Primary liver cancer, including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), remains a significant contributor to cancer-related mortality worldwide. Oxidative stress and lipid peroxidation play a key role in chronic liver diseases and have been shown to be pivotal for tumor initiation and progression. 4-hydroxy-nonenal (4-HNE), one of the major mediators of oxidative stress and a well-established biomarker for lipid peroxidation, can act as a signal transducer, inducing inflammation and exerting carcinogenic effects. However, the role of 4-HNE in primary liver cancer remains poorly explored. In this study, we investigated 4-HNE levels in 797 liver carcinomas, including 561 HCC and 236 iCCA, by immunohistochemistry. We then correlated 4-HNE levels with comprehensive clinical data and survival outcomes. In HCC, lower expression levels of 4-HNE were associated with vascular invasion, a high tumor grade, a macrotrabecular-massive HCC subtype, and poor overall survival. Concerning iCCA, large duct iCCA showed significantly higher 4-HNE levels when compared to small duct iCCA. Yet, in iCCA, 4-HNE levels did not correlate with known prognostic parameters or survival outcomes. To conclude, in HCC but not in iCCA, low amounts of 4-HNE predict unfavorable survival outcomes and are associated with aggressive tumor behavior. These findings provide insights into the role of 4-HNE in liver cancer progression and may enable novel therapeutic strategies. Liver cancer is the fourth most common cause of cancer-related death worldwide, with hepatocellular carcinoma (HCC) being the most frequent tumor type, followed by intrahepatic cholangiocarcinoma (iCCA) . Both HCOxidative stress, characterized by an imbalance between the production of reactive oxygen species (ROS) and antioxidant systems, can lead to ROS interaction with cellular macromolecules, particularly DNA, membrane phospholipids, and proteins, thereby causing cellular damage and dysfunction . ROS, geLipid peroxidation is one of the cellular consequences of uncontrolled ROS elevation. Among the aldehydes produced during lipid peroxidation, 4-hydroxy-nonenal (4-HNE) is one of the best-studied molecules ,8. It isROS generated under inflammatory conditions play a crucial role in iCCA as well, contributing to the formation of oxidative DNA adducts and promoting inflammation-mediated carcinogenesis . While tThe present immunohistochemical study investigates 4-HNE levels in a large cohort of patients with intrahepatic cholangiocarcinoma and hepatocellular carcinoma and correlates 4-HNE expression with clinical data and survival outcomes.A total of 294 paraffin-embedded formalin-fixed (FFPE) samples from 236 iCCA patients and 871 FFPE samples from 561 HCC patients were collected from routine histopathology cases seen at the Institutes of Pathology, University Medical Center Mainz, between 2006 and 2020. Tissue microarrays (TMAs) with cores of 1–2 mm were constructed. Each patients‘ set of samples encompassed the primary tumor, additional tumor foci, corresponding normal tissue, precursor lesions, relapse tumors, and lymph node or distant metastases, if present.Immunohistochemistry (IHC) was carried out on 4 µm-thick histological sections. Antigen retrieval was undertaken with Tris/EDTA buffer, pH 9 , and cell conditioning solution 1 . TMA slides were incubated with a mouse monoclonal anti-4-hydroxynonenal antibody  and a monoclonal anti-glutamine synthetase antibody . Immunohistochemistry was undertaken with an automated staining system  with the Dako EnVision FLEX staining method (Agilent Technologies). Staining procedures were performed as indicated by the manufacturers’ guidelines. Stained slides were digitized using a whole slide scanner set at 40× magnification . Evaluation of staining intensity involved the application of the semiquantitative immunoreactivity score (IRS), calculated by multiplying the staining intensity (ranging from 0 to 3) by the extent of tumor involvement (ranging from 0 to 4), resulting in score values spanning from 0 to 12 . Glutamip values were reported exclusively if divergent from the non-adjusted values to ensure clarity in the presentation. To examine associations between pairs of variables, the Spearman rank-order correlation coefficient was computed for non-metric and non-normally distributed data, while the Pearson correlation coefficient was employed for metric and normally distributed data.All statistical analyses were carried out using the R environment for statistical computing (version 4.3.0) . The nonn = 536), 4-HNE was strongly detected in the cytoplasm of hepatocytes, displaying a granular staining pattern. However, in cells with primary HCC, 4-HNE levels were significantly lower (n = 39) showed a further reduction of 4-HNE compared to the respective primary HCC (n = 11) and distant metastases (n = 28) compared to primary HCC (n = 536), although this trend did not reach statistical significance  = 0.118, p = 0.785.In line with these findings, further analysis also demonstrated a significant reduction of intratumoral 4-HNE levels in HCC with vascular invasion, a higher tumor grade, GS overexpression, and a macrotrabecular-massive subtype c–f. Age To demonstrate the correlation between different prognostic factors, we performed a Cox regression analysis for overall survival . MultivaIn conclusion, our findings indicate that 4-HNE immunohistochemistry may independently predict overall survival, with low 4-HNE expression being associated with poorer survival outcomes.The strong correlation of 4-HNE levels, as tested by immunohistochemistry, with survival in HCC patients prompted the investigation of whether this relationship is a fundamental characteristic of primary liver carcinomas or a specific feature limited to HCC. To address this, we conducted a study on 294 iCCA cases from 236 patients within a clinically established cohort [The present study aimed to investigate the prognostic role of 4-HNE in liver cancer within a large cohort of 797 primary liver cancers, including 561 HCC and 236 iCCA. Our findings revealed distinct associations and prognostic implications of 4-HNE expression in HCC, while no significant correlations were found in iCCA.We observed a significant decrease in 4-HNE levels during hepatocarcinogenesis, from non-neoplastic liver tissue to primary HCC, relapse tumors, as well as lymph node and distant metastases. This indicates the potential role of 4-HNE in HCC progression and metastasis. In this line, our survival analysis demonstrated that patients with low 4-HNE levels had significantly worse overall survival compared to those with high intratumoral 4-HNE adducts, highlighting the prognostic significance of 4-HNE in HCC. Further exploration of prognostic factors in HCC revealed intriguing correlations with 4-HNE levels. HCC with low intratumoral 4-HNE levels exhibited characteristics associated with aggressive tumor behavior, such as increased vascular invasion and poor tumor differentiation. However, these correlations may also represent dependent variables. To investigate this issue, i.e., to determine whether 4-HNE expression negatively affects patient outcomes independent of other prognostic factors, we performed a multivariate regression analysis. This analysis confirmed the independent prognostic significance of 4-HNE in HCC, paralleling established factors such as vascular invasion , BCLC st4-HNE originates from mitochondria through the process of lipid peroxidation, forming adducts with proteins, DNA, and lipids. Due to the crucial role of ROS, manipulating its mitochondrial generation has been proposed as a potential target for cancer prevention and therapy . For exaFurthermore, we observed a lower frequency of liver cirrhosis and, when present, a lower Child–Pugh score in the HCC group with lower intratumoral 4-HNE expression. It is important to note that our patient cohort consisted of resected HCC with available histology, as per treatment guidelines . As a reOxidative stress induced by ROS is a major mechanism leading to nonalcoholic fatty liver disease (NAFLD) . AdditioOur study reveals an association between decreased levels of 4-HNE and unfavorable survival outcomes in HCC patients, which is in accordance with previous studies conducted on various types of cancer. For instance, in prostate cancer, decreased levels of 4-HNE have been linked to disease progression . SimilarIn a parallel study of mouse HCC induced by the potent hepatocarcinogen diethylnitrosamine, 4-HNE localized to frequent eosinophilic globules, which may correspond to so-called pale bodies/hyalin-like globules  in dysplIn contrast to HCC, in iCCA, our analysis did not reveal any significant association between 4-HNE expression levels and overall survival. While large duct iCCA exhibited slightly higher 4-HNE expression compared to small duct iCCA, this difference was not statistically significant. The association between 4-HNE and patient survival observed in HCC may be specific to HCC rather than a universal characteristic of primary liver carcinomas.In this study, we investigated 4-hydroxy-nonenal (4-HNE) in a large, clinically well-characterized patient cohort with primary liver carcinomas, including 561 hepatocellular carcinomas (HCC) and 236 intrahepatic cholangiocarcinomas (iCCA), using immunohistochemistry. 4-HNE is a key molecule in the complex molecular processes of cellular oxidative stress regulation and may act as a signal transducer, inducing inflammation. Both HCC and iCCA demonstrated differential 4-HNE expression in different tumor subtypes. We found a significant adverse association between low 4-HNE levels and overall survival in HCC but not in iCCA patients. This suggests that reduced lipid peroxidation in HCC may contribute to increased invasiveness and dedifferentiation or indicate the potential for tumor evasion of therapeutic approaches. Interestingly, we also observed that low levels of 4-HNE expression were associated with a higher number of vascular invasions, lower tumor differentiation, and a macrotrabecular-massive HCC subtype. These findings underscore the importance of elucidating the role of 4-HNE in HCC and investigating 4-HNE modulating agents as therapeutic agents, emphasizing the need for further comprehensive and mechanistic studies in this area.


