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.

Macrophage inflammatory and regenerative response periodicity is programmed by cell cycle and chromatin state.

Cell cycle (CC) facilitates cell division via robust, cyclical gene expression. Protective immunity requires the expansion of pathogen-responsive cell types, but whether CC confers unique gene expression programs that direct the subsequent immunological response remains unclear. Here, we demonstrate that single macrophages (MFs) adopt different plasticity states in CC, which leads to heterogeneous cytokine-induced polarization, priming, and repolarization programs. Specifically, MF plasticity to interferon gamma (IFNG) is substantially reduced during S-G2/M, whereas interleukin 4 (IL-4) induces S-G2/M-biased gene expression, mediated by CC-biased enhancers. Additionally, IL-4 polarization shifts the CC-phase distribution of MFs toward the G2/M phase, providing a subpopulation-specific mechanism for IL-4-induced, dampened IFNG responsiveness. Finally, we demonstrate CC-dependent MF responses in murine and human disease settings in vivo, including Th2-driven airway inflammation and pulmonary fibrosis, where MFs express an S-G2/M-biased tissue remodeling gene program. Therefore, MF inflammatory and regenerative responses are gated by CC in a cyclical, phase-dependent manner.

Multiplexed evaluation of mouse wound tissue using oligonucleotide barcoding with single-cell RNA sequencing.

Despite its rapidly increased availability for the study of complex tissue, single-cell RNA sequencing remains prohibitively expensive for large studies. Here, we present a protocol using oligonucleotide barcoding for the tagging and pooling of multiple samples from healing wounds, which are among the most challenging tissue types for this application. We describe steps to generate skin wounds in mice, followed by tissue harvest and oligonucleotide barcoding. This protocol is also applicable to other species including rats, pigs, and humans. For complete details on the use and execution of this protocol, please refer to Stoeckius et al. (2018),1 Galiano et al. (2004),2 and Mascharak et al. (2022).3.

Multiomic analysis reveals conservation of cancer-associated fibroblast phenotypes across species and tissue of origin.

Cancer-associated fibroblasts (CAFs) are integral to the solid tumor microenvironment. CAFs were once thought to be a relatively uniform population of matrix-producing cells, but single-cell RNA sequencing has revealed diverse CAF phenotypes. Here, we further probed CAF heterogeneity with a comprehensive multiomics approach. Using paired, same-cell chromatin accessibility and transcriptome analysis, we provided an integrated analysis of CAF subpopulations over a complex spatial transcriptomic and proteomic landscape to identify three superclusters: steady state-like (SSL), mechanoresponsive (MR), and immunomodulatory (IM) CAFs. These superclusters are recapitulated across multiple tissue types and species. Selective disruption of underlying mechanical force or immune checkpoint inhibition therapy results in shifts in CAF subpopulation distributions and affected tumor growth. As such, the balance among CAF superclusters may have considerable translational implications. Collectively, this research expands our understanding of CAF biology, identifying regulatory pathways in CAF differentiation and elucidating therapeutic targets in a species- and tumor-agnostic manner.

Multi-omic analysis reveals divergent molecular events in scarring and regenerative wound healing.

Regeneration is the holy grail of tissue repair, but skin injury typically yields fibrotic, non-functional scars. Developing pro-regenerative therapies requires rigorous understanding of the molecular progression from injury to fibrosis or regeneration. Here, we report the divergent molecular events driving skin wound cells toward scarring or regenerative fates. We profile scarring versus YAP-inhibition-induced wound regeneration at the transcriptional (single-cell RNA sequencing), protein (timsTOF proteomics), and tissue (extracellular matrix ultrastructural analysis) levels. Using cell-surface barcoding, we integrate these data to reveal fibrotic and regenerative "molecular trajectories" of healing. We show that disrupting YAP mechanotransduction yields regenerative repair by fibroblasts with activated Trps1 and Wnt signaling. Finally, via in vivo gene knockdown and overexpression in wounds, we identify Trps1 as a key regulatory gene that is necessary and partially sufficient for wound regeneration. Our findings serve as a multi-omic map of wound regeneration and could have therapeutic implications for pathologic fibroses.

Xenogeneic skin transplantation promotes angiogenesis and tissue regeneration through activated Trem2+ macrophages.

