Murine Lung Cancer Model Research Articles

Macrophage Elastase Induces TRAIL-mediated Tumor Cell Death through Its Carboxy-Terminal Domain.

Macrophage elastase (matrix metalloproteinase [MMP]-12) is a potent protease that contributes to the lung destruction that accompanies cigarette smoking; it simultaneously inhibits lung tumor angiogenesis and metastasis by catalyzing the formation of antiangiogenic peptides. Recent studies have revealed novel nonproteolytic functions of MMP12, including antimicrobial activity through a peptide within its C-terminal domain (CTD). To determine whether the MMP12 CTD contributes to its antitumor activity in lung cancer. We used recombinant MMP12 peptide fragments, including its catalytic domain, CTD, and a 20 amino acid peptide within the CTD (SR20), in an in vitro system to delineate their effects on non-small cell lung cancer cell proliferation and apoptosis. We translated our findings to two murine models of lung cancer, including orthotopic human xenograft and KrasLSL/G12D mouse models of lung cancer. We show that SR20 triggers tumor apoptosis by up-regulation of gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4, sensitizing cells to an autocrine loop of TRAIL-mediated cell death. We then demonstrate the therapeutic efficacy of SR20 against two murine models of lung cancer. The MMP12 CTD initiates TRAIL-mediated tumor cell death through its conserved SR20 peptide.

CCL21 Combined with PD-1 Blockade Cooperatively Inhibits Tumor Growth in KRAS Murine Model of NSCLC

Recent studies of the programmed death-1 (PD-1) checkpoint inhibitors in NSCLC patients reveal an approximately 20% observed response rate. Durable response appears to be often associated with baseline high level PD-L1 expression as well as pre-existing T cell infiltration into the tumor. One potential approach to extending the effectiveness of checkpoint inhibitors is to enhance T cell responses by in situ vaccination, taking advantage of the full repertoire of available tumor antigens. In preclinical studies as well as a recent clinical trial, we have discovered that CCL21-secreting dendritic cells have the capacity to induce both local T cell recruitment and systemic immune responses. We hypothesized that in situ vaccination with CCL21 could restore tumor antigen presentation and T cell infiltration into the tumor, thereby sensitizing non-responsive NSCLC tumors to checkpoint blockade. We evaluated the efficacy of CCL21 and a PD-1 inhibitor as monotherapy and in combination in the syngeneic KRAS murine model of lung cancer. Intratumoral administration of CCL21 resulted in an increased number of CD8+ T cells in the tumor without significantly changing the number of regulatory T cells. The anti-tumor response was more pronounced in the anti-PD-1/CCL21 combination therapy compared to the monotherapy groups. Combination therapy with anti-PD-1 and intratumoral CCL21 cooperatively inhibits tumor growth in vivo to a greater extent than monotherapy.

Stealth liposomes for the delivery of zoledronic acid into tumors enhance the anticancer activity of the drug

Zoledronic acid (ZOL) is a third generation aminobisphosphonate, commonly used for the treatment of bone metastases. Several studies have shown a direct in vitro antitumor activity of ZOL but a clear evidence of clinical activity is still lacking. Unfortunately, the use of ZOL as an anticancer agent in extraskeletal tissues is limited probably because it is rapidly removed from the blood and tends to accumulate in the bone. On these bases, we developed stealth liposomes encapsulating ZOL (Lipo- ZOL) to improve the pharmacokinetic profile of the drug. Compared to free ZOL, Lipo-ZOL induced a stronger inhibition of growth in two different cancer cell lines H460 and CG5. Moreover, Lipo-ZOL also significantly caused a larger inhibition of tumor growth and increased the overall survival in murine models of human lung and breast cancer, in comparison with free ZOL. These results suggest the use of Lipo-ZOL as a potential anticancer agent in patients with neoplastic disease.

