Lung Cancer Mouse Research Articles

HMGB1 secretion by resveratrol in NSCLC: A pathway to ferroptosis-mediated platelet activation suppression.

Cancer-associated venous thromboembolism (CAT) is a frequent and serious complication in cancer patients. Resveratrol, a natural compound with reported anti-tumor effects, is not fully understood in its role regarding CAT in lung cancer. This study aims to explore resveratrol's potential to diminish platelet activation induced by lung adenocarcinoma cells and uncover the underlying mechanisms. Clinical data on coagulation function in non-small cell lung cancer (NSCLC) patients were gathered. A549 human lung cancer cells and Lewis mouse lung cancer cells were employed to assess tumor-induced platelet activation and the impact of resveratrol on this process. Western blotting analyzed protein expression, electron microscopy examined extracellular vesicle (EV) morphology, flow cytometry measured platelet activation, reactive oxygen species (ROS), and exocrine protein expression, while ELISA quantified secretory proteins. Tumor control and platelet function were studied in tumor-bearing mice. We identified that hematological profiles of NSCLC patients frequently manifest a hypercoagulable state relative to healthy controls and lung cancer cells could instigate platelet activation, yet resveratrol could attenuate this phenomenon induced by lung cancer. Resveratrol stimulates lung cancer cells to release HMGB1-enriched EVs, promoting platelet ferroptosis and inhibiting platelet activation through increased ROS, lipid peroxidation, and disrupted cystine transporters. In vivo studies confirm that resveratrol inhibits lung cancer cell growth and suppresses tumor-induced platelet activation in mice. Our studies revealed that resveratrol alleviated lung cancer-induced ferroptosis associated platelet activation. This suggests a potential pharmacological approach for preventing and treating both lung cancer and CAT.

RadioFlow Cytometry Reveals That [18F]FDG Uptake in K-RAS Lung Cancer Is Driven by Immune Cells: An Analysis on a Single-Cell Level.

Tumor metabolism is a hallmark of cancer, yet cellular heterogeneity within the tumor microenvironment presents a significant challenge, as bulk analysis masks the diverse metabolic profiles of individual cell populations. This complexity complicates our understanding of [18F]FDG uptake by distinct cell types in the tumor microenvironment. This study aims to investigate [18F]FDG uptake at the single-cell level in the lung of Kirsten rat sarcoma virus-driven cancer mouse models using the novel technique radio-flow cytometry (radioFlow). Methods: Two Kirsten rat sarcoma virus-driven lung cancer mouse models were injected with [18F]FDG for small-animal PET/CT and subsequent fluorescence-activated cell sorting of the lung. For radioFlow, the sorted cell fractions were then measured in a γ-counter and their radioactivity was normalized to the number of cells. Results: RadioFlow analysis of the lung tissue of both models showed a robust cell type-specific uptake pattern across experiments. Our key findings indicate that the [18F]FDG PET signal predominantly derives from immune cells (CD45+, F4/80-, 78.3% ± 6.6%; macrophage, 13.9% ± 4.3%), whereas tumor cells contributed only with 2.8% ± 1.0%, similar to the uptake of structural cells (CD45-; tumor cells, 5.0% ± 2.3%). Normalization showed that macrophages exhibited the highest glucose metabolism in both tumor models (57% ± 8%), followed by the remaining immune cells (27% ± 3%). Conclusion: These findings highlight the critical influence of immune cell metabolism on [18F]FDG imaging, emphasizing the need to account for immune contributions when interpreting [18F]FDG imaging in cancer.

Broad-spectrum efficacy of CEACAM6-targeted antibody-drug conjugate with BET protein degrader in colorectal, lung, and breast cancer mouse models.

