Vaccines against Japanese encephalitis (JE) have been available for decades. Currently, most JE-endemic countries have vaccination programs for their at-risk populations. Even so, JE remains the leading recognized cause of viral encephalitis in Asia. In 2018, the U.S. Centers for Disease Control and Prevention and PATH co-convened a group of independent experts to review JE prevention and control successes, identify remaining scientific and operational issues that need to be addressed, discuss opportunities to further strengthen JE vaccination programs, and identify strategies and solutions to ensure sustainability of JE control during the next decade. This paper summarizes the key discussion points and recommendations to sustain and expand JE control. JEV is transmitted by mosquitoes and is sustained in an enzootic cycle with pigs and wading birds as amplifying hosts. Human cases typically occur in children and in rural areas where people live and work in proximity to elements in the transmission cycle, such as near rice fields or domestic pigs. There is a risk of JEV transmission in 24 countries in Asia and the Western Pacific, either nationally or in endemic areas2. While some regions have nearly eliminated JE through comprehensive immunization programs, the incidence in other areas remains high3.Despite the availability of safe and effective vaccines against Japanese encephalitis (JE) for several decades Table , JE viru1. JEV infection can rapidly progress to encephalitis, with an estimated case fatality rate of 20–30%5. Common symptoms include sudden onset of high fever, chills, headache, myalgias, mental confusion, and convulsions in pediatric patients6. Among survivors, about 30–50% have long-term neurologic sequelae with intellectual or physical disabilities8. Although there is no specific treatment for JE, supportive care improves outcome. JE burden estimates suffer from poor surveillance resulting from the difficulty of diagnosing JE without cerebrospinal fluid (CSF) samples and because cases generally occur in rural areas where surveillance and laboratory capacity might be limited.While most human JEV infections are asymptomatic, severe disease occurs in about 1 per 250 JEV infections9. However, poor recognition of the burden of disease, prioritization of other vaccines and public health interventions over JE vaccination, and the high cost and multiple-dose regimen of the older, inactivated mouse brain-derived vaccine limited JE vaccine introduction. Since 2003, PATH and multiple stakeholder organizations have worked to expand the use of WHO-prequalified JE vaccines primarily through the support of Gavi, the Vaccine Alliance10. Substantial impact on JE-associated deaths and disability has been shown in countries administering JE vaccine in childhood vaccination schedules. For example, large vaccination campaigns targeting variable age ranges were implemented in 31 districts in Nepal from 2006 through 2011. JE incidence after the campaigns was 78% lower than the pre-campaign incidence, resulting in over 3000 JE cases estimated to have been prevented through 201412.The World Health Organization (WHO) has long recommended JE vaccination programs in areas where JE is a public health problemIn 2018, the U.S. Centers for Disease Control and Prevention and PATH co-convened a group of independent experts to review JE prevention and control successes, identify remaining scientific and operational issues that need to be addressed, discuss opportunities to further strengthen JE vaccination programs, and identify strategies and solutions to ensure sustainability of JE control during the next decade. This paper summarizes the key discussion points and recommendations to sustain and expand JE control Support country efforts for surveillance and vaccine introduction, and quality assurance for laboratory testingMonitor JEV geographic spread as land use and climate change might affect mosquito distributionOther Country Programmatic SupportTroubleshoot vaccine supply issuesWhen possible, integrate surveillance and vaccination programs into other programs for efficiency and sustainabilityConduct community advocacy and communication for maximal vaccine acceptanceProvide guidance for the adverse event following immunization investigations of special interest for JEResearch PrioritiesMonitor duration of protection following a single dose of live JE vaccinesAssess vaccine safety in pregnant and immunocompromised persons vaccinated with live JE vaccinesStandardize and validate plaque reduction neutralization test reagents and proceduresEnsure reliable supply of JE diagnostic kitsFacilitate the development of JE treatmentsEstimate JE vaccine impact globally to highlight the success of JE vaccine programs and the need to sustain progress madeStudy safety and immunogenicity of JE vaccine co-administration with any new vaccines that might be co-administered with JE vaccine12.The first inactivated mouse brain-derived vaccines were developed in the 1930s. Vaccine manufacturers in several Asian countries developed these vaccines primarily for domestic use. Studies of early JE vaccines have shown that human illnesses can be eliminated with high vaccination coverage even though JEV continues to circulate in the enzootic cycle. For example, in Japan, universal vaccination with inactivated mouse brain-derived vaccine virtually eliminated JE in the early 1990s13. Recognizing the potential impact with greater use of CD-JEV, in 2003, PATH partnered with the Chengdu Institute of Biological Products (CDIBP) to export CD-JEV internationally. CDIBP and PATH developed a new manufacturing facility compliant with good manufacturing practices (GMP) and greater production capacity, which opened in 2012. As part of this partnership, CDIBP also made CD-JEV available to low- and lower middle-income countries at a low public sector price to facilitate countries’ abilities to fund JE vaccine introduction.In the 1980s, virologists in China developed SA 14-14-2 (CD-JEV), a live attenuated JE vaccine grown on primary hamster kidney cells. This vaccine was introduced province by province and markedly reduced JE incidence in China14. In a landmark vaccine efficacy study of mouse brain-derived JE vaccine conducted in Thailand, more than 65,000 children were vaccinated15. Due to the high cost of clinical trials, any subsequent efficacy trial would be expensive and likely infeasible due to the need to vaccinate and clinically follow so many children. However, defining an immune correlate of protection allowed manufacturers to develop new JE vaccines for commercial use without large and logistically challenging vaccine efficacy trials. Several Asian and European manufacturers developed inactivated Vero cell culture-based JE vaccines that had a superior safety profile and were easier to manufacture than mouse brain vaccine, but, like earlier inactivated vaccines, still required multiple doses and periodic boosting to maintain immunity. Another manufacturer developed a recombinant chimeric vaccine (JE-CV) by inserting the E and prM genes of SA 14-14-2 JEV into a yellow fever 17D vaccine backbone1. Thus, in a few decades, the JE vaccine portfolio expanded to multiple products using a variety of vaccine technologies.Defining an immune correlate of protection  in 2005 was a critical step in JE vaccine development16. Prequalification is needed for vaccines procured through UNICEF, procured using Gavi support, or intended for use in WHO and other United Nations programs. Biological E. Ltd’s JEEV, a Vero cell culture-based vaccine, was prequalified in 2013, but initially was indicated for adults only. Later in 2013, CD-JEV became the first JE vaccine prequalified for children and the first Chinese vaccine to be WHO prequalified, an accomplishment that may help additional Chinese-manufactured vaccines enter the global markets.WHO prequalification is a process to validate a product’s quality, safety, and efficacy, and to ensure a manufacturer is compliant with WHO and international quality standards3. Guidance and training materials were developed to support country decision-making and vaccine introduction as well as JE surveillance and measurement of vaccine impact19. With products available and funding mechanisms in place, accelerated JE control became one of eight immunization goals in the Regional Framework for Implementation of the Global Vaccine Action Plan in the Western Pacific, highlighting JE as a regional priority and defining operational targets for accelerated control of JE to be achieved by 203020. As of June 2020, 15 countries have national or subnational public health vaccination programs in JE-endemic areas. These include Australia (Outer Torres Strait Islands), Malaysia (Sarawak), Japan, Republic of Korea, Thailand, Cambodia, Lao PDR, Myanmar, Indonesia , Philippines (three high-incidence regions), China, India (about 40–50% of districts), Nepal, Sri Lanka, and Vietnam  antibody in the CSF21. Important progress in improving JE surveillance and defining disease burden came with the development of a range of JE diagnostic kits25. While three commercial JE IgM ELISA assays were previously available in JE-endemic countries, only the InBios JE DetectTM IgM antibody capture ELISA is still being manufactured. CSF can be tested with the InBios JE assay alone, but, because of cross reactivity, serum samples must also be tested with a dengue IgM assay and results interpreted according to a CDC-devised algorithm26.Access to diagnostics is important because JE cannot be clinically differentiated from other viral causes of acute central nervous system infection such as enterovirus 7127. While this finding suggests that serum IgM is unlikely to be related to vaccination administered at least six months prior, it does not negate the importance of CSF collection for proper JE diagnosis.Even when diagnostics are available, interpreting serum IgM results in areas with co-circulation of other flaviviruses can be complicated because antibodies against other flaviviruses often cross react with JEV. In addition, it is well-recognized that IgM following JEV infection can be detectable for months or years, so there is sometimes uncertainty when JE IgM is detected in serum whether it truly reflects JEV as the cause of a patient’s presentation or could be persisting IgM following a previous asymptomatic infection or mild febrile illness unrecognized as JE. A recent analysis of IgM following vaccination with CD-JEV in a clinical trial suggests serum IgM is common at 28 days following vaccination but rare at 6 months following vaccination 28.To improve and standardize JE diagnosis in JE-endemic countries, PATH worked with partners to establish WHO JE Laboratory Networks in WHO’s South-East Asia and Western Pacific Regions during 2006–2008. These networks have substantially increased the frequency and quality of JE laboratory testing and continue to provide support to countries for confirmatory testing, quality assurance, and other JE testing issues29. Based on the reported case fatality rate of 20–30% and long-term neurological sequelae among 30–50% of survivors, it was estimated that JE caused 13,600–20,400 fatalities each year and long-term neuropsychological sequelae in 14,300–27,200 people. In contrast to the estimate of case numbers, only 4402 JE cases were reported through the WHO-UNICEF Joint Reporting Form on Immunization in 2018, suggesting substantial under-reporting2. In a vaccine impact assessment conducted in Nepal using JE and acute encephalitis syndrome (AES) surveillance data, AES incidence was 59% lower than expected had no JE vaccination campaign occurred, with the absolute reduction in AES cases substantially greater than the reduction in confirmed JE cases, suggesting many AES cases were undiagnosed JE cases and again highlighting the unrecognized burden of JE11.Despite progress with JE diagnostics and laboratory testing, because of the difficulty diagnosing JE, reported JE case counts are generally considered