Pancreatic cancer remains one of the most challenging malignancies to treat with standard approaches. Emerging treatment approaches incorporating innovative surgical techniques and novel systemic therapies may help to improve outcomes for pancreas cancer patients. Although immunotherapy has proven to be less effective for the treatment of pancreas cancer relative to more immunogenic tumor types, multiple immune system stimulating agents are under active investigation for pancreatic ductal adenocarcinoma, either alone or in combination with other therapeutic agents. The tumor microenvironment of pancreatic cancer is also an attractive therapeutic target given that it is believed to be highly immunosuppressive and encapsulated by a dense stroma. This review article aims to summarize pre-clinical and clinical studies, including ongoing clinical trials, which attempt to incorporate novel treatment approaches for pancreas cancer. Emerging treatment approaches may help to significantly improve outcomes for this tough to treat disease.Pancreatic Ductal Adenocarcinoma (PDAC) is one of the deadliest malignancies among all cancers. Despite curative intent, surgery and the use of standard cytotoxic chemotherapy and radiation therapy, PDAC remains treatment-resistant. In recent years, more contemporary treatment modalities such as immunotherapy via checkpoint inhibition have shown some promise in many other malignancies, yet PDAC still eludes an effective curative treatment. In investigating these phenomena, research has suggested that the significant desmoplastic and adaptive tumor microenvironment (TME) of PDAC promote the proliferation of immunosuppressive cells and act as major obstacles to treatment efficacy. In this review, we explore challenges associated with the treatment of PDAC, including its unique immunosuppressive TME. This review examines the role of surgery in PDAC, recent advances in surgical approaches and surgical optimization. We further focus on advances in immunotherapeutic approaches, including checkpoint inhibition, CD40 agonists, and discuss promising immune-based future strategies, such as therapeutic neoantigen cancer vaccines as means of overcoming the resistance mechanisms which underly the dense stroma and immune milieu of PDAC. We also explore unique signaling, TME and stromal targeting via novel small molecule inhibitors, which target KRAS, FAK, CCR2/CCR5, CXCR4, PARP and cancer-associated fibroblasts. This review also explores the most promising strategy for advancement in treatment of pancreatic cancer by reviewing contemporary combinatorial approaches in efforts to overcome the treatment refractory nature of PDAC. Pancreatic ductal adenocarcinoma, PDAC, which accounts for greater than 90% of pancreatic tumors, remains a dreadful disease characterized by poor prognosis. Pancreas cancer accounts for 3% of all cancers in the Unites States (US) and 7% of all cancer deaths. Accordingly, the American Cancer Society estimates approximately 60,430 Americans will be diagnosed with pancreatic cancer and about 48,220 will die from pancreatic cancer in 2021 ,6,7. AltAlthough significant advances have been made in exploring and developing a range of novel systemic therapies for the treatment of pancreas cancer, surgical resection remains an essential component of curative intent treatment. Accordingly, innovation in surgical approaches may help to improve long-term outcomes for pancreas cancer patients. Modern advances in technical approaches to multiple aspects of pancreatic cancer surgery have been pursued in recent years. For patients with borderline resectable and locally advanced pancreas cancer, surgical resection, particularly an R0 resection, and optimal surgical approach may have the best chance of improving patient outcomes. In this patient population, techniques that optimize surgical resection and vessel reconstruction may lead to the most significant improvements in outcomes . MultiplInnovations in surgical techniques have allowed for more aggressive resection combined with reconstruction of adjacent blood vessels. For example, the Appleby procedure is a relatively well-established surgical technique that is particularly helpful for patients with locally advanced disease. This procedure involves en bloc resection of the celiac trunk and common hepatic artery along with distal pancreatosplenectomy. Data suggest that the Appleby procedure is associated with a lower risk of hemorrhage . FurtherAnother emerging area of surgical innovation in pancreas cancer treatment is the concept of the mesopancreas. A well-established body of literature has established the benefit of total mesorectal excision for the treatment of rectal cancer. This success has led surgeons to consider a similar anatomic construct for the treatment of pancreas cancer. The mesopancreas has been defined as a firm and well-vascularized structure extending from the posterior surface of the pancreatic head to behind the mesenteric vessels. Given that the mesopancreas has been found to be a common site of positive resection margins for patients with pancreas cancer involving the pancreatic head, total mesopancreas resection has been considered as a potential approach to improving clinical outcomes. A recent single institutional analysis demonstrated that total mesopancreas resection was associated with a significantly higher rate of margin negative resection and number of lymph nodes resected, as well as a lower recurrence rate and longer disease free survival . A more Irreversible electroporation (IRE) is a technique that relies on high voltage microsecond electrical pulses to induce cell membrane porosity leading to cell death. This technique has been applied intraoperatively in patients with locally advanced pancreas cancer. Compared with thermal ablation techniques, including radiofrequency ablation, microwave ablation, and cryoablation, IRE has the benefits of more well-defined areas of tumor damage with relative sparing of adjacent connective tissues and minimal impact of blood flow on its effects. Clinical studies have demonstrated that use of IRE results in significant improvements in both local and distant progression-free survival, as well as overall survival .Increasing emphasis has also been placed on clinical selection and optimizing medical management for patients being considered for surgical resection for pancreas cancer. Although advances in surgical techniques have resulted in improved post-operative mortality for patients undergoing pancreaticoduodenectomy, associated morbidity remains high. Sarcopenia is an emerging predictor of post-operative morbidity after pancreas cancer resection. A recent retrospective analysis demonstrated that sarcopenia is associated with both advanced age and preoperative albumin levels and was a significant predictor of post-operative complications . FurtherDespite advances in surgical techniques, treatment approaches, and patient optimization, disease recurrence and treatment resistance mediated by the TME and stroma of pancreas cancer remain both a serious challenge and simultaneously an avenue for therapeutic targeting as well as novel treatment advances.Normal cells live in harmony with their surrounding environment, communicating with stromal cells in ways that support survival and healthy growth even in the face of pathogens. In comparison, cancer cells have the capacity to interact with immune cells and stromal cells enhance their survival and growth. By manipulating cellular activity and selectively altering cell populations, cancer cells provide themselves opportunities to thrive and rapidly proliferate. Typically, this is accomplished by tipping the balance between pro-tumor and anti-tumor forces within the TME. This adaptive characteristic noted in PDAC plays a significant role in tumor development, serves as a primary factory in immune escape, and has been an obstacle in treatment strategies . In ordeVarious studies in PDAC have shown an upregulation of regulatory T-cells (Tregs) and immature DC’s infiltrating resected tissue regardless of stage ,20,21,22The presence of tumor promoting cells and cytokine-related inflammatory changes are thought to significantly contribute to the limited success of modern treatment strategies ,37,38,39Hypoxia is also believed to play a significant role in the TME of pancreas cancer. Hypoxia occurs in this environment as a result of increased oxygen consumption in the setting of rapid tumor cell proliferation and poor vascularization in a dense desmoplastic stromal environment. The cellular physiologic response to hypoxia is driven primarily by hypoxia-inducible factors (HIFs), which serve as active transcription factors under hypoxic conditions. Corresponding gene transcription has downstream effects on multiple pathways involved in tumorigenesis and cancer progression, including metabolic reprogramming, autophagy, maintenance of stemness, epithelial to mesenchymal transition (EMT), angiogenesis, tumor invasion, migration, and metastasis .All of the elements of the TME, from immunosuppressive myeloid cells to stromal fibroblasts, as well as a lack of high quality effector T cell, pose a significant obstacle to traditional cytotoxic therapy efficacy. However, they are also rational targets for advances in treatment. Immunomodulation, in the TME and beyond, may also prove to be an effective therapeutic strategy in PDAC.Immunotherapy can help to alleviate immunosuppressive changes observed in the TME. By reprogramming the cells of the TME, immunotherapy has the potential to trigger anti-tumor immune response. There have been a variety of immune-modulating therapies incorporated into cancer treatment in recent years, including the use of monoclonal antibody checkpoint inhibitors and cancer vaccines.Since FDA approval of the first immune checkpoint inhibitor, ipilimumab, the use of checkpoint inhibitors has proved to be among the most promising breakthroughs in modern cancer treatment. Ipilimumab, initially approved for treatment of stage III/IV melanoma, achieves therapeutic action by blocking the cytotoxic T lymphocyte antigen 4 (CTLA-4) from binding to its ligands B7.1 and B7.2, effectively functioning as an inhibitor of IL-2 production and T-cell differentiation . CTLA-4,As research in immunotherapy advanced, various other immune checkpoints were found to be of significant clinical importance, including most notably the programmed death receptor-1 (PD-1) and its ligand PD-L1. In 2014, the PD-1/PD-L1 blocking antibodies pembrolizumab and nivolumab were approved for the treatment of stage III/IV melanoma after showing efficacy ,60. SincResistance to single checkpoint inhibition has been well documented in PDAC. This phenomenon tends to occur due to upregulation of secondary compensatory pathways within the TME. In efforts to overcome this burden, studies investigating the use of dual checkpoint inhibitors have been conducted with the hope of identifying synergistic effects