Lewis Lung Carcinoma Cell Line Research Articles

Abstract 6952: The role of interleukin-11 (IL11) in cancer-associated cachexia (CAC)

Abstract Cancer-associated cachexia (CAC), characterized by loss of skeletal muscle and involuntary body weight loss, is responsible for increased chemotherapy toxicity, reduced quality of life, and impaired survival in cancer patients. While interleukin-11 (IL11), one of the IL6 family cytokines, was originally thought to be involved in hematopoiesis, studies increasingly suggest its involvement in tumorigenesis including chemoresistance, metastasis, and survival of cancer cells. However, the precise role of IL11 in CAC has not been determined. Our lab has previously shown that IL11 treatment suppressed the myogenic differentiation of C2C12 myoblasts, suggesting that IL11 may interfere with differentiation and subsequent regeneration of muscle. In this study, we investigated the role of IL11 in cancer-induced muscle wasting using cell culture and mouse models. The mRNA expression of IL11 was highly up-regulated in C26 and Lewis lung carcinoma (LLC) cells, two well-known cachectic mouse cancer cell lines, while its expression was low in a non-cachectic MC38 mouse colon cancer cell line. Knockdown of IL11 in C26 cells using two independent lentiviral shRNAs significantly reduced the mRNA levels of the cytokines involved in CAC including GDF15, LIF, and VEGFA, while the mRNA level of IL6 was not significantly altered. Additionally, IL11 knockdown significantly reduced the mRNA and protein levels of ATF4, CHOP, and TRIB3, suggesting that IL11 may be responsible for the activation of unfolded protein response, an adaptive mechanism for growth, survival, and angiogenesis of cancer. Knockdown of IL11 in C26 and LLC cell lines also significantly reduced the volume of tumors in syngeneic mouse models, while over-expression of IL11 in MC38 cells increased tumor size. Moreover, the knockdown of IL11 in cachectic cancer cells partially prevented the loss of muscle and body weight in tumor-bearing mice, although the amelioration of muscle loss by IL11 knockdown may be associated with the decreased tumor burden. Together, these results indicate that IL11 may contribute to muscle wasting and provide novel insight into the previously unestablished role of IL11 in CAC. Citation Format: Kimberly Drinkwater, Ashlyn Meyers, Samin Kargari, Natalie Crisan, Aaron Mody, Minsub Shim. The role of interleukin-11 (IL11) in cancer-associated cachexia (CAC) [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 6952.

Age-Related Features of the Response of Cancer Stem Cells and T Cells in Experimental Lung Cancer.

The responses of tumor stem cells and various populations of CD4 and CD8 T cells of young and aged C57BL/6 mice were studied in a lung cancer model. Using Lewis lung carcinoma cell line, an orthotopic model of lung cancer was modeled. Cancer stem cells, circulating tumor cells, and various populations of CD4 and CD8 T cells in the blood and lung tissue were studied by cytometry. We revealed age-related differences in the content of various populations of CD4 and CD8 T cells in the blood and lungs of intact young and aged mice. Age-related features of the reaction of various populations of cancer stem cells and CD4 and CD8 T cells in the blood and lungs of animals in the Lewis lung carcinoma were shown.

Whole‑exome sequencing reveals Lewis lung carcinoma is a hypermutated Kras/Nras–mutant cancer with extensive regional mutation clusters in its genome

Lewis lung carcinoma (LLC), as a widely used preclinical cancer model, has still not been genetically and genomically characterized. Here, we performed a whole–exome sequencing analysis on the LLC cell line to elucidate its molecular characteristics and etiologies. Our data showed that LLC originated from a male mouse belonging to C57BL/6L (a transitional strain between C57BL/6J and C57BL/6N) and contains substantial somatic SNV and InDel mutations (> 20,000). Extensive regional mutation clusters are present in its genome, which were caused mainly by the mutational processes underlying the SBS1, SBS5, SBS15, SBS17a, and SBS21 signatures during frequent structural rearrangements. Thirty three deleterious mutations are present in 30 cancer genes including Kras, Nras, Trp53, Dcc, and Cacna1d. Cdkn2a and Cdkn2b are biallelically deleted from the genome. Five pathways (RTK/RAS, p53, cell cycle, TGFB, and Hippo) are oncogenically deregulated or affected. The major mutational processes in LLC include chromosomal instability, exposure to metabolic mutagens, spontaneous 5–methylcytosine deamination, defective DNA mismatch repair, and reactive oxygen species. Our data also suggest that LLC is a lung cancer similar to human lung adenocarcinoma. This study lays a molecular basis for the more targeted application of LLC in preclinical research.

