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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

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 activities, such as promoting increased metastatic incidence compared to p53 loss, often mediated by the interaction of mutant p53 with a set of transcription factors. These interactions are largely context specific. In order 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 to p53-null mice, suggesting gain of function. 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. Additionally, analyses of p53R248W ChIP 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.

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

Table S1 shows predicted off target sites for p53TD mutant mice. Table S2 shows upregulated PPAR target genes in p53AD vs p53-null. Table S3 shows ATAC-seq and RNA-seq congruent genes in p53AD vs p53 null. Table S4 shows p53half site analysis in PPAR target genes Table S5 shows control sequences used for p53 half site analysis Table S6 shows altered genes in p53AD-derived thymic lymphomas Table S7 shows primers used for genotyping of p53 mutant mice Table S8 shows primers used for qRT-PCR Table S9 shows antibodies used for flow cytometry Figure S1 shows extended data for generation of knock-in mouse models and features of p53TD mutant proteins and mice. Figure S2 shows homozygous mutant mice survival curves as well as tumor data. Figure S3 shows exome sequencing results for thymic lymphomas and hemangiosarcomas derived from p53 monomer and dimer mutant mice. Figure S4 shows characterization of stress responses of p53TD mutants. Figure S5 shows RNA-sequencing results for p53AD and p53RP, and Mdm2 genetic deletion rescue experiments. Figure S6 shows RNA-seq results of basal p53AD vs p53-null thymi, upstream regulator analysis, and ATAC-seq results for specific loci. Figure S7 shows extended data for p53 dimer mutant novel activities (co-IP, motif analysis, other dimer mutants activities, and tumor data) Figure S8 shows ferroptosis and scRNA-seq extended data Figure S9 shows diagram summarizing findings within this study.

Inferior vena cava filters (IVCFs) constructed with poly-p-dioxanone (PPDO) are promising alternatives to metallic filters and their associated risks and complications. Incorporating high-Z nanoparticles (NPs) improves PPDO IVCFs’ radiopacity without adversely affecting their safety or performance. However, increased radiopacity from these studies are insufficient for filter visualization during fluoroscopy-guided PPDO IVCF deployment. This study focuses on the use of bismuth nanoparticles (BiNP) as radiopacifiers to render sufficient signal intensity for the fluoroscopy-guided deployment and long-term CT monitoring of PPDO IVCFs. The use of polyhydroxybutyate (PHB) as an additional layer to increase the surface adsorption of NPs resulted in a 2-fold increase in BiNP coating (BiNP-PPDO IVCFs, 3.8%, BiNP-PPDO+PHB IVCFs, 6.2%), enabling complete filter visualization during fluoroscopy-guided IVCF deployment and, 1 week later, clot deployment. The biocompatibility, clot-trapping efficacy, and mechanical strength of the control PPDO (load-at-break, 6.23±0.13 kg), BiNP-PPDO (6.10±0.09 kg), and BiNP-PPDO+PHB (6.15±0.13 kg) IVCFs did not differ significantly over a 12-week monitoring period in pigs. These results indicate that BiNP-PPDO+PHB can increase the radiodensity of a novel absorbable IVCF without compromising device strength. Visualizing the device under conventional radiographic imaging is key to allow safe and effective clinical translation of the device.

Supplementary Data from 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.

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.

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.

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.

The purpose of this systematic review and meta-analysis was to determine if raising grandchildren is related to diminished mental health well-being in custodial grandparents compared to contemporar...

In collaboration with the American College of Veterinary Pathologists

Absorbable inferior vena cava filters (IVCFs) offer a promising alternative to metallic retrievable filters in providing protection against pulmonary embolism (PE) for patients contraindicated for anticoagulant therapy. However, because absorbable filters are not radiopaque, monitoring of the filter using conventional X-ray imaging modalities (e.g. plain film radiographs, computed tomography [CT] and fluoroscopy) during deployment and follow-up is not possible and represents a potential obstacle to widespread clinical integration of the device. Here, we demonstrate that gold nanoparticles (AuNPs) infused into biodegradable filters made up of poly-p-dioxanone (PPDO) may improve device radiopacity without untoward effects on device efficacy and safety, as assessed in swine models for 12 weeks. The absorbable AuNP-infused filters demonstrated significantly improved visualization using CT without affecting tensile strength, in vitro degradation, in vivo resorption, or thrombus-capturing efficacy, as compared to similar non-AuNPs infused resorbable IVCFs. This study presents a significant advancement to the development of imaging enhancers for absorbable IVCFs.

