University Of Colorado Denver

NIH Research Projects · FY 2026 · 2023-01

Project Summary Melanoma immunotherapy with GPR182 blockade Immune checkpoint inhibitor therapy has greatly improved survival of patients with late-stage melanoma. However, over half of patients do not benefit from this therapy. One of the main hurdles is that many melanoma tissues lack effector CD8+ T cell infiltrates. Chemokines such as CXCL9 and CXCL10 play an important role in regulating effector T cell infiltration into the tumors. Atypical Chemokine Receptors are a group of GPCR proteins that are expressed in non-immune cells to actively regulate chemokines by endocytosis. Our studies uncovered GPR182 as a novel ACKR receptor selectively upregulated in peritumoral lymphatics. Our preliminary results indicated that genetic deletion of this molecule in mice led to increased effector T cell infiltration and thereby the retardment of tumor growth in several mouse melanoma models. We further found that GPR182 interacts with chemokines broadly in vitro and blockade of CXCR3 completely abolished improved antitumor immunity in GPR182- deficient mice. Here we hypothesize that GPR182 inhibits anti-tumor immune response by limiting chemokine availability and targeting this pathway offers a novel approach to converting immunologically cold melanoma to hot ones. We will dissect the molecular interaction between GPR182 and chemokines, and also examine the chemokine endocytosis by GPR182 with tumors. The mechanisms by which GPR182 inhibits antitumor T cell response will be investigated. Finally, the in vivo antitumor effect of a GPR182 monoclonal antibody, which blocks the interaction between GPR182 and chemokines, will be assessed. By the completion of these studies, we will identify a new strategy of inflaming immunologically cold melanoma and will have a better understanding of the immunomodulatory role of the lymphatics in melanoma.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY Most cellular functions are carried out by multisubunit protein complexes. Transcribe and translate as much as you want, but odds are if your protein doesn’t assemble properly with other proteins, the gene might as well be off. As the final step in expression of most genes, protein complex assembly is hugely understudied, and it has only recently become clear that the textbook model of diffusion-limited collisions between individual molecules is inadequate. Septin proteins are found as cytoskeletal filaments in many eukaryotic cells and participate in a wide variety of cellular functions. The building blocks of septin filaments are rod-shaped septin complexes composed of distinct septin subunits. Multiple septins “compete” to occupy the same position in, and confer specialized properties to, septin complexes, but it is not fully understood how specific subunits are “chosen” to assemble the functionally appropriate complexes. How individual septins occupy specific positions within complexes is one of the oldest questions in septin biology. Disease-causing septin mutations highlight the importance of answering this question. Our lab is among the leaders in this field and in the next five years we want to use the powerful tools we have developed in budding yeast to address specific knowledge gaps. For other cytoskeletal proteins, achieving the conformation competent for complex assembly requires help from molecular chaperones. How does chaperone-assisted de novo septin folding fit into the pathway of septin complex assembly? Our recent work established the step-wise pathway of septin hetero-octamer assembly, and identified septin-interacting chaperones that engage a septin-septin interaction interface and are necessary for efficient septin folding. We will use a combination of genetics, cell biology and biochemistry to determine how chaperone action sets the stage for septin-septin encounters during complex assembly. Recent studies show that assembly of many complexes occurs co-translationally, and we find chaperone requirements for efficient septin translation. To what extent is septin complex assembly co-translational? We will investigate septin- chaperone and septin-septin interactions in the context of active translation. We previously identified key residues in septin-septin interaction interfaces that mediate “partner recognition” during assembly and dictate the subunit composition within complexes. An enduring mystery is how the two subunits at the “ends” of yeast septin hetero-octamers always match. How do allosteric conformational changes across septin-septin interaction interfaces direct the specificity of septin complex subunit composition? We will determine the mechanistic basis of this phenomenon and determine the phenotypic consequences of inappropriate “mixing” of septin subunits within complexes. Finally, it is not known how, once made, a septin complex is remodeled to incorporate new subunits during cellular differentiation. We will determine the molecular mechanism by which yeast septin complexes are remodeling during gametogenesis. These studies will provide valuable insights into how cells assemble and modify septin complexes, with broader implications for multisubunit assembly in general.

NIH Research Projects · FY 2025 · 2022-12

Project Summary Changes to synaptic architecture underlie the cellular basis for learning and memory and impaired synaptic function and plasticity are observed in numerous brain diseases and disorders including epilepsy, autism, and schizophrenia. The synthesis and precise delivery of AMPA-type glutamate receptors (AMPARs) to the postsynaptic membrane is a critical mechanism for proper basal synaptic function and plasticity. How new receptors are delivered to specific synapses and how receptor trafficking is influenced by neural activity remain important questions. A major limitation for addressing these questions is the current reliance on non- physiological, overexpressed receptors to study neuronal synaptic protein trafficking pathways. To overcome this hurdle, I have developed and validated a new toolkit that allows me to label endogenous AMPARs and control their trafficking. I propose to leverage these new tools to investigate where, when, and how new AMPARs are delivered to specific synapses under basal conditions and following diverse forms of synaptic plasticity.

NIH Research Projects · FY 2026 · 2022-12

PROJECT SUMMARY The Neuroscience Colorado Research Experience (N-CORE) is a summer internship and educational program that gives undergraduate students from historically underrepresented backgrounds (URM) authentic neuroscience research experiences in state-of-the-art biomedical research labs at the University of Colorado Anschutz Medical School Campus (CU Anschutz). N-CORE's overarching goal is to cultivate a better understanding of neuroscience and biomedical research, engender scientific enthusiasm, and promote retention in scientific fields. N-CORE accomplishes this through a summer program that combines high- quality mentored research experiences, educational programming, professional development opportunities, and longitudinal mentoring. N-CORE interns receive instruction on core neuroscience concepts, scientific rigor and reproducibility, scientific communication, and professional development. At the end of the summer internship, all interns present their research at a campus-wide Undergraduate Research Symposium. Emphasis will be placed on ensuring an inclusive environment that fosters diversity in education and research and all Program faculty mentors will receive training in mentoring diverse students. After the internship, intern alumni are automatically enrolled in our Pre-doctoral Mentorship Program, where they will receive customized guidance on profession development and career opportunities. N-CORE brings together the expertise, enthusiasm, and institutional support to succeed in its goals. The N-CORE Program Directors bring over a decade of experience in managing a summer research program for URM undergraduate students and have built a strong alliance with the CU Anschutz Neuroscience Graduate Program (NGP), an NIH-supported T32 training program. Critically, N-CORE also brings on-board NGP graduate and MD/PhD students in the leadership and operations of N-CORE. These students will help interns quickly integrate into the CU Anschutz research community. In this proposal, we seek to build on our collective past successes and expertise to create a well-supported, sustainable summer research experience program for URM undergraduate students that will foster a better understanding of neuroscientific biomedical and clinical research and build a community for like-minded individuals interested in continuing along a scientific or medical career path.