Co-encapsulation of fisetin and cisplatin into liposomes: Stability considerations and in vivo efficacy on lung cancer animal model

Lung cancer is a highly vascularized tumor for which a combination between an antitumor agent, cisplatin, and an antiangiogenic molecule, fisetin, appears a promising therapeutic approach. In order to deliver both chemotherapies within the tumor, to enhance fisetin solubility and decrease cisplatin toxicity, an encapsulation of both drugs into liposomes was developed. Purification and freeze-drying protocols were optimized to improve both the encapsulation and liposome storage. The cytotoxicity of the encapsulated chemotherapies was evaluated on Lewis lung carcinoma (3LL) cell lines. The antitumor effect of the combination was evaluated in vivo on an ectopic mouse model of Lewis Lung carcinoma. The results showed that fisetin and cisplatin co-loaded liposomes were successfully prepared. Freeze-drying allowed a 30 days storage limiting the release of both drugs. The combination index between liposomal fisetin and liposomal cisplatin on 3LL cell line after 24 h of exposure showed a clear synergism: CI = 0.7 for the co loaded liposomes and CI = 0.9 for the mixture of cisplatin loaded and fisetin loaded liposomes. The co-encapsulating formulation showed in vivo efficacy against an ectopic murine model of Lewis Lung carcinoma with a probable reduction in the toxicity of cisplatin through co-encapsulation with fisetin.

Antimetastatic lung cancer therapy using alkaloid Piperlongumine noncovalently bound to С60 fullerene

A novel nanoformulation, C60 fullerene loaded with a plant alkaloid Piperlongumine (PL) molecules (C60-PL nanocomplex), as a potential drug for the treatment of highly metastatic lung cancer was created and characterized by using ultrasonic technology, computer simulation, atomic force and scanning tunneling microscopy. The aim of the study was to evaluate the antimetastatic potential of PL alone and the C60-PL nanocomplex using Lewis lung carcinoma (LLC) cell line as a model. Evidence has been obtained that the 2:1C60-PL nanocomplex is a potent agent capable of effectively reducing the survival, migration and invasion of LLC cells in vitro, as well as tumor growth and metastasis in vivo compared to free PL. These effects in cell behavior were shown to be associated with an increased Bax expression and high level of cleaved PARP confirming the proapototic potential of C60-PL nanocomplex as well as down-regulation of the mRNA of epithelial-mesenchymal transition regulator Twist1 and cancer stem cell marker CD44, a reduced level of phosphorylated mTOR and adaptor protein Ruk/CIN85. Histological analysis of the lung tissue of LLC-bearing mice showed that in animals that received the C60-PL nanocomplex, the regression of metastases prevailed over their growth. The obtained results allow to conclude that the proposed C60-PL nanocomplex represents a promising drug for the treatment of metastatic lung cancer.

Cytotoxic and Antitumor Effects of the Hydroalcoholic Extract of Tagetes erecta in Lung Cancer Cells.

Among all cancers, lung cancer is the one with the highest mortality rate, and it also has limited therapeutics. Antitumor agents based on medicinal plants have gained importance as a source of bioactive substances. Tagetes erecta is a plant of great cultural value, and recent reports have suggested its cytotoxic effects in tumor cells. Our objective was to evaluate the antitumor activity of Tagetes erecta extract in a lung carcinoma model. Hydroalcoholic extracts were obtained from fresh flowers and leaves of T. erecta; both extracts did not exert toxicity on Artemia salina. We observed cytotoxic effects induced by the floral extract in Lewis lung carcinoma (LLC) and breast tumor cell line (MCF7), but not by the leaf extract. In vivo, a xenograft lung carcinoma model was performed with LLC cells implanted on C57BL/6 mice, which showed that the floral extract reduced tumor growth and improved the effect of etoposide. Microscopic analysis of tumors showed a reduction in mitoses and an increase in necrotic areas with the extract and the etoposide. The main phytochemical compounds found are 2,3-dihydro-benzofuran, octadecanoic acid, benzenacetic acid, oleic acid, linoleic acid, and acetic acid. We conclude that the hydroalcoholic extract of T. erecta flowers has cytotoxic effects in lung carcinoma cells and enhances the effect of etoposide.