between the anti-PD-L1 antibody durvalumab used in combination with the anti-CTLA-4 antibody tremelimumab. Unfortunately, as demonstrated in a phase II trial, dual checkpoint inhibition failed to show therapeutic response in PDAC, and subsequent trials has continued to explore this approach .p = 0.06). This study also showed a borderline statistically significant improvement in 1 year overall survival  along with, and increase of activated myeloid dendritic cells and tumoral M1 macrophages associated with the use of standard of care chemotherapy with or without sotigalimab [In contrast to the blockade of pathways, an alternative approach to reprograming the TME is via the use of monoclonal antibodies as agonists. Among the targets of agonist therapy is CD-40, which is a costimulatory molecule present on antigen-presenting cells such as macrophages. In PDAC mouse models, CD-40 agonist enhanced the TME by promoting inflammatory action . When acigalimab .Another method of augmenting cells of the TME of PDAC is to enhance anti-tumor immune activity is using therapeutic vaccines. Like viral vaccinations, these vaccines can function by administering pancreatic tumor antigens to stimulate patient anti-tumor immune response by providing an opportunity for the immune system to recognizing variations between tumor cells and normal pancreatic cells. Current research in the field of therapeutic vaccines is studying the utility of numerous types of vaccines, most notably whole-cell vaccines and antigen-specific vaccines. Among the whole-cell therapeutic vaccines, GVAX was developed for the treatment of PDAC and consists of two allogenic cell lines that stimulate therapeutic action by inciting immune response against a wide range of PDAC antigens, ultimately leading to secretion of granulocyte-macrophage colony stimulating factor ,71,72. GAlgenpantucel-L is another whole cell vaccine under investigation. This vaccine is composed of irradiated allogenic cancer lines that have been modified to express murine α-1,3-galactosyltransferase, leading to accumulation of alpha-galactosyl residues on the cell surface . In theoMore recently, researchers have been able to further identify pancreatic tumor antigens, and peptide vaccines specifically targeting these antigens are being developed. Among these are the KRAS peptide vaccines for the treatment of pancreatic cancer. An estimated 90% of patients with pancreatic cancer have mutant KRAS specific to tumor cells ,80,81,82In addition to the paradigm shifting success of immunotherapy, over the last decade, molecular targeted therapies have also emerged as promising treatments for a range of cancer types. By directly targeting molecular pathways including those involved in tumor growth, carcinogenesis, and drug resistance, targeted therapies have offered a treatment modality that may serve as an adjuvant to chemotherapy, radiation therapy, and immunotherapy. This type of molecular targeting has been approached with the use of multiple types of agents including small molecule inhibitors (SMIs), which have a significantly smaller molecular size and weight than antibodies. SMIs have advantages in oral bioavailability, high tumor penetration, and relatively favorable toxicity profiles secondary to shorter half-life . While mFAK is among the intracellular pathways that have shown promise as a target in cancer treatment, in addition to other well-known signaling molecules including RAS-RAF-MEK1/2-ERK1/2, CCR2/CCR5, and CXCR4. Often upregulated in malignancies, FAK elicits effects including but not limited to, altering intracellular and intercellular processes involving cellular adhesion, apoptosis and survival . FAK pla+ and CD4+ T cell infiltration and a decreased TAM and Treg infiltration following the combination therapy. Currently, the sequential combination of chemotherapy, followed by anti-PD-1 and FAKi is being investigated in the neoadjuvant setting allowing for comprehensive analysis of the TME and the implications of FAKi on it (NCT03727880).Recently, a Phase I/II study of the combination of gemcitabine, pembrolizumab and defactinib in metastatic PDAC showed the combination of anti-PD1 pembrolizumab and FAKi defactinib with chemotherapy to be safe, without significant increase in toxicities compared to what we know with chemotherapy . NeverthSelectively altering chemokine signaling pathways is another approach to targeted therapy made possible by using SMIs. Among these is the targeting of CCR2 and CCR5, which are expressed on MDCS and are known to function as a monocyte recruiting signals to the TME when bound to their ligands, CCL2/CCL5 . Mouse mWhile CCR2/CCR5 can regulate the density of TAMs in the TME, another signaling protein present on MDSCs, CXCR2, is known to control chemotaxis of tumor-associated neutrophils ,105. Thop = 0.004) but not overall  survival [Certain solid tumors have shown to be deficient in various DNA repair mechanisms, such as BRCA mutations in breast cancer. These deficiencies have been exploited with platinum-based agents with some success. However, tumor cells may mitigate effects of these treatments by utilizing poly-adenosine-diphosphate-ribose polymerase (PARP) an enzyme which possesses the molecular function of detecting damage in DNA and promoting repair . The usesurvival .KRAS is another significant therapeutic target in PDAC given the very high associated rate of mutation . HistoriMultiple hypoxia-based therapeutic strategies have also been evaluated for PDAC. For example, metronomic gemcitabine has been shown to improve perfusion and reduce hypoxia in PDAC, in part due to associated functional blood vessel density. Additional vascular normalizing agents, including cediranib and nelfinavir, are also being evaluated in ongoing phase I/II clinical trials . FurtherThe peritumoral stroma is a specific component of the PDAC TME for which targeted therapeutics may help to significantly improve patient outcomes.The cancer stromal environment plays a key role in cancer cell survival and tumor growth in patients with PDAC. A number of molecular pathways that play a role in the cancer stromal environment have been identified as potential therapeutic targets for PDAC. One example is the CXCL12-CXCR4 chemokine signaling pathway. CXCR4, when bound to its ligand CXCL12, can manipulate the TME via crosstalk between stromal cells and immune cells . InhibitBecause of the central role that fibroblasts are believed to play in the stromal environment, CAFs are the target of multiple agents. Many such agents specifically target fibroblast activation protein (FAP). For example, sibrotuzumab is a humanized monoclonal anti-FAP antibody, which was evaluated in a single arm study of patients with metastatic colorectal cancer . AlthougPrior studies have also attempted to disrupt the ECM, which contributes to the desmoplastic barrier in the TME of PDAC. A significant component of the ECM is hyaluronan, which is a non- sulfated glycosaminoglycan. In fact, higher levels of tumor hyaluronan have been associated with poorer prognosis among patients with PDAC. Previous studies evaluating the use of hyaluronidase have demonstrated associated improvement in survival in multiple tumor types . A retroFAK inhibition has been shown to modulate the immune milieu and fibrosis within the TME and is accordingly being pursued in clinical studies as a potential therapeutic target along with standard chemotherapy and/or immunotherapy particularly in pancreatic cancer. Increased levels of FAK in the TME have been shown to correlate with poorer prognosis in multiple cancer types ,139. ThePDAC remains a challenging disease to treat despite advances in systemic therapy. Currently, the best treatment that results in proven clinical benefit is combination systemic chemotherapy. The phase III MPACT and PRODIGE-4/ACCORD-11 studies unequivocally showed the benefit of combination gemcitabine plus Nab-paclitaxel and FOLFIRINOX vs. single agent gemcitabine, with median survival of 8.5 and 11.1 months, respectively. These chemotherapeutic options represent the frontline agents for metastatic PDAC. Although modern chemotherapy approaches have resulted in improved outcomes, long-term survival remains poor. Combinatorial approaches with chemotherapy, radiation, immunotherapy, and/or targeted therapies may result in improved outcomes for patients with this challenging to treat disease. Given the promise of immunotherapy in improving outcomes in multiple tumor types, many emerging treatment strategies involve immune stimulation as part of the therapeutic approach. The TME generally and the tumor stroma specifically are promising targets for combinatorial therapies in patients with PDAC .Promising preclinical evidence has opened the door for clinical studies investigating FAK inhibitors in combination with chemotherapy and immunotherapy. Preclinical studies have demonstrated that FAK inhibition can potentiate the effect of anti-PD-1 therapy in pancreas cancer . In a phMultiple preclinical and clinical studies have also demonstrated potential benefit for use of other agents targeting tumor stroma in combination with immunotherapy, such as those that target CXCR2. For example, AZD5069 is a CXCR2 antagonist that blocks neutrophil migration and reduces circulating neutrophil counts. AZD5069 is currently being tested in a phase I/II trial along with gemcitabine/nab-paclitaxel and durvalumab in patients with metastatic PDAC (NCT02583477). Similarly, BMS-813160, which is a CCR2/CCR5 antagonist, is being tested in combination with nivolumab and either irinotecan/5-FU/leucovorin or gemcitabine/nab-paclitaxel in patients with advanced pancreas cancer in an effort toto assess safety and efficacy (NCT03184870).Another emerging systemic treatment combination involves the use of devimistat, which targets enzymes involved in the mitochondrial tricarboxylic acid (TCA) cycle. These alterations in the TCA induced by devimistat are believed to result in changes in mitochondrial enzyme activity and oxidation/reduction state that lead to apoptosis, necrosis, and autophagy in pancreas cancer cells. A single institution phase I trial of devimistat and FOLFIRINOX demonstrated safety and potential efficacy of this regimen among patients with metastatic pancreatic cancer . A largeCombination therapy with radiation and immunotherapy also has a promising role for improving outcomes for patients with PDAC. It has long been recognized that T cells play an important role in the anti-tumor efficacy of radiation therapy as tumor-bearing mice lacking T cells need significantly more RT dose to achieve similar tumor control . TargetiAdditionally, efficacy signals appreciated with PARPi and previous knowledge that DNA damage enhances radiation induced Type I interferon, provided similar rationale for exploring combination with PARPi, RT and immunotherapy. Preclinical studies by Zhang et al., were able to show  that in Another extremely unique and promising approach in advanced pancreatic