Over the last six decades, populations of the bumblebees Bombus sylvicola and Bombus balteatus in Colorado have experienced decreases in tongue length, a trait important for plant-pollinator mutualisms. It has been hypothesized that this observation reflects selection resulting from shifts in floral composition under climate change. Here we used morphometrics and population genomics to determine whether morphological change is ongoing, investigate the genetic basis of morphological variation, and analyse population structure in these populations. We generated a genome assembly of B. balteatus. We then analysed whole-genome sequencing data and morphometric measurements of 580 samples of both species from seven high-altitude localities. Out of 281 samples originally identified as B. sylvicola, 67 formed a separate genetic cluster comprising a newly-discovered cryptic species ("incognitus"). However, an absence of genetic structure within species suggests that gene flow is common between mountains. We found a significant decrease in tongue length between bees collected between 2012-2014 and in 2017, indicating that morphological shifts are ongoing. We did not discover any genetic associations with tongue length, but a SNP related to production of a proteolytic digestive enzyme was implicated in body size variation. We identified evidence of covariance between kinship and both tongue length and body size, which is suggestive of a genetic component of these traits, although it is possible that shared environmental effects between colonies are responsible. Our results provide evidence for ongoing modification of a morphological trait important for pollination and indicate that this trait probably has a complex genetic and environmental basis.

An increasing number of grandparents are assuming full-time parenting responsibilities for grandchildren when birth parents are unable to do so. This raises concern given the body of research that ...

An AISI 1045 steel modified with vanadium (V) and niobium (Nb) was studied to evaluate microstructural conditioning prior to and throughout a rapid heat treat process. In order to accomplish this, both computational and physical simulation techniques have been employed with the goal of assessing the microstructural evolution in a medium-carbon bar steel during the rapid austenitization and quenching procedures involved in an induction hardening process. The appropriate thermal profiles for induction hardening were obtained through finite element modeling using Flux 2D software. Physical simulations of the induction hardening process were carried out using a Gleeble® 3500. Analysis of prior austenite grain size is complemented by observation of nanoscale carbonitride precipitation via transmission electron microscopy, scanning transmission electron microscopy, and high-energy synchrotron small-angle x-ray scattering. Through a combination of characterization techniques, this study presents a deeper understanding of nano- and microstructural changes occurring in a microalloyed steel during an induction hardening process.

Immunotherapies such as anti-CTLA-4 immune checkpoint blockade (ICB) have revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by off-target tissue damage or immune-related adverse events (irAEs). At present, there is limited understanding of irAE mechanisms, hampering development of approaches to mitigate their damage. We addressed this problem by generating animal models of intestinal irAE. Our results show that disruption of homeostatic immunity by genetic predisposition to intestinal inflammation or acute gastrointestinal infection sensitizes mice to anti-CTLA-4-mediated intestinal toxicity. Inflammation-prone mice treated with anti-CTLA-4 showed neutrophil accumulation, systemic interleukin-6 (IL-6) release, and dysbiosis. Significantly, IL-6 blockade combined with antibiotic treatment improved anti-CTLA-4 therapeutic efficacy and reduced intestinal irAEs. Immune signatures were validated in biopsies from patients who developed colitis during ICB, supporting the utility of our models. This study provides new pre-clinical models, mechanistic insight into irAEs, and potential approaches to enhance ICB efficacy while mitigating irAEs. Citation Format: Yifan Zhou, Yusra B. Medik, Bhakti Patel, Daniel B. Zamler, Sijie Chen, Thomas Chapman, Sarah Schneider, Rachel L. Babcock, Taylor T. Chrisikos, Laura M. Kahn, Allison M. Dyevoich, Elizabeth M. Park, Alexandria P. Cogdill, Daniel H. Johnson, Sarah B. Johnson, Khalida M. Wani, Debora A. Ledesma, Courtney W. Hudgens, Jingjing Wang, Md Abdul Wadud Khan, Aron Y. Joon, Weiyi Peng, Haiyan S. Li, Reetakshi Arora, Ximing Tang, Maria Gabriela Raso, Xuegong Zhang, Wai Chin Foo, Michael T. Tetzlaff, Gretchen E. Diehl, Karen Clise-Dwyer, Elizabeth M. Whitley, Matthew M. Gubin, James P. Allison, Patrick Hwu, Nadim J. Ajami, Adi Diab, Jennifer A. Wargo, Stephanie S. Watowich. Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5545.

