Murine Lung Cancer Model Research Articles

MA17.11 High Sensitivity to PD-1 Blockade Therapy After Ld1 Depletion in KRAS-Driven Lung Cancer Through CD8+/CD3+ Tumor Infiltration and PD-L1 Induction

PD-1/PDL-1 inhibitors are approved for non-small cell lung cancer (NSCLC). However, many patients do not benefit and therapeutic combinations are under investigation. We have previously described Id1, involved in proliferation, angiogenesis and immunosuppression, as a prognostic factor in lung adenocarcinoma (LUAD) (Ponz-Sarvise, Clin Cancer Res 2011), Id1’s role in lung cancer metastasis (Castanon, Cancer Letters 2017) and more recently shown that Id1 sustains mutant KRAS-driven progression and metastasis in NSCLC (Roman, Cancer Res 2019). In a previous syngeneic murine lung cancer model with depleted levels of Id1 using Id1-/- and Id1 wildtype C57BL/6 mice inoculated with Lewis Lung Carcinoma (LLC), we tested a combined therapeutic strategy targeting PD-1 and Id1, showing impaired tumor growth and increased survival (Gil-Bazo, presented at WCLC 2018). Here we study a combined strategy targeting PD-1 and Id1 in a KRAS-mutant murine LUAD model and the immune-related mechanisms involved. First, a correlation between Id1 and PD-L1 mRNA expression was studied in mutant and wild-type KRAS LUAD cohorts from The Cancer Genome Atlas data set (TCGA). Secondly, a syngeneic tumor model using Balb/c mice through subcutaneous injection of KRAS-mutant LUAD (Lacun3) cells and Id1-silenced Lacun3 (Id1sh) cells. In vitro, proliferation was measured in both cell lines through MTS assays. IFNg-induced PD-L1 expression in both cell lines and flow cytometry was used to evaluate its mechanistic effects on the immune response. After tumor cells injection, mice were treated with an anti-PD-1 (RMP-1-14) monoclonal antibody or PBS, i.p. Tumor volumes according to Id1 status in tumor cells and the treatment administered were quantified. Vectra 3.0™ multispectral microscopy was used to characterize the tumor associated immune cells in paraffin-embedded tissues from our previous syngeneic murine lung cancer model using Id1-/- and Id1 wildtype C57BL/6 mice inoculated with LLC in which the combined blockade had been reported as effective. Immune marker antibodies were used to study expression of CD3, CD4 and CD8. An inverse, moderate and statistically significant correlation between Id1 and PD-L1 expression in mutant and wild-type KRAS LUAD cohorts from TCGA was found in both cohorts (-0.367 and -0.351, respectively, p<0.001), indicating that Id1 depletion may lead to PD-L1 expression induction. In vitro assays showed that Id1 silencing reduced Lacun3 cells proliferation (p<0.001). Up-regulation of surface PD-L1 expression occurred in Id1sh cells, but not in Lacun3 cells, after receiving IFNg (p=0.0022). Mechanistically, in the syngeneic murine model, Id1 inhibition in the injected cells, combined with anti-PD-1 treatment, significantly induced a tumor growth impairment (p<0.001). An intense CD8+ and CD3+ immune cell infiltration was observed in LLC Id1-/- C57BL/6 mice treated with anti-PD1 (p<0.05 for CD3+ TILS), compared the control groups, possibly explaining the dramatic tumor growth impairment previously shown on the treated animals. Id1 silencing may induce PD-L1 overexpression according to in silico and in vitro results. Id1 and PD-1 combined blockade in our KRAS-mutant syngeneic murine LUAD model significantly impaired tumor growth, compared to each strategy alone. A significantly increased CD3+ and CD8+ tumor infiltration and IFNg-induced PD-L1 tumor expression after the combined blockade may explain these findings.

MicroRNA‑30a controls the instability of inducible CD4+ Tregs through SOCS1.

Inducible regulatory T cells (iTregs) are an important subset of Tregs and play a role in the maintenance of peripheral tolerance, and the occurrence of a number of diseases, including tumors and autoimmune diseases. However, the instability of iTregs is a major obstacle for their potential application in clinical trials. The underlying mechanism of iTreg instability remains largely unknown. In the present study, the expression level of microRNA (miRNA/miR)‑30a in murine iTregs was evaluated using reverse transcription‑quantitative PCR. miR‑30a mimics and a miR‑negative control (NC) were transiently transfected into iTregs using Nucleofector technology. The effects of miR‑30a on the suppressive function of murine iTregs invitro and invivo were investigated using MTT, adoptive cell transfer (ACT) and flow cytometry assays, as well as a murine model of lung cancer. In the present study, it was identified that the expression level of miR‑30a was lower in murine iTregs invitro compared with natural (n)Tregs. Furthermore, compared with miR‑NC, miR‑30a mimics impaired the suppressive function of murine iTregs on murine CD4+ Tcell proliferation invitro, which was accompanied by the altered expression of cytotoxic Tlymphocyte‑associated antigen4 and glucocorticoid induced tumor necrosis factor receptor, as well as transforming growth factor‑β and interleukin‑10. It was also observed that, compared with miR‑NC, miR‑30a mimics abrogated the suppressive effects of murine iTregs on murine CD8+ Tcell function invivo, producing an effective antitumor effect in mice bearing 3LL lung cancer cells in the ACT assay. From a mechanistic point, the expression level of suppressor of cytokine signaling 1, a putative target of miR‑30a, was elevated, altering the activation of the Akt and STAT1 pathway in the miR‑30a mimic transfected group compared with the miR‑NC group, reducing the suppressive function of murine iTregs. The present study identified a role for miR‑30a in the instability of iTregs and provided a novel insight into the development of therapeutic strategies for promoting T‑cell immunity via the regulation of iTreg instability by targeting specific miRNAs.

