Mitochondrial defects and metabolic vulnerabilities in Lynch syndrome-associated MSH2-deficient endometrial cancer.

Lynch syndrome (LS) is defined by inherited mutations in DNA mismatch repair genes, including MSH2, and carries 60% lifetime risk of developing endometrial cancer (EC). Beyond hypermutability, specific mechanisms for LS-associated endometrial carcinogenesis are not well understood. Here, we assessed the effects of MSH2 loss on EC pathogenesis using a novel mouse model (PR-Cre Msh2 flox/flox , abbreviated Msh2KO), primary cell lines established from this model, human tissues, and human EC cell lines with isogenic MSH2 knockdown. Beginning at eight months of age, 30% of Msh2KO mice exhibited endometrial atypical hyperplasia (AH), a precancerous lesion. At 12 to 16 months of age, 47% of Msh2KO mice exhibited either AH or ECs with histologic features similar to human LS-related ECs. Transcriptomic profiling of EC from Msh2KO mice revealed a transcriptomic signature for mitochondrial dysfunction. Studies in vitro and in vivo revealed mitochondrial dysfunction based upon two mechanisms: marked mitochondrial content reduction, along with pronounced disruptions to the integrity of retained mitochondria. Human LS-related ECs also exhibited mitochondrial content reduction compared with non-LS-related ECs. Functional studies revealed metabolic reprogramming of MSH2-deficient EC cells in vitro , including reduced oxidative phosphorylation and increased susceptibility to glycolysis suppression. We are the first to identify mitochondrial dysfunction and metabolic disruption as a consequence of MSH2 deficiency-related EC. Mitochondrial and metabolic aberrations should be evaluated as novel biomarkers for endometrial carcinogenesis or risk stratification and could serve as targets for cancer interception in women with LS. Significance: This is the first study to report mitochondrial dysfunction contributing to MSH2-deficient endometrial cancer development, identifying a noncanonical pathway for MSH2 deficient carcinogenesis, which also imparts vulnerability to metabolic targeting.

A rat model of multicompartmental traumatic injury and hemorrhagic shock induces bone marrow dysfunction and profound anemia.

BACKGROUND: Severe trauma is associated with systemic inflammation and organ dysfunction. Preclinical rodent trauma models are the mainstay of postinjury research but have been criticized for not fully replicating severe human trauma. The aim of this study was to create a rat model of multicompartmental injury which recreates profound traumatic injury. METHODS: Male Sprague-Dawley rats were subjected to unilateral lung contusion and hemorrhagic shock (LCHS), multicompartmental polytrauma (PT) (unilateral lung contusion, hemorrhagic shock, cecectomy, bifemoral pseudofracture), or naïve controls. Weight, plasma toll-like receptor 4 (TLR4), hemoglobin, spleen to body weight ratio, bone marrow (BM) erythroid progenitor (CFU-GEMM, BFU-E, and CFU-E) growth, plasma granulocyte colony-stimulating factor (G-CSF) and right lung histologic injury were assessed on day 7, with significance defined as p values <0.05 (*). RESULTS: Polytrauma resulted in markedly more profound inhibition of weight gain compared to LCHS (p = 0.0002) along with elevated plasma TLR4 (p < 0.0001), lower hemoglobin (p < 0.0001), and enlarged spleen to body weight ratios (p = 0.004). Both LCHS and PT demonstrated suppression of CFU-E and BFU-E growth compared to naïve (p < 0.03, p < 0.01). Plasma G-CSF was elevated in PT compared to both naïve and LCHS (p < 0.0001, p = 0.02). LCHS and PT demonstrated significant histologic right lung injury with poor alveolar wall integrity and interstitial edema. CONCLUSIONS: Multicompartmental injury as described here establishes a reproducible model of multicompartmental injury with worsened anemia, splenic tissue enlargement, weight loss, and increased inflammatory activity compared to a less severe model. This may serve as a more effective model to recreate profound traumatic injury to replicate the human inflammatory response postinjury.

Multicompartmental Trauma Induces Persistent Inflammation and Organ Injury.

