Murine Squamous Cell Carcinoma Research Articles

Transforming growth factor-β signals promote progression of squamous cell carcinoma by inducing epithelial-mesenchymal transition and angiogenesis

At present, the molecular mechanisms driving the progression and metastasis of oral squamous cell carcinoma (OSCC) remain largely uncharacterized. The activation of transforming growth factor-β (TGF-β) signaling in the tumor microenvironment has been observed in various types of cancer and has been implicated their progression by enhancing the migration and invasion of epithelial cancer cells. However, its specific roles in the oral cancer progression remain unexplored. In this study, we examined the effects of TGF-β signaling on the murine squamous cell carcinoma, SCCVII cells in vitro and in vivo. The incubation of SCCVII cells with TGF-β induced the activation of TGF-β signals and epithelial-mesenchymal transition (EMT). Notably, the motility of SCCVII cells was increased upon the activation of the TGF-β signaling. RNA sequencing revealed upregulation of genes related to EMT and angiogenesis. Consistent with these in vitro results, the inhibition of TGF-β signals in SCCVII cell-derived primary tumors resulted in suppressed angiogenesis. Furthermore, we identified six candidate factors (ANKRD1, CCBE1, FSTL3, uPA, TSP-1 and integrin β3), whose expression was induced by TGF-β in SCCVII cells, and associated with poor prognosis for patients with head and neck squamous cell carcinoma. These results highlight the role of TGF-β signals in the progression of OSCC via multiple mechanisms, including EMT and angiogenesis, and suggest novel therapeutic targets for the treatment of OSCC.

Induction of Immunological Antitumor Effects by the Combination of Adenovirus-Mediated Gene Transfer of B7-1 and Anti-Programmed Cell Death-1 Antibody in a Murine Squamous Cell Carcinoma Model.

The goal of this study was to evaluate the antitumor immune effects of B7-1 gene expression in addition to immune checkpoint inhibitor against squamous cell carcinoma. A murine SCC cell line, KLN205, was infected with adenoviral vector carrying B7-1 (AdB7). Infected cells were injected subcutaneously in the flanks of DBA/2 mice. Three weeks after implantation, anti-mouse PD-1 antibody (antiPD1) was intraperitonially administrated twice a week for a total of six times. CD80 was significantly overexpressed in the AdB7-infected tumors. IFN-gamma in the T cells in the spleen was significantly increased and tumor size was significantly reduced in the mice treated with both AdB7 and antiPD1. Targeted tumors treated with both AdB7 and antiPD1 exhibited significantly increased cell densities of total immune cells as well as Ki-67+ CD8+ T cells and decreased regulatory T cells. These results suggest that the B7-1 gene transfer may enhance the antitumor effect of anti-PD1 antibody against SCC.

Ilex kudingcha’s phenolic-rich fraction inhibits murine squamous cell carcinoma (SCC7) through restoring adaptive immunity and Th1-polarized immune balance in vitro and in vivo

The Ilex kudingcha methanol extract ethanol supernatent (ESIKME) was analyzed for its phenolic compounds using high-performance liquid chromatography. Murine squamous cell carcinoma (SCC)-7 was treated in vitro and in vivo. ESIKME was found abundant in 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid and modulated the immune response toward Th1-polarized immune balance through changing the splenocytes’ Th1/Th2 cytokine secretion profile in vitro. ESIKME had a significant effect against SCC7 cells in vitro. ESIKME administration by daily gavage for 26 days did not cause any apparent toxicity to SCC7-bearing mice. There were relatively lower tumor weights in the medium dose group (KM, 100 mg/kg b.w./day). KM administration slightly restored the B-cell and T-cell immunity through increasing serum non-specific antibody levels and the splenocytes’ proliferative ability in the experimental mice. KM administration tended to increase Th1-polarized immune balance through increasing (IL-2+TNF-⍺)/IL-10 (Th1/Th2) secretion ratios using splenocytes isolated from the experimental mice. Th1/Th2 cytokine secretion ratios by splenocytes were negatively correlated with tumor weights, revealing that ESIKME inhibits SCC7 through restoring adaptive immunity and Th1-polarized immune balance. Our results are the first to prove that ESIKME, which is a phenolic-rich fraction, inhibits SCC7 through partly restoring adaptive immunity and Th1-polarized immune balance in the tumor-bearing mice.