cancer is the PANOVA-3 (NCT03377491) study. This clinical trial explores the use of Tumor Treating Fields (TTF) in locally advanced PDAC patients. TTF are electrical currents that alter the spindle apparatus and cytokinesis impacting cell mitosis. In preliminary pre-clinical studies, TTF led to the killing of rapidly dividing cancer cells, including glioblastoma cells and PDAC, where for the former there is already FDA approval . The iniThe most promising strategy for future therapeutic change in the treatment paradigm for pancreatic cancer is the Precision Promise study (NCT04229004). Precision Promise is a multi-center, Phase 2/3 platform trial designed to evaluate multiple regimens in advanced pancreatic cancer. The goal of this novel platform study is to efficiently evaluate multiple experimental therapies compared to standard of care therapy in first and second-line patients. An adaptive design of this study allows for drug approaches that are working to move forward within the context of the study, whereas less efficacious treatment modalities are removed. This is all analyzed in real time across 15 clinical trial sites. Importantly, new arms to this study which can impact cancer cells, modulate the TME and stroma are being added presently. Additionally, this study incorporates translational molecular mining and biomarker exploration to help not only inform therapy testing, but ultimately impact patient outcomes.Pancreatic adenocarcinoma remains a difficult to treat disease. Traditional forms of therapy including chemotherapy, radiation therapy and surgery though commonly employed in a multimodal fashion still have significant limitations in therapeutic potential for pancreas cancer. As a result, clinical outcomes and survival remain poor. The last two decades have brought about novel and significant advancements in therapeutics across numerous malignancies, chiefly in the domain of immunotherapy, from checkpoint inhibitors to vaccine therapy. Yet the testing of these agents in pancreatic cancer has been met with little clinical success. Promising biologic changes noted on correlative bench science in analyzed tumor specimens treated with novel agents has not consistently translated into clinical success. However, preclinical and clinical work has highlighted that the tumor microenvironment serves as not only a significant hurdle to therapeutic success of novel agents, but also serves as an attractive target for therapy. Immunosuppressive elements, such as myeloid cells, the highly desmoplastic stroma with abundance of cancer-associated fibroblasts or intracellular elements of these cells that promote disease progression, are being consistently evaluated as targets for therapy. There are some important factors to consider in developing novel and hopefully successful therapeutic approaches for pancreatic cancer in the future. First, tumor microenvironment modulation should be considered a critical and adjunctive approach in any treatment paradigm. Secondly, single agent or single therapeutic approaches are unlikely to be met with success. Combinatorial approaches using standard therapies, including surgery or surgical advances for PDAC along with novel small molecular targeting and immunotherapy, are more likely to lead to therapeutic success. Furthermore, combination approaches or agents which simultaneously address the dense stroma and immunomodulation, such as FAK inhibition are even more likely to lead to improved clinical outcomes. Meaningful clinical outcomes may result from combination immunotherapy with stromal targeting agents in pancreas cancer in an effort to overcome immunosuppression in the TME by targeting multiple immunosuppressive pathways simultaneously. Thirdly, sequencing of therapies will also be important to consider in multiagent approaches, as immune and TME priming are important to immunotherapy efficacy in a tumor type considered to be immunologically inactive. Ultimately, novel platform studies, like Precision Promise, which are assessing multiple combinatorial approaches are the most likely to lead to best treatment approach in pancreatic cancer care. Using correlative translational science from these clinical trials looking at novel combinatorial approaches to guide the development of better targeting agents and novel combinations in an iterative fashion will lead to the greatest advances in the treatment of pancreatic ductal adenocarcinoma.


Pulmonary surfactant is a complex and highly surface-active material. It covers the alveolar epithelium and consists of 90% lipids and 10% proteins. Pulmonary surfactant lipids together with pulmonary surfactant proteins facilitate breathing by reducing surface tension of the air-water interface within the lungs, thereby preventing alveolar collapse and the mechanical work required to breathe. Moreover, pulmonary surfactant lipids, such as phosphatidylglycerol and phosphatidylinositol, and pulmonary surfactant proteins, such as surfactant protein A and D, participate in the pulmonary host defense and modify immune responses. Emerging data have shown that pulmonary surfactant lipids modulate the inflammatory response and antiviral effects in some respiratory viral infections, and pulmonary surfactant lipids have shown promise for therapeutic applications in some respiratory viral infections. Here, we briefly review the composition, antiviral properties, and potential therapeutic applications of pulmonary surfactant lipids in respiratory viral infections. Pulmonary surfactant is a complex and highly surface-active material that are found in the fluid lining of the alveolar surface of the lungs . It formIn addition to lowering surface tension and preventing alveolar collapse at end-expiration, pulmonary surfactant functions as a modulator of immune responses . PreviouIn pulmonary surfactant lipids, the most abundant constituents are glycerophospholipids , 3. Surfvia the inhibition of multiple proinflammatory mediators. DPPC inhibits lipopolysaccharide (LPS)-induced cytokine production by airway epithelial cells and monocytes, and DPPC supplementation in mice attenuates lung inflammation. However, PONPC [1-palmitoyl-2-(9-oxononanayl)-PC], another component of PC, can increase the production of nitric oxide and cytokines in macrophages via the upregulation of TLR4 and Myd88 gene expression  infection, indirect evidence indicates that SARS-CoV-2 infection may induce alterations in the composition of pulmonary surfactant lipids in three ways. Firstly, SARS-CoV-2 infects ATII cells by binding angiotensin-converting enzyme 2 , and thevia intranasal inoculation can prevent some respiratory viral infections, and this may provide potential therapeutic applications for respiratory viral infections. This suggests potential therapeutic applications of pulmonary surfactant lipids for preventing or treating respiratory viral infections. We will briefly review these topics in the followings.Altered pulmonary surfactant lipid composition not only influences surface tension-related properties but also impacts the progress of inflammation following viral infections. Importantly, recent studies have shown that supplementation with several pulmonary surfactant lipids, such as PGs ] and PIs Paramyxoviridae family, and that is a leading cause of acute respiratory tract infections in early childhood , the antiviral effects of PI and POPG are achieved by their binding to RSV to block virus attachment to epithelial cells. However, it is unclear why PI and POPG have such a high affinity for RSV and how they bind to RSV. Moreover, it has not been determined whether the high affinity of PG and POPG is specific for RSV, or if this phenomenon applies to other viruses. It is also unknown if the antiviral mechanisms of PG and PI are the same. We believe these mechanisms should to be further explored as this information may be important for developing an effective strategy for controlling RSV infection.in vitro  formation RNA virus , and RNAAfter respiratory viral infection, viruses can interfere with the synthesis and secretion of pulmonary surfactant; this can cause an increase in surface tension, leading to alveolar collapse and ARDS , 39. ARDRespiratory viral infections are accompanied by an aggressive proinflammatory cytokine response that is directly related to the severity of the disease . Thus, tImportantly, recent studies have shown that POPG and PI supplementation can combat RSV and influenza infection by blocking viral replication . AlthougHowever, some pulmonary surfactant lipids such as PEs may facilitate RNA virus infection. Thus, not all pulmonary surfactant lipids can be used to treat viral infections. As pulmonary surfactant lipids contain many species and subclasses, further studies need to be performed to explore the potential functions of each pulmonary surfactant lipid in respiratory viral infections.It is worth noting that, pulmonary surfactant proteins also possess anti-viral and anti-inflammatory properties, especially SP-A and SP-D . Therefo®) was used and it consisted of a mixture of phospholipid , 5% cholesterol, 1% surfactant proteins (SP-B and SP-C), very low levels of free fatty acid, lyso-phosphatidylcholine, water and 0.3% calcium ; and sponsored by Qing Lan Project, and Natural Science Foundation of Nanjing University of Chinese Medicine (No. NZY82004204), The Open Projects of the Discipline of Chinese Medicine of Nanjing University of Chinese Medicine Supported by the Subject of Academic priority discipline of Jiangsu Higher Education Institutions , Natural Science Foundation of Jiangsu Province of China (Grant number BK20180825); Graduate Student Scientific Research Innovation Projects in Jiangsu Province (SJCX21-0696).