Carbon fiber offers numerous material benefits including reduced wear, high strength-to-weight ratio, a similar elastic modulus to that of bone, and high biocompatibility. Carbon fiber implants are increasingly used in multiple arenas within orthopaedic surgery, including spine, trauma, arthroplasty, and oncology. In the orthopaedic oncologic population, the radiolucency of carbon fiber facilitates post-operative imaging for tumor surveillance or recurrence, the monitoring of bony healing and union, and radiation mapping and delivery. Orthopaedic surgery and materials science are closely intertwined, as the success of many orthopaedic devices are contingent on the material properties of the components used. Specifically in orthopaedic oncology, advancements in implant materials and design are crucial to preservation of function and quality of life in oncology patients after the tumor resection or treatment of metastatic disease. Reconstructive implants in orthopaedic oncology ideally maintain limb function and bone strength, allow for full bone healing, minimize risk of infection and implant failure, and facilitate the specific needs of oncology patients, including surveillance imaging, the visualization of bony union or healing, and radiation mapping and delivery. Historically, metallic implants, frequently used for such reconstructive purposes, have been associated with the tradeoff of imaging artifact. Carbon fiber has more recently been making inroads within orthopaedic surgery and oncology, as its material properties have particular advantages in comparison to metallic materials. As a material, its benefits have long been recognized and applied to numerous industrial needs, ranging from aerospace to civil engineering . Within Carbon fiber was first discovered in 1860 and used for light bulb filaments, which typically involved baking cotton threads or bamboo slivers at high temperatures to carbonize them. This was followed by the development of high-performance carbon fibers by using either rayon or polyacrylonitrile as the material to be carbonized, yielding carbon fibers with very high tensile strength and elasticity, while maintaining a high strength-to-weight ratio. Iterative improvements in the production process for carbon fiber, as well as the incorporation of carbon fiber into composite materials, led to widespread commercial application, including use in sporting goods, aerospace applications such as in heat shields or aircraft brakes, aircraft frames such as in sailplanes or military aircraft, race cars, and structural reinforcement of structures such as bridges. Perhaps the most high profile example of a carbon fiber product is the Boeing 787 aircraft, whose structure is 50% carbon fiber .When employed in composite materials within the medical domain, carbon fiber is typically combined with several types of resin matrices, one of the most common of which is carbon fiber–polyether ether ketone (CF–PEEK). There are numerous properties of carbon fiber that make it ideal for orthopaedic applications. It is highly biocompatible and chemically inert , generat3/Mc (million cycles), lower than that of ceramic on cross-linked, ultra-high-molecular-weight polyethylene (UHMWPE), metal on cross-linked UHMWPE, or ceramic or metal on conventional UHMWPE [The ability to withstand fatigue strain is yet another benefit of carbon fiber implants. Traditional implants demonstrate higher failure rates, especially in pathologic fractures, often due to non-union or hardware failure . By contl UHMWPE . This isl UHMWPE ,19. As al UHMWPE .One of the most important advantages of carbon fiber implants over metallic implants is their radiolucency. On both magnetic resonance imaging (MRI) and computed tomography (CT), carbon fiber has minimal scatter or susceptibility artifact, respectively . This ra3 compared to 937 ± 322 mm3, respectively) [With regard to CT imaging, carbon fiber demonstrates similar Hounsfield units compared to biological material, allowing for more accurate dose calculations . In one ctively) . As mighctively)  and reductively) ,28. In actively) . FurtherGiven the numerous material strengths of carbon fiber, it is no surprise that their use in orthopaedic implants has been increasingly studied across nearly all orthopaedic subspecialties in multiple applications. Since the 1990s, carbon fiber implants have been used for spinal surgical procedures such as posterior lumbar interbody fusion  and anteOrthopedic trauma surgery has more recently explored the use of carbon fiber plates in a variety of applications, including intramedullary nailing, proximal humerus or distal radius plating, and dynamic compression plating. When compared to their corresponding metallic implant counterparts, carbon fiber implants have been found to have similar performance in four-point bending, torsional, and bending fatigue, as well as reduced particle generation in wear testing . ClinicaCarbon fiber implants have promising applications particularly with regard to spinal tumor and spinal metastatic surgery. A large comparison of 78 patients with spinal metastases who underwent fixation with CF–PEEK versus titanium implants noted similar rates of post-operative clinical complications and hardware failure. Of note, the CF–PEEK group had longer operative times and higher blood loss, which might suggest a learning curve with these newer implants . AnotherSpecifically with regard to radiotherapy in the spinal oncologic setting, precise radiation planning and delivery is critical to avoid damage to the neural structures in close proximity. In simulations utilizing carbon-fiber-reinforced polyether ether ketone and titanium implants (CFP–T), CFP–T implants allowed the complete visualization of the spinal cord on MRI and allowed for minimal differences in radiation dose between planned and measured dose distributions .In this section, we present five cases from our department utilizing carbon fiber implants to highlight their unique advantages in the treatment of orthopaedic oncology patients.We present a case of an otherwise healthy 54-year-old male who was referred to our center for management of a progressively enlarging, palpable, non-tender mass on his anterolateral leg. The pre-operative MRI demonstrated a large, heterogeneously enhancing, solid soft-tissue mass that was T1 hypointense and T2 hyperintense A. The maThe patient subsequently underwent wide excision. In order to maximize negative margins, a partial corticectomy of the anterolateral tibia was performed. A carbon fiber intramedullary tibial nail was prophylactically placed in order to prevent fracture after cortical resection B. Post-oWe present a case of a 63-year-old female who presented for evaluation of medial-sided left knee pain. The patient had a history of a low-grade cartilaginous neoplasm, treated eight years prior with intralesional curettage and bone grafting at an outside institution. No surgical adjuvants were used during the index operation. She continued to have medial-sided knee pain, and three years after her index operation, underwent knee arthroscopy and medial meniscectomy for a meniscal tear. Surveillance radiographs performed by her prior surgeon demonstrated concerning changes in the medial femoral condyle that were further assessed on MRI A. PercutWe present a case of an otherwise healthy 57-year-old female with right thigh pain for three months. Plain films A and magWe present a case of a 36-year-old female with no past medical history who presented with 3 months of left shoulder pain, paresthesias, and pain with shoulder movement. She previously received a steroid injection to the shoulder without significant relief. Biopsy conducted at an outside institution was concerning for high-grade osteosarcoma, and she was referred to our care. MR images A demonstWe present the case of a 41-year-old female with a past medical history of heart failure, endometriosis, and hypothyroidism, who was previously treated at the age of 14 for osteosarcoma of the right proximal tibia and underwent intercalary allograft reconstruction. She presented to our care with allograft failure. With regard to her oncologic history, she initially presented to care at the age of 14 with 3–4 months of progressive swelling below the right knee. She underwent an open biopsy, which revealed high-grade osteosarcoma; she had a negative metastatic work-up. She underwent neoadjuvant and adjuvant chemotherapy with cisplatin and doxorubicin alternating with high-dose methotrexate, and bleomycin/cytoxan/dacarbazine. She then underwent intercalary resection of the right tibia with allograft reconstruction using conventional metallic plate and screw fixation A. Her poShe presented to our care with increasing pain and decreased ability to bear weight in the right lower extremity. She had normal inflammatory markers and normal serum white blood cell count. Radiographs and CT scan imaging were concerning for allograft failure B. A subs4 months after surgery, she sustained a mechanical fall. A unicortical fracture at the allograft and host bone interface was identified on radiographs without evidence of hardware complication D. Her frIn these cases, carbon fiber implants were applied for an range of applications in orthopaedic oncology patients with examples of the radiologic properties of carbon fiber facilitating post-operative surveillance of tumor recurrence and bony healing. Although preliminary studies in the orthopaedic oncologic population have demonstrated promising early results , future There are several important factors that must be considered in the use of carbon fiber implants in orthopaedics. Firstly, in comparison to metallic implants, carbon fiber has a lower load to failure, suggesting titanium or steel may carry a lower risk of implant failure in patients expected to have a high functional status post-operatively . SecondlAn additional consideration is the potential for undetected implant failure given the radiolucency of carbon fiber implants, which has been reported in case studies . FinallyCarbon fiber implants have numerous promising advantages for use in orthopaedic oncology, including favorable material and radiologic properties. Additional studies are needed as these implants are more widely utilized in the orthopaedic oncologic population to better characterize long-term implant survival and complications as well as the clinical outcomes associated with their use.