Medulloblastoma is the most common malignant paediatric brain tumour, representing 20% of all paediatric intercranial tumours. Current aggressive treatment protocols and the use of radiation therapy in particular are associated with high levels of toxicity and significant adverse effects, and long-term sequelae can be severe. Therefore, improving chemotherapy efficacy could reduce the current reliance on radiation therapy. Here, we demonstrated that systems-level analysis of basal apoptosis protein expression and their signalling interactions can differentiate between medulloblastoma cell lines that undergo apoptosis in response to chemotherapy, and those that do not. Combining computational predictions with experimental BH3 profiling, we identified a therapeutically-exploitable dependence of medulloblastoma cells on BCL-XL, and experimentally validated that BCL-XL targeting, and not targeting of BCL-2 or MCL-1, can potentiate cisplatin-induced cytotoxicity in medulloblastoma cell lines with low sensitivity to cisplatin treatment. Finally, we identified MCL-1 as an anti-apoptotic mediator whose targeting is required for BCL-XL inhibitor-induced apoptosis. Collectively, our study identifies that BCL-XL and MCL-1 are the key anti-apoptotic proteins in medulloblastoma, which mediate distinct protective roles. While BCL-XL has a first-line role in protecting cells from apoptosis basally, MCL-1 represents a second line of defence that compensates for BCL-XL upon its inhibition. We provide rationale for the further evaluation of BCL-XL and MCL-1 inhibitors in the treatment of medulloblastoma, and together with current efforts to improve the cancer-specificity of BCL-2 family inhibitors, these novel treatment strategies have the potential to improve the future clinical management of medulloblastoma. Medulloblastoma is a malignant embryonal tumour of the cerebellum that primarily affects children . A rare Four molecular subgroups of medulloblastoma have been identified—Wnt, SHH, Group 3 and Group 4. These are associated with different molecular and cytogenetic features, affected demographics and metastatic potential –12. The c and SMAC  from the rest of the panel, where most proteins were downregulated compared to other cell lines Fig. . InteresTo investigate the apoptosis response of cell lines that demonstrated a response to cisplatin in the cell viability assay, we carried out Annexin-V/PI staining. This identified that, while four cell lines  underwent apoptosis at clinically-relevant cisplatin concentrations (10–20 µM) , ONS76 aWe, therefore, performed systems-level analyses of the 14 quantified apoptotic proteins. We first applied a PCA-based method that integrates apoptosis pathway-level knowledge by calculating functional groups using arithmetic operations representing protein interactions  Fig. 2B2B. This Having identified that functional grouping of apoptosis regulatory proteins provided power to predict cisplatin sensitivity, we were interested in exploring what proportion of patient tumours may be predicted to be chemosensitive. Using publicly available mRNA measurements from 266 medulloblastoma tumour samples , we geneNext, we were interested in identifying which functional groups contributed most to the separation of the cell lines. Visualising the magnitude and sign of each functional group’s contribution to the second and third PCs Fig.  demonstrη) representing the amount of stress-induced BH3-only proteins required to permeabilize the mitochondrial outer membrane [p = 0.0063)  and upstream (BCL-2 + BCL-XL + MCL-1 and BID) proteins as determinants of cisplatin sensitivity, we applied two ODE-based computational models of the downstream (APOPTO-CELL) and upstream (DR_MOMP) apoptosis pathways to investigate the mechanisms underlying these differential responses Fig. . APOPTO-membrane . DR_MOMP63) Fig. . This suCollating PCA and DR_MOMP findings suggested that the BCL-2 proteins play a key role in mediating cisplatin sensitivity in medulloblastoma cells. We applied DR_MOMP to predict whether BCL-2 family inhibitor could sensitise the ONS76 and UW228 cell lines to cisplatin treatment, and identified that inhibitors of BCL-XL (WEHI-539) and BCL-2 (ABT-199) were predicted to most strongly reduce the stress dose required to induce apoptosis in both cell lines Fig. .Fig. 4InWe next employed BH3 profiling as an in vitro technique to determine the cell lines’ anti-apoptotic dependencies. We observed that HRK (BCL-XL specific) and BAD (BCL-2 and BCL-XL specific) both induced ~60–85% mitochondrial depolarisation compared with the positive control, FCCP Fig. . This suHaving identified that BCL-XL inhibition potentiates cisplatin toxicity, we wanted to understand how cisplatin affects expression of the BCL-2 protein family, and the observed BCL-XL dependency of these cells. Cisplatin treatment in ONS76 cells increased BCL-XL and MCL-1 levels, and decreased BCL-2 levels Fig. , indicatNext, we investigated if cisplatin altered the anti-apoptotic dependencies in the medulloblastoma cell lines. Dynamic BH3 profiling showed that apoptotic priming increased following treatment with cisplatin, as indicated by the response to the BIM BH3 peptide, though not to significant extents Fig. . No signTo confirm the altered interaction of pro-death proteins with BCL-XL in the presence of cisplatin we performed an immunoprecipitation of BCL-XL in the two medulloblastoma cell lines following treatment with cisplatin or cisplatin and WEHI-539. Cisplatin treatment somewhat increased interactions between BAK and BCL-XL in both cell lines Fig. , and betHaving shown that BCL-XL is a critical anti-apoptotic protein in protecting medulloblastoma cells from apoptosis upon treatment, we were interested in investigating why BCL-XL targeting alone failed to elicit apoptosis. Notably, a similar lack of efficacy was noted for ABT-199 and S63845 treatment (Supplementary Fig. Therefore, we hypothesised that combining WEHI-539 with S63845 would be toxic to the cells. This combination synergistically reduced viability Fig. , and triInvestigating the mechanism of combination treatment-induced death, we demonstrated that apoptosis induced by combination treatment was caspase-dependent, as pre-treatment with the caspase inhibitor Q-VD-OPh prevented co-treatment-induced caspase cleavage and apoptosis (Supplementary Fig. Despite increased understanding of the molecular and genetic events underlying medulloblastoma development, a lack of effective treatment options leads to poor prognosis for some patient groups . In addiApoptosis induction is the principal means by which chemotherapy mediates its effects, and here we aimed to identify patterns of apoptosis protein expression associated with cisplatin sensitivity, and targetable therapeutic vulnerabilities within the apoptosis pathway. We began by characterising a panel of medulloblastoma cell lines with respect to their cisplatin sensitivity and expression of apoptosis signalling proteins, and identified heterogeneous protein expression and cisplatin response across all cell lines. The prevalence of chemosensitive cell lines in our study supports the idea that many medulloblastoma tumours are intrinsically chemosensitive . We alsoPrevious research comprehensively demonstrated that, as defective apoptosis may underlie chemoresistance , quantitWe next showed that modelling the interactions of the BCL-2 protein family upstream of MOMP could also accurately predict sensitivity of medulloblastoma cells to cisplatin-induced apoptosis, with similar results previously observed in colorectal and breast cancer , 48. AntDespite the observed BCL-XL dependence of medulloblastoma, we found that WEHI-539 treatment alone failed to effectively induce apoptosis, but that co-inhibition of MCL-1 successfully resulted in cell death. This finding is in alignment with increasing evidence that cancer cells derived from solid malignancies frequently depend on two BCL-2 family members , with BCInvestigating the underlying mechanisms, we utilised dynamic BH3 profiling to demonstrate that WEHI-539 treatment triggers an exploitable MCL-1 dependence, explaining how WEHI-539 and S63845 induce death only when combined. That MCL-1 antagonism is required for apoptosis mediated by BCL-XL inhibition was hinted at in early papers reporting the activity of ABT-737, where ABT-737 induced apoptosis only in tumours with low MCL-1 levels, and MCL-1 downregulation sensitised resistant cells .However, ubiquitous expression of BCL-2 family proteins limits the clinical application of certain BH3 mimetics, as some non-cancer cells are intrinsically dependent on BCL-XL or MCL-1 . For exaCollectively, our data demonstrate that the cisplatin sensitivity of medulloblastoma cell lines is associated with their expression of apoptosis signalling proteins. Signalling upstream of MOMP plays a central role in mediating apoptosis susceptibility, with BCL-XL identified as a key resistance factor. Targeting BCL-XL sensitises cells to cisplatin-induced cytotoxicity, suggesting that such an approach could enhance chemotherapy response and remove the reliance of current treatment paradigms on radiation therapy Fig. . MCL-1 iSupplementary figures and tablesOriginal Data FileReproducibility checklist


The emergence of lethal coronaviruses follows a periodic pattern which suggests a recurring cycle of outbreaks. It remains uncertain as to when the next lethal coronavirus will emerge, though its eventual emergence appears to be inevitable. New mutations in evolving SARS-CoV-2 variants have provided resistance to current antiviral drugs, monoclonal antibodies, and vaccines, reducing their therapeutic efficacy. This underscores the urgent need to investigate alternative therapeutic approaches. Sigma receptors have been unexpectedly linked to the SARS-CoV-2 life cycle due to the direct antiviral effect of their ligands. Coronavirus-induced cell stress facilitates the formation of an ER-derived complex conducive to its replication. Sigma receptor ligands are believed to prevent the formation of this complex. Repurposing FDA-approved drugs for COVID-19 offers a timely and cost-efficient strategy to find treatments with established safety profiles. Notably, diphenhydramine, a sigma receptor ligand, is thought to counteract the virus by inhibiting the creation of ER-derived replication vesicles. Furthermore, lactoferrin, a well-characterized immunomodulatory protein, has shown antiviral efficacy against SARS-CoV-2 both in laboratory settings and in living organisms. In the present study, we aimed to explore the impact of sigma receptor ligands on SARS-CoV-2-induced mortality in ACE2-transgenic mice. We assessed the effects of an investigational antiviral drug combination comprising a sigma receptor ligand and an immunomodulatory protein. Mice treated with sigma-2 receptor ligands or diphenhydramine and lactoferrin exhibited improved survival rates and rapid rebound in mass following the SARS-CoV-2 challenge compared to mock-treated animals. Clinical translation of these findings may support the discovery of new treatment and research strategies for SARS-CoV-2. Sigma receptors are widely expressed intracellular chaperone proteins that typically reside on the endoplasmic reticulum (ER) membrane . Sigma rPreviously, we investigated the role of sigma-1 and sigma-2 receptors in mediating antiviral activity against SARS-CoV-2 in vitro . These sIn this study, we investigated the role of highly specific sigma receptor ligands on SARS-CoV-2-induced mortality in