Ferroptosis Inhibition with Deferoxamine Alleviates Radiation-Induced Fibrosis.

Background Radiation-induced fibrosis (RIF) is a debilitating sequelae of radiation therapy that has been shown to improve with topical treatment with the iron chelator deferoxamine (DFO). We investigated whether DFO exerts this effect through attenuation of ferroptosis, a recently described iron-dependent pathway of cell death. Methods Adult C57BL/6J mice were treated with topical DFO or ferrostastin-1 (Fer-1) and irradiated with 30 Grays of ionizing radiation to the dorsal skin to promote development of chronic RIF. Immunofluorescent staining with 4-hydroxynonenal (4-HNE) antibody was carried out directly following irradiation to assess ferroptosis activity. Perfusion testing with laser Doppler was performed throughout the healing interval. Eight weeks following radiation, dorsal skin was harvested and analyzed histologically and biomechanically. Results Immunohistochemical staining demonstrated lower presence of 4-HNE in non-irradiated skin, DFO-treated skin, and Fer-1-treated skin compared to irradiated, untreated skin. DFO resulted in histological measurements (dermal thickness and collagen content) that resembled normal skin, while Fer-1 treatment yielded less significant improvements. These results were mirrored by analysis of extracellular matrix ultrastructure and biomechanical testing, which recapitulated the ability of topical DFO treatment to alleviate RIF across these parameters while Fer-1 resulted in less notable improvement. Finally, perfusion levels in DFO treated irradiated skin were similar to measurements in normal skin, while Fer-1 treatment did not impact this feature. Conclusions Ferroptosis contributes to the development of RIF and attenuation of this process leads to reduced skin injury. DFO further improves RIF through additional enhancement of perfusion not seen with Fer-1.

Hematoxylin and Eosin Architecture Uncovers Clinically Divergent Niches in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) represents one of the only cancers with an increasing incidence rate and is often associated with intra- and peri-tumoral scarring, referred to as desmoplasia. This scarring is highly heterogeneous in extracellular matrix (ECM) architecture and plays complex roles in both tumor biology and clinical outcomes that are not yet fully understood. Using hematoxylin and eosin (H&E), a routine histological stain utilized in existing clinical workflows, we quantified ECM architecture in 85 patient samples to assess relationships between desmoplastic architecture and clinical outcomes such as survival time and disease recurrence. By utilizing unsupervised machine learning (ML) to summarize a latent space across 147 local (e.g. fiber length, solidity) and global (e.g. fiber branching, porosity) H&E-based features, we identified a continuum of histological architectures that were associated with differences in both survival and recurrence. Further, we mapped H&E architectures to a CO-Detection by indEXing (CODEX) reference atlas, revealing localized cell- and protein-based niches associated with outcome-positive vs. outcome-negative scarring in the tumor microenvironment. Overall, our study utilizes standard H&E staining to uncover clinically relevant associations between desmoplastic organization and PDAC outcomes, offering a translatable pipeline to support prognostic decision-making and a blueprint of spatial-biological factors for modeling by tissue engineering methods.

Understanding wound healing in obesity.

Obesity has become more prevalent in the global population. It is associated with the development of several diseases including diabetes mellitus, coronary heart disease, and metabolic syndrome. There are a multitude of factors impacted by obesity that may contribute to poor wound healing outcomes. With millions worldwide classified as obese, it is imperative to understand wound healing in these patients. Despite advances in the understanding of wound healing in both healthy and diabetic populations, much is unknown about wound healing in obese patients. This review examines the impact of obesity on wound healing and several animal models that may be used to broaden our understanding in this area. As a growing portion of the population identifies as obese, understanding the underlying mechanisms and how to overcome poor wound healing is of the utmost importance.

Deferoxamine topical cream superior to patch in rescuing radiation-induced fibrosis of unwounded and wounded skin.

