Stepping Up Summer Fun: the Cancer Research - Scholarship and Training Experience in Population Sciences (C-STEPS) Program.

Over the last two decades, strides in cancer prevention, earlier detection, and novel treatments have reduced overall cancer mortality; however, cancer health disparities (CHD) persist among demographically diverse and intersecting populations. The development of a culturally responsive workforce trained in interdisciplinary, team-based science is a key strategy for addressing these cancer disparities. The Cancer Research - Scholarship and Training Experience in Population Sciences (C-STEPS) program at the University of New Mexico Comprehensive Cancer Center is designed to increase and diversify the biomedical and behavioral research workforce by providing specialized and experiential curricula that highlight team-oriented cancer control and population science. Undergraduate students interested in CHD and in pursuing STEM-H (science, technology, engineering, mathematics, and health) graduate or professional degrees are eligible for the program. C-STEPS students are paired with a UNM faculty mentor, who guides the student's 10-week summer research experience. They receive mentorship and support from three layers-faculty, near-peers (graduate students), and peers (undergraduates who have completed the C-STEPS program previously). Students generate five products, including a capstone presentation, grounded in the research they conduct with their faculty mentors. Since its founding in 2021, C-STEPS has trained three cohorts with a total of 32 students. The C-STEPS program provides a unique team-science approach with multilayer mentoring to create a sustainable pipeline for the development of students interested in STEM-H fields and CHD research. The capstone project led to 47% of students presenting their work at conferences, and two publishing their manuscripts in peer-reviewed journals. Overall, 89% of students were either "satisfied" or "very satisfied" with the program and the same percentage recommended the program to other undergraduates.

Bone marrow homing and engraftment of human hematopoietic stem and progenitor cells is mediated by a polarized membrane domain.

Manipulation of hematopoietic stem/progenitor cells (HSPCs) ex vivo is of clinical importance for stem cell expansion and gene therapy applications. However, most cultured HSPCs are actively cycling, and show a homing and engraftment defect compared with the predominantly quiescent noncultured HSPCs. We previously showed that HSPCs make contact with osteoblasts in vitro via a polarized membrane domain enriched in adhesion molecules such as tetraspanins. Here we show that increased cell cycling during ex vivo culture of HSPCs resulted in disruption of this membrane domain, as evidenced by disruption of polarity of the tetraspanin CD82. Chemical disruption or antibody-mediated blocking of CD82 on noncultured HSPCs resulted in decreased stromal cell adhesion, homing, and engraftment in nonobese diabetic/severe combined immunodeficiency IL-2γ(null) (NSG) mice compared with HSPCs with an intact domain. Most leukemic blasts were actively cycling and correspondingly displayed a loss of domain polarity and decreased homing in NSG mice compared with normal HSPCs. We conclude that quiescent cells, unlike actively cycling cells, display a polarized membrane domain enriched in tetraspanins that mediates homing and engraftment, providing a mechanistic explanation for the homing/engraftment defect of cycling cells and a potential new therapeutic target to enhance engraftment.

Therapeutic plasma exchange is feasible and tolerable in severely injured patients with trauma-induced coagulopathy.

Objectives: Trauma-induced coagulopathy (TIC) occurs in a subset of severely injured trauma patients. Despite having achieved surgical hemostasis, these individuals can have persistent bleeding, clotting, or both in conjunction with deranged coagulation parameters and typically require transfusion support with plasma, platelets, and/or cryoprecipitate. Due to the multifactorial nature of TIC, targeted interventions usually do not have significant clinical benefits. Therapeutic plasma exchange (TPE) is a non-specific modality of removing and replacing a patient's plasma in a euvolemic manner that can temporarily normalize coagulation parameters and remove deleterious substances, and may be beneficial in such patients with TIC. Methods: In a prospective case series, TPE was performed in severely injured trauma patients diagnosed with TIC and transfusion requirement. These individuals all underwent a series of at least 3 TPE procedures performed once daily with plasma as the exclusive replacement fluid. Demographic, injury, laboratory, TPE, and outcome data were collected and analyzed. Results: In total, 7 patients received 23 TPE procedures. All patients had marked improvements in routine coagulation parameters, platelet counts, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activities, inflammatory markers including interleukin-6 concentrations, and organ system injuries after completion of their TPE treatments. All-cause mortality rates at 1 day, 7 days, and 30 days were 0%, 0%, and 43%, respectively, and all patients for whom TPE was initiated within 24 hours after injury survived to the 30-day timepoint. Surgical, critical care, and apheresis nursing personnel who were surveyed were universally positive about the utilization of TPE in this patient population. These procedures were tolerated well with the most common adverse event being laboratory-diagnosed hypocalcemia. Conclusion: TPE is feasible and tolerable in severely injured trauma patients with TIC. However, many questions remain regarding the application of TPE for these critically ill patients including identification of the optimal injured population, ideal time of treatment initiation, appropriate treatment intensity, and concurrent use of adjunctive treatments. Level of evidence: Level V.

