Liver Cancer Vascularity Driven by Extracellular Matrix Stiffness: Implications for Imaging Research.

BACKGROUND: Extracellular matrix stiffness represents a barrier to effective local and systemic drug delivery. Increasing stiffness disrupts newly formed vessel architecture and integrity, leading to tumor-like vasculature. The resulting vascular phenotypes are manifested through different cross-sectional imaging features. Contrast-enhanced studies can help elucidate the interplay between liver tumor stiffness and different vascular phenotypes. PURPOSE: This study aims to correlate extracellular matrix stiffness, dynamic contrast-enhanced computed tomography, and dynamic contrast-enhancement ultrasound imaging features of 2 rat hepatocellular carcinoma tumor models. METHODS AND MATERIALS: Buffalo-McA-RH7777 and Sprague Dawley (SD)-N1S1 tumor models were used to evaluate tumor stiffness by 2-dimensional shear wave elastography, along with tumor perfusion by dynamic contrast-enhanced ultrasonography and contrast-enhanced computed tomography. Atomic force microscopy was used to calculate tumor stiffness at a submicron scale. Computer-aided image analyses were performed to evaluate tumor necrosis, as well as the percentage, distribution, and thickness of CD34+ blood vessels. RESULTS: Distinct tissue signatures between models were observed according to the distribution of the stiffness values by 2-dimensional shear wave elastography and atomic force microscopy ( P < 0.05). Higher stiffness values were attributed to SD-N1S1 tumors, also associated with a scant microvascular network ( P ≤ 0.001). Opposite results were observed in the Buffalo-McA-RH7777 model, exhibiting lower stiffness values and richer tumor vasculature with predominantly peripheral distribution ( P = 0.03). Consistent with these findings, tumor enhancement was significantly greater in the Buffalo-McA-RH7777 tumor model than in the SD-N1S1 on both dynamic contrast-enhanced ultrasonography and contrast-enhanced computed tomography ( P < 0.005). A statistically significant positive correlation was observed between tumor perfusion on dynamic contrast-enhanced ultrasonography and contrast-enhanced computed tomography in terms of the total area under the curve and % microvessel tumor coverage ( P < 0.05). CONCLUSIONS: The stiffness signatures translated into different tumor vascular phenotypes. Two-dimensional shear wave elastography and dynamic contrast-enhanced ultrasonography adequately depicted different stromal patterns, which resulted in unique imaging perfusion parameters with significantly greater contrast enhancement observed in softer tumors.

Immune modulation by molecularly targeted photothermal ablation in a mouse model of advanced hepatocellular carcinoma and cirrhosis.

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.

Influence of injection technique, drug formulation and tumor microenvironment on intratumoral immunotherapy delivery and efficacy.

BACKGROUND: Intratumoral 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. METHODS: Intratumoral 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. RESULTS: Variations 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). CONCLUSIONS: Injection technique, drug formulation and tumor stiffness play key roles in the accurate delivery of intratumoral immunotherapeutics.

Molecularly targeted photothermal ablation improves tumor specificity and immune modulation in a rat model of hepatocellular carcinoma.

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.

What Are the Effects of Irreversible Electroporation on a Staphylococcus aureus Rabbit Model of Osteomyelitis?

