Self-assemblies from prodrugs composed of antimicrobial peptides: a revolution in local lung cancer treatment, with microbiota as a main actor.

Our previous studies have demonstrated that Plasmodium immunotherapy (infection) has antitumor effects in mice. However, as a new form of immunotherapy, this therapy has a weakness: its specific killing effect on tumor cells is relatively weak. Therefore, we tested whether Plasmodium immunotherapy combined with gemcitabine (Gem), a representative chemotherapy drug, has synergistic antitumor effects. We designed subcutaneously and intravenously implanted murine Lewis lung cancer (LLC) models to test the antitumor effect of Plasmodium chabaudi ASS (Pc) infection in combination with Gem treatment and explored its underlying mechanisms. We found that both Pc infection alone and Gem treatment alone significantly inhibited tumor growth in the subcutaneous model, and combination therapy was more effective than either monotherapy. Monotherapy only tended to prolong the survival of tumor-bearing mice, while the combination therapy significantly extended the survival of mice, indicating a significant synergistic effect of the combination. In the mechanistic experiments, we found that the combination therapy significantly upregulated E-cadherin and downregulated Snail protein expression levels, thus inhibiting epithelial-mesenchymal transition (EMT) of tumor cells, which may be due to the blockade of CXCR2/TGF-β-mediated PI3K/Akt/GSK-3β signaling pathway. The combination of Pc and Gem plays a synergistic role in inhibiting tumor growth and metastasis, and prolonging mice survival in murine lung cancer models. These effects are partially attributed to the inhibition of EMT of tumor cells, which is potentially due to the blockade of CXCR2/TGF-β-mediated PI3K/Akt/GSK-3β/Snail signaling pathway. The clinical transformation of Plasmodium immunotherapy combined with Gem for lung cancer is worthy of expectation.

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.

Flavonoids, like Hesperetin, have been shown to be an ACE2 receptor agonists with antioxidant and pro-apoptotic activity and can induce apoptosis in cancer cells. ACE2 receptors are abundant in lung cancer cells. Here, we explored the application of Hesperetin bound to PegPLGA-coated nanoparticles (Hesperetin nanoparticles, HNPs) and anti-CD40 antibody as an aerosol treatment for lung tumor-bearing mice. The Hesperetin nanoparticles (HNPs) were engineered using a nano-formulation microfluidic technique and polymeric nanoparticles. The in vitro studies were performed in human A549 (ATCC) and murine LL/2-Luc2 (ATCC) lung cancer cell lines. A syngeneic orthotopic murine model of lung cancer was generated in wild (+/+) C57/BL6 background mice with luciferase-positive cell line LL/2-Luc2 cells. Lung tumor-bearing mice were treated via aerosol inhalation with HNP, anti-CD40 antibody, or both. Survival was used to analyze the efficacy of the aerosol treatment. The cohorts were also analyzed for body condition score, weight, and liver and kidney function. Analysis of an orthotopic murine lung cancer model demonstrated a differential uptake of the HNPs and anti-CD40 by the cancer cells. A higher survival rate was observed when the combination of aerosol treatment with HNPs was added with the treatment with anti-CD40 (p < 0.001), as compared to anti-CD40 alone (p < 0.01). Moreover, two tumor-bearing mice survived long-term with the combination treatment, and their tumors were diminished. Subsequently, these two mice were shown to be refractory to the development of subcutaneous tumors, indicating systemic resilience to developing new tumors. Using an inhalation-based administration, we successfully established a treatment model of increased therapeutic efficacy with HNPs and anti-CD40 in an orthotopic murine lung cancer model. Our findings open the possibility of improved lung cancer treatment using nanoparticles like flavonoids and immunoadjuvants.

