SCCVII Tumors Research Articles

NAD+ Metabolism Reprogramming Mediates Irradiation-Induced Immunosuppressive Polarization of Macrophages

Purposeradiotherapy stands as an important complementary treatment for head and neck squamous cell carcinoma (HNSCC), yet it does not invariably result in complete tumor regression. The infiltration of immunosuppressive macrophages is believed to mediate the radiotherapy resistance, which mechanism remains largely unexplored. This study aimed to elucidate the role of immunosuppressive macrophages during radiotherapy and the associated underlying mechanisms. Materials and MethodsMale C3H mice bearing syngeneic SCC-VII tumor were received irradiation (2 × 8Gy). The impact of irradiation on tumor-infiltrating macrophages were assessed. Bone marrow derived macrophages were evaluated in differentiation, proliferation, migration, and inflammatory cytokines after treatment of irradiated tumor culture medium (irCM) and irradiated tumor derived extracellular vesicles (irTEVs). A comprehensive metabolomics profiling of the irTEVs was conducted using liquid chromatography-mass spectrometry, while key metabolites were investigated the mechanism in macrophage in vitro and in vivo. ResultsRadiotherapy on SCC-VII syngeneic graft tumors increased polarization of both M1 and M2 macrophages in tumor microenvironment and drove infiltrated macrophages towards an immunosuppressive phenotype. Irradiation-induced polarization and immunosuppression of macrophages were dependent on irTEVs which delivered an increased amount of nicotinamide (NAM) to macrophages. NAM directly bound to the NF-κB transcriptional activity regulator USP7, through which NAM reduced translocation of NF-κB into the nucleus, thereby decreasing the release of cytokines IL6 and IL8. Increased enzyme activity of nicotinamide phosphoribosyl transferase (NAMPT) which is the rate-limiting enzyme of NAD+ metabolism, contributed to the irradiation-induced accumulation levels of NAM in irradiated HNSCC and irTEVs. Inhibition of NAMPT decreased NAM levels in irTEVs and increased radiotherapy sensitivity through alleviating immunosuppressive function of macrophages. ConclusionsRadiotherapy could induce NAD+ metabolic reprogramming of HNSCC cells, which regulate macrophage towards an immunosuppressive phenotype. Pharmacological targeting NAD+ metabolism might be a promising strategy for radiotherapy sensitization of HNSCC.

Photodynamic Therapy Supported by Antitumor Lipids.

Photodynamic therapy (PDT) destroys tumors by generating cytotoxic oxidants that induce oxidative stress in targeted cancer cells. Antitumor lipids developed for cancer therapy act also by inflicting similar stress. The present study investigated whether tumor response to PDT can be improved by adjuvant treatment with such lipids using the prototype molecule edelfosine. Cellular stress intensity following Photofrin-based PDT, edelfosine treatment, or their combination was assessed by the expression of heat shock protein 70 (HSP70) on the surface of treated SCCVII tumor cells by FITC-conjugated anti-HSP70 antibody staining and flow cytometry. Surface HSP70 levels that became elevated after either PDT or edelfosine rose much higher after their combined treatment. The impact of Photofrin-PDT-plus-edelfosine treatment was studied with three types of tumor models grown in syngeneic mice. With both SCCVII squamous cell carcinomas and MCA205 fibrosarcoma, the greatest impact was with edelfosine peritumoral injection at 24 h after PDT, which substantially improved tumor cure rates. With Lewis lung carcinomas, edelfosine was highly effective in elevating PDT-mediated tumor cure rates even when injected peritumorally immediately after PDT. Edelfosine used before PDT was ineffective as adjuvant with all tumor models. The study findings provide proof-in-principle for use of cancer lipids with tumor PDT.

Abstract 2423: Image-guided interstitial photodynamic therapy with palliative radiotherapy for ablating extrabronchial malignant central airway obstruction