BSBM-11 COMBINING IMMUNOTHERAPY AND WHOLE-BRAIN RADIATION THERAPY IN A NOVEL SYNGENEIC LUNG CANCER BRAIN METASTASIS PRECLINICAL MODEL: DOES TIMING OF ADMINISTRATION MATTER?

Abstract Approximately 50% of all brain metastasis cases originate from primary lung tumors. Whole-brain radiation therapy (WBRT) is a common treatment for patients with multiple brain metastases, but patients rarely survive longer than one year after diagnosis. Recent studies demonstrate improved outcomes with combinatorial radiotherapy and immunotherapy, but the optimal timing of immunotherapy administration is unclear. Eliciting a robust immune response post-immunotherapy is vital for therapeutic efficacy. Our work shows that the BBB is disrupted and proinflammatory cytokines are increased in the brain 12 hours post-WBRT in immunocompetent but not immunocompromised mice. Additionally, efflux transporter activity is decreased at this time point. This study utilizes a novel Lewis Lung Carcinoma cell line, LLC-Br, which was developed in our laboratory to preferentially metastasize to the brain. Mice were treated with immune checkpoint inhibitors (ICIs) before or after WBRT then we evaluated tumor progression, survival, and blood-tumor barrier (BTB) permeability. We hypothesized administration of ICI following WBRT would increase survival, decrease tumor burden, and increase BTB permeability in our preclinical syngeneic lung cancer brain metastasis model.

Exploring the effects of tissue structural cell and tumor cell heparan sulfate on immune system trafficking, activation, and intercommunication via amphiregulin

Abstract Heparan sulfate (HS), a glycan subtype, is ubiquitously present on the surface of structural tissue cells and tumor cells during homeostasis, disease, and cancer, but how it affects immune cell trafficking, activation, and signal transmission in these tissue contexts is poorly understood. Furthermore, amphiregulin (Areg), a growth factor used by immune cells for intercommunication with structural tissue and tumor cells, contains a HS binding domain. We investigated the downstream signaling modalities of Areg-mediated cell activation in the context of HS inhibition. Areg signaling is qualitatively altered, but still present, upon exogenous inhibition or genetic ablation of HS. We further probed the consequences of HS-dependent vs. -independent Areg signaling at the transcriptional level. Next, we used a panel of CRISPR-Cas9-mediated knockout Lewis Lung Carcinoma (LLC) cell lines to interrogate how various aspects of HS (core proteoglycan, HS chain extension, sulfation status) contribute to Areg signaling. We found that the presence of the glypican family of HS-harboring proteoglycans on the surface of cells is critical for Areg signaling. Lastly, we explored the effects of cancer cell HS on immune system trafficking to and activation within tumors. HS-deficient tumors experience similar levels of lymphoid cell trafficking and activation, but show a significant increase in the presence of certain myeloid cell type (neutrophils, monocytes, and macrophages). Additionally, tumor-associated macrophages within HS-deficient tumors show an altered phenotype, with increased maturation markers. Overall, our work has illuminated previously understudied aspects of the relationships between HS, Areg, and the immune system. Supported by a grant from NIH (R21-AI149657)

Abstract 1088: Acquired radiation resistance induces thiol-dependent cisplatin cross-resistance

