade of avb6 in vitro of Panc0403 induced a G2/M phase block (compared with control, 6.5% to 16%) whereas in CfPac1 cells there was a significant increase in apoptosis (comparedwith control, the sub-G1 fraction increased from 8.5% to 46%). Thus avb6 may be an effective therapeutic target for treating PDAC. To test this we grew xenografts of CfPAc1 co-implanted with the human pancreatic stellate cell line PS-1. Histologically the subcutaneous implanted tumours were indistinguishable from orthotopic implanted tumours and showed increased desmoplasia and reduced angiogenesis compared with tumours derived from CfPac1 cells injected alone. Therapy with avb6-blocking antibody (264RAD, ip, 10mg/kg), gemcitabine (ip, 100mg/kg), diluent or both antibody and gemcitabine was given to mice bearing 100mm3 CfPac1/PS1 sc tumours for 6 weeks. While all three therapeutic arms were significantly effective, the greatest therapeutic effect was seen with the 264RAD/Gem combination followed by Gem and then 264RAD alone. In fact combination therapy eliminated tumours in some mice and reduced the volume of all xenografts below 12mm3. Immunohistochemistry showed in 264RAD-treated mice that there was reduced proliferation (Ki67) and growth signaling (pErk) but increased apoptosis (activated Caspase3) in tumour tissue. These data suggest that antibody targeting of avb6 in humans could have a major therapeutic effect and should be considered as part of future treatment regimens for PDAC. 1: Allen MD, et al. Altered Microenvironment Promotes Progression of Preinvasive Breast Cancer: Myoepithelial Expression of avb6 Integrin in DCIS Identifies High-risk Patients and Predicts Recurrence. Clin Cancer Res. 2014 Jan 15;20(2):344-57. 2: Kogelberg H, et al. Generation and characterization of a diabody targeting the avb6 integrin. PLoS One. 2013 Sep 4;8(9):e73260. 3: Eberlein C, et al. A human monoclonal antibody 264RAD targeting avb6 integrin reduces tumour growth and metastasis, and modulates key biomarkers in vivo. Oncogene. 2013 Sep 12;32(37):4406-16.

Fluorescent conjugate of sLex-selective bisboronic acid for imaging application

Carbohydrate-based biomarkers such as sialyl Lewis X are known to correlate with cancer formation and progression. By targeting sialyl Lewis X, we have developed a boronolectin-fluorophore conjugate, which was able to selectively label and image xenograft (sc) tumor. This represents the very first example that a small molecule capable of recognizing a carbohydrate biomarker was used for optical imaging application.

CALM study: A phase II study of intratumoral coxsackievirus A21 in patients with stage IIIc and stage IV malignant melanoma.

TPS3128 Background: Coxsackievirus A21 (CVA21: CAVATAK) is a naturally occurring "common cold" virus. CVA21 displays potent oncolytic activity against both in vitro cultures of cancer cells and against in vivo xenografts of human cancers in mouse models of melanoma, prostate cancer, breast cancer and multiple myeloma, all which exhibit high surface ICAM-1 expression, which is used for viral entry. In mouse human melanoma xenograft CVA21 challenge models, progeny virus released from infected cells is capable of targeting adjacent cells, entering the systemic circulation, and destroying micro-metastatic foci. In a phase 1 study, two intralesional injections of CVA21 were shown to reduce or stabilize the growth of injected melanoma lesions. Methods: The CALM study investigates the efficacy and safety of intratumoral CVA21 in approximately 63 pts with treated or untreated unresectable Stage IIIC-IVM1c melanoma. Pts are treated with up to 3 x 108 TCID50 intratumorally on study days 1, 3, 5 and 8 and then every three weeks for a further 6 injections. Key eligibility criteria are ≥ 18 yrs old, ECOG 0-1, and at least 1 injectable cutaneous, sc, or nodal tumor >1.0 cm. The primary endpoint is irPFS at 6 months following tx; secondary endpoints include durable response rate and OS. A 2-stage Simon’s minimax design will be employed. Based on data from previous trials and literature, a target overall irPFS at 6 months of 22.5% versus a projected rate of 10% in the target population is selected. With an alpha level of 5% and 80% of power, a total of 54 evaluable patients will be required to test the null hypothesis that the true irPFS rate is <10% versus the alternative hypothesis that the true irPFS rate is at least 22.5%. Thirty-five patients will be treated at Stage I. If 3 or more objective responses (CR or PR) are achieved by modified RECIST 1.1 criteria in the first 35 patients, then the study will complete enrollment. Results: Currently, 21 patients have been enrolled on the study and treated with CVA21 injections. The treatment has been well tolerated. The Stage I interim efficacy endpoint of > 3 objective responses has been achieved, and the second stage of the study is proceeding with a planned enrollment of a total of 63 patients. Clinical trial information: NCT01227551.

