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

Cancer Autoantibody Biomarker Discovery and Validation Using Nucleic Acid Programmable Protein Array

Jie Wang, PhD, Arizona State University

Currently in the United States, many patients with cancer do not benefit from population-based screening due to challenges associated with the existing cancer screening scheme. Blood-based diagnostic assays have the potential to detect diseases in a non-invasive way. Proteins released from small early tumors may only be present intermittently and are diluted to tiny concentrations in the blood, making them difficult to use as biomarkers. However, they can induce autoantibody (AAb) responses, which can amplify the signal and persist in the blood even if the antigen is gone. Circulating autoantibodies are a promising class of molecules that have the potential to serve as early detection biomarkers for cancers. This PhD thesis aims to screen for autoantibody biomarkers for the early detection of two deadly cancers, basal-like breast cancer and lung adenocarcinoma. First, a method was developed to display proteins in both native and denatured conformations on a protein array. This method adopted a novel protein tag technology, called a HaloTag, to immobilize proteins covalently on the surface of a glass slide. The covalent attachment allowed these proteins to endure harsh treatment without becoming dissociated from the slide surface, which enabled the profiling of antibody responses against both conformational and linear epitopes. Next, a plasma screening protocol was optimized to increase significantly the signal-to-noise ratio of protein array–based AAb detection. Following this, the AAb responses in basal-like breast cancer were explored using nucleic acid programmable protein arrays (NAPPA) containing 10,000 full-length human proteins in 45 cases and 45 controls. After verification in a large sample set (145 basal-like breast cancer cases, 145 controls, 70 non-basal breast cancer) by enzyme-linked immunosorbent assay (ELISA), a 13-AAb classifier was developed to differentiate patients from controls with a sensitivity of 33% at 98% specificity. A similar approach was also applied to the lung cancer study to identify AAbs that distinguished lung cancer patients from computed tomography–positive benign pulmonary nodules (137 lung cancer cases, 127 smoker controls, 170 benign controls). In this study, two panels of AAbs were discovered that showed promising sensitivity and specificity. Six out of eight AAb targets were also found to have elevated mRNA levels in lung adenocarcinoma patients using TCGA data. These projects as a whole provide novel insights into the association between AAbs and cancer, as well as general B cell antigenicity against self-proteins.

Comment: There are two widely supported models for cancer development and progression—the clonal evolution (CE) model and the cancer stem cell (CSC) model. Briefly, the former claims that most or all cells in a tumor contribute to its maintenance; as newer and more aggressive clones develop by random mutation, they become responsible for driving growth. The range of different mutational profiles generated is assumed to be large enough to account for disease recurrence after therapy (due to rare resistant clones) and metastasis (clones arising with the ability to travel to distant sites). The CSC model instead asserts that a small number of mutated stem cells are the origin of the primary cell mass, drive metastasis through the intermittent release of undifferentiated, highly mobile progeny, and account for recurrence due to a generally quiescent metabolic profile conferring potent resistance to chemotherapy. In either case, the immunological visibility of an early tumor may be highly sporadic. Clones arising early in CE differ little in proteomic terms from healthy host cells; those that do trigger a response are unlikely to have acquired robust resistance to immune attack, so are destroyed quickly in favor of their stealthier brethren. Likewise, CSCs share some of the immune privilege of normal stem cells and, due to their inherent ability to produce differentiated progeny with distinct proteomic signatures, are partially protected from attacks on their descendants. Consequently, such well-hidden cells may remain in the body for years to decades. The ability of the immune system to retain a memory of past threats, however, provides an opportunity for clinicians to access a patient's proteomic history indirectly. Such assessment may reveal prior encounters with rapidly extinguished precancerous clones or CSC progeny, absent at the time of screening but indicative of a concealed threat. The autoantibody panel developed in this study for basal-like breast cancer exhibits exceptional specificity despite a comparatively small training set. Given its ease of application, this suggests great promise for a more exhaustively trained classifier as a population-level screening tool.

