S100A9 upregulated by IFNGR signaling blockade functions as a novel GVHD suppressor without compromising GVL in mice.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for both malignant and nonmalignant hematologic disorders. However, graft-versus-host disease (GVHD) and malignant relapse limit its therapeutic success. We previously demonstrated that the blockade of interferon-gamma receptor (IFNGR) signaling in donor T cells resulted in a reduction in GVHD while preserving graft-versus-leukemia (GVL) effects. However, the underlying molecular mechanisms remain inconclusive. In this study, we found that S100A9 is a novel GVHD suppressor upregulated when IFNGR is blocked in T cells. Both Ifngr1-/- and S100a9-overexpressing T cells significantly reduced GVHD without compromising GVL, altering donor T-cell trafficking to GVHD target organs in our mouse model of allo-HSCT. In addition, in vivo administration of recombinant murine S100A9 proteins prolongs the overall survival of recipient mice. Furthermore, in vivo administration of anti-human IFNGRα neutralizing antibody (αhGR-Nab) significantly upregulates the expression of S100A9 in human T cells and improved GVHD in our mouse model of xenogeneic human peripheral blood mononuclear cell transplantation. Consistent with S100a9-overexpressing T cells in our allo-HSCT model, αhGR-Nab reduced human T-cell trafficking to the GVHD target organs. Taken together, S100A9, a downstream molecule suppressed by IFNGR signaling, functions as a novel GVHD suppressor without compromising GVL.

3D Printing of Poloxamer 407 Nanogel Discs and Their Applications in Adjuvant Ovarian Cancer Therapy.

Nanogels are attractive biocompatible materials that enable local delivery of multiple drugs. In this study, we demonstrated that 3D printing technology could be used to precisely construct nanogel discs carrying paclitaxel and rapamycin. 3D-printed nanogel disc rounds (12 mm diameter × 1 mm thickness) carrying paclitaxel and rapamycin evaded premature gelation during storage and the initial burst release of the drugs in the dissolution medium. In vivo 3D-printed nanogel discs permitted successful intraperitoneal delivery of paclitaxel and rapamycin in ES-2-luc ovarian-cancer-bearing xenograft mice. They were also shown to be therapeutically effective and capable of preventing postsurgical peritoneal adhesions in the treated xenograft mice.

[(18)F]FHBG PET/CT Imaging of CD34-TK75 Transduced Donor T Cells in Relapsed Allogeneic Stem Cell Transplant Patients: Safety and Feasibility.

Described herein is a first-in-man attempt to both genetically modify T cells with an imagable suicide gene and track these transduced donor T cells in allogeneic stem cell transplantation recipients using noninvasive positron emission tomography/computerized tomography (PET/CT) imaging. A suicide gene encoding a human CD34-Herpes Simplex Virus-1-thymidine kinase (CD34-TK75) fusion enabled enrichment of retrovirally transduced T cells (TdT), control of graft-versus-host disease and imaging of TdT migration and expansion in vivo in mice and man. Analysis confirmed that CD34-TK75-enriched TdT contained no replication competent γ-retrovirus, were sensitive to ganciclovir, and displayed characteristic retroviral insertion sites (by targeted sequencing). Affinity-purified CD34-TK75(+)-selected donor T cells (1.0-13 × 10(5))/kg were infused into eight patients who relapsed after allogeneic stem cell transplantation. Six patients also were administered 9-[4-((18)F)fluoro-3-hydroxymethyl-butyl]guanine ([(18)F]FHBG) to specifically track the genetically modified donor T cells by PET/CT at several time points after infusion. All patients were assessed for graft-versus-host disease, response to ganciclovir, circulating TdT cells (using both quantitative polymerase chain reaction and [(18)F]FHBG PET/CT imaging), TdT cell clonal expansion, and immune response to the TdT. This phase 1 trial demonstrated that genetically modified T cells and [(18)F]FHBG can be safely infused in patients with relapsed hematologic malignancies after allogeneic stem cell transplantation.

IFNγR signaling mediates alloreactive T-cell trafficking and GVHD.

