Lung Carcinoma Tumors Research Articles

Cisplatin and Albumin-Based Gold–Cisplatin Nanoparticles Enhance Ablative Radiation Therapy–Induced Antitumor Immunity in Local and Distant Tumor Microenvironment

Ablative radiation therapy (RT) is an important strategy to eliminate primary tumor and can potentially induce the abscopal effect. Human serum albumin nanoparticle (NP) was used for controlled release of cisplatin to decrease cisplatin's systemic toxicity, and gold (Au) was added to increase RT-induced immunogenic cell death and potentiate the abscopal antitumor immunity. The designed albumin-based cisplatin-conjugated AuNPs were administered concurrently with ablative RT. C57BL/6 mice implanted with syngeneic murine Lewis lung carcinoma or murine MB49 tumor models were treated with ablative RT (12 Gy per fraction for 2 fractions, total 24 Gy), cisplatin, or Au-cisplatin NPs. Combining ablative RT with cisplatin or Au-cisplatin NPs both destroyed the primary tumor effectively and elicited immunogenic cell death accompanied by release of danger-associated molecular patterns. This enhanced recruitment of effector tumor-infiltrating immune cells, including natural killer T cells and CD8+ T cells, and elicited an increased percentage of professional antigen-presenting CD11c+ dendritic cells. Transient weight loss, accompanying hepatotoxicity, nephrotoxicity, and hematopoietic suppression, was observed as a systemic adverse event in the cisplatin but not the Au-cisplatin NPs group. Cisplatin and Au-cisplatin NPs both showed equivalent ability to reduce metastatic potential when combined with ablative RT, confirmed by suppressed unirradiated flank tumor growth and decreased metastatic lung tumor burden, which translated to improved survival. Mobilization and abundance of effector tumor-infiltrating immune cells including CD8+ T cells and dendritic cells were observed in the distant lung tumor microenvironment after ablative RT with cisplatin or Au-cisplatin NPs, demonstrating increased antitumor immunotherapeutic activity as an abscopal effect. Compared with cisplatin, the albumin-based Au-cisplatin NPs exhibited equivalent but no superior antitumor immunotherapeutic activity while reducing systemic adverse events and can be safely administered concurrently with ablative RT. Alternative NP formulations may be designed to further improve anticancer outcomes.

Expression Analysis of Five Different Long Non-Coding Ribonucleic Acids in Nonsmall-Cell Lung Carcinoma Tumor and Tumor-Derived Exosomes.

Long non-coding ribonucleic acids (LncRNAs) are recently known for their role in regulating gene expression and the development of cancer. Controversial results indicate a correlation between the tissue expression of LncRNA and LncRNA content of extracellular vesicles. The present study aimed to evaluate the expression of different LncRNAs in non-small cell lung cancer (NSCLC) patients in tumor tissue, adjacent non-cancerous tissue (ANCT), and exosome-mediated lncRNA. Tumor and ANCT, as well as serum samples of 168 patient with NSCLC, were collected. The GHSROS, HNF1A-AS1, HOTAIR, HMlincRNA717, and LINCRNA-p21 relative expressions in tumor tissue, ANCT, and serum exosomes were evaluated in NSCLC patients. Among 168 NSCLC samples, the expressions of GHSROS (REx = 3.64, p = 0.028), HNF1A-AS1 (REx = 2.97, p = 0.041), and HOTAIR (REx = 2.9, p = 0.0389) were upregulated, and the expressions of HMlincRNA717 (REx = -4.56, p = 0.0012) and LINCRNA-p21 (REx = -5.14, p = 0.00334) were downregulated in tumor tissue in contrast to ANCT. Moreover, similar statistical differences were seen in the exosome-derived RNA of tumor tissues in contrast to ANCT samples. A panel of the five lncRNAs demonstrated that the area under the curve (AUC) for exosome and tumor was 0.937 (standard error: 0.012, p value < 0.0001). LncRNAs GHSROS, HNF1A-AS1, and HOTAIR showed high expression in tumor tissue and exosome content in NSCLC, and a panel that consisted of all five lncRNAs improved diagnosis of NSCLC.

Trypanosoma cruzi Antigenic Proteins Shared with Acute Lymphoblastic Leukemia and Neuroblastoma.