Roflumilast treatment inhibits lung carcinogenesis in benzo(a)pyrene-induced murine lung cancer model

Roflumilast, a potent and selective inhibitor of phosphodiesterase-4 (PDE4), has been used in treatment of COPD. PDE4 inhibitor is associated with inhibition of chronic airway inflammation, oxidative stress, and mesenchymal markers in B(a)P-induced lung tumors. The aim of this study was to assess whether roflumilast alone or added to inhaled budesonide might have dose-dependent inhibition on lung carcinogenesis induced by carcinogen B(a)P in mice. Female A/J mice were given a single dose of benzo(a)pyrene. Administration of roflumilast (1mg/kg or 5mg/kg) via oral gavage and aerosolized budesonide (2.25mg/ml) began 2 weeks post-carcinogen treatment and continued for 26 weeks. Tumor load was determined by averaging the total tumor volume in each group. Benzo(a)pyrene induced an average tumor size of 9.38 ± 1.75 tumors per mouse, with an average tumor load of 19.53 ± 3.81mm3. Roflumilast 5mg/kg treatment decreased (P < 0.05) tumor load per mouse compared to the B(a)P group. Roflumilast 5mg/kg treatment significantly increased the levels of cAMP in tumors with adjacent lung tissues (P < 0.05). The expression level of PDE4D gene was decreased by roflumilast 5mg/kg treatment, significantly (P < 0.05). Compared to the B(a)P exposure group, expression levels of HIF-1α and VEGFA were attenuated by roflumilast 5mg/kg treatment (P < 0.05). High-dose roflumilast can attenuate lung carcinogenesis in B(a)P-induced murine lung cancer model. The chemopreventive effect of roflumilast might be associated with inhibition of increased cAMP-mediated inflammatory process and markers of angiogenesis in tumor tissues.

Abstract 168: Loss of function mutation in TIMP2 gene accelerates tumorigenesis and mortality in murine model of lung cancer through EGFR signaling

Abstract The aim of this study is to determine the effect of the tissue inhibitor of metalloproteinase-2 (TIMP-2) on lung tumorigenesis and investigate the underlying molecular mechanisms using an orthotopic mouse model with a loss of function mutation in the Timp2 gene (T2M). T2M and wildtype (WT) control mice were given 1x106 Lewis lung carcinoma cells transfected with luciferase (LL/2-Luc-M38, Caliper) in 50μL PBS via intratracheal installation. IVIS imaging revealed a higher tumor burden in T2M mice compared to WT littermates, suggesting that loss of function of the Timp2 gene enhances tumor growth (p&amp;lt;0.05). We also conducted a Kaplan-Meier analysis to determine the effect of this mutation on mortality following cancer development. We found that LL/2 tumor-induced mortality was significantly higher in T2M mice compared to WT controls (p = 0.01). Histologic analysis and H&amp;E staining of lung tissue sections revealed a significant increase in the number of tumor nodules of T2M mice compared to WT controls (p &amp;lt;0.01). CD31 staining showed a significant increase in microvessel density (MVD) in T2M mice compared to WT controls (p&amp;lt;0.01). Given that VEGF is a primary driver of tumor neovascularization, we determined mRNA levels of VEGF expression in healthy and tumor bearing mice by qRTPCR analysis. Interestingly, basal VEGF expression levels were increased in lungs of both non-tumor bearing and tumor bearing T2M mice compared to WT controls (p&amp;lt;0.01). VEGF is the direct downstream target of HIF-2α, a transcription factor implicated in tumor hypoxia and expression correlates with decreased overall survival in non-small cell lung cancer (NSCLC) patients. Similarly, an examination of TIMP-2 and HIF-2α mRNA expression in a small cohort of patient samples revealed decreased TIMP-2 and increased HIF2α in NSCLC tumors compared to surrounding non-tumor lung tissue. Hypoxia induces EGFR signaling through HIF-2α. Thus, we determined the effect of mutated T2M on the EGFR signaling pathway. We found increased levels of EGFR phosphorylation as well as downstream ERK and Akt activation in tumor bearing and non-tumor bearing T2M lungs compared to WT controls. In conclusion, these findings offer new avenues for TIMP-2 research in regulating hypoxic mediators within the tumor microenvironment through EGFR signaling, suggesting TIMP-2 as a novel bio-therapeutic for lung cancer therapy. * Corresponding author This work is supported by center for cancer research, intramural research program, NCI/NIH research grants ZIA BC011204 &amp; ZIA SC 009179 to WGSS Citation Format: Sarvesh Kumar, Sandra Jensen, David Peeney, Ananda Chowdhury, Beiyang Wei, William G. Stetler-Stevenson. Loss of function mutation in TIMP2 gene accelerates tumorigenesis and mortality in murine model of lung cancer through EGFR signaling [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 168. doi:10.1158/1538-7445.AM2017-168