Condition-Specific Differential Sub-Network Analysis for Biological Systems

Deepali Jhamb, PhD, Indiana University

Biological systems behave differently under different conditions. Advances in sequencing technology over the last decade have led to the generation of enormous amounts of condition-specific data. However, these measurements often fail to identify low-abundance genes and proteins that can be biologically crucial. In this work, a novel text-mining system was first developed to extract condition-specific proteins from the biomedical literature. The literature-derived data was then combined with proteomics data to construct condition-specific protein interaction networks. Furthermore, an innovative condition-specific differential analysis approach was designed to identify key differences, in the form of sub-networks, between any two given biological systems.

The framework developed here was implemented to understand the differences between limb regeneration-competent Ambystoma mexicanum and regeneration-deficient Xenopus laevis. This study provides an exhaustive systems-level analysis to compare regeneration competent and deficient sub-networks to show how different molecular entities inter-connect with each other and are rewired during the formation of an accumulation blastema in regenerating axolotl limbs. This study also demonstrates the importance of literature-derived knowledge, specific to limb regeneration, to augment the systems biology analysis. Our findings show that although the proteins might be common between the two given biological conditions, they can have a high dissimilarity based on their biological and topological properties in the sub-network. The knowledge gained from the distinguishing features of limb regeneration in amphibians can be used in future to induce regeneration chemically in mammalian systems.

The approach developed in this dissertation is scalable and adaptable to understanding differential sub-networks between any two biological systems. This methodology will not only facilitate the understanding of biological processes and molecular functions that govern a given system, but will also provide novel intuitions about the pathophysiology of diseases/conditions.

Comment: We have long advocated a principle of directly comparing young and old bodies as a means to identify the classes of physical damage that accumulate in the body during aging. This approach circumvents our ignorance of the full etiology of each particular disease manifestation, a phenomenally difficult question given the ethical issues of experimenting on human subjects, the lengthy “incubation time” of aging-related diseases, and the complex inter-connections between their risk factors—innate and environmental. Repairing such damage has the potential to prevent pathology before symptoms appear, an approach now becoming increasingly mainstream. ¹¹ However, a naïve comparison faces a number of difficulties, even given a sufficiently large sample set to compensate for inter-individual variation. Most importantly, the causal significance of a given species cannot be reliably determined from its simple prevalence. ¹² The catalytic nature of cell biology means that those entities whose abundance changes the most profoundly in absolute terms are quite unlikely to be the drivers of that change and may even spontaneously revert to baseline levels in the absence of on-going stimulation. Meanwhile, functionality is often heavily influenced independently of abundance by post-translational modifications that may escape direct detection. Sub-network analysis uses computational means to identify groups of genes and/or proteins that vary in a synchronized way with some parameter, indicating functional connectivity. The application of methods such as those developed here to the comparison of a wide range of younger and older conditions will facilitate the identification of processes—not merely individual factors—that are impaired with age, and thus will help greatly in identifying the optimal points for intervention.

Development of a Light Actuated Drug Delivery-on-Demand System

Chase Linsley, PhD, University of California, Los Angeles

The need for temporal–spatial control over the release of biologically active molecules has motivated efforts to engineer novel drug delivery-on-demand strategies actuated via light irradiation. Many systems, however, have been limited to in vitro proof-of-concept due to biocompatibility issues with the photo-responsive moieties or the light wavelength, intensity, and duration. To overcome these limitations, the objective of this dissertation was to design a light-actuated drug delivery-on-demand strategy that uses biocompatible chromophores and safe wavelengths of light, thereby advancing the clinical prospects of light-actuated drug delivery-on-demand systems. This was achieved by: (1) Characterizing the photothermal response of biocompatible visible light and near-infrared-responsive chromophores and demonstrating the feasibility and functionality of the light actuated on-demand drug delivery system in vitro; and (2) designing a modular drug delivery-on-demand system that could control the release of biologically active molecules over an extended period of time.

Three biocompatible chromophores—Cardiogreen, Methylene Blue, and riboflavin—were identified and demonstrated significant photothermal response upon exposure to near-infrared and visible light, and the amount of temperature change was dependent upon light intensity, wavelength, as well as chromophore concentration. As a proof-of-concept, pulsatile release of a model protein from a thermally responsive delivery vehicle fabricated from poly(N-isopropylacrylamide) was achieved over 4 days by loading the delivery vehicle with Cardiogreen and irradiating with near-infrared light. To extend the useful lifetime of the light-actuated drug delivery-on-demand system, a modular, reservoir-valve system was designed. Using poly(ethylene glycol) as a reservoir for model small molecule drugs combined with a poly(N-isopropylacrylamide) valve spiked with chromophore-loaded liposomes, pulsatile release was achieved over 7 days upon light irradiation. Ultimately, this drug delivery strategy has potential for clinical applications that require explicit control over the presentation of biologically active molecules. Further research into the design and fabrication of novel biocompatible thermally responsive delivery vehicles will aid in the advancement of the light-actuated drug delivery-on-demand strategy described here.