Abstract Background: Patients with extrabronchial tumors that induce malignant central airway obstruction (MCAO) face dire prognoses with significant morbidity and 1-7 months overall survival. These tumors are inoperable. High-dose curative radiotherapy is associated with significant toxicity. Chemotherapy has limited localized benefits and immunotherapy cannot address the immediate need to halt tumor progression. Our preliminary data suggest that image-guided interstitial photodynamic therapy (I-PDT) with porfimer sodium is safe and potentially effective in patients with extrabronchial tumors that induce MCAO and there is a need to further improve the rate of response. Here we present results showing an improved tumor response rate by augmenting I-PDT with palliative radiotherapy (p-XRT). Methods: We investigated the addition of p-XRT prior to I-PDT to sensitize the target tumor to subsequent photoreaction in mouse models. Eight- to twelve-week-old C3H and C57B/6 mice with locally advanced (400-600 mm3) SCC-VII or LLC tumors, respectively, were treated with I-PDT alone or with p-XRT followed by I-PDT. The p-XRT regimen was 10 Gy x2 over two days. At 24-26 h after the second 10 Gy fraction, the I-PDT was administered with 5 mg/kg porfimer sodium activated with 630-nm laser light at 5.0 mW/cm2 and 45 J/cm2 at tumor margins. Photoacoustic imaging and light dosimetry measurements were used to investigate the impact of p-XRT on the tumor microenvironment as it pertains to the response to I-PDT. Tumor progression was monitored over 60 days. Image-based treatment planning was used to translate the preclinical findings into a treatment in clinical settings (8 Gy x1 followed by I-PDT) for patients with extrabronchial MCAO. Results: Preceding I-PDT with p-XRT increases tumor oxygenation for 1-2 days after the last radiation fraction and improves light penetration within mouse tumors. Administering p-XRT 24-48 h before I-PDT significantly (p<0.05, log-rank test) increases the rate of complete tumor response and control from 0% in p-XRT alone or 0-6% in I-PDT alone, to 31-64% for p-XRT followed by I-PDT. In patients, administration of I-PDT with porfimer sodium 48 h after 8Gy x1 p-XRT is feasible and can provide control of extrabronchial MCAO accompanied by symptomatic relief. Conclusions: Augmenting I-PDT with p-XRT improves the rate of tumor response. Murine studies show that p-XRT increases tumor blood oxygenation and light penetration which could contribute to better response to I-PDT. I-PDT with porfimer sodium preceded by p-XRT is feasible with possible benefits in patients with extrabronchial MCAO. Citation Format: Emily Oakley, Sarah Chamberlain, Nathaniel Ivanick, Alan Hutson, Theresa Busch, Gal Shafirstein. Image-guided interstitial photodynamic therapy with palliative radiotherapy for ablating extrabronchial malignant central airway obstruction [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 2423.

Laminated holmium-166-containing electrospun bandages for use against skin cancer

The number of non-melanoma skin cancer (NMSC) cases in the US will increase significantly over the next decade due to a rise in UV exposure. One of the treatment methods used to remove NMSC lesions is radiation therapy. The two types of radiation therapy used in the clinic are external beam therapy and brachytherapy. However, both require specialized on-site instrumentation and for patients to remain immobile. In this work, we studied an alternative radiation therapy – one that does not require expensive on-site equipment and would allow for enhanced patient mobility and, thus, comfort. We prepared sealed source, nylon-laminated holmium-166-containing radiotherapeutic bandages and used them in C3H/HeN mice with murine SCCVII tumor grafts. Overall, tumor sizes were smallest when treated with therapeutically relevant radiation doses via radiotherapeutic bandages (compared to controls), and no histological evidence of toxicity to tissues was observed. Thus, our optimized radiotherapeutic bandage offers a flexible approach to treating NMSC.

Enhanced tumor control with a combination of APG-157 and immune checkpoint inhibitors for head and neck cancer.

2652 Background: Natural botanical drugs containing flavonoids are under clinical investigation for the treatment of malignancies. A phase I clinical study for patients with locally advanced head and neck cancer with a new class of botanical drug APG-157 showed excellent tolerability, systemic absorption via sublingual administration, and enhanced immune cell infiltration into the tumor microenvironment and suppression of T cell inhibitory inflammatory cytokine production. We sought to evaluate the efficacy of tumor control for the combination of APG-157 and checkpoint blockade immunotherapy using a head and neck cancer mouse model. Methods: SCCVII (HPV-) and MEER (HPV+) head and neck cancer murine cell line models were utilized in in vivo tumor growth study. After optimization for route and cell number, we measured tumor growth after treating mice with following groups: i) control, ii) APG-157, iii) anti-PD-1, iv) anti-CTLA-4, v) APG-157 + anti-PD-1, vi) APG-157 + anti-CTLA-4, vii) anti-PD-1 and anti-CTLA-4, viii) APG-157 + anti-PD-1 + anti-CTLA-4. Tumors were collected after 2 injections of immune checkpoint inhibitors and characterized for immunophenotype by multi-color flow cytometry, immunohistochemistry, and RNA-sequence analysis. Fecal microbiome was analyzed by 16S rRNA sequence for SCCVII model to assess the impact of APG-157 on the microbiome as we observed the shift of oral microbiome species in the human trial. Results: We confirmed by serum concentration of APG-157 metabolites that the best route of administration is via oral route by mixing with APG-157 with mouse chow. Tumor growth optimization experiments determined that 100,000 cells of SCCVII is the ideal number of cells to inject and day 5-6 is the time to initiate treatment. Among 8 treatment groups, APG-157 + anti-CTLA-4 demonstrated best tumor control (p = 0.0065 compared to control), followed by anti-PD-1 + anti-CTLA-4 treatment group (p = 0.48 compared to control). The immunophenotype assessment by flow cytometry showed over 30% of CD8+ T cell in APG-157 + anti-CTLA-4 group compared to 4-5% of CD8+ T cell for the control group. GSEA analysis from the SCCVII tumors revealed that APG-157 + anti-CTLA-4 group showed an enriched set of genes for inflammatory response, apoptotic signaling pathways. The fecal microbiome analysis showed a substantial difference of lactobacillus genus among groups, highest in the group of APG-157 + anti-CTLA-4 treatment group. The MEER model (HPV positive) tested with same immunotherapy as single or in combination with APG-157 did not grow the tumor, thus we were unable to collect the tumor for immunophenotyping and other studies. Conclusions: These study results indicate that APG-157 is able to modulate the immune system and fecal microbial species that could potentially lead to improved anti-tumor immunity and warrants further studies to define mechanism and the potential for human clinical trial.