Line-field confocal optical coherence tomography for in vivo visualization of morphological characteristics of squamous cell carcinoma in a murine model.

Non-invasive imaging with line-field confocal optical coherence tomography (LC-OCT) can support the diagnosis of squamous cell carcinoma (SCC) through visualization of morphological characteristics specific to skin cancer. We aimed to visualize prominent morphological characteristics of SCC using LC-OCT in a well-established murine SCC model. Nine hairless mice were exposed to ultraviolet radiation three times weekly for 9 months to induce SCC development. Visible SCC tumors (n = 9) were imaged with LC-OCT and the presence of 10 well-described morphological characteristics of SCC were evaluated in the scans by two physicians with adjudication by a third. Overall, murine morphological characteristics resembled corresponding features previously reported in human SCCs. Interrupted dermal-epidermal junction occurred in 100% of tumors. In epidermis, the most frequently observed characteristics were severe epidermal dysplasia (100%) and tumor budding (89%). Common dermal characteristics included broad strands (100%) and collagen alterations (78%). LC-OCT imaging can be used to non-invasively visualize morphological characteristics specific to SCC in an in vivo preclinical model.

HPV-positive murine oral squamous cell carcinoma: development and characterization of a new mouse tumor model for immunological studies

BackgroundInfection with high-risk human papillomavirus (HPV) strains is one of the risk factors for the development of oral squamous cell carcinoma (OSCC). Some patients with HPV-positive OSCC have a better prognosis and respond better to various treatment modalities, including radiotherapy or immunotherapy. However, since HPV can only infect human cells, there are only a few immunocompetent mouse models available that enable immunological studies. Therefore, the aim of our study was to develop a transplantable immunocompetent mouse model of HPV-positive OSCC and characterize it in vitro and in vivo.MethodsTwo monoclonal HPV-positive OSCC mouse cell lines were established by inducing the expression of HPV-16 oncogenes E6 and E7 in the MOC1 OSCC cell line using retroviral transduction. After confirming stable expression of HPV-16 E6 and E7 with quantitative real-time PCR and immunofluorescence staining, the cell lines were further characterized in vitro using proliferation assay, wound healing assay, clonogenic assay and RNA sequencing. In addition, tumor models were characterized in vivo in C57Bl/6NCrl mice in terms of their histological properties, tumor growth kinetics, and radiosensitivity. Furthermore, immunofluorescence staining of blood vessels, hypoxic areas, proliferating cells and immune cells was performed to characterize the tumor microenvironment of all three tumor models.ResultsCharacterization of the resulting MOC1-HPV cell lines and tumor models confirmed stable expression of HPV-16 oncogenes and differences in cell morphology, in vitro migration capacity, and tumor microenvironment characteristics. Although the cell lines did not differ in their intrinsic radiosensitivity, one of the HPV-positive tumor models, MOC1-HPV K1, showed a significantly longer growth delay after irradiation with a single dose of 15 Gy compared to parental MOC1 tumors. Consistent with this, MOC1-HPV K1 tumors had a lower percentage of hypoxic tumor area and a higher percentage of proliferating cells. Characteristics of the newly developed HPV-positive OSCC tumor models correlate with the transcriptomic profile of MOC1-HPV cell lines.ConclusionsIn conclusion, we developed and characterized a novel immunocompetent mouse model of HPV-positive OSCC that exhibits increased radiosensitivity and enables studies of immune-based treatment approaches in HPV-positive OSCC.

Squamous cell carcinoma-derived G-CSF promotes tumor growth and metastasis in mice through neutrophil recruitment and tumor cell proliferation, associated with poor prognosis of the patients.

Tumor-derived G-CSF is a well-known factor to aggravate disease progression in various types of cancers. In this study, we investigated a role of G-CSF in squamous cell carcinoma (SCC). High expression of G-CSF in the tumor tissues of esophageal SCC (ESCC) patients correlated with poor prognosis. Murine SCC NR-S1M cells produce considerable amount of G-CSF, which expression is correlated with its metastatic potentials. Deletion of G-CSF in NR-S1M cells mitigated tumor growth and metastasis to lymph node and lung of subcutaneous NR-S1M tumors in the mice. Mechanistically, G-CSF enhanced cell proliferation in autocrine manner in vitro, whereas in NR-S1M tumor-bearing mice, accumulation of plasma G-CSF was associated with expansion of peripheral neutrophils, which led to a decreased proportion of CD8+ T cells. Antibody depletion of neutrophils restored the number of CD8+ T cells and modestly suppressed tumor outgrowth, albeit no changes in distant metastasis. We propose that G-CSF produced by NR-S1M cells facilitates tumor progression in mice through bi-functional effects to promote neutrophil recruitment and tumor cell proliferation, which may render poor prognosis to the ESCC patients with high G-CSF expression.