Comment: Our combined comments on this thesis and the next one appear after the next abstract.

Light-Inducible Gene Regulation in Mammalian Cells

Lauren Toth, PhD, Duke University

The growing complexity of scientific research demands further development of advanced gene regulation systems. For instance, the ultimate goal of tissue engineering is to develop constructs that functionally and morphologically resemble the native tissue they are expected to replace. This requires patterning of gene expression and control of cellular phenotype within the tissue-engineered construct. In the field of synthetic biology, gene circuits are engineered to elucidate mechanisms of gene regulation and predict the behavior of more complex systems. Such systems require robust gene switches that can quickly turn gene expression on or off. Similarly, basic science requires precise genetic control to perturb genetic pathways or understand gene function. Additionally, gene therapy strives to replace or repair genes that are responsible for disease. The safety and efficacy of such therapies require control of when and where the delivered gene is expressed in vivo.

Unfortunately, these fields are limited by the lack of gene regulation systems that enable both robust and flexible cellular control. Most current gene regulation systems do not allow for the manipulation of gene expression that is spatially defined, temporally controlled, reversible, and repeatable. Rather, they provide incomplete control that forces the user to choose to control gene expression in either space or time, and whether the system will be reversible or irreversible.

The recent emergence of the field of optogenetics—the ability to control gene expression using light—has made it possible to regulate gene expression with spatial, temporal, and dynamic control. Light-inducible systems provide the tools necessary to overcome the limitations of other gene regulation systems, which can be slow, imprecise, or cumbersome to work with. However, emerging light-inducible systems require further optimization to increase their efficiency, reliability, and ease of use.

Initially, we engineered a light-inducible gene regulation system that combines zinc finger protein technology and the light-inducible interaction between Arabidopsis thaliana plant proteins GIGANTEA (GI) and the light oxygen voltage (LOV) domain of FKF1. Zinc finger proteins (ZFPs) can be engineered to target almost any DNA sequence through tandem assembly of individual zinc finger domains that recognize a specific 3-bp DNA sequence. Fusion of three different ZFPs to GI (GI-ZFP) successfully targeted the fusion protein to the specific DNA target sequence of the ZFP. Due to the interaction between GI and LOV, co-expression of GI-ZFP with a fusion protein consisting of LOV fused to three copies of the VP16 transactivation domain (LOV-VP16) enabled blue-light dependent recruitment of LOV-VP16 to the ZFP target sequence. We showed that placement of three to nine copies of a ZFP target sequence upstream of a luciferase or enhanced green fluorescent protein (eGFP) transgene enabled expression of the transgene in response to blue light. Gene activation was both reversible and tunable on the basis of duration of light exposure, illumination intensity, and the number of ZFP binding sites upstream of the transgene. Gene expression could also be patterned spatially by illuminating the cell culture through photomasks containing various patterns.

Although this system was useful for controlling the expression of a transgene, for many applications it is useful to control the expression of a gene in its natural chromosomal position. Therefore, we capitalized on recent advances in programmed gene activation to engineer an optogenetic tool that could easily be targeted to new, endogenous DNA sequences without re-engineering the light inducible proteins. This approach took advantage of CRISPR/Cas9 technology, which uses a gene-specific guide RNA (gRNA) to facilitate Cas9 targeting and binding to a desired sequence, and the light-inducible heterodimerizers CRY2 and CIB1 from Arabidopsis thaliana to engineer a light-activated CRISPR/Cas9 effector (LACE) system. We fused the full-length (FL) CRY2 to the transcriptional activator VP64 (CRY2FL-VP64) and the amino-terminal fragment of CIB1 to the amino, carboxyl, or amino and carboxyl terminus of a catalytically inactive Cas9. When CRY2-VP64 and one of the CIBN/dCas9 fusion proteins are expressed with a gRNA, the CIBN/dCas9 fusion protein localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription. Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins. We achieved light-dependent activation of the IL1RN, HBG1/2, or ASCL1 genes by delivery of the LACE system and four gene-specific gRNAs per promoter region. For some gene targets, we achieved equivalent activation levels to cells that were transfected with the same gRNAs and the synthetic transcription factor dCas9-VP64. Gene activation was also shown to be reversible and repeatable through modulation of the duration of blue light exposure, and spatial patterning of gene expression was achieved using an eGFP reporter and a photomask.