Primary tumors induce neutrophil extracellular traps with targetable metastasis promoting effects.

Targeting the dynamic tumor immune microenvironment (TIME) can provide effective therapeutic strategies for cancer. Neutrophils are the predominant leukocyte population in mice and humans, and mounting evidence implicates these cells during tumor growth and metastasis. Neutrophil extracellular traps (NETs) are networks of extracellular neutrophil DNA fibers that are capable of binding tumor cells to support metastatic progression. Here we demonstrate for the first time that circulating NET levels are elevated in advanced esophageal, gastric and lung cancer patients compared to healthy controls. Using pre-clinical murine models of lung and colon cancer in combination with intravital video microscopy, we show that NETs functionally regulate disease progression and that blocking NETosis through multiple strategies significantly inhibits spontaneous metastasis to the lung and liver. Further, we visualize how inhibiting tumor-induced NETs decreases cancer cell adhesion to liver sinusoids following intrasplenic injection - a mechanism previously thought to be driven primarily by exogenous stimuli. Thus, in addition to neutrophil abundance, the functional contribution of NETosis within the TIME has critical translational relevance and represents a promising target to impede metastatic dissemination.

Abstract 3950: Rovalpituzumab tesirine enhances the anti-tumor efficacy of PD-1 blockade in a murine model of small cell lung cancer with endogenous Dll3 expression

Abstract Rovalpituzumab tesirine (Rova-T) is an antibody drug conjugate (ADC) comprised of a DLL3-targeted antibody conjugated to a potent cytotoxic pyrrolobenzodiazepine (PBD) toxin that is currently being investigated clinically in phase 3 trials for small cell lung cancer (SCLC). Cytotoxic DNA damaging agents including PBDs may elicit immunologic cell death (ICD), a form of apoptosis that enhances immune activation (Rios-Doria J 2017). Blockade of PD-1, an immune checkpoint receptor, using Nivolumab in 3L+ SCLC patients, showed an objective response rate (ORR) of 11.9%, with ORR higher in patients with high tumor mutation burden (Hellmann MD 2018). Here we report that sub-efficacious doses of Rova-T in combination with anti-PD-1 mAb resulted in enhanced anti-tumor activity in KP1, a murine SCLC syngeneic tumor model (Park KS 2011) that endogenously expresses Dll3, compared to Rova-T alone or anti-PD-1 mAb alone. In contrast, a single cycle of cisplatin + etoposide (C/E) standard of care chemotherapy alone showed some tumor burden reduction, but no additive activity was seen for combination C/E + anti-PD1 mAb. Rova-T alone, anti-PD1 alone, and the combination increased CD4+ and CD8+ T-cell infiltration in the tumor, as measured by gene expression analysis, flow cytometry and immunostaining of tumors after treatment. A multiplex immunofluorescence panel showed that the increased lymphocytes are restricted to the surrounding stroma in tumors treated with Rova-T or anti-PD1 alone. Unique to the Rova-T + anti-PD1 treated tumors, lymphocytes were found throughout the tumor, and they expressed granzyme B, a marker of activation. CD8 but not CD4 T-cell depletion before and during in vivo treatment with Rova-T + anti-PD1 reduced the anti-tumor efficacy, supporting a role for cytotoxic CD8 T cells in the combination efficacy. In addition to enhancing T-cell infiltration, Rova-T alone and Rova-T + anti-PD1 increased expression of PD-L1, the immune suppressive ligand of PD-1, on tumor cells. Mice treated with a single efficacious dose of Rova-T alone or Rova-T + anti-PD1 showed durable tumor suppression (&amp;gt;50 days) and did not develop tumors when re-injected with 1 million tumor cells on the opposite flank, indicating that treatment conferred immunologic memory and sustained anti-tumor immunity. Together, these data provide pre-clinical rationale for the ongoing combination SCLC clinical trials of Rova-T and Nivolumab (NCT03026166) and Rova-T and ABBV-181 (NCT03000257). Citation Format: Philip M. Vitorno, Chen-Hua Chuang, Christine Moore, Tolga Turan, Ronald Ferrando, Aaron Nichols, Shravanthi Madhavan, Laura R. Saunders. Rovalpituzumab tesirine enhances the anti-tumor efficacy of PD-1 blockade in a murine model of small cell lung cancer with endogenous Dll3 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3950.