INTRODUCTION: Previous preclinical models of multicompartmental injury have investigated its effects for durations of less than 72 h and the long-term effects have not been defined. We hypothesized that a model of multicompartmental injury would result in systemic inflammation and multiorgan dysfunction that persists at 1 wk. METHODS: Male and proestrus female Sprague-Dawley rats (n = 16/group) underwent polytrauma (PT) (unilateral right lung contusion, hemorrhagic shock, cecectomy, bifemoral pseudofractures) and were compared to naive controls. Weight, hemoglobin, plasma neutrophil gelatinase-associated lipocalin, and plasma toll-like receptor 4 were evaluated on days two and seven. Bilateral lungs were sectioned, stained and assessed for injury at day seven. Comparisons were performed in Graphpad with significance defined as ∗P <0.05. RESULTS: Rats who underwent PT had significant weight loss and anemia at day 2 (P = 0.001) compared to naïve rats which persisted at day 7 (P = 0.001). PT rats had elevated plasma neutrophil gelatinase-associated lipocalin at day 2 compared to naïve (P <0.0001) which remained elevated at day 7 (P <0.0001). Plasma toll-like receptor 4 was elevated in PT compared to naïve at day 2 (P = 0.03) and day 7 (P = 0.01). Bilateral lungs showed significant injury in PT cohorts at day 7 compared to naïve (P <0.0004). PT males had worse renal function at day seven compared to females (P = 0.02). CONCLUSIONS: Multicompartmental trauma induces systemic inflammation and multiorgan dysfunction without recovery by day seven. However, females demonstrate improved renal recovery compared to males. Long-term assessment of preclinical PT models are crucial to better understand and evaluate future therapeutic immunomodulatory and anti-inflammatory treatments.

Unique Transcriptional Profiles Underlie Osteosarcomagenesis Driven by Different p53 Mutants.

Missense mutations in the DNA binding domain of p53 are characterized as structural or contact mutations based on their effect on the conformation of the protein. These mutations show gain-of-function (GOF) activities, such as promoting increased metastatic incidence compared with p53 loss, often mediated by the interaction of mutant p53 with a set of transcription factors. These interactions are largely context specific. To understand the mechanisms by which p53 DNA binding domain mutations drive osteosarcoma progression, we created mouse models, in which either the p53 structural mutant p53R172H or the contact mutant p53R245W are expressed specifically in osteoblasts, yielding osteosarcoma tumor development. Survival significantly decreased and metastatic incidence increased in mice expressing p53 mutants compared with p53-null mice, suggesting GOF. RNA sequencing of primary osteosarcomas revealed vastly different gene expression profiles between tumors expressing the missense mutants and p53-null tumors. Further, p53R172H and p53R245W each regulated unique transcriptomes and pathways through interactions with a distinct repertoire of transcription factors. Validation assays showed that p53R245W, but not p53R172H, interacts with KLF15 to drive migration and invasion in osteosarcoma cell lines and promotes metastasis in allogeneic transplantation models. In addition, analyses of p53R248W chromatin immunoprecipitation peaks showed enrichment of KLF15 motifs in human osteoblasts. Taken together, these data identify unique mechanisms of action of the structural and contact mutants of p53. SIGNIFICANCE: The p53 DNA binding domain contact mutant p53R245W, but not the structural mutant p53R172H, interacts with KLF15 to drive metastasis in somatic osteosarcoma, providing a potential vulnerability in tumors expressing p53R245W mutation.

Dimeric p53 Mutant Elicits Unique Tumor-Suppressive Activities through an Altered Metabolic Program.

Cancer-related alterations of the p53 tetramerization domain (TD) abrogate wild-type (WT) p53 function. They result in a protein that preferentially forms monomers or dimers, which are also normal p53 states under basal cellular conditions. However, their physiologic relevance is not well understood. We have established in vivo models for monomeric and dimeric p53, which model Li-Fraumeni syndrome patients with germline p53 TD alterations. p53 monomers are inactive forms of the protein. Unexpectedly, p53 dimers conferred some tumor suppression that is not mediated by canonical WT p53 activities. p53 dimers upregulate the PPAR pathway. These activities are associated with lower prevalence of thymic lymphomas and increased CD8+ T-cell differentiation. Lymphomas derived from dimeric p53 mice show cooperating alterations in the PPAR pathway, further implicating a role for these activities in tumor suppression. Our data reveal novel functions for p53 dimers and support the exploration of PPAR agonists as therapies. SIGNIFICANCE: New mouse models with TP53R342P (monomer) or TP53A347D (dimer) mutations mimic Li-Fraumeni syndrome. Although p53 monomers lack function, p53 dimers conferred noncanonical tumor-suppressive activities. We describe novel activities for p53 dimers facilitated by PPARs and propose these are "basal" p53 activities. See related commentary by Stieg et al., p. 1046. See related article by Choe et al., p. 1250. This article is highlighted in the In This Issue feature, p. 1027.

