Murine Lung Cancer Model Research Articles

Evaluating the Combined Anticancer Response of Checkpoint Inhibitor Immunotherapy and FAP-Targeted Molecular Radiotherapy in Murine Models of Melanoma and Lung Cancer.

Simple SummaryAlthough newer cancer medicines that help the immune system recognize and attack cancer cells have improved responses to therapy, most patients ultimately have cancer recurrence. Additional therapies and therapy combinations are needed so that responses can last longer or indefinitely. Molecular targeted radiotherapy is another kind of therapy that targets radioactive particles directly to cancer in the hopes of killing cancer cells to stop tumor growth with limited side effects. Prior studies have shown that targeted radiotherapies activate the immune system and can work together with immunotherapy to improve response. Here, we tested a promising new therapy targeting fibroblast activation protein (FAP) with a therapeutic radionuclide 177Lu alone and with immunotherapy in mouse models of melanoma and lung cancer. The FAP-targeted radiotherapy reduced tumor growth in both models and melanoma, resulting in tumor regression. We saw increased tumor cell death in dual-treated tumors. We also found that myeloid cells were affected by the combined therapy to a greater degree than the additive effect of either therapy. These results demonstrate that this is a promising new therapy regimen and requires further preclinical and clinical study to better understand the molecular mechanisms underpinning response. Immunotherapy has dramatically improved outcomes for some cancer patients; however, novel treatments are needed for more patients to achieve a long-lasting response. FAP-targeted molecular radiotherapy has shown efficacy in both preclinical and clinical models and has immunomodulatory effects. Here, we studied if combined immunotherapy and radiotherapy could increase antitumor efficacy in murine models of lung cancer and melanoma and interrogated the mechanisms by which these treatments attenuate tumor growth. Using LLC1 and B16F10 murine models of lung cancer and melanoma, respectively, we tested the efficacy of 177Lu-FAPI-04 alone and in combination with immunotherapy. Alone, 177Lu-FAPI-04 significantly reduced tumor growth in both models. In animals with melanoma, combined therapy resulted in tumor regression while lung tumor growth was attenuated, but tumors did not regress. Combined therapy significantly increased caspase-3 and decreased Ki67 compared with immunotherapy alone. Flow cytometry demonstrated that tumor-associated macrophages responded in a tumor-dependent manner which was distinct in animals treated with both therapies compared with either therapy alone. These data demonstrate that 177Lu-FAPI-04 is an effective anticancer therapy for melanoma and lung cancer which mediates effects at least partially through induction of apoptosis and modulation of the immune response. Translational studies with immunotherapy and 177Lu-FAPI-04 are needed to demonstrate the clinical efficacy of this combined regimen.

Anti-PD-1 antibody-activated Th17 cells subvert re-invigoration of antitumor cytotoxic T-lymphocytes via myeloid cell-derived COX-2/PGE2.

Anti-PD-1 antibody-mediated activation of type 17T-cells undermines checkpoint inhibitor therapy in the LSL-KrasG12D murine lung cancer model. Herein, we establish that the Th17 subset is the primary driver of resistance to therapy demonstrate that the ontogeny of dysplasia-associated Th17 cells is driven by microbiota-conditioned macrophages; and identify the IL-17-COX-2-PGE2 axis as the mediator of CD8+ cytotoxic T-lymphocyte de-sensitization to checkpoint inhibitor therapy. Specifically, anti-PD-1 treatment of LSL-KrasG12D mice, in which CD4+ T-cells were deficient for RORc, resulted in a 60% increase in CTL cytotoxicity and a 2.5-fold reduction in tumor burden confirming the critical role of Th17 cells in resistance to therapy. Lung-specific depletion of microbiota reduced Th17 cell prevalence and tumor burden by 5- and 2.5-fold, respectively; establishing a link between microbiota and Th17 cell-driven tumorigenesis. Importantly, lung macrophages from microbiota sufficient, but not from microbiota-deficient, mice polarized naïve CD4+ T-cells to a Th17 phenotype, highlighting their role in bridging microbiota and Th17 immunity. Further, treatment with anti-PD-1 enhanced COX-2 and PGE2 levels, whereas neutralization of IL-17 diminished this effect. In contrast, inhibition of COX-2 rescued CTL activity and restored tumor suppression in anti-PD-1-treated mice, revealing the molecular basis of IL-17-mediated resistance to checkpoint blockade. Clinical implications of these findings are discussed.

