Commentary on Some Recent Theses Relevant to Combating Aging: August 2015

Engineering Virus-Mimicking Protein Nanoparticles for Cancer Immunotherapy

Nicholas Molino, PhD, University of California, Irvine

The immune system represents a powerful resource for the eradication of cancer. To harness the full potential of this sophisticated network to overcome the low immunogenicity of tumor cells, a sufficiently strong cytotoxic CD8 T cell (CTL)-mediated adaptive immune response is required, which is partly orchestrated by the professional antigen-presenting cells of the innate immune system, most notably the dendritic cell (DC). Protein nanoparticles represent a potentially exceptional vaccination platform for cancer, because they have the ability to mimic viral infections, which are known potent inducers of CTL immunity.

We have been exploring the E2 protein nanoparticle as a delivery platform for antigens and immune-stimulating compounds. The E2 nanoparticle was successfully packaged internally with endolysosomal-releasable immune-activating DNA (CpG) and surface functionalized with MHC I-restricted peptide epitopes. The virus-mimicking nanoparticle induced DC activation at a 25-fold lower concentration compared to free CpG and induced a three-fold increase in cross-presentation of attached epitopes, compared to free forms of peptides or activators. Furthermore, we demonstrated that co-delivery of melanoma-associated epitopes and immune-activating CpG with E2 enhanced the antigen-specific CTL proliferation index by 1.5-fold with a concomitant five-fold increase in interferon-γ (IFN-γ) cytokine secretion, compared to unbound peptide and CpG. Remarkably, a single immunization with the multifunctional E2 nanoparticle increased the frequency of melanoma-specific CTL in vivo (120-fold increase in the lymph node and 30-fold increase in the spleen), and the CTL generated showed approximately three times the lytic capacity toward a gp100-expressing melanoma cell line, compared to unbound peptide and CpG immunization.

We were also able to tune cellular and immunological interactions toward the E2 nanoparticle with surface display of poly(ethylene glycol) (PEG) polymers, where PEGylation through various methods (native surface amines or a recombinantly introduced cysteine) was shown to decrease cell uptake by greater than 50% of both mouse and human cell lines. PEGylation was also shown to mediate moderate increases in complement activation (∼35% C5a production, compared to a known activator), a humoral innate immune mechanism, whereas E2 itself did not cause complement activation. Surface display of CpG on PEGylated E2 nanoparticles was shown specifically to increase cellular uptake by antigen-presenting cells.

Fluorescently labeled E2 was shown to drain preferentially to the lymph nodes after subcutaneous administration, and surface PEGylation allowed further diffusion to more distal locations and blood-draining organs. In contrast, surface display of CpG caused increased proximal lymph node accumulation and demonstrated superior retention, with ∼10-fold increase in LN fluorescence after 48 hr over the other nanoparticles. Within the lymph nodes, ∼50% of the nanoparticles were associated with antigen-presenting cells, including DCs.

Altogether, our results demonstrate the potential of the E2 protein nanoparticle as a versatile virus-mimicking immunomodulatory cancer vaccine platform. We have developed a nanoparticle biomaterial for DC targeting, lymph node retention, and superior induction of CTL-mediated responses against cancer.

Comment: The vertebrate immune system comprises a dizzying array of interconnected processes, reflecting the similarly huge range of methods by which pathogens have attempted to evade it over millions of years of evolution. Our understanding of these processes and their interactions remains sorely incomplete; for evidence, one need only look at the range of adjuvants required to render many vaccines therapeutically potent, often with little or no mechanistic explanation. In general, approaches that engage the system holistically—recreating as many different features of a natural infection as possible, if perhaps in deliberately exaggerated ways—appear to be more effective than those that rely on a narrower mode of action. E2 is an engineered protein, derived from a bacterial pyruvate dehydrogenase complex, which self-assembles into a 60-unit cage capable of encapsulating a range of small molecules. The resulting nanoparticle is of a similar size and composition to a virion and, as this work demonstrates, can trigger a greatly enhanced functional immune response when compared to the same antigens presented in free solution. Excessively strong immune reactions to a pathogen can themselves be dangerous to the host, a phenomenon observed in patients treated with soluble tumor necrosis factor (TNF) receptor apheresis, ¹¹ but in the case of cancer this is definitely a “problem we'd like to have.”

