Patients diagnosed with cancer often undergo considerable psychological distress, and the induction of the psychological stress response has been linked with a poor response to chemotherapy. The psychological stress response is mediated by fluctuations of the hormones glucocorticoids (GCs) and catecholamines. Binding to their respective receptors, GCs and the catecholamines adrenaline/noradrenaline are responsible for signalling a wide range of processes involved in cell survival, cell cycle and immune function. Synthetic GCs are also often prescribed as co-medication alongside chemotherapy, and increasing evidence suggests that GCs may induce chemoresistance in multiple cancer types. In this review, we bring together evidence linking psychological stress hormone signalling with resistance to chemo- and immune therapies, as well as mechanistic evidence regarding the effects of exogenous stress hormones on the efficacy of chemotherapies. Activation of these systems results in host priming responses including vasoconstriction and tachycardia, as well as metabolic catabolism and redirection of resources to stressed sites, designed to allow for a “fight-or-flight” response. However, in the recent evolution of humans, what promotes the initiation of the stress response has changed dramatically, while the actual stress response has not. Modern environmental stressors identified include socioeconomic burden, social isolation and negative life events as opposed to life-threatening survival situations. As such this presents an evolutional disparity between stimuli and response, where responses designed for a “fight-or-flight” such as an increase in appetite to fuel increase energy expenditure, are chronically overstimulated. This exposure to sustained stress hormone signalling has been linked to an increased risk of diseases such as hypertension, immune dysfunction and cancer. The main objective of the present review is to describe the available knowledge linking psychological stress hormone signalling with resistance to chemo- and immune therapies in human and mouse models. A secondary goal is to discuss the mechanistic evidence regarding the effects of exogenous stress hormones on the efficacy of chemotherapies.The psychological stress response is designed to facilitate a response to perceived threat in order to maintain homeostatic equilibria. In humans, the stress response is controlled by fluctuations in hormonal secretions, primarily glucocorticoids and catecholamines, under the control of the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system (SNS). The glucocorticoid cortisol, an important hormone released under stressful conditions, is synthesised from cholesterol in the adrenal cortex. It is released when corticotropin-releasing hormone in the hypothalamus triggers the anterior pituitary to release adrenocorticotropic hormone, in response to complex stress signalling from neurons and somatic cells. This in turn stimulates the release of cortisol by the adrenal cortex. Cortisol can then act upon a number of systems to regulate homeostatic mechanisms by binding to its cytoplasmic glucocorticoid receptor (GR), present on nearly every cell in the body. Activation of the GR translocation to the nucleus where gene activation and transcription can be negatively or positively modulated. The GR is able to bind to specific glucocorticoid response elements (GREs) in gene promotor regions of DNA, and facilitate biphasic transcriptional activation (transactivation). GRs are also able to interact with transcription factors such as AP-1 and NF-κB, inhibiting their transcriptional ability, thus supressing gene expression by a mechanism known as transrepression. In this way GC through the GR can regulate a number of crucial genes controlling metabolism, survival, and apoptosis. GCs can also suppress immune responses and activate anti-inflammatory pathways by downregulating expression of certain interleukins (ILs), thus decreasing immune cell numbers and activation. This is thought to be a protective mechanism, directing the body’s resources away from non-essential processes. The role of GC as anti-inflammatories and their role in suppressing the immune system has been extensively reviewed elsewhere. Synthetic corticosteroids are regularly administered to cancer patients alongside conventional chemotherapies to combat hypersensitivities and reduce inflammation, as well as acting as antiemetics to relieve nausea and vomiting. In lymphoid cells, GC are pro-apoptotic and are regularly used as an anti-cancer therapy, with the potent synthetic GC dexamethasone used regularly with success. However, evidence suggests they may have opposing effects in some solid tumours, contributing to resistance to chemotherapy Glucocorticoids (GCs) are regulated though the HPA axis and function in many roles, including roles in the circadian rhythm as well as mediating adaptive responses under the stress system. Activation of β-adrenergic receptors stimulates synthesis of cyclic adenosine monophosphate (cAMP) which acts as an effector in a range of cellular processes. As a result of cAMP upregulation activation of protein kinase A (PKA) can occur, which in turn allows phosphorylation of a number of PKA-receptive proteins involved in cell survival, proliferation and gene transcription. Induction of gene transcription occurs as a result of PKA-induced phosphorylation of transcription factors responsible for the transcription of genes promoting stress response elements, such as cell differentiation and metabolismThe catecholamines adrenaline (A) and noradrenaline (NA) are produced in the adrenal medulla by chromaffin cells and released into circulation when stressors activate the SNS. Neural fibres of the SNS are able to release these neurotransmitters into all the major organ systems within seconds, allowing for rapid physiological responses. Once released the effects of catecholamines are mediated by α- or β-adrenergic receptors (ARs), which exist in subtypes  and are distributed throughout tissues accordingly. α-adrenergic receptors primarily mediate vasoconstriction and contraction of smooth muscle and are present on vascular muscle. β-adrenergic receptors also regulate muscle contraction with β1-adrenergic receptors located on myocardial muscle acting to increase blood pressure and heart rate thus increasing blood flow to skeletal muscles. β2-adrenergic receptors present on bronchial smooth muscles facilitate muscle relaxation and vasodilation and also stimulate glucose metabolismStress hormones mediate a wide range of biological processes in the cancer setting, and as such the effects of stress hormone signalling on chemotherapies is thought to be due to various mechanisms and dependent on the chemotherapeutic agent. The effects of stress hormone signalling on the efficacy of various anti-cancer treatments is reviewed below.. Several cancers have shown good responses to taxane treatment, however acquired resistance is common following long term exposure. Stress hormones have been shown to mediate effects on the cell cycle in breast cancer cells, upregulating cyclin-dependent-kinase 1 (CDK-1), which lead to cell cycle progression through the G2/M stage. In this way stress hormones can reduce the efficacy of paclitaxel which acts on rapidly proliferating cells by inhibiting paclitaxel-induced G2/M arrest. This mechanism has been further corroborated by an in vivo study demonstrating that the induction of psychological stress promotes tumour growth in mice treated with paclitaxel, negating its anti-tumour effect.Taxanes such as Paclitaxel and Docetaxel are widely used chemotherapeutic agents capable of disrupting microtubule formation and arresting the cell cycle, in turn inducing apoptosis, and exposure to both GCs and catecholamines has been shown to increase levels of ROS/RNS in breast cancer cell lines. The repair of damaged DNA has also been shown to be affected by stress hormones through interference with checkpoint kinase signalling, facilitating circumnavigation of cell cycle checkpoints which function to halt the cell and allow for repair. In breast cancer cells treated with stress hormones the expression of DNA damage response proteins was upregulated, specifically the Kinase ATR which initiates a signalling cascade activating p21 to halt the cell cycle. This allows the cell time to repair the damaged DNA. The CDK inhibitor p21 functions to inhibit progression into the S phase of the cell cycle, and it is proposed that the arrest of cells caused by the addition of stress hormones prevents the action of chemotherapies such as paclitaxel that act on dividing cells. The effects of GCs specifically on cell cycle arrest and the subsequent effect on cytotoxic anti-cancer treatments is reviewed extensively in. GCs can also affect p53, indeed restraint stress decreased levels and function of p53 in mice and promoted the growth of human xenograft tumors in a p53-dependent manner. P53 induced P21, Noxa and the pro-apoptotic gene PUMA were all decreased in response to restraint stress. The same group showed that this mechanism occurred through induction of serum- and glucocorticoid-induced protein kinase (SGK1), which in turn increases MDM2 activity and decreases p53 function potentially leading to DNA damage.It is also proposed that the induction of DNA damage can induce resistance to chemotherapies in breast cancer. Following DNA damage, activation of the serine/threonine kinases, ATM (ataxia-telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related), termed DNA sensors, may occurMDR-1, which encodes for a membrane transporter (P-glycoprotein) that can transport chemotherapeutics such as paclitaxel out of cells. Adrenaline was shown to upregulate multi-drug resistance 1 (MDR-1) in breast cancer cells, and reduce sensitivity to paclitaxel through a reduction of accumulation of the drug within the cell. In a mouse model of psychological stress the concentrations of both cortisol and noradrenaline were increased in the plasma, and MDR-1 was significantly expressed in the primary tumour after 26 days. Tumour regression induced by doxorubicin treatment was further abolished by the induction of psychological stress, and this was reversible by knockdown of MDR-1. These findings were corroborated in a further study using colon adenocarcinoma cells. Stimulation of the β-adrenergic receptor by adrenaline increased expression of MDR-1 and function of P-glycoprotein, and enhanced chemoresistance to 5-fluorouracil treatment.Several mechanisms have been proposed to explain the development of resistance to paclitaxel, including overexpression of multi-drug resistance (MDR) genes such as in vitro have been shown to induce resistance to chemotherapies. In xenograft models of breast and ovarian cancer, pre-treatment with dexamethasone inhibited the therapeutic efficacy of paclitaxel and allowed tumour growth to continue. It was hypothesised that GCs may prevent cells entering an apoptotic pathway after mitotic arrest, and as such interfere with chemotherapy-induced apoptotic cell death.Administration of GCs ,28. Additional experiments revealed stimulation of the GR by dexamethasone prior to chemotherapy was able to reduce the cytotoxic potential of common chemotherapeutic agents including