ACE2-transgenic mice. K18-hACE2 mice were selected for this study because they express human ACE2 receptors, effectively simulating the human response to SARS-CoV-2 and allowing evaluation of the therapeutic effect of sigma receptor ligands . These mThese data are relevant to the diminishing efficacy of available COVID drugs and potential strategies to improve efficacy, including the use of sigma receptor ligands. The potential of combinatorial therapies targeting multiple viral replication stages for enhanced efficacy against future coronaviruses and reduced susceptibility to declining efficacy compared to current COVID drugs is discussed.K18-hACE2 transgenic mice were challenged with SARS-CoV-2 strain UF-1. Mice were treated daily or twice daily with either saline, a sigma-1 and sigma-2 receptor binding ligand (AZ66), a sigma-2 receptor binding ligand (CM398), or an investigational antiviral drug combination consisting of an off-target sigma receptor ligand  and an immunomodulatory protein  . All micIn an additional survival study, K18-hACE2 transgenic mice were challenged with the SARS-CoV-2 strain UF-1. Mice were treated with saline (mock treated) or the investigational diphenhydramine/lactoferrin combination. The survival study presented in 50) in treated mice 48 h after viral challenge compared to mock-treated mice , a viral protease inhibitor drug combination, has flaws that could jeopardize its widespread use. Paxlovid patients commonly experience “COVID rebound”, a phenomenon in which SARS-CoV-2 replication will initially decrease, but then return to normal, causing a rebound in symptomatic disease . One theory behind the mechanism of Paxlovid rebound is that virions are pressured to develop Paxlovid-resistant protease mutations, thus, creating Paxlovid-resistant strains of SARS-CoV-2. While the theory has not been confirmed in clinical settings, two studies (one preprint and one peer-reviewed) showed that SARS-CoV-2 treated with Paxlovid in vitro resulted in mutations at the same protease positions ,21. ThesSARS-CoV-2-specific nucleotide analogs that function by inducing random viral mutations can also result in onward transmission of mutated viruses. A 2023 article found a specific class of phylogenetic branches that emerged in 2022 following the introduction of molnupiravir . ElevateThe most exquisitely specific COVID therapies developed to date are likely to be monoclonal antibodies. When newly developed, monoclonal antibodies exhibit dramatic antiviral effects and acceptable safety profiles. However, the use of monoclonal antibodies to treat COVID has halted because mutations in new variants resulted in a profoundly decreased efficacy ,28. It sDue to the ever-decreasing drug efficacy from drug-induced and spontaneous mutations, continuing to develop variant-specific SARS-CoV-2 antiviral drugs may not be the optimal approach. The strategies most likely to succeed against future SARS-CoV-2 variants, and other new coronaviruses yet to emerge, should inhibit the virus life cycle at multiple steps common to known coronaviruses.Repurposing of FDA-approved drugs is a promising strategy for the rapid identification of treatments due to known safety profiles, robust supply chains, and short deployment time frames. In an effort to identify drugs that can inhibit a SARS-CoV-2 infection, a landmark study generated a protein interaction map as the basis for target identification and drug repurposing . The stuThe study found that many pharmacological agents that displayed antiviral activity were known or predicted ligands of the sigma-1 and sigma-2 receptors, which are ER membrane proteins that participate in the modulation of cell stress. Coronaviruses, including SARS-CoV-2, replicate in a membranous compartment derived from the ER  30]. Th. Th30]. It was demonstrated that specific FDA-approved antihistamines exhibit off-target sigma receptor binding activity and antiviral activity against SARS-CoV-2 . For exaIn this study, we showed that highly specific sigma receptor ligands protected ACE2-transgenic mice from SARS-CoV-2-induced mortality.Finding drug combinations with broad activity against coronaviruses is an important approach to strengthen the toolkit for SARS-CoV-2 and future coronaviruses. For SARS-CoV-2, all variants replicate using the same ER-derived compartment. The same is true of all coronaviruses , therefoAntiviral combinations targeting highly conserved viral mechanisms have proven their success in treating drug-resistant viruses. For example, HIV and hepatitis C patients are treated with drug combinations instead of single drugs, with each drug targeting distinct pathways involved in the virus life cycle ,34. For One such drug is diphenhydramine, an over-the-counter antihistamine that is a potentially useful off-target sigma receptor ligand in an antiviral drug combination due to its wide accessibility and long safety record . DiphenhBoth diphenhydramine and lactoferrin are safe for oral consumption by adult humans. Effective doses of diphenhydramine for its indicated use  are 25 to 50 mg every 4 to 6 h as needed, not to exceed 300 mg/day . LactofeLactoferrin has been tested in the clinic for the treatment of COVID because of its antiviral activity, immunomodulatory properties, and safety profile. Such studies have garnered highly varied and controversial results, ranging from no observed effect to symptom resolution in all treated patients ,45,46,47An investigational antiviral drug combination effective against SARS-CoV-2 in vitro has been described in which each component is an approved agent inhibiting distinct mechanisms of the virus life cycle. Diphenhydramine, an off-target sigma receptor ligand, is thought to exert antiviral activity by inhibiting the host cell stress response and thereby inhibiting the formation of the ER-derived coronavirus replication compartment. Diphenhydramine was combined with lactoferrin which is thought to exert indirect antiviral activity via immunomodulation as well as direct antiviral activity by binding heparan sulfate proteoglycans (HSPGs)  and angiDiphenhydramine and lactoferrin were tested in combination to determine any enhanced antiviral activity. Independently, diphenhydramine and lactoferrin reduced SARS-CoV-2 replication by approximately 30% in vitro. Strikingly, in combination, diphenhydramine and lactoferrin exhibited a synergistic effect in vitro, resulting in a 99% reduction in SARS-CoV-2 replication in monkey and human cell lines  6]. In . In 6]. It follows that the in vivo results in this study of SARS-CoV-2-challenged ACE2-transgenic mice treated with the diphenhydramine/lactoferrin combination exhibited the slowest decline in mass compared to any other group, nonzero survival rates compared to untreated mice, and a rapid rebound in mass in surviving mice. However, a direct antiviral effect was not observed in vivo. This may be explained by the effects of the combination on immunomodulation. COVID is a multiphasic disease in which initial symptomatic manifestations are caused by the “viral phase”, and later symptomatic manifestations, including mortality, are caused by the “inflammatory phase”, which includes lethal cytokine storms . Thus, iAdditionally, therapeutic effect may have been achieved by direct antiviral effect in non-lung sites of the animal, or antiviral effect after the viral phase of the disease which may not have been observed at the time of the predetermined endpoint study.2 environment also as previously described. Vero E6 cells were obtained from ATCC . Viral stocks were harvested and quantified using TCID50 dilution titering in Vero E6 cells.SARS-CoV-2 work was performed in a biosafety level 3 (BSL-3) lab using practices and procedures approved by the University of Florida Institutional Biosafety Committee. SARS-CoV-2 strain UF-1 was obtained from a patient at University of Florida Shands Hospital  as previously described ,50. SARSSigma ligands were produced in the McCurdy lab at the University of Florida  as previously described ,52,53,544 plaque-forming units (PFU) of SARS-CoV-2. Each day thereafter, mice were observed for moribundity, and the mass of each mouse was recorded. Treatments were delivered by an IP injection of the drugs suspended in 100 μL of saline, or saline alone. Treatments were given once or twice daily depending on the experiment, and the doses were titrated based on previously reported drug pharmacokinetic profiles [Animal work was carried out in the Animal Biosafety Level 3 (ABSL3) laboratory and was approved by the University of Florida Institutional Animal Care and Use Committee (IACUC) under IACUC protocol number 202111322. Six- to eight-week-old female K18-hACE2 mice were purchased from The Jackson Laboratory . K18-hACE2 mice are transgenic for the human ACE2 receptor and express it from the keratin 18 promoter to direct ACE2 expression to epithelia including airway epithelia. The mouse strain has been extensively characterized as a SARS-CoV-2 animal model, as their tissue-wide expression of human ACE2 ensures fulminant acute infections ,11,55. Mprofiles ,53. MiceThe model of the SARS-CoV-2 spike protein binding to ACE2 is based on the crystal structure of the SARS-CoV-2 spike protein complexed with ACE2  (PDB 6M1The current toolkit of COVID-specific antivirals is losing efficacy over time. Thus, further development of highly variant-specific antiviral drugs could be questioned. One option is to phase out currently available post-exposure COVID antiviral drugs due to the risks outlined in There are benefits to updating the COVID antiviral toolkit with broader-spectrum antiviral combination drugs targeting multiple distinct mechanisms in the life cycle that are shared by all coronaviruses. This strategy will likely increase drug efficacy while decreasing the risk that drug-induced mutations, natural mutations, or new coronaviruses will render such an antiviral combination ineffective. Ideally, new antiviral drug combinations will function by making host cells nonpermissive to replication, a strategy that has proven to be effective in preventing antiviral resistance . Sigma receptor ligands offer a promising approach to host-targeted antiviral activity against coronaviruses by inhibiting the formation of the coronavirus replication complex from the host endoplasmic reticulum. SARS-CoV-2-challenged ACE2-transgenic mice demonstrated improved survival when treated with highly specific sigma receptor ligands. Mice that survived were shown to rapidly rebound in mass following treatment. The sigma-2 receptor ligand CM398 showed a stronger effect than the sigma-1 receptor ligand AZ66.A current candidate fitting these criteria most likely to be effective for a new coronavirus antiviral combination may be the diphenhydramine/lactoferrin combination . Mice trBoth components of the combination exhibit direct antiviral activity in vitro as well as protective effects in vivo, are FDA-approved, economical, and have long histories of safety. Clinical investigation of the diphenhydramine/lactoferrin combination is warranted to determine benefits for COVID patients. The collection of clinical data pertaining to the efficacy or absence thereof resulting from the employment of the diphenhydramine/lactoferrin combination would be significantly advantageous if communicated by physicians. Such data could help to further inform coronavirus drug development strategies while potentially enabling the widespread employment of the diphenhydramine/lactoferrin combination if proven effective in clinical settings.