Abstract 3334: The FGFR4-388Arg variant exerts pro-tumorogenic effects in lung cancer by inducing an EMT phenotype

Abstract The FGFR4-388Arg allele, where an arginine substitutes a glycine in the 388 codon of the FGFR4 gene, has been associated to poorer patient outcome in different tumor types. In lung cancer, the variant has been related to shorter overall survival in adenocarcinoma (ADC) patients, while no relationship with squamous cell carcinoma prognosis (SCC) has been reported. Mechanistically, the FGFR4-388Arg variant has been related to increased MAPK pathway activation and to EMT in prostate cancer in vitro models. Furthermore, the variant has been associated with higher STAT3 signaling in murine models of breast and lung cancer. However, so far the molecular biology for this FGFR4 variant in lung cancer patients has not been addressed. We overexpressed the FGFR4-388Gly and FGFR4-388Arg variants in lung cancer cell lines from different histologic backgrounds and performed tumorogenicity surrogate assays and downstream signaling activation assessment. In the generated stable cell lines, we also determined the expression of EMT markers. Furthermore, we determined the FGFR4 variant and the FGFR4 and N-cadherin mRNA expression in a cohort of NSCLC patients (N=65) and correlated these data to patient outcome. We reported that FGFR4-388Arg increases tumorogenicity in lung cancer cell lines. Mechanistically, these functional effects seem to be mediated by MAPK and STAT3 overactivation and by the induction of an EMT phenotype, which includes N-cadherin increased expression. In fact, this induction of N-cadherin protein expression by the FGFR4-388Arg variant seems to be responsible for the pro-oncogenic effects reported. In NSCLC patient tumor samples, the FGFR4-388Arg variant correlates with higher N-cadherin expression and with poorer survival. In conclusion, the FGFR4-388Arg variant is a potential prognostic biomarker in NSCLC, including ADC and SCC patients. This variant increases tumorogenesis through the activation of MAPK and STAT3 signaling pathways and through the promotion of an EMT phenotype. Citation Format: Álvaro Quintanal-Villalonga, Rocío Suárez, Laura Ojeda-Márquez, Santiago Ponce-Aix, Amancio Carnero, Luis Paz-Ares, Irene Ferrer, Sonia Molina-Pinelo. The FGFR4-388Arg variant exerts pro-tumorogenic effects in lung cancer by inducing an EMT phenotype [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 3334. doi:10.1158/1538-7445.AM2017-3334

MafB silencing in macrophages does not influence the initiation and growth of lung cancer induced by urethane.

An increased number of tumor-associated macrophages (TAMs) that exhibit the M2 macrophage phenotype is related to poorer prognosis in cancer patients. MafB is a transcription factor regulating the differentiation of macrophages. However, involvement of MafB for the development of TAMs is unknown. This study was designed to investigate the role of MafB in a murine urethane-induced lung cancer model. Urethane was injected intraperitoneally into wild-type and dominant-negative MafB transgenic mice. Twenty-four weeks later, mice were sacrificed and their lungs removed for pathological analysis. The numbers and mean areas of lung cancer were evaluated. In addition, the numbers of Mac-3-positive macrophages were evaluated in each tumor. The numbers and mean areas of lung cancer induced by urethane administration were not significantly different between wild-type and dominant-negative MafB transgenic mice. The numbers of TAMs in lung cancer tissue were not significantly different between the two groups. MafB silencing using dominant-negative MafB did not influence the initiation and growth of lung cancer in mice exposed to urethane. These data suggest that MafB may not be related to the development of TAMs.

CD38 as a PET Imaging Target in Lung Cancer.

