Established Lewis Lung Carcinoma Research Articles

Overcoming resistance to STING agonist therapy to incite durable protective antitumor immunity

BackgroundActivating the Stimulator of Interferon Genes (STING) adaptor incites antitumor immunity against immunogenic tumors in mice, prompting clinical trials to test STING activators. However, STING signaling in the tumor microenvironment...

Cryptotanshinone has curative dual anti-proliferative and immunotherapeutic effects on Lewis lung carcinoma

Abstract Lung cancer is the leading cause of cancer-related mortality, with very limited effective therapeutics. Screening of a variety of Traditional Chinese Medicines (TCMs) for capacity to inhibit the proliferation of human lung cancer A549 cells and to induce the maturation of human dendritic cells (DCs) led to the identification of cryptotanshione (CT), a compound purified from the TCM Salvia miltiorrhiza Bunge. CT can be chemically synthesized. Here CT was shown to inhibit the proliferation of mouse Lewis lung carcinoma (LLC) cells by upregulating p53 and downregulating cyclin B1 and Cdc2 and consequently inducing G2/M cell-cycle arrest of LLC cells. In addition, CT promoted phenotypic maturation of mouse and human DCs with upregulation of costimulatory and MHC molecules, and stimulated DC to produce TNFa, IL-1b and IL-12p70, but not IL-10. CT-induced maturation of DCs depended on MyD88 and also involved the activation of NF-kB, p38, and JNK. CT was effective in the treatment of established LLC tumors and, when used in combination with low-dose anti-PD-L1, can cure LLC-bearing mice with the induction of subsequent anti-LLC specific immunity. Our data indicate that CT has novel unique antitumor and immunotherapeutic activities that may provide a new promising therapeutic for the treatment of human lung cancers.

Targeting macrophages for cancer therapy disrupts bone homeostasis and impairs bone marrow erythropoiesis in mice bearing Lewis lung carcinoma tumors

Macrophages are represented in all tissues by phenotypically distinct resident populations that show great functional diversity. Macrophages generally play a protumoral role, and they are attractive targets for cancer therapy. In this study, we found that CD169+ macrophages depletion inhibited the growth of established Lewis lung carcinoma tumors in mice. Benefits must be weighed against potential adverse effects in cancer therapy. Here, we investigated the adverse effects of CD169+ macrophages depletion on bone and bone marrow in mice bearing Lewis lung carcinoma tumors. Our studies showed that depletion of CD169+ macrophages in LLC tumor-bearing mice disrupted bone homeostasis, including bone weight loss and bone mineral density decrease. Further studies revealed that bone marrow erythropoiesis was severely impaired after depletion of CD169+ macrophages in LLC tumor-bearing mice. Our findings suggest that depletion of macrophages for cancer therapy may be associated with potential adverse effects that need to be recognized, prevented, and optimally managed.

Abstract 3675: Novel immune oncology strategy for targeted cytotoxic lymphocyte activation