The clinical goal of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is to minimize GVHD while maintaining GvL. Here, we show that interferon γ receptor-deficient (IFNγR(-/-)) allogeneic Tconv, which possess normal alloreactivity and cytotoxicity, induce significantly less GVHD than wild-type (WT) Tconv. This effect is mediated by altered trafficking of IFNγR(-/-) Tconv to GVHD target organs, especially the gastrointestinal (GI) tract. We show that the chemokine receptor CXCR3 is induced via IFNγR-mediated signaling and partially contributes to the trafficking of WT Tconv to GVHD target organs. Indeed, CXCR3(-/-) Tconv recapitulate the reduced GVHD potential of IFNγR(-/-) Tconv in a minor-mismatched GVHD model. Most importantly, IFNγR(-/-) (and CXCR3(-/-)) Tconv mediate a robust and beneficial GvL effect. In addition, we show that IFNγR(-/-) regulatory T cells (Tregs) are fully suppressive in vitro although defective in suppressor function in vivo and that WT Tregs suppress GVHD in vivo only when allogeneic Tconv produce interferon γ (IFNγ), suggesting that the IFNγR signaling pathway is the major mechanism for both Tregs and Tconv to migrate to GVHD target organs. Finally, pharmacologic inhibition of IFNγR signaling with inhibitors of JAK1/JAK2, which are mediators of IFNγR signaling, results in the decreased expression of CXCR3 and reduced GVHD and improved survival after allo-HSCT and this effect is mediated by altered trafficking of Tconv to GVHD target organs.

Development of New CD38 Targeted Peptides for Cancer Imaging.

PURPOSE: Multiple myeloma (MM) affects over 35,000 patients each year in the US. There remains a need for versatile Positron Emission Tomography (PET) tracers for the detection, accurate staging, and monitoring of treatment response of MM that have optimal specificity and translational attributes. CD38 is uniformly overexpressed in MM and thus represents an ideal target to develop CD38-targeted small molecule PET radiopharmaceuticals to address these challenges. PROCEDURES: Using phage display peptide libraries and pioneering algorithms, we identified novel CD38 specific peptides. Imaging bioconjugates were synthesized using solid phase peptide chemistry, and systematically analyzed in vitro and in vivo in relevant MM systems. RESULTS: The CD38-targeted bioconjugates were radiolabeled with copper-64 (64Cu) with100% radiochemical purity and an average specific activity of 3.3 - 6.6 MBq/nmol. The analog NODAGA-PEG4-SL022-GGS (SL022: Thr-His-Tyr-Pro-Ile-Val-Ile) had a Kd of 7.55 ± 0.291 nM and was chosen as the lead candidate. 64Cu-NODAGA-PEG4-SL022-GGS demonstrated high binding affinity to CD38 expressing human myeloma MM.1S-CBR-GFP-WT cells, which was blocked by the non-radiolabeled version of the peptide analog and anti-CD38 clinical antibodies, daratumumab and isatuximab, by 58%, 73%, and 78%, respectively. The CD38 positive MM.1S-CBR-GFP-WT cells had > 68% enhanced cellular binding when compared to MM.1S-CBR-GFP-KO cells devoid of CD38. Furthermore, our new CD38-targeted radiopharmaceutical allowed visualization of tumors located in marrow rich bones, remaining there for up to 4 h. Clearance from non-target organs occurred within 60 min. Quantitative PET data from a murine disseminated tumor model showed significantly higher accumulation in the bones of tumor-bearing animals compared to tumor-naïve animals (SUVmax 2.06 ± 0.4 versus 1.24 ± 0.4, P = 0.02). Independently, tumor uptake of the target compound was significantly higher (P = 0.003) compared to the scrambled peptide, 64Cu-NODAGA-PEG4-SL041-GGS (SL041: Thr-Tyr-His-Ile-Pro-Ile-Val). The subcutaneous MM model demonstrated significantly higher accumulation in tumors compared to muscle at 1 and 4 h after tracer administration (SUVmax 0.8 ± 0.2 and 0.14 ± 0.04, P = 0.04 at 1 h; SUVmax 0.89 ± 0.01 and 0.09 ± 0.01, P = 0.0002 at 4 h). CONCLUSIONS: The novel CD38-targeted, radiolabeled bioconjugates were specific and allowed visualization of MM, providing a starting point for the clinical translation of such tracers for the detection of MM.

Induction of complementary immunogenic necroptosis and apoptosis cell death pathways inhibits cancer metastasis and relapse.

