In the past decade, bacteria‐based cancer immunotherapy has attracted much attention in the academic circle due to its unique mechanism and abundant applications in triggering the host anti‐tumor immunity. One advantage of bacteria lies in their capability in targeting tumors and preferentially colonizing the core area of the tumor. Because bacteria are abundant in pathogen‐associated molecular patterns that can effectively activate the immune cells even in the tumor immunosuppressive microenvironment, they are capable of enhancing the specific immune recognition and elimination of tumor cells. More attractively, during the rapid development of synthetic biology, using gene technology to enable bacteria to be an efficient producer of immunotherapeutic agents has led to many creative immunotherapy paradigms. The combination of bacteria and nanomaterials also displays infinite imagination in the multifunctional endowment for cancer immunotherapy. The current progress report summarizes the recent advances in bacteria‐based cancer immunotherapy with specific foci on the applications of naive bacteria‐, engineered bacteria‐, and bacterial components‐based cancer immunotherapy, and at the same time discusses future directions in this field of research based on the present developments. Bacteria‐based cancer immunotherapy has shown significant potential in the realm of tumor therapy, mainly because of bacterial specific targeting and preferential colonization of tumors, the abundant natural antigen library of bacteria, and the great engineerability. These high‐quality properties of bacteria have been and are being researched extensively, allowing the discovering of best immunotherapies. TIME undergoes dynamic changes along with the rapid growth of tumor, acting as a hotbed for tumor's rapid proliferation and invasion and a helper for tumor immune escape. In fact, it is the tumor's hypoxic environment that provides a solid foundation for this hotbed. The oxygen concentration in the tumor tissue is only 7–28 mm Hg (1–4%), while it is 40–60 mm Hg (5–8%) for the normal tissue. Such a hypoxic environment not only poses a huge challenge to tumor radiotherapy and chemotherapy but also directly facilitates the formation of tumor immunosuppressive microenvironment. Therefore, the extreme hypoxia of the tumor and the immunosuppressive microenvironment are two major features of malignant tumors, which can be regarded as the key for developing tumor treatments.The ongoing exploration of the tumor microenvironment (TME) in recent years has gradually unveiled a fact that the tumor immunosuppressive microenvironment is another big hurdle that needs to be overcome in cancer treatment besides those already known adverse factors that severely limit the therapeutic effect like the tumor interstitial pressure, hypoxia, nutritional deprivation, inadequate blood perfusion, immature lymphatic system, etc.1 The  Although the delivery efficiency of nanoparticles can be improved in this way, its total enrichment in tumor remains at a relatively low level. Compared to inactive nanoparticles, bacteria have a variety of complex physiological functions , making them good therapeutic agents or vectors that have potential applications in cancer therapy. As early as the late nineteenth century, William Coley injected heat‐killed streptococcal organisms in conjunction of Serratia marcescens (“Coley's Toxins”) into tumor patients to treat their sarcomas and other malignancies, and observed tumor ablation. Since then, William Coley was regarded as a trailblazer in bacteria‐based cancer immunotherapy. Afterward, researchers found that certain obligate anaerobes and facultative anaerobes could preferentially colonize the hypoxic/necrotic zone inside the solid tumor after systemic administration. Although the mechanism behind bacteria's efficiency in targeting tumors has not yet found a consensus on the answer, it is agreed that bacteria's tumor‐targeting ability is inseparable from the hypoxia property of tumor based on the analysis of the distribution of different strains in the body. Clarifications on this issue have been provided by many researchers. Neil S. Forbes reported that more than 10 000‐fold of Salmonella typhimurium (S. typhimurium) could be accumulated in tumors compared with other organs after one week of systemic injection. Several mechanisms were proposed to explain the excellent specificity: a) chaotic vasculature trapped bacteria in the tumor; b) attraction to specific TME; c) preferential replication in TME. Although a pool of studies has claimed to see a great tumor‐colonization of bacteria after intravenous administration, the initial phase of bacteria escape from vessels to tumor site still remains a mystery. Sara Leschner observed a dramatic increase in tumor necrosis factor‐α (TNF‐α) in blood after the intravenous injection of Salmonella enterica serovar typhimurium in an ectopic transplantable tumor model, inducing the disruption of vessels in tumors. As a result, bacteria were flushed into the tumor with the influx of blood and were trapped inside ever since. More convincingly, authors neutralized TNF‐α in the serum of tumor‐bearing mice and observed retardation of blood influx together with a delay of bacterial tumor‐colonization. Therefore, TNF‐α and the induced hemorrhage inside the tumor play a major part at the early stage of bacterial invasion of the tumor.As an alternative idea, some researchers focused on the tumor hypoxia‐tropic nanoparticles to realize a preferential engagement in hypoxic tumor tissues.5 AlthFigure  For example, the components of bacteria including peptidoglycan, lipopolysaccharide (LPS), lipoteichoic acid (LTA), flagellum, DNA, RNA, etc., could be identified by pattern recognition receptors (PRRs) on dendritic cells (DCs), macrophages, and neutrophils, which subsequently triggered the corresponding immune response. Among them, LPS, found in the outer membrane of Gram‐negative bacteria, is a typical strong immunogenic microbial‐associated molecular patterns (MAMPs), which is mainly combined with Toll‐like receptors (TLRs) on the immune cell membranes. The increase of LPS would cause the overexpression of interleukin‐6 (IL‐6), activation of nuclear factor kappa B (NF‐κB) signaling and TLRs pathway, and phosphorylation of signal transducer and activator of transcription 3 (STAT3). These pathways could promote the maturation of DCs and the proliferation of immune cells, thereby enhancing anti‐tumor immunity. Similarly, bacterial flagellum would activate TLR5‐mediated innate immune response. For Gram‐positive bacteria without LPS and flagellum, such as Bifidobacterium, it could activate macrophages, natural killer (NK) cells, DCs and B lymphocytes by peptidoglycan, extracellular polysaccharides and DNA. Then, these effector cells could be promoted to produce immune active substances, such as IL‐1, IL‐6, IL‐12, TNF‐α, IFN‐γ, and nitric oxide (NO), and finally facilitate the anti‐tumor effect. Therefore, it seems that bacteria and cancer immunotherapy will inevitably be combined . The hypoxic microenvironment in tumor and the nutrients released from the dead tumor cells facilitate the massive proliferation of anaerobic bacteria. Moreover, the immunosuppressive TME prevents the immune system from clearing the bacteria in the tumor at the early stage. Then, these multiplied bacteria will activate the host's immune system, causing a large amount of immune cells to infiltrate within tumors.In the late 19th century, the anti‐tumor effect of Coley's toxins made people realize for the first time the huge potential of bacteria in tumor treatment.19 Alt+) oxidase and elevated intracellular Ca2+ amount. Both of the two mechanisms can form reactive oxygen species (ROS), which is a highly cytotoxic free radical. ROS can initiate the immunogenic death of tumor cells, then activate CD8+ T cells to eliminate residual tumor cells, and finally prevent metastases. Meanwhile, Listeria can infect bone marrow‐derived suppressor cells (MDSCs) at the tumor site, causing a significant quantity decrease in MDSC and subsequently transforming the immunosuppressive microenvironment to an immunostimulatory status. Moreover, IL‐12 can be produced upon the transformation of the remaining infected MDSCs into immunostimulatory phenotype, which can improve the T cells’ and NK cells’ response.It is worth noting that the underlying mechanisms in the immune system for bacteria and host to interact with each other depend on bacterial strains and tumor models. For example, Listeria infection can directly kill tumor cells through the activation of nicotinamide adenine dinucleotide phosphate  from polymorphonuclear neutrophils. Studies have reported that matrix metalloproteinase 8 (MMP‐8) played a vital role in this release process. Moreover, the early expansion of Clostridium in solid tumors could lead to intratumoral infiltration of granulocytes and macrophages, and the increased secretion of chemokines would further trigger the adaptive immunity and recruit immune cells  to the tumor site.Salmonella is another type of bacteria that has been widely studied in the field of cancer therapy; its interaction with the immune system has been clarified as well. After infecting tumor cells, Salmonella directly destroys tumor cells either by initiating autophagy pathway or inducing apoptosis. Furthermore, pathogen‐associated molecular patterns (PAMPs) such as flagellin and LPS released from Salmonella can be captured and recognized by antigen‐presenting cells (APCs). Flagellin can promote the maturation of APCs and up‐regulate pro‐inflammatory cytokines  and co‐stimulatory molecules  by binding and activating TLR5 and Nod‐like receptors (NLRs) on APCs. These inflammatory mediators can subsequently stimulate the secretion of interferon‐gamma (IFN‐γ) and the T helper type 1 (Th1) cell‐mediated immune response. In this process, macrophages and DCs secrete pro‐inflammatory IL‐1β and TNF‐α under the stimulation of LPS‐induced TLR4 signaling and tumor cell debris. Flagellin has been found to be capable of activating NK cells to produce IFN‐γ through a TLR‐independent pathway involving IL‐18 and Myeloid differentiation factor 88 (MyD88), and the produced IFN‐γ will lower the frequency of CD4+ CD25+ regulatory T cells (Tregs) in the TME. Studies have shown that Salmonella infection can lead to the up‐regulation of connexin 43 (Cx43) in human and murine melanoma cells  was tested on 24 patients with metastatic melanoma and one patient with metastatic renal carcinoma. Patients received 106 to 109 cfu m−² of VNP20009 through systematic administration and then were evaluated for dose‐related toxicities, selective replication within tumors, and anti‐tumor effects. The maximum‐tolerated dose was determined as 3.0  ×  108 cfu m−². And some tumor colonization was observed at the highest tolerated dose. Although dose‐related increase of several proinflammatory cytokines  was observed, no patients experienced objective tumor regression. The clinical results suggest that additional methods were required to enhance the efficacy and reduce the toxicity. With the advancement of synthetic biology, bacteria have been engineered for attenuation to ensure safety. After decades of rapid development, this transformative approach not only enables researchers to focus on the attenuation but also adopts a variety of engineering methods to develop intelligent bacteria for a wide range of applications.Although native anaerobic and facultative anaerobic bacteria are excellent at colonization in tumor and killing tumor cells through immune cells’ infiltration, the single functionality and ineffective therapeutic action seriously limit their development and