Radiosensitisation of SCCVII tumours and normal tissues in mice by the DNA-dependent protein kinase inhibitor AZD7648

Background and purposeInhibitors of DNA-dependent protein kinase (DNA-PK) are effective radiation sensitisers in preclinical tumours, but little is known about risks of normal tissue radiosensitisation. Here, we evaluate radiosensitisation of head and neck squamous cell carcinoma (HNSCC) cells by DNA-PK inhibitor AZD7648 under oxia and anoxia in vitro, and tumour (SCCVII), oral mucosa and small intestine in mice. Materials and methodsRadiosensitisation of human (UT-SCC-54C) and murine (SCCVII) HNSCC cells by AZD7648 under oxia and anoxia was evaluated by clonogenic assay. Radiosensitisation of SCCVII tumours in C3H mice by oral AZD7648 (75 mg/kg) was determined by ex vivo clonogenic assay 3.5 days post-irradiation, with evaluation of normal tissue surrogate endpoints using 5-ethynyl-2′-deoxyuridine to facilitate detection of regenerating crypts in the ileum and repopulating S-phase cells in the ileum and oral mucosa of the same animals. ResultsAZD7648 potently radiosensitised both cell lines, with similar sensitiser enhancement ratios for 10% survival (SER10) under oxia and anoxia. AZD7648 diffused rapidly through multicellular layers, suggesting rapid equilibration between plasma and hypoxic zones in tumours. SCCVII tumours were radiosensitised by AZD7648 (SER10 2.5). AZD7648 also enhanced radiation-induced body weight loss and suppressed regenerating intestinal crypts and repopulating S-phase cells in the ileum and tongue epithelium with SER values similar to SCCVII tumours. ConclusionAZD7648 is a potent radiation sensitiser of both oxic and anoxic tumour cells, but also markedly radiosensitises stem cells in the small intestine and oral mucosa.

Optimization of Whole Tumor Cell Vaccines by Interaction with Phagocytic Receptors.

The principal event in the function of whole-cell cancer vaccines is the ingestion of vaccine-delivered tumor antigen-containing material, which is performed by the patient’s antigen-presenting cells (APCs) through the employment of their phagocytic receptors. The goal of the present study was to identify the phagocytic receptors critical for the therapeutic efficacy of whole-cell cancer vaccines. The model of photodynamic therapy (PDT)-generated vaccines based on mouse SCCVII tumors was utilized, with in vitro expanded SCCVII cells treated by PDT serving as the vaccine material used for treating mice bearing established SCCVII tumors. The therapeutic impact, monitored as delayed progression of vaccinated tumors, was almost completely eliminated when antibodies specifically blocking the activity of LOX-1 scavenger receptor were administered to mice 30 min before vaccination. Similar, but much less pronounced, impacts were found with antibodies neutralizing the activity of CR3/CR4 receptors recognizing complement-opsonized vaccine cells, and with those blocking activating Fcγ receptors that recognized IgG antibody-based opsonins. A strikingly contrary action, a greatly enhanced tumor control by the vaccine, was found by blocking immune inhibitory receptor, FcγRIIB. The reported findings establish, therefore, an attractive strategy that can be effectively exploited for potent therapeutic enhancement of PDT-generated (and probably other) whole-cell tumor vaccines.