Multicompartmental traumatic injury induces sex-specific alterations in the gut microbiome.

BACKGROUND: Previous preclinical studies have demonstrated an altered gut microbiome after traumatic injury; however, the impact of sex on dysbiosis remains unknown. We hypothesized that the "pathobiome" phenotype induced by multicompartmental injuries and chronic stress is host sex specific with unique microbiome signatures. METHODS: Male and proestrus female Sprague-Dawley rats (n = 8/group) aged 9 weeks to 11 weeks were subjected to either multicompartmental injury (PT) (lung contusion, hemorrhagic shock, cecectomy, bifemoral pseudofractures), PT plus 2 hours daily chronic restraint stress (PT/CS) or naive controls. Fecal microbiome was measured on Days 0 and 2 using high-throughput 16S rRNA sequencing and Quantitative Insights Into Microbial Ecology bioinformatics analyses. Microbial alpha-diversity was assessed using Chao1 (number of different unique species) and Shannon (species richness and evenness) indices. Beta-diversity was assessed using principle coordinate analysis. Intestinal permeability was evaluated by plasma occludin and lipopolysaccharide binding protein. Histologic evaluation of ileum and colon tissues was scored for injury by a blinded pathologist. Analyses were performed in GraphPad and R, with significance defined as p < 0.05 between males versus females. RESULTS: At baseline, females had significantly elevated alpha-diversity (Chao1, Shannon indices) compared with males ( p < 0.05) which was no longer present 2 days postinjury in PT and PT/CS. Beta-diversity also differed significantly between males and females after PT ( p = 0.01). At Day 2, the microbial composition in PT/CS females was dominated by Bifidobacterium , whereas PT males demonstrated elevated levels of Roseburia ( p < 0.01). The PT/CS males had significantly elevated ileum injury scores compared with females ( p = 0.0002). Plasma occludin was higher in PT males compared with females ( p = 0.004); plasma lipopolysaccharide binding protein was elevated in PT/CS males ( p = 0.03). CONCLUSION: Multicompartmental trauma induces significant alterations in microbiome diversity and taxa, but these signatures differ by host sex. These findings suggest that sex is an important biological variable that may influence outcomes after severe trauma and critical illness.

Alendronate conjugate for targeted delivery to bone-forming prostate cancer.