Topical patch delivery of deferoxamine (DFO) has been studied as a treatment for this fibrotic transformation in irradiated tissue. Efficacy of a novel cream formulation of DFO was studied as a RIF therapeutic in unwounded and excisionally wounded irradiated skin. C57BL/6J mice underwent 30 Gy of radiation to the dorsum followed by 4 weeks of recovery. In a first experiment, mice were separated into six conditions: DFO 50 mg cream (D50), DFO 100 mg cream (D100), soluble DFO injections (DI), DFO 1 mg patch (DP), control cream (Vehicle), and irradiated untreated skin (IR). In a second experiment, excisional wounds were created on the irradiated dorsum of mice and then divided into four treatment groups: DFO 100 mg Cream (W-D100), DFO 1 mg patch (W-DP), control cream (W-Vehicle), and irradiated untreated wounds (W-IR). Laser Doppler perfusion scans, biomechanical testing, and histological analysis were performed. In irradiated skin, D100 improved perfusion compared to D50 or DP. Both D100 and DP enhanced dermal characteristics, including thickness, collagen density and 8-isoprostane staining compared to untreated irradiated skin. D100 outperformed DP in CD31 staining, indicating higher vascular density. Extracellular matrix features of D100 and DP resembled normal skin more closely than DI or control. In radiated excisional wounds, D100 facilitated faster wound healing and increased perfusion compared to DP. The 100 mg DFO cream formulation rescued RIF of unwounded irradiated skin and improved excisional wound healing in murine skin relative to patch delivery of DFO.

Understanding Tendon Fibroblast Biology and Heterogeneity.

Tendon regeneration has emerged as an area of interest due to the challenging healing process of avascular tendon tissue. During tendon healing after injury, the formation of a fibrous scar can limit tendon strength and lead to subsequent complications. The specific biological mechanisms that cause fibrosis across different cellular subtypes within the tendon and across different tendons in the body continue to remain unknown. Herein, we review the current understanding of tendon healing, fibrosis mechanisms, and future directions for treatments. We summarize recent research on the role of fibroblasts throughout tendon healing and describe the functional and cellular heterogeneity of fibroblasts and tendons. The review notes gaps in tendon fibrosis research, with a focus on characterizing distinct fibroblast subpopulations in the tendon. We highlight new techniques in the field that can be used to enhance our understanding of complex tendon pathologies such as fibrosis. Finally, we explore bioengineering tools for tendon regeneration and discuss future areas for innovation. Exploring the heterogeneity of tendon fibroblasts on the cellular level can inform therapeutic strategies for addressing tendon fibrosis and ultimately reduce its clinical burden.

A tension offloading patch mitigates dermal fibrosis induced by pro-fibrotic skin injections.

Skin fibrosis is a clinical problem with devastating impacts but limited treatment options. In the setting of diabetes, insulin administration often causes local dermal fibrosis, leading to a range of clinical sequelae including impeded insulin absorption. Mechanical forces are important drivers of fibrosis and, clinically, physical tension offloading at the skin level using an elastomeric patch significantly reduces wound scarring. However, it is not known whether tension offloading could similarly prevent skin fibrosis in the setting of pro-fibrotic injections. Here, we develop a porcine model using repeated local injections of bleomycin to recapitulate key features of insulin-induced skin fibrosis. Using histologic, tissue ultrastructural, and biomechanical analyses, we show that application of a tension-offloading patch both prevents and rescues existing skin fibrosis from bleomycin injections. By applying single-cell transcriptomic analysis, we find that the fibrotic response to bleomycin involves shifts in myeloid cell dynamics from favoring putatively pro-regenerative to pro-fibrotic myeloid subtypes; in a mechanomodulatory in vitro platform, we show that these shifts are mechanically driven and reversed by exogenous IL4. Finally, using a human foreskin xenograft model, we show that IL4 treatment mitigates bleomycin-induced dermal fibrosis. Overall, this study highlights that skin tension offloading, using an FDA cleared, commercially available patch, could have significant potential clinical benefit for the millions of patients dependent on insulin.

Successful topical treatment of human biofilms using multiple antibiotic elution from a collagen-rich hydrogel.

