Lung Tumor Burden Research Articles

High Intensity Interval Training Increases Natural Killer Cell Number and Function in Obese Breast Cancer-challenged Mice and Obese Women.

High intensity interval training (HIIT) boosts natural killer (NK) cell number and activity in normal weight breast cancer patients; however, whether this occurs in obese individuals is not well established. The goal of this study was to determine whether HIIT effectively boosts NK cells as a therapeutic strategy against breast cancer in an obese mouse model and in overweight/obese women. Diet induced female C57Bl/6 obese mice were assigned to undergo HIIT for four weeks or remain sedentary. Female participants were subjected to a six weeks HIIT protocol. HIIT mice acclimatized to treadmill running were subsequently injected with 5 × 105 polyoma middle T (MT) breast cancer cells intravenously. NK cell number and activation were monitored using flow cytometry, and tumor burden or lipid content evaluated from histological lung and liver tissues, respectively. In both mice and humans, circulating NK cell number and activation (CD3−NK1.1+CD27+ and CD3−CD56+, respectively) markedly increased immediately after HIIT. HIIT obese mice had reduced lung tumor burden compared to controls following MT challenge, and had diminished hepatic lipid deposition despite minimal body weight loss. Our findings demonstrate that HIIT can benefit obese individuals by enhancing NK cell number and activity, reducing tumor burden, and enhancing metabolic health.

Abstract 237: Increased NSCLC tumorigenesis in mice fed a high fat diet is associated with increased plasma IGF-1 levels and PD-1 expression in CD4+ tumor-infiltrating lymphocytes

Abstract Lung cancer is the leading cause of cancer-related mortality worldwide, and 85% of lung cancer cases are associated with tobacco use. Activating mutations in K-ras have been identified in ~25% of tobacco-associated lung adenocarcinomas. Using mouse models of K-ras-driven lung tumorigenesis, we previously demonstrated that deletion of the IGF-1 gene or reduction of systemic IGF-1 levels using the antidiabetic drug metformin markedly reduced tumor burden. Preclinical and clinical studies suggest that diet composition is the best predictor of IGF-1 levels. Therefore, we hypothesized that diets high in fat or carbohydrate would promote lung tumorigenesis by increasing systemic IGF-1 levels. To assess the effect of diet on systemic IGF-1 levels, 9 week old C57Bl/6J and A/J mice were fed standard cereal, high-carbohydrate, or high-fat (HFD) diets for 12 weeks. Compared to cereal-fed control mice, plasma IGF-1 and insulin levels were increased in both strains of mice fed a HFD, but not in mice fed a diet high in carbohydrate. This was not due to obesity, as only the C57Bl/6J mice fed a HFD had an increase in body weight. We then investigated the effect of HFD on lung tumorigenesis using two mouse models. In the first, C57Bl/6LA2 mice, which harbor a mutation in K-ras, were fed either cereal diet or HFD for 10 weeks following weaning. Lung tumor burden in the mice fed HFD was increased 2.7-fold compared to littermates fed cereal diet. In the second model, the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1 butanone (NNK) was given by intraperitoneal injection to A/J mice beginning at 6 weeks of age. This carcinogen causes lung tumor development by inducing K-ras mutations. After 3 weekly injections of NNK, the mice were randomized to cereal diet or HFD for ten weeks. Mice fed HFD had a 60% increase in lung tumor burden. In both models, there was no relationship between the weight of the mice and lung tumor burden. Immunohistochemical analysis of the proliferation marker Ki-67 showed no significant difference in expression between tumors from mice fed a HFD or a cereal diet. Therefore, we evaluated tumor-infiltrating lymphocytes (TIL) from mice on both diets. Interestingly, both immunohistochemical analysis and flow cytometry demonstrated a 50% reduction in the number of TIL in mice fed HFD compared to mice fed a cereal diet. Additionally, HFD was associated with a 2-fold increase in PD-1+ CD4+ TIL, and the PD-1 staining intensity in these lymphocytes was significantly greater than in mice fed a cereal diet. These results may suggest that a high fat diet increases lung tumorigenesis by increasing systemic IGF-1 levels and by creating an immune-permissive tumor microenvironment. Citation Format: Regan M. Memmott, Krista Pearman, Joell Gills, Tony Tullot, Valerie Wong, Benjamin Singer, Kristin Lastwika, Franco D'Alessio, Phillip Dennis, Jeffrey William Norris. Increased NSCLC tumorigenesis in mice fed a high fat diet is associated with increased plasma IGF-1 levels and PD-1 expression in CD4+ tumor-infiltrating lymphocytes [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 237. doi:10.1158/1538-7445.AM2017-237

Abstract 2679: A promoting role for the epithelial MyD88/IRAK4/NF-kB signaling in K-ras mutant lung tumorigenesis

