Non-treated Tumors Research Articles

Proposal for a Multifactorial Prognostic Score That Accurately Classifies 3 Groups of Gastric Carcinoma Patients With Different Outcomes After Neoadjuvant Chemotherapy and Surgery

We have developed a multifactorial histopathological prognostic score (PRSC) for patients with gastric cancer treated with neoadjuvant chemotherapy before surgery for the accurate discrimination of patient subgroups with differing outcomes. For patients with gastric cancer who undergo multimodal treatment, the postoperative staging classifications used for nontreated tumors may not accurately predict patient prognosis. We evaluated 428 gastric carcinoma specimens after a cisplatin-based chemotherapy. The factors for the Union for International Cancer Control/American Joint Committee on Cancer (UICC/AJCC) ypT-category, ypN-category, and histopathological tumor regression were assigned a value from 1 to 3 as follows: ypT0 to 2 = 1 point; ypT3 = 2 points; ypT4 = 3 points; ypN0 = 1 point; ypN1 to 2 = 2 points; ypN3a to 3b = 3 points; less than 10% residual tumor per tumor bed = 1 point; 10% to 50% residual tumor per tumor bed = 2 points; and greater than 50% residual tumor per tumor bed = 3 points. A 3-tiered PRSC based on the sum value was established (group A: 3-4 points; group B: 5-7 points; group C: 8-9 points) and was found to correlate with patient prognosis. The PRSC showed a clear discrimination of 3 significantly different prognostic groups (group A: 76 patients; group B: 210 patients; group C: 142 patients; P < 0.001). In multivariate analyses, including the completeness of resection, tumor diameter, lymphatic vessel invasion, tumor grading, and Lauren classification, the PRSC was the only independent prognostic factor for overall survival (hazard ratio [HR] = 2.03; 95% confidence intervals [CI], 1.49-2.78; P < 0.001). It was slightly superior to the UICC/AJCC staging system (HR = 1.66; 95% CI, 1.20-2.27; P = 0.002) when analyzed with tumor regression as an additional independent factor (HR = 1.27; 95% CI, 1.01-1.62; P = 0.044) included in the analysis. The proposed PRSC reveals the most accurate prediction of survival for patients with gastric carcinoma after neoadjuvant chemotherapy followed by surgery. The PRSC clearly identifies 3 subgroups with different prognoses and may be helpful for therapeutic decisions.

Longitudinal Imaging Studies of Tumor Microenvironment in Mice Treated with the mTOR Inhibitor Rapamycin

Rapamycin is an allosteric inhibitor of mammalian target of rapamycin, and inhibits tumor growth and angiogenesis. Recent studies suggested a possibility that rapamycin renormalizes aberrant tumor vasculature and improves tumor oxygenation. The longitudinal effects of rapamycin on angiogenesis and tumor oxygenation were evaluated in murine squamous cell carcinoma (SCCVII) by electron paramagnetic resonance imaging (EPRI) and magnetic resonance imaging (MRI) to identify an optimal time after rapamycin treatment for enhanced tumor radioresponse. Rapamycin treatment was initiated on SCCVII solid tumors 8 days after implantation (500–750 mm3) and measurements of tumor pO2 and blood volume were conducted from day 8 to 14 by EPRI/MRI. Microvessel density was evaluated over the same time period by immunohistochemical analysis. Tumor blood volume as measured by MRI significantly decreased 2 days after rapamycin treatment. Tumor pO2 levels modestly but significantly increased 2 days after rapamycin treatment; whereas, it decreased in non-treated control tumors. Furthermore, the fraction of hypoxic area (pixels with pO2<10 mm Hg) in the tumor region decreased 2 days after rapamycin treatments. Immunohistochemical analysis of tumor microvessel density and pericyte coverage revealed that microvessel density decreased 2 days after rapamycin treatment, but pericyte coverage did not change, similar to what was seen with anti-angiogenic agents such as sunitinib which cause vascular renormalization. Collectively, EPRI/MRI co-imaging can provide non-invasive evidence of rapamycin-induced vascular renormalization and resultant transient increase in tumor oxygenation. Improved oxygenation by rapamycin treatment provides a temporal window for anti-cancer therapies to realize enhanced response to radiotherapy.