Abstract Purpose. Resistance to radiation, both intrinsic and acquired, remains a significant clinical challenge. It is therefore import to identify the underlying molecular and cellular features involved in this resistance. Materials and Methods. We generated genetically-matched counterparts of various degrees of radiation sensitivity in non-small cell lung carcinoma (NSCLC) tumor models to define characteristics of acquired resistance through repeated exposures to conventional X-ray radiation. Results. As assessed by cell viability and clonogenic assays murine Lewis lung carcinoma (LLC) and human A549 cell lines acquired an approximate 1.5 - 2-fold increase in radiation resistance (RR) as compared to its parental counterpart. Morphologically, we found that A549-RR exhibited a greater nucleus-to-cytosol (N/C) ratio as compared to its respective counterpart. Since the N/C ratio has been linked to the differentiation state, we next investigated the epithelial-to-mesenchymal transition (EMT) phenotype and cellular plasticity. We found that A549 have a greater radiation-induced plasticity, as measured by E-cadherin, vimentin and double positive (DP) modulation, as compared to LLC. Additionally, migration was suppressed in A549-RR, as compared to A549. Subsequently, we confirmed that the LLC-RR and A549-RR are also more resistant to radiotherapy than their isogenic-matched counterpart in vivo. Remarkably, we found that the acquired radiation resistance also induced resistance to first-line chemotherapeutic cisplatin, but not carboplatin or oxaliplatin. This cross-resistance was attributed to induced elevated thiol levels. Decreasing the amount of thiols with buthionine sulfoximine (BSO), a gamma-glutamylcysteine synthetase inhibitor, sensitized the resistant cells to cisplatin, to levels similar as parental cells. Conclusions. By generating genetically-matched radiation resistant NSCLC tumor models we were able to identify and overcome cisplatin cross-resistance. This is an important finding arguing for multi-modality treatment regimens with the potential of improving clinical outcomes in the future. Citation Format: Samir V. Jenkins, Shruti Shah, Azemat Jamshidi-Parsian, Amir Mortazavi, Gunnar Boysen, Kieng B. Vang, Robert J. Griffin, Narasimhan Rajaram, Ruud P. Dings. Acquired radiation resistance induces thiol-dependent cisplatin cross-resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1088.

Exosomes secreted from cardiomyocytes suppress the sensitivity of tumor ferroptosis in ischemic heart failure

Heart failure (HF) patients in general have a higher risk of developing cancer. Several animal studies have indicated that cardiac remodeling and HF remarkably accelerate tumor progression, highlighting a cause-and-effect relationship between these two disease entities. Targeting ferroptosis, a prevailing form of non-apoptotic cell death, has been considered a promising therapeutic strategy for human cancers. Exosomes critically contribute to proximal and distant organ-organ communications and play crucial roles in regulating diseases in a paracrine manner. However, whether exosomes control the sensitivity of cancer to ferroptosis via regulating the cardiomyocyte-tumor cell crosstalk in ischemic HF has not yet been explored. Here, we demonstrate that myocardial infarction (MI) decreased the sensitivity of cancer cells to the canonical ferroptosis activator erastin or imidazole ketone erastin in a mouse model of xenograft tumor. Post-MI plasma exosomes potently blunted the sensitivity of tumor cells to ferroptosis inducers both in vitro in mouse Lewis lung carcinoma cell line LLC and osteosarcoma cell line K7M2 and in vivo with xenograft tumorigenesis model. The expression of miR-22-3p in cardiomyocytes and plasma-exosomes was significantly upregulated in the failing hearts of mice with chronic MI and of HF patients as well. Incubation of tumor cells with the exosomes isolated from post-MI mouse plasma or overexpression of miR-22-3p alone abrogated erastin-induced ferroptotic cell death in vitro. Cardiomyocyte-enriched miR-22-3p was packaged in exosomes and transferred into tumor cells. Inhibition of cardiomyocyte-specific miR-22-3p by AAV9 sponge increased the sensitivity of cancer cells to ferroptosis. ACSL4, a pro-ferroptotic gene, was experimentally established as a target of miR-22-3p in tumor cells. Taken together, our findings uncovered for the first time that MI suppresses erastin-induced ferroptosis through releasing miR-22-3p-enriched exosomes derived from cardiomyocytes. Therefore, targeting exosome-mediated cardiomyocyte/tumor pathological communication may offer a novel approach for the ferroptosis-based antitumor therapy.