Potent antitumor effects of the conditioned medium of bone marrow-derived mesenchymal stem cells via IGFBP-4.

Cell transfer therapy using mesenchymal stem cells (MSCs) has pronounced therapeutic potential, but concerns remain about immune rejection, emboli formation, and promotion of tumor progression. Because the mode of action of MSCs highly relies on their paracrine effects through secretion of bioactive molecules, cell-free therapy using the conditioned medium (CM) of MSCs is an attractive option. However, the effects of MSC-CM on tumor progression have not been fully elucidated. Herein, we addressed this issue and investigated the possible underlying molecular mechanisms. The CM of MSCs derived from human bone marrow greatly inhibited the in vitro growth of several human tumor cell lines and the in vivo growth of the SCCVII murine squamous cell carcinoma cell line with reduced neovascularization. Exosomes in the MSC-CM were only partially involved in the inhibitory effects. The CM contained a variety of cytokines including insulin-like growth factor binding proteins (IGFBPs). Among them, IGFBP-4 greatly inhibited the in vitro growth of these tumors and angiogenesis, and immunodepletion of IGFBP-4 from the CM significantly reversed these effects. Of note, the CM greatly reduced the phosphorylation of AKT, ERK, IGF-1 receptor beta, and p38 MAPK in a partly IGFBP4-dependent manner, possibly through its binding to IGF-1/2 and blocking the signaling. The CM depleted of IGFBP-4 also reversed the inhibitory effects on in vivo tumor growth and neovascularization. Thus, MSC-CM has potent inhibitory effects on tumor growth and neovascularization in an IGFBP4-dependent manner, suggesting that cell-free therapy using MSC-CM could be a safer promising alternative for even cancer patients.

The In Vitro and In Vivo Anticancer Effect of Photomed for Photodynamic Therapy: Comparison with Photofrin and Radachlorin.

To overcome the limitation of conventional cancer treatments, photodynamic therapy (PDT) has been introduced as another treatment option. PDT provides a non-invasive, non-surgical way with reduced toxicity. To improve the antitumor efficacy of PDT, we synthesized a novel photosensitizer, a 3-substituted methyl pyropheophorbide-a derivative (Photomed). The purpose of the study was to evaluate the antitumor effect of PDT with Photomed comparing with the clinically approved photosensitizers Photofrin and Radachlorin. The cytotoxicity assay against SCC VII cells (murine squamous cell carcinoma) was performed to determine whether Photomed is safe without PDT and whether Photomed is effective against cancer cells with PDT. An in vivo anticancer efficacy study was also performed using SCC VII tumor-bearing mice. The mice were divided into small-tumor and large-tumor groups to identify whether Photomed-induced PDT is effective for not only small tumors but also large tumors. From in vitro and in vivo studies, Photomed was confirmed to be (1) a safe photosensitizer without laser irradiation, (2) the most effective photosensitizer with PDT against cancers compared to Photofrin and Radachlorin and (3) effective with PDT in treating not only small tumors but also large tumors. In conclusion, Photomed may contribute as a novel, potential photosensitizer for use in PDT cancer treatment.

Differential Regulation of Innate Lymphoid Cells in Human and Murine Oral Squamous Cell Carcinoma