Diffusion-weighted MRI for detection and differentiation of musculoskeletal tumorous and tumor-like lesions in pediatric patients

MRI is the diagnostic mainstay for detection and differentiation of musculoskeletal tumors. However, a projection regarding the biological dignity of lesions based on standard MRI sequences remains difficult and uncertain. This study was undertaken to analyse whether diffusion-weighted MRI (DWI) can distinguish between benign and malignant musculoskeletal tumorous and tumor-like lesions in pediatric patients. MR examinations of 44 consecutive pediatric patients (26 girls, mean age 11±6 years) including standard sequences and DWI (b=50/800 s/mm(2)) at 1.5 or 3 Tesla were retrospectively evaluated. The study group contained 10 patients with non-treated malignant tumors and 34 patients with benign lesions. Size, relative signal intensity and apparent diffusion coefficient (ADC, unit ×10(-3) mm(2)/s) were determined in one lesion per patient. Mean ADC was 0.78±0.45×10(-3) mm(2)/s in patients with malignant tumors and 1.71±0.75 ×10(-3) mm(2)/s in patients with benign lesions (P<0.001). Relative operating characteristics (ROC) analysis showed a sensitivity of 90% and a specificity of 91% for malignancy, based on an ADC cut-off value of ≤1.03. On logistic regression, mean ADC and lesion size accounted for 62% of variability in benign vs. malignant tumors. For malignant tumors, the signal intensity ratio was higher on DWI than on T1w post-contrast images (P<0.002). Two cases of local tumor recurrence were diagnosed by DWI only. DWI shows promising results for determination of biological dignity in musculoskeletal tumors. Mean ADC ≤1.03×10(-3) mm(2)/s is a strong indicator of malignancy at the first diagnosis. The use of DWI for early diagnosis of tumor recurrence in comparison with standard MRI sequences should be evaluated in prospective studies.

Fast microbubble dwell-time based ultrasonic molecular imaging approach for quantification and monitoring of angiogenesis in cancer.

PURPOSE: To develop and test a fast ultrasonic molecular imaging technique for quantification and monitoring of angiogenesis in cancer. MATERIALS AND METHODS: A new software algorithm measuring the dwell time of contrast microbubbles in near real-time (henceforth, fast method) was developed and integrated in a clinical ultrasound system. In vivo quantification and monitoring of tumor angiogenesis during anti-VEGF antibody therapy was performed in human colon cancer xenografts in mice (n=20) using the new fast method following administration of vascular endothelial growth factor receptor 2 (VEGFR2)-targeted contrast microbubbles. Imaging results were compared with a traditional destruction/replenishment approach (henceforth, traditional method) in an intra-animal comparison. RESULTS: There was excellent correlation (R(2)=0.93; P<0.001) between the fast method and the traditional method in terms of VEGFR2-targeted in vivo ultrasonic molecular imaging with significantly higher (P=0.002) imaging signal in colon cancer xenografts using VEGFR2-targeted compared to control non-targeted contrast microbubbles. The new fast method was highly reproducible (ICC=0.87). Following anti-angiogenic therapy, ultrasonic molecular imaging signal decreased by an average of 41±10%, whereas imaging signal increased by an average of 54±8% in non-treated tumors over a 72-hour period. Decreased VEGFR2 expression levels following anti-VEGF therapy were confirmed on ex vivo immunofluorescent staining. CONCLUSIONS: Fast ultrasonic molecular imaging based on dwell time microbubble signal measurements correlates well with the traditional measurement method, and allows reliable in vivo monitoring of anti-angiogenic therapy in human colon cancer xenografts. The improved work-flow afforded by the new quantification approach may facilitate clinical translation of ultrasonic molecular imaging.

CD40L expression by CD8+ T cells is essential for successful rejection of SV40 T antigen expressing tumor cells in the absence of CD4+ T cells (162.38)

Abstract Cytotoxicity is considered to be the main effector function of CD8+ T cells in the immune system. However, we demonstrate here that CD40L, one of the central effector molecules of CD4+ helper T cells, is expressed by a substantial part of human and murine CD8+ T cells after activation. Human central as well as effector memory CD8+ T cells comprise up to 50% CD40L+ cells after polyclonal activation. These CD40L+ CD8+ T cells display a non-cytotoxic phenotype and resemble functionally various distinct CD4+ T helper cell subtypes with respect to expression of IL2 and IFNγ or IL4 as well as with respect to their potential to induce activation of B cells and maturation of DCs in vitro. To investigate the functional relevance of CD40L expression by CD8+ T cells we analyzed a murine anti-tumor response. During the whole course of the response 50% of the tumor-specific CD8+ T cells expressed CD40L. While application of CD40L competent wt CD8+ T cells to Rag1ko mice challenged with the tumor cells prevented the establishment of solid tumors, injection of CD40Lko CD8+ T cells resulted in a non-controlled tumor progression similar to non-treated tumors. Our results disclose an essential functional relevance of CD40L expressed by CD8+ T cells. Particularly in situations where CD4+ T-cell help is diminished, MHC-II antigen presentation is reduced or limited danger signals are present, CD40L+ CD8+ T cells may exert essential helper responsibilities for a competent cellular immune response.