Interactions of light-sensitive drugs and materials with Cerenkov radiation-emitting radiopharmaceuticals generate cytotoxic reactive oxygen species (ROS) to inhibit localized and disseminated cancer progression, but the cell death mechanisms underlying this radionuclide stimulated dynamic therapy (RaST) remain elusive. Using ROS-regenerative nanophotosensitizers coated with a tumor-targeting transferrin-titanocene complex (TiO2-TC-Tf) and radiolabeled 2-fluorodeoxyglucose (18FDG), we found that adherent dying cells maintained metabolic activity with increased membrane permeabilization. Mechanistic assessment of these cells revealed that RaST activated the expression of RIPK-1 and RIPK-3, which mediate necroptosis cell death. Subsequent recruitment of the nuclear factors kappa B and the executioner mixed lineage kinase domain-like pseudo kinase (MLKL) triggered plasma membrane permeabilization and pore formation, respectively, followed by the release of cytokines and immunogenic damage-associated molecular patterns (DAMPs). In immune-deficient breast cancer models with adequate stroma and growth factors that recapitulate the human tumor microenvironment, RaST failed to inhibit tumor progression and the ensuing lung metastasis. A similar aggressive tumor model in immunocompetent mice responded to RaST, achieving a remarkable partial response (PR) and complete response (CR) with no evidence of lung metastasis, suggesting active immune system engagement. RaST recruited antitumor CD11b+, CD11c+, and CD8b+ effector immune cells after initiating dual immunogenic apoptosis and necroptosis cell death pathways in responding tumors in vivo. Over time, cancer cells upregulated the expression of negative immune regulating cytokine (TGF-ß) and soluble immune checkpoints (sICP) to challenge RaST effect in the CR mice. Using a signal-amplifying cancer-imaging agent, LS301, we identified latent minimal residual disseminated tumors in the lymph nodes (LNs) of the CR group. Despite increased protumor immunogens in the CR mice, RaST prevented cancer relapse and metastasis through dynamic redistribution of ROS-regenerative TiO2 from bones at the early treatment stage to the spleen and LNs, maintaining active immunity against cancer progression and migration. This study reveals the immune-mechanistic underpinnings of RaST-mediated antitumor immune response and highlights immunogenic reprogramming of tumors in response to RaST. Overcoming apoptosis resistance through complementary necroptosis activation paves the way for strategic drug combinations to improve cancer treatment.

LRP6 overexpression defines a class of breast cancer subtype and is a target for therapy.

The Wnt/beta-catenin signaling pathway is activated in breast cancer, a leading cause of cancer mortality in women. Because mutations in the key intracellular components of this pathway are rare, identifying the molecular mechanisms of aberrant Wnt activation in breast cancer is critical for development of pathway-targeted therapy. Here, we show that expression of the Wnt signaling coreceptor LRP6 is up-regulated in a subpopulation of human breast cancers. LRP6 silencing in breast cancer cells reduces Wnt signaling, cell proliferation, and in vivo tumor growth. In vivo administration of an LRP6 antagonist, Mesd, markedly suppressed growth of MMTV-Wnt1 tumors without causing undesirable side effects. These results demonstrate that Wnt activation at the cell surface contributes to breast cancer tumorigenesis. Together, our studies highlight LRP6 as a potential therapeutic target in breast cancer, and introduce Mesd as a promising antitumor agent for treating breast cancer subtypes with Wnt activation at the cell surface.

Breast Cancer Mutations HER2V777L and PIK3CAH1047R Activate the p21-CDK4/6-Cyclin D1 Axis to Drive Tumorigenesis and Drug Resistance.