Abstract 2710: Depletion of CXCR2-dependent myeloid-derived suppressor cells (MDSCs) overcomes anti-PD-1 resistance in a murine model of LKB1-deficient non-small cell lung cancer (NSCLC) with high mutational load

Abstract Checkpoint inhibitors such as PD-L1/PD-1 blockade have rapidly integrated into the paradigm of NSCLC treatment. However, a majority of patients do not benefit from monotherapy with checkpoint inhibitors. High tumor mutational burden (TMB), along with pre-existing intratumoral T cell infiltration and baseline high PD-L1 expression, predicts response to checkpoint blockade. Furthermore, a recent retrospective study identified LKB1 alterations as the most prevalent genomic driver of resistance to PD-1 axis inhibitors in KRAS-mutant lung adenocarcinoma. In this study, we investigate the mechanisms underlying LKB1-mediated immunosuppression in NSCLC. We show that loss of LKB1 in human bronchial epithelial cells (HBECs) and NSCLC cells leads to increased secretion of CXCR2 ligands, including CXCL1, CXCL2, CXCL3, CXCL5 and CXCL8. These CXCR2 ligands are also elevated in LKB1-deficient tumors from patient-derived xenografts and genetically-engineered murine models. We find abundant tumor infiltrating MDSCs in murine Lkb1-deficient NSCLC, consistent with the capacity for CXCR2 ligands to recruit MDSCs. MDSCs mediate potent immune suppressive activities at multiple levels including release of immunosuppressive cytokines, recruitment of regulatory T cells (Tregs), inhibition of CD8 T cell tumor infiltration and upregulation of PD-L1 expression. Although MDSC depletion activates interferon gamma signaling and decreases systemic Tregs in murine KrasK12D;Tp53-/-;Lkb1-/- (KPL) tumors, it does not sensitize KPL tumors to anti-PD-1 therapy. One of the major challenges in the preclinical assessment of lung cancer immunotherapy is that the commonly utilized murine models lack the mutational burden of human NSCLC. To assess this combination therapy in the context of a mutational burden that more accurately reflects the clinical disease, we generated tumors with high TMB by exposing KPL cells in vitroto the tobacco carcinogen N-methyl-N-nitrosourea. In the context of high TMB, MDSC depletion demonstrates remarkable anti-tumor effects in combination with anti-PD-1 therapy. Finally, we delineate the regulation of CXCR2 ligands by LKB1 which is dependent on the MARK-mediated NF-κB pathway. In conclusion, we find that LKB1 deficiency leads to increased CXCR2 ligand production and tumor infiltrating MDSCs. MDSC depletion enhances the efficacy of anti-PD-1 blockade in LKB1-deficient tumors bearing high TMB. Citation Format: Rui Li, Ramin Salehi-Rad, Stephanie Ong, Milica Momcilovic, Bin Liu, Raymond Lim, Linh Tran, Ziling Huang, Zhe Jing, Manash Paul, Kostyantyn Krysan, Stacy Park, John Minna, Michael Teitell, David Shackelford, Steven Dubinett. Depletion of CXCR2-dependent myeloid-derived suppressor cells (MDSCs) overcomes anti-PD-1 resistance in a murine model of LKB1-deficient non-small cell lung cancer (NSCLC) with high mutational load [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2710.

Abstract 1499: Recolonizing microbiota may impact tumor response to PD-1 inhibition following antibiotic and radiotherapy treatment in a bioluminescent orthotopic model of murine lung cancer

Abstract Mounting clinical and preclinical evidence demonstrates an important role for the intestinal microbiome in mediating efficacy of immune checkpoint inhibitor (ICI) antibody therapy across a number of tumor contexts. We sought to determine the effects of microbiome modulation on ICI therapy in a clinically recapitulative orthotopic murine lung cancer model. The microbiome of C57Bl/6NHsd mice was sterilized with antibiotic (ampicillin, streptomycin and colistin) for ten days, resulting in a 99.9% mean decrease in fecal aerobic and anaerobic bacterial load in comparison to naïve mice, handled under specific pathogen free (SPF) conditions. Luciferase-expressing murine Lewis lung carcinoma cells (LL/2-Red-FLuc) were surgically implanted into the left lung parenchyma of all animals. Animals were treated with locoregional radiotherapy (2x 9Gy fractions) targeted to the left lung. The gastrointestinal microbiome was reconstituted via oral gavage Q3D of ~1e09/dose commensal A. muciniphila (A. muc) and E. hirae (E. hir) over five doses or sterile saline as control and animals were randomized within commensal/saline treatment groups to equivalent mean tumor burden as measured by Lumina Series III In-Vivo Imaging System (IVIS). Animals were then administered anti-murine PD-1 or isotype control (0.25 mg) antibody (Ab) treatments Q3D over four doses via intraperitoneal injection. Tumor growth was monitored by IVIS over the course of the study, and ex-vivo IVIS was performed on lungs at endpoint (Day 12 post-tumor implantation). Tumor growth of microbiota non-reconstituted antibiotic-sterilized animals was only slightly inhibited by anti-PD-1 therapy. Animals administered commensal A. muc and E. hir and treatments also displayed slightly inhibited tumor growth kinetics, similar to those observed under saline/anti-PD-1 therapy. Fecal microbial sequencing and immunophenotypic analyses are ongoing. This study demonstrates the utility and ongoing development of a clinically recapitulative contextually accurate preclinical murine lung cancer model to assess the effects of specific microbiota in mediating the efficacy of anti-tumor immunotherapy. Citation Format: Benjamin G. Cuiffo, Caitlin S. Parello, Chelsea Ritchie, Nicholas Rivelli, Alexandra Kury, Sallyann Vu, Gavin Gagnon, Veronica Ritchie, Kasey Reardon, Catarina Costa, Samantha Rogers, Gregory D. Lyng, Stephen T. Sonis. Recolonizing microbiota may impact tumor response to PD-1 inhibition following antibiotic and radiotherapy treatment in a bioluminescent orthotopic model of murine lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1499.