application. Besides, some toxic and side effects have been observed after systemic injection of native bacteria.39 Wit2.22.2.1Immunotherapeutic agents include monoclonal antibodies, chemokines, and cytokines, etc. Due to their large molecular weight and good water solubility, it is particularly difficult even for nano carriers to deal with the targeted delivery of these proteins. The rapid development of synthetic biology enables researchers to creatively develop engineered bacteria that produce and express multiple immunotherapeutics. These bacteria can efficiently and safely express immunotherapeutic agents in the local tumor, thereby triggering a strong anti‐tumor immune response. This kind of immune stimulation usually results from the joint action of two aspects. One is that the expressed immunotherapeutic agent specifically targets a certain part of the immune system, while the other is that the bacteria supplement the immune stimulation through PRRs, thereby generating maximum anti‐tumor immunity. Another benefit of this method is to reduce the off‐target of the therapeutic agent, hence reducing the toxic side effects.Escherichia coli (E. coli) strain to release nanobody antagonist of CD47 (CD47nb), which is an anti‐phagocytic receptor highly expressed in some human cancer types. The E. coli strain used in this work comprised a synchronized lysis circuit (SLC) in which lyse bacteria released its therapeutic products once reaching a critical density  via transforming the high‐copy plasmid carrying programmed death‐ligand 1 (PD‐L1) nanobody and cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) nanobody sequences to locally and controllably express PD‐L1 and CTLA‐4 antagonists  by activating MyD88 after dimerization. In the last step, it activates NF‐κB and regulates cells from cellular transcription. Therefore, flagellin can also be used as an immune adjuvant, mainly based on the ability to activate DCs and non‐intrinsic immune cells . The interaction of flagellin and TLR5 results in the expression of varied proinflammatory cytokines, NO, H2O2, chemokines, and host defense proteins. It is revealed that engineered Salmonella in the TME exerts a strong anti‐tumor efficacy through the activation of the TLR4 and TLR5 pathways. The engineered flagellin‐secreting Salmonella has a greater inhibitory effect on tumors than free Salmonella, indicating that TLR4 and TLR5 pathways have a synergistic effect on tumor growth inhibition. Moreover flagellin is also closely related to tumor immunosuppressive microenvironment. The inhibitory signals produced from tumor cells in the TME effectively prevent the surveillance of the immune system. MDSCs can recruit regulatory T cells and inhibit the activation of NK cells and effector T cells. Intratumoral injection of flagellin can effectively reduce the number of MDSCs in the tumor. In TME, tumor‐associated M2‐like macrophages promote tumor growth by inhibiting DCs maturation, down‐regulating the expression of major histocompatibility complex (MHC) molecules, and recruiting regulatory T cells. Interestingly, it has been proved that flagellin can reverse the tumor immunosuppressive microenvironment into immune‐responsive TIME by the polarization of M2‐like macrophages to M1‐type macrophages. Jin Hai Zheng et al. reported that an engineered attenuated S. typhimurium strain could successfully suppress the tumor growth and metastasis in murine colon and melanoma models through a secretion of Vibrio vulnificus flagellin B (FlaB) .50 As  STING can be activated by binding to cyclic dinucleotides (CDNs) and initiating a potent pro‐inflammatory cytokines production, including type I IFNs. Numerous studies have shown that STING agonists exhibit potent anti‐tumor activity, opening the way for developing STING‐based cancer immunotherapy. However, although the STING agonists can activate the STING pathway to trigger an anti‐tumor immune response in cancer treatment, it still has some shortcomings, such as off‐target effects and toxicity. Therefore, the use of synthetic biology technology to engineer bacteria to produce and release STING agonists after colonization on tumor sites has become a preferred solution. Also, as a good source of immune stimulation through PRRs, bacteria can activate complementary immune pathways to provide better curative effects. In a recent report, Daniel S. Leventhal and co‐workers engineered a strain of E. coli Nissle (referred to as SYNB1891) to express STING‐agonist cyclic diAMP (CDA) . These results confirmed the great potential of SYNB1891 in triggering an anti‐tumor immune response. Moreover, two distinct murine tumor models (B16F10 and A20) and two different genetic backgrounds mice (C57BL/6 and BALB/c) were carried out and verified that SYNB1891 could trigger effective anti‐tumor immunity and immunological memory.Like TLRs, as an important part of the innate immune system, the stimulator of interferon genes (STING) pathway has received elevated attention since its discovery.58 STIS. typhimurium to express LIGHT, which is a TNF‐family cytokine that has been demonstrated to generate promising anti‐tumor activity. In several mouse tumor models, authors observed satisfactory tumor suppression with no significant toxicity. And the TME analysis demonstrated that the efficacy was associated with the massive inflammatory cell infiltration into tumors including the elevated frequency of DCs. Based on this novel strategy, authors further armed attenuated S. typhimurium with the ability to synthesize IL‐18, which is an IFN‐γ‐inducing factor with multiple functions related to the proliferation and activation of immune cells. In preclinical mouse cancer models, the endowment of IL‐18‐producing ability greatly increased S. typhimurium’s anti‐tumor activity. The mechanism was associated with increased frequency of tumor infiltrating CD3+/CD4+ T cells and DX5+ NK cells. Meanwhile, several immunostimulatory and inflammatory cytokines were brought into play in tumor suppression, including GM‐CSF, IL‐1β, and TNFα. These preclinical findings show that cytokine‐producing bacteria could elicit strong immune response with no side effects, suggesting a novel strategy for bacteria‐based cancer immunotherapy.Cytokines are widely studied due to their intrinsic functions to promote the activation and proliferation of immune cells to achieve anti‐tumor effects. However, systemic administration of cytokines causes serious side effects, limiting its clinical application. Among different routes to deliver cytokines to the tumor site, engineered tumor‐targeting bacteria to express cytokines have multiple advantages such as high‐level specificity but less side effects. Markus Loeffler and co‐workers engineered attenuated activity.62 In 2.2.2 Nanomaterials can achieve a variety of functions in cancer therapy due to different synthesis methods and encapsulated drugs. Therefore, compared with the genetic modification of bacteria to secrete therapeutic agents, it seems more direct to integrate nanomaterials with other diverse functions in the outer membrane of bacteria. The biggest benefit brought by this is the easy preparation and the low cost; however, there still exist some shortcomings.In addition to the use of genetic technology to engineer bacteria, the integration of bacteria and nanomaterials for multifunctional synergistic therapy is considered to be a promising treatment.40c Na To prevent the early detachment of nanomaterials in the body fluid environment and the loss of the expected versatility on the collaborative treatment platform, a strong connection between the nanomaterials and bacteria is particularly important. Therefore, researchers mostly use stable amide bonds to connect the two bodies to prevent functional materials from falling off in advance. Furthermore, the proper function should be selected to integrate with the ontology of bacteria to produce a synergy rather than antagonism. The response to external stimuli allows the integrator to act on its orders and achieve a controlled treatment. Light irradiation, magnetic field, and ultrasonic are the commonly used externally applied stimuli. Among them, light irradiation is the most‐widely used and mature stimulus in tumor treatment.First of all, bacteria can target tumors partly because the outer membrane of bacteria is rich in outer membrane proteins, some of which are used to sense the surrounding environment and perform chemotactic movement of certain specific molecular signals. When integrating nanomaterials into the outer membrane of bacteria, the chemotactic function of the bacteria will be inevitably affected if a dense and continuous shell is formed to completely enclose the bacteria, thereby weakening the targeting ability of bacteria. Another consideration lies in the strength of the connection between nanomaterials and bacterial membranes. The engineered bacteria need to face a complex physiological environment, such as high‐speed blood flow, high salinity, a variety of metal ions, and abundant fibrin.65 To  Applying engineering methods to integrate phototherapy nanoparticles with bacteria can effectively achieve precise tumor targeting and powerful primary tumor ablation. Moreover, the sequent anti‐tumor immunity could achieve multi‐functional combination therapy to maximize the therapeutic effect of tumors. However, although the tumor's hypoxia microenvironment provides an excellent condition for bacterial intrinsic tumor targeting ability, it greatly limits the therapeutic effect of PDT. The photosynthetic bacteria were previously applied for cardiovascular disease because of its highly efficient oxygen generation. The light‐controllable growth and photosynthesis of bacteria could relieve tumor hypoxia for enhanced immunogenic PDT. Lanlan Liu et al. engineered photosynthetic bacteria  by integrating photosensitizer‐encapsulated nanoparticles on the bacterial surface via amide bonds  that involved the up‐regulated expression of calreticulin (CRT) on the cell surface. The CRT exposure on the membrane of tumor cells can promote the activation and maturation of DCs. Interestingly, not only CRT but also ROS produced by PDT could accelerate the maturation of DCs. In addition to producing oxygen locally, Syne as immunogenic bacteria can trigger moderate immune activation through upregulating the percentage of MHC II and increasing the production of IL‐12p40. Meanwhile, improved tumor hypoxia promotes the infiltration of effector T cells to the tumor since T cells tend to avoid hypoxic areas and head to neighboring normoxic regions in TME. Photosynthesis‐boosted immunogenic PDT shows a great ability to ameliorate the TIME by elevating the percentage of immune effector cells such as CD8+ T cells, CD4+ T cells, and NK cells, and decreasing the number of immunosuppressive cells such as Tregs, MDSCs, and M2‐like tumor‐associated macrophages (TAM) which are important for suppressing anti‐tumor immunity.Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), has gained increasing attention due to its precision and non‐invasion.66 App Due to the good biocompatibility and biodegradability, photosensitizer polydopamine (pDA) is a promising candidate for PTT. To achieve the combination of hypoxic tumor targeting of bacteria and photothermal behavior of pDA, researchers have integrated pDA into the surface of anaerobe Salmonella strain VNP20009 to make it a thicker artificial outer membrane  to block programmed cell death protein 1 (PD‐1). AUNP‐12 has been proven to perform well on the antagonism of PD‐1 by subcutaneous administration and showed a safe toxicological profile. Although peptide antagonists have better tumor penetration properties, short‐term retention in tumor tissues limits their application. Therefore, the authors applied subcutaneous injection of AUNP‐12‐loaded phospholipid‐based phase separation gel (PPSG) near the tumor to achieve long‐term sustained release of the antagonist. With gel sustained‐release system, the immune checkpoint inhibitor could stay in the tumor tissue for up to 42 days, enhancing the anti‐tumor effect of triple combination therapy. Compared with the VNP20009 treatment alone, the amount of TNF‐α at the tumor in the triple therapy group was 7.9× that of the bacterial treatment group. Moreover, every element in the triple therapy contributed to the immune activation of the tumor. With the release of tumor‐associated antigens, the maturation of DCs, the production of proinflammatory cytokines, the recruitment and functioning of T cells, and the promotion of NK cells, the triple therapy including pDA engineered bacteria and immune checkpoint blockade achieved full‐stage promotion of anti‐tumor immunity and exhibited effective suppression of advanced melanoma.Some researchers also tried to engineer the bacteria with PTT photosensitizer. PTT, as a non‐invasive cancer treatment, can cause strong tumor ablation and simultaneously induce heat shock proteins (HSP) produced by tumor cells, which is a family of proteins with moderate immunostimulant function.74 DueIn addition to genetic engineering, the hybridization of bacteria and nanomaterials is another strategy to functionalize bacteria. The appropriate modification of nanomaterials on the surface of live bacteria would not affect the intrinsic tumor‐targeting ability of bacteria. The additional functions  achieved by the introduction of nanomaterials have a synergistic effect on bacterial therapy and enhance the therapeutic effect. Usually, such combination therapy can simultaneously enhance several aspects of anti‐tumor immune activation, leading to the reversal of the tumor immunosuppressive microenvironment and maximizing anti‐tumor immunity. Although the combination of phototherapy with bacteria‐based cancer immunotherapy has made great achievements, the intrinsic drawbacks of phototherapy including the insufficient light penetration depth and the potential phototoxicity to the skin need to be considered in future studies.2.32.3.1 OMVs mainly consist of cellular components of the bacterial periplasm and the outer membrane, including membrane lipids, proteins, LPS, peptidoglycans (PG), and virulence factors. Some intracellular components may also be wrapped, such as some intracellular proteins, DNA, RNA, ions, metabolites, signaling molecules, and enzymes. The mechanism of OMVs production remains unclear. There are currently three hypotheses widely recognized: 1) the accumulation of phospholipid in the outer leaflet of the bacterial outer membrane and regulation by VacJ/Yrb ATP‐binding cassette transport system for most Gram‐negative bacteria result in the OMVs formation; 2) the cross‐linking of the outer membrane and the PG layer. The cell wall of Gram‐negative bacteria is composed of outer membrane and PG, while lipoprotein crosslinks them with covalent bonds to maintain the integrity of the envelope structure. When decomposition or abnormal synthesis of the PG layer happens, a small part of the outer membrane would dissociate from the PG layer and protrude out of the cell to form OMVs; 3) the accumulation of the misfolded proteins and abnormal envelope components in the periplasm would promote the formation of OMVs. These unexpected substances may reduce the integrity of the envelope, thereby separating the PG layer and the outer membrane layer. As a result, the buds are extruded outward to form OMVs. OMVs containing a large number of MAMPs  can interact with host PRRs to induce innate immune responses. Owing to the abundant natural adjuvant components in OMVs, systemic injection of OMVs or siRNA‐packaged OMVs from a mutant E. coli strain has been reported to up‐regulate the production ofTNF‐α, IL‐6, IFN‐γ, and anti‐tumor cytokines CXCL10, which are closely related to the anti‐tumor immunity. Among a variety of MAMPs, the LPS stands out like a star. It is composed of lipid A, core polysaccharide and O‐antigen. O‐antigen is a kind of polysaccharide exposed on the surface of the bacterial outer membrane, which is an epitope of bacterial cell antigens. Lipid A is regarded as the active biological center of LPS; it can cause strong inflammation and regulate immune response such as stimulating immune cells to produce antibodies against multiple antigens. However, current studies have shown that excess LPS would cause immunosuppressive reactions, and blocking lipid A can remove endotoxin activity and reduce immunosuppression. OMVs as immune adjuvants need to appropriately reduce the toxicity of LPS, that is, knocking out the activity of lipid A through genetic modification to achieve the purpose of attenuation. Oh Youn Kim et al. knocked out the msbB gene encoding endotoxin in E. coli to avoid the immunosuppression caused by lipid A, and found that the use of G‐bacteria OMVs in mouse colon cancer model could inhibit tumor growth. The outer membrane protein of vesicles stimulated NK cells and T cells to produce INF‐γ, which in turn has growth inhibitory effect on tumor. In some cases, naturally‐produced OMVs were used directly as carriers for drug delivery. For example, immunomodulatory molecules, photosensitizers, and chemo drugs can be loaded in OMVs and then transported to tumor, achieving a combination of immunotherapy and chemotherapy/phototherapy. Qi Chen et al. coated DSPE‐PEG‐RGD‐ hybridized bacterial OMVs on drug‐loaded polymer micelles to produce an innovative nanomedicine for effective cancer immunotherapy and metastasis prevention , further realizing remarkable inhibition of pulmonary metastasis. Therefore, in order to enrich the functions of OMVs and achieve more effective tumor suppression, two schemes have been generally adopted. The first solution is to hybridize some functionalized lipid polymers or other biological membranes to OMVs to achieve new functions or to improve their inherent performance. The second method is to take advantage of the high loading capacity of OMVs to realize the anti‐tumor immune activation of other treatment methods and OMVs per se by delivering therapeutic agents  to the tumor site. Synergistic therapy could further improve the anti‐tumor efficacy. In a recent report, Qi Chen et al. designed and constructed a hybrid eukaryotic‐prokaryotic nanoplatform by fusing melanoma cytomembrane vesicles with attenuated Salmonella OMVs  have plenty of specific antigens on the surface that can be recognized by APCs and benefit its maturation, which in turn can trigger the anti‐tumor immune response. Furthermore, the homologous targeting ability of CCMVs allows more vesicles to reach the tumor site, thus triggering a stronger immune response. In order to inherit the excellent characteristics of CCMVs and OMVs, the authors designed a fusion eukaryotic‐prokaryotic vesicle (EPV) which exhibited great potential to suppress the tumor growth based on DCs immune activation and the CTL‐derived tumor‐specific immunity. Due to the limited tumor‐eradication capacity of monoimmunotherapy, the combination with other therapeutics would be more effective. The authors made the best use of EPV's great loadability to enclose a photothermal agent inside the EPV, achieving thermal ablation of tumor mass. The immunogenic death of tumor cells caused by thermal ablation can provide supplementary antigens to the immune system and further improve the anti‐tumor immune response.Outer‐membrane vesicles (OMVs) are nano‐sized spherical vesicles produced by Gram‐negative bacteria.78 OMVFigure  This approach would trigger the following four sequential steps: to produce tumor‐specific antigens by radiation therapy; to capture cancer neoantigens and enhance the uptake of antigens in DCs by BNP; to initiate the anti‐tumor T‐cell response; and finally to activate both innate and adaptive immunity. Bacterial membranes are abundant in PAMPs such as TLR agonists that initiate innate immunity and DCs activation. The maleimide group modification on bacterial membranes endows a better capturability for ICD‐induced tumor‐specific antigens, thus strengthening the activation of anti‐tumor immunity. In murine melanoma or neuroblastoma, the results showed that combining BNP with radiation therapy could lead to a remarkable activation of DCs and effector T cells, significant tumor regression, and anti‐tumor immune memory, indicating that BNP has a great ability to facilitate in situ immune identification of a radiated tumor with a potent immune activation.The superiority of membrane vehicles is not just reflected in their strong carrying capacity; the membrane can see more functions available by simply carrying out some basic modifications. For example, Ravi B. Patel and colleagues developed a bacterial membrane‐coated nanoparticle (BNP) consisting of an immunostimulator PC7A/CpG polyplex core coated with maleimide groups‐modified bacterial membrane. BNP was used as an in situ vaccine in combination with radiation therapy for cancer treatment Figure 13.[91 Owing to the strong chemotactic and phagocytic function, neutrophils can quickly cross the blood vessel composed of endothelial cells (ECs) when pathogens cause infection, subsequently follow the concentration gradient of chemokines to arrive the non‐vascular area and finally migrate to the inflamed area, thus clearing the pathogens through phagocytosis and intracellular killing. One characteristic of tumors is inflammation, which makes neutrophils actively chemotax to tumors. Therefore, neutrophils possess natural advantages as a carrier for nanoparticles or drugs. Due to the short life cycle of neutrophils, they are extremely difficult to be engineered in vitro and injected back into the blood. An alternative solution can be the use of neutrophils and pathogens with high affinity loaded nanoparticles in the blood circulation. As a bacterial product, OMVs completely inherit the protein on the surface of the bacterial membrane. Naturally, it is also a pathogen that can be recognized by neutrophils. Min Li and colleagues designed pathogen mimicking nano‐pathogenoids (NPNs) containing PAMPs by cloaking NPs with OMVs, which can be recognized by PRRs on neutrophils  and macrophage‐inflammatory protein‐2 (MIP‐2), NPNs could be carried to migrate to tumor. Then, under the stimulation of inflammation in tumor, neutrophils would release the loaded nanoparticles that were internalized by the surrounding tumor cells to kill the tumor cells. Generally speaking, OMVs are more like an efficient carrier for cancer therapeutic agents. Indeed, it showed a wide range of application scenarios in loading anticancer agents. On the other hand, more novel applications may be developed by exploiting the potential of OMVs to interact with the human immune system due to the rich and completely inherited bacterial outer membrane information on the surface of OMVs. It is promising that these applications may exert the full potential of OMVs and spawn more effective tumor immunotherapeutics.Apart from reaching the tumor through enhanced permeability and retention (EPR) effect, OMVs can also hitchhike the immune cells to transport them to tumor by immune response. For instance, neutrophils are important natural immune cells in mammals. On the frontline of host defender, neutrophils sense and ingest pathogens by recognizing PAMPs, followed by initiating the innate immune response to prevent the invasion of pathogenic microorganisms.92 Owi2.3.2 The anti‐tumor bacterial toxins can be divided into two categories: conjugated on the surface antigen of tumor cells or conjugated on the ligand. Due