In this study, fruit flies (Drosophila melanogaster) were exposed to an estimated daily human E171 consumption concentration for 20 generations. Exposure to E171 resulted in: a change in normal developmental and reproductive dynamics, reduced fecundity after repetitive breeding, increased genotoxicity, the appearance of aberrant phenotypes and morphologic changes to the adult fat body. Marks of adaptive evolution and directional selection were also exhibited. The larval stages were at a higher risk of sustaining damage from E171 as they had a slower elimination rate of TiO2 compared to the adults. This is particularly worrisome, since among the human population, children tend to consume higher daily concentrations of E171 than do adults. The genotoxic effect of E171 was statistically higher in each subsequent generation compared to the previous one. Aberrant phenotypes were likely caused by developmental defects induced by E171, and were not mutations, since the phenotypic features were not transferred to any progeny even after 5 generations of consecutive crossbreeding. Therefore, exposure to E171 during the early developmental period carries a higher risk of toxicity. The fact that the daily human consumption concentration of E171 interferes with and influences fruit fly physiological, ontogenetic, genotoxic, and adaptive processes certainly raises safety concerns.

Over the last six decades, populations of the bumblebees Bombus sylvicola and Bombus balteatus in Colorado have experienced decreases in tongue length, a trait important for plant-pollinator mutualisms. It has been hypothesized that this observation reflects selection resulting from shifts in floral composition under climate change. Here we used morphometrics and population genomics to determine whether morphological change is ongoing, investigate the genetic basis of morphological variation, and analyze population structure in these populations. We generated a genome assembly of B. balteatus. We then analyzed whole-genome sequencing data and morphometric measurements of 580 samples of both species from seven high-altitude localities. Out of 281 samples originally identified as B. sylvicola, 67 formed a separate genetic cluster comprising a newly-discovered cryptic species (“incognitus”). However, an absence of genetic structure within species suggests that gene flow is common between mountains. We found a significant decrease in tongue length between bees collected between 2012-2014 and in 2017, indicating that morphological shifts are ongoing. We did not discover any genetic associations with tongue length, but a SNP related to production of a proteolytic digestive enzyme was implicated in body size variation. We identified evidence of covariance between kinship and both tongue length and body size, which is suggestive of a genetic component of these traits, although it is possible that shared environmental effects between colonies are responsible. Our results provide evidence for ongoing modification of a morphological trait important for pollination and indicate that this trait likely has a complex genetic and environmental basis.

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.

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

Grafts based on biodegradable polymer scaffolds are increasingly used in tissue-engineering applications as they facilitate natural tissue regeneration. However, monitoring the position and integrity of these scaffolds over time is challenging due to radiolucency. In this study, we used an electrospinning method to fabricate biodegradable scaffolds based on polycaprolactone (PCL) and iodixanol, a clinical contrast agent. Scaffolds were implanted subcutaneously into C57BL/6 mice and monitored in vivo using longitudinal X-ray imaging and micro-computed tomography (CT). The addition of iodixanol altered the physicochemical properties of the PCL scaffold; notably, as the iodixanol concentration increased, the fiber diameter decreased. Radiopacity was achieved with corresponding signal enhancement as iodine concentration increased while exhibiting a steady time-dependent decrease of 0.96% per day in vivo. The electrospun scaffolds had similar performance with tissue culture-treated polystyrene in supporting the attachment, viability, and proliferation of human mesenchymal stem cells. Furthermore, implanted PCL-I scaffolds had more intense acute inflammatory infiltrate and thicker layers of maturing fibrous tissue. In conclusion, we developed radiopaque, biodegradable, biocompatible scaffolds whose position and integrity can be monitored noninvasively. The successful development of other imaging enhancers may further expand the use of biodegradable scaffolds in tissue engineering applications.