Abstract High-dose interleukin 2 (IL2) therapy was the first FDA-approved immunotherapy, and still has an exceptionally high true long-term cure rate for metastatic cancer. However, IL2 therapy has fallen out of favor due to systemic toxicity and off-target side effects. We have recently described a novel fusion cytokine comprised of a mutant form of IL2 with inhibited α-receptor binding and an NKG2D-binding viral decoy protein called Orthopox Major Histocompatibility Complex Class I-like Protein (OMCP). Unlike wild-type IL2, our fusion cytokine (OMCP-mutIL2) binds to and activates only NKG2D expressing cytotoxic lymphocytes. This targeted IL2 activity results in superior efficacy and decreased toxicity over wild-type IL2 therapy. Here, we present data which further establishes the superior efficacy of OMCP-mutIL2 therapy as measured by biomarkers such as systemic cytokine production and tumor lymphocyte infiltration. Additionally, we present data which evaluates OMCP-mutIL2 therapy as a potentiator of PD1 checkpoint inhibitor therapy. To evaluate tumor lymphocyte infiltration mice with established Lewis Lung Carcinoma (LLC) flank tumors were treated for three days with either saline, wild type IL2, or OMCP-mutIL2 and tumor infiltrating lymphocytes evaluated flow cytometrically. A trend for increased tumor infiltrating NK cells was evident for both IL2 and OMCP-mutIL2 treated groups over saline controls, and OMCP-mutIL2 tended to increase tumor infiltrating CD8+ T cells over either IL2 or saline controls was evident as well. Additionally, tumor infiltrating CD8+ T cells in IL2-treated mice had a higher level of exhaustion over OMCP-mutIL2 treated mice as measured by PD-1 expression. Tumor infiltrating CD4+ T cells also showed a trend toward a Foxp3 regulatory phenotype in IL2 treated mice over OMCP-mutIL2. To study cytokine serum profile mice were treated with high dose OMCP-mutIL2 with or without NK depletion and serum cytokine levels measured by cytometric bead array. Serum cytokine analysis of mice treated with supra-therapeutic doses of OMCP-mutIL2 did not reveal an increase in pro-inflammatory cytokines TNF-α, IFN-γ, and IL-6. A significant increase in serum levels of G-CSF was evident in NK cell sufficient mice treated with OMCP-mutIL2. Combination immunotherapy was initiated by the addition of PD-1 blockade (clone RMPI-14) to OMCP-mutIL2 therapy in a lung LLC tumor model. Combination therapy led to near complete control of LLC growth in the lung compared to OMCP-mutIL2 or PD-1 blockade alone. The data presented here suggest that in addition to our previously published data suggesting increased safety of OMCP-mutIL2 over high-dose IL2 therapy, OMCP-mutIL2 therapy may be functionally superior to classic high-dose IL2 therapy. Continued development of OMCP-mutIL2 is ongoing. Citation Format: Eric Lazier, Sarah Hein, John Westwick, Dan Watkins, Alexander S. Krupnick. Novel immune oncology strategy for targeted cytotoxic lymphocyte activation [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 3675. doi:10.1158/1538-7445.AM2017-3675

Humanized HLA-DQ8αβ transgenic mice treated with SEG/SEI superantigens exhibit long-term CD4+-mediated anti-tumor responses.

Abstract SEG and SEI are primordial S. aureus superantigens (SAgs) notably unaccompanied by human neutralizing antibodies that have hampered the use of classic SAgs as cancer therapeutics. Here we show SEG/SEI presented from a HLA-DQ8αβ (HLA-DQA*0301 and HLA-DQB*0302) tg platform outperform C57BL/6 mice in achieving long term survival in vaccinated and established Lewis lung carcinoma (LLC) and B16-F10 melanoma. Vaccination of DQ8 tg mice with LLC or B16-F10 melanoma followed by SEG/SEI immunization and subsequent tumor challenge resulted in 100% and 83% survival for >150 days respectively, compared to a median survival of 14 days in unvaccinated DQ8 tg controls (p<0.001). Likewise, SEG/SEI treatment of established melanoma in DQ8 tg mice resulted in 100% survival for 70 days compared to a median survival of 20 days in untreated DQ8 tg controls. Similar vaccinations/challenges in C57BL/6 mice produced a median survival of 23–28 days. SEG/SEI-activated splenocytes from DQ8 tg mice displayed a TH-1/TH-17 cytokine phenotype with a threefold greater T cell proliferative and CD4+-mediated cytotoxicity responses, compared to C57BL/6 mice. These surprising data delineate a clear pathway to translation of an SEG/SEI-HLA-DQ8αβ tg platform for melanoma/lung cancer prophylaxis and treatment.

Pro‐opiomelanocortin gene delivery suppresses the growth of established Lewis lung carcinoma through a melanocortin‐1 receptor‐independent pathway