Bone is the primary metastasis site for lethal prostate cancer, often resulting in poor prognosis, crippling pain, and diminished functioning that drastically reduce both quality of life and survivability Uniquely, prostate cancer bone metastasis induces aberrant bone overgrowth, due to an increase of osteoblasts induced by tumor-secreted bone morphogenetic protein 4 (BMP4). Conjugating drugs to substances that target the tumor-induced bone area within the metastatic tumor foci would be a promising strategy for drug delivery. To develop such a strategy, we conjugated a near infrared (NIR) fluorescent probe, the dye Cy5.5, to serve as a surrogate for drugs, with alendronate, which targets bone. Characterization, such as infrared spectroscopy, confirmed the synthesis of the Cy5.5-ALN conjugate. The maximum absorbance of free Cy5.5, which was at 675 nm, did not change upon conjugation. Alendronate targeted the bone component hydroxyapatite in a dose-dependent manner up to 2.5 µM, with a maximum of 85% of Cy5.5-ALN bound to hydroxyapatite, while free Cy5.5 alone had 6% binding. In in vitro cell binding studies, Cy5.5-ALN bound specifically with mineralized bone matrix of differentiated MC3T3-E1 cells or 2H11 endothelial cells that were induced to become osteoblasts through endothelial-to-osteoblast transition, the underlying mechanism of prostate-cancer-induced bone formation. Neither Cy5.5-ALN nor free Cy5.5 bound to undifferentiated MC3T3-E1 or 2H11 cells. Bone-targeting efficiency studies in non-tumor-bearing mice revealed accumulation over time in the spine, jaw, knees, and paws injected with Cy5.5-ALN, and quantification showed higher accumulation in femurs than in muscle at up to 28 days, while the free Cy5.5 dye was observed circulating without preferential accumulation and decreased over time. There was a linear relationship with fluorescence when the injected concentration of Cy5.5-ALN was between 0.313 and 1.25 nmol/27 g of mouse, as quantified in mouse femurs both in vivo and ex vivo. Ex vivo evaluation of bone-targeting efficiency in nude mice was 3 times higher for bone-forming C4-2b-BMP4 tumors compared to non-bone-forming C4-2b tumors (p-value <0.001). Fluorescence microscopy imaging of the tumors showed that Cy5.5-ALN co-localized with the bone matrix surrounding tumor-induced bone, but not with the viable tumor cells. Together, these results suggest that a drug-ALN conjugate is a promising approach for targeted delivery of drug to the tumor-induced bone area in the metastatic foci of prostate cancer.

Multicompartmental traumatic injury and the microbiome: Shift to a pathobiome.

BACKGROUND: Previous animal models have demonstrated altered gut microbiome after mild traumatic injury; however, the impact of injury severity and critical illness is unknown. We hypothesized that a rodent model of severe multicompartmental injuries and chronic stress would demonstrate microbiome alterations toward a "pathobiome" characterized by an overabundance of pathogenic organisms, which would persist 1 week after injury. METHODS: Male Sprague-Dawley rats (n = 8 per group) were subjected to either multiple injuries (PT) (lung contusion, hemorrhagic shock, cecectomy, and bifemoral pseudofractures), PT plus daily chronic restraint stress for 2 hours (PT/CS), or naive controls. Fecal microbiome was measured on days 0, 3, and 7 using high-throughput 16S rRNA sequencing and Quantitative Insights Into Microbial Ecology 2 bioinformatics analysis. Microbial α diversity was assessed using Chao1 and Shannon indices, and ß diversity with principle coordinate analysis. Intestinal permeability was evaluated by plasma occludin; ileum and descending colon tissues were reviewed for injury. Analyses were performed in GraphPad (GraphPad Software, La Jolla, CA) and R (R Foundation for Statistical Computing, Vienna, Austria), with significance defined as p < 0.05. RESULTS: There were significant alterations in ß diversity at day 3 and between all groups. By day 3, both PT and PT/CS demonstrated significantly depleted bacterial diversity (Chao1) ( p = 0.01 and p = 0.001, respectively) versus naive, which persisted up to day 7 in PT/CS only ( p = 0.001). Anaerostipes and Rothia dominated PT and Lactobacillus bloomed in PT/CS cohorts by day 7. Plasma occludin was significantly elevated in PT/CS compared with naive ( p = 0.04), and descending colon of both PT and PT/CS showed significantly higher injury compared with naive ( p = 0.005, p = 0.006). CONCLUSIONS: Multiple injuries with and without chronic stress induces significant alterations in microbiome diversity and composition within 3 days; these changes are more prominent and persist for 1 week postinjury with stress. This rapid and persistent transition to a "pathobiome" phenotype represents a critical phenomenon that may influence outcomes after severe trauma and critical illness.

Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by immune-related adverse events (irAEs). Limited understanding of irAE mechanisms hampers development of approaches to mitigate their damage. To address this, we examined whether mice gained sensitivity to anti-CTLA-4 (αCTLA-4)-mediated toxicity upon disruption of gut homeostatic immunity. We found αCTLA-4 drove increased inflammation and colonic tissue damage in mice with genetic predisposition to intestinal inflammation, acute gastrointestinal infection, transplantation with a dysbiotic fecal microbiome, or dextran sodium sulfate administration. We identified an immune signature of αCTLA-4-mediated irAEs, including colonic neutrophil accumulation and systemic interleukin-6 (IL-6) release. IL-6 blockade combined with antibiotic treatment reduced intestinal damage and improved αCTLA-4 therapeutic efficacy in inflammation-prone mice. Intestinal immune signatures were validated in biopsies from patients with ICB colitis. Our work provides new preclinical models of αCTLA-4 intestinal irAEs, mechanistic insights into irAE development, and potential approaches to enhance ICB efficacy while mitigating irAEs.