Chronic non-healing wounds significantly strain modern healthcare systems, affecting 1-2% of the population in developed countries with costs ranging between $28.1 and $96.8 billion annually. Additionally, it has been established that chronic wounds resulting from comorbidities, such as peripheral vascular disease and diabetes mellitus, tend to be polymicrobial in nature. Treatment of polymicrobial chronic wounds with oral and IV antibiotics can result in antimicrobial resistance, leading to more difficult-to-treat wounds. Ideally, chronic ulcers would be topically treated with antibiotic combinations tailored to the microbiome of a patient's wound. We have previously shown that a topical collagen-rich hydrogel (cHG) can elute single antibiotics to inhibit bacterial growth in a manner that is nontoxic to mammalian cells. Here, we analyzed the microbiology of cultures taken from human patients diagnosed with diabetes mellitus suffering from chronic wounds present for more than 6 weeks. Additionally, we examined the safety of the elution of multiple antibiotics from collagen-rich hydrogel in mammalian cells in vivo. Finally, we aimed to create tailored combinations of antibiotics impregnated into cHG to successfully target and treat infections and eradicate biofilms cultured from human chronic diabetic wound tissue. We found that the majority of human chronic wounds in our study were polymicrobial in nature. The elution of multiple antibiotics from cHG was well-tolerated in mammalian cells, making it a potential topical treatment of the polymicrobial chronic wound. Finally, combinations of antibiotics tailored to each patient's microbiome eluted from a collagen-rich hydrogel successfully treated bacterial cultures isolated from patient samples via an in vitro assay.

Establishing a Xenograft Model with CD-1 Nude Mice to Study Human Skin Wound Repair.

BACKGROUND: A significant gap exists in the translatability of small-animal models to human subjects. One important factor is poor laboratory models involving human tissue. Thus, the authors have created a viable postnatal human skin xenograft model using athymic mice. METHODS: Discarded human foreskins were collected following circumcision. All subcutaneous tissue was removed from these samples sterilely. Host CD-1 nude mice were then anesthetized, and dorsal skin was sterilized. A 1.2-cm-diameter, full-thickness section of dorsal skin was excised. The foreskin sample was then placed into the full-thickness defect in the host mice and sutured into place. Xenografts underwent dermal wounding using a 4-mm punch biopsy after engraftment. Xenografts were monitored for 14 days after wounding and then harvested. RESULTS: At 14 days postoperatively, all mice survived the procedure. Grossly, the xenograft wounds showed formation of a human scar at postoperative day 14. Hematoxylin and eosin and Masson trichome staining confirmed scar formation in the wounded human skin. Using a novel artificial intelligence algorithm using picrosirius red staining, scar formation was confirmed in human wounded skin compared with the unwounded skin. Histologically, CD31 + immunostaining confirmed vascularization of the xenograft. The xenograft exclusively showed human collagen type I, CD26 + , and human nuclear antigen in the human scar without any staining of these human markers in the murine skin. CONCLUSION: The proposed model demonstrates wound healing to be a local response from tissue resident human fibroblasts and allows for reproducible evaluation of human skin wound repair in a preclinical model. CLINICAL RELEVANCE STATEMENT: Radiation-induced fibrosis is a widely prevalent clinical phenomenon without a well-defined treatment at this time. This study will help establish a small-animal model to better understand and develop novel therapeutics to treat irradiated human skin.

A Review of Radiation-Induced Vascular Injury and Clinical Impact.

ABSTRACT: The number of cancer survivors continues to increase because of advances in therapeutic modalities. Along with surgery and chemotherapy, radiotherapy is a commonly used treatment modality in roughly half of all cancer patients. It is particularly helpful in the oncologic treatment of patients with breast, head and neck, and prostate malignancies. Unfortunately, among patients receiving radiation therapy, long-term sequalae are often unavoidable, and there is accumulating clinical evidence suggesting significant radiation-related damage to the vascular endothelium. Ionizing radiation has been known to cause obliterative fibrosis and increased wall thickness in irradiated blood vessels. Clinically, these vascular changes induced by ionizing radiation can pose unique surgical challenges when operating in radiated fields. Here, we review the relevant literature on radiation-induced vascular damage focusing on mechanisms and signaling pathways involved and highlight microsurgical anastomotic outcomes after radiotherapy. In addition, we briefly comment on potential therapeutic strategies, which may have the ability to mitigate radiation injury to the vascular endothelium.

Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing.

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data, represents a breakthrough in our ability to discern tumor cell heterogeneity-a primary focus of translational cancer research at this time. However, the quality of sequencing data acquired using this advanced modality is highly dependent on the quality of the input material. Digestion conditions need to be optimized to maximize cell yield without sacrificing quality. This is particularly challenging in the context of solid tumors with dense desmoplastic matrices that must be gently broken down for cell release. Freshly isolated cells from solid tumor tissue are more fragile than those isolated from cell lines. Additionally, as the cell types isolated are heterogeneous, conditions should be selected to support the total cell population. Finally, nuclear isolation conditions must be optimized based on these qualities in terms of lysis times and reagent types/ratios. In this article, we describe our experience with nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor specimens. We provide recommendations for tissue digestion, storage of single-cell suspensions (if desired), and nuclear isolation and assessment.