The Voltage-Gated Hv1 H+ Channel Is Expressed in Tumor-Infiltrating Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are key determinants of the immunosuppressive microenvironment in tumors. As ion channels play key roles in the physiology/pathophysiology of immune cells, we aimed at studying the ion channel repertoire in tumor-derived polymorphonuclear (PMN-MDSC) and monocytic (Mo-MDSC) MDSCs. Subcutaneous tumors in mice were induced by the Lewis lung carcinoma cell line (LLC). The presence of PMN-MDSC (CD11b+/Ly6G+) and Mo-MDSCs (CD11b+/Ly6C+) in the tumor tissue was confirmed using immunofluorescence microscopy and cells were identified as CD11b+/Ly6G+ PMN-MDSCs and CD11b+/Ly6C+/F4/80-/MHCII- Mo-MDSCs using flow cytometry and sorting. The majority of the myeloid cells infiltrating the LLC tumors were PMN-MDSC (~60%) as compared to ~10% being Mo-MDSCs. We showed that PMN- and Mo-MDSCs express the Hv1 H+ channel both at the mRNA and at the protein level and that the biophysical and pharmacological properties of the whole-cell currents recapitulate the hallmarks of Hv1 currents: ~40 mV shift in the activation threshold of the current per unit change in the extracellular pH, high H+ selectivity, and sensitivity to the Hv1 inhibitor ClGBI. As MDSCs exert immunosuppression mainly by producing reactive oxygen species which is coupled to Hv1-mediated H+ currents, Hv1 might be an attractive target for inhibition of MDSCs in tumors.

Antibacterial and biofilm prevention metabolites from Acanthophora spicifera

Abstract Acanthophora spicifera harbors a diverse array of secondary metabolites with therapeutic potential. The aim of this study is to isolate and characterize secondary metabolites from A. spicifera and then evaluate the antiproliferation, antibacterial, and biofilm prevention properties, followed by an analysis of molecular docking experiments. By employing chromatographic analysis and NMR spectroscopy, the isolated compounds were, the known flavonol, 8-hydroxyquercetagetin (1), three recognized steroids cholest-4-ene-3,6-dione (2), cholest-5-en-3β-ol (3), and 5α-cholestane-3,6-dione (4), and 2-bromohexadecanoic acid (5). These compounds exhibited antimicrobial effects against various Gram-negative and Gram-positive bacteria with inhibition zones ranging from 6.5 ± 0.2 to 17.2 ± 0.12 mm and 7.0 ± 0.4 to 15.3 ± 0.60 mm, respectively. Compounds 1 and 2 inhibited biofilm formation in P. aeruginosa and S. aureus. Compounds 1–4 demonstrated binding affinity values between −7.5 and −9.4 kcal/mol to protein 1A0G. These binding affinity values were akin to that of amoxicillin, implying that one potential antibacterial mechanism of action of these compounds may involve the inhibition of bacterial cell wall synthesis. All compounds showed no toxicity against Artemia salina and weak activity against Lymphoma and Lewis lung carcinoma cell lines with LD50 > 100 μg/mL.

Tumor cell-derived conditioned medium induced pro-tumoral phenotypes in macrophages through calcium-nuclear factor κB interaction

BackgroundThe malignant behaviors of lung cancers are affected by not only cancer cells but also many kinds of stromal cells in tumor microenvironment (TME), including macrophages. Macrophages have been proven to extensively influence tumor progression through several mechanisms, among which switching of macrophages from pro-inflammatory phenotypes (M1-like) to anti-inflammatory phenotypes (M2-like) mediated by transcription factors such as nuclear factor κB (NF-κB) is the most crucial event. The regulation of NF-κB has been well studied, however some details remain fuzzy.MethodsMouse primary bone marrow-derived macrophages (BMDMs) were cultured in Lewis lung carcinoma cell line LL-2-derived conditioned medium (LL-2-CM). Proliferation, migration, and polarization of BMDMs were tested by CCK8, scratch test, transwell, and flow cytometry. Secretion of several cytokines were detected by ELISA or cytometric bead array. To further explore the underlying mechanisms, BMDMs cultured in LL-2-CM were harvested for RNA-seq. Cytosolic calcium was detected by calcium probe Fluo-4-AM. Western blot was applied to exam the activation of NF-κB signal. BAPTA-AM was applied to sequestrate cytosolic calcium to further investigate the relationship between calcium and NF-κB signal. The polarization, calcium alteration, and NF-κB signal activation were further validated in BMDMs treated by CMT-64-derived conditioned medium (CMT-64-CM).ResultsLL-2-CM promoted proliferation, migration, and M2-like polarization of BMDMs and inhibited M1-like polarization of BMDMs. However two pro-inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-mathrm{alpha } (TNF-mathrm{alpha }) were secreted. RNA-seq indicated that LL-2-CM activated both canonical and non-canonical NF-κB signal in BMDMs. Western blot showed that canonical NF-κB was temporarily elicited and attenuated at 24 h, while non-canonical NF-κB was consistently activated. At the same time, expression of genes that regulate cytosolic calcium ion concentration were down regulated, which caused diminution of cytosolic calcium in BMDMs treated with LL-2-CM. The decreased cytosolic calcium, M2-like polarization, and NF-κB activation was also observed in CMT-64-CM treated BMDMs. On the contrary, elevated cytosolic calcium was observed during M1-like polarization of BMDMs elicited by lipopolysaccharide (LPS). Interestingly, administration of calcium chelator, BAPTA-AM, impeded activation of canonical NF-κB and expression of M1-like marker induced by LPS, which further confirmed the relationship between cytosolic calcium and canonical NF-κB signal.ConclusionsIn summary, lung cancer cell-derived conditioned medium promoted migration, proliferation, and M2-like polarization of BMDMs. The suppressed M1-like polarization was achieved through mitigating canonical NF-κB pathway via diminishing cytosolic calcium concentration. As far as we know, our work firstly revealed that cytosolic calcium is the key during inhibition of canonical NF-κB and M1-like polarization in macrophages by tumor cells.