paclitaxel and doxorubicin in breast cancer cell lines. It is proposed that activation of the GR induces expression of serum/glucocorticoid regulated kinase 1 (SGK-1), which protects cells from chemotherapy-induced apoptosis, and this was shown to be reversible by blockade of the GR.However, little was understood about the mechanism by which GCs may exert anti-apoptotic effects in cancer cells challenged by chemotherapy. Activation of the GR has previously been shown promote cell survival signalling pathways and inhibit pro-apoptotic signalling in mammary epithelial cells. MAPK activation can be induced by chemotherapy agents and leads to rapid induction of apoptosis. In this study paclitaxel was shown to induce MAPK signalling and subsequent apoptosis in breast cancer cells. However, MAPK phosphatase-1 (MKP-1), an inactivator of MAPK has been shown to be transcriptionally regulated through activation of the GR. Pre-treatment with dexamethasone rapidly induced MKP-1, which was able to inhibit MAPK signalling and promote cell survival.Further investigation into molecular signalling downstream of the GR revealed GCs may also contribute to cell survival through interference with mitogen-activated protein kinase (MAPK) signalling. One dose of dexamethasone was administered to patients during intra-operative biopsies, and samples of tumour collected pre and post infusion. Treatment with dexamethasone significantly increased the expression of SGK-1 and MKP-1 in ovarian tissue compared to control samples. This finding was in concurrence with the in vitro data obtained from ovarian cancer cell lines, which showed that expression of both SGK-1 and MKP-1 was rapidly induced by activation of the GR and could be reversed through GR blockade. Importantly this finding revealed that clinically relevant pharmacological doses of dexamethasone had significant effects on the expression of anti-apoptotic genes in tumour tissue, which may persist during chemotherapy, potentially reducing the efficacy of treatments.Drawing together these two anti-apoptotic mechanisms, and to confirm the effect of GCs on the regulation of anti-apoptotic genes, the expression of SGK-1 and MKP-1 was evaluated in a clinical setting in ovarian cancer patients. The effects of GC on paclitaxel resistance are summarised in In another study of Triple Negative Breast Cancer (TNBC) resistance to paclitaxel was also induced by activation of the GR, and this was shown to occur as a result of activation of the transcription co-activator YAP. The GC mediated activation of YAP also promotes the expansion of cancer stem cells (CSCs), which are both highly metastatic and often resistant to chemotherapy. Platinum-based therapies exert their effects through induction of apoptosis by cross-linking purine bases on DNA, interfering with DNA repair mechanisms and inducing irreparable DNA damage. However, resistance to platinum therapies is a major problem, and is thought to arise as a result of alteration to platinum transport mechanisms, DNA repair processes or apoptotic signalling.Other treatments for cancer include platinum-based chemotherapies, a class of agents used in the treatment of nearly half of all cancers. The most commonly used drugs include cisplatin, carboplatin and oxaliplatin and are used against ovarian, testicular, bladder and colon cancers amongst others. In ovarian carcinoma cells, catecholamines were able to reduce the efficacy of cisplatin and paclitaxel by inhibition apoptosis, and this effect could be modulated through inhibition of the β-adrenergic receptor. Mechanistically, catecholamines induced the expression of DUSP1, a MAPK phosphatase linked to the promotion of chemoresistance, through direct action of the β-2 adrenergic receptor on the promoter, which in turn protects ovarian cancer cells from apoptosis. In a xenograft model of ovarian cancer, stimulation of the adrenergic response by restraint stress significantly increased tumour burden and the expression of DUSP1. Restraint stress combined with docetaxel significantly impaired the efficacy of docetaxel and reduced docetaxel-induced apoptosis, and this could be negated by blockade of the β-AR. Knockdown of DUSP1 expression in a subsequent ovarian cancer xenograft model significantly restored the efficacy of chemotherapy - indicating that the DUSP1 expression induced by adrenergic activation has a functional role in the protection from apoptosis. The findings highlight a potential use for pharmacological inhibition of the β-AR using β-blockers to improve the efficacy of chemotherapy in the treatment of ovarian cancer patients, who report in significantly higher levels of symptoms associated with chronic stress. β-blockers have been used successfully in the treatment of multiple cancers for many years, and more recently have been shown to correlate with increased overall survival in patients with ovarian cancer.Stimulation of the adrenergic response has been shown to have adverse effects on the progression of ovarian cancer. In another model of ovarian carcinoma, the cytotoxic effects of cisplatin were also reduced by treatment with the synthetic glucocorticoid dexamethasone, which inhibited cisplatin-mediated apoptosis. It was hypothesised that dexamethasone may increase the adhesive ability of ovarian carcinoma cells, and indeed dexamethasone was shown to upregulate the expression of certain integrins which play a key role in regulating adhesion to the extracellular matrix (ECM). Inhibition of integrin signalling was able to reverse the protective effect of dexamethasone and restore the efficacy of chemotherapeutic treatment.Activation of the GR has also been shown to promote cell survival signalling in high grade serous ovarian carcinoma cells and inhibit carboplatin-induced cell death. Subsequent antagonism of the GR using an inhibitor was able to enhance the effect of platinum-based chemotherapy on tumour growth in a xenograft model of ovarian carcinoma. Furthermore, the induction of KLF5 contributed to dexamethasone mediated chemoresistance in vivo. Dexamethasone treatment promoted resistance to docetaxel, whereas KLF5-knockdown cells formed tumours that were sensitive to docetaxel even in the presence of dexamethasone. Dexamethasone induced cisplatin resistance via KLF-5 to promote cell proliferation can occur via many mechanisms including through regulation of cyclin D, fibroblast growth factor binding protein 1, microsomal prostaglandin E2 synthase-45.Similar results with GCs have been observed in cell lines representing TNBCs. Dexamethasone was able to decrease sensitivity to cisplatin, as well induce expression of Krüppel-like factor 5 (KLF5), a transcription factor that has been shown to promotes survival and proliferation in basal breast cancers.In the treatment of cervical cancer, GCs may also play a role in response to therapy. Infection with certain strains of human papillomavirus (HPV) is strongly linked to an increased risk of cervical cancer. It is though that this occurs as a result of viral oncoproteins interfering with the normal functioning of the tumour suppressor p53. In HPV-positive cervical cancer cells glucocorticoids not only downregulated p53, but also a microRNA (miR-145) associated with tumour suppression and shown to be downregulated in cervical cancer tissues. The downregulation of miR-145 by cortisol correlated with an increase in resistance to the antitumor drug mitomycin C, and this was shown be a direct result of glucocorticoid-mediated expression of a viral oncoprotein. NF-κB is an important signal transducer that regulates certain apoptotic signalling processes, and inactivation of NF-κB can therefore promote apoptosis. In breast cancer cells a similar result was also observed, with glucocorticoid treatment inhibiting activation of NF-κB, promoting apoptosis and increasing chemosensitivity. This directly contradicts another mechanism suggested by Fan et al. which suggests that paclitaxel promotes the release and translocation of NF-κB, which acts on apoptosis-related gene expression, promoting cell death. GCs meanwhile upregulate the expression of an inhibitor of NF-κB, which prevents this, leading to chemoresistance . These conflicting arguments therefore indicate that the effect of glucocorticoids on chemotherapy- induced apoptosis mediated by NF-κB is largely dependent on the cell type and stimulus.However, dexamethasone has also been shown to enhance the cytotoxicity of cisplatin in cervical cancer cells, through the inhibition of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)et al. also investigated the effects of GCs on solid cancer cell lines alongside lymphoid cells. Human lung and cervical cancer carcinoma cells were implanted as xenografts, and weekly does of cisplatin were given alongside dexamethasone added to the drinking water. It was subsequently observed that dexamethasone inhibits the efficacy of cisplatin cytotoxicity in the lung cancer xenograft model, and this was replicated in vitro in a human cervical cancer line. Blockade of the GR using RU486 was found to significantly reverse the GC-mediated reduction of apoptosis. The induction of apoptotic signalling by chemotherapy agents and irradiation is mediated by caspase cleavage and the cross-linking of death receptors. On a mechanistic level, treatment with dexamethasone interfered with key apoptotic molecules induced by cisplatin, downregulating caspase-8 and caspase-9, as well as death receptor ligands CD95-L (Fas ligand) and TRAIL. In a later study this result was corroborated, with dexamethasone inhibiting TRAIL-induced apoptosis in thyroid cancer cells. The opposite effect was observed in lymphoid cells, indicating the anti-apoptotic effects of GCs are specific to solid tumours. Over-expression of caspase-8 and 9 was able to sensitise dexamethasone treated lung carcinoma cells to cisplatin in vivo, significantly reducing tumour growth compared to dexamethasone/cisplatin treated. Furthermore, in hepatic cells, Yang and colleagues have shown that dexamethasone can negatively affect cisplatin induced apoptosis by increasing anti apoptotic genes BCL-2 and BCL-xl.Herr . Dexamethasone also inhibited cisplatin-induced apoptosis and promoted cell growth in a panel of ovarian and pancreatic cancer cell lines, and similar results were observed with gemcitabine and dexamethasone co-treatment. In primary ovarian cancer cells isolated from fresh surgical samples or pleural effusions, dexamethasone prevented cell death and increased viability of cells treated with chemotherapeutics and γ-irradiation. The same effect was also observed in primary tumour cells isolated from pancreatic carcinomas. When implanted in vivo treatment with dexamethasone negated the therapeutic effect of cisplatin, promoting rapid growth of the pancreatic and ovarian cancer xenografts. Taken together it is likely that dexamethasone can negatively affect the efficacy of cisplatin via the GR with effects on both cell proliferation (KLF-5) and apoptotic mechanisms, e.g., elevation of anti-apoptosis genes by dexamethasone.In a lung carcinoma cell line cell treated with cisplatin and dexamethasone, not only were the cells protected from apoptosis, they were able to recover and continue to proliferate after 3 weeks. The cytotoxic effects of Doxorubicin are mediated