In metastatic breast cancer, HER2-activating mutations frequently co-occur with mutations in PIK3CA, TP53, or CDH1. Of these co-occurring mutations, HER2 and PIK3CA are the most commonly comutated gene pair, with approximately 40% of HER2-mutated breast cancers also having activating mutations in PIK3CA. To study the effects of co-occurring HER2 and PIK3CA mutations, we generated genetically engineered mice with the HER2V777L; PIK3CAH1047R transgenes (HP mice) and studied the resulting breast cancers both in vivo as well as ex vivo using cancer organoids. HP breast cancers showed accelerated tumor formation in vivo and increased invasion and migration in in vitro assays. HP breast cancer cells were resistant to the pan-HER tyrosine kinase inhibitor, neratinib, but were effectively treated with neratinib plus the HER2-targeted antibody-drug conjugate trastuzumab deruxtecan. Proteomic and RNA-seq analysis of HP breast cancers identified increased gene expression of cyclin D1 and p21WAF1/Cip1 and changes in cell-cycle markers. Combining neratinib with CDK4/6 inhibitors was another effective strategy for treating HP breast cancers, with neratinib plus palbociclib showing a statistically significant reduction in development of mouse HP tumors as compared to either drug alone. The efficacy of both the neratinib plus trastuzumab deruxtecan and neratinib plus palbociclib combinations was validated using a human breast cancer patient-derived xenograft with very similar HER2 and PIK3CA mutations to the HP mice. Further, these two drug combinations effectively treated spontaneous lung metastasis in syngeneic mice transplanted with HP breast cancer organoids. This study provides valuable preclinical data to support the ongoing phase 1 clinical trials of these drug combinations in breast cancer. SIGNIFICANCE: In HER2-mutated breast cancer, PIK3CA mutation activates p21-CDK4/6-cyclin D1 signaling to drive resistance to HER2-targeted therapies, which can be overcome using CDK4/6 inhibitors.

In vivo administration of hypomethylating agents mitigate graft-versus-host disease without sacrificing graft-versus-leukemia.

Regulatory T cells (Tregs) suppress graft-versus-host disease (GVHD) while preserving a beneficial graft-versus-leukemia (GVL) effect. Thus, their use in allogeneic stem cell transplantation (SCT) provides a promising strategy to treat GVHD. However, 3 obstacles prevent their routine use in human clinical trials: (1) low circulating number of Tregs in peripheral blood, (2) loss of suppressor function after in vitro expansion, and (3) lack of Treg-specific surface markers necessary for efficient purification. FOXP3 is exclusively expressed in Tregs and forced expression in CD4(+)CD25(-) T cells can convert these non-Tregs into Tregs with functional suppressor function. Here, we show that the FDA-approved hypomethylating agents, decitabine (Dec) and azacitidine (AzaC), induce FOXP3 expression in CD4(+)CD25(-) T cells both in vitro and in vivo. Their suppressor function is dependent on direct contact, partially dependent on perforin 1 (Prf1), but independent of granzyme B (GzmB), and surprisingly, Foxp3. Independence of Foxp3 suggests that genes responsible for the suppressor function are also regulated by DNA methylation. We have identified 48 candidate genes for future studies. Finally, AzaC treatment of mice that received a transplant of major histocompatibility complex mismatched allogeneic bone marrow and T cells mitigates GVHD while preserving GVL by peripheral conversion of alloreactive effector T cells into FOXP3(+) Tregs and epigenetic modulation of genes downstream of Foxp3 required for the suppressor function of Tregs.

CS1 CAR-T targeting the distal domain of CS1 (SLAMF7) shows efficacy in high tumor burden myeloma model despite fratricide of CD8+CS1 expressing CAR-T cells.

Despite improvement in treatment options for myeloma patients, including targeted immunotherapies, multiple myeloma remains a mostly incurable malignancy. High CS1 (SLAMF7) expression on myeloma cells and limited expression on normal cells makes it a promising target for CAR-T therapy. The CS1 protein has two extracellular domains - the distal Variable (V) domain and the proximal Constant 2 (C2) domain. We generated and tested CS1-CAR-T targeting the V domain of CS1 (Luc90-CS1-CAR-T) and demonstrated anti-myeloma killing in vitro and in vivo using two mouse models. Since fratricide of CD8 + cells occurred during production, we generated fratricide resistant CS1 deficient Luc90- CS1- CAR-T (ΔCS1-Luc90- CS1- CAR-T). This led to protection of CD8 + cells in the CAR-T cultures, but had no impact on efficacy. Our data demonstrate targeting the distal V domain of CS1 could be an effective CAR-T treatment for myeloma patients and deletion of CS1 in clinical production did not provide an added benefit using in vivo immunodeficient NSG preclinical models.

Chemosensitization of acute myeloid leukemia (AML) following mobilization by the CXCR4 antagonist AMD3100.

