Tumor-bearing Lungs Research Articles

Abstract A018: Endothelial pyrimidine synthesis deficiency promotes tumor growth

Abstract The tumor microenvironment plays a crucial role in cancer progression and treatment response. Systemically administered metabolic cancer therapies target not only malignant but also stromal cells, including immune cells - the only stromal population previously addressed in this context. However, how such treatments impact other non-malignant cell types in tumors remains poorly understood. Here we show that inhibition of de novo pyrimidine synthesis in endothelial cells accelerates tumor growth and alters tumor immune repertoire. We found that whole-body de novo pyrimidine synthesis deficiency in mice, caused by the inducible whole-body ablation of DHODH, accelerates the growth rate of orthotopic lung tumors. Single-cell transcriptomic analysis of tumor-bearing lungs revealed that DHODH deficiency in the stroma impacts multiple cell populations, including immune and, surprisingly, endothelial cells. To explore the endothelial-specific effects, we generated a mouse model with inducible DHODH deficiency restricted to endothelial cells. The endothelium-specific model recapitulates the accelerated lung tumor growth observed in the whole-body DHODH deficiency model. Single-cell transcriptomics analysis of tumor-bearing lungs in the endothelium-specific model pointed to changes in the immune repertoire, particularly an enrichment of monocytes. We confirmed these results on the protein level using spectral flow cytometry, and we are currently in the process of uncovering the mechanism by which endothelial deficiency of pyrimidine synthesis affects the immune landscape of tumors. Inhibitors of pyrimidine de novo synthesis have been tested in clinical trials but failed due to their low efficiency. Our findings indicate that systemic treatment targeting pyrimidine synthesis may be hampered by its pro-tumorigenic effects in the endothelium, highlighting the unexpected role of endothelial metabolism in this context. Citation Format: Petra Hyrossova, Isidora Milisav, Silvia Novais, Mirko Milosevic, Jakub Rohlena, Katerina Rohlenova. Endothelial pyrimidine synthesis deficiency promotes tumor growth [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A018.

Inhalable chitosan-coated nano-assemblies potentiate niclosamide for targeted abrogation of non-small-cell lung cancer through dual modulation of autophagy and apoptosis

Lung carcinoma, particularly non-small-cell lung cancer (NSCLC), accounts for a significant portion of cancer-related deaths, with a fatality rate of approximately 19 %. Niclosamide (NIC), originally an anthelmintic drug, has attracted attention for its potential in disrupting cancer cells through various intracellular signaling pathways. However, its effectiveness is hampered by limited solubility, reducing its bioavailability. This study investigates the efficacy of NIC against lung cancer using inhalable hybrid nano-assemblies with chitosan-functionalized Poly (ε-caprolactone) (PCL) as a carrier for pulmonary delivery. The evaluation encompasses various aspects such as aerodynamic and physicochemical properties, drug release kinetics, cellular uptake, biocompatibility, cell migration, autophagic flux, and apoptotic cell death in A549 lung cancer cells. Increasing NIC dosage correlates with enhanced inhibition of cell proliferation, showing a dose-dependent profile (approximately 75 % inhibition efficiency at 20 μg/mL of NIC). Optimization of inhaled dosage and efficacy is conducted in a murine model of NNK-induced tumor-bearing lung cancer. Following inhalation, NIC-CS-PCL-NA demonstrates significant lung deposition, retention, and metabolic stability. Inhalable nano-assemblies promote autophagy flux and induce apoptotic cell death. Preclinical trials reveal substantial tumor regression with minimal adverse effects, underscoring the potential of inhalable NIC-based nano-formulation as a potent therapeutic approach for NSCLC, offering effective tumor targeting and killing capabilities.

MRI-based ventilation and perfusion imaging to predict radiation-induced pneumonitis in lung tumor patients at a 0.35 T MR-Linac

Background and purpose:Radiation-induced pneumonitis (RP), diagnosed 6–12 weeks after treatment, is a complication of lung tumor radiotherapy. So far, clinical and dosimetric parameters have not been reliable in predicting RP. We propose using non-contrast enhanced magnetic resonance imaging (MRI) based functional parameters acquired over the treatment course for patient stratification for improved follow-up. Materials and methods:23 lung tumor patients received MR-guided hypofractionated stereotactic body radiation therapy at a 0.35T MR-Linac. Ventilation- and perfusion-maps were generated from 2D-cine MRI-scans acquired after the first and last treatment fraction (Fx) using non-uniform Fourier decomposition. The relative differences in ventilation and perfusion between last and first Fx in three regions (planning target volume (PTV), lung volume receiving more than 20Gy (V20) excluding PTV, whole tumor-bearing lung excluding PTV) and three dosimetric parameters (mean lung dose, V20, mean dose to the gross tumor volume) were investigated. Univariate receiver operating characteristic curve - area under the curve (ROC–AUC) analysis was performed (endpoint RP grade≥1) using 5000 bootstrapping samples. Differences between RP and non-RP patients were tested for statistical significance with the non-parametric Mann–Whitney U test (α=0.05). Results:14/23 patients developed RP of grade≥1 within 3 months. The dosimetric parameters showed no significant differences between RP and non-RP patients. In contrast, the functional parameters, especially the relative ventilation difference in the PTV, achieved a p-value<0.05 and an AUC value of 0.84. Conclusion:MRI-based functional parameters extracted from 2D-cine MRI-scans were found to be predictive of RP development in lung tumor patients.