Abstract K-ras mutation is the most common oncogenic alterations associated with lung cancer development. Unfortunately, all attempts to develop therapies directly targeting K-ras have been failed thus far, clearly stating the need for new strategies targeting downstream effectors and/or cooperating pathways of K-ras to overcome lung cancer displaying such a molecular profile. Using a conditional K-ras mutated lung cancer mouse model, CC-LR (CCSPcre/LSL-K-rasG12D) we previously showed that K-ras mutated lung tumorigenesis is associated with lung inflammation due to activation of NF-κB pathway and increased expression of its downstream targets in the lung. Here we have shown that lack of NF-κB activity in the airway epithelium by selectively targeting IKKβ, which is required for NF-kB activation, significantly reduces lung tumor burden (3.4-fold) and changes the inflammatory cells and mediators in the bronchoalveolar lavage fluid (BALF) of CC-LR mice. Immunohistochemically staining of lung tissues with specific markers, Ki-67 and CD-31, demonstrated significantly lower tumor cell proliferation and angiogenesis in CC-LR mice with lack of epithelial NF-kB activity. To further dissect the role of NF-κB pathway in this process, CC-LR mice were crossed with MyD88f/f mice to develop a mouse with lack of MyD88 (an adaptor protein upstream to IKKβ) in the airway epithelial cells (CC-LR/MyD88Δ/Δ mice). As we had hypothesized, the resulting tumor numbers in the lungs were significantly lower (1.9-fold) in CC-LR mice with lack of MyD88 in the airway epithelial cells compared to control CC-LR mice. Tumor reduction in CC-LR-MyD88Δ/Δ mice was also associated with decreased tumor cell proliferation and angiogenesis compared to control CC-LR mice. Surprisingly, unlike to lack of epithelial NF-kB activity, absence of MyD88 in the airway epithelium did not change the BALF inflammatory cell component of CC-LR mice. We then targeted another upstream signaling molecule to NF-kB, IRAK4, which is down stream of MyD88 by crossing CC-LR mice to IRAK4 knock out mice. Similar to lack of MyD88, we found a significant reduction in lung tumor number (1.8-fold) with no changes in BALF inflammatory cell component in CC-LR mice with lack of IRAK4 compared to the control CC-LR mice. Taken these together, we conclude that there is an essential role for MyD88/IRAK4/NF-kB pathway activation in promotion of K-ras mutant lung cancer. Citation Format: Susana Castro, Soudabeh Daliri, Maria Miguelina De La Garza, Amber M. Cumpian, Misha Umer, Diana Del Bosque, Sabah Akbani, Scott E. Evans, Seyed Javad Moghaddam. A promoting role for the epithelial MyD88/IRAK4/NF-kB signaling in K-ras mutant lung tumorigenesis [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 2679. doi:10.1158/1538-7445.AM2017-2679

IGFBP3 Modulates Lung Tumorigenesis and Cell Growth through IGF1 Signaling.

Insulin-like growth factor binding protein 3 (IGFBP3) modulates cell growth through IGF-dependent and -independent mechanisms. Reports suggest that the serum levels of IGFBP3 are associated with various cancers and that IGFBP3 expression is significantly decreased in cisplatin (CDDP)-resistant lung cancer cells. Based on these findings, we investigated whether Igfbp3 deficiency accelerates mouse lung tumorigenesis and if expression of IGFBP3 enhances CDDP response by focusing on the IGF1 signaling cascade. To this end, an Igfbp3-null mouse model was generated in combination with KrasG12D to compare the tumor burden. Then, IGF-dependent signaling was assessed after expressing wild-type or a mutant IGFBP3 without IGF binding capacity in non-small cell lung cancer (NSCLC) cells. Finally, the treatment response to CDDP chemotherapy was evaluated under conditions of IGFBP3 overexpression. Igfbp3-null mice had increased lung tumor burden (>2-fold) and only half of human lung cancer cells survived after expression of IGFBP3, which corresponded to increased cleaved caspase-3 (10-fold), inactivation of IGF1 and MAPK signaling. In addition, overexpression of IGFBP3 increased susceptibility to CDDP treatment in lung cancer cells. These results, for the first time, demonstrate that IGFBP3 mediates lung cancer progression in a KrasG12D mouse model. Furthermore, overexpression of IGFBP3 induced apoptosis and enhanced cisplatin response in vitro and confirmed that the suppression is in part by blocking IGF1 signaling.Implications: These findings reveal that IGFBP3 is effective in lung cancer cells with high IGF1 signaling activity and imply that relevant biomarkers are essential in selecting lung cancer patients for IGF1-targeted therapy. Mol Cancer Res; 15(7); 896-904. ©2017 AACR.

Harnessing innate anti-tumour immunity using a Klebsiella-derived therapeutic to reduce tumour burden and improve outcomes in mouse models of lung cancer

Abstract Tumour regression and increased survival has been associated with certain acute microbial infections. Immune dysfunction contributes to the development and progression of lung cancer, and therapies that re-constitute anti-tumor immune responses provide an important means to effectively treat malignancies and improve health outcomes. We hypothesized that stimulating the innate immune system with bacterial-derived immunomodulators could induce protective anti-cancer immune responses. A Klebsiella-derived drug product, QBKPN (Qu Biologics), was used to specifically stimulate the innate immune niche in the lungs in established mouse models of lung cancer. Repeated subcutaneous administration with QBKPN significantly reduced lung tumor burden and increased survival. The protective action of QBKPN required prior exposure to Klebsiella through either environmental exposure or lung infection. However, this QBKPN-mediated anti-tumour response was independent of adaptive immunity, as the protective effect remained in RAG2-knockout mice. QBKPN intervention was characterized by a rapid, acute-like systemic immune response, including increased circulatory inflammatory cytokines and innate immune cells, leading to recruitment of immune effector cells into the lung tissue, including macrophages and natural killer (NK) cells. In addition to recruitment of innate immune cells, QBKPN increased markers of classically activated macrophages and increased production of NK cell effector molecules. Together, these data suggest that QBKPN, a Klebsiella-derived immunomodulator, causes activation and recruitment of macrophages and NK cells into the lungs, reducing cancer tumour burden and improving survival outcomes.