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Quiescence has been observed in stem cells (SCs), including adult SCs and cancer SCs (CSCs). Conventional chemotherapies mostly target proliferating cancer cells, while the quiescent state favors CSCs escape to chemotherapeutic drugs, leaving risks for tumor recurrence or metastasis. The tumor microenvironment (TME) provides various signals that maintain resident quiescent CSCs, protect them from immune surveillance, and facilitates their recurrence potential. Since the TME has the potential to support and initiate stem cell-like programs in cancer cells, targeting the TME components may prove to be a powerful modality for the treatment of chemotherapy resistance. In addition, an increasing number of studies have discovered that CSCs exhibit the potential of metabolic flexibility when metabolic substrates are limited, and display increased robustness in response to stress. Accompanied by chemotherapy that targets proliferative cancer cells, treatments that modulate CSC quiescence through the regulation of metabolic pathways also show promise. In this review, we focus on the roles of metabolic flexibility and the TME on CSCs quiescence and further discuss potential treatments of targeting CSCs and the TME to limit chemotherapy resistance. Cancer stem cells (CSCs) are identified in most types of liquid and solid tumors and contribute to tumor onset, chemotherapy resistance, recurrence, and metastasis.When the bulk of the tumor cells are eliminated by adjuvant treatments, CSCs may survive in a reversible quiescent state.In both adult stem cells and CSCs, low ROS levels are frequently associated with a protective intracellular environment and with the perseverance of stem cell quiescence/dormancy.CSCs can reprogram their metabolism to flexibly adapt to environmental changes, which is considered critical for them to enhance the antioxidant compensative capacity and sustain their self-renewal ability.What gives rise to the emergence of quiescent CSCs?What kind of metabolic flexibility favors the maintenance of quiescent CSCs?Which are the therapeutic regimens that quiescent cancer cells might be resistant to and in which manner?Does the future lie in combining chemotherapies that target proliferative cancer cells treatment that target quiescent CSCs?Many of the current chemotherapies are limited to merely targeting proliferative cancer cells. The residual population of chemotherapy-resistant tumor cells capable of regenerating the cancer disease is thought to be enriched in CSCs . CSCs haRecent studies have shown that CSCs depend on different metabolic pathways compared to differentiated tumor cells, and the metabolic activities directly participate in the CSC quiescent/proliferative states transition or support tumor progression . CSCs caThe CSC quiescence is also associated with the tumor microenvironment (TME), the environment around a tumor, including the surrounding blood vessels, immune cells, fibroblasts, signaling molecules, and the extracellular matrix , 11. TheAs early as the 1970s, work on the hematologic malignancies predicted that slow-cycling leukemic stem cells cause tumor relapse , 15. InvQuiescent stem cells are poised for activation by specific energetically favorable mechanisms that are compatible with the low metabolic state of quiescence and that allow for rapid and global responses needed for activation . The NotEpigenetic modifications consist of heritable changes in gene function without alteration of DNA sequence. Epigenetic modifications include DNA methylation, chromatin remodeling, and noncoding RNAs . As cancClinical evidence on the existence of the quiescent state of tumor cells came from the transmission of cancer from transplant organ donors to immunosuppressed recipients , 33. UndHow do quiescent CSCs acquire immune tolerance? It has been proved that dormant cancer cells could evade immune surveillance by reducing antigenicity in lymphoma, fibrosarcoma, and T-lymphoma –37. In aHypoxia has been identified as a hallmark of cancer . HypoxiaTP53 was inactivated. Through the HIF1α/USP22 positive feedback loop of TP53 inactivation, hypoxic TME promotes stemness features (CD44+ and CD24+) and glycolysis in HCC cells, ultimately resulting in sorafenib resistance [As demonstrated in many studies, a hypoxic environment induces the accumulation of HIF subunits in mesenchymal and cancer cells  that binsistance . In colosistance . In addisistance . In addisistance –55.Driven by these mechanisms, a hypoxic TME is frequently associated with a more aggressive tumor phenotype. In addition, understanding the mechanism by which the hypoxic TME affects the quiescence of cancers may provide effective therapeutic opportunities.As an indispensable factor in the TME, the ECM contributes to the induction of CSCs and the initiation of tumors. Interestingly, Wang et al. observed that the resistance to EGFR tyrosine kinase inhibitors (TKIs) was conferred to lung cancer cells that were originally sensitive to TKIs after culturing them on decellularized ECM or coculturing them with ECM donor cells . AzzaritLaminin is a glycoprotein ECM component of the connective tissue basement membrane that promotes cell adhesion. Rohn et al. isolated rat hepatic stellate cells and then seeded them onto uncoated polystyrene (PS) or PS coated with either laminin-521 (PS/LN-521) or laminin-211 (PS/LN-211). PS/LN-521 improved hepatic stellate cells adhesion and better-preserved retinoid stores, as well as quiescence-associated and stem cell-associated phenotypes than PS alone . MoreoveGiven the important role of the ECM on cancer stemness, cancer-associated fibroblasts (CAFs), as the primary source of ECM production in tumors, are also worthy of our comprehensive discussion. CAFs affect tumor progression and resistance via many mechanisms, including morbid secretion of collagens, fibronectins, and ECM-degrading proteases, production of angiogenic factors, and various proinflammatory cytokines and chemokines , 61. Her+GPR77+CAFs and breast cancer cells effectively improved the engraftment formation in patient-derived xenograft models. Most importantly, the CAF subset specifically defined by CD10 and GPR77 expression was observed to be correlated with chemoresistance and poor survival in multiple cohorts of breast and lung cancer patients [+-GFP+) and primary CAFs into NOD/SCID mice, and as expected, CAFs enhanced the oncogenicity of CSCs by activating Notch3-LSD1 signaling in vivo [Su et al. showed that co-injection of CD10patients . Liu et  in vivo . Similar in vivo –67.Tissue formation and development originating from stem cells are orchestrated by a complex network of both chemical and physical properties, but recently researchers have started to focus on the effects of matrix stiffness on CSC and chemoresistance.Matrix stiffness (rigidity of extracellular matrix) is mainly depending on the composition and organization of ECM . Huang’sTraditional views of the TME solely based on cell–cell or cell–ECM interactions may not thoroughly explain the induction, selection, or preferential maintenance of CSC stemness. Thus, gaining a comprehensive understanding of the interplay between CSCs and their microenvironments may be essential for advancing CSC research and applications  to ROS-high proliferative epithelial-like ones (E-BCSCs) . MoreoveMany studies have revealed that CSCs are prone to exhibiting a glycolytic phenotype compared with their descendants. Indeed, aerobic or anaerobic glycolysis contributes to maintaining CSC phenotypes under specific conditions, such as hypoxia and nutrient limitation –82. As wAs for the impact of glycolysis on CSC quiescence, glycolysis is considered crucial for CSCs to sustain their antioxidant compensative capacity, enhance stemness, and improve self-renewal ability , 86–92. Although many studies have reported that CSCs tend to shift from OXPHOS to glycolysis when facing a nutritional or oxygen supply shortage, OXPHOS is also reported to equip certain CSCs with increased survival from metabolic austerity . OXPHOS Vlashi et al. first observed that GSCs and their progenitor cells are less glycolytic than differentiated glioma cells. Their laboratory previously reported that GSCs have lower 26S proteasome activity than nontumorigenic cells , and theIndeed, OXPHOS acts as a far more efficient source of ATP production than glycolysis. These OXPHOS-dependent CSCs make efficient use of specific limited nutrients, allowing them to obtain a selective advantage in certain TMEs.Overall, most CSC primarily relies on either glycolysis or OXPHOS . HoweverLiao et al. introduced L-asparaginase, an enzyme that catalyses the conversion of glutamine to glutamate, into the culture medium of human non-small-cell lung carcinoma (NSCLC)and pancreatic cancer cells to mimic the effect of decreasing glutamine. Mechanistically, glutamine exhaustion results in an enhancement of intracellular ROS levels through attenuation of the cellular levels of reduced GSH (a derivative of glutamine), ultimately leading to a decreased proportion of CSCs in the tumor in vivo . In addiResident quiescent CSCs made the prognosis of patients treacherous after chemotherapeutic treatment –112. Eve+/CXCR4+ cells both in vivo and in vitro. ATRA alone slightly decreases the percentage of CD133+ cells without affecting tumor growth, further demonstrating that ATRA only sensitizes CSCs rather than killing them directly.The idea of ‘waking up’ CSCs into a susceptive state to therapies arose from the observation that chemo-resistant leukemic cells became susceptive when they were induced from an undifferentiated state into a differentiated one by the use of all-trans retinoic acid (ATRA) . The sucAs mentioned above, quiescence is a mechanism whereby CSCs can be poised into a low metabolic state, therefore, interference with intracellular metabolism shows good practical value and application prospect. 2-deoxy-D-glucose (2-DG) is a glucose molecule that has the 2-hydroxyl group replaced by hydrogen, and it interferes with d-glucose metabolism. There is an increasing focus on using 2-DG to ameliorate resistance to cytotoxic therapies. For example, in triple-negative breast cancer, the more aggressive Hs578Ts(i)8 variant with a significantly increased proportion of CSC phenotype showed an enhanced ability to resist anoikis than its parental cells . FurtherPrevious studies have proved that CSCs possess relatively low intracellular ROS levels, especially in those quiescent ones. To be more specific, the increase of ROS by glycolysis disruption may lead to the differentiation into non-CSCs and the loss of stemness markers –121. TheOligomycin and rotenone are both mitochondrial OXPHOS Complex inhibitors. Gale et al. reported that combining ATP synthase inhibitor oligomycin A with trastuzumab led to regression of trastuzumab-resistant breast HER2+ tumors in vivo . MatassaBesides oligomycin A, rotenone also drew some focuses. Cruz-Bermúdez et al. demonstrated that metabolic flexibility from glycolysis to OXPHOS was responsible for cisplatin resistance in NSCLC, and strikingly, the chemoresistance could be reversed by OXPHOS inhibition using metformin or rotenone .Yang et al. used disulfiram (DSF), an inhibitor of ALDH enzyme activity, to induce quiescent-dominated CSCs into proliferative-dominated states and enhance the cytotoxic effect of cisplatin in breast cancer . SimilarSince the TME has the potential to support and initiate stem cell-like programs in cancer cells, targeting the TME components may prove to be a powerful modality for the prevention of chemotherapy resistance. CAFs remodel the tumor ECM and architecture of the TME, leading to poor infiltration of traditional chemotherapies and increased drug resistance. There is an innovative method of preventing chemoresistance by forcing activated CAFs back into quiescence. Sherman et al. documented that vitamin D receptor was over-activated in pancreatic CAFs that could drive tumorigenesis. Importantly, reversion to the quiescent state of CAFs using calcipotriol  witnessed induced stromal remodeling, increased intratumoral gemcitabine infiltration, reduced pancreatic tumor volume, and a 57% sharp increase in survival compared to chemotherapy alone . Aside fConsidering the presence of CSCs after traditional chemotherapy, treatments for CAFs may have better outcomes when they are combined with traditional chemotherapy.There is now solid evidence to support the hypothesis that quiescent CSCs give rise to the refractoriness of chemotherapies in many cancer types. New insights into CSC biology suggest that strategies merely inhibiting CSC stemness characteristics might not suffice to counteract tumor recurrence. The TME maintains the principle properties of CSCs, protects them from immune surveillance, and facilitates their relapse potential. The TME does not only provide various signals that maintain resident quiescent CSCs but also instructs progenitor cells to revert into a stem cell state when the originals are lost . Thus, t-18-fluoro-2-deoxy-D-glucose (F-18-FDG) positron emission tomography, magnetic resonance imaging, and optical imaging based on the fluorescent protein and principal properties of CSCs, the peritumoral microenvironment can be monitored in real-time and serve as a reflection of the metabolic phenotype of CSCs [Recently, via molecular imaging methods, such as F of CSCs . With th


