Animal Tumor Models Research Articles

Antitumor Vaccines Based on Dendritic Cells: From Experiments using Animal Tumor Models to Clinical Trials

The routine methods used to treat oncological diseases have a number of drawbacks, including non-specific action and severe side effects for patients. Furthermore, tumor diseases are associated with a suppression of the immune system that often leads to the inefficiency of standard treatment methods. The development of novel immunotherapeutic approaches having specific antitumor action and that activate the immune system is of crucial importance. Vaccines based on dendritic cells (DCs) loaded with tumor antigens ex vivo that can activate antitumor cytotoxic T-cell responses stand out among different antitumor immunotherapeutic approaches. This review is focused on analyzing different methods of DC-based vaccine preparation and current research in antitumor DC-based vaccines using animal tumor models and in clinical trials.

Enzymatic Inactivation of Endogenous IgG by IdeS Enhances Therapeutic Antibody Efficacy.

Endogenous plasma IgG sets an immunologic threshold that dictates the activity of tumor-directed therapeutic antibodies. Saturation of cellular antibody receptors by endogenous antibody limits antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Here, we show how enzymatic cleavage of IgG using the bacterial enzyme IdeS can be utilized to empty both high and low affinity Fcγ-receptors and clear the entire endogenous antibody pool. Using in vitro models, tumor animal models as well as ex vivo analysis of sera collected during a previous clinical trial with IdeS, we show how clearing of competing plasma antibody levels with IdeS unblocks cellular antibody receptors. We show that therapeutic antibodies against breast cancer (trastuzumab), colon cancer (cetuximab), and lymphomas (rituximab and alemtuzumab) can be potentiated when endogenous IgG is removed. Overall, IdeS is shown to be a potent tool to reboot the human antibody repertoire and to generate a window to preferentially load therapeutic antibodies onto effector cells and thereby create an armada of dedicated tumor-seeking immune cells. Mol Cancer Ther; 16(9); 1887-97. ©2017 AACR.

Micro‐CT study of Bomirski melanoma growing in hamster eye

PurposeThe Bomirski Hamster Melanoma (BHM) cell line has been successfully used as an eye tumor animal model in the studies on tumor vascular network, metastasis and radiation treatment. Visualization and modeling of neoangiogenesis is crucial for further ocular tumor research. This work presents visualization and image analysis of BHM eyeballs using X‐ray microtomography (micro‐CT).MethodsMicro‐CT is a nondestructive preclinical imaging method with isotropic micron resolution suitable for small ex‐vivo samples. This technique provides 3D distribution of linear attenuation coefficient realized as a stack of cross‐sectional images. The micro‐CT scanning and analysis protocol was optimized for simultaneous visualization of normal ocular tissues, and pathological vasculature of BHM. Micro‐CT image contrast was enhanced using iodine (Lugol's solution) staining technique. In this investigation the Bruker SkyScan 1272 micro‐CT system was used.ResultsThe iodine staining method provided well contrasted images presenting a globe anatomy in fine details. Advanced malignancy, with the tumor occupying the entire anterior chamber and the pathological disorganized irregular tumor vascular network was also visualized. Image analysis and processing allowed to distinguish grey level range corresponding to the particular globe and tumor structures. Quantitative analysis of BHM vessel size distribution and complexity level was performed.ConclusionsMicro‐CT enhanced with Lugol solution staining method has a great potential in preclinical eye tumor research using small animal models.

Nano-sized drug carriers: Extravasation, intratumoral distribution, and their modeling

Navigating intratumoral drug distribution has proven to be one of the most challenging aspects of drug delivery. The barriers are significant and varied; increased diffusional distances, elevated interstitial fluid pressure, regions of dense extracellular matrix and high cell density, and overall heterogeneity. Such a long list imposes significant requirements on nano-sized carriers. Unfortunately, other capabilities are eclipsed by the distribution requirements. A drug can do no good until it reaches its target. Numerous strategies to improve drug distribution have been developed, taking account of various unique characteristics of solid tumors, including some mechanisms that are still not fully understood. Most of these strategies were from small animal tumor models which are our primary tool for understanding cancer physiology. The small animal tumor model is the most versatile and effective means of understanding tumor transport, but its prevalence belies some of its weaknesses. Tumors grown under lab conditions are developed much more quickly than naturally developed cancers, potentially impacting tumor heterogeneity, blood vessel development, extracellular matrix organization, cell diversity, and many other features of structure and physiology that may impact transport. These problems come in addition to the difficulties of making precise measurements within a living tumor. Resolving these problems is best done by improving our analysis methods, and by finding complementary models that can clarify and expound the details. In this review, we will first discuss some of the strategies employed to improve transport and then highlight some of the new models that have recently been developed in the Bae lab and how they may aid in the study of tumor transport in the future.