The CXCR4-SDF-1 axis plays a central role in the trafficking and retention of normal and malignant stem cells in the bone marrow (BM) microenvironment. Here, we used a mouse model of acute promyelocytic leukemia (APL) and a small molecule competitive antagonist of CXCR4, AMD3100, to examine the interaction of mouse APL cells with the BM microenvironment. APL cells from a murine cathepsin G-PML-RARalpha knockin mouse were genetically modified with firefly luciferase (APL(luc)) to allow tracking by bioluminescence imaging. Coculture of APL(luc) cells with M2-10B4 stromal cells protected the leukemia cells from chemotherapy-induced apoptosis in vitro. Upon injection into syngeneic recipients, APL(luc) cells rapidly migrated to the BM followed by egress to the spleen then to the peripheral blood with death due to leukostasis by day 15. Administration of AMD3100 to leukemic mice induced a 1.6-fold increase in total leukocytes and a 9-fold increase of circulating APL blast counts, which peak at 3 hours and return to baseline by 12 hours. Treatment of leukemic mice with chemotherapy plus AMD3100 resulted in decreased tumor burden and improved overall survival compared with mice treated with chemotherapy alone. These studies provide a proof-of-principle for directing therapy to the critical tethers that promote AML-niche interactions.

Dissection of platelet and myeloid cell defects by conditional targeting of the beta3-integrin subunit.

The purpose of this work was to determine platelet and myeloid cell-specific requirements for beta3-containing integrins in hemostasis, bone resorption, and tumor growth. LoxP-flanked mice were generated to study the conditional deletion of beta3-integrin in platelets [knockout in platelets (KOP)] and myeloid cells [knockout in myeloid (KOM)]. Using the beta3KOP and beta3KOM strains of mice, we studied the role of beta3-integrin in hemostasis, bone resorption, and subcutaneous tumor growth. Tissue-specific deletion of platelet beta3-integrins in beta3KOP mice did not affect bone mass but resulted in a severe bleeding phenotype. No growth difference of tumor xenografts or in neoangiogenesis were found in beta3KOP mice, in contrast to the defects observed in germline beta3(-/-) mice. Conditional deletion of myeloid beta3-integrins in beta3KOM mice resulted in osteopetrosis but had no effect on hemostasis or mortality. Tumor growth in beta3KOM mice was increased and accompanied by decreased macrophage infiltration, without increase in blood vessel number. Platelet beta3-integrin deficiency was sufficient to disrupt hemostasis but had no effect on bone mass or tumor growth. Myeloid-specific beta3-integrin deletion was sufficient to perturb bone mass and enhance tumor growth due to reduced macrophage infiltration in the tumors. These results suggest that beta3-integrins have cell-specific roles in complex biological processes.-Morgan, E. A., Schneider, J. G., Baroni, T. E., Uluçkan, O., Heller, E., Hurchla, M. A., Deng, H., Floyd, D., Berdy, A., Prior, J. L., Piwnica-Worms, D., Teitelbaum, S. L., Ross, F. P., Weilbaecher, K. N. Dissection of platelet and myeloid cell defects by conditional targeting of the beta3-integrin subunit.

Generation of a highly inducible Gal4-->Fluc universal reporter mouse for in vivo bioluminescence imaging.

Full understanding of the functional complexity of the protein interactome requires mapping of biomolecular complexes within the cellular environment over biologically relevant time scales. New approaches to imaging interacting protein partners in vivo will allow the study of functional proteomics of human biology and disease within the context of living animals. Herein, we describe a universal transgenic reporter mouse strain that expresses firefly luciferase (Fluc) under the regulatory control of a concatenated Gal4 promoter (Tg(G4F(+/-))). Using an adenovirus to deliver a fused binding-domain-activator chimera (Gal4BD-VP16), induction of bioluminescence in Tg(G4F(+/-)) tissues of up to 4 orders of magnitude was observed in fibroblasts, liver, respiratory epithelia, muscle, and brain. The Tg(G4F(+/-)) reporter strain allowed noninvasive detection of viral infectivity, duration of the infection as well as viral clearance in various tissues in vivo. To demonstrate protein-protein interactions in live mice, the well characterized interaction between tumor suppressor p53 (fused to Gal4BD) and large T antigen (TAg) (fused to VP16) was visualized in vivo by using a two-hybrid strategy. Hepatocytes of Tg(G4F(+/-)) mice transfected with p53/TAg demonstrated 48-fold greater induction of Fluc expression in vivo than noninteracting pairs. Furthermore, to demonstrate the feasibility of monitoring experimental therapy with siRNA in vivo, targeted knockdown of p53 resulted in markedly reduced light output, whereas use of a control siRNA had no effect on protein interaction-dependent induction of Fluc. Thus, this highly inducible Gal4-->Fluc conditional reporter strain should facilitate imaging studies of protein interactions, signaling cascades, viral dissemination, and therapy within the physiological context of the whole animal.