Few studies have been developed to map the vascular structures feeding peripheral nerves, with the majority using cadaveric models and inadequate sample sizes. Preliminary evidence, while limited, indicates that the mapping of these vessels may allow or preclude certain procedures in nerve reconstruction due to the location of essential arterial inflow to the vasa nervorum. This review evaluates the evidence regarding historical, current, and emerging techniques for visualizing these vascular structures in vivo and considers their potential application in peripheral nerve vasculature. While the exact mechanism is still unknown, it has been shown that vascular supply to peripheral nerves is crucial to both normal function and regeneration after injury. When repairing peripheral nerves, surgeons aim to preserve intact anatomy and avoid mobilizing a nerve in a way that separates it from perforating vessels when possible. Knowing the location of these vessels may allow surgeons to avoid vascular damage and improve regeneration of injured/repaired nerves. As imaging technology continues to evolve, novel methods have emerged to create detailed images of vasculature in vivo.Early reports detailing the interplay between nerve injury and vascular supply were presented by Rydevik et al, Rydevik and Lundborg, and Lundborg in the 1970s.One possible explanation for persistent nervous deficiencies is a lack of vascular perfusion. It is well documented that angiogenesis is intimately connected with reinnervation, arborization, and growth of peripheral nerves.Given that inflammatory changes, axon growth, and cell regeneration require a large number of nutritive substances and oxygen, it is not surprising that damaged microcirculation can hamper neural regeneration and functional recovery.Given the apparently integral role of vascularization in the maintenance and regeneration of nervous tissue, physicians and scientists have attempted to identify the vascular connections that feed peripheral nerves. In what follows, historical methods of observation will be evaluated, as well as current and emerging imaging techniques that may be of use in visualizing these structures.Few cadaveric studies have been performed to map the connections of peripheral arteries to the vasa nervorum.Characterization of vascular structures has historically been done using techniques such as: digital subtraction angiography (DSA), ultrasound (US), magnetic resonance imaging (MRI), and laser angiography. DSA provides high temporal (≤1 second frame time) and high spatial resolution (≤1 mm) but is an invasive procedure that uses iodinated contrast material and radiation to acquire projected images. US was later used as a noninvasive method of visualizing blood flow to nerves but has limitations including inability to penetrate tissue, small imaging field, and highly operator-dependent examination quality. An MRI protocol using T1- and T2-weighted imaging is currently the preferred method as it is able to characterize the nervous structures and surrounding tissue noninvasively with high spatial resolution. A newer subset of MRI protocols termed MR angiography (MRA) has shown utility in evaluating vascular structures in vivo.Ultrasonography is a low-cost tool that has shown utility in imaging neuro vasculature. In recent years, the spatial resolution of US has improved dramatically, and its applications have evolved to include imaging of the peripheral nervous system.One study of human subjects attempted to observe the collateral vessels in the knee in patients with Buerger's disease using simple Doppler US. Researchers were able to identify collateral vessels, originating from vasa nervorum of the tibial nerve in instances of popliteal artery occlusion.One disadvantage of US techniques is the need for a skilled operator which can have dramatic effects on image quality and precision.MR neurography (MRN) protocols have been developed to provide insights into the fascicular structure of nerves. While this has been useful in evaluating nerve injuries, fascicular structure is not necessarily determinative of functional outcome.Though the literature is limited, MRI has been used to map vascular supply to peripheral nerves. One study using 3-T MRI with fat-suppressed, contrast-enhanced T1 images was able to identify intraneural vessels in 26% of patients, and even identified some perforating vessels along the sciatic nerve.While 7-T MRI improves resolution, it still does not clearly delineate the nerves and vessels in close proximity.There are two MR perfusion methods currently in use: dynamic susceptibility contrast MRI (DSC-MRI) and arterial spin labeling (ASL). DSC requires injection of a contrast agent and subsequent acquisition of a fast time series of images that track the contrast through vascular structures. ASL applies a spatially selective RF excitation pulse to a region containing large arteries and allowing this “labeled” blood to flow into the imaging plane. As T1 decay of the label takes place, it is followed by subtraction of labeled images from unlabeled control images, producing a measured signal that is proportional to blood flow. Typically, multiple images must be averaged to achieve a low SNR.MR perfusion is commonly used to study cerebrovascular disorders including: acute stroke, subacute and chronic ischemia, stenotic-occlusive disease, moyamoya disease, and vasospasm secondary to subarachnoid hemorrhage. MR perfusion can assess collateral flow, duration of occlusion and hypoperfusion, and presence and/or extent of recanalization.One early study used MR perfusion to evaluate median nerve circulation. However, their analysis was mostly qualitative, enhanced  nerves were classified as edemic and unenhanced  nerves as ischemic.Recently, Bäumer et al used dynamic contrast-enhanced MR perfusion to evaluate human peripheral nerve perfusion in 102 patients (43 control and 59 neuropathy).While MR perfusion is advantageous for generalized perfusion of a local area, other techniques, such as MRA, are often used as a baseline to further categorize results.Time-of-flight (TOF) techniques distinguish between flowing blood and stationary tissues by manipulating the magnitude of the magnetization, such that the magnitude of the magnetization from the moving spins is large and the magnetization from the stationary spins is small. This creates a large signal from moving blood spins and a small signal from stationary tissue spins.TOF-MRA has been used clinically to visualize the hemodynamics of intracranial arteries, particularly in patients suspected of having intracranial aneurisms.More recently, Shibukawa et al developed a novel time-resolved MRA called four-dimensional (4D) TOF-MRA using saturation pulse.TOF-MRA shows some benefit in mapping vasculature due to its sensitivity to blood flow and direction. Unfortunately, TOF imaging requires a long acquisition times and images are often obscured by artifacts.Phase-contrast (PC) techniques distinguish between flowing blood and stationary tissues by manipulating the phase of the magnetization, such that the phase of the magnetization of the stationary spins is 0 and the phase of moving spins is non-0. In phase difference images, faster moving spins produce a larger signal, and spins moving in one direction are assigned a bright (white) signal, whereas spins moving in the opposite direction are assigned a dark (black) signal. Thus, the vascular anatomy can be visualized, as well as the speed and direction of the blood flow.Recent advances in PC-MRA acquisition and reconstruction have allowed for high-resolution images to be obtained with shorter imaging times. Nearly all variations of PC-MRA utilize parallel imaging directly with techniques, such as SENSE or GRAPPA, but also with indirect methods such as localized coils. In the case of Cartesian  imaging, scan time is reduced by a factor of 2 to 4. Unfortunately, this still fails to adequately reduce scan times for clinically relevant coverage and spatial resolution (<1 mm).Another method for reducing scan times utilizes non-Cartesian sampling trajectories which either collect more data per excitation  or allow greater under sampling of the imaging volume without obscuring artifacts . Non-Cartesian imaging also reduces echo time, improving imaging of complex flow, and producing higher spatial resolution.In 2004, Bilecen and colleaguesIn a clinical setting, PC-MRA can show vascular morphology and gives quantitative measurements of blood velocity. Velocity data allows observers to derive hemodynamic parameters such as flow volume, relative wall shear stress, streamlines, vorticity, and pressure gradients. PC-MRA has combined anatomic vessel wall imaging, lumen visualization, and physiologic data to augment the characterization of intracranial arterial stenosis, aneurysms, vascular malformations, and dural sinus pathology.CE techniques distinguish between blood and stationary tissues by manipulating the magnitude of the magnetization  and by intravenously injecting a contrast agent into the vascular system to selectively shorten the T1 of the blood. By implementing a T1-weighted imaging sequence during the first pass of the contrast agent, images can be produced that show arteries with striking contrast relative to surrounding stationary tissues and veins.CE-MRA was introduced for clinical use approximately 25 years ago, with early iterations providing 3- to 4-mm spatial resolution in 30 seconds of acquisition timeframes. Since then, improvements in spatial resolution and acquisition time have allowed high-resolution, 3D time-resolved studies.In 2007, CE-MRA was used to determine the level and side of the suspected spinal dural arteriovenous fistula (SDAVF) and the feeding arteries in spinal arteriovenous malformations (SAVMs). These findings were