Chronic pain affects ~10–20% of the U.S. population with an estimated annual cost of $600 billion, the most significant economic cost of any disease to-date. Neuropathic pain is a type of chronic pain that is particularly difficult to manage and leads to significant disability and poor quality of life. Pain biomarkers offer the possibility to develop objective pain-related indicators that may help diagnose, treat, and improve the understanding of neuropathic pain pathophysiology. We review neuropathic pain mechanisms related to opiates, inflammation, and endocannabinoids with the objective of identifying composite biomarkers of neuropathic pain. In the literature, pain biomarkers typically are divided into physiological non-imaging pain biomarkers and brain imaging pain biomarkers. We review both types of biomarker types with the goal of identifying composite pain biomarkers that may improve recognition and treatment of neuropathic pain. Pain is a protective evolutionary function that involves “unpleasant sensory and emotional experiences associated with, or resembling that associated with, actual or potential tissue damage” . Acute pain is an adaptive and essential survival behavior. Chronic pain is a pathological condition that poses a significant clinical, economic and social burden , 2. ChroNeuropathic pain is defined as pain that is “initiated or caused by a primary lesion or dysfunction in the nervous system” . NeuropaPain is a subjective sensory experience that cannot be directly measured nor quantified. Although pain is subjective and influenced by many physiological and psychological factors, measuring biomarkers of neuropathic pain provides an opportunity to identify objective markers of peripheral nerve damage and other pathology contributing to neuropathic pain. If used in combination, biomarkers related to pain mechanisms  offer the possibility to develop objective pain-related indicators that may improve diagnosis, treatment, and understanding of pain pathophysiology –11. The Toward the goal of identifying composite biomarkers for investigating neuropathic pain mechanisms and improving diagnosis and treatment response, we present a review of non-imaging and imaging pain biomarkers related to various neuropathic pain mechanisms, including opiate, inflammation, endocannabinoid mechanisms. In this review, we review mechanisms for neuropathic pain in general, but we focus on pain biomarkers for different types of peripheral neuropathies. Although various reviews of pain biomarkers exist, we focus on creating composite biomarkers through machine learning approaches that can most accurately identify people with neuropathic pain.Endogenous opioids are necessary for the expression of pain relief  and painOpioid receptors are expressed by central and peripheral neurons, by neuroendocrine , immune, and ectodermal cells –25. All Beta-endorphin levels in the CSF, blood and saliva have been investigated as possible pain biomarkers. Plasma Beta-endorphin has been used to investigate age responses to experimental pain . PatientFunctional brain imaging performed on patients with non-neuropathic primary dysmenorrhea with mu-opioid receptor A118G polymorphism has been used to investigate pain sensitivity and opioid-analgesic treatment related to function in the descending pain modulatory system. Specifically, the functional connectivity of the descending pain modulatory system dependence upon mu-opioid receptor A118G polymorphisms was investigated. This study found that patient groups with different alleles for the A118G polymorphisms exhibited varying functional connectivity between the anterior cingulate cortex and periaqueductal gray .in vivo at the molecular level. Such important opioid function information can be obtained through positron emission tomography and can be used to investigate pain opioid mechanisms , the role of neuroinflammation regarding the initiation and maintenance of neuropathic pain has evolved tremendously over the last decade. Pro-inflammatory cytokines have been implicated in the generation of neuropathic pain states at both peripheral and central nervous system sites , 37. NeuPeripheral nerve injury causes neuroinflammation in the spinal cord , 46–48. Concentrations of CSF proinflammatory cytokines are increased in multiple neuropathic pain states . Most stCytokines have also been demonstrated to be potent mediators of pain in peripheral neuropathy. In one peripheral neuropathy study, gene expression of pro- and anti-inflammatory cytokines was shown to be increased in patients compared to controls . AnotherAnother key pro-inflammatory neuropeptide, Substance P, is known to initiate biological inflammatory effects . In pain* relaxometry. These imaging techniques can be used to image human neuroinflammation which have potential to impact patient care in the foreseeable future , endocannabinoids , and synthetic cannabinoids. Similar to the opioid system, versions of the endocannabinoid (ECB) system have been found in the vast majority of species with a nervous system . In parteceptors .The two most prevalent endocannabinoid ligands that bind endocannabinoid receptors are anandamide (AEA) and 2-arachidonoylglycerol (2-AG) . The 2-AThere are two G protein-coupled ECB receptors (CBR1 and CBR2). CBR1 receptors are highly expressed on presynaptic neurons in the brain, spinal cord, and dorsal root ganglion. CBR2 receptors are primarily expressed in immune cells  . AEA maiThe CBR1 receptor is the most abundant G protein-coupled receptor in the brain and one of the most abundant in both the peripheral and central nervous system. CBR1 are expressed primarily on presynaptic peripheral and central nerve terminals. CBR1 is the central receptor responsible for the behavioral and psychotropic effects of the “high” caused by THC , 72. TheThe CBR2 receptor is largely present on peripheral immune cells and participates in regulation of the immune system. The principal endogenous ligand for the CBR2 receptor is 2-AG. In the brain, stimulation of CBR2 receptors does not produce cannabis-like effects , 74. TheIn neural and non-neural systems, in response to tissue injury or excessive nociception, the ECB system generally suppresses inflammation, suppresses sensitization, and suppresses pain , 72, 77;ECB biomarkers have been used to monitor neuropathic pain. Increases in circulating AEA concentrations occur in patients with neuropathic pain complex regional pain syndrome (CRPS) when compared to controls without pain . Increasvia multiple mechanisms  is associated with neuropathic pain in a variety of clinical conditions. One study found that reductions in the diversity and increases in the ratios of two microbial species (ratios of Blautia and Clostridium to Lachnospira) may contribute to HIV-associated neuropathic pain . This maHLA genes, COMT, OPRM1, TNFA, IL6, and GCH1, were found to have an association with neuropathic pain in more than one study. In the meta-analysis, polymorphisms in HLA-DRB1*13, HLA-DRB1*04, HLA-DQB1*03, HLA-A*33, and HLA-B*44 were associated with significantly increased risk of developing neuropathic pain, whereas HLA-A*02 reduced risk of neuropathic pain.In a systematic review and meta-analysis of 29 studies on potential genetic variants associated with neuropathic pain identified , 28 geneTo detect genetic associations, particularly those of small effect size, a study must be sufficiently statistically powered to detect those differences. Most genetic studies of neuropathic pain have typically analyzed cohorts with <1,000 cases, which has resulted in only suggestive associations , 104. OnMicroRNA are small non-coding RNA molecules that contain about 22 nucleotides and are found in plants, animals, and some viruses . IdentifMicroRNA-mRNA interactions allow for modification of gene expression by controlling translation in response to signaling events. Disease states or perturbations in cellular homeostasis can lead to aberrant microRNA expression . NumerouIt is well-established that microRNAs are present in the serum and plasma of humans and stable (as part of RNase resistant molecular complexes or within vesicles or exosomes) such that retrospective studies can be performed using banked samples , 113. HoThe discovery of stable microRNAs in circulation has generated enormous interest in exploring their utility as potential non-invasive biomarkers , 114. ThDysregulations in microRNAs have been reported in several pain disorders in humans in both affected tissues and the circulation , 117: NePresence of circulating microRNAs within exosomes opens up novel avenues for targeting treatments for chronic pain conditions. Such approaches can provide insights on the molecular underpinnings regarding therapeutic targets, treatment doses, and patient eligibility for different treatments , 123.Selectively inhibiting or supplementing a microRNA contributing to pathogenesis is being pursued as a therapeutic strategy for a variety of disorders. Studies from rodent pain models and from patients have now implicated a role for microRNAs in mediating various aspects of pain processing. These non-coding RNAs can provide mechanistic insights into the pathways modulated and could serve as therapeutic targets . Drug trMicroRNA modulated inflammation has a major role in the induction and maintenance of neuropathic pain. Inflammation-regulating microRNA profiles in patients with peripheral neuropathies have been characterized. In patients with polyneuropathies of different etiologies the expression of miR-21-5p, miR-146a, and miR-155 were upregulated. In painful neuropathies, tissues from skin biopsies from the lower leg, where neuropathic changes are most common, had reduced miR-146a and miR-155 expression compared to the thigh; furthermore, peripheral neuropathies are associated with aberrant microRNA expression in the sural nerve and in the skin . In suraAllostatic load indices, an index that measures the effects on the body by chronic stress, have been demonstrated to predict morbidity and mortality. Allostatic load has been studied in pain with allostatic load model covariates including age, sex, education, smoking status, alcohol consumption, activity level, depression, and common comorbid health conditions. Allostatic load pain models have shown a positive relationship between pain severity and allostatic load .Cortisol is a proposed stress-related pain biomarker . DHEA anAllopregnanolone is a neuroactive steroid derived from progesterone that is synthesized within the nervous tissue. Allopregnanolone interacts with GABA-A receptors making it important in neuroprotection particularly in cases of ischemia and peripheral neuropathy. Plasma allopregnanolone immunoreactivity has been associated with decreased pain sensitivity in humans which may be mediated by hypothalamic-pituitary-axis function . AllopreBiomarkers in saliva may be useful as they are easily measurable without requiring a needlestick or invasive methods . SalivarQuantitate sensory testing and skin punch biopsy results are potential peripheral neuropathic pain biomarkers, in particular for diabetic peripheral neuropathy , 142. MaSkin conductance responses and alterations in electrocardiograms have been used to predict pain level ratings with high sensitivity and moderate specificity . PupillaOrnithine levels have also been found to be elevated in patients with persistent muscle pain . SignifiNeurotrophic factors  and erythropoietin with the erythropoietin receptor are up-regulated in patients with peripheral neuropathy . NeurotrIn the past decade, the focus of brain imaging investigation of pain mechanisms has shifted from investigating individual regions of the brain to investigating brain circuits, see One of the most established chronic pain brain imaging biomarkers is reduced regional gray matter volumes in brain regions related to pain processing –164. OneIt has been shown that in multiple chronic pain conditions that there is increased connectivity between the salience network and the default mode network and decreased activity in the default mode network –171. ThiPain rumination occurs when people negatively dwell on their pain experience. Interestingly, this negative cognitive dwelling on the pain experience appears as increased activity in the default mode network . This inIt is plausible to envision that healthy mind wandering away from thinking about the pain experience is the opposite of pain rumination , 173. ThIncreased functional connectivity between the medial prefrontal cortex–nucleus accumbens at the beginning of back pain predicts that patients will go on to develop chronic back pain; while patients with decreased connectivity in this circuit went on to recover from back pain , 175. StThere is substantial overlap between the circuits involved in human placebo analgesia and those that mediate multiple forms of context-based modulation of pain behavior in rodents, including forebrain-brainstem pathways and opioid and cannabinoid systems in particular. This suggests that placebo effects are a set of adaptive mechanisms that shape nociceptive signaling , 178. SuIndividuals have a set point around which different biological attributes can fluctuate transiently into different states. However, if one remains in a different state other than their set point for a considerable period , this different state is considered a new set point. In pain research it is important to consider trait and state pains to gain an understanding of not only an individual's current pain state but also more broadly to their trait pain, which may be more reflective of their general condition . ResilieTwo reviews on mechanisms and imaging biomarkers for diabetic neuropathic pain review that diabetic peripheral neuropathy and associated pain have structural and functional central nervous system changes in the spinal cord, subcortex, and cortex , 185. DiDiabetic neuropathic pain is associated with increased regional brain gray matter volume loss localized to brain regions involved in somatosensory perception ; furtherStructural brain imaging has revealed changes to the brain associated with HIV peripheral neuropathy. Total cortical volume is smaller with HIV distal neuropathic pain . In factMore recent multi-modal brain imaging research has revealed structural brain changes associated with HIV peripheral neuropathy . HIV perHIV-associated distal neuropathic pain is associated with decreased fMRI resting state functional connectivity in the default mode network and increased functional connectivity in the salience network . DecreasIn the setting of experimental heat pain, significant interaction has been found within the right anterior insula during expectation of experimental pain offset in that a group with HIV distal neuropathic pain compared group without HIV distal neuropathic pain exhibited increased insula activation in the feet (with painful neuropathic pain) compared to the hand (without neuropathic pain) . These fAnterior cingulate cerebral perfusion and gray matter density correlate with chemotherapy-induced peripheral neuropathy symptoms including pain . PatientA population of mixed small-fiber peripheral neuropathy  was used to investigate how dysfunction of skin nerves led to abnormal recruitment of pain-related brain regions, suggesting that the brain may be affected in SFN. Greater volume reduction in pain-processing regions, particularly the bilateral anterior cingulate cortices was associated with greater depletion of intraepidermal nerve fibers. There was significant reduction in functional connectivity from the anterior cingulate cortex to the insula pain-processing cortex that is linearly correlated with the severity of intraepidermal nerve fiber depletion . SimilarA population of Charcot-Marie-Tooth patients had abnormal diffusion tensor imaging findings indicative of significant cerebral white matter abnormalities. Diffusion tensor imaging abnormalities were correlated with clinical disability, suggesting that there is comorbidity of central nervous system damage with peripheral neuropathy in Charcot-Marie-Tooth patients . A populAs discussed in the introduction, if used in combination, biomarkers related to pain mechanisms offer the possibility to develop objective pain-related indicators that may help diagnosis, treatment, and understanding of pain pathophysiology , 10. OneModeling pain brain mechanisms can be achieved using multi-modal brain imaging including functional magnetic resonance imaging, structural magnetic resonance imaging, diffusion tensor magnetic resonance imaging, electroencephalography, EMG, and PET , 14, 218Multiple analytic approaches have been used to investigate composite pain biomarkers: (i) composite algorithms have been investigated , (ii) unAs detailed above, chronic pain and neuropathic pain impact multiple organ systems. Advancing the value of pain biomarkers depends on (1) selection of measurements and metrics that are the most mechanistically valid and informative, and (2) combining the selected measurements such that they mechanistically and statistically maximize accurate classification. Advancement of measurement accuracy is vital and the subsequent steps of the approach are entirely contingent upon the success of this step. This literature for the domains discussed in this manuscript is too voluminous for a single review. In the above reviewed literature, we attempted principally to focus on which biological systems and which biomarkers should be the focus of measurement. For effective application of measurements of these domains it is important to discuss approaches for measurement selection.In Two key methods for statistical reduction of variables are (1) selecting top ranking variables and (2) creation of composite variables by factor or component-based analysis. Random Forest, as depicted in via QST, skin conductance, pupil dilation, fatty acid pain biomarkers (ornithine and linoleic acid derivatives), neurotrophic factors, and serum neurotransmitters.A summary of pain biomarkers discussed in this review article are provided in via modulation in brain circuits: acute pain machine learning measures of chronic pain, pain rumination, pain mind wandering, placebo mechanisms, pain traits and states, and resilience. HIV peripheral neuropathy changes in the brain include reduced total cortical gray matter and reduced posterior cingulate cortex volume in particular, white matter degeneration, altered resting state networks, and aberrant expectation of pain relief.Brain imaging pain biomarkers for measuring pain can be evaluated using three different MRI brain methods: gray matter structural imaging, white matter diffusion tensor imaging, and functional brain activation. Brain circuits related to pain mechanisms include an ascending brain circuit, a descending pain modulation circuit, the default mode circuit, the executive network brain circuit, and finally the salience network. Pain mechanisms in the brain can be measured By focusing on a broad array of mechanisms and biomarkers, we can uncover important mechanistic connections and interactions across systems. Neuropathic pain is a debilitating condition that has primary, and cascading affects across body systems. Assessment and understanding in an appropriately comprehensive approach are challenging due to the vast and diverse literature and the complexity measurement. This review aims to facilitate navigation of this literature and the appropriate selection of biomarkers for future research.All authors have contributed to the writing and scientific direction of this manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Tertiary lymphoid structures (TLS) are anatomic entities that are similar to, but distinct from, secondary lymphoid structures  that allow for a host’s own immune system to respond in a more targeted and efficacious way. TLS are increasingly recognized as markers of prognosis in cancer patients and are now being implicated as direct mediators of immunotherapy efficacy. The inherent properties of TLS, as well as their cellular constituents, are being elucidated across tumor types, with commonalities becoming more apparent. Given the importance of TLS in a patient’s response to malignancy, the ability to induce TLS promises to be an advantageous therapeutic avenue and already appears feasible in preclinical models.Since its first application in the treatment of cancer during the 1800s, immunotherapy has more recently become the leading edge of novel treatment strategies. Even though the efficacy of these agents can at times be predicted by more traditional metrics and biomarkers, often patient responses are variable. TLS are distinct immunologic structures that have been identified on pathologic review of various malignancies and are emerging as important determinants of patient outcome. Their presence, location, composition, and maturity are critically important in a host’s response to malignancy. Because of their unique immunogenic niche, they are also prime candidates, not only to predict and measure the efficacy of immunotherapy agents, but also to be potentially inducible gatekeepers to increase therapeutic efficacy. Herein, we review the mechanistic underpinnings of TLS formation, the data on its relationship to various malignancies, and the emerging evidence for the role of TLS in immunotherapy function. Over the last two decades, immunotherapy has come to the forefront of cancer treatment. The number of immune modulating agents and modalities being brought to the bedside is rapidly accelerating along with burgeoning activity to identify tumors and patients who would most benefit from these novel approaches. Within the complex immune contexture of cancer, tumor-infiltrating lymphocytes (TIL) may be programmed and produced in the periphery from secondary lymphoid organs (SLO) and potentially access the tumor microenvironment (TME), thereby receiving support by tertiary lymphoid structures (TLS). SLO and TLS are broadly characterized as sites wherein a host immune response can be activated, modulated, and perpetuated. SLO encompass classically known structures, including lymph nodes, Mucosa-Associated Lymphoid Tissue (MALT), and Peyer’s patches. TLS share many structural characteristics to SLO; however, it is their differences in composition, development within the TME, and role in immunotherapy outcomes that are proving critical.+), the HEV-like structures in TLS are flat compared with the plump cuboidal endothelial cells found in SLO [TLS are fundamentally non-orthotopic accumulations of variably organized lymphoid tissue arising in the setting of, and as a response to, immune-driven inflammation. While this immune state can be the sequalae of malignancy, other processes, including infection, graft rejection, and autoimmunity, may manifest TLS . TLS appd in SLO . Similard in SLO ,5. Precld in SLO , VEGF bld in SLO , and viad in SLO . A variad in SLO . DespiteIt is within this immunologic role that TLS appear to not only be associated with patient prognostication, but also in predicting treatment response and possibly even being the target of novel therapeutic strategies clinically.