Abstract
Theses reviewed in this issue include “A Rapid Lipid-Based Approach for Normalization of Quantum Dot-Detected Biomarker Expression on Extracellular Vesicles in Complex Biological Samples,” “Cancer Dynamics Under a Chemotherapeutic Stress Gradient Using a Microfluidic In Vitro Tumor Environment,” “Cells Exhibiting Strong P16ink4a Promoter Activation In Vivo Display Features of Senescence,” “HLA-G Dimer Prolongs Kidney Allograft Survival by Inhibiting CD8+ T Cell Activation and Granzyme B Expression,” “Lysophosphatidic Acid Suppression of CD8 T Cell Signaling and Function,” and “Targeting Therapeutic T Cells to the Bone Marrow Niche.”
In this column we continue the series, begun in issue 10(1), of surveys highlighting a small selection of recently completed doctoral theses with particular relevance to the fields covered by Rejuvenation Research. Although it has become common for thesis work to appear in the general academic literature, it remains valuable to scan the thesis databases for important advances that one might otherwise overlook.
Footnotes
A Rapid Lipid-Based Approach for Normalization of Quantum Dot-Detected Biomarker Expression on Extracellular Vesicles in Complex Biological Samples
Meryl Rodrigues, PhD, Arizona State University
Extracellular vesicles (EVs), particularly exosomes, are of considerable interest as tumor biomarkers since tumor-derived EVs contain a broad array of information about tumor pathophysiology including its metabolic and metastatic status. However, current EV based assays cannot distinguish between EV biomarker changes by altered secretion of EVs during diseased conditions like cancer, inflammation, etc. that express a constant level of a given biomarker, stable secretion of EVs with altered biomarker expression, or a combination of these two factors.
This issue was addressed by developing a nanoparticle and dye-based fluorescent immunoassay that can distinguish among these possibilities by normalizing EV biomarker level(s) to EV abundance, revealing average expression levels of EV biomarker under observation. In this approach, EVs are captured from complex samples (e.g., serum), stained with a lipophilic dye and hybridized with antibody-conjugated quantum dot probes for specific EV surface biomarkers. EV dye signal is used to quantify EV abundance and normalize EV surface biomarker expression levels.
EVs from malignant (PANC-1) and nonmalignant pancreatic cell lines (HPNE) exhibited similar staining, and probe-to-dye ratios did not change with EV abundance, allowing direct analysis of normalized EV biomarker expression without a separate EV quantification step. This EV biomarker normalization approach markedly improved the ability of serum levels of two pancreatic cancer biomarkers, EV EpCAM, and EV EphA2, to discriminate pancreatic cancer patients from nonmalignant control subjects.
The streamlined workflow and robust results of this assay are suitable for rapid translation to clinical applications and its flexible design permits it to be rapidly adapted to quantitate other EV biomarkers by the simple swapping of the antibody-conjugated quantum dot probes for those that recognize a different disease-specific EV biomarker utilizing a workflow that is suitable for rapid clinical translation.
It was not until 1971 that a study of the flagellate alga Ochromonas danica (in which the modern term “extracellular vesicle” was also coined, amid a bewildering array of other terminology in use at the time) convincingly demonstrated the release of EVs, as now defined, from healthy cell membranes. A comprehensive review published in the same year collated findings from the preceding decade to establish that the phenomenon seemingly extended far beyond unicellular organisms—a conclusion backed up in a growing range of in vitro and in vivo scenarios by further publications throughout the 1970s.
Despite that evidence of their significance, the study of EVs remained a somewhat niche interest until the turn of the millennium, hampered by the considerable technical difficulties in isolating and convincingly characterizing such tiny crumbs of life. The tone of this study changed markedly in 2006, when several laboratories successively reported not only the presence of parent cell-specific RNA in EVs, but also their capacity in at least some cases to transfer it in a functional form to recipient cells—a largely unanticipated mode of intercellular communication qualitatively distinct from classical receptor/ligand signaling, and one correctly anticipated to have clinical relevance most crucially in cancer.
Over the past 15 years the field has exploded, attracting hundreds of millions of dollars of funding and laying the foundations for numerous diagnostic and therapeutic applications. Despite this, the methodological challenges that have dogged the field for half a century remain obstacles to efficient progress. This thesis addresses one of the most prominent of those obstacles, through the introduction (and initial clinical validation) of an elegant and widely applicable assay for previously indistinguishable simultaneous changes in EV quantity and composition.