Oral squamous cell carcinomas (OSCC) remain a major healthcare burden in Asian countries. In Pakistan alone, it is the most common cancer in males and second only to breast cancer in females. Alarmingly, treatment options for OSCC remain limited. With this context, investigations made to explore the inflammatory milieu of OSCC become highly relevant, with the hope of practicing immunotherapeutic approaches to address this highly prevalent tumor. We investigated the newly identified innate lymphoid cells (ILCs) and associated cytokines in well-defined human oral squamous cell carcinoma (OSCC) as well as in a 7,12-dimethylbenz[a]anthracene (DMBA)-induced murine model of OSCC using flow cytometry and quantitative real-time polymerase chain reaction (qPCR). We further went on to explore molecular circuitry involved in OSCC by developing a murine model of OSCC and using an α-Thy1 antibody to inhibit ILCs. Amongst the ILCs that we found in human OSCC, ILC3 (23%) was the most abundant, followed by ILC2 (17%) and ILC1 (1%). Mice were divided into four groups: DMBA (n = 33), DMBA+antibody (Ab) (n = 30), acetone (n = 5), and control (n = 5). In murine OSCC tissues, ILC1 and ILC3 were down-infiltrated, while ILC2 remained unchanged compared to controls. Interestingly, compared to the controls (DMBA group), mice treated with the α-Thy1 antibody showed fewer numbers of large tumors, and a larger percentage of these mice were tumor-free at this study's end point. We present novel data on the differential expansion/downsizing of ILCs in OSCC, which provides a pivotal basis to dive deeper into molecular circuitry and the OSCC tumor niche to devise novel diagnostic, therapeutic, and prognostic strategies to prevent/treat oral cancers.

Histone Deacetylase Inhibitor Panobinostat Benefits the Therapeutic Efficacy of Oncolytic Herpes Simplex Virus Combined with PD-1/PD-L1 Blocking in Glioma and Squamous Cell Carcinoma Models.

Combination therapy has been widely explored for oncolytic virus (OV), as it can be met with tumor resistance. The HDAC inhibitor (HDACi) panobinostat is a potent pan-deacetylase inhibitor which blocks multiple cancer-related pathways and reverses epigenetic events in cancer progression. In this study, oncolytic activity in vitro and antitumor therapeutic efficacy in vivo when combined with oHSV and panobinostat were investigated. (1) Treatment with panobinostat enhanced oHSV propagation and cytotoxicity in human glioma A172 and squamous cell carcinoma SCC9 cells. (2) Combined treatment with oHSV and panobinostat enhanced virus replication mediated by the transcriptional downregulation of IFN-β- and IFN-responsive antiviral genes in human glioma A172 and squamous cell carcinoma SCC9 cells. (3) Panobinostat treatment induced upregulation of PD-L1 expression in both glioma and squamous cell carcinoma cells. (4) A significantly enhanced therapeutic efficacy was shown in vivo for the murine glioma CT-2A and squamous cell carcinoma SCC7 models when treated with a combination of oHSV, including PD-1/PD-L1 blockade and HDAC inhibition. Consequently, these data provide some new clues for the clinical development of combination therapy with OVs, epigenetic modifiers, and checkpoint blockades for glioma and squamous cell carcinoma.

PPARgamma agonism inhibits progression of premalignant lesions in a murine lung squamous cell carcinoma model.

The N-nitroso-trischloroethylurea (NTCU)-induced mouse model of squamous lung carcinoma recapitulates human disease from premalignant dysplasia through invasive tumors, making it suitable for preclinical chemoprevention drug testing. Pioglitazone is a peroxisome proliferator-activated receptor γ (PPARγ) agonist shown to prevent lung tumors in preclinical models. We investigated pioglitazone's effect on lesion development and markers of potential preventive mechanisms in the NTCU model. Female FVB/N mice were exposed to vehicle, NTCU or NTCU + oral pioglitazone for 32 weeks. NTCU induces the appearance of basal cells in murine airways while decreasing/changing their epithelial cell makeup, resulting in development of bronchial dysplasia. H&E and keratin 5 (KRT5) staining were used to detect and grade squamous lesions in formalin fixed lungs. mRNA expression of epithelial to mesenchymal transition (EMT) markers and basal cell markers were measured by qPCR. Dysplasia persistence markers desmoglein 3 and polo like kinase 1 were measured by immunohistochemistry. Basal cell markers KRT14 and p63, club cell specific protein and ciliated cell marker acetylated tubulin were measured by immunofluorescence. Pioglitazone treatment significantly reduced squamous lesions and the presence of airway basal cells, along with increasing normal epithelial cells in the airways of NTCU-exposed mice. Pioglitazone also significantly influenced EMT gene expression to promote a more epithelial, and less mesenchymal, phenotype. Pioglitazone reduced the presence of squamous dysplasia and maintained normal airway cell composition. This work increases the knowledge of mechanistic pathways in PPARγ agonism for lung cancer interception and provides a basis for further investigation to advance this chemoprevention strategy.