Application of Biophysical Techniques to Cellular and Molecular Oncology.

Dysregulated cellular processes drive malignant transformation, tumor progression, and metastasis, and affect responses to therapies [...].

Photoactivatable mCherry for high-resolution two-color fluorescence microscopy.

The reliance of modern microscopy techniques on photoactivatable fluorescent proteins prompted development of mCherry variants that are initially dark but become red fluorescent after violet-light irradiation. Using ensemble and single-molecule characteristics as selection criteria, we developed PAmCherry1 with excitation/emission maxima at 564/595 nm. Compared to other monomeric red photoactivatable proteins, it has faster maturation, better pH stability, faster photoactivation, higher photoactivation contrast and better photostability. Lack of green fluorescence and single-molecule behavior make monomeric PAmCherry1 a preferred tag for two-color diffraction-limited photoactivation imaging and for super-resolution techniques such as one- and two-color photoactivated localization microscopy (PALM). We performed PALM imaging using PAmCherry1-tagged transferrin receptor expressed alone or with photoactivatable GFP-tagged clathrin light chain. Pair correlation and cluster analyses of the resulting PALM images identified < or =200 nm clusters of transferrin receptor and clathrin light chain at < or =25 nm resolution and confirmed the utility of PAmCherry1 as an intracellular probe.

Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure.

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.

High-precision estimation of emitter positions using Bayesian grouping of localizations.

Single-molecule localization microscopy super-resolution methods rely on stochastic blinking/binding events, which often occur multiple times from each emitter over the course of data acquisition. Typically, the blinking/binding events from each emitter are treated as independent events, without an attempt to assign them to a particular emitter. Here, we describe a Bayesian method of inferring the positions of the tagged molecules by exploring the possible grouping and combination of localizations from multiple blinking/binding events. The results are position estimates of the tagged molecules that have improved localization precision and facilitate nanoscale structural insights. The Bayesian framework uses the localization precisions to learn the statistical distribution of the number of blinking/binding events per emitter and infer the number and position of emitters. We demonstrate the method on a range of synthetic data with various emitter densities, DNA origami constructs and biological structures using DNA-PAINT and dSTORM data. We show that under some experimental conditions it is possible to achieve sub-nanometer precision.

High-density mapping of single-molecule trajectories with photoactivated localization microscopy.

We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of molecules, sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.

Acute myeloid leukemia: Therapy resistance and a potential role for tetraspanin membrane scaffolds.

Acute myeloid leukemia (AML) is characterized by the disruption of myeloid differentiation and accumulation of blast cells in the bone marrow. While AML patients respond favorably to induction chemotherapy, long-term outcomes remain poor due to a high rate of chemoresistance. Advances with targeted therapies, which can be used in combination with conventional chemotherapy, have expanded therapeutic options for patients. However, remission is often short-lived and followed by disease relapse and drug resistance. Therefore, there is a substantial need to improve treatment options by identifying novel molecular and cellular targets that regulate AML chemosensitivity. Membrane scaffolds such as the tetraspanin family of proteins often serve as signaling mediators, translating extracellular signaling cues into intracellular signaling cascades. In this review, we discuss the conventional and targeted treatment strategies for AML and review chemoresistance mechanisms with a focus on the tetraspanin family of membrane scaffold proteins.

Tetraspanin CD82 regulates S1PR1-mediated hematopoietic stem and progenitor cell mobilization.

