Enhancing standard of care chemotherapy efficacy using DNA-dependent protein kinase (DNA-PK) inhibition in preclinical models of Ewing sarcoma.

Disruption of DNA damage repair via impaired homologous recombination is characteristic of Ewing sarcoma (EWS) cells. We hypothesize that this disruption results in increased reliance on non-homologous end joining (NHEJ) to repair DNA damage. In this study, we investigated if pharmacological inhibition of the enzyme responsible for NHEJ, the DNA-PK holoenzyme, alters the response of EWS cells to genotoxic standard of care chemotherapy. We used analyses of cell viability and proliferation to investigate the effects of clinical DNA-PK inhibitors (DNA-PKi) in combination with six therapeutic or experimental agents for EWS. We performed calculations of synergy using the Loewe Additivity Model. Immunoblotting evaluated treatment effects on DNA-PK, DNA damage, and apoptosis. Flow cytometric analyses evaluated effects on cell cycle and fate. We used orthotopic xenograft models to interrogate tolerability, drug mechanism, and efficacy in vivo. DNA-PKi demonstrated on-target activity, reducing phosphorylated DNA-PK levels in EWS cells. DNA-PKi sensitized EWS cell lines to agents that function as topoisomerase 2 (TOP2) poisons and enhanced the DNA damage induced by TOP2 poisons. Nanomolar concentrations of single agent TOP2 poisons induced G2M arrest and little apoptotic response, while adding DNA-PKi mediated apoptosis. In vivo, the combination of AZD-7648 and etoposide had limited tolerability but resulted in enhanced DNA damage, apoptosis, and EWS tumor shrinkage. The combination of DNA-PKi with standard of care TOP2 poisons in EWS models is synergistic, enhances DNA damage and cell death, and may form the basis of a promising future therapeutic strategy for EWS.

Analysis of Nanoparticles' Potential to Induce Autoimmunity.

Autoimmune responses are characterized by the presence of antibodies and lymphocytes specific to self or so-called autoantigens. Among such autoantigens is DNA; therefore, screening for antibodies recognizing single- and/or double-stranded DNA is commonly used to detect and classify autoimmune diseases. While autoimmunity affects both sexes, females are generally more affected than males, which is recapitulated in some animal models. A variety of factors, including genetic predisposition and the environment, contribute to the development of autoimmune disorders. Since certain drug products may also contribute to the development of autoimmunity, understanding a drug's potential to trigger an autoimmune response is of interest to immunotoxicology. However, models to study autoimmunity are limited, and it is generally agreed that no model can accurately predict autoimmunity in humans. Herein, we present an in vivo protocol utilizing the SJL/J mouse model to study nanoparticles' effects on the development of autoimmune responses. The protocol is adapted from the literature describing the use of this model to study chemically induced lupus.

Analysis of Nanoparticles' Effects on Drug-Induced Psoriasis.

Psoriasis, an auto-inflammatory disorder, has major manifestations in the skin but can affect other organs. Currently, this condition has no cure, and the treatments include anti-inflammatory medications. Nanoparticles are widely used for drug delivery and have found successful applications in therapy for cancer and infectious diseases. Nanoparticles can also be used to deliver anti-inflammatory drugs to sites of inflammation. Moreover, some nanotechnology platforms possess intrinsic anti-inflammatory properties and may benefit the therapy of inflammation-driven disorders. Herein, we present a protocol to study nanotechnology concepts' anti-inflammatory properties in a chemically-induced psoriasis model.

Distinct uptake and elimination profiles for trastuzumab, human IgG and biocytin-TMR in experimental HER2+ brain metastases of breast cancer.