to be underestimates of true JE incidence. A 2011 estimate of global JE burden, in which high-quality surveillance data from limited areas in Asia were extrapolated to other endemic regions, calculated that 67,900 JE cases occurred annually, of which 75% occurred in children <15 years of age31. Still, there continues to be substantial variability in approach, quality, and extent of surveillance in JE-endemic countries, based on local capacity and resources and the need for integration into pre-existing systems2.In addition to having better burden of disease estimates, JE surveillance is critical to inform vaccine introduction, monitor vaccination programs and identify alternative causes of encephalitis, evaluate vaccine effectiveness in different epidemiological settings, and monitor JEV expansion into new areas. WHO first published JE surveillance standards in 2008, and they were updated in 201832. While vaccination has contributed to a relative shift in cases to older age groups in some countries, other countries without vaccination programs have also shown a substantial proportion of cases in adults, raising the question of the need for adult vaccination33. JE vaccination programs in Nepal that targeted all age groups showed the rate of AES in individuals ≥15 years after vaccination was 77% lower than expected, suggesting an unrecognized burden of JE in this age group. While strong pediatric vaccination programs are the foundation of a JE vaccination program, some countries, based on the burden of disease and prioritization, may consider vaccinating adults as well.JE has been considered a disease of children in JE-endemic countries; immunity to JE by natural infection is widespread by adulthood and generally believed to be lifelong, at least in the context of natural boosting in endemic areas35. As a mosquito-borne disease, JE incidence is highly variable based on temperature, elevation, and rainfall. JEV is transmitted primarily by the mosquito Culex tritaeniorhynchus. There have been limited reports of JEV outside of Asia. In Italy, one pool of Culex pipiens was found to contain JEV as determined by RT-PCR of a small JEV RNA fragment, although no virus isolation was performed. In 2016, a patient with confirmed yellow fever infection in Angola was found to be co-infected with a JEV genotype III virus based on deep sequencing technology37. Further data are needed to confirm JEV transmission outside of Asia; however, with the global presence of Culex vectors and amplifying hosts, it is possible JEV could emerge outside of the traditional geographic boundaries due to population movement into nonendemic areas that results in changes in local farming practices and animal husbandry, expanded rice irrigation to meet population demand, and possibly global climate change. Although less likely, the transport of infected mosquito vectors or their infected eggs, the importation of viremic vertebrate hosts, or changes in bird migration patterns due to global climate change has the potential to expand the range of JEV39.Mosquito-borne transmission is of greatest concern, although transplacental transmission and transmission through blood transfusion have been documented40. JE vaccines fall into four categories: (1) live attenuated vaccine ; (2) live recombinant (chimeric) vaccine ; (3) inactivated Vero cell-derived vaccine; and (4) inactivated mouse brain-derived vaccine. WHO recommends both CD-JEV and JE-CV be administered on a single-dose schedule starting at age 8 months and 9 months, respectively, although the JE-CV vaccine package insert recommends a pediatric booster dose. WHO recommends administering Vero cell JE vaccines per the package insert, which is generally 2–3 doses with a booster dose at variable time intervals by product. CD-JEV holds the greatest global market share with more than 300 million doses to date administered outside of China. WHO recommends countries move away from mouse brain vaccines given the favorable profile of second-generation products, and no mouse brain vaccines are WHO prequalified3.There have been ~15 JE vaccine products commercialized in Asia, including several manufactured for use only in the country of manufacture44. Studies using CD-JEV vaccine generally showed seroprotection  in more than 90% of vaccinees 28 days after primary vaccination, although one study using WHO-prequalified CD-JEV found lower seroprotection rates48. Most case-control studies of CD-JEV vaccine demonstrated vaccine effectiveness at around 95% up to five years post-vaccination, although other studies have found lower point estimates56. Poor ascertainment of JE cases and accurate vaccination status is a challenge for these studies. A vaccine effectiveness study of the WHO-prequalified CD-JEV vaccine is currently underway. Safety of CD-JEV has been assessed both in clinical trials and through post-licensure passive reporting of adverse events and the vaccine has been found to have a robust safety profile57. Reports of encephalitis post-vaccination with CD-JEV are rare and a causal relationship with vaccination has not been established59. The WHO Global Advisory Committee on Vaccine Safety reviewed data on CD-JEV and determined it has a strong safety profile based on available data60.PATH chose CDIBP’s CD-JEV to target for global use because it could be administered as a single-dose vaccine and marketed at an affordable price in low-income countries in Asia. Data from China supported high vaccine effectiveness with a strong safety profile and PATH led additional clinical trials in other Asian countries in accordance with international standards, as did other manufacturers using CD-JEV as a comparison vaccine61. Several countries co-administer CD-JEV vaccine with the measles-containing vaccines, which has been shown convincingly not to cause immune interference62.Countries use different vaccines in country-supported JE immunization programs : 11 countries use CD-JEV , Laos, Myanmar, Nepal, Philippines, South Korea, Sri Lanka, Thailand), four use JE-CV vaccine , and three use Vero cell vaccines . Vietnam continues to use mouse brain vaccines. Brunei and North Korea have used JE-CV and CD-JEV, respectively, but do not have sustained programs, and Singapore has determined routine vaccination is not required based on surveillance data. Bangladesh, Bhutan, Pakistan, Papua New Guinea, Russia, and Timor Leste do not have JE immunization programs, but have recent or past evidence of JEV transmissionDespite great advances in JE vaccination and control over the last 15 years, there continue to be challenges that put these gains at risk and may impede further progress. These challenges fall into the categories of (1) maintaining high JE vaccine coverage, (2) maintaining a stable JE vaccine supply, and (3) maintaining surveillance and vaccination data.66. Careful diagnoses and surveillance are needed to monitor JE geographic spread as human migration, land use and climate change may affect mosquito distribution in the future. Furthermore, general vaccine hesitancy among parents and healthcare providers is on the rise, including in countries affected by JE69. In 2019, WHO named vaccine hesitancy as one of the top 10 threats to global health70. Confidence in vaccination programs is essential to maintain high JE vaccine coverage.Because of the enzootic cycle of JEV involving birds and mosquitoes, JEV elimination from the environment is impossible. Sustained high vaccination coverage must be maintained to prevent human cases. There is also evidence JEV is moving into new geographic areas, including evidence of occasional urban transmission; consequently target areas where vaccination is needed may shift and growCDIBP’s CD-JEV is the most widely used JE vaccine and several low- and middle-income countries rely on it. This reliance on a single product from one site operated by a lone manufacturer presents numerous threats to vaccine supply. Catastrophic events, such as infection among the pathogen-free colony of hamsters that are a source of cells for vaccine virus growth, natural disasters , or human disease outbreaks  could threaten the global vaccine supply. A vaccine supply continuity plan to help ensure CD-JEV global supply and availability is needed, using methods such as off-site bulk storage and identification of alternative sources of manufacturing materials.There are issues of supply and demand due to the need for manufacturers to grow virus for either live or inactivated vaccines. Thus, countries must assess their need for vaccines many months in advance of usage and are unable to acquire WHO-prequalified JE vaccines in a rapid ad hoc fashion, which makes ordering vaccines as a response to a JE outbreak unfeasible. None of the three WHO-prequalified vaccine producers have stockpiles of finished products with which to respond to requests during outbreaks. For example, the time required to produce and release a batch of CD-JEV can be up to nine months; as a result, countries must order at least nine months in advance. Another challenge with CD-JEV is the short shelf life of 24 months . This means CD-JEV must be used expeditiously once it arrives in countries.WHO currently recommends that CD-JEV be given as a single-dose vaccine without need for a booster dose. India and China, large consumers of CD-JEV, administer the vaccine as a two-dose primary series for programmatic reasons, which has an impact on vaccine supply. Because CDIBP has established a public sector price for specific JE-endemic countries, CD-JEV is currently the most viable vaccine product for many low- and lower middle-income countries. The cost and/or multiple-dosing regimens of other JE vaccine products have made them less desirable in many settings. Nonetheless, diversity is needed in the global supply of JE vaccine.Other important challenges exist with respect to disease surveillance and vaccine monitoring. Poor surveillance for JE disease has made the burden difficult to quantify in many countries, hindering decisions about vaccine introduction. When JE vaccination has been introduced, poor surveillance has limited availability of data to demonstrate impact and support advocacy efforts to maintain vaccine programs. Lack of immunization registries makes determination of vaccination status difficult over the longer term; monitoring vaccine failures and determining vaccination status in vaccine effectiveness studies are both critical for studying the duration of vaccine protection and the assessment of the eventual need of a booster dose. Challenges with the laboratory component of surveillance include suboptimal coordination between laboratory and epidemiology staff in some countries, and general challenges such as sample quality on arrival in laboratories, and difficulties with sample transport internationally for testing at reference laboratories. The risk of an insufficient supply of commercial JE IgM kits because of the limited market is a large threat to routine JE diagnostics. There remains no ready alternative if the only company currently producing a commercial IgM ELISA discontinues production. Critically, any reduction in support to countries from the regional JE laboratory networks could threaten the gains made in capacity for, and quality of, JE diagnosis.71. It is important to have robust investigations of adverse events following immunization (AEFI) without creating unsupported concerns. Investigation of post-vaccination encephalitis has not been systematic and requires good epidemiologic and laboratory resources to investigate properly. Enhanced AEFI surveillance following the initiation of a JE vaccine campaign is important. Working with stakeholders to maximize communication efforts while at the same time minimizing misinformation in the community is critical.Vaccine safety concerns, especially around severe adverse events such as encephalitis, can trigger vaccine hesitancy. This was seen in India when a cluster of suspected encephalitis cases were reported shortly after a JE vaccination