Orthogonal targeting of osteoclasts and myeloma cells for radionuclide stimulated dynamic therapy induces multidimensional cell death pathways.

Rationale: Multiple myeloma (MM) is a multifocal malignancy of bone marrow plasma cells, characterized by vicious cycles of remission and relapse that eventually culminate in death. The disease remains mostly incurable largely due to the complex interactions between the bone microenvironment (BME) and MM cells (MMC). In the "vicious cycle" of bone disease, abnormal activation of osteoclasts (OCs) by MMC causes severe osteolysis, promotes immune evasion, and stimulates the growth of MMC. Disrupting these cancer-stroma interactions would enhance treatment response. Methods: To disrupt this cycle, we orthogonally targeted nanomicelles (NM) loaded with non-therapeutic doses of a photosensitizer, titanocene (TC), to VLA-4 (α4ß1, CD49d/CD29) expressing MMC (MM1.S) and αvß3 (CD51/CD61) expressing OC. Concurrently, a non-lethal dose of a radiopharmaceutical, 18F-fluorodeoxyglucose ([18F]FDG) administered systemically interacted with TC (radionuclide stimulated therapy, RaST) to generate cytotoxic reactive oxygen species (ROS). The in vitro and in vivo effects of RaST were characterized in MM1.S cell line, as well as in xenograft and isograft MM animal models. Results: Our data revealed that RaST induced non-enzymatic hydroperoxidation of cellular lipids culminating in mitochondrial dysfunction, DNA fragmentation, and caspase-dependent apoptosis of MMC using VLA-4 avid TC-NMs. RaST upregulated the expression of BAX, Bcl-2, and p53, highlighting the induction of apoptosis via the BAK-independent pathway. The enhancement of multicopper oxidase enzyme F5 expression, which inhibits lipid hydroperoxidation and Fenton reaction, was not sufficient to overcome RaST-induced increase in the accumulation of irreversible function-perturbing α,ß-aldehydes that exerted significant and long-lasting damage to both DNA and proteins. In vivo, either VLA-4-TC-NM or αvß3-TC-NMs RaST induced a significant therapeutic effect on immunocompromised but not immunocompetent MM-bearing mouse models. Combined treatment with both VLA-4-TC-NM and αvß3-TC-NMs synergistically inhibited osteolysis, reduced tumor burden, and prevented rapid relapse in both in vivo models of MM. Conclusions: By targeting MM and bone cells simultaneously, combination RaST suppressed MM disease progression through a multi-prong action on the vicious cycle of bone cancer. Instead of using the standard multidrug approach, our work reveals a unique photophysical treatment paradigm that uses nontoxic doses of a single light-sensitive drug directed orthogonally to cancer and bone cells, followed by radionuclide-stimulated generation of ROS to inhibit tumor progression and minimize osteolysis in both immunocompetent murine and immunocompromised human MM models.

Intracranial glioma xenograft model rapidly reestablishes blood-brain barrier integrity for longitudinal imaging of tumor progression using fluorescence molecular tomography and contrast agents.