P2.12-05 Cancer and Atopy: Parallel Drivers? IL-4 Blockade Synergizes with PD-L1 Blockade to Reverse Type-2Mediated Immunosuppression

Type-2 cytokines, such as IL-4, alter macrophage immunogenicity in vitro leading to the M1/M2 paradigm, however, the role of IL-4 in-vivo in cancer remains unclear. Using high dimensional immune profiling of human NSCLC lesions we previously described several functionally distinct populations of macrophages, and a tumor-enriched dendritic cell program of concomitant immunosuppression and activation which we termed the mregDC, and we identified analogous cells in a murine model of lung cancer (Maier et al, Nature 2020, Casanova-Acebes et al, Nature, 2021, Leader et al, Cancer Cell 2021).

EP16.01-028 Immunomodulatory Effects of Cryoablation Combined With Immune Checkpoint Inhibitors in a Murine Lung Cancer Model

As part of our research on cryoablation, a combination with immune checkpoint inhibitors (ICIs), such as inhibitors of the programmed cell death 1 (PD-1) receptor and its ligand, programmed cell death 1-ligand 1 (PD-L1), to disrupt the immunosuppressive tumor microenvironment has proven a beneficial effect on enhancing immune response. We specifically addressed myeloid-derived suppressor cells (MDSCs) and T-cell immunity, which are thought to play an important role in the formation of the tumor microenvironment.

EP16.01-024 Differential Efficacy of Combined GITR Co-stimulation with PD-1 Blockade in Preclinical Non-small Cell Lung Cancer Models

Lung cancer remains as the most lethal cancer disease in Hong Kong. Although blockade of inhibitory immune checkpoints achieved remarkable clinical outcomes, continuous efforts are being made to explore novel therapeutic strategies. We have previously found that anti-programmed cell death protein 1 (anti-PD-1) induced systemic and intra-tumoral upregulation of an immunoregulatory molecule, glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) in a subcutaneous murine lung cancer model.

Exceptional tumour responses to fasting-mimicking diet combined with standard anticancer therapies: A sub-analysis of the NCT03340935 trial

BackgroundCyclic fasting or calorie-restricted, low-carbohydrate, low-protein diets, collectively referred to as fasting-mimicking diets (FMDs), demonstrated additive or synergistic antitumour effects when combined with chemotherapy, targeted therapies, or immunotherapy in several preclinical in vivo models, including murine models of breast cancer, lung cancer, and colorectal cancer. However, no data on the antitumour efficacy of cyclic FMD in patients with cancer have been published so far. Here, we aim at reporting on patients with advanced cancer achieving complete and long-lasting tumour remissions with cyclic FMD in combination with standard anticancer therapies in the context of the phase Ib NCT03340935 trial. Patients and methodsThe NCT03340935 trial enrolled 101 patients with different tumour types, and it showed that a severely calorie-restricted FMD regimen is safe and feasible in patients with cancer receiving concomitant standard-of-care antineoplastic therapies. In addition, cyclic FMD resulted in positive metabolic and immunologic modifications, thus recapitulating the biological effects that in preclinical models were found to mediate the antitumour effects of fasting/FMD. ResultsOf the 101 patients enrolled in the NCT03340935 trial, we identified five patients with advanced, poor prognosis solid neoplasms (n = 1: extensive stage small cell lung cancer; n = 1: metastatic pancreatic adenocarcinoma; n = 1: metastatic colorectal cancer; n = 2: metastatic triple-negative breast cancer), who achieved complete and long-lasting tumour responses when treated with a combination of cyclic FMD and standard systemic treatments in the context of the NCT03340935 trial. ConclusionThese excellent responses prompt the initiation of clinical trials to investigate cyclic FMD in combination with standard antitumour therapies in specific clinical contexts.

Abstract 2124: ATRA mediated inhibition of myeloid-derived suppressor cells overcomes resistance to immune checkpoint inhibition in murine models of LKB1-deficient non-small cell lung cancer