Estimating the mechanical energy of histotripsy bubble clouds with high frame rate imaging

Mechanical ablation with the focused ultrasound therapy histotripsy relies on the generation and action of bubble clouds. Despite its critical role for ablation, quantitative metrics of bubble activity to gauge treatment outcomes are still lacking. Here, plane wave imaging was used to track the dissolution of bubble clouds following initiation with the histotripsy pulse. Information about the rate of change in pixel intensity was coupled with an analytic diffusion model to estimate bubble size. Accuracy of the hybrid measurement/model was assessed by comparing the predicted and measured dissolution time of the bubble cloud. Good agreement was found between predictions and measurements of bubble cloud dissolution times in agarose phantoms and murine subcutaneous SCC VII tumors. The analytic diffusion model was extended to compute the maximum bubble size as well as energy imparted to the tissue due to bubble expansion. Regions within tumors predicted to have undergone strong bubble expansion were collocated with ablation. Further, the dissolution time was found to correlate with acoustic emissions generated by the bubble cloud during histotripsy insonation. Overall, these results indicate a combination of modeling and high frame rate imaging may provide means to quantify mechanical energy imparted to the tissue due to bubble expansion for histotripsy.

Abstract SY26-01: Targeting natural ligands for TCR engineering

Abstract Neoantigens (NeoAg) offer the potential to directtherapeutic T cell responses to immunogenic mutations exclusively expressed bytumors. Most strategies for their identification select potentialligands for CD8+ T cells rather than for CD4+ T cells,despite the extensive literature showing functional cooperation between thesesubsets. To better understand CD4-CD8synergy in anti-tumor immunity, we have applied a novel functional approach toidentify the subset of expressed mutations targeted as NeoAg by natural CD4+and CD8+ T cell responses to a poorly-immunogenic spontaneoussquamous cell tumor, SCC VII. We demonstrate that a single mutation (Cltc H129>Q) that is recognized byboth subsets is capable of eradicating large established SCCVII tumors in thesetting of therapeutic vaccination provided the relevant epitopes whenphysically linked. Single-cell TCR analysis revealed that the endogenous CD4+T cell response against this epitope comprises both low-affinity CD4+-dependentand high-affinity CD4+-independent receptors, with the latterdisplaying superior capacity in promoting prophylactic and therapeutic CD8+T cell-mediated immunity against the MHC class I+ class II−tumors in the setting of adoptive cellular therapy. These findings demonstratethat linked CD4+ T cell help enables potent therapeuticNeoAg-specific vaccination and provides instructive insights into the qualityof TCRs mediating this effect. Citation Format: Stephen P. Schoenberger. Targeting natural ligands for TCR engineering [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr SY26-01.

Effects of a combined treatment regimen consisting of Hsp90 inhibitor DS-2248 and radiation in vitro and in a tumor mouse model

BackgroundHeat shock protein 90 (HSP90) is a molecular chaperone that is responsible for the conformational maintenance of several client proteins that play important roles in DNA damage repair, apoptosis following radiation, and resistance to radiation therapy. DS-2248 (tricyclic pyrazolopyrimidine derivative) is a newly-developed, orally available inhibitor of HSP90 with low adverse effects. We investigated the combined effects of radiation and DS-2248 in vitro and in vivo.MethodsSCCVII squamous cell carcinoma cells and tumors transplanted in C3H/HeN mice were used. In vitro combined effects of X-ray radiation and DS-2248 were investigated using a colony assay. Phosphorylated histone H2AX (γH2AX) was quantified after 2-Gy irradiation with or without 24-hour pretreatment with DS-2248. The mice bearing SCCVII tumors received oral DS-2248 10 times over 2 weeks and received local irradiation with doses of 1, 2, 3, and 4 Gy delivered 6 times over 2 weeks. Then, tumor volumes were measured.ResultsRadiation plus pretreatment with 50 nM DS-2248 for 24 hours produced synergistic effects on SCCVII cells. γH2AX foci persisted after radiation for longer periods (6 and 24 hours) in DS-2248-treated cells than in control cells. In vivo, the combined effects appeared to be additive when 5 or 10 mg/kg DS-2248 was combined with total radiation doses of 6–18 Gy, but the effect was considered supra-additive when 15 mg/kg of DS-2248 was combined with a total dose of 24 Gy.ConclusionsThe combined effects of DS-2248 and radiation were additive at low drug and radiation doses, but may have been supra-additive at higher doses. Inhibition of slow repair of DNA double strand breaks (i.e., homologous recombination) was considered to contribute to this combined effect.