NIH Research Projects · FY 2026 · 2022-12

PROJECT SUMMARY Cachexia is defined by abnormal loss of body weight and muscle mass that occurs secondary to chronic diseases, such as cancer. It is estimated that musculoskeletal complications affect up to 80% of patients diagnosed with cancer, dramatically impacting patient survival. Thus, there is an urgent need to develop novel treatments for cachexia-related musculoskeletal symptoms. Our recently published observations showing that cancer cachexia can present with bone loss, even in the absence of direct metastases to bone, suggest that tumor-derived soluble factors may play a critical role in the onset of such skeletal phenotype. In this regard, our preliminary findings suggest that receptor activator of NFkB ligand (RANKL), a factor involved in osteoclast- induced bone resorption, plays a causative role in cancer-associated musculoskeletal complications. In our published and preliminary studies we found that patients affected with ovarian cancer present cachexia, as well as elevated RANKL and CTX-I, a marker of bone resorption. Similarly, mice bearing ES-2 ovarian tumors present with muscle and bone loss, along with high RANKL and bone resorption, also consistent with high positivity for the osteoclast marker, TRAP, as well as dramatic osteocyte death. Contrarily, mice carrying C26 tumors, characterized by low RANKL expression, substantially maintain their bone mass, despite evidence of muscle wasting. Interestingly, myotubes exposed to recombinant RANKL undergo atrophy, similar to mice infected with AAV-RANKL or bearing C26 cells overexpressing RANKL, whereas the use of anti-RANKL neutralizing antibodies preserves myotube size in C2C12 myotubes co-cultured with ES-2 cells and counteracts bone and muscle loss in ES-2 tumor hosts. The objective of this proposal is to define the mechanisms by which RANKL-expressing tumors participate in bone and muscle loss in cachexia. Our central hypothesis is that tumor-derived RANKL participates in the activation of bone resorption, and triggers mechanisms adversely affecting muscle mass. In Aim 1, we will determine the effects of tumor-derived RANKL on bone loss in the absence of bone metastases. We hypothesize that tumor-derived RANKL directly activates bone resorption. In Aim 2, we will elucidate the mechanism(s) responsible for RANKL-induced muscle wasting. We hypothesize that activation of the RANKL/RANK-dependent pathway in skeletal muscle is sufficient to induce atrophy and exacerbate cachexia. In Aim 3, we will validate antiresorptive therapies to preserve muscle size and function in mice bearing RANKL-expressing tumors. We hypothesize that tumor-derived RANKL, along with IL-6 consequential to bone resorption, negatively impact muscle. The findings from the proposed studies will define the mechanistic effects of RANKL in cachexia and identify RANKL as a new therapeutic target for the treatment of musculoskeletal complication associated with non- metastatic RANKL-expressing cancers. These results will also open new avenues for cachexia research.

NIH Research Projects · FY 2025 · 2022-12

PROJECT SUMMARY Development of the central nervous system (CNS) requires precise intercellular ‘crosstalk’ between neural cells and non-neural cells. Fibroblasts in the meninges are a non-neural cell that substantially influences brain development, in large part via secreted factors that guide neuronal migration and neurogenesis. This is underscored by significant defects in CNS development observed in mice and humans with mutations in FOXC1, a transcription factor expressed by meningeal fibroblasts but not any neural cells. However much remains unknown about how factors from the meninges activate or inhibit molecular signaling pathways in neural cells to influence developmental processes. Filling these gaps in knowledge would significantly improve our understanding of the homeostatic function of the meninges and the pathology that occurs in neurodevelopmental disorders. Foxc1 mutants do not have normal meningeal fibroblasts over the forebrain, have increased apical progenitor self-renewal and reduced neuron production leading to neocortical lengthening, linked in part to lack of meninges derived retinoic acid. However, it is not known what aberrant signaling pathways in Foxc1 mutant apical progenitors promote increased self-renewal and how this is connected to a reduction of meninges derived factors like retinoic acid. The objective of this proposal is to investigate how meninges derived factors regulate molecular signaling to control neurogenesis in the neocortex. Using spatial transcriptomics on embryonic Foxc1- KO tissue sections, I have identified elevated Notch signaling, known to promote stem cell self-renewal, in Foxc1- KO neocortical progenitors. In Aim 1, I will use global and targeted approaches to inhibit Notch signaling in Foxc1-KO animals and test if this improves neocortical neurogenesis in these mutants. In Aim 2, I will test is meningeal derived retinoic acid or other factors produced by the meninges modulate Notch signaling in neocortical progenitors to promote neurogenesis. Results from this project will provide important insight into molecular mechanisms for meninges-brain signaling required for normal development.

NIH Research Projects · FY 2025 · 2022-12

Project Summary/Abstract Pre-clinical Rheumatoid Arthritis (RA) is a period in which systemic autoimmunity develops in the years prior to onset of clinically apparent disease. Here, we aim to define the mechanisms leading to pre-clinical autoimmunity, which could inform the design of therapies to block these pathways and prevent RA. Our group has been integral in identifying and investigating the “mucosal origins” hypothesis of RA, utilizing the collagen-induced arthritis (CIA) model to demonstrate the requirement of the microbiome for disease development. We recently identified a bacterial-derived tryptophan metabolite, indole, as a key regulator of autoimmunity in this model. Depletion of either the microbiome (using broad-spectrum antibiotics) or dietary tryptophan is protective against CIA, and supplementation with indole in either setting reverses this protection. This indole-CIA model provides, for the first time, a model in which a single bacterial metabolite is sufficient for disease development. Based on our preliminary findings that Th17 cells and inflammatory cytokine production (especially IL-6) are elevated in this indole-CIA model, I hypothesize that indole is a bioactive metabolite that promotes CIA by skewing Th17 cell differentiation and stimulating antigen presenting cells (APCs) to produce IL-6. The experiments outlined in this proposal are designed to define the effects of indole on CIA. Aim 1 will determine the effect of indole on CD4 effector T cell responses, both in the indole-CIA model and in a model of antigen-specific T cell responses utilizing OT-II transgenic mice. Aim 2 will then identify the source of and requirement for indole-mediated IL-6 production. I will utilize combined single cell RNA/ATACseq to identify the cell subsets and signaling pathways in which indole induces IL-6 production. Finally, I will test the requirement for dendritic cell (DC)-mediated IL-6 production in indole-CIA. Successful completion of these aims will (1) define the cell subsets and functions affected by indole in the indole-CIA model, which will allow for future mechanistic studies to better define and modulate this process, (2) define the role of DC-derived IL-6 in CIA, which has never been shown, and (3) generate hypotheses to later identify the signaling pathways affected by indole stimulation, which will be necessary to understand the mechanisms by which indole incites autoimmunity. Altogether, these findings will fill a critical knowledge gap regarding the role of the microbiome in the development of inflammatory arthritis. Furthermore, through these studies I will become an expert in mucosal immunology, learn to design and test rigorous hypotheses, and become proficient in cutting-edge bioinformatic techniques through analysis of the RNA/ATACseq data generated in Aim 2. The training gained through this proposal will enable me to be well- positioned for a research fellowship in rheumatology and apply skills needed for a successful K08 in the future.