Daratumumab (Darzalex, Janssen Biotech) is a clinically approved antibody targeting CD38 for the treatment of multiple myeloma. However, CD38 is also expressed by other cancer cell types, including lung cancer, where its expression or absence may offer prognostic value. We therefore developed a PET tracer based upon daratumumab for tracking CD38 expression, utilizing murine models of non-small cell lung cancer to verify its specificity. Daratumumab was prepared for radiolabeling with 89Zr (t1/2 = 78.4 h) through conjugation with desferrioxamine (Df). Western blot, flow cytometry, and saturation binding assays were utilized to characterize CD38 expression and binding of daratumumab to three non-small cell lung cancer cell lines: A549, H460, and H358. Murine xenograft models of the cell lines were also generated for further in vivo studies. Longitudinal PET imaging was performed following injection of 89Zr-Df-daratumumab out to 120 h postinjection, and nonspecific uptake was also evaluated through the injection of a radiolabeled control IgG antibody in A549 mice, 89Zr-Df-IgG. Ex vivo biodistribution and histological analyses were also performed after the terminal imaging time point at 120 h postinjection. Through cellular studies, A549 cells were found to express higher levels of CD38 than the H460 or H358 cell lines. PET imaging and ex vivo biodistribution studies verified in vitro trends, with A549 tumor uptake peaking at 8.1 ± 1.2%ID/g at 120 h postinjection according to PET analysis, and H460 and H358 at lower levels at the same time point (6.7 ± 0.7%ID/g and 5.1 ± 0.4%ID/g, respectively; n = 3 or 4). Injection of a nonspecific radiolabeled IgG into A549 tumor-bearing mice also demonstrated lower tracer uptake of 4.4 ± 1.3%ID/g at 120 h. Immunofluorescent staining of tumor tissues showed higher staining levels present in A549 tissues over H460 and H358. Thus, 89Zr-Df-daratumumab is able to image CD38-expressing tissues in vivo using PET, as verified through the exploration of non-small cell lung cancer models in this study. This agent therefore holds potential to image CD38 in other malignancies and aid in patient stratification and elucidation of the biodistribution of CD38.

Exosomes from Plasmodium-infected hosts inhibit tumor angiogenesis in a murine Lewis lung cancer model

Previous research to investigate the interaction between malaria infection and tumor progression has revealed that malaria infection can potentiate host immune response against tumor in tumor-bearing mice. Exosomes may play key roles in disseminating pathogenic host-derived molecules during infection because several studies have shown the involvement and roles of extracellular vesicles in cell–cell communication. However, the role of exosomes generated during Plasmodium infection in tumor growth, progression and angiogenesis has not been studied either in animals or in the clinics. To test this hypothesis, we designed an animal model to generate and isolate exosomes from mice which were subsequently used to treat the tumor. Intra-tumor injection of exosomes derived from the plasma of Plasmodium-infected mice provided significantly reduced Lewis lung cancer growth in mice. We further co-cultured the isolated exosomes with endothelial cells and observed significantly reduced expression of VEGFR2 and migration in the endothelial cells. Interestingly, high level of micro-RNA (miRNA) 16/322/497/17 was detected in the exosomes derived from the plasma of mice infected with Plasmodium compared with those from control mice. We observed that overexpression of the miRNA 16/322/497/17 in endothelial cell corresponded with decreased expression of VEGFR2, inhibition of angiogenesis and inhibition of the miRNA 16/322/497/17 significantly alleviated these effects. These data provide novel scientific evidence of the interaction between Plasmodium infection and lung cancer growth and angiogenesis.

Modulation of Bax and mTOR for Cancer Therapeutics.

A rationale exists for pharmacologic manipulation of the serine (S)184 phosphorylation site of the proapoptotic Bcl2 family member Bax as an anticancer strategy. Here, we report the refinement of the Bax agonist SMBA1 to generate CYD-2-11, which has characteristics of a suitable clinical lead compound. CYD-2-11 targeted the structural pocket proximal to S184 in the C-terminal region of Bax, directly activating its proapoptotic activity by inducing a conformational change enabling formation of Bax homooligomers in mitochondrial membranes. In murine models of small-cell and non-small cell lung cancers, including patient-derived xenograft and the genetically engineered mutant KRAS-driven lung cancer models, CYD-2-11 suppressed malignant growth without evident significant toxicity to normal tissues. In lung cancer patients treated with mTOR inhibitor RAD001, we observed enhanced S184 Bax phosphorylation in lung cancer cells and tissues that inactivates the propaoptotic function of Bax, contributing to rapalog resistance. Combined treatment of CYD-2-11 and RAD001 in murine lung cancer models displayed strong synergistic activity and overcame rapalog resistance in vitro and in vivo Taken together, our findings provide preclinical evidence for a pharmacologic combination of Bax activation and mTOR inhibition as a rational strategy to improve lung cancer treatment. Cancer Res; 77(11); 3001-12. ©2017 AACR.