Toosendanin demonstrates promising antitumor efficacy in osteosarcoma by targeting STAT3

Signal transducer and activator of transcription 3(STAT3) is an emerging target for cancer therapy. In this study, we identify Toosendanin (TSN) is an effective inhibitor of STAT3, leading to the impediment of various oncogenic processes in osteosarcoma. TSN selectively inactivates phospho-STAT3 (Tyr-705); subsequent molecular docking and in vitro SPR analysis uncover TSN directly binds to the SH2 domain of STAT3. Consequently, TSN blocks STAT3 dimerization and impairs the complex formation of STAT3 and epidermal growth factor receptor (EGFR). In an animal tumor model study, TSN is well tolerated, inhibits osteosarcoma growth and metastasis. In another osteosarcoma patient-derived xenografts (PDX) model, we find TSN triggers strong inhibitory effects on patient-derived tumors. Further studies show that TSN also displays activity against other solid tumors. Our preclinical work therefore supports that TSN acts as a novel inhibitor of STAT3 that blocks tumorigenesis in ostoesarcoma.

Development of zebrafish medulloblastoma-like PNET model by TALEN-mediated somatic gene inactivation.

Genetically engineered animal tumor models have traditionally been generated by the gain of single or multiple oncogenes or the loss of tumor suppressor genes; however, the development of live animal models has been difficult given that cancer phenotypes are generally induced by somatic mutation rather than by germline genetic inactivation. In this study, we developed somatically mutated tumor models using TALEN-mediated somatic gene inactivation of cdkn2a/b or rb1 tumor suppressor genes in zebrafish. One-cell stage injection of cdkn2a/b-TALEN mRNA resulted in malignant peripheral nerve sheath tumors with high frequency (about 39%) and early onset (about 35 weeks of age) in F0 tp53e7/e7 mutant zebrafish. Injection of rb1-TALEN mRNA also led to the formation of brain tumors at high frequency (58%, 31 weeks of age) in F0 tp53e7/e7 mutant zebrafish. Analysis of each tumor induced by somatic inactivation showed that the targeted genes had bi-allelic mutations. Tumors induced by rb1 somatic inactivation were characterized as medulloblastoma-like primitive neuroectodermal tumors based on incidence location, histopathological features, and immunohistochemical tests. In addition, 3′ mRNA Quanti-Seq analysis showed differential activation of genes involved in cell cycle, DNA replication, and protein synthesis; especially, genes involved in neuronal development were up-regulated.

The relationship between ¹⁸F-FDG uptake and oncogene in human laryngeal squamous cell carcinoma transplantation tumor animal model

Objective:The aim of this study is to explore the relationship between [F18] fluoro 2 deoxy d glucose ¹⁸F-FDG) uptake and oncogene expression in human laryngeal squamous cell carcinoma (LSCC) transplantation tumor.Method:Ten nude mice were randomly divided into two groups, 5 mice of each group. One group was injected subcutaneously with Hep-2 cells to establish xenograft model, and the other one was injected with HIF-RNAi-Hep-2 cells. All animals were conducted with positron emission tomography computed tomography PET scan. The average tumor T/N ratio was calculated. After PET scanning, tumor tissue was picked off for immunohistochemical examination.Result:The tumor imaging were both clear and there was no significant difference between these two groups (P> 0.05). The expression of hypoxia inducible factor 1 alpha (HIF-1α), glucose transporter protein-1 (GLUT-1) and vascular endothelial growth factor (VEGF) were high in Hep-2 group. The expression of HIF-1α was lower in HIF-RNAi-Hep-2 group (P< 0.05). But there was no significant difference about the expression of GLUT-1 and VEGF between these two groups (P> 0.05).Conclusion:The oncogenic pathway activated in LSCC is related with the uptake of ¹⁸F-FDG. The glycolysis gene and vascular factor maybe play a critical role.

Investigation of non-thermal plasma effects on lung cancer cells within 3D collagen matrices

Recent breakthroughs in plasma medicine have identified a potential application for the non-thermal plasma in cancer therapy. Most studies on the effects of non-thermal plasma on cancer cells have used traditional two-dimensional (2D) monolayer cell culture. However, very few studies are conducted employing non-thermal plasma in animal models. Two dimensional models do not fully mimic the three-dimensional (3D) tumor microenvironment and animal models are expensive and time-consuming. Therefore, we used 3D collagen matrices that closely resemble the native geometry of cancer tissues and provide more physiologically relevant results than 2D models, while providing a more cost effective and efficient precursor to animal studies. We previously demonstrated a role for non-thermal plasma application in promoting apoptotic cell death and reducing the viability of A549 lung adenocarcinoma epithelial cells cultured upon 2D matrices. In this study, we wished to determine the efficacy of non-thermal plasma application in driving apoptotic cell death of A549 lung cancer cells encapsulated within a 3D collagen matrix. The percentage of apoptosis increased as treatment time increased and was time dependent. In addition, the anti-viability effect of plasma was demonstrated. Twenty-four hours post-plasma treatment, 38% and 99% of cell death occurred with shortest (15 s) and longest treatment time (120 s) respectively at the plasma-treated region. We found that plasma has a greater effect on the viability of A549 lung cancer cells on the superficial surface of 3D matrices and has diminishing effects as it penetrates the 3D matrix. We also identified the nitrogen and oxygen species generated by plasma and characterized their penetration in vertical and lateral directions within the 3D matrix from the center of the plasma-treated region. Therefore, the utility of non-thermal dielectric barrier discharge plasma in driving apoptosis and reducing the viability of lung cancer cells in 3D collagen matrix indicates a therapeutic potential that warrants further research.