Background. Research studies indicate that immunization with protein extracts of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, prevents the appearance of tumors in 60% of mice injected with the murine lung carcinoma tumor line. The molecular basis of this process is unknown, although the presence of specific antigens in tumor cells and on the surface of T. cruzi suggests an antiparasitic immune response, with an effective cross-reaction against cancer cells, hence the importance to identify the antigens involved and determine their potential as target cells in anticancer therapy. Aim. This study aimed to determine the presence of antigenic proteins of T. cruzi shared with acute lymphoblastic leukemia and neuroblastoma cells. Material and methods. To achieve this, polyclonal antibodies against T. cruzi were developed in rabbits, and reactivity was determined with protein extracts of acute lymphoblastic leukemia cells and neuroblastoma. The immunodetection of five different strains of T. cruzi against anti-T. cruzi polyclonal antibodies was also performed. Conclusion. The study allows the knowledge of the immunological interactions between cancer and parasites to be expanded and, therefore, contributes to the design of more and better projects that improve the therapeutic strategies applied in oncology.

Zonal Intratumoral Delivery of Nanoparticles Guided by Surface Functionalization.

Delivery of small molecules and anticancer agents to malignant cells or specific regions within a tumor is limited by penetration depth and poor spatial drug distribution, hindering anticancer efficacy. Herein, we demonstrate control over gold nanoparticle (GNP) penetration and spatial distribution across solid tumors by administering GNPs with different surface chemistries at a constant injection rate via syringe pump. A key finding in this study is the discovery of different zone-specific accumulation patterns of intratumorally injected nanoparticles dependent on surface functionalization. Computed tomography (CT) imaging performed in vivo of C57BL/6 mice harboring Lewis lung carcinoma (LLC) tumors on their flank and gross visualization of excised tumors consistently revealed that intratumorally administered citrate-GNPs accumulate in particle clusters in central areas of the tumor, while GNPs functionalized with thiolated phosphothioethanol (PTE-GNPs) and thiolated polyethylene glycol (PEG-GNPs) regularly accumulate in the tumor periphery. Further, PEG functionalization resulted in larger tumoral surface coverage than PTE, reaching beyond the outer zone of the tumor mass and into the surrounding stroma. To understand the dissimilarities in spatiotemporal evolution across the different GNP surface chemistries, we modeled their intratumoral transport with reaction-diffusion equations. Our results suggest that GNP surface passivation affects nanoparticle reactivity with the tumor microenvironment, leading to differential transport behavior across tumor zones. The present study provides a mechanistic understanding of the factors affecting spatiotemporal distribution of nanoparticles in the tumor. Our proof of concept of zonal delivery within the tumor may prove useful for directing anticancer therapies to regions of biomarker overexpression.

Pulmonary delivery of size-transformable nanoparticles improves tumor accumulation and penetration for chemo-sonodynamic combination therapy.

Very little is currently known about how inhaled nanomedicine for lung cancer treatment overcomes biological barriers hampering the tumor availability of drug and nanoparticles. Here, we developed a size-transformable nanocarrier (~ 119nm) in which small-size nanoparticles (~ 28nm) were loaded in the large nanocarrier after the addition of modified hyaluronan and could be released upon size-transformation at tumor tissue. Subsequently, the pulmonary and tumor pharmacokinetics of the two nanocarriers containing 7-ethyl-10-hydroxycamptothecin (SN38) and a covalently linked fluorescent sonosensitizer were comparatively investigated after intratracheal instillation to mice bearing orthotopic Lewis lung carcinoma tumors. The results showed that both instilled nanoparticles seemed to transport drug to tumor by direct access and transcytosis of nanoparticles, and diffusion of the released drug with the latter accounting for a great proportion of the drug tumor bioavailability. Relative to the small-size nanocarrier, the size-transformable counterpart appeared to restrict the mucociliary and absorption clearances from the lung and the clearance from the tumor interstitium to circulation, leading to increases in lung and tumor bioavailability of SN38 by 58.5% and 199%, respectively. In addition, the size-transformable nanoformulation conferred deep tumor penetration and sustained levels of both sonosensitizer and SN38 within tumors and simultaneously exerted sonodynamic- and chemo-therapies. Overall, the pulmonary delivery of size-transformable nanocarrier could co-deliver sonosensitizer and drug to deep tumor sites with enhanced tumor accumulation to realize combination therapy in lung cancer.

Abstract 3136: Molecular characterization of the effects of cancer-derived exosomes on murine lung cancer tumors