Knockdown of Importin 7 Inhibits Lung Tumorigenesis in K-rasLA1 Lung Cancer Mice.

Background/Aim: Lung cancer shows the highest estimated deaths in both males and females in the Unites States. Importin 7 is overexpressed in lung adenocarcinoma tissues. In this study, we aimed to demonstrate the anticancer effect of importin 7 down-regulation, especially in lung cancer. Materials and Methods: Glycerol propoxylate triacrylate spermine (GPT-SPE) is a biocompatible carrier used for aerosol gene delivery. Repeated aerosol delivery of GPT-SPE/shImportin 7 complexes was performed to 10-week-old male K-ras LA1 mice (a murine lung cancer model) twice a week for 4 weeks (8 times) in a nose-only exposure chamber. Results: Aerosol delivery of GPT-SPE/shImportin 7 inhibits lung cancer in K-ras LA1 mice compared to control and scramble control groups. Moreover, importin 7-down-regulated stable cell-line demonstrates suppression of proliferation through Akt inhibition and apoptosis. Conclusion: Down-regulation of importin 7 significantly suppresses lung cancer in vitro and in vivo.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Differential PI3Kδ Signaling in CD4+ T-cell Subsets Enables Selective Targeting of T Regulatory Cells to Enhance Cancer Immunotherapy.

To modulate T-cell function for cancer therapy, one challenge is to selectively attenuate regulatory but not conventional CD4+ T-cell subsets [regulatory T cell (Treg) and conventional T cell (Tconv)]. In this study, we show how a functional dichotomy in Class IA PI3K isoforms in these two subsets of CD4+ T cells can be exploited to target Treg while leaving Tconv intact. Studies employing isoform-specific PI3K inhibitors and a PI3Kδ-deficient mouse strain revealed that PI3Kα and PI3Kβ were functionally redundant with PI3Kδ in Tconv. Conversely, PI3Kδ was functionally critical in Treg, acting there to control T-cell receptor signaling, cell proliferation, and survival. Notably, in a murine model of lung cancer, coadministration of a PI3Kδ-specific inhibitor with a tumor-specific vaccine decreased numbers of suppressive Treg and increased numbers of vaccine-induced CD8 T cells within the tumor microenvironment, eliciting potent antitumor efficacy. Overall, our results offer a mechanistic rationale to employ PI3Kδ inhibitors to selectively target Treg and improve cancer immunotherapy. Cancer Res; 77(8); 1892-904. ©2017 AACR.

Loss of function mutation in TIMP2 gene accelerates tumorigenesis and mortality in murine model of lung cancer through EGFR signaling

The aim of this study is to determine the effect of the tissue inhibitor of metalloproteinase‐2 (TIMP‐2) on lung tumorigenesis and investigate the underlying molecular mechanisms using an orthotopic mouse model with a loss of function mutation in the Timp2 gene (T2M). T2M and wildtype (WT) control mice were given 1×106 Lewis lung carcinoma cells transfected with luciferase (LL/2‐Luc‐M38, Caliper) in 50μL PBS via intratracheal installation. IVIS imaging revealed a higher tumor burden in T2M mice compared to WT littermates, suggesting that loss of function of the Timp2 gene enhances tumor growth (p&lt;0.05). We also conducted a Kaplan‐Meier analysis to determine the effect of this mutation on mortality following cancer development. We found that LL/2 tumor‐induced mortality was significantly higher in T2M mice compared to WT controls (p = 0.01). Histologic analysis and H&amp;E staining of lung tissue sections revealed a significant increase in the number of tumor nodules of T2M mice compared to WT controls (p &lt;0.01). CD31 staining showed a significant increase in microvessel density (MVD) in T2M mice compared to WT controls (p&lt;0.01). Given that VEGF is a primary driver of tumor neovascularization, we determined mRNA levels of VEGF expression in healthy and tumor bearing mice by qRTPCR analysis. Interestingly, basal VEGF expression levels were increased in lungs of both non‐tumor bearing and tumor bearing T2M mice compared to WT controls (p&lt;0.01). VEGF is the direct downstream target of HIF‐2a, a transcription factor implicated in tumor hypoxia and expression correlates with decreased overall survival in non– small cell lung cancer (NSCLC) patients. Similarly, an examination of TIMP‐2 and HIF‐2a mRNA expression in a small cohort of patient samples revealed decreased TIMP‐2 and increased HIF2a in NSCLC tumors compared to surrounding non‐tumor lung tissue. Hypoxia induces EGFR signaling through HIF‐2a. Thus, we determined the effect of mutated T2M on the EGFR signaling pathway. We found increased levels of EGFR phosphorylation as well as downstream ERK and Akt activation in tumor bearing and non‐tumor bearing T2M lungs compared to WT controls. In conclusion, these findings offer new avenues for TIMP‐2 research in regulating hypoxic mediators within the tumor microenvironment through EGFR signaling, suggesting TIMP‐2 as a novel bio‐therapeutic for lung cancer therapy.Support or Funding InformationThis work is supported by center for cancer research, intramural research program, NCI/NIH research grants ZIA BC011204 &amp; ZIA SC 009179 to WGSS