Finally, we engineered a light-activated genetic “on” switch (LAGOS) that provides permanent gene expression in response to an initial dose of blue light illumination. LAGOS is a lentiviral vector that expresses a transgene only upon Cre recombinase–mediated DNA recombination. We showed that this vector, when used in conjunction with a light-inducible Cre recombinase system, could be used to express MyoD or the synthetic transcription factor VP64-MyoD in response to light in multiple mammalian cell lines, including primary mouse embryonic fibroblasts. We achieved light-mediated up-regulation of downstream myogenic markers myogenin, desmin, troponin T, and myosin heavy chains I and II as well as fusion of C3H10T1/2 cells into myotubes that resembled a skeletal muscle cell phenotype. We also demonstrated LAGOS functionality in vivo by engineering the vector to express human VEGF₁₆₅ and human ANG1 in response to light. HEK 293T cells stably expressing the LAGOS vector and transiently expressing the light-inducible Cre recombinase proteins were implanted into mouse dorsal window chambers. Mice that were illuminated with blue light had increased micro-vessel density compared to mice that were not illuminated. Analysis of human vascular endothelial growth factor (VEGF) and human ANG1 levels by enzyme-linked immunosorbent assay (ELISA) revealed statistically higher levels of VEGF and ANG1 in illuminated mice compared to non-illuminated mice.

In summary, the objective of this work was to engineer robust light-inducible gene regulation systems that can control genes and cellular fate in a spatial and temporal manner. These studies combine the rapid advances in gene targeting and activation technology with natural light-inducible plant protein interactions. Collectively, this thesis presents several optogenetic systems that are expected to facilitate the development of multicellular cell and tissue constructs for use in tissue engineering, synthetic biology, gene therapy, and basic science both in vitro and in vivo.

Comment: Although it is easy to characterize technological progress as following in the wake of scientific discoveries, the reverse is almost equally true; advances in technique open the door to types of experiment previously intractable or impossible. Such is currently the case for the field of optically controlled biotechnology, which has exploded into prominence, particularly over the last half-decade. Light of an appropriate wavelength can penetrate mammalian tissues to a depth of up to a couple of centimeters, rendering much of the living body accessible to optical study and control—still more if the detector/source is integrated into an endoscopic or fiber optic probe. Techniques borrowed from the semiconductor industry allow patterns of illumination to be controlled down to the nanometer scale, ideal for addressing individual cells. The highly controlled time course of such experiments, as compared to traditional means of gene activation, such as the addition of a chemical agent to the medium, eliminates confounding variables, and simplifies data analysis. Furthermore, this level of immediate control opens the door to closed-loop systems where the activity of entities under optical control can be continuously tuned in relation to some parameter(s). In the first of these two illuminating theses, a vehicle is developed that permits light-driven release of a small molecule. Such a system could be employed to target a systemically administered antibiotic or anti-neoplastic agent to a site of infection or cancer while sparing other bodily tissues from toxicity. Because most modern drugs cannot be produced in the body, even given arbitrarily good control of cellular biochemistry, this technique will have lasting value in numerous clinical contexts. In the second thesis, the level of precision achieved is even more profound; the CRISPR/Cas9 system has received much recent attention ¹³ in its own right for its capacity to target arbitrary genetic sequences without an arduous protein-engineering step. The LACE system described stands to permit genetic manipulation with almost arbitrarily good spatial, temporal, and genomic site-specific control, using only means available to a typical university laboratory.