The dual effect of mesenchymal stem cells on tumour growth and tumour angiogenesis

IntroductionUnderstanding the multiple biological functions played by human mesenchymal stem cells (hMSCs) as well as their development as therapeutics in regenerative medicine or in cancer treatment are major fields of research. Indeed, it has been established that hMSCs play a central role in the pathogenesis and progression of tumours, but their impact on tumour growth remains controversial.MethodsIn this study, we investigated the influence of hMSCs on the growth of pre-established tumours. We engrafted nude mice with luciferase-positive mouse adenocarcinoma cells (TSA-Luc+) to obtain subcutaneous or lung tumours. When tumour presence was confirmed by non-invasive bioluminescence imaging, hMSCs were injected into the periphery of the SC tumours or delivered by systemic intravenous injection in mice bearing either SC tumours or lung metastasis.ResultsRegardless of the tumour model and mode of hMSC injection, hMSC administration was always associated with decreased tumour growth due to an inhibition of tumour cell proliferation, likely resulting from deep modifications of the tumour angiogenesis. Indeed, we established that although hMSCs can induce the formation of new blood vessels in a non-tumoural cellulose sponge model in mice, they do not modify the overall amount of haemoglobin delivered into the SC tumours or lung metastasis. We observed that these tumour vessels were reduced in number but were longer.ConclusionsOur results suggest that hMSCs injection decreased solid tumour growth in mice and modified tumour vasculature, which confirms hMSCs could be interesting to use for the treatment of pre-established tumours.

Abstract 233: Therapeutic efficacy of cancer stem cell-based vaccine in an adjuvant setting.