Enriching the Housing Environment for Mice Enhances Their NK Cell Antitumor Immunity via Sympathetic Nerve-Dependent Regulation of NKG2D and CCR5.

Mice housed in an enriched environment display a tumor-resistant phenotype due to eustress stimulation. However, the mechanisms underlying enriched environment-induced protection against cancers remain largely unexplained. In this study, we observed a significant antitumor effect induced by enriched environment in murine pancreatic cancer and lung cancer models. This effect remained intact in T/B lymphocyte-deficient Rag1-/- mice, but was nearly eliminated in natural killer (NK) cell-deficient Beige mice or in antibody-mediated NK-cell-depleted mice, suggesting a predominant role of NK cells in enriched environment-induced tumor inhibition. Exposure to enriched environment enhanced NK-cell activity against tumors and promoted tumoral infiltration of NK cells. Enriched environment increased the expression levels of CCR5 and NKG2D (KLRK1) in NK cells; blocking their function effectively blunted the enriched environment-induced enhancement of tumoral infiltration and cytotoxic activity of NK cells. Moreover, blockade of β-adrenergic signaling or chemical sympathectomy abolished the effects of enriched environment on NK cells and attenuated the antitumor effect of enriched environment. Taken together, our results provide new insight into the mechanism by which eustress exerts a beneficial effect against cancer. Cancer Res; 77(7); 1611-22. ©2017 AACR.

ABT-737 Synergizes with Cisplatin Bypassing Aberration of Apoptotic Pathway in Non-small Cell Lung Cancer

A subset of non-small cell lung cancer (NSCLC), which does not have a druggable driver mutation, is treated with platinum-based cytotoxic chemotherapy, but it develops resistance triggered by DNA damage responses. Here, we investigated the effect of activation of STAT3 by cisplatin on anti-apoptotic proteins and the effectiveness of a co-treatment with cisplatin and a BH3 mimetic, ABT-737. We analyzed the relationship between cisplatin and STAT3 pathway and effect of ABT-737, when combined with cisplatin in NSCLC cells and K-ras mutant mouse models. The synergism of this combination was evaluated by the Chou-Talalay Combination Index method. In vivo activity was evaluated by micro-CT. In NSCLC cells, there was a time and dose-dependent phosphorylation of SRC-JAK2-STAT3 by cisplatin, followed by increased expression of anti-apoptotic molecules. When the expression of the BCL-2 protein family members was evaluated in clinical samples, BCL-xL was most frequently overexpressed. Dominant negative STAT3 suppressed their expression, suggesting that STAT3 mediates cisplatin mediated overexpression of the anti-apoptotic molecules. ABT-737 displaced BCL-xL from mitochondria and induced oligomerization of BAK. ABT-737 itself showed cytotoxic effects and a combination of ABT-737 with cisplatin showed strong synergistic cytotoxicity. In a murine lung cancer model, co-treatment with ABT-737 and cisplatin induced significant tumor regression. These findings reveal a synergistic cytotoxic and anti-tumor activity of ABT-737 and cisplatin co-treatment in preclinical models, and suggest that clinical trials using this strategy may be beneficial in advanced NSCLC.

Antitumor Effect of Nanoparticle 131I-Labeled Arginine-Glycine-Aspartate-Bovine Serum Albumin-Polycaprolactone in Lung Cancer.