A longitudinal MRI study on lymph nodes histiocytosis of a xenograft cancer model.

BackgroundEfforts are continuously made to detect and investigate the pivotal processes and interplay between the response of sentinel lymph node and malignant cells from a primary tumor. Conversely, some frequently used tumor animal models, such as human cancer xenografts, rarely feature metastasis. Therefore, lymph node alterations are seldom assessed. We consider that studying lymph node response could contribute to the understanding of host reaction to cancer. In the present study, we explored the presence of regional lymph node alterations in parallel with tumor growth using a pancreatic tumor xenograft model which does not develop metastasis.Methods and findingsWe established an animal cancer model by the subcutaneous inoculation of PANC-1 (a metastatic human pancreatic cancer cell line) in the left upper flank of athymic nude mice. Tumor animals, along with controls (n = 7 / group) were subjected to Magnetic Resonance Imaging (MRI) in order to follow tumor growth and brachial and axillary lymph nodes alterations over several weeks. Further histological analyses were performed at the end of the study.The individual average of the different lymph nodes sizes was 15–40% larger in the tumor animals compared to control animals at week 8 to week 20. The tumor size and lymph node size were not correlated. Histological analysis of the lymph nodes showed paracortical histiocytosis. No metastasis to lymph nodes could be detected by histology. In tumor bearing animals, histiocytosis was associated with isolated apoptotic bodies and migration of human tumoral cells was confirmed by specific immunostaining of human origin markers.ConclusionsThe lack of metastasis as well as the pathological manifestation of the lymph node alteration in this pre-clinical model established here parallels findings in patients with sinus histiocytosis that is correlated with improved survival.

Abstract 4500: Inhibition of PERK by small molecule inhibitors enhances the response to ionizing radiation in vitro and animal tumor models

Abstract Endoplasmic reticulum (ER) is a well organized membranous network, responsible for synthesis and folding of secretory and membrane proteins, lipid and sterol biosynthesis and intracellular calcium storage. Perturbations in ER lumen caused by depletion of Ca2+ levels, hypoxia, nutrient deprivation, affect the ER homeostasis, a condition known as an ER stress. In order to cope with this stress, cells have developed an orchestrated biochemical response, termed Unfolded Protein Response (UPR). Hypoxia, an ER stress inducer, is an important and unique characteristic of the solid tumor microenvironment, promotes resistance to radiotherapy and contributes to poor patient prognosis. Cancer cells overcome these stress conditions through activation the PERK-eIF2α arm of the UPR, leading to inhibition of global protein synthesis and cell survival. We hypothesize that the use of small molecule PERK inhibitors will decrease the survival of hypoxic cells and eventually sensitize them to irradiation. In this study we used highly radioresistant melanoma and head and neck squamous cell carcinoma cell lines B16F10-ova and SQ20B, under nomroxia and hypoxia, respectively. We used two specific PERK inhibitors (PERKi) and tested for their efficacy both in vitro and in vivo. Induction of ER stress in both cell lines, either by treatment with the pharmacologic agents thapsigargin (TG) and tunicamycin (TN), or by exposure to hypoxic conditions (0.2%), was evaluated by activation of PERK and its downstream targets. Pre-treatment of stressed B16F10-ova and SQ20B cells for 2h with PERKi (1μM), caused complete inhibition of PERK-eIF2a-ATF4-CHOP arm of the UPR. Moreover, both cell lines pre-treated with PERKi presented a significant decrease in survival fraction under ER-stress induced by TG (0.5μM) as well as under hypoxia (0.2%) along with increasing doses of irradiation (IR). For the in vivo experiments, nude and C57BL/6 mice were treated with either 100mg/kg of PERKi by oral gavage, twice a day for 3weeks, or IR or a combination of PERKi and IR. Mice treated with PERKi showed a small but not significant delay of tumor growth in both backgrounds of mice. However, combined treatment with IR (12 and 15Gy) caused a significant reduction of tumor volume as well as a delayed tumor growth which was significantly more pronounced in the immunocompetent mice. Data from flow cytometry analysis of one of the the PERKi+IR treated tumors from C57BL/6 mice showed that there is a significant infiltration of CD3+ cells as well as significant higher number of CD3+CD8+ cells compared to the untreated mice or mice treated with either PERKi or IR alone. Collectively, our results show that PERKi effectively reduces the survival of hypoxic B16F10-ova and SQ20B cells and sensitize them to IR in vitro and in tumors. Further experiments will clarify whether PERKi have a dual role on reducing the hypoxic fraction and direct recruitment of the cytotoxic T-cells. Citation Format: Ioannis Verginadis, Joel Encarnación, Souvik Dey, Constantinos Koumenis. Inhibition of PERK by small molecule inhibitors enhances the response to ionizing radiation in vitro and animal tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4500. doi:10.1158/1538-7445.AM2017-4500