Despite remarkable advances in cancer treatment, most solid cancers remain difficult to cure. We recently developed an antibody-drug conjugate (ADC, 84-EBET) for pancreatic cancer by using the carcinoembryonic-antigen-related cell-adhesion molecule 6 (CEACAM6) antibody #84.7 and the bromodomain and extra-terminal (BET) protein degrader EBET. Here, we showed the overexpression of CEACAM6 in colorectal, lung, and breast cancers (CRC, LC, BC) and the broad-spectrum efficacy of 84-EBET in mouse models of these cancers. In vitro assays using cancer organoids and cell lines of CRC, LC, and BC revealed that 84-EBET was more potent than ADCs with known approved payloads-DXd, SN38, and monomethyl auristatin E (MMAE)-or standard chemotherapies. In mouse studies, a single injection of 84-EBET induced marked regression of CRC-, LC-, and BC-patient-derived xenograft tumors and cell-line-derived xenograft tumors. Moreover, in mouse syngeneic CRC, LC, and BC models resistant to PD-1 antibody, the combination of 84-EBET and PD-1 antibody induced complete regression of most tumors. Mechanistically, 84-EBET degraded BRD4 protein in both cancer and stromal cells via bystander efficacy. It decreased stromal inflammatory phenotypes and increased activated T-cell numbers in tumors. These results demonstrate that delivering BET protein degraders to tumors and their microenvironments via a CEACAM6-targeted ADC may be effective against a wide range of solid cancers.

Evaluation of anticancer efficacy of survivin si-RNA functionalized combined drug-loaded mesoporous silica nanoparticles in a lung cancer mouse model.

Lung cancer continues to be the leading cause of mortality globally. Nanotechnology-mediated targeted drug delivery approach is one of the promising strategies for the treatment of lung cancer. Due to their multifactorial role, mesoporous silica nanoparticles (MSNs), have attracted a lot of attention for drug delivery. The emerging dual-drug co-delivery approach has drawn much attention due to circumventing various drug-resistant mechanisms in tumor cells. Further, functionalization of si-RNA (survivin) to the dual drugs (etoposide plus carfilzomib) or (docetaxel plus carfilzomib) loaded MSNs can be a potential tool to inhibit gene expression specifically. In the present study, we investigated the comparative therapeutic efficacy of co-delivered anticancer drugs functionalized with survivin siRNA in MSNs for lung cancer. According to our findings, this kind of combination therapy has inhibited the function of the survivin protein while promoting increased therapeutic efficacy due to synergistic pharmacological activity, and found si-RNA- (etoposide plus carfilzomib) to be a better candidate for lung cancer treatment in the future.

A novel oncolytic Vaccinia virus armed with IL-12 augments antitumor immune responses leading to durable regression in murine models of lung cancer.

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.

Rabeprazole inhibits lung cancer progression by triggering NLRP3/CASP-1/caspase-dependent pyroptosis.

Gastric acid-related diseases could be treated using proton pump inhibitors (PPIs), which have been found to have anti-tumor ability. Rabeprazole is a type of PPI whose effect and mechanism in lung cancer remained to be clarified. Lung cancer cells and lung cancer mice were treated with different concentrations of Rabeprazole and then cell proliferation was detected by CCK-8 and colony formation assays. Pyroptosis was assessed by morphological observation and Lactate dehydrogenase (LDH) release assays. Western blot, immunofluorescence and immunohistochemistry were adopted to detect the expressions of GSDMD and NLRP3. Reactive oxygen species (ROS) level, lysosomal damage and autophagic flux were measured by flow cytometry. Rabeprazole suppressed lung cancer cell proliferation and lung tumor growth in mice in a concentration-dependent manner. Lung cancer cells treated with Rabeprazole showed typical pyroptosis morphology and significantly increased LDH release. Rabeprazole upregulated the expression of GSDMD, NLRP3, and cleaved-Caspase 1, but such an effect was partially blocked by Z-LLSD-FMK. In lung cancer cells treated with Rabeprazole and lung cancer mice injected with Rabeprazole, the expressions of GSDMD, NLRP3 and caspase-1 were promoted, ROS-stained cells were increased significantly, lysosomal damage was aggravated, and autophagic flux was noticeably reduced. Rabeprazole activated NLRP3/caspase 1/GSDMD cascade by promoting ROS accumulation and lysosomal destruction, thereby inducing pyroptosis to fulfill its anti-tumor effect on lung cancer.

Ansofaxine suppressed NSCLC progression by increasing sensitization to combination immunotherapy.