Abstract Lung cancer remains the deadliest form of cancer, claiming the lives of 1.8 million worldwide in 2020. While treatment options have improved with the advent of immune checkpoint inhibitors (ICI), many patients do not respond to immunotherapy or develop resistance following initial response. A significant subset of patients harbor somatic mutations in both Kirsten rat sarcoma virus (KRAS) and Liver kinase 1 [LKB1, also known as serine/threonine kinase 11 (STK11)] genes. LKB1 loss has been identified as a driver of primary resistance to ICI therapy. Tumors from patients that possess these mutations are characterized by an increase in the population of myeloid derived suppressor cells (MDSCs). MDSCs are thought to play a critical role in promoting tumor progression by blocking the tumor killing function of cytotoxic T cells in the tumor microenvironment (TME). Our studies revealed all-trans retinoic acid (ATRA), a metabolite derived from vitamin A, sensitized murine models of non-small cell lung cancer (NSCLC) with LKB1 and KRAS co-mutations to PD-1 blockade. Combination therapy with anti-PD-1 and ATRA improved local and systemic T cell proliferation and generated systemic tumor-specific immunity. We observed that ATRA augmented anti-PD-1 efficacy in additional murine NSCLC models. We demonstrated that ATRA suppresses tumor growth through the inhibition of immunosuppressive MDSCs in the TME, and preliminary data indicates that ATRA increases intracellular reactive oxygen species (ROS) and IFN signaling in MDSC. Our findings implicate MDSCs as a potential mediator of immunosuppression in Lkb1-deficient NSCLC and provide a rationale for utilizing ATRA in combination with anti-PD-1 therapy in patients with Lkb1-deficient NSCLC refractory to ICIs. * B. Liu and S. M. Dubinett contributed equally to this work Citation Format: William P. Crosson, Rui Li, Ramin Salehi-Rad, Raymond J. Lim, Jensen W. Abascal, Bitta P. Kahangi, Edgar Perez Reyes, Camelia Dumitras, Zhe Jing, Kostyantyn Krysan, Linh M. Tran, Bin Liu, Steven M. Dubinett. ATRA mediated inhibition of myeloid-derived suppressor cells overcomes resistance to immune checkpoint inhibition in murine models of LKB1-deficient non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2124.

Abstract 4212: In situvaccination with Flt3l gene-modified CD103+type 1 conventional dendritic cells (cDC1) in murine models of non-small cell lung cancer (NSCLC)

Abstract A major hurdle in treatment of Non-Small Cell Lung Cancer (NSCLC) with anti-PD-1 immune checkpoint blockade (ICB) therapy is a lack of response (primary resistance) and relapse after an initial response (acquired resistance). Recent studies reveal that responses to PD-1/PD-L1 blockade are associated with high tumor mutational burden (TMB), increased CD8+ T cell infiltration and high baseline PD-L1 expression within the tumor microenvironment (TME), while impaired tumor antigen presentation and the immunosuppressive TME have been associated with resistance to ICB. One approach to overcome anti-PD-1 resistance is to intratumorally vaccinate NSCLC tumors with gene modified conventional dendritic cells (cDC), specifically the type I conventional DC (cDC1) lineage. Recent studies have established that generation of an anti-tumor immune response driven by CD8+ T cells requires the cross presentation of tumor associated antigens and that cDC1s are the primary cross presenting APC subtype in vivo, which can license CD8+ T cells to initiate an adaptive anti-tumor immune response. In addition, previous studies have shown that intratumoral administration of the FMS-like tyrosine kinase 3 ligand (FLT3L) protein can expand endogenous CD103+ cDC1 cells in the TME and augment anti-tumor immune responses to ICB therapy. Here, we engineered murine CD103+ cDC1 cells to constitutively secrete soluble FLT3L (FLT3L_cDC1) and performed in situ vaccination studies on anti-PD1 resistant murine models of NSCLC with LKB1-deficiency and elevated TMB that better represents human disease. The FLT3L_cDC1 cells showed enhanced anti-tumor efficacy compared to non-modified cDC1 cells and synergized with anti-PD-1 ICB to inhibit tumor growth. Our data suggests intratumoral injection of FLT3L_cDC1 cells may represent a promising strategy to potentiate the efficacy of ICB and improve outcomes for patients with primary resistance PD-1/PD-L1 monotherapy. *B. Liu and S.M Dubinett contributed equally to this work Citation Format: Jensen W. Abascal, Raymond J. Lim, Ramin Salehi-Rad, Zhe Jing, William P. Crosson, Bitta P. Kahangi, Edgar Perez Reyes, Diana Reyimjan, Jessie Zhu, Linh M. Tran, Manash Paul, Kostyantyn Krysan, Bin Liu, Steven M. Dubinett. In situvaccination with Flt3l gene-modified CD103+type 1 conventional dendritic cells (cDC1) in murine models of non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4212.