Hematopoietic stem and progenitor cell (HSPC) mobilization into the blood occurs under normal physiological conditions and is stimulated in the clinic to enable the isolation of HSPCs for transplantation therapies. In the present study, we identify the tetraspanin CD82 as a novel regulator of HSPC mobilization. Using a global CD82 knockout (CD82KO) mouse, we measure enhanced HSPC mobilization after granulocyte-colony stimulating factor (G-CSF) or AMD3100 treatment, which we find is promoted by increased surface expression of the sphingosine 1-phosphate receptor 1 (S1PR1) on CD82KO HSPCs. Additionally, we identify a disruption in S1PR1 internalization in CD82-deficient HSPCs, suggesting that CD82 plays a critical role in S1PR1 surface regulation. Finally, combining AMD3100 and anti-CD82 treatments, we detect enhanced mobilization of mouse HSPCs and human CD34+ cells in animal models. Together, these data provide evidence that CD82 is an important regulator of HSPC mobilization and suggests exploiting the CD82 scaffold as a therapeutic target to enhance stem cell isolation.

Canonical NF-κB Promotes Lung Epithelial Cell Tumour Growth by Downregulating the Metastasis Suppressor CD82 and Enhancing Epithelial-to-Mesenchymal Cell Transition.

BACKGROUND: The development of non-small cell lung cancer (NSCLC) involves the progressive accumulation of genetic and epigenetic changes. These include somatic oncogenic KRAS and EGFR mutations and inactivating TP53 tumour suppressor mutations, leading to activation of canonical NF-κB. However, the mechanism(s) by which canonical NF-κB contributes to NSCLC is still under investigation. METHODS: Human NSCLC cells were used to knock-down RelA/p65 (RelA/p65KD) and investigate its impact on cell growth, and its mechanism of action by employing RNA-seq analysis, qPCR, immunoblotting, immunohistochemistry, immunofluorescence and functional assays. RESULTS: RelA/p65KD reduced the proliferation and tumour growth of human NSCLC cells grown in vivo as xenografts in immune-compromised mice. RNA-seq analysis identified canonical NF-κB targets mediating its tumour promoting function. RelA/p65KD resulted in the upregulation of the metastasis suppressor CD82/KAI1/TSPAN27 and downregulation of the proto-oncogene ROS1, and LGR6 involved in Wnt/ß-catenin signalling. Immunohistochemical and bioinformatics analysis of human NSCLC samples showed that CD82 loss correlated with malignancy. RelA/p65KD suppressed cell migration and epithelial-to-mesenchymal cell transition (EMT), mediated, in part, by CD82/KAI1, through integrin-mediated signalling involving the mitogenic ERK, Akt1 and Rac1 proteins. CONCLUSIONS: Canonical NF-κB signalling promotes NSCLC, in part, by downregulating the metastasis suppressor CD82/KAI1 which inhibits cell migration, EMT and tumour growth.

Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes.

Accurate determination of the relative positions of proteins within localized regions of the cell is essential for understanding their biological function. Although fluorescent fusion proteins are targeted with molecular precision, the position of these genetically expressed reporters is usually known only to the resolution of conventional optics ( approximately 200 nm). Here, we report the use of two-color photoactivated localization microscopy (PALM) to determine the ultrastructural relationship between different proteins fused to spectrally distinct photoactivatable fluorescent proteins (PA-FPs). The nonperturbative incorporation of these endogenous tags facilitates an imaging resolution in whole, fixed cells of approximately 20-30 nm at acquisition times of 5-30 min. We apply the technique to image different pairs of proteins assembled in adhesion complexes, the central attachment points between the cytoskeleton and the substrate in migrating cells. For several pairs, we find that proteins that seem colocalized when viewed by conventional optics are resolved as distinct interlocking nano-aggregates when imaged via PALM. The simplicity, minimal invasiveness, resolution, and speed of the technique all suggest its potential to directly visualize molecular interactions within cellular structures at the nanometer scale.

Tetraspanin Scaffold Proteins Function as Key Regulators of Hematopoietic Stem Cells.

Hematopoietic stem and progenitor cells (HSPCs) are responsible for the development, maintenance, and regeneration of all the blood forming cells in the body, and as such, are critical for a number of patient therapies. For successful HSPC transplantation, stem cells must traffic through the blood and home to the bone marrow (BM) microenvironment or "niche," which is composed of soluble factors, matrix proteins, and supportive cells. HSPC adhesion to, and signaling with, cellular and extracellular components of the niche provide instructional cues to balance stem cell self-renewal and differentiation. In this review, we will explore the regulation of these stem cell properties with a focus on the tetraspanin family of membrane proteins. Tetraspanins are molecular scaffolds that uniquely function to distribute proteins into highly organized microdomains comprising adhesion, signaling, and adaptor proteins. As such, tetraspanins contribute to many aspects of cell physiology as mediators of cell adhesion, trafficking, and signaling. We will summarize the many reports that identify tetraspanins as markers of specific HSPC populations. Moreover, we will discuss the various studies establishing the functional importance of tetraspanins in the regulation of essential HSPC processes including quiescence, migration, and niche adhesion. When taken together, studies outlined in this review suggest that several tetraspanins may serve as potential targets to modulate HSPC interactions with the BM niche, ultimately impacting future HSPC therapies.