Context matters - Daxx and Atrx are not robust tumor suppressors in the murine endocrine pancreas.

Genome sequencing has revealed the importance of epigenetic regulators in tumorigenesis. The genes encoding the chromatin remodeling complex DAXX:ATRX are frequently mutated in pancreatic neuroendocrine tumors; however, the underlying mechanisms of how mutations contribute to tumorigenesis are only partially understood, in part because of the lack of relevant preclinical models. Here, we used genetically engineered mouse models combined with environmental stress to evaluate the tumor suppressor functions of Daxx and Atrx in the mouse pancreas. Daxx or Atrx loss, alone or in combination with Men1 loss, did not drive or accelerate pancreatic neuroendocrine tumorigenesis. Moreover, Daxx loss did not cooperate with environmental stresses (ionizing radiation or pancreatitis) or with the loss of other tumor suppressors (Pten or p53) to promote pancreatic neuroendocrine tumorigenesis. However, owing to promiscuity of the Cre promoter used, hepatocellular carcinomas and osteosarcomas were observed in some instances. Overall, our findings suggest that Daxx and Atrx are not robust tumor suppressors in the endocrine pancreas of mice and indicate that the context of a human genome is essential for tumorigenesis. This article has an associated First Person interview with the first author of the paper.

Differential Gain-of-Function Activity of Three p53 Hotspot Mutants In Vivo.

The majority of TP53 missense mutations identified in cancer patients are in the DNA-binding domain and are characterized as either structural or contact mutations. These missense mutations exhibit inhibitory effects on wild-type p53 activity. More importantly, these mutations also demonstrate gain-of-function (GOF) activities characterized by increased metastasis, poor prognosis, and drug resistance. To better understand the activities by which TP53 mutations, identified in Li-Fraumeni syndrome, contribute to tumorigenesis, we generated mice harboring a novel germline Trp53R245W allele (contact mutation) and compared them with existing models with Trp53R172H (structural mutation) and Trp53R270H (contact mutation) alleles. Thymocytes from heterozygous mice showed that all three hotspot mutations exhibited similar inhibitory effects on wild-type p53 transcription in vivo, and tumors from these mice had similar levels of loss of heterozygosity. However, the overall survival of Trp53R245W/+ and Trp53R270H/+ mice, but not Trp53R172H/+ mice, was significantly shorter than that of Trp53+/- mice, providing strong evidence for p53-mutant-specific GOF contributions to tumor development. Furthermore, Trp53R245W/+ and Trp53R270H/+ mice had more osteosarcoma metastases than Trp53R172H/+ mice, suggesting that these two contact mutants have stronger GOF in driving osteosarcoma metastasis. Transcriptomic analyses using RNA sequencing data from Trp53R172H/+, Trp53R245W/+, and Trp53R270H/+ primary osteosarcomas in comparison with Trp53+/- indicated that GOF of the three mutants was mediated by distinct pathways. Thus, both the inhibitory effect of mutant over wild-type p53 and GOF activities of mutant p53 contributed to tumorigenesis in vivo. Targeting p53 mutant-specific pathways may be important for therapeutic outcomes in osteosarcoma. SIGNIFICANCE: p53 hotspot mutants inhibit wild-type p53 similarly but differ in their GOF activities, with stronger tumor-promoting activity in contact mutants and distinct protein partners of each mutant driving tumorigenesis and metastasis.

Effect of bead size and doxorubicin loading on tumor cellular injury after transarterial embolization and chemoembolization in a rat model of hepatocellular carcinoma.