Abstract 5744: Preclincial therapy of androgen independent prostate cancer using combinationLu-177-BB2r targeting peptides and docetaxel

Abstract Introduction: The bombesin receptor subtype 2 (BB2r) is an attractive target for the selective delivery of peptide receptor targeted radiation therapy for androgen independent prostate cancer (AIPC). Earlier work from our group has focused on the use of the BB2r agonist 177Lu-177-DOTA-8-AOC-BBN(7-14)NH2 administered concurrently with docetaxel to achieve tumor control in flank xenograft models of AIPC. We now present a direct comparison of 177Lu-BB2r agonist verses 177Lu-BB2r antagonist demonstrating the superiority of employing the BB2r antagonist, RM2, for the treatment of AIPC using a SCID mouse model. Methods: DOTA-8-AOC-BBN(7-14)NH2 was synthesized in house. RM2 was commercially synthesized. 177Lu radiolabeled peptides were prepared and purified using analytical HPLC resulting in a high specific activity preparation. The AIPC tumor bearing mice were created following bilateral flank inoculation of 1X106 PC-3 cells. Pharmacokinetic analysis and Micro-SPECT imaging of 177Lu-RM2 was conducted out to 14 days post administration. Survival study design compared a 177Lu-BB2r agonist /docetaxel arm with a 177Lu-BB2r antagonist /docetaxel arm including a non-treated tumor bearing control arm. Multi-modal therapy consisting of docetaxel (8mg/kg; administered weekly) and 177Lu-BB2r peptide therapy (37MBq/25g; administered weekly) was delivered for six consecutive weeks. Subjects were removed from study based on evidence of morbidity and body condition score. Results: Pharmacokinetic analysis and Micro-SPECT imaging of 177Lu-RM2 demonstrated prolonged tumor retention with minimal non-target tissue accumulation observed within 24 hours post administration. Survival analysis at Day 100 demonstrated that 67% of the mice in the multi-modal BB2r antagonist therapy arm (chemo + targeted 177Lu radiation therapy) were responding to treatment as compared to a 17% survival of the combination BB2r agonist/chemotherapy arm only arm at the same time point. 177Lu-RM2 antagonist therapy demonstrated tumor regression and complete control whereas 177Lu agonist therapy tumor regression was short lived with complete regrowth observed 45 days following last treatment. Conclusions: Our preclinical data suggests that 177Lu-BB2r antagonist therapy is superior to 177Lu-BB2r agonist therapy when used concurrently with docetaxel employing a preclinical model of AIPC. The use of 177Lu-BB2r antagonists permits accurate SPECT imaging of therapeutic agent localization at prolonged times post injection. More extensive evaluation of 177Lu-BB2r antagonist therapy for AIPC are warranted to exploit the potential of these agents for clinical use. 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 5744. doi:1538-7445.AM2012-5744