Depression negatively impacts the prognosis of various cancers, including lung cancer, by influencing antitumor immune responses and impairing immune cell function. Antidepressants may modulate the tumor immune microenvironment, enhancing immunotherapy efficacy. However, the specific mechanisms remain unclear. This study investigates the effects of the antidepressant Ansofaxine on immune therapy in non-small cell lung cancer (NSCLC) mice with comorbid depression. Chronic unpredictable mild stress (CUMS) and Lewis lung cancer cells (LLC) model was established in mice. Ansofaxine and a combination of triple immunotherapy (anti-PD-1, anti-TNFR2, and anti-PTP1B) were treated in mice to monitor tumor growth and survival rates. Flow cytometry and immunohistochemistry were employed to analyze the dynamics of the immune system, while ELISA kits were used to quantify neurotransmitter levels. Depression accelerated NSCLC progression, evidenced by increased tumor volume, spleen size, and reduced survival rates. Flow cytometry analysis demonstrated a reduction in the population of immune effector cells, with an increase in the proportion of immunosuppressive cells. Ansofaxine inhibited LLC cell proliferation and migration, enhancing apoptosis more effectively than venlafaxine and fluoxetine. Combined with triple immunotherapy, Ansofaxine improved survival rates and enhanced immune responses, increasing CD8+ T cell proportions and decreasing Tregs. Ansofaxine also restored serum serotonin and norepinephrine levels in depressed mice, reduced corticosterone, and decreased PD-L1 and TNFR2 expression in tumor tissues. The findings suggest that Ansofaxine may represent a promising therapeutic approach for NSCLC patients with comorbid depression, potentially enhancing both mental well-being and cancer-related outcomes.

Total nutritional double emulsion-based supplement enriched with low-molecular-weight oyster peptides: A promising nutritional strategy for alleviating cachexia in chemotherapy-treated Lewis lung cancer mice

Total nutritional double emulsion-based supplement enriched with low-molecular-weight oyster peptides: A promising nutritional strategy for alleviating cachexia in chemotherapy-treated Lewis lung cancer mice

Superior Anti-Tumor Response After Microbeam and Minibeam Radiation Therapy in a Lung Cancer Mouse Model

Objectives: The present study aimed to compare the tumor growth delay between conventional radiotherapy (CRT) and the spatially fractionated modalities of microbeam radiation therapy (MRT) and minibeam radiation therapy (MBRT). In addition, we also determined the influence of beam width and the peak-to-valley dose ratio (PVDR) on tumor regrowth. Methods: A549, a human non-small-cell lung cancer cell line, was implanted subcutaneously into the hind leg of female CD1-Foxn1nu mice. The animals were irradiated with sham, CRT, MRT, or MBRT. The spatially fractionated fields were created using two specially designed multislit collimators with a beam width of 50 μm and a center-to-center distance (CTC) of 400 μm for MRT and a beam width of 500 μm and 2000 μm CTC for MBRT. Additionally, the concept of the equivalent uniform dose (EUD) was chosen in our study. A dose of 20 Gy was applied to all groups with a PVDR of 20 for MBRT and MRT. Tumor growth was recorded until the tumors reached at least a volume that was at least three-fold of their initial value, and the growth delay was calculated. Results: We saw a significant reduction in tumor regrowth following all radiation modalities. A growth delay of 11.1 ± 8 days was observed for CRT compared to the sham, whereas MBRT showed a delay of 20.2 ± 7.3 days. The most pronounced delay was observed in mice irradiated with MRT PVDR 20, with 34.9 ± 26.3 days of delay. Conclusions: The current study highlights the fact that MRT and MBRT modalities show a significant tumor growth delay in comparison to CRT at equivalent uniform doses.

Cryoablation plus chemotherapy regimen enhance anti-tumor immune response in a mouse model of Lewis lung cancer

Background Cryoablation (cryo) is a local anti-tumor method and activation of immunity is one of its mechanisms, but it is affected by many factors. Numerous studies have proved that combination therapy based on cryo can activate immunity more effectively and synergistically. Cryo combined with chemotherapy(chemo) has been proven to improve the quality of life and prolong survival of tumor patients, but the immune effect is still unclear. Methods C57B/L6 mouse lung cancer subcutaneous transplanted tumor model was established and sacrificed at two time points after intervention. We observed the effects of cryo + chemo on survival time, tumor growth, and dynamic changes of immune cells and cytokines. Results Cryo + chemo could not only significantly prolong the survival period of mice, inhibit tumor growth and reduce the proliferation activity of tumor cells, but also promote immune response more effectively. Cryo + chemo could increase the number of CD4+ T cells both in spleen and tumor microenvironment, decrease the infiltration of Treg cells in the tumor microenvironment. Besides, cryo + chemo could increase the expression levels of IL-2 and IFN-γ, and reduce the levels of TGF-β. However, it is worth noting that the immune-promoting effect gradually decreases over time. Conclusions Cryo + chemo can effectively inhibit the growth of lung cancer, prolong the survival period and activate the immune response, providing a theoretical basis for the combined treatment. How to maintain the immune response for a long time is the next problem to be solved.