Abstract Lung cancer is the second most diagnosed type of cancer and is the leading cause of cancer-related deaths in both men and women. An estimated 235,760 new cases of lung cancer and 131,880 lung cancer deaths are expected this year accounting for 12% of new cancer cases and 22% of cancer deaths in the United States. Exosomes are secreted vesicles that contain selectively packaged biomaterials (protein, lipids, and RNA) and serve as a form of extracellular communication between cells in the tumor microenvironment. Previous studies have shown the effects of cancer-derived exosomes on specific cell types or gross tumor response in vivo yet, the competitive nature and dynamics of exosomal uptake in the tumor microenvironment remains largely unknown. This work seeks to identify molecular responses induced by cancer-derived exosomes on cells residing in the tumor microenvironment. In order to control tumor cell exposure to cancer-derived exosomes, heterogeneous ex vivo cultures derived from subcutaneous Lewis lung carcinoma (LLC) tumors in C57BL6 mice were exposed to fluorescently dyed LLC exosomes for up to 72 hours. Flow cytometric analysis was performed to identify preferential uptake of cancer exosomes into the stromal subpopulations of tumor cells. Increased uptake was observed throughout the duration of the exposure and preferential uptake was observed into neutrophils, macrophages, and endothelial cells while cancer cells, fibroblasts, and lymphocytes were underrepresented in the exosome uptaken population. Additionally, single cell RNA-sequencing was performed 24 hours post-exosomal exposure to identify transcriptional changes promoted by cancer exosome uptake. We found tumor-derived cancer cells to up-regulate transcripts associated with proliferation and down-regulated genes associated with interferon signaling in response to exosome uptake. These preliminary findings suggest that cancer-derived exosomes have targeted effects on specific cell types in the tumor microenvironment. Specifically in cancer cells, exosomal uptake can elicit protumor (cancer proliferation and immune suppression via decreased interferon signaling) effects that may serve as mediators of disease progression. An understanding of both cancer and host-derived exosomal response to disease progression may identify novel biomarkers and potential therapeutic targets previously undiscovered using canonical discovery methods. This study received funding from LLNL LDRD grant 21-LW-028. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). LLNL-ABS-829271 Citation Format: Nicholas R. Hum, Nicole F. Leon, Aimy Sebastian, Kelly A. Martin, Gabriela G. Loots. Molecular characterization of the effects of cancer-derived exosomes on murine lung cancer tumors [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 3136.

Abstract 6056: Activation of Sting in response to partial-tumor radiation exposure

Abstract Purpose: It has been shown that ionizing radiation can mediate antitumor immunity via the activation of the cytosolic DNA sensor cGAS/STING pathway. Therefore, the purpose of this study was to determine whether STING activation is involved in partial volume radiation therapy (RT). Materials/Methods: We investigated 67NR murine orthotopic breast tumors in Balb/c mice and LLC cells injected in the flank of C57Bl/6, cGAS, or STING KO mice. RT was delivered to 50% or 100% of the tumor volume using a 2X2 cm collimator on a microirradiator allowing precise irradiation. Tumors were collected at different time points post-RT and assessed for different measurements. Results: We previously showed that a single dose of radiation delivered to half of the tumor (50% RT) activated an anti-tumor immune response comparable to the response in a fully-irradiated tumor in the immunogenic 67NR murine breast carcinoma tumor model and in the less immunogenic and more radioresistant Lewis lung carcinoma (LLC) tumor model. We have also demonstrated that this immune response was due to the infiltration of CD8+ T cells along with an increased expression of ICAM adhesion molecules. Treatment with either anti-CD8 or anti-ICAM antibodies abrogated the hemi-RT response. Furthermore, a significant abscopal effect was observed after partial irradiation with a single dose of 10Gy in a bilateral 67NR tumors model. In this study, we tested whether the hemi-irradiation-mediated immune response involves the cGAS/STING canonical pathway, or a non-canonical activation of STING, in the 67NR or LLC tumor models. It has been reported that STING can be activated, independently of cGAS, via non-canonical activation of STING, involving ATM and TRAF6, among other factors. We found significant activation of the cGAS/STING pathway in the hemi-irradiated tumors as compared to control and to 100% exposed 67NR tumors. Interestingly, the increased expression of the cGAS/STING pathway was found in the hemi-irradiated tumor but, also in the non-irradiated part of this hemi-irradiated tumor. In the LLC model, a non-canonical activation of STING was involved. Using both cGAS and STING KO mice, we demonstrated that the partial exposure RT-mediated immune response is dependent on STING activation in the host while cGAS is dispensable. Conclusions: Upstream pathways responsible for STING activation are tumor type-specific. Identifying the upstream pathways responsible for STING activation in the partial RT-mediated immune response in different tumor types would improve this therapy and its potential combination with immune checkpoint blockade. Citation Format: Mickael Mathieu, Prerna R. Nepali, Sadna Budhu, Simon N. Powell, John Humm, Joseph Deasy, Adriana Haimovitz-Friedman. Activation of Sting in response to partial-tumor radiation exposure [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 6056.