Abstract 1570: Frameshift peptides as prophylactic cancer vaccines antigens

Abstract Background: The goal of the project is to develop a universal, prophylactic cancer vaccine. We are integrating bioinformatics, genetics, proteomics and immunological methods to identify tumor DNA-encoded components for the development of an effective vaccine. In a previous study, we used bioinformatic and genetics analyses to identify a list of abberant transcripts associated with tumor cells that encode frameshift-derived (FS) neo-peptides. These FS peptides are a result of various gene mutations and abnormal splicings in tumor cells that would cause expression of gene sequences in alternate reading frames. The transcript encoding one of these vaccine component candidates, SMC1A (Structural Maintenance of Chromosomes 1A) FS was detected in numerous mouse and human tumor cell lines and primary human breast and pancreatic tumors. One dose genetic immunization with a plasmid expressing SMC1A FS significantly delayed tumor growth in C57BL/6 mice transplanted with the B16F10 melanoma cell line. To exam protection of SMC1A FS in a more realistic prophylactic model, we investigated the protection of SMC1A FS in two mouse spontaneous breast tumor models. Results: We similarly detected the transcript corresponding to the SMC1A FS peptide in tumors from spontaneous mouse breast tumor models: FVB/N-NeuT and BALB-NeuT. The SMC1A FS expression increases with the expression of Rat Her-2 in mouse mammary gland. Wild type and mutated Rat Her 2 are the oncogenes that induce spontaneously breast tumor development in FVB/N-NeuT and BALB-NeuT respectively. Both anti-SMC1A FS IgG and SMC1A FS peptide induced IFN-γ released splenocytes were detected in non-treated tumor bearing FVB/N-NeuT mice. One dose genetic immunization of mice with an SMC1A FS expression plasmid significantly reduced spontaneous breast tumor development in FVB/N-NeuT mouse as compared to that in non-treated mice. Genetic immunization followed by a protein boost (prime/boost regimen) in BALB-NeuT mice also significantly reduced tumor development and the reduction is associated with antibody reactivity to SMC1A FS. Discussion: The protection of SMC1A FS prophylactic immunization in spontaneous mouse tumor models indicates SMC1A S can be a potential prophylactic cancer vaccine candidate to prevent a variety of tumors types. We are applying the same basic approach to accumulate sufficient antigens to constitute a broadly protective, prophylactic cancer vaccine. (This research is supported by an Innovation Award from the DoD and The Keck Foundation to SAJ.) 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 1570. doi:1538-7445.AM2012-1570

Abstract 2806: BRAF and MEK inhibitor profiling across 240 tumor cell lines to correlate with sensitivity and resistant biomarkers

Abstract Activated kinases that induce cell proliferation have been attractive targets for targeted cancer therapy development. Cancer cells could become dependent on tumor-specific activated kinases and this tendency has been coined oncogene addiction. Here, we investigated three activated kinase inhibitors: BRAF inhibitor, PLX-4032 analog, and the MEK inhibitors, CI1040 and PD0325901. We established a high throughput cellular approach to profile 240 human tumor cell lines identifying genotype-correlated sensitivity or resistance. Proliferative, apoptotic and cell cycle arrest responses were measured using multiplexed high content screening with automated fluorescence microscopy and image analysis based technology. Growth index was measured using nuclear dye. Activated caspase 3 antibodies were used for the apoptosis induction detection. Phospho-histone H3 antibodies were used to measure the cell cycle block. We generated cell line profiles to reveal drug sensitivity and resistance patterns that may correlate with the clinical genotype responses. Cell lines with BRAFV600E mutations showed overlapping sensitivity to all three MEK and BRAF inhibitors. RAS mutations confer resistance to the BRAF inhibitor and confer sensitivity to both MEK inhibitors. In addition, we used Alphascreen technology to measure phosphoERK across the 240 non-treated tumor cell line panel. We found that the majority of PLX-4032 sensitive cell lines expressed high levels of phosphoERK. In addition, we investigated BRAF, MEK and EGFR inhibitor combinations to evaluate potential synergies. This preclinical approach can be used to predict mechanisms of susceptibility or resistance to these agents which in turn could be used for the optimization of targeted cancer therapeutics. 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 2806. doi:1538-7445.AM2012-2806

Combination with bortezomib enhances the antitumor effects of nanoparticle-encapsulated thiostrepton

Bortezomib is well-known for inducing cell death in cancer cells, specifically through the mechanism of proteasome inhibition. Thiostrepton, a thiazole antibiotic, has also been described for its proteasome inhibitory action, although differing slightly to bortezomib in the proteasomal site to which it is active. Previously we had shown the synergic effect of bortezomib and thiostrepton in breast cancer cells in vitro, where sub-apoptotic concentrations of both proteasome inhibitors resulted in synergic increase in cell death when combined as a treatment. Here, we administered such a combination to MDA-MB-231 xenograft tumors in vivo, and found that the effect of complementary proteasome inhibitors reduced tumor growth rates more efficiently than compared with when administered alone. Increased induction of apoptotic activity in tumors was found be associated with the growth inhibitory activity of combination treatment. Further examination additionally revealed that combination-treated tumors exhibited reduced proteasome activity, compared with non-treated and single drug-treated tumors. These data suggest that this drug combination may be useful as a therapy for solid tumors.