580 Biodegradable bioadhesive nanoparticle delivery of chemotherapy for the treatment of cutaneous malignancies

Encapsulation of chemotherapeutic agents within biodegradable nanoparticles (NP) allows for gradual drug release from the polylactic acid core. Bioadhesive, degradable NPs can be retained at the site of administration, maximizing local effects while minimizing systemic side effects. Our bioadhesive NP (BNP) loaded with chemotherapy have previously been proven efficacious in the treatment of murine PDVC57 squamous cell carcinoma (SCC) tumors, with increased intratumoral (i.t.) retention of the active agent.

LB882 Dissecting mechanisms of responsiveness to the combination therapy of radiation and anti-PD-L1/anti-TGFb treatment in murine squamous cell carcinoma models

Squamous cell carcinoma (SCC) is often marked by an immunosuppressive tumor microenvironment, particularly in metastatic/recurrent disease. The purpose of this study is to evaluate the anti-tumor efficacy of and mechanisms of response to the combination therapy of radiotherapy (RT) and anti-PD-L1/anti-TGFb treatment using SCC models. We transplanted tumor cells derived from either carcinogen-induced SCCs or spontaneous SCCs of K15.Kras12D/Smad4-/- mice to syngeneic mouse recipients and subjected them to RT and anti-PD-L1/anti-TGFb therapy.

Molecular characterization of gene expression changes in murine cutaneous squamous cell carcinoma after 5-aminolevulinic acid photodynamic therapy

5-Aminolevulinic acid-based photodynamic therapy (ALA-PDT) is an effective therapy in treating inoperable cutaneous squamous cell carcinoma (cSCC) in China. However, its exact mechanism in treating cSCC is still unclear. Here, we analyzed expression profiles of lncRNAs and mRNAs of cSCC after ALA-PDT in a mouse model using Mouse Transcriptome Assay 1.0 chip. A total of 592 and 828 differentially expressed genes were identified in comparisons of ALA-PDT after 3 h vs. control, ALA-PDT after 6 h vs. control respectively. Regarding the lncRNAs analysis, a total of 185 and 410 differentially expressed lncRNAs were sought out. Bioinformatics analyses including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway, pathway relation network, and gene set enrichment analysis revealed that the pathways of ALA-PDT in treating cSCC were mainly associated with immune regulation, NF-κB signaling pathway, TLR signaling pathway, PI3K-Akt signaling pathway, TNF signaling pathway, and MAPK signaling pathway, which was validated by western blot analysis. Furthermore, a lncRNA-mRNA co-expression network revealed 118 network pairs comprising 36 lncRNAs and 68 mRNAs. These findings provide a basis for investigations into the treatment mechanisms of ALA-PDT in treating cSCC.

TCR-engineered neoantigen-specific CD4+ T cells mediate immunotherapy of a class II-negative murine squamous cell carcinoma

Abstract The expansion of neoantigen (NeoAg)-specific T cells often accompanies clinical responses to immunotherapies such as immune checkpoint blockade (ICB), adoptive cellular therapy (ACT), and personalized cancer vaccines (PCV), highlighting their importance for antitumor immunity. While the functions of NeoAg-specific CD8+ T cells have been characterized, the role of NeoAg-specific CD4+ T cells is less well understood. To study the antitumor mechanisms of NeoAg-specific CD4+ T cells, we sorted single CD4+ T cells specific for a mutated clathrin heavy chain epitope (Cltc H129Q) expressed by the murine squamous cell carcinoma VII (SCC VII) tumor. T cell receptor (TCR) sequencing analysis revealed the presence of four distinct TCR clonotypes and expression of these TCRs in primary cells was sufficient to confer preferential recognition of Cltc H129Q as compared to the corresponding wildtype Cltc epitope. Despite differences in TCR avidity, both low and high avidity CD4+ T cells were capable of proliferating to similar degrees in response to tumor antigen in vivo and enhancing primary tumor immunity in a CD8+ T cell- and CD40L-dependent manner. ACT with Cltc H129Q specific CD4+ T cells also synergized with cyclophosphamide treatment to reduce disease burden in mice with established tumors. Overall, these findings illuminate the mechanistic role of NeoAg-specific CD4+ T cells in tumor immunity, provide insights into the impact of TCR avidity on the helper functions of CD4+ T cells, and highlight the therapeutic potential of ACT with TCR-engineered CD4+ T cells.