Inflammasome Independent Leukotriene B4 Production Drives Crystalline Silica Induced Sterile Inflammation in Lungs

Abstract Occupational exposure to Crystalline Silica (CS) is a major public health concern with millions of workers continuously exposed to CS. Deposition of fine silica particles in the lungs can lead to severe lung inflammation and cancer. Lipid chemokine Leukotriene B4 (LTB4) and IL-1b that mediate leukocyte recruitment to the site of injury, are the key mediators of CS-induced sterile inflammation. Our previous studies have shown that CS exposure increases lung tumor burden in K-rasLA1 mice and that is abrogated in the LTB4 receptor, BLT1 deficient mice. Here, we show that phagocytosis of CS sequentially activates a series of pathways leading to LTB4, IL-1b and CXC chemokine production. CS uptake induces rapid formation of lipid bodies termed “lipidosome” independent of inflammasome activation. Studies with inhibitors suggested that CS-induced lipidosome formation and LTB4 production is linked to the phagocytosis pathway, an observation confirmed by confocal microscopy. We further demonstrated the SiRNA knockdown of cPla2, JNK or Rab40c prevents the formation of lipidosome and production of LTB4. Lastly, we demonstrate using an air pouch model in mice that JNK inhibitor, BI-78D3 is effective in reducing CS induced sterile-inflammation in-vivo. In summary, our studies highlight a novel inflammasome independent and JNK activation dependent lipidosome pathway leading to the production LTB4 as a major regulator of CS-induced sterile inflammation.

Targeted Treatment of Metastatic Breast Cancer by PLK1 siRNA Delivered by an Antioxidant Nanoparticle Platform

Metastatic breast cancer is developed in about 20% to 30% of newly diagnosed patients with early-stage breast cancer despite treatments. Herein, we report a novel nanoparticle platform with intrinsic antimetastatic properties for the targeted delivery of Polo-like kinase 1 siRNA (siPLK1). We first evaluated it in a triple-negative breast cancer (TNBC) model, which shows high metastatic potential. PLK1 was identified as the top therapeutic target for TNBC cells and tumor-initiating cells in a kinome-wide screen. The platform consists of a 50-nm mesoporous silica nanoparticle (MSNP) core coated layer-by-layer with bioreducible cross-linked PEI and PEG polymers, conjugated with an antibody for selective uptake into cancer cells. siRNA is loaded last and fully protected under the PEG layer from blood enzymatic degradation. The material has net neutral charge and low nonspecific cytotoxicity. We have also shown for the first time that the MSNP itself inhibited cancer migration and invasion in TNBC cells owing to its ROS- and NOX4-modulating properties. In vivo, siPLK1 nanoconstructs (six doses of 0.5 mg/kg) knocked down about 80% of human PLK1 mRNA expression in metastatic breast cancer cells residing in mouse lungs and reduced tumor incidence and burden in lungs and other organs of an experimental metastasis mouse model. Long-term treatment significantly delayed the onset of death in mice and improved the overall survival. The platform capable of simultaneously inhibiting the proliferative and metastatic hallmarks of cancer progression is unique and has great therapeutic potential to also target other metastatic cancers beyond TNBC. Mol Cancer Ther; 16(4); 763-72. ©2017 AACR.

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis.

Metastasis, the spread and growth of malignant cells at secondary sites within a patient's body, accounts for > 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Pharmacological Inhibition of Myocardin-related Transcription Factor Pathway Blocks Lung Metastases of RhoC-Overexpressing Melanoma.

Melanoma is the most dangerous form of skin cancer with the majority of deaths arising from metastatic disease. Evidence implicates Rho-activated gene transcription in melanoma metastasis mediated by the nuclear localization of the transcriptional coactivator, myocardin-related transcription factor (MRTF). Here, we highlight a role for Rho and MRTF signaling and its reversal by pharmacologic inhibition using in vitro and in vivo models of human melanoma growth and metastasis. Using two cellular models of melanoma, we clearly show that one cell type, SK-Mel-147, is highly metastatic, has high RhoC expression, and MRTF nuclear localization and activity. Conversely, SK-Mel-19 melanoma cells have low RhoC expression, and decreased levels of MRTF-regulated genes. To probe the dependence of melanoma aggressiveness to MRTF transcription, we use a previously developed small-molecule inhibitor, CCG-203971, which at low micromolar concentrations blocks nuclear localization and activity of MRTF-A. In SK-Mel-147 cells, CCG-203971 inhibits cellular migration and invasion, and decreases MRTF target gene expression. In addition, CCG-203971-mediated inhibition of the Rho/MRTF pathway significantly reduces cell growth and clonogenicity and causes G1 cell-cycle arrest. In an experimental model of melanoma lung metastasis, the RhoC-overexpressing melanoma cells (SK-Mel-147) exhibited pronounced lung colonization compared with the low RhoC-expressing SK-Mel-19. Furthermore, pharmacologic inhibition of the MRTF pathway reduced both the number and size of lung metastasis resulting in a marked reduction of total lung tumor burden. These data link Rho and MRTF-mediated signaling with aggressive phenotypes and support targeting the MRTF transcriptional pathway as a novel approach to melanoma therapeutics. Mol Cancer Ther; 16(1); 193-204. ©2016 AACR.