Abstract B23: Selective inhibition of a long non-coding RNA (lncRNA), MALAT1 by antisense oligonucleotides results in significant anti-tumor effects in a variety of preclinical cancer models

Abstract An increasing body of evidence supports the notion that long noncoding RNAs (lncRNAs) play important roles in diseases including cancer. MALAT1 (also called NEAT2) was originally identified in tumors of highly metastatic non-small cell lung cancer (NSCLC), however, the functional role of MALAT1 in carcinogenesis has remained largely unknown. However, MALAT1 is highly expressed in various types of cancer and is implicated in the regulation of alternative splicing. To determine if MALAT1 constitutes an important driver of tumor growth in vivo, we developed potent antisense oligonucleotides (ASOs) that were able to achieve strong inhibition of MALAT1 in the tumor cells of a variety of preclinical models following systemic delivery. MALAT1 ASOs were well tolerated in rodents at doses leading to &amp;gt;95% inhibition of MALAT1 RNA in the liver. Inhibition of MALAT1 expression in tumor and tumor-associated stromal cells was determined by both q-RT-PCR using species-specific probe/primer sets and/or in situ ‘viewRNA’ technology, where target knockdown can be visualized on a cell by cell basis. Systemic administration of mouse MALAT1 ASOs resulted in a decrease in the numbers of polyps and proliferation index (BrdU (+)) in the small intestine of Apcmin mouse model of colon cancer and correlated well with MALAT1 inhibition in the polyps. Mouse MALAT1 ASOs were also effective in a DEN-induced HCC model, where ASO treatment reduced the target RNA ~ 90% in tumor cells with a concomitant decrease in tumor numbers, while control ASO had no effects on either measure. In addition, higher expression of MALAT1 in non-treated tumors compared to adjacent normal hepatocytes was also clearly visualized by the in situ ‘view RNA’ method. Furthermore, MALAT1 ASO significantly delayed tumor growth in C26 colon cancer and reduced tumor size in TRAMP mouse model of prostate cancer, where the target RNA was decreased by 80% in tumor cells. In a human NSCLC patient-derived xenograft model, both significant MALAT1 RNA reduction and a delay in tumor growth were achieved after MALAT1 ASO treatment. The effects of MALAT1 downregulation by ASO were not limited to the inhibition of tumor growth alone. MALAT1 ASO treatment not only inhibited the growth of primary tumors in the EBC-1 human NSCLC xenograft model, but also resulted in a decrease in lung metastasis as measured by micro CT scanning. Furthermore, cross-species MALAT1 ASOs greatly improved the survival of animals bearing Hep3B human hepatocellular carcinoma (HCC) tumor orthotopically (48.5 days with control ASO vs 88 days with MALAT1 ASO, p=0.005). Taken together, these results demonstrate previously undiscovered roles of MALAT1 as an important regulator in vivo tumor growth and metastasis and suggest that selective inhibition of MALAT1 by ASO could have therapeutic value for the cancer treatment. Citation Format: Jeff Hsu, Guobin He, Gourab Bhattacharjee, Tianyuan Zhou, Chris May, Xiaokun Xiao, Gene Hung, Brett P. Monia, A. Robert MacLeod, Youngsoo Kim. Selective inhibition of a long non-coding RNA (lncRNA), MALAT1 by antisense oligonucleotides results in significant anti-tumor effects in a variety of preclinical cancer models [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B23.

Enhanced Cancer Immunotherapy Using STAT3-Depleted Dendritic Cells with High Th1-Inducing Ability and Resistance to Cancer Cell-Derived Inhibitory Factors

STAT3 signaling constitutes an important negative feedback mechanism for the maintenance of immune homeostasis, a suppressive signal for the Th1 immune response in murine macrophages, and a cancer immune evasion signal in various immune cells. The strategy for STAT3 signal inhibition should be considered, because these features could impede effective cancer immunotherapy. We have evaluated the effects of STAT3 inactivation in dendritic cells (DCs) on immune responses in mice and humans. DCs derived from LysMcre/STAT3(flox/flox) mice displayed higher cytokine production in response to TLR stimulation, activated T cells more efficiently, and were more resistant to the suppression of cytokine production by cancer-derived immunosuppressive factors compared with DCs from control littermates. Antitumor activities of STAT3-depleted and control DCs were compared by intratumoral administration of gp70 Ag peptide-pulsed DCs in the therapeutic MC38 tumor model. Intratumoral administration of STAT3-depleted DCs significantly inhibited MC38 tumor growth of both injected and nontreated remote tumors. The inhibition was accompanied by an increase in gp70-specific T cell response as well as in systemic Th1 immune response. STAT3-depleted human DCs with adenoviral STAT3 short hairpin RNA were also capable of producing more cytokines with TLR stimulation and more resistant to cancer-derived factors, and they induced tumor Ag-specific T cells more efficiently than control DCs. The identified role of DC STAT3 signaling in both in vivo therapeutic tumor models in mice and in vitro-specific T cell induction in humans indicates that STAT3-inactivated DCs may be a promising approach for cancer immunotherapy.