Abstract IA16: Augmenting anti-PD1 activity via an HLA-agnostic, mutation-burden independent, personalized neoantigen vaccine strategy

Abstract Neoantigens (neoAg) offer a unique opportunity for directing a patient’s adaptive immune system against tumors while avoiding damage to normal tissues. Current methods for their accurate identification and therapeutic targeting suffer from three main drawbacks: 1) they predict rather than confirm neoAg, 2) their “hit rate” is often too low to be useful in tumors of low to moderate mutational burden such as HNSCC, and, 3) they fail to inform on whether a given mutation is a natural target that can be recognized by CD8+ T cells on tumor cells or by CD4+ T cells on local antigen-presenting cells (APC). This last aspect is perhaps most critical, as a T cell that recognizes a peptide but not a target cell is of little therapeutic value to a cancer patient. We have developed a new unbiased functional approach to neoAg identification that combines bioinformatic analysis of genomic sequence data with functional T-cell assays from a patient’s own PBMC and tumor-infiltrating lymphocytes (TIL). It is based on the concept that, although in silico algorithms may seek to model one or another features of a given mutation’s predicted immunogenicity, the intact immune system can provide proof as to which mutations have been targeted by T cells, and does so according to defined rules of antigen processing and presentation that operate in both cross-presenting antigen-presenting cells (APC) that prime the response and in the tumor cells that express the source antigen. Our approach does not rely on in silico prediction of MHC binding nor any other speculative aspect of peptide immunogenicity and has minimal tissue and peripheral blood requirements. Our preliminary data show it can identify neoAg at a 10x higher rate than current methods and reveals both CD4+ and CD8+ responses. We have performed this analysis for the 4 neoAg identified for the syngeneic murine SCC VII model, and find that the prime/boost vaccination protocol results in significant but incomplete protection from challenge with live SCC VII tumor cells, thus demonstrating that, as a pool, these peptides induce T cells capable of tumor recognition. Furthermore, we have performed preliminary experiments demonstrating that, although SCC VII tumor responds to both PD-1 and CTLA4 blockade immunotherapy, the growing tumors are eradicated much faster in mice that had a pre-existing neoAg-specific T cell response induced in them prior to challenge and treatment. Mice that were vaccinated twice with neoAg peptides + polyI:C but did not receive immune checkpoint blockade showed some initial tumor control that was subsequently lost, leading to progressive growth. A phase 1b clinical trial is now enrolling subjects with advanced cancer that will produce a personalized vaccine for each patient based on our neoantigen identification methodology. The trial combines the vaccine with the anti-PD1 antibody, pembrolizumab, and enrolls patients into 2 consecutive cohorts to elucidate the effects on neoepitope specific T-cell responses of the vaccine versus anti-PD1 targeting. This presentation will summarize our efforts to identify neoantigens, efficacy of a neoantigen-specific vaccine in the murine model, and the current phase 1b study rationale and design. Citation Format: Ezra Cohen. Augmenting anti-PD1 activity via an HLA-agnostic, mutation-burden independent, personalized neoantigen vaccine strategy [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr IA16.

Tumor membrane vesicle-based vaccine immunotherapy inhibits tumor growth and metastasis of squamous cell carcinoma of head and neck in mice

Abstract Head and neck cancer is a leading cause of cancer related deaths accounting for approximately 3% of all cancer related mortalities in the US. Currently, there is no cure for the advanced squamous cell carcinoma of the head and neck (SCCHN) thus development of efficacious therapies is urgently needed. To test whether vaccine-induced immunity inhibits tumor growth, we investigated efficacy of a tumor membrane-based vaccine (TMV) immunotherapy in murine SCCHN models. The MOC1, MOC2 and SCC VII tumors grown subcutaneously in syngeneic mice were harvested to generate TMVs. TMVs were then protein transferred with glycolipid-anchored immunostimulatory molecules GPI-B7.1 and GPI-IL-12 to generate the TMV vaccine. Mice were vaccinated with TMV vaccine after tumor cell challenge (therapeutic) and tumor growth was monitored every 3 days. Survival was then assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. The TMV vaccine inhibited tumor growth and improved the survival of mice challenged with MOC1 or SCC VII tumor cells. Interestingly, combination of anti-PD1 antibody with TMV vaccine decreased MOC1 tumor growth. However, MOC2 (poorly immunogenic tumor) did not respond to anti-PD1 or anti-CTLA-4 antibody therapy but significantly inhibited by TMV vaccine. MOC2 cells metastasize to the lungs in control mice but it is significantly inhibited in mice administered with TMV vaccine. TMV vaccine increased the infiltration of CD4+ and CD8+ T cells into the tumors suggesting that TMV vaccine converts cold tumors into hot tumors. These observations suggest that TMV vaccines can be harnessed to develop an effective immunotherapy for squamous cell carcinoma of the head and neck.