NIH Research Projects · FY 2025 · 2022-12

Abstract Oral squamous cell carcinoma (OSCC) patients have a dismal survival rate due to distant metastases that escape primary care. Transforming growth factor beta (TGFβ) is a well-known driver of metastasis, modulator of immune cell activity, and regulator of extracellular matrix (ECM) genes. In breast cancer, a stiff ECM is generally attributed to excess type I collagen deposition and crosslinking that promotes cancer cell metastasis and cancer stem cell (CSC) expansion. In OSCC, a consensus has not been reached on whether a stiff or soft ECM increases metastatic potential and CSC expansion. The impact of OSCC ECM composition on immune cell trafficking is also unknown. Our laboratory has produced murine OSCC cell lines derived from Keratin15+ stem cells with Smad4 loss and KrasG12D mutation. Despite having the same genetic background, these cell lines have different metastatic ability, suggesting microenvironmental factors or cell intrinsic differences may mediate metastasis. RNAseq analysis revealed that metastatic OSCC cells have increased levels of laminins and laminin- binding integrins but downregulated type I collagen genes. Additionally, while OSCC cells cultured on stiff ECM demonstrate increased invasion, those cultured on a soft ECM display increased CSC characteristics. Treating metastatic OSCC cells with a TGFβ inhibitor reduced migration and invasion. Comprehensive immune profiling using flow cytometry revealed that metastatic tumors have decreased numbers of CD8+ T infiltrating lymphocytes (TILs) compared to non-metastatic OSCC tumors. These CD8+ TILs are instrumental for a robust anti-tumor immune response and the success of immune checkpoint inhibitor (ICI) therapy. Identifying responders to ICI and TGFβ inhibitors and sensitizing non-responders to these therapies persist as a major obstacle. Defining how OSCC cells interact with the ECM to promote their dissemination and enhancing current prognostic markers are both crucial to improve patient care and prolong survival. The goal of this proposal is to use unique murine models and human patient samples to define how ECM components and rigidity influence OSCC CSCs metastasis to improve the efficacy of emerging, targeted therapeutics to inhibit OSCC metastatic outgrowth. The hypothesis of this proposal is that elevated TGFβ signaling in OSCC induces increased integrin expression and a laminin/collagen imbalance in the ECM, altering ECM stiffness and modulating OSCC metastasis, OSCC CSCs, CD8+ TIL motility and ICI responsiveness. I will 1) evaluate TGFβ-dependency and function of laminins and associated integrins in OSCC metastasis; 2) determine how laminins contribute to ECM rigidity and subsequent impact on OSCC CSC characteristics and motility; and 3) assess if inhibiting laminin deposition enhances ICI. This study will provide insight into how enhanced laminin-binding integrin expression and laminin deposition facilitates OSCC metastasis, CSC characteristics, and CD8+ TIL exclusion. It will also identify predictive prognostic biomarkers for ICI and therapeutic targets for treating metastatic OSCC.

NIH Research Projects · FY 2025 · 2022-10

PROJECT SUMMARY The central nervous system has an intrinsic pain modulatory system that regulates nociceptive processing through descending projections from the brainstem to the spinal cord dorsal horn. The ventrolateral periaqueductal gray (vlPAG) integrates sensory information with input from higher cortical and subcortical areas, and sends projections to the rostral ventromedial medulla (RVM) that are relayed to the dorsal horn of the spinal cord. Both the vlPAG and RVM are heterogenous with respect to participating in multiple behavioral circuits. The proposed studies build on extensive previous work from the Heinricher laboratory that has defined the output from the RVM, showing that bidirectional pain control from this region is mediated by two physiologically defined cell classes, “ON-cells” and “OFF-cells,” that respectively facilitate and inhibit dorsal horn nociceptive transmission under different conditions. The Ingram laboratory has expertise studying opioid actions within the PAG and RVM, as well as adaptations in both areas with persistent inflammation. Proposed viral optogenetic strategies will map and define the vlPAG circuit that regulates RVM ON-cells involved in the facilitation of pain and elucidate underlying cellular mechanisms that shift the balance of RVM output from inhibition of pain to facilitation of pain with persistent inflammation. The combined expertise of the two laboratories will focus on identified PAG-RVM synapses using optogenetic stimulation of RVM terminals originating from the PAG. In vitro brain-slice recordings (Ingram lab) will examine the heterogeneity of PAG output to the RVM and PAG-RVM synapses, as well as cellular mechanisms of synaptic plasticity induced in persistent inflammation. These studies will use a fluorescent label for the μ-opioid receptor to differentiate presumed ON-cells from other classes in the slice to determine whether PAG terminals directly synapse on ON-cells, OFF-cells, or both, as well as what neurotransmitters are released. In vivo single-cell recording studies (Heinricher lab) will determine how inflammation-induced changes in PAG-RVM synapses control excitability of specific populations of RVM neurons and establish the link between these changes and pain behaviors. A better understanding of molecular, cellular, and circuit-level mechanisms that underlie pain is essential if we are to develop better treatments. By carefully mapping the descending projections from PAG to RVM during the development of persistent inflammation, and by tying these to defined RVM outputs and behavior, we can begin to determine the interactions in this complex network, and gain new insights into how pain-modulating systems are recruited and modulated in acute and chronic pain.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Posterior capsular opacification (PCO) is a major vision-impairment problem that emerges after cataract surgery and Nd:YAG laser posterior capsulotomy is required to restore vision. During PCO, the lens epithelial cells (LECs) that remain tethered to the anterior capsule after cataract surgery proliferate, migrate to the posterior capsule where they undergo epithelial-to-mesenchymal transition (EMT) and secrete extracellular matrix, leading to fibrous tissue formation along with wrinkling of the posterior capsule. Transforming growth factor-2 (TGFβ2) has been proposed as a major driver of EMT. We have previously demonstrated that the advanced glycation end products (AGEs) present in aged lens capsules promote the TGFβ2-mediated EMT of LECs. In our preliminary studies for this proposal, we have discovered that capsule-AGEs through binding to RAGE receptor promote senescence of LECs. We have also observed that senescent LECs promote EMT of nonsenescent LECs through paracrine mechanisms. Our data also suggest a greater synthesis of TGFβ2 in senescent cells than nonsenscent cells. Based on these observations, we propose a novel hypothesis that capsule AGEs induce senescence in LECs, which promotes the EMT of LECs during posterior capsule opacification. This hypothesis is further supported by our recent observation of senescent cells in the posterior capsules of psuedophakic human donor eyes. In Aim 1, we will test the hypothesis that lens capsule AGEs induce senescence of LECs through formation of reactive oxygen species in cells cultured on AGE- modified extracellular matrix. In Aim 2, we will test the hypothesis that senescent cells promote the EMT of human LECs through paracrine mechanisms. The secretion of TGFβ2 and IL-6 from senescent cells and their ability to induce EMT in nonsenescent cells will be investigated. In Aim 3, we will test the hypothesis that inhibition/downregulation of RAGE prevents LEC senescence and PCO-like changes in human capsular bags. Together, the proposed studies are expected to expand our understanding of the molecular mechanisms of lens fibrosis and aid in the development of novel drugs for treating PCO.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY/ABSTRACT Influenza infection causes high morbidity and mortality each year in the United States. Influenza vaccination of children is one of the most effective strategies for reducing the burden of disease among individuals and communities, yet coverage levels remain suboptimal. Hospitals are a promising setting for reaching a large number of high-risk children. Recent studies, however, indicate that many influenza vaccine-eligible children fail to receive this needed vaccine during hospitalization. Local data suggest that multimodal approaches leveraging health information technology may increase influenza vaccine uptake among hospitalized children. This project will build upon this existing evidence to establish a standardized pediatric inpatient influenza vaccination program that can be readily disseminated and successfully implemented across diverse health systems. In Aim 1 of this proposal, the study team will engage parents, nurses, providers and other key stakeholders from Seattle Children’s Hospital, Children’s Hospital Colorado, and Lurie Children’s Hospital of Chicago to create a best practice implementation guide for the inpatient influenza vaccination program. Core components will include creating a multidisciplinary leadership team, maintaining end-user engagement, utilizing novel electronic health record and data analytic tools, and enhancing evidence-based education and communication of parents, nurses, and providers. The program will be piloted at the 3 lead sites and refined as needed based upon evaluation metrics. In Aim 2, the study team will conduct a cluster randomized trial using a Sequential Multiple Assignment Randomized Trial adaptive design at 12 health systems nationally to test the effectiveness of the influenza vaccination program in improving influenza vaccination rates of hospitalized children. In the first intervention season, sites will be randomized to implement the standardized influenza vaccination program using the aforementioned implementation guide or usual care. In the second intervention season, the usual care sites and lower-performing intervention sites will be re-randomized to the standardized or intensified version of the program. The latter will include a learning collaborative, with monthly meetings of site leaders as well as one-on-one meetings with the study team to facilitate successful implementation. In the third intervention season, all sites will continue their influenza vaccination program to assess sustainability. In Aim 3, the study team will use mixed methods and the Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) framework to evaluate the program, including identification of key barriers, facilitators, and considerations for future dissemination. This information will be used to optimize and expand the program. This evidence-based program, which leverages novel clinical decision support tools in one of the most utilized electronic health record systems and optimizes hospital infrastructure, has the potential to markedly increase influenza vaccination of high-risk children. It also has broad public health implications, as the program could be readily adapted and expanded to include other needed vaccines, patient populations, and healthcare settings.