BACKGROUND: The aim of this study is an improved understanding of drug distribution in brain metastases. Rather than single point snapshots, we analyzed the time course and route of drug/probe elimination (clearance), focusing on the Intramural Periarterial Drainage (IPAD) pathway. METHODS: Mice with JIMT1-BR HER2+ experimental brain metastases were injected with biocytin-TMR and either trastuzumab or human IgG. Drugs/probes circulated for 5 min-48h, followed by perfusion. Brain sections were stained for human IgG, vascular basement membrane proteins laminin or collagen IV, and periarterial α-SMA. A machine learning algorithm was developed to identify metastases, metastatic microenvironment, and uninvolved brain in confocally scanned brain sections. Drug/probe intensity over time and total imaged drug exposure (iAUC) were calculated for 27,249 lesions and co-immunofluorescence with IPAD- vascular matrix analyzed in 11,668 metastases. RESULTS: In metastases, peak trastuzumab levels were 5-fold higher than human IgG but 4-fold less than biocytin-TMR. The elimination phase constituted 85-93% of total iAUC for all drugs/probes tested. For trastuzumab, total iAUC during uptake was similar to the small molecule drug probe biocytin-TMR, but slower trastuzumab elimination resulted in a 1.7-fold higher total iAUC. During elimination trastuzumab and IgG were preferentially enriched in the α-SMA+ periarterial vascular matrix, consistent with the IPAD clearance route; biocytin-TMR showed heterogeneous elimination pathways. CONCLUSIONS: Drug/probe elimination is an important component of drug development for brain metastases. We identified a prolonged elimination pathway for systemically administered antibodies through the periarterial vascular matrix that may contribute to the sustained presence and efficacy of large antibody therapeutics.

Engineering CD276/B7-H3-targeted antibody-drug conjugates with enhanced cancer-eradicating capability.

CD276/B7-H3 represents a promising target for cancer therapy based on widespread overexpression in both cancer cells and tumor-associated stroma. In previous preclinical studies, CD276 antibody-drug conjugates (ADCs) exploiting a talirine-type pyrrolobenzodiazepine (PBD) payload showed potent activity against various solid tumors but with a narrow therapeutic index and dosing regimen higher than that tolerated in clinical trials using other antibody-talirine conjugates. Here, we describe the development of a modified talirine PBD-based fully human CD276 ADC, called m276-SL-PBD, that is cross-species (human/mouse) reactive and can eradicate large 500-1,000-mm3 triple-negative breast cancer xenografts at doses 10- to 40-fold lower than the maximum tolerated dose. By combining CD276 targeting with judicious genetic and chemical ADC engineering, improved ADC purification, and payload sensitivity screening, these studies demonstrate that the therapeutic index of ADCs can be substantially increased, providing an advanced ADC development platform for potent and selective targeting of multiple solid tumor types.

Organoids and metastatic orthotopic mouse model for mismatch repair-deficient colorectal cancer.

Background: Genome integrity is essential for the survival of an organism. DNA mismatch repair (MMR) genes (e.g., MLH1, MSH2, MSH6, and PMS2) play a critical role in the DNA damage response pathway for genome integrity maintenance. Germline mutations of MMR genes can lead to Lynch syndrome or constitutional mismatch repair deficiency syndrome, resulting in an increased lifetime risk of developing cancer characterized by high microsatellite instability (MSI-H) and high mutation burden. Although immunotherapy has been approved for MMR-deficient (MMRd) cancer patients, the overall response rate needs to be improved and other management options are needed. Methods: To better understand the biology of MMRd cancers, elucidate the resistance mechanisms to immune modulation, and develop vaccines and therapeutic testing platforms for this high-risk population, we generated organoids and an orthotopic mouse model from intestine tumors developed in a Msh2-deficient mouse model, and followed with a detailed characterization. Results: The organoids were shown to be of epithelial origin with stem cell features, to have a high frameshift mutation frequency with MSI-H and chromosome instability, and intra- and inter-tumor heterogeneity. An orthotopic model using intra-cecal implantation of tumor fragments derived from organoids showed progressive tumor growth, resulting in the development of adenocarcinomas mixed with mucinous features and distant metastasis in liver and lymph node. Conclusions: The established organoids with characteristics of MSI-H cancers can be used to study MMRd cancer biology. The orthotopic model, with its distant metastasis and expressing frameshift peptides, is suitable for evaluating the efficacy of neoantigen-based vaccines or anticancer drugs in combination with other therapies.