Abstract 1281: Antibiotics aerosolization shrinks intratumoral Tregs and impairs lung tumor growth by perturbing the taxonomic structure of tumor-associated microbiota

Abstract The lung has been traditionally regarded as a sterile environment. Emerging evidence has overturned this belief unveiling the existence of a specific lung microbiota that plays a pivotal role in modulating lung immune tolerance. Recent research also sheds light on how the tissue microbiota is significantly altered by the presence of a growing tumor. Here, we investigated the compositional changes in lung-colonizing bacterial populations associated with the growth of LLC1 lung carcinoma cells in C57BL/6 mice, by analyzing the lung 16S rRNA profiling. While overall microbial diversity and structure were similar in tumor-bearing and healthy lungs, abundance analysis of amplicon sequence variants (ASVs) revealed that 48 ASVs were differentially modulated at significant level (FDR ≤ 0.05). Notably, local treatment with aerosolized vancomycin/neomycin antibiotics starting 4 days after tumor injection caused a different representation of 47 ASVs (FDR ≤ 0.05), mostly reducing those ASVs specifically emerging in the lung by the tumor growth, belonging to Bacterioidales, Clostridiales, Lactobacillales and Enterobacteriales. A different microbial composition was also suggested by the results of Partial Least Squares Discriminant Analysis (PLS-DA) that clustered lung tumor samples as saline or antibiotic-aerosolized. Based on the reported ability of some bacteria belonging to these orders to induce immunosuppressive cells in the gut, we examined the impact of antibiotic aerosol on tumor-associated Tregs in the lung. The presence of the tumor promoted the appearance of Tregs expressing CD69 and PD-1 activation markers, but vancomycin/neomycin aerosol treatment strongly dropped Treg frequency in tumor-bearing lungs, compared to saline. In a therapeutic setting, aerosolization of antibiotics starting 4 days after LLC1 tumor intravenous injection significantly reduced the number of lung metastases (p=0.0116). This effect was associated with an increased cytotoxic activity of immune infiltrating effector cells suggesting that the modulation of the lung microbiota impairs tumor growth and enhances antitumor immune response. Some cultivable bacterial strains, that were in agreement with metataxonomic profile results, were isolated from tumor-bearing lungs, but not from healthy lungs. Ongoing experiments suggest an effect of their in vivo transfer in the lung by aerosol on tumor growth and the ability to induce Treg expansion in vitro. Overall, this study unveils that the growth of the murine LLC1 tumor shapes the lung microbial composition by favoring the expansion of microbes with potential immunosuppressive activity eventually affecting the tumor progression. Moreover, our findings unravel the therapeutic potential of localized antibiotics to revert the immunosuppressive microenvironment by targeting tumor-associated bacteria. Citation Format: Giancarla Bernardo, Valentino Le Noci, Elena Montanari, Emerenziana Ottaviano, Giorgio Gargari, Elisa Borghi, Simone Guglielmetti, Federico Ambrogi, Tiziana Triulzi, Elda Tagliabue, Serenella M. Pupa, Michele Sommariva, Lucia Sfondrini. Antibiotics aerosolization shrinks intratumoral Tregs and impairs lung tumor growth by perturbing the taxonomic structure of tumor-associated microbiota [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1281.

Abstract 3862: Identification, profiling and clonotypic tracking of tumor-reactive T cells derived from bronchoalveolar lavage in lung cancer patients

Abstract Immunotherapeutic strategies for disinhibiting T cell responses have revolutionized lung cancer treatment. However, the identification of tumor-reactive T cells and the acquisition of adequate tissue for molecular and immune profiling remains a challenge that hinders our ability to adequately characterize the mechanisms of immunotherapy response versus resistance. To address this gap, we are investigating the use of bronchoalveolar lavage (BAL) as an alternative to blood or tumor biopsy samples to identify tumor reactive T cells. We collected cells from a prospective bronchoscopic biorepository of patients undergoing lung cancer evaluation that were subjected to bilateral BAL, phlebotomy, and tissue sampling. We then employed flow cytometry and single-cell multi-omic analyses to identify, characterize and clonotypically track tumor-reactive T cells obtained from BAL, blood and tumor samples from lung cancer patients. Analysis of BAL samples revealed a significant enrichment of terminally exhausted CD8+CD39+CD103+ T cells in BAL from tumor-bearing lungs compared to non-tumor bearing lung BAL and blood PBMCs in 14 patients. We then used single-cell sequencing-based multi-omics to deeply profile bilateral BAL cells, blood PBMCs and tumor tissues from 5 of these patients. By tracking T cell receptor (TCR) sequences across patient-matched samples, we found BAL derived T cell clonotypes that expressed markers of terminal exhaustion, such as CD39 and CXCL13 in the tumor, which strongly implicates them as being tumor reactive and relatively abundant in BAL. Further validation through functional assays is being used to confirm the tumor reactivity of these clones. These data demonstrate the feasibility of capturing tumor-specific T cells during bronchoscopy, providing promising opportunities for patient outcome characterization and exploration of novel therapies. This relatively non-invasive and practical test during routine bronchoscopy offers a cost-effective and safe alternative to identifying tumor-reactive T cells from surgical specimens or biopsies. Citation Format: Heeju Ryu, Wei Sun, Michelle A. Wurscher, Viswam S. Nair, Evan W. Newell. Identification, profiling and clonotypic tracking of tumor-reactive T cells derived from bronchoalveolar lavage in lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3862.