Studies have shown that flavonoids like Hesperetin, an ACE2 receptor agonist with antioxidant and pro-apoptotic activity, can induce apoptosis in cancer cells. ACE2 receptors are abundant in lung cancer cells. Here, we explored the application of Hesperetin bound to PLGA-coated nanoparticles (Hesperetin-nanoparticles, HNPs), and anti-CD40 antibody as an aerosol treatment for lung tumor-bearing mice. In-vitro and in-vivo studies were performed in human A549 (ATCC) and murine LLC1 (ATCC) lung cancer cell lines. Hesperetin Nanoparticles (HNP) of about 60nm diameter were engineered using a nano-formulation microfluidic technique. A syngeneic orthotopic murine model of lung adenoma was generated in wild (+/+) C57/BL6 background mice with luciferase-positive cell line LLC1 cells. Lung tumor-bearing mice were treated via aerosol inhalation with HNP, anti-CD40 antibody, or both. Survival was used to analyze the efficacy of aerosol treatment. Cohorts were also analyzed for body condition score, weight, and liver and kidney function. Analysis of an orthotopic murine lung cancer model demonstrates a differential uptake of the HNP and anti-CD40 by cancer cells relative to normal cells. A higher survival rate, relative to untreated controls, was observed when aerosol treatment with HNP was added to treatment via anti-CD40 (p<0.001), as compared to CD40 alone (p<0.01). Moreover, 2 out of 9 tumor-bearing mice survived long term, and their tumors diminished. These 2 mice were shown to be refractory to subsequent development of subcutaneous tumors, indicating systemic resilience to tumor formation. We successfully established increased therapeutic efficacy of anti-CD40 and HNP in an orthotopic murine lung cancer model using inhalation-based administration. Our findings open the possibility of improved lung cancer treatment using flavonoids and immuno-adjuvants.

BackgroundNew pharmacologic targets are urgently needed to treat or prevent lung cancer, the most common cause of cancer death for men and women. This study identified one such target. This is the canonical Wnt signaling pathway, which is deregulated in cancers, including those lacking adenomatous polyposis coli or β-catenin mutations. Two poly-ADP-ribose polymerase (PARP) enzymes regulate canonical Wnt activity: tankyrase (TNKS) 1 and TNKS2. These enzymes poly-ADP-ribosylate (PARsylate) and destabilize axin, a key component of the β-catenin phosphorylation complex.MethodsThis study used comprehensive gene profiles to uncover deregulation of the Wnt pathway in murine transgenic and human lung cancers, relative to normal lung. Antineoplastic consequences of genetic and pharmacologic targeting of TNKS in murine and human lung cancer cell lines were explored, and validated in vivo in mice by implantation of murine transgenic lung cancer cells engineered with reduced TNKS expression relative to controls.ResultsMicroarray analyses comparing Wnt pathway members in malignant versus normal tissues of a murine transgenic cyclin E lung cancer model revealed deregulation of Wnt pathway components, including TNKS1 and TNKS2. Real-time PCR assays independently confirmed these results in paired normal-malignant murine and human lung tissues. Individual treatments of a panel of human and murine lung cancer cell lines with the TNKS inhibitors XAV939 and IWR-1 dose-dependently repressed cell growth and increased cellular axin 1 and tankyrase levels. These inhibitors also repressed expression of a Wnt-responsive luciferase construct, implicating the Wnt pathway in conferring these antineoplastic effects. Individual or combined knockdown of TNKS1 and TNKS2 with siRNAs or shRNAs reduced lung cancer cell growth, stabilized axin, and repressed tumor formation in murine xenograft and syngeneic lung cancer models.ConclusionsFindings reported here uncovered deregulation of specific components of the Wnt pathway in both human and murine lung cancer models. Repressing TNKS activity through either genetic or pharmacological approaches antagonized canonical Wnt signaling, reduced murine and human lung cancer cell line growth, and decreased tumor formation in mouse models. Taken together, these findings implicate the use of TNKS inhibitors to target the Wnt pathway to combat lung cancer.