Neoadjuvant Immunoradiotherapy in a Tobacco-Signature Preclinical Oral Squamous Cell Carcinoma Model

<h3>Purpose/Objective(s)</h3> PD-1 inhibition (PD1i) has demonstrated no benefit for locally advanced HNSCC, and emerging neoadjuvant PD1i window of opportunity yield promising but limited responses. Our preclinical work demonstrates that ablating tumor draining lymphatics compromises the response to immune-oncology (IO) therapy. In concert, a recently completed phase I trial investigating neoadjuvant immunoradiotherapy (NIRT) – 8Gy to gross tumor volume plus PD1i followed by surgery – for treatment-naïve HPV- HNSCC demonstrated a 60% major pathologic (>25%) response and clinical to pathologic downstaging of 100% (NCT03247712). Accordingly, we hypothesize that lymphatic-preserving immune oncology (IO) therapy can potentiate PD1i and promote antitumor immunity by enhancing immunosurveillance along the tumor-immune-lymphatic axis and reversing the suppressive tumor immune microenvironment (TIME). <h3>Materials/Methods</h3> To explore this, we employed our recently characterized tobacco-signature, orthotopic murine oral squamous cell carcinoma (OSCC) models, one of which matches the immune infiltrate and PD1i response of human OSCC and the other of which is immune-cold with no response to PD1i. Using these models, we delivered sequences of NIRT along with procedures to ablate tumor-draining lymph nodes (tdLNs) or lymphatic channels to characterize the immune mechanisms that optimize antitumor immunity and therapy response. <h3>Results</h3> We define a NIRT scheme that spares tumor lymphatics and leads to complete and durable immunity in our immune-responsive OSCC preclinical model. By ablating tumor-draining lymphatics, we find that tdLNs are critical for the NIRT response. Moreover, we interrupt draining lymphatics to the sentinel lymph node (SLN), mapped with an IRDye-tagged tilmanocept, to explore immunosurveillance across the tumor-immune-lymphatic axis. Lymphatic channel ablation blocks the tumor NIRT response and leads to a restriction of cytotoxic, tumor-antigen specific CD8 T cells from the TIME. Within tdLNs, we find that conventional type I dendritic cells are necessary for the NIRT response and enhanced following NIRT. Lastly, we find that NIRT reverses local immunosuppression by reducing myeloid derived suppressor cells within the TIME, potentiating PD1i and leading to complete response in our immune-cold model. <h3>Conclusion</h3> We demonstrate that targeting tumors with stereotactic radiation and PD1i while sparing draining lymphatics enhances anticancer immunity by promoting regional immunosurveillance and repolarizing local suppressive TIMEs, resulting in significantly improved responses. Overall, this work represents a paradigm-shift in the design of IO therapies, which can immediately inform the design of next-generation immune oncology trials for HNSCC.

Harnessing radiotherapy-induced NK-cell activity by combining DNA damage–response inhibition and immune checkpoint blockade

BackgroundDespite therapeutic gains from immune checkpoint inhibitors (ICI) in many tumor types, new strategies are needed to extend treatment benefits, especially in patients failing to mount effective antitumor T-cell responses....

Abstract 13400: STAT3 ASO-Loaded Microbubbles With Pulsed Ultrasound for Enhanced Therapeutic Effect in Head and Neck Tumors

