Untreated Control Tumors Research Articles

Impact of mutant IDH (mIDH) inhibition on DNA hydroxymethylation, tumor cell function, and tumor immune microenvironment (TIME) in resected mIDH1 lower-grade glioma (LGG).

2008 Background: Somatic mutations in IDH1 and IDH2 occur in ̃80% and ̃4% of LGGs, respectively, promoting tumorigenesis via increased levels of the oncometabolite D-2-hydroxyglutarate (2-HG). Vorasidenib (VOR; AG-881) is an oral, brain-penetrant, dual inhibitor of mIDH1/2; ivosidenib (IVO; AG-120) is a first-in-class oral inhibitor of mIDH1. In this ongoing perioperative study, treatment with IVO/VOR reduced 2-HG levels in resected tumors vs untreated control tumors in patients (pts) with LGG (NCT03343197; Mellinghoff SNO 2019). We assessed the biological impact of 2-HG suppression on tumors and TIME. Methods: Pts (n = 49) with recurrent, non-enhancing, mIDH1-R132H LGG eligible for resection were randomized to IVO (500 mg QD/250 mg BID), VOR (10/50 mg QD), or no treatment, for 4 weeks preoperatively. Tumor tissue samples collected at surgery were assessed in genomic (n = 42), transcriptomic (n = 42), and immunohistochemistry (IHC; n = 43) analyses. Unpaired t-test was used for statistical comparisons. Results: Optimal 2-HG suppression (post-treatment 2-HG below the upper limit of 2-HG levels in a reference set of 15 wild-type [wt] IDH samples) was observed in 23 of 40 pts, including 9 (90%) pts receiving VOR 50 mg QD and 6 (50%) receiving IVO 500 mg QD. Of samples with valid biomarker data, those with optimal 2-HG suppression (n = 21) showed upregulation of neural differentiation-related gene expression, but downregulation of stemness-related gene expression, vs those with suboptimal 2-HG suppression (post-treatment 2-HG above upper limit of wt IDH 2-HG levels; n = 17; p < 0.01). IHC analysis of the proliferation marker Ki-67 showed a ̃2-fold decrease in Ki-67–positive cells in samples with optimal 2-HG suppression (mean 2.7%; n = 22) vs those with suboptimal suppression (5.8%; n = 16; p < 0.05). Epigenetic analysis revealed a ̃2-fold increase in mean 5-hydroxymethylcytosine (5hmC) levels in samples with optimal (0.36%; n = 17) vs suboptimal 2-HG suppression (0.2%; n = 15; p < 0.05), suggesting reversal of TET2 inhibition. IHC analysis of TIME revealed increases in mean CD3+ and CD8+ tumor-infiltrating lymphocyte levels in samples with optimal (1.05% [CD3]/0.22% [CD8]; n = 22) vs suboptimal 2-HG suppression (0.44% [CD3]/0.07% [CD8]; n = 16; p < 0.05). Optimal 2-HG suppression was associated with upregulation of gene expression related to type I interferon signaling and antigen presentation (p < 0.01). Conclusions: These data suggest that both tumor-intrinsic and -extrinsic mechanisms underlie 2-HG suppression by VOR and IVO. VOR, and IVO to a lesser extent, increased 5hmC, promoted cellular differentiation, and inhibited tumor cell proliferation; both also increased T-cell infiltration, activated interferon signaling, and increased antigen presentation capability. These data support development of VOR in combination with immunotherapy. Clinical trial information: NCT03343197.

Abstract 6185: In vivo screening in HPV positive and HPV negative head and neck squamous cell carcinoma

Abstract Background: The pathogenesis of head and neck squamous cell carcinoma (HNSCC) largely occurs through two pathways, human papillomavirus (HPV) mediated or chemical carcinogenesis secondary to alcohol and tobacco use. HPV status is an important prognostic factor and marker for sensitivity to radiation therapy in HNSCC patients. HPV negative HNSCC has been shown to have increased radioresistance compared to HPV positive HNSCC leading to poorer prognosis. The goal of this work is to utilize in vivo screening technology to identify key differential targets between HPV positive and negative tumors for both therapeutic purposes as well as to identify differential pathways between these two malignancies. Methods: Pooled, barcoded shRNA libraries focused on DNA damage repair (DDR) and currently targetable genes were transduced into three HPV positive (UM-SCC-147, UD-SCC-2, UPCI:SCC152) and three HPV negative (UM-SCC-22A, Cal-27, HN31) cell lines. Each cell line was injected into the mouse flank and allowed to grow to ~100mm3. Tumors were divided into control and radiation groups; the latter being treated with 6-10 Gy at 2 Gy/treatment. For each tumor type radiation was titrated to generate an approximate 20% reduction in tumor size. Tumors were then allowed to grow for an additional 2 weeks and then harvested. Barcodes from each tumor were sequenced and compared to both the reference input cells as well as untreated control tumors. Barcodes observed at decreased frequency in treated tumors indicated a potential target for therapeutic sensitization. Both p-values as well as percentile median fold change for each gene were calculated. These values were averaged for the three HPV negative and three HPV positive cell lines and used for analysis. Results: Multiple genes in the DDR and FDA libraries were identified from the HPV negative and HPV positive groups that may be associated with radioresistance in vivo. Example genes that were identified primarily in the HPV negative group include BTK, TXN, TTK, while those in the HPV positive group include RNF168, RAD50, ESR1. Inhibition of these genes may lead to increased cell death following radiation therapy through diminished DNA repair of radiation-induced damage. Conclusion: Our study identifies targetable genes that may contribute to radioresistance in both HPV negative and HPV positive head and neck squamous cell carcinoma and warrants further analysis. Citation Format: Mohamed K. Abdelhakiem, Liangpeng Yang, Tongxin Xie, Manish Kumar, David Molkentine, Aakash Sheth, Mitchell Frederick, Jeffrey Myers, Curtis R. Pickering, Heath D. Skinner. In vivo screening in HPV positive and HPV negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6185.