Embolic agents used in transarterial embolization for intermediate stage hepatocellular carcinoma reduce blood flow into tumors and can deliver anticancer drugs. Tumor blood supply can be interrupted using doxorubicin-eluting beads (DEB-TACE) or non-loaded beads (TAE) of different calibers. In this preclinical study, we characterized the extent of remaining stressed tumor cells after treatment, hypoxia within the surviving tumor regions, and inflammatory immune cell infiltrates after embolization with 40-60 or 70-150 µm with non-loaded or doxorubicin-loaded beads at 3 and 7 days after treatment. TAE-treated tumors had more stressed and surviving tumor cells after 3 days, irrespective of bead size, compared with DEB-TACE-treated tumors. Hypoxic stress of residual cells increased after treatment with 70-150 µm beads without or with doxorubicin. Treatment with DEB-TACE of 70-150 µm resulted in increased inflammation and proliferation in the adjacent parenchyma. Inflammatory cell infiltrates were reduced at the periphery of tumors treated with 40-60 µm DEB-TACE.

Canine osteosarcoma checkpoint expression correlates with metastasis and T-cell infiltrate.

BACKGROUND: Immune-targeted therapies are being successfully implemented into cancer clinical practice. In particular checkpoint inhibitors are employed to modulate the immune microenvironment of solid tumors. We sought to determine the expression of PD-L1, HVEM, and B7H3 in human and canine osteosarcoma, and correlate expression with clinical features and tumor infiltrating lymphocytes in naturally-occurring canine osteosarcoma. METHODS: Flow cytometry was used to measure ligand surface expression of five human and three canine cell lines. Immunohistochemistry was utilized for expression of ligands and lymphocyte markers in thirty-seven treatment-naïve canine osteosarcoma patients. RESULTS: All cell lines expressed all three ligands at variable levels in both species. Metastatic lesions were associated with higher expression of all three ligands in patient tumor samples. PD-L1 expression strongly correlated with B7H3 and HVEM expression, while HVEM and B7H3 were weakly correlated. Whereas peritumoral T-cell expression positively correlated with PD-L1 and HVEM tumor expression, the presence of T-cells intratumorally were rare. Furthermore, intratumor penetration by T-cells was greatest in metastatic lesions, despite log-fold increases in peritumoral T-cells. In summary, PD-L1, HVEM, and B7H3 are expressed in osteosarcoma, with metastatic disease lesions expressing higher levels. We show for the first time that these ligands expressed on osteosarcoma cells positively correlate with each other and the presence of peritumoral T cell infiltration. Furthermore, osteosarcoma appears to be an intratumoral immune desert with significant resistance to effector T cells. Multiple agents targeting checkpoints are in clinical practice, and may have immune modulating benefit in osteosarcoma.

A Genetic Mouse Model Recapitulates Immune Checkpoint Inhibitor-Associated Myocarditis and Supports a Mechanism-Based Therapeutic Intervention.

Immune checkpoint inhibitors (ICI) targeting CTLA4 or PD-1/PD-L1 have transformed cancer therapy but are associated with immune-related adverse events, including myocarditis. Here, we report a robust preclinical mouse model of ICI-associated myocarditis in which monoallelic loss of Ctla4 in the context of complete genetic absence of Pdcd1 leads to premature death in approximately half of mice. Premature death results from myocardial infiltration by T cells and macrophages and severe ECG abnormalities, closely recapitulating the clinical and pathologic hallmarks of ICI-associated myocarditis observed in patients. Using this model, we show that Ctla4 and Pdcd1 functionally interact in a gene dosage-dependent manner, providing a mechanism by which myocarditis arises with increased frequency in the setting of combination ICI therapy. We demonstrate that intervention with CTLA4-Ig (abatacept) is sufficient to ameliorate disease progression and additionally provide a case series of patients in which abatacept mitigates the fulminant course of ICI myocarditis. SIGNIFICANCE: We provide a preclinical model of ICI-associated myocarditis which recapitulates this clinical syndrome. Using this model, we demonstrate that CTLA4 and PD-1 (ICI targets) functionally interact for myocarditis development and that intervention with CTLA4-Ig (abatacept) attenuates myocarditis, providing mechanistic rationale and preclinical support for therapeutic clinical studies.See related commentary by Young and Bluestone, p. 537.This article is highlighted in the In This Issue feature, p. 521.

Correlation of molecular and morphologic effects of thermoembolization in a swine model using mass spectrometry imaging.