Abstract B020: Charting the cellular heterogeneity of primary and metastatic PDAC microenvironment

Abstract Cancer-associated fibroblasts (CAFs) are important regulators of tumor progression and therapeutic response in PDAC, with several functionally diverse populations now identified. However, while CAF populations with similar transcriptional characteristics have been identified across different tissues, emerging data suggests that these populations may diverge functionally. For example, whereas antigen presenting CAFs (apCAFs) have been suggested to promote immune evasion in PDAC, apCAFs have been associated with anti-tumor immunity in lung cancer. Thus, high resolution mapping of the microenvironment in both primary and metastatic tumors is required to define tissue specific and conserved subsets for functional interrogation. We deployed cytometry by time of flight (CyTOF) to annotate stromal and immune subsets in healthy and tumor bearing murine pancreas, liver and lung. Spatial proximity of tumor and host cells was further annotated using sLP-mCherry, a secreted lipo-permeable form of mCherry. In total, we annotated 50.3 million cells across normal and tumor bearing tissues. Stromal fibroblast populations were highly divergent across both healthy and tumor bearing tissues. Commonly used markers were expressed in a manner dominated by tissue of origin, with less than 10% of fibroblasts and CAFs displaying common phenotypes across tissues. For example, aSMApos/PDGFRalow myCAFs were abundant in lung and liver, Ly6cpos/PDGFRapos/aSMAlow iCAFs were more abundant in the pancreas and MHCIIpos/CD74pos apCAFs were more dominant in lung. In contrast, most immune subsets, with exception of alveolar macrophages and Kupfer cells, were consistently observed across all tissues analysed albeit at different abundances. Tumors arising from epithelial and mesenchymal PDAC cells induce unique changes in CAF subset abundances where epithelial tumors are enriched for myCAFs and mesenchymal tumors induce more apCAFs. These observations were consistent across tumor bearing liver and lung. MyCAFs and PD-L1 or PDL-2 expressing macrophages were generally more proximal to epithelial tumor cells, whereas tumors originating from mesenchymal PDAC cells were enriched for antigen-experienced T cells. Taken together these analyses reveal tissue specific effects on the emerging microenvironment, with shared properties emerging from immune and mesenchymal niche composition. Citation Format: Adrian Blanco-Gomez, Catherine Felton, Antonia Banyard, Claus Jorgensen. Charting the cellular heterogeneity of primary and metastatic PDAC microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B020.

Abstract A177: In vivo TuBa-seq growth profiling identifies a differential role of the Tbx2 subfamily in oncogene-negative versus Kras-driven lung cancers

Abstract Lung adenocarcinoma (LUAD) is a complex cancer driven by diverse combinations of oncogenic and tumor suppressive events. The TBX2 subfamily genes, including TBX2, TBX3, TBX4, and TBX5, are known to play a crucial role in lung development as master transcriptional regulators. Our previous clinical and in vitro studies found that their expression is significantly suppressed in LUADs, suggesting a potential tumor suppressor role through modulating tumor methylation patterns in early tumorigenesis. However, the role of TBX2 subfamily genes in other cancer types is paradoxical, with both oncogenic and tumor suppressive effects being reported. Herein, we employed the Tumor Barcoding with Ultradeep Sequencing (Tuba-seq) and CRISPR/Cas9-mediated genome editing technologies to investigate the putative tumor suppressive role of TBX2 genes functionally and quantitatively and their downstream targets in genetically engineered mouse models of LUAD. Lung tumors were initiated by intratracheal intubation with pooled lentiviral-Cre vectors targeting each gene independently with two different sgRNAs per gene. To enable the unique identification of each tumor and its corresponding sgRNA, we utilized Lenti-sgRNA/Cre vectors harboring barcodes specific to each targeted gene. Initial assessment of tumor burden was carried out through fluorescence microscopy, lung weight measurements, and histological analysis. To simultaneously determine the number of neoplastic cells corresponding to each gene knockout, the barcoded region was amplified from genomic DNA extracted from bulk tumor-bearing lung samples and sequenced. Quantification of tumor sizes was carried at 6 and 20 weeks after tumor initiation. Hyperplasia, adenomas, and/or early adenocarcinomas within the lungs were detected among different genetic backgrounds and throughout the study time intervals. Specifically, the inactivation of Tbx2, Tbx3, Tbx4, Tbx5, and Cdh2, increased tumor initiation and growth in the oncogene-negative mouse model (n=8). However, strikingly, inactivation of Tbx3, Tbx4, and Cdh2 suppressed tumorigenesis in a KRAS-driven genetic background while Tbx2, Tbx5, and Tnfaip3, consistently exhibited tumor suppressive effects (n=9). Further investigation is needed to understand these newly discovered interactions between the TBX2 subfamily critical developmental pathway and Ras signaling in modulating lung cancer progression. These insights emphasize the importance of considering tumor suppressor phenotypic heterogeneity and their context-dependent roles when developing targeted therapeutic approaches for lung adenocarcinoma and other cancers. Citation Format: Athar Khalil, Mira Rahm, Zachary Faber, Madeline Bedrock, Xiangzhen Wei, Bindi Patel, Christopher Mcfarland. In vivo TuBa-seq growth profiling identifies a differential role of the Tbx2 subfamily in oncogene-negative versus Kras-driven lung cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A177.