Abstract 4208: In situ vaccination of non-small cell lung cancer with CXCL9/10-engineered dendritic cells combined with PD-1 blockade

Abstract Lung cancer is the most common cause of cancer death worldwide with approximately 85% of patients having non-small cell lung cancer (NSCLC). Checkpoint blockade immunotherapy has evolved the current treatment landscape with robust and durable responses in approximately 20% of patients with progressive, locally advanced or metastatic NSCLC, as well as in treatment-naïve advanced disease. Inefficient tumor antigen presentation, diminished T cell infiltration into tumor and LKB1-inactivating mutations contribute to the mechanisms of resistance to programmed death-ligand 1 (PD-L1) or programmed cell death protein 1 (PD-1) blockade in NSCLC. LKB1 deficiency also facilitates an immunosuppressive environment that promotes tumor growth. One approach to overcome this immunosuppressive tumor microenvironment (TME) is to utilize in situ vaccination with gene-modified functional antigen presenting cells (APCs) to enhance tumor antigen presentation and promote tumor-specific T cell activation. It has been shown that the chemokines CXCL9 and CXCL10 (CXCL9/10) are important signaling molecules secreted by CD103+ dendritic cells (DCs) to orchestrate the anti-tumor immunity. Thus, we hypothesize that intratumoral (IT) injection of CXCL9/10-secreting DCs (CXCL9/10-DCs) can potentiate the response to anti-PD-1 monotherapy in LKB1-deficient NSCLC by promoting antigen presentation and inducing T cell infiltration. Our preliminary data shows that CXCL9/10-DC injection modulates systemic immunity and generates anti-tumor responses that are augmented by the combination treatment with anti-PD1. In an in vivo murine model of Lkb1-null lung cancer bearing high tumor mutational burden (TMB), CXCL9/10-DC facilitates recruitment of effector T cells into the TME and tumor-specific T cell activation. These studies will enhance our understanding of anti-tumor immunity mediated by in situ DC vaccination and facilitate the improvement of novel vaccine therapies. B. Liu and S. M. Dubinett contributed equally to this work. Citation Format: Raymond J. Lim, Ramin Salehi-rad, Linh M. Tran, Camelia Dumitras, Jensen W. Abascal, William P. Crosson, Tejas S. Patel, Samantha Man, Rui Li, Kostyantyn Krysan, Bin Liu, Steven M. Dubinett. In situ vaccination of non-small cell lung cancer with CXCL9/10-engineered dendritic cells combined with PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4208.

Abstract 3565: Viroimmunotherapy for solid tumors results in local and abscopal anti-cancer effects and the remodeling of tumor microenvironment

Abstract Oncolytic viruses are a promising experimental treatment for solid tumors. Recently, several phase 1 clinical trials have reported encouraging therapeutic effects of oncolytic viruses in adult and pediatric patients with malignant gliomas. To further improve the therapeutic outcome of viroimmunotherapy, we have developed Delta-24-RGDOX (DNX-2440), a replication competent adenovirus encompassing the T-cell activator OX40L in the genetic backbone of Delta-24-RGD. We have previously reported the effect of Delta-24-RGDOX in murine brain tumors supporting the translation of this new agent to treat patients with recurrent malignant gliomas (NCT03714334). In the work presented here, we have tested the therapeutic effect of Delta-24-RGDOX in murine syngeneic models of breast (4T1), gastric (M12) and lung cancer (LLC and CMT 167). We found that Delta-24-RGDOX infected all cancer cell lines efficiently. In addition, infection of cells was followed by the expression of the ectopic ligand in vitro and in vivo. Because the elicitation of an anti-tumor immunity is part of the mechanisms underlying the therapeutic effect of oncolytic viruses, we examined whether infection of tumors led to the reshaping of the tumor microenvironment. We observed that Delta-24-RGDOX infection was followed by increased frequencies of tumor infiltrating lymphocytes, particularly CD8+ T cells and NK cells. In addition, the CD8+/CD4+ ratio was increased in Delta-24-RGDOX-treated tumor versus PBS-treated tumors. Interestingly, abscopal modifications were observed in breast cancer brain metastases with increased frequency of CD8+ T cells at the distal, untreated site. Delta-24-RGDOX treatment induced an anti-cancer effect in orthotopically implanted breast cancer and subcutaneously implanted lung and gastric tumors, as well as in metastatic niches. In summary, our data showed that treatment of solid tumors with Delta-24-RGDOX induces robust remodeling of the tumor microenvironment and produces anti-tumor effects leading to decrease in tumor volume, along with a delay in the development and in the reduction of the number of metastases. These data suggest that Delta-24-RGDOX should be tested in the clinical setting in patients with metastatic breast, gastric and lung cancers. Citation Format: Arie C. Van Wieren, Sagar Sahoni, Teresa Nguyen, Ashley Ossimetha, Yisel Rivera-Molina, Hong Jiang, Dong Ho Shin, Debora Kim, Xuejun Fan, Yanhua Yi, Natalie M. Melendez-Vazquez, Filipa Godoy-Vitorino, Joy Gumin, Frederick F. Lang, Marta M. Alonso, Juan Fueyo, Candelaria Gomez-Manzano. Viroimmunotherapy for solid tumors results in local and abscopal anti-cancer effects and the remodeling of tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3565.

