Bilateral Tumor Model Research Articles

Biodistribution and imaging of fluorescently-tagged iron oxide nanoparticles in a breast cancer mouse model.

Iron oxide nanoparticle (IONP) hyperthermia is an emerging treatment that shows great potential as a cancer therapy both alone and in synergy with conventional modalities. Pre-clinical studies are attempting to elucidate the mechanisms of action and distributions of IONP in various in vitro and in vivo models, however these studies would greatly benefit from real-time imaging of IONP locations both in cellular and in mammalian systems. To this end, fluorescently-tagged IONP (fIONP) have been employed for real time tracking and co-registration of IONP with iron content. Starch-coated IONP were fluorescently-tagged, purified and analyzed for fluorescent signal at various concentrations. fIONP were incubated with MTGB cells for varying times and cellular uptake analyzed using confocal microscopy, flow cytometry and inductively-coupled plasma mass spectrometry (ICP-MS). fIONP were also injected into a bilateral mouse tumor model for radiation modification of tumor tissue and enhanced fIONP deposition assessed using a Xenogen IVIS fluorescent imager. Results demonstrated that fIONP concentrations in vitro correlated with ICPMS iron readings. fIONP could be tracked in vitro as well as in tissue samples from an in vivo model. Future work will employ whole animal fluorescent imaging to track the biodistribution of fIONP over time.

Preferential Replication of Systemically Delivered Oncolytic Vaccinia Virus in Focally Irradiated Glioma Xenografts

Radiotherapy is part of the standard of care in high-grade gliomas but its outcomes remain poor. Integrating oncolytic viruses with standard anticancer therapies is an area of active investigation. The aim of this study was to determine how tumor-targeted ionizing radiation (IR) could be combined with systemically delivered oncolytic vaccinia virus. U-87 glioma xenografts were grown subcutaneously or orthotopically. Oncolytic vaccinia viruses GLV-1h68 and LIVP 1.1.1 were injected systemically and IR was given focally to glioma xenografts. In a bilateral tumor model, glioma xenografts were grown in both flanks, oncolytic vaccinia was injected systemically and radiation was delivered specifically to the right flank tumor, whereas the left flank tumor was shielded. Viral replication and tumor regression, after systemic injection, was analyzed and compared in irradiated and nonirradiated glioma xenografts. Systemically administered oncolytic vaccinia virus replicated to higher titers in preirradiated U-87 xenografts than in nonirradiated glioma xenografts. This increased oncolytic viral replication correlated with increased tumor xenograft regression and mouse survival in subcutaneous and orthotopic U-87 glioma models compared with monotherapies. The ability of focal IR to mediate selective replication of oncolytic vaccinia was shown in a bilateral glioma model in which systemically administered oncolytic vaccinia replicated preferentially in the irradiated tumor compared with the nonirradiated tumor in the same mouse. These findings show a potential clinical role of focal IR in sensitizing irradiated tumor sites for preferential vaccinia virus-mediated oncolysis.

Abstract 1537: Combined Tbet and IL-12 gene therapy elicits and recruits superior anti-tumor immunity in vivo

Abstract We have recently shown that intra-tumor (i.t.) injection of syngenic dendritic cells (DC) engineered to express the transcription factor Tbet (TBX21) promotes protective Type-1 T cell-mediated immunity via a mechanism that is largely interleukin (IL)-12p70-independent. Since IL-12 is a classical promoter of Type-1 immunity, the current study was undertaken to determine whether gene therapy using combined Tbet and IL-12 cDNA would yield improved anti-tumor efficacy based on the complementary/synergistic action of these biologic modifiers. Mice bearing established s.c. tumors injected with DC concomitantly expressing ectopic Tbet and IL-12 (i.e. DC.Tbet/IL12) displayed superior i.) rates of tumor rejection and extended overall survival, ii.) cross-priming of Tc1 reactive against antigens expressed within the tumor microenvironment, and iii.) infiltration of CD8+ T cells into treated tumors in association with elevated locoregional production of CXCR3 ligand chemokines. In established bilateral tumor models, i.t. delivery of DC.Tbet/IL12 into a single lesion led to slowed growth or regression at both tumor sites. Furthermore, DC.Tbet/IL12 pulsed with tumor antigen-derived peptides and injected as a therapy distal to the tumor site prevented tumor growth and activated robust antigen-specific Tc1 responses. These data support the translational use of combined Tbet and IL-12p70 gene therapy in the cancer setting. In order to target multiple gene pathways which should provide a more beneficial clinical outcome, we have begun combinational immunotherapy with DC.Tbet/IL12 and chemotherapeutic drugs targeting multiple receptor tyrosine kinases (i.e. sunitinib). 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 1537. doi:1538-7445.AM2012-1537