Desmoplastic stromal signatures predict patient outcomes in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related death. Hallmarks include desmoplasia with variable extracellular matrix (ECM) architecture and a complex microenvironment with spatially defined tumor, stromal, and immune populations. Nevertheless, the role of desmoplastic spatial organization in patient/tumor variability remains underexplored, which we elucidate using two technologies. First, we quantify ECM patterning in 437 patients, revealing architectures associated with disease-free and overall survival. Second, we spatially profile the cellular milieu of 78 specimens using codetection by indexing, identifying an axis of pro-inflammatory cell interactions predictive of poorer outcomes. We discover that clinical characteristics, including neoadjuvant chemotherapy status, tumor stage, and ECM architecture, correlate with differential stromal-immune organization, including fibroblast subtypes with distinct niches. Lastly, we define unified signatures that predict survival with areas under the receiver operating characteristic curve (AUCs) of 0.872-0.903, differentiating survivorship by 655 days. Overall, our findings establish matrix ultrastructural and cellular organizations of fibrosis linked to poorer outcomes.

Allele-specific expression reveals genetic drivers of tissue regeneration in mice.

In adult mammals, skin wounds typically heal by scarring rather than through regeneration. In contrast, "super-healer" Murphy Roths Large (MRL) mice have the unusual ability to regenerate ear punch wounds; however, the molecular basis for this regeneration remains elusive. Here, in hybrid crosses between MRL and non-regenerating mice, we used allele-specific gene expression to identify cis-regulatory variation associated with ear regeneration. Analyzing three major cell populations (immune, fibroblast, and endothelial), we found that genes with cis-regulatory differences specifically in fibroblasts were associated with wound-healing pathways and also co-localized with quantitative trait loci for ear wound-healing. Ectopic treatment with one of these proteins, complement factor H (CFH), accelerated wound repair and induced regeneration in typically fibrotic wounds. Through single-cell RNA sequencing (RNA-seq), we observed that CFH treatment dramatically reduced immune cell recruitment to wounds, suggesting a potential mechanism for CFH's effect. Overall, our results provide insights into the molecular drivers of regeneration with potential clinical implications.

Understanding Fibroblast Heterogeneity in Form and Function.

Historically believed to be a homogeneous cell type that is often overlooked, fibroblasts are more and more understood to be heterogeneous in nature. Though the mechanisms behind how fibroblasts participate in homeostasis and pathology are just beginning to be understood, these cells are believed to be highly dynamic and play key roles in fibrosis and remodeling. Focusing primarily on fibroblasts within the skin and during wound healing, we describe the field's current understanding of fibroblast heterogeneity in form and function. From differences due to embryonic origins to anatomical variations, we explore the diverse contributions that fibroblasts have in fibrosis and plasticity. Following this, we describe molecular techniques used in the field to provide deeper insights into subpopulations of fibroblasts and their varied roles in complex processes such as wound healing. Limitations to current work are also discussed, with a focus on future directions that investigators are recommended to take in order to gain a deeper understanding of fibroblast biology and to develop potential targets for translational applications in a clinical setting.

Medical Biology of Cancer-Associated Fibroblasts in Pancreatic Cancer.

Pancreatic cancer is one of the deadliest forms of cancer with one of the lowest 5-year survival rates of all cancer types. A defining characteristic of pancreatic cancer is the existence of dense desmoplastic stroma that, when exposed to stimuli such as cytokines, growth factors, and chemokines, generate a tumor-promoting environment. Cancer-associated fibroblasts (CAFs) are activated during the progression of pancreatic cancer and are a crucial component of the tumor microenvironment (TME). CAFs are primarily pro-tumorigenic in their activated state and function as promoters of cancer invasion, proliferation, metastasis, and immune modulation. Aided by many signaling pathways, cytokines, and chemokines in the tumor microenvironment, CAFs can originate from many cell types including resident fibroblasts, mesenchymal stem cells, pancreatic stellate cells, adipocytes, epithelial cells, endothelial cells, and other cell types. CAFs are a highly heterogeneous cell type expressing a variety of surface markers and performing a wide range of tumor promoting and inhibiting functions. Single-cell transcriptomic analyses have revealed a high degree of specialization among CAFs. Some examples of CAF subpopulations include myofibrotic CAFs (myCAFs), which exhibit a matrix-producing contractile phenotype; inflammatory CAFs (iCAF) that are classified by their immunomodulating, secretory phenotype; and antigen-presenting CAFs (apCAFs), which have antigen-presenting capabilities and express Major Histocompatibility Complex II (MHC II). Over the last several years, various attempts have been undertaken to describe the mechanisms of CAF-tumor cell interaction, as well as CAF-immune cell interaction, that contribute to tumor proliferation, invasion, and metastasis. Although our understanding of CAF biology in cancer has steadily increased, the extent of CAFs heterogeneity and their role in the pathobiology of pancreatic cancer remains elusive. In this regard, it becomes increasingly evident that further research on CAFs in pancreatic cancer is necessary.