Abstract 361: MicroRNA expression in murine spontaneous non-small cell lung cancer and human cell lines post-treatment of PRIMA-1

Abstract A variety of cancers have been correlated with having a mutation on the TP53 gene, including lung cancer. Lung cancer has been one of the deadliest cancers in the United States. TP53-dependent reactivation and induction of massive apoptosis (PRIMA-1) changes mutant TP53’s conformation, resulting in the restoration of DNA binding and activation of p53 target genes. PRIMA-1 methylated analog, PRIMA-1Met (APR-246, Eprenetapopt) have been used in clinical trials to target cancers containing mutant TP53. It has been reported that PRIMA-1 induced apoptosis through the regulation of microRNAs. Our research group has previously developed a transgenic mice model with a mutant form of TP53 being expressed lung specifically, to closely model human lung cancer development. The aim of the current study is to investigate the effects of PRIMA-1 on miRNA expressions in a murine spontaneous lung cancer mice model, human lung cancer cell lines H211 and H1155. The transgenic FVB/N mice lung tumor model was developed using the lung specific human surfactant protein C (SPC) promoter to control the expression of mutant TP53(273H) following the standard injection method. Tumor bearing mice were identified with a micro-CT and were treated with the PRIMA-1 at a dose of 100mg/kg. Additionally, we treated two human cell lines H211 (containing mutant TP53 R248Q) and H1155 (containing mutant TP53 R273H) with the PRIMA-1. The miRNA expression profile was evaluated with the NanoString nCounter array analysis and qt-PCR array analysis. We found that three miRNAs were up-regulated (>1.5-fold change) in the human lung cancer cells H211, H1155, and murine spontaneous non-small cell lung cancers model post treatment of PRIMA-1. The result of the study showed that miR-200c, miR-181c, and miR-183 was up-regulated in the two human cell lines and the transgenic mice lung tumors after treatment of PRIMA-1. While there was no significant downregulation in miRNA expression in these human lung cancer cells and the murine spontaneous lung cancer model. However, the overall miRNA expression is lower in murine spontaneous lung tumors when compared to the overall miRNA expression of the cell lines post PRIMA-1 treatment. This difference is due to model specific expression. Further investigation is needed to better understand the effects of PRIMA-1 in regulation of micro-RNA expression in the treatment of lung cancers containing mutant p53. Citation Format: Melanie Perez, Shaylyn Grier, Li Gao, Wenrui Duan. MicroRNA expression in murine spontaneous non-small cell lung cancer and human cell lines post-treatment of PRIMA-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 361.

Abstract 1980: Cigarette smoke induced oxidative phosphorylation and mitochondrial biogenesis enhance response to immune therapy in NSCLC patients

Abstract Background: The treatment regimen of non-small cell lung cancer (NSCLC) has drastically changed owing to the superior anti-cancer effects generated by the immune-checkpoint blockade (ICB). However, only a subset of patients experience benefit after receiving ICBs. There are several treatments including combination of PD-1 blockade and low dose chemotherapy, but only few robust synergetic effects were reported. Therefore, it is of paramount importance to increase the response rate by elucidating the underlying molecular mechanisms and identifying novel therapeutic targets to enhance the efficacy of IBCs in non-responders. Methods: We analyzed the progression-free survival (PFS) and overall survival (OS) of 295 NSCLC patients who received anti-PD-1 therapy by segregating them with multiple clinical factors including sex, age, race, smoking history, BMI, tumor grade and subtype. Tumor samples from 48 patients were sequenced and their genomic profiles were extracted for analyses. We identified key signaling pathways and mutations that are enriched in those patients with distinct responses to ICB by gene set enrichment analysis (GSEA) and mutational analyses. The efficacy of combination treatment with mitochondrial activator, all-trans retinoic acid (ATRA) and PD-1 antibody is evaluated in LL/2 and CMT167 murine lung cancer model. Results: We found that smoking status was the only parameter that was correlated with the ICB responses compared to sex, age, race, BMI, tumor grade and subtype. Former and current smokers have a higher response rate to anti-PD-1 treatment than non-smokers. GSEA results revealed that oxidative phosphorylation (OXPHOS) and mitochondrial related pathways are significantly enriched in both responders and smokers, suggesting a potential role of cellular metabolism in regulating immune response to ICB. We also demonstrated that ATRA which increased mitochondrial function significantly by MitoSox and MitoTrack enhanced the efficacy of anti-PD-1 treatment in vivo. Our clinical and bioinformatics based analyses revealed a connection between smoking induced metabolic switch and the response to immunotherapy, which can be the basis for developing novel combination therapies that are beneficial to never smoked NSCLC patients. Conclusion: In this study, we discovered that smokers respond better to immune therapy than non-smokers due to elevated OXPHOS capacity and mitochondrial activity, which could be a potential target to boost the response to immune therapy in NSCLC patients who never smoked. Citation Format: Yuezhu Wang, Jimmy Ruiz, Yin Liu, Margaret Smith, Gregory Kucera, Umit Topaloglu, Michael Chan, Stacey O’Neill, Jing Su, Fei Xing. Cigarette smoke induced oxidative phosphorylation and mitochondrial biogenesis enhance response to immune therapy in NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1980.