+ HEV that were only noted to be found within the TLS and not in any other areas of examined breast tissue. TLS were specifically found to be present in nearly one-third of that cancer cohort [CXCL13-producing T follicular helper (Tfh) cells and germinal centers. A derivative 8-gene signature, which included detection of the aforementioned CXCL13 and Tfh, was found to be associated with improved disease-free survival in systemic-adjuvant naïve patients and improved tumor response to those treated with neoadjuvant chemotherapy [The role of immune cell infiltration on patient prognosis was extensively queried in breast cancer and indeed studies demonstrate the importance of the microenvironment in determining clinical outcomes. Early work indicated that immune infiltration in breast cancer in the form of increased inflammation was key in the outcomes of breast cancer patients. Histopathologic examination of breast cancer patients who had undergone surgical resection found that diffuse intratumoral inflammation was associated with histologic grade . Those pr cohort . Additiootherapy .+, TLS were associated with improved disease-free survival independent of TIL levels. Most intriguing, this study was conducted in a patient population in the pre-targeted therapy area and almost no patients received trastuzumab [Interestingly, the role of TLS in the initiation/maintenance of the host anti-tumor milieu could also be related to the structural organization and composition, rather than just the ability of lymphocytes to enter the solid tumor. In a cohort of breast tumors which underwent primary surgical resection, TLS were found to be associated with high lymphocyte infiltration in the whole patient cohort; however, in a subset of patients who were HER2stuzumab . This instuzumab . Althougstuzumab ,18.+, CD8+, CD45R0+, and FOXP3+, were noted to be important contributors to patient prognostication [Malignancy-instigated immune responses are well described in colorectal cancer and the presence of TIL and specific TIL subpopulations can be important prognosticators. Initial studies demonstrated that the location of immune cell infiltration and specific cellular populations could prognosticate patients even beyond traditional metrics . Indeed,tication . When moCCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21, CXCL9, CXCL10, CXCL11, and CXCL13) correlated with the presence of TLS and in a highly selected subgroup with improved patient prognosis and overall survival [CXCL13 and CCL21, as well as the presence of PNAd+ HEV. In fact, the HEV characterized were only noted to be localized to these lymphoid aggregates and not with other tumoral or non-tumoral tissue, lending credence to their role in TLS function. Importantly, in a subset analysis, patients who had earlier disease (stage II) versus later disease (stage III) significantly correlated with disease-free interval indicating that there may be a time during which TLS play a crucial role in immune-mediated tumor control, but that after a certain threshold, immune escape renders them ineffective  [In resected specimens of colorectal adenocarcinoma, marked peri-tumoral TLS were identified in a particularly distinct stage of organ maturation. A derivative gene signature that encompassed the expression of TLS determinant chemokines  . This leulation) . Their sulation) . The TLSCCL18, 19 and 21, CXCL9-11 and 13 and CCL 2, 3, 5 and 8, was an accurate prognosticator for HCC patients, independent of traditional clinical factors. Given that HCC tends to arise in the setting of chronically inflamed parenchyma, the role of TLS in tumorigenesis in this setting is distinct from that which was seen in other tumor types. As opposed to peritumoral TLS, intratumoral TLS appear to play a different role in tumor progression and prognosis [While a preponderance of data across tumor types characterizes TLS as key in the antitumoral coordination of the host immune system, investigations in hepatocellular carcinoma (HCC) provide insight into the mechanisms of such responses. The location of TLS in relation to the tumor appears to be extraordinarily important in their ability to incite effective immune-regulated tumor control. Indeed in non-cancerous liver parenchyma, the presence of TLS allowed for a local microniche wherein malignant progenitor cells could appear and eventually progress into tumors . These prognosis . In a corognosis .+ DC). Interestingly, TLS had no discernable presence within normal lung parenchyma distant from the tumor. Additionally, when intratumoral TLS detected by LAMP+ DC were present within these early-stage resected specimens, there was a univariate correlation to improved disease-free survival, disease-specific survival, and overall survival [Within lung cancer, the presence of TLS was identified as a progression of lymphoid aggregates that previously was only described in neonatal tissue. In this setting, it was termed bronchus associated lymphoid tissues (BALT) and these were known to involute by adulthood . Howeversurvival . TLS preIn NSCLC, intratumoral TLS may also support the production of memory/effector cells that remain within the host and continue to provide immune-derived tumor control even after the offending nidus had been resected. This theoretical mechanism is further bolstered by a follow-up examination of resected NSCLC patients, including those who were treatment naïve and those that received systemic neoadjuvant therapy . HistopaUrothelial bladder cancer has a long-standing connection with immunology, beginning with the treatment of malignancy with Bacilus Calmette-Guerin (BCG). Additionally, there was a focus on the immune infiltration patterns of bladder cancers, especially given the recognition that low grade variants and high grade variants may be distinct clinical entities with significant impact on patient prognosis. In an early study examining immune cell infiltration in a cohort of both high grade  and low grade bladder cancer , TLS were only present in 25% of NMIBC cases, while they were present in 75% of MIBC cases . Moreove+ HEV. This early data also attempted to identify similar aggregates in primary lesions using follicular DC expression (defined by CD21L) as a molecular proxy for TLS, and found no presence [+ HEVs [TLS in melanoma were originally described in metastatic disease where samples of cutaneous progression demonstrated ectopic lymphoid aggregation, as well as the presence of PNAdpresence . However [+ HEVs . Additio [+ HEVs ,38.In a study of patients undergoing melanoma metastasectomy, TLS were identified in 47% of the samples and were associated with a lower risk of recurrence and improved overall survival following resection . Further+ HEV was identified in 68% of tumors, but not seen at all in distant, normal cutaneous samples [+, CD3+ and CD8+). Specific chemokine signatures associated with antitumor T-helper activity correlated with HEV density, while anti-tumor/tumor escape T-regulatory cell density did not appear to be associated. Taken in aggregate, this provides indirect evidence of the role of HEVs in immune-system priming driven by TLS.The study of HEV within melanoma also provided an important vantage of the interplay between the immune system and melanoma tumor control. In a study of primary resected melanoma, MECA-79 samples . Additio samples . HEV denThe presence of TLS appears to be correlated with patient prognosis in variable tumor types. While the aforementioned work in breast cancer indicates that TLS may be useful prognosticators in the setting of traditional chemotherapy, responses to novel immunotherapies may be more relevant given the important immunologic niche that TLS may serve.+) were noted to be localized to just outside the TLS, indicating that the TLS may be a focus of activation for the systemic immune response to the tumor [+ infiltration or TLS presence had improved survival; those with both had the best survival; and those with neither had the worst. Additionally, this study was able to derive a gene signature using key determinants of TLS formation in melanoma and tumors were subsequently able to be stratified as TLS high, intermediate, and low. This signature was then applied to the biopsy specimens of patients who went on to receive CTLA4 blockade. TLS-high signature patients demonstrated a significantly improved overall survival in a stepwise fashion compared with those with TLS intermediate or TLS low signatures after treatment with CTLA4 blockade. This finding was reciprocated in an additional cohort of melanoma patients and highlights the importance of TLS in modulating the immune response to tumors and allowing the more efficacious application of checkpoint blockade.In one study of resected melanoma specimens, TLS were identified and T-cell subpopulations  the secretion of chemotactic cytokines  [TLS share many of the structural foundations of SLO, including (1) the presence of HEV-like structures, 2) distinct cell clusters characterized by T-cell rich areas, LAMP distinct+ HEV. Subsequently, the number of HEV-associated endothelial cells (HEC), as well as specific TIL, was noted to increase when treated with combined CTLA-4 and PD-1 blockade. When a sample of pre-treatment metastatic lesions was examined, the presence of HEV was higher in patients who responded to combined CTLA-4 and PD-1 blockade and this response was likely a driver for the observed improvement in both progression-free survival and overall survival seen with those that had been treated with combined therapy [The crux of TLS functionality may lie in the presence of HEV-like structures. Immune surveillance is dependent on the ability of lymphocytes to transition to and from the blood. This circulation allows relatively rare naïve, antigen-specific lymphocytes to broadly surveil the host organism. This movement is dependent on specialized blood vessels, HEVs, which were well characterized in relation to peripheral lymph node function  but were therapy .Speed—TLS may allow for rapid lymphocyte priming by circumventing the extraneous trafficking needed for SLO function; (2) Efficiency—localized lymphoid contexture is fostered by the approximation of lymphocyte and recognized tumor-associated antigens; (3) Control—TLS allow for immune response refinement by aggregating immune cells and malignant targets; and (4) Survival—lymphocyte homeostasis is modulated by TLS-cell population factors [Collectively, TLS may provide the immune-activating functionality of SLO, while providing distinct benefits that foster antitumor activity outlined succinctly by Schumacher and Thommen: (1)  factors . Indeed, factors .Consequently, the ability to generate inducible TLS (“iTLS”) presents a potentially attractive avenue for therapeutic intervention, whereby TLS could be introduced into the tumor microenvironment and allow the host immune system to either intuitively prime itself to an antitumor state or allow for the more efficacious application of immunotherapy agents, such as checkpoint blockade . One sucAs the role of the tumor microenvironment in regulating patient prognosis and therapeutic response is now more evident, immune augmentation is quickly becoming the first line approach for tumor control and even eradication. TLS are unique, immunologically advantageous anatomic structures that allow for improved patient prognostication and, perhaps most importantly, are potentially an underutilized avenue for therapeutic intervention.