Preclinical Evaluation of the FGFR-Family Inhibitor Futibatinib for Pediatric Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Despite decades of clinical trials, the overall survival rate for patients with relapsed and metastatic disease remains below 30%, underscoring the need for novel treatments. FGFR4, a receptor tyrosine kinase that is overexpressed in RMS and mutationally activated in 10% of cases, is a promising target for treatment. Here, we show that futibatinib, an irreversible pan-FGFR inhibitor, inhibits the growth of RMS cell lines in vitro by inhibiting phosphorylation of FGFR4 and its downstream targets. Moreover, we provide evidence that the combination of futibatinib with currently used chemotherapies such as irinotecan and vincristine has a synergistic effect against RMS in vitro. However, in RMS xenograft models, futibatinib monotherapy and combination treatment have limited efficacy in delaying tumor growth and prolonging survival. Moreover, limited efficacy is only observed in a PAX3-FOXO1 fusion-negative (FN) RMS cell line with mutationally activated FGFR4, whereas little or no efficacy is observed in PAX3-FOXO1 fusion-positive (FP) RMS cell lines with FGFR4 overexpression. Alternative treatment modalities such as combining futibatinib with other kinase inhibitors or targeting FGFR4 with CAR T cells or antibody-drug conjugate may be more effective than the approaches tested in this study.

The beneficial effects of commensal E. coli for colon epithelial cell recovery are related with Formyl peptide receptor 2 (Fpr2) in epithelial cells.

BACKGROUND: Formyl peptide receptor 2 (Fpr2) plays a crucial role in colon homeostasis and microbiota balance. Commensal E. coli is known to promote the regeneration of damaged colon epithelial cells. The aim of the study was to investigate the connection between E. coli and Fpr2 in the recovery of colon epithelial cells. RESULTS: The deficiency of Fpr2 was associated with impaired integrity of the colon mucosa and an imbalance of microbiota, characterized by the enrichment of Proteobacteria in the colon. Two serotypes of E. coli, O22:H8 and O91:H21, were identified in the mouse colon through complete genome sequencing. E. coli O22:H8 was found to be prevalent in the gut of mice and exhibited lower virulence compared to O91:H21. Germ-free (GF) mice that were pre-orally inoculated with E. coli O22:H8 showed reduced susceptibility to chemically induced colitis, increased proliferation of epithelial cells, and improved mouse survival. Following infection with E. coli O22:H8, the expression of Fpr2 in colon epithelial cells was upregulated, and the products derived from E. coli O22:H8 induced migration and proliferation of colon epithelial cells through Fpr2. Fpr2 deficiency increased susceptibility to chemically induced colitis, delayed the repair of damaged colon epithelial cells, and heightened inflammatory responses. Additionally, the population of E. coli was observed to increase in the colons of Fpr2-/- mice with colitis. CONCLUSION: Commensal E. coli O22:H8 stimulated the upregulation of Fpr2 expression in colon epithelial cells, and the products from E. coli induced migration and proliferation of colon epithelial cells through Fpr2. Fpr2 deficiency led to an increased E. coli population in the colon and delayed recovery of damaged colon epithelial cells in mice with colitis. Therefore, Fpr2 is essential for the effects of commensal E. coli on colon epithelial cell recovery.

Cross-neutralizing protection of vaginal and oral mucosa from HPV challenge by vaccination in a mouse model.

The species and tissue specificities of HPV (human papillomavirus) for human infection and disease complicates the process of prophylactic vaccine development in animal models. HPV pseudoviruses (PsV) that carry only a reporter plasmid have been utilized in vivo to demonstrate cell internalization in mouse mucosal epithelium. The current study sought to expand the application of this HPV PsV challenge model with both oral and vaginal inoculation and to demonstrate its utility for testing vaccine-mediated dual-site immune protection against several HPV PsV types. We observed that passive transfer of sera from mice vaccinated with the novel experimental HPV prophylactic vaccine RG1-VLPs (virus-like particles) conferred HPV16-neutralizing as well as cross-neutralizing Abs against HPV39 in naïve recipient mice. Moreover, active vaccination with RG1-VLPs also conferred protection to challenge with either HPV16 or HPV39 PsVs at both vaginal and oral sites of mucosal inoculation. These data support the use of the HPV PsV challenge model as suitable for testing against diverse HPV types at two sites of challenge (vaginal vault and oral cavity) associated with the origin of the most common HPV-associated cancers, cervical cancer and oropharyngeal cancer.