campaign in 2014, though on review the cases were determined to be unrelated to vaccinationDecisions about vaccine introduction in resource-limited settings frequently require prioritization of multiple public health interventions for diseases that affect populations. Without good JE burden data, it has been difficult to contextualize the importance of JE vaccination relative to other high-priority vaccines competing for government resources, such as rotavirus and human papillomavirus vaccines. Continued advocacy is necessary for keeping JE on the list of priority vaccines for countries to consider.48. Seroprotection rates 28 days after vaccination for recipients of the new facility vaccine were 80.2–84.5% compared to 86.3% among recipients of the vaccine made in the older facility. Although this was not statistically different, seroprotection rates were noted to be higher  in earlier clinical studies of CD-JEV72. Because plaque reduction neutralization tests in these studies were performed at different times, comparing results is difficult. Still, while the findings of the lot-to-lot consistency trial supported the use of “newer facility” CD-JEV, continued monitoring is required on the vaccine’s duration of protection, which potentially could have implications for booster doses.Much of the earlier research on CD-JEV effectiveness and immunogenicity was done using vaccines produced in an older CDIBP facility that did not meet good manufacturing practice (GMP) standards. To license and prequalify CD-JEV made in the newer, GMP-compliant facility, a non-inferiority study compared three lots of “new facility” vaccine with one lot of vaccine from the older facility40. Data on vaccine effectiveness studies and vaccine failure monitoring are important to inform the need for booster doses for all WHO-prequalified JE vaccines. Studies are in progress  that will provide longer-term vaccine effectiveness data  and inform the duration of protection of a single dose of CD-JEV. Studies of antibody persistence alone are insufficient as undetectable neutralizing antibody might not indicate the absence of protective immunity. For example, data from a trial of JE-CV indicated that at two years post-primary vaccination some children who did not seroconvert with a primary JE-CV dose exhibited an anamnestic response following a receipt of a booster dose40. Similar findings were seen in a study of the other live JE vaccine, CD-JEV. Children who did not seroconvert initially or no longer had seroprotective antibody four years after immunization were given a booster dose of CD-JEV that resulted in a strong anamnestic response73. Anamnestic responses following revaccination with live JE vaccines suggest that despite undetectable antibodies, vaccines might be protected from clinical disease. Should booster doses of CD-JEV ultimately be recommended, this would have additional implications for vaccine supply and programmatic implementation. The anamnestic response has not been studied for inactivated JE vaccines.With reasonable immunogenicity of CD-JEV over a sustained period of time and an absence of reported vaccine failures with programmatic use, WHO does not recommend JE vaccine booster doses unless future evidence warrants it3.Furthermore, data on safety and immunogenicity following vaccination of special populations, such as pregnant women and immunocompromised persons, are missing for both live vaccines, CD-JEV and JE-CV. Research priorities across vaccine platforms are available in WHO’s position paper on JE vaccines75. Two genotype V JEV isolates have been identified, one from a human and one from a mosquito, and genotype V sequences from Culex mosquitoes also have been recently identified76. Additional evidence is required to determine if this genotype is emerging. If so, the question has arisen if currently available vaccines might be less effective, based on neutralizing antibody data and the 9% amino acid difference between genotype V and genotype III viruses. This situation should be monitored.All licensed vaccine viruses are genotype III JEV. While studies suggest these vaccines provide good protection against multiple genotypes, there are questions pertaining to future vaccine effectiveness if a new JEV genotype emerges77. There is a potential role of inflammation as a contributor to poor clinical outcomes from JEV infections, thus anti-inflammatory agents in combination with antiviral agents might be effective; this deserves further study77.Although the expert meeting focused on JE vaccination, there are other research gaps related to JE and treatment. While disability following encephalitis has long been recognized, the extent of sequelae following mild, non-neurologic JE illness is poorly understood. Such data could provide a better understanding of the full burden of JE morbidity and a more comprehensive understanding of the benefits of vaccination. Better information on the comparative severity of JE in adults and children would also be useful. Finally, as there is no specific treatment for JE currently available, investigation of possible JE treatments is another important avenue for further research. Limited clinical trials to date have not yielded any successful compounds, although promising targets existThe challenges defined above highlight that despite major achievements in JE control, efforts are required to continue and sustain progress. Experts at the meeting identified key recommendations in the following categories: (1) vaccine supply, (2) global coordination and policy optimization, (3) JE surveillance and diagnostics, (4) country programmatic support, and (5) research priorities.An adequate supply of JE vaccine is essential to JE control and is currently at risk as discussed above. A reliable JE vaccine supply requires vaccination schedules be optimized, countries improve lead-time for ordering JE vaccine, and vaccine manufacturers develop contingency plans for events such as natural disasters or other disruptions of vaccine supply. JE vaccine manufacturers with pre-qualified products currently in limited use globally are encouraged to make their products available and affordable to affected countries. At a minimum, country licensure of more than one product will allow product switching if the need arises.A coalition of key partners should be established that is responsible for exchange of information, best practices, and coordinated actions among JE control programs. This coalition would serve as a technical resource for countries and should maintain a web-based library of materials to support vaccine introduction and monitoring. As national and international partners have highlighted the utility of biannual WHO JE biregional  meetings to share experiences and lessons learned, these meetings should continue under the coordination of the coalition. Biregional meetings are important for information exchange and discussion of new or emerging issues. Advocacy for JE vaccine introduction is still needed to ensure that the momentum achieved through the efforts of PATH, WHO and its regional offices, and other partners, WHO prequalification of JE vaccines, and Gavi support, is not lost. As additional data become available (see Research Priorities), re-evaluation of global JE vaccine policies  might be warranted.The need for strong surveillance and diagnostics does not end when a vaccine is introduced in the country. Many JE-endemic countries will still need support in the areas of JE surveillance, diagnostics, and vaccination program monitoring after JE vaccine has been introduced. JE surveillance support will help decision-making for countries planning to introduce JE vaccine to understand disease burden and target vaccination geographically and by age. In countries with JE vaccination, JE surveillance and immunization registries are important to help identify program deficiencies, assess long-term vaccine protection, and monitor the geographic spread of JE. JE vaccination program monitoring should be improved through vaccine coverage surveys and better completion of the WHO/UNICEF Joint Reporting Form on Immunization.WHO Regional offices need to be supported as they directly assist countries with these issues—surveillance and vaccine introduction, quality assurance for laboratory testing, and troubleshooting vaccine supply issues. To maximize efficiencies across programs and ensure stable funding, efforts in surveillance, diagnostics, and vaccine coverage monitoring should be combined for JE and other vaccine-preventable diseases; this will also help integrate JE into routine immunization activities. Community advocacy and communication must be supported for maximal vaccine acceptance, especially during vaccination campaigns. To help address vaccine safety concerns and vaccine hesitancy, all reported serious AEFIs should be investigated promptly and properly, but with the care that the investigations do not create inappropriate concern that they are caused by vaccination if other etiologies are more likely. Readily available resources to guide AEFI investigations, particularly of encephalitis post-vaccination, would be helpful.The potential need for a JE vaccine booster dose to achieve life-long protection against JE. Longer-term vaccine effectiveness studies and documentation of vaccine failures through careful JE surveillance will be the most convincing data to indicate if booster doses are required for CD-JEV or JE-CV and the frequency of boosting for inactivated JE vaccines.Vaccine safety in special populations. To address research gaps on vaccine safety in immunocompromised persons and pregnant women who are knowingly or unknowingly vaccinated with CD-JEV and JE-CV, vaccine registries for these populations are needed.Laboratory and diagnostic issues. Additional diagnostic tests are urgently needed. Market shaping for JE ELISA kits will likely be needed through Gavi and other agencies, as other manufacturers have not shown interest in the development of similar kits due to the limited market. The use of new technologies and platforms for diagnostics could hold promise in the future but would require investments. Standardized and validated plaque reduction neutralization test reagents and procedures may allow comparison of immunogenicity results across laboratories.JE treatment. To help facilitate JE treatment research, expert consultation is needed with the aim of identifying the next drugs that should be tested in humans as well as to discuss the design of therapeutic trials, including types of studies, suitable locations, and outcomes to be measured.JE vaccine impact. To document the importance of JE vaccination and the need for sustained efforts, it will be important to conduct a retrospective impact study of the number of JE cases, disabilities, and deaths averted and to estimate the total cost savings from JE vaccination. Mathematical modeling of expected JE cases in the absence of vaccination could be a potential approach to overcome the deficiencies in JE surveillance. Such studies could consolidate country support for existing JE immunization programs and place JE in proper context alongside other vaccine-preventable diseases to support decision-making for JE vaccine introduction.Safety and immunogenicity of JE vaccine co-administration with new vaccines. Ongoing safety and immunogenicity studies of co-administration of JE vaccines with newly introduced vaccines  should be conducted when there is a programmatic desire to co-administer vaccines.JE control in Asia through immunization programs is a public health success. Together with affected country drive to develop new vaccines and control JE, international investments into an Asian domestic vaccine manufacturer enabled an affordable product and expanded access to this life-saving vaccine. However, more work is needed to secure the gains made and accelerate control of JE by expanding the use of JE vaccines in JE-endemic areas that do have immunization programs or do not have adequate programs.