Skin allo- and xenotransplantation are the standard treatment for major burns when donor sites for autografts are not available. The relationship between the immune response to foreign grafts and their impact on wound healing has not been fully elucidated. Here, we investigated changes in collagen architecture after xenogeneic implantation of human biologic scaffolds. We show that collagen deposition in response to the implantation of human split-thickness skin grafts (hSTSGs) containing live cells recapitulates normal skin architecture, whereas human acellular dermal matrix (ADM) grafts led to a fibrotic collagen deposition. We show that macrophage differentiation in response to hSTSG implantation is driven toward regenerative Trem2+ subpopulations and found that hydrogel delivery of these cells significantly accelerated wound closure. Our study identifies the preclinical therapeutic potential of Trem2+ macrophages to mitigate fibrosis and promote wound healing, providing a novel effective strategy to develop advanced cell therapies for complex wounds.

Integrated spatial multiomics reveals fibroblast fate during tissue repair.

In the skin, tissue injury results in fibrosis in the form of scars composed of dense extracellular matrix deposited by fibroblasts. The therapeutic goal of regenerative wound healing has remained elusive, in part because principles of fibroblast programming and adaptive response to injury remain incompletely understood. Here, we present a multimodal -omics platform for the comprehensive study of cell populations in complex tissue, which has allowed us to characterize the cells involved in wound healing across both time and space. We employ a stented wound model that recapitulates human tissue repair kinetics and multiple Rainbow transgenic lines to precisely track fibroblast fate during the physiologic response to skin injury. Through integrated analysis of single cell chromatin landscapes and gene expression states, coupled with spatial transcriptomic profiling, we are able to impute fibroblast epigenomes with temporospatial resolution. This has allowed us to reveal potential mechanisms controlling fibroblast fate during migration, proliferation, and differentiation following skin injury, and thereby reexamine the canonical phases of wound healing. These findings have broad implications for the study of tissue repair in complex organ systems.

Preventing Engrailed-1 activation in fibroblasts yields wound regeneration without scarring.

Skin scarring, the end result of adult wound healing, is detrimental to tissue form and function. Engrailed-1 lineage-positive fibroblasts (EPFs) are known to function in scarring, but Engrailed-1 lineage-negative fibroblasts (ENFs) remain poorly characterized. Using cell transplantation and transgenic mouse models, we identified a dermal ENF subpopulation that gives rise to postnatally derived EPFs by activating Engrailed-1 expression during adult wound healing. By studying ENF responses to substrate mechanics, we found that mechanical tension drives Engrailed-1 activation via canonical mechanotransduction signaling. Finally, we showed that blocking mechanotransduction signaling with either verteporfin, an inhibitor of Yes-associated protein (YAP), or fibroblast-specific transgenic YAP knockout prevents Engrailed-1 activation and promotes wound regeneration by ENFs, with recovery of skin appendages, ultrastructure, and mechanical strength. This finding suggests that there are two possible outcomes to postnatal wound healing: a fibrotic response (EPF-mediated) and a regenerative response (ENF-mediated).

Proceed with Caution: Mouse Deep Digit Flexor Tendon Injury Model.

The purpose of this study was to determine the feasibility of using mouse models for translational study of flexor tendon repair and reconstruction. METHODS: Quantitative data detailing the gross anatomy, biomechanical characteristics, and microscopic structure of the deep digit flexor tendon (DDF) of the mouse hindpaw were obtained. Histological characterization of the DDF and the anatomy of the digit in the mouse hindpaw are detailed. Biomechanical testing determined the load-to-failure, stress, elastic modulus, and the site of tendon failure. RESULTS: In gross anatomy, the origins and insertions of the mouse deep digit flexor tendon are similar to those of the human digit, surrounded by a synovial sheath that is only 1- to 2-cells thick. A neurovascular network runs on each side of the digit outside the synovial sheath, but does not clearly penetrate it. The thickness of the DDF is 0.14 ± 0.03 mm and the width is 0.3 ± 0.03 mm. The thickness of the DDF is less than that of 9-0 nylon needle. The mean failure force of the deep flexor tendon was 2.79 ± 0.53N. CONCLUSIONS: The gross anatomy of the mouse hindpaw digit is similar to that of the human digit except for key differences seen in the synovial sheath and vascular supply. The dimensions of the mouse DDF make it challenging to create a clinically translatable repair model using currently available surgical techniques. Despite the similarities between the human and mouse anatomy, and the powerful basic science tools available in murine models, mice are an unreliable model for assessing flexor tendon injury and repair.

Molecular Targeting of H/MDM-2 Oncoprotein in Human Colon Cancer Cells and Stem-like Colonic Epithelial-derived Progenitor Cells.