Non-Linear Laser Wave–Mixing Detection for Capillary Electrophoresis and Multi-Channel Arrays for Biomedical and Environmental Applications

Eric Maxwell, MS, San Diego State University

Degenerate four-wave mixing is demonstrated as a highly sensitive non-linear spectroscopic detection method for small-molecule and protein biomedical and environmental targets. This is achieved through refractive index change within an absorbing liquid medium, which produces a laser-like signal beam. This signal has high spatial resolution and may be collected with high efficiency against a nearly 100% dark background. A cubic dependence on laser power and a square dependence on analyte concentration allow for high signal intensity in trace analysis applications. In this work, the degenerate four-wave mixing technique is coupled with capillary electrophoresis, immunoprecipitation, or color-forming reactions to provide specificity. Coupling with multi-channel capillary arrays has also been explored as a means of high-throughput sample analysis. The veterinary drugs malachite green and crystal violet are shown to be detectable at concentrations as low as 6.9 × 10⁻¹⁰ M (2.5 × 10⁻¹⁹ mol) and 8.3 × 10⁻¹¹ M (3.0 × 10⁻²⁰ mol), respectively (S/N = 2). Capillary electrophoresis is used in conjunction with a two-laser degenerate four-wave mixing detector to allow simultaneous identification of both analytes. For another small-molecule target, ammonium nitrate, sample preparation with diphenylamine is used to produce a colored compound capable of absorbing 635-nm light. This explosive component is of significant forensics interest due to its use in the manufacture of improvised explosive devices. The limit of detection for ammonium nitrate in 1-mm ID capillary cells is determined to be 1.5 × 10⁻⁹ M (5.2 × 10⁻¹⁸ mol). The detection limit in a 1.5-mm thin-film cell is found to be 3.2 × 10⁻⁷ M (2.0 × 10⁻¹⁵ mol) for S/N = 2. In addition to small-molecule environmental targets, a detector for the cancer biomarker carcinoembryonic antigen is demonstrated with a combination of magnetic immunoprecipitation, multi-channel capillary arrays, and degenerate four-wave mixing. Isolated protein samples are reacted with bicinchoninic acid (BCA) to yield a colored product, and absorbance of a 532-nm laser produces a wave-mixing signal. Multiple samples may be processed rapidly though computer-controlled positioning of the multi-channel capillary array. The limit of detection for carcinoembryonic antigen is determined to be 3.3 × 10⁻¹² M (0.59 ng/mL), with a corresponding mass detection limit of 1.2 × 10⁻²¹ mol (0.22 fg) for S/N = 2.

Comment: Detecting tumors as early as possible in their development is crucial to successful treatment. For many cancers (notably excluding lung), detection at Stage I corresponds to a 5-year survival chance of close to 100%, whereas detection at Stage III implies 5-year survival no better than 60% and often closer to 20%. Unfortunately, Stage I cancers are normally asymptomatic and too small to be detected by current imaging strategies. Consequently much effort is being dedicated to the identification of blood-borne biomarkers—abnormal molecules (or abnormal levels thereof) that indicate the presence of an early-stage tumor somewhere in the body. ¹² A fundamental challenge of this approach is the extremely low concentrations at which such changes manifest; one model from 2011 indicated that a typical tumor can grow for around a decade, reaching a size of ∼25 mm and a population of around 2 billion cells, before its shedding of a well-established biomarker becomes substantial enough to register in serum using contemporary techniques. ¹³ Clearly, new and improved analytical methods will be required alongside the discovery of additional biomarkers for early detection to take its place at the forefront of cancer prevention. The technique of degenerate four-wave mixing explored in this study is among the most promising candidates, with detection limits already comparable to those achievable via the much better-studied (and more optimized) approach of laser-induced fluorescence spectroscopy.

Optogenetically Engineered Chemokine Receptors in T Cell Migration and Cancer Immunotherapy

Yuexin Xu, PhD, University of Rochester

Chemokine receptors are promising therapeutic targets because chemokine-mediated cell migration plays key roles in mediating leukocyte differentiation, survival, and tissue-specific homing. However, chemokine-sensing mechanisms are largely unknown due to the lack of proper tools to control and measure chemokine signals in live animals. Therapeutically, effective trafficking of the cytotoxic T lymphocytes (CTLs) to tumor sites is one of the main barriers to achieving successful melanoma remission. Local induction of chemokine signaling to bring more CTLs to a tumor site induces tumor cell survival, proliferation, and angiogenesis and promotes tumor metastasis. To address these issues, we developed a strategy for optically controlling chemokine-mediated T cell trafficking. The intracellular loops of Gαt-coupled rhodopsin were replaced with those of the Gi-coupled chemokine receptor CXCR4. Photoactivatable CXCR4 (PA-CXCR4) transmitted intracellular CXCR4 signals in response to 505-nm light. Local activation of PA-CXCR4 induced T cell polarization and directional migration ("phototaxis") both in vitro and in vivo. Stimulating mouse ear melanomas with light was sufficient to recruit PA-CXCR4–expressing tumor-targeting CTLs and improved the efficacy of adoptive T cell transfer immunotherapy, with a significant reduction in tumor growth. These findings suggest that the use of photoactivatable chemokine receptors allows remotely controlled leukocyte trafficking with outstanding spatial resolution in tissue and may be feasible in other cell transfer therapies.