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 808-nm fibered laser at 1.5 W for 3 min. 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 Conclusions Doxorubicin-loaded HAuNS is a promising therapeutic agent for dual ablation/chemoembolization treatment of liver cancer.

Germline telomere maintenance defects are associated with an increased incidence of inflammatory diseases in humans, yet whether and how telomere dysfunction causes inflammation are not known. Here, we show that telomere dysfunction drives pATM/c-ABL-mediated activation of the YAP1 transcription factor, up-regulating the major pro-inflammatory factor, pro-IL-18. The colonic microbiome stimulates cytosolic receptors activating caspase-1 which cleaves pro-IL-18 into mature IL-18, leading to recruitment of interferon (IFN)-γ-secreting T cells and intestinal inflammation. Correspondingly, patients with germline telomere maintenance defects exhibit DNA damage (γH2AX) signaling together with elevated YAP1 and IL-18 expression. In mice with telomere dysfunction, telomerase reactivation in the intestinal epithelium or pharmacological inhibition of ATM, YAP1, or caspase-1 as well as antibiotic treatment, dramatically reduces IL-18 and intestinal inflammation. Thus, telomere dysfunction-induced activation of the ATM-YAP1-pro-IL-18 pathway in epithelium is a key instigator of tissue inflammation., How telomere dysfunction is directly linked to inflammation in humans is currently unclear. Here the authors reveal that telomere dysfunction drives activation of the YAP1 transcription factor, up-regulating the pro inflammatory factor, pro-IL-18 thus revealing a link between telomere dysfunction and initiation of intestinal inflammation.

Grafts based on biodegradable polymer scaffolds are increasingly used in tissue-engineering applications as they facilitate natural tissue regeneration. However, monitoring the position and integrity of these scaffolds over time is challenging due to radiolucency. In this study, we used an electrospinning method to fabricate biodegradable scaffolds based on polycaprolactone (PCL) and iodixanol, a clinical contrast agent. Scaffolds were implanted subcutaneously into C57BL/6 mice and monitored in vivo using longitudinal X-ray imaging and micro-computed tomography (CT). The addition of iodixanol altered the physicochemical properties of the PCL scaffold; notably, as the iodixanol concentration increased, the fiber diameter decreased. Radiopacity was achieved with corresponding signal enhancement as iodine concentration increased while exhibiting a steady time-dependent decrease of 0.96% per day in vivo. The electrospun scaffolds had similar performance with tissue culture-treated polystyrene in supporting the attachment, viability, and proliferation of human mesenchymal stem cells. Furthermore, implanted PCL-I scaffolds had more intense acute inflammatory infiltrate and thicker layers of maturing fibrous tissue. In conclusion, we developed radiopaque, biodegradable, biocompatible scaffolds whose position and integrity can be monitored noninvasively. The successful development of other imaging enhancers may further expand the use of biodegradable scaffolds in tissue engineering applications.

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.

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-naive 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.

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.

Hydrogen sulfide (H2 S) is a highly neurotoxic gas. Acute exposure can lead to neurological sequelae among survivors. A drug for treating neurological sequelae in survivors of acute H2 S intoxication is needed. Using a novel mouse model we evaluated the efficacy of cobinamide (Cob) for increasing survival of, and reducing neurological sequalae in, mice exposed to sublethal doses of H2 S. There were two objectives: (1) to determine the dose-response efficacy of Cob and (2) to determine the effective therapeutic time window of Cob. To explore objective 1, mice were injected intramuscularly with Cob at 0, 50, or 100 mg/kg at 2 min after H2 S exposure. For objective 2, mice were injected intramuscularly with 100 mg/kg Cob at 2, 15, and 30 min after H2 S exposure. For both objectives, mice were exposed to 765 ppm of H2 S gas. Cob significantly reduced H2 S-induced lethality in a dose-dependent manner (P