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.

Tumor necrosis factor links chronic obstructive pulmonary disease and K-ras mutant lung cancer through induction of an immunosuppressive pro-tumor microenvironment

ABSTRACTTumor necrosis factor (TNF) is known as an important regulator of tumor microenvironment and inflammation. TNF levels are markedly elevated in the bronchoalveolar lavage fluid (BALF) of patients with chronic obstructive pulmonary disease (COPD), which is an independent risk factor for lung cancer. We have previously shown that COPD-like airway inflammation promotes lung cancer in a K-ras mutant mouse model (CC-LR mouse). This was associated with a significant increase of neutrophils in BALF, accompanied by a marked increase in TNF level, suggesting a link between COPD, TNF, and lung cancer promotion. Therefore, we first overexpressed TNF in the airway epithelium of CC-LR mice, which promoted lung cancer by ∼2-fold. This was associated with increased numbers of Ki67 and CD31 positive cells in lung tumors of CC-LR/TNF-Tg mice. We also found a robust increase in NF-κB activation, and numbers of neutrophils and myeloid-derived suppressor cells (MDSCs) in lung. Accordingly, we depleted MDSCs in CC-LR/TNF-Tg mice, which lead to significant tumor suppression emphasizing on the role of TNF-induced MDSCs in K-ras induced lung tumorigenesis. Finally, we targeted TNF expression by crossing CC-LR mice with TNF knock-out mice (CC-LR/TNF-KO), which resulted in a significant decrease in lung tumor burden in the absence or presence of COPD-like airway inflammation. Interestingly, there were less MDSCs and lower Ki67 and CD31 expression in the lung of the CC-LR/TNF-KO mice. We conclude that TNF links COPD to lung cancer promotion by induction of an immunosuppressive MDSC response, and subsequent amplification of proliferation and angiogenesis in tumors.

Abstract 1459: Revealing the underlying causes of the gender disparity in melanoma: Role of testosterone

Abstract Introduction: At advanced age, men have increasingly higher incidence of melanoma compared with women, as well as continuously decreasing levels of testosterone. With the low rate of survival for distant metastatic patients with melanoma, especially in men, studies aimed at elucidating the immunological and hormonal mechanisms underlying the gender disparity are warranted. Procedures: To investigate the gender disparity observed between men and women affected by melanoma, we used an experimental murine model where B16 melanoma cells were injected i.v. into C57BL/6 mice and lung colonization was assessed at day 14. The roles of neutrophils and NK cells were then investigated by specific cell depletion using anti-Ly6G(1A8) and anti-NK1.1 monoclonal antibodies, respectively. Depleting antibodies or control IgG were injected every 3 days, starting one day before melanoma cell injection. To elucidate the role of hormones, ovariectomy or castration surgeries were performed 4 weeks before B16 injection and 60 day slow release pellets were inserted at the time of surgery. Findings were reproduced with a second murine melanoma cell line (YUMM1.7) derived from a genetically engineered mouse harboring human mutations in BrafV600, as well as loss of Cdkn2a and PTEN. Data: Compared with males, naïve female mice injected with melanoma (B16 or YUMM1.7) cells have higher lung tumor burden, reduced neutrophil infiltration, and decreased NK cell activation. Ovariectomy of female mice did not affect lung tumor burden, indicating that estrogen and progesterone are not protective. Interestingly, castrated males revealed increased lung metastasis and worse survival rates compared with sham male mice, suggesting a protective role for testosterone. Further, testosterone replacement in castrated mice significantly reduced the elevated lung tumor burden to equal levels observed in sham males. Depletion of NK cells greatly increased tumor burden in both male and female mice in a gender independent manner. In contrast, neutrophil depletion increased lung tumor burden only in male mice and had no significant affect in female mice, indicating a potential gender difference in neutrophils. Moreover, neutrophil depletion in male mice also reduced NK activation and IFN-γ production, suggesting a stimulatory relationship between neutrophils and NK cells. Conclusion: Our data indicates that both neutrophils and NK cells are important for the initial response against melanoma colonization of the lung. Neutrophil depletion, as well as our castration data, indicate that there is a gender specific differential response, which may contribute to the gender disparity seen in human melanoma incidence and survival. Further, testosterone replacement at physiological levels may benefit a subset of melanoma patients. Citation Format: Janet Markman, Daiko Wakita, Timothy Crother, Moshe Arditi. Revealing the underlying causes of the gender disparity in melanoma: Role of testosterone. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1459.