In vivo targeting of cell death using a synthetic fluorescent molecular probe

A synthetic, near-infrared, fluorescent probe, named PSS-794 was assessed for its ability to detect cell death in two animal models. The molecular probe contains a zinc(II)-dipicolylamine (Zn(2+)-DPA) affinity ligand that selectively targets exposed phosphatidylserine on the surface of dead and dying cells. The first animal model used rats that were treated with dexamethasone to induce thymic atrophy. Ex vivo fluorescence imaging and histological analysis of excised organs showed thymus uptake of PSS-794 was four times higher than a control fluorophore that lacked the Zn(2+)-DPA affinity ligand. In addition, the presence of PSS-794 produced a delayed and higher build up of dead and dying cells in the rat thymus. The second animal model employed focal beam radiation to induce cell death in tumor-bearing rats. Whole-body and ex vivo imaging showed that the amount of PSS-794 in a radiation-treated tumor was almost twice that in a non-treated tumor. The results indicate that PSS-794 may be useful for preclinical optical detection of tumor cell death due to therapy.

Abstract 2525: Intravenous infusions of TriN 2755, a new alkylating agent, inhibit the growth of human MDA-MB-231 breast tumor in nude rats

Abstract Background: The new cytotoxic anti-neoplastic agent TriN 2755, containing a triazene unit with alkylating properties, exhibited a potent antitumor activity in a large panel of human xenograft tumor models in mice when dosed by IV bolus injection. In rat, dog and mini pig toxicity studies, TriN 2755 demonstrated a favourable safety profile. Here, we present efficacy data of TriN 2755 intravenously injected in the MDA-MB-231 tumor bearing Nude rat model. Material and methods: The breast tumors were induced subcutaneously by injecting 2×107 of MDA-MB-231 cells in 200 µl of RPMI 1640 containing matrigel (50:50, v:v). Tumor-bearing nude rats received a single 4-hour IV infusion of TriN 2755 at 30, 80 or 240 mg/kg/infusion or IV bolus injection of TriN 2755 at 160, 240 or 360 mg/kg/injection twice weekly for 4 consecutive weeks. Moreover, two groups of rats received a single 4-hour IV infusion of TriN 2755 at 240 mg/kg/infusion twice weekly for 4 consecutive weeks in combination with repeated weekly IV bolus injection of CPT-11 at 40 mg/kg/inj for 4 consecutive weeks or Docetaxel at 2.5 mg/kg/inj for 3 consecutive weeks. The treatment started when the mean tumor volumes reached about 400-800 mm3, and the antitumor activity of TriN 2755 was assessed as tumor volume inhibition relative to a vehicle control group (T/C value in %). Tolerability was analysed by recording mortality and body weight loss. Results: Repeated IV infusions of TriN 2755 were well tolerated by female nude rats bearing subcutaneous (SC) MDA-MB-231 tumors at all tested doses (no death, no body weight loss). The histopathological analysis of liver, kidneys, and bone marrow of treated rats revealed no treatment related changes. Based upon TriN 2755 antitumor efficacy criteria at 80 and 240 mg/kg when injected by IV infusion, and at 160, 240 or 360 mg/kg when injected by IV bolus displayed a marked, and dose-dependent antitumor activity against SC MDA-MB-231 tumor (Optimal T/C% values were 37, and 7%, 21, 19 and 12%, respectively) Tumor regressions were observed after two weeks of treatment for all rats treated with TriN 2755 administered by IV bolus or IV infusion of 240mg/kg/infusion. Tumor cells derived from TriN 2755 IV bolus treated rats had an impaired colony formation compared to non-treated tumors in vitro. Moreover, an enhancement of the antitumor activity of drugs alone was observed in the SC MDA MB-231 tumor bearing nude rats model when TriN 2755 at 240 mg/kg/infusion was combined with CPT-11 at 40 mg/kg/inj or Docetaxel at 2.5 mg/kg/inj. Conclusions: These results demonstrate that TriN 2755 dosed by IV infusion and IV bolus displayed a marked antitumor activity alone, and efficacy of IV infusion was increased by combination with CPT-11 or Docetaxel in SC nude rats human breast tumor model. TriN 2755 is currently under evaluation in phase I clinical trials for the treatment of solid tumors. 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 2525. doi:10.1158/1538-7445.AM2011-2525