Hydrogen sulfide (H2 S) is a highly neurotoxic gas. It is the second most common cause of gas-induced deaths. Beyond mortality, surviving victims of acute exposure may suffer long-term neurological sequelae. There is a need to develop countermeasures against H2 S poisoning. However, no translational animal model of H2 S-induced neurological sequelae exists. Here, we describe a novel mouse model of H2 S-induced neurotoxicity for translational research. In paradigm I, C57/BL6 mice were exposed to 765 ppm H2 S for 40 min on day 1, followed by 15-min daily exposures for periods ranging from 1 to 6 days. In paradigm II, mice were exposed once to 1000 ppm H2 S for 60 minutes. Mice were assessed for behavioral, neurochemical, biochemical, and histopathological changes. H2 S intoxication caused seizures, dyspnea, respiratory depression, knockdowns, and death. H2 S-exposed mice showed significant impairment in locomotor and coordinated motor movement activity compared with controls. Histopathology revealed neurodegenerative lesions in the collicular, thalamic, and cortical brain regions. H2 S significantly increased dopamine and serotonin concentration in several brain regions and caused time-dependent decreases in GABA and glutamate concentrations. Furthermore, H2 S significantly suppressed cytochrome c oxidase activity and caused significant loss in body weight. Overall, male mice were more sensitive than females. This novel translational mouse model of H2 S-induced neurotoxicity is reliable, reproducible, and recapitulates acute H2 S poisoning in humans.

Osteoporosis is a common health problem severely affecting the quality of life of many people, especially women. Current treatment options for osteoporosis are limited due to their association with several side-effects and moderate efficacy. Therefore, novel therapies for osteoporosis are needed. This study tested the feasibility of adipose tissue-derived mesenchymal stem cell (ATMSC)-based human alpha-1 antitrypsin (hAAT, SERPINA1) gene therapy for the prevention of bone loss in an ovariectomized (OVX) mouse model. Eight-week-old female C57BL6 mice underwent ovariectomy and were treated with hAAT (protein therapy), ATMSC (stem-cell therapy), ATMSC + hAAT (combination of ATMSC and hAAT therapy), and ATMSCs infected with lentiviral vectors expressing hAAT (ATMSC-based hAAT gene therapy). The study showed that lenti-hAAT vector-infected ATMSCs (ATMSC-LV-hAAT) produced high levels of hAAT. Transplantation of these cells significantly decreased OVX-induced serum levels of interleukin 6 and interleukin 1 beta, and receptor activator of nuclear factor kappa B gene expression levels in bone tissue. Immunohistological analysis revealed that transplanted cells migrated to the bone tissue and secreted hAAT. Importantly, bone microstructure analysis by microcomputerized tomography showed that this treatment significantly protected against OVX-induced bone loss. The results suggest a novel strategy for the treatment of osteoporosis in humans.

Purpose To evaluate trifluoroacetic acid (TFA) as a theranostic chemical ablation agent and determine the efficacy of TFA for both noninvasive imaging and tissue destruction. Materials and Methods Fluorine-19 magnetic resonance imaging (19F-MRI) was optimized at 7 T using a custom-built volume coil. Fluorine images were acquired with both rapid acquisition with relaxation enhancement and balanced steady-state free precession (bSSFP) sequences with varying parameters to determine the optimal sequence for TFA. The theranostic efficacy of chemical ablation was examined by injecting TFA (100 μL; 0.25, 0.5, 1.0, and 2.0M) into ex vivo porcine liver. 19F and proton MRI were acquired and superimposed to visualize distribution of TFA in tissue and quantify sensitivity. Tissue damage was evaluated with gross examination, histology, and fluorescence microscopy. Results The optimal 19F-MRI sequence was found to be bSSFP with a repetition time of 2.5 ms and flip angle of 70°. The minimum imageable TFA concentration was determined to be 6.7 ± 0.5 mM per minute of scan time (0.63×0.63×5.00 mm voxel), and real-time imaging (temporal resolution of at least 1 s-1) was achieved with 2M TFA both in vitro and in ex vivo tissue. TFA successfully coagulated tissue, and damage was locally confined. In addition to hepatic cord disruption, cytoskeletal collapse and chromatin clumping were observed in severely damaged areas in tissues treated with 0.5M or higher TFA concentrations. Conclusions TFA was determined to be a theranostic agent for chemical ablation of solid tissue. Ablation was both efficacious and imageable in ex vivo healthy tissue, even at low concentrations or with high temporal resolution.