Regulation of gene expression by chromatin structure has been under intensive investigation, establishing nuclear organization and genome architecture as a potent and effective means of regulating developmental processes. The substantial growth in our knowledge of the molecular mechanisms underlying retinogenesis has been powered by several genome-wide based tools that mapped chromatin organization at multiple cellular and biochemical levels. Studies profiling the retinal epigenome and transcriptome have allowed the systematic annotation of putative cis-regulatory elements associated with transcriptional programs that drive retinal neural differentiation, laying the groundwork to understand spatiotemporal retinal gene regulation at a mechanistic level. In this review, we outline recent advances in our understanding of the chromatin architecture in the mammalian retina during development and disease. We focus on the emerging roles of non-coding regulatory elements in controlling retinal cell-type specific transcriptional programs, and discuss potential implications in untangling the etiology of eye-related disorders. The retina has been an excellent system to study neurogenesis, due to its simplified anatomical structure, accessibility and well-defined cell types . The verRetinal differentiation initiates when multipotent retinal progenitor cells (RPCs) exit the cell cycle and differentiate into neurons or glia in a temporally conserved order under the control of gene regulatory networks and signaling pathways  Wetts a. Early rUnlike development in the mammalian cortex, retinal cell types are born in waves during which the periods of neuron generation overlap considerably  Marquar. Hence, The mechanisms that determine RPC competence are rooted in the ability of progenitor cells to integrate signaling pathways and the activities of complex networks of transcription factors (TFs) that drive cell fate decisions at the genomic level . ChromatChromatin regulators and retinal lineage-specific programs.Nuclear DNA is wrapped around a disc of highly conserved proteins (histones) to form the nucleosome, the basic unit of chromatin. Histones are classified into core histones , the principal components of the nucleosome, and linker histones (H1), which bind the nucleosome at the cross point of DNA entry/exit sites . AccessiGiven the association between chromatin pathways and regulation of gene expression, genetic studies have focused on investigating the roles of chromatin remodelers and histone modifying complexes during retinal development, a topic that has been reviewed recently . BrieflyThe function of chromatin remodelers that govern nucleosome mobilization has been investigated as well . For insChromatin-associated complexes can change subunit composition during development, indicative of cell-type-specific roles . Indeed,via activities of chromatin regulators, a function yet to be explored in the retina.More recently, chromosome confirmation capture (3C) techniques revealed that manipulation of chromatin regulators such as SWI/SNF and the polycomb repressive complexes can lead to changes in compartment-level chromatin organization . These iGenome-wide profiling of histone marks and chromatin associated proteins greatly facilitated the in depth probing of chromatin signature dynamics during developmental stages of mouse and human retina, revealing non-random genomic localization of histone marks and association with gene expression . In progThe retinal enhancer landscape exhibits exquisite reconfiguration concomitant with changes in gene expression during retinal developmental transitions: whereas cis-regulatory elements of progenitor genes lose their activities, enhancers targeting differentiation genes are gradually activated . Mechanivia recruitment of stage- and cell-type specific TFs and/or by the utilization of cell-type exclusive enhancers. Retina-specific enhancer elements with temporally restricted activities have been identified as the case with those nearby Otx2, a transcription factor expressed in a subset of progenitor cells and marks bipolar cells and photoreceptors  performed on rod nuclei indicates that while Sox2 coding region is located in euchromatin, its long-range putative enhancers reside in heterochromatin, thus likely inaccessible to the action of repressive complexes  was developed to distinguish groups (states) of co-occurring chromatin marks across the genome ; 2012. AMapping of the epigenomic marks and regions of chromatin accessibility has emerged as a powerful tool to annotate retinal putative regulatory elements, particularly enhancers . Given tEnhancers are stretches of non-coding DNA elements that spatially and temporally regulate transcription by acting as platforms to recruit transcription factors and transcriptional machinery, irrespective of sequence orientation . Enhancevia looping, to contact their cognate gene promoters in 3D space , Nrl, Otx2 and Prdm1 (photoreceptors), Atoh7 (ganglion cells), Onecut1 and Thrb , and Pax6 , among others  to profile the mouse developing retina, leading to the identification of developmentally regulated enhancer elements near the transcription factors Neurog2, Otx2 and Olig2, . By far,To date, most of the studies surveying retinal open chromatin regions used bulk tissues as input, which renders the determination of cell type specific deployment of regulatory elements in rare retinal cell types challenging. To overcome this limitation, ATAC-Seq, and sometimes ChIP-Seq, experiments have been performed on purified cells from transgenic mice carrying cell type-specific reporter genes, and as a result, epigenomic data from enriched rods, cones, bipolar cells and Müller glia are now available .The advent of single cell technologies, methods that circumvent heterogeneity and allow the investigation of rare cell populations at high resolution, has revolutionized the field, and a large cohort of studies focusing on surveying the adult and developing retinal single cell transcriptome has been performed . Still, in vivo experimental characterization of those elements remains necessary to validate their functions. In theory, a regulatory element should recapitulate its cognate gene’s spatial and temporal expression pattern, and when mutated should lead to alteration in gene expression. A large body of work has been directed toward investigating enhancer activities in the retina using reporter assays, which test the ability of a candidate enhancer sequence to activate a reporter gene . Electroporation of the mouse developing retina has been the main method for construct introduction into the retina, testing enhancer activity one element at a time , that are selectively enriched near genes important for stem cell identity . Super-eGiven the emerging interest in SEs roles in regulating tissue-specific gene expression, dynamically regulated SEs in mouse and human developing retina have been annotated . StudiesDefining cis-regulatory elements is crucial to understand disease mechanisms, as variations in DNA sequences linked to inherited human disorders often lie in non-coding regions . RetinalGlobal alterations in retinal enhancer landscape have been observed in patients with retinal degenerative diseases. A recent study profiled the genome-wide chromatin accessibility in patients with dry age-related macular degeneration (AMD), a disease characterized by a progressive loss of photoreceptors, and revealed a genome-wide quantitative reduction in chromatin accessibility associated with advanced stages of the disease, particularly in the macular region . Of notede novo regulatory landscape, causing gene deregulation. In one such instance, a cohort of patients with autosomal-dominant retinitis pigmentosa suffered a structural rearrangement that led to a repositioning of retina-specific regulatory landscape nearby GDPD1, a gene involved in lipid metabolism. The ectopic activation of GDPD1 driven by the newly created enhancer region likely leads to de-regulation of lipid metabolism, an essential process for phototransduction (Retinal diseases can be associated with genomic rearrangements that lead to the formation of a sduction . This wovia loop formation that involves homo-dimerization of CTCF at the loop anchors (Thanks to the rapid development of 3C techniques, the mammalian 3D genome conformation has been profiled at high resolution, illuminating that chromatin is organized into compartments in which multiple levels of DNA-DNA preferential interactions exist . At the  anchors  (Rao et  anchors . These i anchors . The ori anchors .Research investigating high order chromatin of the developing retina remains limited, and available data is primarily collected from bulk mouse retina and purified rod photoreceptors . OverallGenomic technologies have enabled the identification and cataloging of putative regulatory elements yet defining their cognate genes remains challenging . Promotevia CTCF-mediated looping, to regulate promoter activities, bypassing proximally located genes (It is now broadly accepted that enhancers can act over great genomic distances, ed genes . The speed genes . CTCF ised genes . The gened genes . Interesed genes . Still, ed genes . Thus thThe chromatin spatial architecture is commonly shared among animal nuclei, where inactive heterochromatin is preferentially sequestered to the nuclear periphery while active euchromatin occupies the nuclear interior . The strDespite the stark structural differences between inverted and conventional nuclei, Hi-C data indicate that the hierarchical chromatin compartmentalization is qualitatively similar . AdditioThe establishment of inverted nuclei occurs during rod photoreceptors terminal differentiation and is completed by postnatal day 28 in mice . During Genomic studies thus far have provided insights into modulation of retinal development by chromatin structure, yet the field is still in its infancy and a tremendous amount of work is needed to gain a comprehensive understanding on how epigenetics shape retinal development and are associated with retinal diseases. As sequencing technologies and computational analyses continue to rapidly evolve, it is likely that more high resolution data from retinal cell types will be available in the near future.What are the long-range interactions that occur among cis-regulatory elements during retinal development and how essential are they to retinal development and homeostasis? Are these interactions disrupted in ocular diseases? If so in what way? What are the factors that govern nuclear organization in retinal neurons? How does nuclear architecture influence gene expression during retinal cell type specification? Do liquid-phase separation properties of nuclear compartments influence retinal transcriptional programs? These are some of the outstanding questions that are likely to help elucidating how chromatin influence transcriptional regulation in the retina.Animal models have been immensely valuable in understanding molecular mechanisms underlying human biology and diseases but more studies investigating chromatin structure in human native and diseased retina are needed. This is particularly important to advance therapeutic strategies aiming at stimulating regeneration and/or preventing degeneration in the mammalian retina.