Abstract 4330: A high-fat diet promotes tumorigenesis in two mouse models of K-ras-driven lung cancer

Abstract Lung cancer is the leading cause of cancer-related mortality worldwide, and 85% of lung cancer cases are associated with tobacco use. Within this group, activating mutations in K-ras have been identified in ∼25% of lung adenocarcinomas. Using a mouse model of k-ras-driven lung tumorigenesis, we previously demonstrated that deletion of the IGF-1 gene or reduction of systemic IGF-1 levels using the antidiabetic drug metformin markedly reduced tumor burden. Since preclinical and clinical studies suggest that diet composition is the best predictor of IGF-1 levels, we hypothesized that diets high in fat or carbohydrate would promote lung tumorigenesis by increasing systemic IGF-1 levels. To assess the effect of diet on systemic IGF-1 levels, 9 week old C57Bl/6J and A/J mice were fed standard cereal, high-carbohydrate, or high-fat (HFD) diets for 12 weeks. At the conclusion of the study plasma, liver, and lung samples were collected. Compared to the cereal-fed control mice, IGF-1 and insulin levels were increased in both strains of mice only with HFD. Average body weight only increased for the C57Bl/6J group that was fed HFD. We investigated the effect of HFD on lung tumorigenesis using two mouse models of lung cancer. In the first, C57Bl/6LA2 mice, which are genetically modified with a K-ras mutation present in human smokers, were fed either cereal diet or HFD for 10 weeks following weaning. Lung tumor burden in the mice fed HFD was increased 2.7-fold compared to littermates fed cereal diet. In the second model, the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1 butanone (NNK) was given by IP injection to A/J mice beginning at 6 weeks of age. This carcinogen causes lung tumor formation by inducing K-ras mutations. Following once weekly injections of NNK for 3 weeks, the mice were randomized to cereal diet or HFD. 10 weeks later, mice fed HFD were found to have a 60% increase in lung tumor burden. In both studies, there was no relationship between the final body weight of the mice and tumor burden. These studies show that HFD promotes lung tumor growth resulting from a mutation commonly observed in smokers. Therefore, dietary modification may slow the progression of tumorigenesis resulting from smoking-related genetic changes through IGF-1. Chemopreventative drugs, like metformin, may also have greater efficacy in a HFD model due to the increased insulin and IGF-1 associated with this model. Finally, understanding the molecular mechanisms by which HFD promotes tumor growth may help to identify new targets for cancer prevention. Citation Format: Jeffrey Norris, Krista Pearman, Regan Memmott, Kristin Lastwika, Joell Gills, Phillip Dennis. A high-fat diet promotes tumorigenesis in two mouse models of K-ras-driven lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4330.

Abstract 1665: Piperlongumine (PPLGM) has potent tumor-radiosensitizing properties in a mouse model of lung cancer

Abstract Background: Although over half of all lung cancer patients receive radiation therapy as part of their treatment plan, the therapeutic window of radiation is quite small, and it is difficult to deliver tumoricidal doses of radiation without the development of severe adverse effects in the nearby normal tissues. An agent that could sensitize lung cancer tissue to radiotherapy while sparing damage to normal tissues is still an unmet need. Piperlongumine (PPLGM), a natural product of the plant Piper longum has shown potent anti-carcinogenic properties through an ROS-inducing mechanism. Our hypothesis is that PPLGM will sensitize tumor cells to radiation-induced cell death in an orthotopic murine model of lung cancer. Methods: 7-week old female C57BL/6 mice were injected intravenously with 1×106 Lewis lung carcinoma (LLC) cells. Mouse cohorts (n = 30/group) were administered PPLGM (1 mg/kg of body weight) or vehicle daily by oral gavage initiated 7 days post-LLC injection. At 14 days post-LLC injection, 15 mice per treatment cohort were exposed to 12 Gy thoracic X-ray radiation treatment (XRT). Administration of PPLGM or vehicle continued 7 days post-XRT. Mice were euthanized 14 days post-XRT and evaluated for total lung weight and tumor burden using morphometric analysis. Additionally, we assessed the potential for a dual role of PPLGM (0.1-2.5μM) as a radiosensitizer of cancer cells (LLC) and a radioprotector of normal lung cells (pulmonary microvascular endothelial cells (PMVEC)) via clonogenic survival assays. Results: Using LC/MS/MS we were able to detect significantly elevated levels of PPLGM above baseline in mouse plasma and lung tissue. We observed a significant (p<0.01) increase in lung weight (from 0.14 g to 0.36 g), an indicator of tumor burden, that was significantly (p<0.01) reduced by exposure to a tumoricidal dose of XRT. Importantly, while thoracic XRT-alone reduced tumor burden by 48%, administration of PPLGM+XRT radiosensitized tumor and enhanced tumor killing by XRT, leading to a significant (p<0.05) 56% reduction in lung weight. Morphometric analysis of lung sections confirmed that PPLGM-alone significantly (p<0.01) reduced tumor area relative to vehicle, irrespective of radiation exposure. While exposure to XRT significantly (p<0.01) reduced tumor volume by 65% (from 44.4 ± 7.6% to 15.4 ± 4.5% tumor area), lung sections from mice exposed to PPLGM+XRT significantly (p<0.05) potentiated radiation-induced tumor eradication (reduction in tumor area to 5.6 ± 3.0%) above XRT-only exposure. Furthermore, PPLGM significantly (p<0.05) radiosensitized LLC dose-dependently to gamma-radiation killing, while trending towards radioprotecting normal lung cells. Conclusion: These findings provide evidence that PPLGM is a potent radiosensitizing agent that could aid in the eradication of irradiated tumors in the clinical setting. Funding: Funded in part by NIH-1R21CA178654-01 Citation Format: Anastasia Velalopoulou, Ralph A. Pietrofesa, Raymond Luke, Katie Reindl, Melpo Christofidou-Solomidou. Piperlongumine (PPLGM) has potent tumor-radiosensitizing properties in a mouse model of lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1665.