Identification and characterization of CD8+ T helper cells based on CD40L expression (155.1)

Abstract CD8+ T cells are primarily regarded as cytotoxic cells. We have identified a substantial subset of CD40L expressing non-cytotoxic T-cells among primary human CD8+ T cells. CD40L+ CD8+ T cells exert diverse characteristic Th-cell functions such as activation of B cells, induction of DC maturation and secretion of cytokines such as IL-2, IFNγ, TNFα or IL-4. Up to 25% of total human central and effector memory CD8+ T cells express CD40L including cells specific for pathogens such as influenza, CMV and EBV, as well as yellow fever virus specific cells after primary vaccination. At next we assessed the functional relevance of CD40L expression on CD8+ T cells during a anti-tumor response. We challenged Rag1-/- mice with SV40 Tag expressing tumor cells and injected in parallel wt or CD40L-/- CD8+ T cells. Only application of CD40L competent wt CD8+ T cells prevented the establishment of tumors, whereas injection of CD40L-/- CD8+ T cells resulted in non-controlled tumor progression similar to non-treated tumors, although tumor-specific T cells were primed in these mice. Our results disclose an essential functional relevance of CD40L expressed by CD8+ T cells. Especially, in situations of reduced CD4+ T-cell help and MHC-II antigen presentation and/or limited danger signals CD40L+ CD8+ T cells may exert essential helper responsibilities for immunity and thus are potent candidate T cells to execute or support effective anti-tumor or anti-pathogen immune therapies.

Optimization of hyperthermia and dendritic cell immunotherapy for squamous cell carcinoma

The aims of this study were to explore the feasibility of a novel combination therapy comprising hyperthermia (HT) and dendritic cell (DC) application for squamous cell carcinoma (SCC), and to optimize the conditions of this therapy. In vitro, the correlation between maturation of DCs by co-culture with SCCVII cells and HT was investigated. DCs did not mature in simple HT (43 °C for 30 min) with SCCVII cells. On the other hand, DC maturation occurred in additional mild HT (mHT: 41 °C for 30 min) with simple HT. To assess whether additional mHT was effective, in vivo combined treatment was performed using tumor-bearing C3H/HeJ mice. A more suppressive effect of tumor growth was observed, and cytotoxic T cell infiltration was significantly increased by adding mHT compared to conventional only simple HT with DCs. These phenomena also occurred in non-treated contralateral tumors as well as treated ones. Our data suggest that the combination of 43 °C preheated simple HT SCCVII tumors and additional 41 °C heat mHT promotes DC maturation, resulting in suppression of tumor growth systemically and lifetime prolongation in mice. A three-step process of additional mHT after local HT and intratumoral immature DC (iDC) injection could be a more effective and novel method for the treatment of SCC.

Chemotherapeutic agents in low noncytotoxic concentrations increase immunogenicity of human colon cancer cells

We have recently reported that chemotherapeutic agents in ultra low noncytotoxic concentrations may block the ability of tumor cells to suppress functional activation of dendritic cells (DCs). HCT-116 human colon cancer cells were treated with 0.5 nM paclitaxel (PAC) or 2 nM doxorubicin (DOX) with the aim of defining the immunogenic changes induced by ultra low noncytotoxic concentrations of antineoplastic chemotherapeutic agents. Genetic alterations were screened by DNA microarray that revealed increased expression of genes involved in antigen processing and presentation, including the heat-shock protein, calmodulin, and proteasome 26 genes. As the proteins encoded by these genes are involved in the cytosolic route of antigen processing machinery, we next evaluated whether PAC and DOX in noncytotoxic concentrations changed expression of MHC class I antigen processing machinery (APM) components in three different colon cancer cell lines. Our results showed that PAC and DOX increased the intracellular expression of APM proteins, including calmodulin, LMP2, LMP7, TAP1 and tapasin. The biological significance of modulation of antigen processing and presentation proteins in tumor cells by ultra low nontoxic concentrations of chemotherapeutic drugs was revealed when non-treated and treated tumor cells were used as a source of tumor antigens for the generation of tumor-specific cytotoxic T cells (CTLs) in vitro. We demonstrated that (i) DCs that engulf tumor cells pretreated with noncytotoxic concentrations of chemotherapeutic agents induced CTLs with a higher cytotoxic potential than DCs loaded with nontreated tumor cells, and (ii) CTLs induced by tumor lysate-pulsed DCs killed live tumor cells more efficiently if these tumor cells were pretreated with noncytotoxic concentrations of chemotherapeutic drugs. These results demonstrate that chemomodulation of human tumor cells with noncytotoxic concentrations of chemotherapeutic agents increases tumor immunogenicity and results in the generation of more efficient DC vaccines and CTLs, which can be used for cell-based anticancer immunotherapies.