Self-healing elastomers refer to a class of synthetic polymers that possess the unique ability to autonomously repair from internal and external damages. In recent years, significant progress has been made in the field of self-healing elastomers. In particular, intrinsic self-healing elastomers have garnered a great deal of attention. This mini-review outlines recent advancements in the mechanisms, preparation methods, and properties of various intrinsic self-healing elastomers based on non-covalent bond systems, reversible covalent bond systems, and multiple dynamic bond composite systems. We hope that this review will prove valuable to researchers in order to facilitate the development of novel strategies and technologies for preparing high-performance self-healing elastomers for advanced applications. Elastomers possess high extensibility and the capability to recover their original shape after the removal of external forces and are therefore extensively applied in various industries, including manufacturing, medical instruments, and automobile manufacturing. However, in practical applications, elastomers are often susceptible to environmental or external stresses which can lead to unexpected damage, cracks, and even macroscopic fractures, severely degrading their functionality and lifespan . Self-heSelf-healing can be divided into two kinds: extrinsic self-healing and intrinsic self-healing. Extrinsic self-healing elastomers rely on pre-embedded repairing reagents  in the matrix ,10,11,12A new generation of intrinsic self-healing elastomers has emerged based on the mechanisms of non-covalent and covalent bonds or their combinations . In thisNon-covalent interactions are intrinsically reversible due to their lability. Introducing non-covalent cross-links into the molecular design of elastomers can result in increased mechanical strength without sacrificing extensibility, toughness, and tensile strength . Non-cov−3) and high tensile strength (44 MPa) after self-healing. This may be attributed to the relatively low entanglement and the dynamic exchange of H-bond pairs. Generally, dynamic dense hydrogen bonding interactions contribute to extraordinary toughness and recoverability. On the other hand, Yoshie et al. demonstrated that entropy-driven strong H-bonds enabled the creation of a vicinal diol-functionalized polymer with mechanical robustness, along with functionalities based on the dynamicity of the H-bonds . T. T3+ cooover 90% . In addibilities ,41,42,43bilities ,45,46.Compared with the non-covalent interactions illustrated above, cyclodextrins (CDs) have emerged as promising host monomers that can construct reversible cross-linked network in elastomers via host–guest interactions . For exaOther than the host–guest interaction, shape memory has also been verified to facilitate self-healing since physical damage usually occurs during the deformation process. In blended polymer complexes, polycaprolactone (PCL) can act as a healing agent capable of diffusing and rearranging between cracks during the annealing process to heal scratches at 80 °C for 30 min as well as a semicrystalline thermoplastic d 28]. I. I28]. INon-covalent bonds have inherent limitations due to their weak bond energy, which can easily lead to poor structural stability and mechanical properties ,55. In cDiels−Alder (DA) reactions are thermoreversible and can be applied to a wide range of elastomers. The integration of DA chemistry and rubber products has been extensively researched . Most ofOwing to the distinct imine exchange processes, imine bonds have been established as a form of reversible covalent bond . SiliconDisulfide bonds are easily broken, after which a new covalent bond is formed through a translocation exchange reaction to achieve self-healing in elastomers under mild conditions ,79. The A type of bio-based epoxy elastomer composed of a commercial bio-based epoxy resin (ESO) and an aromatic disulfide-containing agent (DTSA) benefits from dynamic aromatic disulfide bonds that can rapidly self-heal even at room temperature due to fast translocation exchange . AnotherSelf-healing PUs based on disulfide bonds have attracted significant research efforts in recent years ,85. Liu 2) [Other dynamic covalently cross-linked systems have also emerged . For exa2) .Otherwise, via transesterification reactions, the composite materials can be recycled and self-healed at elevated temperatures by undergoing dynamic reshuffling and rearranging the network topology . The obtIn addition, a dynamic urea-bond-based cross-linking polyurea using polydimethylsiloxane as the soft segment was developed as a self-healing elastomer . The dynIn general, it is hard to balance the self-healing properties and mechanical properties of materials which are essentially incompatible. At present, combining multiple interactions has become the main solution to overcome the contradiction between the mechanical properties and self-healing properties of elastomers, which has created the so-called fourth generation of self-healing materials .Covalent bonds possess higher energy compared to non-covalent bonds, contributing to the mechanical performance of a material. Therefore, it is crucial to enhance their reversibility to achieve high healing efficiencies by introducing non-covalent bonds or other reversible interactions ,98.The study of disulfide-bond-assisted H-bonding self-healing materials began systematically in 2017 ,100. H-bSignificantly, H-bonds are commonly used to support covalent bonds . HoweverCompared to H-bonds, ionic bonds not only have a higher bond energy but also a higher association energy due to their high propensity to segregate into ionic aggregates . A dual-In addition to dual-network structures, novel and facile methods incorporating multiple dynamic bonds (more than two types of bonds) have been proposed . For insFurthermore, two types of dynamic reversible covalent bonds can also produce synergistic self-healing effects. The introduction of imine bonds and boroxine bonds into PDMS networks can easily fabricate a polysiloxane elastomer with a maximum healing efficiency of up to 97.8% . IntegraMultiple non-covalent interactions refer to the interaction of two or more non-covalent bonds between molecules. Compared with a single non-covalent-interaction cross-linking material, multiple dynamic-interaction cross-linking polymer complexes provide a simpler method for enhancing the mechanical properties and structures of repairable elastomers.The most common non-covalent interaction is H-bonding, which can be combined with other interactions including ionic interactions , coordinSelf-healing polymers based on ionic bonds and hydrogen bonds can achieve excellent mechanical strength while maintaining a high healing efficiency. Wang et al. utilized hydrogen bonding to prepare amide oligomers with a certain elasticity and then introduced zinc–carboxylate interactions in the oligomers . The con2+-ligand coordination which can realize fast solid–liquid separation during photocuring and 3D printing and exhibits high stretchability, a shape-programming ability, and a self-healing capacity [Regarding H-bonds and metal–ligand coordination, Qiu and Liu et al. constructed a dual physical network of H-bonds and Zncapacity . This thConnecting H-bonds and host–guest interactions led to the construction of double networks . The twoIn addition, H-bonds and hydrophobic associations show great potential in enhancing the mechanical performance and self-healing ability of elastomers, as indicated by Mooney–Rivlin equation calculations . Further(1)The cost of self-healing elastomers is too high due to the high raw material cost, complex synthesis steps, and reaction conditions. In particular, the reaction intermediates typically involve highly active functional groups, such as amines, isocyanates, and free radicals, which will undoubtedly limit their shelf life and prohibit their applications.(2)So far, the repair conditions of self-healing materials are too harsh. Generally, achieving a self-healing function at room temperature is difficult, and it requires heat at a certain temperature or electromagnetic radiation to achieve self-healing. Moreover, flow and closure are the foundations that make healing possible. Thus, it is vital to put fractured structures back together, which is not currently possible without manual intervention.(3)The repair speeds of self-healing elastomers have not yet met the needs of practical applications.(4)With increases in use time and repair time, the physical properties of self-healing materials will weaken. Meanwhile, their healing speed and efficiency will be greatly reduced due to the water molecules or dust particles that may occupy the positions where reversible bonds form and break between polymer chains.(5)It is a challenge to ally robust mechanical performance with virtuous self-healing abilities in elastomers.This mini-review focuses on the latest advances in intrinsic self-healing elastomers, especially the various strategies used to construct new self-healing elastomers via different mechanisms. The self-healing elastomers prepared in this manner have a wide range of applications, including 3D printing , optical(1)Introducing self-healing properties into commercially available polymer materials without changing the original properties of the polymer material, such as PDMS, rubbers, and PUs.(2)Optimizing the position, concentration, and structural arrangement of the dynamic chemical bonds in the polymer network to realize the transition from elastomer to liquid after dynamic bond breakage so that the flow of the polymer segment can also completely fill the damaged area.(3)Improving the mobility of molecular chains and the dynamic exchange rate of dynamic bonds to shorten the healing time.(4)Integrating different chemical groups into polymer networks with fine control of the proportions and positions responsible for self-healing and other functions during synthesis or through post-modification.(5)Increasing cross-linking density and endowing the dynamic properties of cross-linking points, which may improve the mechanical properties of self-healing materials, or alternatively constructing a dual network structure composed of both non-covalent and covalent bonds into the material. Dynamic covalent bonds cause a significant improvement in mechanical properties, while non-covalent interactions, as the primary sacrificial bond, provide substantial improvements in healing efficiency. In addition, it is advisable to choose reversible covalent bonds with higher bond energies combined with auxiliary supramolecular interactions, controlling the structure of the soft segment and the soft/hard segment ratio or constructing a microphase separation system and introducing an enhanced phase. All these methods aim to achieve self-healing elastomers with both high mechanical strength and superior healing efficiency.(6)Specifically, it is beneficial to establish mechanisms by mimicking organisms found in plants and human skin when designing self-healing elastomers.(7)Exploring multiple combinations in search of further positive effects.(8)Using fillers or other additives as carriers for additional repair mechanisms may also be a promising option.In view of the above limitations, future works on self-healing elastomers should focus on the following aspects:In summary, ideal self-healing elastomers should possess a quick healing time, high efficiency, robust mechanical properties, and autonomous healing without external energy and be fully restored  after repair. Additionally, developing multifunctional self-healing elastomers will be a future research hotspot. We believe that the present serious limitations will be resolved in the near future, and that diverse high-performance self-healing elastomers with various properties have excellent prospects.


Hospital-onset (HO) antibiotic-resistant (AR) infections increased in 2020. We examined what proportion of HO AR infection increases could be attributed to patients with COVID-19.HO cases were identified using PINC-AI microbiology data from a cohort of 240 hospitals in 2019-2021. The data were divided into a pre-pandemic period (Jan 2019-Jan 2020) and a pandemic period (Apr 2020-Dec 2021). We built a predictive model for HO AR infections using multivariable logistic regression including patient and facility demographics. The predictive model was built using pre-pandemic period data and applied to pandemic period patients not diagnosed with COVID-19 and pandemic period patients with COVID-19 (COVID cohort). The excess number of HO AR infections (observed – expected) was determined for each cohort and the proportion of excess attributable to the COVID cohort was calculated. We then repeated the predictive model process adding patient clinical characteristics in the model. Recycled predictions were used to estimate confidence intervals.Pseudomonas  and CRE  all had similar proportions of excess (Table). VRE had the lowest proportion of excess . After adjusting for clinical characteristics, the proportion of excess attributable to the COVID cohort decreased to 0% for MRSA, CRE, and VRE. CRAsp had the highest proportion of excess after adjusting ; ESBL  and MDR Pseudomonas  had lower but non-zero proportions of excess. Rates of excess cases attributable to the COVID cohort can be seen in the Figure.Before adjusting for clinical characteristics, MRSA had the highest proportion of excess HO AR cases attributable to the COVID cohort . ESBL , CRAsp , MDR Patients with COVID-19 accounted for 20-100% of excess HO AR infections, varying by pathogen. In COVID-19 patients, increases in HO AR infections were largely due to patients’ clinical characteristics such as length of stay and comorbidities. In non-COVID-19 patients, increases in HO AR infections were not entirely explained by clinical characteristics, and might be due to factors such as lapses in infection prevention practices.All Authors: No reported disclosures