OX40 enhances T cell immune response to PD-1 blockade therapy in non-small cell lung cancer

Immune-checkpoint blockade is widely studied for cancer therapy. Although the co-inhibitory receptor Programmed death-1(PD-1) blockade benefits some non-small cell lung cancer (NSCLC) patients, a large portion of NSCLC patients still fail to respond to this immunotherapy, and the underlying mechanism is unclear. Thus, a synergistic therapy to enhance the effect of PD-1 is urgently needed to improve the poor outcome of NSCLC patients. Here, we demonstrated that effector memory T cells were increased and T cell response became stronger in PD-1 immunotherapy responders (n = 20) but not in non-responders (n = 10). The expression of co-stimulatory receptor OX40 was upregulated on T cells following PD-1 immunotherapy and was positively associated with the percentage of PD-1+T cells and the responsiveness of T cells. Combination treatment of antagonistic anti-PD-1 and agonistic anti-OX40 antibodies (Abs) promoted the proliferation and cytokines production of T cells from PBMCs of non-responders ex vivo. Consistently, anti-PD-1 and anti-OX40 therapy synergistically augmented T cell response in an in vivo mouse lung cancer model. Our study confirmed the antitumor effects of anti-PD-1/OX40 combination in lung cancer patients as well as in the murine lung cancer model, and the results provide a rationale for clinical trials evaluating the therapeutic effect of this combination of antibodies for NSCLC immunotherapy.

Nanoparticle based depletion of Myeloid-derived Suppressor cells in Tuberculosis

Abstract The bacterial pathogen Mycobacterium tuberculosis (Mtb) successfully evades host innate immune responses to cause tuberculosis (TB) disease. Recent studies showed that myeloid-derived suppressor cells (MDSCs) are recruited to the lung after Mtb infection and harbor Mtb. While MDSC-mediated negative regulation of tumor micro-environments in cancer has been extensively studied, in-depth analyses of the phenotype, functions and transcriptional signatures of MDSCs in TB are lacking. In this study we sought to characterize MDSCs in the lungs of Mtb-infected mice and develop tools for depleting MDSCs as a strategy for host-directed therapy. We determined MDSCs in the aerosol mouse model of TB and in human blood using flow cytometry. MDSCs are recruited into the lung as early as 2 weeks post-infection and expresses immunomodulatory mediators associated with immune suppression such as iNOS, IDO and arginase. We recently reported the development of novel synthetic nanoparticle antibodies (SNAbs) that specifically target and deplete MDSCs via antibody-like mechanisms. We used SNAbs to deplete Mtb-induced MDSCs in vitro and in human PBMCs ex vivo. Interestingly, we found that SNAbs efficiently depleted MDSCs in both models. To test the efficacy of SNAb-mediated depletion of MDSCs in mouse lungs in vivo, we first used a murine lung cancer model and found that intra-tracheal delivery of SNAbs successfully depleted MDSCs in vivo. Ongoing studies include evaluating the ability of SNAbs to deplete lung MDSCs following Mtb infection of mice and to assess the impact on lung T cell functions, pathology and Mtb burden. These studies will establish a platform to investigate MDSCs as a target for host-directed therapies that improve TB treatment efficacy. Supported by grant from NIH (1R01AI155023-01A1)

Tumor-associated Tregs obstruct antitumor immunity by promoting T cell dysfunction and restricting clonal diversity in tumor-infiltrating CD8+ T cells

BackgroundAccumulation of regulatory T cells (Treg) has been described to often correlate with poor prognosis in many solid tumors. How Treg presence impinges on limited functionality and clonal composition of...