Pro-opiomelanocortin (POMC) is the precursor of several neuropeptides, such as corticotropin, melanocyte-stimulating hormone and the endogenous opioid (β-endorphin). Our previous studies have indicated that POMC gene delivery inhibited the progression and metastasis of B16-F10 melanoma via the α- melanocyte-stimulating hormone/melanortin-1 receptor (MC-1R) pathway. In the present study, the therapeutic efficacy of POMC gene therapy was evaluated in mice bearing established Lewis lung carcinoma (LLC) models both in vitro and in vivo. We also investigated the MC-1R-independent mechanism underlying POMC gene therapy. We found that POMC gene delivery significantly inhibited the growth and colony formation in MC-1R-deficient LLC cells. In addition, POMC gene transfer effectively suppressed the growth of established LLC in mice. The inhibitory mechanisms underlying POMC gene delivery were attibuted to be inhibition of proliferation and the induction of apoptosis. Moreover, POMC gene delivery attenuated tumor β-catenin signaling by reducing protein levels of β-catenin and its downstream proto-oncogenes, including cyclin D1 and c-myc. Lastly, POMC gene delivery induced a significant suppression of tumor vasculature. These results support the existence of an MC-1R-independent pathway for POMC gene therapy, which further expands the therapeutic spectrum of POMC therapy for multiple types of cancer.

Inhibitory effect of ginsenoside Rg3 combined with gemcitabine on angiogenesis and growth of lung cancer in mice

BackgroundGinsenoside Rg3, a saponin extracted from ginseng, inhibits angiogenesis. The combination of low-dose chemotherapy and anti-angiogenic inhibitors suppresses growth of experimental tumors more effectively than conventional therapy or anti-angiogenic agent alone. The present study was designed to evaluate the efficacy of low-dose gemcitabine combined with ginsenoside Rg3 on angiogenesis and growth of established Lewis lung carcinoma in mice.MethodsC57L/6 mice implanted with Lewis lung carcinoma were randomized into the control, ginsenoside Rg3, gemcitabine and combination group. The quality of life and survival of mice were recorded. Tumor volume, inhibitive rate and necrosis rate were estimated. Necrosis of tumor and signals of blood flow as well as dynamic parameters of arterial blood flow in tumors such as peak systolic velocity (PSV) and resistive index (RI) were detected by color Doppler ultrasound. In addition, expression of vascular endothelial cell growth factor (VEGF) and CD31 were observed by immunohistochemstry, and microvessel density (MVD) of the tumor tissues was assessed by CD31 immunohistochemical analysis.ResultsQuality of life of mice in the ginsenoside Rg3 and combination group were better than in the control and gemcitabine group. Combined therapy with ginsenoside Rg3 and gemcitabine not only enhanced efficacy on suppression of tumor growth and prolongation of the survival, but also increased necrosis rate of tumor significantly. In addition, the combination treatment could obviously decrease VEGF expression and MVD as well as signals of blood flow and PSV in tumors.ConclusionGinsenoside Rg3 combined with gemcitabine may significantly inhibit angiogenesis and growth of lung cancer and improve survival and quality of life of tumor-bearing mice. The combination of chemotherapy and anti-angiogenic drugs may be an innovative and promising therapeutic strategy in the experimental treatment of human lung cancer.

Combination of Fasl and GM‐CSF confers synergistic antitumor immunity in an in vivo model of the murine Lewis lung carcinoma

Gene transfer of Fas ligand (FasL) to tumor cells has been demonstrated to inhibit tumor growth in vivo, and neutrophils are primarily responsible for this immunoprotection. The granulocyte-macrophage colony stimulating factor (GM-CSF) secreted by tumor vaccine can recruit dendritic cells (DCs) for efficient antigen presentation to T cells that generate the tumor-specific response. To investigate whether the combination of FasL and GM-CSF can efficiently suppress tumor growth, we have established Lewis lung carcinoma (LLC-1) cells that are transduced with GM-CSF (LLC/GM-CSF), FasL (LLC/FasL) or both genes (LLC/FasL/GM-CSF) to test their tumorigenic potential in vivo. Mice inoculated with LLC/GM-CSF display high survival rates along with reduction of tumor growth. In contrast, none of the mice injected with LLC/FasL or LLC/FasL/GM-CSF develop tumors. Specific memory immune response and delayed LLC-1 tumor growth are found in mice immunized with LLC-1/FasL or LLC-1/FasL/GM-CSF. Furthermore, therapeutic effects are observed only when LLC-1/FasL/GM-CSF tumor vaccine is employed to retard growth of preexisting LLC-1 tumors. Tumor growth is also completely suppressed in mice injected with a mixture of LLC-1 and LLC-1/FasL/GM-CSF. In addition, IL-12 production, cytotoxic T-cell activity and IgG against LLC-1 are manifested in mice injected with LLC/FasL/GM-CSF. Our data show that FasL-induced pathway triggers expression of proinflammatory cytokines, including IL-1 beta, IL-6, MIP-2 and MCP-1, while GM-CSF-dependent pathway promotes functional maturation and activation of DCs. Taken together, the results indicate that dual gene-based delivery with FasL and GM-CSF may serve as a more effective tumor vaccine to suppress lung cancer cell growth in vivo.