Magnetic Resonance Imaging As an Adjunct to Conventional Mammography Screening for Cancer in Dense Breast Tissue

Rachel Connett, PhD, Walden University

Diagnostic methods to effectively image dense breast tissue (DBT) can pose challenges for breast cancer screening. While conventional mammography is the gold standard for breast cancer screening, this technique has a low sensitivity to DBT and can miss about 78% of cancers in DBT, but magnetic resonance imaging (MRI) has a high sensitivity for imaging DBT and produces a smaller number of false positives. The purpose of this study was to determine the extent to which conventional mammograms can miss breast cancer in women with DBT and to determine if an adjunct method of imaging DBT might detect breast cancers that are missed by mammography alone. Quantitative data were collected from a sample of 300 randomly selected participants using surveys. SPSS statistical software was used to analyze the data with the factor analysis method. Qualitative data were collected by telephone interviews from 10 women who were patients of a breast cancer center. NVivo software was used to analyze the data with the thematic analysis method. All analyses were guided by theoretical framework of von Bertalanffy's general systems theory, Miller's living systems theory, and the theory of intelligent medical diagnosis. Key results determined that a significant number of women with DBT had breast cancer that was undetected by mammograms; results also showed that women with DBT can benefit from breast cancer screening by adding an adjunct screening method (e.g., MRI). This study may contribute to social change by making the breast cancer screening community aware of the potential benefit of adding MRI as an adjunct to conventional screening so that more breast cancers are detected in the early stages of the disease.

Comment: First applied to patients in 1930, mammography uses low-energy X-rays to examine the breast for patches of abnormal density that may be diagnostic of tumors and associated calcifications. Regular mammographic screening of asymptomatic women was historically effective in reducing cancer mortality and remains widely recommended by national health authorities, despite high rates of false positives and negatives. Yet damningly, a recent Cochrane meta-analysis of 600,000 women found that there is now no significant effect of mammographic screening per se on mortality after 10 years. ¹⁴ Furthermore, for every screened patient whose life was saved by early detection, around 10 are treated unnecessarily and more than 200 experience the stress of an initial false-positive finding. The loss of clinical value is accounted for by several factors—notably improvements in other diagnostic methods and wider public adoption of breast self-assessment, as well as new treatments that are effective later in disease progression (reducing the value of very early detection). The term “dense” refers to the non-fatty tissues of the breast (milk glands and ducts and supportive tissue). The proportion and distribution of these tissues varies naturally, and approximately 50% of women are classified as having “dense breast tissue (DBT)” at examination; this is most common in patients under the age of 50. DBT is independently associated with a slightly increased risk of breast cancer, but also closely resembles neoplastic tissue on a mammogram and can obscure it from detection. Thus, it accounts for much of the high false-positive/negative rate of conventional mammography. Breast MRI essentially eliminates these issues, but is difficult to access for many patients due to higher costs and logistical issues; portable MRI machines remain very uncommon, whereas mobile mammography clinics have been instrumental in enabling population-level screening. It is generally subsidized only for those with an elevated genetic risk of radiation injury. We are hopeful that this study, in highlighting a large group of women for whom adjunct breast MRI is of substantial diagnostic value, will encourage more widespread use of the technique in screening and ultimately drive down costs.

Targeting T Cells for the Immune-Modulation of Human Diseases

Regina Lin, PhD, Duke University

Dysregulated inflammation underlies the pathogenesis of a myriad of human diseases ranging from cancer to autoimmunity. As coordinators, executers, and sentinels of host immunity, T cells represent a compelling target population for immune-modulation. In fact, the antigen-specificity, cytotoxicity, and promise of long-lived of immune-protection make T cells ideal vehicles for cancer immunotherapy. Interventions for autoimmune disorders, on the other hand, aim to dampen T cell–mediated inflammation and promote their regulatory functions. Although significant strides have been made in targeting T cells for immune modulation, current approaches remain less than ideal and leave room for improvement. In this dissertation, I seek to improve on current T cell-targeted immunotherapies, by identifying and pre-clinically characterizing their mechanisms of action and in vivo therapeutic efficacy.