SIGNIFICANCE: The blood-brain barrier (BBB) is a major obstacle to detecting and treating brain tumors. Overcoming this challenge will facilitate the early and accurate detection of brain lesions and guide surgical resection of tumors. AIM: We generated an orthotopic brain tumor model that simulates the pathophysiology of gliomas at early stages; determine the BBB integrity and breakdown over the time course of tumor progression using generic and cancer-targeted near-infrared (NIR) fluorescent molecular probes. APPROACH: We developed an intracranial tumor xenograft model that rapidly reestablished BBB integrity and monitored tumor progression by bioluminescence imaging. Sham control mice were injected with phosphate-buffered saline only. Fluorescence molecular tomography (FMT) was used to quantify the uptake of tumor-targeted and passive NIR fluorescent imaging agents in orthotopic glioma (U87-GL-GFP PDE7B H217Q cells) tumor model. Cancer-induced and transient (with focused ultrasound, FUS) disruption of BBB integrity was monitored with NIR fluorescent dyes. RESULTS: Stereotactic injection of 50,000 cells into mouse brain allowed rapid reestablishment of BBB integrity within a week, as determined by the inability of both tumor-targeted and generic NIR imaging agents to extravasate into the brain. Tumor-induced BBB disruption was observed 7 weeks after tumor implantation. FUS achieved a similar effect at any time point after reestablishing BBB integrity. While tumor uptake and retention of the passive NIR dye, indocyanine green, was negligible, both actively tumor-targeting agents exhibited selective accumulation in the tumor region. The tumor-targeting molecular probe that clears rapidly from nontumor brain tissue exhibits higher contrast than the analogous vascular-targeting agent and helps delineate tumors from sham control. CONCLUSIONS: We highlight the utility of FMT imaging for longitudinal assessment of brain tumors and the interplay between the stages of BBB disruption and molecular probe retention in tumors, with potential application to other neurological diseases.

Osteotropic Radiolabeled Nanophotosensitizer for Imaging and Treating Multiple Myeloma.

Rapid liver and spleen opsonization of systemically administered nanoparticles (NPs) for in vivo applications remains the Achilles' heel of nanomedicine, allowing only a small fraction of the materials to reach the intended target tissue. Although focusing on diseases that reside in the natural disposal organs for nanoparticles is a viable option, it limits the plurality of lesions that could benefit from nanomedical interventions. Here we designed a theranostic nanoplatform consisting of reactive oxygen (ROS)-generating titanium dioxide (TiO2) NPs, coated with a tumor-targeting agent, transferrin (Tf), and radiolabeled with a radionuclide (89Zr) for targeting bone marrow, imaging the distribution of the NPs, and stimulating ROS generation for cell killing. Radiolabeling of TiO2 NPs with 89Zr afforded thermodynamically and kinetically stable chelate-free 89Zr-TiO2-Tf NPs without altering the NP morphology. Treatment of multiple myeloma (MM) cells, a disease of plasma cells originating in the bone marrow, with 89Zr-TiO2-Tf generated cytotoxic ROS to induce cancer cell killing via the apoptosis pathway. Positron emission tomography/X-ray computed tomography (PET/CT) imaging and tissue biodistribution studies revealed that in vivo administration of 89Zr-TiO2-Tf in mice leveraged the osteotropic effect of 89Zr to selectively localize about 70% of the injected radioactivity in mouse bone tissue. A combination of small-animal PET/CT imaging of NP distribution and bioluminescence imaging of cancer progression showed that a single-dose 89Zr-TiO2-Tf treatment in a disseminated MM mouse model completely inhibited cancer growth at euthanasia of untreated mice and at least doubled the survival of treated mice. Treatment of the mice with cold Zr-TiO2-Tf, 89Zr-oxalate, or 89Zr-Tf had no therapeutic benefit compared to untreated controls. This study reveals an effective radionuclide sensitizing nanophototherapy paradigm for the treatment of MM and possibly other bone-associated malignancies.

Calcium carbonate nanoparticles stimulate tumor metabolic reprogramming and modulate tumor metastasis.

AIM: CaCO3 nanoparticles (nano-CaCO3) can neutralize the acidic pHe of solid tumors, but the lack of intrinsic imaging signal precludes noninvasive monitoring of pH-perturbation in tumor microenvironment. We aim to develop a theranostic version of nano-CaCO3 to noninvasively monitor pH modulation and subsequent tumor response. MATERIALS & METHODS: We synthesized ferromagnetic core coated with CaCO3 (magnetite CaCO3). Magnetic resonance imaging (MRI) was used to determine the biodistribution and pH modulation using murine fibrosarcoma and breast cancer models. RESULTS: Magnetite CaCO3-MRI imaging showed that nano-CaCO3 rapidly raised tumor pHe, followed by excessive tumor-associated acid production after its clearance. Continuous nano-CaCO3 infusion could inhibit metastasis. CONCLUSION: Nano-CaCO3 exposure induces tumor metabolic reprogramming that could account for the failure of previous intermittent pH-modulation strategies to achieve sustainable therapeutic effect.