BACKGROUND/AIM: We have tested whether the anticancer peptide, PNC-27, that kills cancer cells but not normal cells by binding to cancer cell membrane HDM-2 forming pores, kills CD44+ colon cancer stem cells. MATERIALS AND METHODS: Flow cytometry determined the CD44 and HDM-2 expression on six-colon cancer cell lines and one normal cell line (CCD-18Co). MTT, LDH release, annexin V binding and caspase 3 assays were used to assess PNC-27-induced cell death. Bioluminescence imaging measured PNC-27 effects on in vivo tumor growth. RESULTS: High percentages of cells in all six tumor lines expressed CD44. PNC-27 co-localized with membrane HDM-2 only in the cancer cells and caused total cell death (tumor cell necrosis, high LDH release, negative annexin V and caspase 3). In vivo, PNC-27 caused necrosis of tumor nodules but not of normal tissue. CONCLUSION: PNC-27 selectively kills colon cancer stem cells by binding of this peptide to membrane H/MDM-2.

Wounds Inhibit Tumor Growth In Vivo.

OBJECTIVE: The aim of this study was to determine the interaction of full thickness excisional wounds and tumors in vivo. SUMMARY OF BACKGROUND DATA: Tumors have been described as wounds that do not heal due to similarities in stromal composition. On the basis of observations of slowed tumor growth after ulceration, we hypothesized that full thickness excisional wounds would inhibit tumor progression in vivo. METHODS: To determine the interaction of tumors and wounds, we developed a tumor xenograft/allograft (human head and neck squamous cell carcinoma SAS/mouse breast carcinoma 4T1) wound mouse model. We examined tumor growth with varying temporospatial placement of tumors and wounds or ischemic flap. In addition, we developed a tumor/wound parabiosis model to understand the ability of tumors and wounds to recruit circulating progenitor cells. RESULTS: Tumor growth inhibition by full thickness excisional wounds was dose-dependent, maintained by sequential wounding, and relative to distance. This effect was recapitulated by placement of an ischemic flap directly adjacent to a xenograft tumor. Using a parabiosis model, we demonstrated that a healing wound was able to recruit significantly more circulating progenitor cells than a growing tumor. Tumor inhibition by wound was unaffected by presence of an immune response in an immunocompetent model using a mammary carcinoma. Utilizing functional proteomics, we identified 100 proteins differentially expressed in tumors and wounds. CONCLUSION: Full thickness excisional wounds have the ability to inhibit tumor growth in vivo. Further research may provide an exact mechanism for this remarkable finding and new advances in wound healing and tumor biology.

Prrx1 Fibroblasts Represent a Pro-fibrotic Lineage in the Mouse Ventral Dermis.

Fibroblast heterogeneity has been shown within the unwounded mouse dorsal dermis, with fibroblast subpopulations being identified according to anatomical location and embryonic lineage. Using lineage tracing, we demonstrate that paired related homeobox 1 (Prrx1)-expressing fibroblasts are responsible for acute and chronic fibroses in the ventral dermis. Single-cell transcriptomics further corroborated the inherent fibrotic characteristics of Prrx1 fibroblasts during wound repair. In summary, we identify and characterize a fibroblast subpopulation in the mouse ventral dermis with intrinsic scar-forming potential.

Evaluation of Outcomes Following Surgery for Locally Advanced Pancreatic Neuroendocrine Tumors.

Importance: Although outcome of surgical resection of liver metastases from pancreatic neuroendocrine tumors (PNETs) has been extensively studied, little is known about surgery for locally advanced PNETs; it was listed recently by the European neuroendocrine tumor society as a major unmet need. Objective: To evaluate the outcome of patients who underwent surgery for locally aggressive PNETs. Design, Setting, and Participants: This retrospective single-center case series reviewed consecutive patients who underwent resection of T3/T4 PNETs at a single academic institution. Data collection occurred from 2003 to 2018. Data analysis was performed in August 2019. Main Outcomes and Measures: Disease-free survival (primary outcome) and overall mortality (secondary outcome) were assessed with Kaplan-Meier analysis. Recurrence risk (secondary outcome, defined as identification of tumor recurrence on imaging) was assessed with Cox proportional hazard models adjusting for covariates. Results: In this case series, 99 patients with locally advanced nondistant metastatic PNET (56 men [57%]) with a mean (SEM) age of 57.0 (1.4) years and a mean (SEM) follow-up of 5.3 (0.1) years underwent surgically aggressive resections. Of those, 4 patients (4%) underwent preoperative neoadjuvant treatment (including peptide receptor radionuclide therapy and chemotherapy); 18 patients (18%) underwent pancreaticoduodenectomy, 68 patients (69%) had distal or subtotal pancreatic resection, 10 patients (10%) had total resection, and 3 patients (3%) had other pancreatic procedures. Additional organ resection was required in 86 patients (87%): spleen (71 patients [71%]), major blood vessel (17 patients [17%]), bowel (2 patients [2%]), stomach (4 patients [4%]), and kidney (2 patients [2%]). Five-year disease-free survival was 61% (61 patients) and 5-year overall survival was 91% (91 patients). Of those living, 75 patients (76%) had an Eastern Cooperative Oncology Group score of less than or equal to 1 at last followup. Lymph node involvement (HR, 7.66; 95% CI, 2.78-21.12; P < .001), additional organ resected (HR, 6.15; 95% CI, 1.61-23.55; P = .008), and male sex (HR, 3.77; 95% CI, 1.68-8.97; P = .003) were associated with increased risk of recurrence. Functional tumors had a lower risk of recurrence (HR, 0.23; CI, 0.06-0.89; P = .03). Required resection of blood vessels was not associated with a significant increase recurrence risk. Conclusions and Relevance: In this case series, positive lymph node involvement and resection of organs with tumor involvement were associated with an increased recurrence risk. These subgroups may require adjuvant systemic treatment. These findings suggest that patients with locally advanced PNETs who undergo surgical resection have excellent disease-free and overall survival.