Ke-Chih Lin, PhD, Princeton University
In this dissertation, we use the microfluidic cancer-on-chip system we have developed to explore cancer population dynamics and how cancer acquires drug resistance. The microfluidic cell culture device, the “evolution accelerator” (EA), generates an in vitro landscape of stress heterogeneity across a tumor population. The system allows for high-magnification real-time observations of different cancer cell lines and downstream analysis of cell phenotype as a function of position on the stress landscape. With the EA technology, we investigate the adaptation and evolution dynamics in prostate cancer cell metapopulations under a stress landscape of a chemotherapeutic drug (docetaxel). High-resolution time-lapse scanning provides abundant information about the change in cell morphology, population dynamics, cell motility and cell migration over time on a cellular level.
We further implement this technology to study quantitatively the emergence of polyploid, mesenchymal and stem-like cancer cells in the context of complex heterogeneous yet controllable in vitro environments with a spatially-varying drug concentration. Within our microfluidic stress landscape, we observe: (1) a previously-unobserved surprisingly large number of polyploid giant cancer cells (PGCCs) which emerged in a highly stressful region in response to chemotherapy; (2) the transition of the epithelial to the mesenchymal state; (3) the stem-like characteristics of PGCCs.
We argue that the elevated emergence of PGCCs in a high drug environment is due to migration of diploid epithelial cells from regions of low drug concentration, where they proliferate, to regions of high drug concentration, where they rapidly convert to PGCCs. The coexistence of the emerging drug-resistance PGCCs and the altruistic proliferative diploid cells may serve as a survival strategy for the cancer population. This suggests the clinical value of identifying vulnerabilities of PGCCs that might be considered critical targets.
Finally, we present a microfluidic device, the static diffuser, which, unlike earlier work using continuous flows, generates a long-term chemical gradient within tumor microenvironment based on a static diffusion mechanism. Due to the simplicity of the experimental setup, the system allows not only well-controlled continuous microscopic studies of the interaction among various cell types, but also parallel experimentation for up to 18X time-resolved downstream cellular assays. As a proof of concept, we report the co-culture of human bone marrow stromal cell line (HS-5) and bone-metastatic prostate cancer cell line (PC3) using the static diffuser.
Taken together, the experimental platform and cancer studies presented in this dissertation show the power of sophisticated in vitro environments to enable the discovery of new pathways and mechanisms underlying evolution of drug resistance in cancer.
The actuators of the process by which the metastatic phenotype arises in vivo is still unclear, but recent studies have placed heavy suspicion on a formerly underemphasized subpopulation of cancerous cells—the polyploid giants. (The term “polyaneuploid” has very recently been suggested to distinguish these cells from naturally polyploid cells, including the early blastomere and mature osteoclasts, emphasizing that essentially all cancers are initially aneuploid—thus their “polyploidy” is relative to an aneuploid baseline, a distinction of great biological importance.)
Polyploid giant cancer cells (PGCCs), which as the name suggests are enlarged cells bearing either multiple nuclei or a single giant nucleus containing multiple complete genomes, have actually been noted in clinical specimens for over a century. Historically associated in particular with virally induced cancers, they have now been identified in malignancies arising from virtually any class of genotoxic stress, against which they are exceedingly resilient. Two major classes of mechanisms have been proposed for the genetic amplification observed, to wit endoreplication—through failure of some postinterphase stage of mitosis, or reactivation of the earliest embryonic program—and multicellular assembly, through cell fusion or phagocytosis of neighboring cells (such as, perhaps, the diploid cells observed in this dissertation).
It is entirely possible that multiple routes are relevant in vivo, and work such as that described in this thesis is required to establish their relative contributions. Studies of PGCCs were initially confounded in part by the strong requirement for stressful conditions not merely to initiate but also to sustain their phenotype; most such cells become dormant in the context of typical in vitro colony formation assays, giving the false impression that they are not still viable and metabolically active.
Polyploidy confers very obvious potential advantages on the cells involved, particularly by complementing deleterious mutations—thus increasing resistance to most chemo- and radiotherapeutic techniques, certainly by comparison with the doses calibrated as sufficient for nonpolyploid cells—and by enabling a facile transition to a quiescent or reversibly senescent state.