Comment: This exciting thesis illustrates how the burgeoning field of optogenetics is employing the precision of modern optical technology to introduce unprecedented spatial and temporal control into biological systems. T lymphocytes can be highly effective in destroying transformed cells, and so, of course, a growing cancer quickly adapts to repel them by every means available. In adoptive T cell immunotherapy, it is typical for only 1% of the transferred lymphocytes to be found in the tumor. Even those that are appropriately localized are often confined to the tumor periphery and blocked from accessing the more clinically significant core regions by some combination of dysregulated adhesion factors, mutant signaling molecules, and the sheer bulk of intervening cells. Here, the common structural–functional architecture of the G protein–coupled receptors is exploited to place a potent T cell migration stimulus, CXCR4 signaling, under rhodopsin-mediated optical control. Cells that have been modified in this way migrate with enthusiasm toward increasingly intense light, thus obviating those defenses of the tumor that rely on misdirecting or expelling the arriving immune cells. Although applied here in the most accessible case, melanoma, there is no reason for the fundamental technique to be limited to the skin. Endoscopes can be used to deliver light to much of the body, while even in dense tissue implanted “beacon” microdevices could in principle perform a similar role. We greatly look forward to the application of similar methods to directing the migration and behavior of stem and progenitor cells, or the growth of axons in the central nervous system. Optically guided tissue regeneration represents a flexible and powerful solution to the problem of reconstructing complex biological macrostructures under conditions wildly different from those in utero.

Polymeric Micelles for Combination Drug Delivery in Cancer Therapy

Sang Zen, PhD, University of Wisconsin–Madison

Single small-molecule drug therapy is often hindered by drug resistance that develops in cancer cells. Delivery of small interfering RNA (siRNA) to cells via drug delivery systems (DDS) is one effective approach to silence these resistance genes, often successfully re-sensitizing cancer cells to anti-cancer drugs. Polymeric nanoparticles have been widely explored to protect and deliver siRNA therapeutically. Combination delivery of siRNA and chemotherapeutic drugs is effective against different molecular targets and can increase the sensitization of cancer cells to chemotherapy, thereby overcoming drug resistance.

A three-layer (trilayer) polymeric micelle system based on the self-association of the triblock co-polymer poly(ethylene glycol)-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl] aspartamide}-b-poly([varepsilon]-caprolactone) (PEG-b-PAsp[DET]-b-PCL) was synthesized to deliver rapamycin (RAP) and siRNA targeting the Y-box binding protein-1 (siYB-1). The trilayer micelle is composed of (1) a hydrophilic poly(ethylene glycol) (PEG) block constituting the outer layer to improve pharmacokinetics, (2) an intermediate compartment composed of the cationic poly{2-[(2-aminoethyl)amino] ethyl aspartamide} (PAsp[DET]) segment for interacting with siYB-1, and (3) an inner hydrophobic poly([varepsilon]-caprolactone) (PCL) compartment for encapsulation of RAP. PEG and PCL are both approved by the Food and Drug Administration (FDA), and PAsp(DET) is a non-toxic pH-responsive cationic poly(amino acid)-based polymer. We showed that PCL can encapsulate RAP with high loading efficiencies, and PAsp(DET) interacts with siRNA for efficient transfection/knockdown with negligible cytotoxicity. Enhanced therapeutic efficacy of RAP/siYB-1 micelles was demonstrated in cell cultures and in a PC3 xenograft nude mouse model of human prostate cancer.

We also developed a micelle formulation based on the self-association of PEG-b-PCL to deliver another anti-cancer drug, citral. Citral is found in the essential oils of many plants. Although it is composed of a random mixture of the two terpenoid isomers geranial (trans-citral) and neral (cis-citral), few studies have directly compared the in vivo anti-tumor properties of these isomers.