Characterization of Diabetic and Non-Diabetic Foot Ulcers Using Single-Cell RNA-Sequencing.

Background: Recent advances in high-throughput single-cell sequencing technologies have led to their increasingly widespread adoption for clinical applications. However, challenges associated with tissue viability, cell yield, and delayed time-to-capture have created unique obstacles for data processing. Chronic wounds, in particular, represent some of the most difficult target specimens, due to the significant amount of fibrinous debris, extracellular matrix components, and non-viable cells inherent in tissue routinely obtained from debridement. Methods: Here, we examined the feasibility of single cell RNA sequencing (scRNA-seq) analysis to evaluate human chronic wound samples acquired in the clinic, subjected to prolonged cold ischemia time, and processed without FACS sorting. Wound tissue from human diabetic and non-diabetic plantar foot ulcers were evaluated using an optimized 10X Genomics scRNA-seq platform and analyzed using a modified data pipeline designed for low-yield specimens. Cell subtypes were identified informatically and their distributions and transcriptional programs were compared between diabetic and non-diabetic tissue. Results: 139,000 diabetic and non-diabetic wound cells were delivered for 10X capture after either 90 or 180 min of cold ischemia time. cDNA library concentrations were 858.7 and 364.7 pg/µL, respectively, prior to sequencing. Among all barcoded fragments, we found that 83.5% successfully aligned to the human transcriptome and 68% met the minimum cell viability threshold. The average mitochondrial mRNA fraction was 8.5% for diabetic cells and 6.6% for non-diabetic cells, correlating with differences in cold ischemia time. A total of 384 individual cells were of sufficient quality for subsequent analyses; from this cell pool, we identified transcriptionally-distinct cell clusters whose gene expression profiles corresponded to fibroblasts, keratinocytes, neutrophils, monocytes, and endothelial cells. Fibroblast subpopulations with differing fibrotic potentials were identified, and their distributions were found to be altered in diabetic vs. non-diabetic cells. Conclusions: scRNA-seq of clinical wound samples can be achieved using minor modifications to standard processing protocols and data analysis methods. This simple approach can capture widespread transcriptional differences between diabetic and non-diabetic tissue obtained from matched wound locations.

Fibroblast Heterogeneity in and Its Implications for Plastic and Reconstructive Surgery: A Basic Science Review.

Fibroblasts' integral role in tissue development, maintenance, and disease represents a fast-growing field of basic science research. Although fibroblasts were long thought to be a homogeneous cell population, recent research has illuminated the unforeseen complexity of these cells, giving rise to the rapidly expanding research field of "fibroblast heterogeneity." Fibroblasts play a critical role in states of tissue fibrosis such as skin scarring, which affects hundreds of millions of patients annually and causes severe aesthetic, developmental, and functional morbidity. Beyond scarring, major organ fibrosis is an enormous public health concern responsible for nearly half of all deaths in the United States. Because fibrosis is a conserved response to tissue damage in all organs, the study of fibroblasts throughout the body may help us to understand their role in the conditions most relevant to plastic and reconstructive surgery-for instance, skin scarring (eg, from burns, traumatic lacerations, or surgical incisions), "pathological" scarring (hypertrophic scars, keloids), and capsular contracture. Here, we present a basic science review of fibroblast heterogeneity in wound healing, cancer, organ fibrosis, and human dermal architecture. The field of fibroblast heterogeneity is young, and many of the insights discussed have yet to be translated clinically. However, plastic surgeons stand in a unique position to bridge these discoveries into clinical realities. We hope this information can spur readers to consider both what questions in plastic surgery can be studied from the lens of fibroblast heterogeneity, and how these preclinical insights can be translated to improving care of our patients.