Abstract 530: Phosphatidylserine-targeting antibody combined with chemoradiation enhances systemtic anti-tumor immunity

Abstract The discovery of immune checkpoint blockades have resulted in significant clinical benefits for patients with metastatic cancers including non-small cell lung cancer (NSCLC). Nevertheless, a large proportion of patients do not respond to such therapy. Strategies to enhance the therapeutic efficacy of cancer immunotherapy among a larger group of patients are urgently needed. Increasing the recognition of cancer cells by immune system may enhance response to immunotherapies. Tumor-associated macrophage (TAM) is the most abundant immune cell in most solid tumors, and is critical in bridging innate and adaptive immune system functions. Cancer cells overexpress multiple immune suppressive molecules including phosphatidylserine (PS) to inhibit immunological functions of TAMs. PS expression can be enhanced by cancer therapeutics such as radiation to induce immune suppression. Our results show that radiation increased expression of PS on lung cancer cells. The combination of PS blocking antibody with radiation decreased immune-suppressive phagocytosis and enhanced antigen presentation of macrophages. In murine lung cancer models, combination treatment of PS blockade and chemoradiation more effectively controlled tumor growth and prolonged survival than each treatment used alone. In a bilateral lung cancer tumor model, addition of PS blockade to chemoradiation promoted cytotoxic T cell infiltration and induced tumor growth inhibition of the non-irradiated tumors. Together, our results demonstrate that PS-targeting antibodies may be combined with chemoradiation to enhance intrinsic tumor immunogenicity and activate systemic anti-tumor immune responses. This approach may provide new routes for improving the efficacy of modern immunotherapeutics. Citation Format: Yifan Wang, Zhigang Liu, Weiye Deng, Betty Y. Kim, Steven H. Lin, Wen Jiang. Phosphatidylserine-targeting antibody combined with chemoradiation enhances systemtic anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 530.

Abstract 3214: In situ vaccination with CCL21-modified dendritic cells (CCL21-DC) combined with checkpoint blockade in murine models of NSCLC

Abstract Studies that target the PD-1/PD-L1 axis of immune checkpoints have demonstrated clinical responsesin approximately 20% of non-small cell lung cancer (NSCLC) patients.However, majority of the patients do not benefit from the initial treatment.Mechanistic studies reveal that responses to PD-1/PD-L1 blockade are associated with high tumor mutational burden (TMB) andincreased CD8+T cell tumor infiltration and high baseline tumor PD-L1 expression. Furthermore, recent studies have identified Stk11/Lkb1 loss as a major driver of primary resistance to PD-1 blockade in inKRAS-mutant lung adenocarcinoma (LUAC), a major subtype of NSCLC. One potential approach to enhance the effectiveness of checkpoint inhibitors is to enhance tumor antigen presentation and tumor-specific T cell immune responses by in situvaccination, taking advantage of the full repertoire of available tumor antigens. In preclinical and clinical studies, we have demonstrated that CCL21-secreting dendritic cells (CCL21-DC) have the capacity to induce the infiltration of DC and T cells into the TME and to promote tumor-specific T cell activation both locally and systemically. This response, however, was accompanied by the upregulation of PD-L1 expression in the tumor. We hypothesize that in situvaccination with CCL21-DC could restore tumor antigen presentation and T lymphocyte infiltration into the tumor, thereby sensitizing non-responsive NSCLC tumors to checkpoint blockade. To test this hypothesis, we first established novel genetically-engineered murine models (GEMMs) of lung cancer that bear the common driver mutations (KPL; KrasG12D;Tp53-/-;Lkb1-/-) and varying mutational loads to accurately reflect the clinical disease. This was achieved by exposing KPL cells in vitroto the tobacco carcinogen N-methyl-N-nitrosourea (MNU) with various duration. Using these models, we demonstrated that (1) KPL with higher TMB is more sensitive to anti-PD-1 monotherapy as compare to low TMB, consistent with previous studies; (2) Intratumoral (IT) administration of CCL21-DC in combination with anti-PD-1 resulted in significantly enhanced anti-tumor response compared to either monotherapy alone. Further work is underway to evaluate the efficacy of the combination therapy with IT CCL21-DC and PD-1 inhibition in multiple murine models of NSCLC. In addition, mechanistic studies will be performed to understand the anti-tumor effect mediated by the CCL21-DC and anti-PD-1 combination therapy, including immune phenotyping of TME and functional studies of DC and T cells. As we prepare to initiate our clinical trial that combines IT CCL21-DC with PD-1 inhibition in advanced NSCLC patients with low baseline PD-L1 expression, we anticipate these preclinical models will serve as a platform to enhance our understanding of the molecular mechanisms of response and resistance to immunotherapy. Citation Format: Raymond J. Lim, Ramin Salehirad, Bin Liu, Rui Li, Linh M. Tran, Kostyantyn Krysan, Stephanie Ong, Zi L. Huang, David B. Shackelford, Sherven Sharma, Steven M. Dubinett. In situ vaccination with CCL21-modified dendritic cells (CCL21-DC) combined with checkpoint blockade in murine models of NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3214.