Abstract 4678: Pilot trial of talazoparib (BMN 673), an oral PARP inhibitor, in patients with advanced solid tumors carrying deleterious BRCA mutations

Abstract Inhibition of poly (ADP-ribose) polymerase (PARP) sensitizes tumor cells to DNA damage that would normally be repaired through the base excision repair pathway. PARP inhibitors are active clinically against BRCA-deficient ovarian cancers. The PARP inhibitor talazoparib produces cytotoxicity in human cancer cell lines and animal models of tumors that harbor mutations that compromise DNA repair pathways. In this study, single agent talazoparib (1000 µg/day) was administered to patients with deleterious BRCA1 or BRCA2 mutations and advanced solid tumors in 28 day cycles. The primary objective of the trial was to examine pharmacodynamic (PD) effects of talazoparib; the secondary objective was to determine response rate in patients whose tumors carry BRCA mutations. Mandatory paired tumor biopsies were obtained pre-treatment and 3-6 hrs post-treatment on cycle 1 day 8. Optional biopsies were collected at the time of progression. One core from each time point was analyzed for PARP inhibition by a validated ELISA assay while the other core was used for IFA analysis of γH2AX. A total of 9 patients (pts) were enrolled and treated before this trial was closed due to lack of drug availability: [prostate (3), ovarian (2), breast (2), uterine sarcoma (1), pancreatic (1)]. Median age was 63 (range: 33-73 yrs); male-to-female ratio was 4:5; and the median number of prior treatments was 6 (range: 1-12). All 9 pts were evaluable for PD endpoints. One pt progressed during the first cycle of treatment; 8 pts were evaluable for clinical response. Mean time on study for evaluable pts was 8 cycles (range: 2-18); 5 of 8 (62%) pts experienced a documented partial responses [ovarian (2), prostate (2), breast (1)] lasting between 4 and 12 cycles (median: 6 cycles). Two pts had stable disease for 4 to 6 cycles, and one progressed after 2 cycles. The agent was well tolerated; the most frequent adverse events were hematologic including grade (gr) 4 anemia (1) and thrombocytopenia (1), and gr 3 anemia (2), neutropenia (1), lymphopenia (1). Decreases in PAR levels (&amp;gt;75%) were observed in all cycle 1 day 8 biopsy pairs, documenting a primary PD effect. Increased γH2AX expression was observed for 4/6 pts in post-dose biopsies; pre-treatment γH2AX levels, measured as %Nuclear Associated Protein (NAP), increased from a mean ± SD of 1.33 ± 1.08 to a post-treatment %NAP mean of 5.60 ± 0.78; (p=0.018), supporting a role for drug-enhanced DNA double strand breaks in the mechanism of action of talazoparib for BRCA mutant tumors. In summary, talazoparib demonstrated significant clinical activity as a single agent in patients with BRCA-deficient tumors and produced substantial reductions in tumor PAR levels in matched pre and post-treatment tumor biopsies. Citation Format: Robert S. Meehan, Alice P. Chen, Geraldine O’Sullivan Coyne, Shivaani Kummar, Jiuping Ji, Rasa Vilimas, Lamin Juwara, Robert J. Kinders, Katherine Ferry-Galow, Deborah Wilsker, Yiping Zhang, Angie B. Dull, Tony Navas, Lihua Wang, Ralph E. Parchment, James H. Doroshow. Pilot trial of talazoparib (BMN 673), an oral PARP inhibitor, in patients with advanced solid tumors carrying deleterious BRCA mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4678. doi:10.1158/1538-7445.AM2017-4678

Abstract 4558: Immunogenicity and antitumor efficacy of live attenuated Salmonella typhimurium-based oral T-cell vaccines VXM01m, VXM04m and VXM06m