The anti-tumor properties of drug formulations were evaluated on the 4T1 xenograft mouse model. Geranial was found to be the more potent isomer of citral, and western blotting of tumor tissues confirmed that autophagy and not apoptosis was the major mechanism of tumor growth inhibition in p53-null 4T1 cells.

Comment: Multi-drug resistance (MDR) is one of the most challenging phenotypes exhibited by cancer and a major obstacle to the effectiveness of chemotherapy. It is mediated by two adenosine triphosphate (ATP)-dependent protein pumps, P-glycoprotein and multi-drug resistance protein (MRP), which evolved to actively expel a wide range of harmful foreign substances from the interior of cells (often directly into the excretory pathway, as in the intestinal endothelium or the proximal tubule of the kidney). In cancer, one or both of these proteins usually becomes highly up-regulated in response to the selection pressure imposed by cytotoxic chemotherapy. P-glycoprotein in particular is already elevated in many stem cell populations, and there is now convincing evidence that the functional transporter can be disseminated in microparticles to nearby cells, making it unfortunately easy for any given cancer to develop the phenotype. Although the significance of these proteins has been recognized since the late 1980s/early 1990s, they are intrinsically difficult to target with small-molecule inhibitors, and clinical trial results to date have been disappointing. In this study, RNA interference is employed to first knock down levels of Y-box-binding protein-1, a key activator of P-glycoprotein transcription, rendering the cells vulnerable to conventional drug therapy delivered subsequently by the same compound micelle. Because siRNAs are not exported by either pump, this mechanism works even in the presence of established resistance. Of course YB-1 is not the sole activator of P-glycoprotein/MRP transcription, and it seems likely that clinically relevant tumors treated as described would soon find other means to reactivate drug resistance. Employing multiple siRNA species (or other agents with similar function ^14,15 ) simultaneously to target other activators, or the pump genes themselves, might however suffice to shut down MDR long enough to achieve complete remission.

Progerin Expression Disrupts Critical Adult Stem Cell Functions That Mediate Tissue Repair Primarily Through a Mechanism That Permanently Farnesylates Lamin

Laurin Pacheco, PhD, University of Miami

Vascular disease is a leading cause of death worldwide. Vascular repair, essential for tissue maintenance, is critically reduced during vascular disease and aging. Efficient vascular repair requires functional adult stem cells unimpaired by aging or mutation.

One protein candidate for reducing stem cell–mediated vascular repair is progerin, a truncated and permanently farnesylated splice variant of lamin A. Progerin expression significantly increases during aging and is detected in aged, atherosclerotic tissues. Mutations triggering progerin over-expression cause the premature aging disorder Hutchinson–Gilford progeria syndrome (HGPS), in which patients die at approximately 13 years of age due to atherosclerosis. Non-specific farnesylation inhibitors are currently in clinical trials for treatment of HGPS.

Progerin affects tissues rich in cells derived from mesenchymal stromal cells (MSCs). It has been hypothesized that progerin promotes atherosclerosis by disrupting stem cell functions. Studies using heterogeneous, multipotent MSCs have led to discrepant results. To understand the mechanisms of progerin expression better, we examined exogenous progerin effects using a well-defined, immature, homogeneous subpopulation of MSCs, marrow-isolated adult multilineage-inducible (MIAMI) cells.

Progerin decreased cellular proliferation, migration, and self-renewal gene expression. Progerin increased abnormal nuclear morphology, membrane rigidity, and glycolysis. Progerin selectively altered protein expression and subcellular organization, and cytokine secretion; effects on differentiation were inconsistent. Inhibiting farnesylation ameliorated progerin effects on nuclear morphology, proliferation, gene expression, migration, and membrane rigidity, with distinct consequences on cytokine secretion. These results identify novel mechanisms by which progerin alters critical stem cell functions required for tissue repair, offering promising treatment targets for future therapies.