Tumor Membrane Vesicle Vaccine Augments the Efficacy of Anti-PD1 Antibody in Immune Checkpoint Inhibitor-Resistant Squamous Cell Carcinoma Models of Head and Neck Cancer.

Immune checkpoint inhibitor (ICI) immunotherapy improved the survival of head and neck squamous cell carcinoma (HNSCC) patients. However, more than 80% of the patients are still resistant to this therapy. To test whether the efficacy of ICI therapy can be improved by vaccine-induced immunity, we investigated the efficacy of a tumor membrane-based vaccine immunotherapy in murine models of HNSCC. The tumors, grown subcutaneously, are used to prepare tumor membrane vesicles (TMVs). TMVs are then incorporated with glycolipid-anchored immunostimulatory molecules GPI-B7-1 and GPI-IL-12 by protein transfer to generate the TMV vaccine. This TMV vaccine inhibited tumor growth and improved the survival of mice challenged with SCCVII tumor cells. The tumor-free mice survived for several months, remained tumor-free, and were protected following a secondary tumor cell challenge, suggesting that the TMV vaccine induced an anti-tumor immune memory response. However, no synergy with anti-PD1 mAb was observed in this model. In contrast, the TMV vaccine was effective in inhibiting MOC1 and MOC2 murine oral cancer models and synergized with anti-PD1 mAb in extending the survival of tumor-bearing mice. These observations suggest that tumor tissue based TMV vaccines can be harnessed to develop an effective personalized immunotherapy for HNSCC that can enhance the efficacy of immune checkpoint inhibitors.

N-dihydrogalactochitosan-supported tumor control by photothermal therapy and photothermal therapy-generated vaccine

Photothermal therapy (PTT) is recently clinically established cancer therapy that uses near-infrared light for thermal ablation of solid tumors. The biopolymer N-dihydrogalactochitosan (GC) was shown in multiple reports to act as a very effective adjunct to tumor PTT. In the present study, mouse tumor model SCCVII (squamous cell carcinoma) was used with two protocols, in situ tumor PTT and therapeutic PTT vaccine for tumors, for investigating the effects of GC. The results reveal that GC can potentiate tumoricidal action of PTT through both direct and indirect mechanisms. In addition to previously known capacity of GC for activating immune effector cells, the indirect means is shown to include reducing the populations of immunoregulatory T cells (Tregs) in PTT-treated tumors. Testing the effects of GC on PTT-treated SCCVII tumor cells in vitro uncovered the existence of a direct mechanism evident by reduced colony survival of these cells. Fluorescence microscopy demonstrated increased binding of fluorescein-labeled GC to PTT-treated compared to untreated SCCVII cells that can be blocked by pre-exposure to annexin V. The results of additional in vitro testing with specific inhibitors demonstrate that these direct mechanisms do not involve the engagement of death surface receptors that trigger extrinsic apoptosis pathway signaling but may be linked to pro-survival activity of caspase-1. Based on the latter, it can be suggested that GC-promoted killing of PTT-treated cells stems from interference of GC bound to damaged membrane components with the repair of these structures that consequently hinders cell survival.

Abstract 4097: TMV vaccine inhibits growth of squamous cell carcinoma of head and neck in mice

Abstract Introduction: Head and neck cancer is a leading cause of cancer related deaths accounting for approximately 3% of all cancer related mortalities in the US. Currently, there is no cure for the advanced squamous cell carcinoma of the head and neck (SCCHN) thus development of efficacious therapies is urgently needed. To test whether vaccine-induced immunity inhibits tumor growth, we investigated efficacy of a tumor membrane-based vaccine immunotherapy in a murine SCCHN (SCC VII) model. Materials and Methods: The SCC VII tumors grown subcutaneously in C3H/HeJ mice were harvested to generate tumor membrane vesicles (TMVs). TMVs were then protein transferred with glycolipid-anchored immunostimulatory molecules mouse GPI-B7.1 and mouse GPI-IL-12 to generate the TMV vaccine. Mice were vaccinated with TMV vaccine either before (prophylactic) or after SCC VII tumor cell challenge (therapeutic) and tumor growth was monitored every 3 days. Survival was then assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. Results: SCCVII cells express MHC I, CD44, CD47 and respond to IFN-γ in vitro. The therapeutic TMV vaccine inhibited tumor growth and improved the survival of mice challenged with SCCVII tumor cells. Tumor-free mice remained protective from rechallenge with SCCVII cells. Conclusions: These observations suggest that tumor tissue-based vaccines can be harnessed to develop an effective immunotherapy for SCCHN. Funding: Supported by Head and Neck SPORE pilot funding from Emory Winship Cancer Institute. Citation Format: Ramireddy Bommireddy, Luis Munoz, Chrstopher D. Pack, Sampath Ramachandiran, Shaker JC Reddy, Janet Kim, Georgia Chen, Nabil F. Saba, Dong M. Shin, Periasamy Selvaraj. TMV vaccine inhibits growth of squamous cell carcinoma of head and neck in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4097.