Killed Klebsiella pneumoniae treatment reduces tumor burden in murine melanoma

Abstract Acute infection in a cancer-bearing organ has been associated with improved prognosis and enhanced survival, although the anti-cancer immune mechanisms remained undefined. In collaboration with Qu Biologics, we hypothesized that stimulating pathogen-specific immunity, using bacteria that have previously infected specific organs, may induce protective anti-cancer immune responses in those target organs. We evaluated Klebsiella-mediated anti-cancer efficacy in metastatic-like B16 melanoma using heat-killed Klebsiella pneumoniae (Qu Biologics’ drug product QBKPN Site Specific Immunomodulator [SSI]). Subcutaneous injection of QBKPN SSI significantly reduced tumor burden. Furthermore, subcutaneous treatment with heat-killed E. coli, (QBECO SSI) reduced lung tumor burden but not as substantially as with QBKPN. This suggests that subcutaneous immune induction using a lung specific-pathogen activates a lung-specific antitumor response. We next asked whether pre-exposure to K. pneumoniae would improve anti-tumor efficacy. We exposed mice to live K. pneumoniae via intratracheal infection prior to subcutaneous injections with QBKPN SSI. Pre-exposure to K. pneumoniae significantly enhanced QBKPN SSI-induced anti-tumor immunity and control of metastatic-like B16 melanoma in the lungs. The anti-tumor efficacy in exposed mice correlated with an influx of monocytes and neutrophils, but did not correlate with an influx of T cells into the lungs. Collectively, these data suggest pre-exposure to K. pneumoniae may induce tissue resident memory cells that mediate tumor cytolysis, and thus explain the lack of T cell infiltration into the lungs.

Site-Specific Immunomodulators (SSIs) are novel immunotherapies for cancer

Abstract The immune system recognizes and eliminates malignant cells, and maintains an intricate balance between immune activation and cancer. Historically, spontaneous tumor regression has been observed in some patients experiencing acute microbial infections. Building upon this, it was discovered that repeated subcutaneous injection of novel immunotherapies called Site-Specific Immunomodulators (SSIs), derived from killed bacteria known to cause infection in a particular organ or tissue, may be effective at treating cancer growing in the same site. We hypothesize that SSIs stimulate the body’s immune system to reverse immune suppression and dysfunction in the tumor microenvironment, enabling effective anti-cancer immune responses. To test this hypothesis, we evaluated the efficacy of a lung-specific SSI, QBKPN, in preclinical lung cancer models. Repeated subcutaneous administration of QBKPN significantly reduced lung tumor burden at day 16 post-inoculation (p<0.0001) in the Lewis Lung Carcinoma model, improving median survival by 10 days (p<0.005). Similar results were obtained using the aggressive and poorly-immunogenic B16 lung cancer model. Anticancer efficacy was associated with site-specific infiltration of newly recruited monocytes and neutrophils to the lung and mobilization of antigen presenting cells in the lung-draining lymph node. These data complement the positive results from our compassionate use clinical data, and provide strong evidence that SSIs may improve therapeutic outcomes for cancer patients. QBKPN is currently studied in a Phase 2a clinical trial of non-small cell lung cancer in collaboration with the BC Cancer Agency (Trial NCT02256852).

Inhibition of Lysyl Oxidase and Lysyl Oxidase-Like Enzymes Has Tumour-Promoting and Tumour-Suppressing Roles in Experimental Prostate Cancer.

Lysyl oxidase (LOX) and LOX-like (LOXL) enzymes are key players in extracellular matrix deposition and maturation. LOX promote tumour progression and metastasis, but it may also have tumour-inhibitory effects. Here we show that orthotopic implantation of rat prostate AT-1 tumour cells increased LOX and LOXLs mRNA expressions in the tumour and in the surrounding non-malignant prostate tissue. Inhibition of LOX enzymes, using Beta-aminopropionitrile (BAPN), initiated before implantation of AT-1 cells, reduced tumour growth. Conversely, treatment that was started after the tumours were established resulted in unaffected or increased tumour growth. Moreover, treatment with BAPN did not suppress the formation of spontaneous lymph node metastases, or lung tumour burden, when tumour cells were injected intravenously. A temporal decrease in collagen fibre content, which is a target for LOX, was observed in tumours and in the tumour-adjacent prostate tissue. This may explain why early BAPN treatment is more effective in inhibiting tumour growth compared to treatment initiated later. Our data suggest that the enzymatic function of the LOX family is context-dependent, with both tumour-suppressing and tumour-promoting properties in prostate cancer. Further investigations are needed to understand the circumstances under which LOX inhibition may be used as a therapeutic target for cancer patients.