Dietary low-fiber promotes resistance to immune checkpoint inhibitor immunotherapy in the LSL-KrasG12D Lung Cancer Model

Abstract Dietary fiber interaction with the gut microbiota impact gut microbiota composition and is likely to play a large role in affecting the efficacy of immune checkpoint inhibitors (ICIs) therapy. Here we show that a diet with low-fiber promoted lung tumor development and reduced tumor-bearing animal long-term survival in both prophylactic early disease and established late disease of murine LSL-KrasG12D lung cancer model, which displays low neoantigen burden and is refractory to anti-PD-1 antibody. In this model, the administration of anti-PD-1 in the presence of IL-17 neutralization resulted in tumor suppression, indicating that limited intrinsic neoantigen alone was not the primary determinant of resistance to ICIs therapy. Interestingly, dietary low-fiber led to reduced anti-tumor effect of anti-PD-1 and anti-IL-17 partially in gut microbiota-dependent manner. Fecal microbiota transplantation from mice fed low-fiber diet also enhanced lung tumor burden. An altered immune response with significantly reduced gut lamina propria RORgt+IL-17A+ Th17 cells, lung Foxp3+Treg cells, enhanced lung Th17 cells, but no alteration of lung gdTCR+ cells were found in mice fed low-fiber diet. Furthermore, a low-fiber diet led to significant changes in metabolites including a high level of serine in the plasma of animals. Mice fed normal chow with drinking water containing serine increased lung tumorigenesis. Our study demonstrates a functional interconnection between dietary fiber and spontaneous lung tumor development. Supported by grant from NIH/NIGMS(P20-GM135004)

Resistance to Anti-PD-1 Therapy is mediated via the Microbiota-Th17-Prostoglandin E2 Axis in the LSLKrasG12D Lung Cancer Model

Abstract We previously demonstrated that anti-PD-1 antibody mediated activation of type 17 T-cells undermined checkpoint inhibitor therapy in the LSLKrasG12D murine lung cancer model. In this study we confirm that the Th17 subset is the primary driver of resistance to anti-PD-1; demonstrate that the ontogeny of dysplasia-associated Th17 cells is driven by microbiota-conditioned macrophages; and identify IL-17-COX-2/Prostoglandin E2 (PGE2) axis as the mediator of CD8+ cytotoxic T-lymphocyte (CTL) de-sensitization to checkpoint inhibitor therapy. Specifically, anti-PD-1 treatment of CD4+ T-cell RORc-deficient LSLKrasG12D mice resulted in a 50% increase in CTL cytotoxicity and a 2.2-fold reduction in tumor burden confirming the critical role of Th17 cells in resistance to therapy. Lung-specific depletion of microbiota reduced Th17 cell prevalence and tumor burden by ~2 and ~1.5 fold, respectively, establishing a link between microbiota and the Th17-driven tumorigenesis. Importantly, interstitial macrophages from microbiota sufficient, but not from microbiota-deficient, mice polarized naïve CD4+ T-cells towards a Th17 phenotype, highlighting their role in bridging microbiota and lung Th17 immunity. Neutralization of IL-17 diminished COX-2/PGE2 levels, whereas inhibition of COX-2 rescued CTL activity and restored tumor suppression in anti-PD-1 antibody-treated mice, revealing the molecular basis of IL-17-driven anti-PD-1 resistance. The clinical implications of these findings are discussed. Supported by grant from DOD grant (to N.E., OGMB190737) and in part through the NIH/NIGMS P20-GM135004 (1P20GM135005-01)

Glucose-Restricted Diet Regulates the Tumor Immune Microenvironment and Prevents Tumor Growth in Lung Adenocarcinoma.