As COVID-19 pandemic public health measures are easing globally, the emergence of new SARS-CoV-2 strains continue to present high risk for vulnerable populations. The antibody-mediated protection acquired from vaccination and/or infection is seen to wane over time and the immunocompromised populations can no longer expect benefit from monoclonal antibody prophylaxis. Hence, there is a need to monitor new variants and its effect on vaccine performance. In this context, surveillance of new SARS-CoV-2 infections and serology testing are gaining consensus for use as screening methods, especially for at-risk groups. Here, we described an improved COVID-19 screening strategy, comprising predictive algorithms and concurrent, rapid, accurate, and quantitative SARS-CoV-2 antigen and host antibody testing strategy, at point of care (POC). We conducted a retrospective analysis of 2553 pre- and asymptomatic patients who were tested for SARS-CoV-2 by RT-PCR. The pre-screening model had an AUC (CI) of 0.76 (0.73–0.78). Despite being the default method for screening, body temperature had lower AUC (0.52 [0.49–0.55]) compared to case incidence rate (0.65 [0.62–0.68]). POC assays for SARS-CoV-2 nucleocapsid protein (NP) and spike (S) receptor binding domain (RBD) IgG antibody showed promising preliminary results, demonstrating a convenient, rapid (<20 min), quantitative, and sensitive (ng/mL) antigen/antibody assay. This integrated pre-screening model and simultaneous antigen/antibody approach may significantly improve accuracy of COVID-19 infection and host immunity screening, helping address unmet needs for monitoring vaccine effectiveness and severe disease surveillance. As the fight against COVID-19 enters its fourth year, public health mandates around the pandemic started moving towards surveillance of new variants of concern (VOC), vaccination efficacy, and protection of vulnerable populations. While over 13 billion vaccine doses were administered worldwide so far ,3. FurthDuring this new phase of pandemic response, screening and surveillance of the at-risk population can help contain transmission rates, guide vaccination strategy, and aid the prevention of severe disease and associated morbidity and mortality. Additionally, a screening strategy may help guide convalescent plasma donations, a key therapy for immunocompromised patients who are susceptible to refractory infection . With riReal-time reverse transcriptase polymerase chain reaction (RT-PCR) remained the current gold standard method for SARS-CoV-2 detection, especially in the first 5 days of infection with viral load peaking around day 4 ,20. WhilWhereas molecular diagnostic tests such as nucleic acid based testing including RT-PCR and antigen tests can only reveal whether a person is currently infected with SARS-CoV-2, antibody tests detect the body’s humoral immune response  following viral exposure and/or vaccination. Antibody responses can appear by day 5–7 post infection exposure, and it can persist in the bloodstream for many months after infection ,23. SeroMolecular testing combined with serological assays can improve the overall diagnosis of SARS-CoV-2 ,31. HoweIn the current study, we explored whether pre-screening patients using convenient non-laboratory data can predict COVID-19 status in patients without symptoms. This paper also demonstrated a quantitative strategy for concurrent COVID-19 and host antibody screening, suitable for use in POC settings, that has potential to be assisted simultaneously with the newly developed pre-screening method reported here. A preliminary assay validation was performed for this duplex COVID-19 test including a combination SARS-CoV-2 NP antigen and host IgG antibody, covering the entire diagnostic timeline of the disease with a single multiplexed test. This device integrated a lab-on-a-chip microfluidics platform facilitating automated liquid sample handling, with an easy, simple and sensitive assay readout and AI-assisted screening.Pre-screening algorithms were developed from a retrospective analysis of asymptomatic or pre-symptomatic patient encounters resulting in a COVID-19 RT-PCR positive test. Data were collected across clinics and hospitals within the Family Health Centers (FHC) network at New York University (NYU) Langone from 1 January to 25 June 2020, although the first known positive case in the state of New York was detected 1 March 2020. Test positivity prior to this date was assumed 0. Data were analyzed at the encounter level rather than the patient level because many patients had multiple encounters. Symptomatic patient encounters, in which one or more primary COVID-19 symptoms  was present, were excluded. Physiological predictors were evaluated at two levels . County-level testing data were acquired from the New York State Department of Health (New York State Statewide COVID-19 Testing 2020). For each patient, a local positivity rate was calculated . Similarly, case incidence rate was calculated as the local 7-day average cases per 100,000. Consistent with NYU’s institutional review board (IRB) policy and federal regulations, this study did not involve human participants and did not require IRB review. The data set is available from the authors upon reasonable request and with permission of FHC at NYU Langone. Currently, with the changing dynamics of COVID pandemic scenario and infectivity across society, the COVID community clinics are no longer performing community surveillance data collection.t-test. Proportions were compared using the chi-squared test . On. On35]. A retrospective analysis determined whether pre-screening could effectively rule out COVID-19 negative patients . Many patients had multiple encounters, and we based our analysis on a total of 3477 patient encounters resulting in RT-PCR tests at NYU Langone Health FHCs. Patient encounters with one or more primary symptoms  were excluded (n = 924 encounters). The remaining 2553 asymptomatic or pre-symptomatic patient encounters either tested negative (n = 2059 encounters) or positive (n = 494 encounters) for SARS-CoV-2 by RT-PCR .p = 0.005 and 0.021). Those with Hispanic ethnicity accounted for 56.6% of the positives vs. 38.7% negatives (p < 0.001). Comorbid condition rates were similar in those that tested positive vs. negative. At the patient encounter level, all physiological measurements discriminated between the RT-PCR-positive and negative groups at their respective cutoffs . The local positivity rate was higher for patients testing positive (32.8%) vs. negative (17.7%) (p < 0.001). Similarly, the local case incidence rate was higher for COVID-19 positives vs. negatives . Details of daily changes in positivity rates and case incidence rates from New York State Department of Health are detailed in Pre-screening models for COVID-19 were developed and internally validated . The locFurthermore, the diagnostic performance for models discriminating COVID-19 positive vs. negative (RT-PCR) in pre- and asymptomatic individuals is demonstrated in Despite being the de facto method for COVID-19 screening to date, temperature was found to be relatively ineffective at distinguishing which pre- or asymptomatic patients were infected. However, temperature checks may still play an important role in detecting symptomatic individuals who unknowingly present with a fever. Likewise, measurements of oxygen saturation did not show significant improvements over temperature despite its potential importance in monitoring disease progression in confirmed COVID-19 cases. One unexpected finding of this analysis was that when and where a person was screened was the most important factor in predicting COVID-19 status. The local test positivity rate and case incidence rate were the strongest predictors of COVID-19 status, outperforming physiological and demographic factors. This result demonstrates the significance of time- and location-specific spread data within communities in estimating the pre-test probabilities for COVID-19 screening. This result may be especially relevant for large clinical centers which see an influx of patients from a broader geographic region.Combining test positivity with recorded race and ethnicity changed the performance (AUC 0.76); however, the inclusion of racial and ethnic information was dependent on information collected at time of patient registration and may not generalize well to less diverse populations . While cWhile the pre-screening algorithm determined who is at elevated risk with 90% sensitivity , the comThe conceptual basis for simultaneous COVID-19 NP antigen and host IgG antibody assay sequence is shown schematically in As the multiplexed immunoassay progressed towards the antibody detection steps, during stage 7, the antibody detection reagent (conjugated to Alexa Fluor 488) was introduced via the left reagent pad through buffer flow pushing the reagent over the bead array (Stage 7), followed by incubation (Stage 8) and final wash (Stage 9). In the presence of SARS-CoV-2 IgG antibody in the sample, the post-assay completion image (the second image captured during the entire assay) showed the antibody capture beads fluorescing as a result of the antibody immune-complex formation (Stage 9C).This combination test modality allows for the placement of positive and negative control beads alongside the antigen and antibody reactive beads. Both antibody and antigen assays can be completed through the spatial placement of specific reactive bead ensembles within the array. Likewise, the 250–300 μm, in-house fabricated, analyte capturing agarose beads (conjugated with complementary protein) were placed in the 4 × 5 format bead array, each with a vertically tapering well.To demonstrate initial validation, standard curves for antigen (SARS-CoV-2 NP) and antibody (anti-S RBD IgG) were completed with 4-fold serially diluted analyte-spiked sample buffer, covering a range of high viral antigen and immune response load  to very low loads (2 ng/mL) . StandarDiagnostics at the POC are critical for successfully mitigating COVID-19 transmission risk in asymptomatic and pre-symptomatic populations. Expanding access to in situ testing capabilities adds significant convenience to the risk management infrastructure much needed in communities with vulnerable individuals, such as retirement homes, cancer care centers, and critical care clinics. While the current gold-standard RT-PCR and ELISA detection techniques are highly valuable, the added time, cost, and demand-supply chain are major bottlenecks for processing the growing needs for testing. Convenient antigen testing combined with rapid antibody-based testing has much potential in addressing these testing bottlenecks. In contrast to traditional immunochromatographic strip and ELISA techniques, the multiplexed microfluidics-based assay developed here has the potential to achieve high sensitivity in a convenient format with noninvasive sampling while maintaining high specificity.Furthermore, viral antigen detection, in conjunction with screening host antibodies, may be a helpful strategy to achieve early detection of SARS-CoV-2 infection. NP, utilized in this work, is one of the most predominantly expressed and abundant protein in SARS-CoV-2 virions, ideal for early diagnostic detection, detectable up to 1 day before the appearance of clinical symptoms . ImportaThe concurrent assay developed here also utilized the anti-S RBD IgG to reflect the high utility and specificity of antibody-mediated protection associated with vaccination and/or infection. Most major COVID-19 vaccines elicit specific immune responses against the S protein of SARS-CoV-2, preventing the virion from host cell binding, fusion, and entry . AdditioOur concurrent assay described a fully functional multiplexing of COVID-19 infection and host immune response biomarkers. A significant challenge with multiplexing is cross-reactivity between capture and detecting reagents, particularly in combining immunoassay formats. These issues can be mitigated through optimization of reagent sources, subtypes, blocking strategies, assay flow rates, and volumes. Additionally, limitations of this testing strategy included obtaining negative results in patients during their incubation period who later become infectious.Multiple qualitative POC antigen or separately antibody detection strategies were approved by emergency used authorization by the FDA, significantly easing the testing bottlenecks across the pandemic timeline. Though the work in this manuscript represents a POC, quantitative, and concurrent antigen and antibody testing strategy, embodying a significant advancement over the antigen only or antibody only tests which are mostly non/semi-quantitative POC applications.While this current work served to demonstrate initial method validation and a promising implementation for high-risk settings; requiring rapid, cost-effective, convenient, and accurate screening results, future work will involve further assessment of qualitative performance (sensitivity and specificity) and blinded validation of the combinatorial format with real patient samples confirmed by RT-PCR and lab-based serological testing methods.A quantitative concurrent antigen/antibody screening platform may also be useful for assessment of COVID-19 convalescent plasma donor fitness. The passive transfer of anti–SARS-CoV-2 neutralizing antibodies from the plasma of recently recovered individuals to patients with severe COVID-19 is associated with a decrease in incidences of disease progression, hospitalization, and mortality . PatientAs the public health demands for COVID-19 pandemic change, there is a strong need to rapidly adapt testing strategies, monitoring both the infection and immune status of patients, especially directed towards the vulnerable populations. To facilitate public health policy decisions, governments across the globe use estimates of transmission rates, case numbers, and fatality rates. The assessment of infection can help mitigate transmission, while insights on virus-specific antibody titer levels can help recognize changes in the antibody-mediated protection affected by new, rapidly evolving SARS-CoV-2 variants and vaccination response. The pre-screening algorithm alongside simultaneous multiplexing capabilities and its streamlined workflow described in this paper represent important steps towards building an infrastructure necessary in this next phase of pandemic, continue protecting the at-risk individuals, and provide information to clinicians and public health policy makers to facilitate this process.