Abstract 1508: Primary tumors induce neutrophil extracellular traps with targetable metastasis promoting effects

Abstract Targeting the dynamic tumor immune microenvironment can provide effective therapeutic strategies for cancer. Neutrophils (polymorphonuclear neutrophils, PMNs) are the predominant circulating leukocyte population in humans and are vital to fight infection. Despite mounting evidence that PMNs can promote tumor progression, depleting PMNs is not a viable therapeutic option. Neutrophil extracellular traps (NETs) are networks of extracellular neutrophil DNA fibers that are capable of trapping tumor cells and promoting their growth and their metastasis. Targeting NETs can therefore be a potentially successful therapeutic option to block the tumor promoting functions of PMNs. Here we demonstrate that circulating NET levels are elevated in esophageal, gastric and lung cancer patients compared to healthy controls. This increase correlates with disease stage and NET levels are independent predictors of advanced stage. Using pre-clinical murine models of lung and colon cancer, we observe elevated NET levels in tumor bearing mice compared to non-tumor bearing mice; these levels correlated with tumor size. NET levels significantly decrease following tumor resection or treatment with DNase1, a NET degrader, or neutrophil elastase inhibitor (NEi), a NET inhibitor. NET levels do not rise following tumor inoculation in peptidyl arginine deiminase-IV knock out (PAD4-/-) mice; PAD4 being an enzyme essential for citrullination of histones, a crucial step in NET release. Moreover, PMNs from tumor bearing mice are more primed for NETosis than PMNs from non-tumor bearing mice or NEi-treated or PAD4-/- tumor bearing mice. Finally, elevated in vivo hepatic adhesion and spontaneous liver and lung metastases are observed in tumor bearing mice compared to DNase1- or NEi-treated or PAD4-/- tumor bearing mice. Therefore, inhibiting NETs represents a promising strategy to impede metastatic dissemination in several types of cancer patients. Citation Format: Roni F. Rayes, Jack G. Mouhanna, Ioana Nicolau, Phil Vourtzoumis, Carson Wong, Jules Eustache, France Bourdeau, Betty Giannias, Aya Siblini, Emma Lee, Veena Sangwan, Simon Rousseau, Daniela Quail, Logan Walsh, Nicholas Bertos, Jonathan Cools-Lartigue, Lorenzo E. Ferri, Jonathan D. Spicer. Primary tumors induce neutrophil extracellular traps with targetable metastasis promoting effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1508.

Abstract 1094: Lipopolysaccharide-induced chronic inflammation promotes lung tumorigenesis in the context of an immunosuppressive microenvironment

Abstract Clinical and epidemiological evidence suggest that chronic infection and inflammation increase risk of lung cancer. Pseudomonas aeruginosa infection is frequently found in patients with chronic obstructive pulmonary disease (COPD) and is associated with increased lung inflammation and acute exacerbations. However, the mechanism of chronic bacterial infection-induced lung inflammation in promoting lung tumorigenesis remains unclear. To elucidate this mechanism, we established a murine lung cancer model by treating mice with or without recurrent lipopolysaccharides (LPS) from Pseudomonas aeruginosa in combination with nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Interestingly, combined LPS and NNK exposure significantly increased tumor number, tumor incidence, and tumor area compared to NNK treatment alone. In addition, the inflammatory cell counts in the bronchoalveolar lavage (BAL) including macrophages, neutrophils, and lymphocytes were significantly increased in the LPS/NNK treatment group. The BAL fluid of chemokines/cytokines, as analyzed by luminex assays, revealed higher levels of IL-17, CXCL10, GM-CSF, G-CSF, MIP-1a, and KC in LPS/NNK than in NNK treatment group. Flow cytometry analysis of the mouse lung tissue revealed that combined LPS and NNK exposure significantly increased CD4+ T cells including Th1, Th17, and Tregs and myeloid-derived suppressor cells recruitment in the lung. Real-time polymerase chain reaction of mouse lung tissue showed T cell exhaustion related genes, including Pdcd1, Ctla-4, Tim-3, Lag-3, and Foxp3, were significantly upregulated in the LPS/NNK treatment than NNK treatment. Moreover, immunohistochemical staining of LPS/NNK-exposed lung tumors showed higher PD-L1 expression than NNK-exposed lung tumors. Our data suggest that chronic LPS exposure-promoted and NNK-induced lung tumorigenesis is associated with immunosuppressive tumor microenvironment. The changes include recruitment of Tregs and MDSCs, increased T cell exhaustion, and upregulated PD-1/PD-L1 pathway, which may be used as the therapeutic target for chronic inflammation-associated lung cancer treatment. Citation Format: Chia-Hsin Liu, Kong Chen, Yuanpu Di. Lipopolysaccharide-induced chronic inflammation promotes lung tumorigenesis in the context of an immunosuppressive microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1094.

