We know a great deal about the biochemistry of cells because they can be isolated and studied. The biochemistry of the much more complex in vivo environment is more difficult to study because the only ways to quantitate concentrations is to sacrifice the animal or biopsy the tissue. Either method disrupts the environment profoundly and neither method allows longitudinal studies on the same individual. Methods of measuring chemical concentrations in vivo are very valuable alternatives to sacrificing groups of animals. We are developing microscopic magnetic nanoparticle (mNP) probes to measure the concentration of a selected molecule in vivo. The mNPs are targeted to bind the selected molecule and the resulting reduction in rotational freedom can be quantified remotely using magnetic spectroscopy. The mNPs must be contained in micrometer sized porous shells to keep them from migrating and to protect them from clearance by the immune system. There are two key issues in the development of the probes. First, we demonstrate the ability to measure concentrations in the porous walled alginate probes both in phosphate buffered saline and in blood, which is an excellent surrogate for the complex and challenging in vivo environment. Second, sensitivity is critical because it allows microscopic probes to measure very small concentrations very far away. We report sensitivity measurements on recently introduced technology that has allowed us to improve the sensitivity by two orders of magnitude, a factor of 200 so far.

Active molecular targeting has become an important aspect of nanoparticle development for oncology indications. Here, we describe molecular targeting of iron oxide nanoparticles (IONPs) to the folate receptor alpha (FOLRα) using an engineered antibody fragment (Ffab). Compared to control nanoparticles targeting the non-relevant botulinum toxin, the Ffab-IONP constructs selectively accumulated on FOLRα-overexpressing cancer cells in vitro, where they exhibited the capacity to internalize into intracellular vesicles. Similarly, Ffab-IONPs homed to FOLRα-positive tumors upon intraperitoneal administration in an orthotopic murine xenograft model of ovarian cancer, whereas negative control particles showed no detectable tumor accumulation. Interestingly, Ffab-IONPs built with custom 120 nm nanoparticles exhibited lower in vitro targeting efficiency when compared to those built with commercially sourced 180 nm nanoparticles. In vivo, however, the two Ffab-IONP platforms achieved equivalent tumor homing, although the smaller 120 nm IONPs were more prone to liver sequestration. Overall, the results show that Ffab-mediated targeting of IONPs yields specific, high-level accumulation within cancer cells, and this fact suggests that Ffab-IONPs could have future utility in ovarian cancer diagnostics and therapy.

Combinatorial use of iron oxide nanoparticles (IONPs) and an alternating magnetic field (AMF) can induce local hyperthermia in tumors in a controlled and uniform manner. Heating B16 primary tumors at 43 degrees C for 30 min activated dendritic cells (DCs) and subsequently CD8(+) T cells in the draining lymph node (dLN) and conferred resistance against rechallenge with B16 (but not unrelated Lewis Lung carcinoma) given 7 days post hyperthermia on both the primary tumor side and the contralateral side in a CD8(+) T cell-dependent manner. Mice with heated primary tumors also resisted rechallenge given 30 days post hyperthermia. Mice with larger heated primary tumors had greater resistance to secondary tumors. No rechallenge resistance occurred when tumors were heated at 45 degrees C. Our results demonstrate the promising potential of local hyperthermia treatment applied to identified tumors in inducing anti-tumor immune responses that reduce the risk of recurrence and metastasis. (C) 2014 Elsevier Inc. All rights reserved.

Local tumour hyperthermia for cancer treatment is currently used either for ablation purposes as an alternative to surgery or less frequently, in combination with chemotherapy and/or radiation therapy to enhance the effects of those traditional therapies. As it has become apparent that activating the immune system is crucial to successfully treat metastatic cancer, the potential of boosting anti-tumour immunity by heating tumours has become a growing area of cancer research. After reviewing the history of hyperthermia therapy for cancer and introducing methods for inducing local hyperthermia, this review describes different mechanisms by which heating tumours can elicit anti-tumour immune responses, including tumour cell damage, tumour surface molecule changes, heat shock proteins, exosomes, direct effects on immune cells, and changes in the tumour vasculature. We then go over in vivo studies that provide promising results showing that local hyperthermia therapy indeed activates various systemic anti-tumour immune responses that slow growth of untreated tumours. Finally, future research questions that will help bring the use of local hyperthermia as systemic immunotherapy closer to clinical application are discussed.