It is well established that PGCCs can give rise to progeny through the reversal of the polyploid state (a process that again echoes early embryonic development). Although it is unclear what proportion of their progeny are viable, those that do survive are highly enriched in characteristics of metastasis—crucially including an enhanced capacity for PGCC reformation. Thus these cells provide, in effect, a facultative switch between low-mutation proliferative growth and high-mutation stress-resistant adaptation.
That capacity, plus their tendency toward inactivity in vitro, may now explain a great deal of the disconnection between bench and bedside results in trials of antimetastatic agents; indeed, it is beginning to seem that therapeutically targeting PGCCs may be the single most promising strategy to combat metastasis in the general case. Of course, their resilience and genetic flexibility render this inherently onerous; however, one intriguing idea is to target polyploidy itself, perhaps through the use of a synthetic biology construct that induces cell death by a bio-orthogonal mechanism in cases wherein the copy number of selected genetic sequences exceeds the physiological maximum.
Jie-Yu Liu, PhD, The University of North Carolina at Chapel Hill
The activation of cellular senescence throughout the lifespan promotes tumor suppression, whereas the persistence of senescent cells contributes to aspects of aging. This theory has been limited, however, by an inability to identify and isolate individual senescent cells within an intact organism. Toward that end, we generated a murine reporter strain by “knocking-in” a fluorochrome, tandem-dimer Tomato (tdTom), into exon 1α of the endogenous p16INK4a locus. We used this allele (p16tdTom) for the enumeration, isolation and characterization of individual p16INK4a -expressing cells (tdTom+).
The half-life of the knocked-in transcript was shorter than that of the endogenous p16INK4a mRNA, and therefore reporter expression better correlated with p16INK4a promoter activation than p16INK4a transcript abundance. The frequency of tdTom+ cells increased with serial passage in cultured murine embryonic fibroblasts from p16tdTom/+ mice. In adult mice, tdTom+ cells could be readily detected at low frequency in many tissues, and the frequency of these cells increased with aging.
Using an in vivo model of peritoneal inflammation, we compared the phenotype of cells with or without activation of p16INK4a and found that tdTom+ macrophages exhibited some features of senescence, including reduced proliferation, senescence associated beta-galactosidase (SA-β-gal) activation and increased mRNA expression of a subset of transcripts encoding SA-secretory phenotype (SASP) phenotype. These results indicate that cells harboring activation of the p16INK4a promoter accumulate with aging and inflammation in vivo, and display characteristics of senescence.
Critically, the depletion of p16INK4a-expressing cells was shown a decade ago to rapidly reverse numerous aspects of age-related decline (initially in a progeroid mouse model, but subsequently also in wild-type strains), whilst the gene's locus has been associated with human age-related diseases in multiple genome-wide association studies. Accordingly, research into cellular senescence and its remediation is now proceeding at breakneck speed. We highlight this particular dissertation as the first demonstration of a system capable of identifying senescence in vivo down to the single-cell level; although not directly translatable to clinical use, this technique will without doubt be of tremendous use in assessing the effectiveness of senolytic drugs and other proposed therapeutic avenues, as well as in fundamental research exploring the drivers of natural senescence.
Ashwin Ajith, PhD, Augusta University
Solid organ transplantation is the preferred therapy for many patients diagnosed with end stage organ failure, however allograft rejection is a significant barrier for graft survival. Patient care involves heavy immunosuppressive drug treatment leading to elevated risk for cancer and other opportunistic infections. Hence there is a need to develop effective alternative approaches to minimize graft rejection. We focused on human leukocyte antigen G (HLA-G), a nonclassical HLA class Ib molecule critically involved in the maintenance of maternal tolerance to semi-allogeneic fetal tissues during pregnancy and has emerged as a potential therapeutic target to control allograft rejection.
We demonstrate here that the level of soluble HLA-G dimer was higher in a group of 90 patients with a functioning renal allograft compared with 40 patients who rejected (RJ) their transplants. The HLA-G dimer level was not affected by demographic status. One of the potential mechanisms in tissue organ allograft rejection involves the induction of granzymes and perforin, which are the main effector molecules expressed by CD8+ cytotoxic T lymphocytes and function to destroy allogeneic transplants.