Abstract So far, the majority of cancer stem cell (CSC) studies have been confined to human tumors inoculated into severely immunosuppressed hosts (e.g. SCID mice), where adaptive immune responses are absent, and such hosts are therefore not suitable for the immunologic evaluation of CSCs. The lack of immunocompetent syngeneic animal tumor models where stem cells can be isolated has been the primary obstacle to evaluating the immunogenicity of CSCs. We recently reported the identification of CSC-enriched populations using ALDEFLUOR/ALDH as a marker in murine melanoma D5 and squamous cell cancer SCC7, and evaluated their immunogenicity in two genetically distinct syngeneic immunocompetent hosts. Enriched CSCs are immunogenic and significantly more effective as an antigen source in inducing protective anti-tumor immunity than unsorted tumor cells or purified non-CSCs. We further found that selective targeting of CSCs by CSC-primed antibodies and T cells represents the mechanisms involved in CSC vaccine-conferred protective immunity. If a CSC vaccine is to be clinically relevant, it needs to be evaluated in a therapeutic model. In this study, we examined two models for this purpose. The first model involves the treatment of micrometastatic disease. We initiated vaccination 24 hrs after s.c. inoculation of D5 tumor cells in the syngeneic immunocompetent host (B6 mice), and repeated the vaccination one week later. Comparison was made among the group with PBS-injected controls; mice vaccinated with dendritic cells (DC) pulsed with the lysate of ALDHhigh D5 cells (CSC-TPDC) vs. ALDHlow D5 cells (ALDHlow -TPDC). We found that CSC-TPDC inhibited the tumor growth more than ALDHlow-TPDC and PBS controls, and significantly prolonged the survival of the tumor-bearing mice. The second model involves the treatment of established tumors using CSC-TPDC vaccine as an additional strategy to radiation therapy (RT). Day 7 D5 sc tumors were treated with localized RT with repeat treatments on D8. Vaccine therapy commenced on D8. This combination therapy was repeated twice with one week apart. CSC-TPDC plus RT mediated D5 tumor regression more than ALDHlow-TPDC + RT, RT alone and PBS controls, and prolonged the survival of the tumor-bearing mice. Importantly, we observed metastasis of the D5 tumors to the lung in all the groups except for the one treated with CSC-TPDC plus RT. The findings may help develop novel approaches for cancer treatment by utilizing an autologous cancer stem cell-based vaccine to selectively and immunologically target cancer stem cells. Citation Format: Lin Lu, Huimin Tao, Martin Egenti, Max S. Wicha, Alfred E. Chang, Qiao Li. Therapeutic efficacy of cancer stem cell-based vaccine in an adjuvant setting. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 233. doi:10.1158/1538-7445.AM2013-233

Abstract P2-05-09: Identification and validation of a miRNA signature associated with breast cancer progression

Abstract Introduction: MiRNAs are aberrantly expressed in both the circulation and tissue of patients with breast tumours, however little is currently known about the relationship between circulating and tissue miRNAs. The aim of this study was to quantify circulating and tumour tissue miRNA expression in a murine model of breast cancer and identify novel circulating markers of disease progression. Materials and Methods: Athymic nude mice (n = 20) received either a mammary fat pad (n = 8, MFP), or subcutaneous (n = 7, SC) injection of MDA-MB-231 cells. Controls received no tumour cells (n = 5). Tumour volume was monitored weekly and blood sampling performed at weeks 1, 3 and 6 following tumour induction (total n=60). Animals were sacrificed at week 6 and tumour tissue (n = 15), lungs (n = 20) and enlarged lymph nodes (n = 3) harvested. RNA were extracted from all samples (n = 98) and relative expression of miRNAs previously associated with tumour progression were quantified using RQ-RCR. A microRNA array was also preformed comparing animals with early (weeks 1, n=5) versus late (week 6, n=5) disease and results were analysed using RQ-PCR in all blood samples (n = 60). Reverse transcription for the relevant targets and endogenous controls was carried out and expression was quantified using RQ-PCR. Results: MiR-10b expression was significantly higher in MFP compared to SC tumours (p < 0.05), with the highest levels in diseased lymph nodes (p < 0.05). MiR-10b was undetectable in the circulation. MiR-221 expression was significantly increased in tumour tissue (p < 0.001). MiR-195 and miR-497 were significantly decreased in tumour tissue (p < 0.05), and also in the circulation of animals 3 weeks following tumour induction (p < 0.05). At both tissue and circulating level, a significant correlation was observed between miR-497 and miR-195 (r = 0.61, p < 0.001; r = 0.41, p < 0.01 respectively). Microarray analysis revealed the level of 47 miRs to be significantly changed between week 1 and week 6 as disease progressed in a murine model, 10 of which (miR A-J) were significantly altered and internally validated within the murine model. Conclusions: This study highlights the importance of miRNAs in breast cancer, with each displaying distinct roles in circulation and tissue. Novel circulating miRNAs which have never previously been associated with breast cancer have been identified, with quantification of circulating levels in breast cancer patients currently ongoing. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-05-09.