NIH Research Projects · FY 2024 · 2022-09

Project Summary Effectively treating pain while safely prescribing opioid pain medicines is a public health priority. The CDC opioid prescribing guidelines are an evidence-based approach to decreasing unnecessary opioid exposures, high-risk opioid use and abuse. The 2016 version produced modest improvements, but were sometimes misapplied for unsafe opioid tapering; highlighting the challenges of implementation and changing provider behavior. With guideline updates expected soon, there is a need for prospective trials to identify strategies to efficiently and effectively deliver CDC guideline recommended practices while documenting their impact on patient centered outcomes (e.g., pain control, morbidity and mortality). Clinical decision support (CDS) is a promising implementation strategy to both operationalize evidence-based practices and maximize the value of routinely collected data. We will use electronic health record (EHR) embedded CDS to modify clinical behavior toward CDC guideline-concordant recommendations. We propose a hybrid effectiveness-implementation trial using accepted implementation science frameworks PRISM (Practical, Robust Implementation and Sustainability Model) and RE-AIM (Reach, Effectiveness, Adoption, Implementation and Maintenance) to consider the multilevel contextual factors that influence implementation success. By developing a data-driven learning health system with the ability to evaluate patient outcomes through linkages to Prescription Drug Monitoring Program, insurance claims and death data, this project will evaluate: (1) The effectiveness of CDC guideline-concordant prescribing by describing the association between guideline concordant actions and patient outcomes. (2) The user-centered design of EHR based CDS to facilitate CDC guideline concordant actions and promote non-opioid approaches to pain management. (3) The evaluation of the implementation and effectiveness of CDS strategies to deliver guideline- concordant care in a pragmatic cluster randomized trial in a large, integrated health system. We will systematically assess key implementation outcomes and then evaluate the effectiveness of CDS on patient outcomes (vs usual care). This pragmatic research will address the need to link provider prescribing actions to individual patient outcomes. Using established implementation science approaches to evaluate CDS as an implementation strategy is innovative and important. Our results will provide robust data to document the effectiveness of CDC guideline concordant prescribing and evaluate an emerging, scalable implementation strategy using existing data to decrease morbidity and mortality from the opioid crisis.

NIH Research Projects · FY 2025 · 2022-09

Intermuscular adipose tissue (IMAT) is marbled within skeletal muscle and appears to play a key role in the age-induced risk of type 2 diabetes and sarcopenia. What is not known is how IMAT promotes decreased muscle insulin sensitivity and sarcopenia. There is a critical need to address this gap in knowledge to understand how IMAT contributes to the risk of aging-induced sarcopenia and diabetes to inform intervention strategies. The overall objective for this project is to determine the impact of aging and exercise training on IMAT secretion of fibronectin and myostatin and the cellular composition of IMAT. Our central hypothesis is that IMAT secretion of fibronectin promotes muscle insulin resistance, and IMAT secretion of myostatin promotes sarcopenia, both of which are intensified by aging and diminished by exercise. The rationale that underlies the proposed research is that clarifying the extent to which aging and exercise training alter the IMAT secretome and cell composition will inform development of interventions to modify IMAT and improve muscle mass, strength, and insulin sensitivity in older individuals. We propose two specific aims: Specific Aim 1. Determine the impact of age and exercise training on IMAT secretion of fibronectin, IMAT fibroblast composition, and the importance of fibronectin in the IMAT secretome to decrease insulin sensitivity in vitro. Preliminary data inform our working hypothesis that IMAT secretion of fibronectin increases with age due to greater fibroblast content, decreases muscle insulin sensitivity, and is attenuated after exercise training. In vitro experiments will measure the extent to which IMAT fibronectin secretion explains IMAT-induced muscle insulin resistance. We propose a coordinated effort between Colorado and Florida MoTrPAC clinical centers. Both sites will generate IMAT and subcutaneous adipose tissue conditioned media from fresh tissue, followed by conditioned media analyses and testing of its direct metabolic effects in vitro in Colorado. IMAT will also be analyzed using single nuclei RNAseq to measure cell composition. Specific Aim 2 – Evaluate the extent to which age and exercise training alter IMAT secretion of myostatin, IMAT lymphocyte composition, and the potency of myostatin in the IMAT secretome to promote sarcopenia in vitro. We hypothesize that the IMAT secretome promotes sarcopenia via myostatin signaling that increases with age due to greater IMAT lymphocyte content and is attenuated after exercise training. In vitro experiments will determine the degree to which IMAT myostatin secretion explains IMAT-induced sarcopenia outcomes. The proposed research is innovative because it represents a new and substantive departure from the status quo by testing specific IMAT secreted paracrine signals rather than clinical associations with IMAT content. These contributions will be significant by identifying the first IMAT paracrine signals impacting muscle insulin sensitivity and sarcopenia revealing IMAT as a novel target to combat aging-induced sarcopenia and metabolic dysfunction.