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters. Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation. Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters. Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation. Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Abstract P4-02-03: Gene Copy Number Alterations Related to mRNA and miRNA Expression in Endocrine Resistant Breast Cancers

Abstract Background Endocrine resistance is a major clinical issue in breast cancer. Multiple studies provide data relating molecular features of primary tumours on their response to endocrine treatment. However, there is a paucity of data on molecular profiles of breast tumours at time when endocrine resistance has already manifested. Aim Provide multilayer molecular profiling of a series of breast tumours at time of relapse or progression on endocrine treatment. Results High density microarray-comparative genomic hybridisation (aCGH) analysis was performed in 39 endocrine treated breast tumours at time of progression or relapse. 25 of these tumours were also profiled using whole genome mRNA and miRNA arrays (HT-12 mRNA array and DASL Sentrix Array Matrix micro-RNA array, Illumina). Overall the aCGH profiles of resistant tumours were similar to aCGH profiles of un-selected luminal breast tumours and cell lines published earlier. The most common gene copy gain areas (&amp;gt;10M of length) were located in 1q23.1-44, 5p13.1-33, 8p11.23-q24.3, 16p12.1-13.3, 17q21.33-24.1 and 20q13.2-13.33, while the most common losses were in 1p33-36.32, 8p12-23.3, 11q22.3-24.1, 16q12.2-24.3 and 17p11.2-13.3. Unsupervised hierarchical clustering by gene copy number profiles split of the resistant tumours into 3 groups. As expected, on average mRNAs and microRNAs were higher expressed in tumours carrying gains of their genes and were decreased in tumours carrying the losses. Copy number gains in this series were associated with the upregulation of multiple RNAs, including C8orf76, TATDN1, CYC1, ZBTB10, C16orf63, THUMPD1, TFB2M, miR-454, miR-301a and miR-296-3p/5p. Losses were associated with the downregulation of RNAs including ACADVL, BNIP3L, UBE4A, REXO2, ATP5L, PPP2CB, NBL1 and PCM1. Of the above genes UBE4A and ATP5L are closely co-localised in 11q23.3; miR454-miR301a are located closely in 17q22. Such associations suggest that changes in expression of these genes in breast cancers may be driven by alterations in the gene copy number. Analysis of the recurrent amplification regions on this dataset is underway. Conclusions aCGH profiles of endocrine resistant tumours at time of relapse or progression are similar to those found in un-selected and non-treated luminal tumours. The study provides the first dataset integrating gene copy number with mRNA and microRNA gene expressions in clinical specimens of endocrine resistant breast cancers. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-03.

Melanoma‐targeted chemo‐thermo‐immuno (CTI)‐therapy using N‐propionyl‐4‐S‐cysteaminylphenol‐magnetite nanoparticles elicits CTL response via heat shock protein‐peptide complex release

Melanogenesis substrate, N-propionyl-4-S-cysteaminylphenol (NPrCAP) is specifically taken up by melanoma cells and inhibits their growth by producing cytotxic free radicals. By taking advantage of this unique chemical agent, we have established melanoma-targeting intracellular hyperthermia by conjugating NPrCAP with magnetite nanoparticles (NPrCAP/M) upon exposure to an alternating magnetic field (AMF). This treatment causes cytotoxic reaction as well as heat shock responses, leading to elicitation of antitumor immune response, which was proved by tumor rechallenge test and CTL induction. We found the level of heat shock protein 72 (Hsp72) to be increased in the cell lysate and culture supernatant after intracellular hyperthermia. Melanoma-specific CD8(+) T-cell response to dendritic cells loaded with hyperthermia-treated tumor lysate was enhanced when compared with non-treated tumor lysate. When heat shock protein, particularly Hsp72, was immuno-depleted from hyperthermia-treated tumor cell lysate, specific CD8(+) T-cell response was abolished. Thus, it is suggested that antitumor immune response induced by hyperthermia using NPrCAP/M is derived from the release of HSP-peptide complex from degraded tumor cells. Therefore, this chemo-thermo-immuno (CTI)-therapy might be effective not only for primary melanoma but also for distant metastasis because of induction of systemic antimelanoma immune responses.