BACKGROUND: The treatment of osteomyelitis can be challenging because of poor antibiotic penetration into the infected bone and toxicities associated with prolonged antibiotic regimens to control infection. Irreversible electroporation (IRE), a percutaneous image-guided ablation technology in which the targeted delivery of high-voltage electrical pulses permanently damages the cell membrane, has been shown to effectively control bacterial growth in various settings. However, IRE for the management of bone infections has yet to be evaluated. QUESTIONS/PURPOSES: We aimed to evaluate IRE for treating osteomyelitis by assessing (1) the efficacy of IRE to suppress the in vitro growth of a clinical isolate of S. aureus, alone or combined with cefazolin; and (2) the effects of IRE on the in vivo treatment of a rabbit model of osteomyelitis. METHODS: S. aureus strain UAMS-1 expanded in vitro to the log phase was subjected to an electric field of 2700 V/cm, which was delivered in increasing numbers of pulses. Immediately after electroporation, bacteria were plated on agar plates with or without cefazolin. The number of colony-forming units (CFUs) was scored the following day. ANOVA tests were used to analyze in vitro data. In a rabbit osteomyelitis model, we inoculated the same bacterial strain into the radius of adult male New Zealand White rabbits. Three weeks after inoculation, all animals (n = 32) underwent irrigation and débridement, as well as wound culture of the infected forelimb. Then, they were randomly assigned to one of four treatment groups (n = eight per group): untreated control, cefazolin only, IRE only, or combined IRE + cefazolin. Serial radiography was performed to assess disease progression using a semiquantitative grading scale. Bone and soft-tissue specimens from the infected and contralateral forelimbs were collected at 4 weeks after treatment for bacterial isolation and histologic assessment using a semiquantitative scale. RESULTS: The in vitro growth of S. aureus UAMS-1 was impaired by IRE in a pulse-dependent fashion; the number of CFUs/mL was different among seven pulse levels, namely 0, 10, 30, 60, 90, 120, and 150 pulses. With the number of CFUs/mL observed in untreated controls set as 100%, 10 pulses rendered a median of 50.2% (range 47.1% to 58.2%), 30 pulses rendered a median of 2.7% (range 2.5% to 2.8%), 60 pulses rendered a median of 0.014% (range 0.012% to 0.015%), 90 pulses rendered a median of 0.004% (range 0.002% to 0.004%), 120 pulses rendered a median of 0.001% (range 0.001% to 0.001%), and 150 pulses rendered a median of 0.001% (range 0.000% to 0.001%) (Kruskal-Wallis test: p = 0.003). There was an interaction between the effect of the number of pulses and the concentration of cefazolin (two-way ANOVA: F [8, 30] = 17.24; p < 0.001), indicating that combining IRE with cefazolin is more effective than either treatment alone at suppressing the growth of S. aureus UAMS-1. Likewise, the clinical response in the rabbit model (the percentage of animals without detectable residual bacteria in the bone and surrounding soft tissue after treatment) was better in the combination group than in the other groups: control, 12.5% (one of eight animals); IRE only, 12.5% (one of eight animals); cefazolin only, 25% (two of eight animals); and IRE + cefazolin, 75% (six of eight animals) (two-sided Fisher's exact test: p = 0.030). CONCLUSIONS: IRE effectively suppressed the growth of S. aureus UAMS-1 and enhanced the antibacterial effect of cefazolin in in vitro studies. When translated to a rabbit osteomyelitis model, the addition of IRE to conventional parenteral antibiotic treatment produced the strongest response, which supports the in vitro findings. CLINICAL RELEVANCE: Our results show that IRE may improve the results of standard parenteral antibiotic treatment, thus setting the stage for models with larger animals and perhaps trials in humans for validation.

Sterile α Motif Domain Containing 9 Is a Novel Cellular Interacting Partner to Low-Risk Type Human Papillomavirus E6 Proteins.

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.

E6 proteins from low-risk human papillomavirus types 6 and 11 are able to protect keratinocytes from apoptosis via Bak degradation.

Infection of epithelial surfaces with low-risk human papillomavirus (HPV) types 6 and 11 causes troublesome clinical diseases, such as recurrent respiratory papillomatosis, that carry a significant cost burden to the healthcare system. Despite this, less has been studied at the molecular level for the low-risk HPV types when compared with their high-risk counterparts. Recent studies have shown the ability of the HPV E6 protein to degrade the pro-apoptotic family member Bak in high-risk and betapapillomavirus HPV types, which confers a cytoprotective advantage on E6-expressing cells. It is unknown whether low-risk E6 expression disrupts the apoptosis pathway and confers a cytoprotective advantage as a result of Bak degradation. We tested the abilities of 6E6 and 11E6 to degrade Bak and protect keratinocytes from UV-initiated apoptosis. Both low-risk 6E6 and 11E6 proteins were able to degrade activated Bak following UV treatment of keratinocytes. The degradation of Bak in 6E6- and 11E6-expressing cells occurred through the proteasomal pathway, and protected them from apoptosis, specifically through the intrinsic pathway to the same extent as their high-risk HPV16 E6 counterpart. In conclusion, we have found a new, critical and conserved function of low-risk HPV E6 proteins, i.e. the ability to degrade Bak, which gives them a cytoprotective advantage over normal, uninfected cells by specifically disrupting the intrinsic pathway of apoptosis.

Genetic dysregulation in recurrent respiratory papillomatosis.

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.