Recent technological and conceptual advances will allow the elucidation of the physiological, ecological, and chemical contributions of understudied marine fungi to coral holobiont and reef ecosystem functioning and health and may help provide an outlook for reef management actions.Tropical coral reefs are hotspots of marine productivity, owing to the association of reef-building corals with endosymbiotic algae and metabolically diverse bacterial communities. However, the functional importance of fungi, well-known for their contribution to shaping terrestrial ecosystems and global nutrient cycles, remains underexplored on coral reefs. We here conceptualize how fungal functional traits may have facilitated the spread, diversification, and ecological adaptation of marine fungi on coral reefs. We propose that functions of reef-associated fungi may be diverse and go beyond their hitherto described roles of pathogens and bioeroders, including but not limited to reef-scale biogeochemical cycles and the structuring of coral-associated and environmental microbiomes  Conceptual perspective on known and proposed functions of understudied marine fungi on coral reefs and in reef-building corals, their main ecosystem engineers. Tropical coral reefs are highly diverse and productive ecosystems shaped by their main ecosystem engineers, reef-building corals. Corals are “holobionts,” multicellular animal hosts associated with a diverse suite of prokaryotes and microeukaryotes . We further discuss the potential spectrum of interactions of fungi with other organisms on and around reefs, ranging from mutualism and commensalism to parasitism. Based on these comparisons, we hypothesize that fungi may play a pivotal role in the health and ecological functioning of coral reefs, and in reef-building coral holobionts in particular. Finally, we conclude our work with future research directions that we hope will stimulate the advancement of research of fungi on coral reefs.Fungi in the marine realm, and on coral reefs in particular are understudied compared to terrestrial and freshwater ecosystems , respectively, as well as by prokaryotes and fungi , Hyalorhinocladiella , and Physalospora spp. . Within the Eurotiomycetes, notable representatives are Aspergillus spp. or Penicillium spp. .Few studies have investigated the community dynamics of coral-associated fungal communities in response to environmental change. While it appears that coral-associated fungal communities might be host-specific  is considered a potential barrier to fungal growth  associated with Pacific yew trees , which feed into specialized biosynthetic pathways involving large multimodular enzymes such as polyketide synthases PKSs, nonribosomal peptide synthetase NRPSs, prenyltransferases, and terpene cyclases  and the use of genome mining approaches to identify BGCs has tremendously increased our knowledge in recent years  in the unicellular parasitic Microsporidia to around 2 Gb in Pucciniales (rust fungi), in the same order of magnitude as the human genome Stajich . Fungal via hybridization occurs sexually or asexually . The observed gene duplications may reflect the requirements of a more demanding environment, such as a marine habitat, which selects for the retention of duplicated genes even when resulting changes in encoded protein activities are very slight . This in turn affects the fitness and capabilities of fungal cells after germination, stress and antifungal resistance, mycotoxin and secondary metabolite production, and virulence  during certain stages in the fungal life cycle may produce phenotypic variation in response to fluctuating or changing environments, which may be conducive to survival and acclimation. Conidiation, the formation of conidiophores from vegetative hyphae, is one such critical stage. Wang et al.  reportedFinally, little is known about host–fungi symbioses and their underlying molecular mechanisms of symbiosis establishment and maintenance on coral reefs. Major changes in host–symbiont gene (co-) expression reflecting genetic reprogramming and modulation of molecular crosstalk may facilitate novel associations, as reported for arbuscular mycorrhizal fungi symbioses . Thereby, marine fungi may help mobilize organic C in the ocean via the remineralization of recalcitrant high molecular weight detritus, thereby diverting energy to higher trophic levels through saprobic , and sulfur (S) in marine systems  or potentially even the tissues organic S compounds (including sulfides and methanethiol) are readily metabolized by different marine fungal isolates, suggesting a tentative contribution to coral reef S cycling .In conclusion, fungi may contribute to coral reef biogeochemical cycling, albeit at likely varying spatial scales and levels of biological organization Figs  and 4. Evia the generation of more accessible, intermediate decomposition products of recalcitrant organic matter, which bacteria (or other organisms) cannot access on their own  as QS molecules capable of regulating the production of antimicrobial metabolites  regulate morphological differentiation and mycotoxin production , a concerted, density-dependent cell-to-cell signaling process, is one of the most widely studied chemical communication strategies in fungi Hogan . UnicellAspergillus-infested beetle larvae, which in turn is used by parasitoid wasps as a cue to detect unfavorable environments  are ubiquitous in a wide range of organisms, play major roles in biological processes, e.g. regulating inflammation and cellular homeostasis in metazoans. Oxylipins are major mediators of cross-kingdom talk and host–fungal interactions that encompass predator–prey, mutualistic, and pathogenic and biological processes  are small compounds that diffuse easily through water and gas and play a critical role in biosphere–atmosphere interactions, in plant signaling, and as infochemicals in multitrophic interactions  highly depends upon these complex multipartite interactions  of affected tissues and gall formation. Affected tissue samples contained high loads of septate fungal hyphae, and culturing efforts identified the putative agent of the disease, a soil-dwelling saprobic fungus affiliated to A. sydowii demonstrated a correlation of the secondary metabolites sydowinol, sydowinin A and B, and sydowic acid with strain pathogenicity  were laden with fungal hyphae following a marine heat wave resulting in “coral bleaching.” Opportunistic coral-associated saprobic fungi may be able to overcome weakened immune defenses of their vulnerable host, analogous to human secondary fungal infection in the aftermath of viral disease , as observed in phytoplankton–chytrid pathosystems  have yet to be characterized, it may be possible they divert photosynthate from coral-associated algae  parasitic fungi in coral reef food webs.One of the best studied examples of aquatic fungal interactions are parasitic associations of chytrids with phytoplankton  yet to be discovered. The lack of examples of such mutualistic associations of marine fungi with other biota may be due to a bias toward the study of fungal pathogens and opportunists, a trend noticeable also in the study of terrestrial crop and human pathogen emergence.via fungal chemical communication and defense, microbiome structuring as well as biogeochemical cycling, thereby extending beyond their previously reported roles as pathogens and opportunists. In the following, we briefly summarize major challenges and open questions in the study of reef-associated fungi and propose a multidisciplinary toolbox to help address these questions.Taken together, we anticipate that functions of reef-associated fungi are likely diverse, spanning a spectrum of interkingdom interactions that may mediate processes at different levels of biological organization, from cell–cell interactions to ecosystem-scale effects. These may be facilitated vice versa, how do fungal interactions on coral reefs shape their (a)biotic environment from the cellular to the ecosystem level?Diverse knowledge gaps remain regarding the ecology of marine fungi in coral reef environments. Broadly, outstanding questions include: how does the taxonomic and functional diversity of fungal communities in corals and on coral reefs distribute across space and time; which ecological guilds and types of (symbiotic) interactions do occur in coral holobionts and on the reef; what are the mechanistic underpinnings of microbe–microbe and host–microbe interactions that fungi engage with in coral holobionts and on coral reefs; which (a)biotic drivers govern, maintain, and alter fungal communities and interactions; These questions could be addressed by employing a multipronged approach which targets different levels of biological organization for an integrated view of fungal functions in complex holobionts and the ecosystem Fig. :SymPortal, which resolves “defining” intragenomic variants for ITS2-type profiles of coral-associated endosymbiotic algal communities  is challenged by e.g. the pervasive amount of host-derived nucleic acids, which constitute a major hurdle for culture-independent sequencing approaches, as well as well-known limits to microbial cultivability  prior to unlabeled coculture, and subsequently preserved and prepared for correlative electron microscopy and SIMS analysis to visualize (sub)cellular assimilation and distribution of isotopic labels and metabolic interactions between cells. Such approaches, especially in combination with correlative fluorescence in situ hybridization  and omics applications as outlined above, would allow for the assessment of interactions in natural microbe–microbe assemblages or in clearly defined, synthetic communities of reduced complexity by targeting specific microbial functional groups or combinations of taxa. Notably, cultures of major coral-associated algal symbionts are available at dedicated microbial culture collections and can be readily maintained in stable cultures. Similarly, the availability of well-characterized coral bacterial isolates is steadily increasing, facilitating functional studies of microbe–microbe interactions in vitro  could be addressed in a combination of different culture-dependent and -independent applications. High-throughput OSMAC applications on microbial cocultures , as detailed for the study of complex multipartite host–microbe interactions , ex situ , or in situ  experiments, as recently established for rate measurements of selected prokaryotic or holobiont-level metabolisms, such as dinitrogen fixation or oxygen fluxes . Such approaches may hold the promise to address the work outlined above in a) to c), to help elucidate fungal contributions to reef-scale biogeochemical cycling.Finally, assessing the roles that fungi play at the community and ecosystem-levels and their spatio-temporal dynamics will be critical to advance the knowledge of fungal functions in coral reef ecosystems. Understanding and predicting how changes in coral reef fungal communities might translate into community-scale cascade effects and shifts should become a major goal. These include but are not limited to the (a) determination of (a)biotic drivers of fungal distribution and abundance in space and time, (b) quantification of fungi-specific activities at the community level such as their involvement in nutrient and energy transfer within and between the benthic and the pelagic reef environment, and (c) modeling of community-level budgets (e.g. C or N budgets) as well as ecological networks under consideration of fungal activity. In terrestrial environments, climate has been often reported as one of the strongest drivers affecting fungal community composition  from simpler ones, which requires energy, whereas the latter is the breakdown of complex molecules into simpler ones (i.e. catabolites) releasing energy.The former describes a cumulative effect, i.e. less than additive, i.e. less than the sum of effects . The latter defines a cumulative effect greater than the additive sum of effects.Aspergillus. These fungi can infect a wide range of hosts, ranging from coral to humans.A disease caused by fungi of the genus Processes