DNA-based gene therapy and vaccine development has received plenty of attention lately. DNA replicons based on self-replicating RNA viruses such as alphaviruses and flaviviruses have been of particular interest due to the amplification of RNA transcripts leading to enhanced transgene expression in transfected host cells. Moreover, significantly reduced doses of DNA replicons compared to conventional DNA plasmids can elicit equivalent immune responses. DNA replicons have been evaluated in preclinical animal models for cancer immunotherapy and for vaccines against infectious diseases and various cancers. Strong immune responses and tumor regression have been obtained in rodent tumor models. Immunization with DNA replicons has provided robust immune responses and protection against challenges with pathogens and tumor cells. DNA replicon-based COVID-19 vaccines have shown positive results in preclinical animal models. Plasmid DNA-based delivery of transgenes has been used for gene therapy , which tPlasmid DNA has largely been administered as intramuscular, intradermal, and subcutaneous injections ,11. The Self-replicating RNA viruses including alphaviruses, flaviviruses, measles viruses, and rhabdoviruses have been frequently used for cancer therapy and vaccine development . In the The self-replicating vector systems contain the genes for the viral non-structural proteins and the gene of interest (GoI), which are in vitro transcribed from an SP6 or T7 RNA polymerase promoter . ElectroAlthough DNA-based systems provide superior safety levels compared to the application of recombinant particles, the inferior delivery compared to viral vectors has had a negative impact on their application . The nee6 copies of subgenomic RNA are accumulated per cell leading to massive transgene expression. Regarding self-replicating RNA viruses, alphaviruses , and flaecto) [ecto induced binding and neutralizing antibodies. Superior vaccine potency was obtained for DREP-S, eliciting high titers of SARS-CoV-2-specific IgG antibodies in mice receiving a single injection [In the context of DNA replicon vectors mainly alphaviruses have been utilized for vaccine development against infectious diseases . Among aecto) . Both DRnjection .Mycobacterium tuberculosis p85 antigen elicited strong p85-specific immune responses in mice [M. tuberculosis. Application of VEE DNA replicons for the expression of a fusion protein of the M. tuberculosis α-crystallin (Acr) and Ag85B antigens induced strong CD4+ and CD8+ T-cell responses in mice and prevented bacterial growth in lungs and spleen of mice after challenge with aerosol-based M. tuberculosis [Clostridium botulinum neurotoxin, the serotype A (BoNT/A) Hc gene was co-expressed with the granulocyte-macrophage colony-stimulating factor (GM-CSF) from SFV DNA replicons [Bacillus anthracis A16R strain in mice. In the context of protozoans, SFV DNA replicon-based expression of the nucleoside triphosphate hydrolase-II of Toxoplasma gondii (TgNTPase-II) provided partial protection against acute infection and toxoplasmosis in mice [Bacterial infections and toxins have also been targeted with DNA replicons. For example, SIN DNA expressing the  in mice . Moreoverculosis . In the eplicons . Mice imeplicons . Moreoveeplicons . Initialeplicons . Furthereplicons . Relatedeplicons . Both SF in mice .Phlebotomus papatasi and the Leishmania major stress inducible protein 1 (PpSP15-LmSTI1) was expressed from SFV RNA replicons, SFV DNA replicons, and conventional plasmid DNA vectors [In the context of parasites, the fusion protein of the SP15 protein from the insect  vectors . In thisM. tuberculosis heat shock protein 70 (Hsp70) from SFV DNA replicons [+ T-cell-mediated immune responses and enhanced the antitumor activity in mice.DNA replicons have been frequently evaluated for cancer therapeutics and vaccines . For exaeplicons . The fuseplicons . Moreoveeplicons . In anoteplicons . It geneIn the case of breast cancer, intramuscular administration of SIN DNA replicons expressing the HER2/neu gene generated robust immune responses and reduced tumor incidence and tumor mass in mice . MoreoveIn the context of melanoma, SIN DNA replicons expressing the self/tumor antigen tyrosine-related protein-1 (TRP1) in mice demonstrated that, unlike conventional DNA vaccines, DNA replicon-based vaccines can break tolerance and provide immunity against melanomas . Double-+/CD8+ T cells and IFN-γ mediated the antitumor and protective effects, and the survival rate was significantly improved in mice with implanted B16 tumors. Related to brain tumors, co-expression of human gp100 and mouse interleukin-18 (IL-18) from SIN DNA replicons in mice with implanted B16-gp100 brain tumors showed both protective and therapeutic efficacy . Both CD+ T-cell responses compared to 1 µg of conventional DNA vaccine, equivalent to a 625-fold higher molar dose [M. tuberculosis [Comparative studies between DNA replicon-based vaccines and conventional DNA vaccines have indicated that significantly lower doses of DNA replicons are needed to elicit robust immune responses ,37. For lar dose . This ovlar dose . Althouglar dose . The strrculosis  was not The feasibility of DNA replicon-based vaccines has been verified by direct comparison not only to conventional DNA plasmid-based vaccines but also to recombinant viral particles and RNA replicons. For example, the medium (M) and small (S) gene segments of the envelope glycoproteins or nucleocapsid protein of the Seoul virus (SEOV), belonging to the hantavirus family, were compared for expression from a conventional DNA vector, a SIN DNA replicon, and recombinant SIN particles in Syrian hamsters . All vecIn the context of mRNA-based vaccines, there is no doubt that recent success seen for vaccines developed against SARS-CoV-2 leading to emergency use authorization and global mass vaccinations has been superior to any DNA vaccine development so far . RelatedDNA replicons have demonstrated protective and therapeutic efficacy in animal models for infectious diseases and various cancers, as summarized in Aspects of superiority of using DNA replicons are their easy, inexpensive, and rapid handling for large-scale production. In comparison to the degradation-sensitive RNA replicons, the double-stranded structure of DNA replicons is stable, which allows easier handling, transport, and storage of DNA-based vaccines. Furthermore, it has been demonstrated that DNA plasmids are not integrated into the host genome at any level of concern. In comparison to viral particles, there is no safety risk of using DNA-based material. However, as indicated in comparative studies, expression levels from DNA replicons are often inferior to those obtained from RNA replicons or recombinant viral particles, which might be related to the requirement of delivery of DNA to the cell nucleus. Generally, delivery improvement through LNP and lipoplex formulations as well as improved nuclear translocation by the introduction of nuclear localization signals need to be explored to be able to provide more efficient DNA replicons for future gene therapy and vaccine exploration.


Adipose-derived stem cells (ADSC) therapy shows promise as an effective treatment for dystrophinopathy. Fibro-/adipogenic progenitors (FAPs) play an essential role in the myogenesis of muscle satellite cells and contribute to muscle fibrosis and adipocyte infiltration. The interleukin 4 (IL-4) pathway acts as a switch that regulates the functions of FAPs. The interaction between FAPs and engrafted cells remains unclear. In this study, we used a co-culture system to investigate possible crosstalk between the FAPs of dystrophic mice and ADSC overexpressing IL4 (IL4-ADSC) and control ADSC. Systemic transplantation of IL4-ADSC and control ADSC in dystrophic mice was conducted for 16 weeks, after which motor function and molecular improvements were evaluated. Overexpression of IL4 in ADSC significantly promoted myogenesis in vitro, increasing the expression of Pax7, Myogenin, and MyHC. Co-culture indicated that although myoblasts derived from control ADSC promoted adipogenic and fibrogenic differentiation of FAPs, FAPs did not significantly affect myogenesis of ADSC-derived myoblasts. However, overexpression of IL4 in ADSC inhibited their myotube-dependent promotion of FAPs differentiation on the one hand and promoted FAPs to enhance myogenesis on the other. Dystrophic mice administered with IL4-ADSC-derived myoblasts displayed significantly better motor ability, more engrafted cells showing dystrophin expression, and less muscle fibrosis, intramuscular adipocytes, and macrophage infiltration than mice administered control-ADSC-derived myoblasts. In conclusion, IL4 activation enhanced the therapeutic potential of ADSC transplantation in dystrophic mice, possibly by improving the myogenesis of IL4-ADSC and altering the crosstalk between engrafted stem cells and resident FAPs. DMD gene leading to defective dystrophin cause DMD. Progressive pathological changes in muscles due to fibrosis and fatty infiltration are hallmarks of advanced DMD . Oil Red O staining revealed that FAPs (Co-cult with VEH-ADSC) form more lipid-laden adipocytes compared to mono-cultured FAPs and FAPs (Co-cult with IL4-ADSC)  were dramatically increased compared to those in mono-cultured IL4-ADSC Fig. A and C. We speculated that FAPs may secrete cytokines that promote the myogenesis of IL4-ADSC-derived myoblasts. Therefore, we investigated the mRNA expression levels of several common myogenesis-promoting factors  and IL4 in co-cultured FAPs and mono-cultured FAPs  significantly improved terminal myogenesis caused by IL4-ADSC-derived myoblasts; (ii) the obviously enhanced effect of IL4-ADSC-derived myoblast transplantation on muscle fibrosis via activation of the IL4 pathway in FAPs, compared to that of VEH-ADSC-derived myoblasts; (iii) significantly less infiltration of muscle by mdx + IL4-ADSC compared to that of mdx + VEH-ADSC, due to IL4 activation inhibiting the adipogenic differentiation of FAPs promoted by ADSC-derived-myoblasts. For the first time, we report that overexpression of IL4 effectively improves the effect of ADSC therapy on muscle fat infiltration in mdx mice, thereby providing a new concept in ADSC therapy that may help enhance muscle fat infiltration in mdx mice.We found that IL4 expression in the TA of 6-month-old mdx mice was increased compared to that of C57/BL6 mice, which was consistent with previous reports indicating that the regenerative stage of mdx mouse limb muscles was possibly associated with infiltration by T cells or eosinophils , 37. EleThis study was affected by certain limitations. Firstly, we simply focused on the relationship between engrafted cells and FAPs, and failed to investigate the benefits conferred by the in vivo study to other mechanisms. Further research should focus on elucidating more complicated interactions that may exist between other cell types. Furthermore, the improved post-transplantation therapeutic effects exerted by IL4-ADSC on mdx mice cannot be said to have benefited from the altered crosstalk between engrafted IL4-ADSC and FAPs or from the improved myogenic impact of IL4-ADSC themselves, although the in vitro co-culture study of IL4-ADSC and FAPs does offer some evidence. Besides, many studies show the detrimental effects of promoting the activity and generation of this cytokine, as it is an immunosuppressor of several cell activities. In this regard, IL4 has been shown to worsen the outcome of important diseases, such as cancer, virus, and parasitic diseases , 41. ThuTo the best of our knowledge, this is the first study to determine the extra benefits conferred by a combination therapy involving IL4 administration and cell therapy on the positive effects exerted by muscle micro-environment cells on the regenerative therapy of DMD. In conclusion, IL4 overexpression improves the therapeutic potential of ADSC transplantation in mdx mice, possibly by altering crosstalk between engrafted stem cells and resident FAPs and improving the myogenesis efficacy of IL4-ADSC.