to the high expression of specific antigens on the surface of tumor cells, bacterial toxins that specifically target these antigens, such as Diphtheria toxin (DT) and Clostridium perfringens enterotoxin (CPE), and Pseudomonas exotoxin (PE) can be used for targeting and killing cancer cells. Among them, DT has been widely used in tumor treatment both in mice and humans because of its relatively desiable anti‐tumor effect. This may benefit from its strong cytotoxicity or the simultaneously induced anti‐tumor immunity. Silvio Buzzi and colleagues employed a non‐toxic cross‐reacting material 197 (CRM197) to treat a group of cancer patients. CRM197 is a nontoxic mutant of DT that possesses similar immunological properties as DT. Similar to DT, CRM197 targets heparin‐binding epidermal growth factor (HB‐EGF), which is commonly overexpressed in cancer cells. The authors found that subcutaneous injection of CRM197 caused inflammatory‐immunological reactions, triggering biological anti‐tumor response. Through the experimental results, the authors speculated that neutrophils and TNF‐α were involved in the anti‐tumor process. Thus, bacterial toxins not only show a great lethal effect on cancer cells but also initiate anti‐tumor immunity. The fusion proteins composed of bacterial toxins and targeting antibody fragments are also called immunotoxins. The fused targeting antibody fragment can target cancer cells, leading to a more potent lethal effect of bacterial toxin fragment on target cells. Immunotoxins that contain bacterial toxins which achieve potent cytotoxicity by blocking protein translation have been proved to be effective in treating some hematological malignancies. In a previous study, Ontak, a fusion protein composed of DT and anti‐IL‐2, was studied for treating chronic lymphocytic leukaemia (CLL) based on the overexpression of high‐affinity IL‐2 receptors on CLL cells, and satisfactory results were achieved. Similar to chemotherapy, repeated administration is necessary for immunotoxins treatment to maintain the optimal cell lethal concentration. However, the retreatment is limited to its immunogenetics, which is also known as the formation of anti‐drug antibodies (ADA). After treatment with immunotoxin, many patients will have a rapid immune response and the formation of ADA, which will neutralize the efficacy of immunotoxins and prohibit multiple administrations. To solve this problem, some researchers have tried to combine immunotoxin with chemo drugs or modify bacterial toxins to evade recognition of the immune system. These methods have achieved effective immunogenetic reduction. A more direct and well‐accepted method is to delete or mutate T cell epitopes by rational design of recombinant proteins to achieve immunogenetic reduction. Ronit Mazor et al. reported a novel immunotoxin consisting of disulfide stabilized Fv of anti‐Tac antibody and PE38 with nine point mutations both in domains II and III. Moreover, they demostrated that domain II is essential to CD25‐mediated cell killing, which is different from CD22‐mediated internalization. Compared with LMB‐2 (anti‐Tac(Fv)‐PE38), the newly constructed immunotoxin LMB‐142 shows both a strong cytotoxic activity in vitro and a five‐time lower nonspecific toxicity in mice.Bacterial toxins are a class of highly toxic proteins that are produced and released by bacteria with certain functions. Bacterial toxins have been proven to be a tool for cancer treatment due to high toxicity.95 The As the vanguard of the immunosuppressive microenvironment, Tregs promote the growth of tumors. This has prompted researchers to develop many immunotherapies for Tregs, including their function suppression and consumption. One method of depleting Tregs is to reposition bacterial toxins to take advantage of their efficient cytotoxicity to kill Tregs directly. This approach involves replacing the natural binding domain of bacterial toxins with known ligands of Treg receptors, transferring the killing mechanism of toxins to the cells rich in Treg receptors, thereby eliminating them instead. Benefits include the alleviation of immunosuppression of TME and the minimization of the toxicity associated with bacterial toxins to non‐target cells.In addition to using immunotoxins to treat T‐cell malignancies, Tregs depleting fusion protein toxins create a paradigm shift in cancer immunotherapy for other solid tumors as well. Tregs are an important population of T cells, which are regarded as the brake of the effector T cell‐mediated immune response. Besides, Tregs play important roles in immune tolerance, prevention of autoimmune diseases, and suppression of anti‐tumor immunity.103 As Studies have shown that the abundant CD25 on Tregs can consume IL‐2 in the local environment, while the lack of cytokines will cause the apoptosis of activated effector T cells. Therefore, CD25 can be selected as an ideal targeting site. Laurene S. Cheung and co‐workers developed a new generation of IL‐2 receptor‐targeted diphtheria fusion toxin, which has a good anti‐tumor effect related to the reduction of Tregs. In fact, this new‐generation fusion toxin has also seen a good synergy effect together with anti‐PD‐1 to treat melanoma. The risk of vascular leakage and production issues  limit the clinical application of denileukin diftitox (Ontak), which is a fusion protein composed of the bacterial toxin DT and anti‐IL‐2. The authors reported a production method that employed Corynebacterium diphtheriae to directly replicate biologically‐active and fully‐folded s‐DAB‐IL‐2 as a monomer into the culture medium. Furthermore, the authors prepared a more advanced fusion protein s‐DAB‐IL‐2(V6A) with a single amino acid mutation (V6A). Compared with s‐DAB‐IL‐2, V6A reduced vascular leak in vitro by 50× and lethality in mice by 3.7×. In mouse melanoma model, significant suppression in tumor growth was observed for both s‐DAB‐IL‐2(V6A) monotherapy and the combination therapy with anti‐PD‐1. The authors analyzed and confirmed that the excellent therapeutic effect was related to the depletion of Tregs and the enhancement of effector T cells.The high expression of Foxp3 in Tregs leads to a high‐level CD25 expression on the surface of Tregs, forming a heterotrimeric high‐affinity IL‐2 receptors.105 StAlthough bacterial toxins have been widely studied on imposing the great toxicity on tumor cells for directly killing, it is also an effective method for fusion with Tregs targeted proteins for Tregs depletion and anti‐tumor response enhancement. More hopefully, the successful combination with immune checkpoint blockade makes it possible for bacterial toxins to be applied in cancer immunotherapy.2.3.3 They can survive in oxygen‐rich tissues for a long time without germination. When encountering a suitable environment, such as the hypoxic/necrotic area of the tumor core, spores will germinate and multiply. There is no severe hypoxic environment in normal human tissues, which means that spores will not show toxicity to normal human organs. Researchers have tried to inject Clostridium histolyticum spore suspension into the tumor and observed effective suppression of transplanted mouse sarcomas without obvious systemic toxicity. Not only limited to intratumoral injection, some researchers observed the death of mice due to tetanus within 48 h after intravascular injection of Clostridium spores in tumor‐bearing mice. The healthy mice with the same treatment were asymptomatic for 40 days. This confirms that spores exhibit tumor‐specific germination even with vascular administration. Clostridium novyi (C. novyi) has been widely investigated because of its extreme sensitivity to oxygen and its excellent mobility owing to the numerous peritrichous flagella. These two characteristics lead to tumor enrichment of C. novyi even with only a small amount of spore germination. Considering the toxicity, the major systemic toxin (α‐toxin) gene of C. novyi was removed, and a new attenuated C. novyi‐NT was created which has a better prospect for application because of its lower systemic toxicity. Nishant Agrawal et al. observed that systemic injection of C.novyi‐NT spores in fully immune tumor‐bearing mice could achieve tumor regression and have long‐term effects  and tissue inhibitor of metalloproteinases 1 (TIMP‐1) to attract tumoral infiltration of various immune cells. At the very beginning, it was mainly a neutrophil response, but led to the participation of monocytes subsequently. The inflammatory response can inhibit the spread of bacterial infections and provide a second layer of control besides the first layer provided by an anaerobic environment. Inflammation may also directly cause tumor cells’ destruction through the production of ROS, proteases and other enzymes. Moreover, inflamation induces an effective cellular immune response, which continues to destroy the remained tumor cells that have not been killed by the bacteria. The authors observed that 30% of tumor‐bearing mice were cured, which is a quite gratifying result. In a later report, injection of C. novyi‐NT spores into the naturally occurring tumors on dogs induced a strong immune response. The authors observed that intratumoral administration of C. novyi‐NT spores resulted in enhanced phagocytosis and increased NK cell‐like function. Intravenous injection of C. novyi‐NT spores will lead to LPS‐triggered TNF‐α production, LTA‐triggered IL‐10 production, and also increasing of NK cell‐like function. This indicates that the administration of C. novyi‐NT spores can induce long‐term changes in immune cell function. In another report, John T. Heaps and colleagues injected engineered Clostridial spores into blood vessels to effectively suppress and cure human colon carcinoma in the mouse xenograft model. The engineered spores can germinate and be activated after reaching the hypoxic necrosis area of the tumor core, and then release the prodrug‐converting enzyme (PCE) that can convert non‐toxic prodrug molecule into a strong cytotoxic form at the tumor site, a process further causing tumor cells into death.As a dormant form of bacteria, spores are highly resistant.108 Th3 Since both bacteria and their components display inherent stimulation to the host immune system, thereby producing a positive anti‐tumor immune response, the bacteria‐based tumor immunotherapy has developed rapidly. From bacteria attenuation, transformation engineering, and individual component extraction and application to synergistic therapy, researchers have tried a variety of solutions to obtain the optimal efficacy. The latest research also shows that bacteria play a vital role in both the occurrence and development of tumors. Although the triangle interactions between bacteria, tumors, and the immune system are not yet clear, it is hopeful that with the deepening of research, researchers can make the best use of the great adaptability of bacteria to regulate the interaction of the other two to achieve better efficacy.Further clarifications on the interaction between tumor and the human immune system has unveiled that immunotherapy is one of the most promising approaches to cure cancer.113 Si Due to the specific colonization of bacteria in the tumor, the chance of immunotherapeutics reaching the target is greatly improved, thus reducing the repeated administration of free drugs and reducing the possible side effects. However, bacteria are complex therapeutic agents with vitality rather than precise formulations. The targeting effect of bacteria is variable, and there may be differences in efficacy between patients with different tumors during treatment. Besides, the level of immunotherapeutics expressed by bacteria may vary. Too low expression levels may result in poor efficacy, while