Abstract 5944: Euglena water extract attenuates lung tumorigenesis induced by tobacco-specific carcinogen through modulation of gut microbiota metabolites

Abstract Lung cancer is the leading cause of cancer-related mortality in the world. Our previous studies have shown that the oral administration of Euglena water extract significantly inhibits the growth of grafted lung carcinoma tumors in mice. Euglena gracilis, a single-celled alga used as a nutritional dietary supplement, possesses a broad range of medicinal properties including anticancer activity against a few types of cancers. Most studies describing this anticancer activity have used xenograft cancer mouse models, but not carcinogen-induced cancer models. Since tobacco smoke carcinogen-induced lung carcinoma in mice mimics human lung cancer development, in this study, we investigated the anti-cancer properties and the underlying mechanism of the E. gracilis water extract against lung tumorigenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). A/J mice (n=10 per treatment group) were used and the treatment regimen consisted of: (i) PBS control, (ii) Euglena water extract 2 weeks before NNK injection (pre-NNK) and (iii) this extract 10 weeks after NNK injection (post-NNK). We examined the metabolite landscape of the gut microbiota by a high-throughput metabolomics approach. The Euglena water extract treatment greatly attenuated NNK-induced tumorigenesis in lungs of A/J mice. A Partial Least-Squares Discriminant Analysis demonstrated a separation of the fecal metabolites between the control and treatment groups. Using the untargeted metabolomics, we identified 20 potential metabolites that were differentially expressed in the Euglena extract (pre- and post-NNK) treatment groups as compared to the control group. Specifically, succinate, malate, triethanolamine, acetylserine were increased with the Euglena water extract treatment in comparison to the control. These metabolites are involved in the biosynthesis of short-chain fatty acids (SCFA). Furthermore, using the targeted analysis of SCFA, we observed a significant increase in acetic acid in the feces of mice treated with Euglena water extract both before and after NNK exposure; however, butyric and propionic acids were significantly higher in only post-treated mice. Moreover, in vitro treatment of both human and murine lung cancer cells with SCFA significantly suppressed their proliferation. The present study indicated that treating with the Euglena water extract both pre- and post-NNK exposure greatly inhibits NNK-induced lung tumorigenesis in mice even after early tumorigenesis has been established. This study also found that Euglena water extract increases the levels of SCFA and their precursors in the feces of the treated mice indicating a role of gut microbiota in suppressing the NNK-induced tumorigenesis. This study was supported by 2017 EUGLENA-RC2 (MT and JC), Kansas State University Johnson Cancer Research Center (MT and JC), and NIH grant P20 GM103418 (MT). Citation Format: Deepa Upreti, Susumu Ishiguro, Ayaka Nakashima, Kengo Suzuki, Jeffrey Comer, Masaaki Tamura. Euglena water extract attenuates lung tumorigenesis induced by tobacco-specific carcinogen through modulation of gut microbiota metabolites [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 5944.

MRI and US imaging reveal evolution of spatial heterogeneity of murine tumor vasculature

The tumor microenvironment, especially the vasculature, undergoes dynamic remodeling during tumor growth and progression. The aim of this study was to investigate changes in the structure and function of tumor microenvironment (TME), with a special focus on vasculature, during the growth of the Lewis Lung Carcinoma tumor (LLC). We have used several MRI techniques and ultrasound imaging of live animals to assess how heterogenous TME features change in time.Lewis lung carcinoma bearing C57BL/6 mice were examined for three weeks. During this time, assessment of tumor vasculature was performed with Time of Flight (TOF) angiography, Dynamic Contrast Enhanced (DCE) MRI and Power Doppler Ultrasound. Additionally, diffusion and perfusion were analyzed using Diffusion Weighted MRI (DWI).Consecutive measurements of the same animals revealed an approximately twofold decrease in the density of LLC vessels in time. Heterogeneity of vasculature was best uncovered by changes in histogram based DCE analysis and revealed deterioration of tumor vessels during its progression. The tumor vasculature became less dense and with slower blood flow, with larger and more permeable vessels. As a rule, tumor tissue perfusion and diffusion parameters decreased in time, but locally increase was observed. Time- and spatial heterogeneity of tumor microenvironment, including vasculature, was revealed by 3D imaging, demonstrating that local changes are often contradictory to parameters averaged over the whole tumor volume.

Ixovex-1, a novel oncolytic E1B-mutated adenovirus

There is a great demand for improved oncolytic viruses that selectively replicate within cancer cells while sparing normal cells. Here, we describe a novel oncolytic adenovirus, Ixovex-1, that obtains a cancer-selective replication phenotype by modulating the level of expression of the different, alternatively spliced E1B mRNA isoforms. Ixovex-1 is a recombinant adenovirus that carries a single point mutation in the E1B-93R 3’ splice acceptor site that results in overexpression of the E1B-156R splice isoform. In this paper, we studied the characteristics of this novel oncolytic adenovirus by validating its in vitro behaviour in a panel of normal cells and cancer cells. We additionally studied its anti-tumour efficacy in vivo. Ixovex-1 significantly inhibited tumour growth and prolonged survival of mice in an immune-deficient lung carcinoma tumour implantation model. In complementation experiments, overexpression of E1B-156R was shown to increase the oncolytic index of both Ad5wt and ONYX-015. In contrast to prior viruses of similar type, Ixovex-1 includes a functional E3B region for better in vivo efficacy. Throughout this study, the Ixovex-1 virus has been proven to be superior in competency compared to a virus with multiple deletions.