through multiple mechanisms; the intercalation of DNA which forms doxorubicin-DNA adducts that induce cell death, and the disruption of topoisomerase-II activity, thereby preventing DNA resealing and subsequently preventing replication in rapidly dividing cells. Furthermore, the oxidization of Doxorubicin to a semi-quinone by NAD(P)H-oxioreductases also generates free radicals increasing oxidative stress and promoting protein, lipid and DNA damage,57. Although highly effective, and considered a mainstay of chemotherapy regimens, Doxorubicin treatment also triggers many side effects including acute cardiotoxicity, vomiting, hair loss and inflammation. As such it is often prescribed alongside dexamethasone to treat hypersensitivity.Doxorubicin is a non-selective anthracycline widely used in the treatment of breast, bladder and ovarian cancer as well as types of leukaemia and lymphoma, often in combination with other therapies. Resistance to Doxorubicin has been associated with a number of factors related to its mechanisms of action, including MDR-associated proteins, alterations in DNA repair and topoisomerase activity and increased detoxification capacity,60,61. The ability of a cell to increase protection from oxidative stress is particularly relevant in the context of Doxorubicin resistance as oxidative stress plays a role in its mechanism of action. An increase in intracellular reduced glutathione which has anti-oxidant capacities has been observed in cells with reduced Doxorubicin sensitivity. Furthermore, studies have positively correlated expression of genes associated with increase antioxidant capacity with chemoresistance - with particular focus on nuclear factor erythroid 2-related factor (NRF2), a transcription factor that controls cellular redox homeostasis and detoxification by regulating several antioxidant response related genes.Resistance to doxorubicin poses a significant problem in the treatment of aggressive breast cancers, and since there is currently no marker to predict the efficacy of Doxorubicin, it is often prescribed without a full understanding of the therapeutic gainNRF2 is upregulated in several cell lines resistant to chemotherapy drugs including cisplatin and etoposide, as well as Doxorubicin resistance breast cancer lines, and its inhibition is capable of restoring sensitivity,64. Tamoxifen resistance was also reversed by the blockade of NRF2 in an MCF-7 derived cell line which possessed an enhanced antioxidant expression profile. Furthermore, the inhibition of downstream targets of NRF2 such as heme oxygenase-1 (HO-1) have been shown to be able to decrease cell viability in leukaemia cells. In NRF2 knockout mice studies showed an increased susceptibility to tumour formation in response to exposure to carcinogens, supporting the current consensus that the NRF2 pathway functions a cell survival pathway protecting against oxidative stress that can be deregulated in tumourigenic transformation.Expression of  and reduce the cytotoxic potential of common chemotherapeutic agents in breast and ovarian cancer cell lines,41. Multiple mechanisms have been proposed to explain the pro-survival effects of GCs including increased cell adhesion and inhibition of apoptosis via the induction of anti-apoptotic proteins,68. Dexamethasone has also been shown to modulate immunological effects in patients receiving chemotherapy, increasing the activation of regulatory T cells which are capable of suppressing anti-tumour responses. Dexamethasone has been show to mediate conflicting effects on Doxorubicin specifically, acting as a chemosensitiser in vivo enhancing the inhibition of tumour growth by Doxorubicin, and promoting chemoresistance in cancer cell lines and models.GCs have been shown promote cell survival signalling pathways in mammary epithelial cells. Activation of the GR can inhibit NRF2 activity through repression of transcription of the NRF2 gene, and subsequently suppress the antioxidant response including the expression of antioxidant response controlled genes. Conversely GCs have also been shown to increase levels of damaging free radicals in the context of breast cancer in a rapid non-genomic manner. In this way, GCs may serve to increase oxidative stress which can promote decreased sensitivity to Doxorubicin through upregulation of the antioxidant response. In addition, GC-increases in oxidative stress may lead to low levels of DNA damage, decreases on DNA repair mechanisms and genome instability leading to a pro survival phenotype.GCs are also known to play a role in oxidative stress in cancer. Through genomic and non-genomic pathways the GR is involved in the transactivation and transrepression of multiple anti-inflammatory responses that suppress oxidative stress. Cortisol and noradrenaline have not only been shown to induce DNA damage, but also affect DNA repair,73. In precancerous 3T3 cells, we have shown that stress hormones decrease UV induced DNA repair as measured by the comet assay. The addition of stress hormones increased the expression of DNA damage response proteins Chk1 and Chk2, as well as CDC25a, a member of the CDC25 family, levels of which would be expected to decrease in response to damage. Both GC and NA have been shown to reduce the rate of DNA repair in TNBC; cells treated with cortisol and assessed for DNA damage after a 20 minute repair period showed high levels of DNA damage inferring a lack of repair. These effects may represent further possible mechanisms by which stress hormones may contribute to resistance to platinum-based therapies.Alterations to DNA repair mechanisms are known to strongly influence the acquisition of resistance to many therapies including platinum-based therapies, due to the need for recognition and removal of platinum-DNA adducts. Activation of the DNA repair machinery slows down or halts the transition of cells through the cell cycle, and as such the ability of cells to repair the damaged DNA dictates sensitivity or resistance to platinum-based drugs. Taken together, these results show that adrenergic stress has an effect in shaping the T cell phenotype in the context of tumour microenvironment. Also, the pharmacological blockade of the adrenergic stress signaling pathway improves to gain a pro-inflammatory effector phenotype (IFN-γ+CD8+) and reduces the anergy phenotype (PD-1high). For these reasons, a strategic treatment with a combination of propranolol and immune therapies such as PD-1/PD-L1 inhibitors has potential.Recent animal studies have focused the attention on adrenergic stress effects on the efficiency of immune therapies. In particular, the role of stress on immune checkpoint therapy and on the immune tumour microenvironment has been shown using syngeneic models of breast cancer and melanoma. In this study, mice were exposed to mild, chronic cold stress and housed at 22 °C instead of 30 °C, causing the activation of the adrenergic signaling pathway. To block the adrenergic signaling, mice were treated with propranolol, a non-selective β-blocker. The stress effect on the immune response against cancer was assessed by evaluating the adaptive cell-mediated immune response. In particular, the presence of active CD8+ T cells was assessed in the tumour of stressed mice as well as the ratio between IFN-γ+ CD8+ and CD4+ cells. The tumour microenvironment was also studied considering the cancer-mediated immunosuppression via PD-1/PD-L1 interaction and mice were treated with a PD-1/PD-L1 inhibitor. Cold-stressed mice undergoing a treatment with propranolol showed a reduced tumour growth and an enhanced CD8+ T cell-mediated tumour immune response compared to control group housed at standard temperature. Moreover, PD-1 expression was increased in CD8+ tumour infiltrating lymphocytes (TILs) of cold-stressed mice with a decrease in the frequency in the propranolol treated group. This finding suggests that PD-1 signalling is one of the routes used by the adrenergic stress to cause immunosuppression in the tumour microenvironment and PD-1 blockade is significantly improved when cold-stressed mice are treated with propranolol.The influence of β-adrenergic signalling on T-cell targeting immune therapies was also shown in a mouse model of lymphoma. In this study, mice treated with isoprenaline, a non-selective β-adrenergic agonist, showed an acceleration in the growth of lymphoma which was reversed when the treatment was administered in mice not expressing β-adrenergic receptors (β1β2AR-KO). The effect on immune therapies was evaluated by treating mice with monoclonal antibodies against 4-1BB and PD-1 showing a significant inhibition of lymphoma growth in the 4-1BB treated group. However, when mice were also treated with isoprenaline both α4-1BB and αPD-1 immune therapies were less effective. To further investigate the effect on the cell-mediated response, mice received tumour specific CD8+ T cells with adoptive transfer prior tumour inoculation and the functional status of these cells was assessed after the isoprenaline and immune treatments. Isoprenaline significantly decreased the IFN-γ production and the cytotoxicity that T cells acquired with the α4-1BB treatment. This consideration leads to the hypothesis that T cell infiltration into the tumour represents an indication of how successful the immunotherapy is likely to be. Since stress hormones modulate T cell migration, stress can also have a role in the T cell trafficking and infiltration into the tumour. As a final consideration, stress can affect the immune response directly or indirectly by affecting the tumour itself. In this much more complex scenario stress can create an immune hostile tumour microenvironment or regulate the tumour escape process. For these reasons, the role of stress on tumour growth, spread and angiogenesis has an important impact on how it can cooperate in undermining the tumour immune response and the efficacy of immunotherapies Although these first studies confirm the idea that adrenergic stress weakens the efficacy of immune therapies by inhibiting the pro-inflammatory and anti-tumour response more work is necessary for a better understanding of the molecular mechanisms modulating the immune response against cancer. It is known that stress affects the cell-mediated immunity by regulating T cell subtypes and activation status. However, the literature is deficient in addressing some important aspects of the stress effect in cancer immunity that can undermine the immunotherapy potential. For instance, the efficacy of immunotherapies such as PD-1 and PD-L1 inhibitors is correlated with the immune infiltration (TILs) status of the tumour. In a study on metastatic melanoma, tumours from patients with high TILs, also known as “hot tumours”, showed a higher PD-L1 expression and a better outcomeIdentification of the role of stress hormone signalling in cancer drug resistance highlights the need for greater understanding of biobehavioural factors in the treatment of cancers. Furthermore, the use of interventions including stress hormone receptor antagonism by both well-characterised and novel inhibitors may provide useful tools to complement current therapeutic strategies. Research into better anti-emetic therapies to reduce the use of synthetic glucocorticoids as co-treatments may also prove beneficial.