Lung cancer is a leading cause of cancer deaths worldwide. Despite advances in chemotherapy, radiation therapy, and surgery, lung cancer continues to have a low 5-year survival rate, highlighting a dire need for more effective means of prevention, diagnosis, prognosis, and treatment. Mouse models that recapitulate the clinical features of advanced human lung cancer are critical for testing novel therapeutic approaches. This unit describes a highly reproducible, easy-to-establish orthotopic murine model of lung cancer, provides methods for in vivo imaging and monitoring of tumor growth, and discusses the usefulness of this model for translational lung cancer research and the development of therapeutic strategies.

Oncolytic vaccinia viruses (VVs) are potent stimulators of the immune system and induce immune-mediated tumor clearance and long-term surveillance against tumor recurrence. As such they are ideal treatment modalities for solid tumors including lung cancer. Here, we investigated the use of VVL-m12, a next-generation, genetically modified, interleukin-12 (IL-12)-armed VV, as a new therapeutic strategy to treat murine models of lung cancer and as a mechanism of increasing lung cancer sensitivity to antibody against programmed cell death protein 1 (α-PD1) therapy. The cytotoxicity and replication of VVL-m12, VVL-h12 and control VVs were assessed in lung cancer cell lines. Subcutaneous lung cancer mouse models were established to investigate the anti-tumor activity of the viruses after intratumoral delivery in an immunocompetent disease model. Synergy with α-PD1 or a VV armed with soluble PD-1 (VV-sPD1) was investigated and functional mechanisms behind efficacy probed. Tumor-targeted VVL-m12 replicated to high levels, was cytotoxic in lung cancer cell lines. VVL-m12 demonstrated superior antitumor efficacy in subcutaneous lung cancer models compared with other VVs examined. Importantly, rational combination of VVL-m12 and PD-1 blockade worked synergistically to significantly enhance survival of animals and safely cured lung cancer with no evidence of recurrence. VVL-m12 therapy induced increased intratumoral infiltration of CD4+ and CD8+ T cells and was able to clear tumor at early time points via increased induction and infiltration of effector T cells and central memory T cells (TCM). In addition, VVL-m12 increased dendritic cell activation, induced polarization of M2 macrophages towards an M1 phenotype, and inhibited tumor angiogenesis in vivo. These results demonstrate that VVL-12 has strong potential as a safe and effective antitumor therapeutic for lung cancer. Importantly, VVL-12 can sensitize lung cancers to α-PD1 antibody therapy, and the combined regime creates a highly effective treatment option for patients.

Variable expression of the coxsackie and adenovirus receptor (CAR) has limited gene transfer efficacy to many types of tumors. Consequently, tropism-modified adenoviruses have been developed for enhanced infectivity. To the authors' knowledge, targeting approaches for nonsmall cell lung cancer (NSCLC) have not been comprehensively evaluated. The current hypothesis was that modified adenoviruses could be used for increasing gene transfer to and killing of NSCLC cells in vitro and in vivo. Ten NSCLC cell lines were analyzed to represent the different NSCLC histologies. Because clinical tumors may differ from established cell lines, 6 clinical specimens fresh from patients were analyzed. For in vivo studies, a novel orthotopic murine model of advanced lung cancer was developed. Because tumor response is difficult to quantitate in orthotopic models, noninvasive imaging of green fluorescent protein (GFP) was utilized as a surrogate for tumor size measurements. Adenoviruses whose capsids were modified with RGD-4C, the serotype 3 knob, or polylysine displayed increased gene transfer to NSCLC cell lines and clinical samples in comparison to serotype 5 viruses. Conditionally replicating oncolytic adenoviruses (CRAds) with the same modifications showed enhanced therapeutic efficiency in vitro and in vivo. The median survival of mice treated with Ad5.pK7-Delta24 or Ad5-Delta24RGD increased 37% (P<.01). GFP imaging allowed noninvasive individualized detection of response and recurrence. Targeting of adenovirus to heterologous receptors can improve killing of NSCLC cells. Utilization of clinical samples and an orthotopic model of advanced lung cancer may provide clinically relevant translational data.