Abstract A135: Phosphodiesterase-5 inhibition augments postoperative natural killer cell antitumor immunity by reducing myeloid-derived suppressor cell function

Abstract Background and Objective: Cancer surgery while necessary for primary tumor removal, has been shown to induce immune suppression and promote metastases in preclinical models and human cancer surgery patients. We have previously shown that cancer surgery induces natural killer (NK) cell dysfunction and promotes postoperative metastases formation. Activating the immune system and reversing immune suppression have emerged as promising ways to treat cancer and they can be safely employed in the perioperative period. In this study, we investigated the role of Myeloid-Derived Suppressor Cells (MDSC) as the mediators of postoperative NK cell impairment and subsequent metastases and evaluated the ability to target MDSC with a phosphodiesterase-5 (PDE-5) inhibitor in the clinically relevant postoperative period. Methods: The suppression of NK cell cytotoxic activity by MDSC from control and surgically stressed mice was compared both in vitro and in vivo. Further, NK cell function and lung tumor burden was assessed following perioperative PDE-5 administration. In human cancer patients, MDSC expansion and function were assessed prior to and following surgical resection. Results: MDSC expansion was observed following surgery in mouse melanoma and breast tumor models of surgical stress and this correlates with NK cell suppression postoperatively. Co-cultures of NK cells with MDSC from control and surgical stressed mice resulted in their functional suppression with surgically stressed MDSC, but not with control MDSC. Adoptive tranfser of MDSC from surgically stressed donor mice into recipient mice resulted in impaired in vivo clearance of NK cell sensitive RMA-S tumor cells. Further, surgically stressed mice depleted of MDSC, demonstrated enhanced ability to clear RMA-S tumor cells and fewer lung tumour metastases. Using the clinically relevant PDE-5 inhibitor (sildenafil) to target MDSC suppressive pathways, we demonstrated recovery of NK cell in vitro and in vivo function, followed by reduction of lung tumor metastases. In human cancer surgery patients, MDSC expansion and suppression of NK cells in co-culture with autologous MDSC was observed in the early postoperative period. Conclusions and Significance: MDSC are increased in both number and suppressive activity of NK cells following surgery. MDSC represent a hurdle to successful treatment of postoperative metastatic disease, but this effect is reversible using PDE-5 inhibitors. A clinical trial of perioperative PDE-5 inhibitors is currently being undertaken. Citation Format: Lee-Hwa Tai, Almohanad A. Alkayyal, Christiano Tanese de Souza, Amanda Lynn Leslie, Shalini Sahi, Sean Bennett, Jiqing Zhang, John C. Bell, Rebecca A. Auer. Phosphodiesterase-5 inhibition augments postoperative natural killer cell antitumor immunity by reducing myeloid-derived suppressor cell function. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A135.

Evan's Syndrome Associated with Pembrolizumab Therapy in Metastatic Non-Small Cell Lung Cancer

IntroductionPembrolizumab is a humanized monoclonal antibody that disrupts programmed cell death 1 (PD-1) receptor signaling and unmasks native cytotoxic T-cell activity against tumor cells. Early clinical trials show that pembrolizumab induces a prolonged and sometimes dramatic antitumor response in advanced melanoma, renal cell carcinoma, and non-small cell lung cancer (NSCLC) (Topalian et al., 2012; NEJM: 366;26). Consequences of immunomodulation include immunopathologies of skin, endocrine, and/or gastrointestinal organs (Patnaik et al., 2015; Clin Cancer Res: 21;15). Herein we describe the first reported case of Evan's syndrome following the use of pembrolizumab.CaseA 67-year old man with 70-pack year smoking history presented to hospital with right-sided weakness and gait ataxia. Radiologic studies of head, thorax, abdomen, and limbs revealed left-sided frontal lobe mass, pulmonary lesions, multiple liver metastases, and left lower extremity venous thrombosis. The patient underwent cranial metastasectomy, which confirmed NSCLC primary neoplasm, followed by cranial irradiation, dexamethasone treatment, and indefinite enoxaparin. First and second-line chemotherapy with gemcitabine-carboplatin and pemetrexed failed to control his disease; he was therefore enrolled in the MK 3475-001 clinical trial and received investigational pembrolizumab (10mg/kg every 2 weeks).Following 4 cycles of pembrolizumab therapy, repeat CT scans showed a marked reduction in cerebral, liver, and lung tumor burden. Treatment continued with transient interruption of therapy at cycle 8 due to delayed side effects of cranial radiation. An additional 10 cycles (18 cycles total) of therapy were delivered, after which the patient was admitted to hospital for new-onset fatigue, exertional dyspnea, and jaundice. Laboratory evaluation showed a hemoglobin of 6.2 g/dL, reticulocyte count 239 ×103/µL, and platelet count 113 × 103/µL, with elevated total bilirubin of 1 mg/dL, LDH 398 U/L, haptoglobin level less than 20 mg/dL, and normal creatinine and liver enzymes. Peripheral blood film revealed microspherocytes and thrombocytopenia, indirect and direct testing revealed a pan-reactive IgG autoantibody, while serologic testing was negative for infectious and rheumatologic diseases. Diagnosis of warm-autoimmune hemolytic anemia was made and the patient was initiated on prednisone, azathioprine, cyclophosphamide, and IVIG therapy in conjunction with erythropoietin injections and transfusion support. Hemoglobin reached a nadir of 3.2 g/dL before recovering to 11.6 g/dL at 2 weeks into admission, followed by discharge on daily oral prednisone at 105 mg and cyclophosphamide 100 mg.Sustained hematologic stability permitted gradual prednisone taper to a dose of 20 mg. Fourteen weeks post-admission, the patient returned to hospital with severe bruising and epistaxis. Laboratory evaluation showed hemoglobin of 11.6 g/dL and platelet count of 6 × 109/L, concerning for ITP and fulfilling criteria for Evan's syndrome. Salvage therapy commenced with weekly rituximab and re-initiation of high-dose prednisone, until platelets recovered to 146 × 109/L following the fourth/final dose of rituximab. Prednisone taper was re-trialed with complete discontinuation of steroids after 24 weeks. Despite a persistently positive Coombs test, the patient maintained normal hemoglobin and platelet counts 40 weeks after rituximab therapy. Opportunistic Aspergillus fumigatus pulmonary infection followed salvage therapy, but was successfully treated with voriconazole over 12 weeks. CT staging scans at 48 weeks post-discontinuation of pembrolizumab demonstrated stable metastatic disease.ConclusionReversal of cancer-induced immune suppression through PD-1 inhibition has the potential to provoke immunodysregulation against multiple hematologic lineages. Though autoimmune hyperactivation persists for months after pembrolizumab cessation, immune-related hemolysis and thrombocytopenia remain amenable to treatment with corticosteroids and rituximab. Despite immune downregulation with corticosteroids and rituximab, pembrolizumab appears to exhibit sustained immune control over advanced malignancy in this case. [Display omitted] [Display omitted] DisclosuresOff Label Use: Rituximab to treat refractory ITP. Butler:Merck: Consultancy.