Introduction: Microsatellite instability (MSI), caused by defects in DNA mismatch repair genes, including MLH1 and MSH2, occur in 30% of endometrial cancers (EC). High mutational loads associated with MSI have been associated with greater immunogenicity, and an increase in immune inhibitory factors such as PD-1 and PD-L1. While immunotherapy with anti-PD-L1 has shown promising results in MSI ECs, up to 40% did not respond, suggesting there are alternate immunosuppressive mechanisms. To improve understanding of response/resistance to immunotherapy, we evaluated immune activation and evasion using a novel immunocompetent mouse model of MSI EC. Methods: Endometrial lesions were evaluated in mice with MSH2 loss targeted to the endometrium (PRCre+MSH2flox/flox). When aged to 12-16 months, 22% develop hyperplasia and 18% develop spontaneous EC. MSI status was confirmed by PCR. Immune infiltration and function were assessed by immunohistochemistry (IHC) and Nanostring digital spatial profiling. Tumors were evaluated for tumor infiltrating lymphocytes (TIL) based on CD8a expression and found to separate into TILHIGH and TILLOW groups and further characterized to determine immune activation/resistance mechanisms using transcriptome analysis with Affymetrix Clariom D assay and validation by RT-PCR. A gene signature score approach was used to quantify immune-related expression changes for tumors from PRCre+MSH2flox/flox mice and MSI EC patient data from TCGA. Cell lines were generated from tumors and used to evaluate a syngeneic orthotopic mouse model for immunotherapy studies, where immune infiltration was characterized. Results: All endometrial tumors were MSI and included endometrioid, serous, and mixed histologies. Tumors showed varying degrees of CD8+ T cell infiltration, irrespective of histology. TILHIGH tumors had increased infiltration of dendritic cells and Type I interferon response, including genes IFI203 (fold-change, FC=18.5), IFI204 (FC=11.1) and CXCL9 (FC=5.7). There was no significant difference in type II interferon response. TILLOW tumors had low expression of IFN-associated genes. TILHIGH tumors also exhibited increased immune exhaustion markers such as TIM3 (FC=2.4) and LAG3 (FC=3.2). Tumoral PD-L1 expression was negative ( Conclusions: Elucidating immune activation and evasion mechanisms is critical for improving immunotherapies. The PRCre+MSH2flox/flox mouse model reflects the spectrum of immunogenicity observed in patients. A panel of primary cell lines generated from this model are an important tool that enable studies of immunotherapy efficacy and resistance mechanisms, in both immunogenic and non-immunogenic tumors. Citation Format: Brenda Melendez, Emily Hinchcliff, Nisha Gokul, Xiaoping Su, Daniel McGrail, Elizabeth Whitley, Russell Broaddus, Rosemarie Schmandt, Karen Lu, Melinda Yates. Identifying mechanisms of immune evasion in microsatellite instable endometrial cancer mouse models [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PR008.

Grandparents raising grandchildren build strong foundations for their grandchildren. Despite grandparents’ significant contributions to their grandchildren’s future and society in general, there is a limited understanding of the unique needs and service utilization of grandparents raising grandchildren in various contexts. This symposium is focused on the needs of and services for the grandparent population at the regional, national, and international levels. Stucki will present findings from an examination of types and locally available services for grandparents raising grandchildren in Appalachia by sub-region. Musil and colleagues will discuss the service need utilization and unmet service needs of a nationwide sample of 284 grandmothers living with/ raising grandchildren and the relationships between service use/need and resilience, resourcefulness, perceived stress, reward, and appraisals of their current living environment for themselves and their grandchildren. Lastly, Lee will describe research findings from her comparative transnational research on needs and experiences of grandparents raising grandchildren in Malawi (n=29), South Korea (n=23), and the U.S. (n=23). Unique needs and cultural interpretation of intergenerational caregiving in each country will be presented. The symposium discussion will address diverse needs of grandparents raising grandchildren and strategies to meet those needs at regional, national, and international levels. Grandparents as Caregivers Interest Group Sponsored Symposium.