Combined Tbet and IL12 Gene Therapy Elicits and Recruits Superior Antitumor Immunity In Vivo

We have recently shown that intratumor (i.t.) injection of syngenic dendritic cells (DC) engineered to express the transcription factor Tbet (TBX21) promotes protective type-1 T cell-mediated immunity via a mechanism that is largely interleukin (IL)-12p70-independent. Since IL-12 is a classical promoter of type-1 immunity, the current study was undertaken to determine whether gene therapy using combined Tbet and IL-12 complementary DNA (cDNA) would yield improved antitumor efficacy based on the complementary/synergistic action of these biologic modifiers. Mice bearing established subcutaneous (s.c.) tumors injected with DC concomitantly expressing ectopic Tbet and IL12 (i.e., DC.Tbet/IL12) displayed superior (i) rates of tumor rejection and extended overall survival, (ii) cross-priming of Tc1 reactive against antigens expressed within the tumor microenvironment, and (iii) infiltration of CD8(+) T cells into treated tumors in association with elevated locoregional production of CXCR3 ligand chemokines. In established bilateral tumor models, i.t. delivery of DC.Tbet/IL12 into a single lesion led to slowed growth or regression at both tumor sites. Furthermore, DC.Tbet/IL12 pulsed with tumor antigen-derived peptides and injected as a therapy distal to the tumor site prevented tumor growth and activated robust antigen-specific Tc1 responses. These data support the translation use of combined Tbet and IL-12p70 gene therapy in the cancer setting.

Tumor Protection Following Vaccination With Low Doses of Lentivirally Transduced DCs Expressing the Self-antigen erbB2

Gene therapy strategies may accelerate the development of prophylactic immunotherapy against cancer. We synthesized a lentiviral (LV) vector encoding a kinase-deficient form of erbB2 (erbB2tr) to transduce murine dendritic cells (DCs) efficiently. Murine erbB2 models a clinically relevant tumor-associated self-antigen; its human homolog (HER-2/neu) is overexpressed in breast cancer and in 80% of metastatic prostate cancers. Following one infection, approximately 47% of DCs overexpressed erbB2tr. To determine whether low doses of transduced DCs could protect mice from prostate cancer cells, we performed prime/boost vaccinations with 2 x 10(3) or 2 x 10(5) erbB2tr-transduced DCs. Six weeks after vaccination, mice were simultaneously bilaterally challenged with the aggressive RM-1 prostate cancer cell line and an erbB2tr-expressing variant (RM-1-erbB2tr). Whereas control mice developed both tumors, all recipients of 2 x 10(5) erbB2tr-transduced DCs developed only wild-type RM-1 tumors. One-third of mice vaccinated with just 2 x 10(3) erbB2tr-transduced DCs also demonstrated erbB2tr-specific tumor protection. Protection against RM-1-erbB2tr tumors was associated with sustained levels of anti-erbB2tr antibody production and also correlated with erbB2tr-specific Th1 cytokine secretion. Depletion of CD4(+), CD8(+), or natural killer (NK) cells prior to tumor challenge underscored their role in mediating tumor protection. We conclude that administration of DCs expressing a self-antigen through efficient LV-based gene transfer activates cellular and humoral immunity, protecting host animals against specific tumor challenge.

Local and Distant Immune-Mediated Control of Colon Cancer Growth with Fusogenic Membrane Glycoproteins in Combination with Viral Oncolysis

We evaluated whether the expression of measles virus fusogenic membrane glycoproteins H and F (MV-FMG), encoded by a herpes simplex virus type 1 (HSV-1) amplicon vector, can serve with or without viral oncolysis (G47Delta) and facultative irinotecan chemotherapy, alone or in combination with the monoclonal epidermal growth factor receptor (EGFR) inhibitory antibody cetuximab, as a platform for inducing tumor-specific immune responses against colon cancer. We demonstrated in vitro that MV-FMG expression in murine cells resulted in cell-cell fusion and synergistically enhanced the cytotoxicity of irinotecan alone or in combination with cetuximab. In a bilateral syngeneic subcutaneous MC38 and Colon26 tumor model in C57BL/6 and BALB/c mice we assessed both the effect on directly vector-treated tumors and the effect on contralateral, not directly vector-treated tumors. We demonstrated that the combination of three treatment components with or without cetuximab resulted in the best volume reduction of both directly vector-treated and not directly vector-treated tumors as well as pronounced infiltration of both tumor types with natural killer cells, macrophages, and T cells. T cells of these animals exhibited strong ex vivo cytotoxic activity against the tumor cells, indicating that the antineoplastic effect on untreated tumors was mediated by an antitumor immune response. Preexisting immunity against HSV-1 or measles virus had no detrimental effect on overall treatment efficacy. Our data indicate that MV-FMG expression in combination with viral oncolysis with or without clinically relevant chemotherapy for colon cancer treatment warrants further investigation.