Untargeted metabolomics profiling in a mouse model of lung cancer treated with thermal ablation

ABSTRACT Thermal ablation is widely used in the treatment of lung cancer and is beneficial for the overall survival of patients in clinic. However, there is barely a priority in which ablation system should be chosen under different periods of tumor progression in lung cancer. The present study investigated different modes of thermal ablation systems in mice with transplanted Lewis lung carcinoma tumors and their various biological effects in local regions using untargeted metabolomics. The results showed that thermal ablation could significantly suppress tumor growth and the differentially expressed metabolites of tumors after ablation relative to untreated tumors concentrated on organic compounds, organic acids and derivatives, nucleosides, nucleotides, and lipids. The upregulated metabolites indicated an inflammatory reaction in the ablation groups at an early stage after ablation. Steroid hormone and tryptophan metabolism, which are associated with immune responses, were modulated after both cryoablation and hyperthermal ablation. Characteristically, the results also indicated that cryoablation suppressed glucose oxidation and carbohydrate metabolism of tumor, while hyperthermal ablation suppressed lipid metabolism of tumor. In conclusion, thermal ablation could inhibit tumor growth under either freezing or heating modes with characteristic different biological effects on tumors.

High farnesoid X receptor expression predicts favorable clinical outcomes in PD‑L1low/negative non‑small cell lung cancer patients receiving anti‑PD‑1‑based chemo‑immunotherapy.

Anti-programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1)-directed immunotherapy has revolutionized the treatment of advanced non-small cell lung cancer (NSCLC). However, predictive biomarkers are still lacking, particularly in identifying PD-L1low/negative patients who will benefit from immunotherapy. It was previously reported that farnesoid X receptor (FXR) downregulated PD-L1 expression in NSCLC, and that FXRhighPD-L1low mouse Lewis lung carcinoma tumors showed an increased susceptibility to PD-1 blockade compared with mock tumors. At present, whether the FXRhighPD-L1low phenotype predicts clinical response to immunotherapy in patients with NSCLC remains unclear. Herein, a retrospective study was conducted to examine the expression levels of FXR, PD-L1 and CD8+ T cells by immunohistochemistry in a cohort of 149 patients with NSCLC receiving anti-PD-1-based chemo-immunotherapy. The results revealed that high FXR and PD-L1 expression levels were associated with higher objective response rates (ORR) in all patients. High PD-L1 expression also indicated superior progression-free survival (PFS). Interestingly, an inverse correlation was identified between FXR and PD-L1 expression in specimens with NSCLC. Subgroup analysis revealed that high FXR expression was associated with a higher ORR, as well as longer PFS and overall survival (OS) in PD-L1low patients. Cox multivariate analysis revealed that high FXR expression was an independent predictor for PFS and OS in PD-L1low patients. Tumor microenvironment evaluation revealed a statistically significant decrease of infiltrating CD8+ T cells in FXRhigh specimens with NSCLC. Overall, the present study proposed an FXRhighPD-L1low signature as a candidate predictor of response to anti-PD-1-based chemo-immunotherapy in PD-L1low/negative patients with NSCLC, providing evidence that could be used to broaden the patients benefitting from immunotherapy.

Fish Oil, Plant Polyphenols, and Their Combinations Have No Tumor Growth Promoting Effects on Human Lung and Colon Carcinoma Xenograft Mice

The goal of this study was to evaluate if combinations of ingredients with known anti-cachexia benefits (Fish oil-FO with either curcumin or Green tea extract-GTE), have adverse effects on tumor growth, using human carcinoma xenograft mice models. FO (EPA/DHA 360 mg/kg bw), GTE (90 mg/kg bw), and curcumin (180 mg/kg bw) were administered orally, alone or in combination, to nude mice bearing either A549 human non-small cell lung carcinoma or SW620 human colon carcinoma tumors. Bodyweight, tumor growth, survival time, and other clinical endpoints were assessed. The ingredients either alone or in combinations were well tolerated in both lung and colon tumor-bearing mice. There were no significant group differences between individual or combination treatments for tumor growth (A549 or SW620) as measured by the median time in days to endpoint of tumor volume (TTE). TTE results indicate that these ingredients (alone or combinations) did not adversely impact tumor growth. No significant differences in body weights or survival were observed between controls and treatment groups indicating no adverse health effects of the ingredients. In conclusion, FO, GTE or curcumin administered as monotherapies and in combination were well tolerated and displayed no adverse effects on tumor growth in mouse xenograft models of lung and colon cancer.