<i>In vivo</i> antitumor effects of bis-benzimidazole derivatives in mouse melanoma and lung cancer models

BACKGROUND: The development of new antitumor agents on the basis of benzimidazoles opens new opportunities for treatment of the malignant tumors, including those refractory to conventional therapies. The benzimidazole derivatives demonstrate wide spectrum of biological activities, specifically antineoplastic effects, and high-level cytotoxicity toward human’s tumor cell lines. The exploration of antineoplastic effects of new synthetic benzimidazole derivatives in in vivo models of tumor growth will help the development of the effective doses and cancer treatment regimens with the use of such compounds. AIM: To assess the antineoplastic effects of the benzimidazole derivatives — monomeric compound MB2Py(Ac) and dimeric compound DB2Py(3) — in the continuous cellular models of Lewis lung carcinoma (LLC) and melanoma B16 in mice. MATERIALS AND METHODS: In vivo assessment of antineoplastic effects was performed in the continuous mouse models of Lewis lung carcinoma (LLC) and B16 melanoma after a single intravenous injection of MB2Py(Ac) and DB2Py(3). Irinotecan was used as a comparator drug. Antitumor effects were measured via standard parameters, such as tumor growth inhibition (TGI%) and tumor growth rate (TGR). RESULTS: The study compounds DB2Py(3) and MB2Py(Ac) used in the study doses and regimen demonstrated mild antitumor effect in the model of murine solid tumors (TGI 50%). In melanoma B16 model, the maximum tumor growth inhibition was 15% and 38.5%, respectively, for MB2Py(Ac) and DB2Py(3). In the lung carcinoma model, the mild effect was observed, e.g. 8% for MB2Py(Ac) and 23.4% for DB2Py(3). The effect of the comparator drug, irinotecan, was more expressive, although short-term. In particular, TGI achieved 52.5% for melanoma B16 and 34.5% for Lewis lung carcinoma (LLC). In general, melanoma B16 was more sensitive to the effects of both bis-benzimidazoles and irinotecan, compared to the lung carcinoma LLC. CONCLUSION: This study shows that the dimeric compound DB2Py(3) was the most promising among three study substances in the models of melanoma and lung carcinoma in mice. Nevertheless, its high toxicity imposes the necessity of further optimization for the clinical use. The substance MB2Py(Ac) demonstrated lowest efficacy, although it may be also explored in the modified treatment regimens.

High throughput application of the NanoBiT Biochemical Assay for the discovery of selective inhibitors of the interaction of PI3K-p110α with KRAS

The NanoBiT Biochemical Assay (NBBA) was designed as a biochemical format of the NanoBiT cellular assay, aiming to screen weak protein-protein interactions (PPIs) in mammalian cell lysates. Here we present a High Throughput Screening (HTS) application of the NBBA to screen small molecule and fragment libraries to identify compounds that block the interaction of KRAS-G12D with phosphatidylinositol 3-kinase (PI3K) p110α. This interaction promotes PI3K activity, resulting in the promotion of cell growth, proliferation and survival, and is required for tumour initiation and growth in mouse lung cancer models, whilst having little effect on the health of normal adult mice, establishing the significance of the p110α/KRAS interaction as an oncology drug target. Despite the weak binding affinity of the p110α/KRAS interaction (KD = 3 μM), the NBBA proved to be robust and displayed excellent Z’-factor statistics during the HTS primary screening of 726,000 compounds, which led to the identification of 8,000 active compounds. A concentration response screen comparing KRAS/p110α with two closely related PI3K isoforms, p110δ and p110γ, identified selective p110α-specific compounds and enabled derivation of an IC50 for these hits. We identified around 30 compounds showing greater than 20-fold selectivity towards p110α versus p110δ and p110γ with IC50 < 2 μM. By using Differential Scanning Fluorimetry (DSF) we confirmed several compounds that bind directly to purified p110α. The most potent hits will be followed up by co-crystallization with p110α to aid further elucidation of the nature of the interaction and extended optimisation of these compounds.