Hepatocellular carcinoma is a growing worldwide problem with a high mortality rate. This malignancy does not respond well to chemotherapy, and most patients present late in their disease at which time surgery is no longer an option. Over the past three decades, minimally invasive methods have evolved to treat unresectable disease and prolong survival. Intra-arterial embolization techniques are used for large or multiple tumors but have distressingly high levels of local recurrence and can be costly to implement. A new method called thermoembolization was recently reported, which destroys target tissue by combining reactive exothermic chemistry with an extreme local change in pH and ischemia. Described herein are experiments performed using this technique in vivo in a swine model. A microcatheter was advanced under fluoroscopic guidance into a branch of the hepatic artery to deliver a targeted dose of dichloroacetyl chloride dissolved in ethiodized oil into the liver. The following day, the animals were imaged by computed tomography and euthanized. Assessing the reaction product distribution and establishing a correlation with the effects are important for understanding the effects. This presented a significant challenge, however, as the reagent used does not contain a chromophore and is not otherwise readily detectable. Mass spectrometry imaging was employed to determine spatial distribution in treated samples. Additional insights on the biology were obtained by correlating the results with histology, immunohistochemistry, and immunofluorescence. The results are encouraging and may lead to a therapy with less local recurrence and improved overall survival for patients with this disease.

Combining Chemistry and Engineering for Hepatocellular Carcinoma: Nano-Scale and Smaller Therapies.

Primary liver cancer, or hepatocellular carcinoma (HCC), is a major worldwide cause of death from carcinoma. Most patients are not candidates for surgery and medical therapies, including new immunotherapies, have not shown major improvements since the modest benefit seen with the introduction of sorafenib over a decade ago. Locoregional therapies for intermediate stage disease are not curative but provide some benefit. However, upon close scrutiny, there is still residual disease in most cases. We review the current status for treatment of intermediate stage disease, summarize the literature on correlative histopathology, and discuss emerging methods at micro-, nano-, and pico-scales to improve therapy. These include transarterial hyperthermia methods and thermoembolization, along with microfluidics model systems and new applications of mass spectrometry imaging for label-free analysis of pharmacokinetics and pharmacodynamics.

Doxorubicin-loaded hollow gold nanospheres for dual photothermal ablation and chemoembolization therapy.

BACKGROUND: Doxorubicin-loaded hollow gold nanospheres (Dox@HAuNS) are a promising technology for simultaneous trans-arterial tumor-targeted chemotherapy delivery and thermal ablation. We evaluated the efficacy of intra-arterial delivery of Dox@HAuNS followed by photothermal ablation (PTA) in a rabbit model of liver cancer. Adult New Zealand white rabbits (N=25) were inoculated with VX2 tumors into the left lobe of the liver. The animals were then randomized to sham surgery (N=5), PTA only (N=3), Dox@HAuNS only (N=5), HAuNS + PTA (N=5), and Dox@HAuNS + PTA (N=7). Nanoparticles were delivered as an emulsion with Lipiodol (Guerbet, France) via a trans-arterial approach. Following nanoparticle delivery, PTA was performed using an 808nm fibered laser at 1.5W for 3 minutes. Thermography during PTA demonstrated a sustained elevation in tumoral temperature in both HAuNS + laser and Dox@HAuNS + laser treatment groups relative to animals that underwent laser treatment without prior nanoparticle delivery. RESULTS: There was a significant decrease in tumor volumes in all three treatment arms relative to control arms (P = 0.004). Concentrations of intratumoral doxorubicin were significantly greater in animals treated with laser compared to those that were not treated with laser (P< 0.01). CONCLUSIONS: Doxorubicin-loaded HAuNS is a promising therapeutic agent for dual ablation/chemoembolization treatment of liver cancer.

Clonidine restores vascular endothelial growth factor expression and improves tissue repair following severe trauma.