NIH Research Projects · FY 2025 · 2022-09

Charlotte Farewell, PHD, MPH is a behavioral health scientist whose overarching career goal is to adapt, implement and evaluate well-being promotion programs that are delivered during critical periods to promote perinatal mental health. The research she proposes entitled, “Mothers Optimizing Resources Everyday (MORE)”, aligns with numerous NIMHD research strategies including strengthening analytical models to identify critical periods of malleability that are based in life course perspectives to mitigate the intergenerational transmission of poor health, advancing understanding of how protective resources impact health, and building the science of adapting behavioral health interventions to enhance population well-being. Dr. Farewell is an Assistant Professor of Community and Behavioral Health at the Colorado School of Public Health. The proposed career development plan includes four training goals to enhance her trajectory towards becoming an independent investigator: 1. Advance expertise in latent growth modeling with a specific focus on modeling growth trajectories; 2. Gain skills in adaptation of perinatal mental health-related research and practice; 3. Enhance training in evidence-based psychological capital interventions; and 4. Increase proficiency in dissemination and implementation science and integration of care. Perinatal mood disorders are prevalent. Women who are able to acquire and maintain personal and social resources are better situated to cope with the demands associated with the transition to motherhood. Psychological capital interventions (PCIs) are evidence-based approaches that bolster personal resources and positively impact mental health and well-being. Adaptation of a multi-level PCI that seeks to foster combinations of personal and social resources across the perinatal period may serve as an important yet underexplored strategy to optimize perinatal mental health and well-being. Aim 1 will investigate point-estimates and trajectories of psychological capital across the perinatal period in 300 perinatal women. Aim 2 will identify cross-sectional and longitudinal associations between psychological capital trajectories, social resources and mental health outcomes. Aim 3 will consist of interviews, mixed methods data integration, and an iterative adaptation process, resulting in a multi-level psychosocial intervention for perinatal women. The innovation of the proposed research is threefold: 1) identification of personal and social protective factors associated with perinatal mental health outcomes, 2) application of a life course perspective and advanced longitudinal modeling to investigate the heterogeneity of psychological capital across critical, malleable periods and associations with mental health outcomes, and 3) adaptation of PCIs that offer support without pathologizing, thus increasing access to mental health supports. Interventions at the psychosocial level that focus on enhancing multi-level resources to better cope with stress may be cost-efficient and effective strategies to reduce poor mental health across the perinatal period. This project will provide necessary preliminary data for an R01 to implement and evaluate MORE delivered during specific critical periods in order to reduce the intergenerational transmission of poor health.

NIH Research Projects · FY 2025 · 2022-09

Lymphocytes must correctly localize to mount effective immune responses. To do this, lymphocytes migrate through tissue environments with very different biophysical characteristics, including extravasation from blood vessels and crawling through cell-packed or extracellular matrix-rich tissues. To navigate through these diverse environments, lymphocytes squeeze through constrictions and migrate in low- or high-adhesive environments. However, there is a key gap in the understanding of how specific cytoskeletal effectors regulate force generation, shape changes, and cell-cell or cell-matrix interactions during lymphocyte migration in different settings. Given their relevance to immune function, primary T lymphocytes are a highly significant model to investigate cytoskeletal regulation of the varying modes of amoeboid cell motility in three-dimensional (3D) environments. Formin family proteins are cytoskeletal effectors involved in mediating actin network remodeling. Formin-like-1 (FMNL1) and Diaphanous-homologue-1 (mDia1) are the two most highly expressed Formins in T cells. We recently determined that FMNL1 is required for T cell transendothelial migration (TEM) and trafficking to inflamed tissues. Interestingly, our preliminary data support that FMNL1 and mDia1 have distinct functions in T cell migration through confined environments. Our goal is to achieve a more comprehensive understanding of the mechanisms by which the cytoskeleton enables migration through diverse tissue environments by determining the mode of action of Formin proteins in T cell motility. We will investigate the mechanisms by which Formins generate mechanical forces during T cell migration, the contribution of Formins in promoting T cell nucleus passage through constrictions, and how Formins regulate T cell motility in vivo. We will also determine if FMNL1 and mDia1 act independently or in concert with the Arp2/3 complex and/or Myosin-IIA. Our hypothesis is that to promote migration through complex environments FMNL1 mediates force generation from the back of the T cell while mDia1 creates force and membrane protrusions at the cell front. To test this hypothesis, we will use a multi-faceted approach, including genetic/mutational approaches and advanced imaging techniques in complementary model systems in vitro and physiological environments in vivo. Aim 1: Determine the mechanisms by which FMNL1 and mDia1 promote T cell transendothelial migration. Aim 2: Determine how FMNL1 and mDia1 regulate T cell motility within 3D environments with diverse biophysical characteristics. Aim 3: Define how Formins regulate T cell extravasation and interstitial motility in vivo. Overall, our studies are significant in that they will advance our knowledge of T cell migration by providing new data to determine the mechanism of action of Formins in T cell motility and if they cooperate with Myosin-IIA to promote migration through environments with varied biophysical characteristics. Thus, this work has the potential to provide important insight into novel ways to therapeutically modulate lymphocyte migration, such as in autoimmune settings and inflammatory diseases.