Muscle metabolic stress determines cancer cachexia severity in mice.

Objectives: To determine the metabolic effects of cancer-conditioned media on myotube metabolism and to understand whether the variability of these effects is associated with cancer cachexia progression. Materials and methods: We established single-cell clones from murine Lewis lung carcinoma (LLC) cells and generated conditioned media from each clonal line. Differentiated primary mouse myotubes were incubated with conditioned media derived from each individual clonal cell line. After initial analysis, we selected a specific LLC clonal cell line that failed to induce metabolic stress in myotubes for further investigation in vitro and in vivo. Results: Short-term incubation with conditioned media from 10/34 LLC clonal cells failed to affect oxygen consumption rate (OCR) in myotubes. Incubation with parental LLC-conditioned media decreased protein content and changed the expression of key regulators of muscle function in myotubes, but the incubation of conditioned media from a selected clone that failed to affect OCR in myotubes also did not affect protein content and expression of muscle regulators. Mice injected with parental LLC cells had a significantly reduced body mass and muscle wasting compared to the mice injected with cells derived from this selected LLC clone. Conclusion: Factors secreted by LLC cells induce metabolic stress in primary myotubes and induce cancer cachexia in mice. However, a selected clonal LLC cell line that failed to induce metabolic stress in myotubes also promoted weaker catabolism in mice. These novel findings establish that early disruption of muscle oxidative metabolism is associated with cancer cachexia progression.

Designing fisetin nanocrystals for enhanced in cellulo anti-angiogenic and anticancer efficacy

We report the formulation, characterization, colloidal stability, and in vitro efficiency of Fisetin nanocrystals stabilized by poloxamer P407. Such nanocrystals present a nanometer scale (148.6 ± 1.1 nm) and a high homogeneity (polydispersity index of 0.17 ± 0.01), with a production yield of 97.0 ± 2.5%. The engineered formulations of nanocrystals suspension (pH of 7.4 ± 0.1), stabilized via steric repulsion, are stable for several days in aqueous environment (Milli Q water, NaCl 10 mM or mannitol 5% w/v), for few days in HEPES buffered saline (HBS) (20 / 150 mM) under sink conditions, and in culture medium. After freeze drying in 5% w/v mannitol, the nanocrystal formulations can be stored at −80 °C for at least 120 days. Drug release experiments displayed a 98.7 ± 5.1% cumulative release over 3 days in HBS. Compared to the free drug, the nanocrystal formulations showed an improved cytotoxicity highlighted by the decrease of the half maximal inhibitory concentration for both murine Lewis lung carcinoma (3LL) and human endothelial (EA.hy926) cell lines. In addition, after incubation with Fisetin nanosuspensions, significant changes in the cell morphology for both cell lines were observed, showing an improved anti-angiogenic effect of nanocrystals formulation compared to the free drug. Overall, Fisetin formulated as nanocrystals showed enhanced biopharmaceutical properties and in vitro activity, offering a wide range of indications for challenging applications in the clinic.