Whereas stem cell lineages are of enormous importance in animal development, their roles in plant development have only been appreciated in recent years. Several specialized lineages of stem cells have been identified in plants, such as meristemoid mother cells and vascular cambium, as well as those located in the apical meristems. The initiation of axillary meristems (AMs) has recently gained intensive attention. AMs derive from existing stem cell lineages that exit from SAMs and define new growth axes. AMs are in fact additional rounds of SAMs, and display the same expression patterns and functions as the embryonic SAM, creating a fractal branching pattern. Their formation takes place in leaf-meristem boundaries and mainly comprises two key stages. The first stage is the maintenance of the meristematic cell lineage in an undifferentiated state. The second stage is the activation, proliferation, and re-specification to form new stem cell niches in AMs, which become the new postembryonic “fountain of youth” for organogenesis. Both stages are tightly regulated by spatially and temporally interwound signaling networks. In this mini-review, I will summarize the most up-to-date understanding of AM establishment and mainly focus on how the leaf axil meristematic cell lineage is actively maintained and further activated to become CLV3-expressed stem cells, which involves phytohormonal cascades, transcriptional regulations, epigenetic modifications, as well as mechanical signals. Plants display incredible organogenetic ability and developmental plasticity throughout the whole life cycle, which comes from the sustained activity of stem cells in apical meristems. In addition, stem cells contained in apical meristems are protected from harsh conditions including drought, high salinity, high temperatures, and UV irradiation accompanying the movement from aquatic to terrestrial habitats. Therefore, these stem-cell harboring structures comprise the “fountain of youth” for indeterminate plant growth  and reprWUSCHEL (WUS) and CLVATA3 (CVL3), we consider that new stem cells have emerged.Stem cells are formed not only during embryogenesis, but also in postembryonic development when axillary meristems (AMs) initiate. Stem cells are specified in the center of newly established AMs. AM formation involves cell fate determination, meristematic cell maintenance, and meristem organization. AMs form in the leaf axil located at the leaf base, giving rise to lateral branches as new growth axes and conferring seed plants a ramifying and fractal architecture. Because of the activity of AMs, the shoot is self-replicating and scalable at different levels of branching, generating a fractal geometry pattern. The reiterative AM initiation from boundary regions suggests that it is a highly robust and precisely controlled developmental process. The establishment of AMs combined with the subsequent outgrowth of AMs determines the number and activity of lateral branches and therefore significantly influences crop yield. Each AM has the same developmental potential as the shoot apical meristem (SAM) and is required for new lateral organ formation on lateral branches. Once the AM begins to express marker genes such as de novo in AMs. Whether AMs originate as detached meristems or de novo induced ones had remained as a long-debating question in developmental biology (SHOOTMERISTEMLESS (STM) encodes an important KNOTTED-like transcription factor required for both embryonic shoot apical meristem (SAM) formation and AM formation in leaf axils, suggesting that SAM and AM share a common molecular regulatory mechanism. AM formation can be divided into two steps, meristematic cell lineage maintenance and stem cell activation , gibberellins (GA) and possibly brassinosteroids (BR).Arabidopsis found that AM initiation is severely compromised in a dominant gain-of-function allele of MONOPTEROS (MP), encoding a class A Auxin Response Factor  module . The croCUP-SHAPED COTYLEDON (CUC) and LATERAL ORGAN FUSION 1 (LOF1) genes  genes  that pro1) genes , which, Arabidopsis thaliana, tomato (Solanum lycopersicum), rice (Oryza sativa), and maize (Zea mays) revealed a handful of transcription factors involved in axillary meristem initiation. These transcription factors include CUC, LATERAL SUPPRESSOR (LAS), REGULATOR OF AXILLARY MERISTEMS (RAX), REVOLUTA (REV), and REGULATOR OF AILLARY MERISTEM FORMATION (ROX) in Arabidopsis and tomato, and their orthologs in monocots  and PRC2 are actively involved in STM locus modification in mature leaf cells and mediate transcriptional repression  and its closely related Jagged Lateral Organs (JLO)  are also actively modulated in the boundaries. Consistent with their consequential occurrence, in mutants defective in boundary formation, AM initiation may also be disrupted, suggesting that these two developmental processes are often regulated by shared factors. For instance, in af axils . Similar mutants . The fus mutants . Neverthns (JLO) . JLO conignaling .LAS or CUC promoters, boundaries are significantly widened and boundary cell morphology has greatly changed, connecting cell fate shift and boundary geometry reshaping, both resulting from auxin induction , Goblet (CUC ortholog), Blind/Potato Leaf (C) (RAX ortholog), auxin and cytokinin (STM expression and even ectopic meristem formation between leaflets.Boundary domain establishment not only affects AM formation, it is also recruited in compound leaf development in species such as tomato, another example of fractal pattern. In tomato compound leaves, boundaries between leaflets are characterized by suppressed cell division and reduced cell growth  and shapytokinin . Ectopicytokinin , suggestytokinin  and thatIn summary, recent breakthroughs in the field of AM initiation have highlighted the importance of meristematic cell lineage maintenance in branching, and establish AM initiation as an excellent system to study cell lineage, which has broad applications in stem cell biology. There are still many open questions to be addressed before the molecular and mechanical signals can be united to answer the question of indeterminacy maintenance and activation.It is obvious that many regulators in SAM development also regulate AM formation, with a few awaiting further verification and characterization. For example, the state of nutrients such as nitrate and phosphorus, patterns stem cell dynamics and SAM activity . As planArabidopsis.Furthermore, given the incompleteness of GRNs governing meristematic cell lineage maintenance and activation, more genetic components are likely to emerge with the aid of single-cell RNA-seq, which helps to construct GRNs at the cellular-resolution. Forward and reverse genetic screenings are also necessary and critical to identify more genetic components, which have long been missing in the model plant YW wrote and edited the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Biological functions arise from protein interactions, which are reflected in the natural variation of proteome configurations across individual cells. Emerging single-cell proteomics methods may decode this variation and empower inference of biological mechanisms with minimal assumptions. Biological functions arise from protein interactions, which are reflected in the natural variation of proteome configurations across individual cells. This Perspective article argues that emerging single-cell proteomics methods may decode this variation and empower inference of biological mechanisms with minimal assumptions. We normally think of experiments as procedures carefully designed by scientists and engineers. However, some of the best experiments arise from natural processes. For example, evolution and gene segregation during reproduction can be seen as natural experiments that have revealed much about protein functions and about genetic associations with diseases . A salieSingle-cell proteomics will open a window to a vast data trove of natural experiments: the proteome configurations that reflect molecular interactions within and between our cells. Indeed, signals received by cells in our bodies are transmitted via networks of proteins, and the relationships among these proteins reflect the underlying regulatory process. The transmission often involves protein cleavages, additions of chemical groups , and binding interactions among different protein partners. These molecular events shape the covariation among the involved proteins, and thus the protein configurations of single cells may report on biological functions and regulatory networks . TherefoOne such example is the possibility of inferring cell type–and cell state–specific protein complexes from covariation of protein levels across single cells. Covariation among the subunits of protein complexes is well established from analyzing bulk samples composed of mixed cellular states. Such covariation was among the first demonstrated results from Single-Cell ProtEomics by Mass Spectrometry (SCoPE-MS) . CovariaAnother example of learning from single-cell variation is the inference of transcriptional regulatory interactions. These are among the most studied regulatory interactions over the last 2 decades because of the wealth of transcriptomic measurements . NonetheAnalysis of single-cell protein variation may also enable a long elusive goal: the quantitative characterization of the direct protein interactions that weave the signal transduction networks in our cells. While genomics identifies many causal genetic associations, these associations are indirect . Such inBy contrast, single-cell measurements of proteins may enable inference of direct regulatory interactions with minimal assumptions, as shown in Learning from the patterns of single-cell protein variation requires accurate single-cell protein measurements. Indeed, ideas described here have previously surfaced in the literature , but theTherefore, realizing these possibilities requires that we advance the technology. First, we must make the state-of-the-art methods widely accessible via detailed protocols, computational resources, and community standards . Second,In addition to increasing the accessibility and accuracy, single-cell proteomic technologies will develop toward measuring protein dynamics, activities, and localization. For example, protein synthesis and degradation dynamics may be encoded into the proteomes of single cells by pulsing at different time points amino acids labeled with heavy isotopes. These encoded dynamics can be decoded at the end of the experiment by MS measurements of the abundances of proteins labeled with each isotopic composition. Protein localization may be measured by physically isolating cellular organelles, while posttranslational modifications (PTMs) may be analyzed by using PTM-enriched isobaric carriers ,9. Such


Advanced Science, tries to fill this gap. In their comprehensive, data-filled paper, the group of Michaela Kress  investigated iPSC-derived human nociceptor (iDN) development in the dish. Their study provides novel insights into and new data for (i) global gene expression during stem cell differentiation into sensory neurons, (ii) novel miRNA candidates regulating distinct stages of iDN differentiation, (iii) disease onset or susceptibility windows for disease-associated gene sets, and (iv) bundles all this information in the searchable online tool NOCICEPTRA.Induced pluripotent stem cells (iPSCs) are an established cellular system to study healthy and diseased conditions in a human context in vitro. Sensory neurons and signaling mechanisms mediating the perception of pain share many similarities but also substantial differences between mice and humans, and thus, translating the findings from rodents to humans is not always straightforward . AlthougUsing a small molecule–based differentiation method modified from Chambers et al. , they fiUsing trajectory analysis, they were able to identify five different iDN differentiation stages: A pluripotency stage which similarly to the early differentiation stage  continuously decreases during differentiation by measures of marker gene expression, while the nociceptor genes are upregulated during nociceptor and maturation stages. Interestingly, there are two complementary groups which seemingly inverse gene regulation: early/late maturation genes and those in the early neural progenitor group. Thus, the presented data convincingly recapitulate the stages of nociceptor differentiation as described for mice , 3, 6 suSeveral recent studies performed single cell transcriptomic analysis on DRGs of non-human primates (macaque)  and humaBeyond mRNA transcriptomic analysis, Zeidler et al. set out to also identify the expression trajectories of small non-coding microRNAs (miRNAs) whose many regulatory functions we only start to understand. Zeidler and colleagues establish a link between their findings from coding and non-coding RNA analysis: they identified hub genes that are most likely important (if not the most important) regulators of the modules, and the authors additionally performed a miRNA:mRNA target analysis for those hub genes identified. Thereby they identified miRNAs which e.g. regulate pre- and post-synapse development via hub genes, which can potentially also act as master switches, and miRNAs with impact on neurite outgrowth modulation. This interesting approach could in the future also be expanded to long non-coding RNAs (lncRNA), as they are of increasing importance for nociceptor differentiation. The author’s data set already covers lncRNAs and could easily be implemented within their online tool NOCICEPTRA as a searchable RNA.To make the complex data and their analysis pipelines accessible and manageable for other researchers, the authors developed the mentioned online tool NOCICEPTRA to analyze and visualize time trajectories of genes and miRNAs of interest. This valuable tool allows for general exploration of gene and miRNA expression, hub modules and pathways as well as disease susceptibility windows with the option to specifically zoom into genes of interest, their expression trajectory during nociceptor differentiation and potential regulation by miRNAs. With the increasing availability of big data, such tools become more and more important and will hopefully in the future also include state-of-the art single cell and single nuclear sequencing data and approaches.