Evaluation of Nonmodified Wireframe DNA Origami for Acute Toxicity and Biodistribution in Mice.

Wireframe DNA origami can be used to fabricate virus-like particles for a range of biomedical applications, including the delivery of nucleic acid therapeutics. However, the acute toxicity and biodistribution of these wireframe nucleic acid nanoparticles (NANPs) have not been previously characterized in animal models. In the present study, we observed no indications of toxicity in BALB/c mice following a therapeutically relevant dosage of nonmodified DNA-based NANPs via intravenous administration, based on liver and kidney histology, liver and kidney biochemistry, and body weight. Further, the immunotoxicity of these NANPs was minimal, as indicated by blood cell counts and type-I interferon and pro-inflammatory cytokines. In an SJL/J model of autoimmunity, we observed no indications of NANP-mediated DNA-specific antibody response or immune-mediated kidney pathology following the intraperitoneal administration of NANPs. Finally, biodistribution studies revealed that these NANPs accumulate in the liver within one hour, concomitant with substantial renal clearance. Our observations support the continued development of wireframe DNA-based NANPs as next-generation nucleic acid therapeutic delivery platforms.

High-throughput and targeted drug screens identify pharmacological candidates against MiT-translocation renal cell carcinoma.

BACKGROUND: MiT-Renal Cell Carcinoma (RCC) is characterized by genomic translocations involving microphthalmia-associated transcription factor (MiT) family members TFE3, TFEB, or MITF. MiT-RCC represents a specific subtype of sporadic RCC that is predominantly seen in young patients and can present with heterogeneous histological features making diagnosis challenging. Moreover, the disease biology of this aggressive cancer is poorly understood and there is no accepted standard of care therapy for patients with advanced disease. Tumor-derived cell lines have been established from human TFE3-RCC providing useful models for preclinical studies. METHODS: TFE3-RCC tumor derived cell lines and their tissues of origin were characterized by IHC and gene expression analyses. An unbiased high-throughput drug screen was performed to identify novel therapeutic agents for treatment of MiT-RCC. Potential therapeutic candidates were validated in in vitro and in vivo preclinical studies. Mechanistic assays were conducted to confirm the on-target effects of drugs. RESULTS: The results of a high-throughput small molecule drug screen utilizing three TFE3-RCC tumor-derived cell lines identified five classes of agents with potential pharmacological efficacy, including inhibitors of phosphoinositide-3-kinase (PI3K) and mechanistic target of rapamycin (mTOR), and several additional agents, including the transcription inhibitor Mithramycin A. Upregulation of the cell surface marker GPNMB, a specific MiT transcriptional target, was confirmed in TFE3-RCC and evaluated as a therapeutic target using the GPNMB-targeted antibody-drug conjugate CDX-011. In vitro and in vivo preclinical studies demonstrated efficacy of the PI3K/mTOR inhibitor NVP-BGT226, Mithramycin A, and CDX-011 as potential therapeutic options for treating advanced MiT-RCC as single agents or in combination. CONCLUSIONS: The results of the high-throughput drug screen and validation studies in TFE3-RCC tumor-derived cell lines have provided in vitro and in vivo preclinical data supporting the efficacy of the PI3K/mTOR inhibitor NVP-BGT226, the transcription inhibitor Mithramycin A, and GPNMB-targeted antibody-drug conjugate CDX-011 as potential therapeutic options for treating advanced MiT-RCC. The findings presented here should provide the basis for designing future clinical trials for patients with MiT-driven RCC.

Evaluation of non-modified wireframe DNA origami for acute toxicity and biodistribution in mice.

