BackgroundChemoradiotherapy (CRT) remains one of the most common cancer treatment modalities, and recent data suggest that CRT is maximally effective when there is generation of an anti-tumoral immune response. However, CRT has also been shown to promote immunosuppressive mechanisms which must be blocked or reversed to maximize its immune stimulating effects.MethodsTherefore, using a preclinical model of human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC), we developed a clinically relevant therapy combining CRT and two existing immunomodulatory drugs: cyclophosphamide (CTX) and the small molecule inducible nitric oxide synthase (iNOS) inhibitor L-n6-(1-iminoethyl)-lysine (L-NIL). In this model, we treated the syngeneic HPV-HNSCC mEER tumor-bearing mice with fractionated (10 fractions of 3 Gy) tumor-directed radiation and weekly cisplatin administration. We compared the immune responses induced by CRT and those induced by combinatory treatment (CRT + CTX/L-NIL) with flow cytometry, quantitative multiplex immunofluorescence and by profiling immune-related gene expression changes.ResultsWe show that combination treatment favorably remodels the tumor myeloid immune microenvironment including an increase in anti-tumor immune cell types (inflammatory monocytes and M1-like macrophages) and a decrease in immunosuppressive granulocytic myeloid-derived suppressor cells (MDSCs). Intratumoral T cell infiltration and tumor antigen specificity of T cells were also improved, including a 31.8-fold increase in the CD8+ T cell/ regulatory T cell ratio and a significant increase in tumor antigen-specific CD8+ T cells compared to CRT alone. CTX/LNIL immunomodulation was also shown to significantly improve CRT efficacy, leading to rejection of 21% established tumors in a CD8-dependent manner.ConclusionsOverall, these data show that modulation of the tumor immune microenvironment with CTX/L-NIL enhances susceptibility of treatment-refractory tumors to CRT. The combination of tumor immune microenvironment modulation with CRT constitutes a translationally relevant approach to enhance CRT efficacy through enhanced immune activation.Electronic supplementary materialThe online version of this article (10.1186/s40425-018-0485-9) contains supplementary material, which is available to authorized users.

Background Immune checkpoint inhibitors (ICIs) for solid tumors, including those targeting programmed cell death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), have shown impressive clinical efficacy, however, most patients do not achieve durable responses. One major therapeutic obstacle is the immunosuppressive tumor immune microenvironment (TIME). Thus, we hypothesized that a strategy combining tumor-directed radiation with TIME immunomodulation could improve ICI response rates in established solid tumors. Methods Using a syngeneic mouse model of human papillomavirus (HPV)-associated head and neck cancer, mEER, we developed a maximally effective regimen combining PD-1 and CTLA-4 inhibition, tumor-directed radiation, and two existing immunomodulatory drugs: cyclophosphamide (CTX) and a small-molecule inducible nitric oxide synthase (iNOS) inhibitor, L-n6-(1-iminoethyl)-lysine (L-NIL). We compared the effects of the various combinations of this regimen on tumor growth, overall survival, establishment of immunologic memory, and immunologic changes with flow cytometry and quantitative multiplex immunofluorescence. Results We found PD-1 and CTLA-4 blockade, and radiotherapy alone or in combination, incapable of clearing established tumors or reversing the unfavorable balance of effector to suppressor cells in the TIME. However, modulation of the TIME with cyclophosphamide (CTX) and L-NIL in combination with dual checkpoint inhibition and radiation led to rejection of over 70% of established mEER tumors and doubled median survival in the B16 melanoma model. Anti-tumor activity was CD8 + T cell-dependent and led to development of immunologic memory against tumor-associated HPV antigens. Immune profiling revealed that CTX/L-NIL induced remodeling of myeloid cell populations in the TIME and tumor-draining lymph node and drove subsequent activation and intratumoral infiltration of CD8 + effector T cells. Conclusions Overall, this study demonstrates that modulation of the immunosuppressive TIME is required to unlock the benefits of ICIs and radiotherapy to induce immunologic rejection of treatment-refractory established solid tumors. Electronic supplementary material The online version of this article (10.1186/s40425-019-0698-6) contains supplementary material, which is available to authorized users.

Aim: Glycoconjugated C 60 derivatives are of particular interest as potential cancer targeting agents due to an upregulated metabolic glucose demand, especially in the case of pancreatic adenocarcinoma and its dense stroma, which is known to be driven by a subset of pancreatic stellate cells. Materials & methods: Herein, we describe the synthesis and biological characterization of a hexakis-glucosamine C 60 derivative (termed 'Sweet-C 60 '). Results: Synthesized fullerene derivative predominantly accumulates in the nucleus of pancreatic stellate cells; is inherently nontoxic up to concentrations of 1 mg/ml; and is photoactive when illuminated with blue and green light, allowing its use as a photodynamic therapy agent. Conclusion: Obtained glycoconjugated nanoplatform is a promising nanotherapeutic for pancreatic cancer.