APT102, a novel adpase, cooperates with aspirin to disrupt bone metastasis in mice.

Platelets contribute to the development of metastasis, the most common cause of mortality in cancer patients, but the precise role that anti-platelet drugs play in cancer treatment is not defined. Metastatic tumor cells can produce platelet alphaIIb beta3 activators, such as ADP and thromboxane A(2) (TXA(2)). Inhibitors of platelet beta3 integrins decrease bone metastases in mice but are associated with significant bleeding. We examined the role of a novel soluble apyrase/ADPase, APT102, and an inhibitor of TXA(2) synthesis, acetylsalicylic acid (aspirin or ASA), in mouse models of experimental bone metastases. We found that treatment with ASA and APT102 in combination (ASA + APT102), but not either drug alone, significantly decreased breast cancer and melanoma bone metastases in mice with fewer bleeding complications than observed with alphaIIb beta3 inhibition. ASA + APT102 diminished tumor cell induced platelet aggregation but did not directly alter tumor cell viability. Notably, APT102 + ASA treatment did not affect initial tumor cell distribution and similar results were observed in beta3-/- mice. These results show that treatment with ASA + APT102 decreases bone metastases without significant bleeding complications. Anti-platelet drugs such as ASA + APT102 could be valuable experimental tools for studying the role of platelet activation in metastasis as well as a therapeutic option for the prevention of bone metastases.

P-glycoprotein deficiency at the blood-brain barrier increases amyloid-beta deposition in an Alzheimer disease mouse model.

Accumulation of amyloid-beta (Abeta) within extracellular spaces of the brain is a hallmark of Alzheimer disease (AD). In sporadic, late-onset AD, there is little evidence for increased Abeta production, suggesting that decreased elimination from the brain may contribute to elevated levels of Abeta and plaque formation. Efflux transport of Abeta across the blood-brain barrier (BBB) contributes to Abeta removal from the brain. P-glycoprotein (Pgp) is highly expressed on the luminal surface of brain capillary endothelial cells and contributes to the BBB. In Pgp-null mice, we show that [I]Abeta40 and [I]Abeta42 microinjected into the CNS clear at half the rate that they do in WT mice. When amyloid precursor protein-transgenic (APP-transgenic) mice were administered a Pgp inhibitor, Abeta levels within the brain interstitial fluid significantly increased within hours of treatment. Furthermore, APP-transgenic, Pgp-null mice had increased levels of brain Abeta and enhanced Abeta deposition compared with APP-transgenic, Pgp WT mice. These data establish a direct link between Pgp and Abeta metabolism in vivo and suggest that Pgp activity at the BBB could affect risk for developing AD as well as provide a novel diagnostic and therapeutic target.

Factors affecting human T cell engraftment, trafficking, and associated xenogeneic graft-vs-host disease in NOD/SCID beta2mnull mice.

OBJECTIVE: Graft-vs-host disease (GVHD) is the major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Models of immunodeficient mice that consistently and efficiently reconstitute with xenoreactive human T cells would be a valuable tool for the in vivo study of GVHD, as well as other human immune responses. MATERIALS AND METHODS: We developed a consistent and sensitive model of human GVHD by retro-orbitally injecting purified human T cells into sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID)-beta2m(null) recipients. In addition, we characterized for the first time the trafficking patterns and expansion profiles of xenoreactive human T cells in NOD/SCID-beta2m(null) recipients using in vivo bioluminescence imaging. RESULTS: All NOD/SCID-beta2m(null) mice conditioned with 300 cGy total body irradiation and injected with 1 x 10(7) human T cells exhibited human T-cell engraftment, activation, and expansion, with infiltration of multiple target tissues and a subsequent >20% loss of pretransplantation body weight. Importantly, histological examination of the GVHD target tissues revealed changes consistent with human GVHD. Furthermore, we also showed by in vivo bioluminescence imaging that development of lethal GVHD in the NOD/SCID-beta2m(null) recipients was dependent upon the initial retention and early expansion of human T cells in the retro-orbital sinus cavity. CONCLUSION: Our NOD/SCID-beta2m(null) mouse model provides a system to study the pathophysiology of acute GVHD induced by human T cells and aids in development of more effective therapies for human GVHD.