Predicting potential DNA binding motifs is a critical part of understanding gene expression across all domains of life. Here, we report the development of Delila-PY, an easy-to-use pipeline to utilize the Delila suite to identify DNA binding motifs. Predicting potential DNA binding motifs is a critical part of understanding gene expression across all domains of life. Here, we report the development of Delila-PY, an easy-to-use pipeline for utilizing the Delila suite of software to identify DNA binding motifs. Proteins binding to DNA to regulate transcription are a key part of growth and responding to environmental stimuli. Identification of the DNA sequences bound by these proteins can elucidate members and function of the regulons. There exist several tools that can identify these DNA motifs, including MEME (using expectation maximization) and BioProspector (using Gibbs sampling) , 2. The 3–Delila-PY requires the GenBank file , 10 for delila (note that delila is a specific program within the Delila-PY software suite) to generate a book of sequences for subsequent use. To maximize the information content in the DNA motif, the pipeline runs three cycles. The first cycle uses the malign and malin programs to search for the alignment with the highest information content among all input sequences. The results of the malin program are fed back to the delila program to generate an updated book file. In the second cycle, the alignment book is passed to programs  to calculate the information content on each sequence. Delila-PY removes poorly matching sequences . Then, a new file is generated and fed back to the delila program to generate an updated book file. The final cycle generates a DNA motif . Delila-PY produces a DNA sequence logo in postscript and PDF formats, the position weight matrix (PWM) (Delila-PY processes data through the Delila software  3–7). T–7. T3–7)–ix (PWM)  values oRhodobacter sphaeroides transcription factor FnrL using locations from ChIP-chip data . All scripts used in Delila-PY are publicly available on GitHub (https://github.com/GLBRC/delila).The Delila-PY Docker image can be found on the Docker hub (recommended use;


This executive summary of a national living guideline aims to provide rapid evidence based recommendations on the role of drug interventions in the treatment of hospitalized patients with COVID-19.The guideline makes use of a systematic assessment and decision process using an evidence to decision framework (GRADE) as recommended standard WHO (2021). Recommendations are consented by an interdisciplinary panel. Evidence analysis and interpretation is supported by the CEOsys project providing extensive literature searches and living (meta-) analyses. For this executive summary, selected key recommendations on drug therapy are presented including the quality of the evidence and rationale for the level of recommendation.3 should not be used in COVID-19 routine care.The guideline contains 11 key recommendations for COVID-19 drug therapy, eight of which are based on systematic review and/or meta-analysis, while three recommendations represent consensus expert opinion. Based on current evidence, the panel makes strong recommendations for corticosteroids  and prophylactic anticoagulation  as standard of care. Intensified anticoagulation may be considered for patients with additional risk factors for venous thromboembolisms (VTE) and a low bleeding risk. The IL-6 antagonist tocilizumab may be added in case of high supplemental oxygen requirement and progressive disease . Treatment with nMABs may be considered for selected inpatients with an early SARS-CoV-2 infection that are not hospitalized for COVID-19. Convalescent plasma, azithromycin, ivermectin or vitamin DFor COVID-19 drug therapy, there are several options that are sufficiently supported by evidence. The living guidance will be updated as new evidence emerges. The ongoing COVID-19 pandemic causes an enormous pace and volume of research output of varying quality and reliability which is challenging for clinical decision making. To provide a clinical treatment guideline that includes a profound evaluation of rapidly changing evidence, the scientific medical societies conduct a living treatment guideline for hospitalized patients with COVID-19 moderated by the Association of the Scientific Medical Societies in Germany (AWMF) in collaboration with the COVID-19 Evidence Ecosystem Project (CEOsys). CEOsys is an association of 20 German university hospitals whose goal is to compile, summarize, and assess the quality of study results within a structured approach and meta-analysis. Evidence profiles of treatment strategies provided by CEOsys form the basis for a structured decision process using the GRADE  Evidence to Decision Framework . The folThe AMWF S3 guideline aims to provide a comprehensive overview of evidence based recommendations on therapeutic strategies for hospitalized patients with COVID-19. The guideline addresses physicians involved in the inpatient care of patients with COVID-19 but is also intended to provide information for individuals and organizations directly or indirectly involved in this topic. This compendium highlights the key pharmacologic treatment recommendations of the guideline that are most important for clinical care.The guideline group comprises actually 23 delegates from 15 participating scientific medical societies and organizations, as well as a patient representative .The guidhttps://app.magicapp.org), a digital platform and evidence ecosystem committed to support rapid recommendations and living guidelines during the COVID-19 pandemic .Evidence: Six RCTs with a total 7595 randomized patients were included .Evidence: Nine RCTs with a total of 6481 patients were included to evaluate tocilizumab .Evidence: A total of four RCTs were included in the assessment [P < 0.001]. There was a numerical reduction in all-cause mortality at day 29  .Evidence: Four randomized controlled trials with a total of 931 patients .Evidence: One RCT of 314 hospitalized patients was available. A clinical benefit of bamlanivimab in patients hospitalized for moderate to severe COVID-19  was not shown in the ACTIV3/TICO trial . In theRationale for the level of recommendation: Based on available evidence, the guideline group strongly recommends against bamlanivimab monotherapy in patients hospitalized for COVID-19. There is currently insufficient evidence on combinational therapy with multiple nMABs, so no recommendation can be formulated.In early SARS-CoV-2-infected hospitalized patients without respiratory COVID-19 symptoms (< 72 h after first positive NAAT and/or < 7 days since symptom onset), with at least one risk factor for a severe course of disease, SARS-CoV-2 specific monoclonal antibodies may be administered ⇔.Evidence: Currently, there are insufficient data to evaluate the use of neutralizing SARS-CoV-2 monoclonal antibodies (nMABs) in hospitalized patients. Results from outpatient trials cannot be adequately extrapolated to  infected inpatients not hospitalized for COVID-19. This recommendation, therefore, represents consented expert opinions considering available evidence on the efficacy and tolerability of nMABs in general as well as personal off-label use experience.Rationale for the level of recommendation: Interim analyses of ongoing phase II trials indicate that early (≤ 7 days after symptom onset) administration of combination therapies with bamlanivimab and etesevimab, as well as casirivimab and imdevimab might be beneficial in outpatients with mild to moderate COVID-19 disease  and at least one risk factor for disease progression. Early administration of nMABs resulted in a significant reduction in viral load, and  reduction in hospitalization rate or subsequent physician contacts .Evidence: Thromboembolic events are common complications of moderate to severe COVID-19 predominantly affecting the venous system and to a lesser extend arterial vessels .*For example: intermediate, half-therapeutic dose of LMWH. No universal definition exists for intermediate dose of unfractionated heparin (UFH). A dose of 250 IU/kg over 24 h and/or a 1.5-fold prolongation of the baseline aPTT value can serve as guidance.Evidence: Observational data suggest that prophylactic dose anticoagulation may not be sufficiently effective for VTE prophylaxis in patients with COVID-19, especially those that require ICU treatment .Evidence: Based on in vitro antiviral effects, azithromycin has been evaluated as therapeutic agent for the treatment of COVID-19 in four randomized controlled trials including a total of 8759 patients .Evidence: Numerous preprints are available online reporting solely on viral load reduction rather than clinically relevant endpoints or with significant methodological issues and a high risk of bias (exemplary .Evidence: Due to the lack of randomized controlled trials (February 2021) only one study with a total of 76 patients could be included into the assessment [3 are frequently observed in severely ill patients requiring intensive care treatment and may correlate with disease severity [3 levels seem to be a relatively common phenomenon in COVID-19. This observation does not indicate a causal relationship and does not justify vitamin D3 supplementation in this setting. After completion of the primary evidence analysis, another study was published [sessment . In thisseverity . In analublished . In thisRationale for the level of recommendation: Considering available evidence, the guideline group strongly recommends against the use of vitamin D3 to treat hospitalized patients with COVID-19. Besides the lack of evidence, the guideline group took into account the generally widespread availability of vitamin D3 and low costs, the intention to avoid wrong incentives for self-medication and potential adverse effects that can result from overdosing. Due to the lack of therapeutic consequences, routine determination of serum vitamin D3 in COVID-19 is also not recommended which is in line with guidelines released by the German Nutrition Society [ Society .Despite theoretical considerations that combinational therapy might improve efficacy of COVID-19 treatment regimens , only feReliable evidence for clinical efficacy in hospitalized patients with moderate to severe COVID-19 is available for dexamethasone. The use of dexamethasone in patients requiring oxygen  and patients with severe/critical disease requiring ventilation (WHO 6–9) significantly reduces mortality . The interleukin-6 antagonist tocilizumab demonstrated a statistically significant but low absolute effect on mortality and progression to invasive ventilation in our meta-analysis for patients with moderate to severe COVID-19 . The guideline panel recommends to consider tocilizumab for patients with higher level of supplemental oxygen requirement and progressive disease but not for those with more than 24 h of mechanical ventilation. Evidence regarding antiviral treatment with remdesivir is conflicting and currently no fundamental recommendation is made. The use of remdesivir in patients with invasive ventilation or without oxygen supplementation in not reasonable based on current clinical evidence. If remdesivir is used in patients with oxygen supplementation, an early treatment and 5-day course of remdesivir seems the most rational approach. The use of pharmacological thromboprophylaxis is strongly recommended for all patients hospitalized with COVID-19 while an intensified anticoagulation may be considered in patients with additional risk factors for venous thromboembolisms (VTE) and a low bleeding risk (expert opinion). In the setting of SARS-CoV-2 infection and hospitalization due to other indications than COVID-19 and/or nosocomial SARS-CoV-2 infection, an early treatment (< 72 h after first positive PCR and/or < 7 days since symptom onset) with SARS-CoV-2 neutralizing monoclonal antibodies (nMABs) can be considered as it might reduce the risk of disease progression in patients with at least one risk factor for severe COVID-19 (expert opinion). Other agents cannot be recommended for use outside clinical trials and eligible clinical settings (Table