(2018) TGF-β1 programmed myeloid-derived suppressor cells (MDSC) acquire immune-stimulating and tumor killing activity capable of rejecting established tumors in combination with radiotherapy, OncoImmunology, 7:10, e1490853,

Caspase-8 ( CASP8 ) is one of the most frequently mutated genes in head and neck squamous carcinomas (HNSCCs), and CASP8 mutations are associated with poor survival. The distribution of these mutations in HNSCCs suggests that they are likely to be inactivating. Inhibition of CASP8 has been reported to sensitize cancer cells to necroptosis, a regulated cell death mechanism. Here, we show that knockdown of CASP8 renders HNSCCs susceptible to necroptosis by a second mitochondria-derived activator of caspase (SMAC) mimetic, birinapant, in combination with pan-caspase inhibitors Z-VAD-FMK or emricasan and radiation. In a syngeneic mouse model of oral cancer, birinapant, particularly when combined with radiation, delayed tumor growth and enhanced survival under CASP8 loss. Exploration of molecular underpinnings of necroptosis sensitivity confirmed that the level of functional receptor-interacting serine/threonine protein kinase 3 (RIP3) determines susceptibility to this mode of death. Although an in vitro screen revealed that low RIP3 levels rendered many HNSCC cell lines resistant to necroptosis, patient tumors maintained RIP3 expression and should therefore remain sensitive. Collectively, these results suggest that targeting the necroptosis pathway with SMAC mimetics, especially in combination with radiation, may be relevant therapeutically in HNSCC with compromised CASP8 status, provided that RIP3 function is maintained.

[60]Fullerene is a highly versatile nanoparticle (NP) platform for drug delivery to sites of pathology owing to its small size and both ease and versatility of chemical functionalization, facilitating multisite drug conjugation, drug targeting, and modulation of its physicochemical properties. The prominent and well-characterized role of the enhanced permeation and retention (EPR) effect in facilitating NP delivery to tumors motivated us to explore vascular transport kinetics of a water-soluble [60]fullerene derivatives using intravital microscopy in an immune competent murine model of breast adenocarcinoma. Herein, we present a novel local and global image analysis of vascular transport kinetics at the level of individual tumor blood vessels on the micron scale and across whole images, respectively. Similar to larger nanomaterials, [60]fullerenes displayed rapid extravasation from tumor vasculature, distinct from that in normal microvasculature. Temporal heterogeneity in fullerene delivery to tumors was observed, demonstrating the issue of nonuniform delivery beyond spatial dimensions. Trends in local region analysis of fullerene biokinetics by fluorescence quantification were in agreement with global image analysis. Further analysis of intratumoral vascular clearance rates suggested a possible enhanced penetration and retention effect of the fullerene compared to a 70 kDa vascular tracer. Overall, this study demonstrates the feasibility of tracking and quantifying the delivery kinetics and intratumoral biodistribution of fullerene-based drug delivery platforms, consistent with the EPR effect on short timescales and passive transport to tumors.

In this work, we develop a drug-mimicking nanofibrous peptide hydrogel that shows long-term bioactivity comparable to a small-molecule inhibitor of inducible nitric oxide synthase (iNOS). The iNOS inhibitor, N 6-(1-iminoethyl)-l-lysine (l-NIL), is a positively charged amino acid whose structure could be readily integrated into the framework of a positively charged multidomain peptide (MDP) through the modification of lysine side chains. This new l-NIL-MDP maintains the self-assembling properties of the base peptide, forming β-sheet nanofibers, which entangle into a thixotropic hydrogel. The l-NIL-MDP hydrogel supports cell growth in vitro and allows syringe-directed delivery that persists in a targeted location in vivo for several weeks. Multiple characterization assays demonstrate the bioactivity of the l-NIL-MDP hydrogel to be comparable to the l-NIL small molecule. This includes iNOS inhibition of macrophages in vitro, reduced nitrotyrosine immunostaining in murine subcutaneous histology, and reduced serum levels of vascular endothelial growth factor in vivo. This study expands the toolbox of available peptide hydrogel scaffold designs that can modify biological activity without the need for any additional small-molecule drugs, proteins, or cells.

Immunotherapeutic treatments in head and neck cancer clinical trials include cancer vaccines targeting foreign viral antigens or mutational neoantigens derived from cancer-expressed proteins. Anti-tumor immune responses place cancer cells under selective pressure to lose or downregulate target antigens; therefore, vaccination against virus-or host-"driver" oncogenes are proposed as a strategy to overcome immune escape. Herein, we demonstrate the impact of immunogenic viral antigens on anti-tumor response and immune editing in MOC2-E6E7, a syngeneic murine oral cancer cell line expressing HPV-16 E6 and E7 oncoproteins. Using orthotopic syngeneic models, we observed in vivo tumor growth kinetics of MOC2-E6E7 is delayed in immunocompetent mice compared to parental MOC2 tumors. In contrast, tumor growth remained similar in Rag1 -/mice lacking adaptive immunity. MOC2-E6E7 tumors demonstrated an "inflamed" or immune-activated tumor microenvironment and greater infiltration of CD8 + T cells compared to MOC2. By real-time PCR, we detected downregulation of E6 and E7 genes in MOC2-E6E7 tumors only in immunocompetent mice, suggesting the loss of ectopic viral antigen expression due to immune editing. We then assessed the efficacy of a biomaterials-based mesoporous silica rod (MSR) cancer vaccine targeting HPV-16 E7 in our model. Vaccination induced robust infiltration of antigen-specific CD8 + T cells, which led to tumor growth delay and modestly prolonged survival in MOC2-E6E7 tumors. Increased efficacy was seen in a separate head and neck cancer tumor model, mEER, which obligately expresses E7 antigen. Collectively, our data highlight the need for both immunogenicity and 'driver' status of target antigens to be considered in cancer vaccine design.