Therapeutic co-assemblies for synergistic NSCLC treatment through dual topoisomerase I and tubulin inhibitors

Camptothecin (CPT) and podophyllotoxin (PPT) function as topoisomerase (TOP) I and tubulin inhibitors, respectively, with potent anticancer effects in a variety of cancers. Despite its promise, the clinical applicability of the combination of CPT and PPT faces challenges, including potential side effect and limited therapeutic efficacy. In this study, we designed co-assembly nanomedicines with the different weight (w/w) ratios of amphiphilic Evans blue conjugated CPT prodrug (EB-ss-CPT) and PPT molecules, denoted as ECT Nano. The co-assembly of EB-ss-CPT and PPT without other excipients has nearly 100% drug loading efficiency and high drug loading content of PPT of up to 74.29 ± 0.90 wt%. Notably, the ECT Nano (1:2) equipped with the ability to inhibit TOP I activity and tubulin polymerization, which provided a highly efficient strategy to improve synergistic efficacy and decrease side toxicity in non-small cell lung cancer mouse model. This work represents a step forward to the development of practical applications for dual TOP I and tubulin inhibitors and especially hopeful to the rational design of combination nanomedicine for therapeutic purposes.

Inhibition of lung tumorigenesis by transient reprogramming in cancer cells

Oncogenic transformation and Oct4, Sox2, Klf4 and c-Myc (OSKM)-mediated induction of pluripotency are two independent and incompatible cellular fates. While continuous expression of OSKM can convert normal somatic cells into teratogenic pluripotent cells, it remains speculative what is the impact of transient OSKM expression in cancer cells. Here, we find that OSKM expression limits the growth of transformed lung cells by inducing apoptosis and senescence. We identify Oct4 and Klf4 as the main individual reprogramming factors responsible for this effect. Mechanistically, the induction of cell cycle inhibitor p21 downstream of the reprogramming factors acts as mediator of cell death and senescence. Using a variety of in vivo systems, including allografts, orthotopic transplantation and KRAS-driven lung cancer mouse models, we demonstrate that transient reprogramming by OSKM expression in cancer cells impairs tumor growth and reduces tumor burden. Altogether, our results show that the induction of transient reprogramming in cancer cells is antitumorigenic opening novel potential therapeutic avenues in oncology.

Effect of Brassica rapa L. Polysaccharide on Lewis Lung Cancer Mice by Inflammatory Regulation and Gut Microbiota Modulation.

Lung cancer is the leading cause of cancer-related fatalities globally, related to inflammatory and gut microbiota imbalance. Brassica rapa L. polysaccharide (BP) is a functional compound, which is utilized by the gut microbiota to regulate immunity and metabolism. However, the effect of BP on lung cancer and whether it affects the "gut-lung" axis remains unclear. This study explored the intervention of BP in Lewis lung cancer (LLC) mice and its effect on the gut microbiota. The results revealed that BP reduced tumor weight and downregulated the expression of Ki67 protein. Additionally, BP reduced the content of inflammatory factors and growth factors, promoting tumor cell apoptosis and inhibiting the growth of LLC. The intervention of BP suppressed intestinal inflammation, preserved intestinal barrier integrity, and augmented the level of beneficial microbiota, such as Blautia and Bifidobacterium. Furthermore, BP significantly increased the production of short-chain fatty acids (SCFAs), particularly butyrate and propionate. A correlation analysis showed significant correlations among the gut microbiota, SCFAs, inflammatory factors, and tight junction proteins. A functional analysis indicated that BP promoted amino acid metabolism and fatty acid metabolism. These findings suggested that BP had the potential to act as prebiotics to prevent disease and improve lung cancer progression by regulating the gut microbiota.

Multifunctional approach with LHRH-mediated PLGA nanoconjugate for site-specific codelivery of curcumin and BCL2 siRNA in mice lung cancer