Abstract Significant progresses have been recently accomplished in immuno-oncology and in the development of cancer immunotherapies. However, novel solutions are necessary to overcome the peripheral tolerance and the immunosuppressive tumor microenvironment that prevent the eradication of cancer. VAXIMM is developing first-in-kind Salmonella typhi Ty21a-based oral T-cell vaccination platform for the initiation of anti-tumor cellular immune responses via a unique mode-of-action. This study summarizes the immunogenicity and preclinical anti-cancer efficacy for Salmonella typhimurium murine vaccines VXM01m, VXM04m and VXM06m which encode murine vascular endothelial growth factor receptor 2 (VEGFR2), mesothelin (MSLN) and Wilm's tumor 1 (WT1) protein antigens, respectively. Immunokinetic studies were performed in healthy animals treated with 1010 CFU/dose of either the control Salmonella typhimurium empty vector VXM0m_empty, or Salmonella typhimurium VXM01m, VXM06m or VXM04m, and antigen-specific T cells were detected with MHC class I pentamers by flow cytometry in the spleen. The anti-tumor efficacy of VXM01m and VXM04m was evaluated in the Panc02 syngeneic model of pancreatic adenocarcinoma overexpressing MSLN, and the anti-cancer activity of VXM06m was evaluated in the FBL-3 disseminated model of erythroleukemia expressing WT1, in a prime-boost setting. Treatment with each of the vaccines VXM01m, VXM04m and VXM06m induce a peak antigen-specific systemic immune response 7 to 10 days after the last vaccination. Treatment of Panc02 tumor-bearing mice with VXM01m and VXM04m single agents resulted in a significant reduction in the tumor growth rate, compared to the control group, with a median T/C of 37.6% and 19.4% respectively, 35 days after tumor challenge. Treatment of mice bearing FBL-3 leukemia with VXM06m generated a rapid and sustained anti-tumor effect with 100% (10 out of 10) of surviving animals 175 days after leukemia challenge. In contrast, treatment with VXM0_empty did not show any anti-cancer effect, with a median survival of 45 days and 0% (0 out of 10) of cancer regression. VXM01m, VXM04m and VMX06m were tolerated at the effective doses and have demonstrated consistent anti-cancer activities with substantial T cell responses in different animal tumor models. This study provides further evidence that VAXIMM’s versatile oral T-cell vaccination platform can be used to stimulate anti-tumor immunity against various tumor-associated antigens. Further studies of VAXIMM's cancer vaccine candidates, as monotherapy as well as in combination, are warranted. Citation Format: Sébastien Wieckowski, Lilli Podola, Marco Springer, Iris Kobl, Zina Koob, Caroline Mignard, Amine Adda Berkane, Ming Wei, Albrecht Meichle, Klaus Breiner, Matthias Schroff, Philipp Beckhove, Heinz Lubenau. Immunogenicity and antitumor efficacy of live attenuated Salmonella typhimurium-based oral T-cell vaccines VXM01m, VXM04m and VXM06m [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4558. doi:10.1158/1538-7445.AM2017-4558

Abstract 5215: Time dependent blood-brain barrier modulation by vascular endothelial growth factor to improve CNS drug delivery

Abstract Introduction: Efforts to transiently increase blood brain barrier permeability would facilitate improved CNS delivery of chemotherapy agents. Previous studies demonstrated that vascular endothelial factor (VEGF) increases permeability in traumatic brain injury and ischemic brain disease. Exogenously administered VEGF increases barrier permeability in the non-diseased CNS. In our study, we investigated the role for exogenous VEGF therapy to modulate blood-brain barrier permeability Methods: For in vitro studies, confluent mouse brain endothelial cells (BEND.3) were treated with VEGF165. Trans-endothelial electrical resistance measured cell impedance changes and immunofluorescence was used to assess qualitative expression of tight and adherens junction proteins at key time points. Western blotting and qPCR were done to evaluate the effect of VEGF on expression of protein and RNA associated with tight and adherens junctions. Results: Confluent endothelial cells treated with rVEGF165 (200ng/mL) evaluated by TEER show a significant drop in cell impedance approximately 6 hours post treatment, compared to control (more than 10-fold) reflecting increased permeability of the monolayer. Treatment with progressively higher doses of VEGF (200, 400, and 600ng/mL) showed a progressively greater drop in cell impedance. Immunofluorescence of endothelial cells treated with rVEGF165 (200ng/mL) shows disassembly of both tight junctions and adherens junctions between 4 and 8 hours post-treatment compared to controls. Western blots and qPCR demonstrate that rVEGF165 treatment induces down-regulation of genes associated with tight junctions, such as Claudin5, beginning as early as 4 hours post treatment and continuing up to 24 hours post treatment. Conclusion: Our results suggest that VEGF is capable of inducing transient breakdown of the blood-brain barrier, which may facilitate improved CNS delivery of chemotherapeutic agents. Our in vitro data warrants further investigations of the applicability of VEGF therapy to induce endothelial cell permeability in animal tumor models, potentially improving delivery of chemotherapeutic drugs. Citation Format: Matthew McCord. Time dependent blood-brain barrier modulation by vascular endothelial growth factor to improve CNS drug delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5215. doi:10.1158/1538-7445.AM2017-5215