CD8⁺ cytotoxic T cells have potent anti-tumor activity and therefore are leading candidates for use in cancer immunotherapy. The application of CD8⁺ T cells for clinical use has been limited by the susceptibility of ex vivo–expanded CD8⁺ T cells to become dysfunctional in response to immunosuppressive microenvironments. To enhance the efficacy of adoptive cell transfer therapy (ACT), we established a novel microRNA (miRNA)-targeting approach that augments CTL cytotoxicity and preserves immunocompetence. Specifically, we screened for miRNAs that modulate cytotoxicity and identified miR-23a as a strong functional repressor of the transcription factor Blimp-1, which promotes CTL cytotoxicity and effector cell differentiation. In a cohort of advanced lung cancer patients, miR-23a was up-regulated in tumor-infiltrating CD8⁺ T cells, and its expression correlated with impaired anti-tumor potential of patient CD8⁺ T cells. We determined that tumor-derived transforming growth factor-β (TGF-β) directly suppresses CD8⁺ T cell immune function by elevating miR-23a and down-regulating Blimp-1. Functional blockade of miR-23a in human CD8⁺ T cells enhanced granzyme B expression; and in mice with established tumors, immunotherapy with just a small number of tumor-specific CD8⁺ T cells in which miR-23a was inhibited robustly hindered tumor progression. Together, our findings provide a miRNA-based strategy that subverts the immunosuppression of CD8⁺ T cells that is often observed during adoptive cell transfer tumor immunotherapy and identify a TGF-β-mediated tumor immune-evasion pathway

Having established that miR-23a-inhibition can enhance the quality and functional resilience of anti-tumor CD8⁺ T cells, especially within the immune-suppressive tumor microenvironment, we went on to interrogate the translational applicability of this strategy in the context of chimeric antigen receptor (CAR)-modified CD8⁺ T cells. Although CAR T cells hold immense promise for ACT, CAR T cells are not completely curative due to their in vivo functional suppression by immune barriers—such as TGF-β—within the tumor microenvironment. Because TGF-β poses a substantial immune barrier in the tumor microenvironment, we sought to investigate whether inhibiting miR-23a in CAR T cells can confer immune competence to afford enhanced tumor clearance. To this end, we retrovirally transduced wild-type and miR-23a–deficient CD8⁺ T cells with the EGFRvIII-CAR, which targets the PepvIII tumor-specific epitope expressed by glioblastomas (GBM). Our in vitro studies demonstrated that while wild-type EGFRvIII-CAR T cells were vulnerable to functional suppression by TGF-β, miR-23a abrogation rendered EGFRvIII-CAR T cells immune-resistant to TGF-β. Rigorous preclinical studies are currently underway to evaluate the efficacy of miR-23a-deficient EGFRvIII-CAR T cells for GBM immunotherapy.

Last, we explored novel immune-suppressive therapies by the biological characterization of pharmacological agents that could target T cells. Although immune-suppressive drugs are classical therapies for a wide range of autoimmune diseases, they are accompanied by severe adverse effects. This motivated our search for novel immune-suppressive agents that are efficacious and lack undesirable side effects. To this end, we explored the potential utility of subglutinol A, a natural product isolated from the endophytic fungus Fusarium subglutinans. We showed that subglutinol A exerts multimodal immune-suppressive effects on activated T cells in vitro. Subglutinol A effectively blocked T cell proliferation and survival, while profoundly inhibiting pro-inflammatory interferon-γ (IFN-γ) and interleukin-17 (IL-17) production by fully differentiated effector Th1 and Th17 cells. Our data further revealed that subglutinol A might exert its anti-inflammatory effects by exacerbating mitochondrial damage in T cells, but not in innate immune cells or fibroblasts. Additionally, we demonstrated that subglutinol A significantly reduced lymphocytic infiltration into the footpad and ameliorated footpad swelling in the mouse model of Th1-driven delayed-type hypersensitivity. These results suggest the potential of subglutinol A as a novel therapeutic for inflammatory diseases.

Comment: Immunotherapy is among the most promising approaches to cancer treatment, having the specificity and scope to selectively target transformed cells wherever they may reside within the body and the potential to install a permanent defense against disease recurrence. By the time a typical cancer is clinically diagnosed, however, it has already found means to survive a prolonged period of potential immune attack. The mechanisms by which tumors evade immune surveillance are beginning to be elucidated, ^15,16 and include both direct suppression of effector cells and progressive editing of the host's immune repertoire to disfavor future attack. It is inherently difficult to interfere with these defenses directly, due to the selection pressures in genetically heterogeneous neoplastic tissue. Much effort is thus being focused on methods for rendering therapeutically delivered immune cells resistant to their effects. The cytokine TGF-β is paradoxically known to function as both a tumor suppressor in healthy tissue and as a tumor-derived species associated with multiple cancer-promoting activities, including enhanced immune evasion. This work identifies the pathway by which TGF-β compromises cytotoxic T cell function in the tumor microenvironment, and demonstrates an effective method for blocking this signal. In many clinical cases, however, editing of the patient's immune repertoire has already removed or rendered anergic those immune cells able to recognize their cancer. Thus, the finding that blocking TGF-β signaling also appears to enhance the effectiveness of CAR-modified T cells—engineered with an antibody fragment targeting them with high affinity to a particular tumor-associated epitope—is a welcome addition to these already promising results.