Elucidating the fundamental fibrotic processes driving abdominal adhesion formation.

Adhesions are fibrotic scars that form between abdominal organs following surgery or infection, and may cause bowel obstruction, chronic pain, or infertility. Our understanding of adhesion biology is limited, which explains the paucity of anti-adhesion treatments. Here we present a systematic analysis of mouse and human adhesion tissues. First, we show that adhesions derive primarily from the visceral peritoneum, consistent with our clinical experience that adhesions form primarily following laparotomy rather than laparoscopy. Second, adhesions are formed by poly-clonal proliferating tissue-resident fibroblasts. Third, using single cell RNA-sequencing, we identify heterogeneity among adhesion fibroblasts, which is more pronounced at early timepoints. Fourth, JUN promotes adhesion formation and results in upregulation of PDGFRA expression. With JUN suppression, adhesion formation is diminished. Our findings support JUN as a therapeutic target to prevent adhesions. An anti-JUN therapy that could be applied intra-operatively to prevent adhesion formation could dramatically improve the lives of surgical patients.

Management of Ileal Neuroendocrine Tumors with Liver Metastases.

PURPOSE: Assessment of treating metastatic ileal neuroendocrine tumors (NETs) with complete resection of primary tumor, nodal and liver metastases, plus administration of long-acting somatostatin analogues (SSAs). METHODS: A prospective database was queried for patients with ileal or pancreatic NETs with pathology-confirmed liver metastases and tumor somatostatin receptors. Patients did not have MEN-1 and had no previous treatment. The impacts of SSA treatment on the primary outcome of survival and secondary outcome of progression-free survival were assessed with Kaplan-Meier analysis. Log rank test was used to compare overall and progression-free survival among groups. RESULTS: Seventeen ileal NET patients and 36 pancreatic NET patients who underwent surgical resection between 2001 and 2018, who had pathology-confirmed liver metastases and confirmed tumor somatostatin receptors, did not have MEN-1, and had no previous treatment were identified. Median follow-up for patients with ileal NETs was 80 months (range 0-197 months) and 32 months (range 1-182 months) for pancreatic NETs. Five-year survival was 93% and 72% for ileal and pancreatic NET, respectively. Progression-free 5-year survival was 70% and 36% for ileal and pancreatic NET, respectively. Overall 5-year survival for pNETs was greater in those patients treated with SSA (79%) compared to those who underwent surgery alone (34%, p < 0.01). The average ECOG score was low for surviving patients with ileal (0.15) and pancreatic NET (0.73) indicating a good quality of life. CONCLUSIONS: Resection of primary lymph node and liver metastatic ileal or pancreatic NETs followed with continued SSAs is associated with an excellent progression-free and overall survival and minimal side effects.

Doxycycline Reduces Scar Thickness and Improves Collagen Architecture.

OBJECTIVE: To investigate the effects of local doxycycline administration on skin scarring. BACKGROUND: Skin scarring represents a major source of morbidity for surgical patients. Doxycycline, a tetracycline antibiotic with off-target effects on the extracellular matrix, has demonstrated antifibrotic effects in multiple organs. However, doxycycline's potential effects on skin scarring have not been explored in vivo. METHODS: Female C57BL/6J mice underwent dorsal wounding following an established splinted excisional skin wounding model. Doxycycline was administered by local injection into the wound base following injury. Wounds were harvested upon complete wound closure (postoperative day 15) for histological examination and biomechanical testing of scar tissue. RESULTS: A one-time dose of 3.90 mM doxycycline (2 mg/mL) within 12 hours of injury was found to significantly reduce scar thickness by 24.8% (P < 0.0001) without compromising tensile strength. The same effect could not be achieved by oral dosing. In doxycycline-treated scar matrices, collagen I content was significantly reduced (P = 0.0317) and fibers were favorably arranged with significantly increased fiber randomness (P = 0.0115). Common culprits of altered wound healing mechanics, including angiogenesis and inflammation, were not impacted by doxycycline treatment. However, engrailed1 profibrotic fibroblasts, responsible for scar extracellular matrix deposition, were significantly reduced with doxycycline treatment (P = 0.0005). CONCLUSIONS: Due to the substantial improvement in skin scarring and well-established clinical safety profile, locally administered doxycycline represents a promising vulnerary agent. As such, we favor rapid translation to human patients as an antiscarring therapy.