Introduction: Signal transducer and activator of transcription 3 ( STAT3 ) is an oncogenic transcription factor implicated in carcinogenesis, tumor progression, and drug resistance in many cancers, including head and neck squamous cell carcinoma ( HNSCC ). STAT3 antisense oligonucleotide ( ASO ) specifically targeting the nucleotide sequence of STAT3 has therapeutic promise, but effective delivery remains challenging. We developed microbubble ( MB ) delivery vehicles loaded with STAT3 ASO and hypothesized that compared to intravenous ( i.v. ) delivery, ultrasound-targeted microbubble cavitation ( UTMC )-mediated ASO delivery confers superior tumor growth inhibition. Methods: Lipid MBs loaded with STAT3 ASO (S3MB) or mutant ASO (Mut-MB) were prepared. First, murine squamous carcinoma cells (SCC-7) in contact with MBs were exposed to pulsed ultrasound (1 MHz, 0.5 MPa, 100 μs pulse duration, 1 ms pulse interval) for 10 sec to establish efficacy with respect to cytotoxicity, apoptosis, and gene knockdown. Thereafter, the anti-tumor effect of 3 treatments with STAT3 ASO (10 μg) delivered i.v. or via UTMC x 15 min ( f= 1MHz, 0.7MPa, 100 cycles x 5 every 2 sec) was compared in vivo in a murine SCC-7 xenograft model followed up to 17 days. Results: S3MB + UTMC conferred more cytoxocitiy, apoptosis, and downregulation of transcripts for STAT3 and its target genes (Bcl-xL and Cyclin D1) compared to Mut-MB + UTMC in vitro (p&lt;0.05). In tumor bearing mice, compared to i.v. STAT3 ASO (n=8), Mut-MB+UTMC (n=5) or no treatment (n=8), S3MB + UTMC (n=9) significantly attenuated tumor burden, prolonged doubling time, and improved survival (p&lt;0.05). While i.v. STAT3 ASO had no effect on tumor expression of STAT3, Bcl-x, and cyclin D1 at the transcript and protein levels, its delivery via UTMC significantly reduced tumor expression of these genes (p&lt;0.01). Conclusion: At a dose of 10 μg, STAT3 ASO significantly inhibited murine HNSCC tumor growth and downregulated STAT3 and its target genes when delivered via UTMC, compared with intravenous administration alone. Our platform offers a promising imaged-guided, targeted delivery strategy for nucleotide-based therapeutics in cancer applications.

Extracellular acidity in tumor tissue upregulates programmed cell death protein1 expression on tumor cells via proton-sensing G protein-coupled receptors.

Acidity in the tumor microenvironment has been reported to promote cancer growth and metastasis. In our study, we examined a potential relation between extracellular acidity and expression level of the immune checkpoint molecule programmed cell death protein1 (PD-L1) in murine squamous cell carcinoma (SCC) and melanoma cell lines. PD-L1 expression in the tumor cells was upregulated by culturing in a low pH culture medium. Tumor-bearing mice were allowed to ingest sodium bicarbonate, resulting in neutralization of acidity in the tumor tissue, a decrease in PD-L1 expression in tumor cells and suppression of tumor growth in vivo. Proton-sensing G protein-coupled receptors, T-cell death-associated gene 8 (TDAG8) and ovarian cancer G-protein-coupled receptor 1 (OGR1), were upregulated by low pH, and essentially involved in the acidity-induced elevation of PD-L1 expression in the tumor cells. Human head and neck SCC RNAseq data from the Cancer Genome Atlas also suggested a statistically significant correlation between expression levels of the proton sensors and PD-L1 mRNA expression. These findings strongly suggest that neutralization of acidity in tumor tissue may result in reduction of PD-L1 expression, potentially leading to inhibition of an immune checkpoint and augmentation of antitumor immunity.

HIF-1α affects sensitivity of murine squamous cell carcinoma to boron neutron capture therapy with BPA

Purpose To examine whether hypoxia and Hif-1α affect sensitivity of murine squamous cell carcinoma cells to boron neutron capture therapy (BNCT). Materials and methods SCC VII and SCC VII Hif-1α-deficient mouse tumor cells were incubated under normoxic or hypoxic conditions, and cell survival after BNCT was assessed. The intracellular concentration of the 10B-carrier, boronophenylalanine-10B (BPA), was estimated using an autoradiography technique. The expression profile of SLC7A5, which is involved in the uptake of BPA, and the amount of DNA damage caused by BNCT with BPA were examined. A cell survival assay was performed on cell suspensions prepared from tumor-bearing mice. Results Hypoxia ameliorated SCC VII cell survival after neutron irradiation with BPA, but not BSH. Hypoxia-treated SCC VII cells showed decreased intracellular concentrations of BPA and the down-regulated expression of the SLC7A5 protein. BPA uptake and the SLC7A5 protein were not decreased in hypoxia-treated Hif-1α-deficient cells, the survival of which was lower than that of SCC VII cells. More DNA damage was induced in SCC VII Hif-1α-deficient cells than in SCC VII cells. In experiments using tumor-bearing mice, the survival of SCC VII Hif-1α-deficient cells was lower than that of SCC VII cells. Conclusion. Hypoxia may decrease the effects of BNCT with BPA, whereas the disruption of Hif-1α enhanced sensitivity to BNCT with BPA.