Functional and marker MicroPET imaging in a murine model of orthotopic lung cancer

ABSTRACTLung-specific overexpression of prostacyclin synthase (PGIS) decreases tumor initiation in murine lung cancer models. Prostacyclin analogs prevent lung tumor formation in mice and reverse bronchial dysplasia in former smokers. However, the effect of prostacyclin on lung cancer progression has not been well studied. We investigated the effects of pulmonary PGIS overexpression in an orthotopic immunocompetent mouse model of lung cancer using two murine lung cancer cell lines. Pulmonary PGIS overexpression significantly inhibited CMT167 lung tumor growth, increased CXCL9 expression, and increased CD4+ tumor-infiltrating lymphocytes. Immunodepletion of CD4+ T cells abolished the inhibitory effect of pulmonary PGIS overexpression on CMT167 lung tumor growth. In contrast, pulmonary PGIS overexpression failed to inhibit growth of a second murine lung cancer cell line, Lewis Lung Carcinoma (LLC) cells, and failed to increase CXCL9 expression or CD4+ T lymphocytes in LLC lung tumors. Transcriptome profiling of CMT167 cells and LLC cells recovered from tumor-bearing mice demonstrated that in vivo, CMT167 cells but not LLC cells express MHC class II genes and cofactors necessary for MHC class II processing and presentation. These data demonstrate that prostacyclin can inhibit lung cancer progression and suggest that prostacyclin analogs may serve as novel immunomodulatory agents in a subset of lung cancer patients. Moreover, expression of MHC Class II by lung cancer cells may represent a biomarker for response to prostacyclin.

P1.03-42 Angiotensin System Inhibitors Improve Survival in Stage IIIA NSCLC and Show Anti-Tumor Effect in Combination with Chemotherapy in a Model of NSCLC

Lung microbiota and gut microbiota are closely related to lung cancer. Studies have shown that the dysbiosis, i.e., the significantly altered composition and structure of gut and lung microbiota, usually occurs in patients with lung cancer. With the introduction of "Gut-Lung Axis", an increasing attention has been paid to the close relationship between the lung and gut microbiota in human body. A deeper insight into this relationship would facilitate understanding the mechanisms behind the carcinogenesis and development of lung cancer. This article summarizes the composition of lung and gut microbiota in patients with lung cancer and the possible interaction mechanisms, highlighting the importance of the immune system in the Gut-Lung Axis. The effects of lung and gut microbiota on the clinical treatment of lung cancer were summarized, based on which the authors propose that the lung and gut microbiota can be used as novel targets for early diagnosis and treatment of lung cancer.

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Evaluation of cyclooxygenase-2 inhibition in an orthotopic murine model of lung cancer for dose-dependent effect

Human lung cancer carries high genetic alterations, expressing high tumor-specific neoantigens. Although orthotopic murine lung cancer models recapitulate many characteristics of human lung cancers, genetically engineered mouse models have fewer somatic mutations than human lung cancer, resulting in scarce immune cell infiltration and deficient immune responses. The endogenous mouse lung cancer model driven by Kras mutation and Trp53 deletion (KP model) has minimal immune infiltration because of a scarcity of neoantigens. Fine-tuning tumor antigenicity to trigger the appropriate level of antitumor immunity would be key to investigating immune responses against human lung cancer. We engineered the KP model to express antigens of OVA peptides (minOVA) as neoantigens along with ZsGreen, a traceable fluorescent conjugate. The KP model expressing minOVA exhibited stronger immunogenicity with higher immune cell infiltration comprised of CD8+ T cells and CD11c+ dendritic cells (DCs). Consequently, the KP model expressing minOVA exhibits suppressed tumor growth compared to its origin. We further analyzed tumor-infiltrated DCs. The majority of ZsGreen conjugated with minOVA was observed in the conventional type 2 DCs (cDC2), whereas cDC1 has minimal. These data indicate that tumor immunogenicity regulates host immune responses, and tumor neoantigen is mostly recognized by cDC2 cells, which may play a critical role in initiating antitumor immune responses in an orthotopic murine lung cancer model.