EGFR, ALK, KRAS, and ROS1. Tyrosine kinase inhibitors (TKIs) and angiogenesis inhibitors have shown better therapeutic responses and lower toxicity compared to systemic chemotherapy. However, resistance to these therapies remains a challenge. Immune checkpoint inhibitors (ICIs) targeting PD-1 or PD-L1 have changed the treatment paradigm for NSCLC without actionable genetic alterations, but combining targeted therapy and ICIs does not provide survival benefits. The review focuses on the tumor immune microenvironment and its role in applying ICIs for this subpopulation of lung ADC patients.The development of targeted therapies has led to personalized medicine for advanced non-small cell lung cancer (NSCLC), particularly in lung adenocarcinoma (ADC) with actionable genetic alterations, such as EGFR), anaplastic lymphoma kinase (ALK), Kirsten rat sarcoma virus (ALK) and c-ros oncogene 1 (ROS1) treated with tyrosine kinase inhibitors (TKIs) incurred lesser treatment toxicity but better therapeutic responses compared with systemic chemotherapy. Angiogenesis inhibitors targeting vascular endothelial growth factor (VEGF) have also shown an increase in overall survival (OS) for NSCLC patients. However, acquired resistance to these targeted therapies remains a major obstacle to long-term maintenance treatment for lung ADC patients. The emergence of immune checkpoint inhibitors (ICIs) against programmed cell death protein 1 (PD-1) or programmed cell death-ligand 1 (PD-L1) has changed the treatment paradigm for NSCLC tumors without actionable genetic alternations. Clinical studies have suggested, however, that there are no survival benefits with the combination of targeted therapy and ICIs. In this review, we will summarize and discuss the current knowledge on the tumor immune microenvironment and the dynamics of immune phenotypes, which could be crucial in extending the applicability of ICIs for this subpopulation of lung ADC patients.The rapid development of targeted therapy paved the way toward personalized medicine for advanced non-small cell lung cancer (NSCLC). Lung adenocarcinoma (ADC) harboring actionable genetic alternations including epidermal growth factor receptor ( EGFR), anaplastic lymphoma kinase (ALK), Kirsten rat sarcoma virus (KRAS) and c-ros oncogene 1 (ROS1), tyrosine kinase inhibitors (TKIs) are primarily recommended as first-line treatment. On the other hand, for NSCLC patients whose tumors lack druggable driver alternations, immunotherapy in the form of immune checkpoint inhibitors (ICIs) has emerged as a durable treatment option for patients with high tumor programmed cell death—ligand 1 (PD-L1) expression. However, subgroup analysis of various clinical studies has shown that druggable-mutation-positive NSCLC may have an inferior response to first- or second-line ICIs treatment [In the last century, surgery, radiotherapy and chemotherapy were almost the only therapeutic options for the treatment of lung cancer. The median overall survival (mOS) for the disease remained as low as a few months. Nowadays, with the advances in targeted therapy and immunotherapy, the mOS of advanced non-small cell lung cancer (NSCLC) leaps to more than a year . Howeverreatment ,3, with EGFR) (~50%) and a smaller proportion with Kirsten rat sarcoma virus (KRAS) mutations (~10–20%), anaplastic lymphoma kinase (ALK) (~5%) or ROS proto-oncogene 1 (ROS1) rearrangement (<5%). KRAS mutations, however, could be found in about 30% of NSCLC in Caucasian populations. Indeed, some genetic variants are more common in certain geographic regions, such as TP53 mutations are higher in Iranian patients with lung cancer [EGFR mutations, lorlatinib and brigatinib for ALK re-arrangement, sotorasib and adagrasib for KRAS G12C mutations, and entrectinib for ROS1 rearrangement, are currently US-FDA approved next-line treatment for NSCLC patients with respective resistance following primary therapy. However, selection pressure from specific targeted therapy can induce gradual genetic and epigenetic adaptation in heterogenous clones of cancer cells, leading to an enrichment of the best-fit subpopulation of cancer cells within the tumor microenvironment [EGFR T790M, ALK L1196M) or gene amplification (MET). Such an assumption of a single hit resistance mechanism may allow for the further emergence of unidentified mechanisms of drug resistance. The need to search for long-lasting and low-toxicity treatment strategies remains.The rapid development of tyrosine kinase inhibitors (TKIs) in the past two decades drove toward precision medicine with the advancement of molecular diagnostic tools . By inhig cancer . NSCLC pironment . HoweverBesides oncogenic mutations, another significant therapeutic approach involves directing treatment toward growth factors that promote tumor angiogenesis. By sprouting new vasculature towards the tumor mass to sustain its active metabolism and metastasis, tumor angiogenesis is essential for tumor growth and metastasis . Cancer The development of immunotherapy, especially immune-checkpoint inhibitors (ICIs), rapidly emerged as the cornerstone of the management of NSCLC. Tumor mutations lead to aberrant protein-coding that eventually results in the expression of tumor antigens, which could be recognized, processed and presented by major histocompatibility complexes (MHC) on antigen-presenting cells (APCs), such as dendritic cells (DCs), to cytotoxic T cells in lymph nodes. Primed and activated T cells will then seek and kill tumor cells by recognizing tumor antigens. Although tumor cells can be eliminated by the immune system, certain subpopulations of tumor cells manage to escape immunosurveillance by the body. Indeed, for recognition of tumor cells by effector T cells, programmed cell death protein 1 (PD-1) is critical in counteracting the tumor immune responses. Upon binding with programmed death-ligand 1 (PD-L1) on tumor cells, PD-1 serves as a negative signal which normally keeps T cells from attacking normal cells. Some cancer cells can express high levels of PD-L1, which supports their escape from immune attack. ICIs targeting PD-1 and PD-L1 reinvigorate T cells that were inactivated by the PD-1/PD-L1 signaling. It has been proven that PD-1/PD-L1 blockade therapy significantly stimulates the anti-tumor effects of cytotoxic T lymphocytes in different solid tumors, including advanced-stage lung cancer. Clinically, first-line ICIs treatment attained a mOS of 16.2 months and 50% of 2-year survival in non-oncogene-driven NSCLC patients . HoweverIn recent years, tumor PD-L1 expression  has been the most commonly used predictive biomarker for anti-PD-1 treatment. A PD-L1 TPS level of higher than 50% in tumor cells is regarded as the most promising biomarker for anti-PD-1 and anti-PD-L1 treatments in those patients who could benefit from immunotherapy. In KEYNOTE-024, pembrolizumab prolonged primary endpoint, median progression-free survival (PFS), and median overall survival (OS) compared with platinum-based chemotherapy in 305 patients with PD-L1 TPS score of ≥50% . KEYNOTEEGFR-mutated NSCLC patients did not derive benefit from using immunotherapy compared with chemotherapy. In KEYNOTE-010, 86 patients (8.3%) were EGFR-mutant, and 6 (0.6%) were ALK-positive; patients with these oncogenic driver mutations did not achieve prolonged OS in response to pembrolizumab compared to docetaxel. In CheckMate057, 82 patients  were EGFR-mutant, and 21 (4%) were ALK-positive. Subgroup analyses of OS revealed that patients with EGFR mutation, having received or received an additional line of TKI, did not benefit from nivolumab compared with docetaxel .The combination of TKIs and ICIs remains an attractive treatment strategy to explore. Results from the CheckMate057 and KEYNOTE-010 demonstrated a statistically significant improvement in the OS in NSCLC patients treated with nivolumab or pembrolizumab in comparison to patients receiving standard second-line docetaxel-based chemotherapy ,20. HoweEGFR mutations in NSCLC could induce up-regulation of PD-1 expression through activation of the ERK signaling pathway, which may mediate the immune escape [EGFR-mutated lung tumors [EGFR mutation status and PD-L1 expression suggested the indirect and complicated correlation between the two factors [ALK fusion protein [KRAS mutations [Early studies suggested that e escape ,22. Furtg tumors . The het factors . Indeed, factors , indicat protein  and KRASutations  in NSCLCutations . These oThe battling of immunity against malignant cells is a tightly regulated multi-step process, which is determined by the counteracting and highly dynamic effects of different immune cells, along with their interactions and responses to the malignant cells. The tumor immune cycle proposed by Daniel Chen and Ira Mellman has become the conceptual framework in cancer immunotherapy research .+ T cells through the binding of T cell receptors (TCRs), along with B7-CD28 binding, which results in full activation of CD8+ T cells [The framework described that persistent accumulation of DNA damage in a cell that is not properly repaired could lead to genomic instability, which may affect normal cellular functions and ultimately lead to malignant transformation. The unrepaired DNA generates neoantigens, which can be recognized by antigen-presenting cells (APC) through major histocompatibility class I (MHC-I) molecules on malignant cells. These neoantigens released by malignant cells could be recognized by the immune system. Dead and dying malignant cells release neoantigens and damage-associated molecular patterns  that are captured by and activate conventional dendritic cells. Activated dendritic cells travel back to lymph nodes where they process and present MHC-I-bound-neoantigens to naïve CD8 T cells . Activat T cells . Part ofThe tumor immune cycle is a dynamic process that can be disrupted by various mechanisms, such as immune evasion by tumor cells, inhibition of T cell activation, or suppression of immune responses. In NSCLC patients who respond effectively to ICIs therapy, precise execution of tumor immune cycles by restoring harmony to the immune system is typically noted. This observation also indicates that low immunogenicity and insensitivity to ICI in oncogene-driven lung ADC suggested the presence of unknown barriers during the tumor immune cycle.The dynamic interactions between tumor and immune cells determine the development of tumor immunity and response to treatments. Recent studies have shown that cancer cells, particularly in oncogene-driven lung ADC, can affect their normal counterparts to create an immunosuppressive TIME. The different cell types within the TIME engage in reciprocal communication that involves a network of growth factors, cytokines, extracellular vesicles, and adhesion molecules. These complex interactions play fundamental roles in promoting tumor progression and can significantly affect the response of tumors to ICIs. The rapid growth of high-throughput single-cell sequencing technologies has resolved the detailed varieties of subpopulations of immune cells, which allows for substantial improvement in the characterization of diverse immune cells .