that connect the sea floor (i.e. the benthic zone) and the water column (i.e. the pelagic zone) through the exchange of energy, mass, or nutrients. It plays a prominent role in nutrient cycling and energy transfer in aquatic food webs, and thereby ecosystem processes. BGC: tightly linked sets of (mostly) nonhomologous genes participating in a common, discrete metabolic pathway. The genes are arranged in physical proximity to each other on the genome, and their expression is often coregulated. Common in bacterial and fungal genomes and most widely known for the production of secondary metabolites.Carbohydrate-active enzymes which build and break down complex carbohydrates and glycoconjugates for a large body of biological roles (collectively studied under the term of Glycobiology).Movement of a cell or organism in response to an environmental diffusible chemical substance.Unicellular or mycelic, aerobic zoosporic fungi that operate as saprotrophs and pathogens in freshwater, brackish, and marine habitats.A phylum within the animal kingdom which includes jellyfish, anemones, and reef-building corals. Cnidaria are simple, multicellular organisms characterized by two main cell layers (ectodermis and endodermis) and an apparatus consisting of stinging cells (cnidocytes) for prey capture and defense.The ability of several fungi to switch between a multicellular hyphal and unicellular yeast morphology and growth form. The mechanism underlying this biological reorganization process depends on external  triggers.via the DMSP demethylation or DMSP cleavage pathways.An organosulfur compound produced in vast quantities by phytoplankton and seaweeds, and known to have osmoprotectant and antioxidant function. DMSP is an important carbon source for many marine bacteria, which can break it down Any group of species that exploit the same resources, or that exploit different resources in related ways. Among fungi, common guilds are decomposers (saprobic), pathogens, endophytes, and mycorrhiza.A group of organisms including cyanobacteria, fungi, algae, and bacteria that dwell in the pore space of rocks and similar substrates, such as coral skeletons.A molecular technique that uses fluorescent probes that bind to only particular parts of a nucleic acid (DNA or RNA) sequence with a high degree of sequence complementarity. Widely used in the field of microbial ecology to identify taxa and to visualize the distribution and proportion of specific taxa within environmental samples.Matrix Assisted Laser Desorption Ionization coupled to Time-Of-Flight mass spectrometry.The observed shift in ecosystem trophic structure toward higher microbial biomass and energy use. On coral reefs, causes of microbialization include overfishing and eutrophication.Refers to the behavior, precise control, and manipulation of fluids geometrically constrained to a small scale. In (micro)biology, it offers a powerful approach to control the complete cellular environment, thereby enabling the study of microbial community microscale organization, cellular behavior, adaptation, or gene expression.Mycorrhizae are soil-borne fungi closely associated with the roots of terrestrial plants. Arbuscular mycorrhiza colonize the intercellular spaces of plant roots . Arbuscular mycorrhizae are considered vital endosymbionts of plant holobionts, as they enhance productivity.Sequencing is the process of determining the order of nucleotides in entire genomes or targeted regions of DNA or RNA. Next-generation sequencing (NGS) is a technology that offers ultrahigh throughput, scalability, and speed, and includes applications such as metabarcoding (deep sequencing of target regions) for whole community studies, whole genome sequencing, RNA sequencing, or the assessment of genome-wide DNA methylation and DNA–protein interactions.The former is the imbalance between the systemic manifestation of reactive oxygen species (ROS) and a biological system's ability to readily detoxify the reactive intermediates or to repair the damage resulting in cellular components, including proteins, lipids, and the DNA. Oxidative stress responses encompass the production of antioxidant enzymes , which aim to strike a balance between ROS production and consumption.Lipids, often bioactive, generated by the oxidation of polyunsaturated fatty acids (PUFAs).Compounds with a high vapor pressure and low water solubility. Although VOCs gas can be emitted from different solids and liquids, in this manuscript we only refer to VOCs of biogenic origin (i.e. produced by living organisms).Study of all the VOCs that are produced by a biological matrix (organism and ecosystem).One Strain MAny Compounds is an approach, which by altering cultivation parameters , aims to activate silent BGCs and expand or modify the metabolite production fingerprints of microbial strains.Live microorganisms with beneficial qualities for a host/recipient organism. Probiotic microorganisms help to restore recipient health by antagonistic action against pathogenic microbes, or enhance performance and growth by providing nutritional benefits to the recipient.Describes the bioavailability for or timescales of degradation of organic matter by organisms, which is reflected in the timescales by which this matter is respired. Organic matter follows a spectrum of recalcitrant  to labile .A technique used to analyze the composition of solid surfaces and thin films, which permits the spatial mapping of atoms or molecules. In correlation with electron microscopy increasingly used in biological research to create isotopic maps of histological sections, which can be used to visualize the assimilation and translocation of nutrients within complex symbiotic systems, such as corals.15N2 can be used to find out which microbes are active nitrogen fixers.A technique in microbial ecology for tracing uptake of nutrients by microbes. A substrate is enriched with a heavier stable isotope, i.e. consumed by the organisms to be studied. Biomarkers with the heavier isotopes incorporated into them can be separated from biomarkers containing the more naturally abundant lighter isotope by buoyant density centrifugation. As an example, Process of making an RNA copy (mRNA) of a gene's DNA.fuac028_Supplemental_FileClick here for additional data file.


Nanoparticles have now long demonstrated capabilities that make them attractive to use in biology and medicine. Some of them, such as lipid nanoparticles (SARS-CoV-2 vaccines) or metallic nanoparticles (contrast agents) are already approved for their use in the clinic. However, considering the constantly growing body of different formulations and the huge research around nanomaterials the number of candidates reaching clinical trials or being commercialized is minimal. The reasons behind being related to the “synthetic” and “foreign” character of their surface. Typically, nanomaterials aiming to develop a function or deliver a cargo locally, fail by showing strong off-target accumulation and generation of adverse responses, which is connected to their strong recognition by immune phagocytes primarily. Therefore, rendering in negligible numbers of nanoparticles developing their intended function. While a wide range of coatings has been applied to avoid certain interactions with the surrounding milieu, the issues remained. Taking advantage of the natural cell membranes, in an approach that resembles a cell transfer, the use of cell-derived surfaces has risen as an alternative to artificial coatings or encapsulation methods. Biomimetic technologies are based on the use of isolated natural components to provide autologous properties to the nanoparticle or cargo being encapsulated, thus, improving their therapeutic behavior. The main goal is to replicate the (bio)-physical properties and functionalities of the source cell and tissue, not only providing a stealthy character to the core but also taking advantage of homotypic properties, that could prove relevant for targeted strategies. Such biomimetic formulations have the potential to overcome the main issues of approaches to provide specific features and identities synthetically. In this review, we provide insight into the challenges of nano-biointerfaces for drug delivery; and the main applications of biomimetic materials derived from specific cell types, focusing on the unique strengths of the fabrication of novel nanotherapeutics in cancer therapy. In the context of biomedical applications, nanobiotechnology allows the manipulation of materials at molecular levels, which aims to produce non-toxic bioactive nanodevices that have specificity toward a desired tissue and location. The advantages of using nanomaterials as drug delivery systems (DDS) are related to their small size, which allows them to cross biological barriers and small capillaries thus reaching targets of interest such as tissues, tumors, or individual cells Fig. A 1]. Fu. Fu1]. F(EGF) [Based on the mechanisms through which the diseased tissue is reached, the DDS are broadly classified into passive and active targeting  Passive (EGF) , and fol(EGF) , among o(EGF) , nuclei (EGF) , or lyso(EGF) .Despite all the above-mentioned advantages, still many challenges that have limited the widespread success of conventional NP-based drug delivery systems  need to be faced. Therefore, research efforts remain aiming at engineering drug nanocarrier biointerfaces that provide the desired features to cross heterogeneous biological barriers and reach the target therapeutic sites. Biomimetic nanocarriers have the potential to improve circulation times, to transport across membranes, to improve solubility and stability of encapsulated cargos. These delivery nanoplatforms offer smart designs with sophisticated and organized self-assembled architectures with functional diversities and integrated stability . EmployiIn addition to the general in vivo improvements in terms of avoided sequestration by the reticuloendothelial system (RES), prolonged circulation time, and specific cell or tissue targeting, nanomaterials' internalization pathways must be taken into account, as intracellular delivery that require endosomal escape is a fundamental prerequisite for obtaining successful DDS . In most Intracellular delivery can be achieved by a range of carrier-based or membrane-disruption-based techniques Fig. . PhysicaAs an alternative to mechanical methods, nanocarrier-mediated delivery nanosystems can be designed to respond to the microenvironment changes that trigger the drug release in the target site. Some infection and inflammation-derived conditions, associated with different pathologies, can be exploited as stimuli to promote the disassembly of the nanocarrier, such as variations in pH , 30, oxyHowever, once the nanocarriers are taken up by cells via endocytosis, the cargo released from the endosome vesicles is required. Several endosomal escape strategies have been developed based on the endosome rupture promoted by membrane destabilizing agents such as pH-sensitive membrane-perturbing peptides and polymers –39. ThesAn alternative solution to achieve intracellular delivery is represented by the direct fusion of a specific nanocarrier with the plasma membrane of cells. This approach is inspired by the natural mechanism viruses use to deliver their genetic material into the cytosol while transfecting their hosts. The mechanisms behind the viral genome release vary among the several family types of viruses. Some envelope viruses, such as herpes simplex virus type I (HSV-I), enter into host cells either by fusion with the plasmatic membrane or intracellularly after internalization via endocytosis , 46. AmoOn this note, virus-inspired systems used for drug delivery have been developed, namely, (i) viral gene vectors, (ii) virus-like particles (VLPs), and (iii) virosomes. Generally, viral vectors are used to deliver therapeutic genes and are mainly applied as engineered vaccines. Retroviruses, adenovirus, adeno-associated virus, herpesvirus, and poxvirus have been selected as gene delivery vehicles thank to their capability to carry and deliver foreign genes . The virSesbania mosaic virus coat protein engineered