The aim of the present study is to investigate the biologic effects of internal irradiation and the therapeutic effectiveness of 131I-labeled arginine-glycine-aspartate (RGD)-bovine serum albumin (BSA)-polycaprolactone (PCL) (131I-RGD-BSA-PCL) in murine lung cancer models. The target binding and cellular uptake of NCI-H460 lung cancer cells overexpressing integrin αvβ3 were observed by confocal microscopy. Flow cytometry was used to assay apoptosis. The biologic effects of internal irradiation and the therapeutic efficacy of 131I-RGD-BSA-PCL were investigated in murine lung cancer models; tumor size, body weight, histopathologic findings, and SPECT/CT imaging findings were also monitored. In vitro uptake studies performed using confocal microscopy showed that, after 1 hour of incubation with RGD-BSA-PCL or BSA-PCL, visible fluorescence was present in the cells, and after 8 hours, the florescent signal did not disappear. The mean (± SE) tumor uptake level (i.e., the percentage of the injected dose per gram of tissue [% ID/g]) of 131I-labeled BSA-PCL (131I-BSA-PCL) at 24 and 72 hours after injection was 11.06% ± 2.15% ID/g and 3.83% ± 0.87% ID/g, respectively, which is significantly higher than the uptake levels noted for other organs (p < 0.05). The level of tumor uptake of 131I-RGD-BSA-PCL at 24 and 72 hours after injection was 39.49% ± 6.06% ID/g and 6.97% ± 1.43% ID/g, respectively, which is significantly higher than that of 131I-labeled liposome (p < 0.05). The decrease in body weight in the group treated with 131I-RGD-BSA-PCL was only 3.5% of the original body weight and was much lower than noted in the group receiving saline (i.e., 21.5% of original body weight). The median survival time for the therapeutic groups was prolonged to 27 days and 23 days after treatment with 131I-RGD-BSA-PCL and 131I-BSA-PCL, respectively. RGD-BSA-PCL has excellent cellular binding in vitro in a non-small cell lung cancer xenograft model. Furthermore, 131I-RGD-BSA-PCL was evaluated as an imaging agent and is an interesting candidate for targeting therapies in the non-small cell lung cancer xenograft model.

Abstract A42: Combination immunotherapy of an autochthonous murine lung cancer model expressing human CEA as a tumor-associated self-antigen

Abstract While cancer immunotherapies like checkpoint inhibitors have resulted in unprecedented clinical success, they only benefit a subset of patients. To improve therapeutic outcomes for greater numbers of patients, one strategy is to rationally combine different immunotherapy modalities. We recently demonstrated that attaching albumin-binding lipophilic tails to peptide antigens or molecular adjuvants (creating amphiphile vaccines) results in enhanced T-cell responses. Additionally, we observed significant tumor regression upon combining tumor-antigen targeting antibodies with extended half-life IL-2 (exPK-IL-2) in mouse models of melanoma and prostate cancer. In the present work, we combined both approaches to treat a spontaneous model of lung adenocarcinoma expressing carcinoembryonic antigen (CEA), an oncofetal protein expressed in some human lung cancers. KrasLSL-G12D/+;;p53fl/fl mice were crossed with transgenic mice expressing human-CEA to generate a CEA-tolerant background. Lung tumors were induced by infection with a lentivirus expressing Cre recombinase and human CEA. Due to the extended latency of tumor initiation in this model, we also generated a CEA-expressing cell line from these mice to test the efficacy of different combination immunotherapy regimens. We discovered that weekly treatments combining a CEA-targeting amphiphile-vaccine, exPK-IL-2, and an anti-CEA antibody with checkpoint inhibitors (anti-PD-1 and -CTLA4) resulted in sustained tumor regression in 50% of mice bearing established tumors. We are currently testing this combination immunotherapy on autochthonous lung tumors. Our results suggest that breaking tolerance to a tumor-associated self-antigen (CEA) and combining immunotherapies to recruit both innate and adaptive immune effectors can have a potent therapeutic effect in intractable tumors like lung cancer. Citation Format: Kavya Rakhra, Eric F. Zhu, Wuhbet Abraham, Kelly D. Moynihan, Naveen Mehta, Karl D. Wittrup, Darrell J. Irvine. Combination immunotherapy of an autochthonous murine lung cancer model expressing human CEA as a tumor-associated self-antigen. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A42.

