Murine Squamous Cell Carcinoma Research Articles

Mild hyperthermia upregulates PD-L1 in the tumor microenvironment and enhances antitumor efficacy of PD-L1 blockade in murine squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) has a low five-year survival rate because of its high rate of recurrence and metastasis. After surgical resection or radiation, the main treatments for HNSCC, patients sometimes experience functional or aesthetic disorders. Therefore, there is a great demand for the development of non-surgical treatment strategies to improve clinical outcomes and patients' quality of life. One such non-surgical treatment is mild hyperthermia (mHT). Many studies have investigated combination treatments with mHT and immune checkpoint inhibitors in preclinical settings. However, there have been no detailed reports on the effects of mHT on immune checkpoint molecules. Here, we investigated the effects of mHT on the tumor microenvironment (TME), particularly on programmed cell death receptor-1 (PD-1)/programmed cell death ligand-1 (PD-L1), in SCCVII cells and a squamous cell carcinoma mouse model. First, we found that PD-L1 mRNA levels and surface PD-L1 expression significantly increased after mHT. Second, a single tumor model was used to determine the effect of HT on the TME. mHT enhanced the accumulation of CD4+ and CD8+ T cells, elevated PD-L1 expression in the TME, and decreased the PD-1 positive rate of CD4+ T cells. Finally, using a bilateral tumor model, we found that anti-PD-L1 monotherapy and combination therapy resulted in longer survival than the isotype control or mHT monotherapy. Moreover, the combination therapy resulted in a significantly higher survival rate than anti-PD-L1 monotherapy. In conclusion, our findings elucidate changes in PD-L1 expression in the TME and strengthen the rationale for mHT and PD-L1 blockade combination therapy.

Supplementation with a phenolic-rich fraction from Ilex kudingcha improves inflammatory stress, gastrocnemius weights, and foreleg muscle grip strength in murine squamous cell carcinoma (SCC7)-bearing mice

Ethanol supernatant of Ilex kudingcha methanol extract (ESIKME) was identified to be rich in phenolic compounds and inhibited murine squamous cell carcinoma (SCC)-7 growth through slightly restoring adaptive immunity and Th1-inclination immune balance in the SCC7-bearing mice. To further evaluate the inhibitory effects and mechanisms of ESIKME against SCC7-induced inflammation status, the effects of ESIKME on inflammatory stress and related cachexia induced by SCC7 cells were investigated in vitro and in vivo. Chemical components of ESIKME were measured using an advanced LC-MS/MS analysis. SCC7 cells were treated with mouse splenocyte (SCM) and macrophage conditioned media (MCM) cultured without/with ESIKME in vitro. SCC7-bearing mice were orally administered with ESIKME for 26 days in vivo. Cachexia-related biomarkers in the SCC7-bearing mice were analyzed. Most of the detected compounds in ESIKME were polymeric or oxidized phenolic compounds. Either SCM or MCM without ESIKME significantly inhibited SCC7 cell growth. The high-dose ESIKME-administered group (400 mg/kg b.w./day) had significantly higher body weights, feed efficiency, and energy efficiency than the dietary control (DC) group, suggesting non-adverse toxic effects for ESIKME supplementation in the SCC7-bearing mice. The DC group had a higher TNF-α and TNF-α/IL-10 secretion ratio but lower anti-inflammatory IL-10 cytokine secretions by macrophages than the vehicle control group. ESIKME administration dose-dependently and significantly reversed these adverse statuses. Medium dose ESIKME (100 mg/kg b.w./day) decreased tumor sizes but increased gastrocnemius weights and foreleg muscle grip strength. Our results suggest that ESIKME alleviated inflammatory stress and related cachexia in vitro and in vivo.

Effect of CBD on anti-tumor immune response and interaction with GPR55 on MAPK signaling in head and neck squamous cell carcinoma.

6060 Background: Head and neck squamous cell carcinoma (HNSCC) is a lethal malignancy and comprises two distinct etiology: human papillomavirus (HPV)+ve and -ve. Marijuana use is associated with HPV+ oropharyngeal infection and conflicting data show positive and negative associations of daily marijuana use with HPV+ and tobacco-associated HPV- HNSCC. However, cannabinoid use continues to increase in the US general population for recreational purposes as well as in cancer patients for palliative care. G-protein coupled receptors (GPCRs), including cannabinoid GPCRs, stimulated by specific ligands interact with multiple G proteins creating diversity and complexity of signaling. In this study, we aimed to investigate the CBD-induced GPCR coupling and its effect on anti-tumor activity by modulating immune response in HNSCC by using pre-clinical models. Methods: The anti-cancer effect of CBD was measured by cell proliferation, apoptosis and migration analysis. Next, TGF-α shedding assay was performed to identify the CBD-induced GPCR coupling with different cannabinoid receptors. The cannabinoid receptor GPR55 was subjected to silencing by siRNA and western blot analysis was performed to evaluate its role in activation of p38 MPAK pathway. Next, the anti-tumor immune response of CBD was evaluated by using an immunocompetent syngeneic C57BL/6 mEER RasG12D+/- HPV E6/E7 transformed tongue epithelial cell model that serves as a model for HPV+ HNSCC and the 4MOSC1 orthotopic, syngeneic, 4NQO (4-nitroquinoline-1 oxide)-induced murine oral tongue squamous cell carcinoma C57BL/6 mouse model that serve as HPV- HNSCC model as well as in immune-deficient Rag1 -/- and athymic nude mouse followed by measurement of immune cell infiltration by IHC analysis. Results: We found that 10μM of CBD treatment inhibited cell proliferation and migration in HNSCC cells by promoting apoptosis. Interestingly, we observed that CBD resulted in GPCR coupling for Gαi with GPR55 and Gαq/o and Gαq/s for CNR1, CNR2 and GPR55. Furthermore, we silenced GPR55 and observed that 10μM of CBD treatment inhibits the activation of p38 MAPK pathway whereas it promotes activation in non-silenced HNSCC cells. Next, we observed that treatment with 10μM of CBD significantly inhibited tumor growth compared to control in both HPV- and HPV+ models in immune-intact animals whereas, no significant difference in the tumor growth was observed in immune-deficient (Rag1 -/- and athymic nude) mouse, indicating role of CBD in modulating tumor immune microenvironment. We also observed CBD treatment enhanced CD4+ and CD8+ T cell infiltration into the primary tumors of HPV+ and HPV- mice models, implicating a downstream cytotoxic T cell response. Conclusions: Our study suggests that CBD promotes anti-tumor activity by modulating the immune microenvironment and interacts with GPR55 to activate intrinsic MAPK signaling pathway.

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....