Understanding the Role of Adipocytes and Fibroblasts in Cancer.

ABSTRACT: Cancer is currently the second leading cause of death in the United States. There is increasing evidence that the tumor microenvironment (TME) is pivotal for tumorigenesis and metastasis. Recently, adipocytes and cancer-associated fibroblasts (CAFs) in the TME have been shown to play a major role in tumorigenesis of different cancers, specifically melanoma. Animal studies have shown that CAFs and adipocytes within the TME help tumors evade the immune system, for example, by releasing chemokines to blunt the effectiveness of the host defense. Although studies have identified that adipocytes and CAFs play a role in tumorigenesis, adipocyte transition to fibroblast within the TME is fairly unknown. This review intends to elucidate the potential that adipocytes may have to transition to fibroblasts and, as part of the TME, a critical role that CAFs may play in affecting the growth and invasion of tumor cells. Future studies that illuminate the function of adipocytes and CAFs in the TME may pave way for new antitumor therapies.

Cas9-mediated knockout of Ndrg2 enhances the regenerative potential of dendritic cells for wound healing.

Chronic wounds impose a significant healthcare burden to a broad patient population. Cell-based therapies, while having shown benefits for the treatment of chronic wounds, have not yet achieved widespread adoption into clinical practice. We developed a CRISPR/Cas9 approach to precisely edit murine dendritic cells to enhance their therapeutic potential for healing chronic wounds. Using single-cell RNA sequencing of tolerogenic dendritic cells, we identified N-myc downregulated gene 2 (Ndrg2), which marks a specific population of dendritic cell progenitors, as a promising target for CRISPR knockout. Ndrg2-knockout alters the transcriptomic profile of dendritic cells and preserves an immature cell state with a strong pro-angiogenic and regenerative capacity. We then incorporated our CRISPR-based cell engineering within a therapeutic hydrogel for in vivo cell delivery and developed an effective translational approach for dendritic cell-based immunotherapy that accelerated healing of full-thickness wounds in both non-diabetic and diabetic mouse models. These findings could open the door to future clinical trials using safe gene editing in dendritic cells for treating various types of chronic wounds.

Del1 Is a Growth Factor for Skeletal Progenitor Cells in the Fracture Callus.

Failure to properly form bone or integrate surgical implants can lead to morbidity and additional surgical interventions in a significant proportion of orthopedic surgeries. While the role of skeletal stem cells (SSCs) in bone formation and repair is well-established, very little is known about the factors that regulate the downstream Bone, Cartilage, Stromal, Progenitors (BCSPs). BCSPs, as transit amplifying progenitor cells, undergo multiple mitotic divisions to expand the pool of lineage committed progenitors allowing stem cells to preserve their self-renewal and stemness. Del1 is a protein widely expressed in the skeletal system, but its deletion led to minimal phenotype changes in the uninjured mouse. In this paper, we demonstrate that Del1 is a key regulator of BCSP expansion following injury. In Del1 knockout mice, there is a significant reduction in the number of BCSPs which leads to a smaller callus and decreased bone formation compared with wildtype (WT) littermates. Del1 serves to promote BCSP proliferation and prevent apoptosis in vivo and in vitro. Moreover, exogenous Del1 promotes proliferation of aged human BCSPs. Our results highlight the potential of Del1 as a therapeutic target for improving bone formation and implant success. Del1 injections may improve the success of orthopedic surgeries and fracture healing by enhancing the proliferation and survival of BCSPs, which are crucial for generating new bone tissue during the process of bone formation and repair.