Worldwide malaria incidence and cancer mortality are inversely associated

BackgroundInvestigations on the effects of malaria infection on cancer mortality are limited except for the incidence of Burkitt’s lymphoma (BL) in African children. Our previous murine lung cancer model study demonstrated that malaria infection significantly inhibited tumor growth and prolonged the life span of tumor-bearing mice. This study aims to assess the possible associations between malaria incidence and human cancer mortality.MethodsWe compiled data on worldwide malaria incidence and age-standardized mortality related to 30 types of cancer in 56 countries for the period 1955–2008, and analyzed their longitudinal correlations by a generalized additive mixed model (GAMM), adjusted for a nonlinear year effect and potential confounders such as country’s income levels, life expectancies and geographical locations.ResultsMalaria incidence was negatively correlated with all-cause cancer mortality, yielding regression coefficients (log scale) of −0.020 (95%CI: −0.027,-0.014) for men (P < 0.001) and-0.020 (95%CI: −0.025,-0.014) for women (P < 0.001). Among the 29 individual types of cancer studied, malaria incidence was negatively correlated with colorectum and anus (men and women), colon (men and women), lung (men), stomach (men), and breast (women) cancer.ConclusionsOur analysis revealed a possible inverse association between malaria incidence and the mortalities of all-cause and some types of solid cancers, which is opposite to the known effect of malaria on the pathogenesis of Burkitt’s lymphoma. Activation of the whole immune system, inhibition of tumor angiogenesis by Plasmodium infection may partially explain why endemic malaria might reduce cancer mortality at the population level.

MA09.01 Dual Blockade of PD-1 and C5a/C5aR Synergistically Protects against Non-Small Cell Lung Cancer Tumor Growth

Immunotherapy based on PD-1/PD-L1 immune checkpoint inhibitors has emerged as a powerful tool for the treatment of lung cancer. To further enhance the antitumor efficacy of individual treatments, numerous ongoing studies are trying to identify synergistic combinations that simultaneously block more than one immunomodulatory pathway. C5aR1 is a G protein-coupled receptor activated by C5a, an anaphylatoxin released during the activation of the complement system, a major component of innate immunity. We have previously shown in a murine model of lung cancer that pharmacological blockade of C5aR1 reduces cancer progression by reversing the immunosuppressive microenvironment. Thus, we hypothesized that a combined inhibition of C5aR1 and PD-1 may have a synergistic effect in the treatment of lung cancer. We characterized the immunosuppressive activity of C5aR1 and evaluated the therapeutic efficacy of the dual administration of PD-1 and C5a/C5aR1 antagonists in syngeneic non-small cell lung cancer mouse models. The RMP1-14 monoclonal antibody was used to block PD-1, and a PEG-modified L-aptamer, which binds to complement C5 and C5a, was used to inhibit the C5a/C5aR1 interaction. KrasG12D/+ mice deficient for C5aR (KrasG12D/+;C5aR1Δ/Δ) had a lower lung tumor burden and survived longer than KrasG12D/+;C5aR1wt/wt littermates. Interestingly, KrasG12D/+;C5aR1Δ/Δ mice showed a significant reduction of myeloid-derived suppressor cells (MDSCs), a subpopulation of immune cells that profoundly influences the effectiveness of cancer immunotherapies. We therefore evaluated whether C5a/C5aR blockade may enhance the efficacy of anti-PD-1 therapy by reversing the immunosuppressive microenvironment. In the Kras/Tp53 mutant 393P syngeneic lung cancer model, the combination of C5a and PD-1 blockade dramatically reduced in vivo tumor growth, as compared to the effect of each treatment alone. Similarly, this combination showed a remarkable synergistic antitumor effect in Lewis lung carcinoma (3LL)-bearing mice. Survival analysis confirmed the benefit of the combined treatment. Finally, the therapeutic combination significantly diminished the in vivo metastatic capacity of the highly aggressive Lacun3 lung cancer cell line in syngeneic BALB/c mice, as compared to the effect of anti-PD-1 or anti-C5a drugs as monotherapy. Our study supports the notion that the efficacy of anti-PD-1 therapy is limited by the immunosuppressive tumor microenvironment. In this context, C5a/C5aR1 blockade concomitant to anti-PD1 therapy obliterates the resistance mechanisms mediated by MDSCs, improving antitumor immune responses. These findings provide a framework for the clinical evaluation of this therapeutic strategy.