Abstract 5606: Characterization of temporal proteomic dynamics of murine breast and melanoma tumor microenvironments without and with pulsed focused ultrasound

Abstract Focused ultrasound (FUS) ablation has been successfully used for thermal ablation of solid malignancies. However, non-ablative pulsed-FUS (pFUS) has received less attention but could be less destructive anti-tumor therapy by stimulating a pro-inflammatory immune response within the tumor microenvironment (TME). Clinically, such an approach could be useful to treat peripheral disease (e.g. micro-metastases) in otherwise normal-appearing tissues. Here, we explored the alterations of cytokines, chemokines and trophic factors (CCTF) and cell adhesion molecules (CAM) in the TME of mouse flank tumor models following pFUS treatment at different time points of tumor progression. Mice were bilaterally inoculated with subcutaneous B16 melanoma or 4T1 breast tumors into the hind limbs and allowed to reach diameters of ~5 mm (n=6/group/timepoint) before performing experiments (Day 1). Separate groups of mice were subjected to pFUS (1 MHz; Peak negative pressure = 6 MPa; Duty cycle = 10%, 100 pulses per site) at Days 1, 5, or 10 and euthanized 24 hr post-pFUS for proteomic analyses. Separate groups of time-matched tumor-bearing mice that did not receive pFUS were also euthanized at the same time points both to assess relative changes due to pFUS at each time point and provide information regarding the natural history of TME progression in each tumor type. Proteomic analyses in untreated control tumors revealed an overall shift toward immunosuppressive (pro-tumor) TME over the 10 days. When comparing proteomic effects of pFUS treatment in compared to untreated time-matched controls, tumors treated on Day 1 exhibited greater upregulation of pro-inflammatory (anti-tumor) CCTF and CAM and pFUS had less pronounced effects on the TME when administered in groups of mice treated at Days 5 or 10. To further examine the acute effects of pFUS at on CCTF and CAM over 72hr. pFUS treatment in elicited unique CCTF and CAM profiles for the two tumor types, but both could be considered shifted towards anti-tumoral TME, especially within the first 24 hrs. These data suggest that pFUS can potentially modulate TME in a therapeutically beneficial way. However, they reveal two important caveats that require substantial investigation: 1) Different tumor types have unique molecular responses and imply pFUS may be more suitable for certain tumor types; 2) Natural histories encompass dynamic TME changes as part of disease progression and appropriate timing of pFUS in each tumor type will be necessary to maximizing the potential therapeutic benefits of pFUS. Citation Format: Gadi Cohen, Parwathay Chandran, Lauren E. Tomlinson, Maggie E. Sundby, Rebecca M. Lorsung, Scott R. Burks, Joseph A. Frank. Characterization of temporal proteomic dynamics of murine breast and melanoma tumor microenvironments without and with pulsed focused ultrasound [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5606.

Establishment of a Patient-Derived Xenograft Tumor From Hepatitis C-Associated Liver Cancer and Evaluation of Imatinib Treatment Efficacy.

Chronic hepatitis C virus (HCV) infection is one of the major causal factors for hepatocellular carcinoma (HCC). The treatment options for HCC are limited for lack of a convenient animal model for study in HCV infection and liver pathogenesis. This study aimed to develop a patient-derived xenograft (PDX) tumor in mice by using a tumor from a patient with HCV-associated HCC and evaluating this model's therapeutic potential. After resection of the primary tumor from the patient liver, excess viable tumor was implanted into highly immunodeficient mice. A mouse xenograft tumor line was developed, and the tumor was successfully passaged for at least three rounds in immunodeficient mice. The patient's primary tumor and the mouse xenografts were histologically similar. Genetic profiling by short-tandem-repeat analysis verified that the HCC-PDX model was derived from the HCC clinical specimen. HCV RNA present in the patient liver specimen was undetectable after passage as xenograft tumors in mice. Human albumin, α1 -antitrypsin, glypican-3, α-smooth muscle actin, and collagen type 1A2 markers were detected in human original tumor tissues and xenograft tumors. Both the patient primary tumor and the xenograft tumors had a significantly higher level of receptor tyrosine kinase (c-Kit) mRNA. Treatment of HCC-PDX xenograft tumor-bearing mice with the c-Kit inhibitor imatinib significantly reduced tumor growth and phospho-Akt and cyclin D1 expression, as compared with untreated control tumors. Our results demonstrated establishment of an HCV-associated HCC-PDX model as a powerful tool for evaluating candidate drugs. Information on molecular changes in cancer-specific gene expression facilitates efficient targeted therapies and treatment strategies.