Inhalable siRNA Nanoparticles for Enhanced Tumor-Targeting Treatment of KRAS-Mutant Non-Small-Cell Lung Cancer.

Kirsten rat sarcoma (KRAS) is the most commonly mutated oncogene in lung cancers. Gene therapy is emerging as a promising cancer treatment modality; however, the systemic administration of gene therapy has been limited by inefficient delivery to the lungs and systemic toxicity. Herein, we report a noninvasive aerosol inhalation nanoparticle (NP) system, termed "siKRAS@GCLPP NPs," to treat KRAS-mutant non-small-cell lung cancer (NSCLC). The self-assembled siKRAS@GCLPP NPs are capable of maintaining structural integrity during nebulization, with preferential distribution within the tumor-bearing lung. Inhalable siKRAS@GCLPP NPs show not only significant tumor-targeting capability but also enhanced antitumor activity in an orthotopic mouse model of human KRAS-mutant NSCLC. The nebulized delivery of siKRAS@GCLPP NPs demonstrates potent knockdown of mutated KRAS in tumor-bearing lungs without causing any observable adverse effects, exhibiting a better biosafety profile than the systemic delivery approach. The results present a promising inhaled gene therapy approach for the treatment of KRAS-mutant NSCLC and other respiratory diseases.

RHAMM regulates MMTV-PyMT-induced lung metastasis by connecting STING-dependent DNA damage sensing to interferon/STAT1 pro-apoptosis signaling

BackgroundRHAMM is a multifunctional protein that is upregulated in breast tumors, and the presence of strongly RHAMM+ve cancer cell subsets associates with elevated risk of peripheral metastasis. Experimentally, RHAMM impacts cell cycle progression and cell migration. However, the RHAMM functions that contribute to breast cancer metastasis are poorly understood.MethodsWe interrogated the metastatic functions of RHAMM using a loss-of-function approach by crossing the MMTV-PyMT mouse model of breast cancer susceptibility with Rhamm−/− mice. In vitro analyses of known RHAMM functions were performed using primary tumor cell cultures and MMTV-PyMT cell lines. Somatic mutations were identified using a mouse genotyping array. RNA-seq was performed to identify transcriptome changes resulting from Rhamm-loss, and SiRNA and CRISPR/Cas9 gene editing was used to establish cause and effect of survival mechanisms in vitro.ResultsRhamm-loss does not alter initiation or growth of MMTV-PyMT-induced primary tumors but unexpectedly increases lung metastasis. Increased metastatic propensity with Rhamm-loss is not associated with obvious alterations in proliferation, epithelial plasticity, migration, invasion or genomic stability. SNV analyses identify positive selection of Rhamm−/− primary tumor clones that are enriched in lung metastases. Rhamm−/− tumor clones are characterized by an increased ability to survive with ROS-mediated DNA damage, which associates with blunted expression of interferon pathway and target genes, particularly those implicated in DNA damage-resistance. Mechanistic analyses show that ablating RHAMM expression in breast tumor cells by siRNA knockdown or CRISPR-Cas9 gene editing blunts interferon signaling activation by STING agonists and reduces STING agonist-induced apoptosis. The metastasis-specific effect of RHAMM expression-loss is linked to microenvironmental factors unique to tumor-bearing lung tissue, notably high ROS and TGFB levels. These factors promote STING-induced apoptosis of RHAMM+ve tumor cells to a significantly greater extent than RHAMM−ve comparators. As predicted by these results, colony size of Wildtype lung metastases is inversely related to RHAMM expression.ConclusionRHAMM expression-loss blunts STING-IFN signaling, which offers growth advantages under specific microenvironmental conditions of lung tissue. These results provide mechanistic insight into factors controlling clonal survival/expansion of metastatic colonies and has translational potential for RHAMM expression as a marker of sensitivity to interferon therapy.