Nanosecond electrochemotherapy using bleomycin or doxorubicin: Influence of pulse amplitude, duration and burst frequency

Electroporation is a pulsed electric field (PEF) induced phenomenon, which effectiveness varies dependent on pulse parameters. This work focuses on nano-electrochemotherapy with bleomycin and doxorubicin to derive protocols as effective as European Standard Operating Procedures on Electrochemotherapy (ESOPE), which employ conventional microsecond range pulses. As a model, murine Lewis lung carcinoma (LLC1) cell line was used. The effects of pulse duration (100–500 ns), PEF amplitude (6–10 kV/cm) and pulse repetition frequency (10 kHz, 100 kHz, 1 MHz) were studied. A total of 75 ns protocol variations have been used. For detection of cell permeabilization, Yo-Pro-1 and flow cytometry were employed. Cell viability was evaluated 24-, 48-, or 72-hours post-electroporation. Nanosecond parametric protocols resulting in comparable treatment efficiency as ESOPE (1.3 kV/cm × 100 μs × 8) have been proposed. It was shown that high-frequency nanosecond electrochemotherapy with bleomycin or doxorubicin could be an alternative for established ESOPE protocols.

Abstract 5630: Intratumoral regulatory T cell clones originate from the draining lymph nodes regulatory T cells

Abstract Forkhead box P3 (Foxp3)-positive intratumoral regulatory T (Treg) cells play critical roles in suppressing anti-tumor immune responses. It is reported that human tumors accumulate more Tregs than peripheral blood. However, it remains unclear whether intratumoral Tregs are primarily derived from Tregs in tumor-draining lymph nodes (dLNs) or are converted from conventional CD4+ T cells (Tconvs) in the transforming growth factor-beta -rich tumor microenvironment.Because each T-cell clone has its unique T-cell receptor (TCR), tracking the TCRs of the tumor Treg repertoire in the dLN Treg, dLN Tconv, and tumor Tconv enables us to identify the primary source of intratumoral Tregs. GFP+ hCD2+ CD25high and GFP- hCD2+ CD25int Tregs, which we defined as mature and immature Tregs, respectively, are distinguished from Tconvs in Foxp3-GDL Tg × Foxp3hCD2 Treg reporter mice having the Foxp3-GFP-diphtheria toxin receptor transgene and Foxp3-human CD2/CD52 knocked-in locus. On day 14 after subcutaneous injection of the Lewis lung carcinoma cell line, CD4+ Treg subsets and Tconvs in the tumor and dLN were collected for TCR sequencing, and the TCR repertoire overlap between these populations was quantified.We found that approximately 70%-80% of the Treg repertoire in the tumor overlapped with that in the dLN. Overlap between tumor Treg and tumor Tconv or dLN Tconv was at most 30% in tumor Tregs, significantly smaller than the tumor Treg-dLN Treg overlap. Moreover, single-cell TCR/RNA sequencing analysis exhibited that the tumor and dLN Treg clusters shared a significant number of T-cell clones, with few clones shared with tumor or dLN Tconv clusters. To examine whether dLN Tregs were the source of tumor Tregs even when they were actively repopulating, GFP+ mature Tregs were transiently depleted by administrating diphtheria toxin to tumor bearing mice. Mature tumor Tregs were completely depleted by day 2 and recovered rapidly through day 5 after DT administration. Analysis of the TCR repertoire of repopulated Treg and Tconv subsets revealed that repertoire overlap between tumor Treg and dLN or tumor Tconv was small and that tumor Treg-dLN Tregs overlap remained dominant, proposing that the repopulated intratumoral Tregs were also mainly derived from dLN Tregs. These results demonstrated that dLN-tumor mobilization was the primary source of intratumoral Tregs and that Tregs converted from Tconvs comprised only a small fraction of the intratumoral Tregs in our model. This finding suggests that Treg clones that suppress anti-tumor responses in the tumor also inhibit the priming of tumor-reactive T cells in the dLN, which can limit the efficacy of tumor site-specific Treg depletion therapy. Citation Format: Hiroyasu Aoki, Haruka Shimizu, Shigeyuki Shichino, Mikiya Tsunoda, Haru Ogiwara, Shohei Hori, Kouji Matsushima, Satoshi Ueha. Intratumoral regulatory T cell clones originate from the draining lymph nodes regulatory T cells [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 5630.