THER-16. INTERACTION OF IMMUNE SYSTEM WITH ONCOLYTIC MEASLES VIRUS THERAPY IN AN MV-SENSITIVE IMMUNOCOMPETENT MURINE MODEL OF MEDULLOBLASTOMA

Abstract INTRODUCTION: Oncolytic viruses selectively replicate in tumor cells, and may attract immune cells into the microenvironment to stimulate a systemic antitumor response. We have shown efficacy of oncolytic measles virus (MV) against medulloblastoma (MB) in preclinical models. To study the effect of immune system on MV therapy, we have developed the first immunocompetent MV-sensitive murine model of Group 3 MB (CSCG model) and showed that intratumoral MV treatment of tumor-bearing mice significantly prolonged survival. Herein, we study the interaction of MV therapy and tumor immune microenvironment in this model. METHODS: Naive and MV-preimmunized mice were injected with 200,000 CSCG tumor cells in caudate putamen and tumor-bearing mice were treated identically with intratumoral MV injections (n=5 per group). Tumor growth was monitored by bioluminescence imaging and RT-PCR was performed to assess infiltrating CD4+ and CD8+ T cells in MV-treated and untreated control tumors. RESULTS: RT-PCR of untreated tumors shows an average of 43.4 ±1.45-fold and 13.24 ±1.34-fold increase in infiltrating CD8+T cells and CD4+T cells, respectively, compared to normal brain (n=3). Tumors that responded to MV therapy, but recurred had a dramatic decrease in infiltrating CD8+T cells (2.66 ±0.8-fold higher than normal brain). No significant difference in survival was observed between MV-naive and MV-immunized mice (p= 0.773), indicating that anti-measles immunity does not affect MV efficacy, an important point given that almost all patients have been vaccinated with MV. Long-term survivors, both naïve and MV-immune, remained tumor-free for at least 150 days post treatment. These mice (n=3) were re-challenged with a 3-fold higher number of tumor cells than the original injection, but tumor failed to grow for at least 50 days post-injection. CONCLUSION: This finding strongly suggests MV treatment induces anti-tumor immunity. Ongoing studies are being performed to dissect this immune response and to explore the possibility of enhancing it.

Diffusion-Weighted MRI Is Insensitive to Changes in the Tumor Microenvironment Induced by Antiangiogenic Therapy

Antiangiogenic treatment (AAT) used in combination with radiation therapy or chemotherapy is a promising strategy for the treatment of several cancer diseases. The vascularity and oxygenation of tumors may be changed significantly by AAT, and consequently, a noninvasive method for monitoring AAT-induced changes in these microenvironmental parameters is needed. The purpose of this study was to evaluate the potential usefulness of diffusion-weighted magnetic resonance imaging (DW-MRI). DW-MRI was conducted with a Bruker Biospec 7.05-T scanner using four diffusion weightings and diffusion sensitization gradients in three orthogonal directions. Maps of the apparent diffusion coefficient (ADC) were calculated by using a monoexponential diffusion model. Two cervical carcinoma xenograft models (BK-12, HL-16) were treated with bevacizumab, and two pancreatic carcinoma xenograft models (BxPC-3, Panc-1) were treated with sunitinib. Pimonidazole and CD31 were used as markers of hypoxia and blood vessels, respectively, and fraction of hypoxic tissue (HFPim) and microvascular density (MVD) were quantified by analyzing immunohistochemical preparations. MVD decreased significantly after AAT in BK-12, HL-16, and BxPC-3 tumors, and this decrease was sufficiently large to cause a significant increase in HFPim in BK-12 and BxPC-3 tumors. The ADC maps of treated tumors and untreated control tumors were not significantly different in any of these three tumor models, suggesting that the AAT-induced microenvironmental changes were not detectable by DW-MRI. DW-MRI is insensitive to changes in tumor vascularity and oxygenation induced by bevacizumab or sunitinib treatment.

Abstract 832: An AXL-specific antibody-drug conjugate shows preclinical anti-tumor activity in non-small cell lung cancer, including EGFR-inhibitor resistant NSCLC

Abstract Enhanced AXL expression has been observed in tumor tissues obtained from non-small cell lung (NSCLC) patients, both in drug-treatment naïve patients and in patients with acquired resistance to EGFR tyrosine kinase inhibitors (EGFRi). In this study, we have evaluated the antitumor activity of the novel AXL-targeting antibody-drug conjugate (ADC) AXL-107-MMAE (HuMax-AXL-ADC) in non-small cell lung cancer (NSCLC) in vitro and in vivo. The antitumor activity of AXL-107-MMAE (4 mg/kg) was evaluated in vivo in a mouse patient-derived xenograft (PDX) clinical trial (1 mouse per group), using a collection of 57 NSCLC-derived PDX models, encompassing the typical NSCLC histological subtypes and mutational genotypes. AXL-107-MMAE induced responses, defined by a decrease in relative tumor growth compared to an untreated control tumor, in 35/57 (61%) of PDX models. Potent anti-tumor activity, i.e. tumor stasis or tumor regression, was observed in 16 out of 57 (28%) models. Potent anti-tumor activity was associated with higher AXL RNA expression compared to models showing intermediate response or non-responders (p<0.001). The therapeutic activity of AXL-107-MMAE (2 and 4 mg/kg) was confirmed in a panel of NSCLC PDX or cell line-derived xenograft models using 6-8 mice per group. AXL-107-MMAE induced dose-dependent, single agent anti-tumor activity in 8 out of 9 models. These included 2 EGFR-mutant PDX models that were resistant to the EGFR inhibitor erlotinib, including one model containing L858R/T790M EGFR mutations and one model with a L858R EGFR mutation. To specifically evaluate opportunities for AXL-107-MMAE in EGFR-mutant, treatment-resistant NSCLC, we established a panel of EGFR-mutant NSCLC cell lines with acquired resistance to the EGFRi erlotinib, gefitinib, or osimertinib. In general, cell lines showed enhanced AXL protein expression upon acquiring resistance to EGFRi. Moreover, the AXL-expressing, EGFRi-resistant cell lines were efficiently killed by AXL-107-MMAE in vitro, whereas the parental, EGFR-TKI sensitive NSCLC cell lines, which expressed little or no AXL, did not respond to treatment with AXL-107-MMAE. In summary, we show that AXL-107-MMAE has therapeutic activity as a single agent in the majority of NSCLC-derived models evaluated in vivo, representing different NSCLC histological and mutational subtypes. Moreover, AXL-107-MMAE induced efficient cytotoxicity in NSCLC cell lines that showed enhanced AXL expression upon acquired resistance to EGFRi. Finally, AXL-107-MMAE induces anti-tumor activity in EGFR-mutant, EGFRi resistant NSCLC PDX models. Citation Format: Louise A. Koopman, Maarten L. Janmaat, Kirstine Jacobsen, Mikkel Green Terp, Elke Gresnigt-van den Heuvel, Ulf Forssman, Andreas Lingnau, Paul W. Parren, Henrik Ditzel, Esther C. Breij. An AXL-specific antibody-drug conjugate shows preclinical anti-tumor activity in non-small cell lung cancer, including EGFR-inhibitor resistant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 832.