Heterogeneity within a tumour increases its ability to adapt to constantly changing constraints, but adversely affects a patient’s prognosis, therapy response and clinical outcome. Intratumoural heterogeneity results from a combination of extrinsic factors from the tumour microenvironment and intrinsic parameters from the cancer cells themselves, including their genetic, epigenetic and transcriptomic traits, their ability to proliferate, migrate and invade, and their stemness and plasticity attributes. Cell plasticity constitutes the ability of cancer cells to rapidly reprogramme their gene expression repertoire, to change their behaviour and identities, and to adapt to microenvironmental cues. These features also directly contribute to tumour heterogeneity and are critical for malignant tumour progression. In this article, we use breast cancer as an example of the origins of tumour heterogeneity  and of tumour cell plasticity , as well as considering interclonal cooperativity and cell plasticity as sources of cancer cell heterogeneity. We review current knowledge on the functional contribution of cell plasticity and tumour heterogeneity to malignant tumour progression, metastasis formation and therapy resistance. A combinatorial evaluation of the histopathology of the primary tumour and of the expression pattern of hormone receptors  and epidermal growth factor receptor 2 (HER2/Neu), together with further genomic and transcriptomic profiling, classifies breast cancer as one of the following molecular subtypes: normal breast-like, luminal A (ER+/PR+ and Ki67-low), luminal B , HER2-enriched (HER2+), basal-like and claudin-low.10 These bidirectional interactions induce stochastic and dynamic changes during malignant cancer progression and, therefore, require cancer cells to constantly adapt to new environmental cues and challenges. The ability to rapidly react by genetic and epigenetic reprogramming appears to involve a cellular status of high plasticity, as will be described below.15Generally speaking, inter- and intra-tumoural heterogeneity are a consequence of differences in a number of cancer-cell-intrinsic parameters, such as genetic profile, interplay between the genome, epigenome/transcriptome and proteome, migration and invasion capabilities, proliferation, stemness and intrinsic cell plasticity Fig. . As well16 In 2000, DNA microarray profiling demonstrated for the first time distinctive gene expression patterns for normal breast, luminal-like, HER2+ and basal-like subtypes, thus already highlighting the presence of considerable intertumoural heterogeneity;6 a claudin-low subtype that specifically expresses markers of epithelial-to-mesenchymal transition (EMT) and stemness, as well as stromal and immune-related signatures, was subsequently added to this classification.8 In 2002, microarray gene expression analysis of 117 breast tumours identified three expression signatures that were predictive of therapy response: a signature indicative of poor prognosis consisting of genes important for cell-cycle progression, invasion, metastasis and angiogenesis; an ER signature; and a signature that identifies carriers of BRCA1 mutations.17In addition to opening up new avenues for the reconstruction of the temporal and spatial evolution of cancer, genome sequencing has also provided novel insights into heterogeneity between tumours, even of the same pathological subtype. Additional technological advances in novel whole omics profiling also offer the opportunity to better comprehend the enormous complexity and interplay of the genetic, epigenetic, transcriptomic and proteomic variations that ultimately cause phenotypic heterogeneity Fig. .13,16 In19 For example, Curtis and colleagues have described the genomic and transcriptomic landscape of 2000 human breast cancer samples by using an integrated analysis of copy number variations and gene expression patterns and they have identified additional subtypes of breast cancer.20 A similar analysis of triple-negative breast cancers (TNBC) have revealed a high heterogeneity within this one type of breast cancer, indicating that patients need to be analysed at an individual level.21 At around the same time, the Cancer Genome Atlas Network combined various methods to draw the molecular portraits of human breast cancers in a more holistic way, revealing a tremendous amount of variation even within individual breast cancer subtypes.22 In 2017, an analysis of somatic copy number alterations led to a newer classification of breast cancers into integrative clusters associated with prototypical patterns of single-nucleotide variants and distinct clinical outcome.23 Clinically relevant, these molecular classifications are associated with patient prognosis and possible treatment options. Hence, the genomic and transcriptomic analysis has to be refined for individual patients to match the observed high heterogeneity between tumours of individual patients even within one specific breast cancer subtype.Around 2010, a research goal has been to simultaneously sequence the genome and the transcriptome of human breast cancer samples, including single-cell sequencing.In this review, we summarise recent progress in the identification of the molecular and cellular determinants of tumour heterogeneity with a focus not only on genetic aberrations, but rather on epigenetic and transcriptomic differences defining specific tumour cell phenotypes and their functional contribution to malignant tumour progression. A major focus is given to the role of EMT and cell plasticity in generating intratumoural heterogeneity and the varying contributions to tumour cell invasion, metastasis formation and the development of therapy resistance.24 This somatic heterogeneity ultimately results in the formation of subclones with different biological capabilities, which will follow Darwin’s principle of ‘survival of the fittest’. As early as 1976, Peter Nowell described the concept of clonal evolution, whereby subclones of cells with a growth advantage in a certain ecosystem will persist, survive and ultimately expand, whereas less fit subclones are unable to compete against the others and become extinct.26Whole genome and exome sequencing of human tumour samples has unveiled the existence of extensive intratumoural genetic heterogeneity Fig. .9,21,22,28 Similarly, Nik-Zainal and co-workers reconstructed the evolutionary tracks of 21 breast cancer cases,30 demonstrating that, due to the early occurrence of driver mutations and alterations in cancer genes, genetic variation arises, selective pressures are shifted, and various subclones expand. Along these lines, DNA barcoding and massive parallel sequencing of samples from xenograft transplantation mouse models of human breast cancer confirmed the complex heterogeneity of clonal subpopulations: some subclones remain stable, some expand, and others vanish over time. As a result, dramatic changes in clone numbers and sizes and in the emergence of new subclones within and between primary and secondary tumours occur during breast cancer progression.33 Barcoding experiments have also revealed that the dominant clones of primary tumours do not necessarily contribute to tumour metastasis, distinguishing the cells that are shed from primary tumours but that do not yet form metastasis (shedders) from actual metastasis-seeding cells (seeders), indicating that therapeutic strategies need to target selected subpopulations.35Advances in DNA sequencing technologies eventually made it possible to infer the evolution of human tumours based on genomic alterations. For example, multi-region DNA sequencing of primary and metastatic pancreatic tumour samples allowed the reconstruction of distinct phylogenetic trees that describe clonal evolution in this type of cancer.37 In addition, a more recent study based on topographical genomic copy number profiling of 1293 single cells from ten breast cancer patients with ductal carcinoma in situ has led to the proposal of a fourth, so-called ‘multi-clonal invasion’, model.38 Here, genomic evolution within the mammary ducts gives rise to multiple tumour subpopulations, which subsequently co-migrate into the underlying tissue to form an invasive carcinoma. Regardless of the evolutionary path, a tumour might take, genetic heterogeneity and subclonal diversification enhance a primary tumour’s robustness and ultimately complicate patient prognosis, therapy response and clinical outcome.Data obtained from a variety of genomic profiling experiments of cancer cells from primary tumours and from distant sites subsequently allowed three distinct evolutionary patterns of primary tumour and metastasis to be inferred: a simple linear evolution model; a parallel evolution model with early metastatic spread; and a model assuming late seeding from either a single or multiple subclone(s) within the primary tumour.39 To investigate the topographical distribution of subclones, Yates and colleagues representatively sampled surgically excised invasive primary breast tumours in a multiple biopsy approach41. Most of the tumours exhibited at least one mutation that was confined to one to three neighbouring tissue regions, indicating a locally constrained expansion of subclones. Only a few tumours displayed clonal intermingling throughout the whole tumour tissue.40 In a similar study, when multifocal breast cancer lesions with comparable tumour grades and ER and HER2 status were subjected to whole genome and targeted sequencing,41 genetically similar lesions were topographically located closer to each other than genetically distinct lesions, indicating a process of clonal evolutionary outgrowth. Interestingly, despite histopathological homogeneity, considerable inter-lesion heterogeneity was found in one-third of the patients for oncogenic mutations in PIK3CA, TP53, GATA3 and PTEN, the most frequent driver mutations in breast cancer.41 These results already indicate that the simple genomic analysis of a sample taken from an undefined topographical position might not adequately molecularly characterise a tumour.Cell-intrinsic traits not only evolve temporally throughout tumour progression, but also spatially within a primary tumour and its secondary lesions Fig. .39 To in44The genetic, epigenetic, transcriptomic and proteomic traits of a cell are not fixed determinants. Triggered by stimuli from the microenvironment and by stochastic changes in gene expression, cancer cells can transition between phenotypic states through cell plasticity. This condition of cellular phenotypic uncertainty, yet high reactivity to external and internal cues, is often referred to as a state of high cell plasticity. It greatly contributes to tumour heterogeneity. The functional and molecular definition of cellular plasticity is context-dependent, yet cell plasticity is a broadly acceptable explanation of various biological and cancer-related processes.45 During EMT in cancer progression, epithelial cancer cells lose their cell–cell adhesions, dedifferentiate and acquire a migratory and invasive mesenchymal phenotype. This process has been associated with metastasis and drug resistance. Rather than being a binary switch, however, EMT/MET allows cells to transition between a spectrum of phenotypic hybrid states,47 although little is known about this heterogeneous spectrum, the stability of these cell states or their functional characteristics. Pastushenko and colleagues characterised six populations of distinct EMT states in transgenic mouse models of skin squamous cell carcinoma and of breast cancer.48 These EMT states exhibited different functional characteristics regarding migration, invasion, metastasis and stemness, although they all showed a similar tumour-propagating cell capacity. Interestingly, they also