+ cytotoxic T lymphocytes (CTLs) play a crucial role in the tumor immune cycle by recognizing and eliminating cancer cells. Upon activation through the engagement of TCR on CD8+ T cells with neoantigens presented by MHC-1 on APCs and target cells, followed by secondary activation signal by CD28 and CD80 co-stimulatory signals, CD8+ T cells can directly kill tumor cells by releasing cytotoxic molecules, such as perforin and granzyme B. Perforin is a cytolytic pore-forming protein which allows entry of granzyme serine protease to activate the caspase cascade and eventually induces apoptosis of target cells. However, CD8+ T cells can also have negative regulatory roles in the tumor immune cycle. For instance, they can become terminally exhausted (progenitor-exhausted), which could limit their ability to recognize and kill tumor cells, but also actively participate in tumor progression by maintaining the cancer stemness [+ T cell responses were induced by EGFR-TKI. They showed that EGFR inhibition is a prerequisite for the subsequent ICI treatment [CD8stemness . This ocstemness  or chronstemness , or inhistemness . Howeverreatment .+ helper T cells are important regulatory cells providing help to other immune cells, including CD8+ T cells, to mount an effective anti-tumor response. A recent study showed that CD4+PD-1+CXCL13+ cells (or previously classified as putative TFH cells) are direct targets of anti-PD-1 antibodies, which act as a potential hub for APCs in the TIME [+ T cells can differentiate into different subsets, including Th1, Th2, Th17, and Treg cells, that have distinct functions in tumor immunity. Th1 cells produce cytokines, such as IFN-γ, that activate CD8+ T cells and promote cytotoxicity against tumor cells [+ and CD8+ T cells. They play a critical role in preventing autoimmunity and maintaining self-tolerance but can also inhibit effective anti-tumor immunity. Co-treatment of IL-6 with anti-PD-1 could enhance the activation of tumor-infiltrating Treg and Th17 in EMT-associated TKI-resistant EGFR-mutant NSCLC [+ helper T cells can have both positive and negative regulatory roles in the tumor immune cycle, depending on their subset and the context of the tumor microenvironment [CD4the TIME . CD4+ T or cells ,40. Theyor cells  and inteor cells . Th2 celor cells . They pror cells . Th17 ceor cells . Salazaror cells . Preclinor cells . Regulatnt NSCLC . Therefoironment .+ Treg [Although T cells remain the focus in immune-oncology, increasing evidence suggests that intratumoral or peritumoral B cells are associated with the response to immunotherapy. B cells in many tumors are often organized in tertiary lymphoid structures (TLS). The significance of active versus inactive TLS remains a key open question . Tumor-i+ Treg . IL-35-p+ Treg . TIBs ca+ Treg . This maNK cells are capable of recognizing and killing cancer cells without the need for prior sensitization or activation, making them an important first line of defense against cancer. NK cells can recognize and directly kill cancer cells through the recognition of stress-induced ligands on the surface of cancer cells, which are up-regulated in response to cellular stress. These ligands can bind to the activating receptor NKG2D on NK cells, leading to the activation and killing of cancer cells . NK cellCumulative evidence suggested that TAM are key determinant of T cell excluded tumor phenotypes observed in oncogene-driven NSCLC ,62. HoweTAN could be recruited to the tumor microenvironment in response to inflammatory signals, but the role of TANs in tumor immunity is complex and can be both pro-tumorigenic and anti-tumorigenic depending on the context . TANs haMDSCs are a heterogeneous population of immature myeloid cells that can suppress the immune response and promote tumor growth . MDSCs cDC are specialized antigen-presenting cells that can capture and process antigens from tumor cells and present them to T cells, initiating an immune response against cancer cells . ConventThe role of mast cells in tumor development remains controversial in NSCLC . Mast ceAccording to the spatial distribution relative to the tumoral area and the activation status of immune cells, as mentioned above, there are three recognized sub-types of tumor immune phenotypes, immune-deserted tumor, immune-excluded tumor, and immune-enriched tumor.LKB1 exhibit a poor response to anti-PD1 therapy despite a high tumor mutational burden (TMB) due to reduced expression of the immunoproteasome, impairing antigen presentation. However, a lower proteasome activity can lead to enhanced autophagy as a compensatory mechanism. Inhibition of autophagy by targeting ULK1 has been shown to restore antigen presentation and synergize with PD-1 antibody blockade, promoting tumor regression in LKB1-mutant mice. [Immune-deserted tumors are a specific tumor immune phenotype category characterized by a lack of tumor-infiltrating lymphocytes (TILs) in both the tumor and stromal areas and low antigen presentation. This phenotype can result from several factors, including a low gradient of chemokines required for dendritic cell recruitment . The altnt mice. . Different mice. .Immune-excluded tumors are a tumor immune phenotype characterized by the presence of cytotoxic T cells within the tumor stroma but not infiltrating the tumor area. This phenotype can result from several factors, including the expression of chemokines/cytokines, such as CCL2, IL6, IL10, and TGFβ, as well as the presence of cell types associated with immune suppression or tolerance, such as Treg cells . Despite+ cells often showing an exhausted and dysfunctional state [An immune-enriched tumor phenotype is characterized by the presence of various TILs, including CD4- and CD8-expressing effector T cells, as well as inhibitory Tregs, myeloid-derived suppressor cells, suppressor B cells, and CAFs. In NSCLC, immune-enriched tumors are associated with a smoking history and a high TMB  but alsoal state . PD-L1 sal state . In addial state . Despiteal state .Oncogenic driver mutations do not just promote abnormal cell growth and division, leading to the development of tumors; in addition, oncogenes also equip tumor cells with the ability to modulate the recognition and assault by the immune system, contributing to the development of an immunosuppressive TIME that facilitates tumor growth and progression . The eff+T cells and DC cells expand, and T cell infiltration increases with a concomitant decrease in immunosuppressive cells, such as FoxP3+ Tregs and M2-like polarization of macrophages [EGFR mutation, the application of erlotinib increases infiltrating T lymphocytes and NK cells other than B cells. Increasing MHC-II expression in antigen-presenting cells, such as dendritic cells and macrophages, can enhance antigen-presenting activity. TKIs can reduce the levels of immune checkpoints in TIME. Studies suggested PD-L1 expression was downregulated by inhibiting the EGFR pathway after 4 weeks of gefitinib treatment [+ T cells [In short-term TKIs treatment, CD8rophages . TILs anrophages . After arophages . Meanwhirophages . The sigrophages . EGFR inrophages . In animreatment . EGFR-TKreatment  in NSCLCreatment ,105. Trereatment . The inc T cells . Overall T cells . More imEGFR-mutated lung ADC promotes tumor development. EGFR mutation increases PD-L1 expression in cancer cells, which can promote the exhaustion and apoptosis of CD8+ T cells. As a proinflammatory chemokine, decreased CXCL10 reduces the migration and recruitment of CD8+ T cells and lowers the infiltration of CD8+ T cells. Elevated production of CCL5 and CCL22 promotes the migration of Treg cells and further attenuates the function and proliferation of CD8+ T cells. EGFR mutation also induces upregulation of MDSCs, increases IDO in DCs, and enhances M2-like polarization and migration of TAMs, which abrogates CD8+ T cell and cytotoxic NK cell-mediated tumor killing. EGFR mutation also leads to a decrease in the activation of B cells, lowering the expression of MHC-I and -II on B cells, and decreasing the activation and cytotoxicity of NK cells, while increasing cytotoxicity of NKT cells. EGFR-TKIs block the activation of downstream signaling in the EGFR pathway. ICIs alter the immune response and reverse the immunosuppressive microenvironment in EGFR-mutated ADC. TIME, tumor immune microenvironment environment; EGFR, epidermal growth factor receptor; TKIs, tyrosine kinase inhibitors; ADC, adenocarcinoma; MDSC, myeloid-derived suppressor cell; NK, natural killer; NKT, natural killer T; IDO, indoleamine 2, 3-dioxygenase; DC, dendritic cell; MHC, major histocompatibility complex; IFN-γ, interferon-gamma; TNF-α, tumor necrosis factor alpha; PFN, perforin; GzmB, Granzyme B; NSCLC, non-small cell lung cancer.Conventional technologies used for characterizing tumor immune phenotypes are limited by modest analysis parameters and large quantities of specimens required. Advances in various technologies, including spatial single-cell transcriptomic analysis, imaging mass cytometry, and quantitative multiplex imaging.+CD33+PD-L1+ TAMs in tumors from hyper-progressor patients in comparison with patients not experiencing hyper-progression strongly suggests the spatial distribution of TAMs plays an important role in determining the outcome of patients with NSCLC treated with ICIs [The spatial single-cell transcriptomic analysis is a technique used to study gene expression patterns within individual cells in a tissue sample while also preserving their spatial context. This technique combines in situ hybridization, imaging mass cytometry, and spatially resolved RNA sequencing, which allows researchers to map the location of cells within a tissue and analyze their gene expression profiles at the same time. Spatial transcriptomic profiling of lung tumors and brain metastases suggested that the tumor microenvironment of the brain is distinguished by diminished antigen presentation and impaired B/T cell activity, heightened neutrophils and M2-type macrophages, immature microglia, reactive astrocytes . Additioith ICIs .2 core of formalin-fixed paraffin-embedded tumor tissue) [Imaging mass cytometry combines laser ablation to vaporize tissue samples and ionize the resulting particles, which are then analyzed using mass spectrometry to determine the presence and quantity of specific proteins or molecules in individual cells. Like spatial single-cell transcriptomic analysis, this method allows the analysis of up to 40 protein markers on a tissue section while preserving the tissue architecture and enables the in-situ characterization of tumor immune contexture. A recent study using imaging mass cytometry coupled with deep learning allows the prediction of recurrence with high accuracy with routine surgical resection (a 5-µm section of a single 1-mm tissue) .+ and CD8+ T cells are scored on a scale from 0 to 4 based on the area occupied by the immune cells in the invasive margin and center of the tumor. However, with the advancement of multiplex immunofluorescence imaging, precise characterization of the immune landscape of the tumor or even quantitative analysis of the TIME is now feasible [Quantitative multiplex immunofluorescence image analysis is a powerful tool used to characterize tumor immune phenotypes by simultaneously detecting multiple cell markers in a single tissue sample. This technique allows for the identification of different cell populations and, most importantly, provides detailed information about the spatial distribution and abundance of different immune cell populations within the tumor microenvironment. The previously established Immunoscore analyzed only the expression of CD3 and CD8 by immunohistochemistry in the invasive margin and center of the tumor . The denfeasible . A studyfeasible .Both targeted therapy and immunotherapy have shown promising therapeutic efficacy in defined groups of advanced-stage or locally advanced lung cancer patients. However, a combination of targeted therapy and immunotherapy did not show synergistic antitumor effects both in vitro and in clinical trials. Further characterization of TIME in oncogene-driven NSCLC suggested a generally weak immunogenic TIME. Innovative preclinical studies explored different entry points in TIME  to foste