BackgroundIntratumoral delivery of immunotherapeutics represents a compelling solution to directly address local barriers to tumor immunity. However, we have previously shown that off-target delivery is a substantial problem during intratumoral injections; this can lead to diminished drug efficacy and systemic toxicities. We have identified three variables that influence intratumoral drug delivery: injection technique, drug formulation and tumor microenvironment. The purpose of this study was to characterize the impact of modifications in each variable on intratumoral drug delivery and immunotherapy efficacy.MethodsIntratumoral injections were performed in a hybrid image-guided intervention suite with ultrasound, fluoroscopy and CT scanning capabilities in both rat and mouse syngeneic tumor models. Intratumoral drug distribution was quantified by CT volumetric imaging. The influence of varying needle design and hydrogel-based drug delivery on the immune response to a stimulator of interferon genes (STING) agonist was evaluated using flow cytometry and single cell RNA sequencing. We also evaluated the influence of tumor stiffness on drug injection distribution.ResultsVariations in needle design, specifically with the use of a multiside hole needle, led to approximately threefold improvements in intratumoral drug deposition relative to conventional end-hole needles. Likewise, delivery of a STING agonist through a multiside hole needle led to significantly increased expression of type I interferon-associated genes and ‘inflammatory’ dendritic cell gene signatures relative to end-hole STING agonist delivery. A multidomain peptide-based hydrogel embedded with a STING agonist led to substantial improvements in intratumoral deposition; however, the hydrogel was noted to generate a strong immune response against itself within the target tumor. Evaluation of tumor stroma on intratumoral drug delivery revealed that there was a greater than twofold improvement in intratumoral distribution in soft tumors (B16 melanoma) compared with firm tumors (MC38 colorectal).ConclusionsInjection technique, drug formulation and tumor stiffness play key roles in the accurate delivery of intratumoral immunotherapeutics.

Thermal ablation is a standard therapy for patients with hepatocellular carcinoma (HCC). Contemporary ablation devices are imperfect, as they lack tumor specificity. An ideal ablation modality would generate thermal energy only within tumoral tissue. Furthermore, as hyperthermia is known to influence tumor immunity, such a tumor-specific ablation modality may have the ability to favorably modulate the tumor immune landscape. Here we show a clinically relevant thermal ablation modality that generates tumor-specific hyperthermia, termed molecularly targeted photothermal ablation (MTPA), that is based upon the excellent localization of indocyanine green to HCC. In a syngeneic rat model, we demonstrate the tumor-specific hyperthermia generated by MTPA. We also show through spatial and transcriptomic profiling techniques that MTPA favorably modulates the intratumoral myeloid population towards tumor immunogenicity and diminishes the systemic release of oncogenic cytokines relative to conventional ablation modalities.

Low-risk type human papillomavirus (HPV) 6 and 11 infection causes recurrent respiratory papillomatosis (RRP) and genital warts. RRP is the most common benign tumor of the larynx in children with frequent relapses. Repeated surgeries are often needed to improve vocal function and prevent life-threatening respiratory obstruction. Currently, there are no effective treatments available to completely eliminate these diseases, largely due to limited knowledge regarding their viral molecular pathogenesis. HPV E6 proteins contribute to cell immortalization by interacting with a variety of cellular proteins, which have been well studied for the high-risk type HPVs related to cancer progression. However, the functions of low-risk HPV E6 proteins are largely unknown. In this study, we report GST-pulldown coupled mass spectrometry analysis with low-risk HPV E6 proteins that identified sterile alpha motif domain containing 9 (SAMD9) as a novel interacting partner. We then confirmed the interaction between HPV-E6 and SAMD9 using co-immunoprecipitation, proximity ligation assay, and confocal immunofluorescence staining. The SAMD9 gene is down-regulated in a variety of neoplasms and deleteriously mutated in normophosphatemic familial tumoral calcinosis. Interestingly, SAMD9 also has antiviral functions against poxvirus. Our study adds to the limited knowledge of the molecular properties of low-risk HPVs and describes new potential functions for the low-risk HPV E6 protein.

Immunotherapy is a promising new treatment approach for hepatocellular carcinoma (HCC), but there are numerous barriers to immunotherapy in HCC, including an immunosuppressive microenvironment and the “immunotolerance” of the liver. Hyperthermia treatment modalities are standard of care for early stage HCC, and hyperthermia is known to have immunomodulatory effects. We have developed a molecularly targeted photothermal ablation (MTPA) technology that provides thermally tunable, tumor-specific heat generation. The purpose of this study was to evaluate the morphologic and immunologic effects of MTPA in an immunotherapy-resistant syngeneic mouse model of HCC in a background of toxin-induced cirrhosis. We found that the anatomic, cellular, and molecular features of this model recapitulate the characteristics of advanced human HCC. MTPA as a monotherapy and in combination with immune checkpoint therapy significantly increased intratumoral CD3+ and activated CD8+ T cells while decreasing regulatory T cells relative to control or immune checkpoint therapy alone based on immunohistochemistry, flow cytometry, and single cell RNA sequencing data. Furthermore, we identified evidence of MTPA’s influence on systemic tumor immunity, with suppression of remote tumor growth following treatment of orthotopic tumors. The results of this study suggest that tumor-specific hyperthermia may help overcome resistance mechanisms to immunotherapy in advanced HCC.

Objectives/Hypothesis Recurrent respiratory papillomatosis (RRP) is a devastating disease, caused by infection of the upper aerodigestive tract with human papillomavirus types 6 and 11. There is no cure for RRP, and surgical removal is the mainstay of treatment. The purpose of this project was to compare genes of cell cycle, apoptosis, and inflammatory cytokines in laryngeal papilloma versus normal tissue for a better understanding of the molecular mechanisms of the disease to discover novel therapies. Study Design Basic science research study. Methods Papilloma tissue was obtained from patients requiring surgical debridement. For comparison, normal mucosa was obtained from the excised uvula of patients undergoing uvulopalatopharyngoplasty. Total RNA was extracted from both groups and then probed using customized reverse transcriptase real time polymerase chain reaction gene arrays. Results The custom arrays examine expression of 84 separate genes within the cell cycle, apoptosis, and inflammatory cytokine pathways. Our findings based on 11 papilloma samples run in comparison to normal mucosa shows that the MCL–1 gene of the apoptosis pathway is significantly downregulated. cytokine genes IL1-A, IL-8, IL-18, and IL-31 are also significantly dysregulated. Conclusions Genes of cell cycle and apoptosis are generally upregulated and downregulated, respectively, as expected in papilloma tissue, with MCL-1 achieving significance when compared to normal tissue. The finding of particular interest is that inflammatory cytokine genes were significantly downregulated, including IL1-A, IL-18, and IL-31. This finding may explain why patients infected with the virus are unable to mediate a T-cell immune clearance of their disease.