excessive expression levels may cause autoimmune diseases. The long‐term stable production of bacteria in the host is very important. Moreover, some uncontrollable mutations may bring toxicity to patients in the process of bacteria proliferation. Fortunately, with the rapid development of synthetic biology, solutions to these potential problems may be available and worth of exploration. Researchers can precisely control the production and release behavior of immunotherapeutics by controlling gene copy number, promoter strength, bacterial metabolic rate, and initial bacteria injection dose. For patients with different tumors and at different stages, a personalized plan is preferred by adjusting the expression intensity of the therapeutics to yield the optimized treatment effect.Current engineered bacteria exhibit new functions that enable them to locally produce and release immune checkpoint blockers or other immunotherapeutics in tumors.61 Due Therefore, the immune cells infiltrated in the tumor may have spatially distributed heterogeneity, resulting in an unsatisfactory anti‐tumor effect. Some other tumor treatment methods (such as radiotherapy) have a better therapeutic effect on areas where the outer layer of the tumor has good oxygen perfusion. The combination of these therapies focusing on different areas may produce better treatment results.Although the administration of bacteria into the body can cause a variety of immune cell responses to produce anti‐tumor efficacy, most of the bacteria are colonized in the core necrosis area of the tumor with less distribution in the outermost area of the tumor.115 ThLactobacillus and Bifidobacterium etc.) to pathogenic bacteria. Meanwhile, most probiotics are less virulent than pathogenic bacteria, even compared with attenuated strains, which completely eliminates the worry of potential toxicity on patients.Some extracted components of bacteria are used as immunostimulators. Although they can effectively elicit anti‐tumor immune activation, their single action site in the entire anti‐tumor immune response link extremely limits the maximum efficacy. Despite that the increased dose or rounds of injection can better suppress tumor growth, some new problems come out. One of the problems is that the extraction of certain components of bacteria with high purity is a very time‐consuming task and may limit large‐scale applications. Another problem is that patients may not accept multiple injections as easily as oral administration. Therefore, replacement of oral dosage forms will help patients better accept multiple administrations. As a promising treatment approach, oral administration has attracted increasingly more attention in recent years not only because of the most natural and easiest administration, but also its self‐assistance that eliminates the requirement for nursing. However, it still remains a challenge as the complex physiological conditions  in gastrointestinal tract may digest or deactivate drugs or bacteria before they reach the action site. In order to avoid these problems, the therapeutics is usually wrapped in some protective materials and made into capsules to improve the utilization rate and also achieve sustained release. As another alternative and creative strategy, some researchers took full advantage of bioengineering technology to retrofit orally‐taken bacteria to serve as “living factory” to produce “products” in the gut, which may play beneficial roles in microbiome‐induced anti‐tumor immunity. Different from tumor‐targeting bacteria, orally‐taken bacteria prefer probiotics (such as bacteria.116 MeThe complexity of bacteria as a living body determines the difficulties and risks of transforming them into weapons to fight against tumor. However, complexity is precisely the most fascinating part of bacteria, which enables scientist to fine‐tune the various functions of different strains to realize anti‐tumor activities unachievable via other therapies. This complexity is also reflected in the interactions between bacteria and the human immune system. On top of that, with the in‐depth understanding of their relationship, bacteria‐based cancer immunotherapy will provide a steady stream of power to fight against cancer.The authors declare no conflict of interest.


HDC) mutation is a rare genetic cause with high penetrance in patients with TS. HDC‐knockout (KO) mice have similar behavioral and neurochemical abnormalities as patients with TS. Therefore, HDC‐KO mice are considered a valuable TS pathophysiological model as it reveals the underlying pathological mechanisms that cannot be obtained from patients with TS, thus advancing the development of treatment strategies for TS and other tic disorders. This review summarizes some of the recent research hotspots and progress in HDC‐KO mice, aiming to deepen our understanding of brain mechanisms relevant to TS. Furthermore, we encapsulate the possible brain nerve cell changes in HDC‐KO mice and their potential roles in TS to provide multiple directions for the future research on tics.Tourette syndrome (TS) is caused by complex genetic and environmental factors and is characterized by tics. Histidine decarboxylase ( Tourette syndrome (TS) is caused by complex genetic and environmental factors and is characterized by tics. Histidine decarboxylase (HDC) mutation is a rare genetic cause with high penetrance in patients with TS. HDC‐knockout (KO) mice have similar behavioral and neurochemical abnormalities as patients with TS. Therefore, HDC‐KO mice are considered a valuable TS pathophysiological model as it reveals the underlying pathological mechanisms that cannot be obtained from patients with TS, thus advancing the development of treatment strategies for TS and other tic disorders. This review summarizes some of the recent research hotspots and progress in HDC‐KO mice, aiming to deepen our understanding of brain mechanisms relevant to TS. Furthermore, we also encapsulate the possible brain nerve cell changes in HDC‐KO mice and their potential roles in TS, to provide multiple directions for the future research on tics. We have investigated the role of the histidine decarboxylase gene (HDC) in TS susceptibility in the Chinese Han population  mice as a pathophysiological model of TS are discussed. Subsequently, recent research using animal models is summarized from two different aspects of HDC‐KO mice: the change of dopamine and its receptor and the change of histamine and its receptor. Finally, we express our opinion about the possible brain nerve cell changes in HDC‐KO mice and their potential roles in TS. These findings highlight the importance of HDC‐KO mice for TS research and provide a new perspective for future research. We believe that our study makes a significant contribution to the literature because we demonstrate the role of the HDC‐KO mouse model in enhancing the understanding of TS pathophysiology, which is an important area for future research with direct implications for the clinical management of selected phenotypes.The histidine decarboxylase gene  may function as inhibitory autoreceptors  regulates dopamine (DA) negatively  ones. Striatonigral neurons expressing the D1 dopamine receptor (D1R) and striatopallidal neurons expressing the D2 dopamine receptor (D2R) provide excitatory and inhibitory feedback to the cortex, respectively. The dynamic imbalance between the two pathways is considered core to the TS pathogenesis  pathway is activated in striatal dMSNs. MAPK is phosphorylated by rsk‐mediated s235/236 rpS6, and the number of phosphorylated rps6‐s235/236‐positive cells increase in the dorsal striatum of HDC‐KO mice  can be divided into D1 receptor‐expressing (dMSNs) and DHDC‐KO mice and identify new potential targets for the treatment of tic disorders , histamine H2 receptor (H2R), histamine H3 receptor (H3R), and histamine H4 receptor (H4R), located presynaptically (H3R and H4R) and postsynaptically  to perform a range of regulatory functions. The changes in histamine levels in the brain are closely related to the central nervous system  in the posterior hypothalamus, and histamine‐positive fibers are projected into the cortex, hippocampus, basal ganglia, thalamus, and almost all other areas of the central nervous system  showed a decrease in the H4R mRNA level  mice, a recent study analyzed the different effects of HHDC‐KO mice treated with RAMH, MAPK signal is activated in dMSNs. Phosphorylation of MSK‐T581 decreases in dMSNs, whereas that of MSK‐T581, the target of MAPK, is not affected in iMSNs. RAMH has no significant effect on rpS6 phosphorylation at s235/236 but decreases the phosphorylation of AKT‐T308 in iMSNs. Further, RAMH has no significant effect on the phosphorylation of AKT‐T308 in dMSNs  are key players in controlling excitatory inputs in the cortex and thalamus, thus maintaining the excitatory‐inhibitory balance in the striatum  of HDC‐KO mice. Igf‐1 has a neuroprotective and limiting effect on inflammatory responses and is an important mediator of homeostatic microglial function. Therefore, the decrease in igf‐1 expression in the microglia of HDC‐KO mice might lead to impaired neuroprotection and dysregulation of the response to proinflammatory stimuli, which contribute to enhanced susceptibility to neuroinflammation after an environmental challenge  has received increasing research attention. Some studies have showed that the imbalance in H3R in the basal ganglia leads to the correlational phenomenology in HDC−/− mice. The H3R in the striatum of HDC‐KO mice was upregulated and still had constitutive activity without the ligand  in a concentration‐dependent manner. To identify their targets, OPC was treated with H3R reverse agonist and medium antagonist. The results showed that the total number of OPC cells is not changed, indicating that both the H3R reverse agonist and medium antagonist had no effect on OPC proliferation. Although medium antagonists had no effect on OPC differentiation, H3R reverse agonists promoted OPC differentiation in a dose‐dependent manner. This difference suggests that H3R reverse agonist positively regulates OPC differentiation. Western blot analysis showed that H3R is moderately expressed in oligodendrocytes, which decreased on the first day of OPC differentiation, but was upregulated on the third and fifth days and throughout the differentiation process. H3R knockout resulted in the increased expression of myelin related glycoprotein (MAG) and myelin basic protein, markers of oligodendrocyte differentiation, whereas its overexpression resulted in their low expression and decreased number of mature oligodendrocytes. Therefore, increasing H3R expression in the absence of histamine is enough to inhibit the differentiation of oligodendrocytes. These findings suggest that the constitutive activity of H3R negatively regulates the differentiation of oligodendrocytes and cAMP‐response element binding protein (CREB), and that the H1.4.4Astrocytes play an important role in the physiological functions of the central nervous system. They provide nutritional support for migrating neurons and guidance for the formation and maintenance of neural pathways. Furthermore, astrocytes participate in the control of cerebral blood flow, energy metabolism, and ionic constant pressure, as well as in synaptic formation and maturation. Previous research suggests that astrocytic signals play an important role in complex rodent behaviors, such as memory and circadian time‐keeping  in astrocytes stimulated by the proinflammatory cytokine IL‐6 and has a superposition effect with the proinflammatory cytokine IL‐1β (Lipnik‐Stangelj, 2HDC variation plays an important role in genetic susceptibility to TS. Subsequently, HDC‐KO mice is a good animal model that contributes to substantial progress in the understanding of TS pathophysiology. The parallel TS‐related behavioral and neurochemical abnormalities in humans with HDC W317X mutation and HDC‐KO and heterozygous mice provide favorable evidence for the causal association between HDC deficiency and TS. The observed basal ganglia abnormalities in HDC‐deficient mice, which involve the histaminergic regulation of dopaminergic modulation and information processing, are important factors in TS pathogenesis. Furthermore, we also focused on the possible alterations in the information processing of nerve cells in the brain of HDC‐KO mice, which contribute to pathophysiological changes associated with TS. Future studies on histaminergic regulation and dopamine modulation of striatal circuits and their potential role in nerve cells will be important to further our understanding of brain mechanisms relevant to TS.In summary, TS is a developmental neuropsychiatric disorder associated with genetic factors in most cases. All authors claim that there are no conflicts of interest.SL and XZ designed the project conception. LX, CZ, MZ, FC, and CG performed the literature search and analysis. LX wrote the manuscript with contribution from SL and XZ. All authors read and approved the final manuscript.https://publons.com/publon/10.1002/brb3.2511The peer review history for this article is available at