Three-Dimensional Printing of Antibiotic-Laden Calcium Phosphates for Treatment of Orthopedic Implant-Associated Bone Infections

Jason Inzana, PhD, University of Rochester

Bone fracture fixation devices, such as plates and screws, account for over two-thirds of all surgical implant–associated infections. The current standard of care for chronic cases is surgical debridement of the infected tissue followed by extensive courses of systemic and local antibiotics. Local antibiotics are typically delivered via poly(methyl methacrylate) (PMMA) bone cement. However, PMMA is not biodegradable, which impairs antibiotic delivery and requires that the PMMA be removed in a follow-up surgery. Furthermore, some antibiotics are incompatible with PMMA polymerization, which constrains the surgeon's treatment choices. In this thesis, antibiotic bone graft substitutes were designed to overcome many of the limitations of PMMA and study the efficacy of local rifampin delivery for treating an implant-associated bone infection. Rifampin is a key antibiotic for implant-associated infections and is incompatible with PMMA. Three-dimensional printing processes were optimized to fabricate calcium phosphate scaffolds using modular strategies that enabled the addition of antibiotics during three-dimensional printing. These scaffolds are biocompatible, biodegradable, and were shown to facilitate bone regeneration in an aseptic mouse model of long bone healing. A new mouse model of implant-associated bone infection was developed to accommodate the clinical treatment algorithm that includes extensive surgical debridement and placement of an antimicrobial spacer. Many of the important features of implant-associated bone infections that make it a challenging clinical problem, such as biofilm formation and osseous destruction, were evident. Using this novel murine model, concomitant local delivery of rifampin and vancomycin from the three-dimensional printed scaffolds significantly reduced the bacterial load and infection-induced bone destruction compared to vancomycin-laden PMMA. Increasing the rifampin dose and extending the release through a biodegradable polymeric coating further enhanced the efficacy of the three-dimensional printed scaffolds. The fabrication advantages of three-dimensional printing enabled bone graft substitutes with superior biofunctionality to the current standard of care, whereas the high reproducibility and easy customization for medical image-guided, patient-specific therapies will facilitate future translation to larger animal models and human studies.

Comment: Despite reduced levels of physical activity, older patients are disproportionately affected by trauma-related infections—having on average lower bone density, ¹⁷ a higher risk of accidental injury ¹⁸ due to sensorimotor impairments, ¹⁹ weaker immune responses to pathogens, and an impaired wound healing process resulting from elevated systemic inflammation. In the current era of increasingly widespread antibiotic resistance, the elderly population not only faces an elevated risk of death from infection, but also acts to increase that risk for the populace as a whole, serving as unwilling incubators for new resistant strains. This situation seems unlikely to change substantially for the better before the arrival of a mature rejuvenation biotechnology able to reverse the aforementioned regenerative impairments. However, this study demonstrates that there is still plenty of room for improvement in the techniques used to manage bone infections. PMMA has been used in skeletal repair since the 1940s and is ubiquitous in modern surgical practice, having excellent tissue compatibility and mechanical properties well suited to the job of bridging between an injury site of arbitrary dimensions and a mass-manufactured implant. In the absence of a viable alternative, its downsides have been accepted as unavoidable. Yet the rapidly advancing techniques of three-dimensional printing, combined with modern imaging, offer a new opportunity to replace PMMA in many clinical contexts. Grafts shaped precisely to the patient's requirements eliminate the need for “cementing,” whereas the use of a biodegradable material throughout the implant improves healing and eliminates the risks of subsequent removal surgery. Once it is demonstrated in human trials that such implants also exceed PMMA's performance in preventing infection, little will remain in favor of the traditional method.