Unique Glycoform-Dependent Monoclonal Antibodies for Mouse Mucin 21.

Mucin 21(Muc21)/epiglycanin is expressed on apical surfaces of squamous epithelia and has potentially protective roles, which are thought to be associated with its unique glycoforms, whereas its aberrant glycosylation is implicated in the malignant behaviors of some carcinomas. Despite the importance of glycoforms, we lack tools to detect specific glycoforms of mouse Muc21. In this study, we generated two monoclonal antibodies (mAbs) that recognize different glycoforms of Muc21. We used membrane lysates of Muc21-expressing TA3-Ha cells or Chinese hamster ovary (CHO)-K1 cells transfected with Muc21 as antigens. Specificity testing, utilizing Muc21 glycosylation variant cells, showed that mAb 1A4-1 recognized Muc21 carrying glycans terminated with galactose residues, whereas mAb 18A11 recognized Muc21 carrying sialylated glycans. mAb 1A4-1 stained a majority of mouse mammary carcinoma TA3-Ha cells in vitro and in engrafted tumors in mice, whereas mAb 18A11 recognized only a subpopulation of these. mAb 1A4-1 was useful in immunohistochemically detecting Muc21 in normal squamous epithelia. In conclusion, these mAbs recognize distinct Muc21 epitopes formed by combinations of peptide portions and O-glycans.

Toward Localized In Vivo Biomarker Concentration Measurements.

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.

Antibody-mediated targeting of iron oxide nanoparticles to the folate receptor alpha increases tumor cell association in vitro and in vivo.

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.

Local tumour hyperthermia as immunotherapy for metastatic cancer.

Abstract 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.

Stimulating antitumor immunity with nanoparticles.

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.

Local hyperthermia treatment of tumors induces CD8(+) T cell-mediated resistance against distal and secondary tumors.

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°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°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. FROM THE CLINICAL EDITOR: Local heating of tumors via iron oxide NPs and an alternating magnetic field led to activation of anti-cancer CD8 T cells, which resulted in resistance against re-challenge and greater resistance to secondary tumors. Similar local heating-based strategies may become an important weapon in enhancing tumor elimination via a naturally existing but attenuated immune response.

Quantification of magnetic nanoparticles with low frequency magnetic fields: compensating for relaxation effects.

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.

Phagocytes mediate targeting of iron oxide nanoparticles to tumors for cancer therapy.

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.

Immune-mediated regression of established B16F10 melanoma by intratumoral injection of attenuated Toxoplasma gondii protects against rechallenge.

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.