CFOD-TS, was constructed using cohort St (N = 78) to predict DFS based on CFOD-TS features. The prognostic performance of MCFOD-TS was validated on cohort Sv (N = 219), a prospective clinical trial dataset (ECOG 2197). MCFOD-TS was prognostic of DFS in both St and Sv, independent of clinicopathological variables. Additionally, the molecular pathways regarding cell cycle regulation were identified as being significantly associated with MCFOD-TS derived risk scores. Our results also found that collagen fiber organization was more ordered in patients with short DFS. Our study provided a H&E image-based pipeline to derive a potential prognostic biomarker for early stage ER+ IBC without the need of special collagen staining or advanced microscopy techniques.Collagen fiber organization has been found to be implicated in breast cancer prognosis. In this study, we evaluated whether computerized features of Collagen Fiber Orientation Disorder in Tumor-associated Stroma (CFOD-TS) on Hematoxylin & Eosin (H&E) slide images were prognostic of Disease Free Survival (DFS) in early stage Estrogen Receptor Positive (ER+) Invasive Breast Cancers (IBC). A Cox regression model named M The standard treatment regimen for early stage ER+ Invasive Breast Cancer (IBC) patients is breast conserving surgery followed by hormonal therapy. While chemotherapy is able to significantly reduce the breast cancer associated mortality rate2, most early stage ER+ IBC patients do not receive added benefit from adjuvant chemotherapy3. Oncotype dx (Odx)4 is a 21-gene expression assay to assess the likelihood of cancer recurrence and the need for adjuvant chemotherapy for early stage IBC5. High Odx risk category indicates a high risk of recurrence and low Odx category suggests a low recurrence risk. The intermediate Odx category, however, has a more ambiguous prognostic/predictive meaning. While the Odx test is now routinely used in the clinical setting, Odx and other similar multi-gene assays tend to be time-consuming, high-cost, tissue-destructive and are not widely available in many countries.Breast cancer is the second leading cause of cancer death among women in the United States, with approximately 80% of these cancers being Estrogen Receptor Positive (ER+) and 64% being early stage 9. Collagen is the most abundant ECM component in breast cancer, which provides tensile strength and structural support to the tumor tissues10. Collagen fiber organization has been shown to be different between IBC and benign breast tumor, as well as between more and less aggressive IBC in both in-vivo/vitro models14. Using Second Harmonic Generation (SHG) based microscopy, Golaraei et al.15 observed an ultrastructure alteration in collagen fiber architecture in breast cancers, as compared to normal breast tissue. Friedl et al.16 observed that the focal sites of breast cancer cell micro-invasion often co-existed with stiff aligned collagen fibers. In addition, some studies17 have shown that organized collagen architecture in breast cancer was associated with a worse prognosis, as evidenced by SHG and Laser Scanning Microscopy (LSM) techniques.Numerous studies have demonstrated that the interaction between tumor cells and extracellular matrix (ECM) plays a critical role in breast cancer progression and metastasis19, techniques not routinely used in pathology labs or in clinical practice.Taken together, these studies strongly suggest that collagen fiber organization is associated with prognosis of breast cancer. However, the reported studies have employed advanced imaging techniques such as SHG or LSM microscopy20, there has been substantial interest in exploring the role of computationally extracted quantitative histomorphometric attributes based on Hematoxylin & Eosin (H&E) stained Whole Slide Image (WSI) in diagnosis and prognosis across multiple cancer types25. In the context of breast cancer, histomorphometric features relating to measurements of shape, texture, and orientation of individual cancer nuclei both from the tumor epithelium23 and tumor-associated stroma have been implicated in cancer progression and patient survival26.With the advent of digital pathology27, compared to the SHG/LSM microscopy-based biomarkers. Specifically, the automated pipeline consists of image processing techniques combined with a machine learning model to segment tumor-associated stroma on H&E slide images, from which we subsequently detected the collagen fiber orientations. Subsequently, we utilized entropy theory28 to calculate CFOD-TS, in order to quantitatively characterize the degree of disorder of the collagen fiber orientations at the tumor leading edge as well as across the entire tumor region. Following that, 78 patients (number of DFS event = 34) with early stage ER+ IBC identified from The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA BRCA) were used as the training set (St) to construct a Cox proportional regression model (MCFOD-TS) to predict DFS based on the CFOD-TS features. 219 breast cancer patients (number of DFS event = 64) from the completed Eastern Cooperative Oncology Group 2197 (ECOG 2197) were employed (Sv) for validating MCFOD-TS. ECOG 219729 is a randomized phase III clinical trial to compare the effectiveness of two combination chemotherapy regimens in treating early stage IBC (no significant difference observed in cancer outcome between the two regimens)29. The prognostic significance of MCFOD-TS was also validated on intermediate (indeterminate) Odx category as well as the subgroup of lymph node negative (LN−) and lymph node positive (LN+) patients in Sv. Additionally, we explored the relationship between CFOD-TS characterized phenotype and the underlying genotype utilizing the genomic data in St.We present a digital pathology-based pipeline that is able to prognosticate DFS in early stage ER+ IBC by automatically quantifying the Collagen Fiber Orientation Disorder in Tumor-associated Stroma (CFOD-TS) directly from digitized routine H&E image. Our computational pathology-derived biomarker could be more readily transitioned into clinical practice by taking advantage of the rapid increase in the availability of WSI scanningt and Sv are provided in Table t, 34 patients (45%) had an event (recurrence or death) with median DFS being 1379 days versus 65 patients (30%) with an event and median DFS of 3517 days in Sv. All patients in Sv received adjuvant chemotherapy while treatment related information was only available for 6 patients in St, with 4 of them receiving adjuvant chemotherapy. Other than tumor size and age, no clinicopathological variables were found to be significantly different between St and Sv. Sv was considered a rigorous validation set of MCFOD-TS given that the relative homogeneity in terms of treatment regimen  and cancer clinical characteristics enabled the outcome to be more reflective of the inherent aggressiveness of breast cancer.Clinicopathological variables for St (N = 78) and integrated into MCFOD-TS. The number of top features was determined as approximately 10% of the patient number in St. All 8 CFOD-TS features were found to be significantly (p < 0.05 from WRST) lower in patients with short DFS (with an event and DFS < 5 years) compared to those with long DFS (DFS > = 5 years) in St+v (St + Sv). The eight identified features consisted of the following: CFOD-TS in the FOV of 50, 100, 250 μm at the tumor leading edge and CFOD-TS in FOV of 75, 125, 150, 200, 225 μm across the whole tumor region. Notably, among the eight identified top features, CFOD-TS features derived from relatively small FOVs were found to be more distinguishable  between short and long DFS patient groups  on St and HR = 2.64  on Sv using a log-rank test. In the subgroup survival analysis on Sv     Fig.  and LN+ 28) Fig. . Further87) Fig. .Fig. 3PrM for DFS. In the univariate analysis setting, other than lymph node status in Sv, neither clinicopathological variables nor Odx risk category was identified as being significantly prognostic of DFS on St and Sv. Patients in RMH had significantly worse DFS  compared to those in RML. In the multivariate analysis controlling the effects of the clinicopathological variables, RM was found independently prognostic of DFS with HR = 3.45  on St and HR = 3.82  on Sv. The prognostic significance of lymph node status in Sv still held after adjusting the impact of other clinicopathological variables.Table MH and RML. Among the GO identified pathways with the 1062 genes as the input, ten molecular pathways  and Regulation of Cell Cycle (RCC) were identified to be significantly associated with MCFOD-TS risk scores, with the Pearson correlation coefficients respectively being −0.35 (p = 0.0017) and −0.31 (p = 0.0069). CCA30 is a checkpoint in the cell cycle before proceeding to duplication and division. CCA was found to be overexpressed in MCFOD-TSL patients, suggesting that the mechanism facilitating DNA repair or cell cycle progression was differentially regulated in low risk compared to high risk patients. Similarly, RCC31 was also found to be negatively associated with MCFOD-TS derived risk scores, suggesting a more dysregulated cell division process in RMH patients.Out of the 60488 annotated genes for TCGA BRCA, 1062 genes  based on the quantitative measurement of collagen fiber organization, these studies have typically involved the use of advanced microscopy imaging technologies like Second Harmonic Generation (SHG) or Laser Scanning Microscopy (LSM) system. Those technologies, however, are far from translation into the routine clinical pathology workflow. With the advent of digital pathology, there has been substantial research interest in investigating computationally extracted quantitative histomorphometric attributes such as shape and spatial arrangement of cancer nuclei and immune cells on H&E images for breast cancer prognosis32. However, to the best of our knowledge, no study has attempted to quantitatively measure collagen fiber organization directly on standard H&E slide images and relate these measurements to breast cancer outcome.Increasing evidence has emerged toCFOD-TS) with CFOD-TS features to predict DFS, with a continuous risk score as the output. MCFOD-TS was validated to be prognostic on an independent, archived clinical trial dataset (ECOG 2197) independent of clinicopathologic variables e.g. tumor grade, tumor size. MCFOD-TS was also found to be prognostic on the patients in the subgroups of LN−, LN+ and intermediate Odx risk category. Additionally, the molecular pathways relevant to cell cycle regulation were identified as associated with the collagen fiber organization phenotype. We found the aligned and ordered organization of collagen fibers was indicative of a worse prognosis in terms of DFS for early stage ER+ IBC.The objective of this study is to quantitatively measure the organization of collagen fibers from routine H&E slide images alone and investigate its association with the outcome (DFS) for early stage ER+ IBC. Utilizing a set of imaging processing techniques and a state-of-the-art deep learning model, we were able to quantitatively characterize Collagen Fiber Orientation Disorder in Tumor-associated Stroma (CFOD-TS) solely with routine H&E stained images, without the need of special collagen staining or advanced imaging and microscopy techniques. Following