A variety of strategies, have been applied to cancer treatment and the most recent one to become prominent is immunotherapy. This interest has been fostered by the demonstration that the immune system does recognize and often eliminate small tumors but tumors that become clinical problems block antitumor immune responses with immunosuppression orchestrated by the tumor cells. Methods to reverse this tumor-mediated immunosuppression will improve cancer immunotherapy outcomes. The immunostimulatory potential of nanoparticles (NPs), holds promise for cancer treatment. Phagocytes of various types are an important component of both immunosuppression and immunostimulation and phagocytes actively take up NPs of various sorts, so NPs are a natural system to manipulate these key immune regulatory cells. NPs can be engineered with multiple useful therapeutic features, such as various payloads such as antigens and/or immunomodulatory agents including cytokines, ligands for immunostimulatory receptors or antagonists for immunosuppressive receptors. As more is learned about how tumors suppress antitumor immune responses the payload options expand further. Here we review multiple approaches of NP-based cancer therapies to modify the tumor microenvironment and stimulate innate and adaptive immune systems to obtain effective antitumor immune responses.

Quantifying the number of nanoparticles present in tissue is central to many in vivo and in vitro applications. Magnetic nanoparticles can be detected with high sensitivity both in vivo and in vitro using the harmonics of their magnetization produced in a sinusoidal magnetic field. However, relaxation effects damp the magnetic harmonics rendering them of limited use in quantification. We show that an accurate measure of the number of nanoparticles can be made by correcting for relaxation effects. Correction for relaxation reduced errors of 50% for larger nanoparticles in high relaxation environments to 2%. The result is a method of nanoparticle quantification suitable for in vivo and in vitro applications including histopathology assays, quantitative imaging, drug delivery and thermal therapy preparation.

Nanotechnology has great potential to produce novel therapeutic strategies that target malignant cells through the ability of nanoparticles to get access to and be ingested by living cells. However its specificity for accumulation in tumors, which is the key factor that determines its efficacy, has always been a challenge. Here we tested a novel strategy to target and treat ovarian cancer, a representative peritoneal cancer, using iron oxide nanoparticles (IONPs) and an alternating magnetic field (AMF). Peritoneal tumors in general are directly accessible to nanoparticles administered intraperitoneally (IP), as opposed to the more commonly attempted intravenous (IV) administration. In addition, tumor-associated immunosuppressive phagocytes, a predominant cell population in the tumor microenvironment of almost all solid tumors, and cells that are critical for tumor progression, are constantly recruited to the tumor, and therefore could possibly function to bring nanoparticles to tumors. Here we demonstrate that tumor-associated peritoneal phagocytes ingest and carry IONPs specifically to tumors and that these specifically delivered nanoparticles can damage tumor cells after IONP-mediated hyperthermia generated by AMF. This illustrates therapeutic possibilities of intraperitoneal (IP) injection of nanoparticles and subsequent ingestion by tumor-associated phagocytes, to directly impact tumors or stimulate antitumor immune responses. This approach could use IONPs combined with AMF as done here, or other nanoparticles with cytotoxic potential. Overall, the data presented here support IP injection of nanoparticles to utilize peritoneal phagocytes as a delivery vehicle in association with IONP-mediated hyperthermia as therapeutic strategies for ovarian and other peritoneal cancers.