DCE-MRI of Sunitinib-Induced Changes in Tumor Microvasculature and Hypoxia: A Study of Pancreatic Ductal Adenocarcinoma Xenografts

The purpose of this study was dual: to investigate (a) whether sunitinib may induce changes in tumor microvasculature and hypoxia in pancreatic ductal adenocarcinoma (PDAC) and (b) whether any changes can be detected by DCE-MRI. Sunitinib-treated and untreated control tumors of two PDAC xenograft models (BxPC-3 and Panc-1) were subjected to DCE-MRI before the imaged tumors were prepared for quantitative analysis of immunohistochemical preparations. Pimonidazole was used as a hypoxia marker, and fraction of hypoxic tissue (HFPim), density of CD31-positive microvessels (MVDCD31), and density of αSMA-positive microvessels (MVDαSMA) were measured. Parametric images of Ktrans and ve were derived from the DCE-MRI data by using the Tofts pharmacokinetic model. BxPC-3 tumors showed increased HFPim, decreased MVDCD31, unchanged MVDαSMA, and increased vessel maturation index (VMI = MVDαSMA/MVDCD31) after sunitinib treatment. The increase in VMI was seen because sunitinib induced selective pruning rather than maturation of αSMA-negative microvessels. Even though the microvessels in sunitinib-treated tumors were less abnormal than those in untreated tumors, this microvessel normalization did not improve the function of the microvascular network or normalize the tumor microenvironment. In Panc-1 tumors, HFPim, MVDCD31, MVDαSMA, and VMI were unchanged after sunitinib treatment. Median Ktrans increased with increasing MVDCD31 and decreased with increasing HFPim, and the correlations were similar for treated and untreated BXPC-3 and Panc-1 tumors. These observations suggest that sunitinib may induce significant changes in the microenvironment of PDACs, and furthermore, that Ktrans may be an adequate measure of tumor vascular density and hypoxia in untreated as well as sunitinib-treated PDACs.

T cell infiltration into Ewing sarcomas is associated with local expression of immune-inhibitory HLA-G.

Ewing sarcoma (EwS) is an aggressive mesenchymal cancer of bones or soft tissues. The mechanisms by which this cancer interacts with the host immune system to induce tolerance are not well understood. We hypothesized that the non-classical, immune-inhibitory HLA-molecule HLA-G contributes to immune escape of EwS. While HLA-Gpos suppressor T cells were not increased in the peripheral blood of EwS patients, HLA-G was locally expressed on the tumor cells and/or on infiltrating lymphocytes in 16 of 47 pretherapeutic tumor biopsies and in 4 of 12 relapse tumors. HLA-G expression was not associated with risk-related patient variables or response to standard chemotherapy, but with significantly increased numbers of tumor-infiltrating CD3+ T cells compared to HLA-Gneg EwS biopsies. In a mouse model, EwS xenografts after adoptive therapy with tumor antigen-specific CAR T cells strongly expressed HLA-G whereas untreated control tumors were HLA-Gneg. IFN-γ stimulation of EwS cell lines in vitro induced expression of HLA-G protein. We conclude that EwS cells respond to tumor-infiltrating T cells by upregulation of HLA-G, a candidate mediator of local immune escape. Strategies that modulate HLA-G expression in the tumor microenvironment may enhance the efficacy of cellular immunotherapeutics in this cancer.

673. Therapeutic Efficacy of Retroviral Replicating Vector (RRV) -Mediated Prodrug Activator Gene Therapy for Pancreatic Cancer