NIH Research Projects · FY 2025 · 2022-09

Background: We seek to further our understanding of normal mammary gland biology and determine how pregnancy associated changes in the mammary gland may contribute to development of breast cancers. Though pregnancy is well-known to provide a protective effect against breast cancer risk, all women who give birth experience a transient increase in breast cancer risk following each pregnancy. The magnitude and length of this elevated risk is largely determined by the woman's age at first birth and for women over 30 at the time of first pregnancy the protective effect likely never occurs. Postpartum breast cancers (PPBC), defined as breast cancers diagnosed within 5-10 years of last childbirth, are more than twice as likely to become metastatic and result in death. This devastating diagnosis affects ~12,000 women annually. In 2006, for the first time, the number of women having children in their 30s was greater than the number under 25. Thus, PPBC cases are rising. It is thought that modern childbearing practices contribute to the increased risk for developing PPBC. We have shown that tumor cells and normal adjacent mammary epithelial cells (MECs) in PPBC patients exhibit high levels of SEMA7A expression, and SEMA7A expressing tumors are more metastatic. Thus, we believe that understanding the mechanisms underlying SEMA7A signaling in the mammary gland will lead to novel insights into aggressive PPBC. Our recent preliminary data reveal SEMA7A+ live MECs during postpartum involution suggesting that SEMA7A may promotes cell survival during postpartum glandular regression. Consistent with our hypothesis, SEMA7A promotes cell survival in cultured MECs and our preliminary data suggest a reduction in MECs, as well as lymphatic endothelial cells (LECs) and immune cell populations, in SEMA7A-/- (KO) mice. Additional data from KO mice reveal a decrease in programmed death ligand-1 (PD-L1+), a molecule that we have shown to be important for immunosuppression during involution, on mammary macrophages, LECs and MECs. As the epithelial cell apoptosis that occurs during involution likely results in presentation of self-antigens, an immune-tolerant tissue microenvironment may be necessary to prevent activation of the immune system by self-antigens and to simultaneously support survival of remaining cells in the mammary and stromal compartments. Our overarching hypothesis is that SEMA7A expression promotes cell survival and a transiently tolerant microenvironment in the mammary gland; furthermore, aberrant sustained upregulation of SEMA7A may pre-dispose women to breast malignancy. Aim1 will define the role of SEMA7A in epithelial cell survival during mammary gland involution. Aim2 will decipher SEMA7A mediated mechanisms of immune tolerance in the mammary microenvironment during postpartum involution. Relevance: To establish clinical relevance, we will also perform multi-color immunostaining on normal mammary tissues from recently lactating women.

NIH Research Projects · FY 2025 · 2022-09

Project Summary NOD1 and NOD2 are Pattern Recognition Receptors that sense fragments of bacterial peptidoglycans, and are able to detect perturbations in cellular processes such as the regulation of the actin cytoskeleton and disturbance in endoplasmic reticulum (ER) homeostasis. Under different stressful conditions, such as bacterial infections, protein misfolding and perturbations in calcium homeostasis, the ER is unable to maintain homeostasis and activates the unfolded protein response (UPR). Within the UPR three transmembrane receptors, IRE1α, PERK and ATF6, are activated and regulate biological processes such as inhibition of protein translation, autophagy, and inflammation to reestablish cellular homeostasis. NOD1 and NOD2 have been implicated in ER stress- induced inflammation, by acting downstream of IRE1α in the UPR to induce inflammatory responses. This link between the UPR and NOD1/2 signaling is of particular interest in intestinal inflammation since mutations in genes associated with the UPR (XBP1) and innate immune signaling (NOD2) have been associated with intestinal epithelial cell (IEC) dysfunction in intestinal inflammatory diseases. Salmonella Typhimurium (S. Tm) is a gram-negative bacterium that induces robust inflammation, partially dependent on NOD1/2 activation, of the intestinal epithelium resulting in gastroenteritis. As a survival mechanism, S. Tm has adapted to these inflammatory conditions in the intestinal tract by utilizing products of inflammation as a nutrient source to outcompete the resident microbiota. Considering the importance of ER stress in intestinal inflammation and the fact that S. Tm is a major cause of gastroenteritis, it is surprising that ER stress in the context of Salmonella infections is significantly underexplored. Furthermore, it is currently unknown whether S. Tm can exploit ER stress-induced inflammation resulting in luminal expansion. Our objectives are to investigate the link between ER stress and inflammation in the S. Tm-induced colitis model. Our central hypothesis is that activation of the ER stress response in the gastrointestinal tract contributes significantly to S. Tm-induced inflammation, luminal expansion and pathology. We will test our hypothesis by pursuing the following three aims. 1) Determine the contribution of calcium flux and UPR activation to NOD1/2 signaling. In in vitro experiments we will determine which branches of the UPR, in conjunction with dysregulation of calcium signaling, contributes to NOD1/2 activation. 2) Determine the role of CHOP in the S. Tm-induced colitis model. Using Chop-/- mice and conditional mutant mice we will investigate the role of CHOP in S. Tm-induced inflammation and outgrowth. 3) Determine the role of NOD1/2 and IRE1α in response to S. Tm. We will investigate the role of IRE1α in Nod1/2-/- mice in the S. Tm-induced colitis model. Characterizing the mechanisms downstream of the UPR that orchestrate ER stress-induced responses is necessary to safely modulate this process for the development of future therapeutics and will significantly increase our understanding in Salmonella pathogenesis.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Post-transcriptional mechanisms play a fundamental role in regulating gene expression at the protein level, and are frequently implicated in stress response, aging, and diseases. The goal of this project is to develop and apply multi-omics methods to examine the post-transcriptional mechanisms that regulate protein composition of multiple tissues and their ability to respond to proteostatic stressors. In recent work, our team has developed mass spectrometry and multi-omics methods that are designed to elucidate the protein isoform composition and spatiotemporal dynamics. Building on these progresses, we will focus here on the roles of three post-transcriptional mechanisms known to influence protein translation in stress response. Specifically, Aim 1 will integrate proteomics and transcriptomics data to identify the role of alternative splicing in modulating principal isoform abundance, creating alternative proteoforms, and influencing protein localization in mammalian tissues. Aim 2 will determine the differential expression, localization, and targets of RNA-binding proteins in proteostatic stress responses including paraquat in vivo as well as doxorubicin and hydrogen peroxide in vitro. Finally, Aim 3 will examine the configuration and interactome of the translation apparatus including the core ribosome and an increasing number of known ribosome-associated proteins, which have emerged as important factors that can fine-tune the translational efficiency of individual transcripts and the associated protein synthesis rates. The proposed experiments will interrogate the relationships between post-transcriptional regulation and stress response, and at the same time generate novel data sets including isoform-resolved, spatiotemporal atlases of the normal, stressed, and aged/senescent proteomes. We anticipate the results will lead to novel insights into basic cellular processes of stress response and resilience that will be relevant to studies of multiple systems.