Tetraspanin CD82 drives acute myeloid leukemia chemoresistance by modulating protein kinase C alpha and ß1 integrin activation.

A principal challenge in treating acute myeloid leukemia (AML) is chemotherapy refractory disease. As such, there remains a critical need to identify key regulators of chemotherapy resistance in AML. In this study, we demonstrate that the membrane scaffold, CD82, contributes to the chemoresistant phenotype of AML. Using an RNA-seq approach, we identified the increased expression of the tetraspanin family member, CD82, in response to the chemotherapeutic, daunorubicin. Analysis of the TARGET and BEAT AML databases identifies a correlation between CD82 expression and overall survival of AML patients. Moreover, using a combination of cell lines and patient samples, we find that CD82 overexpression results in significantly reduced cell death in response to chemotherapy. Investigation of the mechanism by which CD82 promotes AML survival in response to chemotherapy identified a crucial role for enhanced protein kinase c alpha (PKCα) signaling and downstream activation of the ß1 integrin. In addition, analysis of ß1 integrin clustering by super-resolution imaging demonstrates that CD82 expression promotes the formation of dense ß1 integrin membrane clusters. Lastly, evaluation of survival signaling following daunorubicin treatment identified robust activation of p38 mitogen-activated protein kinase (MAPK) downstream of PKCα and ß1 integrin signaling when CD82 is overexpressed. Together, these data propose a mechanism where CD82 promotes chemoresistance by increasing PKCα activation and downstream activation/clustering of ß1 integrin, leading to AML cell survival via activation of p38 MAPK. These observations suggest that the CD82-PKCα signaling axis may be a potential therapeutic target for attenuating chemoresistance signaling in AML.

Establishment and characterization of OS 99-1, a cell line derived from a highly aggressive primary human osteosarcoma.

Osteosarcoma is the most common form of primary bone cancer. In this study, we established a human osteosarcoma cell line (OS 99-1) from a highly aggressive primary tumor. G-banding karyotype analysis demonstrated a large number of clonal abnormalities, as well as extensive intercellular heterogeneity. Through the use of immunologic, molecular, and biochemical analyses, we characterized protein and gene expression profiles confirming the osteogenic nature of the cells. Further evaluation indicated that OS 99-1 cells maintain the capacity to differentiate in an in vitro mineralization assay as well as form tumors in the in vivo chicken embryo model. This cell line provides a useful tool to investigate the molecular mechanisms contributing to osteosarcoma and may have the potential to serve as a culture system for studies involving bone physiology.

Ovarian Tumor Microenvironment Signaling: Convergence on the Rac1 GTPase.

The tumor microenvironment for epithelial ovarian cancer is complex and rich in bioactive molecules that modulate cell-cell interactions and stimulate numerous signal transduction cascades. These signals ultimately modulate all aspects of tumor behavior including progression, metastasis and therapeutic response. Many of the signaling pathways converge on the small GTPase Ras-related C3 botulinum toxin substrate (Rac)1. In addition to regulating actin cytoskeleton remodeling necessary for tumor cell adhesion, migration and invasion, Rac1 through its downstream effectors, regulates cancer cell survival, tumor angiogenesis, phenotypic plasticity, quiescence, and resistance to therapeutics. In this review we discuss evidence for Rac1 activation within the ovarian tumor microenvironment, mechanisms of Rac1 dysregulation as they apply to ovarian cancer, and the potential benefits of targeting aberrant Rac1 activity in this disease. The potential for Rac1 contribution to extraperitoneal dissemination of ovarian cancer is addressed.