Pancreatic ductal adenocarcinoma is one of the most lethal cancers, thus new therapeutic strategies for this disease are urgently needed. Retroviral replicating vectors (RRV)-mediated prodrug activator gene therapy with Toca 511 (vocimagene amiretrorepvec), an optimized RRV encoding yeast cytosine deaminase (yCD) which converts the prodrug 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU), is showing promising clinical activity in patients with recurrent high grade glioma, and is now being evaluated in a multi-center Phase II/III clinical trial. In the present study, we evaluated the therapeutic efficacy of RRV-mediated prodrug activator gene therapy in preclinical models of pancreatic cancer. We first examined the replication kinetics of RRV expressing the GFP reporter gene (RRV-GFP) in murine (Pan02) and human (MIAPaCa-2, BxPC-3, PANC-1 and SUIT-2) pancreatic cancer cell lines by flow-cytometric analysis and quantitative PCR. In all of these pancreatic cancer lines, RRV-GFP inoculated at MOI=0.05 (~5% initial transduction levels) showed rapid viral replication subsequently resulting in high levels of transduction, with the majority of pancreatic cancer lines reaching >90% GFP-positive cells over time. Next, we tested in vitro cytotoxicity by MTS assay after prodrug treatment of pancreatic cancer cells (Pan02 and MIAPaCa-2) transduced with Toca 511. In RRV-transduced pancreatic cancer cells, significant (~90%) cytotoxicity was induced by exposure to 0.1-1.0 mM 5-FC prodrug for 4 days, compared to untransduced and RRV-GFP transduced controls. We then evaluated in vivo therapeutic efficacy of Toca 511/5-FC prodrug activator gene therapy in Pan02 pancreatic tumor models established subcutaneously in immunocompetent syngeneic C57BL/6 mice. While 5-FC treatment alone showed no obvious inhibition of tumor growth as well as the nontreated control group, the majority (n=7/8) of Toca 511-transduced tumors showed complete regression after 5-FC treatment. Notably, systemic biodistribution studies showed no detectable RRV signals in genomic DNA from normal tissues of treated mice. In orthotopic models using luciferase-marked MIAPaCa-2 tumors established in the pancreas in nude mice, significant inhibition of bioluminescent signals was observed by optical imaging after 5-FC administration in mice with Toca 511-infected tumors, as compared to untreated control tumors. Thus, RRVs are highly efficient vehicles for delivering prodrug activator genes such as yCD to pancreatic cancer cells, thereby achieving significant cell killing upon pro-drug administration. Further preclinical studies are ongoing toward translating this novel therapeutic strategy into clinical trials for patients with pancreatic cancer.

Sunitinib to upregulate IFNg-STAT1 signaling and to increase indoleamine 2,3-dioxygenase (IDO) expression in renal cell carcinoma (RCC).

614 Background: Recently we reported that antiangiogenic therapy-treated primary RCC demonstrated increased infiltration of CD4 and CD8 T cells and higher expression of the immune checkpoint ligand PD-L1, all of which were associated with therapy resistance. IFNg-STAT1 signaling pathway drives the expression of both immunosuppressive and immunostimulatory molecules that are involved in lymphocyte trafficking, antigen presentation and immune checkpoints. The purpose of this study was to assess the impact of antiangiogenic therapy on IFNg signaling and on a broader tumor specific immune profile. Methods: We performed immunohistochemical staining of tissue microarrays (TMAs) generated from 40 sunitinib treated primary RCC from patients treated on clinical trial and compared stains to untreated control tumors. We used Vectra imaging to spectrally unmix antibody signal and to quantitate expression levels. We used immunoblot and real-time PCR to investigate the changes in protein and mRNA, respectively. We assessed for statistical significance using an unpaired Student t test. Results: We found that sunitinib treated RCC tissues demonstrated STAT1 phosphorylation (1.2% vs. 4.8%; P<0.001) or nuclear localization (7.4% vs. 15.6%; P=0.005), which was inversely related to patient survival. Furthermore, sunitinib treatment magnified IFNg-induced STAT1 activity by increasing the activity of janus kinase 2 (JAK2) and decreasing the protein levels of inhibitors PIAS and SOCS. On the one hand, sunitinib increased the transcription of VCAM1, CCL4, HLA-B and CD80, which facilitate T cell infiltration or antigen presentation; on the other hand, sunitinib increased transcription of the T cell negative regulator IDO1 (1 vs. 3.2, P<0.001) and CTLA4 (1 vs. 3.1, P=0.01). Conclusions: These results indicate that sunitinib both positively and negatively regulates the tumor immune microenvironment via modulation of STAT1 mediated signaling. IDO upregulation is a key driver of T-cell nutrient deprivation and can be targeted therapeutically. These findings will help us devise precise interventions to overcome tumor cell specific immunosuppressive signaling.

Radiation-Guided Peptide Delivery in a Mouse Model of Nasopharyngeal Carcinoma.

Purpose. This study aimed to evaluate the characteristics of the HVGGSSV peptide, exploring radiation-guided delivery in a mouse model of nasopharyngeal carcinoma. Methods. Mice with CNE-1 nasopharyngeal carcinoma were assigned to two different groups treated with Cy7-NHS and Cy7-HVGGSSV, respectively. Meanwhile, each mouse received a single dose of 3 Gy radiation. Biological distribution of the recombinant peptide was assessed on an in vivo small animal imaging system. Results. The experimental group showed maximum fluorescence intensity in irradiated tumors treated with Cy7-labeled HVGGSSV, while untreated (0 Gy) control tumors showed lower intensity levels. Fluorescence intensities of tumors in the right hind limbs of experimental animals were 7.84 × 107 ± 1.13 × 107, 1.35 × 108 ± 2.66 × 107, 4.05 × 108 ± 1.75 × 107, 5.57 × 108 ± 3.47 × 107, and 9.26 × 107 ± 1.73 × 107 photons/s/cm2 higher compared with left hind limb values at 1, 2, 15, 24, and 48 h, respectively. Fluorescence intensities of tumor in the right hind limbs of the experimental group were 1.66 × 108 ± 1.71 × 107, 1.51 × 108 ± 3.23 × 107, 5.38 × 108 ± 1.96 × 107, 5.89 × 108 ± 3.57 × 107, and 1.62 × 108 ± 1.69 × 107 photons/s/cm2 higher compared with control group values at 1, 2, 15, 24, and 48 h, respectively. Fluorescence was not specifically distributed in the control group. Compared with low fluorescence intensity in the heart, lungs, and tumors, high fluorescence distribution was found in the liver and kidney at 48 h. Conclusions. HVGGSSV was selectively bound to irradiated nasopharyngeal carcinoma, acting as a targeting transport carrier for radiation-guided drugs that are mainly metabolized in the kidney and liver.