In situ tumor vaccination with adenovirus vectors encoding measles virus fusogenic membrane proteins and cytokines

To evaluate whether intratumoral expression of measles virus fusogenic membrane glycoproteins H and F (MV-FMG), encoded by an adenovirus vector Ad.MV-H/F, alone or in combination with local coexpression of cytokines (IL-2, IL-12, IL-18, IL-21 or GM-CSF), can serve as a platform for inducing tumor-specific immune responses in colon cancer. We used confocal laser scanning microscopy and flow cytometry to analyze cell-cell fusion after expression of MV-FMG by dye colocalization. In a syngeneic bilateral subcutaneous MC38 and Colon26 colon cancer model in C57BL/6 and BALB/c mice, we assessed the effect on both the directly vector-treated tumor as well as the contralateral, not directly vector-treated tumor. We assessed the induction of a tumor-specific cytotoxic T lymphocyte (CTL) response with a lactate dehydrogenase (LDH) release assay. We demonstrated in vitro that transduction of MC38 and Colon26 cells with Ad.MV-H/F resulted in dye colocalization, indicative of cell-cell fusion. In addition, in the syngeneic bilateral tumor model we demonstrated a significant regression of the directly vector-inoculated tumor upon intratumoral expression of MV-FMG alone or in combination with the tested cytokines. We observed the highest anti-neoplastic efficacy with MV-FMG and IL-21 coexpression. The degree of tumor regression of the not directly vector-treated tumor correlated with the anti-neoplastic response of the directly vector-treated tumor. This regression was mediated by a tumor-specific CTL response. Our data indicate that intratumoral expression of measles virus fusogenic membrane glycoproteins is a promising tool both for direct tumor treatment as well as for tumor vaccination approaches that can be further enhanced by cytokine coexpression.

Overcoming Self-Tolerance: Long-Term Protection Against Specific erbB2-Expressing Prostate Tumors Using Low Doses of Lentivirus-Transduced DCs.

Anti-tumor immunotherapy is difficult to achieve in vivo in part due to naturally occurring peripheral tolerance. Our laboratory has developed a potent immunotherapy strategy that uses unusually low doses of dendritic cells (DCs) to break self-tolerance in a mouse model of prostate cancer. We constructed a lentiviral vector (LV) encoding a truncated form of the true self-antigen murine erbB2 (LV/erbB2tr). This protein is the murine form of HER-2/neu, a tumor associated antigen (TAA) upregulated in 20% of primary prostate tumors and 80% of metastatic cases. With LV/erbB2tr, we achieved highly efficient gene transfer into DCs that were generated from donor murine bone marrow, as up to 47% of DCs over-expressed erbB2tr following one infection (MOI of 3). Similar transduction levels were achieved using a control enGFP LV. The purity of transduced DCs was confirmed by flow cytometry for CD11c, CD80, CD86, and I-Ab expression. We then tested the ability of two low dose inoculations of these transduced DCs to overcome self-tolerance to erbB2 and protect against specific challenge in a bilateral tumor model. Mice were vaccinated twice, two weeks apart by administration of either 2 × 105 or 2 ×103 cells (i.p.). Six weeks later, the mice were injected on one hind flank with the highly aggressive murine tumor cell line RM1 and on the opposite flank with erbB2tr-transduced RM1 (RM1-erbB2tr). We saw complete protection from RM1-erbB2tr tumors in 100% of the mice receiving 2 × 105 erbB2tr-transduced DCs (n=6). Control RM1 tumors were not rejected. No systemic auto-immune responses to the self-antigen were observed, as all mice survived and the procedure was well tolerated. Furthermore, our strategy was effective even using 100-fold fewer DCs in the inoculations. In the mice receiving 2 ×103 erbB2tr-transduced DCs, 33% (2/6 animals) were completely protected from developing RM1-erbB2tr tumors and 2 others showed near-complete protection. At regular points throughout the experiment, serum levels of anti-erbB2tr antibodies were measured. In mice receiving the 2 ×105 DC dose, a peak antibody response was seen 1 week after the second immunization (4.6-fold above controls). Long-term antibody responses were detected beyond 6-weeks post-vaccination. T cell responses are being assessed by cytokine (IFN-γ, IL-2, IL-4, IL-5, and IL-10) secretion assays. Cytokine specificity data are pending. We speculated that the antigen-specific anti-tumor effect might have been modulated, in part, by apoptosis of regulatory T (Treg) cells that help maintain peripheral tolerance. Indeed, CD4+CD25+Foxp3+ Treg cells were consistently reduced in mice vaccinated with erbB2tr-transduced DCs, as shown by flow cytometry. Together these experiments show that we can overcome tolerance to an endogenously-expressed TAA, even using very low doses of erbB2-transduced DCs. Future studies will focus on testing our DC therapy in mice with established prostate tumors for a more clinically applicable model.