presented with different degrees of intrinsic cell plasticity, exemplified by their potential to spontaneously transition to another EMT state when subcutaneously transplanted or intravenously injected into mice. Notably, whereas only transitions between EMT hybrid states occurred in the subcutaneously transplanted primary tumours, cells in the lung could also revert back to an epithelial state,48 indicating that cancer cell plasticity is determined both by cell-intrinsic characteristics and the microenvironment.EMT and its reverse process, mesenchymal-to-epithelial transition (MET), constitute particularly well-studied programmes of high cell plasticity, occurring, for example, during fundamental processes such as embryonic development.49 A diverse selection of EMT markers is often used to assess EMT/MET in tumour progression and metastasis formation.51 However, although EMT markers and EMT transcription factors can be useful when adjusted to a specific cancer model, they have fallen short in capturing the dynamic nature of hybrid EMT states.52 Indeed, EMT is a process that is potentially inducible in different cell types and also differentiation stages.45 For instance, triple-negative breast cancer (TNBC) is considered to exemplify a highly de-differentiated, mesenchymal cellular phenotype, but TNBC cells can also undergo EMT-like changes and transiently enhance cellular plasticity, which, in turn, contributes to metastasis and drug resistance.54 Thus, a mesenchymal cancer cell can also be triggered to undergo further EMT-like changes, thereby displaying phenotypic plasticity. These observations raise the question of how hybrid, intermediate stages of EMT and a linear transition process of EMT can be reconciled. Can we identify a plasticity signature and capture a plasticity moment induced or manifested by reiterated processes of EMT and MET, and are these processes not linear at all?Lineage-tracing studies in vivo and 3D imaging have provided a first glimpse into EMT dynamics during early and late stages of malignant tumour progression.58 However, stemness is not a unique feature of the mesenchymal phenotype and plasticity between differentiated epithelial cancer cells and epithelial CSCs has also been observed.60 Similarly, breast cancer cells can transition between luminal, basal and progenitor-like cell states,63 further underscoring the potential of cell plasticity to affect cancer cell phenotype and malignancy and thus also increasing the complexity of clinical care.Another source of tumour heterogeneity lies in the various degrees of cell plasticity that might be driven by the extent of (de)differentiation of cancer cells. The cancer stem cell (CSC) model proposes that tumour growth and progression are driven by CSCs, which have the ability to self-renew and differentiate, thereby fostering the bulk of tumour cells. Mounting evidence supports the idea that CSCs might dynamically arise by the dedifferentiation of ‘regular’ cancer cells. In particular, EMT has been linked to the generation of breast CSCs.64 indeed, similar to c-Myc the introduction of an oncogenic PIK3CA mutation into mammary epithelial cells of basal or luminal lineage induced multipotency and led to the formation of heterogeneous multi-lineage tumours.63 However, the tumours were phenotypically different, depending on whether they originated from a basal or a luminal cell: transformed basal cells mainly formed benign differentiated tumours, whereas transformation of luminal cells induced aggressive tumours.63 These data indicate that the epigenetic landscape of the originating cancer cell might have a long-lasting effect on its (de)differentiation potential and plasticity.The roots of intrinsic cell plasticity are poorly understood. Studies of breast cancer indicate that the extent to which cancer cells undergo EMT might be dependent on the mutational landscape. For example, larger numbers of tumour cells that had undergone EMT were found in MMTV-Myc-driven mammary tumours as compared with MMTV-PyMT-driven or MMTV-Neu-driven mammary tumours. This increased number might be due to the pleiotropic oncogenic functions of c-Myc as compared with the single pathway activations by polyoma middle T (PyMT) or the Neu oncogene;65A heterogeneous population of cells facilitates the interaction of diverse subpopulations of cancer cells with each other—predominantly through interclonal cooperativity rather than competition—and with cells in the tumour microenvironment (TME) by physical interaction or via paracrine signalling networks to drive cancer progression and metastasis. The studies described below offer a glimpse into the interclonal cooperativity that occurs between breast cancer subclones as well as between breast cancer cells and stromal cells and the TME, and the impact of these interactions on malignant breast cancer progression. However, many more critical interactions of cancer cells exist—for instance, with other types of immune cell and cells of the vascular system that contribute to tumour progression; these interactions have been reviewed elsewhere Fig. .65HRas mutations. The cancer-driving HRas-mutant basal cells were deficient for Wnt signalling, but recruited the HRas-wildtype luminal cells to secrete Wnt1 to drive biclonal primary tumour growth.67 However, breast cancer subclones can cooperate not only with neighbouring clones in the same primary tumour, but also with cancer cells in metastatic lesions. Kim et al.68 identified a bidirectional spread of metastatic cancer cells from the primary tumour to the metastatic sites as well as from the metastatic lesions back to the primary tumour. This so-called ‘self-seeding’ does not require further adaptation by the tumour cells and appears to be directed by the interleukin (IL)-6/8-mediated attraction of circulating tumour cells (CTCs) from primary tumours and metastases back to primary tumours. It might promote tumour growth, angiogenesis and stromal recruitment.68 Similarly, whole genome sequencing of matched primary and metastatic prostate cancer samples has revealed interclonal cooperation between subpopulations of cells of primary tumours and metastatic lesions and the polyclonality of metastatic lesions at different metastatic sites,69 i.e. metastasis may also present high tumour heterogeneity and interclonal cooperativity within a metastatic lesion. As further described below, breast cancer cells with luminal and basal epithelial phenotypes, and possibly epithelial/mesenchymal features, might disseminate collectively, giving rise to polyclonal metastases.71Tumour heterogeneity is frequently manifested by interclonal cooperativity or dependency. For example, in a transgenic mouse model of Wnt1-driven mammary tumorigenesis (MMTV-Wnt1), a heterogenous mixture of basal and luminal cancer cells demonstrates interclonal cooperation to support their own tumour growth. Despite the initial expression of Wnt1 by bipotent progenitor cells, these produce basal and luminal cells with luminal tumour cells expressing Wnt1, while basal and luminal cells gained 76 Conversely, even a minor subpopulation of cancer cells might drive tumour growth by inducing microenvironmental changes77—for example, by activating angiogenesis or by recruiting fibroblasts or cells of the immune system to the primary TME or by changing the composition and/or the physicochemical consistency of the TME. These bidirectional heterotypic interactions between cancer cells and the cells of the TME are critical for tumour progression.73As well as homotypic interactions between cancer cells themselves, extensive heterotypic cooperation of cancer cells with various types of cell in the TME, including endothelial cells, fibroblasts and cells of the immune system, occurs. These interactions jointly dictate the pace of tumour progression, the occurrence of metastasis and a patient’s therapy response Fig. .72–76 Co78 As further discussed below, fibroblasts might also act as leaders in cancer cell migration and invasion.81 Finally, cooperation between fibroblasts and breast cancer cells might contribute to metastatic colonisation.82 Here, infiltrating CSCs induce fibroblasts to secrete periostin, an ECM protein, which promotes metastatic colonisation by enhancing Wnt signalling in the tumour cells.In particular, interactions between cancer cells and cancer-associated fibroblasts (CAFs) during breast cancer metastasis have been well studied. For example, fibroblast-derived caveolin-1 causes increased fibroblast contraction, matrix alignment and stiffening of the microenvironment, which, in turn, promotes breast cancer cell elongation, directional migration, and metastasis.85 TAMs can also induce early dissemination from the primary tumour by producing Wnt186 and play a critical role in guiding the intravasation of invasive cancer cells into the bloodstream87  have also been shown to contribute to malignant tumour progression at multiple levels. Macrophages are, for example, recruited by the chemokine ligand 2 (CCL2) secreted by cancer cells and, once at the tumour site, they are reprogrammed from an M1 anti-tumour phenotype to an M2 pro-tumorigenic phenotype, which promotes the secretion of pro-angiogenic, pro-invasive and immunosuppressive factors.89 Interestingly, the physical association of neutrophils and breast cancer cells in heterotypic CTC clusters drives cell-cycle progression in the circulation and enhances metastatic seeding.90 Thus, the interaction of breast cancer cells and neutrophils contributes to the metastatic process at multiple stages -D systemically prime pulmonary stromal cells to secrete neutrophil-stimulating factors. This leads to the attraction of pro-tumorigenic neutrophils, which enhance the ability of breast cancer cells to disseminate.ges Fig. .92 Notably, in some tumours there is an increase in immune cell infiltrations (‘hot tumours’), while in others immune cells are actively repressed from infiltrating the TME (‘cold tumours’) by the downregulation of immune cell-attracting cues and homing receptor systems. Most importantly, tumours might escape immunosurveillance by activating checkpoint blockades by the expression of PD1, PD-L1, CTLA4 and others by tumour cells and immune cells to prevent the activation of cytotoxic T cells for tumour cell killing. These molecules are the targets of current immunotherapies.93 Finally, non-cellular cues from the TME, including tumour hypoxia or changes in the stiffness of the ECM, critically affect cancer cell behaviour and phenotypes. These changes in the physicochemical parameters of the TME have been repeatedly shown to support malignant tumour progression and to play a critical role in increasing cell plasticity during malignancy and thus in tumour heterogeneity.94Major progress has been made in investigating how cancer cells escape immune surveillance during malignant progression. In fact, it appears that programmes underlying cancer cell malignancy, such as EMT, also include various molecular changes that support immune tolerance, an observation that is important for improving current immunotherapies96 Similarly, single-cell analysis of early and late metastatic breast cancer cells has revealed dramatic differences in gene expression: whereas early metastatic cells presented with a basal/stem cell-like signature and expressed genes associated with a de-differentiated, EMT-like phenotype, cells in established metastases showed a more differentiated, luminal-like and proliferative signature.53 Whole exome sequencing of matched primary tumour and metastasis from individual patients with a variety of cancer types, including of the breast, revealed that polyclonal seeding occurs frequently in untreated patients, while it is rare in therapy-treated patients. Hence, specific mutations that