NIH Research Projects · FY 2025 · 2022-09

Epidemiological evidence shows that paternal preconception exposures to environmental perturbations, such as stress and adverse childhood experiences (ACEs), are associated with changes in reproductive outcomes, offspring gestational development, and ultimately, offspring health and disease. Studies in animal models have implicated the germ cell transfer of small non-coding RNA (sncRNA), including miRNA and tRNA fragments, in programming these effects. We recently published our foundational work which allowed us to construct a scaffold to initially assess the composition of, and dynamic changes in, sncRNA (including miRNA, piRNA, and tRNA) in sperm samples from a young, healthy and relatively homogenous student cohort. This repeated-measures design allowed us to define in humans the between- and within-participant variation in the most abundant sperm sncRNA content over time. In addition, by utilizing complex modeling of the relationships between individual sncRNA and perceived stress states preceding each sperm donation, we were able to identify specific sncRNA responsive to the dynamics of prior stress. Ultimately, our model identified highly expressed miRNA common to all subjects, including miR-34c-5p and miR-16-5p, and three miRNA, including miR-181a-5p and let-7f-5p, that fit strict criterion for dynamic expression within- and between-subjects, and were associated with prior perceived stress. To test our hypothesis, the following Aims are provided: 1) in Aim 1 to test our current sperm sncRNA framework within a larger and more representative cohort of students, we will examine the outcomes identified in our first study, including perceived stress across 6 months of sperm collection and test the sncRNA populations for expression, variance, and responses to prior perceived stress; 2) in Aim 2 to test the additional influence of subject ACEs in our model, as one of the major influences on adult current stress perception, for effects on sperm sncRNA in low vs high ACE-exposed males; and 3) in Aim 3 to substantiate a causal importance of the sperm-associated miRNA previously identified in our model that were consistently expressed at high levels across subjects, or dynamically expressed in association with prior perceived stress within- and between- subjects. We will utilize mouse zygotic microinjection of miRNA inhibitors to specifically reduce levels of normally highly expressed sperm-associated miRNA, miR-22-3p, miR-16-5p, and miR-34c-5p, and miRNA mimics to specifically elevate individual miRNA normally lowly expressed, but dynamically environmentally responsive, miR-181a-5p, miR-4454, or let-7f-5p. Outcomes will examine the impact of these microinjections on in vitro preimplantation embryonic development, in vivo embryonic and fetal development, and transcriptomic changes by RNA sequencing of E7.5 embryos.

NIH Research Projects · FY 2025 · 2022-09

Asthma remains one of the top three causes of hospitalization in children in the United States. Interventions that rapidly reverse bronchial obstruction and decrease the need for continuous beta-agonist therapy will not only directly benefit the patient, but also decrease the consumption of acute care resources. Bilevel positive airway pressure (BPAP) is a form of non-invasive positive pressure ventilation that may stent open airways, improve mucous clearance, recruit alveoli, and increase responsiveness to continuous beta-agonist therapy in the small distal airways. However, BPAP is not currently part of the NIH guidelines for the management of pediatric acute asthma secondary to the lack of quality pediatric clinical trials. The goal of this prospective, randomized, double blind, controlled trial is to determine if early initiation of BPAP is effective and safe in pediatric patients presenting to a pediatric emergency department with acute moderate to severe asthma exacerbations who fail first line therapy.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT By the age of 3 years, Latino children are disproportionately affected by deficient sleep (short sleep duration, poorly timed sleep) and obesity. However, few studies have considered predictors of deficient sleep and its relationship with the disparate prevalence of early childhood obesity, specifically in toddlers (12 to 39 months) from Mexican American families. Because sleep is influenced by many different factors, this study will be one of the first to evaluate socioecological predictors, including environmental (e.g., societal, neighborhood, household), sociocultural (e.g., acculturation, beliefs), and parental factors (e.g., sleep-related parenting practices) in this population. While the relationship between deficient sleep and obesity has been found in children and adolescents, research with toddlers is limited by a lack of long-term studies and the use of parent report of child sleep (instead of an objective measure of sleep). Further, toddlers from Mexican American families are underrepresented in this research, despite being one of the largest growing ethnic minority populations in the U.S. The proposed study will address these knowledge gaps, answering critical questions about how different factors impact toddler sleep, and in turn whether toddler sleep contributes to the disparate prevalence of obesity in toddlers from Mexican American families. We have brought together a multidisciplinary team with substantial expertise in pediatric sleep, obesity, and socioecological contributors to health behaviors to (1) identify environmental, sociocultural, and parental factors contributing to deficient sleep, and (2) determine the relationship between sleep and weight status over a 2-year period in toddlers from Mexican American families. In order to ensure culturally-relevant measurement, we will use qualitative methods (focus groups) with Mexican American parents to adapt measures of parental sleep beliefs and sleep-related parenting practices as needed. We will then enroll 380 Mexican American families (mothers, fathers, other primary caregivers residing in the home) with 12 to 15 month old children living in a large metropolitan area to participate in 3 annual assessments. At each assessment, questionnaire and anthropometric data will be collected, and parent and toddler sleep will be measured by 7 consecutive days/nights of actigraphy (a wrist- watch sized device that objectively measures sleep). This innovative study will provide an in-depth evaluation of the ecology in which parent and toddler sleep are embedded, and the relationship of sleep with toddler weight status in the target population. To ensure the child’s entire family system is considered, mothers, fathers, and other primary caregivers in the home will be included. Altogether, study findings will contribute to the future development of family-focused, culturally-tailored, and contextually-informed early prevention programs focused on sleep and weight status in this underserved population. This study is a critical next step toward reducing health disparities and improving health outcomes among toddlers from Mexican American families.