Stangoe et al. describe the successful management of a rare cause of tracheostomy tube ‘ball‐valve’ obstruction . The autconsider partial airway obstruction” while continuing to assess the airway. Appropriate next steps when faced with an inability to ventilate a patient who has a tracheostomy in situ are cuff deflation; endoscopic evaluation if the clinical situation allows; removal of the tracheostomy tube; all followed by re‐assessment and increasingly invasive attempts to oxygenate by either airway.The NTSP took a transparent approach to guideline development, detailing the methodologies , testingWe commend Dr Stangoe and colleagues for their effective management of this highly challenging airway emergency. We encourage readers to always consider any emergency algorithm in conjunction with the supporting information, full manuscript and educational resources.


In the 2016/2017 influenza season, England was in its fourth season of the roll‐out of a live‐attenuated influenza vaccine (LAIV) targeted at healthy children aged two to less than 17 years. For the first time, all healthy children aged 2 to 8 years were offered LAIV at national level in 2016/2017. Since the commencement of the programme in 2013/2014, a series of geographically discrete pilot areas have been in place where quadrivalent LAIV was also offered to all school age children. In 2016/2017, these were children aged 8 to 11 years, other than those targeted by the national programme.We evaluated the overall and indirect impact of vaccinating primary school age children, on the population of England, by measuring vaccine uptake levels and comparing cumulative disease incidence through various influenza surveillance schemes, in targeted and non‐targeted age groups in pilot and non‐pilot areas in 2016/2017.Our findings indicate that cumulative primary care influenza‐like consultations, primary and secondary care swab positivity, influenza confirmed hospitalisations and emergency department attendances in pilot areas were overall lower than those observed in non‐pilot areas; however, significant differences were not always observed in both targeted and non‐targeted age groups. Excess mortality was higher in pilot areas compared with non‐pilot areas.These results are similar to earlier seasons of the programme indicating the importance and continuing support of vaccinating all primary school children with LAIV to reduce influenza related illness across the population, although further work is needed to understand the differences in excess mortality. This annual programme aimed to protect children against infection and indirectly protect those in other age groups, in particular those who are at higher risk of severe disease.Epidemiological and mathematical modelling studies have supported the concept that vaccinating children can play a key role in reducing the burden of influenza across all ages; however, there has only been limited evidence on the impact of such programmes at national level in other countries to date.The 2016/2017 influenza season was the fourth season of introducing the LAIV vaccine in the UK. England extended the childhood vaccine programme nationally to include all healthy children aged 2 to 4 years as well as to children of school years 1–3 (aged 5 to 8 years).The 2016/2017 season was dominated by the circulation of influenza A(H3N2), the impact of which predominately affects older persons. There were increased care home outbreaks, hospital admissions and excess mortality in this age group.This study aimed to evaluate the uptake, direct and indirect impact of the childhood LAIV programme in England in the 2016/2017 influenza season using established influenza surveillance schemes.2Most of the pilot areas which started vaccinating children of primary school age in the 2013/2014 season continued with the programme up to and including the 2016/2017 season.2.1In the 2016/2017 pilot programme, the children recommended to receive vaccination were defined as those born between 1 September 2005 and 31 August 2011 (aged 5 to 11 years) who resided in five geographically discrete pilot areas in England: Greater Manchester (Bury), Leicestershire and Lincolnshire , London (Havering), Essex (Southend‐On‐Sea and Thurrock) and Northumberland Tyne & Wear , as shown in Figure An established and standardised web‐based portal, Immform, was used to collate and report vaccine administration data by locally commissioned data providers, to Public Health England (PHE).The end of season vaccination uptake was calculated as the number of children in the target population who received at least one dose of influenza vaccine in the period from 1 September 2016 until 31 January 2017. Healthy children and those children identified to be at‐risk with no contraindication against the LAIV vaccine were offered the quadrivalent LAIV vaccine. Children identified to be at‐risk in whom LAIV was contraindicated, were offered the quadrivalent inactivated vaccine instead.2.2The 2016/2017 LAIV impact study ran from weeks 402 016 to 142 017, by which time influenza transmission in the community had returned to well below baseline levels.To denote the overall impact of the childhood LAIV programme, cumulative disease incidence in non‐pilot areas was compared against the cumulative disease incidence in pilot areas for a number of virological and clinical respiratory end points in primary and secondary care surveillance schemes, during the period of the study. In addition, the impact of the LAIV programme on excess all‐cause mortality was also studied.As the dominant circulating influenza A subtype during the study period 2016/2017 was influenza A(H3N2) which is known to affect the older age groups, the previously described 17+ age group was split into two further age groups.2.3Various established influenza surveillance schemes were used to measure the impact of the LAIV programme in the 2016/2017 season, as described below and summarised in Table 2.4Surveillance in primary care was carried out through quantifying weekly general practice (GP) consultations for influenza‐like illness (ILI) and acute bronchitis in children less than 5 years old through GP practices that are part of the Royal College of General Practitioners (RCGP) Research and Surveillance Centre (RSC) Weekly Returns Service sentinel GP network.Respiratory swabbing in primary care is undertaken through GP practices within the RCGP RSC network as well as an additional sentinel swabbing network, the Specialist Microbiology Network (SMN).2.5Surveillance in secondary care was undertaken through four surveillance schemes.The UK Severe Influenza Surveillance System (USISS) sentinel scheme comprised a network of 23 National Health Service (NHS) hospital trusts across England (20 in non‐pilot areas and three in pilot areas) in the 2016/2017 season. The USISS mandatory scheme comprised all intensive care (ICU) and high dependency units (HDU) in England (137 in non‐pilot areas and 11 in pilot areas) for the 2016/2017 season.The weekly number of laboratory confirmed influenza hospital admissions and ICU/HDU admissions, collated through the two USISS (sentinel and mandatory) schemes were used to calculate confirmed influenza hospital and ICU/HDU admission rates by the specified age groups and pilot and non‐pilot areas, using estimated hospital catchment population per 100 000 population as the denominator.The Emergency Department Syndromic Surveillance System (EDSSS), as previously described,Respiratory swabbing in secondary care is monitored through the Respiratory DataMart system (RDMS), as over 90% of samples from this scheme are collated from patients in secondary care settings.2.6Excess all‐age all‐cause mortality and all‐age respiratory deaths (defined as ICD‐10J codes) were estimated as the observed weekly number of deaths (corrected for delays in reporting) compared to the expected number of deaths based on historical trends to denote significant excess mortality in both pilot and non‐pilot areas. This was conducted using the European Monitoring of Excess Mortality for Public Health Action (EuroMOMO) standard algorithm2.7The methods used to evaluate and measure the impact of the LAIV vaccine on the various surveillance schemes were the same as those used in previous seasons.Surveillance schemes where the cumulative disease incidence rates were observed, including the RCGP RSC, USISS sentinel and mandatory schemes, the number of weekly disease incidences between weeks 402 016 to 142 017 were summed over the average weekly population at risk, per 100 000 population. Separate calculations were performed for overall and age group specific rates in non‐pilot and pilot areas, where exact Poisson confidence intervals (CI) were calculated.For the primary care GP swabbing and the RDMS, cumulative influenza swab positivity was calculated by summing the number of positive samples over the total number of samples tested between weeks 402 016 and 142 017, by age group and pilot area.For the syndromic surveillance ED scheme, cumulative proportions of respiratory ED attendances were calculated by summing the total number of respiratory coded ED attendances over the total number of ED attendances with a diagnosis between weeks 402 017 and 142 017, by age group and pilot area.To measure the impact of the LAIV programme, the non‐pilot areas were set as reference, and odds ratios and 95% CI were calculated by age‐group and surveillance scheme. For each scheme, adjusting for clustering at the reporting unit level , data were converted to binomial individual level and random effects logistic regression undertaken.For excess mortality monitoring, cumulative excess mortality rates were calculated by summing the difference between observed and expected weekly deaths over the time of the study by pilot area and using the resident population respective to non‐pilot or pilot areas.2.8Real‐time PCR methods were used to detect circulating influenza A, B and other respiratory viruses for influenza laboratory confirmation of samples from the various primary and secondary care schemes.33.1An estimated 172 175 primary school children aged 5 to 11 years of age in five pilot areas received at least one dose of influenza vaccine during the vaccination campaign period (1 September 2016 to 31 January 2017), with the estimated total target