Abstract A51: Chemotherapy rescues hypoxic tumor cells and induces reoxygenation and repopulation - an effect that is inhibited by the hypoxia-activated pro-drug TH-302

Abstract Introduction: Hypoxia is associated with tumor treatment failure. Tumor cells closest to blood vessels are well nourished and rapidly proliferating but hypoxic cells located farther away are slowly-proliferating. Chemotherapy targets rapidly proliferating cells and therefore may spare hypoxic cells because of poor drug distribution to them. Intervals between administrations of chemotherapy allow normal tissues to recover but might also allow re-oxygenation and resumed proliferation of formerly hypoxic cells due to a better supply of nutrients to them. Tumor cell repopulation following radiation therapy is well-documented; but repopulation following chemotherapy and its effect on hypoxia has not been studied. Hypoxia activated pro-drugs (i.e. TH-302) may inhibit tumor repopulation by killing hypoxic cells. Here we evaluate repopulation following chemotherapy and the effect of TH-302 on changes in hypoxia over time in human tumor xenografts. Methods: Human breast (MCF7) tumor bearing mice were administered two specific markers of hypoxia (pimonidazole [pimo] and EF5). Hypoxia-labeled tumor cells were recognized in tumor sections (in relation to DioC7+ve functional tumor blood vessels) using immunohistochemistry. Proliferating cells were identified by an antibody to Ki67. Mice were treated with pimo and: saline, doxorubicin, TH-302 or TH-302 + doxorubicin; EF5 was given after a variable interval of 24, 48, 72, 96 or 120 hours and tumors collected. Changes in the hypoxia, proliferation and oxygen status of formerly hypoxic (pimo+ve) cells were quantified by their Ki67 status and uptake of EF5 as a function of time. Results: Following doxorubicin treatment, the proportion of hypoxic cells in the entire tumor decreased from 1.5% (pimo+ve) prior to injection to 0.7% (EF5+ve) at 24 hours. The percentage of pimo+ve formerly hypoxic cells that are no longer hypoxic (i.e. EF5 – ve) at 24 hours was 90% after doxorubicin compared to 27% in controls, indicating rescue of previous hypoxic cells that would have died in the absence of treatment. Proliferation of these pimo+ve cells that were cycling (Ki67+ve) increased from 7% to 15.0% at 24 hours and then slowly decreased. TH-302 + doxorubicin combination inhibited repopulation by reducing Ki-67 to 1%. Conclusions: Chemotherapy (doxorubicin) treatment paradoxically contributes to tumor treatment failure (tumor cell repopulation) at all time points investigated by causing formerly hypoxic cells to become oxygenated and proliferate above those levels found in the untreated control tumors. TH-302 treatment led to increased DNA damage and inhibited hypoxic cell proliferation and tumor repopulation, thereby increasing chemotherapeutic outcome. Supported by a research grant from the Canadian Institutes for Health Research. Citation Format: Jasdeep K. Saggar, Ian F. Tannock. Chemotherapy rescues hypoxic tumor cells and induces reoxygenation and repopulation - an effect that is inhibited by the hypoxia-activated pro-drug TH-302. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A51. doi:10.1158/1538-7445.CHTME14-A51

Longitudinal evaluation of the metabolic response of a tumor xenograft model to single fraction radiation therapy using magnetic resonance spectroscopy

Proton magnetic resonance spectroscopy (MRS) was used to evaluate the metabolic profile of human glioblastoma multiform brain tumors grown as xenografts in nude mice before, and at multiple time points after single fraction radiation therapy. Tumors were grown over the thigh in 16 mice in this study, of which 5 served as untreated controls and 11 had their tumors treated to 800 cGy with 200 kVp x-rays. Spectra were acquired within 24 h pre-treatment, and then at 3, 7 and 14 d post-treatment using a 9.4 T animal magnetic resonance (MR) system. For the untreated control tumors, spectra (1–2 per mouse) were acquired at different stages of tumor growth. Spectra were obtained with the PRESS pulse sequence using a 3 × 3 × 3 mm3 voxel. Analysis was performed with the LCModel software platform. Six metabolites were profiled for this analysis: alanine (Ala), myo-inositol (Ins), taurine (Tau), creatine and phosphocreatine (Cr + PCr), glutamine and glutamate (Glu + Gln), and total choline (glycerophosphocholine + phosphocholine) (GPC + PCh). For the treated cohort, most metabolite/water concentration ratios were found to decrease in the short term at 3 and 7 d post-treatment, followed by an increase at 14 d post-treatment toward pre-treatment values. The lowest concentrations were observed at 7 d post-treatment, with magnitudes (relative to pre-treatment concentration ratios) of: 0.42 ± 24.6% (Ala), 0.43 ± 15.3% (Ins), 0.68 ± 27.9% (Tau), 0.52 ± 14.6% (GPC+PCh), 0.49 ± 21.0% (Cr + PCr) and 0.78 ± 24.5% (Glu + Gln). Control animals did not demonstrate any significant correlation between tumor volume and metabolite concentration, indicating that the observed kinetics were the result of the therapeutic intervention. We have demonstrated the feasibility of using MRS to follow multiple metabolic markers over time for the purpose of evaluating therapeutic response of tumors to radiation therapy. This study provides supporting evidence that metabolite/water concentration ratios have the potential to be used as biomarkers for the assessment of the response to therapy.