Using genomics, molecular and cellular analyses of cells from T-cell–mediated RJ and nonrejected kidney transplant patients, cells from leukocyte Ig-like receptor B1 (LILRB1) transgenic mice, humanized mice, and genetically engineered HLA-G dimer, we demonstrated a novel mechanism by which HLA-G dimer inhibits activation and cytotoxic capabilities of human CD8+ T cells. This mechanism implicated the downregulation of Granzyme B expression and the essential involvement of LILRB1. Thus, HLA-G dimer has the potential to be a specific and effective therapy for prevention of allograft rejection and prolongation of graft survival.
Unlike the ubiquitous and ultrapolymorphic HLA-A and HLA-B genes involved in presenting intracellular peptide samples to the immune system, very few alleles of HLA-G exist in the human population, and its sequence incorporates modifications that enable a potent immunosuppressive function—dampening reactions to interferons, and interacting with inhibitory receptors on leukocytes to trigger their apoptosis or reprogram them into a suppressive mode. Unsurprisingly, the proposal to employ the variant to mitigate or even abolish immunological rejection of clinically transplanted tissue followed very shortly after its discovery, although it was initially unclear whether this would be an option for male as well as female patients.
Fortunately, although cell-bound HLA-G is restricted to the cells of the maternal–fetal interface (barring rather frequent spontaneous reactivation in cancers), soluble HLA-G dimers have since been identified in the blood of adults of both genders—although at higher levels in females, and with significant variation in concentrations associated with polymorphisms of the gene—suggesting the potential for universal applicability. This dissertation is exemplary of recent study associating higher soluble HLA-G with better outcomes in a growing range of transplant patients, and identifies a novel mechanism mediating that effect.
Lysophosphatidic Acid Suppression of CD8 T Cell Signaling and Function
Divij Mathew, PhD, University of Colorado Denver
Immunotherapies have demonstrated the utility of targeting the immune system for eradication of tumors, yet a majority of patients remain refractory to such treatments. Therefore, further advances in our understanding of T cell activation and inhibitory signals are required to find alternative combinational strategies to treat cancers. Lysophosphatidic acid (LPA) is a bioactive lipid that has been characterized to promote tumor growth via distinct mechanisms and thus displays multiple “hallmarks” of tumorgenesis, including enhancing tumor-promoting inflammation, metastasis, and angiogenesis.
Despite LPA receptors (LPA1-6) being expressed on all immune cells, the impact of LPA on immune cells remains unclear. Work from our lab has previously shown that LPA signals via LPA5 expressed by CD8 T cells to suppress TCR-mediated intracellular calcium mobilization. Thus, we propose that aberrant expression of LPA by diverse tumors serves also to dampen adaptive immune responses, thereby creating an immune suppressive tumor microenvironment.
Here, we demonstrate that CD8 T cells express three of the six known LPA receptors, and that LPA signaling through LPA5 on CD8+ T cells has not only a profound inhibition of Ca2+ release after TCR stimulation, but also impedes ERK and Nur77 TCR induced signaling pathways. Importantly, we now demonstrate that LPA signaling also decreases the cytolytic function of CD8 T cells by inhibiting granule exocytosis. Both adoptive transfer of Lpar5-/- CD8+ T cells and implantation of tumors into Lpar5-/- hosts leads to greater control of tumor growth, underscoring an immunosuppressive signal mediated by LPA through LPA5 on CD8 T cells.
Finally, human CD8 T cells also express the same LPA receptors as in the mouse and similarly fail to efficiently release Ca2+ upon TCR stimulation in the presence of LPA. Pharmaceutical inhibition of human LPA5 restores Ca2+ flux, suggesting a similar mechanism seen in mice. Thus, our data illuminates a novel lipid-receptor interaction that suppresses CD8 T cell function in both human and murine cells.
Under normal circumstances, LPA negatively regulates its own production, but cytokine signaling can defeat this mechanism; under sustained inflammatory conditions, LPA can accumulate to very high levels despite the ongoing action of its natural degradation pathway through the lipid phosphate phosphatase (LPP) family of extracellular phosphatases. Thanks to extensive preclinical studies, a number of agents have now been identified with the capacity to inhibit ATX activity, block LPA signaling, or enhance LPA degradation, and therapeutics of the first two classes have entered clinical trials for idiopathic pulmonary fibrosis, a classic disease of chronic inflammation. Disappointingly, no similar candidates have yet been advanced to address the elevated LPA signaling seen in malignant disease.