the CFOD-TS feature extraction, we constructed a Cox regression model . Most of the patients (20/28) in intermediate Odx were identified as low risk by MCFOD-TS and among the 8 patients in RMH group, 7 patients recurred or died along the course of follow-up. While Odx test has been validated to be prognostic on ECOG 21975, Odx test did not show prognostic significance in the univariate analysis for DFS  is a multi-gene test which is prognostic and predictive of benefit of adjuvant chemotherapy for early stage IBC34. For example, multiple investigators35 have shown that in mice model, cancer cells preferentially invaded along aligned collagen fibers into the adjacent stroma, and focal sites of breast cancer cell micro-invasion often co-existed with stiff aligned collagen fibers. Also, Bredfeldt et al17. and Conklin et al11. found that the presence of straightened, aligned collagen fibers at a local tumor boundary observed on SHG images was indicator of poor prognosis for IBC patients. One of the advantages of our present pipeline is that we expanded the samples from tumor cores that previous studies17 have utilized in the collagen fiber organization analysis to WSIs, which harbor more comprehensive tumor characteristic information. A number of hypotheses37 have been proposed to demonstrate the mechanism behind how the aligned collagen fibers facilitated tumor migration and invasion. In the aspect of physical mechanism, given that the cancer cells have been observed to migrate rapidly along aligned collagen fibers, it has been assumed that collagen realignment could eliminate the physical barrier impeding the moving cells16 and generate tube-like trails of least resistance to promote cell movement34. For example, Riching et al34. found that by performing a microchannel migration assay to track the tumor cells traveling in a 3D collagen gel, the tumor cells were able to travel greater distances in aligned collagen fiber gels compared to the randomly organized collagen gels within the same time period. This observation suggests that the efficiency of tumor migration is enhanced in aligned fibers, possibly by limiting protrusions along the fibers. Some researchers39 also identified molecular involvement in the facilitated tumor cell movement along the aligned collagen fibers. For example, Wang et al.38 found that the cancer cells on highly aligned ECM possessed organized Focal Adhesions (FAs). Highly aligned FAs and associated F-actin stress fibers in turn resulted in the localization of active Rac1 which further stabilized the cell protrusions along the direction of ECM alignment.Quantitative measurement Fig.  and qualThe CFOD-TS feature calculated from a small FOV was found to be more discriminating between patients with long and short DFS compared to the features derived from a larger FOV. A possible explanation for this is that the fibers in a small FOV are more likely to constitute a micro-environment, functioning as a unit. The fibers mutually and directly interact with the tumor, and further facilitate tumor migration through the alignment in the orientations, which in turn is quantified as low CFOD-TS. However, a large FOV (big tumor neighborhood) could actually comprise multiple independent fiber-composed micro-environments. In this case, the calculated CFOD-TS not only reflects the fiber orientation alignment within each individual micro-environment as in the case of a small FOV, but also captures the relative orientation alignment among the multiple micro-environments. However, considering that the multiple micro-environments within a larger FOV affect the tumor behavior independently, the corresponding calculated CFOD-TS from the large FOV might end up being less prognostic. Additional experiments were also performed to allow for more intuition with respect to the extracted CFOD-TS features. A high correlation between CFOD-TS at the tumor leading edge and across the whole tumor region  as in our study, Verma et al41. built a prognostic model to classify patients into low- versus high-risk groups for recurrence by leveraging the nuclear morphometric signatures from H&E slide images, with HR = 2.41 . In addition, they found that by combining the nuclear-morphology-based prognostic model with the Odx risk category, 20% more patients in low risk could be correctly identified compared to using Odx test alone. Similarly, Lu et al.21 found that nuclear texture heterogeneity as well as the nuclear orientation disorder were associated with overall survival for early stage IBC patients. Apart from the handcraft features, deep learning based neural network models have also been used to directly prognosticate cancer outcome based on pathology slide images. Wulczyn et al.40 developed and trained a deep learning system consisting of a weakly supervised approach and survival loss function using H&E slide images in TCGA. The risk scores generated from the deep learning classifier were validated as being significantly associated with DFS in IBC, after adjusting for the impact of age and stage. However, different from previous studies in computerized histomorphology analysis, our study quantitatively measured collagen fiber orientation organization on H&E slide images and associated with the breast cancer outcome. Our study demonstrated that CFOD-TS, which is not typically examined to assess disease prognosis in pathological practice, contains potential valuable prognostic information that could be captured by visual inspection on H&E slides.A number of studies have been conducted to interrogate the prognostic value of computerized tumor morphometric features on H&E slide images for IBC, either based on quantitative handcraft histomorphometric featuresCFOD-TS derived risk scores. CCA30 is often used by cells to facilitate DNA repair and cell cycle progression. A down-regulation of CCA pathway was found in the high-risk patients identified by MCFOD-TS, which indicated a dysregulated tumor division process. Similarly, RCC31 was also found to be down-regulated among the MCFOD-TS identified high-risk patients, which further suggested a defected regulation of division or duplication process of cancer cells. The mechanism of ECM interacting with the molecular processes within tumor cells has been demonstrated in previous studies43. Mouw et al. found that the increased ECM stiffness led to reduced expression of the tumor suppressor phosphatase43; meanwhile, the increase in ECM stiffness was also identified as one of the causes of collagen fiber realignment42. Those findings provide an insight into the possible molecular mechanism behind the association between the suppressed cell cycle regulation and model-derived collagen orientation risk score developed in our study.In this work, we also explored the molecular underpinning of CFOD-TS on TCGA BRCA dataset with available mRNA sequencing data. Two biological pathways including Cell Cycle Arrest (CCA) and Regulation of Cell Cycle (RCC) were identified as being significantly associated with the MWe do acknowledge that the study did have its limitations. Since we did not have access to specialized collagen-specific stained slide images, we were unable to definitively validate the segmentation performance of our algorithm for identifying collagen fibers on the H&E images. However, we were still able to accurately and quantitatively measure the collagen fiber orientation disorder by capturing the directional pattern of the collagen fibers on H&E slide images  without requiring stringent discernment on each individual collagen fiber. Another limitation pertaining to this study is the relatively small data size due to the restrictive patient inclusion criterion to retain the homogenous nature of the cohorts. The purpose of narrowing the scope down to relative homogenous datasets in terms of tumor stage is to eliminate the co-effect of the tumor stage on the CFOD-TS aside from the effect from inherent tumor aggressiveness. Also, unlike the Odx test, which was prospectively validated in both prognostic significance and treatment benefit prediction, our study was retrospectively validated on a single clinical trial dataset and only validated in terms of prognostic ability. Future work will entail validating the pathology image-based prognostic pipeline in larger, additional independent pan-stage cohorts, and also in terms of its predictive benefit for adjuvant chemotherapy. Additionally, we will expand our validation into the pathology images scanned under different parameters (e.g. microns-per-pixel) and for other molecular subtypes, such as triple negative breast cancer. Furthermore, we will look to expand our CFOD-TS analysis into tumor adjacent non-oncologic tissue in future studies. Specifically, we will use the non-oncologic tissue as a reference to investigate the disruption of CFOD-TS by the tumor cells, and study how the disruption in the organization is associated with the tumor aggressiveness.In summary, this study quantitatively measured the collagen fiber organization in breast tumor-associated stroma solely using routine H&E stained images and demonstrated its prognostic significance in terms of DFS for early stage ER+ IBC.t: N = 78) and ECOG 2197 (Sv: N = 219). A flow diagram describing the inclusion and exclusion criterions on each of the datasets is provided in Supplementary Fig. Two cohorts were used in this study: TCGA BRCA  IBC tissue (×40 magnification (mag): ~0.25 μm (um)/pixel). The corresponding clinicopathological and outcome data were downloaded from Genomic Data Commons (GDC) data portal. In order to keep relative consistency with respect to tumor stage with Sv, only the patients with Stage II  or high risk (tumor size > = 1 cm) LN− tumors were recruited into this cohort. All the patients with an DFS event (recurrence or death) meeting the inclusion criteria, matched with a set of censored patients (no DFS event) from TCGA BRCA were used to constitute St.Sv was used as an independent validation set to evaluate the model performance. The ECOG 2197 trial was a prospective, randomized, clinical trial from 1998 to 2007 that recruited patients with IBC (1–3 positive LN/LN− with tumor size > = 1 cm) to compare the patient’s outcome under two different chemotherapy regimens29. 