with Staphylococcus aureus protein A (SpA) to deliver multiple antibodies [VLPs and virosomes are interesting alternatives and more applicable to protein delivery. VLPs are self-assembled capsules that mimic the capsid structure, while virosomes are liposome-like vesicles made of a phospholipid bilayer that is modified to incorporate surface glycoproteins of viruses . Since ttibodies .The expectations for translational medical solutions based on nanotechnology are high and the proof-of-concept reports are increasing in the literature. Some first generation of nanomedicines is currently commercialized. These nanoformulations such as Doxil® -loaded liposomes, Janssen Biotech, Inc., Horsham, PA), Abraxane® , AmBisome , Onivyde®, Marqibo®, and Nanotherm® were approved by the Food and Drug Administration (FDA) and are applied in clinics. The receAmong nanomaterials, liposomes have shown to be an effective DDS with fewer side effects thanks to their biodegradability, biocompatibility, and easy-to-manipulate size and surface . LiposomBesides liposomes, a range of other nanomaterial-based formulations is also being studied as potential candidates for vaccine development. For example, self-assembling protein NPs , polymerParticles found in nature including cells, viruses, or extracelluar vesicles (EVs) are highly complex and heterogeneous. The biointerfacing capabilities of these natural particles are mainly determined by their membrane layer. The information integrated at their surface has key inherent class properties such as “don’t-eat-me” signals, targeting specific sites, modulating the immune system response, etc. A deeper understanding of cell-cell communication and signaling has led to engineering cell-derived systems as better DDS for wide application in disease treatments, particularly in immunotherapy. For instance, a recent strategy being developed as potential immunotherapy is based on the use of autologous cells modified ex vivo for reinfusion in the patient (adoptive cell therapy), which reduces issues regarding innate immune recognition and clearance. Cell therapies hold great potential for efficacy engineered cellular immunotherapies in cancer. Chimeric antigen receptor (CAR)-T cell therapy, which targets specific cell surface antigen, has been remarkably effective towards certain pathologies such as leukemias and lymphomas .Clearly one of the most important challenges for nanomedicine is to mimic the multicompartmental architecture of cells and the complexity of the cellular membrane. Inspired by this approach, the use of cell-derived membranes in the form of nanomaterials has been proposed. In the last decade, there has been a considerable interest to develop bioinspired nanomaterials derived from biological entities already present in nature. Thanks to their biointerfacing capabilities, cellular structures  or invasive pathogens  can be exploited to design a new class of nanomaterials to overcome the limitations of synthetic NPs . Among tRecent progress in cell-derived nanosystems has further broadened the nanomedicine tools for advanced therapeutic approaches in imaging, phototherapy, detoxification, immune modulation, and drug delivery . In partThe complexity and dynamism of the cell membrane go beyond just being a passive lipid bilayer envelope. Cell membrane proteins and carbohydrates  are active components of the cellular machinery, and they are the first responders to what surrounds the cell. The cell membrane regulates signaling, transport, and immune response, which has inspired the idea of taking advantage of their intrinsic properties as an integral part of therapies and biomimetic nanoformulations, still exploiting the physical properties related to the synthetic nanomaterial.Biomimetic nanosystems based on the use of cellular structures have shown numerous advantages compared to synthetic nanomaterials. The use of entities that the body does not recognize as foreign agents, avoids rapid recognition by the RES, leading to a slower clearance and avoiding immune response. Furthermore, there are countless receptor-ligand, binding, and adhesion interactions that can be taken advantage of to achieve high targeting efficiency without applying elaborate functionalization methods. Cell membrane receptor profiles and characteristics are vital in performing therapeutic functions that can be translated onto the membrane coating with no loss in their native functionalities.On this note, recent studies have been focusing on the possibility to develop DDS using whole cells, cell-derived membranes, or EVs as drug carriers .Whole cell-based nanocarriers have been developed using a different kind of cells such as red blood cells (RBCs) , plateleHowever, a limitation in the use of whole cells as nanocarriers is the possibility that drugs loaded on their surface or inside them may be cytotoxic and damage the cell membrane or the whole cell , which pIn addition to cells, EVs have been recently reported as promising active targeted nanocarriers. These are a heterogeneous group of cell-derived membranous structures comprising exosomes and microvesicles. In particular, exosomes originating from the endosomal system are capable of delivery of various cargos, including proteins, lipids, and nucleic acids acting as communicators that mediate signaling between cells . Thanks Most of the targeting and biointerfacing abilities of a cell can be attributed to its plasma membrane. Hence, current efforts are focused on the development of core-shell structures by using plasma membrane of different cell sources as biomimetic coating for synthetic NPs (see Table co-glycolic acid) (PLGA) NPs combined with RBCM purified from fresh RBCs showed a half-life significantly longer than PEGylated-PLGA (39.6 vs. 15.8 h) with an in-blood retainment even 72 h long after injection [don’t eat me signal and is responsible for the RBCM ability to escape the recognition by the immune system and minimize premature blood clearance, phenomena observed in PEG functionalized NPs [Thanks to their interesting properties, such as prolonged blood circulation time, absence of nuclei, and abundance in the body, RBCs represent the more exploited source of cell membrane coatings. Zang and co-workers were pioneers to explore RBC membrane (RBCM) camouflaged NPs for cancer therapy. In their first study, biomimetic NPs composed of poly combined with checkpoint antibody treatment has promise as a potential clinical treatment for breast cancer [RBCMs have recently been used for cancer immunotherapy, leveraging host anti-cancer immune reactions  Liang ett cancer .Another type of cell membrane that can be extremely useful in biomimetic nanotechnology development is the platelets-derived one. The platelets originate in the bone marrow and are involved in numerous vital process as homeostasis, tissue repair, and thrombosis as well as inflammation and adaptive and innate immune responses , 100 PlaSimultaneously, equipped with an acid-responsive encapsulation matrix, PM-NVs can be digested after endocytosis and enhanced the DOX accumulation at the nuclei for activation of the intrinsic apoptosis pathway .ανβ3, overexpressed in tumor blood vessels, while TNF-α, an inflammation-induced cytokine, is applied to trigger tumor vascular damage. The second nanocarrier is a PM-coated acid-responsive dextran nanostructure loaded with the chemotherapeutic agent Paclitaxel (PTX). The study showed that PM proteins such as CD36, CD42d, P-Selectin, and CD40L were efficiently transferred with their origin membranes and enriched the NP coating. Thanks to the specific interaction between the L-Selectin and the CD44 receptor of the tumoral cells, the authors demonstrated that the presence of the PM gives to the NPs the ability to target the myeloma cells with high internalization, high intracellular drug localization, and decreased side effect [The same group, to improve the drug accumulation in tumors, developed a new strategy combining two nanocarriers. For the first one, they used Arg-Gly-Asp (RGD) peptide to decorate tumor necrosis factor α (TNF-α) loaded nanovesicles. The RGD peptide selectively binds the integrins such as e effect .Biomimetic coating for NPs has recently also been obtained from membranes extracted from white blood cells (WBCs), which are recruited into the tumor site in relation to chronic inflammation. First studies showed that this approach can enhance immune evasion and inflammation targeting . ‘LeukolThanks to the presence of lymphocyte function-associated antigen 1 (LFA1 or CD11a), coated NPs bind actively the TNFα-activated endothelium as evidenced by clustering of endothelial intracellular adhesion molecule 1 (ICAM-1). Furthermore, a transwell chamber assay showed the high suitability of LLVs to cross the layer of inflamed endothelium .Leukocyte membrane coating was also used for designing nanoformulations for imaging and PTT. Xuan et al.  successfMesenchymal cells (MSCs) are multipotent stem cells with the ability to differentiate into other types of cells such as adipocytes, fibroblasts, osteoblasts, chondroblasts, and pericytes. Furthermore, the MSCs migrate to the injured and inflamed tissue under environmental conditions such as hypoxia, and interaction with Toll-like receptors or cytokines. Since the tumor is considered to be a chronic inflammation disease, MSCs membrane coated NPs strategy has been harnessed as a biomimetic approach for targeted delivery of cancer drugs to tumors , 135. ToBesides RBCs, platelets, WBCs, and MSCs, cancer cells present exciting advantages to be exploited as membrane coating against tumors. Camouflaging strategies based on their use take advantage of innate homotypic aggregation properties and the immune escape ability.Homotypic targeting is the intrinsic ability of cancer cell coating nanoformulations to interact preferentially and strongly with the same cells from which they are originated. This feature provides a unique asset for any specific targeted drug delivery strategies against cancer. The homotypic affinity between cancer cells can be attributed to the interaction between galectin-3 and carcinoembryonic antigen expressed on cancer cells . Fang etA similar approach was applied by Sun and colleagues developing a 4T1 cell membrane-coated paclitaxel-loaded polymeric core as biomimetic (CPPNs) DDS against breast cancer and its metastasis in the lungs . 