Abstract LB-202: Immunotherapy with TMV vaccine inhibits metastatic tumor growth in an immune checkpoint resistant murine lung cancer model

Abstract Lung cancer is the second most common cancer among men and women and the leading cause of cancer deaths in the world. Despite the advancements in diagnostic technologies and the development of novel treatments in the recent years such as immune checkpoint inhibitor (ICI) therapy, the prognosis of lung cancer patients is still poor, and the survival rate is low. The patient-to-patient heterogeneity is the major problem in treating lung cancer, which results in differential responses to ICI therapy. In this study, we investigated the efficacy of a vaccine immunotherapy prepared by modification of tumor membrane vesicles (TMVs) prepared from the tumors to express immunostimulatory molecules in combination with ICI therapy in a partially ICI resistant murine lung cancer model. We demonstrate that prophylactic vaccination of TMV adjuvanted with immunostimulatory molecules prevents the CMT-167 tumor growth in mice and helps the mice develop immunological memory to the tumor cell challenge. In a therapeutic setting, the TMV vaccine inhibited tumor growth and induced primary tumor regression in 40% of mice and significantly reduced the metastatic tumor burden. However, the combination of TMV vaccination with anti-PD-1 mAb doesn’t have a synergistic or additive effect on the primary or metastatic tumor growth. Overall, these studies provide insight into using the TMV vaccine immunotherapy for treating lung cancer. Funding: Supported by the Lung Cancer seed grant from the Winship Cancer Institute of Emory University Citation Format: Lei Huang, Dr. Selvaraj's lab. Immunotherapy with TMV vaccine inhibits metastatic tumor growth in an immune checkpoint resistant murine lung cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-202.

SHOC2 phosphatase-dependent RAF dimerization mediates resistance to MEK inhibition in RAS-mutant cancers

Targeted inhibition of the ERK-MAPK pathway, upregulated in a majority of human cancers, has been hindered in the clinic by drug resistance and toxicity. The MRAS-SHOC2-PP1 (SHOC2 phosphatase) complex plays a key role in RAF-ERK pathway activation by dephosphorylating a critical inhibitory site on RAF kinases. Here we show that genetic inhibition of SHOC2 suppresses tumorigenic growth in a subset of KRAS-mutant NSCLC cell lines and prominently inhibits tumour development in autochthonous murine KRAS-driven lung cancer models. On the other hand, systemic SHOC2 ablation in adult mice is relatively well tolerated. Furthermore, we show that SHOC2 deletion selectively sensitizes KRAS- and EGFR-mutant NSCLC cells to MEK inhibitors. Mechanistically, SHOC2 deletion prevents MEKi-induced RAF dimerization, leading to more potent and durable ERK pathway suppression that promotes BIM-dependent apoptosis. These results present a rationale for the generation of SHOC2 phosphatase targeted therapies, both as a monotherapy and to widen the therapeutic index of MEK inhibitors.

Plasmodium infection inhibits the expansion and activation of MDSCs and Tregs in the tumor microenvironment in a murine Lewis lung cancer model

BackgroundA major challenge in the development of effective cancer immunotherapy is the ability of tumors and their microenvironment to suppress immune cells through immunosuppressive cells such as myeloid -derived suppressor cells and regulatory T cells. We previously demonstrated that Plasmodium infection promotes innate and adaptive immunity against cancer in a murine Lewis lung cancer model but its effects on immunosuppressive cells in the tumor microenvironment are unknown.MethodsWhole Tumors and tumor-derived sorted cells from tumor-bearing mice treated with or without plasmodium infected red blood cells were harvested 17 days post tumor implantation and analyzed using QPCR, western blotting, flow cytometry, and functional assays. Differences between groups were analyzed for statistical significance using Student’s t-test.ResultsHere we found that Plasmodium infection significantly reduced the proportions of MDSCs and Tregs in the lung tumor tissues of the treated mice by downregulating their recruiting molecules and blocking cellular activation pathways. Importantly, CD8+ T cells isolated from the tumors of Plasmodium-treated mice exhibited significantly higher levels of granzyme B and perforin and remarkably lower levels of PD-1.ConclusionWe reveal for the first time, the effects of Plasmodium infection on the expansion and activation of MDSCs and Tregs with a consequent elevation of CD8+T cell-mediated cytotoxicity within the tumor microenvironment and hold great promise for the development of effective immunotherapeutic strategies.

Physiological levels of chromogranin A prevent doxorubicin-induced cardiotoxicity without impairing its anticancer activity.