population being 285 735 primary school aged children. This resulted in an overall vaccine uptake of 60.3%.The uptake ranged from 44.5% to 73.4% by pilot site Figure . Uptake In 2 to 4 year olds, the influenza vaccine uptake delivered through GPs was 44.4% (569 GP practices) in primary school pilot areas compared to 37.6% (6867 GP practices) in non‐pilot areas.In children of school year groups 1–3, the influenza vaccine uptake was 62.0% (90 322/145 664 children) in primary school pilot areas compared to 54.9% (1 032 905/1 880 516 children) in non‐pilot areas.3.2From weeks 402 016 to 142 017, cumulative GP ILI consultation rates and swab positivity in primary and secondary (RDMS) care were lower in pilot areas compared with rates in non‐pilot areas across all age groups.Such differences were however less marked in secondary care schemes in particular ED respiratory attendances (EDSSS) and ICU/HDU flu confirmed rates (USISS) in the older age groups (17–64 and 65+ years) Figures  and 3.Cumulative all‐age all‐cause excess mortality by season was significantly higher in pilot areas both pre‐introduction (2011/2012 and 2012/2013) and post‐introduction (2013/2014 to 2016/2017) of the childhood vaccination programme, as previously observed. By way of contrast, cumulative all‐age respiratory related excess mortality was significantly higher in pilot areas in the seasons pre‐introduction of the programme; however, in the post‐introduction period of the programme, excess mortality was significantly lower in 2013/2014 and 2015/2016 , provided a mixed pattern. Cumulative ICU/HDU admission and hospitalisation rates for influenza were similar in primary school pilot compared to non‐pilot areas for the two seasons prior to the start of the childhood vaccination programme, whereas for the three seasons since, they have been consistently lower. The differences were less marked for GP ILI consultation rates, where pre‐introduction ILI rates were generally lower in pilot compared to non‐pilot areas, which may reflect underlying differences between the pilot and non‐pilot areas Figure .3.3For primary care schemes, significant reductions were observed in cumulative GP ILI consultation rates in adults in pilot areas compared to non‐pilot areas when adjusting for clustering, as a result of vaccinating primary school aged children Table . Non‐sigFor the secondary care schemes, non‐significant reductions were seen in all but one indicator (USISS mandatory—ICU admissions) in 5 to 10 year old children in pilot areas compared to non‐pilot areas Table . Non‐sig4This study evaluates the uptake and impact of the childhood LAIV influenza vaccine programme in its fourth season of its phased introduction in England. The programme continued to target children of primary school age in discrete geographical pilot areas in the 2016/2017 season, in addition to its national implementation. As in previous seasons, higher or similar levels of vaccine uptake amongst children of primary school age continue to be reported in the targeted pilot population. We also demonstrate that populations with vaccinated children of primary school age are associated with reductions, although not always significant, in the incidence of influenza for a range of surveillance schemes in comparisons to populations without vaccinated children of primary school age. We also observed higher all‐cause and respiratory excess deaths in pilot compared to non‐pilot areas—both before and after introduction of LAIV.Also, as in previous seasons, good vaccine uptake levels were observed with the targeted population achieving 7.1% higher uptake than the non‐targeted population. Amongst the targeted population, almost 55% of pilot areas achieved a vaccine uptake in excess of 60%. This reinforces the choice of a school based administration to achieve high childhood vaccination uptake.Findings from historical trends for GP ILI consultations, hospitalisations and ICU/admissions suggest significant differences post‐programme introduction in GP ILI consultations in all age groups; however, these differences should be interpreted with caution due to pre‐existing differences between the pilot and non‐pilot areas prior to the programme. Nonetheless, as seen previously, reductions remain evident in targeted populations and there continues to be evidence that an indirect effect on the <5 year olds is present. This is also highlighted in a recent study by Benjamin‐Chung et al.,Although there was no evidence of significant adjusted vaccine effectiveness for the >65 year old vaccine programme for the 2016/2017 season,Our finding that excess all‐cause mortality was significantly higher in pilot areas is one that has been noted in previous seasons and can be likely explained by the pre‐existing higher all‐cause excess seen in the pilot areas prior to the introduction of the LAIV programme, which may reflect differences in the underlying health and socio‐demographic profile of the populations in pilot and non‐pilot areas.A number of strengths have been highlighted from this study. First, this study uses data collected from a wide range of well‐established surveillance systems which cover healthcare service utilisation across the disease spectrum of influenza. Second, the methods used in this study to assess the uptake and impact of the childhood programme have been developed over the past three seasons which has enabled us to confidently assess its findings. Third, vaccine uptake is measured at population level which allows for direct comparisons to previous published studies.There are some potential limitations to this study, including potential differential reporting. New GP practices and hospitals are recruited each season in pilot and non‐pilot areas which may contribute to higher differential reporting and in turn introduce less sensitive case detection amongst these new entities compared to long‐standing participating practices. There was only one emergency department site in the pilot areas compared to 25 in the non‐pilots areas, although this is taken into account in the model. As the national vaccination programme continues to roll out to children in other school years and the gap between pilot and non‐pilot areas decreases, the ability to carry out such comparisons is diminishing, which is evident when looking at the historical comparisons.In conclusion, this study is the fourth of its kind in a series of evaluations of the LAIV programme to find continuing positive outcomes in support of the roll out of the national childhood LAIV programme. Other approaches to estimate the population impact of the programme may need to be considered as the narrowing differences between uptake between pilot and non‐pilot areas means that the methods used in this study may not be suitable in future seasons. Lack of impact of the programme on excess all‐cause mortality also warrants further investigation.The authors declare no conflict of interest.Fiona Warburton: Conceptualization; data curation; formal analysis; methodology. Nick Andrews: Conceptualization; formal analysis; methodology. nicola boddington: Data curation. Hongxin Zhao: Data curation. Joanna Ellis: Data curation. Elise Tessier: Data curation. Matthew Donati: Data curation. Alex Elliot: Data curation. Helen Hughes: Data curation. Rachel Byford: Data curation. Gillian Smith: Data curation. Manasa Tripathy: Data curation. Simon De Lusignan: Data curation. Maria Zambon: Data curation. Richard Pebody: Conceptualization; methodology; supervision.https://publons.com/publon/10.1111/irv.12898.The peer review history for this article is available at


N/dS, also known as ω). Nowadays, modern sequencing technologies and the large amount of already available sequence data allow the retrieval of thousands of orthologous genes across large numbers of species. Nonetheless, the tools available to explore selection regimes are not designed to automatically process all genes, and their practical usage is often restricted to the single‐copy ones which are found across all species considered . This approach limits the scale of the analysis to a fraction of single‐copy genes, which can be as low as an order of magnitude in respect to those which are not consistently found in all species considered . Here, we present a workflow named BASE that—leveraging the CodeML framework—eases the inference and interpretation of gene selection regimes in the context of comparative genomics. Although a number of bioinformatics tools have already been developed to facilitate this kind of analyses, BASE is the first to be specifically designed to allow the integration of nonubiquitous genes in a straightforward and reproducible manner. The workflow—along with all relevant documentation—is available at github.com/for‐giobbe/BASE.Inferring the selective forces that orthologous genes underwent across different lineages can help us understand the evolutionary processes that have shaped their extant diversity and the phenotypes they underlie. The most widespread metric to estimate the selection regimes of coding genes—across sites and phylogenies—is the ratio of nonsynonymous to synonymous substitutions  while those which are not found across some of them  are often overlooked, due to the lack of automated approaches to include them. Yet, disregarding such a large portion of genes may potentially conceal important evolutionary processes. For this reason, we developed a novel workflow—named BASE—which allows the integration of nonubiquitous genes in a straightforward and reproducible manner. Quantifying its nature and strength is a key step to understand the diverse evolutionary histories of orthologous genes across different species and clades. Statistical models of molecular evolution have proven to be fundamental approaches to investigate such processes and can be divided into those based on comparing divergence and segregating polymorphism—such as the MK test and its extensions , which is calculated as the ratio of nonsynonymous substitution rates  to synonymous substitution rates . This metric is fundamental to investigate the extent to which selection modulates sequence evolution of the protein‐coding portions of genes. While dS are assumed to evolve neutrally, dN are expected to be exposed to selection, as they change the amino acid structure of proteins. Despite the fact that some of these assumption have been challenged  or not found in some of them . The latter case can be either due to biological or technical causes, but nonetheless a large number of single‐copy genes in comparative genomics datasets consist of nonubiquitous genes. As an exploratory example, we retrieved the proportion of ubiquitous and nonubiquitous genes from 18 published datasets, which have been generated for comparative genomic or phylogenomic purposes. While we tried to consider datasets varying in total gene number and taxonomic level, this overview has to be considered far from comprehensive: The outcome of orthology inferences is rarely included in publications, and thus, we largely relied on authors’ personal communications. Despite the partial nature of this analysis, it shows how comparative genomics datasets consistently include a large portion of nonubiquitous genes , evolutionary model parameters are inferred across alignment sites and tree branches for the different genes, while the subsequent step (“extract”) allows to retrieve the different metrics associated with specific branches or clades in the species tree. CodeML provides in large part the statistical and computational framework to perform these analyses and is at the core of the workflow, whose general description is reported in Figure 2.1p values are automatically adjusted using a false‐discovery‐rate (FDR) correction; then, the codeml output relative to the best‐fit model is selected for each gene, and LRT outcome is summarized in a table. When leveraging models which assume specific clades and/or branch rates, the user can specify labels in a custom input file and BASE will automatically use them to annotate the trees using phangorn R package  a folder containing protein‐coding genes alignments in fasta format; (b) two CodeML control files describing two nested models—where one is a specific case of the other; all the parameters in control files can be customized, supporting branch, site, and branch‐site models; moreover, other optional files can be required depending on analysis specifications: (c) a species tree in newick format, which can be multifurcating but has to include all the species present across the different gene alignments; and (d) a labeling scheme when the user wants to analyze models that assume specific clades and/or branch rates. The workflow initially checks the alignments for the presence of stop codons using Transeq of the EMBOSS package  the output folder generated by the previous step or a folder containing CodeML outputs generated by means other than BASE; and (b) a list of all branches and/or clades of interest, defined by their associated species. In the first place, this step will annotate internal nodes of each tree to match the output of CodeML and will list all species associated with each branch of the phylogeny. Subsequently, the pipeline will create a table for each branch/clade specified by the user, containing the ds Figure : (A) ress Figure .2.3Even if the focal feature is the integration into selection analyses of single‐copy genes which are not found across all considered species, BASE also includes two other major technical implementations. The first one concerns analyses with a high number of parameters and which may encounter local likelihood optima: A good—yet often overlooked—practice is to run analyses multiple times with varying starting values in order to obtain the global optimum. BASE allows to seamlessly carry out a user‐specified number of replicate analyses, incorporating random omega starting values: The replicate which has the "best" likelihood value will be then used for further analyses. A second issue concerning phylogeny‐based selection analyses is the possibility of discordance between gene trees and species tree. This circumstance can underlie a wide range of technical and biological phenomena—such as sequence misalignment, nonorthology, and incomplete lineage sorting—which can ultimately bias evolutionary rate inference. In order to account for such possibility, when a fixed species tree is specified BASE will report its normalized Robinson–Foulds distances with each gene tree, calculated using ete3 ; Investigation (lead); Methodology (lead); Software (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Angelo Alberto Ruggeri: Conceptualization (supporting); Investigation (supporting); Methodology (supporting); Software (supporting); Validation (supporting); Writing‐original draft (supporting); Writing‐review & editing (supporting). Giovanni Piccinini: Conceptualization (supporting); Investigation (supporting); Methodology (supporting); Software (supporting); Validation (supporting); Writing‐original draft (supporting); Writing‐review & editing (supporting). Andrea Luchetti: Conceptualization (supporting); Methodology (supporting); Supervision (lead); Writing‐review & editing (supporting).https://github.com/for‐giobbe/BASE.This article has earned an Open Data Badge for making publicly available the data necessary to reproduce its results; BASE along with the relative tutorials are hosted on GitHub:


In the early 1900s, German chemist, Paul Ehrlich commenced developing drugs to treat infectious diseases and coined the term ‘chemotherapy’ defining it as the use of chemicals to treat disease. He developed the first alkylating agents, to treat cancer.a magic bullet with a single drug able to kill tumour cells,a magic bomb combining surgery, radiotherapy and chemotherapy to achieve higher levels of anti‐tumour efficacy,monoclonal antibodies to receptors over‐expressed on tumour cell surface to reduce toxic side effects and achieve tumour‐targeted therapy,synthetic nanoparticles and polymers to package drugs and deliver them to tumour cells to reduce toxic side effects and achieve higher anti‐tumour efficacy,molecularly targeted drugs that targeted specific oncogenic proteins to avoid killing normal cells,checkpoint inhibitor immunotherapy to reactivate the patient’s own immune system,Chimeric antigen receptors to give T cells the ability to target a tumour‐specific protein and T cell activating function in a single receptor.During the past 100 years, the major ‘cancer cure’ concepts evolved from;While some spectacular cures were observed with each of these approaches, most patients experienced tumour relapse and eventually succumbed to the disease. These advances provided an incremental advance in the treatment of cancer and almost all of them were associated with moderate to severe toxicity. Throughout this time, there was a major effort to discover antigens that were tumour‐specific with a hope that tumour‐targeted therapies could be developed with minimal toxicity to normal tissues. This effort has yet to bear fruit.et al., Most cancers are diagnosed when patients are at Stage III or IV. It is well known that tumour heterogeneity at this stage is extensive and most tumour cells have already elaborated multiple drug resistance and immune‐suppression mechanisms. Cell proliferation assays on tumour cells isolated from biopsies show near complete resistance to most available chemotherapeutic drugs  could alleviate the symptoms of asthma or chronic obstructive pulmonary disease, penicillin and other antibiotics could cure bacterial infections. The same principle has been applied to cancer therapy for example, when it was discovered that cytotoxic T cells were being suppressed by PD1 ligands secreted by tumour cells, it was thought that anti‐PD1 inhibitor monoclonal antibody would reactivate CD8+ T cells and would effect a cure. While anti‐PD1 inhibitors do help in some cancers, the disease is far too complex with thousands of molecular events in action simultaneously to allow tumours to escape the immune system and aggressively overcome host defences  PNU‐159682, a metabolite of the anthracycline nemorubicin, a highly potent DNA topoisomerase I inhibitor which is over 2000‐fold more toxic than conventional drug doxorubicin, (ii) Duocarmycin which is a DNA minor groove‐binding alkylating agent, (iii) Maytansine, a benzoansamacrolide, a highly potent microtubule‐targeted compound that induces mitotic arrest and kills tumour cells at sub‐nanomolar concentrations etc. Unfortunately, these drugs cannot be administered in patients as free chemotherapy since they are too toxic and would kill a person due to rapid and widespread killing of normal cells. These drugs are being developed as antibody‐drug conjugates but even then, they are seriously toxic in patients.If it were possible to safely administer these drugs into cancer patients so that the drug is specifically taken up inside cancer cells and not normal cells, then it should be possible to kill even the most drug‐resistant cancer cells. Bacterial minicells which are anucleate nanoparticles produced as a result of inactivating the genes that control normal bacterial cell division   they are retained in the normal blood circulation since the gaps between endothelial cells lining the blood vessels is less than 2 nm. However, it is known that cancer cells require access to blood vessels for growth and metastasis and hence they over‐express pro‐angiogenic factors which leads to the development of disorganized blood vessel networks that are fundamentally different from normal vasculature. Tumour vasculature is typified by aberrant structural dynamics and vessels that are immature and hyperpermeable Siemann, . The fenThe EDVs being 400 nm rapidly fall out of these fenestrations and enter into the tumour microenvironment and since they carry the bispecific antibody on the EDV surface, the anti‐EGFR component binds to EGFR on the tumour cell surface. This provokes macropinocytosis and the EDVs are taken into the early endosomes, followed by lysosomes and broken down in these organelles releasing the drug PNU‐159682. The drug enters into the tumour cell cytoplasm and the nucleus and intercalates with the chromosomal DNA resulting in tumour cell apoptosis. In the event that a tumour type does not express EGFR for example liver cancer, which expresses asialoglycoprotein, then the bispecific antibody can be changed to anti‐asialoglycoprotein while the anti‐O‐polysaccharide component remains constant. Similarly, HER‐2 positive breast cancers can be targeted via anti‐HER2/anti‐O‐polysaccharide bispecific antibody.et al., et al., This is the first time a super‐cytotoxic drug has been administered into human cancer patients with no toxicity. The double membrane structure of the EDVs prevented leakage of the drug in general circulation, the large size of the EDVs allow it to avoid the normal tissues that are surrounded by normal sealed blood vessels, the tumour‐associated leaky vasculature allows the EDV to enter specifically into the tumour microenvironment, the bispecific antibody targeting of the EDVs allow it to enter specifically into tumour cells and the lysosomal degradation machinery allowed the EDV to be broken down intracellularly and release the drug that could overcome drug resistance and for the first time, kill tumour cells that are highly drug resistant with no toxicity. All 170 cancer patients treated so far were end‐stage palliative care patients who had run out of all treatment options. Highly significant anti‐tumour efficacy has been observed in mesothelioma  such as LPS by professional phagocytes (APCs) being macrophages and dendritic cells (DCs) which are present in the lymph nodes, liver and spleen. Recognition of PAMPS results in the APCs releasing ‘alarm signals’ like ATP  release ‘find‐me’ signals such as low levels of nucleotides ATP and UTP, fractalkine, lysophosphatidyl choline, or sphingosine 1‐phosphate, which attract APCs to the sites of death within the tissue Gregory, . The apoet al., This cascade of events continues to escalate as more APCs are attracted to the new dying tumour cells, engulf them, go to the draining lymph nodes and activate more CD8+ cytotoxic T cells. This cascade then results in tumour antigen‐specific CD8+ effector memory T cells that provide long‐term immunity to the specific cancer (Sagnella The polar sites of septum formation in Gram −ve and Gram +ve bacteria are thought to be vestigial sites of cell division left over from bacterial evolution and it is possible that the minicell may have been the primordial cell during the path of bacterial evolution. The minicell may therefore be considered to be a very early ancestor of bacteria that existed millions to possibly billions of years ago.Today, it re‐emerges to offer a way forward in the intractable problem of cancer treatment. These minicells offer solutions required for treatment of late‐stage cancers with little toxicity, both by killing cancer cells and stimulating a robust anti‐tumour immune response, in effect, allowing the patient’s own immune cells to help in the heavy lifting of getting the patient back on his/her feet. No drug or immunotherapy comes near the minicell as a one‐stop‐shop for cancer treatment and for the next 15 years, the bacterial minicell is likely to radically change how cancer is treated.No funding information provided.The authors have no conflict of interest to declare.