Combination tumor immunotherapy with radiotherapy and Th1 cell therapy against murine lung carcinoma

Mice bearing established Lewis lung carcinoma (LLC) expressing model tumor antigen, ovalbumin (OVA) (LLC-OVA) marginally responded to local radiotherapy, but none of the mice was cured. In contrast, treatment of the tumor-bearing mice with intratumoral injection of tumor-specific T helper type 1 (Th1) cells and tumor antigen (OVA) after radiotherapy dramatically prolonged the survival days and induced complete cure of the mice at high frequency (80%). Radiation therapy combined with Th1 cells or OVA alone showed no significant therapeutic activity against LLC-OVA. Such a strong therapeutic activity was not induced by intratumoral injection of Th1 cells plus OVA. Compared with other treatment, radiation therapy combined with Th1 cells and OVA was superior to induce the generation of OVA/H-2(b) tetramer(+) tumor-specific cytotoxic T lymphocyte (CTL) with a strong cytotoxicity against LLC-OVA in draining lymph node (DLN). Moreover, the combined therapy is demonstrated to inhibit the growth of tumor mass, which grew at contralateral side. These results indicated that radiotherapy combined with Th1 cell/vaccine therapy induced a systemic antitumor immunity. These findings suggested that combination therapy with radiotherapy and Th1 cell/vaccine therapy may become a practical strategy for cancer treatment.

Efficacy of GM-CSF-producing Tumor Vaccine after Docetaxel Chemotherapy in Mice Bearing Established Lewis Lung Carcinoma

In this report, we evaluated the efficacy of a GM-CSF-producing tumor vaccine given before and after docetaxel in mice bearing established lung tumors. Mice bearing established 3LL tumors were treated with docetaxel and tumor vaccines transduced with either control or GM-CSF adenoviral vectors. Docetaxel (5-20 mg/kg) treatment alone had only a minimal effect on growth of established 3LL tumors in vivo, although docetaxel was cytotoxic to 3LL cells in vitro. When mice bearing established 3LL tumors were pretreated with docetaxel followed by vaccination with irradiated GM-CSF- transduced 3LL tumor cells, significant tumor regression and prolonged survival were observed compared with chemotherapy alone. Delaying docetaxel treatment until after tumor vaccination abrogated the vaccine's anti-tumor effects. Mice that survived treatment were able to resist a lethal rechallenge of 3LL tumor cells. Memory CTL specific for an epitope (MUT-1) derived from 3LL were detected in surviving mice. Docetaxel induced a mild lymphodepletion in mice, both CD4 and CD8 subsets were reduced in LN and spleens. Interestingly, docetaxel also diminished the number of memory CD8+ T cells (CD122+) and possible CD4+ CD25+ Foxp3+ natural Treg cells. Docetaxel treatment did not affect antigen-driven proliferation of naive T cells but significantly promoted survival of activated T cells. Thus, augmentation of vaccine induced antitumor immunity in docetaxel-treated mice primarily due to the enhanced survival of antigen-experienced T cells.

Cytokine‐containing gelfoam implants at a postsurgical tumor excision site to stimulate local immune reactivity