The clinical use of doxorubicin (Doxo), a widely used anticancer chemotherapeutic drug, is limited by dose-dependent cardiotoxicity. We have investigated whether chromogranin A (CgA), a cardioregulatory protein released in the blood by the neuroendocrine system and by the heart itself, may contribute to regulation of the cardiotoxic and antitumor activities of Doxo. The effects of a physiologic dose of full-length recombinant CgA on Doxo-induced cardiotoxicity and antitumor activity were investigated in rats using in vivo and ex vivo models and in murine models of melanoma, fibrosarcoma, lymphoma, and lung cancer, respectively. The effect of Doxo on circulating levels of CgA was also investigated. In vivo and ex vivo mechanistic studies showed that CgA can prevent Doxo-induced heart inflammation, oxidative stress, apoptosis, fibrosis, and ischemic injury. On the other hand, CgA did not impair the anticancer activity of Doxo in all the murine models investigated. Furthermore, we observed that Doxo can reduce the intracardiac expression and release of CgA in the blood (i.e., an important cardioprotective agent). These findings suggest that administration of low-dose CgA to patients with low levels of endogenous CgA might represent a novel approach to prevent Doxo-induced adverse events without impairing antitumor effects.-Rocca, C., Scavello, F., Colombo, B., Gasparri, A. M., Dallatomasina, A., Granieri, M. C., Amelio, D., Pasqua, T., Cerra, M. C., Tota, B., Corti, A., Angelone, T. Physiological levels of chromogranin A prevent doxorubicin-induced cardiotoxicity without impairing its anticancer activity.

Deletion of Atm in Tumor but not Endothelial Cells Improves Radiation Response in a Primary Mouse Model of Lung Adenocarcinoma.

Stereotactic body radiotherapy is utilized to treat lung cancer. The mechanism of tumor response to high-dose radiotherapy (HDRT) is controversial, with competing hypotheses of increased direct tumor cell killing versus indirect effects on stroma including endothelial cells. Here we used dual recombinase technology in a primary murine lung cancer model to test whether tumor cells or endothelial cells are critical HDRT targets. Lenti-Cre deleted one or two copies of ataxia-telangiectasia mutated gene (Atm; KPAFL/+ or KPAFL/FL), whereas adeno-FlpO-infected mice expressed Cre in endothelial cells to delete one or both copies of Atm (KPVAFL/+ or KPVAFL/FL) to modify tumor cell or endothelial cell radiosensitivity, respectively. Deletion of Atm in either tumor cells or endothelial cells had no impact on tumor growth in the absence of radiation. Despite increased endothelial cell death in KPVAFL/FL mice following irradiation, tumor growth delay was not significantly increased. In contrast, a prolonged tumor growth delay was apparent in KPAFL/FL mice. Primary tumor cell lines lacking Atm expression also demonstrated enhanced radiosensitivity as determined via a clonogenic survival assay. These findings indicate that tumor cells, rather than endothelial cells, are critical targets of HDRT in primary murine lung cancer. SIGNIFICANCE: These findings establish radiosensitizing tumor cells rather than endothelial cells as the primary mechanism of tumor response to high-dose radiotherapy, supporting efforts to maximize local control by radiosensitizing tumors cells.See related commentary by Hallahan, p. 704.

MCC1019, a selective inhibitor of the Polo-box domain of Polo-like kinase 1 as novel, potent anticancer candidate

Polo-like kinase (PLK1) has been identified as a potential target for cancer treatment. Although a number of small molecules have been investigated as PLK1 inhibitors, many of which showed limited selectivity. PLK1 harbors a regulatory domain, the Polo box domain (PBD), which has a key regulatory function for kinase activity and substrate recognition. We report on 3-bromomethyl-benzofuran-2-carboxylic acid ethyl ester (designated: MCC1019) as selective PLK1 inhibitor targeting PLK1 PBD. Cytotoxicity and fluorescence polarization-based screening were applied to a library of 1162 drug-like compounds to identify potential inhibitors of PLK1 PBD. The activity of compound MC1019 against the PLK1 PBD was confirmed using fluorescence polarization and microscale thermophoresis. This compound exerted specificity towards PLK1 over PLK2 and PLK3. MCC1019 showed cytotoxic activity in a panel of different cancer cell lines. Mechanistic investigations in A549 lung adenocarcinoma cells revealed that MCC1019 induced cell growth inhibition through inactivation of AKT signaling pathway, it also induced prolonged mitotic arrest—a phenomenon known as mitotic catastrophe, which is followed by immediate cell death via apoptosis and necroptosis. MCC1019 significantly inhibited tumor growth in vivo in a murine lung cancer model without affecting body weight or vital organ size, and reduced the growth of metastatic lesions in the lung. We propose MCC1019 as promising anti-cancer drug candidate.

Electrochemotherapy with cisplatin increases survival and induces immunogenic responses in murine models of lung cancer and colorectal cancer

Electrochemotherapy is an emerging treatment modality for cancer patients which can effectively reduce tumour burden and induce immunogenic cell death. Electrochemotherapy is most commonly used with bleomycin as the drug of choice, here we examine the efficacy of electrochemotherapy with cisplatin. Electrochemotherapy with cisplatin was found to effectively reduce tumour growth in a range of murine models and induce significant intratumoural recruitment of myeloid and humoral immune cells. Following the observations of immune system mobilisation, we have shown an ability of electrochemotherapy to reduce metastatic potential as determined by tumour burden in the lung, and to exert an abscopal effect by reducing growth at distal untreated secondary tumours.