4T1 coaMany other results regarding the exciting specific interaction capabilities leveraged by cancer cell coating have recently been reported in the literature , 144 BesBesides mammalian cells, also pathogens cells can be a source of membranes for NP coating. Many studies have drawn attention to bacteria as a novel delivery system for various biomedical applications . While mE. coli as a model pathogen to coat AuNPs and produce an antibacterial vaccine [+ DCs cells of the bacterial membrane-coated AuNPs compared to the OMVs. In addition, both humoral (antibody titers) and cellular (levels of interferon-γ IFNγ and interleukin 17 IL-17) responses in vivo against E. coli were more pronounced. These findings were followed by other research lines aimed to use OMVs as immunotherapeutic agents against tumor.Zhang’s group applied  vaccine . Specifi vaccine , 154. ThE. coli accumulate in tumor tissues in vivo, and elicit strong activation of IFNγ-mediated long-term anti-tumor immune response [In 2017, Kim et al. reported that OMVs from response . Recentlresponse . They shDespite the growing number of research aimed at developing these new biomimetic systems, the mechanisms involved in biointerfacing with the biological environment, i.e., how these membrane coatings interact with cells at the molecular level, still require in-depth studies. In addition, because in most cases the cargo consists of drugs that need to reach the cytosolic target, it is crucial to investigate the intracellular delivery mechanisms.Trafficking processes, access to barriers and into specific cellular locations is governed by active biological recognition. Despite synthetic nanocarriers can be designed with variable features such as size, shape, surface charge and functional molecules properties, and responsiveness to deliver a specific therapeutic; a very significant element of the limitations experience so far in actively targeted nanomedicines, derives from the fact that insufficient tools have been available to address the complex role of biological interactions.Natural nanostructures are highly structurally and functionally heterogeneous and complex. They present key inherent class properties to develop precision nanomedicines. For instance, the complexity and dynamism of the cellular membrane can be translated to nanocarriers offering the capability of overcoming heterogeneous biological barriers. Such biomimetic interfaces have emerged to overcome the main drawbacks inherent to synthetic nanomaterials. Cell membrane-derived nanostructures hold great promise as bio-inspired synthetic nanocarriers for vaccines and drug-delivery systems. These cell-derived nanocarriers can offer a powerful toolbox for cancer treatment, combining their intrinsic properties with existing therapeutic strategies, such as phototherapy, immunotherapy, gene therapy, and chemotherapy . Cancer These tunable biomimetic properties combined with adequate organic or inorganic nanomaterials could offer new synergistic benefits of cell-based delivery nanosystems in the fields of bioimaging and therapy with a special focus on cancer treatment.For instance, coating inorganic NPs with the right biomimetic nanocarriers can be exploited for photodynamic therapy (PDT) and PTT in cancer treatment. PTT can be used to kill cancer cells utilizing light energy of the NIR wavelength range to generate localized heat. Combining cell-derived carriers with the tunable features of AuNPs, which also have strong interactions with light, make them efficient alternatives for use in PTT . PDT useImmunotherapies have a huge potential for clinical success in cancer treatment. The use of immune cell-derived nanocarriers will allow for enhancing immune responses by delivering immunomodulators and activating T cells or by delivering adjuvants and tumor antigens to DCs for tumor vaccination. Recent research has been focused on delivering tumor neoantigens with the aim to activate the patient’s immune system to recognize and kill tumor cells (tumor vaccination) .Biomimetic nanocarriers have the potential to improve cell targeting and precision delivery of drugs in vivo, can be surface engineered, and have a large capacity for cargo encapsulation and stabilization. It has been demonstrated that cell membrane coating of nanocarriers reduces immune activation, increases blood circulating time, and shows tumor-homing capacity. Most of the studies reported their in vivo application in mouse models where they had shown good biocompatibility and safety. However successful clinical applications require understanding how they behave at the cellular level. Fundamental investigations of biomimetic nanocarriers and the resulting interactions within the tissues and cells are not completely elucidated yet and need to be more thoroughly and systematically analyzed. On one hand, identifying critical motifs responsible for targeting surface markers and cell receptors is essential for the rational design of efficient nanocarriers to target specific tissues. On the other hand, a better understanding of the cell internalization mechanisms opens new opportunities for intracellular applications that require an endosomal escape. Investigate and predicting these bio-molecular mechanisms require the implementation of more sophisticated and reliable three-dimensional cell culture models  closer to natural conditions. Recreating a 3D microenvironment as cell-cell and cell-extracellular matrix interactions offers a bridge between in vitro 2D cell culture and animal testing before moving to clinical translation. These new platforms will allow understanding the complexities of in vivo cellular behavior of cell-derived nanocarriers. As it is described along this review cell membrane-coated nanocarriers have immense potential to enhance drug delivery mechanisms however critical limitations need to be overcome before being translated to clinical trial stages (see Table Cell-derived nanocarriers are suitable candidates for cancer therapy but it also offers the versatility to develop personalized nanomedicines. A variety of cellular sources can be exploited to design a library of biologically derived membranes with unique properties. They also can be loaded with a combination of cargoes, either several drugs or a combination of drugs and inorganic NPs. However, the use of natural components continues to raise safety concerns such as potential immunogenicity. Overall, we expect this emerging cell membrane-coated technology capable of mimicking natural scenarios will open a new class of nanocarriers for targeted cancer therapy and other biomedical applications.


Pulmonary arterial hypertension (PAH), characterized as a resting mean pulmonary artery pressure greater than 25 mmHg, is due to the narrowing of the pulmonary arteries, which can be idiopathic, inherited, or drug-related. Alkylating agents, including cyclophosphamide, are a risk factor for developing the pulmonary veno-occlusive disease. Drug-induced PAH is extremely rare. A 59-year-old female with newly diagnosed invasive ductal carcinoma of the right breast and high-grade ductal carcinoma in situ of the left breast was initiated treatment with doxorubicin and cyclophosphamide. About one week after receiving the first cycle, the patient developed worsening lower extremity edema and shortness of breath. She was then hospitalized, and a transthoracic echocardiogram and coronary angiogram revealed PAH. The team then changed the breast cancer treatment regimen to Taxol and carboplatin, and PAH was resolved in a follow-up echocardiogram after five months. This report has described the first case of PAH directly related to cyclophosphamide and doxorubicin. It is imperative to promptly recognize this rare but important side-effect as early diagnosis and response can potentially reverse the disease progression. Pulmonary arterial hypertension (PAH) is characterized as a resting mean pulmonary artery pressure >25mmHg. It happens when the pressure in the blood vessels leading from the heart to the lungs is elevated. The World Health Organization (WHO) divides pulmonary hypertension into five categories. Group one is known as PAH due to narrowing of the pulmonary arteries, which can be idiopathic, hereditary, or drug-related. Group two is due to left heart systolic and diastolic dysfunction or valvular disease. Group three is secondary to pulmonary causes such as chronic obstructive pulmonary disease (COPD) or hypoxemia. Group four is due to obstruction of the pulmonary circulation such as malignancy, benign mass, or inflammation. Lastly, group five's etiology is unclear/multifactorial such as secondary to hematological diseases or metabolic disorders .Although pulmonary hypertension can be categorized into five different types, the principal indicator of all morphologies of pulmonary hypertension is increasing exercise dyspnea. It is usually escorted by tiredness and fatigue. Due to the non-specificity of associated symptoms, establishing a diagnosis can be delayed for months to years. Due to the delay in recognition, progression of the disease leads to eventually dyspnea on bending down and syncope mainly after or during exertion. Frequent syncopal episodes on slight exertion are associated with higher mortality and cardiac decompensation. The rise in right heart cardiac pressures is associated with venous congestion, abdominal ascites, and lower extremity edema .Pulmonary hypertension continues to be an untreatable ailment. The gold standard to diagnose pulmonary hypertension is right heart catheterization. In patients with idiopathic, heritable, or drug-related PAH, right heart catheterization is supplemented by vasoreactivity testing to recognize the responders who may benefit from medication with high-dose calcium antagonists. However, treatment with calcium works out in fewer than 5% of patients with PAH and must be monitored closely once treatment is initiated. The goal of the treatment is to slow down the progression of the disease and stabilize the patient without signs of right heart failure. Treatment in newly diagnosed PAH with low or intermediate risk can be initiated on a combination compromising an endothelin receptor antagonist (ERA) with a phosphodiesterase-5 (PDE5) inhibitor or a soluble guanylate cyclase (sGC) stimulator. We can use a three-layered blend of an ERA, a PDE5 inhibitor, an sGC stimulator, and an intravenously administered prostacyclin analog for the high-risk patient category. If treatment response remains inadequate, patients should be evaluated for lung transplant without delay .In conclusion, pulmonary hypertension is most associated with left-sided heart failure or secondary to lung diseases. Other causes are not as commonly seen and thought to be rare. As such, we present this unique case of chemotherapy-induced pulmonary hypertension.A 59-year-old female with newly diagnosed invasive ductal carcinoma of the right breast and high-grade ductal carcinoma in situ of the left breast was initiated treatment with doxorubicin and cyclophosphamide. About one week after receiving the first cycle, the patient developed worsening lower extremity edema and shortness of breath. She was then hospitalized, and a transthoracic echocardiogram (TTE) revealed moderately reduced right ventricular function, enlarged right ventricle with severe pulmonary hypertension, and estimated pulmonary artery systolic pressure of 79.5 mmHg, assuming a right atrial pressure of 10 mmHg as seen in Videos Coronary angiogram revealed severe pulmonary hypertension without response to nitric oxide, mild to moderate elevation in left ventricular end-diastolic pressure, pulmonary capillary wedge pressure, and normal coronary anatomy Videos , 4.TTE obtained about one month before initiating chemotherapy revealed normal right ventricular size and function, mild pulmonary hypertension, estimated pulmonary artery systolic pressure of 44.8 mmHg, assuming right atrial pressure of 3 mmHg. The team then changed the breast cancer treatment regimen to Taxol and carboplatin, of which the patient completed 12 cycles; complications were noted to be fatigue and pancytopenia. Breast MRI revealed interval decrease in size of right breast carcinoma and resolution of surrounding satellite nodules , and exposure to cyclophosphamide leads to pulmonary venous remodeling in experimental models which, in turn, develops pulmonary hypertension . PVOD isThe metabolism of cyclophosphamide in the lung is partly responsible for its pulmonary toxicity. Data suggest that cyclophosphamide and its metabolites cause peroxidative injury of the membrane lipids . DoxorubWe have described the first case of PAH directly related to cyclophosphamide and doxorubicin. Since our patient received both drugs and both were discontinued when the patient developed PAH, we cannot establish which chemotherapeutic agent was responsible. However, there is more data on the pulmonary toxicity of cyclophosphamide than doxorubicin ,11. InteThe uniqueness of our case stems from the fact that after discontinuation of the chemotherapeutic agents, our patient's pulmonary hypertension resolved, showing that this effect might be reversible. It is imperative to recognize this rare but important side-effect as early diagnosis and response can potentially reverse the disease progression.Pulmonary hypertension remains challenging to treat. The most common cause of pulmonary hypertension is left-sided heart failure, but it can occur secondary to chemotherapeutic drugs. Our patient developed pulmonary hypertension after one cycle of chemotherapy with doxorubicin and cyclophosphamide, confirmed with a right heart catheterization. It resolved after the discontinuation of these drugs. In conclusion, chemotherapeutic drugs are often the offending agent in causing pulmonary complications, and when using these treatment regimens, the potential side effects should be kept in mind.