A Multimodal Molecular Imaging Study Evaluates Pharmacological Alteration of the Tumor Microenvironment to Improve Radiation Response.

: Hypoxic zones in solid tumors contribute to radioresistance, and pharmacologic agents that increase tumor oxygenation prior to radiation, including antiangiogenic drugs, can enhance treatment response to radiotherapy. Although such strategies have been applied, imaging assessments of tumor oxygenation to identify an optimum time window for radiotherapy have not been fully explored. In this study, we investigated the effects of α-sulfoquinovosylacyl-1,3-propanediol (SQAP or CG-0321; a synthetic derivative of an antiangiogenic agent) on the tumor microenvironment in terms of oxygen partial pressure (pO2), oxyhemoglobin saturation (sO2), blood perfusion, and microvessel density using electron paramagnetic resonance imaging, photoacoustic imaging, dynamic contrast-enhanced MRI with Gd-DTPA injection, and T2*-weighted imaging with ultrasmall superparamagnetic iron oxide (USPIO) contrast. SCCVII and A549 tumors were grown by injecting tumor cells into the hind legs of mice. Five days of daily radiation (2 Gy) combined with intravenous injection of SQAP (2 mg/kg) 30 minutes prior to irradiation significantly delayed growth of tumor xenografts. Three days of daily treatment improved tumor oxygenation and decreased tumor microvascular density on T2*-weighted images with USPIO, suggesting vascular normalization. Acute effects of SQAP on tumor oxygenation were examined by pO2, sO2, and Gd-DTPA contrast-enhanced imaging. SQAP treatment improved perfusion and tumor pO2 (ΔpO2: 3.1 ± 1.0 mmHg) and was accompanied by decreased sO2 (20%-30% decrease) in SCCVII implants 20-30 minutes after SQAP administration. These results provide evidence that SQAP transiently enhanced tumor oxygenation by facilitating oxygen dissociation from oxyhemoglobin and improving tumor perfusion. Therefore, SQAP-mediated sensitization to radiation in vivo can be attributed to increased tumor oxygenation. SIGNIFICANCE: A multimodal molecular imaging study evaluates pharmacological alteration of the tumor microenvironment to improve radiation response.

Fast and sensitive dynamic oxygen-enhanced MRI with a cycling gas challenge and independent component analysis.

There is a critical need for non-invasive imaging biomarkers of tumor oxygenation to assist in patient stratification and development of hypoxia targeting therapies. Using a cycling gas challenge and independent component analysis (ICA), we sought to improve the sensitivity and speed of existing oxygen enhanced MRI (OE-MRI) techniques to detect changes in oxygenation with dynamically acquired T1 W signal intensity images (dOE-MRI). Mice were implanted with SCCVII, HCT-116, BT-474, or SKOV3 tumors in the dorsal subcutaneous region and imaged at 7T. T1 W images were acquired during a respiratory challenge with alternating 2-minute periods of air and 100% oxygen for three cycles. Data were analyzed with ICA and oxygenation maps were generated and compared to corresponding histology sections stained for hypoxia (pimonidazole) and blood vessels (CD31). Cycling air-oxygen-air gas challenges were well tolerated and ICA permitted extraction of the oxygen-enhancing component in all imaged tumors from four different models. Comparison with synthetic response functions showed that dOE-MRI does not require any a-priori knowledge of the physiological response. The fraction of O2 -negative dOE-MRI voxels that correlate inversely with the ICA gas-cycling component correspond well with the histological hypoxic fraction in SCCVII tumors (r=0.91, p=0.0016) but did not correlate in HCT-116 tumors (r=0.13, p=0.81). Using ICA and adding a cycling gas challenge extends the sensitivity of OE-MRI and allows the oxygenation status of tumors to be assessed in as little as six minutes. These findings support further development of OE-MRI as a biomarker of tumor oxygenation.