Quantitative proteomic analysis of bronchoalveolar lavage fluids from patients with small cell lung cancers.

Small cell lung cancer (SCLC) is one of the malignant cancers with aggressive progression and poor prognosis. Bronchoalveolar lavage fluid (BALF) has been arising recently as a potential source of biomarkers for lung cancers. In this study, we performed quantitative BALF proteomic analysis to identify potential biomarkers for SCLC. BALF were collected from tumor-bearing lungs and non-tumor lungs of five SCLC patients. Then, BALF proteomes were prepared for a TMT-based quantitative mass spectrometry analysis. Differentially expressed proteins (DEP) were identified when considering individual variation. Potential SCLC biomarker candidates were validated by immunohistochemistry (IHC). A public database of multiple SCLC cell lines was used to evaluate the correlation of these markers with SCLC subtypes and chemo-drug responses. We identified 460 BALF proteins in SCLC patients and observed considerable individual variation among the patients. Immunohistochemical analysis and bioinformatics resulted in the identification of CNDP2 and RNPEP as potential subtype markers for ASCL1 and NEUROD1, respectively. In addition, CNDP2 was found to be positively correlated with responses to etoposide, carboplatin, and irinotecan. BALF is an emerging source of biomarkers, making it useful for the diagnosis and prognosis of lung cancers. We characterized the proteomes of paired BALF samples collected from tumor-bearing and non-tumor lungs of SCLC patients. Several proteins were found elevated in tumor-bearing BALF, and especially CNDP2 and RNPEP appeared to be potential indicators for ASLC1-high and NEUROD1-high subtypes of SCLC, respectively. The positive correlation of CNDP2 with chemo-drug responses would help to make decisions for treatment of SCLC patients. These putative biomarkers could be comprehensively investigated for a clinical use towards precision medicine.

Circadian disruption enhances HSF1 signaling and tumorigenesis in Kras-driven lung cancer.

Disrupted circadian rhythmicity is a prominent feature of modern society and has been designated as a probable carcinogen by the World Health Organization. However, the biological mechanisms that connect circadian disruption and cancer risk remain largely undefined. We demonstrate that exposure to chronic circadian disruption [chronic jetlag (CJL)] increases tumor burden in a mouse model of KRAS-driven lung cancer. Molecular characterization of tumors and tumor-bearing lung tissues revealed that CJL enhances the expression of heat shock factor 1 (HSF1) target genes. Consistently, exposure to CJL disrupted the highly rhythmic nuclear trafficking of HSF1 in the lung, resulting in an enhanced accumulation of HSF1 in the nucleus. HSF1 has been shown to promote tumorigenesis in other systems, and we find that pharmacological or genetic inhibition of HSF1 reduces the growth of KRAS-mutant human lung cancer cells. These findings implicate HSF1 as a molecular link between circadian disruption and enhanced tumorigenesis.

Abstract 936: Developing a sex-biased model for LKB1-mutant lung adenocarcinoma

Abstract Liver kinase B1 (LKB1) is the third most frequently mutated gene in lung adenocarcinoma (LUAD), and LKB1-mutant lung cancers are resistant to existing targeted therapy or immune checkpoint blockade therapy. In the TCGA-LUAD dataset, LKB1 was found to be more frequently mutated in males than in females even after correcting for potential confounders (male versus female: 22.8% versus 11.3%, p &amp;lt; 6.9x 10-4, FDR = 0.033). Similar trends can be found in all other large LUAD datasets. However, the mechanistic basis of this bias is unknown because the existing mouse models of lung cancers either did not exhibit sex bias or showed female mice being more susceptible to the development of lung cancers. Here, we demonstrate that our lentivirus-inducing Kras/LKB1-mutant genetically engineered mouse model (GEMM) and tail-vein injection syngeneic model exhibit sex bias against the tumor formation in the female host. In our KRASLSL-G12D/LKB1fl/fl/Rosa-Luc GEMM mice, the lung tumor formation was monitored by bioluminescence imaging (BLI) and validated by immunohistochemistry of tumor bearing-lung. The sex bias of time to lung adenocarcinoma formation in males and females was determined by the Kaplan-Meier method. 97% (31/32) male GEMM developed LUAD after the induction, whereas only 58% (15/26) female GEMM developed LUAD (p=0.0001, log-rank). The tail-vein injection method used the WRJ388-AD1 cell line, which was derived from WRJ388. Tumor growth in the chest area was monitored by BLI and evaluated by the mixed-effects model. Sex bias preference for developing lung tumors in the male host reached significance on day 14 post-injection (p=0.0037) and lasted during the study (day 21, p=0.0021). On day 21, with the cutoff as 1x106 p/sec/cm2/sr in lung region, 100% male GEMM formed lung tumor while only 36% female GEMM formed lung tumor after the injection. Collectively, this KRAS/LKB1-mutant lung adenocarcinoma exhibits sex-biased tumor formation and growth, which are consistent with clinical data among LUAD patients. This model will provide researchers with opportunities to understand the mechanistic basis of this sex bias and to develop novel personalized therapies. Citation Format: Yijian Fan, Rui Jin, Xiuju Liu, Yuan Liu, Chunzi Huang, Mellissa Gilbert-Ross, Adam Marcus, Wei Zhou. Developing a sex-biased model for LKB1-mutant lung adenocarcinoma [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 936.