Mesoporous silicon nanoparticles loaded with salinomycin for cancer therapy applications

Mesoporous silicon nanoparticles loaded with salinomycin, a promising anticancer drug, were investigated as potential nanocontainers for cancer therapy. The drug loading and release were studied by means of the infrared spectroscopy, which revealed a gradual release of the drug with rate dependent on the mass ratio between the nanoparticles and salinomycin. In vitro studies revealed that the prepared nanoparticles loaded with salinomycin resulted in a notable cytotoxic effect on human glioblastoma cells, MCF-7 breast cancer cells and multidrug resistant MCF-7/MDR1 cells. In vivo experiments showed a strong suppression of the Lewis lung carcinoma tumor growth for 10–30 days after intraperitoneal administration of the nanocontainers. The obtained results indicate that salinomycin-loaded mesoporous silicon nanoparticles are prospective for mild chemotherapy of both primary cancer tumors and metastasis. The revealed efficiency against chemotherapy-resistant cancer stem cells and possibility of the intraperitoneal administration are very promising for further clinical applications of the prepared nanocontainers.

Activation of Sting in Response to Partial-Tumor Radiation Exposure

It has been shown that ionizing radiation can mediate antitumor immunity via the activation of the cytosolic DNA sensor cGAS/STING pathway. Therefore, the purpose of this study, was to determine whether STING activation is involved in partial volume radiation therapy (RT).We investigated 67NR murine orthotopic breast tumors in Balb/c mice and LLC cells injected in the flank of C57Bl/6, cGAS or STING KO mice. RT was delivered to 50% or 100% of the tumor volume using a 2 × 2 cm collimator on a microirradiator allowing precise irradiation. Tumors were collected at different time points post-RT and assessed for different measurements.We previously showed that a single dose of radiation delivered to half of the tumor (50% RT) activated an anti-tumor immune response comparable to the response in a fully-irradiated tumor in the immunogenic 67NR murine breast carcinoma tumor model and in the less immunogenic and more radioresistant Lewis lung carcinoma (LLC) tumor model. We have also demonstrated that this immune response was due to the infiltration of CD8+ T cells along with an increased expression of ICAM adhesion molecules. Treatment with either anti-CD8 or anti-ICAM antibodies abrogated the hemi-RT response. Furthermore, a significant abscopal effect was observed after partial irradiation with a single dose of 10Gy in a bilateral 67NR tumors model. In this study we tested whether the hemi-irradiation-mediated immune response involves the cGAS/STING canonical pathway, or a non-canonical activation of STING, in the 67NR or LLC tumor models. It has been reported that STING can be activated, independently of cGAS, via non-canonical activation of STING, involving ATM and TRAF6, among other factors. We found a significant activation of the cGAS/STING pathway in the hemi-irradiated tumors as compared to control and to 100% exposed 67NR tumors. Interestingly, the increased expression of the cGAS/STING pathway was found in the hemi-irradiated tumor but, also in the non-irradiated part of this hemi-irradiated tumor. In the LLC model, a non-canonical activation of STING was involved. Using both cGAS and STING KO mice, we demonstrated that the partial exposure RT-mediated immune response is dependent on STING activation in the host while cGAS is dispensable.Upstream pathways responsible for STING activation are tumor type specific. Identifying the upstream pathways responsible for STING activation in the partial RT-mediated immune response in different tumor types would improve this therapy and its potential combination with immune checkpoint blockade.

Predicting Response to Immunotherapy in Non-Small Cell Lung Cancer — From Bench to Bedside