Ultrasound-mediated nanoparticle drug delivery in an in vivo tumor model

One of the exciting possible future clinical uses of ultrasound is site-targeted drug delivery using focused acoustic energy. In an earlier study, it was shown that ultrasound exposure promoted lipid delivery from nanoparticles into melanoma cells in culture [Crowder et al., UMB 2005]. In order to extend the study to in vivo situations, a novel, combined ultrasound therapeutic and imaging system was developed. The system consisted of a therapeutic transducer (−6-dB BW 1.1 to 2.3 MHz; natural focus 8 cm; f-number 1) with a centrally located and coregistered imaging transducer connected to a commercial ultrasound imaging system. The system provided capability for arbitrary acoustic excitation, electronic axial steering, and mechanical translation of the therapy transducer as well as real-time acquisition of images. The device was tested on bilateral murine tumor models (MDA435). Tumor images provided real-time feedback for treatment planning. The animals were injected with perfluorooctyl bromide nanoparticles targeted to αvβ3 integrin and with a radioactive complex. Initial results showed that application of therapeutic ultrasound affected the delivery of the radioactive complex as measured by radioactivity 24 h postinjection. Experimental results and experiences will be presented detailing the importance of imaging for in vivo treatment guidance and planning.

Dendritic cell immunotherapy with poly(d,l‐2,4‐diaminobutyric acid)‐mediated intratumoral delivery of the interleukin‐12 gene suppresses tumor growth significantly

A conventional DC-based immunotherapy has been tested clinically for treatment of patients with advanced cancer but requires modification to further improve the clinical results. In this study, we evaluated the in vivo antitumor effects of DC therapy, non-viral-mediated IL-12 gene therapy, and a combination of the two in a murine bilateral subcutaneous tumor model. DC therapy alone and IL-12 gene therapy alone suppressed tumor growth at the injected sites. However, the antitumor effect on the distant contralateral tumor was insufficient. When DC therapy and IL-12 gene therapy were carried out simultaneously, tumor growth was significantly suppressed bilaterally (P < 0.001). Cytolytic activity was augmented significantly in mice given the combination treatment compared to in mice treated with either DC or IL-12 gene therapy alone (P < 0.05). Microvessel density of both tumors was significantly lower in mice subjected to the combination therapy than in mice treated otherwise (P < 0.05). Furthermore, no side-effects were observed in the treated mice. DC therapy combined with non-viral-mediated intratumoral IL-12 gene delivery has a synergistic antitumor effect not only on targeted tumors but also on contralateral distant tumors and may be of great potential as a therapeutic treatment for patients with advanced cancer.

Reovirus oncolysis of human breast cancer.

We have previously shown that human reovirus replication is restricted to cells with an activated Ras pathway, and that reovirus could be used as an effective oncolytic agent against human glioblastoma xenografts. This study examines in more detail the feasibility of reovirus as a therapeutic for breast cancer, a subset of cancer in which direct activating mutations in the ras proto-oncogene are rare, and yet where unregulated stimulation of Ras signaling pathways is important in the pathogenesis of the disease. We demonstrate herein the efficient lysis of breast tumor-derived cell lines by the virus, whereas normal breast cells resist infection in vitro. In vivo studies of reovirus breast cancer therapy reveal that viral administration could cause tumor regression in an MDA-MB-435S mammary fat pad model in severe combined immunodeficient mice. Reovirus could also effect regression of tumors remote from the injection site in an MDA-MB-468 bilateral tumor model, raising the possibility of systemic therapy of breast cancer by the oncolytic agent. Finally, the ability of reovirus to act against primary breast tumor samples not propagated as cell lines was evaluated; we found that reovirus could indeed replicate in ex vivo surgical specimens. Overall, reovirus shows promise as a potential breast cancer therapeutic.