A novel, synthetic TLR7/8 agonist demonstrates monotherapy efficacy and synergy with anti-PD-1 in syngeneic mouse tumor models

Abstract Cancer is the second leading cause of death in the United States, with more than half a million Americans succumbing to this disease every year. To date, immunotherapies such as anti-PD-1 and anti-CTLA-4 have shown some advancement toward treating certain cancer types, but efficacy is typically reserved for a small subset of cancer patients. Recent clinical trials have shown that melanoma and renal cell carcinoma patients had some of the highest rates of response to checkpoint treatment, but even then, the rate of response was only 20% and 22%, respectively. Furthermore, immunotherapies can lead to severe adverse reactions due to their ability to activate non-specific immune responses. Inimmune has developed and tested a novel TLR7/8 agonist as an immunotherapy for cancer. In vivo mouse studies demonstrated that IV treatment with our novel, synthetic TLR7/8 agonist in combination with anti-PD-1 resulted in increased efficacy in eliminating tumors or slowing tumor growth over anti-PD-1 treatment alone in B16F10 and MC38 tumor models in C57BL/6 mice. Furthermore, lead formulations of our novel, synthetic TLR7/8 agonist demonstrated efficacy as a monotherapy in BALB/c mice inoculated with a highly tumorigenic Lewis Lung Carcinoma (LLC) cell line. Taken together, our data indicate that this novel, synthetic TLR7/8 agonist was tolerated systemically, functioned as a monotherapy, and synergized well with anti-PD-1 treatment in various mouse syngeneic tumor models. As we move this compound to Phase 1 clinical trials, these data suggest that our TLR7/8 agonist can be dosed safely as a monotherapy or in combination with checkpoint therapy and may drive efficacious anti-tumor responses in humans.

Effects of Time Delay Between Unipolar Pulses in High Frequency Nano-Electrochemotherapy.

this work focuses on bleomycin electrochemotherapy using new modality of high repetition frequency unipolar nanosecond pulses. As a tumor model, Lewis lung carcinoma (LLC1) cell line in C57BL mice (n = 42) was used. Electrochemotherapy was performed with intertumoral injection of bleomycin (50 μL of 1500 IU solution) followed by nanosecond and microsecond range electrical pulse delivery via parallel plate electrodes. The 3.5 kV/cm pulses of 200 and 700 ns were delivered in a burst of 200 at frequencies of 1 kHz and 1 MHz. For comparison of treatment efficiency, a standard 1.3 kV/cm x 100 μs x 8 protocol was used. It was shown that it is possible to manipulate the efficacy of unipolar sub-microsecond electrochemotherapy solely by the time delay between the pulses. the results suggest that the sub-microsecond range pulses can be as effective as the protocols in European Standard Operating Procedures on Electrochemotherapy (ESOPE) using 100 μs pulses.

Tumor-derived exosomes drive pre-metastatic niche formation in lung via modulating CCL1+ fibroblast and CCR8+ Treg cell interactions.

Since the lung is one of the most common sites for cancer metastasis, it could provide a suitable microenvironment for pre-metastatic niche (PMN) formation to facilitate tumor cell colonization. Regulatory T cells (Tregs) are an immunosuppressive cell type found ubiquitously in tumors and may play a crucial role in PNM formation. In this study, we investigated tumor-derived exosome (TDE)-induced Treg differentiation in the lung PMN as well as the underlying mechanisms. TDEs were isolated from the Lewis lung carcinoma cell line (LLC-exo) and their effects on mouse pulmonary fibroblasts was investigated in vitro as well as on lung tumor formation and metastasis in a pre-injected mouse model. Immune cell populations in the lung were analyzed by flow cytometry. Expression of CCL1 and CCR8 was evaluated by immunofluorescence staining, qRT-PCR and Western blot analyses. Cytokine expression was measured using mouse cytokine arrays and ELISA. The number of CD4+ FoxP3+ Tregs was significantly increased in lungs in a LLC-exo pre-injected mouse model. Lung fibroblasts secreted increased amounts of CCL1 after co-culture with LLC-exo, which induced Treg differentiation by activating its specific receptor CCR8, ultimately contributing to the establishment of an immunologically tolerant PMN. Moreover, inhibiting the release of LLC-exo by GW4869, or blocking the CCL1-CCR8 axis using AZ084, suppressed Tregs differentiation and tumor metastasis in the lung. Collectively, our study provides a novel mechanism by which Tregs are activated to form an immunologically tolerant PMN and demonstrates a critical link among lung fibroblasts, Tregs and metastatic tumor cells.