Immune checkpoint inhibitor (ICI) therapy has emerged as the standard of care for patients with advanced non-small cell lung cancer (aNSCLC). Immuno-monotherapy (ImT) offers a better side effect profile than combination ICIs, but treatment response rates remain less than 25%. Predicting response to ImT could spare potential responders from adverse events and direct non-responders toward alternative treatments. Radiomics, a datamining technique that extracts patterns from medical imaging, has been incorporated into predictive models for many cancers. We hypothesized that a model combining pre-ImT radiomics, clinical, and laboratory data can predict patient response and guide clinical decision-making.A murine model was designed to search for texture features and blood biomarkers predictive of ImT response. Nineteen C57BL/6 mice had Lewis Lung Carcinoma tumors injected into bilateral flanks. Pre-ImT CTs and bloodwork were obtained on day 7 post-implant. On day 8, 15 mice underwent radiotherapy (RT) to the right flank tumor (24 Gy in 3 daily fractions), followed by daily anti-PDL-1 ImT. Tumors on both flanks were assessed for treatment response. Bilateral response was seen in 4 mice. Pre-ImT CTs were mined for texture features correlating with systemic response. Next, 117 consecutive aNSCLC patients treated with nivolumab monotherapy were identified in an IRB-approved database. Baseline clinical features were assessed, including laboratory data and RT history. For all patients, the primary tumor in the last pre-ImT CT was segmented per RTOG guidelines. These volumes were analyzed for texture features associated with overall survival (OS). Univariate and multivariate cox regression were used to identify pre-ImT imaging, clinical, and laboratory parameters associated with OS. Areas under the receiver operating characteristic curve (AUC) were estimated to assess accuracy to predict greater than median OS.Identical texture features (surface-to-mass ratio, average Gray, and 2D kurtosis) and neutrophil-to-lymphocyte (NLR) correlated with OS in humans (P = 0.041, < 0.001) and systemic response in mice (P < 0.001, = 0.01). A murine model using NLR and radiomics predicted systemic response with AUC of 1. A human model incorporating baseline ECOG, sex, pre-ImT NLR and radiomics had an AUC for predicting greater than median OS (308 days) of 0.808. Half of patients had a history of RT, but location, dose, fractionation, and timing were heterogeneous and RT was not significant.For patients with aNSCLC treated with ImT, a combined model of pre-ImT radiomics, NLR, ECOG, and sex is predictive of OS. Texture features and NLR correlated with treatment response in both human and murine cohorts, suggesting generalizability. RT history was too heterogeneous to factor significantly as a predictor of OS. Future directions include identification of genomic biomarkers and model validation on an external patient cohort.

Effects of combination treatment with durvalumab plus tremelimumab on the tumor microenvironment in non-small-cell lung carcinoma

The rapid development of immune checkpoint blockade (ICB) therapies has revolutionized the cancer treatment landscape and brightened the long-term forecast for many cancer patients. However, the specific genomic and proteomic changes in tumors treated with different ICB treatments have yet to be fully characterized. We treated four non-small-cell lung carcinoma (NSCLC) tumor digests ex vivo with the anti-PD-L1 antibody durvalumab (D) alone or in combination with the anti-CTLA-4 antibody tremelimumab (T) to explore changes in gene and protein expression associated with these ICB therapies. All four tumors showed a robust increase in interferon gamma (IFN-γ) production (100–300% higher than isotype control) in both D- and D + T-treated tumors. Three of the four tumors showed additional increases in IFN-γ production with D + T compared with D (40–70%). A substantial reduction in interleukin 10 (IL-10) was also found in three of the four tumors (reduced to 4–8%) in response to D and D + T. Conventional CD4 + /CD8 + populations and T cell activation markers increased after D and D + T treatment. D and D + T upregulated multiple IPA pathways involving T cell activation. D + T resulted in additional upregulation of Th1/Th2 pathways through a different set of genes, as well as greater reduction in genes involved in epithelial-mesenchymal transition (EMT), angiogenesis, and cancer stemness. Our results demonstrated that D + T augmented the effects of D in the microenvironment of this set of NSCLC tumors. The specific impact of D + T on the regulation of EMT, angiogenesis, and cancer stemness warrants further evaluation in a larger set of tumors.

Expression of tdTomato and luciferase in a murine lung cancer alters the growth and immune microenvironment of the tumor.

Imaging techniques based on fluorescence and bioluminescence have been important tools in visualizing tumor progression and studying the effect of drugs and immunotherapies on tumor immune microenvironment in animal models of cancer. However, transgenic expression of foreign proteins may induce immune responses in immunocompetent syngeneic tumor transplant models and augment the efficacy of experimental drugs. In this study, we show that the growth rate of Lewis lung carcinoma (LL/2) tumors was reduced after transduction of tdTomato and luciferase (tdTomato/Luc) compared to the parental cell line. tdTomato/Luc expression by LL/2 cells altered the tumor microenvironment by increasing tumor-infiltrating lymphocytes (TILs) while inhibiting tumor-induced myeloid-derived suppressor cells (MDSCs). Interestingly, tdTomato/Luc expression did not alter the response of LL/2 tumors to anti-PD-1 and anti-CTLA-4 antibodies. These results suggest that the use of tdTomato/Luc-transduced cancer cells to conduct studies in immune competent mice may lead to cell-extrinsic tdTomato/Luc-induced alterations in tumor growth and tumor immune microenvironment that need to be taken into consideration when evaluating the efficacy of anti-cancer drugs and vaccines in immunocompetent animal models.