Abstract 1664: Inhibition of 4T1 mammary tumor growth in BALB/c mice by subcutaneous and intraperitoneal injection of a 4T1 whole cell vaccine containing IL-2 and GM-CSF as adjuvants

Abstract Developing animal tumor models for human cancer vaccines creates a tool to investigate the mechanism of action, variations in formulation, dosing schedules, and combinations with other forms of cancer therapy. In this study we developed a mouse whole cell mammary cancer vaccine model with both subcutaneous and intraperitoneal injection of the vaccine. The vaccine, containing 4T1 mouse mammary cancer cells (1,000,000 cells), IL-2 (0.2 µg) and GM-CSF (0.1 µg) in a total volume of 150 µl, was injected either subcutaneously into the backs of BALB/c mice or intraperitoneally. There were six injections of the vaccine (weeks 1, 2, 3, 7, 11, 15) and one week after the last injection 100,000 4T1 cells from cell culture in a volume of 100 µl were injected into the scapular region of the mice. At 22 days post 4T1 tumor transplantation there was a 37% inhibition of the growth of the 4T1 tumor in the mice receiving the subcutaneous injection of the vaccine and a 42% inhibition of the growth of the 4T1 tumor in the mice receiving the intraperitoneal injection of the vaccine. The inhibition of 4T1 mammary tumor growth in this mouse model with subcutaneous injection of a whole cell vaccine gives further support for the previously reported efficacy in a Phase 1/2 clinical trial of our therapeutic breast cancer vaccine containing autologous and allogeneic breast cancer cells in the adjuvant setting. The inhibition of 4T1 tumor growth with intraperitoneal injection suggests a delivery method that may be applicable to ovarian cancer and cancer patients with malignant ascites. Citation Format: Jonathan F. Head, Jeffrey T. Phillips, Xianpeng Jiang, Robert L. Elliott. Inhibition of 4T1 mammary tumor growth in BALB/c mice by subcutaneous and intraperitoneal injection of a 4T1 whole cell vaccine containing IL-2 and GM-CSF as adjuvants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1664. doi:10.1158/1538-7445.AM2017-1664

Abstract 683A: Mechanism of liver metastasis induced systemic suppression of checkpoint inhibitor response

Abstract Introduction: In stage IV melanoma and NSCLC, PD-1 checkpoint inhibitor therapy is effective and now FDA approved as first-line therapy. However, despite these rationally designed strategies to manipulate immune cells, curative responses remain rare, and the majority of metastatic cancer patients ultimately succumb to their disease. Emerging potential biomarkers of response have required specialized technologies such as next-generation sequencing or flow cytometry, which are difficult to access, costly, and not immediately clinically applicable. To this end, we have previously reported that presence of liver metastasis was significantly associated with the lack of antitumor immune infiltrates at distant biopsy sites as well as poor clinical response to PD-1 blockade. Here, we sought to study the mechanism of liver metastasis related non-response to PD-1 blockade using a syngeneic immunocompetent murine tumor model. Methods: C57BL/6 mice were implanted with a "primary” subcutaneous tumor as well as a secondary “metastatic” tumor in the liver or the lungs (control), and given systemic PD-1 blockade therapy to mimic the clinical observation and study the effects of liver tumor involvement on systemic antitumor immunity. Animals are monitored for survival and tumor growth. The immune infiltrates of both the primary and metastatic sites are phenotypically analyzed. Results: Animals with liver tumor involvement appear to have significantly shortened survival as well as difficulty rejecting tumors, both at the primary and secondary site of implantation, when compared to non-liver involved controls. The lack of response to PD-1 blockade in the liver cohort is correlated with a significant decrease in CTLA4+/PD-1+/CD8+ “exhausted” antigen experienced T cells (TEx) within the tumor microenvironment of the subcutaneous and liver-embedded sites. The deleterious effect on TEx appears to be liver-specific and not tumor cell-line dependent, as delivery of different tumor cells to the liver reproduces the same effect while delivery of tumor cells to other organs does not change the quantity of TEx at the subcutaneous site. Lastly, liver associated deletion of TEx appears to be a an antigen dependent process, as delivery of mismatched tumor cell line to the liver abrogates this effect and restores TEx within the tumor microenvironment at the distant subcutaneous site. Conclusions: Using an immunocompetent checkpoint blockade animal tumor model, we recapitulated the clinically observed difference in response to PD-1 blockade treatment that is associated with liver metastasis. Our studies suggest that the liver is capable of suppressing effective T cell mediated antitumor response systemically, rendering checkpoint blockade less effective. The findings here propose a novel mechanism for which non-responsiveness to PD-1 blockade therapy could occur and may impact the treatment decision for cancer patients with liver metastasis. Citation Format: James C. Lee, Adil Daud, Jeff Bluestone. Mechanism of liver metastasis induced systemic suppression of checkpoint inhibitor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 683A. doi:10.1158/1538-7445.AM2017-683A