BackgroundLung cancer remains a leading cancer type, but current chemotherapy is limited by issues including poor drug delivery, toxicity, and resistance. To address these challenges, we developed a novel PLGA-PEG-LHRH (PPL) nanoconjugate system for improved drug delivery. Curcumin, known for its anticancer and P-gp inhibition properties, was co-loaded with bcl2siRNA (bclsR) to inhibit the bcl2 protein, thus overcoming both resistance mechanisms.ResultsThe PPL conjugate was successfully synthesized and characterized using FTIR, 1H NMR, XRD, XPS and BCA assay. Curcumin and bclsR-loaded PLGA nanoemulsions were prepared by double emulsion solvent evaporation method and characterized. The optimized nanoconjugate had size of 179 ± 16 nm, favorable zeta potential, high drug entrapment, and was confirmed via TEM. Controlled release studies indicated 83% drug release within 24 h. In vitro studies revealed significant cytotoxicity against A549 lung cancer cells, with the nanoconjugate showing IC50 of 8.24 µg/mL compared to 21.26 µg/mL for plain curcumin. Enhanced cellular uptake and effective targeting of A549 cells were observed. Molecular analyses demonstrated significant downregulation of MDR1 and Bcl2 RNA and protein expression, highlighting the nanoconjugates' ability to suppress resistance mechanisms. Pharmacokinetic studies in Wistar rats showed superior plasma drug concentrations, half-life, and AUC for the nanoconjugate versus pure drug suspension. Biodistribution studies showed increased drug accumulation in the lungs. In vivo efficacy studies in Balb/c mice demonstrated higher tumor inhibition ratios for CUR-siRNA PPL NPs (66.89%) and CUR-PPL NPs (59.84%) which was further confirmed with TNFα and p53 levels in blood. Histopathological studies showed good healing in the CUR-siRNA PPL NP- and CUR-PPL NP-treated mice compared to suspension.ConclusionFrom the study, it may be concluded that the PPL nanoconjugate system, loaded with curcumin and bcsR, can be potentially effective, multifunctional targeted approach for lung cancer therapy.Graphical

Characterization of A Bronchoscopically Induced Transgenic Lung Cancer Pig Model for Human Translatability.

There remains a need for animal models with human translatability in lung cancer (LC) research. Findings in pigs have high impact on humans due to similar anatomy and physiology. We present the characterization of a bronchoscopically-induced LC model in Oncopigs carrying inducible KRASG12D and TP53R167H mutations. Twelve Oncopigs underwent 29 injections via flexible bronchoscopy. Eighteen Adenovirus-Cre recombinase gene (AdCre) inductions were performed endobronchially (n=6) and transbronchially with a needle (n=12). Eleven control injections were performed without AdCre. Oncopigs underwent serial contrast-enhanced chest CT with clinical follow-up for 29 weeks. Following autopsy, lung and organ tissues underwent histopathology, immunohistochemistry, and RNA-sequencing with comparative analysis with The Cancer Genome Atlas (TCGA) human LC data. All 18 sites of AdCre injections had lung consolidations on CT imaging. Transbronchial injections led to histopathologic invasive cancer and/or carcinoma in situ (CIS) in 11/12 (91.7%), and invasive cancer (excluding CIS) in 8/12 (66.6%). Endobronchial inductions led to invasive cancer in 3/6 (50%). A soft tissue metastasis was observed in one Oncopig. Immunohistochemistry confirmed expression of Pan-CK+/epithelial cancer cells, with macrophages and T cells infiltration in the tumor microenvironment. Transcriptome comparison showed 54.3% overlap with human LC (TCGA), in contrast to 29.88% overlap of KRAS-mutant mouse LC with human LC. The transgenic and immunocompetent Oncopig model has a high rate of LC following bronchoscopic transbronchial induction. Overlap of the Oncopig LC transcriptome with human LC transcriptome was noted. This pig model is expected to have high clinical translatability to the human LC patient.

Silibinin Induces Both Apoptosis and Necroptosis with Potential Anti-tumor Efficacy in Lung Cancer.