Combination suicide/cytokine gene therapy as adjuvants to a defective herpes simplex virus-based cancer vaccine.

We have used syngeneic, established bilateral subcutaneous tumor models to examine the antitumor activity of herpes simplex virus (HSV) vectors, including the induction of an immune response against non-inoculated distant tumors. In such a model with CT26 murine colon adenocarcinoma, unilateral intratumoral inoculation of replication-deficient HSV-1 tsK inhibited the growth of both the inoculated and noninoculated established tumors. To enhance this limited antitumor immune response, we generated a defective HSV vector, dvIL12-tk encoding both interleukin-12 (IL-12) and HSV thymidine kinase (TK), with tsK as the helper virus. In a 'suicide gene' strategy, ganciclovir (GCV) treatment after intratumoral inoculation of dvlacZ-tk/tsK, encoding E. coli lacZ instead of IL-12, resulted in enhanced antitumor activity. Antitumor activity was also enhanced by local expression of IL-12 from dvIL12-tk/tsK. The combination of IL-12 cytokine therapy with GCV treatment was the most efficacious approach, with significantly greater inhibition of tumor growth than IL-12 or TK + GCV alone. These results illustrate the power of combining different cancer therapy approaches; 'suicide gene' therapy, cytokine therapy, and HSV vector infection. HSV vectors are particularly well suited to this because they can accommodate the insertion of large and multiple gene sequences.

Tumor Growth Inhibition by Intratumoral Inoculation of Defective Herpes Simplex Virus Vectors Expressing Granulocyte–Macrophage Colony-Stimulating Factor

To evaluate the potential of defective herpes simplex virus (HSV) amplicon vectors as in vivo cytokine gene transfer vehicles for active immunotherapy, we generated a defective HSV vector that encodes the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, using a replication-defective HSV as helper virus. A variety of murine tumor cell lines were efficiently infected in vitro with the defective GM-CSF vector (dvGM), and this led to the synthesis and secretion of murine GM-CSF. In an established bilateral subcutaneous tumor model with Harding-Passey murine melanoma, unilateral intratumoral inoculation of dvGM significantly inhibited tumor growth of both the inoculated and noninoculated contralateral tumors. This tumor inhibition was dose-dependent and resulted in increased survival of the dvGM-treated mice. Inoculation of a lacZ-expressing defective vector had no effect on tumor growth. We conclude that this defective HSV vector system offers an effective method for cytokine gene delivery in vivo and that GM-CSF expression in tumors has antitumor activity.

In Situ Cancer Vaccination: An IL-12 Defective Vector/Replication-Competent Herpes Simplex Virus Combination Induces Local and Systemic Antitumor Activity

Intratumoral inoculation of replication-competent, attenuated herpes simplex virus (HSV) mutants inhibits tumor growth by direct cytotoxic viral replication and induction of a tumor-specific immune response. To boost the antitumor response, we describe a defective HSV vector encoding IL-12 as an adjuvant to in situ vaccination by the replication-competent HSV helper virus. The defective HSV vector system consists of defective particles containing tandem repeats of the cytokine genes (p40 and p35) in combination with a HSV helper virus. Heterodimeric IL-12 was expressed and secreted after IL-12 defective vector infection of tumor cells. In a syngeneic, bilateral established tumor model with CT26 murine colon carcinoma, unilateral intratumoral inoculation with an IL-12 defective/replication-competent HSV vector combination significantly reduced tumor growth of the inoculated and noninoculated contralateral tumors. This antitumor effect was significantly greater than with a lacZ-defective/replication-competent HSV vector combination, which itself was significantly greater than the mock inoculation. Efficacy is associated with enhancement of tumor-specific CTL activity, including specificity against the CT26 immunodominant MHC class I restricted Ag AH1, and IFN-gamma production. There was no significant tumor growth inhibition after intratumoral inoculation of s.c. CT26 tumors in athymic mice. We conclude that this defective HSV vector system is an effective method for cytokine gene delivery to tumors in situ and IL-12 expression in tumors synergizes the antitumor activity mediated by the replication-competent HSV helper virus.