The tetraspanin CD82 regulates bone marrow homing and engraftment of hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cell (HSPC) transplantation represents a treatment option for patients with malignant and nonmalignant hematological diseases. Initial steps in transplantation involve the bone marrow homing and engraftment of peripheral blood-injected HSPCs. In recent work, we identified the tetraspanin CD82 as a potential regulator of HSPC homing to the bone marrow, although its mechanism remains unclear. In the present study, using a CD82 knockout (CD82KO) mouse model, we determined that CD82 modulates HSPC bone marrow maintenance, homing, and engraftment. Bone marrow characterization identified a significant decrease in the number of long-term hematopoietic stem cells in the CD82KO mice, which we linked to cell cycle activation and reduced stem cell quiescence. Additionally, we demonstrate that CD82 deficiency disrupts bone marrow homing and engraftment, with in vitro analysis identifying further defects in migration and cell spreading. Moreover, we find that the CD82KO HSPC homing defect is due at least in part to the hyperactivation of Rac1, as Rac1 inhibition rescues homing capacity. Together, these data provide evidence that CD82 is an important regulator of HSPC bone marrow maintenance, homing, and engraftment and suggest exploiting the CD82 scaffold as a therapeutic target for improved efficacy of stem cell transplants.

Tetraspanins Function as Regulators of Cellular Signaling.

Tetraspanins are molecular scaffolds that distribute proteins into highly organized microdomains consisting of adhesion, signaling, and adaptor proteins. Many reports have identified interactions between tetraspanins and signaling molecules, finding unique downstream cellular consequences. In this review, we will explore these interactions as well as the specific cellular responses to signal activation, focusing on tetraspanin regulation of adhesion-mediated (integrins/FAK), receptor-mediated (EGFR, TNF-α, c-Met, c-Kit), and intracellular signaling (PKC, PI4K, ß-catenin). Additionally, we will summarize our current understanding for how tetraspanin post-translational modifications (palmitoylation, N-linked glycosylation, and ubiquitination) can regulate signal propagation. Many of the studies outlined in this review suggest that tetraspanins offer a potential therapeutic target to modulate aberrant signal transduction pathways that directly impact a host of cellular behaviors and disease states.

Chronic lymphocytic leukemia with clinical debut as neurological involvement: a rare phenomenon and the need for better predictive markers.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries. The frequency of symptomatic central nervous system (CNS) involvement is unknown but thought to be a rare phenomenon. Currently there are no known risk factors for CNS involvement. CASE PRESENTATION: We describe a clinically staged low-risk CLL case that presented with symptomatic CNS involvement and progressed rapidly to death. Evaluation of the surface adhesion molecules identified a markedly altered expression pattern of the integrin, CD49d, and the tetraspanin, CD82, in the index case when compared to similar low-risk CLL cases. We found that the early Rai clinical stage CLL patients showed linear correlation for the co-expression of CD82 and CD49d. In contrast, this unique index case with CNS involvement, which has the same Rai clinical stage, had a significantly lower expression of CD82 and higher expression of CD49d. CONCLUSIONS: These data suggest that the expression profile of CD49d and CD82 may represent potential biomarkers for patients with increased propensity of CNS involvement. Moreover, this study illustrates the critical need for a better mechanistic understanding of how specific adhesion proteins regulate the interactions between CLL cells and various tissue sites.

Low-affinity binding in cis to P2Y2R mediates force-dependent integrin activation during hantavirus infection.

Pathogenic hantaviruses bind to the plexin-semaphorin-integrin (PSI) domain of inactive, ß3 integrins. Previous studies have implicated a cognate cis interaction between the bent conformation ß5/ß3 integrins and an arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2Y2R. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant αIIbß3 integrins and (RGD)P2Y2R expressed on cell membranes. Mutation of the RGD sequence to RGE in the P2Y2R removes this interaction. Binding of inactivated and fluorescently labeled Sin Nombre virus (SNV) to the integrin PSI domain stimulates higher affinity for (RGD)P2Y2R on cells, as measured by an increase in the unbinding force. In CHO cells, stably expressing αIIbß3 integrins, virus engagement at the integrin PSI domain, recapitulates physiologic activation of the integrin as indicated by staining with the activation-specific mAB PAC1. The data also show that blocking of the Gα13 protein from binding to the cytoplasmic domain of the ß3 integrin prevents outside-in signaling and infection. We propose that the cis interaction with P2Y2R provides allosteric resistance to the membrane-normal motion associated with the switchblade model of integrin activation, where the development of tensile force yields physiological integrin activation.