Purpose: To measure local biomarker concentrations longitudinally over time. Biomarker concentrations can be used to diagnose disease but the most promising application is in monitoring therapy. For example, the VEGF level provides a very direct evaluation of anti-angiogenic therapies or heat shock proteins provide a direct evaluation of thermal therapies. In this effort, the potential of the proposed technology was evaluated for sensitivity and specificity. The potential sensitivity was also estimated. Methods: Magnetic nanoparticles (NPs) can be detected remotely by measuring their magnetization induced by an alternating magnetic field. Magnetic spectroscopy of Brownian motion (MSB) characterizes the rotational Brownian motion from the shape of the magnetization. Rotational Brownian motion characterizes the local microenvironment surrounding the NPs: temperature, viscosity and chemical binding. We are reporting on a novel application of MSB that will allow the local concentration of biomarkers or drugs to be measured in vivo longitudinally over time. Magnetic NPs in ∼200 micron porous walled containers can be deposited in the vascular bed or into the extracellular space via injection in a lesions' blood supply or directly into the lesion. The NPs are decorated so that the target molecule (either biomarker or drug) binds the NPs together into an aggregate. The aggregate has a much longer relaxation time than the free NPs so the MSB signal changes dramatically. The MSB signal can be used to estimate the quantitative concentration of the biomarker. Results: The method has been explored in vitro for the following analytes (minimum sensitivities): 24 mer DNA (100 pM sensitivity); 20 mer DNA (2 nM); thrombin (4 nM); streptavidin (150 pM); VEGF (4 nM). Conclusion: These proof of concept experiments demonstrate that MSB methods are capable of measuring hormone concentrations of biomarkers and would require only reasonable increases in sensitivity to measure cytokine concentrations. NIH-NCI 1U54CA151662-01; Hopeman Fund Grant: Norris Cotton Cancer Center

Immune recognition of tumors can limit cancer development, but antitumor immune responses are often blocked by tumor-mediated immunosuppression. Because microbes or microbial constituents are powerful adjuvants to stimulate immune responses, we evaluated whether intratumoral administration of a highly immunogenic but attenuated parasite could induce rejection of an established poorly immunogenic tumor. We treated intradermal B16F10 murine melanoma by intratumoral injection of an attenuated strain of Toxoplasma gondii (cps) that cannot replicate in vivo and therefore is not infective. The cps treatment stimulated a strong CD8+ T cell–mediated antitumor immune response in vivo that regressed established primary melanoma. The cps monotherapy rapidly modified the tumor microenvironment, halting tumor growth, and subsequently, as tumor-reactive T cells expanded, the tumors disappeared and rarely returned. The treatment required live cps that could invade cells and also required CD8+ T cells and NK cells, but did not require CD4+ T cells. Furthermore, we demonstrate that IL-12, IFN-γ, and the CXCR3-stimulating cytokines are required for full treatment efficacy. The treatment developed systemic antitumor immune activity as well as antitumor immune memory and therefore might have an impact against human metastatic disease. The approach is not specific for either B16F10 or melanoma. Direct intratumoral injection of cps has efficacy against an inducible genetic melanoma model and transplantable lung and ovarian tumors, demonstrating potential for broad clinical use. The combination of efficacy, systemic antitumor immune response, and complete attenuation with no observed host toxicity demonstrates the potential value of this novel cancer therapy.

Purpose: Our hypothesis was that the weight of magnetic nanoparticles could be accurately estimated from the magnetic spectroscopy of nanoparticle Brownian motion (MSB) signal. Quantification is critical to bio‐sensing and histology and is becoming more important in medical imagining. A nanoparticle assay is important in histology and bio‐sensing where antibody targeted magnetic nanoparticles are used to mark specific protein expression. Quantitative imaging of magnetic nanoparticles is also important in applications including hyperthermia and nanoparticle drug delivery. Two factors make quantitative estimates very difficult to achieve: relaxation effects that change the signal produced by each nanoparticle and non‐nanoparticle iron that confuses mass spectroscopy measurements. We introduce a method only sensitive to nanoparticle iron that compensates for relaxation effects that achieves quantitative estimates of the number of magnetic nanoparticles in a sample. Methods: Samples with varying quantities of iron oxide nanoparticles (100 nm mean hydrodynamic diameter) and varying quantities of glycerol were prepared. The samples contained from 1.46 mg to 0.05 mg nanoparticles and from 0% to 27% glycerol. MSB signals were recorded for each sample. The relaxation time was calculated using previously reported methods. The MSB signals were then shifted in frequency to compensate for the change in relaxation time. The scaling of the normalized MSB signal that approximates the reference sample is the weight of nanoparticles present in the sample. The apparatus used was built for 1.5 mL liquid samples; a system for smaller samples would be more sensitive. Results: The RMS percentage error in the weight of nanoparticles was 4.9%. The RMS error in the weight of nanoparticles was 0.018 mg iron. Other sources of iron such as blood did not bias the estimates. Conclusions: The method presented makes accurate MSB estimates of the weight of nanoparticles. Acknowledgement: NIH‐NCI 1U54CA151662‐01 NIH‐NCI 1U54CA151662‐01