Cisplatin nanoparticles possess stronger anti-tumor synergy with PD1/PD-L1 inhibitors than the parental drug

Programmed cell death protein 1 (PD1)/programmed death-ligand 1 (PD-L1) inhibitors provide an evolution in the field of cancer therapy. This results in unprecedented rates of long-lasting tumor responses, once cancer patients respond to PD1/PD-L1 inhibitors. However, the response rate of most cancers is not greater than 30%, which results in a limited therapeutic efficacy. Therefore, the increase of the therapeutic efficacy of PD1/PD-L1 inhibitors is of utmost importance. Hence, this study demonstrated that the sustained increase of tumor PD-L1 levels induced by long-tumor retaining cisplatin (Cis) nanoparticles improved the therapeutic outcomes of PD1/PD-L1 inhibitors. Cis-loaded poly(L-glutamic acid)-graft-methoxy poly(ethylene glycol) complex nanoparticle (Cisplatin nanoparticle, P-Cis) caused tumor PD-L1 overexpression in a time dependent manner in vitro and amplified tumor PD-L1 signals at 72 h post treatment in vivo. Synergistic tumor inhibition was achieved when P-Cis was combined with PD1/PD-L1 inhibitors, such as BMS-202 and anti-PD1 antibody (aPD1), and a significantly superior tumor inhibition rate was observed in the combination group (P-Cis plus aPD1). In addition, when mice were treated with a single dose of P-Cis plus aPD1, its synergistic anti-tumor effect was much stronger than that of a single dose of Cis plus aPD1, as their Q values were 1.15 and 1.05 in the Lewis lung carcinoma (LLC) tumor model, and 1.92 and 0.95 in the B16F10 tumor model, respectively. The single dose of P-Cis could increase tumor PD-L1 expression at 72 h post injection, while a single-dose of Cis did not, thus the sustained tumor PD-L1 overexpression induced by P-Cis was essential for enhancing aPD1 therapy. The sustained tumor PD-L1 overexpression highlighted the involvement of PD1/PD-L1 pathway in tumor cell proliferation and CD8+ T cell weakening and increased the role and possibility of PD1/PD-L1 inhibitors to block the PD1/PD-L1 pathway. Collectively, this study identified a potential clinical treatment with P-Cis plus PD1/PD-L1 inhibitors. Statement of significanceProgrammed cell death protein 1 (PD1)/programmed death-ligand 1 (PD-L1) inhibitors provide an evolution in the field of cancer therapy. However, the response rate of most cancers is not greater than 30%, which results in a limited therapeutic efficacy. Therefore, the increase of the therapeutic efficacy of PD1/PD-L1 inhibitors is of utmost importance. Here, Cisplatin (Cis) loaded poly(L-glutamic acid)-graft-methoxy poly(ethylene glycol) complex nanoparticle (P-Cis) is found to improve the therapeutic outcomes of PD1/PD-L1 inhibitors via sustained increase of tumor PD-L1 levels, and P-Cis possesses stronger anti-tumor synergy with PD1/PD-L1 inhibitors than the parental drug. This identifies a potential clinical treatment with P-Cis plus PD1/PD-L1 inhibitors.

MicroRNA-324-5p-CUEDC2 Axis Mediates Gain-of-Function Mutant p53-Driven Cancer Stemness.

Regulation of cancer stemness has recently emerged as a new gain-of-function (GOF) property of mutant p53. In this study, we identify miR-324-5p as a critical epigenetic regulator of cancer stemness and demonstrate its role in mediating GOF-mutant p53-driven stemness phenotypes. We report that miR-324-5p is upregulated in human cancer cell lines and non-small cell lung carcinoma (NSCLC) tumors carrying TP53 GOF mutations. Mechanistically, we show that GOF mutant p53 upregulates miR-324-5p expression via c-Myc, an oncogenic transcription factor in cancer cells. Our experimental results suggest that miR-324-5p-induced CSC phenotypes stem from the downregulation of CUEDC2, a downstream target gene of miR-324-5p. Accordingly, CUEDC2 complementation diminishes elevated CSC marker expression in miR-324-5p-overexpressing cancer cells. We further demonstrate that mutant p53 cancer cells maintain a low level of CUEDC2 that is rescued upon miR-324-5p inhibition. Importantly, we identify CUEDC2 downregulation as a novel characteristic feature of TP53-mutated human cancers. We show that activation of NF-κB due to downregulation of CUEDC2 by miR-324-5p imparts stemness in GOF mutant p53 cancer cells. Finally, we provide evidence that TP53 mutations coupled with high miR-324-5p expression predict poor prognosis in patients with lung adenocarcinoma. Thus, our study delineates an altered miR-324-5p-CUEDC2-NF-κB pathway as a novel regulator of GOF mutant p53-driven cancer stemness. IMPLICATIONS: Our findings implicate miRNA-324-5p as a novel epigenetic modifier of human cancer stemness.