NIH Research Projects · FY 2024 · 2022-09

Relationship between maternal and fetal immune responses The fetal immune system starts to develop in the third week of gestation and progresses until birth. During this time, it is greatly influenced by maternal events. HIV-exposed uninfected infants (HEU) provide an excellent example in which neonates at birth have multiple immunologic differences compared with HIV-unexposed uninfected infants (HUU) that persist for months to years and, despite the absence of HIV infection, result in an increased risk of severe infection, hospitalization and death. Other maternal medical conditions can also affect the fetal immune system, such as chronic hepatitis B infection, type 1 diabetes and obesity. The mechanism whereby the maternal and fetal immune systems communicate is not known. We hypothesized that the fetal immune system receives instructions from the mother through small molecules or nanoparticles resulting in common immunologic biases between maternal-fetal dyads. In this study, we will investigate maternal metabolites, cytokines/chemokines, placental hormones and extracellular vesicles (EV) as potential mediators of the cross talk between maternal and fetal immune systems. To test this hypothesis, we will leverage paired CBMC and maternal PBMC from women with and without HIV collected in a previous NIAID- funded study. In the current study, we formulated the following Specific Aims: Aim 1. To determine the breadth of the immunologic characteristics shared by mothers with and without HIV and their fetuses. Using high-dimensional flow cytometry, Cite-seq and ATAC-seq we will determine the phenotypic, transcriptomic and epigenetic profiles of maternal and fetal T cells and antigen presenting cells and identify characteristics shared by mother-fetus dyads both unique and common to HEU and HUU dyads. Aim 2. To identify metabolites, maternal hormones, cytokines/chemokines and/or EV shared by maternal-fetal dyads that distinguish HEU from HUU dyads. We will characterize maternal and fetal metabolomic, proteomic, hormonal and EV plasma profiles to identify the factors that are shared between maternal-fetal dyads. We will then identify those that are specific to HEU compared with HUU dyads. Aim 3. To identify mediators of the communication between the maternal and fetal immune systems. Using a transwell system to simulate the placental barrier, we will examine functional and phenotypic changes in the Treg CBMC induced by maternal factors. Impact. We will study maternal-fetal information transfer that programs the HUMAN fetal immune system. This is a critical step in devising interventions to mitigate fetal immune dysfunctions. By leveraging resources from a prior NIAID-funded study, we will be able to use multi-omics to maximize the depth of these pioneering studies.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Investigations of Proteome Turnover Kinetics Under Cellular Differentiation: Proteins are in a constant flux of continuous synthesis and degradation, both of which contribute to regulate protein levels in the cell. Although this fact has been known for 80 years, currently most biological inquiries are limited to static snapshots of overall transcript and protein levels, whereas knowledge into the dynamic changes of protein turnover remains severely lagging. This ESI MIRA proposal seeks to advance the current understanding of how cellular proteomes remodel during cell state transitions, by incorporating protein turnover kinetics information at key stages of human induced pluripotent stem cell (iPSC) differentiation into distinct cellular lineages. Working with collaborators, we previously developed deuterium stable isotope labeling, mass spectrometry, and kinetic modeling methods to quantitate protein turnover in animal models and in human. In doing so, we found many novel cell states and disease markers may be discovered from integrating orthogonal protein abundance and kinetics information. In the next five years, we propose to: (1) apply these methods to acquire a high temporal density map of human iPSC trilineage differentiation into cardiomyocyte, hepatocyte, and neuroprogenitor cells; (2) interrogate the regulatory principles that govern turnover flux across different differentiation stages; and (3) assess the functional consequences of protein degradation on the success and cell maturity of terminal cell production. Finally, a limitation of current techniques is that the kinetic models used in protein turnover studies largely assume a non-changing protein pool size at steady state, which does not apply to differentiating cells or progressing diseases. We propose to expand current models of analyzing heavy water stable isotope label experiments toward dynamical systems with variable protein pool sizes. If successful, the proposed research would greatly expand the current knowledge of molecular events that take place during human iPSC differentiation, as well as generate publicly available data sets and software tools to advance protein turnover studies in diverse areas.

NIH Research Projects · FY 2025 · 2022-09

An understanding of cancer immune evasion has recently led to revolutionary immunotherapies and a subsequent rush, by both industry and academia, to identify additional mechanisms of immune suppression employed by cancer cells. Since these efforts rely on models of full-fledged cancer, there remains a neglected opportunity to target neoplasms prior to the development of immune evasive character. The lack of models for tracing de novo somatic transformation in vivo has prevented direct characterization of early carcinogenesis, including the first interactions with immune cells. To address this deficiency, a novel mouse model has been developed, which allows fluorescent tracing of individual transformed clones in the skin. A special transplant technique has been used to integrate fluorescent, transformation-inducible keratinocytes into the epidermis of an immunocompetent mouse, where they generate isolated, homeostatic clones. These colonies can be non- invasively imaged at subcellular resolution via intravital confocal microscopy as transformation is induced. This technique provides the first-ever direct visualization of cancer development in situ. Since immunity represents a pivotal barrier to the successful outgrowth of neoplasms, this model was engineered to allow visualization of immune cells, as well. The concept of immunoediting provides a framework for how cancers evolve immune- evasive strategies during their development. Immunoediting includes a prolonged dormancy, termed the “equilibrium phase”, during which immunity prevents tumor outgrowth without destroying the transformed cells. For the first time, this model allows the observation of all three phases of immunoediting: elimination, equilibrium, and escape, and reveals that the normal tissue microenvironment plays a central role in early immune evasion. This novel model also reveals a role for innate immune cells in the early stages of immune surveillance of skin cancer and in the maintenance of the equilibrium phase. During the equilibrium phase, transformed cells may be uniquely sensitive to interventions since their lower numbers and relative homogeneity will hinder development of resistance mechanisms. The ability to visualize de novo transformation in this model allows this hypothesis to be tested. In addition, this model will allow the characterization of mechanisms that mediate the hidden events of immunoediting. New preliminary data reveal that the transition from equilibrium lesions to escape tumors involves the upregulation of TGFβ3 in escape tumors, which concurrently undergo epithelial-mesenchymal transition. The increased levels of TGFβ3 convert NK cells, that can inhibit tumor growth, into intermediate type 1 innate lymphoid cells that cannot inhibit tumor growth. This revised application will further pursue both cellular and molecular mechanisms suggested by these preliminary data. Finally, the ability to visualize immune-mediated dormant lesions may uncover potential biomarkers, which might be translatable for early detection in high-risk human patients, such as immunosuppressed organ transplant recipients, who have a 100-fold increased risk of developing skin cancer.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract: Collecting representative and inclusive data about sexual orientation and gender identity (SOGI) is a critical component of combating the devastating health disparities affecting sexual and gender minority (SGM) older adults. This is particularly crucial for patients living with ADRD, which is widely regarded as a family disease requiring the active caregiver involvement, particularly with advanced disease. Failure to collect and integrate SOGI data to identify patients’ informal support systems may have adverse health consequences for SGM older adults, particularly for those dependent on informal caregivers to provide in-home support and assist with activities of daily living. Improper identification of chosen family and caregivers contributes to incomplete care delivery and disenfranchised grief. Given the historical discrimination experienced by older SGM people, adding SOGI questions without proper training has the potential to harm patients and create staff discomfort rather than foster inclusive interactions. For this career development award, I propose to characterize SOGI data collection challenges from patients and caregivers enrolling in hospice while exploring understudied intersections, such as SGM people living with ADRD, and how they affect staff approaches to delivering person-centered care. These insights will be used to develop and pilot test an intervention to train hospice interdisciplinary team (IDT) staff to sensitively collect and utilize SOGI data to improve communication with SGM patients and caregivers. Candidate, Mentors and Proposed Training: I am an Assistant Professor at the University of Colorado School of Medicine. My long-term goal is to become a leader in communication aging research to reduce health disparities by affecting change for SGM older adults via more effective, personcentered communication. This work will harness my background as a communication health researcher, my previous research with hospices and older SGM communities including those living with ADRD, and my lived experience as a SGM person. Research Plan: My research aims to: 1) characterize barriers and facilitators to communicating with SGM patients and caregivers who have a serious illness, with a special emphasis on those with ADRD; 2) develop an educational intervention for hospice IDT members to improve communication with older SGM patients; and 3) pilot test a novel communication training intervention for feasibility and acceptability of behavior change to engage in meaningful dialogue. My training goals include developing additional expertise in: 1) statistical methods for testing hypotheses and evaluating clinical trial interventions; 2) community-based stakeholder engagement and bioethical intervention development; and 3) clinical trials and intervention development and evaluation. This K01 is critical to my becoming an independent leader in SGM aging research. Implication: This project will not only inform how to improve SOGI data collection but will contribute to communication behavior change to facilitate meaningful dialogue with SGM older adults, including those with ADRD, and their caregivers around the end of life.