might already exist in early primary tumours appear to be selected for in metastasis by therapy, although they might not contribute to metastatic dissemination and outgrowth as such97 and might instead be associated with drug resistance (see below).Whole omics approaches have been increasingly employed to investigate the functional implications of tumour heterogeneity in malignant progression and metastasis. A comparison of gene expression profiles of primary tumours and secondary lesions from different metastatic sites revealed tremendous heterogeneity, which was linked to organotropism. In vivo selection and transcriptomic analysis of bone- and lung-homing breast cancer cell lines have identified numerous genes that are crucial for specific organotropism and site-specific outgrowth in bone, lung and other organs.98 undertook transcriptomic, proteomic and phospho-proteomic profiling of primary tumours and their corresponding metastases in transgenic mouse models of breast cancer. Their data revealed an increase in stress hormones during tumour progression, which resulted in the activation of the glucocorticoid receptor specifically at metastatic sites—this heterogenic expression of glucocorticoid receptor increased tumour heterogeneity and was critical for metastatic colonisation.98Obradovic and colleagues99 Whole genome bisulphite sequencing in combination with RNA sequencing has revealed differences in the epigenetic landscape of single CTCs versus CTC clusters. Of note, CTC clusters exhibited active (hypomethylated) transcriptional binding sites in embryonic stem cell genes, which probably contributes to their enhanced metastatic potential.100Considered to be the precursors of metastases, CTCs have become a major focus of cancer research for the study of tumour heterogeneity and plasticity. Notably, CTC clusters transpire to be more efficient in metastatic seeding and outgrowth than do single CTCs.Taken together, the emergence of whole omics techniques has led to an increasing understanding of tumour heterogeneity and its functional implications, especially in metastasis. However, other, non-genomic, factors are also likely to contribute to metastasis, and more sophisticated combinations of genomic, epigenomic, transcriptomic and proteomic analyses at the single-cell level will be required to further delineate the functional implications of tumour heterogeneity during the metastatic process.102 However, a more complex picture of cell plasticity and metastasis is emerging on the basis of experimental evidence, although the molecular details of how cell plasticity is defined and how specific pathways and factors contribute to EMT, cell plasticity and thus to metastasis remain to be functionally characterised  but none of the conventional mesenchymal cell markers. However, interconversion between K14– and K14+ epithelial cell states has been observed,70 and these leader cells have been shown to exhibit a hybrid epithelial/mesenchymal rather than a full mesenchymal phenotype.108 Together with insights into the hybrid states of EMT in cancer cells, it has been concluded that specific reversible stages of the EMT continuum might confer the cell plasticity required for tumour cell dissemination and thus for metastasis formation (discussed in ref. 47) However, direct evidence for reversible epigenetic changes during the metastatic process in patients remains to be shown.Depending on the microenvironment at the primary tumour site, cancer cells migrate and invade as single cells or as cell collectives.ion Fig. . On the 109 Once in the circulation, CTCs comprising cancer cells that have actively invaded as well as those that have been passively shed represent a heterogeneous population of cancer cells. Single CTCs expressing EMT markers have been widely observed in breast cancer patients and, in particular, the presence of CTCs with hybrid EMT phenotypes correlated with poor prognosis.111 Lineage labelling of cancer cells in breast and squamous cell carcinoma mouse models revealed a dramatic enrichment of cells that have undergone EMT in CTCs compared with primary tumours, indicating a strong contribution of EMT to metastatic dissemination.113 Intriguingly, CTC clusters also consisted of a diverse spectrum of hybrid EMT stage cancer cells as well as fully epithelial and mesenchymal cancer cells, thus supporting a role of the EMT continuum in collective cell invasion.111Migratory cancer cells can eventually actively invade the blood or lymphatic vasculature as individual cells or as multicellular clusters, although, as the neovasculature of the developing tumour is abnormally permeable, a considerable number of cancer cells might also be passively shed into the circulation Fig. .109 Once99 This increase might be due to the physical advantage conferred by a larger size in surviving shear stress and possibly immunosurveillance in the circulation. In addition, however, CTC clusters are also able to seed polyclonal metastases, and critical molecular interactions within the cluster can define distinct cell states that are associated with the dissemination of CTC clusters.114 For example, hypomethylation of the promoters of the stemness regulators OCT4, NANOG, SOX2 and SIN3A was observed in breast cancer CTC clusters, suggesting increased stemness of cancer cells that travel the circulation in clusters.100Compared with single CTCs, multicellular CTC clusters were reported to show an up to 50-fold increased metastatic potential.117 However, the plasticity and stemness associated with distinct EMT and MET states is poorly understood. To date, observations from breast, prostate and squamous cell carcinoma indicate a particularly high degree of plasticity in the intermediate EMT states, which is reflected by efficient metastatic dissemination and outgrowth.118 Notably, in a mouse model of colorectal cancer, the majority of disseminated cells in the circulation did not express Lgr5, a marker of cancer stem cells, but formed distant metastases in which Lgr5-positive cells were subsequently detected. Thus, although the cell plasticity required for metastatic dissemination does not appear to rely on cancer cell stemness, metastatic outgrowth does.119Metastatic colonisation Fig. , which i120 yet needs to be further investigated, since the actual phenotypic state of dormant tumour cells still remains enigmatic. Similarly, the association of distinct EMT hybrid states and metastatic organotropism should be further explored. For instance, Reichert et al. reported that mesenchymal pancreatic ductal adenocarcinoma cells might preferentially form lung metastases over liver metastases when prevented from undergoing MET.121In light of these findings, the spatial and temporal aspects of cell plasticity during colonisation warrant further investigation. In particular, the relationship between cancer cell plasticity and tumour dormancy is of great interest. Cells in less plastic EMT states can persist at the metastatic site for an extended period of time before eventually undergoing MET and growing out as a macrometastasis. Consistent with this notion, experimental evidence linking tumour dormancy and mesenchymal CSCs is increasing,Fsp), vimentin (Vim) or α-smooth muscle actin  in combination with a tdTomato-to-GFP colour-switching reporter.123 Such lineage-tracing systems, combined with a variety of conditional transgenic mouse models of cancer, have been used to study the contribution of EMT to metastasis in a whole organism context (Table 124) Although lineage-traced mesenchymal cancer cells have been reported to be highly enriched in CTCs compared with primary tumours,123 only a minority of metastatic lesions were observed to be derived from cells that had undergone EMT.123 Furthermore, although the experimental induction of reversible EMT has been clearly demonstrated to be sufficient for promoting metastasis,115 the inhibition of EMT by tumour-cell-specific knockout of the key EMT transcription factors Snail1, Zeb1 or Twist1 in various carcinoma models has led to conflicting results.126 These inconsistencies might be explained by tissue-specific functions and overlapping, yet distinct, roles for these EMT-inducing transcription factors, thus precluding the complete inhibition of the EMT programme by the elimination of only one factor.127Even though EMT/MET can contribute to various aspects of the invasion-metastasis cascade, the relevance of EMT in cancer metastasis is still debated. In order to track the fate of cancer cells that have undergone EMT, cell-specific lineage-tracing systems have been developed; these are based on the expression of Cre recombinase under the control of promoters of the mesenchymal marker genes fibroblast-specific protein  EMT contribute to the metastasis of epithelial cells, perhaps by leading collective invasion strands or by promoting ECM degradation, without themselves disseminating or surviving at the metastatic site.What is clear from these experiments is that cancer cells that have undergone EMT are more refractory to conventional chemotherapies and, thus, are enriched in treated primary tumours and metastasis—indeed, EMT-traced tumour cells were highly enriched in tumours of mice treated with conventional chemotherapies.129 This important result is consistent with the notion that EMT is required for local cancer cell invasion, but that MET is required for metastatic outgrowth at the distant site.102Based on current knowledge, the contribution of EMT to metastasis is likely to be context-dependent. Although EMT is sufficient to induce cancer cell dissemination, epithelial cells and cells undergoing partial EMT—especially when travelling as clusters—might establish metastases more efficiently than fully mesenchymal cancer cells. Consistent with this, it has been shown that the loss of E-cadherin—a hallmark of full EMT—promotes local dissemination but at the same time reduces proliferation, survival, metastatic colonisation and metastatic outgrowth.52) and is consistent with the observation that therapy-resistant tumours in patients frequently exhibit highly aggressive phenotypes. To dissect the molecular details that might distinguish the impact of EMT on metastasis from that on therapy resistance, we need to better understand the functional characteristics of distinct EMT states and dynamics of the transition between them. In other words, we need to define the type of cell plasticity that enables metastasis formation or therapy resistance at a molecular or omics level to identify novel therapeutic targets for preventing metastasis and/or therapy resistance.In summary, we need to consider that microenvironmental factors at distinct steps of the metastatic cascade could select for certain EMT states and modes of dissemination. At the same time, cell plasticity allows cancer cells to transition between EMT states. The contribution of a partial or full EMT programme to the metastatic burden might therefore critically depend on microenvironmental and cancer-cell-intrinsic factors and could dynamically change during tumour progression or during the course of therapy. Indeed, the contribution of EMT to therapy resistance has been amply demonstrated in various experimental systems , it is important to evaluate these opportunities and to assess whether an individually selected combination therapy will finally improve patient care.Despite advances in various sequencing techniques, we are still far away from translating our increasing functional and molecular understanding of tumour heterogeneity and cancer cell plasticity into clinical practice. In 2018, more than 620,000 women lost their lives to breast cancer, mostly due to the presence of tumour heterogeneity and its consequences emerging as metastasis formation and therapy resistance.