The incidence of lung cancer is steadily on the rise, posing a growing threat to human health. The search for therapeutic drugs from natural active substances and elucidating their mechanism have been the focus of anti-tumor research. Silibinin (SiL) has been shown to be a natural product with a wide range of pharmacological activities, including anti-tumour activity. In our work, SiL was chosen as a possible substance that could inhibit lung cancer. Moreover, its effects on inducing tumor cell death were also studied. CCK-8 analysis and morphological observation were used to assess the cytotoxic impacts of SiL on lung cancer cells in vitro. The alterations in mitochondrial membrane potential (MMP) and apoptosis rate of cells were detected by flow cytometry. The level of lactate dehydrogenase (LDH) release out of cells was measured. The expression changes of apoptosis or necroptosis-related proteins were detected using western blotting. Protein interactions among RIPK1, RIPK3, and MLKL were analyzed using the co-immunoprecipitation (co-IP) technique. Necrosulfonamide (Nec, an MLKL inhibitor) was used to carry out experiments to assess the changes in apoptosis following the blockade of cell necroptosis. in vitro, SiL was evaluated for its antitumor effects using LLC tumor-bearing mice with mouse lung cancer. With an increased dose of SiL, the proliferation ability of A549 cells was considerably inhibited, and the accompanying cell morphology changed. The results of flow cytometry showed that after SiL treatment, MMP levels decreased, and the proportion of cells undergoing apoptosis increased. There was an increase in cleaved caspase-9, caspase-3, and PARP, with a down-regulation of Bcl-2 and an up-regulation of Bax. In addition, the amount of LDH released from the cells increased following SiL treatment, accompanied by augmented expression and phosphorylation levels of necroptosis-related proteins (MLKL, RIPK1, and RIPK3), and the co-IP assay further confirmed the interactions among these three proteins, indicating the necrosome formation induced by SiL. Furthermore, Nec increased the apoptotic rate of SiL-treated cells and aggravated the cytotoxic effect of SiL, indicating that necroptosis blockade could switch cell death to apoptosis and increase the inhibitory effect of SiL on A549 cells. In LLC-bearing mice, gastric administration of SiL significantly inhibited tumor growth, and H&E staining showed significant damage to the tumour tissue. The results of the IHC showed that the expression of RIPK1, RIPK3, and MLKL was more pronounced in the tumor tissue. This study confirmed the dual effect of SiL, as it can induce both biological processes, apoptosis and necroptosis, in lung cancer. SiL-induced apoptosis involved the mitochondrial pathway, as indicated by changes in caspase-9, Bcl-2, and Bax. Necroptosis may be activated due to the changes in the expression of associated proteins in tumour cells and tissues. It has been observed that blocking necroptosis by SiL increased cell death efficiency. This study helps clarify the anti-tumor mechanism of SiL against lung cancer, elucidating its role in the dual induction of apoptosis and necroptosis. Our work provides an experimental basis for the research on cell death induced by SiL and reveals its possible applications for improving the management of lung cancer.

High mitochondrial DNA levels accelerate lung adenocarcinoma progression.

Lung adenocarcinoma is a common aggressive cancer and a leading cause of mortality worldwide. Here, we report an important in vivo role for mitochondrial DNA (mtDNA) copy number during lung adenocarcinoma progression in the mouse. We found that lung tumors induced by KRASG12D expression have increased mtDNA levels and enhanced mitochondrial respiration. To experimentally assess a possible causative role in tumor progression, we induced lung cancer in transgenic mice with a general increase in mtDNA copy number and found that they developed a larger tumor burden, whereas mtDNA depletion in tumor cells reduced tumor growth. Immune cell populations in the lung and cytokine levels in plasma were not affected by increased mtDNA levels. Analyses of large cancer databases indicate that mtDNA copy number is also important in human lung cancer. Our study thus reports experimental evidence for a tumor-intrinsic causative role for mtDNA in lung cancer progression, which could be exploited for development of future cancer therapies.

Characterizing dynamic tumor-immune interactions in lung adenocarcinoma through orthotopic allograft modeling.

The major clinical challenge in lung cancer immunotherapy is drug resistance. Therefore, establishing efficient orthotopic lung cancer mouse models to explore the mechanisms of drug immunotherapy resistance is highly important. In this study, we generated multiple fluorescently labeled lung adenocarcinoma cell lines from agenetically engineered KPZ mice model. Orthotopic transplantation of the primary 1F3 cell line induced a strong immune response, causing many small tumors to disappear, but some tumors evaded the immune attack and eventually formed large tumors. Tumor microenvironment analysis demonstrated that M2 macrophages play key roles in the immune response. Further mechanistic studies revealed that the chemokine CCL7 promoted the infiltration of M2 macrophages to facilitate immune escape, thereby promoting tumor growth in the orthotopic mouse model. Moreover, CCL7 levels were elevated in human lung cancer biopsies and positively correlated with M2 macrophage infiltration, and high CCL7 levels predicted advanced pathological stage and poor survival in lung cancer patients. Overall, we established a visualized and orthotopic mouse model with fluorescently labeled cells to better dissect the tumor microenvironment of lung cancer and define the critical role of CCL7 in promoting M2 macrophage polarization and tumorigenesis, providing new preclinical tools and potential targets for lung cancer immunotherapy.