Blood vessel hyperpermeability and pathophysiology in human tumour xenograft models of breast cancer: a comparison of ectopic and orthotopic tumours

BackgroundHuman tumour xenografts in immune compromised mice are widely used as cancer models because they are easy to reproduce and simple to use in a variety of pre-clinical assessments. Developments in nanomedicine have led to the use of tumour xenografts in testing nanoscale delivery devices, such as nanoparticles and polymer-drug conjugates, for targeting and efficacy via the enhanced permeability and retention (EPR) effect. For these results to be meaningful, the hyperpermeable vasculature and reduced lymphatic drainage associated with tumour pathophysiology must be replicated in the model. In pre-clinical breast cancer xenograft models, cells are commonly introduced via injection either orthotopically (mammary fat pad, MFP) or ectopically (subcutaneous, SC), and the organ environment experienced by the tumour cells has been shown to influence their behaviour.MethodsTo evaluate xenograft models of breast cancer in the context of EPR, both orthotopic MFP and ectopic SC injections of MDA-MB-231-H2N cells were given to NOD scid gamma (NSG) mice. Animals with matched tumours in two size categories were tested by injection of a high molecular weight dextran as a model nanocarrier. Tumours were collected and sectioned to assess dextran accumulation compared to liver tissue as a positive control. To understand the cellular basis of these observations, tumour sections were also immunostained for endothelial cells, basement membranes, pericytes, and lymphatic vessels.ResultsSC tumours required longer development times to become size matched to MFP tumours, and also presented wide size variability and ulcerated skin lesions 6 weeks after cell injection. The 3 week MFP tumour model demonstrated greater dextran accumulation than the size matched 5 week SC tumour model (for P < 0.10). Immunostaining revealed greater vascular density and thinner basement membranes in the MFP tumour model 3 weeks after cell injection. Both the MFP and SC tumours showed evidence of insufficient lymphatic drainage, as many fluid-filled and collagen IV-lined spaces were observed, which likely contain excess interstitial fluid.ConclusionsDextran accumulation and immunostaining results suggest that small MFP tumours best replicate the vascular permeability required to observe the EPR effect in vivo. A more predictable growth profile and the absence of ulcerated skin lesions further point to the MFP model as a strong choice for long term treatment studies that initiate after a target tumour size has been reached.

Relationship between Circulating and Tissue microRNAs in a Murine Model of Breast Cancer

MiRNAs are key regulators of tumorigenesis that are aberrantly expressed in the circulation and tissue of patients with cancer. The aim of this study was to determine whether miRNA dysregulation in the circulation reflected similar changes in tumour tissue. Athymic nude mice (n = 20) received either a mammary fat pad (n = 8, MFP), or subcutaneous (n = 7, SC) injection of MDA-MB-231 cells. Controls received no tumour cells (n = 5). Tumour volume was monitored weekly and blood sampling performed at weeks 1, 3 and 6 following tumour induction (total n = 60). Animals were sacrificed at week 6 and tumour tissue (n = 15), lungs (n = 20) and enlarged lymph nodes (n = 3) harvested. MicroRNAs were extracted from all samples (n = 98) and relative expression quantified using RQ-PCR. MiR-221 expression was significantly increased in tumour compared to healthy tissue (p<0.001). MiR-10b expression was significantly higher in MFP compared to SC tumours (p<0.05), with the highest levels detected in diseased lymph nodes (p<0.05). MiR-10b was undetectable in the circulation, with no significant change in circulating miR-221 expression detected during disease progression. MiR-195 and miR-497 were significantly decreased in tumour tissue (p<0.05), and also in the circulation of animals 3 weeks following tumour induction (p<0.05). At both tissue and circulating level, a positive correlation was observed between miR-497 and miR-195 (r = 0.61, p<0.001; r = 0.41, p<0.01 respectively). This study highlights the distinct roles of miRNAs in circulation and tissue. It also implicates miRNAs in disease dissemination and progression, which may be important in systemic therapy and biomarker development.