BackgroundLung cancer is the second common cancer type in western countries and has a high mortality. During the development and progression of the tumor, the nutrients in its environment play a central role. The tumor cells depend crucially on glucose metabolism and uptake. Tumor cell metabolism is dominated by the Warburg effect, where tumor cells produce large amounts of lactate from pyruvate under aerobic conditions. We thus reasoned that, reducing carbohydrates in the diet might support anti-tumoral effects of current immunotherapy and additionally target tumor immune escape.ObjectivesThe link between reducing carbohydrates to improve current immunotherapy is not clear. We thus aimed at analyzing the effects of different glucose levels on the tumor development, progression and the anti-tumoral immune response.MethodsWe correlated the clinical parameters of our LUAD cohort with different metabolic markers. Additionally, we performed cell culture experiments with A549 tumor cell line under different glucose levels. Lastly, we investigated the effect of low and high carbohydrate diet in an experimental murine model of lung cancer on the tumor progression and different immune subsets.ResultsHere we found a positive correlation between the body mass index (BMI), blood glucose levels, reduced overall survival (OS) and the expression of Insulin-like growth factor-1 receptor (IGF1R) in the lung tumoral region of patients with lung adenocarcinoma (LUAD). Furthermore, increasing extracellular glucose induced IGF1R expression in A549 LUAD cells. Functional studies in a murine model of LUAD demonstrated that, glucose restricted diet resulted in decreased tumor load in vivo. This finding was associated with increased presence of lung infiltrating cytotoxic CD8+ T effector memory (TEM), tissue resident memory T (TRM) and natural killer cells as well as reduced IGFR mRNA expression, suggesting that glucose restriction regulates lung immunity in the tumor microenvironment.ConclusionsThese results indicate that, glucose restricted diet improves lung immune responses of the host and suppresses tumor growth in experimental lung adenocarcinoma. As glucose levels in LUAD patients were negatively correlated to postoperative survival rates, glucose-restricted diet emerges as therapeutic avenue for patients with LUAD.

ROR\u03b3t agonist enhances anti-PD-1 therapy by promoting monocyte-derived dendritic cells through CXCL10 in cancers

BackgroundThe overall response rate to checkpoint blockade remains unsatisfactory, partially due to the immune-suppressive tumor microenvironment. A retinoic acid-related orphan receptor γt (RORγt) agonist (LYC-55716) is currently used in clinical trials combined with anti-PD-1, but how the Th17 cell transcription factor RORγt enhances antitumor immunity of PD-1 in the tumor microenvironment remains elusive.MethodsThe expression of mRNA was analyzed using qPCR assays. Flow cytometry was used to sort and profile cells. Cell migration was analyzed using Transwell assays. Biacore was used to determine the binding affinity to the RORγt protein. The RORγt GAL4 cell-based reporter gene assay was used to measure activity in the RORγt driven luciferase reporter gene expression.ResultsWe designed a potent and selective small-molecule RORγt agonist (8-074) that shows robust antitumor efficacy in syngeneic tumor models and improves the efficacy of anti‑PD‑1 in a murine lung cancer model. RORγt agonist treatment increased intratumoral CD8+ T cells, which were correlated with CXCL10 and monocyte-derived dendritic cells (MoDCs). In addition, the RORγt agonist promoted Type 17 T cell migration by upregulating CCL20 and CCR6 expression, and Type 17 T cell tumor infiltration. CCL20 induces MoDCs migration, and CXCL10 derived from MoDCs promotes CD8+ T cell migration.ConclusionOur results revealed that the RORγt agonist improved the efficacy of anti-PD-1. The RORγt agonist increased the migration of MoDCs, which increased the local levels of CXCL10, thus promoting CD8+ T cell tumor infiltration. Our findings provide the mechanistic insights implicating the RORγt agonist in immunotherapy and offer a strategy for targeting the RORγt agonist to improve PD-1 antibody efficacy in cancers.

The impact of CCR8+ regulatory T cells on cytotoxic T cell function in human lung cancer

Regulatory T cells (Tregs) suppress the host immune response and maintain immune homeostasis. Tregs also promote cancer progression and are involved in resistance to immune checkpoint inhibitor treatments. Recent studies identified selective CCR8 expression on tumor-infiltrating Tregs; CCR8+ Tregs have been indicated as a possible new target of cancer immunotherapy. Here, we investigated the features of CCR8+ Tregs in lung cancer patients. CCR8+ Tregs were highly activated and infiltration of CCR8+ Tregs in tumors was associated with poor prognosis in lung cancer patients. We also investigated their immune suppressive function, especially the influence on cytotoxic T lymphocyte cell function. The Cancer Genome Atlas analysis revealed that CD8 T cell activities were suppressed in high CCR8-expressing tumors. Additionally, depletion of CCR8+ cells enhanced CD8 T cell function in an ex vivo culture of lung tumor-infiltrating cells. Moreover, CCR8+ Tregs, but not CCR8− Tregs, induced from human PBMCs markedly suppressed CD8 T cell cytotoxicity. Finally, we demonstrated the therapeutic effect of targeting CCR8 in a murine model of lung cancer. These findings reveal the significance of CCR8+ Tregs for immunosuppression in lung cancer, especially via cytotoxic T lymphocyte cell suppression, and suggest the potential value of CCR8-targeted therapy for cancer treatment.