A low molecular weight zinc2+-dipicolylamine-based probe detects apoptosis during tumour treatment better than an annexin V-based probe

Molecular imaging of apoptosis is frequently discussed for monitoring cancer therapies. Here, we compare the low molecular weight phosphatidylserine-targeting ligand zinc2+-dipicolylamine (Zn2+-DPA) with the established but reasonably larger protein annexin V. Molecular apoptosis imaging with the fluorescently labelled probes annexin V (750 nm, 36 kDa) and Zn2+-DPA (794 nm, 1.84 kDa) was performed in tumour-bearing mice (A431). Three animal groups were investigated: untreated controls and treated tumours after 1 or 4 days of anti-angiogenic therapy (SU11248). Additionally, μPET with 18 F-FDG was performed. Imaging data were displayed as tumour-to-muscle ratio (TMR) and validated by quantitative immunohistochemistry. Compared with untreated control tumours, TUNEL staining indicated significant apoptosis after 1 day (P < 0.05) and 4 days (P < 0.01) of treatment. Concordantly, Zn2+-DPA uptake increased significantly after 1 day (P < 0.05) and 4 days (P < 0.01). Surprisingly, annexin V failed to detect significant differences between control and treated animals. Contrary to the increasing uptake of Zn2+-DPA, 18 F-FDG tumour uptake decreased significantly at days 1 (P < 0.05) and 4 (P < 0.01). Increase in apoptosis during anti-angiogenic therapy was detected significantly better with the low molecular weight probe Zn2+-DPA than with the annexin V-based probe. Additionally, significant treatment effects were detectable as early using Zn2+-DPA as with measurements of the glucose metabolism using 18 F-FDG. • The detection of apoptosis by non-invasive imaging is important in oncology. • A new low molecular weight probe Zn2+-DPA shows promise in depicting anti-angiogenic effects. • The small Zn2+-DPA ligand appears well suited for monitoring therapy. • Treatment effects are detectable just as early with Zn2+-DPA as with 18F-FDG.

Efficacy of schedule-dependent metronomic S-1 chemotherapy in human oral squamous cell carcinoma cells

Metronomic chemotherapy is based on administration of anticancer agents at low-doses at close regular intervals with no prolonged breaks, and aims to inhibit vascular endothelial cells as well as tumor cells. Recently, it was suggested that metronomic chemotherapy exerts anti-angiogenic effects by inducing thrombospondin-1 (TSP-1) and early growth response-1 (EGR-1), and antitumor effects by suppressing cancer stem cells. S-1 is a novel orally administered anticancer drug that is a combination of tegafur, 5-chloro-2, 4-dihydroxypyridine and oteracil potassium for maintaining efficacious concentrations of 5-FU and reducing the serious gastrointestinal toxicity associated with 5-FU. In the present study, we tried to determine the suitable administration method of S-1 against oral squamous cell carcinoma as a metronomic chemotherapy. We performed in vivo experiments in which tumor-bearing nude mice were used to examine the antitumor activity of S-1 (6.9 mg/kg). HSC2 tumors were treated with three different regimens, given as 4-week treatment and 2-week rest (4W-2W, 1 cycle); 2-week treatment and 1-week rest (2W-1W, 2 cycles); or alternate days treatment (1D-1D, 6 weeks). A fourth group served as control. Antitumor effects and body weight changes were compared in each group. Expression of TSP-1, EGR-1, CD31 and CD44 in HSC2 tumors was examined by immunohistochemistry. The treated groups showed higher tumor growth inhibition compared to the control group, and the relative tumor growth inhibition was not different between the treated groups. Briefly, each relative tumor growth inhibition was 32.4% (4W-2W), 39.6% (2W-1W) and 37.0% (1D-1D). During treatment periods, body weights were lower in the mice with 4W-2W or 2W-1W than 1D-1D or control. Moreover, reduction of microvessel density and CD44 expression, and induction of TSP-1 and EGR-1 expression was markedly seen in 1D-1D-treated tumors compared to 4W-2W-, 2W-1W-treated tumors or untreated control tumors by immunohistochemistry. These findings suggest that the 1D-1D regimen is more useful than the 4W-2W or 2W-1W regimen as a metronomic chemotherapy.

Proteomics of Genetically Engineered Mouse Mammary Tumors Identifies Fatty Acid Metabolism Members as Potential Predictive Markers for Cisplatin Resistance

In contrast to various signatures that predict the prognosis of breast cancer patients, markers that predict chemotherapy response are still elusive. To detect such predictive biomarkers, we investigated early changes in protein expression using two mouse models for distinct breast cancer subtypes who have a differential knock-out status for the breast cancer 1, early onset (Brca1) gene. The proteome of cisplatin-sensitive BRCA1-deficient mammary tumors was compared with that of cisplatin-resistant mammary tumors resembling pleomorphic invasive lobular carcinoma. The analyses were performed 24 h after administration of the maximum tolerable dose of cisplatin. At this time point, drug-sensitive BRCA1-deficient tumors showed DNA damage, but cells were largely viable. By applying paired statistics and quantitative filtering, we identified highly discriminatory markers for the sensitive and resistant model. Proteins up-regulated in the sensitive model are involved in centrosome organization, chromosome condensation, homology-directed DNA repair, and nucleotide metabolism. Major discriminatory markers that were up-regulated in the resistant model were predominantly involved in fatty acid metabolism, such as fatty-acid synthase. Specific inhibition of fatty-acid synthase sensitized resistant cells to cisplatin. Our data suggest that exploring the functional link between the DNA damage response and cancer metabolism shortly after the initial treatment may be a useful strategy to predict the efficacy of cisplatin.