Genetic Analysis Reveals AMPK Is Required to Support Tumor Growth in Murine Kras-Dependent Lung Cancer Models

AMPK, a conserved sensor of low cellular energy, can either repress or promote tumor growth depending on the context. However, no studies have examined AMPK function in autochthonous genetic mouse models of epithelial cancer. Here, we examine the role of AMPK in murine KrasG12D-mediated non-small-cell lung cancer (NSCLC), a cancer type in humans that harbors frequent inactivating mutations in the LKB1 tumor suppressor-the predominant upstream activating kinase of AMPK and 12 related kinases. Unlike LKB1 deletion, AMPK deletion in KrasG12D lung tumors did not accelerate lung tumor growth. Moreover, deletion of AMPK in KrasG12D p53f/f tumors reduced lung tumor burden. We identified a critical role for AMPK in regulating lysosomal gene expression through the Tfe3 transcription factor, which was required to support NSCLC growth. Thus, AMPK supports the growth of KrasG12D-dependent lung cancer through the induction of lysosomes, highlighting an unrecognized liability of NSCLC.

Inhaled IL-10 Suppresses Lung Tumorigenesis via Abrogation of Inflammatory Macrophage-Th17 Cell Axis.

Intratracheal administration of a novel IL-10 formulation suppressed IL-17-driven, CD4+ T cell-dependent tumorigenesis in the LSL-K-rasG12D murine lung cancer model. Analysis of lung lymphocyte populations demonstrated that antitumor activity of IL-10 was associated with a 5-fold decline in Th17 cell prevalence and a concurrent suppression of inflammatory M1-like macrophage activity. Further phenotypic characterization revealed that macrophages and dendritic cells, but not Th17 cells, expressed IL-10RA on the cell surface with the CD11b+F4/80+CX3CR1+ interstitial macrophages representing the dominant IL-10RA+ subset. Consistent with these observations, in vitro stimulation of sorted CD4+ T cells with IL-10 did not affect their ability to produce IL-17, whereas similar treatment of purified interstitial macrophages resulted in a dramatic M1 to M2 phenotypic switch. Importantly, preconditioning of macrophages (but not of CD4+ T cells) with IL-10 led to potent suppression of CD4+ T cell IL-17 production in an in vitro coculture assay, suggesting that IL-10 suppressed Th17 cell activity primarily via its upstream effects on macrophages. In support of this notion, in vivo macrophage depletion resulted in a 5-fold decline in Th17 cell numbers and a concurrent 6-fold reduction in tumor burden. Collectively, these data demonstrate that in the LSL-K-rasG12D murine lung cancer model, inflammatory macrophage-Th17 cell axis is critical to tumorigenesis and that IL-10 blocks this process primarily via a direct effect on the former. Inhaled IL-10 formulations may be of use in prophylaxis against lung cancer in high-risk patients.

Anti-GITR with Radiation Therapy Enhances Anti-Tumor Abscopal Effects in Anti-PD1 Resistant Murine Model

Although immunotherapy has demonstrated potential to cure metastatic NSCLC, many patients do not respond to such intervention. As a monotherapy, anti-PD1 response rates remain low, around 20%, in advanced 2L NSCLC. Previously, we demonstrated the ability of radiation to partially abrogate anti-PD1 resistance via upregulation of MHC I expression in an anti-PD1 resistant murine lung cancer model, suggesting that radiation therapy (RT) has potential to increase response rates. Herein, we sought to further improve these anti-tumor responses (local and abscopal) by targeting Tregs, which are upregulated by RT, through the addition of anti-GITR therapy. 129 Sv/Ev mice were inoculated with 344SQ resistant (344SQ_R) NSCLC cells into the right leg and, four days later, to the left leg. Tumor-bearing mice were treated with intravenous anti-PD1 and anti-GITR and RT to the primary tumor. Mice were followed for survival and tumor growth was recorded. Spleen, blood, draining lymph node and tumor infiltrating lymphocytes were isolated and analyzed by flow cytometry at various post-treatment time points. CD4 and CD8 depleting antibodies were administered to explore the cellular mechanism of treatment response. To evaluate the ability of triple therapy to generate a memory response, triple therapy mice were rechallenged with S344SQ_R cells 90 days after tumor clearance. Mice treated with triple therapy (anti-GITR, anti-PD1 and RT) had significantly improved survival compared to any dual therapy combination. Of note, anti-GITR therapy depleted Tregs at the tumor site but not in the spleen. Interestingly, half of the triple therapy mice became tumor free. Upon rechallenge with 344SQ_R cells, 100% (4/4) of these mice remained tumor free, showing a durable response. Mechanistically, we confirmed that this was related to generation of CD4 and CD8 T cell memory (CD44hi/CD62Llow). To confirm the observations, we did depletion studies and further demonstrated that the mechanism is largely dependent on CD4, and partially CD8 T cells. The addition of anti-GITR to anti-PD1 and local RT may circumvent a NSCLC anti-PD1 resistant phenotype through enhanced anti-tumor immunity via depletion of Tregs and generation of anti-tumor memory, thereby improving survival.