Abstract LB-B13: The application of high-dose low fraction image-guided irradiation (IGMI) in combination with anti-CTLA4 immunotherapy results in an additive inhibition of tumor growth in syngeneic tumor models

Abstract INTRODUCTION: Radiotherapy (RT) is used across multiple cancer types in a number of clinical settings. The primary mechanism of action is thought to be through irreversible damage to tumor cell or associated stromal DNA, but there is an increasing body of evidence that irradiation also mobilizes tumor-specific immunity. Recent advances in RT with stereotactic and image-guided micro-irradiation (IGMI) have resulted in an increase in tumor specific targeting with a corresponding reduction in associated side-effects. The resultant use of high-dose, reduced fraction dosing regimens (hypofractionation) has resulted in improved clinical response. However, IGMI in the preclinical setting is less common and typically studies utilize whole body irradiation with lead shielding or simple single beam techniques to focus the radiation. Here we report the application of high-dose, low fraction IGMI to demonstrate an additive combination outcome of radiotherapy and immunotherapy and an abscopal effect in a preclinical model. EXPERIMENTAL PROCEDURES: CT26 mouse colon and bioluminescent 4T1 mammary carcinoma cells were implanted subcutaneously into BALB/c mice. Subcutaneous tumor growth was monitored by caliper measurement and bioluminescent imaging (BLI) was carried out to confirm metastatic 4T1 lung tumors. Tumors (∼100-200mm3) were treated with hypofractionated IGMI, anti-CTLA4 therapy or a combination of both. For IGMI, mice were anaesthetized and CBCT images were acquired using the small animal research platform (SARRP; Xstrahl Ltd). Fractionated irradiation (5 × 6Gy) was administered to the isocenter of the tumor using a multi beam approach to spare the surrounding tissue. Bodyweight and clinical signs were monitored daily. At termination the primary and metastatic lung (4T1) tumors were collected and assessed for immune cell infiltration by FACs. RESULTS: Response to the hypofractionated IGMI regimen and anti-CTLA4 either alone or in combination was well tolerated with no adverse effects or significant body weight-loss. Monotherapy with anti-CTLA4 exerted no statistically significant effect on primary or metastatic (4T1) tumor growth and treatment with RT resulted in a modest primary tumor growth inhibition (TGI; 40%). When the two treatments were combined, TGI increased (60%) which was statistically significant compared to RT monotherapy, indicating an additive effect. Additionally, there was a decrease in normalized lung cell counts following combination treatment (4T1) indicating an abscopal response on the lung metastases. This decrease in lung tumor burden corresponded to an increase in both the CD8+ T-cells and the ratio of Teff:Treg in the lung samples. CONCLUSIONS: Hypofractionated IGMI when combined with anti-CTLA4 treatment had minimal side effects and showed statistically significant additive reduction in primary tumor growth. Furthermore, combination treatment resulted in a reduced metastatic lung burden indicating an abscopal effect that appears to be driven by an increase in CD8+ T-cells. The SARRP platform enables clinically relevant radiotherapy regimens to preclinical models facilitating improved evaluation of combinations strategies with immunotherapeutics and progression to the clinic. Citation Format: Andrew Mckenzie, Nektaria Papadopoulou, Yinfei Yin, Shonna Glenn, Russell Garland, Neil Williams, Qian Shi, Rajendra Kumari. The application of high-dose low fraction image-guided irradiation (IGMI) in combination with anti-CTLA4 immunotherapy results in an additive inhibition of tumor growth in syngeneic tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B13.