This dissertation highlights the importance of a distinctly separate tumor-supportive role of LPA signaling through immunosuppression, which further enhances the case for the application of such therapeutics in cancer. Although ATX inhibition will be challenging to sustain in cancer cells, with their inevitable capacity to evolve around such obstacles, the blockade of LPA receptors or the reinforcement of its degradation in tumor stroma (whether through upregulation of the normal enzymatic pathway, or through the introduction of an artificial catalyst) are highly promising potential adjuncts to future cancer therapy.
Targeting Therapeutic T Cells to the Bone Marrow Niche
Anjum Khan, PhD, University College London
Anti-cancer immunotherapies aim to mediate a specific response targeting malignant cells without accompanying damage to normal tissue associated with conventional therapies, but induction of T cell differentiation and exhaustion enables successful tumour progression. In this thesis I will explore different means of enhancing the accumulation and function of therapeutic CD8 T cells, as a means of achieving functional cure through persisting immunological memory. I will show that the key features of T cell memory can be imprinted upon CD8+ T cells by enhancing homing to specific organs, enabling privileged access to cell-mediated factors.
The interaction between the chemokine receptor CXCR4 and the ligand CXCL12/SDF-1 is required for successful homing of haematopoietic stem cells (HSCs) to stromal niches within the bone marrow (BM). The bone marrow is known to be a unique organ for immunological memory, including memory T cells. I hypothesised that replicating this bone marrow homing interaction in CD8+ T cells would preferentially generate memory T cells.
I demonstrate through in vivo imaging and flow cytometric analyses that T cells over-expressing CXCR4 accumulate preferentially in the BM near vascular-associated CXCL12+ cells, retain a less differentiated central memory phenotype despite repeated antigenic stimulation, and produce enhanced effector cytokines on restimulation. Compared to control T cells, these cells demonstrate lower expression of exhaustion and senescence markers, suggesting the capacity for long-term persistence after activation.
I go on to show that numerical accumulation and many of these functional attributes are dependent upon cell-extrinsic expression of IL-15Rα. TCXCR4 demonstrate heightened graft-versus-tumour effects in allogeneic bone marrow transplant models of B-cell lymphoma in comparison to control T cells. I provide evidence that this anti-tumour effect is mediated by enhanced functional capacity rather than numerical accumulation or out-competing immunosuppressive populations. In summary, this strategy offers a tractable means of enhancing T cell engraftment, persistence and function, with potential for cross-platform therapeutic applications including anti-cancer immunotherapy.
Conversely, memory T cells—which are far more persistent in vivo, with stable clones sometimes enduring for decades—are far more difficult to prepare in sufficient volumes for therapeutic application. Despite copious work on the topic (and in marked contrast to the now well-understood pathway followed by B cells), the lineage relationship between effector and memory T cells is still not clear, making the selective production of the latter all the more challenging.
In the linear model, exposure of naive T cells to antigen triggers a progressive differentiation toward the effector state, with the longest-lived memory cell subclasses representing the first stages after loss of naivety. The strongest evidence for this pathway arises from studies of telomere length and telomerase activity, both of which are consistently reduced in effector versus memory cells—implying that the latter have undergone fewer cycles of division.
Conversely, the circular model proposes that naive cells initially proliferate into a large clone of effector cells, a subset of which escape apoptosis after antigen withdrawal and instead developmentally regress to a more naive-like memory phenotype. This model is supported particularly by data showing that multiple genes involved in survival and homing behaviors follow the “on-off-on” expression pattern it predicts, although there is a notable lack of direct evidence for the retrodifferentiation step also required. (The prominent transcriptional and epigenetic similarities between memory cells and both the naive and effector populations have been claimed as evidence by investigators favoring either side, but do not really seem to clearly distinguish between the two models.)
Thankfully, the findings in this thesis suggest that it may be possible to bypass the continued debate over physiological developmental mechanisms during the production of therapeutic cells, and instead directly amplify their memory-like properties by encouraging their residency in the bone marrow microenvironment; excitingly, these results include promising initial evidence of improvements in both viability and therapeutic efficacy.
Acknowledgment
The author is grateful to Dr. Aubrey de Grey for his input, in April of this year, on the selection of these dissertations for discussion.