Wireframe DNA origami can be used to fabricate virus-like particles for a range of biomedical applications, including the delivery of nucleic acid therapeutics. However, the acute toxicity and biodistribution of these wireframe nucleic acid nanoparticles (NANPs) have not previously been characterized in animal models. In the present study, we observed no indications of toxicity in BALB/c mice following therapeutically relevant dosage of unmodified DNA-based NANPs via intravenous administration, based on liver and kidney histology, liver biochemistry, and body weight. Further, the immunotoxicity of these NANPs was minimal, as indicated by blood cell counts and type-I interferon and pro-inflammatory cytokines. In an SJL/J model of autoimmunity, we observed no indications of NANP-mediated DNA-specific antibody response or immune-mediated kidney pathology following the intraperitoneal administration of NANPs. Finally, biodistribution studies revealed that these NANPs accumulate in the liver within one hour, concomitant with substantial renal clearance. Our observations support the continued development of wireframe DNA-based NANPs as next-generation nucleic acid therapeutic delivery platforms.

Comparison of Three Transcytotic Pathways for Distribution to Brain Metastases of Breast Cancer.

Advances in drug treatments for brain metastases of breast cancer have improved progression-free survival but new, more efficacious strategies are needed. Most chemotherapeutic drugs infiltrate brain metastases by moving between brain capillary endothelial cells, paracellular distribution, resulting in heterogeneous distribution, lower than that of systemic metastases. Herein, we tested three well-known transcytotic pathways through brain capillary endothelial cells as potential avenues for drug access: transferrin receptor (TfR) peptide, low-density lipoprotein receptor 1 (LRP1) peptide, albumin. Each was far-red labeled, injected into two hematogenous models of brain metastases, circulated for two different times, and their uptake quantified in metastases and uninvolved (nonmetastatic) brain. Surprisingly, all three pathways demonstrated distinct distribution patterns in vivo. Two were suboptimal: TfR distributed to uninvolved brain but poorly in metastases, while LRP1 was poorly distributed. Albumin distributed to virtually all metastases in both model systems, significantly greater than in uninvolved brain (P < 0.0001). Further experiments revealed that albumin entered both macrometastases and micrometastases, the targets of treatment and prevention translational strategies. Albumin uptake into brain metastases was not correlated with the uptake of a paracellular probe (biocytin). We identified a novel mechanism of albumin endocytosis through the endothelia of brain metastases consistent with clathrin-independent endocytosis (CIE), involving the neonatal Fc receptor, galectin-3, and glycosphingolipids. Components of the CIE process were found on metastatic endothelial cells in human craniotomies. The data suggest a reconsideration of albumin as a translational mechanism for improved drug delivery to brain metastases and possibly other central nervous system (CNS) cancers.In conclusion, drug therapy for brain metastasis needs improvement. We surveyed three transcytotic pathways as potential delivery systems in brain-tropic models and found that albumin has optimal properties. Albumin used a novel endocytic mechanism.

The Combination of Trametinib and Ganitumab is Effective in RAS-Mutated PAX-Fusion Negative Rhabdomyosarcoma Models.

PURPOSE: PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS-754807, but this combination was not pursued clinically due to intolerability in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor, which would be tolerated in murine models and effective in both cell line and patient-derived xenograft models of RAS-mutant FN RMS. EXPERIMENTAL DESIGN: Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a mAb with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of trametinib/ganitumab was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models. RESULTS: Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in four of six models of RAS-mutant RMS. The combination had little effect on body weight and did not produce thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the phosphorylation of AKT induced by MEK inhibition alone. Therapeutic response to the combination was observed in models without a mutation in the PI3K/PTEN axis. CONCLUSIONS: We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. Our data also show that the trametinib/ganitumab combination likely has a favorable tolerability profile. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS.

Metastasis suppressor NME1 in exosomes or liposomes conveys motility and migration inhibition in breast cancer model systems.