219 patients with ER+ IBC were selected to comprise Sv after the inclusion criteria applied on the 256 patients in ECOG 2197, for whom we had access to both the corresponding de-identified slide images and relevant clinical information. The access to the ECOG dataset involved 2-year process including a proposal review first through ECOG and subsequently through Cancer Therapy Evaluation Program (CTEP) in National Cancer Institute (NCI). In ECOG 2197, the effects of treatment and clinical characteristics of cancers on the outcome were relatively controlled thus enabling the outcome to be more reflective of the nature aggressiveness of breast cancer and making Sv an ideal validation set. All the FFPE tissue slides were scanned and digitized using a Philips scanner at 40x mag. Clinicopathological and outcome information were obtained from retrospective chart review.SThe study conformed to HIPAA guidelines was approved by the Institutional Review Board (IRB) at University Hospitals Cleveland Medical Center. IRB No 02-13-42C. The need for written consent from participants was waived due to the use of de-identified retrospective data.44, a quality control tool for digital pathology slides was employed to identify the useful tumor tissue by excluding fat tissues and regions with artifacts e.g. tissue folding, pen marking, and blurriness.Following the acquisition of the digital WSIs, HistoQC45. The cGAN model  model was implemented on the useful tumor tissues on the digitized WSIs identified by HistoQC. The cGAN model is an extension of GAN with both the generator and discriminator being conditioned on auxiliary informationask Fig. . The str48, thus usually presenting with a linear phenotype on H&E slide images of breast cancer. A derivative-of-Gaussian (DtG) based model described in Griffin et al.’s49 work was used to capture the fiber orientation by detecting the linear structures in tumor-associated stroma on H&E slide images. Specifically, the DtG based model classified each pixel on the slide image as being within one of seven image structures . By retrieving the image pixels identified as belonging to a linear fiber (dark Line structure), we were able to capture the directional pattern of the collagen fibers  representative of collagen fiber orientation was denoted as Vi, ranging between 0 degree and 180 degree. The orientations were then discretized to angular bins from 0 to 17. An orientation co-occurrence matrix  was subsequently calculated from the orientation co-occurrence matrix based on entropy theory28 Fig. . The ent28 Fig. .50, henceforth referred to as Cox regression model, relates the time that passes until some event occurs, to multiple covariates. We regularized the Cox regression model by a Least Absolute Shrinkage and Selection Operator (LASSO) penalty function to identify important predictors of DFS. We constructed a Cox regression model named MCFOD-TS on St between CFOD-TS features and DFS. A corresponding coefficient was assigned to each of the features in the final model. A continuous risk score was then calculated from a linear combination of the top features weighted by the corresponding coefficients for each individual patient  vs. low (RML: risk score< = threshold) risk categories. The threshold was selected so as to give the most significant prognosis on St. KM curves were generated to compare the DFS between RMH and RML groups, with the difference assessed by the log-rank test on both St and Sv. Additionally, we performed a dedicated survival analysis for different subgroups in Sv. KM curves were plotted respectively for the subgroup of LN+ (N = 103) and LN− (N = 116) patients. For the set of patients (N = 59) with available Odx scores, we applied the Trial Assigning Individualized Options for Treatment (Rx) (TAILORx) trial recurrence score categorization33  to assign the patients into the low (low recurrence risk), intermediate (ambiguous recurrence risk) and high (high recurrence risk) Odx risk categories. The KM curves were generated for the patients with intermediate Odx category to evaluate whether MCFOD-TS could provide significant prognostic value for the set of patients whose recurrence risk could not be unequivocally interpreted by Odx.Kaplan–Meier (KM) survival analysis was used to analyze the difference of the expected duration of time until an event happens between two defined categories. We converted the continuous risk scores calculated from Mt or Sv. Additionally, we used multivariate Cox regression analysis to evaluate the independent prognostic significance of MCFOD-TS by adjusting the impact of the clinicopathological variables on DFS. Multivariate analysis was performed on the clinicopathological variables which were available for more than two thirds of patients in each cohort (St or Sv).Univariate Cox analysis was performed on clinicopathological variables and Odx risk category to evaluate if any of those clinical routinely examined parameters or the widely used gene test was prognostic of DFS on St). Histogenomics analysis was, however, not conducted on ECOG 2197 (Sv) due to lack of corresponding genomic data. Wilcoxon Rank Sum Test (WRST) was first used to identify the genes significantly differentially expressed between RMH and RML groups. The identified gene set was in turn taken as the input of Gene Ontology (GO)52 to identify the associated GO molecular pathways whose member genes were overrepresented in this gene set. After reviewing the list of the identified GO pathways, we selected the ones potentially representative of biological processes underpinning the collagen fiber organization in tumor-associated stroma. The single-sample Gene Set Enrichment Analysis (ssGSEA)53 was then used to generate an enrichment score for each of the molecular pathways, assessing the activity level of the pathway in which the member genes were coordinately up- or down-regulated. Unlike GSEA working at the level of sample population, ssGSEA was able to calculate a separate enrichment score for each individual patient. The Pearson correlation was then used to assess the correlation between the enrichment score and the MCFOD-TS derived risk score.Histogenomics analysis was performed by investigating the association between biological pathways and CFOD-TS utilizing the normalized Messenger Ribonucleic acid (mRNA) expression data obtained from GDC data portal on TCGA BRCA (SFurther information on research design is available in the Supplementary MaterialsReporting Summary


Background: The perspectives and experiences of people hospitalised with COVID-19 have been under-reported during the coronavirus pandemic. We developed and conducted a COVID-19 patient satisfaction survey in a large university-affiliated secondary healthcare centre in Liverpool, UK, during Europe’s first coronavirus wave (April-June 2020). The survey found that care was rated highly, including among people of Black Asian and Minority Ethnic (BAME) backgrounds. However, sleep-quality and communication about medications and discharge-planning were identified as areas for improvement.  Methods: To improve care for people with COVID-19 admitted to our centre, we designed an educational package for healthcare professionals working on COVID-19 wards. The package, implemented in August 2020, included healthcare worker training sessions on providing holistic care and placement of “Practice Pointers” posters. Patient satisfaction was re-evaluated during the second/third COVID-19 waves in Liverpool (September 2020 - February 2021).Results: Across waves, most (95%) respondents reported that they would recommend our hospital to friends and/or family and rated overall care highly. Comparison of the responses of second/third-wave respondents (n=101) with first-wave respondents (n=94) suggested improved patient satisfaction across most care domains but especially those related to having worries and fears addressed and being consulted about medications and their side-effects.Conclusions: People admitted with COVID-19 to our centre in Liverpool, including those from BAME backgrounds, rated the care they received highly. A simple education package improved the feedback on care received by respondents between the first and second/third waves. These UK-first findings are informing regional strategies to improve person-centred care of hospitalised people with COVID-19. In the UK, there have been in excess of 4.5 million confirmed cases of whom over 10% have required admission to hospital and more than 152,000 have died.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 171 million people worldwide and caused over 3.5 million deaths. To address this, we designed and implemented a satisfaction survey of people with COVID-19 admitted during the first wave of Covid-19 infections (March-June 2020) to our large university hospital in Liverpool, UK. The survey findings  showed that nursing and medical care was rated highly and most respondents (96%) reported that they would recommend our hospital to friends or family. However, the survey also highlighted potential areas for improvement including communication about medications and their side effects and informing patients about discharge plans. To address these shortcomings and improve holistic COVID-19 hospital care, we subsequently implemented a package of complementary interventions on COVID-19 wards in our centre.Despite extensive research into COVID-19 vaccines, diagnostics, and biomedical treatments, evidence concerning the perspectives of people with COVID-19, especially from vulnerable and Black Asian Minority Ethnic (BAME) groups, is negligibleHere we report the satisfaction survey findings from the second/third COVID-19 waves in Liverpool (September 2020 - February 2021) and compare them to those of the first wave..This was an unpowered before-and-after observational quality improvement project (QIP) registered with the local Clinical Effectiveness Department. The COVID-19 patient satisfaction survey was developed in collaboration with our centre’s ‘Patient Experience’ team to ensure it was patient-friendly and suitable for people with learning and reading difficulties. According to the policy activities that constitute research at the Liverpool University Hospitals NHS Trust (LUHFT), this Quality Improvement Project met criteria for operational improvement activities exempt from ethics review. All respondents were consented verbally but, in line with local QIP policy, written consent was not deemed to be requiredThe survey was adapted from existing patient satisfaction surveys and integrated with our centre’s “Friends and Family Test” questions. An open-access version of the survey itself and accompanying standard operating procedure can be found inth March 2020 to 15th June 2020) and second/third waves (21st September 2020 to 6th February 2021). Between these time periods, the first-wave survey findings were presented at Tropical Infectious Disease Unit QIP meetings and centre-wide Patient Experience meetings. A package of complementary interventions was designed and implemented in August-September 2021. The package consisted of: concise feedback and training sessions for healthcare workers on holistic care on the COVID-19 wards; updated COVID-19 patient information leaflets for admission and discharge; and a ‘COVID-19 Practice Pointers Poster’ .An earlier version of this article can be found on medRxiv  from the first wave and 101 (52%) from the second/third wave, were collated . ComparePatient satisfaction was high with overall care rated 4.8/5 on average. Nearly all (95%) respondents reported that they would recommend our hospital to friends and/or family .Compared to first wave respondents, second/third wave patient satisfaction increased across multiple domains of care but, most notably, being involved in care decisions (81% to 95%), able to share worries and fears (77% to 93%), and communication about medications and side effects  and third waves (January 2021) in our centre. Keeping in touch with friends and family was identified as a key determinant of inpatient satisfaction in the ‘UK Patient Association’ survey. Indeed, this was a priority in our Covid-19 Practice Pointers poster .Data are available under the terms of the Looks good. Strongly consider for indexing.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Proning in COVID procedural complications in the ICUI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I enjoyed reading this manuscript and highly recommend it for indexing. As the article discusses, the majority of the medical community's focus has been on the biomedical treatments of COVID-19. In this, the psychosocial impact of this illness on hospitalised patients may have taken a backseat. Identifying areas of improvement through patient satisfaction surveys such as this one should help us provide more comprehensive and fulfilling care. The points that the team identified, such as discussion about medications and side effects, discharge planning, are being observed globally. Improvement projects such as this one can help guide many centers and providers, especially regarding the care of minorities who often get suboptimal care. In the 'conclusion' section of the abstract, it should be BAME backgrounds (not background).Figure 4: Please explain what 'LUFTH' stands for. Readers from outside institutions might not know it.  