Accuracy of a clinical PET/CT vs. a preclinical μPET system for monitoring treatment effects in tumour xenografts

PurposeSmall animal imaging is of growing importance for preclinical research and drug development. Tumour xenografts implanted in mice can be visualized with a clinical PET/CT (cPET); however, it is unclear whether early treatment effects can be monitored. Thus, we investigated the accuracy of a cPET versus a preclinical μPET using 18F-FDG for assessing early treatment effects. Materials and methodsThe spatial resolution and the quantitative accuracy of a clinical and preclinical PET were evaluated in phantom experiments. To investigate the sensitivity for assessing treatment response, A431 tumour xenografts were implanted in nude mice. Glucose metabolism was measured in untreated controls and in two therapy groups (either one or four days of antiangiogenic treatment). Data was validated by γ-counting of explanted tissues. ResultsIn phantom experiments, cPET enabled reliable separation of boreholes≥5mm whereas μPET visualized boreholes≥2mm. In animal studies, μPET provided significantly higher tumour-to-muscle ratios for untreated control tumours than cPET (3.41±0.87 vs. 1.60±.0.28, respectively; p<0.01). During treatment, cPET detected significant therapy effects at day 4 (p<0.05) whereas μPET revealed highly significant therapy effects even at day one (p<0.01). Correspondingly, γ-counting of explanted tumours indicated significant therapy effects at day one and highly significant treatment response at day 4. Correlation with γ-counting was good for cPET (r=0.74; p<0.01) and excellent for μPET (r=0.85; p<0.01). ConclusionClinical PET is suited to investigate tumour xenografts≥5mm at an advanced time-point of treatment. For imaging smaller tumours or for the sensitive assessment of very early therapy effects, μPET should be preferred.

Effect of antiangiogenic therapy on luciferase activity in a cytomegalovirus- or HSP70-promoter-transfected M21 tumor model

We investigated the effect of targeted gene therapy on heat shock protein 70 expression (Hsp70) and protein production (HSP70) in a melanoma tumor model (M21; M21-L). M21 and M21-L cells transfected with a plasmid containing the Hsp70 (Hspa1b) or the cytomegalovirus (CMV) promoter and the luciferase reporter gene were injected into mice; the resulting tumors grew to a size of 650 mm(3). Mice (five per group) were intravenously treated with an Arg-Gly-Asp peptide-nanoparticle/Raf-1 kinase inhibitor protein complex [RGD-NP/RAF(-)] or with a nanoparticle control. Bioluminescence imaging (IVIS®, Xenogen, USA) was performed at 12, 24, 48, and 72 h after the treatment cycle. Western blot analysis of HSP70 protein was performed to monitor protein expression. The size of the treated M21 tumors remained fairly constant (647.8 ± 103.4 mm(2) at the beginning versus 704.8 ± 94.4 mm(3) at the end of the experiment). The size of the M21-L tumors increased, similar to the untreated control tumors. Bioluminescent imaging demonstrated that when transcription was controlled by the CMV promoter, luciferase activity decreased to 17.9% ± 4.3% of baseline values in the treated M21 tumors. When transcription was controlled by the Hsp70 promoter, the highest luciferase activity (4.5 ± 0.7-fold increase over base-line values) was seen 24 h after injection in the M21 tumors; however, no luciferase activity was seen in the M21-L tumors. In accordance with bioluminescent imaging, western blot analysis showed a peak in HSP70 production at 24 h after the injection of the RGD-NP/RAF(-) complex in the M21 tumors; however, no HSP70 protein induction was seen in the M21-L tumors. Thus, targeted antiangiogenic therapy can induce Hsp70 expression and HSP70 protein in melanoma tumors.

Abstract 4098: Triple negative breast cancer: Cisplatin plus TRAIL treatment enhance tumor and cancer stem cell death in vitro and in vivo models

Abstract Women with triple negative breast cancer (TNBC) do not benefit from hormonal or herceptin-based therapies due to loss of target receptors such as ER, PR and HER-2 amplification. Current therapeutic approaches such as chemo- and/ or radiation therapy are in effective because cancer stem cells (CSC) were shown to be resistant to conventional chemo- and/or radiation therapy after treatment; they can regenerate all the cell types in the tumor through their stem cell-like behavior. The goal of this proposal is develop treatment strategy to effectively target tumor as well as CSC without significantly affecting the normal cells thereby improving therapeutic outcome or completely eliminate the tumor. Our data shows: (1) Treatment of TNBC cells, CRL-2335 and MDA-MB-468, with cisplatin plus Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) significantly enhanced cell death (by∼ 60-70%) as compared with untreated cells. (2) Similar treatment had a minimal effect (∼10-15% cell death) on CRL-8799 normal breast cells. (3) Human triple-negative breast cancers have high levels of cancer stem cells in breast carcinoma (∼ 41%) compared to surrounding benign/normal (∼5-10%) areas by CD44 positivity (cancer stem cell staining). (4) CRL-2335 TNBC cells treated with cisplatin plus TRAIL exhibited significant reduction (from ∼45% to ∼9% after treatment) in CSC. Treatment with cisplatin/TRAIL also inhibited the expression of EGFR, p63, survivin, Bcl-2 and Bcl-xL in TNBC cells. Specific inhibition of EGFR and/or p63 protein in TNBC cells by siRNA does not increase TRAIL-induced apoptosis. However, inhibition of survivin by siRNA enhances TRAIL-induced apoptosis. These observations suggested the possibility that Survivin played an important role in cisplatin plus TRAIL-induced apoptosis in TNBC cells. In vivo experiments, treatment of mice with cisplatin plus TRAIL resulted in a significant inhibition of CRL2335 xenograft tumors compared to untreated control tumors. Taken together the data suggests that cisplatin plus TRAIL treatment have the potential of providing a new strategy for improving the therapeutic outcome in TNBC patients. 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 4098. doi:10.1158/1538-7445.AM2011-4098