BACKGROUND: We hypothesized that clonidine and propranolol would increase VEGF and VEGF-receptor expression and promote lung healing following severe trauma and chronic stress. METHODS: Sprague-Dawley rats were subjected to lung contusion (LC), lung contusion/hemorrhagic shock (LCHS), or lung contusion/hemorrhagic shock/daily restraint stress (LCHS/CS). Clonidine and propranolol were administered daily. On day seven, lung VEGF, VEGFR-1, VEGFR-2, and HMGB1 were assessed by PCR. Lung injury was assessed by light microscopy (*p < 0.05). RESULTS: Clonidine increased VEGF expression following LCHS (43%*) and LCHS/CS (46%*). Clonidine increased VEGFR-1 and R-2 expression following LCHS/CS (203%* and 47%*, respectively). Clonidine decreased HMGB1 and TNF-alpha expression following LCHS/CS (22%* and 58%*, respectively.) Clonidine decreased inflammatory cell infiltration and total Lung Injury Score following LCHS/CS. Propranolol minimally affected VEGF and did not improve lung healing. CONCLUSIONS: Clonidine increased VEGF and VEGF-receptor expression, decreased HMGB1 expression, decreased lung inflammation, and improved lung tissue repair.

Effects of trauma, hemorrhagic shock, and chronic stress on lung vascular endothelial growth factor.

BACKGROUND: Vascular endothelial growth factor (VEGF) and its receptors (VEGFR-1 and VEGFR-2) regulate vascular permeability and endothelial cell survival. We hypothesized that hemorrhagic shock (HS) and chronic stress (CS) would increase expression of lung VEGF and its receptors, potentiating pulmonary edema in lung tissue. MATERIALS AND METHODS: Male Sprague-Dawley rats aged 8-9 wk were randomized: naïve control, lung contusion (LC), LC followed by HS (LCHS), and LCHS with CS in a restraint cylinder for 2 h/d (LCHS/CS). Animals were sacrificed on days 1 and 7. Expressions of lung VEGF, VEGFR-1, and VEGFR-2 were determined by polymerase chain reaction. Lung Injury Score (LIS) was graded on light microscopy by inflammatory cell counts, interstitial edema, pulmonary edema, and alveolar integrity (range: 0 = normal; 8 = severe injury). RESULTS: Seven days after LC, lung VEGF and VEGFR-1 were increased, and lung tissue healed (LIS: 0.8 ± 0.8). However, 7 d after LCHS and LCHS/CS, lung VEGF and VEGFR-1 expressions were decreased. VEGFR-2 was also decreased after LCHS/CS. LIS was elevated 7 d after LCHS and LCHS/CS (6.5 ± 1.0 and 8.2 ± 0.8). Increased LIS after LCHS and LCHS/CS was because of higher inflammatory cell counts, increased interstitial edema, and loss of alveolar integrity, whereas pulmonary edema was unchanged. CONCLUSIONS: Elevation of lung VEGF and VEGFR-1 expressions after LC alone was associated with healing of injured lung tissue. Expressions of VEGF, VEGFR-1, and VEGFR-2 were reduced after LCHS and LCHS/CS, and injured lung tissue did not heal. Persistent lung injury after severe trauma was because of inflammation rather than pulmonary edema.

Effects of Dietary Resistant Starch on the Wnt Signaling Pathway and Preneoplastic Cells in the Colons of Azoxymethane-Treated Rats.

Dietary resistant starch (RS) has been suggested to reduce colonic neoplasia. To determine the effects of digestion-resistant cornstarch on colonic carcinogenesis and Wnt signaling in azoxymethane (AOM)-treated F344 rats, diets containing naturally occurring RS from corn lines derived partially from Guat209 (GUAT), AR16035 (AR), or a hybrid (ARxGUAT), containing 34.5 ± 2.0, 0.2 ± 0.1, and 1.9 ± 0.1% RS, respectively, were fed at 55% of the diet. GUAT-fed rats had increased cecal content and tissue weight and decreased cecal pH compared with AR- or ARxGUAT-fed rats. Numbers of aberrant crypt foci (ACF) were not different among diet groups. Increased numbers of crypts/focus were observed in AOM-injected rats fed GUAT compared with rats fed other diets. ß-catenin mRNA expression of the crypts was significantly increased in GUAT-fed rats injected with AOM relative to those injected with saline. These findings suggest that selected dietary RSs may at some level further enhance colonocyte proliferation and differentiation in an AOM-treated colon.