corroborated via comparison with DSA of the same tissue. CE-MRA was able to reliably detect or exclude various types of spinal AV abnormalities and localize the predominant arterial feeder of most spinal AV shunts. These findings indicate that MRA may serve as a noninvasive means of focusing subsequent DSA in these patients.While CE-MRA requires an intravenous contrast to be administered, there are significant benefits to this technique in mapping perfusion of peripheral nerves. High temporal and spatial resolution can be gained from accurate timing of the contrast agent bolus, as well as the low likelihood of flow-related artifacts.OCT is a relatively new imaging technique that uses low coherence light to produce a 2D or 3D image. OCT is an ideal candidate for obtaining a submicrometer resolution, and is commonly used medically to obtain high-resolution images of the retina.A study performed on rats in vivo using OCT aimed to detect cerebral edema status post cerebral artery occlusion, and was able to detect gradual ischemic changes with high accuracy, similar to the results of an MRI.These studies demonstrate that OCT appears reliable for imaging of superficial structures, but few studies have attempted to visualize deeper structures. One such study attempted to evaluate coronary arteries following stent implantation. A high amount of resolution was achieved, but there were still shortcomings of imaging deep vessels. The study mentions that OCT needs complete blood clearance from the lumen and that thrombi casts large shadows of artifact.Optical microangiography (OMAG) is a subset of the OCT technique designed specifically for imaging blood flow in microcirculation. It is based on the principal that moving blood scatters light in such a way that it can be captured and adjusted into a 3D image.This technique is frequently applied to visualizing cutaneous blood flow. OMAG is a reliable biomarker in many pathological situations such as lesions and basal cell carcinoma.None of these studies have evaluated OMAG in the context of peripheral nerve vasculature; however, the ability to visualize microcirculation could be beneficial. Most studies using OMAG, however, view microcirculation at relatively superficial levels.Laser angiography techniques have recently been developed to combine the real-time acquisition of Doppler angiography and precision of MRA techniques, without the highly operator-dependent image quality (US) or long acquisition times (MRA).Laser angiography has been used successfully to monitor tissue perfusion in a variety of settings, including colorectal, plastic, endocrine, ophthalmologic, and vascular surgery.MRI and MRN provide ideal imaging of fascicular structure of nerves. However, fascicular regeneration does not always correlate with return to function.In addition to inherent challenges of size and tissue depth, random variation can be observed in human vascular networks, such as accessory or aberrant arterial supply.Historical imaging techniques used in nervous and vascular tissue were often unable to adequately show vascular structures at this depth with limitations including invasive nature of the procedure, poor resolution, inability to show microscopic structures, or the need for highly skilled operators.US/Doppler, OCT, and laser angiography are most useful in a clinical setting to visualize superficial structures in individual patients. US is currently limited by depth of structures and necessary operator skill. Despite these shortcomings, US is beneficial in intraoperative settings due to its mobility and rapid function. CEUS has been used successfully to measure intraneural vascularity in a variety of nerve pathologies, most commonly compressive neuropathy.Various MR techniques are most useful for mapping common vessels to develop a knowledge base for cases in which direct visualization is untenable. These may also be beneficial for nonacute visualization in individual cases, either pre- or postoperatively. While the MR perfusion has been used to detect intraneural edemic/ischemic conditions in peripheral nerves, this is an indirect method of determining the location of feeder vessels.CE-MRA produces high-resolution images of deep structures with little to no artifact and reduced acquisition time compared with TOF or PC-MRA.Currently, US imaging has demonstrated the greatest clinical utility. When a skilled operator is available, US allows surgeons to evaluate nervous vascularization to corroborate clinical symptoms and direct treatment. Multiple studies have shown that US has improved diagnostic accuracy compared with MRI.While imaging technologies are rapidly advancing, studies evaluating their efficacy in the context peripheral nerve vasculature are limited. Accurate imaging and mapping of these vessels may augment surgeons' ability to perform repairs without compromising nerve perfusion, ultimately resulting in improved postoperative outcomes. The published data, while limited, support further investigation to determine which modality(s) might be best suited to the task of locating arterial inflow to peripheral nerves. Whether by direct visualization in individual cases or by mapping structures conserved across patients, the techniques presented in this review may translate to improve patient outcomes following nerve injury.


As the cornerstone of high-grade glioma (HGG) treatment, radiotherapy temporarily controls tumor cells via inducing oxidative stress and subsequent DNA breaks. However, almost all HGGs recur within months. Therefore, it is important to understand the underlying mechanisms of radioresistance, so that novel strategies can be developed to improve the effectiveness of radiotherapy. While currently poorly understood, radioresistance appears to be predominantly driven by altered metabolism and hypoxia. Glucose is a central macronutrient, and its metabolism is rewired in HGG cells, increasing glycolytic flux to produce energy and essential metabolic intermediates, known as the Warburg effect. This altered metabolism in HGG cells not only supports cell proliferation and invasiveness, but it also contributes significantly to radioresistance. Several metabolic drugs have been used as a novel approach to improve the radiosensitivity of HGGs, including dichloroacetate (DCA), a small molecule used to treat children with congenital mitochondrial disorders. DCA reverses the Warburg effect by inhibiting pyruvate dehydrogenase kinases, which subsequently activates mitochondrial oxidative phosphorylation at the expense of glycolysis. This effect is thought to block the growth advantage of HGGs and improve the radiosensitivity of HGG cells. This review highlights the main features of altered glucose metabolism in HGG cells as a contributor to radioresistance and describes the mechanism of action of DCA. Furthermore, we will summarize recent advances in DCA’s pre-clinical and clinical studies as a radiosensitizer and address how these scientific findings can be translated into clinical practice to improve the management of HGG patients. High-grade gliomas (HGGs) are one of the deadliest and hardest to treat cancers. HGGs are fast-growing glial cell tumors found in the brain and spinal cord. In this review, we will largely be focusing on two types of HGGs: high-grade adult gliomas including glioblastoma multiforme, and pediatric diffuse pontine intrinsic gliomas (DIPG). Despite recent advances in multimodality treatment ,2,3, theDeregulated metabolism is a universal hallmark of cancer. Tumor cells take up and metabolize nutrients such as glucose and glutamine to support key energetic processes, which are actively driven by oncogenes and the tumor microenvironment. This was initially thought to be due to the development of an uncontrolled cellular mass, leading to poor vascularization of the tumor and reduced oxygen supply. However, Otto Warburg observed that glucose was fermented via the glycolytic pathway even in the presence of ample oxygen, which was later referred to as aerobic glycolysis (or the Warburg effect) . Like maDeregulated glucose metabolism can be therapeutically targeted at different levels, from the master regulators that control glucose metabolism  to key enzymes controlling rate-limiting steps in glycolytic pathways. One promising target is the pyruvate dehydrogenase kinase (PDK)/pyruvate dehydrogenase (PDH) axis. The inhibition of PDH activity by PDK is associated with many disorders including cancer. Dichloroacetate (DCA) is an inhibitor of PDK with the capability of maintaining PDH in its unphosphorylated active form . It was originally used as an experimental drug to treat lactic acidosis induced by congenital mitochondrial disorders and has recently been repurposed as an anticancer drug ,9,10,11.In this review, we will discuss the tumor microenvironment and altered glucose metabolism in HGGs. We will review DCA mechanisms of action and how this impacts tumor metabolism, followed by a summary of recent animal and human trials of DCA in HGGs in combination with radiotherapy (RT). The potential to translate these scientific findings into clinical practice is also discussed with the goal of improving the management of patients with HGG.Like most cancer cells, HGG cells have altered cellular metabolism and are capable of shifting their primary metabolic pathways in response to nutrient availability in the surrounding microenvironment . HGG celGlucose metabolism, or glycolysis, converts one molecule of glucose into two molecules each of pyruvate, ATP, and NADH . There aThe Warburg effect can result in decreased pyruvate entering the mitochondria, decreasing OXPHOS. Glycolysis only yields 2 molecules of ATP from glucose, while glycolysis combined with OXPHOS yields up to 38 molecules of ATP, leading to the question of why cells would utilize metabolic pathways with a lower energy yield. It was initially believed that the Warburg effect was occurring due to defective mitochondria, but studies have shown that OXPHOS increases in HGG cells, indicating the presence of functioning mitochondria . It is nAlong with upregulated glycolysis, HGG cells are still capable of utilizing glucose coupled with OXPHOS . Recent Oxygen is required for oxidative metabolism, and hypoxia is common in HGGs ,34. GlioThe Warburg effect can be partially driven by HIF-1α. Cancer cells have increased growth factor signaling, mutations , and accumulation of TCA intermediates (succinate and fumarate), which increase and stabilize HIF-1α even in the presence of sufficient oxygen . FurtherAs glycolysis increases and uncouples from the TCA cycle by producing lactate, glutamine metabolism is reprogrammed to top up and maintain flux of TCA intermediates. HIF activation assists switching from oxidative decarboxylation to reductive carboxylation of glutamine to maintain citrate and acetyl-CoA for lipid synthesis ,47,48. H0 and late S-phase), (iii) alterations of gene expression and microenvironment which counteract the effect of RT, (iv) induction