I have a couple of suggestions: Overall, I feel the article is very well written, is succinct, and should be of interest to health care providers and centers worldwide.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Proning in COVID procedural complications in the ICUI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Dear Dr Paul, Thank you for your constructive and supportive comments. We have made the updates suggested. Yours sincerely, Tom Wingfield  Well written and well executed study. Most limitations identified - excludes those who were extremely ill and either died or went to critical care; pre-dates vaccination which may alter future similar studies (with use of ronapreve for example) in spike protein antibody negative patients.  Relevant study with relevant findings and easily reproducible in other settings with realistic interventions. It would be interesting to see if these outcomes are maintained over time and in non-COVID patients.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Infectious diseases clinician treating patients with COVID-19 since 2020 (in addition to patients with other infections).  Most research is on other areas - bacterial meningitis RCT with adjuvant therapy, CSF and plasma cytokine studies, perinatal HIV cohort studiesI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Thin-film saturable absorbers (SAs) are extensively used in mode-locked fiber laser due to the robust and simple application methods that arise because SAs are alignment-free and self-standing. Single-walled carbon nanotubes (SWCNTs) are the most suitable low dimensional material uesd for SAs because of their high nonlinearity and the wavelength control of absorption based on tube diameters. The most challenging problem with the use of CNT-based thin film SAs is thermal damage caused during high power laser operation, which mainly occurs due to aggregation of CNTs. We have demonstrated improved thermal damage resistance and enhanced durability of a film-type SA based on functionalization of SWCNTs, which were subjected to a mechanical functionalization procedure to induce covalent structural modifications on the SWCNT surface. Increased intertube distance was shown by X-ray diffraction, and partial functionalization was shown by Raman spectroscopy. This physical change had a profound effect on integration with the host polymer and resolved aggregation problems. A free-standing SA was fabricated by the drop casting method, and improved uniformity was shown by scanning electron microscopy. The SA was analyzed using various structural and thermal evaluation techniques . Damage tests at different optical powers were also performed. To the best of our knowledge, a comprehensive analysis of a film-type SA is reported here for the first time. The partially functionalized SWCNT (fSWCNT) SA shows significant structural integrity after intense damage tests and a modulation depth of 25.3%. In passively mode-locked laser operation, a pulse width of 152 fs is obtained with a repetition rate of 77.8 MHz and a signal-to-noise ratio of  75 dB. Stable operation of the femtosecond fiber laser over 200 h verifies the enhanced durability of the fSWCNT SA.The online version contains supplementary material available at 10.1186/s11671-021-03648-2. Passively mode-locked erbium-doped fiber lasers (EDFLs) have been popular in generating ultrashort optical pulses with widespread applications in industrial and scientific fields. Complex technical areas such as high-resolution microscopy, biophotonics, optical signal processing and optical metrology have extensively employed EDFLs due to their simple, reliable operation and consistent ability to operate in the picosecond and femtosecond regimes –5. The wLow-cost manufacturing combined with attractive features such as high nonlinearity has enabled the use of low-dimensional materials such as carbon nanotubes (CNTs) or graphene as alternatives to SESAMs. Film-type SAs with a free-standing capability can be applied to all fiber lasers in the simplest way. However, low-dimensional material-based SA films have also suffered significantly from low thermal stability and a high degradation rate owing to unreliable preparation processes, rendering them unsuitable for durable laser applications. Several studies using microfiber (for evanescent wave interactions) have been performed to overcome these limitations, but polarization sensitivity and high loss problems are caused by side-polished fibers , 9. Addicument}3 –13.Saturable absorption is a nonlinear phenomenon in which photonic materials undergo saturation of optical absorption under high-intensity illumination, which constitutes the basis of passive mode locking , 15. AltAs presented in this paper, SWCNTs were partially functionalized, resulting in an increase in the intertube distance. The improved uniformity of the the functionalized SWCNT (fSWCNT) SA is shown by scanning electron microscopy (SEM) images. Structural and chemical evaluation of SAs was also performed by applying X-ray diffraction (XRD), Raman and Fourier transform infrared (FTIR) techniques. The fSWCNT SA is proven to be thermally stable at high temperatures exceeding 650 n and m are integers. The unique electronic properties of SWCNTs depend on the chiral vector and are divided into metallic  and multiwalled CNTs (MWCNTs) according to the number of layers constituting them. SWCNTs are cylinders composed of a layer of a graphene strip, and the structural characteristics can be defined in terms of the chirality by a chiral vector , poly methyl methacrylate (PMMA) and sodium carboxymethylcellulose (NaCMC) are commonly used for the host polymer of SA films but have several limitations. The low glass transition temperatures of PVA  of the CNTs by transforming the spaterials . Figure mentclass2pt{minimWe set up a femtosecond fiber laser to test the mode locking capability of the fSWCNT-PDMS SAs. In soliton mode locking, when the soliton pulse goes through the fiber cavity, no temporal or spectral changes in the pulses occur as a result of the combined effects of chromatic dispersion and nonlinearity (self-phase modulation). Even though soliton lasers have some limitations in practical applications due to spectral sideband generation, among others issues, they are relatively stable and easy to control.The laser setup is shown in Fig. Erbium (Er) belongs to the group of rare earth materials and was used in the form of the trivalent ion ErTo evaluate the performance of SAs, two kinds of representative indicators, the modulation depth and nonsaturable loss, should be determined. The modulation depth is the maximum change in absorption (or reflection) of SAs that emerges due to incident light. This is the most important parameter: the laser cannot be mode locked in the case of a modulation depth lower than a sufficient value. In the soliton regime, a moderate modulation depth can be used, but in the case of other regimes , a very high value is required for mode locking because of the high instability. The nonsaturable loss is typically an unwanted factor of an SA, and its value is the absorption rate when the SA is saturated at high intensity. In the case of a thin film SA, an excessively high concentration of nanomaterial and a high thickness of the film lead to a high value of the nonsaturable loss.In general, SAs tend to exhibit increasd transmittance under high-intensity light. Therefore, to determine the saturation absorption characteristics, the power incident on the SA must be changed and how the absorption rate changes accordingly measured. A schematic diagram of the nonlinear absorbance measuring system is presented in Fig. Structural analysis of the CNT and fCNT samples was carried out by applying XRD and Raman spectroscopy techniques. XRD was helpful in ascertaining the degree of the difference in the crystallinity of the samples. We analyzed the extent of damage or reorganization of structural bonding in the CNT walls as a result of intensive ultrasonication via Raman spectroscopy.The typical crystalline nature of the CNT structure results in a strong peak at g’s law) . The 10g’s law) . The XRDg’s law) . The  with a central wavelength of 1576 nm. The full-width at half-maximum (FWHM) bandwidth is 19.8 nm. Figure 2 pulse shape, the inferred pulse duration is 152 fs. The time-bandwidth product is 0.373 which means that pulses are close to transform-limited. The pulse train is shown in Fig. The soliton fiber laser demonstrated self-starting single-pulse mode locking between 90 and 140 mW pump power. Figure formance .Fig. 9PuPartially functionalized SWCNTs were uniformly incorporated within a PDMS polymer matrix, and fSWCNT SAs showed high damage resistance with enhanced durability. The mechanical functionalization process solved the problem of CNT aggregation without using any surfactant or effecting chemical functional groups, both of which compromise the inherent nonlinear characteristics in conventional methods. The SAs demonstrate superior thermal stability and damage resistance during TGA and rigorous damage tests, so they ensure high-power ultrashort laser operation by virtue of their superior material characteristics and preparation procedure. Stable mode locking was acheived by placing the fSWCNTs into an EDFL. The ultrashort pulse trains exhibited a 77.82 MHz repetition rate, a 152 fs pulse duration and an SNR of 75 dB in single pulse generation. Stable femtosecond laser operation was also demonstrated for over 200 h, so the proposed fSWCNT SA provides a promising solution for laser applications that require ultrashort pulsed lasers with long-term durability. We demonstrated new approaches for developing damage-resistant polymer SA films and comprehensive analyses for self-standing SA films. Various related studies on SAs that use other low dimensional materials and photonic devices for pulsed fiber lasers with high output energy are expected based on this work.A fiber Bragg grating-stabilized 980 nm pump laser diode  connected to a tabletop laser and a Peltier controller  was used as the laser source. Three kinds of SM optical fibers, SMF-28, HI1060 (high-index) and ER80-4/125 (erbium-doped fiber for 980 nm pumping with emission at 1550 nm), were purchased from Thorlabs. All SMF-28 (transmission from 1260 to 1650 nm), HI-1060 (transmission from 980 to 1650 nm) and ER80-4/125 (80±8 dB/m core absorption peak at 1530 nm) fibers had a diameter of 125 µm. The WDM combines two wavelengths (980 and 1550 nm) into a single fiber output, the isolator (1550 nm center wavelength) prevents reverse transmission of light, and the The fiber laser characterization consisted of measuring the spectral bandwidth, pulse duration, repetition rate and SNR of the output pulses. An indium gallium arsenide (InGaAs) photodetector  was used for optical signal acquisition. The spectral bandwidth was measured with an optical spectrum analyzer , and the pulse duration was measured with an autocorrelator . The repetition rate was measured through a pulse train from an oscilloscope , and the SNR was measured through an RF signal from a spectrum analyzer . The optical power was measured with two kinds of power meters: an integrating sphere photodiode power sensor (PM100D) and a compact fiber photodiode power sensor (S155C), which were both manufactured by Thorlabs.We analyzed the structural aspects of the CNT, fCNT and SA samples using XRD. Both high-resolution powder XRD  and high-resolution thin film XRD  were operated between 5° and 50°  peak in XRD patterns according to ultrasonication time. Figure S2. Normalized saturable absorption of the SWCNT-PDMS SA.