IR792-MCN@ZIF-8-PD-L1 siRNA drug delivery system enhances photothermal immunotherapy for triple-negative breast cancer under near-infrared laser irradiation

BackgroundDespite extensive investigations on photothermal therapy, the clinical application is restricted due to poor stability, low therapeutic efficacy of photothermal therapy agents and its affinity loss in the multistep synthesis of delivery carriers. To address this, we designed an IR792-MCN@ZIF-8-PD-L1 siRNA (IM@ZP) nanoparticle drug delivery system. IM@ZP was prepared by in situ synthesis and physical adsorption, followed by characterization. Photothermal conversion ability of IM@ZP was assessed by irradiation of near-infrared (NIR) laser, followed by analysis of its effect on 4T1 cell viability, maturation of dendritic cells (DCs) and the secretion of related cytokines in vitro, and the changes of tumor infiltrating T cells and natural killer (NK) cells in vivo. Subcutaneous 4T1 tumor-bearing mouse and lung metastasis models were established to investigate the role of IM@ZP in killing tumor and inhibiting metastasis in vivo.ResultsIM@ZP was uniform nanoparticles of 81.67 nm with the characteristic UV absorption peak of IR792, and could effectively adsorb PD-L1 siRNA. Under the irradiation of 808 nm laser, IM@ZP exhibited excellent photothermal performance. IM@ZP could be efficiently uptaken by 4T1 cells, and had high transfection efficiency of PD-L1 siRNA. Upon NIR laser irradiation, IM@ZP effectively killed 4T1 cells, upregulated HSP70 expression, induced DC maturation and increased secretion of TNF-α and IL-6 in vitro. Moreover, in vivo experimental results revealed that IM@ZP enhanced photothermal immunotherapy as shown by promoted tumor infiltrating CD8 + and CD4 + T cells and NK cells, and inhibited tumor growth and lung metastasis.ConclusionTogether, biocompatible IM@ZP nanoparticles result in high photothermal immunotherapy efficiency and may have a great potential as a delivery system for sustained cancer therapy.Graphical

In Situ biomimetic Nanoformulation for metastatic cancer immunotherapy

Metastasis is the leading cause of death in cancer patients. Eliciting anti-tumor immune responses against lung metastasis is hindered by the immunosuppressive microenvironment. This study explored a biomimetic nanoformulation, comprising a nanovaccine (OP) that delivers tumor antigens and adjuvants spatially and temporally in a virus-like manner, and a pulmonary surfactant-biomimetic liposome with an immunomodulator, JQ1 (PS-JQ1). The findings of this study showed that intratracheal administration of OP+PS-JQ1 activated lung immune cells without concomitant excess inflammation, enhanced tumor antigen cross-presentation, generated a significantly high antigen-specific CD8+ T cell response, and reshaped the immunocellular composition in B16 melanoma tumor-bearing lung. OP+PS-JQ1 nanoformulation exhibited a striking immunotherapeutic efficacy, induced local and systemic tumor suppression, improved survival of mice, initiated immune memory that prevents recurrence of secondary tumors. This stable and nontoxic nanoformulation provides a simple, flexible, and robust strategy for augmenting anti-tumor immunity for metastatic cancer. Statement of significanceEgg glue proteins are produced by female insects, which can make the eggs firmly attached to the oviposition sites, not affected by wind and rain. However, genes encoding insect egg glue proteins have not yet been reported, and the molecular mechanism underpinning their adhesion is still unknown. Our study makes a significant contribution to the literature as it identifies the sequence, structure, adhesive property, and mechanism of silkworm egg glue protein. Furthermore, it outlines key insights into the structure-function relationships associated with egg glue proteins. We believe that this paper will be of interest to the readership of your journal as it identifies the first complete sequence of insect egg glue proteins, thereby highlighting their potentials future applications in both the biomedical and technical fields.

Fractionated Radiation Severely Reduces the Number of CD8+ T Cells and Mature Antigen Presenting Cells Within Lung Tumors.