Abstract 2089: Evaluation of fruquintinib, a potent and selective oral VEGFR inhibitor, in combination with targeted therapies or immune checkpoint inhibitors in preclinical tumor models

Abstract The development of therapies targeting tumor angiogenesis, tumor driver gene alterations and tumor immune evasion has made tremendous advancement in improving overall survival. However, efficacy may be limited and resistance often develops rapidly when targeting a single axis of tumorigenesis. Therefore, it is worthwhile to explore rational combination of therapies based on tumor-specific features. Fruquintinib is a potent and selective oral VEGFR inhibitor currently in Phase III clinical trials for non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC). We report here the evaluation of anti-tumor effect of fruquintinib in preclinical animal tumor models in combination with therapies targeting tumor driver gene alterations such as EGFR and c-MET or with immune checkpoints. In NSCLC xenograft models with EGFR activation such as activating mutations, gene amplification or protein overexpression, fruquintinib plus an EGFR tyrosine kinase inhibitor such as gefitinib or theliatinib (HMPL-309) was found to be more efficacious than either monotherapy. For instance, in PC-9 subcutaneous tumor model carrying EGFR exon 19 deletion, single agent treatment with fruquintinib at 2 mg/kg and gefitinib at 5 mg/kg produced the tumor growth inhibition (TGI) of 58% and 63%, respectively, while the combination treatment resulted in a TGI of 100% and tumor regression was observed in 11 of 16 mice treated with combinational therapy. In multiple xenograft models derived from lung cancer or renal cell cancer with c-MET activation (amplification or over-expression), addition of fruquintinib to a c-MET inhibitor savolitinib (AZD6094, HMPL-504) also improved the tumor growth inhibition substantially. At the end of the efficacy studies, CD31 and phosphorylation of EGFR, c-MET, AKT and ERK were analyzed with immunohistochemistry and western blotting method in tumor tissues. The results suggested that the enhanced anti-tumor effect in combination therapy could be attributed to the simultaneous blockade of cell signaling in tumor cells (EGFR or c-MET) and VEGFR suppression in the tumor microenvironment. Up-regulation of the immune inhibitory checkpoints induced by VEGF is one of the important mechanisms for tumor cells to escape immune surveillance. In a syngeneic murine tumor model, co-administration of fruquintinib and anti-PD-L1 antibody was found to provide improved anti-tumor effect compared to fruquintinib or anti-PD-L1 single agent alone. Studies to understand the mechanism responsible for the combination effect are under way. All combinations with fruquintinib described above were well tolerated. The efficacy observed in these models suggested that simultaneous blockade of tumor angiogenesis and tumor cell signaling or immune evasion may be a promising approach in improving treatment outcomes. Citation Format: Yongxin Ren, Qiaoling Sun, Jingwen Long, Shiming Fan, Renxiang Tang, Wei Zhang, Xuelei Ge, Jianxing Tang, Linfang Wang, Dongxia Shi, Hongbo Chen, Min Cheng, Weiguo Qing, Weiguo Su. Evaluation of fruquintinib, a potent and selective oral VEGFR inhibitor, in combination with targeted therapies or immune checkpoint inhibitors in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2089. doi:10.1158/1538-7445.AM2017-2089

Tumor-specific delivery of doxorubicin through conjugation of pH-responsive peptide for overcoming drug resistance in cancer

The major obstacles opposed to doxorubicin (Dox) based chemotherapy are the induction of drug resistance, together with non-specific toxicities for healthy cells. In this study, we prepared a peptide-Dox conjugate aimed at offering Dox molecules a tumor-specific functionality and improving the therapeutic effects of Dox against resistant tumor cells. A slightly acidic pH-sensitive peptide (SAPSP) with high selectivity for cancer cells was attached to Dox to obtain SAPSP-Dox prodrug. The structures and properties of this prodrug were characterized, confirming several merits, including desirable pH-sensitive property, good serum stability and favorable release behavior. Cellular uptake studies demonstrated that SAPSP-Dox was preferably accumulated in cancer cells (Dox-sensitive MCF-7 and Dox-resistant MCF-7/ADR), followed by displaying 26-fold less toxic toward noncancerous MCF-10A cells than free Dox do. The conjugated peptides enabled Dox to escape the efflux effect of P-glycoprotein mediated pump via endocytotic pathway, giving rise to remarkable cytotoxicity and apoptotic effect on MCF-7/ADR cells. Moreover, the superior inhibition efficacy of SAPSP-Dox in vivo was more evident in the both drug-sensitive and drug-resistant xenograft tumor animal models, which enabled Dox to primarily accumulated in tumor. The conjugates also demonstrated a longer half-life in plasma and a lower side effect, for example, reduced cardiac toxicity. Evidence of this study suggests that SAPSP-Dox has the potential to be a potent prodrug for treating drug resistant cancers.