Comment: Although caution is always required in interpreting the symptoms of “premature aging” diseases, there is increasing evidence that the pathogenic mechanism in Hutchinson–Gilford progeria syndrome (HGPS) does reflect the acceleration of a process that also contributes to normal aging. Lamin A is an essential component of the lamina, the protein scaffold that supports and regulates the function of the nuclear membrane. Around 80% of HGPS patients have a point mutation that constitutively activates a cryptic splice site in the lamin A gene, resulting in the truncated form—progerin—which fails to integrate properly into the lamina, remaining attached to the nuclear rim and disrupting the overall structure of the nucleus. Nuclei burdened with significant amounts of progerin become visibly deformed and functionally impaired. Importantly, the splice site in question is not completely inactive even in its wild-type form, implying that low levels of progerin are produced in normal cells; there is also evidence that its expression is elevated in the presence of telomere damage. ¹⁶ Nematodes show almost organism-wide accumulation of lamin changes similar to those seen in HGPS over their life span, arguing for a deeply conserved role in aging. However, until recently there was no convincing mechanism for how the low levels detectable in naturally aged human tissue could contribute to systemic aging pathologies. The stem cell–specific toxicity elucidated in this work provides exactly that explanation. Devising a method for removing accumulated progerin from aged nuclei will be challenging, given its inaccessible location (certainly it is much easier to target waste found in the lysosomal compartment ^{17 –19} or the extracellular space ²⁰ ) and its structural similarity to the abundant lamin A—but it is now clear that this is a challenge we must prepare to meet.

Remote Actuation of Magnetic Nanoparticles in Breast Cancer Cells

Philise Williams, PhD, University of Nebraska Medical Center

There is a great need to develop deliver chemotherapeutics to their target sites within the body and specifically in the treatment of breast cancer. The development of a nanoscale method of mechanical organelle disruption for remote actuation of cell death holds vast possibilities for biomedical nanotechnology. Though many stimuli-based drug delivery systems exist, magnetic nanoparticles have garnered significant interest. In this study, a magnetic nanoparticle- (MNP) based system has been designed with polymer coatings that allow efficient cellular uptake. To accomplish this, polymers were designed on the basis of previous cellular uptake and accumulation results. Polymers were then used to coat magnetic nanoparticle cores and were evaluated in breast cancer cells to determine the best coating for uptake into cancerous cells. After selection of polymer coating, remote actuation of these MNPs by super-low-frequency alternating current (AC) fields allows for cytoskeletal disruption, leading to subsequent cell death without perceptible cellular hyperthermia. This novel technology is also not contingent upon ligand targeting and therefore has allowed us to induce significant cell death in triple-negative breast cancer (TNBC). This cancer currently lacks adequate treatment strategies due to its lack of an effective drug target. Confocal microscopy of BT474 cells, a ductal carcinoma breast cancer, showed disruption of the cytoskeleton as well as activation of apoptosis. These observations suggest that magnetic nanoparticles can serve as a cytotoxic agent in the absence of chemotherapy and that polymer coating can promote uptake of magnetic nanoparticles to cancer cells that were previously rendered as “non-targetable” as well as other types of breast cancer. Additionally, cell death is not seen with the same treatment in breast tissue cells, indicating some level of selectivity of treatment.

Overall, the present study enhanced the understanding of basic principles of magnetic nanoparticle design, formulation, and use in cancer chemotherapy. These particles were found to kill breast cancer cells selectively after remote actuation of internalized particles, and this work could have translational impact in the way breast cancer, and more specifically triple-negative breast cancer, is treated.

Comment: Cancer is perhaps the most difficult disease to treat by biological means, since its great potential for evolutionary adaptation allows almost any distinctive marker or vital metabolic process to be discarded or tweaked to circumvent the effects of a given therapeutic. More straightforwardly physical approaches, having less dependency on the molecular properties of the transformed cells, are thus highly attractive. Although surgical resection has always been a crucial tool in the oncologist's arsenal, it is limited to those scenarios where the cancer mass is well defined and where its removal will not itself constitute a fatal injury. For cancers buried deeply inside the body, achieving access to the target at all may be too dangerous—although remarkable progress is being made in the area of robotic keyhole surgery. There is consequently great interest in “remote-controlled” means of triggering cell death within the body. Magnetic nanoparticles have already been used in this context to induce localized heating within a tissue mass, yet that technique carries an inevitable risk of collateral damage to nearby healthy cells—especially for tumors with a more diffuse boundary (conversely, it does offer the ability to target the less evasive tumor stroma, frequently home to cancer-promoting senescent cells ²¹ ). In this work, a much lower-frequency field is used to agitate similar particles, disrupting delicate cellular structures without generating significant heat. The use of a polymer coating to target cancerous cells specifically is, of course, still vulnerable to mutagenic escape. In this context, it is interesting to note that the magnetic focusing of nanoparticles to locations deep within the body has recently been demonstrated, ²² raising hopes that, with sufficient improvements in imaging technology, a wholly physical (and thus mutation status-agnostic) cancer therapy may one day be realized.