The combination of standard-of-care radiation therapy (RT) with immunotherapy is moving to the mainstream of non-small cell lung cancer treatment. Multiple preclinical studies reported on the CD8+ T cell stimulating properties of RT, resulting in abscopal therapeutic effects. A literature search demonstrates that most preclinical lung cancer studies applied subcutaneous lung tumor models. Hence, in-depth immunologic evaluation of clinically relevant RT in orthotopic lung cancer models is lacking. We studied the therapeutic and immunologic effects of low-dose fractionated RT on lungs from C57BL/6 mice, challenged 2 weeks before with firefly luciferase expressing Lewis lung carcinoma cells via the tail vein. Low-dose fractionation was represented by 4 consecutive daily fractions of image guided RT at 3.2 Gy. We showed reduced lung tumor growth upon irradiation using in vivo bioluminescence imaging and immunohistochemistry. Moreover, significant immunologic RT-induced changes were observed in irradiated lungs and in the periphery (spleen and blood). First, a significant decrease in the number of CD8+ T cells and trends toward more CD4+ and regulatory T cells were seen after RT in all evaluated tissues. Notably, only in the periphery did the remaining CD8+ T cells show a more activated phenotype. In addition, a significant expansion of neutrophils and monocytes was observed upon RT locally and systemically. Locally, RT increased the influx of tumor-associated macrophages and conventional type 2 dendritic cells, whereas the alveolar macrophages and conventional type 1 DCs dramatically decreased. Functionally, these antigen-presenting cells severely reduced their CD86 expression, suggesting a reduced capacity to induce potent immunity. Our results imply that low-dose fractionated RT of tumor-bearing lung tissue shifts the immune cell balance toward an immature myeloid cell dominating profile. These data argue for myeloid cell repolarizing strategies to enhance the abscopal effects in patients with non-small cell lung cancer treated with fractionated RT.

NSG Mice Facilitate ex vivo Characterization of Ewing Sarcoma Lung Metastasis Using the PuMA Model.

Ewing sarcoma (EwS) is a highly malignant bone and soft tissue tumor primarily affecting children and young adults. While most patients initially respond well to conventional front-line therapy, frequent metastasis results in poor 5-year overall survival rates for this disease. Accordingly, there is a critical need to develop better models to understand EwS metastasis. We and others previously used the ex vivo pulmonary metastasis assay (PuMA) to study lung metastasis in solid tumors including osteosarcoma (OS), but this technique has to date not been achievable for EwS. PuMA involves tail vein injection of fluorescent tumor cells into NOD-SCID mice, followed by their visualization in long-term cultures of tumor-bearing lung explants. Here we demonstrate successful implementation of PuMA for EwS cells using NOD-SCID-IL2 receptor gamma null (NSG) immunocompromised mice, which demonstrated high engraftment of EwS cell lines compared to NOD-SCID mice. This may be linked to immune permissiveness required by EwS cells, as increased basal cytotoxicity of EwS cells was observed in NOD-SCID compared to NSG lung sections, possibly due to the absence of natural killer (NK) cell activity in the latter. Together, our data demonstrate the utility of NSG mice for PuMA modeling of EwS lung metastasis.

52350 PKM2 mediates anti-tumor immunity and T cell dysfunction

ABSTRACT IMPACT: T cell dysfunction is a dominant suppressor of anti-tumor immunity, reducing immunotherapeutic efficacy and benefit to patients; our work will identify novel mediators of this process for both therapeutic potential and underlying mechanism, allowing for both potential immediate clinical utility and identification of future targets based on new mechanistic insights. OBJECTIVES/GOALS: T cell dysfunction is a dominant suppressor of anti-tumor immunity, reducing immunotherapeutic efficacy and clinical benefit to the majority of patients. We aim to interrogate a novel mediator of dysfunction identified from transcriptome analyses, pyruvate kinase muscle isozyme isoform 2 (PKM2), for therapeutic utility and underlying mechanism. METHODS/STUDY POPULATION: Transcriptome analyses of CD8+ lymphocytes from tumor-bearing lungs from both murine KrasG12D p53-/- and human non-small cell lung cancer (NSCLC) patients were performed, and differentially expressed genes identified. Flow cytometric analyses for PKM isoform expression and effects of target knockdown on accumulation of dysfunctional characteristics, including checkpoint and transcription factor expression, proliferation, and cytokine production, were performed using an in vitro co-culture of murine antigen-specific T (OT-I) cells and antigen-expressing NSCLC (HKP1-ova) cells. In vivo examination of the same was performed using adoptive transfer of OT-I cells into immunocompetent recipient mice with engraftment of HKP1-ova cells, and subsequent evaluation of mouse survival and T cell phenotypes. RESULTS/ANTICIPATED RESULTS: Transcriptome analyses demonstrated that PKM expression was upregulated in dysfunctional T cells from both murine and human samples. This was confirmed both in vitro with co-culture and in vivo with adoptive transfer approaches, with both activated and dysfunctional OT-I cells expressing higher levels of isoform 2 of PKM than naive OT-I cells. Expression of PKM2 mimicked the kinetics of the transcription factor Tox, a known driver of dysfunction, and knockdown of PKM2 resulted in reduced granzyme B expression, and increased proportions of progenitors with fewer terminally differentiated dysfunctional cells. Knockdown of PKM2 in adoptively-transferred OT-I cells led to enhanced tumor control; results are being extended to other tumor models, and T cells metabolically profiled with PKM2 manipulation. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work identified a novel mediator of dysfunction whose targeting has the potential to enhance anti-tumor immunity. Mechanistically, targeting PKM2 led to altered T cell differentiation to a dysfunctional state, linking metabolic phenotypes to these traits and underlining the importance and therapeutic potential of T cell metabolic pathways.