Characterization of HPV18 E6-specific T cell responses and establishment of HPV18 E6-expressing tumor model

Human papillomavirus (HPV) has been identified as the primary etiologic factor of cervical cancer, and subsets of anogenital and oropharyngeal cancers. HPV18 is the second most prevalent high-risk HPV type after HPV16. Furthermore, HPV18 is responsible for approximately 12% of cervical squamous cell carcinoma and 37% of cervical adenocarcinoma cases worldwide. In this study, we aimed to characterize the HPV18-E6-specific epitope and establish an HPV18 animal tumor model to evaluate the E6-specific immune response induced by our DNA vaccine. We vaccinated naïve C57BL/6 mice with a prototype DNA vaccine, pcDNA3-HPV18-E6, via intramuscular injection followed by electroporation, and analyzed the E6-specific CD8+ T cell responses by flow cytometry using a reported T cell epitope. We then characterized the MHC restriction element for the characterized HPV18-E6 epitope. Additionally, we generated an HPV18-E6-expressing tumor cell line to study the antitumor effect mediated by E6-specific immunity. We observed a robust HPV18-E6aa67-75 peptide-specific CD8+ T cell response after vaccination with pcDNA3-HPV18-E6. Further characterization demonstrated that this epitope was mainly restricted by H-2Kb, but was also weakly presented by HLA-A∗0201, as previously reported. We observed that vaccination with pcDNA3-HPV18-E6 significantly inhibited the growth of HPV18-E6-expressing tumor cells, TC-1/HPV18-E6, in mice. An antibody depletion study demonstrated that both CD4+ and CD8+ T cells are necessary for the observed antitumor immunity. The characterization of HPV18-E6-specific T cell responses and the establishment of HPV18-E6-expressing tumor cell line provide infrastructures for further development of HPV18-E6 targeted immunotherapy.

Potent anti-cancer effects of less polar Curcumin analogues on gastric adenocarcinoma and esophageal squamous cell carcinoma cells

Curcumin and its chalcone derivatives inhibit the growth of human cancer cells. It is reported that replacement of two OH groups in curcumin with less polar groups like methoxy increases its anti-proliferative activity. In this study, we explored benzylidine cyclohexanone derivatives with non-polar groups, to see if they possess increased anti-cancer activity. Novel 2,6-bis benzylidine cyclohexanone analogues of curcumin were synthesized, and their inhibitory effects on gastric adenocarcinoma (AGS) and esophageal squamous cell carcinoma (KYSE30) cancer cells were studied using an MTT assay. Cell apoptosis was detected by EB/AO staining, and cell cycle was analyzed by flow cytometry. Real-time PCR was performed for gene expression analysis. All synthesized analogues were cytotoxic toward gastric and esophageal cancer cells and showed lower IC50 values than curcumin. Treatment with 2,6-Bis-(3-methoxy-4-propoxy-benzylidene)-cyclohexanone (BM2) was 17 times more toxic than curcumin after 48 h incubation. All novel compounds were more effective than curcumin in apoptosis induction and cell cycle arrest at G1 phase. These results suggest that less polar analogues of curcumin have potent cytotoxicity in vitro. However, they need to be investigated further, especially with animal tumor models, to confirm their chemotherapeutic activity in vivo.

IMMU-09. P53 REGULATES IMMUNOGENICITY OF MEDULLOBLASTOMA

Immunotherapy has recently emerged as an important strategy for treating cancer, but has not been tested extensively in pediatric brain tumors. To determine whether immunotherapy would be effective in this context, we need robust animal models of brain tumors that have a fully functional immune system. We recently created genetically engineered mouse models of Group 3 (Myc-driven) medulloblastoma, the most aggressive and lethal form of the disease. These models are generated by transducing cerebellar stem cells with viruses encoding Myc and dominant negative p53 (MP model), or Myc and GFI1 (MG model), and transplanting them into the cerebellum of an adult immunodeficient (ID) mouse. To evaluate whether these models can be used to study immunotherapy, we transplanted tumor cells into syngeneic, immunocompetent (IC) hosts. While MP tumors grew robustly in both ID and IC hosts, MG tumors failed to grow when transplanted into IC mice. Depletion of T cells rescued this effect, supporting the notion that MG tumors are being recognized and rejected by the immune system. Notably, knockdown or inactivation of p53 in MG tumors also allowed them to grow in IC hosts, suggesting that p53 mutations may render tumor cells invisible to the immune system. Ongoing studies are focused on understanding the basis of p53-mediated immune regulation, and on developing strategies to overcome it. These studies will provide insight into the interactions between medulloblastoma cells and the immune system, and identify novel approaches to immunotherapy of this tumor. Since p53 is mutated in a large proportion of human cancers, these studies may also have important implications for immunotherapy of other cancers.