We previously demonstrated increased numbers of CD34(+) progenitor cells in the peripheral blood of tumor bearers. Also demonstrated was the feasibility of chemoattracting these cells by sponge implants containing VEGF. The present study used a murine Lewis lung carcinoma (LLC) model to test if CD34(+) cells that are chemoattracted to a tumor excision site can be differentiated in situ into dendritic cells and whether this leads to increased local immune reactivity. After surgically excising established LLC tumors, mice received at the excision site gelatin sponge implants containing VEGF to chemoattract CD34(+) cells, and/or GM-CSF plus SCF to induce CD34(+) cell differentiation into dendritic cells. In some studies, lysates of GFP-transfected LLC cells (LLC(GFP)) were also included in the implants as a source of tumor antigen. After 2 weeks, implants and local lymph nodes were removed and analyzed. Implants containing VEGF, GM-CSF/SCF or VEGF/GM-CSF/SCF had a higher proportion of CD34(+) cells compared to control implants. However, the number of dendritic cells was higher in implants containing GM-CSF/SCF or VEGF/GM-CSF/SCF than those containing either VEGF or diluent. Regional lymph node from mice containing GM-CSF/SCF or VEGF/GM-CSF/SCF implants showed increased dendritic cell levels. However, when lysates from LLC(GFP) were added to the implants, the highest proportion of dendritic cells associated with GFP was in lymph nodes of mice containing GM-CSF/SCF implants. Lymph node cells from mice with GM-CSF/SCF or VEGF/GM-CSF/SCF had a higher level of proliferation and IFN-gamma secretion in response to in vitro LLC lysate challenge, with the greatest response being from lymph node cells of mice with GM-CSF/SCF implants. These results suggest the feasibility of using GM-CSF/SCF-containing implants to increase dendritic cell levels, uptake of tumor antigens, trafficking to lymph nodes and stimulation of immune reactivity at tumor excision sites with residual tumor.

Polyethylene glycol-diacyllipid micelles demonstrate increased acculumation in subcutaneous tumors in mice.

The purpose of this work is to study the potential of micelles prepared from amphiphilic polyethelene glycol/phosphatidylethanolamine (PEG-PE) conjugates as a particulate drug delivery system capable of accumulation in tumors via the enhanced permeability and retention (EPR) effect. Micelles were prepared from PEGs of different molecular lengths conjugated with PE. The micelles were characterized by fluorescence-based critical micellization concentration (CMC) measure ments, dynamic light scattering, and HPLC. Blood clearance an tumor accumulation of 111In-labeled micelles were studied in mic with subcutaneously established Lewis lung carcinoma (LLC) and EL4 T lymphoma (EL4) tumors. Various versions of PEG-PE conjugates with PEG blocks ranging from 750 to 5000 Da formed very stable low CMC micelles at all concentrations down to 10(-5) M. The size of the micelles varie between 7 and 35 nm depending on the length of the PEG block. Micelles remained intact after prolonged incubation with the blood serum. Upon intravenous administration into mice, the micelles demonstrated circulation longevity, and they efficiently and selectively accumulated in both subcutaneous Lewis lung carcinoma and EL4 T lymphoma tumors. PEG-PE conjugates form very stable, long-circulating micelles. These micelles efficiently accumulate in tumors in vivo an may potentially be used as a tumor-specific delivery system for poorly soluble anticancer drugs.

Accumulation of protein-loaded long-circulating micelles and liposomes in subcutaneous Lewis lung carcinoma in mice.

The purpose of our work was to compare the biodistribution and tumor accumulation of a liposome- or micelle-incorporated protein in mice bearing subcutaneously-established Lewis lung carcinoma. A model protein, soybean trypsin inhibitor (STI) was modified with a hydrophobic residue of N-glutaryl-phosphatidyl-ethanolamine (NGPE) and incorporated into both polyethyleneglycol(MW 5000)-distearoyl phosphatidyl ethanolamine (PEG-DSPE) micelles (< 20 nm) and PEG-DSPE-modified long-circulating liposomes (ca. 100 nm). The protein was labeled with 111In via protein-attached diethylene triamine pentaacetic acid (DTPA), and samples of STI-containing liposomes or micelles were injected via the tail vein into mice bearing subcutaneously-established Lewis lung carcinoma. At appropriate time points, mice were sacrificed and the radioactivity accumulated in the tumor and main organs was determined. STI incorporated into PEG-lipid micelles accumulates in subcutaneously established Lewis lung carcinoma in mice better than the same protein anchored in long-circulating PEG-liposomes. Small-sized long-circulating delivery systems, such as PEG-lipid micelles, are more efficient in the delivery of protein to Lewis lung carcinoma than larger long-circulating liposomes.