of autophagy in cancer cells as a self-protective mechanism, (v) generation of cancer stem cells (CSCs), and (vi) rewiring of metabolic pathways to be more radioresistant [RT is the cornerstone of treatment for many types of cancer including HGGs. RT is used either as a palliative treatment for inoperable HGGs or following surgery to kill residual HGG cells in the tumor bed. The efficacy of RT primarily relies on its ability to damage the DNA of cancer cells and subsequently cause cell death . Despiteesistant ,52,53.Most HGGs will relapse due to the presence of radioresistant GSCs, which are enriched in hypoxic regions of the tumor . Oxygen RT induces changes in the tumor microenvironment and adjacent healthy brain parenchyma. These include changes in cellular bioenergetics, vascularity, and immune activation leading to altered drug metabolism, reduced therapeutic efficacy, and/or radioresistance . MetabolThe PDK/PDH axis is considered the gatekeeper linking cytoplasmic glycolysis to the TCA cycle and OXPHOS in mitochondria . PDH is DCA was identified as a metabolically active moiety of diisopropylammonium dichloroacetate back in 1970 . AlthougThe relapse of HGGs following radiotherapy is due in part to intrinsic or acquired radioresistance mediated by hypoxia and deregulated glucose metabolism. DCA in this setting could represent a strategy to overcome tumor radioresistance by suppressing HIF-1α, altering glucose metabolism and inducing higher levels of ROS in tumor cells. Our own studies have demonstrated the efficacy of DCA to increase radiosensitivity in both adult and pediatric HGG pre-clinical models ,32.There is a growing interest in targeting CSCs, which are one of the main drivers of radioresistance . CSC/GSC50 of DCA is comparable among HGG stem cells (15–40 mM) [50 values are at millimolar levels, concentrations of DCA as low as 0.1 and 1 mM still induce radiosensitization, decrease clonogenic survival, and increase sub-G1 specific apoptosis in human primary glioblastoma cells [Despite the metabolic heterogeneity of HGGs, the cell survival/viability (in vitro) IC5–40 mM) , immorta5–40 mM) ,71. Althma cells . DCA appma cells ,11. Usinma cells .DCA may also have anti-cancer potential when used in combination with radiotherapy and other drugs. In glioblastoma, standard treatment comprises fractionated radiation with concomitant and adjuvant temozolomide chemotherapy . TemozolHGGs are characterized by VEGF-mediated vascularization leading to highly disorganized tumor vasculature, focal regions of tumor hypoxia, and impaired oxygen-dependent RT-induced DNA damage. In glioblastoma patients, bevacizumab, an anti-angiogenic monoclonal antibody that targets VEGF-A, has been explored in conjunction with standard therapy to reduce tumor vascularization and restore normal vessel architecture ,74. UnfoDual-targeting of HGG bioenergetics using DCA in combination with other metabolic blockades has also been trialed to circumvent the plasticity HGG cells possess in adapting their ‘primary’ metabolic pathways following chemoradiation . DCA comIn addition to the direct actions of DCA and other metabolic inhibitors on cancer cells, these drugs alter the tumor immune response via shifts in cytokine profiles, differential transcriptional regulation, abrogation of the immunosuppressive network, and reduction in lactate ,80. GiveWhile DCA leads to radiosensitization of HGG cells in vitro, these results have not always been able to be replicated in vivo. In vitro DCA/ranolazine significantly reduced clonogenicity of HGG cells and induced ROS, DNA damage, autophagy, and apoptosis. Despite these initial results, only a modest ~20–30% increase in in vivo median survival was seen in orthotopic syngeneic murine HGG models . The conZwicker et al. demonstrated DCA can induce radiosensitization in LN18 glioblastoma and WIDR colorectal cells in vitro, but not in vivo . HistopaIn a rat allograft 9 L glioblastoma model, biodegradable wafers were utilized to deliver high-dose DCA within millimeters of the tumor . These wFinally, the response of cells to DCA may be influenced by post-transcriptional regulation of key metabolism gene expression, such as via microRNAs. From bioinformatic databases, miR-144 is predicted to target PDK1, TIGAR, IDH1, and IDH2 , which aThere are five published reports of phase I/II clinical trials using DCA for the treatment of cancer patients . Two triLong-term administration of millimolar concentrations of DCA presents several challenges in the clinical setting. A pilot study in glioblastoma patients  indicateIn the largest clinical trial of DCA, Chu et al.  enrolledIn a “3 + 3” study design in 15 patients , DCA dosing was based on haplotype variation in glutathione transferase zeta 1/maleylacetoacetate isomerase (GSTZ1/MAAI), which participates in DCA and tyrosine catabolism . PatientDue to the potential impact of GSTZ1 genetics on DCA metabolism, an Australian study by Tian et al.  investigIt is worth noting that a reversible peripheral neuropathy was reported from DCA treatment in several species, including humans. However, recent clinical trials indicate that adults are considerably more susceptible to this adverse effect than children. A later study then evaluated the kinetics of DCA and found a striking age-dependent decrease in DCA in its plasma clearance and an increase in its plasma half-life in both murine models and human patients . This agAll of the clinical trials of DCA are early-phase studies and, as such, conclusions about efficacy are limited. There does however appear to be at least some evidence of modest activity, and it can be generally safely delivered.Targeting metabolism of HGG cells with DCA represents a new pharmacological approach to treat cancer. The ability of DCA to reverse the aberrant glucose metabolism has increased interest in this drug, which is already known for its anticancer properties. Recent evidence in vitro and in vivo confirms the capability of DCA to overcome radioresistance in several cancer types including HGGs. The conclusion from the clinical studies is that DCA is generally safe, though the effect of GSTZ1 genetics on pharmacokinetics, toxicities, and efficacy needs to be explored further. The very limited clinical data do support some clinical activity of DCA but are probably best trialed in cancer populations, in combination with other established anti-cancer therapies such as radiation therapy, and in those tumors which are not rapidly progressive due to the time required for DCA to achieve therapeutic levels in serum.There is a growing number of pre-clinical studies designing and testing new formulations of DCA (reviewed by Tararanni and Piccoli ). The go


Antifreeze proteins (AFPs) or thermal hysteresis (TH) proteins are biomolecular gifts of nature to sustain life in extremely cold environments. This family of peptides, glycopeptides and proteins produced by diverse organisms including bacteria, yeast, insects and fish act by non-colligatively depressing the freezing temperature of the water below its melting point in a process termed thermal hysteresis which is then responsible for ice crystal equilibrium and inhibition of ice recrystallisation; the major cause of cell dehydration, membrane rupture and subsequent cryodamage. Scientists on the other hand have been exploring various substances as cryoprotectants. Some of the cryoprotectants in use include trehalose, dimethyl sulfoxide (DMSO), ethylene glycol (EG), sucrose, propylene glycol (PG) and glycerol but their extensive application is limited mostly by toxicity, thus fueling the quest for better cryoprotectants. Hence, extracting or synthesizing antifreeze protein and testing their cryoprotective activity has become a popular topic among researchers. Research concerning AFPs encompasses lots of effort ranging from understanding their sources and mechanism of action, extraction and purification/synthesis to structural elucidation with the aim of achieving better outcomes in cryopreservation. This review explores the potential clinical application of AFPs in the cryopreservation of different cells, tissues and organs. Here, we discuss novel approaches, identify research gaps and propose future research directions in the application of AFPs based on recent studies with the aim of achieving successful clinical and commercial use of AFPs in the future. Long before the term cryopreservation was coined, storage at lower or subzero temperatures has been used for simpler applications like food preservation . With reThe stages in cryopreservation generally involve the selection or harvesting of the sample, freezing, storage in liquid nitrogen and thawing but cryopreservation research has revealed some challenges majorly attributed to ice-induced injury or damage to cells during ice formation, ice nucleation and/or ice recrystallization (IR). To overcome these drawbacks, the addition of cryoprotectants or cryoprotective agents (CPAs) before sample freezing or vitrification has become the mainstay . CPAs arAFPs are impermeable CPAs which have also been referred to as thermal hysteresis proteins and ice structuring or binding proteins based on their mechanism of action  which isThis review delves in-depth into the advances in applications of AFPs in hypothermic storage and cryopreservation. We have studied, documented and discussed with evidence the relevance of antifreeze proteins to cryopreservation of oocytes, sperm cells, embryos, cancer cells, various tissues and organs, foods, etc. The challenges encountered therein and approaches for future research are also explored, which will serve as a guide to the recent trends in AFP application and stir the intellect of researchers towards innovative thinking as we strive to attain clinical approved and commercial AFP usage in cryopreservation protocols. Flavobacterium frigoris ice-binding protein (FfIBP), type III AFP, Leucosporidium ice-binding protein (LeIBP) and Tenebrio molitor AFP have been more extensively studied. TH is mostly determined using differential scanning calorimetry (DSC) [Presented below  is a sumry (DSC)  and AFPsry (DSC) . Ortiz ery (DSC) .Glaciozyma sp. ice-binding protein (LeIBP) [Recently, numerous research has been performed to evaluate the substitutability of AFPs in a bid to achieve better cryopreservation of oocytes and embryos. Cryopreservation of oocytes is a major breakthrough in reproductive medicine, especially considering the ethical, religious or legal controversies surrounding embryo preservation in some regions . Assays  (LeIBP) . To further the research, Lee et al.  performeAnatolica polita-derived AFP to the controlled slow freezing versus vitrification Sheep embryos where vitrification resulted in higher survival and embryo hatching rates, and 10 μg/mL of the AFP promoted hatching of slow-growing vitrified embryos [Scophthalmus maximus) [Sparus aurata) [Furthermore, embryo cryopreservation and the quality of embryos produced with cryopreserved gametes