Macrophage-derived exosomal miR-342-3p promotes the progression of renal cell carcinoma through the NEDD4L/CEP55 axis.

Due to its difficulty in early diagnosis and lack of sensitivity to chemotherapy and radiotherapy, renal cell carcinoma (RCC) remains to be a frequent cause of cancer-related death. Here, we probed into new targets for its early diagnosis and treatment for RCC. microRNA (miRNA) data of M2-EVs and RCC were searched on the Gene Expression Omnibus database, followed by the prediction of the potential downstream target. Expression of target genes was measured via RT-qPCR and Western blot, respectively. M2 macrophage was obtained via flow cytometry with M2-EVs extracted. The binding ability of miR-342-3p to NEDD4L and to CEP55 ubiquitination was studied with their roles in the physical abilities of RCC cells assayed. Subcutaneous tumor-bearing mouse models and lung metastasis models were prepared to observe in vivo role of target genes. M2-EVs induced RCC growth and metastasis. miR-342-3p showed high expression in both M2-EVs and RCC cells. M2-EVs carrying miR-342-3p promoted RCC cell abilities to proliferate, invade and migrate. In RCC cells, M2-EV-derived miR-342-3p could specifically bind to NEDD4L and consequently elevate CEP55 protein expression via suppressing NEDD4L, thereby exerting tumor-promoting effects. CEP55 could be degraded by ubiquitination under the function of NEDD4L, and miR-342-3p delivered by M2-EVs facilitated the RCC occurrence and development by activating the PI3K/AKT/mTOR signaling pathway. In conclusion, M2-EVs promote RCC growth and metastasis by delivering miR-342-3p to suppress NEDD4L and subsequently inhibit CEP55 ubiquitination and degradation via activation of the PI3K/AKT/mTOR signaling pathway, strongly driving the proliferative, migratory and invasive of RCC cells.

Metastasis-associated macrophages constrain antitumor capability of natural killer cells in the metastatic site at least partially by membrane bound transforming growth factor β

BackgroundMetastatic breast cancer is a leading cause of cancer-related death in women worldwide. Infusion of natural killer (NK) cells is an emerging immunotherapy for such malignant tumors, although elimination of...

Suppression of Metastatic Melanoma Growth in Lung by Modulated Electro-Hyperthermia Monitored by a Minimally Invasive Heat Stress Testing Approach in Mice.

Simple SummaryThe lung is the most frequent site of distant melanoma metastases. Metastases of melanoma in the lungs offer a very poor prognosis, with a 5-year survival rate of below 10%. Hyperthermic therapies including modulated electro-hyperthermia (mEHT) in clinical settings have been used to improve the efficacy of radiotherapy, chemotherapy, and immunotherapy of tumors. In this study, we focused primarily on the optimization of mEHT for targeted lung treatment of mice lungs burdened with B16F10 melanoma pulmonary metastases, with a particular focus on elucidating the mechanism of action of mEHT on treated melanoma cells while investigating any potential treatment-related side effects on normal lung tissue. mEHT showed evidence of significant anti-tumor effects as demonstrated by the reduced number of pulmonary metastatic nodules, DNA damage response, downregulation of Ki67 expression, higher immune cell infiltration, and upregulation of p21waf1 expression in mEHT-treated tumors.Modulated electro-hyperthermia (mEHT) is a novel complementary therapy in oncology which is based on the higher conductivity and permittivity of cancerous tissues due to their enhanced glycolytic activity and ionic content compared to healthy normal tissues. We aimed to evaluate the potential of mEHT, inducing local hyperthermia, in the treatment of pulmonary metastatic melanoma. Our primary objective was the optimization of mEHT for targeted lung treatment as well as to identify the mechanism of its potential anti-tumor effect in the B16F10 mouse melanoma pulmonary metastases model while investigating the potential treatment-related side effects of mEHT on normal lung tissue. Repeated treatment of tumor-bearing lungs with mEHT induced significant anti-tumor effects as demonstrated by the lower number of tumor nodules and the downregulation of Ki67 expression in treated tumor cells. mEHT treatment provoked significant DNA double-strand breaks indicated by the increased expression of phosphorylated H2AX protein in treated tumors, although treatment-induced elevation of cleaved/activated caspase-3 expression was insignificant, suggesting the minimal role of apoptosis in this process. The mEHT-related significant increase in p21waf1 positive tumor cells suggested that p21waf1-mediated cell cycle arrest plays an important role in the anti-tumor effect of mEHT on melanoma metastases. Significantly increased CD3+, CD8+ T-lymphocytes, and F4/80+CD11b+ macrophage density in the whole lung and tumor of treated animals emphasizes the mobilizing capability of mEHT on immune cells. In conclusion, mEHT can reduce the growth potential of melanoma, thus offering itself as a complementary therapeutic option to chemo- and/or radiotherapy.