Irradiance controls photodynamic efficacy and tissue heating in experimental tumours: implication for interstitial PDT of locally advanced cancer

BackgroundCurrently delivered light dose (J/cm2) is the principal parameter guiding interstitial photodynamic therapy (I-PDT) of refractory locally advanced cancer. The aim of this study was to investigate the impact of light dose rate (irradiance, mW/cm2) and associated heating on tumour response and cure.MethodsFinite-element modeling was used to compute intratumoural irradiance and dose to guide Photofrin® I-PDT in locally advanced SCCVII in C3H mice and large VX2 neck tumours in New Zealand White rabbits. Light-induced tissue heating in mice was studied with real-time magnetic resonance thermometry.ResultsIn the mouse model, cure rates of 70–90% were obtained with I-PDT using 8.4–245 mW/cm2 and ≥45 J/cm2 in 100% of the SCCVII tumour. Increasing irradiance was associated with increase in tissue heating. I-PDT with Photofrin® resulted in significantly (p < 0.05) higher cure rate compared to light delivery alone at same irradiance and light dose. Local control and/or cures of VX2 were obtained using I-PDT with 16.5–398 mW/cm2 and ≥45 J/cm2 in 100% of the tumour.ConclusionIn Photofrin®-mediated I-PDT, a selected range of irradiance prompts effective photoreaction with tissue heating in the treatment of locally advanced mouse tumour. These irradiances were translated for effective local control of large VX2 tumours.

Co-imaging of the tumor oxygenation and metabolism using electron paramagnetic resonance imaging and 13-C hyperpolarized magnetic resonance imaging before and after irradiation.

To examine the relationship between local oxygen partial pressure and energy metabolism in the tumor, electron paramagnetic resonance imaging (EPRI) and magnetic resonance imaging (MRI) with hyperpolarized [1-13C] pyruvate were performed.SCCVII and HT29 solid tumors implanted in the mouse leg were imaged by EPRI using OX063, a paramagnetic probe and 13C-MRI using hyperpolarized [1-13C] pyruvate. Local partial oxygen pressure and pyruvate metabolism in the two tumor implants were examined. The effect of a single dose of 5-Gy irradiation on the pO2 and metabolism was also investigated by sequential imaging of EPRI and 13C-MRI in HT29 tumors.A phantom study using tubes filled with different concentration of [1-13C] pyruvate, [1-13C] lactate, and OX063 at different levels of oxygen confirmed the validity of this sequential imaging of EPRI and hyperpolarized 13C-MRI. In vivo studies revealed SCCVII tumor had a significantly larger hypoxic fraction (pO2 < 8 mmHg) compared to HT29 tumor. The flux of pyruvate-to-lactate conversion was also higher in SCCVII than HT29. The lactate-to-pyruvate ratio in hypoxic regions (pO2 < 8 mmHg) 24 hours after 5-Gy irradiation was significantly higher than those without irradiation (0.76 vs. 0.36) in HT29 tumor. The in vitro study showed an increase in extracellular acidification rate after irradiation.In conclusion, co-imaging of pO2 and pyruvate-to-lactate conversion kinetics successfully showed the local metabolic changes especially in hypoxic area induced by radiation therapy.

Systemic administration of a TLR7 agonist attenuates regulatory T cells by dendritic cell modification and overcomes resistance to PD-L1 blockade therapy.

Research on immune checkpoint blockade therapy has made great progress in cancer immunotherapy, but the number of patients who benefit from this therapy remains limited. In this study, we examined the effects of monotherapy with systemic low-dose resiquimod, a synthesized TLR7 agonist, and examined its combined effects with PD-L1 blockade in two PD-L1 blockade-resistant tumor models (SCCVII and Colon 26). Resiquimod monotherapy in SCCVII tumors, representing impaired CD8+ T cell function and accelerated regulatory T cells (Tregs) within the tumors, efficiently reduced tumor growth with more recruitment of CD8+ T cells and a reduction of Treg. The results of resiquimod monotherapy in Colon 26, representing impaired Treg recruitment, were inferior to that in SCCVII. Combined resiquimod treatment with PD-L1 blockade exerted clear additional effects, as it was associated with reduced tumor size, attenuation of Tregs, and an increased ratio of CD8+ T cells/Tregs in both tumors. Systemic administration of low-dose resiquimod induced a transient and rapid activation of plasmacytoid and conventional dendritic cells, resulting in enhanced priming of T cells in regional lymph nodes. Experiments with more limited doses of resiquimod that did not yield beneficial effects after single treatment, showed additional effects to PD-L1 blockade and comparable antitumor effects when the frequency of anti-PD-L1 therapy was decreased. Our results suggest that systemic administration of low-dose resiquimod is useful as a companion drug to PD-1/PD-L1 blockade therapy.