is of utmost importance, especially in clinical applications. Li et al. included  embryos . Similar embryos  and turbmaximus)  embryos,maximus) ,74, AFP  aurata)  and shee aurata) , LeIBP i aurata) . To the Researchers have resorted to investigating other potential cryoprotectants like ectoine  since thEffective long-term spermatozoa storage is crucial in reproductive medicine, especially for bioconservation, artificial insemination/in vitro fertilization and to accommodate the high demand of various animals and seafood. For instance, consumption of abalone is considered beneficial to health and of high demand in countries like China and Japan ; human aHaliotis discus hannai  spermatozoa with AFP III [Dendroides canadensis antifreeze protein) and AFGPs have been applied in sperm banking research, e.g. sea bream sperm [Bubalus bubalis) sperm [Acipenser persicus) sperm [Cyprinus carpio) sperm [Macaca fascicularis) sperm [Hossen et al. obtained better outcomes by supplementing cryopreservation of  AFP III . AFPs inam sperm ,86, buffs) sperm ,88,89,90s) sperm , rabbit s) sperm , Persians) sperm , roosters) sperm , common o) sperm , human so) sperm  and crabs) sperm , where ls) sperm . AFP IIIs) sperm  comparedAcipenser ruthenus) sperm [Contrary to the above studies, Xin et al. report no significant improvement using AFP I and III in the cryopreservation of sterlet (s) sperm  and no ps) sperm . This miCryobanking of cancer and stem cells have become an integral part of cell-based therapy  where thLolium perenne glycoproteins were unproductive but the findings attempt to explain the mechanism of cellular ice crystal injury/damage [AFP III supplemented with DMSO was tested on the cryopreservation of monolayer adenocarcinomic human alveolar basal epithelia (A549) cells . The effy/damage .Ictalurus furcatus (blue catfish) [In stem cells, Huelsz-Prince et al.  supplemecatfish) . TestingA potent strategy in combating insulin dependent diabetes mellitus is the introduction of insulin-secreting cells into the hepatic portal vein. Unfortunately, such cells are not always readily available; therefore, an effective means of preserving them where insulin release is not lost has to be developed and standardized. Fish derived AFPs I, II, III and an AFGP have been used in a study  aimed atThe little evidence retrieved of AFP application in cryopreservation of pancreatic cells indicates the opportunity for more studies especially with human derived pancreatic cells. Human ovarian tissue (OT) cryopreservation is beneficial for the restoration of female reproductive capability following ovarian cancer chemotherapy. OT cryopreservation has been performed previously using DMSO , EG 117117 and sA combination of 5% DMSO and 0.1 mg/mL LeIBP has been beneficial in the cryopreservation of hamster ovary cells (CHO-K1) (99). The synergistic effect FfIBP and type III AFP at 10 mg/mL on the vitrification of mouse ovaries was carried out by Kim et al. in 2017 (17). Although the combination offered marked protection of ovarian follicles against cryodamage, the results were not statistically significantly different from when the AFPs were used individually. This suggests the need for more investigations into the synergistic effect of other AFPs at varying concentrations and storage conditions. Moreover, investigation into the clinical application of these findings and further studies to determine the exact mechanism through which IBPs are able to preserve ovarian tissue during the freeze-thaw cycle is recommended. Another approach is the addition of AFPs to the warming stage after vitrification with other cryoprotectants—a strategy tested and proven effective in the vitrification and warming of tissues ,124. KonIn 2021, Kong et al. went further to investigate the effect of LeIBP in inhibiting IR during the warming of vitrified bovine ovarian tissue, which is closely anatomically related to the human OT. Findings revealed reduced changes and apoptosis in follicles, reduced fibrosis and better tissue micro-vascularization. Although the concentrations of AFP used may not have affected the outcome as results did not vary remarkably, 20 mg/mL of LeIBP showed high serum estradiol concentrations indicative of successful OT xenotransplantation post-cryopreservation . A combination of 5% DMSO and 0.1 mg/mL LeIBP has been beneficial in the cryopreservation of hamster ovary cells (CHO-K1) . The synGlaciozyma antractica yeast in the cryopreservation of rat skin grafts and compared its activity with that of glycerol. Subzero storage was done at −4 °C for 72 h followed by 21 days post-transplantation examinations of the skin layers for variations in anatomy and mechanical properties. Results showed that 5 mg/mL of Afp1m is a prospective candidate as it appreciably preserved the skin grafts. In 2019, Khan et al. [Ibrahim et al.  employedn et al.  conducteUsing the rat heart model, hypothermic preservation at 4 °C with marked conservation of hemodynamic and metabolic properties of the organ using 500 μg/mL of codfish AFGP for up to 120 min  and survTransplantation of Pancreatic Islet in the treatment of diabetes is often hindered by organ rejection via auto and/or alloimmune responses. Immunosuppressants are often administered to inhibit these immune responses but adversely, they can be toxic to the transplanted organs. Boris et al.  added a The safety and quality of food are of principal consideration during storage; therefore, agents used in preservation have to be carefully selected to avoid the risk of toxicity/poisoning. An Ice structuring protein (ISP) isolated from wheatgrass juice was found to improve the freezing and thawing of commercially available pasta sauces . More thHippophae rhamnoides (seabuckthorn) seeds on green beans [Cyprinus carpio L.) would usually result in loss of freshness by causing aggregation of microfibrillar proteins and loss of moisture. According to Du et al., the cryoprotective effect of ISP from ryegrass is a potential candidate for mirror carp storage as it minimized the damage [To confirm the effectiveness of this technique, further research into the preservation of flavour, colour, texture and nutritional value following freezing and thawing would be key. Notably, this is seen in studies to evaluate the effect AFP from en beans  and thaten beans  and cheren beans . These se damage . Tenebrio molitor larvae AFP in vegetables [Micropterus salmoides) [Pagrosomus major) [Good quality bread has been produced from dough preserved with recombinant carrot antifreeze protein (132) and barley antifreeze protein . In a lagetables , recombigetables , cold-acgetables , Herringlmoides)  and red s major) , where tHosta capitata meristems [Fish AFPs I and III have been used in an attempt to stimulate the germination of tomato seeds which would generally not happen at temperatures below 10 °C . Compareeristems . Solanum tuberosum) shoot tips to upregulation of low temperature-related genes [Chrysanthemum morifolium (Borami) shoots by AFP III [Lolium perenne AFP genes in transgenic tomato cell lines [Alternatively, Seo et al. attribute the cryopreservative effect of AFP III on potato (ed genes . This in AFP III . Balamurll lines . This isStreptococcus thermophilus. Although the clinical application of AFPs is still limited, with some, e.g., human sperm  and blooD. canadensis shortened the onset of ice nucleation and produced better post thaw viability of embryonic rat thoracic aorta smooth muscle cells when used with DMSO [LeIBP has also aided cryopreservation of cells like fibroblasts, proteoblasts and keratinocytes  and red ith DMSO . Moreoveith DMSO .The AFP discovery and research trend  and TablIt is therefore pertinent to conduct studies focused on structural elucidation, classification and THA determination. Usman et al.  propose The extensive clinical application of AFPs has also been hindered by toxicity and immunogenic responses  as seen To overcome these obstacles, other methods are emerging. These methods include but are not limited to the use of AFP mimetics like nanoparticles  and syntAFP mimetics are materials that confer protection against cryoinjury by imitating the mechanism of action of AFPs and recently, many researchers are investigating these mimetics as a potential replacement for AFPs in the future. Polyvinyl alcohol, metallohelices and polyampholytes mimic AFPs by inhibiting ice recrystallization while ammonium polyacrylate and complexes of zirconium acetate act by inducing thermal hysteresis and shaping ice, respectively. From the understanding of the stages in cryopreservation, it is possible that the use of an AFP mimetic with a singular mechanism of action might not be completely effective throughout the cryopreservation process. Hence, we propose that supplementation with other mimetics of varying mechanisms might yield better results.In the case an AFP’s use is only limited due to immunogenic reactions, it is suggested to research techniques to ensure complete AFP removal post-thaw and also to evaluate the effect of co-administering immunosuppressants. Another potent strategy to overcoming AFP toxicity is to study how they are metabolized and eliminated naturally in AFP utilizing organisms. This might provide insights on how to either simulate this process or restrict AFP use to only systems that are capable of such metabolic reactions. More research on the optimal conditions suitable to avert denaturing of AFPs is also encouraged.This review has also revealed significant progress and potential commercialization of AFP utilization in food cryopreservation. This is probably because most of these food sources especially fruits and vegetables may share genealogy with naturally AFP producing plants hence better tolerance and cryopreservation outcome. Notwithstanding, in-depth safety and toxicity studies have to be conducted before such preserved foods can be consumed. The concept of cryopreservation is applicable in other atypical research fields such as spaceflight experiments involving living samples , cryosleIn conclusion, AFP application in cryopreservation has shown good prospects for future optimization, validation, standardization and large-scale application. There is scientific evidence to support the successful application of AFP III in cryopreservation of human sperm, A549 cells, and HepG2 cells, and AAGP in cryopreservation of human islets. The few cases where indifferent or negative results were obtained for instance the unproductive use of Lolium perenne AFGPs in cryopreservation of HepG2 cells should be considered as pointers to where improvement and further research is needed rather than deterrents. AFP mimetics are gaining enormous research attention, we envision them as the solution to most of the challenges encountered in AFP applications and encourage studies in this direction. This and other novel investigations would permit the use of AFPs and its derivatives as potential ingredients in clinically approved cryopreservation solutions.