Tumor-derived exosomes have documented roles in accelerating the initiation and outgrowth of metastases, as well as in therapy resistance. Little information supports the converse, that exosomes or similar vesicles can suppress metastasis. We investigated the NME1 (Nm23-H1) metastasis suppressor as a candidate for metastasis suppression by extracellular vesicles. Exosomes derived from two cancer cell lines (MDA-MB-231T and MDA-MB-435), when transfected with the NME1 (Nm23-H1) metastasis suppressor, secreted exosomes with NME1 as the predominant constituent. These exosomes entered recipient tumor cells, altered their endocytic patterns in agreement with NME1 function, and suppressed in vitro tumor cell motility and migration compared to exosomes from control transfectants. Proteomic analysis of exosomes revealed multiple differentially expressed proteins that could exert biological functions. Therefore, we also prepared and investigated liposomes, empty or containing partially purified rNME1. rNME1 containing liposomes recapitulated the effects of exosomes from NME1 transfectants in vitro. In an experimental lung metastasis assay the median lung metastases per histologic section was 158 using control liposomes and 15 in the rNME1 liposome group, 90.5% lower than the control liposome group (P = 0.016). The data expand the exosome/liposome field to include metastasis suppressive functions and describe a new translational approach to prevent metastasis.

Mesothelioma Mouse Models with Mixed Genomic States of Chromosome and Microsatellite Instability.

Malignant mesothelioma (MMe) is a rare malignancy originating from the linings of the pleural, peritoneal and pericardial cavities. The best-defined risk factor is exposure to carcinogenic mineral fibers (e.g., asbestos). Genomic studies have revealed that the most frequent genetic lesions in human MMe are mutations in tumor suppressor genes. Several genetically engineered mouse models have been generated by introducing the same genetic lesions found in human MMe. However, most of these models require specialized breeding facilities and long-term exposure of mice to asbestos for MMe development. Thus, an alternative model with high tumor penetrance without asbestos is urgently needed. We characterized an orthotopic model using MMe cells derived from Cdkn2a+/-;Nf2+/- mice chronically injected with asbestos. These MMe cells were tumorigenic upon intraperitoneal injection. Moreover, MMe cells showed mixed chromosome and microsatellite instability, supporting the notion that genomic instability is relevant in MMe pathogenesis. In addition, microsatellite markers were detectable in the plasma of tumor-bearing mice, indicating a potential use for early cancer detection and monitoring the effects of interventions. This orthotopic model with rapid development of MMe without asbestos exposure represents genomic instability and specific molecular targets for therapeutic or preventive interventions to enable preclinical proof of concept for the intervention in an immunocompetent setting.

Targeted multicolor in vivo imaging over 1,000 nm enabled by nonamethine cyanines.

Recent progress has shown that using wavelengths between 1,000 and 2,000 nm, referred to as the shortwave-infrared or near-infrared (NIR)-II range, can enable high-resolution in vivo imaging at depths not possible with conventional optical wavelengths. However, few bioconjugatable probes of the type that have proven invaluable for multiplexed imaging in the visible and NIR range are available for imaging these wavelengths. Using rational design, we have generated persulfonated indocyanine dyes with absorbance maxima at 872 and 1,072 nm through catechol-ring and aryl-ring fusion, respectively, onto the nonamethine scaffold. Multiplexed two-color and three-color in vivo imaging using monoclonal antibody and dextran conjugates in several tumor models illustrate the benefits of concurrent labeling of the tumor and healthy surrounding tissue and lymphatics. These efforts are enabled by complementary advances in a custom-built NIR/shortwave-infrared imaging setup and software package for multicolor real-time imaging.

Aging and CNS Myeloid Cell Depletion Attenuate Breast Cancer Brain Metastasis.

PURPOSE: Breast cancer diagnosed in young patients is often aggressive. Because primary breast tumors from young and older patients have similar mutational patterns, we hypothesized that the young host microenvironment promotes more aggressive metastatic disease. EXPERIMENTAL DESIGN: Triple-negative or luminal B breast cancer cell lines were injected into young and older mice side-by-side to quantify lung, liver, and brain metastases. Young and older mouse brains, metastatic and naïve, were analyzed by flow cytometry. Immune populations were depleted using antibodies or a colony-stimulating factor-1 receptor (CSF-1R) inhibitor, and brain metastasis assays were conducted. Effects on myeloid populations, astrogliosis, and the neuroinflammatory response were determined. RESULTS: Brain metastases were 2- to 4-fold higher in young as compared with older mouse hosts in four models of triple-negative or luminal B breast cancer; no age effect was observed on liver or lung metastases. Aged brains, naïve or metastatic, contained fewer resident CNS myeloid cells. Use of a CSF-1R inhibitor to deplete myeloid cells, including both microglia and infiltrating macrophages, preferentially reduced brain metastasis burden in young mice. Downstream effects of CSF-1R inhibition in young mice resembled that of an aged brain in terms of myeloid numbers, induction of astrogliosis, and Semaphorin 3A secretion within the neuroinflammatory response. CONCLUSIONS: Host microenvironmental factors contribute to the aggressiveness of triple-negative and luminal B breast cancer brain metastasis. CSF-1R inhibitors may hold promise for young brain metastasis patients.

CENP-A overexpression promotes aneuploidy with karyotypic heterogeneity.

Chromosomal instability (CIN) is a hallmark of many cancers. Restricting the localization of centromeric histone H3 variant CENP-A to centromeres prevents CIN. CENP-A overexpression (OE) and mislocalization have been observed in cancers and correlate with poor prognosis; however, the molecular consequences of CENP-A OE on CIN and aneuploidy have not been defined. Here, we show that CENP-A OE leads to its mislocalization and CIN with lagging chromosomes and micronuclei in pseudodiploid DLD1 cells and xenograft mouse model. CIN is due to reduced localization of proteins to the kinetochore, resulting in defects in kinetochore integrity and unstable kinetochore-microtubule attachments. CENP-A OE contributes to reduced expression of cell adhesion genes and higher invasion of DLD1 cells. We show that CENP-A OE contributes to aneuploidy with karyotypic heterogeneity in human cells and xenograft mouse model. In summary, our results provide a molecular link between CENP-A OE and aneuploidy, and suggest that karyotypic heterogeneity may contribute to the aggressive phenotype of CENP-A-overexpressing cancers.

Improvement of RG1-VLP vaccine performance in BALB/c mice by substitution of alhydrogel with the next generation polyphosphazene adjuvant PCEP.

Current human papillomavirus (HPV) vaccines provide substantial protection against the most common HPV types responsible for oral and anogenital cancers, but many circulating cancer-causing types remain for which vaccine coverage is lacking. In addition, all current HPV vaccines rely on aluminum salt-based adjuvant formulations that function through unclear mechanisms with few substitutes available. In an effort to expand the toolbox of available adjuvants suitable for HPV vaccines, we compared the immunogenicity of the RG1-VLP (virus-like particle) vaccine in BALB/c mice when formulated with either the aluminum hydroxide adjuvant Alhydrogel or the novel polyphosphazene macromolecular adjuvant poly[di (carboxylatoethylphenoxy) phosphazene] (PCEP). PCEP-formulated RG1-VLPs routinely outperformed VLP/Alhydrogel in several measurements of VLP-specific humoral immunity, including consistent improvements in the magnitude of antibody (Ab) responses to both HPV16-L1 and the L2 RG1 epitope as well as neutralizing titers to HPV16 and cross-neutralization of pseudovirion (PsV) types HPV18 and HPV39. Dose-sparing studies indicated that RG1-VLPs could be reduced in dose by 75% and the presence of PCEP ensured activity comparable to a full VLP dose adjuvanted by Alhydrogel. In addition, levels of HPV16-L1 and -L2-specific Abs were achieved after two vaccinations with PCEP as adjuvant that were equivalent to or greater than levels achieved with three vaccinations with Alhydrogel alone, indicating that the presence of PCEP resulted in accelerated immune responses that could allow for a decreased dose schedule. Given the extensive clinical track record of polyphosphazenes, these data suggest that substitution of alum-based adjuvants with PCEP for the RG1-VLP vaccine could lead to rapid seropositivity requiring fewer boosts, the dose-sparing of commercial VLP-based vaccines, and the establishment of longer-lasting humoral responses to HPV.